Environmental Mutagenesis 1: 115-196 (1979)

Abstracts of the Tenth Annual Meeting of the Environmental Mutagen Society

(Aa) MUTAGENS I N THE HUMAN ENVIRONMENT

Chaired by: M. L. Meltz SW Foundation for Research and Education and T. K. Rao Oak Ridge National Laboratory

(Aa-1)

OETECTION OF MUTAGENS PRODUCED BY FUNGI ISOLATED FROM TOMATOES. D.R. Stoltz, P.M. Scott", and J. Harwig*, Health Protection Branch, Health and Welfare Canada, Ottawa, Ontario, Canada, K1A OL2. Twenty-seven fungal isolates from local decaying tomatoes were classified as Flamulina (l), Clados orium (l), Mucof (1), C o l l e t o t r i c h u m , B o t r e - o t r i c h u m (3), Pencillium ( 3 ) , Alternaria (5), Fusarium (8) and Epicoccum (1). Pure cultures of fungi were grown on YES medium for 8-13 days. Ethyl acetate extracts of the culture medium filtrates and mycelial mats were screened for mutagenicity with Salmonella typhimurium TA98 and TA100. One Fusarium and all five Alternaria cultures contained mutagenic activity. The Alternaria isolates were investigated further since this genus, although a common plant pathogen and ubiquitous storage mold, is not appreciated as a source of mutagenic mycotoxins. Mutagenicity of extracts of Alternaria culture filtrates and mycelial mats may be attributed, at least partially, to the presence of alternariol and alternariol monomethyl ether. 0192-2521/79/0102-0115512.80 @ 1979 Alan R. Liss, Inc.

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(Aa-2) FAILURE OF IRRADIATED BEEF AND HAM TO MUTATE DROSOPHILA. Sidney M i t t l e r , Northern I l l i n o i s U n i v e r s i t y , DeKalb, I L 60115. Ham t h a t had been i r r a d i a t e d w i t h a dose o f 3.7-5.2 Megarads w i t h e l e c t r o n s produced by t h e LINAC lOMeV e l e c t r o n and beef t h a t had been exposed t o 3 m i l l i o n c u r i e C060 source f o r a dose o f 4.7-7.1 Megrads were t e s t e d f o r m u t a g e n i c i t y u t i l i z i n g Drosophila. Did the r a d a p p o r t i z a t i o n ( r a d i a t i o n process t h a t s t e r i l i z e s food) induce mutagens? The i r r a d i a t e d meat was f i n e l y ground and mixed a t 1 t o 1 r a t i o w i t h Carolina 4-24 Drosophila media. Oregon R and yB/C(l)DX, yf/sc8yt Y stocks were reared on t h i s mixture. The a d u l t males which emerged, having spent t h e i r e n t i r e l i f e on t h e t e s t media were examined f o r t h e l o s s o f t h e X and Y chromosomes, n o n d i s j u n c t i o n and induced sex-linked recessive l e t h a l s . None o f these g e n e t i c aberrations were s i g n i f i c a n t l y increased by feeding on these i r r a d i a t e d meats. (Supported by t h e U.S. Army Medical Research and Development Command Contract DADA 17-71-C-1030.) (Aa-3)

INDUCTION OF MUTAGENICITY AND DNA SINGLE-STRAND BREAKAGE IN MAMMALIAN CELLS BY DIETHYLSTILBESTROL. M.L. Meltz, N. J. Whittam*, and R. L. Purdy*, Southwest Foundation for Research and Education, San Antonio, TX 78284 The genetic effects ef diethylstilbestrol (DES) are being examined in mammalian cells in culture. Using the L5178Y mouse lpphoma cell line heterozygous at the thymidine kinase locus, experiments involving treatment in the presence of 3% horse serum and an Aroclor 1254 induced rat liver S-9 preparation have confirmed the previously reported weak mutagenicity of DES in these cells. The mutagenicity was not evident if 0.5% albumin was substituted for 3% horse serum. Using the alkaline elution technique, we have detected a small amount of DNA single-strand breakage when 0.5% albumin was present during treatment. The data suggest possible cross-linking when an S-9 system is present. In an effort to improve the in vitro metabolic activation system for use in studies of the genetic toxicology of estrogenic hormones, microsomes have been isolated from the liver of a baboon treated with rifampicin and are being substituted for the rat liver S-9 preparation in mutagenesis and DNA single-strand break studies. These microsomes are very active in hydroxylating DES in vitro. This research is being supported by the American Cancer Society and the National Cancer Institute. k ? & 4 I C I T Y OF SUME LIPSTICKS AND THEIR DYES kOK SALMONELLA TYPHIMURIUM TA98. M. R. Green and J . V. Pastewka, i n t r . by M. C. P o i r i e r , National Cancer I n s t i t u t e , NIH, Bethesda, MD 20014. L i p s t i c k s o f various shades and c o l o r s from e i g h t cosmetic f i r m s were t e s t e d f o r mutagenicity. Twenty-four 1 i p s t i c k s were t e s t e d a t concentrat i o n s o f 1-500 pg per p l a t e w i t h t e s t e r s t r a i n Salmonella typhimurium TA98 developed by Ames. E i g h t were mutagenic w i t h o u t microsomal (S-9) a c t i v a t i o n . Dose response e f f e c t s were observed. Eight dyes, 1 i s t e d as 1 i p s t i c k i n g r e d i e n t s by t h e cosmetic f i r m s , were t e s t e d a t concentrations o f 0.1 t o 500 vg per p l a t e w i t h and w i t h o u t S-9. D&C Orange No. 17, a monoazo dye w i t h two n i t r o groups was h i g h l y mutagenic i n t h e absence o f s-9. A much weaker d i r e c t mutagenic e f f e c t was observed f o r D&C Orange No. 5. The mutagenic e f f e c t o f b o t h dyes was decreased o r l o s t i n the presence o f S-9 prepared from l i v e r s o f male Sprague-Dawley r a t s i n j e c t e d w i t h Arochlor 1254. The o t h e r dyes tested, DlCC Red Nos. 3 , 6, 8, 19, 2 2 and 36, were not mutagenic. S i x l i p s t i c k s very s i m i l a r w i t h respect t o ingre-

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117

d i e n t s except f o r t h e presence o r absence o f O&C Orange No. 17 o r No. 5 were tested. Two c o n t a i n i n g D&C Orange No. 1 7 were d i r e c t l y mutagenic. The mutagenic e f f e c t was decreased by t h e presence o f S-9. D&C Orange No. 17 probably accounts f o r t h e mutagenicity o f t h e l i p s t i c k s t e s t e d since t h e number o f r e v e r t a n t s obtained could r e s u l t from a dye content o f l e s s than 1%. The observed weak mutagenic e f f e c t o f D&C Orange No. 5 may be due t o a contaminant since D&C Orange No. 5 i s very s i m i l a r i n s t r u c t u r e t o other dyes which were not mutagenic.

(Aa-5) EFFECT OF ACETAMINOPHEN I N MICE. Raymond A. Popp. Biology D i v i s i o n , Oak Ridge N a t i o n a l L a b o r a t o r y , * Oak Ridge, TN; Cynthia S e s s i o n s , Bioc h e m i s t r y Department, Clemson U n i v e r s i t y , Clemson, SC; and S a l l y Owens, i n t r . by Biology Department, Colorado C o l l e g e , Colorado S p r i n g s , CO. P. Carolyn Gooch. Acetaminophen i s a n o n p r e s c r i p t i o n a n a l g e s i c used f r e q u e n t l y a s a subs t i t u t e f o r a s p i r i n . It i s m e t a b o l i z e d by monooxygenases t o form r e a c t i v e i n t e r m e d i a t e s t h a t d e p l e t e g l u t a t h i o n e i n t h e mouse l i v e r r e s u l t i n g i n s e v e r e l i v e r n e c r o s i s . I n h i g h d o s e s , acetaminophen also produces cataracts i n a d u l t mice and a f f e c t s t h e growth of t h e l e g bones i n r a t s . The purpose o f t h i s s t u d y was t o d e t e r m i n e t h e e f f e c t s o f acetaminophen on t h e d e v e l o p i n g mouse embryo. The LD50 f o r C57BL/lO and (SEC X CBA)F1 mice i s n e a r 500 mg/kg o f acetaminophen whether i t is a d m i n i s t e r e d i n t r a A s i n g l e dose of 250 mg/kg i . p . i n peritoneally or intragastrically. mice a t 1 0 t o 1 4 days of g e s t a t i o n caused n e a r l y 50% e m b r y o t o x i c i t y . A d o s e o f 375 mg/kg a d m i n i s t e r e d i . g . i h mice a t 1 3 and 14 days of g e s t a t i o n caused approximately 40% e m b r y o t o x i c i t y . Among o f f s p r i n g of t r e a t e d mothers, e y e d e f e c t s were o b s e r v e d i n 5% o f t h e s u r v i v o r s ; one o f t h e s e w a s born of a mother g i v e n 50 mg/kg/day a t days 10 through 1 4 of g e s t a t i o n . Thus, acetaminophen h a s been shown t o c a u s e e m b r y o t o x i c i t y and t e r a t o g e n i c e f f e c t s i n mice. (Research sponsored by t h e D i v i s i o n of Biomedical and Environmental Research, U.S. Department o f Energy under c o n t r a c t W-7405-eng-26 w i t h t h e Union Carbide C o r p o r a t i o n . )

(Aa-6) THE MUTAGENICITY OF CIGARETTE SMOKE CONDENSATE I N NEUROSPORA CRASSA. D. M . DeMarini, I l l i n o i s S t a t e U n i v e r s i t y , Normal, I L 61761. I n g e n e r a l , c i g a r e t t e smoke condensate (CSC) is n o t mutagenic i n S a l m o n e l l a i n t h e absence of S-9 mix. However, one r e p o r t i n d i c a t e d t h a t s t a n d a r d CSC i n t h e a b s e n c e of S-9 mix i s weakly mutagenic a t low d o s e s i n TAlOO (Sugimura e t a l . , O r i g i n s of Human Cancer, H i a t t e t a l . , e d . , pg 1561, 1977), and a n o t h e r r e p o r t showed t h a t CSC p r e p a r e d from n i t r a t e t r e a t e d c i g a r e t t e s is mutagenic i n TA1538 i n t h e a b s e n c e of S-9 mix ( K i e r e t a l . , Proc. N a t l . Acad. S c i . U.S.A. 71:4159, 1974). I n t h e p r e s e n t s t u d y , t h e m u t a g e n i c i t y of CSC and s e v e r a l f r a c t i o n s of CSC was i n v e s t i g a t e d i n Neurospora c r a s s a , b o t h i n t h e p r e s e n c e and i n t h e a b s e n c e of S-9 mix. The c o n d e n s a t e s were from t h e U n i v e r s i t y of Forward m u t a t i o n was a s s a y e d a t t h e Kentucky R e f e r e n c e C i g a r e t t e I R I . ad-3 r e g i o n of two-component h e t e r o k a r y o n s . The m u t a g e n i c i t y of CSC w a s i n v e s t i g a t e d by a l i q u i d s u s p e n s i o n test c o n s i s t i n g of 2.5 X 107 c o n i d i a , 0.6 m l b u f f e r o r S-9 mix c o n t a i n i n g Aroclor-induced r a t h e p a t i c microsomes, and 0.15 m l CSC a t v a r i o u s c o n c e n t r a t i o n s . These c o n i d i a w e r e i n c u b a t e d a t 37'C f o r 1 h , washed t w i c e , and a s s a y e d by t h e d i r e c t method developed by d e S e r r e s . I n t h e absence of S-9 mix, d i r e c t a c t i n g N. mutagens of CSC were d e t e c t e d a t c o n c e n t r a t i o n s of a b o u t 1 mg/ml i n c r a s s a . I n t h e p r e s e n c e of S-9 mix, CSC w a s a l s o mutagenic, b u t a t s l i g h t l y higher doses (2.5 8 . 0 mg/ml). [Research s u p p o r t e d by N I H . ]

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118

(Aa-7) MUTAGENICITY OF CIGARETTE SMOKE CONDENSATE AND CIGARETTE SMOKE I N DROSOPHILA MELANOGASTER. A. R . P e s c i t e l l i , I l l i n o i s S t a t e U n i v e r s i t y , Normal. I L 61761. The m u t a g e n i c i t y of c i g a r e t t e smoke c o n d e n s a t e (CSC) h a s been r e p o r t e d i n S a l m o n e l l a typhimurium ( K i e r e t a l . , 1974, P r o c . Nat. Acad. S c i . USA 71:4159; Hutton and Hackney, 1975, Cancer Res. 35:2461) and Saccharomyces I n t h e p r e s e n t work, c e r e v i s i a e (DeMarini, 1978, Mutation R e s . 53:84). t h e m u t a g e n i c i t y of CSC and c i g a r e t t e smoke w a s s t u d i e d i n D r o s o p h i l a m e l a n o g a s t e r . Wild-type (Oregon-R) males were exposed t o t h e a g e n t and mated t o Bast f e m a l e s , and t h e f r e q u e n c i e s of s e x - l i n k e d r e c e s s i v e l e t h a l s (SLRL) were d e t e r m i n e d i n t h e F 2 . The c o n d e n s a t e from a p o p u l a r brand of c i g a r e t t e s w a s c o l l e c t e d on f i l t e r p a p e r w i t h a c i g a r e t t e smoki n g machine. The c o n d e n s a t e w a s d i s s o l v e d i n d i m e t h y l s u l f o x i d e ; t h i s s o l u t i o n i s r e f e r r e d t o a s CSC. Males t h a t w e r e f e d a CSC-sucrose s o l u t i o n as a d u l t s had a small i n c r e a s e i n t h e f r e q u e n c y of SLRL o v e r t h e c o n t r o l f r e q u e n c y , b u t t h i s i n c r e a s e was n o t s i g n i f i c a n t . I n two e x p e r i ments, males t h a t were f e d CSC e q u i v a l e n t t o s i x - h u n d r e d t h s of one c i g a r e t t e p e r 1 0 m l of food a s l a r v a e had a s i g n i f i c a n t l y h i g h e r f r e q u e n A d u l t s t h a t were excy of SLRL t h a n t h e c o n t r o l f r e q u e n c y ( p < 0.05). posed t o two t o t h r e e p u f f s of c i g a r e t t e smoke a day f o r s e v e n d a y s w i t h a smoking machine had a small i n c r e a s e i n t h e f r e q u e n c y of SLRL o v e r t h e c o n t r o l f r e q u e n c y , b u t t h e i n c r e a s e w a s n o t s i g n i f i c a n t . I n two e x p e r i m e n t s , t h e r e was a s i g n i f i c a n t i n c r e a s e i n t h e f r e q u e n c y of SLRL o v e r t h e c o n t r o l f r e q u e n c y ( p < 0.05) when l a r v a e were exposed t o two t o t h r e e p u f f s of c i g a r e t t e smoke p e r d a y . To my knowledge, t h i s is t h e f i r s t r e p o r t of t h e m u t a g e n i c i t y of CSC i n D r o s o p h i l a acd of t h e i n d u c t i o n of m u t a t i o n s by c i g a r e t t e smoke.

(Aa-8) APPLICATION OF MUTAGENICITY TESTING TO URINE SAMPLES OBTAINED FROM ANIMALS Della W . Ramey, J . L. E p l e r , T . K . Rao, EXPOSED TO CIGARETTE SMOKING1. Biology D i v i s i o n w i t h M . P. Maskarinec" A n a l y t i c a l Chemistry D i v i s i o n , Oak Ridge N a t i o n a l L a b o r a t o r y , Oak Ridge, TN 37830. P h y s i o l o g i c a l body f l u i d s such as u r i n e samples o b t a i n e d from dogs exposed t o c i g a r e t t e smoking were a n a l y z e d f o r m u t a g e n i c i t y i n t h e Ames Salmonella/microsomal a c t i v a t i o n aaPay. The samples were p r e - c o n c e n t r a t e d 300-fold u s i n g XAD-2 column f o r t h e i s o l a t i o n and c o n c e n t r a t i o n o f potent i a l c a r c i n o g e n s . Mutagenic r e s p o n s e w a s n o t e d w i t h t h e h i s t i d i n e auxot r o p h i c s t r a i n ~ ~ and 9 m 8 e t a b o l i c a c t i v a t i o n w i t h A r o c l o r i n d u c e d S-9 mix w a s o b l i g a t o r y . Mutagenic a c t i v i t y w a s n o t e d w i t h samples (20-100 u l / p l a t e ) o b t a i n e d from dogs exposed t o h i g h t a r medium n i c o t i n e c i g a r e t t e s , and h i g h t a r low n i c o t i n e c i g a r e t t e s . S i g n i f i c a n t mutagenic a c t i v i t y was a l s o n o t e d w i t h t h e samples o b t a i n e d from dogs exposed t o h i g h t a r , h i g h n i c o t i n e c i g a r e t t e s . Enormous i n c r e a s e i n i n d u s t r i a l a c t i v i t y makes it necessary f o r monitoring i n d i v i d u a l s f o r occupational exposure ( v i a i n h a l a t i o n o r s k i n ) t o v a r i o u s c a r c i n o g e n i c a g e n t s i n t h e working environment. A b i l i t y t o d e t e c t chemical mutagens/carcinogens i n t h e p h y s i o l o g i c a l f l u i d s i n c r e a s e s t h e scope o f b a c t e r i a l m u t a g e n i c i t y a s s a y s f o r i t s a p p l i c a t i o n as a i n d u s t r i a l m o n i t o r i n d e t e c t i n a e a r l y e x p o s u r e s t o m u t a g e n i c / c a r c i n o g e n i c chemicals. 'Research

j o i n t l y sponsored by t h e Environmental P r o t e c t i o n Agency I n t e r a g e n c y Agreement 40-516-75) and t h e D i v i s o n of B i o l o g i c a l and Environmental R e s e a r c h , U.S. Department of Energy, under c o n t r a c t W-74 05-eng-26 w i t h t h e Union C a r b i d e C o r p o r a t i o n .

(IAG-D5-E681;

(Aa-9) MUTAGENICITY STUDIES OF SACCHARIN AND SEVERAL OF ITS IMPURITIES. E . Gocke, K . E c k h a r d t , M.-T. King, D . Wild. Z e n t r a l l a b o r a t o r i u m fur M u t a g e n i t a t s p r u fung d e r Deutschen F o r s c h u n g s g e m e i n s c h a f t , F r e i b u r g , Germany.

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119

Remsen Fahlberg s a c c h a r i n and f o u r of t h e contaminants o f Remsen Fahlberg s a c c h a r i n ( 0 - t o l u e n e s u l f o n a m i d e , p - t o l u e n e s u l f o n a m i d e , o - s u l f amoyl b e n z o i c a c i d , p-sulfamoyl b e n z o i c a c i d ) were t e s t e d f o r m u t a g e n i c i t y i n t h r e e d i f f e r e n t t e s t s y s t e m s : t h e hnes t e s t , t h e Basc t e s t i n Drosophi l a , and t h e m i c r o n u c l e u s t e s t i n mice. S a c c h a r i n and t h e sulfamoyl b e n z o i c a c i d s were i n a c t i v e i n a l l t e s t s , b u t t h e most i m p o r t a n t contami n a n t i n q u a n t i t a t i v e terms, OTS, and i t s isomer PTS were mutagenic i n t h e Ames t e s t and i n t h e Basc t e s t . The r e s u l t s w i l l be d i s c u s s e d w i t h r e g a r d t o t h e r e p o r t e d p o t e n t i a l c a r c i n o g e n i c i t y o f commercial s a c c h a r i n .

(Aa-10) VITAMIN C I S POSITIVE I N THE DNA SYNTHESIS INHIBITION AND SISTER CHROMATID EXCHANGE TESTS. S.M. Galloway* and R.B. P a i n t e r , L a b o r a t o r y o f R a d i o b i o l ogy, U n i v e r s i t y o f C a l i f o r n i a , San Francisco, C a l i f o r n i a 94143. Work supported by USDOE. Ascorbate causes a dose-dependent i n c r e a s e i n s i s t e r chromatid exchanges (SCEs) i n Chinese hamster ovary (CHO) c e l l s and i n human lymphoc y t e s . Moreover, i n t h e DNA s y n t h e s i s i n h i b i t i o n t e s t w i t h HeLa c e l l s , ascorbate g i v e s r e s u l t s t y p i c a l o f DNA-damaging chemicals. Catalase reduces SCE i n d u c t i o n by ascorbate, prevents i t s c y t o t o x i c i t y i n CHO c e l l s , and prevents i t s e f f e c t on HeLa DNA s y n t h e s i s . Ascorbate reduces induct i o n o f SCE i n CHO c e l l s by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by d i r e c t i n a c t i v a t i o n o f MNNG. When ascorbate and MNNG a r e incubated t o g e t h e r w i t h HeLa c e l l s , t h e e f f e c t on DNA s y n t h e s i s i s l e s s t h a n w i t h ascorbate alone, suggesting a d i r e c t chemical r e a c t i o n between t h e two chemicals. (Aa- 11 ) THE MUTAGENIC AND ANTI-MUTAGENIC EFFECTS OF ASCORBATE AND ASCORBATEMETAL MIXTURES. H. F. S t i c h , L. Wei* and R. F. Whiting*, B. C. Cancer Research C e n t r e , 601 West 1 0 t h Avenue, Vancouver, B . C . , Canada.

Many o r g a n i c and i n o r g a n i c compounds w i t h i n c e l l s c a n i n t e r a c t w i t h a s c o r b a t e (Asc) a f f e c t i n g i t s chromosome-damaging and mutagenic capaci t i e s . To g a i n i n s i g h t i n t o t h e p o t e n t i a t i n g o r i n h i b i t i n g e f f e c t s of v a r i o u s compounds on t h e a c t i v i t y of Asc w e examined t h e a c t i o n o f m e t a l s , c a t a l a s e and v a r i o u s t r a p p i n g a g e n t s . Cuft and Mn* enhance t h e c y t o t o x i c as w e l l as chromosome damaging e f f e c t o f A s c f o r c u l t u r e d human and r o d e n t i n h i b i t t h e cytotoxic a c t i o n but p o t e n t i a t e t h e c e l l s . Fe* and Fechromosome damaging e f f e c t of Asc. A t p a r t i c u l a r r a t i o s between a s c o r b a t e 80% t o 100% of a l l examined metaphase p l a t e s showed and F e u o r Fe* m u l t i p l e chromosome exchanges and b r e a k s . The c y t o t o x i c and chromosome damaging a c t i o n s of a s c o r b a t e a p p e a r t o be due t o t h e f o r m a t i o n of H202 and t h e s u b s e q u e n t a p p e a r a n c e of h i g h l y r e a c t i v e r a d i c a l s . The a d d i t i o n of catalase t o . A s c o r Asc/metal m i x t u r e s reduced o r a b o l i s h e d t h e s e effects. On t h e o t h e r hand Asc i n h i b i t e d t h e mutagenic (Salmonella t e s t ) and chromosome damaging ( c u l t u r e d mammalian c e l l s ) a c t i o n of 8 p r o x i m a t e o r u l t i m a t e c a r c i n o g e n s . Whether Asc and A s c p l u s t r a n s i t i o n metals e x e r t i n v i v o a mutagenic o r anti-mutagenic e f f e c t ( o r b o t h ) remains t o be answered.

(Aa-12)

USE OF THE MOUSE IN VIVO SOMATIC MUTATION METHOD TO TEST FOR CAFFEINE MTAGENESIS OR POTENTIATION. Liane B. R u s s e l l , C.S.Montgomery*, and M. Hayes*, Biology D i v i s i o n , Oak Ridge N a t i o n a l Lab., Oak Ridge, TN 37030. E a r l i e r tests f o r mutagenic action of c a f f e i n e i n mouse g e r m c e l l s have been n e g a t i v e f o r e a c h of several e n d p o i n t s , b u t weak e f f e c t s have been rep o r t e d i n a few o t h e r systems. There are a l s o r e p o r t s t h a t c a f f e i n e can, i n some s i t u a t i o n s , p o t e n t i a t e t h e e f f e c t s of o t h e r mutagens by i n h i b i t i n g c e r t a i n t y p e s of r e p a i r . W e have employed t h e i n v i v o s o m a t i c m u t a t i o n

120

Abstracts

method ("spot test") t o test f o r a n e f f e c t of c a f f e i n e , a l o n e o r i n conj u n c t i o n w i t h X r a y s . The r e c e s s i v e s p o t s (RS) s c o r e d by t h i s method detect p o i n t m u t a t i o n s as w e l l as chromosomal l o s s e s i n v o l v i n g s p e c i f i c l o c i . C a f f e i n e w a s a d m i n i s t e r e d , i n one of t h r e e ways, on day 1071, postconcept i o n t o C57BL females pregnant by T males: ( a ) one i . p . i n j e c t i o n , 35mgIkg; (b) two 1.p. i n j e c t i o n s , 35 mg/kg each, s e p a r a t e d by 1 hour; (c) i n the d r i n k i n g w a t e r a t l g / ; , s t a r t i n g a t least 1 day p r i o r t o day 10% p.c. and c o n t i n u i n g through day 11%p.c. Another group (d) was i n j e c t e d w i t h s a l t s o l u t i o n . Each of t h e s e f o u r groups w a s matched w i t h a n o t h e r which, i n a d d i t i o n , r e c e i v e d 100 R a c u t e X r a y s on day 1 0 k i n t h e c a s e of i n j e c t e d groups, one hour a f t e r t h e ( l a s t ) i n j e c t i o n . A l t o g e t h e r 1896 mice, t r e a t e d as embryos, were observed f o r s p o t s . The i n c i d e n c e of RS was s i g n i f i c a n t l y g r e a t e r (PqO.01) i n a l l i r r a d i a t e d t h a n i n a l l n o n i r r a d i a t e d groups. I n t h e absence of i r r a d i a t i o n , RS frequency w a s h i g h e r i n c a f f e i n e than non-caffeine groups, the d i f f e r e n c e , however, b e i n g s i g n i f i c a n t (P 0.02) o n l y when h i s t o r i c a l c o n t r o l s were i n c l u d e d w i t h t h e contempor a r y ones. There was no e v i d e n c e f o r c a f f e i n e p o t e n t i a t i o n of t h e X-ray e f f e c t ; a t f a c e v a l u e , t h e e f f e c t s appear a d d i t i v e . [Research sponsored i n p a r t by EPA (IAGD5-E681) and i n p a r t by t h e Div. o f Biomed. h E n v t l . R e s . , USDOE, under c o n t r a c t W-7405-eng-26 w i t h t h e Union Carbide Corp.]

