153
Mutation Research, 49 (1978) 153--161 © Elsevier/North-Holland Biomedical Press
NON-ENZYMIC ACTIVATION OF POLYCYCLIC AROMATIC HYDROCARBONS AS MUTAGENS
THOMAS L. GIBSON, VERA B. SMART and LELAND L. SMITH Division of Biochemistry, The Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Tx. 77550 (U.S.A.) (Received 26 May 1977) (Revision received 20 September 1977) (Accepted 26 September 1977)
Summary Samples of 22 polycyclic aromatic hydrocarbons and related derivatives were subjected to ~°Co gamma radiation in air, and the irradiated samples were tested for mutagenicity with the Salmonella typhimurium strains TA 98, TA 1535, TA 1537, and TA 1538. Testing was conducted with the bacterial strains alone, thus not fortified with liver-microsomal enzymes or other metabolizing systems. Marked mutagen responses were obtained for several irradiated samples with the TA 98, TA 1537, and TA 1538 strains but not with the TA 1535 strain. Irradiated samples of benzo [a ] anthracene, benzanthrone, benzo[g,h,i]perylene, benzo[a]pyrene, chrysene, fluorene, 9-methylanthracene, 1-methylphenanthrene, 2-methylphenanthrene, and pyrene gave positive mutagenic tests and dose--responses, whereas unirradiated control samples of these were inactive. Acenaphthene, phenanthrene, and phenanthrenequinone exhibited toxicity which interfered with interpretation of mutagenicity testing. Samples of 2-methylanthracene and tetracene were mutagenic with or without irradiation. Alizarin, anthracene, anthraquinone, anthrone, dibenzo[a,h]anthracene, picene, and triphenylene gave negative results. Samples of benzo[a]pyrene adsorbed on silica gel irradiated in air by ~°Co gamma radiation or by 254 nm ultraviolet light and samples adsorbed on filter paper irradiated by visible light yielded preparations mutagenic towards the TA 98, TA 1537, and TA 1538 strains. These results suggest that parent polycyclic aromatic hydrocarbons not themselves mutagenic towards S. typhimurium may be oxidized in air by radiation-induced processes to products whose mutagenicity resembles that of liver-microsomal metabolites of the parent polycyclic aromatic hydrocarbon.
154
Introduction Although much attention is currently being given to the metabolic activation by mammalian tissues of polycyclic aromatic hydrocarbons as mutagenic agents, little interest in such activation by non-enzymic means has developed. In that polycyclic aromatic hydrocarbons found quite broadly in nature, including soils, drinking water, polluted air, cigarette smoke, etc. are exposed to the oxygen of the air and to radiation from many sources the question of radiation-induced oxidations of this class of compounds to active mutagens attracted our interest. We have pursued the matter using as convenient means the Salmonella typhimurium bacterial mutagen testing procedure of Ames et al. [1--3,9]. We report herein our results using this approach, results which demonstrate that several polycyclic aromatic hydrocarbons are indeed transformed into mutagenic agents following irradiation in air with 6°Co gamma radiation. Materials and Methods Samples of anthracene (99.9% gold label), anthrone, anthraquinone, 2methylanthracene, 9-methylanthracene (99%), alizarin (97%), benzanthrone, phenanthrene (98+%), triphenylene (98%), and benzo[g,h,i]perylene were purchased from Aldrich Chemical Co., Milwaukee, WI. Chrysene, fluorene, 1-methylphenanthrene, 2-methylphenanthrene, picene, tetracene, acenaphthene, benzo[a]pyrene, and benzo[a]anthracene were from Analabs, North Haven, CT, and pyrene from Eastman Organic Chemicals, Rochester, NY. The purity of the compounds was checked by ultraviolet spectroscopy, thin-layer or high-pressure liquid chromatography, mass spectrometry or other methods without finding impurities. Benzo[a]pyrene was irradiated under four different conditions: (1) crystals irradiated by 6°Co gamma radiation from a Gammacell 200 (Atomic Energy of Canada Ltd., Ottawa) for 7 days, providing 2.5 X 107 rad, (2) adsorbed (1% by weight) on silica gel (60--200 mesh) irradiated by 6°Co gamma radiation (0.5 X 107 tad), (3) adsorbed (1% by weight) on Silica Gel HF2s4 (Merck, Darmstadt) and irradiated by 254 nm ultraviolet light from two 15-W quartz lamps for 2 days, and (4) adsorbed on Whatman No. 1 filter paper (2.5 mg per 50 sq.cm.), irradiated for 18 days by room light from overhead 40-W fluorescent tubes. Irradiation of the other polycyclic aromatic hydrocarbons was conducted only with 6°Co gamma radiation (2.5 X 107 rad). Irradiated samples were stored under dry nitrogen until tested for mutagenicity. Mutagenicity was determined with the Salmonella typhimurium test of Ames et al., [1--3,9] in which mutant strains of the bacterium nutritionally dependent upon histidine were used. No tissue homogenate, enzyme, or cell preparation was included for intermediate metabolic transformation of irradiated samples. The test strains used included TA 1