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(Aa-13) CONTINUED STUDIES OF IN SITU MUTAGENIC AND BIOCHEMICAL EFFECTS OF ENVIRONMENTAL POLLUTION. Wm. R. Lower and V. K. Drobney", Environmental Trace Substances Research Center, University of Missouri, Columbia, MO 65201. Continued in situ studies for three years with Tradescantia strain4430 and corn strain W22 of an isolated lead smelter show differences in patterns of mutation frequency with distance of from 0 . 3 to 11.4 km from the smelter. These patterns, as graphed responses or their regressions, vary seasonally, yearly and with the activity of the smelter. Not all patterns are linear with distance. Depressed mutation frequencies are observed due to physiological changes in plants at the sites closest to the smelter. These changes are variably estimated in terms of nitrogen fixation, pollen abortion, linear growth, flower production and, most recently as photon absorption and electron transport in the primary event in the production of energy for photosynthesis. This last method is measured as variable fluorescence. Corn, Tradescantia and soybean are used to measure the physiological functions. The estimation of physiological changes are very important both in themselves and add as an aid in interpreting mutation data. Mutation frequencies and physiological changes have been determined at another lead smelter located i n a suburban locality, two urban industrial areas, a petrochemical complex, a reactor and, of course, control sites. Control sites in each general area of study are necessary. In addition, samples of substrate, soil from the above mentioned areas and soil-mud from a domestic water reservoir, have been removed to greenhouses to estimate mutation frequency and other effects free of airborne contaminants. The use of Tobacco Mosaic Virus - Tobacco is also currently under investigation f o r the study of in situ mutagenesis.

(Ab) TESTING METHODS

- MICROBIOLOGICAL AND WHOLE ORGANISM k i r e d by:

B. S. Hass Argonne N a t i o n a l Laboratory

and

J. T . MacGregor Western Regional Research Center, USDA

Abstracts

121

(Ab-1 ) A SALMONELLA MUTANT USEFUL FOR THE DETECTION OF DELETIONS - C.I. Reich* E. Balbinder - AMC Cancer Res. Center, Lakewood CO 80214. A strain of Salmonella typhimurium carrying the amber mutation trpD28 (formerly trpB28) in the early portion of the second gene of the operon plus two conditionally expressed missense mutations on either side of it is unable to grow on minimal medium as well as on the same medium supplemented with anthranilic acid, the first intermediate of tryptophan biosynthesis. This strain produces revertants of two different phenotypes: prototrophs by base substitution, and anthranilic acid requiring auxotrophs. The latter cannot revert to prototrophy and were shown by gene(formtic mapping to contain deletions covering the early portion of and extending into (formerly -) the first structural erly gene of the operon. In preliminary studies employing nitrous acid, we have found that the frequency of deletions as well as the percentage of deletions over total mutations increased with increased times of exposure to the mutagen. At time zero the spontaeous frequency of mutations was of which 1.5% were deletions while after 45 minutes of exposure 8X to nitrous acid the frequency of mutation was of the order of lo-' and the frequency of deletions ranged between 35% and 50% in different experiments. This, then, provides a system to test mutagens as well as potential carcinogens for their ability to induce both point mutations and deletions.

s)

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(Ab-2) COMPARISON OF PROPHAGE INDUCTION AND MUTAGENICITY IN AMES TESTER STRAIN TA1535 BY AFLATOXINS AND NITROSAMINES. L. Wheeler, M. Halula and M. DeMeo, intr. by S. Matsuyama, Wadsworth VA Hospital and UCLA, Los Angeles, CA. 90024. IJe have recently found that the Ames mutagen tester strains are lysogenic for Fels-like prophages. This provides an opportunity to compare prophage induction and histidine reversion under similar conditions in the same tester strain. A liquid suspension method supplemented with rat liver homogenate (S9) was successfully used to quantitate prophage induction in TA1535 by aflatoxin B1, G1 and diethylnitrosamine. For aflatoxin B1 the Induction Index (number of plaques in the treated tube divided by the number of plaques in the control tubes) for 1, 10, 100 and 1000 ng/ml was 5 , 10, 61 and 201 respectively. Aflatoxin G1 was a poor prophage inducer. The Induction Index for 10, 100 and 1000 ng/ml was 1.2, 2.5 and 0.6 respectively. For diethylnitrosamine the Induction Index for 100 pg/ml was 5.3. No histidine reversion responses above the controls were observed with any of the compounds mentioned above. These results suggest prophage induction in the Ames strains may offer a supplemental method of detecting DNA damage in these strains. (Supported by NIH Grant CA22249). (Ab-3) THE BACILLUS SUBTlLlS MULTI-GENE SPORULATION TEST: SENSITIVITY TO KNOWN MUTAGENS AND CARCINOGENS. J T MacGre or and L.E. Sacks;':, Western Regional Research Center, USDA, B e r e l o . The multi-gene forward mutation test based on the sporulation system o f B.subtilis (MacGregor and Sacks, Mut. Res.38(1976)271) detects mutations in any of up to several hundred genes in 2 F 4 5 separate map locations. The test permits either a liquid culture exposure during any part of the growth cycle or a top agar exposure during growth and sporulation on agar plates. The top agar exposure is simple and has given good results with all mutagens tested, except a few which cannot be tested this way because they block sporulation. The test is sensitive to the following prototypes of major classes of mutagens and carcinogens: dimethyl hydrazine,hydrazine sulfate,mitomycin C,decarbamyl mitomycin C, 8-methoxypsoralen (plus longwave UV 1 ight) ,aflatoxin B 1 (M) ,furylfuramide(AF-2) ,UV radiation,nitrogen ~ustard,k-nitroquinoline-N-oxide,lCR-l~l,acriflavin,acridineorange, MNNG, EMS ,-(2,3-d1 bromopropyl )phosphate (M) ,k-n i tro-o-phenylened iami ne (R) ,

122

Abstracts

sodium azide,nitrous acid,ethidium bromide,streptozotocin,dimethylnitrosamine(M),Z-acetylaminofluorene(MR) and its N-acetoxy- and N-hydroxy- derivatives, 2-nitroso- and 2-aminofluorene(M), and benzo(a)pyrene(MR) and i ts trans-7,8-d iol -9.10-epoxy- and trans-9,lO-diol-7,8-epoxy- derivatives Compounds designated (M) required an in vitro metabolic activation system, and those designated ( R ) an excision repair-deficient strain, for detection. Quercetin was not mutagenic in the top agar assay. The standard Ames Salmonella assay (Ames et al., Hut. Res.z(1975)347) is insensitive to at least two major mutagen prototypes (the hydrazines and the psoralen and mitomycin cross1 inking agents) which are readily detected by this test, indicating that currently employed reverse mutation tests may fail to detect some important mutagen classes detectable by this test.

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(Ab-4) COLORIElETRIC PHAGE INDUCTION ASSAY FOR CARCINOGENS AND CANCER CHEMOTHERAPEUTIC AGENTS. R.K. Elespuru and M.B. Yarmolinskyft, Cancer Biology Program and Chemotherapy Fermentation Laboratory, Frederick Cancer Research Center, Frederick, Md. 21701. Rapid screening assays for detection of carcinogens and cancer chemotherapeutic agents as inducers of bacteriophage lambda have been a & gene, fused within an operon under lambda developed. The bacterial & repressor control, is expressed upon induction of the prophage. The lac2 gene product, 6-galactosidase, is detected colorimetrically in a one tube assay or in a spot test on agar, 3 to 5 hrs after addition of test substance. Mutations introduced into the strain allow prolonged synthesis of enzyme, prevent cell lysis, and enhance sensitivity to low doses of inducing agents. Mammalian activating enzymes may be incorporated into the assay when necessary. Possible advantages of this system include 1) rapidity, 2 ) necessity for use of only one strain for the detection of a variety of DNA-chemical interactions, 3 ) use with test samples that are contaminated with microbial agents. nutrients. or toxicsubstances that inhibit growth and phage production. Carcinogens aflatoxin B1, acetoxyacetylaminofluorene. ethylnitrosonitroguanidine. and chemotherapeutic agents bleomycin. daunorubicin, and streptonigrin are good inducers in this system. (Ab-5) THE CHEMOSTAT IN THE ABSENCE OF ANY ACTIVATING AGENT I S A QUANTITATIVE MEASURING DEVICE OF THE MUTAGENICITY OF POLYCYCLIC AROMATIC HYDROCARBONS AND PRODUCTS FROM FOSSIL FUEL ENERGY CONVERSION PROCESS STREAMS. Bruce S. Hass and Robert B. Webb, Argonne National Laboratory, Argonne, IL 60439, The use of E. coli strain B in the chemostat has provided a quantiin the tative measure of the mutageni2Tly of benzo(a)pyrene [B(a)P] absence of any activating agents. Phage T5 resistant mutants in chemostat cultures inrreased linearly with time in the presence of unactivated B(a)P at twice the spontaneous rate of 1pM concentration of B(a)P and On the other hand, SUM at five times the spontaneous rate at 5pM B(a)P. concentration of benzo(e)pyrene showed no increase over the spontaneous rate. No effect of B(a)P was seen in the B (wildtype) or W l O (H) strains. In the absence of activation samples of organic process streams from a coal conversion plant produced four-fold increases in mutation concentration of the rate over spontaneous in the presence of 2 x raw material. This represents an increase comparable to an Ames test response. Data with other polycyclic aromatic hydrocarbons will be discussed. The advantages of the chemostat as a mutation screening device include: (1) ability to apply chronic (and therefore lower) doses of mutagen with no cell killing; ( 2 ) a simultaneous measurement of mutation and cell population; ( 3 ) the possibility of measuring aromatic hydrocarbon mutation in the absence of any activating agent. Work supported by the United States Department of Energy.

Abstracts

123

(Ab-6) A SERIAL DILUTIOH MULTIWELL SUSPENSION ASSAY FOR DNA DAPlAGE IN E. E. N. E. McCarroll*, C. E. Piper, G. 11. Fukui, B. J . Keech*, and G. Gridley*, Hazleton Laboratories America, Inc., Vienna, VA 22180. Detection of D!IA damage in bacteria as an indicator of chemical i n t e r action with genetic material has been limited by the lack of s e n s i t i v i t y and predictability of the t e s t methods currently in use. As an a l t e r n a t e approach we have devised a multiwell suspension assay u t i l i z i n g E. c o l i indicator s t r a i n s WP2, WP2 uvrA, WP67, Cf1611, WP100, W3110 poZA+ and p3478 poZA- f o r detection of chemical ly i nduced preferenti a1 ki 11 of repai r def i c i e n t s t r a i n s . Eleven s e r i a l 1 : 2 dilutions of the t e s t chemicals were assayed against each indicator s t r a i n in plates having 96 "U" bottom wells. After overnight incubation the chemical concentration causing toxi c i t y , as indicated by inhibition of growth, was determined f o r each s t r a i n . The survival r a t i o s of WP2 and derivative s t r a i n WP101) were determined with selected chemicals by enumeration of the organisms Dresent in the appropriate wells. A correlation between known mutagenic o r nonmutagenic a c t i v i t y and preferenti a1 ki 11 in the mi croti t e r system was observed with the 25 compounds tested. A comparative study indicated t h a t the multiwell suspension assay i s more effective t h a n the spot t e s t f o r demonstrating preferential DNA damage induced by promutagens and i s more sensitive in detecting damage caused by d i r e c t acting agents. Dose related s e l e c t i v e toxicity was obtained with 2-anthramine, P-acetylaminofluorene, 8-aminoquinoline, 2-ni trofluorene, daunomycin, and N-methyl-Nnitrosourea. The multiwell suspension assay f o r DNA damage affords rapid assessment o f multiple chemical dilutions, reproducibility, quantitation, and correlation w i t h established mutagenicity t e s t s . (Ab-7) MODIFICATIONS AND IMPROVEMENTS OF THE &. DNA REPAIR ASSAY. Zev Leifer, and Herbert S. Rosenkranz, Dept. of Microbiology, New Y o r r Medical College, Valhalla, NY 10595. The E. coli DNA repair assay using normal (pol A+> and DNA-polymerase deficient (pol Al-) bacteria shows promise as a pre-screen assay for mutagens and carcinogens. The original assay is based on differential inhibition of the pol A1- strain by DNA-modifying agents as evidenced by a larger zone of growth inhibition. However, agents which diffuse poorly or which require metabolic activation present problems in this assay. To overcome this, modifications of the assay were investigated. These depend upon the preferential killing of pol A1- by DNA-modifying agents. Using a suspension assay, it was found that of 7 1 known carcinogens tested, 62% were mutagenic in the Salmonella assay while 75% were positive i n the pol A system. However, when used in tandem accuracy of detection is increased to 79%. An additional assay based upon plate incorporation of the bacteria and test chemical is also being developed, thereby greatly increasing the rapidity and versatility of the assay.

(Ab-8) MICRONUCLEI INDUCED BY ULTRAVIOLET LIGHT AND CHFNICAL MUTAGENS IN MEIOTIC POLLEN MOTHER CELLS OF TRADESCANTIA. Te-Hsiu Ma and Van Allen Anderson*, Department of Biological Sciences, Western Illinois University, Macomb,

IL 61455. Micronuclei (MCN) in tetrad of the meiotic pollen mother cells of Tradescantia have been used as indicators of chromosome damage induced by X-irradiation and chemical mutagens such as ethyl methanesulfonate, sodium azide and 1,2-dibromoethane. In the present study, ultraviolet light and two other chemical mutagens, maleic hydraaide and cyclohexylamifie, were used as the chromosome breaking agents for the "MCN-in-Tetrad" mutagen

124

Abstracts

tests. Plant cuttings of Tradescantia clone #OZ were maintained in water and exposed to approximately 4 X l o b , 8 X lo6 and 1.6 X 107 ergs per cm2 of ultraviolet light generated by a germicidal lamp. The MCN frequencies from these treated pollen mother cells were 14.8, 18.5 and 29.8 MCN/IOO tetrads respectively. Plant cuttings of Tradescantia clone #O3 were maintained in solutions of maleic hydrazide or cyclohexylamine for 8 24 h as treatments. An average of 15.8 MCN/IOO tetrads nas induced by 0.065% of maleic hydrazide. An average of 15.1 and 56.5 MCN/lOO tetrads were induced by 0.25% and 0.5% of cyclohexylamine solutions respectively. The MCN frequencies of control groups of three series of experiments were around 7 MCN/lOO tetrads.

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(Ab-9) GENETIC ACTIVITY OF PRO-MUTAGENS IN LOGARITHMIC PHASE CULTURES OF SACCHAROMYCES CEREVISIAEl. F. W. Larimer, A. A. Hardigree, Della W. Ramey, and J. L. Epler, Biology Division, O a k Ridge National Laboratory, Oak Ridge, TN 37830. The sensitivity of stationary and log phase cultures of Saccharomyces cerevisiae to a variety of pro-mutagens was investigated. A haploid strain was used to monitor forward mutation to canavanine resistance and reversion of the hisl-7 missense marker. A decrease in survival and an increase in mutation frequency was observed in harvested log phase cells when treated in buffer with aflatoxin B1, 2-aminofluorene, benzo(a)pyrene, or 8-nitroquinoline. These compounds were not genetically active in stationary phase cells. Log phase cells grown on glucose or galactose as carbon source were of approximate equal sensitivity; raffinose grown cells were more senr;it,ivein comparison. When the mutagen was administered to l o g phase cells in growth medium, a marked decrease in survival followed, but no increase in mutation frequency was observed. Cell extracts are being examined f o r mutagen metabolizing activities. 'Research jointly sponsored by the Environmental Protection Agency (IAG-D5-E681; Interagency Agreement 40-516-75) and the Division of Biological and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation.

(Ab-10) USE OF THE AMES SALMONELLA/MICROSOME ASSAY TO MONITOR LABORATORY CONTAMINATION BY CHEMICAL MUTAGENS. M. Virginia Peirce, Thomas M. Distler," Sharon L. Eckford, and Vincent F. Simmon, SRI International, Menlo Park, CA 94025 and U.C. Lawrence Livermore Laboratory, Livermore, CA 94550 Procedures to monitor laboratory contamination by chemical mutagens were developed, incorporating the Ames Salmonella/microsome assay. To establish lower limits of detection and response curves for mutagen spills in the laboratory, controlled quantities of known mutagens were distributed on stainless steel, vinyl tile, and polyethylene plastic surfaces at U.C. Lawrence Livermore Laboratory (LLL). After 10 minutes, a filter paper disc was swiped over the spill area and placed in 1 ml of solvent. The sample was then delivered to SRI International where various aliquots of the solvent were tested for mutagenic activity in the Ames Salmonella/ microsome assay. Controls included swipes of "clean" surfaces to establish normal background. Detectable mutagenicity was compared with mutagenicity of standard solutions of each mutagen. Chemicals that were detected as mutagens by these procedures included: benzo(a)pyrene, N-acetylaminofluorene, 8-naphthylamine, MNNG, 2-aminoanthracene, 4-methoxy-2-naphthylamine-HC1, ethylnitrosourea, 4-nitroquinoline oxide, 9-aminoacridine, nitrogen mustard, 8-hydroxyquinoline, and benzidine. The results indicate that these procedures are sensitive enough to be used for routine monitoring of laboratory surfaces for many chemical mutagens, in the presence of normal backgrounds.

Abstracts

125

(Ab-11 ) MUTATIONAL DOSE RESPONSE FOR DROSOPHILA EXPOSED TO GASEOUS 1,2-DIBROMOETHANE." P. G. Kale and J. W. Baum, Brookhaven N a t i o n a l L a b o r a t o r y , Upton, NY, 11973. S e x - l i n k e d recessive l e t h a l m u t a t i o n s were i n d u c e d i n D r o s o p h i l a melanogaster males by g a s e o u s 1 , 2 - d i b r o m o e t h a n e a t c o n c e n t r a t i o n s r a n g i n g S i g n i f i c a n t numbers of m u t a t i o n s c o u l d from 0.2 t o 2 p a r t s p e r m i l l i o n . Pronounced germ c e l l s e n s i t i v i t y be induced a t a l l t h e s e concentrations. d i f f e r e n c e s were o b s e r v e d . F o r low e x p o s u r e s , s p e r m a t i d s and s p e r m a t o c y t e s were a b o u t 10 t o 2 0 times more s e n s i t i v e t h a n s p e r m a t o z o a . The d o s e e f f e c t r e l a t i o n was l i n e a r below 60 ppmehr f o r t h e t h r e e c e l l t y p e s . A t h i g h e r e x p o s u r e s , s t e r i l i t y p r e v e n t e d m u t a t i o n d e t e c t i o n i n spermatoc y t e s and i n s p e r m a t o g o n i a . The l o w e s t e f f e c t i v e e x p o s u r e f o r s p e r m a t o z o a w a s 18 ppm-hr (0.25 ppm f o r 72 h o u r s ) . I n s p e r m a t i d s , t h e lowest e x p o s u r e t e s t e d , 2.3 ppm-hr (0.2 ppm for 11 h o u r s ) i n d u c e d f o u r t i m e s t h e s p o n t a n e o u s m u t a t i o n r a t e . T h e r e f o r e , u s i n g p r o l o n g e d e x p o s u r e p e r i o d s one may b e a b l e t o d e t e c t c o n c e n t r a t i o n s i n t h e r a n g e of p a r t s p e r b i l l i o n . T h u s , Drosophila appears s u i t a b l e as a system f o r d e t e c t i n g very l o w concentrat i o n s o f g a s e o u s m u t a g e n s i n i n d u s t r i a l , a g r i c u l t u r a l , and e n v i r o n m e n t a l atmospheres. *Work s u p p o r t e d t h r o u g h a n i n t e r a g e n c y a g r e e m e n t between t h e E n v i r o n m e n t a l P r o t e c t i o n Agency, R e s e a r c h G r a n t NO. EPA-LAG-27 (!1'207 dnd t h e Departtnent of Energy C o n t r a c t EY-76-C-02-0016.

(Ab-12) DEFINITION AND ASSESSMENT OF ETHYL METHANESULFONATE INDUCED MUTATIONS I N C57BL/6J MICE. R. J . F e u e r s * and J . B. B i s h o p , N a t i o n a l C e n t e r f o r T o x i c o l o g i c a l R e s e a r c h , J e f f e r s o n , A R 72079. S e v e r a l E t h y l M e t h a n e s u l f o n a t e (EMS) i n d u c e d m u t a t i o n s h a v e b e e n d e t e c t e d a n d d e f i n e d u s i n g o u r i n v i v o enzyme parameter a s s e s s m e n t s y s t e m , " M i c r o l e s i o n Assay" ( F e u e r s , e t a l , Mutat. Res. 53:99, 1 9 7 7 ) . The e n s u i n g m o l e c u l a r d e f i n i t i o n of t h e s e m u t a t i o n s w e r e p e r f o r m e d by a number of b i o c h e m i c a l t e c h n i q u e s i n c l u d i n g semi- au t o m a t e d M i c h a e l i s Menten enzyme k i n e t i c s . Comparisons o f the v a r i o u s d a t a f r o m F2 g e n e r a t i o n s t o t h a t o f F3 g e n e r a t i o n s h a v e , i n t u r n , b e e n made. These r e s u l t a n t d a t a were u s e d t o 1) p r o v i d e p r e l i m i n a r y gene class d e f i n i t i o n s a n d 2 ) e s t i m a t e the d e g r e e o f m e t a b o l i c c o n t r o l the measured l o c i are u n d e r . S e v e r a l o f t h e s e m u t a t i o n s are o f classes o t h e r t h a n s t r u c t u r a l and appear t o be u n d e r t h e i n f l u e n c e o f a h i g h l y i n t e g r a t e d c o n t r o l s y s t e m . F u r t h e r , m u t a t i o n s w i t h i n some o f t h e s e i n t e g r a t e d c o n t r o l s y s t e m s s e e m to b e more r e c o v e r a b l e than others. These d a t a a n d t h e i r u s e f u l n e s s i n d e f i n i n g a n d f u r t h e r i n c r e a s i n g t h e o v e r a l l p o t e n t i a l and s e n s i t i v i t y o f the a s s a y w i l l b e d i s c u s s e d .

(Ab-13) INDUCTION OF ENZYMATIC ACTIVITY MUTATIONS BY ETHYL METHANESULFONATE ( E r n ) J. B. B i s h o p , a n d R. J. F e u e r s * , N a t i o n a l C e n t e r f o r T o x i c o l o g i c a l R e s e a r c h , J e f f e r s o n , A R 72079 A mammalian i n v i m m u t a g e n e s i s a s s a y which u t i l i z e s enzyme parameters as g e n e t i c "markers" f o r d e t e c t i n g m u t a t i o n s h a s b e e n d e s c r i b e d

p r e v i o u s l y ( F e u e r s , e t a1 M u t a t . Res. 53:99, 1 9 7 7 ) . W e h a v e employed this a s s a y p r o c e d u r e t o i d e n t i f y m u t a n t s among t h e F1 p r o g e n y from m a t i n g s of u n t r e a t e d C57BL/6J f e m a l e m i c e w i t h C57BL/6J F,, m a l e s g i v e n

126

Abstracts

interperitoneal injections of either 200, 175, 100 or 0 mg/kg EMS. Data on the frequency and types of enzymatic mutations recovered will be presented and comparisons made with other types of mammalian mutational test results. The application of this assay in the regulatory decision making process will also be discussed.

(Ac) GERM CELL

EFFECTS

Chaired by: G. A. Sega Oak Ridge N a t i o n a l L a b o r a t o r y

and

A . J . Wyrobek Lawrence L i v e n n o r e L a b o r a t o r y

(AC-1 ) RESPONSE OF SPERMATOGONIA AND KINETICS OF SPERMATOGENESIS TO 6-MERCAPTOPURINE. E. F. OakberP. Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830. Twelve-week-old 101 x C3H hybrid male mice were given 15 pCi [ 3H]thymidine, followed 30 min later by an injection of 150 mg/kg of 6-merca topurine in 0.03 N NaOH. Other groups received no injections, 15 pCi [ a] thymidine only; 15 PCi [3H]thymidine + 0.25 ml 0.03 N NaOH; and 150 mg/kg of 6-MP only. Animals were killed 72 hrs-17 days after treatment. Number of spermatogonia was determined at 3 and 10 days. Stem cell numbers were unaffected. At 3 days, frequency of A2-A4, In, and B spermatogonia were reduced, respectively, to 80, 50, and 54% of control. These counts represent survival of Al-Az, A2-A3, and A3-A4 gonia. Number of preleptotene spermatocytes was normal at 3 days, suggesting no killing of late Aq-early In spermatogonia. This pattern of response is different from that observed for irradiation and all other chemicals we have investigated. Progression of the labeled cohort of preleptotene stages was normal for two cycles of the seminiferous epithelium, indicating no effect on the kinetics of spermatogenesis. [Research sponsored by the Division of Biomedical and Environmental Research, U. S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation.]

3

(Ac-2) A SENSITIVE HYBRID FOR THE DETECTION OF SPERM HEAD ABNORMALITIES AND ITS POTENTIAL FOR THE DETECTION OF TRANSMLSSIBLE MUTATIONS IN MICE J.C. Topham, ICI Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, UK, S K l O 4TC. The percentage abnormal sperm heads in control BALB/c, CBA and (BALB/c x CBA)Fl mice are about 40%. 4% and 1% respectively. Lncreases in abnormalities in F1 mice above control were seen 5 weeks after I.P. administration of cyclophosphamide 5 x 20 mg/kg (+ 2.7%), ethylmethane sulphonate (EMS) 4 x 250 mg/kg (+ 9.6%). tris(2-methyl-1-aziridinyl) phosphine oxide (METEPA) 5 x 10 mg/kg (+ 11.0%). methylmethane sulphonate (MMS) 3 x 2 5 mg/kg (+ 2.9%), methylcholanthrene 5 x 2 5 rng/kg (+ 15.6%) and Mitomycin C 3 x 0.2 mg/kg (+ 2.3%). 1 4 other compounds including a range of substituted anilines have been studied and the results related to their possible carcinogenic potential. Mating experiments with untreated mice (i.e. F 1 x CBA, F1 x BALB/c and F1 x F 1 ) have demonstrated

Abstracts

127

t h a t t h e c h a r a c t e r i s t i c s which c o n t r o l t h e i n c i d e n c e o f abnormal sperm heads a r e t r a n s m i t t e d t o t h e progeny. An a t t e m p t t o i n c r e a s e t h e i n c i d e n c e of sperm head a b n o r m a l i t i e s i n t h e F1 males i s b e i n g made by t r e a t m e n t o f Fo mice f o r 5 days w i t h EMS, METEPA, Mitomycin C o r cyclophosphamide p r i o r t o mating. Female CBA mice a r e mated immediately a f t e r t r e a t m e n t w i t h u n t r e a t e d BALB/c males. W h i l s t t r e a t e d CBA males a r e mated w i t h u n t r e a t e d BALB/c females 1 , 5 and 8 weeks l a t e r . A l l t h e male progeny a r e k i l l e d when 10-12 weeks o l d f o r t h e a s s e s s m e n t of s p e r m abnormalities.

(Ac-3) IKOUCED INCREASES IN MORPHOLOGICALLY ABERRANT SPERM I N RATS FOLLOWING Co6O IRRADIATION, L. F. Lock and E. R. Soares, Chemical I n d u s t r y I n s t i t u t e o f Toxicology, Research T r i a n g l e Park, NC 27709. Results o f recent studies suggest t h a t the sperm morphology assay (SMA) may be a s e n s i t i v e i n v i v o means o f d e t e c t i n g mutagens i n mice. However, very l i t t l e work has been published on o t h e r mammalian species. Since the r a t i s t h e animal o f choice i n many long-term toxicity/carcinogenicity t e s t s and since the SMA c o u l d be e a s i l y incorporated i n t o such t e s t s , we undertook a study o f the e f f e c t s o f r a d i a t i o n on r a t s as measured by the SMA. Four groups o f 20 each, s e x u a l l y mature, Fischer-344 male r a t s were exposed t o one o f the f o l l o w i n g doses o f r a d i a t i o n : O r , 150r, 300r, o r 500r. Exposure was t o the r e a r q u a r t e r only. A t 5 and 7 weeks post-treatment, sperm were c o l l e c t e d from 10 animals per group. The sperm were d i l u t e d i n p h y s i o l o g i c a l s a l i n e . Three s l i d e s were prepared from each animal and stained w i t h Eosin Y. Six-hundred sperm ( 2 0 0 / s l i d e ) were counted and categorized as morphologically normal o r abnormal f o r each r a t . I n weeks 5 and 7, post- treatment r a d i a t i o n produced a s i g n i f i c a n t dose-correl ated increase i n aberrant sperm. Mean values f o r aberrant sperm obtained a t week 5 f o r O r , 150r, 300r, and 500r were 5.36%, 10.23% (P < .05), 27.46% (P < .01) and 27.86% (P < . O l ) , respectively. A t week 7, r e s u l t s obtained f o r O r , 150r, 300r and 500r were 7.24%, 16.9% (P < .Ol), 29.7% (P < .01) and 60.7% (P < - 0 1 ) aberrant sperm, r e s p e c t i v e l y . Studies are p r e s e n t l y underway i n our l a b o r a t o r y t o determine ( 1 ) the e f f e c t s o f r a d i a t i o n on sperm t r e a t e d as spermatogonial c e l l s and ( 2 ) the e f f e c t s o f a known chemical mutagen on r a t s as measured by the SMA. (Ac-4) RADIATION-INDUCED HERITABLE SPERM ABNORMALITIES IN MICE. A. P. Hugenholtz and W. R. Bruce, Department of Medical B i o p h y s i c s , Univ. of Toronto, Toronto, O n t a r i o M4X 1K9

Exposure o f male mice t o r a d i a t i o n o r t o chemicals i n c r e a s e s t h e i r l e v e l s of abnormally-shaped sperm. We have s u g g e s t e d t h a t t h e s e induced a b n o r m a l i t i e s are t h e consequence of m u t a t i o n s which r e d u c e t h e f i d e l i t y of s p e r m a t o g e n i c d i f f e r e n t i a t i o n . W e have examined t h e progeny of i r r a d i a t e d mice f o r sperm a b n o r m a l i t i e s u s i n g f o u r d i f f e r e n t s t r a i n s of mice, e a r l y and d e l a y e d c o n c e p t i o n s f o l l o w i n g p a r e n t a l i r r a d i a t i o n and r a d i a t i o n d o s e s r a n g i n g from 25 t o 600 r a d . We d e t e c t e d v a r i a n t progeny a t f r e q u e n c i e s which d i s p l a y e d a humped d o s e r e s p o n s e from p r e s t e r i l e conc e p t i o n s . R e c i p r o c a l t r a n s l o c a t i o n s were found i n o n e - t h i r d of t h e v a r i Delayed c o n c e p t i o n s f o l l o w i n g a n t s and showed a s i m i l a r d o s e response. e x p o s u r e of e i t h e r p a r e n t were less e f f e c t i v e a t p r o d u c i n g sons w i t h abn o r m a l i t i e s . Subsequent b r e e d i n g of F1 a n i m a l s from p r e s t e r i l e concept i o n ( p a t e r n a l l y exposed) i n d i c a t e d t h a t t h e sperm a b n o r m a l i t y t r a i t may d i s p l a y dominant, s e x - l i n k e d or recessive i n h e r i t a n c e . Moreover, sperm a b n o r m a l i t i e s and r e c i p r o c a l t r a n s l o c a t i o n s may s e g r e g a t e among t h e F2 males. These f i n d i n g s p r o v i d e s t r o n g e v i d e n c e t h a t induced sperm abnorm a l i t i e s r e f l e c t m u t a t i o n s i n t h e male germ l i n e . S i n c e sperm abnormalit i e s i n t h e F 1 s o n s of mutagenized mice a r e d e t e c t e d a t r e l a t i v e l y h i g h

128

Abstracts

f r e q u e n c i e s (2-8% o f males c o n c e i v e d i n t h e p r e s t e r i l e p e r i o d f o l l o w i n g 300 r a d e x p o s u r e o f t h e i r f a t h e r ) , t h e s e d e f e c t s c o u l d p r o v i d e a n e f f i c i e n t h e r i t a b l e i n v i v o mammalian s y s t e m t o m o n i t o r m u t a g e n e x p o s u r e . Such d e f e c t s would d e t e c t p r e s u m a b l y p o i n t m u t a t i o n s o r s m a l l d e l e t i o n s a s w e l l a s some r e c i p r o c a l t r a n s l o c a t i o n s . ( S u p p o r t e d by The M e d i c a l R e s e a r c h C o u n c i l o f Canada)

(Ac-5) CHANGES I N MURIIIE SPERM HEAD DIMENSIONS INDUCED 3Y LOW DOSES OF X-IRRADIATION. A J Wyrobek, G. Watchmaker", D. B e n n e t t * , D. K r a n z l e r * , a n d D. Moore, II*, B i o m e d i c a l S c i e n c e s Div., Lzwrence L i v e r m o r e Laboratory, L i v e r m o r e , CA. 94550 Chemical mutagens and x - i r r a d i a t i o n p r o d u c e m o r p h o l o g i c a l l y a b n o r m a l sperm t h a t c a n b e e a s i l y d i s t i n g u i s h e d u n d e r t h e m i c r o s c o p e . We are d e v e l o p i n g s e n s i t i v e , o b j e c t i v e methods t o q u a n t i t a t e s u c h c h a n g e s u s i n g d i r e c t measurements of t h e d i m e n s i o n s of t h e s p e r m head. Groups of 3 B6C3F1 mice r e c e i v e d a c u t e , t e s t i c u l a r d o s e s of 0 , 30, 60, 90 o r 1 2 0 r a d s of x - i r r a d i a t i o n . T h i r t y - f i v e d a y s later 500 eosin-Y s t a i n e d e p i d i d y m a l sperm were s c o r e d per mouse. A d o s e d e p e n d e n t i n c r e a s e i n p e r c e n t a b n o r m a l s p e r m w a s f o u n d w i t h a 70 rad d o s e r e q u i r e d t o d o u b l e background F o r e a c h mouse, 50 s p e r m were c h o s e n a t random, phot1,Craphed a n d p r i n t e d (MAG % l o 4 ) . F o r e a c h of t h e 750 s p e r m h e a d s i l h o u e t t e s , 11 p a r a m e t e r s were m e a s u r e d (4 p a r a l l e l t o t h e t a i l a x i s , 3 p e r p e n d i c u l a r t o t h e t a i l a x i s , 2 d i a g o n a l s , area, p e r i m e t e r a n d a c a l c u l a t e d s h a p e f a c t o r ) . F o r each sperm w e t h e n c a l c u l a t e d i t s M a h a l a n o b i a n d i s t a n c e f r o m t h e g r a n d mean of t h e 0 r a d g r o u p a n d c l a s s i f i e d as non-normal t h o s e s p e r m t h a t f e l l o u t s i d e t h e u p p e r 5% C h i - s q u a r e d va lue of t h e 0 r a d g r o u p . The 11 p a r a m e t e r s y i e l d e d a d o s e d e p e n d e n t i n c r e a s e i n t h e p r o p o r t i o n o f nonnormal sperm. T h e r e is no s i g n i f i c a n t i n f o r m a t i o n l o s s when c e r t a i n 4 - r 5 o f t h e 11 parameters w e r e s e l e c t e d . T y p i c a l d o s e s r e q u i r e d t o d o u b l e T h i s q u a n t i t a t i v e method i s c o n s i d e r a b l y b a c k g r o u n d are a b o u t 10 r a d s . more s e n s i t i v e t o r a d i a t i o n i n d u c e d c h a n g e s t h a n i s v i s u a l m o r p h o l o n i c a l assessment. The m e t h s d s h o i i l d be r e a d i l y a d a p t a b l e t o h i g h s p e e d , a u t o (Work p e r f o r m e d b y LLL u n d e r m a t e d , s c a n n i n g - m i c r o s c o p i c image a n a l y s i s . and the Environa u s p i c e s of U.S. DOE u n d e r c o n t r a c t No. W-7h05-ENC-48 m e n t a l P r o t e c t i o n Agency u n d e r c o n t r a c t EPA-IAC-D6-E681-AN.)

(Ac-6)

STRAIN COMPARISON OF TEM INDUCED DOMINANT LETHAL EFFECTS I N SPERMATOZOA TREATED MICE. J . B . FAVOR AND J.W. CRENSHAW, G e o r g i a I n s t i t u t e o f Techn o l o g y , Atyan'ta, GA 30332 The d e m o n s t r a t i o n o f mouse s t r a i n d i f f e r e n c e s i n mutagen s e n s i t i v i t y would b e i m p o r t a n t t o t h e s t u d y of m u t a g e n e s i s by p r o v i d i n g g e n e t i c v a r i a t i o n w i t h which t o s t u d y t h e m u t a t i o n p r o c e s s e s i n a mammalian s y s t e m . A d o s e r e s p o n s e e x p e r i m e n t s t u d y i n g TEM i n d u c t i o n o f d o m i n a n t l e t h a l s i n t h r e e s t r a i n s of mice was c o n d u c t e d i n two r e p l i c a t e s . Twelve week o l d m a l e mice o f s t r a i n s BALB/cByJ, C3H/HeJ, a n d DBAl2.J were e a c h t r e a t e d i . p . w i t h 0 , 0 . 1 , o r 0 . 2 mg TEM/kg body w e i g h t and mated i m m e d i a t e l y a f t e r t r e a t m e n t f o r s e v e n d a y s w i t h v i r g i n f e m a l e s o f t h e same a g e and s t r a i n . F e m a l e s were a l l o w e d t o g i v e b i r t h and wean t h e i r l i t t e r s . F o l l o w i n g t h e w e a n i n g of l i t t e r s , f e m a l e s were d i s s e c t e d , t h e i r u t e r i n e h o r n s i n s p e c t e d , a n d scars c l a s s i f i e d as l i v e and dead i m p l a n t a t i o n s a c c o r d i n g t o t h e p r o c d u r e s of S o a r e s . The dominant l e t h a l e f f e c t s s t u d i e d were p e r c e n t d e a d implant a t i o n s , t o t a l i m p l a n t a t i o n s , and t h e p e r c e n t l a t e d e a t h s ( t h e d i f f e r e n c e b e t w e e n t h e number o f i m p l a n t a t i o n s c a r s i n d i c a t i v e o f l i v e embryos a n d R e s u l t s i n d i c a t e : 1 ) t h a t treatt h e number o f l i v e o b s e r v e d a t b i r t h ) . ment was e f f e c t i v e i n i n c r e a s i n g t h e p e r c e n t d e a d i m p l a n t a t i o n s p e r f e m a l e , a n d t h a t t h e r e w a s a s i g n i f i c a n t i n t e r a c t i o n o f s t r a i n and d o s e d u e t o t h e r e d u c e d r e s p o n s e o f s t r a i n C3H/HeJ t o mutagen d o s e ; a n d 2 ) t h a t t r e a t m e n t w a s e f f e c t i v e i n r e d u c i n g t h e t o t a l number of i m p l a n t a t i o n s

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129

p e r female and i n c r e a s i n g t h e p e r c e n t l a t e d e a t h s w i t h no s i g n i f i c a n t i n t e r a c t i o n of s t r a i n and dose. These p r e l i m i n a r y r e s u l t s s u p p o r t t h e c o n c l u s i o n t h a t , i n mice, s t r a i n d i f f e r e n c e s e x i s t f o r mutagen s e n s i t i v i t y due t o induced e a r l y p o s t i m p l a n t a t i o n l o s s e s as i n d i c a t e d by p e r c e n t dead i m p l a n t a t i o n s .

(Ac-7) SPERMATID HEAD ABNORMALITIES I N TRANSLOCATION HETEROZYGOTES FROM EMS- OR CPA-TREATED SIRES. Rene E. Sotomayor, Biology D i v i s i o n , Oak Ridge N a t i o n a l L a b o r a t o r y , Oak Ridge, TN 37830. Male mice were i n j e c t e d i p w i t h 200 mg/kg of EMS o r 350 mg/kg of cyclophosphamide (CPA). C o n t r o l a n i m a l s r e c e i v e d i n j e c t i o n s o f s a l i n e . T r e a t e d males w e r e caged w i t h normal female mice 5.5 through 8 . 5 days a f t e r EMS t r e a t m e n t and 18 through 2 1 d a y s a f t e r CPA t r e a t m e n t . The same procedure w a s followed f o r t h e c o n t r o l s . The male progeny o b t a i n e d from t h e s e mating i n t e r v a l s were s t u d i e d f o r chromosome a b e r r a t i o n s a t d i a k i n e s i s and s p e r m a t i d head a b n o r m a l i t i e s (SHA). The F1 c o n t r o l s showed 1.4 f 0.7% SHA whereas 1.0 t o 62% SHA w a s observed i n t h e t r e a t e d - d e r i v e d mice. N i n e t y - s i x p e r c e n t of t h e n o n t r a n s l o c a t i o n c a r r i e r s and 50% of t h e t r a n s l o c a t i o n h e t e r o z y g o t e s showed no d i f f e r e n c e w i t h t h e c o n t r o l s . The remaining 50% of t h e t r a n s l o c a t i o n h e t e r z y g o t e s w a s d i v i d e d a s f o l l o w s : 20% had 1 0 . 8 f 1.7, 20% had 29.5 f 5.9 and 10% had 61.4 f 1.0 % SHA. (Research sponsored by t h e D i v i s i o n of Biomedical and Environmental Research, U. S . Department of Energy under c o n t r a c t W-7405-eng-26 w i t h t h e Union Carbide C o r p o r a t i o n . )

(Ac-8) FLOW CYTOMETRIC EVIDENCE FOR RADIATION INDUCED DNA VARIABILITY I N MATURE D. Pinkel", S. Lake*, B.L. G l e d h i l l , and M.A. Van Dills*. Biomedical S c i e n c e s D i v i s i o n , Lawrence Livermore L a b o r a t o r y , Livermore, CA 94550. Mutagenic e x p o s u r e results i n p r o d u c t i o n o f m o r p h o l o g i c a l l y abnormal Flow c y t o m e t r i c sperm and may c a u s e v a r i a b i l i t y i n sperm DNA c o n t e n t . measurements o f e p i d i d y m a l sperm t a k e n from B6C3 F1 mice 5 weeks a f t e r l o c a l x - i r r a d i a t i o n t o t h e testes show a dose-dependent f r a c t i o n o f c e l l s w i t h a l t e r e d DNA s t a i n c o n t e n t . A c r i f l a v i n e - F e u l g e n s t a i n e d sperm n u c l e i are measured i n a f l o w c y t o m e t e r t h a t h y d r o d y n a m i c a l l y o r i e n t s them t o c o n t r o l o p t i c a l effects c a u s e d b y t h e i r f l a t s h a p e and h i g h i n d e x o f refraction. The f l u o r e s c e n t s t a i n i s e x c i t e d by a laser beam o r t h o g o n a l t o Fluorescence i s t h e sample f l o w axis and f l a t s u r f a c e of t h e n u c l e i . d e t e c t e d b o t h i n l i n e w i t h ( f o r w a r d d e t e c t o r ) and o r t h o g o n a l t o t h e laser beam. F l u o r e s c e n c e h i s t o g r a m s from t h e f o r w a r d d e t e c t o r are symmetric and are f i t w e l l by t h e sum of two normal d i s t r i b u t i o n s w i t h t h e same mean, one w i t h a c o e f f i c i e n t of v a r i a t i o n ( C V ) o f a b o u t 3% a n d t h e o t h e r w i t h a CV of a b o u t 8%. These CV's are i n d e p e n d e n t o f dose. The p r o p o r t i o n o f c e l l s i n t h e b r o a d component i s 0%, 30%, and 50% a t d o s e s of 0, 300, and 600 r a d s r e s p e c t i v e l y . Symmetry o f t h e h i s t o g r a m s from t h e f o r w a r d det e c t o r and c o r r e l a t e d changes i n s i g n a l s from t h e two d e t e c t o r s i n d i c a t e t h a t t h e s e changes i n f l u o r e s c e n c e are n o t c a u s e d by r a d i a t i o n i n d u c e d shape changes i n t h e sperm. The i n t e r p r e t a t i o n o f f l u o r e s c e n c e v a r i a t i o n s as DNA c o n t e n t v a r i a t i o n s i s t e n t a t i v e b e c a u s e w e do n o t r e s o l v e t h e exp e c t e d d i f f e r e n c e between t h e X a n d Y b e a r i n g sperm i n t h e s e samples. This d i f f e r e n c e i s s e e n i n b u l l sperm n u c l e i . (Work p e r f o r m e d u n d e r a u s p i c e s o f U,S. Department o f Energy by t h e Lawrence Livermore L a b o r a t o r y , C o n t r a c t and DOEINIEHS I n t e r a g e n c y Agreement No. 22Y01-ES-70031). No. W-7405-#NC-48

MOUSE SPERM.

(Ac-9)

THE SYNERGISTIC EFFECT OF CAFFEINE ON THE UNSCHEDULED DNA SYNTHESIS INDUCED I N EAlUY SPERMATID STAGES OF THE MOUSE BY METHYL METHANESULFONATE.

130

Abstracts

Gary A. Sega and Mark R. K e l l e y * , B i o l o g y D i v i s i o n , Oak Ridge N a t i o n a l L a b o r a t o r y , Oak R i d g e , TN 37830. W e f i r s t d e t e r m i n e d t h a t a 100 mg/kg i p i n j e c t i o n o f male mice w i t h [ 3 H ] c a f f e i n e r e s u l t e d i n a maximum c o n c e n t r a t i o n o f c a f f e i n e i n t h e testes ..?i h a f t e r i n j e c t i o n . S u b s e q u e n t l y , o t h e r males were g i v e n a n i p i n j e c t i o n of c a f f e i n e % h b e f o r e i p i n j e c t i o n s o f v a r i o u s d o s e s of methyl m e t h a n e s u l f o n a t e (MMS) and t e s t i c u l a r i n j e c t i o n s o f [ 3H] t h y m i d i n e (i3HIdT) (2.36 v C i / t e s t i s ) . C o n t r o l males r e c e i v e d Hanks' b a l a n c e d s a l t s o l u t i o n % h b e f o r e t h e MMS a n d [3HldT. While c a f f e i n e d i d n o t p r o d u c e m e a s u r a b l e u n s c h e d u l e d DNA s y n t h e s i s (UDS) i n e a r l y s p e r m a t i d s when u s e d by i t s e l f , i t e n h a n c e d t h e amount o f UDS p r o d u c e d a f t e r e x p o s u r e t o d i f f e r e n t d o s e s o f MMS. The s l o p e o f t h e l e a s t s q u a r e s l i n e a r f i t c u r v e r e l a t i n g MMS a d m i n i s t e r e d d o s e t o t h e amount o f u n s c h e d u l e d u p t a k e o f [3H]dT i n t o e a r l y s p e r m a t i d s w a s 1 . 5 t i m e s g r e a t e r when c a f f e i n e w a s p r e s e n t t h a n when i t w a s a b s e n t . The i n c r e a s e d UDS r e s p o n s e t o MMS i n t h e p r e s e n c e o f c a f f e i n e d o e s n o t a p p e a r t o b e due t o g r e a t e r m e t h y l a t i o n o f germ c e l l DNA b e c a u s e c h e m i c a l d o s i m e t r y s t u d i e s u s i n g a f i x e d [3H]MMS e x p o s u r e o f 75 mg/kg r e s u l t e d i n t h e same amount o € m e t h y l a t i o n o f t e s t i c u l a r DNA w h e t h e r o r n o t c a f f e i n e was p r e s e n t . Other p o s s i b l e r e a s o n s f o r t h e s y n e r g i s m between c a f f e i n e and MMS i n i n d u c i n g UDS i n mouse germ c e l l s a r e u n d e r s t u d y . (Research sponsored by t h e D i v i s i o n of B i o m e d i c a l a n d E n v i r o n m e n t a l R e s e a r c h , U . S. Department o f Energy u n d e r M. R. K e l l e y c o n t r a c t W-7405-eng-26 w i t h t h e Union C a r b i d e C o r p o r a t i o n . ) is a Great Lakes C o l l e g e s A s s o c i a t i o n / A s s o c i a t e d C o l l e g e s of t h e Midwest s t u d e n t , F a l l 1978. from DePauw U n i v e r s i t y . G r e e n c a s t l e , I n d i a n a .

(Ac-10) HYDROLYSIS OF ETHYIATED BASES IN STORED DROSOPHIIA SPEREl AETER DB TREA'IMENT. W.R. Lee, P.M.S. Skinner and F.C. Janca. Department of Zoology and Physiology, Louisiana S t a t e University, Baton Rouge, LA 70803. Previous work has shown t h a t a l k y l groups are removed f r a n DNA by repair enzymes i n a v a r i e t y of metazoan t i s s u e s . Janca, g a l . , have shown a rapid l o s s of e t h y l groups between f e r t i l i z a t i o n and z e second cleavage d i v i s i o n i n Droso h i l a melano aster, but only a slow l o s s of the labeled e t h y l groups was o s e r v e i n t e mature sperm c e l l . This paper reports the rate constant f o r l o s s of e t h y l groups f r a n DNA i n D. melano a s t e r spemtozoa stored i n the seminal receptacles of insemina t e d w d u l t males with t h e i r DNA randomly labeled with 32P p r i o r t o maturation of sperm c e l l s were t r e a t e d with (l-3H) e t h y l methanesu1fonate (DE) by feeding 25mM f o r 24 h r s . DNA was e x t r a c t e d from t h e s t o r e d sperm and the r a t i o 3H/32P determined f r a n samples taken over a 10 day period. The rate constant f o r l o s s of e t h y l groups was found t o be k=1.75 x 10-4 min-1 (half l i f e = 2 . 8 d ) . This r a t e of l o s s of labeled e t h y l groups is i n accordance with the expected rate due t o hydrolysis. From t h e number of i n i t i a l e t h y l a t i o n s per c e l l as determined by Aaron and Lee and the rate constant as determined here the number of a p u r i n i c s i t e s can be canputed, assuming t h a t loss o f . t h e e t h y l group occurs by hydrolysis of t h e a l k y l a t e d base (depurination). Storage of spermatozoa has previously been shown t o lead to increased chramsane breakage. A p l o t of computed a p u r i n i c sites versus dominant l e t h a l s i n Drosophila shows a multi-hit type of curve. For genetic endp o i n t s t h a t depend on the number of ChromcJsome breaks per cell i t is the number of a p u r i n i c sites per c e l l t h a t should be used i n comparisons among species r a t h e r than a l k y l a t i o n s per nucleotide. (Supported by NIEHS ES00320-11 and DOE EX-76-S-05-3728)

--%-&

(Ac-11 ) MONOSPECIFIC ANTIBODY TO MOUSE LACTATE DEHYDROGENASE-X: ITS PURIFICATION AND USE I N LOCALIZING THE ENZYME AND I N THE STUDY OF MUTAGENESIS. A f t a b A. A n s a r i , James Burkhart*, and H e i n r i c h V. M a l l i n g , L a b o r a t o r y of B i o chemical Genetics, N a t i o n a l I n s t i t u t e o f Environmental H e a l t h Sciences, P . 0. Box 12233, Research T r i a n g l e Park, N o r t h C a r o l i n a 27709.

Abstracts

131

The sperm s p e c i f i c enzyme, l a c t a t e dehydrogenase-X (LDH-X) i s an i s o zyme o f LDH t h a t i s encoded i n the gene locus designated c. Antibodies against mouse LDH-X do n o t cross-react w i t h mouse LDH-1 o r LDH-5 b u t show strong c r o s s - r e a c t i o n w i t h r a t LDH-X. I n t h i s paper we describe ( i ) prep a r a t i o n o f a monospecific antibody against mouse LDH-X t h a t does n o t cross-react w i t h r a t LDH-X, ( i i ) use o f t h i s p u r i f i e d antibody i n l o c a l i z i n g t h e enzyme on t h e sperm c e l l surface, and ( i i i ) p o t e n t i a l i t y o f .using t h i s antibody i n d e t e c t i n g mutation i n t h e sperm. The monospecific antibody against mouse LDH-X was prepared from a horse antiserum against t h e enzyme by i t s repeated absorption on a r a t LDH-XSepharose imnunoabsorbent. When used i n f l u o r e s c e n t antibody technique w i t h washed sperm suspension, t h i s antibody gave s t r o n g fluorescence i n the t a i l o f t h e m u s e sperm whereas no fluorescence i n t h e head and midpiece was observed. The whole antiserum against mouse LDH-X, however, gives fluorescence a l l over t h e sperm. This observation was l a t e r found t o be due t o t h e presence i n the serum o f some n a t u r a l antibodies a g a i n s t carbohydrates. These carbohydrate-binding n a t u r a l antibodies are removed d u r i n g absorption of t h e serum on Sepharose imunoabsorbent thus e l i m i n a t i n g t h e non-specific fluorescence i n the head and midpiece regions. (Ac-1 2) DETECTION OF INDIVIDUAL SPERM CONTAINING HEAT-RESISTANT ENZYMES. H. V. M a l l i n q and J. G. Burkhart, Laboratory o f Biochemical Genetics, National I n s t i t u t e o f Environmental Health Sciences, P. 0. Box 12233, Research T r i a n g l e Park, North Carolina 27709. Numerous techniques have been developed f o r l o c a l i z i n g s p e c i f i c enzymes i n c e l l s . The midpiece o f mamnalian sperm contains a l a r g e number o f mitochondria which c o n t a i n many d i f f e r e n t enzymes. Thus f a r we have developed histochemical methods f o r a-glycerol phosphate dehydrogenase (a-GPD) and s u c c i n i c dehydrogenase. The dehydrogenases were l o c a l i z e d by using n i t r o blue tetrazolium. The accumulation o f s t a i n i n the s i n g l e midpiece was measured d i r e c t l y by use o f a Zeiss Axiomat microscope and a photomultip l i e r tube. The accumulation of s t a i n was p r o p o r t i o n a l t o t i m e o f s t a i n i n g up t o 40 minutes. V a r i a t i o n o f substrate concentration, s t a i n i n g temperature, and pH i n d i c a t e d t h a t t h e enzyme had expected enzyme p r o p e r t i e s I n sperm from mice a-GPD can be i n a c t i v a t e d by heat treatment o f the sperm. I n sperm from DBA/2J mice t h e h a l f - l i f e o f a-GPD a t 60°C was 42 minutes and a t 63°C was 18 minutes. I n v i t r o the h a l f - l i f e a t 63°C was 8 minutes, i n d i c a t i n g t h a t t h e r e was o n l y a s l i g h t d i f f e r e n c e i n t h e thermola b i l i t y f o r a-GPD i n s i t u and i n v i t r o . The a-GPD i n sperm from C57BL/6J mice was s l i g h t l y more s e n s i t i v e t o the heat treatment i n s i t u than t h e same enzyme i n DBA/2J mice. A f t e r heating o f sperm from DBAIPJ mice t o 63°C f o r 60 minutes most sperm do n o t s t a i n a t a l l ; however, a few sperm were stained s t r o n g l y . I t i s p o s s i b l e t h a t these sperm could be mutants c o n t a i n i n g thermo-resistant enzymes. The use o f histochemical techniques f o r development of systems f o r study o f mutagenesis i n s i n g l e c e l l s i n whole animals w i l l be discussed.

e.

(Ac-1 3) ESTIMATION OF RATES OF NONDISJUNCTION IN DROSOPHILA FEMALES. A. J. Katz, Department of Biological Sciences, Illinois State University, Normal, IL 61761. One of the simpler methods of detecting nondisjunction in Drosophila involves the exposure to a suspected mutagen o f females homozygous for a readily visible sex-linked recessive mutant gene (Traut, 1964, Mutat. Res. 1: 157-162). The treated females are mated to wild-type males and the F 1 flies are scored for "regular" progeny (wild-type 9 9 and mutant d8) and "exceptional" progeny (mutant 9 9 and wild-type dd). The exceptional flies

132

Abstracts

result from nondisjunction and/or chromosome loss of the X-chromosomes during oogenesis. By employing a mathematical model which describes the fate of primary oocytes during meiosis, one can derive an estimate of the frequency of nondisjunction in treated females (this includes nondisjunction at both anaphase-1 and anaphase-2 of meiosis). One can also derive an estimate of the frequency of chromosome loss during oogenesis (chromosome loss is here defined as the production o f nullo-X eggs by any means other than nondisjunction). The mathematical model allows for the -differential viabilities among the F1 flies and also allows for the numbers of functional gametes from the male parents (i.e., the numbers of X-bearing and Y-bearing sperm) to differ from a 1:l ratio. Both estimates are functions solely of the observed numbers of F1 flies in the four phenotypic classes, and both estimates may be statistically described as unbiased, sufficient and consistent. Approximate standard errors are also available. (Ad) MECHANISMS OF MUTAGENESIS

Chaired by: H. E. Brockman I l l i n o i s State University and

M. A . Conkling National I n s t i t u t e o f Environmental Health Sciences

(Ad-1 ) COMPARISON OF THE MUTAGENIC EFFECTS OF 2-AMINOPURINE AND ACTINOMYCIN D IN EXCISION-REPAIR DEFICIENT AND WILD-TYPE TWO-COMPONENT HETEROKARYONS OF NEUROSPORA CRASSA. F. J. de Serres, H. F. Brockman, C. Y. Hung* and T. M. Ong. National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 and Illinois State University, Normal, IL 61761

A comparison has been made between the mutagenic effects of 2-amino purine (2AP) and Actinomycin-D (Act-D) in heterokaryon 12 (wild-type) and heterokaryon 59 (hornozygous for excision-repair deficient mutation uvs-2) with regard to mutation-induction in the ad-3 region. In both experiments chemicals were tested by using growing cultures of singlecolony isolates. Comparable concentrations of each chemical gave no significant differences between the ad-3 forward-mutation frequencies in heterokaryons 12 and 59. (Mutation Res. 53, 81, 1978) Genetic analysis of the 2 AP-induced ad-3 mutants showed that 88.4% point mutations (43R) and 11.6% multilocus deletion mutations ( M I R ) were induced in mutations and heterokaryon 12, whereas in heterokaryon 59, 32.1% MR 67.9% e I R mutations were found. No difference was fouria between the complementation patterns of 2 @-induced ad-3B mutants. Genetic analysis of the Act D-induced ad-3B mutants showed comparable percentages of adjR and H I R mutations in both heterokaryon 12 (69.7% and 3 0 . 3 % , respectively) and heterokaryon 59 (68.9% and 31.1%, respectively). However a marked difference was found between the complementation patterns of ad-3B mutants. In heterokaryon 12 only 17.2% are complementing and of these 45.0% have nonpolarized complementation patterns; in heterokaryon 59, 57.4% are complementing with 26.0% having nonpolarized patterns. The genetic basis for these striking differences in the spectra of specific locus mutations is as yet unknown.

Abstracts

133

(Ad-2) POTENT MUTAGENICITY OF 6-N-HYDROXYLAMINO PURINE AND 2-AMINO-N6HYDROXYADENINE AT THE AD-3 REGION OF NEUROSPORA CRASSA. H. E. Brockman, c. Y. Hung", F. J. d e S e r r e s , and T. M . Ong, I l l i n o i s S t a t e U n i v e r s i t y , Normal, I L 61761 and N a t i o n a l I n s t i t u t e of Environmental H e a l t h S c i e n c e s , Research T r i a n g l e Park, NC 27709. Base a n a l o g u e s have been used i n f r e q u e n t l y i n m u t a t i o n r e s e a r c h i n e u k a r y o t e s , and o n l y two base analogues-2-aminopurine (2AP) and 5bromodeoxyuridine-have been used e x t e n s i v e l y i n b a c t e r i a and phages. We have r e p o r t e d t h a t ZAP i s a weak mutagen a t the ad-3 r e g i o n i n growing c u l t u r e s of Neurospora crassa. ZAP induced less t h a n a t e n - f o l d i n c r e a s e over t h e s p o n t a n e o u s ad-3 m u t a t i o n f r e q u e n c y , and t h e f r e q u e n c i e s d i d n o t d i f f e r i n heterokaryon-59 (H-59, homokaryotic f o r t h e e x c i s i o n r e p a i r d e f i c i e n t gene uvs-2) and heterokaryon-12 (H-12, uvs-2+) (Brockman, Hung, F r e e s e (1968, Mutation Ong, and d e S e r r e s , 1978, Mutation Res. 53:81). Res. 5:299) r e p o r t e d t h a t 6-N-hydroxylarnino p u r i n e (HAP) i s l e s s mutagenic t h a n ZAP i n two rII m u t a n t s of phage T4. We r e p o r t h e r e t h a t HAP i n d u c e s a h i g h f r e q u e n c y of ad-3 m u t a t i o n i n growing c u l t u r e s of H-12 and H-59. The dose-response c u r v e s i n t h e two s t r a i n s a r e n o t a p p r e c i a b l y d i f f e r e n t . The maximum f r e q u e n c y of ad-3 m u t a t i o n is o v e r 100 times g r e a t e r t h a n t h e spontaneous f r e q u e n c y a t c o n c e n t r a t i o n s t h a t are v e r y much less than t h o s e used i n our e a r l i e r s t u d i e s on 2AP. J a n i o n (1978, Mutation Res. 56:225) r e p o r t e d r e c e n t l y t h a t a n o t h e r p u r i n e a n a l o g u e , 2amino-N6-hydroxyadenine (AHA), i s much more mutagenic t h a n 2AP i n rev e r s i o n tests w i t h c e r t a i n b a c t e r i a l s t r a i n s . Our e x p e r i m e n t s show t h a t AHA i s a l s o a p o t e n t mutagen a t t h e ad-3 r e g i o n i n growing c u l t u r e s of [Research supported by N W -~ N. crassa.

(Ad-3)

GENETIC ANALYSIS OF AD-3 MUTANTS INDUCED BY AF-2 AND OTHER NITROFURANS I N NEUROSPORA CRASSA. T . Ong and F. J . d e S e r r e s . N a t i o n a l I n s t i t u t e f o r O c c u p a t i o n a l S a f e t y and H e a l t h , ALOSH, Morgantown, WV 26505 and N a t i o n a l I n s t i t u t e of Environmental H e a l t h S c i e n c e s , Research T r i a n g l e P a r k , NC 27709 Our p r e v i o u s s t u d i e s have shown t h a t AF-2, SQl8506 and FANFT are p o t e n t mutagens i n Neurospora c r a s s a . AF-2, a n a n t i b a c t e r i a l a g e n t , w a s e x t e n s i v e l y used as a food a d d i t i v e i n Japan. SQ18506 i s a n a n t i s c h i s t o soma1 a g e n t w h i l e FANFT i s a p o t e n t c a r c i n o g e n i n s e v e r a l mammalian s p e c i e s . The g e n e t i c damage produced by t h e s e c h e m i c a l s i n N . c r a s s a h a s been c h a r a c t e r i z e d by a series of g e n e t i c t e s t s . R e s u l t s of t h e g e n e t i c a n a l y s i s of ad-3 mutants induced by t h e s e n i t r o f u r a n s i n d i c a t e t h a t a l l t h r e e a g e n t s induce a h i g h frequency of b a s e - p a i r s u b s t i t u t i o n m u t a t i o n s and a low frequency of m u l t i l o c u s d e l e t i o n s . I n a d d i t i o n , t h e g e n e t i c a l t e r a t i o n s induced by t h e s e compounds probably i n c l u d e frames h i f t m u t a t i o n s and i n t r a g e n i c d e l e t i o n s . Comparison of t h e complem e n t a t i o n p a t t e r n s among t h e n i t r o f u r a n - i n d u c e d ad-3B m u t a n t s and t h o s e induced by o t h e r chemical a g e n t s s u g g e s t s t h a t t h e mechanisms of m u t a t i o n i n d u c t i o n by AF-2, SQl8506 and FANFT i n N . c r a s s a are p r o b a b l y s i m i l a r t o t h o s e of monofunctional a l k y l a t i n g a g e n t s .

(Ad-4) THE CHARACTERIZATION OF STREPTONIGRIN-INDUCED MUTATION AT THE AD-3 REGION I N A TWO-COMPONENT HETEROKARYON OF NEUROSPORA CRASSA. D. L. Weaver, I l l i n o i s S t a t e U n i v e r s i t y , Normal, I L 61761. The a n t i b i o t i c and a n t i n e o p l a s t i c a g e n t s t r e p t o n i g r i n (SN) c a u s e s g r e a t e r l e t h a l i t y i n t h e p r e s e n c e t h a n i n t h e absence of 02 i n b a c t e r i a SN i s a p o t e n t i n d u c e r (White and White, 1968, Mol. Pharmacol. 4:549). of chromosome a b e r r a t i o n s i n c u l t u r e d human l e u k o c y t e s (Cohen e t a l . ,

134

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1963, Proc. Nat. Acad. Sci. USA 50:16) and Vicia faba (Kihlman, 1964, In 1. faba, SN induced more aberrations under an Mutation Res. 1:54). anoxic condition than when 02 was present. In the present work, the killing and mutagenic activities of SN were studied by suspending conidia from a two-component heterokaryon (heterokaryon-12) of Neurospora crassa in a O.lM, pH 7 phosphate buffer and bubbling with 02 or N2 gas 2 hr before and 4 hr during exposure to SN (60, 120, 180, 360, The killing or mutagenic activities of SN were not apand 600 preciably different in the two gases. A sample of ad-3 mutants was analyzed from a dose of SN (360 ~ J Mfor 4 hr) in the presence of 02 that resulted in 29% survival and 135 ad-3 mutants/l06 survivors. A high frequency (25%) of the M m u t a n t s were multilocus deletions. Among the ad-3B mutants, 34% exhibited allelic complementation, and among these complementing ad-3B mutants, 79% had nonpolarized complementation patterns. [Research supported by DOE.]

m).

(Ad-5) LONG-RANGE BASE-PAIR EFFECTS ALTERING 2-AMINOPURINE MUTAGENESIS IN BACTERIOPHAGF T4. M. 4. Conkling. National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.

Earlier studies showed that the 2-aminopurine-induced mutation rate at a particular base pair can be influenced by the base pair adjacent to, o r one additional base pair removed from, the measured site. This study extends to 0.3 map units (about 30 base pairs) the distance at which a single basepair substitution can exert such an effect. The presence of a base-pair gene) reduces the 2substitution (defined as a % mutation in the aminopurine-induced reversion of an rlIA amber mutation approximately three-fold. The mutation also reduces the corresponding UAA + UAG conversion by three-fold. The UAA UGA conversion is reduced about eightfold in the presence of the mutation, while the reversion of the ochre CAA) is not affected. Selection controls show no codon to glutamine (UAA measurable selection against the marker. The possibility that either glutamine or tryptophan revertants of the amber codon would be non-mutant in the absence of the allele, but still mutant in its presence, could explain the observations. Control experiments measuring the efficiencies of plating of various homologous nonsense mutations on the appropriate amino acid-inserting suppressor strains demonstrate this is not the case.

+ J

+

+

(Ad-6) FRP-MESHIFT MUTATIONS I N BACTERIA PRODUCED I N THE DARK BY SEVERAL FUROCOUMARIrJS; ABSENCE OF A C T I V I T Y OF 4,5'8- TRI MET!1YLPSORALEN. M.J. Ashwood-Smith:

S e n i o r NATO Research F e l l o w 1977-78

L a b o r a t o i r e de B i o l o g i e Physico-Chimique, ENSBANA; U n i v e r s i t e de D i j o n , 21000 D i j o n (France). P r e s e n t l y : Department o f B i o l o g y , U n i v e r s i t y o f V i c t o r i a , V i c t o r i a , B.C., Canada V8W 2Y2 L i g h t p h o t o s e n s i t i z e d l e t h a l and mutagenic a c t i o n i s w e l l documented f o r a number o f furocoumarins i n c l u d i n g numerous n a t u r a l l y o c c u r i n g psoral e n s and d e r i v a t i v e s . 8-methoxypsoralen i s a f r a m e s h i f t mutagen i n t h e absence o f l i g h t and I w i s h t o r e p o r t t h a t t h e " p a r e n t compound", p s o r a l e n and t h e n o n - c r o s s l i n k i n g , a n g u l a r furocoumarin, a n g e l i c i n , a r e a l s o weak f r a m e s h i f t mutagens when t e s t e d i n t h e d a r k i n E. c o l i l a c - . In light p h o t o s e n s i t i z e d l e t h a l r e a c t i o n s t h e r a n k o r d e r i n b a c t e r i a and y e a s t covers a range o f a b o u t 17 from a n g e l i c i n as t h e l e a s t a c t i v e , t h r o u g h p s o r a l e n , 8-methoxypsoralen, and 4,5' , 8 - t r i m e t h y l p s o r a l e n (TMP) as t h e most a c t i v e . S u r p r i s i n g l y TMP was found t o be t o t a l l y w i t h o u t a c t i o n as a " d a r k i n d u c i n g " f r a m e s h i f t mutagen. These r e s u l t s , o f i n t e r e s t i n thems e l v e s and perhaps i n terms o f p s o r i a s i s treatment, a l s o suggest t h a t a s i m p l e view o f i n t e r c a l a t i o n as an a p r i o r i p r e r e q u i s i t e f o r l i g h t photos e n s i t i z a t i o n i s t o o s i m p l i s t i c and-perhaps wrong.

Abstracts

135

(Ad-7) LACK OF MUTAGENICITY OF TRYPTOPHAN AND ITS METABOLITES I N SALMONELLA R . B. Baker", Department o f A.S.Shetty TYPHIMURIUM REVERSION ASSAY1. Biology, F l o r i d a A & M U n i v e r s i t y , T a l l a h a s s e e , FL 32307 w i t h J. L. E p l e r and T. K . Rao, Biology D i v i s i o n , Oak Ridge N a t i o n a l L a b o r a t o r y , Oak Ridge, TN 37830.

,

S e v e r a l m e t s b o l i t e s o f t r y p t o p h a n such as h y d r o x y a n t h r a n i l i c a c i d , Xanthurenic a c i d and Kynurenic a c i d have been i m p l i c a t e d as c a u s a t i v e a g e n t s o f human b l a d d e r c a n c e r f o r o v e r two d e c a d e s . A b i l i t y t o c a u s e chromosomal a b e r r a t i o n s by some o f t r y p t o p h a n m e t a b o l i t e s (3-hydroxyant h r a n i l i c a c i d and 3-hydroxjrkynurenin) i n c u l t u r e d human s o m a t i c c e l l s S i n c e most c a r was r e p o r t e d by Kuznetsova (Nature 222 (1969) 484-485). c i n o g e n s c a u s e somatic m u t a t i o n s , we have examined t r y p t o p h a n and i t s m e t a b o l i t e s for t h e i r a b i l i t y t o r e v e r t h i s t i d i n e a u x o t r o p h i c t e s t e r s t r a i n s o f S a l m o n e l l a typhimurium developed by D r . B. N. Ames. The b a s i c set of f i v e s t r a i n s (TAl535, TA1537, TA1538, TA98 and TA100) w a s used i n t h e p r e s e n c e o r a b s e n c e o f A r o c l o r induced rat l i v e r homogenate (S-9 mix) f o r m e t a b o l i c a c t i v a t i o n . Tryptophan and n i n e m e t a b o l i t e s of t r y p t o p h a n v i z : kynurenic a c i d , hydroxykinurenine, k y n u r e n i n e s u l f a t e , a n t h r a n i l i c a c i d , h y d r o x y a n t h r a n i l i c a c i d , c i n n a b a r i c a c i d , x a n t h u r e n i c a c i d , quinol e n i c a c i d and i n d o l e f a i l e d t o induce r e v e r t a n t s e i t h e r d i r e c t l y o r upon m e t a b o l i c a c t i v a t i o n i n a p l a t e - i n c o r p o r a t i o n a s s a y and i n a l i q u i d p r e - i n c u b a t i o n a s s a y ( o n e hour i n c u b a t i o n a t 37OC on s h a k i n g ) . Even though t h e m u t a g e n i c i t y r e s u l t s are i n c o n c l u s i v e i n d e t e r m i n i n g t h e b i o l o g i c a l a c t i v i t y o f t h e s e m e t a b o l i t e s , t h e y once a g a i n emphasize t h e c a u t i o n one h a s t o e x e r c i s e i n drawing c o n c l u s i o n s from s h o r t - t e r m g e n e t i c a s s a y s . R e s e a r c h s u p p o r t e d by N a t i o n a l Cancer I n s t i t u t e g r a n t number CA18817 and N I H , MBS g r a n t number RR 08111 t o F l o r i d a A & M U n i v e r s i t y .

(Ad-8) THE INFLUENCE OF HISTIDINE ON REVERSION RATES I N SALMONELLA TYPHIMURIUM TA 100. J.B. Johnston, i n t r . by D. Schaeffer, U n i v e r s i t . v o f I l l i n o i s , Urbana, I 1 61801. The Salmonella typhimurium s t r a i n s used i n the Ames r e v e r s i o n assay e x h i b i t a s y s t e m a t i c i n c r e a s e o f spontaneous r e v e r s i o n frequency w i t h added h i s t i d i n e , as has been p r e v i o u s l y r e p o r t e d by Ames (E. Yamasaki, and B.N. Ames. 1977.Proc. N a t l . Acad. S c i . USA 74, 3555-3559). With TA 1535 grown i n minimal l i q u i d medium we have X w n t h a t t h e i n c r e a s i n g spontaneous r e v e r s i o n frequency w i t h i n c r e a s i n g h i s t i d i n e r e f l e c t s growth on t h e r e v e r s i o n t e s t p l a t e . TA 100 however e x h i b i t s a b i p h a s i c i n c r e a s e o f spontaneous r e v e r t a n t s w i t h i n c r e a s i n a h i s t i d i n e , a p p a r e n t l y due t o t r a n s i e n t changes i n t h e spontaneous r e v e r s i o n r a t e d u r i n g t h e f i r s t d i v i s i o n s on t h e p l a t e . H i s t i d i n e a l s o i n f l u e n c e s t h e frequency of r e v e r s i o n induced b y rnutaaens. These r e s u l t s w i l l be i n t e r p r e t e d i n terms o f t h e i n t e r a c t i o n o f p r o t e i n s encoded on p l a s m i d and chromosomal DNA. (Ad-9) KINETICS, DECOMPOSITION PRODUCTS AND FACTORS AFFECTING THE MUTAGENIC S. M i l s t e i n * and J . B . G u t t e n p l a n , ACTIVITY OF N-METHYL-N-NITROSOUFSA. New York U n i v e r s i t y D e n t a l C e n t e r , New York, N.Y. 10010. (NMU) i s r e p o r t The r a t e o f decomposition o f N-nitroso-N-methylurea e f i n d t h a t one e d l y dependent on t h e b u f f e r i n which it is d i s s o l v e d . W r e a s o n f o r t h e dependency relates t o t h e b u f f e r c a p a c i t y o f t h e media s i n c e NMU (monitored a t 390 nm) decomposes t o y i e l d a c i d i c p r o d u c t s and i s known t o b e more stable i n m o d e r a t e l y acidic media. The i n i t i a l h a l f l i v e s o f 0.01 M NMU s o l u t i o n s a t 37O i n 0 . 1 M T r i s , p h o s p h a t e o r Hepes were 4,5.5 and 6 min resp. The p H when decomposition w a s complete w a s 7.15 i n Hepes, 7.3 i n p h o s p h a t e and 7.4 i n T r i s . I n water decomposition

136

Abstracts

w a s n o t d e t e c t a b l e a f t e r 1 h r and t h e p H of t h e NMU s o l u t i o n w a s 4.2. When a n aqueous u n b u f f e r e d s o l u t i o n of NMU w a s b r o u g h t t o pH 7 . 4 and m a i n t a i n e d a t t h a t pH by a d d i t i o n of NaOH it w a s found t h a t decomposition The a c i d t h a t w a s g e n e r a t e d t i t r a t e d o f 0.01 M NMU g e n e r a t e d 0.006 M H'. w i t h a p K o f 4.0, c o n s i s t e n t w i t h a report on t h e p r o d u c t i o n of i s o c y a n i c a c i d f r o m NMU. The d e c o m p o s i t i o n s w e r e a l s o c a r r i e d o u t i n s e a l e d I t w a s found v e s s e l s and t h e dead s p a c e a n a l y s e d by g a s chromatography. t h a t N2 was g e n e r a t e d s t o i c h i o m e t r i c a l l y from NMU i n a l l t h r e e b u f f e r s . Mutagenesis i n d u c e d by NMU i n S a l m o n e l l a typhimurium TA 1530 i n a l i q u i d phase a s s a y w a s m o r e e f f i c i e n t a t p H 6.0 t h a n pH 7 . 4 even though decomposition of NMU, presumably t o mutagenic p r o d u c t s w a s more r a p i d a t p H 7.4. T h i s may i n d i c a t e t h a t a s u b s t a n t i a l f r a c t i o n NMU i n d u c e d m u t a g e n e s i s r e s u l t s from i n t r a c e l l u l a r d e c o m p o s i t i o n o f NMU. T h i s work w a s s u p p o r t e d by G r a n t CA-19023 by t h e NCI.

(Ad-10) FORMATION OF CYCLOBUTANE PYRIMIDINE DIMERS I N THE DNA OF ESCHEii'ICHIA COLT EXPOSED TO NEAR-UV LIGHT. John D. C h i l d s , Michael J. E l l i s o n * , and Raymond P i l o n * , Atomic Energy of Canada L i m i t e d , Chalk R i v e r , O n t a r i o , KOJ 1 J 0 , Canada. The f o r m a t i o n of c y c l o b u t a n e p y r i m i d i n e d i m e r s i n DNA h a s o n l y been s t u d i e d in,de_taLl u s i n g far-UV i r r a d i a t i o n . I n t h e s e s t u d i e s t h e a v e r a g e r a t i o of CC:CT:TT formed i n E. cozi DNA w i t h low f l u e n c e s ( ~ 3 0 0J/m2) of We have measured t h e y i e l d 254 nm o r 265 nm i r r a d i a t i o n w a s 0.35:0.65:1.0. of t h e s e t h r e e dimer s p e c i e s d u r i n g i r r a d i a t i o n of E. c o z i - D N A w i t h UV l i g h t from 254 nm t o 320 nm. We found t h a t t h e y i e l d of CT r e l a t i v e t o fT a t low f l u e n c e s i n c r e a s e d from 0.6:l.O a t 254 nm t o ? maximum of 1 . 5 : l a t [The CC d i m e r s r e a c h e d a n 313 nm and t h e n d e c l i n e d t o 0 . 9 : l a t 320 nm. e q u i l i b r i u m between f o r m a t i o n and s p l i t t i n g a t v e r y low f l u e n c e s a t a l l wavelengths and d i d n o t a c c o u n t f o r more t h a n 25% of t h e t o t a l p y r i m i d i n e d i m e r s a t t h e l o w e s t f l u e j c e ( e q u i v a l e n t t o 200 J / m 2 a t 254 nm) a t any_of t h e w a v e l e n g t h s . ] Thus CT d i m e r s a r e n u m e r i c a l l y more i m p o r t a n t t h a n TT d i m e r s i n 313 n m - i r r a d i a t e d E. cozi DNA and p r o b a b l y i n 313 n m - i r r a d i a t e d DNA from o t h e r o r g a n i s m s . T h e i r a c t u a l i m p o r t a n c e in V i V o w i l l depend n o t o n l y on t h e i r r e l a t i v e rate o f f o r m a t i o n b u t on t h e r e l a t i v e rates of removal and t h e r e l a t i v e p o t e n t i a l f o r m u t a t i o n i n d u c t i o n of e a c h dimer species.

-

(Ad-1 1 ) COMPARATIVE EVALUATION OF THREE BACTERIAL DNA REPAIR TESTS. J o h n E . Heinze_,*M. L . A u g u s t i n e * a n d J o h n R . Roheim, Armour a n d Company, Armour R e s e a r c h C e n t e r , S c o t t s d a l e , A Z 85260 T h r e e b a c t e r i a l DNA r e p a i r t e s t s were e v a l u a t e d f o r t h e i r a b i l i t y t o complement t h e Ames S a l m o n e l l a m u t a g e n i c i t y t e s t i n a g e n e t i c t o x i c o l o g y s c r e e n i n g program. The tests exami n e d were t h e E. PolA t e s t ( S l a t e r , e t a l . , C a n c e r R e s . 3 1 : 9 7 0 , 1 9 7 1 1 , t h e B . s u b t i l i s r e c - a s s a y (Kada, e t a l . , Mutat. R e s . 16:165, 1972) and t h e E. c o l i RecBC test ( I c h i n o t s u b o , e t a l . , M u t a t . R e s . 4 6 : 5 3 , 1 9 7 7 ) . The e v a l u a t i o n w a s p e r f o r m e d w i t h 3 0 t e s t carcinogens/noncarcinogens representing a v a r i e t y of chemical s t r u c t u r e s including a number of c a r c i n o g e n s m i s s e d by t h e s t a n d a r d Ames p l a t e t e s t . The r e s u l t s i n d i c a t e t h a t t h e R e c B C t e s t i s n o t r e p r o d u c i b l e enough f o r u s e i n a s c r e e n i n g program. The P o l A t e s t a n d r e c - a s s a y are shown t o be u s e f u l a d d i t i o n s t o a s c r e e n i n g p r o g r a m b e c a u s e b o t h tests are r e p r o d u c i b l e a n d d e t e c t some c a r c i n o g e n s m i s s e d b y t h e Ames t e s t . However, b o t h t e s t s

Abstracts

137

a l s o m i s s some c a r c i n o g e n s d e t e c t e d by t h e Ames t e s t , e m p h a s i z i n g t h e i m p o r t a n c e of u t i l i z i n g a b a t t e r y of t e s t s i n a g e n e t i c t o x i c o l o g y s c r e e n i n g program. The PolA t e s t a p p e a r s t o b e t h e p r e f e r r e d b a c t e r i a l DNA r e p a i r t e s t bec a u s e it d e t e c t s a w i d e r s p e c t r u m o f d i r e c t - a c t i n g c a r c i n o g e n s t h a n t h e rec-assay

.

(Ad-1 2 ) A RECOMBINATIONAL PROCESS IS THE MAJOR PATHWAY FOR BOTH UV-REPAIR AND UVINDUCED MUTAGENESIS IN SCHIZOSACCHAROMYCES POMBE. Norman E. Gentner, Atomic Energy of Canada Limited, Chalk River, Ontario, KOJ 1J0, Canada. A recombinational process is known to be the major UV-repair pathway in the haploid fission yeast S. pombe. Both G1 and G2 phase cells show lethal enhancement (W-sensitization) in the presence of caffeine. We found that in UV-irradiated G2 phase cells, caffeine-sensitive repair began immediately and was completed before DNA synthesis resumed, whereas caffeinesensitive repair in G1 phase cells showed a considerable lag and then occurred concomitantly with DNA synthesis. This suggests that G2 cells perform a prereplicative (i.e. sister chromatid exchange) recombinational process, using the two DNA copies present at the time of UV-exposure. In contrast, G1 phase cells may have to replicate a lesion-containing DNA in order to obtain the necessary second copy with which to perform recombinational repair (i.e. post-replication repair). Both these modes may be manifestations of one recombinational mechanism, since the radl mutation, which causes loss of recombination proficiency, abolishes UV-sensitization by caffeine in both G1 and G2 phase cells. In the absence of caffeine, G2 cells are considerably more UV-resistant than G1 cells, but have similar UV-sensitivity in the presence of caffeine. The prereplicative process therefore is the more important for UV-resistance. Our data further indicates that recombinational repair is the major pathway responsible for W-induced mutagenesis: UV-sensitive mutants which lack a caffeine-sensitive UV-repair process are relatively UV-immutable, whereas mutants which retain UV-sensitization by caffeine are generally highly UV-mutable. The study of W-mutagenesis in G1 and G2 phase cells should indicate the relative error-proneness of the post-replication and prereplicative recombinational pathways.

(Ad-1 3 ) MJTAGENIC EFFECT OF TRANSFORMING RNA AND DNA TUMOR VIRUSES. Nirmal K. Mishra, Food and Drug Administration, Silver Spring, Md. 20910 A number of recent studies have demonstrated the simultaneous mutagenic and transforming effect of various chemical carcinogens. Although several arguments to the contrary have been made, usually the strong correlation for induction of these two genetic events is interpreted as experimental evidence for mutagenic hypothesis of cancer. Accordingly, it may be useful to investigate the mutagenic effect of the transforming tumor viruses. Two prototype RNA and DNA viruses-Kirsten Murine Sarcoma virus (Ki-MSV) and Polyoma virus (Py)--were used to determine the induction of mutation in Fischer rat (F111) cells by ouabain (Oua) and 8-azaguanine(Az) resistance assays. Since Flll cells are permissive for transformation by both Ki-MSV and Py, quantitative soft agar assays were also performed simultaneously. Under assay conditions, where unambiguous cell transformations can be demonstrated by KiMSV and Py, no significant alteration of the mutation frequencies post infection could be demonstrated. On the other hand, 3-Methylcholanthrene (3-Mc) induced transformation and simultaneous mutation with both Oua and Az assays. These results were further confirmed by constitutive temperature shift studies with a temperature sensitive Py (TsPy) mutant. Ts-Py induced temperature sensitive expression f o r cell transformation, but failed to alter the baseline frequencies for Oua

138

Abstracts

or Az-resistant subpopulations. A reconstruction experiment with previously characterized Oua-resistant Flll cells was performed to ascertain the limits of sensitivity of the assay. These results will be discussed in the context of previously published reports on the mutagenic effects on S V - 4 0 , a DNA tumor virus.

(Ad- 14 ) EFFECTS OF pH ON THE MUTAGI3”CI’IY OF SODIUM AZIDE IN NEUROSPORA C W S A AND SUDNELLA TYPHlbKRIUbl. C. R. Tomlinson, I l l i n o i s S t a t e University, Normal, I L 61761. The mutagenicity of sodium azide has been reported i n higher p l a n t s , bacteria, and mammalian c e l l s . The l i t e r a t u r e indicates t h a t an acidic pH i s required f o r sodium azide nutagenicity in barley, t h a t sodium azide reverts only c e r t a i n base-pair s u b s t i t u t i o n mutants of Salmonella typhimurium, t h a t excision repair-deficient s t r a i n s of b a c t e r i a a r e very s e n s i t i v e t o sodium azide mutagenicity, and t h a t DNA r e p l i c a t i o n and/or metabolism may be necessary for sodium azide mutagenicity. I have studied whether sodium azide is mutagenic a t the adenine-3 (ad-3) region of Neurospora crassa and whether sodium azide mutagenicity idependent i n N. crassa a n r t liimurium. fleterokaryon-12 of E. crassa is repair sufTicient , whereas hetero aryon-59 is excision-repair d e f i c l c n t (uvs-2). Conidia from both s t r a i n s were inactivated by sodium azide a t pH 3l i t t l e o r no inactivation was observed a t pll 4 through 8 . In both s t r a i n s , heterokaryotic conidia and the t o t a l population of conidia were inactivated t o the same degree by sodium azide a t pli 3 ; t h i s result i n dicates that sodium azide inactivated conidia by interacting with the cytoplasm rather than the nucleus. Sodium azide a t ptl 3 through 8 did not induce forward mutation a t the region i n r e s t i n g conidia o r growing cultures of e i t h e r heterokaryon. A lack of mutagenicity of sodium azide a t pH 3 through 8 was a l s o observed in an overlay reverse-mutation t e s t using a base-pair s u b s t i t u t i o n ( X 3 ) and a Cranieshift (N31) t e s t e r of y. crassa. The mutagenicity of sodium a z i J e i n s t r a i n s TAlOO and TA1535 of - typhimurium was independent of ptl i n the range of 3 through 8 . S. (Research supported by N l t l ; author’s present address is The University of Texas a t Austin, Austin, ‘TX 78712.)

+

ad-3

(Ba) SYMPOSIUM I : SISTER CHROMATID EXCHANGE

Chaired by: A. V . Carrano Lawrence Livermore Laboratory (Ba-1 )

THE FORMATION OF SCEs. s. Wolff U n i v e r s i t y o f C a l i f o r n i a , San Francisco

(Ba-2)

SISTER CHROMATID EXCHANGE: RELATION TO SINGLE GENE MUTATION, ACUTE AND CHRONIC EXPOSURE I N V I V O , AND HUMAN POPULATION MONITORING. A . V . Carrano Lawrence Livermore Laboratory

(Ba-3)

SISTER CHROMATID EXCHANGE I N VITRO, I N V I V O , AND I N UTERO E. L. Schneider N a t i o n a l I n s t i t u t e on Aging

Abstracts

139

Detection of genetic damage from exposure t o environmental pollutants is of utmost importance: genetic injury may signal increased r i s k of cancer

f o r individuaIs d i r e c t l y exposed or may place their unborn children a t r i s k of b i r t h defects. Most methods of detecting genetic injury are tedious or extremely cumbersome and few have been able t o measure e f f e c t s d i r e c t l y i n humans. The s i s t e r chromatid exchange assay potentially o f f e r s a solution t o t h i s d i l e m a . The objective of t h i s symposim w i l l be t o demonstrate that s i s t e r chroma t i d exchange i s a sensitive and quantitative indicator of chemical exposure and genetic injury. The discussions w i l l focus on the relation of s i s t e r chromatid exchange t o other biologically measureable endpoints and the application of the technique t o quantify genetic damage in v i t r o in animals and i n humans.

(Ca) CHEMICAL MUTAGENS Chaired by: A . D . Burrell I B Y Corporation

and V . F. Simnons S R I International

(Ca-1) MUTAGENICITY OF NEOCARZINOSTATIN IN ESCHERICHIA C m AND SALMONELLA TYPHIMURTUM. Eric Eisenstadt, Michael Wolf*, and Irving H. Goldberg*. Harvard __ University Schools of Public Health and Medicine. Boston, MA 02115 Neocarzinostatin (NCS) is an antibiotic protein of MW 10,700 which causes base-specific strand scissions in DNA in vitro. NCS is mutagenic for 1.& strains B and K12 and 2. typhimurium strains LT2 and LT7. Lipopolysaccharide defective derivatives of these bacteria are 100 times more sensitive than their parental strains to the mutagenic and lethal effects of NCS treatment. The mutagenicity of NCS towards B and K12 was detected by measuring the reversion of ochre mutations. The mutagenicity of NCS towards S. typhimurium was detected by measuring either the induction of fonmrdmutations leading to resistance to Lazetidine-2-carboxylic acid o r by the reversion o € the hisC117 ochre mutation. Two of the mutations carried by the standard Ames tester strains, the hisG46 missense mutation and the hisD3052 frameshift mutation. are not reverted by NCS treatment. NCS induced reversion of the trpochre mutation in E. &c B and of the hisC117 ochre mutation in 2. typhimurium is enhanced in strains hearing the pKMlOl plasmid. Three other his- ochre mutations in 5. typhimurium (hisC342, hisC354, and hisC502) are not measurably reverted following NCS treatment unless pKMlOl is present. Since ochre mutations are, in principle, both revertable and suppressable, we are presently determining the number of true ochre revertants and ochre suppressor mutations induced by NCS treatment. These studies contribute to our understanding of the influence of both specific DNA base sequences and cellular DNA repair capacity on the mutability of specific alleles.

e. so

140

Abstracts

(Ca-2) MUTAGENICITY SCREENING OF ANTHRAQUINONE, BENLANTHRONE AND TRIPHENYLMETHANE DYES IN THE B A C I L L U S S U B T I L I S REC ASSAY: COPJARISON WITH his- REVERSION IN SALMONELLA TYPHIMURIUM TA TESTER STRAINS. R. Driscoll*, P. S. Dietrich and J. P. Brown, Dynapol, 1454 Page Mill Road, Palo Alto, CA 94304.

About 140 dyes including 115 9,lO-anthraquinones (AQs) were screened for DNA damaging ability in the B . subtilis r e c M45/H17 assay of Kada. No mammalian metabolism was involved in the tests. Thirty-eight AQs were found to give significant DNA damaging effects in the B . subtilis rec assay compared to 4 (out of 20) benzanthrones and 2 (out of 8 ) triphenylmethane dyes. Previous testing of these agents in 5 S. typhimurium strains (TA:1535,100,1537,1538,98) yielded 4 6 direct acting AQ mutagens (i.e. not requiring microsomal activation), 7 benzanthrones and no triphenylmethanes. Significantly, a l l except two of the AQs found to damage DNA in B . subtilis, but to be apparently non-mutagenic in the Salmonella/mammalian microsome assay, contained sulfonate or sulfide functional groups. These were: 1-sulfonato-AQ; 1,8-disulfonato-AQ; 1,2-dihydroxy-3-sulfonato-AQ; 1,8-di(2-carboxyethyl sulfide)-AQ; 1-amino-2-sulfonato-AQ; and 1,4di[3-(3-sulfobenzyl)cyclohexylamino]-5-hydroxy-AQ. Significant differences in cell permeability with respect to these anionic dyes between the S. typhimurium and B . subtilis test strains may partially explain this phenomenon.

(;a-3) MUTAGENICITY OF RHODAMINE DYES I N THE SmLLA/MICROSCME TEST -Earle R. N e s t m , Tibor I. Matula, and David J. Kowbel"

Environmental Mutagenesis Section, Lhvimrunental Toxicology Division, Department of National Health and Welfare, Ottawa, CMtario K1A OL2, and Bio-Research Laboratories, Ltd., Senneville, Quebec, Canada Comnercial Fhodamine dyes l3 and 6G induce His+ reversion mutations in Salmonella with metabolic activation by Aroclor-induced, rat liver m e n a t e ( S 9 ) . Rhodanine B induces only frameshift mutations (in strains TAl538 and TA98), whereas Rhodamine 6G induces both frameshift (in strains TAl537, TA1538, and TA98) and base substitution (in strain TAlOO) mutations. Rhodamine 6G is genetically m r e active and more toxic than Rhodmine 9. Rhodamine 6G and Rhodarm'ne B induce doublings of H i s + revertants at doses of 0.02 and 0.52 p l e s per plate, respectively. Since the comnercial dyes are only 90-95% pure, purified dyes were obtained using thin layer chromatography. Further testing of the pure dyes in Salmonella revealed that pure Rhodamine 6G is a mutagen, but pure Rhodamine B is not. Impurities from comnercial Rhodamine B demonstrate the same extent of mutagenicity as the comnercial dye. Cmmercial Rhodamine dyes B and 6G also are reported to have genetic activity in cul turxd CHO cell s (Douglas and Grant, these proceedings) . (Ca-4) GENETIC ACTIVITY OF RHODAMINE DYES IN CHO CELLS. George R. Douglas and Caroline E. Grant", Environmental Mutagenesis Section, Environmental Toxicology Division, Department of National Health and Welfare, Ottawa, Ontario, K1A OL2. The commercially available dyes Rhodamine B and Rhodamine 6G induce single strand DNA damage in CHO cells, as detected by alkalitfe sucrose gradient sedimentation analysis. DNA dtmage (0.17 breaks110 daltons) is evident after treatment with 9 x 10 M Rhodamine B, whereas Rhodamine 6G is approximately 10 times more efficient in inducing equivalent DNA damage. Conversely, Rhodamine B is more toxic than 6G at concentrations

Abstracts

141

pr_o$ucing equivalent DNA daqge. The Do dose of Rhodamine B (9.6 x 10 M) induces 0.2 breaks/lO daltons, whereas equivale_nh DNA damage is induced by Rhodamine 66 at a concentration (1.2 x 10 M) which reduces colony forming ability by-gnly 20% (80% survival). The D dose for Rhodamine 6G is 1.75 x 10 M. Since the mutagenicity of ahodamine B in the Salmonella/microsome test is due mainly to impurities (Nestmann et al, these proceedings) it is possible that the high relative lethality of Rhodamine B is due in part to toxic effects of the pure fraction that are not related to DNA damage.

(Ca-5) POLYAMINE-NITRITE REACTION PRODUCTS WITH MUTAGENIC ACTIVITY. H. Kong* , , and M.L. Murray. It has been reported that polyamines potentiate the mutagenic action of nitrous acid. We have investigated further the chemical and biological properties of nitrite-polyamine reaction products. The mutagenic activity can be extracted into organic solvents and concentrated by evaporation under a stream of nitrogen. At least two mutagenic compounds can be separated from the reacted mixture by thin layer chromatography (TLC). Structural requirements for production of mutagenic activity from the amine reactant seem t o include at least one secondary amine bracketed by two primary amines separated by a chain of carbons relatively unencumbered with other functional groups. Spermine and spermidine are both active; spermidine is more s o . Putrescine, lysine, cadaverine, ornithine and agmatine are not active in our hands. Polyamine-nitrite mixtures reacted at pH 4.2 and then neutralized t o inactivate the mutagen(s) also contain a reversible growth inhibitor. The characteristics of the inhibitory activity suggest a non-covalent interaction between a polyamine-nitrite reaction product and some component or components of the cell.

M. Murphey-Corb

(Ca-6) MUTAGENICITY OF ALIPHATIC NITROSAMINES IN DROSOPHILA MELANOGASTER. Bobbie Brewen and C. E. Nix, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830. Fifteen selected aliphatic nitrosamines, diluted in 2% sucrose, were fed for 48 hours to Drosophila melanogaster males. Toxicity was determined for each compound and at least three concentrations were chosen for mutagenicity testing using X-linked recessive lethals as the end-point. A brood analysis was also carried out for each compound. Eleven of the fifteen compounds were mutagenic. The mutagenic effect ranged from very weak (1.00% SLRL) to very strong (Q 40.00% SLRL). With the exception of dicyanodiethylamine all those compounds that were mutagenic were also carcinogenic in the rat. Of the four which were negative, two have been shown to be weak carcinogens. Comparisons between the X-linked recessive lethal assay and the Salmonella/mammalian microsome assay will be discussed. Brood analysis revealed that most of the compounds were effective mutagens f o r all meiotic and post-meiotic stages of spermatogenesis. The highest yield of lethals was, however, in brood-2 which corresponds to the early spermatid and spermatocyte stages. Research jointly sponsored by the Environmental Protection Agency (IAC-D5-E681; Interagency Agreement 40-516-75) and the Division of Biological and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation.

(Ca-7) CYTOGENETIC STUDIES IN VICIA FABA WITH SOME INSECTICIDES, FUN GI CI DE S AND HE RBI CI DE S H.N.B. Gopalan and G.D.E. Njagi*, Department of Botany, Kenyatta University College, P.O. Box 4 3 8 4 4 , Nairobi, KENYA.

.

142

Abstracts

The e v e r i n c r e a s i n g u s e of p e s t i c i d e s , f u n g i c i d e s , h e r b i c i d e s and food p r e s e r v a t i v e s both i n a z r i c u l t u r e and industry calls f o r a detailed study of t h e i r genetic effects, We h a v e u n d e r t a k e n a n i n t e n s i v e s t u d y o f t h e g e n e t i c e f f e c t s o f some of t h e commonly u s e d c h e m i c a l s u s i n g D r o s o p h i l a , I n t h i s communication we Salmonella and p l a n t test systems. r e p o r t t h e c y t o g e n e t i c e f f e c t s of t h e i n s e c t i c i d e s : summithion, l a n n a t e , c a r b i c r o n , t h i o d a n and k e l t h a n e ; t h e fungicides: b e n l a t e , d i a t h a n e M45 a n d a l a d r i n ; a n d t h e herbicides: l a s s o , r a m r o d , r o u n d up a n d grammoxone on V i c i a A l l t h e a b o v e named p e s t i c i d e s faba root meristematic cells. induce acute m i t o s t a t i c e f f e c t s , formation of pycnotic n u c l e i , and p r e m a t u r e chromosome c o n d e n s a t i o n . Also, carbicron, k e l t h a n e , lasso and ramrod i n d u c e f o r m a t i o n of anaphase bridges and r e l a t i v e l y s c a r c e micronuclei, suggesting t h a t t h e y may p o s e a g e n e t i c h a z a r d t o m a n , p l a n t s a n d a n i m a l s exposed t o those p e s t i c i d e s . Preliminary d a t a from t h e other test systems i n d i c a t e t h a t they do n o t induce sex linked r e c e s s i v e l e t h a l s i n D r o s o p h i l a , b u t a few of them i n c l u d i n g k e l t h a n e and l a s s o i n d u c e r e v e r s i o n m u t a t i o n s i n S a l m o n e l l a t yph imu r ium.

-

(Ca-8) ANALYSIS OF THE MUTAGENIC PROPERTIES OF PESTICIDES INCORPORATING ANIMAL AND PLANT ACTIVATION. ?I. J . P l e w a , E . D. Wagner* a n d J . M. G e n t i l e . U n i v e r s i t y o f I l l i n o i s , U r b a n a , I L 61801 and Hope C o l l e g e , H o l l a n d , M I 49423. We h a v e b e e n c o n d u c t i n g a c o m p reh en si v e a n a l y s i s of t h e m u t a g e n i c p r o p e r t i e s of 3 5 p e s t i c i d e s o r c o m b i n a t i o n o f p e s t i c i d e s . Our s t u d y i s u n i q u e i n t h a t we (1) u s e a p r o k a r y o t e ! S a h o n e l l a typhimurium), a lower e u k a r y o t e (Saccharoqces cerevisiael a n d a h i g h e r e u k a r y o t e (Zea mays) a s g e n e t i c i n d i c a t o r o r g a n i s m s , ( 2 ) i n c o r p o r a t e mammalian a n d g r e e n p l a n t a c t i v a t i o n r e g i m e s , and ( 3 ) e v a l u a t e e a c h p e s t i c i d e u n d e r t h e c o n d i t i o n s o f modern a g r i c u l t u r a l p r a c t i c e . The g e n e r a l c h e m i c a l c l a s s e s t h a t t h e p e s t i c i d e s belong t o are t h e a n i l i d e s , a c e t a n i l i d e s , carbamates, t r i a z i n e s , c h l o r i n a t e d h y d r o c a r b o n s a n d t h e o r g a n i c p h o s p h a t e s . We h a v e d e t e c t e d m o d er at e p o s i t i v e r e s p o n s e s w i t h t h e t r i a z i n e s , a n i l i d e s , an d c h l o r i n a t e d hydrocarbons. T h i s comprehensive approach is u s e f u l because i t p r o v i d e s a l a r g e and c o h e s i v e d a t a b a s e from which c o n c l u s i o n s may b e drawn. We s h a l l present o u r experimental design, the data f o r each p e s t i c i d e o r c o m b i n a t i o n an d o u r g e n e r a l c o n c l u s i o n s t o d a t e .

(ca-9)

-IN VITRO

XJTAGEIJICITY AND GENOTOXICITY ASSAYS OF 38 PZSTICIDES. Vincent F. Simmons, D e n i s C. Poole", Edwarg S . Riccio", Dougias C . Ro b i n so n , Ann 1). i . l i t c h e l 1 and M i c h a e l D. Waters* , SRI I n t e r n a t i o n a l , Plenlo P a r k , CA 9 4 0 2 5 and E n v i r o n m e n t a l P r o t e c t i o n Agency, L e s e a r c h T r i a n g l e P a r k , NC 27711. h'e h av e p r e v i o u s l y r e p o r t e d t h e r e s u l t s o f i n v i t r o a s s a y s of 2 0 p e s t i c i d e s f o r m u t a g e n i c and g e n o t o x i c e f f e c t s . T h i s s t u d y h a s b e e n e x t e n d e d t o i n c l u d e 18 a d d i t i o n a l t e c h n i c a l g r a d e chemicals and f u r t h e r I n v i t r o a s s a y s a p p l i e d t o e a c h of t h e 38 t e s t i n g i s i n progress. p e s t i c i d e s i n c l u d e d reverse m u t a t i o n i n S, typhimurium and E. c o l i , m i t o t i c r e c o m b i n a t i o n i n S, cerevisciae, r e l a t i v e t o x i c i t y i n E. c o l i and B. s u b t i l i s - and u n s c h e d u l e d DNA s y n t h e s i s i n h u m n f i b r o b l a s t s . The f o l l o w i n g chemicals were n e g a t i v e i n the i n v i t r o tests: aspon, b r o m o c i l , c a r b o f u r a n , d i a z i n o n , i)SMA, e n d r i n , e t h i o n , f e n s u l f o t h i o n , f e n t h i o n , f o n o f o s , m a l a t h i o n , methomyl, m e t h o x y c h l o r , NSXA, p h o r a t e , q u i n t o z e n e , s i d u r o n , s i m a z i n e , t r i f u r a l i n . N i n e t e e n of t h e 38 c h e m i c a l s w e r e p o s i t i v e i n o n e o r more t e s t s . Of t h e s e 1 9 , 6 were p o s i t i v e i n one t e s t i n c l u d i n g

Abstracts

143

cacodylic acid, crotoxyphos, disulfoton, monuron, parathion, paratiiionmethyl, 6 were positive in 2 tests including azinphos-methyl, chlorpyrifos, 2,4-D, 2,4-D3, dicamba, dinoseb, monocrotophos, propanil, and only 5 were positive in 4 or more tests. Two of the latter 5 compounds, captan and folpet, have been studied previously in detail. The remaining 3, acephate, demeton and trichlorfon, are under continuing investigation in confirmatory short-term mutagenesis and carcinogenesis bioassays. Testing is also in progress on 20 additional pesticide chemicals. Representative data and current information on structure-activity correlations will be presented.

(Ca-10) EVALUATION OF THE GENETIC ACTIVITY OF NINE CHLORINATED PHENOLS, SEVEN CHLORINATED BENZENES, AND THREE CHLORINATED HEXANES. T. Lawlor* and S. R. Haworth, EG&G Mason Research Institute, Rockville, MD 20852 and P. Voytek? EPA, Washington, DC 20460. 2-Chloro, 3-chloro, 4-chloro, 2.4-dichlor0, 2,3,5-trichloro, 2,4,5-trichloro, 2,4,6-trichloro, 2,3,4,6-tetrachloro, and pentachlorophenol; 1-chloro, l,Z-dichloro, 1,4-dichloro. 1,2,4-trichloro, 1,2,3,5-tetrachloro, 1,2,4,5-tetrachloro, and 1.2,3.4,6-pentachlorobenzene; and alpha, beta, and gamma hexarhlorohexane were evaluated for their ability to cause gene mutations or DNA damage in bacteria. All compounds were tested in the Salmonella plate incorporation mutagenesis assay at five doses on strains TA98, TA100, TA1535, TA1537, and TA1538 both with and without metabolic activation by Aroclor 1254 induced rat liver microsomes. Genotoxicity was determined by DNA repair spot tests and by liquid suspension t e s t s utilizing E. coli strains WPZEA+EA+ 2nd WPlOOEA-recA-. and Salmonella strains TA197S=B+ and T A 1 5 3 8 ~ B - . All DNA repair tests were conducted both with and without metabolic activation. Initial results indicate that none of the compounds tested possessed detectable levels of mutagenic activity in the Salmonella tester strains. However, preferential killing of DNA repair deficient strains was observed among the chlorinated phenols ( 6 / 9 ) , benzenes (2/7), and hexanes (2/3) tested. Due to the low water solubility of the test compounds, the suspension method was superior to the spot test in detecting differential killing of the repair deficient strains. The WPlOOEA-recA- strain was much more sensitive than TA1538xB-. The significance of these results will be discussed.

(Ld-11)

EFFECT OF CHAIN LENCrH ON TtE MUTAGENICITY OF A SERIES OF a,w-ALKYLDIBROMIDES. Ann D. Burrell and Norman Chow*, IRM Corporation, 5600 Cottle Road, General Products Div., San Jose, C . . 95193 A series of a,w-alkyldibromides ranging from 1,Z-dibromoethane to 1,6dibromohexane were investigated for mutagenicity in the Salmonella typhimurium mutation test. All the compounds gave positive results in strain TA1535, indicating base pair substitution, typical of the action of alkylating agents. The addition of 'S9' liver microsomal enzymes appeared to have no effect on the results obtained. 1,2-dihromoethane has beenreported to be positive in several systems, as has 1,s-dibromopentane. Previously, lY3-dibrmopropanehas been reported to be negative in four different assay systems, and there are no published data on 1,4-dibromobutaneand 1,6-dibromohexane. The compounds exhibited a wide range of mutagenic potency with lY4-dibromobutaneexhibiting the greatest activity and 1,3-dibrmopropane the least. The chemicals have been ranked according to mutagenic potency as follows: 1,4- >1,2- >1,5- >1,6- >1,3-. A series of experiments which attempts to explain the order of reactivity will be presented.

144

Abstracts

(Ca-12) THE MUTAGENICITY OF TRIS(2,3-DIBROMOPROPYL)PHOSPHATE. R. M. Valencia and K . Houtchens, University of Wisconsin, Madison, WI 53706.

As reported previously ( t h e s e meetings, 1977). we have found t h a t t h e flame r e t a r d a n t , Tris(2,3-dibromopropyl)phosphate, induces sex-linked r e c e s s i v e l e t h a l s i n t h e germ c e l l s of a d u l t Drosophila melanogaster males exposed by feeding. The response is e s p e c i a l l y g r e a t i n t h e spermat i d s t a g e ( 7 . 8 % from days 5-7 post-mating a f t e r exposure t o 0.1% T r i s ) . W e have obtained some f u r t h e r information on t h e mutagenicity of T r i s i n the course of u s i n g i t a s a " p o s i t i v e control'' compound i n some of our t e s t i n g procedures. It w a s found t o be s t r o n g l y mutagenic when added t o the c u l t u r e medium i n which t h e f l i e s were reared. A t 500 ppm, t h e l e t h a l frequency i n males was, f o r the i n d i c a t e d post-mating days, a s follows: days 1-3, 11.40%; days 4-5, 8.94%; days 6-7. 2.24% and d a y s 8-10, 1.94%. The brood response p a t t e r n confirms t h e s e n s i t i v i t y of spermatids, s i n c e t h i s s t a g e would have been p r e s e n t i n the l a t e l a r v a l s t a g e and would develop i n t o the f i r s t sperm e j a c u l a t e d a f t e r mating.

Preliminary experiments i n d i c a t e t h a t T r i s induces chromosome breakage, l o s s and non-disjunction i n m a l e s and females. Additional e x p e r i ments a r e i n progress o r planned t o confirm and c h a r a c t e r i z e t h e s e e f f e c t s . T r i s i s a l s o proving t o be a u s e f u l t o o l i n methodology experimentation, due t o i t s s t a b i l i t y and e f f e c t i v e n e s s under v a r i e d conditions. This use w i l l be discussed and p e r t i n e n t d a t a w i l l be presented. Recent work supported by FDA and NIEHS.

(Cb) METABOLIC ACTIVATION

Chaired by:

J. S. Felton Lawrence L i v e r m o r e L a b o r a t o r y and

J. P a t r i c k O ' N e i l l Oak Ridge N a t i o n a l L a b o r a t o r y (Cb-1 ) STABILITY OF S-9 PREPARATIONS DURING LONG AND SHORT TERM STORAGE

J . ELLIOTT AND G. TAYLOR. M i c r o b i o l o g y Section, L a b o r a t o r y I n v e s t i g a t i o n s Branch, DHEW, PHS, CDC, NIOSH, ALOSH, 944 Chestnut Ridge Road, Morgantown, W. Va. 26505 A l t h o u g h many i n v e s t i g a t o r s use s t o r e d S-9 p r e p a r a t i o n s i n m u t a g e n i c i t y assays, l i t t l e p u b l i s h e d d a t a i s a v a i l a b l e on t h e d e c l i n e o f b i o l o g i c a l a c t i v i t y i n these p r e p a r a t i o n s . Enzyme assays f o r benzo(a)pyrene hydroxylase, 2 - a c e t y l a m i n o f l u o r e n e hydroxylase, N-demethylase, and epoxide hydrase were performed on S-9 f r a c t i o n s which had been s t o r e d a t -20", -70", o r -180" f o r v a r i o u s t i m e p e r i o d s . S-9 f r a c t i o n s which had been s t o r e d a t t h e above temperatures were a l s o assayed f o r t h e i r a b i l i t y t o form mutagenic compounds u s i n g h i s + r e v e r t a n t s i n S . typhimurium s t r a i n TA 98 and benzo( a)pyrene, d i m e t h y l h y d r a z i n e , o r methoxyphenylenediamine I n a n o t h e r s e t o f experiments e i t h e r benzo(a)pyrene o r sulfate. methoxyphenylenediamine s u l f a t e was s t o r e d w i t h t h e S-9 f r a c t i o n . Enzyme

Abstracts

145

s t a b i l i t y was enhanced when the mutagen was present. I n terms o f absolute a c t i v i t y the enzyme preparations stored i n l i q u i d N2 appear t o be the most s t a b l e . The relevance o f these d a t a t o niutagenicity t e s t i n g w i l l be discussed. (Cb-2) INHIBITORY EFFECT OF HIGH S-9 CONCENTRATIONS ON ACTIVATION OF HYDROCARBONS I N THE L5178Y MOUSE LYMPHOMA ASSAY. S. Thornton+ and M. Hite, Merck I n s t i t u t e , West P o i n t , PA 19486. M u t a t i o n i n d u c t i o n i n t h e L5178Y mouse lymphoma mutagen a s s a y was s t u d i e d as a f u n c t i o n o f t h e c o n c e n t r a t i o n o f an exogenously added r a t l i v e r homogenate a c t i v a t i o n system (S-9). Five p o l y c y c l i c aromatic hydrocarbons (PAHs) were t e s t e d a t c o n s t a n t dosage l e v e l s o v e r f o u r S-9 c o n c e n t r a t i o n s f o r t h e a b i l i t y t o i n d u c e forward m u t a t i o n s a t t h e thymidine k i n a s e (TK) l o c u s . With a l l f i v e compounds, i n c r e a s i n g S-9 c o n c e n t r a t i o n c a u s e d a s i g n i f i c a n t d e c r e a s e i n m u t a t i o n f r e q u e n c y and c o r r e s p o n d i n g i n c r e a s e i n s u r v i v a l . No s i g n i f i c a n t a c t i v a t i o n was observed w i t h o u t exogenous a t i v a t i o n . I n an e f f o r t t o i n t e r p r e t t h i s o b s e r v a t i o n , metabolism o f [ H]benzo(a)pyrene(BaP) was measured by

s

o r g a n i c s o l v e n t e x t r a c t i o n . A f t e r a 30-minute i n c u b a t i o n p e r i o d , o n l y 20% o f t h e r a d i o a c t i v i t y was r e c o v e r e d i n t h e o r g a n i c phase, a t h i g h S-9 c o n c e n t r a t i o n s , whereas 30% remained o r g a n i c s o l v e n t - e x t r a c t a b l e a t low S-9 c o n c e n t r a t i o n s . These d a t a s u g g e s t t h a t a d e c r e a s e o f b i o l o g i c a l e f f e c t i n t h e p r e s e n c e o f h i g h S-9 c o n c e n t r a t i o n s might r e s u l t from e x t e n s i v e metabolism o f t h e PAH t o non-mutagenic m e t a b o l i t e s . + P r e s e n t a d d r e s s : Wistar I n s t i t u t e , P h i l a d e l p h i a , PA 19104.

(Cb-3) THE USE OF A RAT LIVER Sg ACTIVATION SYSTEM I N THE CHO/HGPRT MUTATION INDUCTION ASSAY. J. P a t r i c k O ' N e i l l , Richard Machanoff, and Abraham W. Hsie, Biology D i v i s i o n , Oak Ridge N a t i o n a l L a b o r a t o r y , Oak Ridge, TN 37830. The o p t i m a l c o n d i t i o n s f o r t h e u s e o f a n Sg microsomal p r e p a r a t i o n t o m e d i a t e a c t i v a t i o n of promutagens h a s been i n v e s t i g a t e d i n t h e CHO/HCPRT system. The Sg f r a c t i o n w a s p r e p a r e d from A r o c l o r 1254-induced male Sprague-Dawley r a t l i v e r s , and two model p r o c a r c i n o g e n s b e n z o ( a ) pyrene [B(a)P] and d i m e t h y l n i t r o s a m i n e (DMN) u t i l i z e d f o r t h e i n i t i a l s t u d i e s . With t h e s e two compounds, s e v e r a l d i f f e r e n c e s were found i n t h e c o n d i t i o n s which y i e l d e d maximal m u t a t i o n i n d u c t i o n . Over an Sg p r o t e i n concent r a t i o n r a n g e of 0.1-1.5 mg/ml, t h e m u t a g e n i c i t y o f a c o n s t a n t amount o f DMN i n c r e a s e s w i t h i n c r e a s i n g p r o t e i n , w h i l e t h a t o f B(a)P increases a t low p r o t e i n c o n c e n t r a t i o n s , f o l l o w e d by a d e c r e a s e . T h i s may r e f l e c t a change i n t h e b a l a n c e between a c t i v a t i o n and i n a c t i v a t i o n w i t h d i f f e r e n t compounds, and l i m i t s t h e range of p r o t e i n c o n c e n t r a t i o n s a t which comparisons o f mutagenic potency are v a l i d a t least w i t h t h e s e two compounds. I n a d d i t i o n , DMN r e q u i r e s h i g h c o n c e n t r a t i o n s of CaC12 f o r maximal mutagenic a c t i v i t y , w h i l e B(a)P does n o t . These r e s u l t s complic a t e t h e development o f a s i n g l e Sg p r o t e i n mix which would b e u s e f u l i n r o u t i n e m u t a g e n e s i s s c r e e n i n g and i n t h e q u a n t i f i c a t i o n o f t h e mutagenic (Research s u p p o r t e d by p o t e n t i a l o f compounds f o r c o m p a r a t i v e s t u d i e s . EPA and DOE under c o n t r a c t w i t h Union Carbide C o r p o r a t i o n . ) (Cb-4) THE EFFECT OF SPECIES USED AS THE SOURCE FOR S-9 ON THE MUTAGENICITY OF DIALKYLNITROSAMINES I N THE SALMONELLA PLATE ASSAY. Michael J . P r i v a l , A l b e r t T. Sheldon, Jr., and V a l e r i e 6. King, G e n e t i c l ' G i c o l o g y Branch, Food EL Drug A d n i n i s t r a t i o n , Washington, D.C. 20204. One o f the problems w i t h the standard Salmonella p l a t e incorporation assay (Ames t e s t ) has been t h a t t h e mutag-TGcinogen N-nitrosodimethylamine (DMN) has n o t been p o s i t i v e i n t h i s t e s t . When we used

146

Abstracts

Aroclor-induced mouse o r hamster l i v e r as t h e source o f S-9 r a t h e r than the more comnonly used r a t l i v e r , DMN was found t o be mutagenic. Hamster l i v e r gave b e t t e r a c t i v a t i o n than mouse l i v e r whether t h e animals had been induced w i t h A r o c l o r o r phenobarbital o r were uninduced. Hamster l i v e r S-9 a l s o a c t i v a t e d N-nitrosodiethylamine (DEN) and N-nitrosodi(n-buty1)amine (DBN) more e f f e c t i v e l y than e i t h e r r a t o r mouse l i v e r . DMN i s mutagenic using Aroclor-induced r a t l i v e r S-9 i f the "preincubation" m o d i f i c a t i o n o f t h e p l a t e i n c o r p o r a t i o n assay i s used (Yahagi, e t al., Mutat. Res., 48, 121, 1977). I n t h e preincubation assay, t h e b a c t e X a T C h e X C a T a n d - 9 7 i x are incubated together p r i o r t o the a d d i t i o n o f s o f t agar and p l a t i n g . It has been p o s t u l a t e d t h a t the p l a t e i n c o r p o r a t i o n assay i s negative w h i l e the preincubation assay i s p o s t i v e because DMN metabolites may be i n a c t i v a t e d by r e a c t i n g w i t h t h e agar t h a t i s present a t a l l times i n the p l a t e i n c o r p o r a t i o n assay. When the S-9 concentration i n t h e p r e i n c u b a t i o n assay was adjusted t o t h a t present i n t h e p l a t e i n c o r p o r a t i o n assay, t h e mutagenic response was n o t observed. I t i s concluded t h a t t h e a b i l i t y o f the p r e i n c u b a t i o n assay t o d e t e c t the mutagenicity o f DMN i s due t o the f a c t t h a t t h e components are incubated a t higher concentrations i n t h i s assay than are used i n the p l a t e i n c o r p o r a t i o n assay.

(Cb-5) STRAIN AND SEX DIFFERENCES 13 A STEROID INDUCIBLE MOUSE KIDNEY MICROSOMAL MONOOXYGENASE: ITS RELATIONSHIP TO DIMETHYLNITROSAMINE METABOLISM AND MUTAGENICITY. J . S . F e l t o n , L . E . O l s e n : M.H. Corzett: M.E. Johnson: and J . H . C a r v e r , Biomedical S c i e n c e s Div., Lawrence Livermore Lab., Livermore, CA Determining t h e b i o c h e m i c a l b a s i s f o r t i s s u e s p e c i f i c d i f f e r e n c e s i n d i m e t h y l n i t r o s a m i n e (DMN) metabolism and i t s c o r r e s p o n d i n g m u t a g e n e s i s i s i m p o r t a n t f o r e v e n t u a l u n d e r s t a n d i n g o f t i s s u e and s e x s p e c i f i c c a r c i n o g e n e s i s . Kidney microsomes from b o t h male and female mice o f t h e C3H/HeJ and BALB/CJ s t r a i n s were compared f o r a number o f monooxygenase p r o p e r t i e s . Cytochrome P-450 c o n t e n t w a s 3 f o l d h i g h e r i n C3H male k i d n e y microsomes t h a n i n C3H female k i d n e y microsomes. A s i n g l e 54,000 m o l e c u l a r w e i g h t p r o t e i n , i d e n t i f i a b l e f o l l o w i n g SDS p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s w a s d r a m a t i c a l l y i n c r e a s e d i n m a l e mouse k i d n e y . A r y l hydrocarbon h y d r o x y l a s e ( A H H ) a c t i v i t y d i d n o t d i f f e r between males and f e m a l e s . P h e n o b a r b i t a l and 3 - m e t h y l c h o l a n t h r e n e , b o t h i n d u c e r s o f l i v e r microsomal cytochrome P - ~ ~ O S , i n c r e a s e d AHH a c t i v i t y 1 0 f o l d o v e r c o n t r o l , b u t d i d not i n c r e a s e t h e 54,000 d a l t o n p r o t e i n . I n c o n t r a s t , i m p l a n t i n g t e s t o s t e r o n e p e l l e t s i n f e m a l e s c a u s e d a marked i n c r e a s e i n b o t h t h e P-450 c o n t e n t and t h e 54,000 dalton protein. L i k e w i s e , c a s t r a t i o n o f t h e male r e s u l t e d i n a d e c r e a s e o f cytochrome P-450 c o n t e n t and t h e 54,000 d a l t o n p r o t e i n , b u t n o change i n AHH. The BALB/C s t r a i n showed l e s s k i d n e y cytochrome P-450 and l e s s 54,000 d a l t o n p r o t e i n t h a n t h e C3Y. Both t h e sex and s t r a i n d i f f e r e n c e s c o r r e l a t e w i t h t h e mutagenic r e s p o n s e of DMN i n S a l m o n e l l a b a c t e r i a f o l l o w i n g k i d n e y microsomal a c t i v a t i o n (Weekes and B r u s i c k , Mutation Res. 31:175,1975). There i s a d e f i n i t e c o r r e l a t i o n between DMN-induced mammalian c e l l m u t a g e n e s i s and t h e p r e s e n c e o f t h i s k i d n e y - s p e c i f i c , s t e r o i d - i n d u c i b l e c y t o chrome P-450 i n t h e a c t i v a t i n g m i x t u r e (see n e x t a b s t r a c t ) . ( S u p p o r t e d by a n d U.S. DOE by LLL, Cont.No. W-7405-ENG-48) EPA c o n t r a c t EPA-IAG-06-0439-1

(Cb-6) SEX DIFFERENCES I N MOUSE KIDNEY MICROSOMAL PROTEINS PARALL.EL DMN-INDUCED MUTAGENESIS I N CHINESE HAMSTER OVARY CELLS. J . H . C a r v e r , D.L. Wandres", M.E. Johnson", and J.S. F e l t o n , Biomedical S c i e n c e s Div., Lawrence Livermore L a b o r a t o r y . Livermore, CA 94550 The a b i l i t y of C3H/HeJ mouse k i d n e y microsomes t o a c t i v a t e d i m e t h y l n i t r o s a m i n e (DMN) i n t o mutagenic m e t a b o l i t e s was d e t e r m i n e d , m e a s u r i n g f o r w a r d m u t a t i o n f r e q u e n c i e s at. m u l t i p l e d r u g - r e s i s t a n c e m a r k e r s i n C h i n e s e h a m s t e r o v a r y ( C H O ) c e l l s . Kidney microsomal p r e p a r a t i o n s from male, female, o r t e s t o s t e r o n e - i n d u c e d f e m a l e mice were added w i t h co-

Abstracts

147

factors t o monolayer CHO c e l l s w i t h DMN a t 60 o r 100 mM f o r 2 h r i n serum-free medium. After a p p r o p r i a t e e x p r e s s i o n , t h e e l l s were p l a t e d t o s e l e c t f o r m u t a n t s r e s i s t a n t t o 8-azaadenine (aprt‘?-), 6-thioguanine ( h g p r t - ) , and o u a b a i n ( a l t e r e d Na+-@-ATPase). P r e p a r a t i o n s from male k i d n e y y i e l d e d DMN-induced mutant f r e q u e n c i e s a p p r o x i m a t e l y 10-40 f o l d h i g h e r a t a l l t h r e e l o c i t h a n c o n t r o l o r female v a l u e s ; k i d n e y s from t e s t o s t e r o n e - i n d u c e d females i n c r e a s e d DMN-induced m u t a t i o n from 4 t o 35 f o l d o v e r c o n t r o l s and non-induced females. I n c r e a s i n g t h e microsomal p r o t e i n c o n c e n t r a t i o n frcm 0.5 t o 1.0 mg/ml i n c r e a s e d t h e mutant y i e l d 2-4 f o l d . F o r t h e female p r e p a r a t i o n s , t h e r e were i n d i c a t i o n s a t a l l t h r e e markers f o r DMN-induced mutagenesis (100 mM) at a microsomal C o n c e n t r a t i o n of 1.0 mg/ml, i n d i c a t i n g a p o s s i b l e b a s e - l i n e a c t i v i t y . The d a t a were n o t s t a t i s t i c a l l y s i g n i f i c a n t , and h i g h e r p r o t e i n c o n c e n t r a t i o n s are b e i n g examined. ( T h i s work s u p p o r t e d by t h e U.S. DOE b y LLL under c o n t r a c t . No. W-7405-ENG-48 and by I n t e r a g e n c y Agreements TAG-D5-E681-AN and A 0 wit.h t.he Environmental P r o t e c t i o n Agency. (Cb-7) THE MICROSOMAL METABOLISM OF 2 AND 4-AMINOBIPHENYL. AN EXAMPLE OF THE CRUCIAL ROLE OF METABOLIC ACTIVATION I N DETERMINING THE MUTAGENICPOTENTIAL OF A COMPOUND. Robert E. McMahon, and Judy C. Turner*. The L i l l y R e s e a r c h L a b o r a t o r i e s , I n d i a n a p o l i s , I n d i a n a 46206. The pathways by which a chemical i s m e t a b o l i z e d i n t h e body can be c r u c i a l i n d e t e r m i n i n g whether o r n o t t h a t chemical may o r may n o t possess mutagenic and/or c a r c i n o g e n i c p r o p e r t i e s . I n t h e p r e s e n t s t u d y HPLC procedures have been developed f o r t h e r e l i a b l e and a c c u r a t e a n a l y s i s o f t h e m e t a b o l i c t r a n s f o r m a t i o n s o f 4-aminobiphenyl and o f 2-aminobiphenyl. The p r i n c i p a l m e t a b o l i t e o f t h e microsomal o x i d a t i o n o f 4 - a m i n o b i ~ h e n y l i s T h i s m e t a b o l i t e i s accomparlied however by subN-hydroxyaminobiphenyl. s t a n t i a l q u a n t i t i e s o f 3-hydroxyc4-aminobiphenyl and by l e s s e r q u a n t i t i e s The e x t e n t o f N - h y d r o x y l a t i o n i s o f 4’- and 2’-hydroxy-4-aminobiphenyl. species dependent and i s i n c r e a s e d i n a l l species by a r o c l o r p r e t r e a t m e n t . 4 - h i n o b i p h e n y l i s an a c t i v e f r a m e s h i f t mutagen i n s e v e r a l Salmonella s t r a i n s i n t h e a c t i v a t e d system. N-hydroxy-4-aminobiphenyl i s a c t i v e i n t h e same f r a m e s h i f t organisms w i t h o u t microsomal a c t i v a t i o n i m p l y i n g t h a t i t i s t h e a c t i v e m e t a b o l i t e . Since 4 - n i t r o s o b i p h e n y l i s a c t i v e as a b a s e - s u b s t i t u t i o n mutagen i t i s u n l i k e l y t h a t t h i s m e t a b o l i t e c o n t r i b u t e s I n c o n t r a s t t o 4-aminobit o t h e m u t a g e n i c i t y seen from 4-aminobiphenyl. phenyl, t h e microsomal o x i d a t i o n o f 2-aminobiphenyl does n o t produce t h e N-hydroxy m e t a b o l i t e . A l l o f t h e m e t a b o l i t e s i d e n t i f i e d t o d a t e a r e p h e n o l i c i n n a t u r e . Since however N-hydroxy-2-aminobiphenyl i t s e l f i s a f r a m e s h i f t mutagen i n t h e u n a c t i v a t e d Ames t e s t t h e s u g g e s t i o n i s t h a t t h e i n a c t i v i t y o f 2-aminobiphenyl i s due, n o t t o an i n h e r e n t l a c k o f a c t i v i t y o f 2 - s u b s t i t u t e d b i p h e n y l s , but r a t h e r i s due t o t h e i n a b i l i t y o f l i v e r enzymes t o c o n v e r t 2-aminobi phenyl t o M-hydroxy-2-aminobiphenyl . (Cb-8) A COMPARISON OF MUTATION FREQUENCIES INDUCED BY BENZO(A)PYRENE AT MULTIPLE LOCI I N CHINESE HAMSTER CELLS: METABOLIC ACTIVATION BY SYRIAN HAMSTER EMBRYO CELLS, RAT LIVER HgMOGENATES, OR LIVER MICROSOMAL FRACTIONS. D.L. Wandres: E.P. S a l a z a r , M.G. Knize: and J . H . Carver, Biomed. S c i . Div., Lawrence Livermore Lab., Livermore, CA 94550.

The m u t a g e n i c i t y o f b e n z o ( a ) p y r e n e (BP) t o mammalian c e l l s i n c u l t u r e w a s compared u s i n g a c t i v a t i o n by: ( a ) p r i m a r y c u l t u r e s o f S y r i a n hamster embryo c e l l s ; ( b ) l i v e r homogenate (S-9) from rats i n d u c e d w i t h A r o c k l o r ; and ( c ) microsomal f r a c t i o n s p r e p a r e d by 100,000 g c e n t r i f u g a t i o n o f t h e S-9 material. Hamster embryo c e l l metabolism f a v o r s d i o l f o r m a t i o n , b u t microsomal p r e p a r a t i o n s a l s o m e t a b o l i z e BP t o p h e n o l s and quinones by a competing r e a c t i o n . The S-9 p r e p a r a t i o n c o n t a i n s c o n j u g a t i n g enzymes

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Abstracts

t h a t may be decreased by microsomal p u r i f i c a t i o n . Mutation f r e q u e n c i e s were measured a t m u l t i p l e , d r u g - r e s i s t a n c e m a r ers i n Chinese hamster r e s i g t a n c e t o 6-thioovary c e l l s ( r e s i s t a n c e t o 8-azaadenine, apt-’-; guanine, hgprt-; r e s i s t a n c e t o ouabain, a l t e r e d N a -K -ATPase). The hydrocarbon w a s a c t i v a t e d by a 2 h r exposure i n serum-free medium c o n t a i n i n g BP at 10 pg/ml, t h e a p p r o p r i a t e c o - f a c t o r s , and e i t h e r S-9 homogenate o r microsomal m a t e r i a l . Cell-mediated a c t i v a t i o n w a s c a r r i e d out by a 48 h r exposure t o BP i n t h e presence o f a l e t h a l l y - i r r a d i a t e d f e e d e r l a y e r o f S y r i a n hamster embryo c e l l s . The BP-induced mutation f r e q u e n c i e s w e r e from 6 t o 8 f o l d h i g h e r a f t e r microsomal a c t i v a t i o n t h a n a f t e r S-9 homogenate a c t i v a t i o n . Cell-mediated metabolism produced mutation f r e q u e n c i e s comparable t o t h o s e from microsomal a c t i v a t i o n . This work supported by t h e U.S. Dept. of Energy Contract No. W-7405-ENG48 and by Interagency Agreements IAG-D5-E681-AIi and A 0 with t h e Environmental P r o t e c t i o n Agency.

(Cb-9) VARIATION I N IlESrrXJSE I N THE ADULT RAT LIVER EPITHELIAL CELL/THINE-GUWVINE PW3SPHOKIROSYL TIWJSFERFSE ( ARL/HGPmse) MUTATICN ASSAY WI?H D I F F F m WAEXILIC ACTWATICN-DEPENDW CARCINOGENS. Charles Tong and G a r y M. Xilliams, intr. by John H. Weisburqer, Naylor Dana Inst. for

Disease Prevention, American Health Foundation, Valhalla, New York 10595. The ARL/HGPRIkse assay has begn shown to be useful for measuring 6thioguanine resistant nutant ( X ) induction by netabolic activationdependent and independent carcinogens. In the first ARL line studied, 6, the mtant incidences induced by exposure to several carcinogens requirinq different netabolic activation pathways varied considerably. This result could be due to different levels of enzymes in this line for activatinq and detoxifying different types of carcinogens. If so, the response of different ARL, lines to various carcinogens miqht also differ. merefore, a survey was nade of the effect of 5 types of activation-clependent carcinoqens on 4 ARL, lines. Current data s h m belcw reveal nutaqenesis of some lines by certain carcinoqens but not by others. Thus, variations in metabolism appear to exist between lines, a d it will be necessary to use several lines in screeninq to provide broad activation capability. ~6Yutagenesis mpe of ARL6 ARL14 ARL18 ARL19 Carcinoqen + + NT Mvcotoxin (AFR.1 Aminoazo rye (h) + NT NT polycyclic Aromatic + + Hydrocarbon (DMBA) Nitrosamine (l74N) NT ?wmatic .&nine ( A A F ) + NT NT + ICb-10) COMBINED I N VITRO AND HOST-MEDIATED ASSAYS FOR TRANSFORMATION OF BALB/3T3 CELLS. J . W . B a r n e t t , Jr.* and J. B. Ward, Jr.*. U n i v e r s i t y of Texas Medical Branch, Galveston. Texas 77550.

The high d e g r e e of c o r r e l a t i o n between mutagenic a c t i v i t y and t h e a b i l i t y t o induce morphologic t r a n s f o r m a t i o n of mammalian c e l l s w i t h chemical a g e n t s s u g g e s t s t h a t t h e c r i t i c a l transforming s t e p s i n v o l v e g e n e t i c m u t a t i o n s . S i n c e b i o a c t i v a t i o n i s necessary f o r many chemicals t o e x e r t t h e i r e f f e c t s , w e have developed a n a s s a y t h a t couples t h e host-mediated technique with a q u a n t i t a t i v e t r a n s f o r m a t i o n system. Syngeneic Balb/c mice were i n o c u l a t e d i n t r a p e r i t o n e a l l y w i t h 2-4 x 106 Balb/3T3 c e l l s immediately p r i o r t o subcutaneous i n j e c t i o n of t h e chemical. A f t e r 3-4 hours exposure, c e l l s were recovered by t r y p s i n i n j e c t i o n and withdrawal, and c e l l y i e l d s Recovered c e l l s were c u l t u r e d & were e s t i m a t e d t o range from 5-25%.

Abstracts

149

vitro and grown to near confluence in 2-5 days, at which time they were plated in a standard protocol to determine transformation frequency and plating efficiency. Dose-dependent increases in transformation rates were observed for the direct-acting agent, ethyl methanesulfonate (EMS), both -in vitro and in vivo. 2-Aminofluorene (2AF), which requires activation, had no effect in vitro,but increased transformation frequencies with increasing dosage up to 10 mg/kg were seen in vivo. Significantly higher doses of EMS (372 mg/kg) than 2AF (10 mg/kg) were required to produce comparable increases of 4-6 times control ( - 3 foci/lO4 surviving cells) in the host-mediated assay. Transformation frequencies were twofold over control at 0.1 and 1.0 mg/kg but not significantly different from control at 10 mg/kg. A preliminary experiment with acrylonitrile indicated a weak positive response in the host-mediated assay. Supported in part by Monsanto Fund fellowship and grants from the Environmental Protection Agency and Dow Chemical C o . (Cb-11) USE OF THE SALMONELLA/MICROSOMAL ASSAY IN EVALUATION OF BENZO (A)PYRENE

METABOLITES PRODUCED IN THE ISOLATED PEWUSED RABBIT LUNG PREPARATION R. S. Schoeny, D. Warshawsky, intr. by E. Bingham, Department of Environmental Health, Kettering Laboratory, University of Cincinnati, Cincinnati, Ohio 45267 It is well established that many presumptive carcinogens, such as benzo(a)pyrene (BaP), require metabolism for their tumorigenic or mutagenic activity. Only recently has the metabolic potential of the lung been recognized as a factor in the response of the organism to environmental carcinogens or mutagens. By using the isolated perfused rabbit lung (IPL) system, BaP metabolism can be studied in the absence of the influence of other organs. The use of a whole organ preparation offers these advantages: 1) recovery not only of blood perfusate for analysis but also of pulmonary tissue and macrophages; 2) alternate routes of exposure of the lung to BaP; 3) opportunities for both inducing pretreatment of the whole animal and introduction of materials, such as particulates, with the BaP in the IPL. HPLC analysis has shown organic extracts of perfusate and tissue to contain phenols, diols, quinones, material cochromatographing with the 4,5-epoxide and an as yet unidentified metabolite which is fluorescent and ninhydrin positive. Each sample also contains unmetabolized BaP. Preliminary data indicate that extracts of p u l monary macrophage and lung tissue contain material mutagenic for Salmonella Qphimurium strains TA1538 and TAlOO when assayed with microsomal activation. When unmetabolized BaP is removed from the extracts by thick layer chromatography, there remain components mutagenic for TA1538. Procedures to optimize detection of mutagenic materials generated in the IPL will be discussed. (Cb-12) IMPROVED DETECTION OF -IN VIVO NITROSATION OF MORPHOLINE WITH AN INTRAHEPATIC HOST-?EDIATED ASSAY. Wen-Zong Whong*, Norman Speciner*, Gordon Edwards, Thermo Electron Corp. Waltham, Mass. 02154. The in vivo formation of nitrosomorpholine (NMOR) from intubated precursors, morpholine (MOR) and nitrite, at pH 3 . 4 was measured by an intfahepatic host-mediated mutagenicity assay. Reversions from his- to his were scored in indicator cells (Salmonella typhimurium TA1530) whichwere administered intravenously prior to nitrite and MOR dosing and recovered subsequently from the liver of the host (female CD-I mice). There was no mutagenic activity detected after the oral administration of either precursor alone. However, a dose-mutagenic response was clearly demonstrated when MOR and nitrite were intubated simultaneously. Observations from a time-course study indicated that the processes of MOR nitrosation, NMOR uptake and biotransformation of NMOR to a mutagenic

150

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metabolite took place at a rapid rate. By comparing the mutagenic resnonse following administration of the precursors with that caused by dirrctly administered NMOR it was possible to estimate the amount of MOR nitrosated in mouse stomach. Under optimal conditions, approximately 17% of MOR was converted to NMOR. The limit of NMOR detection by this assay was 0 . 2 pg/g body weight. Sodium ascorbate, a nitrite scavenger and nitrosation inhibitor, caused a complete inhibition of induced reversions at a dose equimolar to nitrite. Sodium thiocyanate, a known catalyst of nitrosation reactions, caused an enhanced in vivo mutagenicity response when gavaged at 120 ug/g together with nitrite (120 pg/g) and XOR ( 4 ug/g). This is the first account of detection of MOR nitrosation by an intraheptic mutagenicity assay; it represents an improvement over the detiction limits reported for other host-mediated assays. (Cb-13) BIOACTIVATION OF DMN I N INTRA-SANGUINOUS HOST-MEDIATED ASSAY I N MALE AND FEMALE M I C E . K. S. Bakshi and D. J. B r u s i c k , L i t t o n B i o n e t i c s , I n c . , Kensington, Md. 20795. P r e v i o u s r e p o r t s have d e s c r i b e d a s e x - r e l a t e d d i f f e r e n c e i n t h e m e t a b o l i c a c t i v a t i o n o f DMN i n t h e mouse k i d n e y i n t h a t o n l y microsomes f r o m s e x u a l l y mature male mice c o u l d a c t i v a t e DMN t o a mutagen. I n t h i s r e p o r t , we compared t h e organ o r sex s p e c i f i c DMN a c t i v a t i o n u s i n g i n v i t r o and i n v i v o i n t r a - s a n g u i n e host-mediated assays. The d a t a f r o m i n v i t r o and i n v i v o s t u d i e s showed t h a t DMN c o u l d be a c t i v a t e d t o a mutagen i n t h e l i v e r s and l u n g s o f b o t h sexes. I n c o n t r a s t o n l y t h e k i d n e y microsomes f r o m s e x u a l l y mature male mice c o u l d be a c t i v a t e d t o a mutagen i n t h e i n v i t r o mutagenesis assay. However, i n HMA, t h e b a c t e r i a recovered f r o m t h e k i d n e y s o f e i t h e r sex were found t o be mutated a t h i g h e r dose l e v e l s , whereas, a t l o w e r dose l e v e l s o n l y t h e b a c t e r i a from male k i d n e y s were found t o be mutated. T h i s suggests t h e p o s s i b i l i t y t h a t t h e a c t i v e m e t a b o l i t e o f DMN, i n case o f t h e female i s produced i n t h e l i v e r o r l u n g s and t h e n b r o u g h t t o t h e kidneys. The m u t a t i o n s i n t h e male k i d n e y c o u l d be a t t r i b u t e d i n p a r t t o t h e metabol i s m o f DMN i n t h e k i d n e y i t s e l f and i n p a r t by t h e s p i l l o v e r p o s s i b l y from l i v e r o r lungs. The s p l e e n i s a r e l a t i v e l y i n e r t o r g a n i n i t s a b i l i t y t o m e t a b o l i z e o r t r a n s f o r m promutagens t o u l t i m a t e mutagens. B u t i n o u r i n v i v o HMA, a l a r g e number o f b a c t e r i a recovered from s p l e e n were found t o have been r e v e r t e d . S i n c e s p l e e n i s n o t a m e t a b o l i c a l l y a c t i v e organ, t h e m u t a t i o n s induced i n s p l e e n c o u l d be a t t r i b u t e d a g a i n t o t h e s p i l l o v e r o f t h e a c t i v e m e t a b o l i t e . These f i n d i n g s have i m p o r t a n t i m p l i c a t i o n s i n t h a t t h e d a t a from i n t r a - s a n g u i n e HMA can be used t o p r e d i c t s p e c i f i c organs a t r i s k from t h e t e s t chemical under i n v e s t i g a t i o n . The organs a t r i s k may o r may n o t be i n v o l v e d i n m e t a b o l i c a c t i v a t i o n o f t h e compound. (Cc) HUMAN STUDIES

Chaired by: R. J. A l b e r t i n i U n i v e r s i t y o f Vermont and H. Rappaport Carnegie-Mellon U n i v e r s i t y (cc-1 ) EFFECT ON HUMAN ILL HEALTH OF AN INCREASE IN THE RATE OF DOMINANT MUTATION. John D. Childs, "intr. by David K. Myers", Atomic Energy of Canada Limited, Chalk River, Ontario, KOJ 1J0, Canada.

Abstracts

151

I n o r d e r t o assess t h e g e n e t i c r i s k from a n e n v i r o n m e n t a l mutagen i t i s i m p o r t a n t t o know n o t o n l y t h e l i k e l y i n c r e a s e i n m u t a t i o n r a t e caused by t h e mutagen b u t t h e e f f e c t of t h e i n c r e a s e on human ill h e a l t h . Dominant m u t a t i o n s are p a r t i c u l a r l y i m p o r t a n t a s an i n c r e a s e i n m u t a t i o n r a t e w i l l be r e f l e c t e d i n t h e f i r s t g e n e r a t i o n f o l l o w i n g e x p o s u r e by an i n c r e a s e i n dominant d i s e a s e s . Furthermore t h e b i r t h f r e q u e n c y i s e x p e c t e d t o r a p i d l y r e a c h a new e q u i l i b r i u m i n d i r e c t p r o p o r t i o n t o t h e i n c r e a s e i n m u t a t i o n r a t e . S e v e r a l committees (e.g. B E I R , UNSCEAR and ICRP) have a t t e m p t e d t o e s t i m a t e t h e l i k e l y i n c r e a s e i n dominant d i s e a s e s i n t h e f i r s t o r second g e n e r a t i o n f o l l o w i n g human exposure t o i o n i z i n g r a d i a t i o n . T h e i r c a l c u l a t i o n s a r e l a r g e l y based on t h e o b s e r v e d number of m u t a t i o n s i n mouse e x p e r i m e n t s and t h e e x t e n t t o which t h e induced i n c r e a s e , i f i t o c c u r r e d i n man, would be r e f l e c t e d i n an i n c r e a s e i n t h e f r e q u e n c y of dominant d i s e a s e . A more d i r e c t a p p r o a c h , which w i l l be d e s c r i b e d i n t h i s p a p e r , i s t o c o l l e c t d a t a p e r t a i n i n g t o t h e f r e q u e n c i e s of each of t h e i m p o r t a n t dominant d i s e a s e s of man, t h e r e p r o d u c t i v e f i t n e s s of t h e a f f e c t e d i n d i v i d u a l s and t h e m u t a t i o n rates f o r e a c h of t h e d i s e a s e s . From t h i s i t i s poss i b l e t o c a l c u l a t e a n e x p e c t e d r a t e of i n c r e a s e i n dominant d i s e a s e f o r any p r o j e c t e d i n c r e a s e i n m u t a t i o n frequency. T h i s approach should b e j u s t a s a p p l i c a b l e t o chemical mutagens i n t h e environment a s t o r a d i a t i o n . (CC-2) DETECTION OF VOLATILE NITROSAMINE I N THE URINE OF NORMAL INDIVIDUALS ON WESTERN DIETS. S. Wang*, T. Kakizoek and W.R. Bruce. O n t a r i o Cancer Ins t i t u t e , 500 Sherbourne S t r e e t , Toronto N4X 1K9, OntariopCanada. The n i t r o s a m i n e s are a major class o f chemical mutagens/carcinogens. We had observed t h a t f e c e s may c o n t a i n v o l a t i l e n i t r o s a m i n e s which have presumably a r i s e n from endogeneous n i t r i t e and amines i n t h e lower g a s t r o i n t e s t i n a l t r a c t ( N a t u r e , 276, 280-281, 1978). We, t h e r e f o r e , wished t o d e t e r m i n e whether o t h e r body f l u i d s c o n t a i n e d t h e s e compounds i n o r d e r t o assess t h e i r importance as e n v i r o n m e n t a l l y r e l a t e d h a z a r d s a s s o c i a t e d w i t h d i e t . U r i n e from normal d o n o r s was e x t r a c t e d w i t h d i c h l o r o m e t h a n e w i t h a c o n v e n t i o n a l l i q u i d - l i q u i d e x t r a c t i o n procedure. V o l a t i l e n i t r o s a m i n e s were d e t e c t e d w i t h a g a s - l i q u i d and h i g h - p r e s s u r e l i q u i d chromatograph i n t e r f a c e d t o a t h e r m a l energy a n a l y s e r . Of 27 i n d i v i d u a l s s t u d i e d , 1 2 had u r i n e c o n t a i n i n g v o l a t i l e n i t r o s a m i n e s a t l e v e l s t o 3.8 p g p e r l i t e r . A more r a p i d , e f f i c i e n t and r e p r o d u c i b l e e x t r a c t i o n method f o r v o l a t i l e n i t r o s a m i n e s i s p o s s i b l e w i t h t h e JETUBE. The e x t r a c t i o n e f f i c i e n c y (8896%) i s h i g h e r t h a n t h a t o b t a i n e d w i t h t h e c o n v e n t i o n a l l i q u i d - l i q u i d ext r a c t i o n method, t h e XAD r e s i n o r t h e C-18 SEPAK. The method should p e r m i t t h e r a p i d assessment of t h e d i e t a r y f a c t o r s a s s o c i a t e d w i t h t h e prod u c t i o n of t h e s e mutagen/carcinogens i n t h e body. ( T h i s work was supp o r t e d by g r a n t s from t h e N a t i o n a l Cancer I n s t i t u t e o f Canada and t h e O n t a r i o Cancer Treatment and Research Foundation.)

(cc-3)

HEROIN-INDUCED REVERSIBLE ALTERATION OP CYTOGENETIC AND DNA REPAIR RESPONSES. D.A. S h a f e r a , J.J. Madden*, and A. F a l e k , G e o r g i a Mental H e a l t h I n s t i t u t e , A t l a n t a , G.4 30306, and Emory U n i v e r s i t y , A t l a n t a , GA.

Chromosome a b e r r a t i o n , s i s t e r c h r o m a t i d exchange (SCE) and DNA exc i s i o n (UDS) r e p a i r s t u d i e s were conducted on s t r e e t h e r o i n u s e r s , normal controls, and on methadone maintenance p a t i e n t s a t various t r e a t m e n t intervals. P e r i p h e r a l b l o o d l e u c o c y t e s were i n v e s t i g a t e d f o r b a s e levels of chromosome damage and SCE, and for SCE and UDS r e s p o n s e t o f a r UV. I n contrast t o c o n t r o l s u b j e c t s , h e r o i n u s e r s were found t o have s i g n i f i c a n t l y h i g h e r chromosome a b e r r a t i o n and SCE l e v e l s w h i l e t h e i r UV induced UDS r e p a i r levels were s i g n i f i c a n t l y lower. Half of t h e h e r o i n a d d i c t s t e s t e d were i n c a p a b l e of r e p a i r i n g UV f l u e n c e s one q u a r t e r as l a r g e as t h o s e r e p a i r e d by t h e control s u b j e c t s ( 2 0 J/m2), After one y e a r , sub-

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jects on methadone maintenance showed a return t o normal chromosome aberration and W repair l e v e l s . With t h e larger control sample (90). age, sex, coffee and alcohol use had no apparent effect on maximal UDS response but smoking tobacco s i g n i f i c a n t l y decreased the UDS mean and variance though f a r less than with heroin use. These s t u d i e s i n d i c a t e a general interaction between cytogenetic response and r e p a i r capacity and suggest that methadone may ameliorate the apparent mutagenic s e n s i t i v i t y induced by heroin. SupDorted i n part by National I n s t i t u t e on Drug Abuse Grant # DA 01451. (Cc-4) LONG1TUOINAL DETERMI t.IATI ON OF 6-THIOGUANIME RESISTANT PER1PHERAL BLOOD LYMPHOCYTE FREQUENCIES I N INDIVIDUALS R E C E I V I N G 8-f4ETH3XYPSOF!ALEN AM0 LOHG SIAVE ULTRAVI3LET LIGHT TREATMENTS (PUVA). G.H. Strauss and R. J . A l b e r t i n i , Univ. o f V t . , B u r l i n g t o n , Vermont 05405. Thioguanine r e s i s t a n t (TGR) p e r i p h e r a l blood lymphocyte (PBL) frequenc i e s s e r i a l l y were determined i n i n d i v i d u a l s r e c e i v i n g PUVA treatments. Previously determined elevated frequencies o f v a r i a n t TFR P B L ' s i n PUVA t r e a t e d p s o r i a t i c and v i t i l i g o p a t i e n t s were made by comparisons w i t h normals and untreated v i t i l i g o p a t i e n t s . I n t h e c u r r e n t study, v a r i a n t frequencies were determined f o r s i x p s o r i a t i c p a t i e n t s b e f o r e and s e r i a l l y d u r i n g PUVA treatments. The TGR PBL frequencies o f 3 p s o r i a t ' c s w i t h normal pretreatment values o f 1 . 5 ~ 1 0 - 4 , 1 . 6 ~ 1 0 - ~ and , 1 . 3 ~ 1 0 - rose t o maxima o f 3.8~10-3, 2 . 9 ~ 1 0 - 3 and 4 . 4 ~ 1 0 - 3 r e s p e c t i v e l y a t some t i m e d u r i n g treatment. I n three p o r i a t i c p t i e n t s w i t h t l e v a t e d pretreatment TG? PBL frequencies o f 5.3x10-', 2 . 0 ~ 1 0 - and 4 . 9 ~ 1 0 - t h e values l s o increased w i t h PUVA reaching maxima o f 2.7~19-3, 4 . 3 ~ 1 0 - and ~ 2.6~10- respectively. Because p s o r i a t i c p a t i e n t s nay show elevated TGR PBL v a r i a n t frequencies w i t h o u t PUVA, t h e e f f e c t s o f PUVA on a normal i n d i v i d u a l were determined. The range o f TGR PBL frequencies f o r one normal determined i n 25 separate ~ 3 . 1 ~ 1 0 - 5 . This t e s t s d u r i n g a one year p e r i o d was found t o be Z X ~ O -t o i n d i v i d u a l was given semi-weekly PUVA treatments f o r 8 weeks and weekly treatments f o r 6 weeks. TGR PBL v a r i a n t frequencies rose t o a maximum o f 2.8~10-3 and subsequently declined. The d e c l i n e was temporally associated w i t h t h e longer i n t e r v a l betwe n treatments. PUVA has been discontinued b u t s e r i a l determination o f T G ' PBL frequencies continue. Concurrent TGR PBL frequencies i n a normal i n d i v i d u a l n o t undergoing PUVA remained normfjl d u r i n g t h i s i n t e r v a l . PUVA treatment i n humans produces e l e v a t i o n i n TG v a r i a n t frequencies. (Support by U.V.M. Surgeons and Hoffmann-LaRoche, Inc

4

3

3

F

(CC-5) INDUCTION OF THIOGUANINE RESISTANCE I N NORMAL AND XEROOERMA PIGMENTOSUM VARIANT HUMAN FIBROBLASTS BY ULTRAVIOLET LIGHT. B . C. Myhr, L i t t o n Bionetics, Inc., Kensington, MD 20795 I n order t o h e l p determine whether p o s t - r e p l i c a t i o n r e p a i r i n human c e l l s i s error-prone, t h e m u t a b i l i t i e s o f normal human f i b r o b l a s t s and xeroderma pigmentosum v a r i a n t c e l l s (XP4BE) by UV 1 i g h t were compared. The c e l l s were reseeded a t various times a f t e r UV i r r a d i a t i o n t o achieve s e l e c t i o n of s i n g l e c e l l s r e s i s t a n t t o 20 cg 6-thioguanine/ml. Approximately 6 t o 12 days ( 4 t o 8 p o p u l a t i o n doublings), depending on t h e UV dose, were necessary f o r complete expression o f r e s i s t a n c e t o 6-thioguaA h i g h e r frequency o f T G r colonies was obtained f o r XP4BE n i n e (TGr). c e l l s f o r comparisons a t equal UV doses o r a t e q u i t o x i c treatments. The frequency o f induced T G r c e l l s i n XP4BE c u l t u r e s was a l i n e a r f u n c t i o n of t h e UV dose which passed through the o r i g i n a t zero dose. I n c o n t r a s t , normal c e l l s gave a l i n e a r response t h a t e x t r a p o l a t e d t o zero induced mutants a t 3 J/m2. The UV doses and induced frequencies o f TGr c e l l s corresponding t o 37% s u r v i v a l o f c l o n i n g a b i l i t i e s were 6.7 J/m2 and 6.2 x 10-5, r e s p e c t i v e l y , f o r normal c e l l s , and 3.75 J / d and 17.3 x f o r XP4BE c e l l s . The induced frequency o f T G r c e l l s was n o t a l i n e a r

Abstracts

153

function of t h e logarithm o f s u r v i v a l for e i t h e r c e l l type. Thus, postr e p l i c a t i o n r e p a i r o f UV-induced lesions appears t o be composed o f more than one process and seems t o be e r r o r - f r e e ( o r n e a r l y SO) i n normal human f i b r o b l a s t s u n t i l a threshold dose o f 3 J/m2 i s exceeded. Errorprone r e p a i r occurs i n XP4BE c e l l s a t a l l UV doses. (CC-6) CHARACTERIZATION OF LYMPHOBLASTOID CULTURES FROM XERODERMA PIGMENTOSUM PATIENTS: UNSCHEDULED DNA SYNTHESIS AND QUANTITATIVE SURVIVAL AFTER EXPOSURE TO CHEMICAL MUTAGENS. H. Rappaport, B. J . Rose* and R. S. Slesinski, Carnegie-Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213. We have been characterizing lymphoblastoid cultures from patients with Xeroderma pigmentosum ( a )obtained from the Human Genetic Mutant Cell Repository in Camden, NJ. These include XP3BE (GMC2248, Group C), XPlZBE (GMC2250, Group A) and XPllBE (GMC2252, Group B). Cultures of XP3BE have been exposed to doses of ethylmethanesulfonate (EMS) and 4-nitroquinoline1-oxide (4NQO). One aliquot of the exposed cell suspension was examined by autoradiography to determine unscheduled DNA synthesis (UDS) and another aliquot was cloned in soft agarose (0.28%) to examine survival in a quantitative manner. The control culture in all experiments was MGL33C19 (L19) a clone derived from PGLC33H originally established by P. Glade. XP3BE is more sensitive to the cytotoxic effects of 4NQ0 (a UV-mimetic chemical) than is L19, while it is not more sensitive to EMS (not UVEMS induces equal levels of UDS in both XP3BE and L19, while mimetic). 4NQO induces lower levels of UDS in XP3BE. Preliminary experiments with XPl2BE and XPllBE indicate that they also respond in this manner. Comparative mutagenesis experiments to determine the frequency of thioguanine-resistant clones induced by 4NQ0 and EMS in the XP lines are in progress.

(CC-7) MEASURING SUBTLE DIFFERENCES IN HUMAN DNA REPAIR SYSTEM. D.F. Minka, P. Yu* and R.M. Antley, Indiana University, Indianapolis, IN 46202. It is now clear that defects in DNA repair such as XF' are related to the precocious development of cancer. The principle of defective DNA repair being etiologically important in the development of cancer suggests the possibility that subtle differences in DNA repair among humans may relate to susceptibility of malignancy. It is the long term purpose of this research to evaluate small differences in the ability of human fibroblast to repair DNA. To approach this question it has been necessary to develop measurement parameters which have sufficient sensitivity to distinguish these subtle differences in repair. Measurement of unscheduled DNA synthesis (Trosko and Yager) has been used to define the dose response curve in normal human fibroblasts. The statistically defined dose response curve accounts for 97% of the variance. The findings indicate that for fluences greater than 100 erg/mm2 the UDS responses cannot be distinguished. Sensitivity as defined by maximum response per erg with minimal experimental variation was greatest at fluences of less than 50 ergs/mm2. An analysis of the data for minimizing the coefficient of variation has been used to statistically define sensitivity. The findings o f this study provide methods for using available techniques for maximizing the possibility of demonstrating subtle but significant differences among humans in their ability to carry out DNA repair. This is Publication //78-56 and is supported in part by PHS P50 GM 21054 and PHS T32 GM 07468. (CC-8) HYPERSENSITIVITY TO ETHYLNITROSOUREA IN CULTURED FIBROBLAST STRAINS FROM PATIENTS WITH ATAXIA TELANGIECTASIA. Malcolm C. Patemon*, Paul J. Smith*, Michael V. Middlestadt*, Blake P. Smith, and N. Torben Bech-Hansen*, Atomic Energy of Canada Limited, Chalk River, Ontario. KOJ 1J0, Canada.

154

Abstracts

Persons inheriting ataxia telangiectasia (AT), a rare disease featuring neurovascular and immune abnormalities, are cancer-prone and respond fatally to conventional radiotherapy. Moreover, diploid fibroblast strains from AT patients exhibit hypersensitivity to inactivation by ionizing radiation. Ile were thus prompted to compare the colony forming ability of 3 AT strains [AT2BE (CRL 1343), AT3B1, AT4BII with two control strains from normal volunteers (GM 38, GM 498) following a 1-hr exposure to varying doses of three radiomimetic chemical mutagens: ethyl-N-nitrosourea (ENU), N-methyl-"nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS). The survival data, expressed as D10 values, are as follows: Strains ENU ( X 10-4 Mj- MNNG (X 10-6 M) MMS (X 1 0 - ~ M) Control 50-55 16 11-12.5 6-9 8-12 AT 25- 30 The AT strains are ~2-fold more sensitive than the control strains to ENU and MNNG but are only marginally sensitive to MMS (~1.2-fold). [The results confirm the abnormal lethal response of AT cells to M"G, reported recently by Scudiero (J. Supramol. Struct., suppl. 2, 83, 1978.11 It is of interest that a major component of the spectrum of reaction products induced in DNA by both ENU and MNNG, but not by MMS, is O6-n1kylguanine, a lesion implicated in nervous system-specific tumorigenesis in rodents. Our earlier radiation studies suggest that certain AT strains are defective in the excision of radio,enic base defects from DNA and it may be that AT cells are likewise defective in the removal of 06-alkylguanine residues. Molecular studies are underway to test this possibility. (CC-9) IN VITRU CO-SENSITIVITY TO NEAR-UV LIGHT AND IONIZING RADIATION IN TWO HUMAN SYNDROMES. Paul J. Smith and Malcolm C. Paterson, "intr. by Donald P. Morrison", Atomic Energy of Canada Limited, Chalk River, Ontario, K O J 1J0, Canada. We have studied the UV and y-ray survival responses of cultured fibroblasts from normal donors, ataxia telangiectasia (AT) patients, and a patient with the recessive skin disorder Rothmund-Thomson Syndrome (RTS). Significant features of AT are: the oculo-cutaneous telangiectasis, i n vivo and i n v i t r o sensitivity to ionizing radiation, and the predisposition of AT patients to cancer. RTS is characterized by telangiectasis, erythema, and juvenille cataracts. The RTS patient studied exhibited sunsensitivity together with basal cell carcinoma of the eyelid. Although all strains studied were found to show normal responses to far-UV light (254 nm), the RTS strain and one out of three AT strains showed a two-fold sensitivity to near-UV light (313 nm) based upon D10 values. Furthermore it was found that the RTS strain was also 1.6-fold more sensitive to oxic y-radiation than normal strains whereas the AT strains were ~3.2-fold more sensitive. Thus, we report mutual co-sensitivity to near-UV light and yradiation in v i t r o for two clinical situations diagnosed as either ionizing radiation sensitive (i.e. AT) or sunlight sensitive (i.e. RTS). The results suggest that components of the DNA damage induced by near-UV light and by y-radiation are qualitatively similar, or are dealt with by overlapping repair pathways. DNA repair studies on DNA strand break rejoining and base damage excision are in progress. We conclude that the long UV wavelengths not absorbed by the atmosphere may have a partially radiomimetic biological effect, therefore modifying the terms in which this ubiquitous carcinogen should be considered.

(CC-lo) ISOLATION O F A HUMAN LYMPHOBLASTOID LINE HETEROZYGOUS AT 1 H E THYMIDINE KINASE LOCUS: POSSIBILITY FOR A PAPID HUMAN CELL MUTATION ASSAY. H . L . Liber, B . W . Penman, T . R . Skopek and W.G. Thilly. Genetic Toxicology Group, M . I . T . , Cambridge, Massachusetts 02139.

Abstracts

155

A thymidine kinase (tk) heterozygote has been isolated f r o m the diploid human lymphoblastoid line HH4. Isolation of the line was accomplished by f i r s t selecting an ICR-191-induced tk-'- homozygote via its r e s i s t a n c e to the thy idine analog trifluorothymidine (TFTdR). An ICR-191 -induced tk+? heterozygote was then selected f r o m this population v i a its r e s i s t a n c e to medium containing aminopterin, cytidine, thymidine, and hypoxanthine (CHAT). Significant increases in TFTdRresistant fraction in the heterozygous line h a v e been observed with several mutagens including ICR-191 and butyl methanesulfonate. Phenotypic expression is complete 48 h o u r s after treatment. The line is presently being used in a study investigating the mutagenic potential of s o o t and s o o t components. (CC-11) MUTAGICNESIS I N HUMAN CELLS. W .G. Thilly, Genetic Toxicology Group, M . J . T . , Cambridge, Massachusetts 02139.

The u s e of diploid human lymphoblasts a s targets in mutation assays has been quantitatively characterized and examined with a wide range of environmental mutagens. A s a scientist interested in thc. mechan i s m s of chemically induced mutation, I wish to apply the a s s a y to studies of the kinetics of the appearance and removal of pre-mutagenic lesions. What a r e the experimental perturbations which teach u s more aDout the mutagenic process via changes in the amount of mutation observed ? (cc-12) BILE AS A SOURCE OF MUTAGENIC METABOLITES PRODUCED I N V I V O AND DETECTED BY SALMONELLA TYPHIMURIUM. T.H. Connor, G. C a n t e l l i F o r t i , P . S i t r a f and M.S. L e g a t o r , U n i v e r s i t y of Texas M e d i c a l Branch, G a l v e s t o n , Texas 77550 S e v e r a l d i r e c t - a c t i n g mutagens o r promutagens r e q u i r i n g a c t i v a t i o n by l i v e r enzymes were a s s a y e d i n a n i n v i v o system. Male S-D r a t s w e r e a n e s t h e t i z e d w i t h u r e t h a n e and t h e common b i l e d u c t was c a n n u l a t e d u s i n g p o l y e t h y l e n e t u b i n g . The b i l e w a s c o l l e c t e d a t one-hour i n t e r v a l s o v e r a s i x - h o u r p e r i o d a f t e r i . v . i n j e c t i o n of t h e c h e m i c a l . A f t e r f i l t r a t i o n , t h e b i l e w a s a s s a y e d f o r mutagenic a c t i v i t y u s i n g Salmonella typhimurium TA1535, TA1537, TA1538, TA100 o r TA98 w i t h and w i t h o u t t h e a d d i t i o n of 6 - g l u c u r o n i d a s e t o t h e a g a r o v e r l a y . Of t h e d i r e c t - a c t i n g mutagens, hycanthone m e t h a n e s u l f o n a t e , 25 mg/kg (TA1538) r e v e r t e d t h e tester s t r a i n w h i l e m e t h y l m e t h a n e s u l f o n a t e , 1 0 mg/kg (TAlOO) d i d n o t . Of t h e mutagens r e q u i r i n g m e t a b o l i c a c t i v a t i o n , t h e f o l l o w i n g r e v e r t e d t h e tester s t r a i n s : cyclophosphamide, 50, 100 mg/kg (TA1535); b e n z o ( a ) p y r e n e 10, 20 mg/kg (TAl538, TA98) ; 2-aminoanthracene, 5 mg/kg (TA1538) ; and 2-aminofluorene, 10 mg/kg (TA1538). A l l o f t h e s e mutagens, e x c e p t cyclophosphamide, were e x c r e t e d a s c o n j u g a t e s o f g l u c u r o n i c a c i d . Chemicals which d i d n o t r e v e r t t h e tester s t r a i n s w e r e : i s o n i a z i d , 100 mg/kg (TA1535) ; dibromochloropropane ( p u r i f i e d ) , 5 mg/kg (TA1535); d i e t h y l s t i l b e s t r o l , 50 mg/kg (TA153a; and s a c c h a r i n ( p u r i f i e d ) 100 mg/kg (TA100, TA98). U r e t h a n e i t s e l f had no e f f e c t on any of t h e f i v e tester s t r a i n s d u r i n g t h e 6-hour p e r i o d . A d d i t i o n a l s t u d i e s are b e i n g c a r r i e d o u t w i t h known and s u s p e c t e d mutagens t o f u r t h e r ev a lu a te t h i s procedure.

Supported i n p a r t by a g r a n t from t h e Environmental P r o t e c t i o n Agency.

( CC-1 3 ) CONVERSION OF HUMAN BILE TO MUTAGENS BY HUMAN-DERIVED STRAINS OF BACTEROIDES FRAGILIS. George E. Karpinsky, Elena C. McCoy and H e r b e r t S. Rosenkranz, Dept. o f Microbiology, New York Medical C o l l e g e , V a l h a l l a , NY 10592.

156

Abstracts

Epidemiologic studies have implicated high meat diets in human colon cancer causation. Such diets have also been shown to result in an enrichment of the anaerobic colonic flora which may further be involved in the etiology of colon cancer as a result of the ability of anaerobic bacteria to participate in the biotransformation of bile components to substances possessing potential carcinogenic activity. An attempt was made to determine whether certain strains of 1.fragilis, a normal constituent of human colonic flora, could alter human bile, in such a way as to produce mutagenic substances. Strains of g . fragilis isolated from humans were selected for their ability to grow in the presence of relatively large concentrations of bile acids (200-250 ug/ml). Human biles from patients free of neoplasias were used. The untreated bile when tested in the Salmonella mutagenicity assay using strains TA1538 and TA100, with and without rat liver S 9 , did not exhibit mutagenicity. When bile (10-20% v/v) was added to growing 1.fragilis and the filtrate collected, genetic activity was demonstrated. This activity was enhanced further in the presence of S9. This finding suggests the possibility that bile-tolerant anaerobic bacteria participate in the bioconversion of bile to substances which may possess carcinogenic potential.

( CC- 14) DETECTION OF SOMATIC MUTATIOXS IJ HUMAN ERYPH3OCYTES. W.L. Bigbee*, E.W. Branscomb", M.L. Mendelsohn, Biomedical Sciences Division, Lawrence Livermore Laboratory, Livermore, CA., and G. Stamatoyannopoulos*, Div. of Medical Genetics, Univ. of Iv'ashington School of Medicine, Seattle, WA. Evidence linking the processes of mutagenesis and carcinogenesis and the existence of cancer prone individuals with DNA repair deficiences suggest a compellint: need for an assay of the level of genetic damage to the somatic tissue of human individuals. We are developing such an assay using a high speed microfluorometric method with a goal to detect somatic mutations in human erythrocytes by labeling abnormal hemoglobins, expressed as a result of point mutations in the DNA of the erythropoietic stem cell population. Labeling can be achieved with fluorescent antibodies specific for point mutant sites on the hemoglobin molecule. Red blood cells can be reacted with the antibody in suspension yielding fluorescent cells which can then be detected at high counting rates in a flow sorter. Quantitative recoveries of labeled cells can be obtained with processing rates exceeding l o 6 cells per second, thus affording the opportunity to count rare mutant cells with high statistical accuracy. I n reconstruction experiments, O I - . ~ heterozygous sickle cell ( A S ) in l o 4 normal cells (AA) is presently detectable; sensitivity must be increased at least 1000 fold to measure expected background rates. While the relationship between the frequency of circulating mutant hemoglobin-containing red cells and the stem cell mutation rate is unclear due to the unknown kinetic details of red cell differentiation and maturation, the demonstration of elevated frequencies of mutant cells in vulnerable individuals may provide presumptive evidence of genetic risk. Work performed under NIEHS contracts with DOE/LLL and with the University of Washington.

(Cd) MECHANISMS AND INTERPRETATION OF MUTAGENICITY Chaired by:

J. B. Favor Georgia Institute o f Technology and P . B. Selby Oak Ridqe National Laboratory

Abstracts

157

(Cd-1) RELATIVE SENSITIVITIES O F FORWARD AND REVERSE MUTATION ASSAYS IN SALMONELLA TYPHIMURIUM. T . R . Skopek, H . L . Liber, D.A. Kaden and W.G. Thilly, Genetic Toxicology Group, M.I. T . , Cambridge, Massachusetts 02139.

Forward mutation to 8-azaguanina r e s i s t a n c e and r e v e r s e mutation to histidine prototrophy were measured in Salmonella typhimurium after t r e a t m e n t with 1 6 mutagens of both base-substitution and frameshift classes. The two approaches were found to be equisensitive for all 16 mutagens --i. e., induction of significant mutation occurred at s i m i l a r concentrations in the forward mutation a s s a y and in the most sensitive of the five Ames t e s t e r s t r a i n s . Two other forward mutation a s s a y s based on r e s i s t a n c e to fluorouracil and azetidine carboxylic acid a r e approximately as sensitive a s the 8-azaguanine s y s t e m . Since only one s t r a i n is needed to detect a variety of mutagens with equal s e n s i tivity to the Ames assay, the 8-azaguanine forward mutation a s s a y may play a valuable r o l e in toxicological screening programs. (Cd-2) THE NEED FOR DEFINING AREAS OF DIVERGENCE BETWEEN MUTAGEN SCREENING TESTS. BENZENE AND PHENYLENEDIAMINE (HAIR DYE), INDUCED SISTER CHROMATID EXCHAEZGE G . Bynum, D. Kram, E. L. Schneider*, Gerontology Research Center, b!IA, NIH, Baltimore, MD 21224. The difficulties in identifying a single model for human mutagenicity, uecessitates defining matrices of mutagen screening tests for which discontinuities and areas of convergence and divergence are well established. Given such information, a matrix or battery of screening tests could be optimized for a compound based upon known test responses to structurallv similar compounds. Existing screening assays give a high percentage of positive findings, resulting in a high degree of data convergence or overlap when a compound is evaluated in multiple tests. Therefore, it is the areas of divergence that are of critical importance in defining testing matrices. Benzene and phenylenediamine were evaluated for SCE induction in vivo and in vitro. These data when contrasted with Ames test data in the literature for these compounds, illustrate areas of divergence f o r Ames and SCE mutagen screening tests. In Vitro In Vivo -Ame s SCE SCE Benzene + + Phenylenediamine + + (+) Mutagenic (-) Non-Mutagenic Benzene and phenylenediamine are, therefore, compounds in which Ames and SCE diverge as screening tests. Data of this nature if systematically developed could be utilized to identify test batteries. Such optimized mutagen screening matrices would enhance cost effectiveness and maximipe the probability of identifying a mutagenic compound. (Cd-3) INDUCTION OF PUTATIVE HISTOCOMPATIBILITY MUTATIONS IN MOUSE SPERMATOGONIA BY TRIETHYLENE MELAMINE (TEM) H. I. Kohn and G . R. Dunn, Dept. of Radiation Therapy, Harvard Medical School, and Shields Warren Radiation Laboratory, New England Deaconess Hospital, Boston, MA 02115.

.

This expeqiment retests the observation that TEM mutates histocompatibility genes (Kohn, Mutation Res. 20:235, 1973). Male BALB/c mice,125150 days old, received either 0, 2.5, 2.9, 3 . 8 or 5.8mgfkg of TEMintraperitoneally as a single dose, or in two equal fractions given one or eight days apart, or in four equal fractions given on days 0, 1, 8 and 9 .

158

Abstracts

Seventy t o n i n e t y d a y s l a t e r t h e y were mated t o C57BL/6 f e m a l e s 125-150 d a y s o l d . The progeny were t e s t e d f o r m u t a t i o n s w i t h o r t h o t o p i c t a i l - s k i n g r a f t s a t 37-43 d a y s of a g e , s u b s e q u e n t l y o b s e r v e d f o r 60 d a y s . The p u t a t i v e m u t a t i o n r a t e s o b s e r v e d t h u s f a r a p p e a r t o depend o n TEM d o s e and on f r a c t i o n a t i o n s c h e d u l e .

(Cd-4) NO INCREASED INCIDENCE OF HISTOCOMPATIBILITY MUTATIONS SEEN I N PROGENY FROM IRRADIATED MOUSE SPERM. G.R.Dunn and H . I . K o h n , Dept. o f R a d i a t i o n Therapy, Harvard Medical S c h o o l , and S h i e l d s Warren R a d i a t i o n L a b o r a t o r y , New England Deaconess H o s p i t a l , Boston, MA 02115. The progeny o f mice whose s p e r m a t o g o n i a had been i r r a d i a t e d showed no i n c r e a s e i n t h e f r e q u e n c y o f m u t a t i o n s of h i s t o c o m p a t i b i l i t y (H) g e n e s ( B a i l e y and Kohn, Genet. R e s . 6:330, 1965; Kohn and Melvold, N a t u r e 259: 209, 1976; Kohn, Melvold and Dunn, M u t a t i o n R e s . 37:237, 1976). Because H-gene m u t a n t s might be e l i m i n a t e d d u r i n g s p e r m a t o g e n e s i s , t h e o c c u r r e n c e o f mutants i n progeny from i r r a d i a t e d sperm h a s been d e t e r m i n e d . Male BALB/c mice, 125-150 d a y s of a g e , r e c e i v e d s i n g l e d o s e s of x-rays t o t h e c a u d a l t h i r d o f t h e body o f 0 , 350, o r 650 r a d s , o r 350+300 r a d s 24 h o u r s a p a r t . Immediately f o l l o w i n g i r r a d i a t i o n , e a c h male w a s mated w i t h two C57B1/6 f e m a l e s f o r one week. The progeny were a s s a y e d f o r H-gene mut a t i o n s by o r t h o t o p i c t a i l - s k i n g r a f t s made a t 37-43 d a y s o f a g e and obs e r v e d f o r 60 d a y s . P u t a t i v e m u t a n t s , d e t e c t e d by unexpected g r a f t rej e c t i o n s , were mated and t h e i r progeny g r a f t - t e s t e d t o prove t r a n s m i s s i o n . No Class I m u t a n t s ( 3 5 s p e c i f i c l o c i ) have been found i n 32,795 g e n e s t e s t e d i n c o n t r o l progeny and o n l y one i n 53,060 g e n e s a t r i s k i n e x p e r i m e n t a l s . I n C l a s s I1 H-genes (number unknown), one mutant h a s been found i n 937 cont'rol progeny and two i n 1516 e x p e r i m e n t a l progeny. The rel a t i o n s h i p of t h e s e r e s u l t s t o o t h e r s t u d i e s w i l l be d i s c u s s e d .

(Cd-5) THE EFFECTS OF MUTAGENIC TREATMENT ON QUANTITATIVE TRAITS I N THREE INBRED STRAINS OF MICE. J. W. Crenshaw and J . B . Favor, Georgia I n s t i t u t e of Technology, A t l a n t a . GA 30332.

-I n v i v o mammalian m u t a g e n i c i t y tests u t i l i z i n g q u a n t a t i v e t r a i t s i n mice would be u s e f u l i n p r e d i c t i n g human r i s k s s i n c e t h e y would s c r e e n f o r c u m u l a t i v e e f f e c t s o f p o i n t m u t a t i o n s o f s l i g h t e f f e c t which t e n d t o persist i n t h e gene p o o l . The e f f e c t s of two d o s e s of TEM ( 0 . 1 and 0.2 mg/kg) were compared w i t h c o n t r o l s i n t h r e e s t r a i n s of mice: BALB/cByJ, C3H/HeJ and DBA/2J. T r e a t e d males were mated w i t h f e m a l e s o f t h e same s t r a i n a t i n t e r v a l s from time of t r e a t m e n t t o produce spermatozoan and spermatognnial t r e a t e d progeny. The number of m a t i n g s s e t i n each d o s e group was adj u s t e d t o compensate f o r dominant l e t h a l e f f e c t s of t r e a t m e n t i n t h e h i g h d o s e g r o u p s i n such a way t h a t s i m i l a r numbers of F1 progeny would b e produced i n a l l groups. A t m a t u r i t y F 1 mice were randomly mated w i t h i n d o s e , s t r a i n and t r e a t m e n t group t o produce F2 progeny. V a r i a t i o n i n e i g h t quant i t a t i v e t r a i t s w e r e compared w i t h i n g e n e r a t i o n and t r e a t m e n t group f o r t r e a t m e n t e f f e c t s . T r a i t s employed were: (1) a g e of development of t h e r i g h t i n g r e s p o n s e , ( 2 ) body weight a t weaning, ( 3 ) t a i l l e n g t h a t s e v e n w e e k s , ( 4 ) h e m a t o c r i t a t seven weeks, ( 5 ) t h e d e f a c a t i o n p o r t i o n of t h e open f i e l d t e s t , ( 6 ) b r a i n weight a t 13% o r more weeks, ( 7 ) p e r c e n t f e m a l e s weaned and (8) number o f young weaned p e r female. S i g n i f i c a n t t r e a t m e n t e f f e c t s were o b s e r v e d f o r a number of t r a i t s i n t h e spermatoman t r e a t e d group, and r e m a r k a b l e d i f f e r e n c e s were found among i n b r e d s t r a i n s with respect t o treatment e f f e c t s . The most i m p r e s s i v e d i f f e r e n c e s were found w i t h r e s p e c t t o t r e a t m e n t e f f e c t s upon t h e t i m e of development o f t h e r i g h t i n g r e s p o n s e , h e m a t o c r i t and t a i l l e n g t h a t seven weeks of a g e . (Cd-6) GREATER GENETIC W A R D FROM RADIATION RESULTS FROM BALANCED RECIPROCAL TRANSLOCATIONS THAN FROM SEGMENTAL ANEUPLOIDS. P. B. S e l b y , Biology D i v i s i o n , Oak Ridge National L a b o r a t o r y ? Oak Ridge, Tenn. 37830

Abstracts

159

At least 3, and perhaps 4, of the 37 dominant skeletal mutations reported by Selby and Selby (1977, Mutat. Res. 43: 357-375) are reciprocal translocations, i.e., the mice have malformations because they are heterozygous for reciprocal translocations. A comparison of this frequency of induction with the frequency of induction of heritable translocations reported by Generoso et.al. (Biology Division Annual Report, 1974. ORNL4993: 136-138), suggests that most reciprocal translocations cause malformations. (This conclusion assumes, as did UNSCEAR for dominant mutations in general in its 1977 report, that the reciprocal translocations affecting the skeleton constitute about one-tenth of the reciprocal translocations affecting all body systems.) Furthermore, the point estimate of the fraction of all translocations that would cause malformations severe enough to be a serious handicap is about one-half, with wide confidence limits. In contrast, in the UNSCEAR Reports of 1972 and 1977, it is assumed that the induced incidence of liveborn (and presumably seriously handicapped) segmental aneuploids is, at most, 12% of the incidence of induced reciprocal translocations. This comparison is one of the ways of showing that the yield of induced damage is probably greater for translocations in the balanced state than for segmental aneuploids. This is an interesting conclusion because past risk estimates that have been based upon cytological data or frequencies of heritable translocations have ignored any component of hazard resulting from reciprocal translocations in the balanced state. Instead, the hazard from induced translocations was thought to result only from segmental aneuploids. Operated by Union Carbide Corporation with the U.S. Department of Energy

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(Cd-7) GENETIC EFFECTS OF LOW DOSE NEUTRON IRRADIATION. D. Crahn, B. H. Frystak*, and C. H. R. Lee*, Division of Biological and Medical Research, Argonne National Laboratory, Argonne, IL 60439. Recent concerns about the potential genetic and somatic effects of extremely low doses of fission neutrons prompted us to evaluate the genetic effects of 1, 2.5, 5, 10, 20, and 40 rads of single whole-body doses of % 0.8 MeV fission neutrons in C57BL/6 x BALB/c F1 male mice. We used the short-term, economical, and accessible end points of dominant lethal mutation frequency in postmeiotic cells, frequency of abnormal sperm head morphology, testis weight loss, and frequency of reciprocal chromosome translocations induced in spermatogonia. These end points may fulfill the need for a model short-term approach to the study of long-term somatic effects. The frequency of dominant lethals is taken as the average over the first five postexposure weeks; abnormal sperm frequencies are the average for weeks five and six; and testis weight is measured at week four. Chromosome aberrations are scored at first meiotic metaphase five to seven weeks after exposure. A limited comparison with 6oCo y-rays is also incorporated in the evaluation. The important result to date is that a response at 1 rad can be detected. Due to as yet inadequate sampling statistics, the responses at 1, 2.5, and 5 rads are not significantly above the control, but generally they increase proportionally to dose. At these low dose levels neutron effects are about 20-fold greater than those following 6oCo y-irradiation. At higher doses, the difference is less than 10-fold. A greater than the linearly predicted response appears at 1 and 2.5 neutron rads, whereas responses to 5 rads and above generally conform to the expected monotonic relationship. (Work supported by the U. S . DOE).

(Cd-8) ANALYSES OF PY)USE LYMPHOMA TK ASSAY RESULTS FOR STATISTICAL SIGNIFICANCE V I A A TWO SAMPLE t TEST. D. E. Amacher*, S.C. Paillet*, D. S. Salsburg*, and Verne A. Ray. Drug Safety Evaluation Dept. Pfizer Central Research, Groton, CT 06340

160

Abstracts

The m u s e lymphoma L5178Y TK+'-+ TIC-'point mutation assay is currently being considered as part of a standard screening battery for determining the mutagenic potential of chemicals. Unfortunately, adequate studies to determine whether this in vitro test can correctly discriminate between mutagens and nonmutagens have been lacking. A more fundamental deficiency in this and other point mutation assays is an objective basis for the interpretation of test results; i.e., what constitutes a positive or negative response. Various criteria for test significance used by others have largely been based on subjective or arbitrary guidelines. After testing and retesting over twenty different chemical compounds or agents, we have concluded that a simple 2 sample t test can be used to compare the number of induced mutants in treated versus control cultures for statistically significant differences. Using data from treatment levels producing 20-100% cell survival, strong mutagens such as ICR 170H, ICR 191G, N-nitroso-methylurea, 3 methylcholanthrene, or 4 nitroquinoline-1-oxide produced significant increases in the numbers of trifluorothymidine-resistant mutants (P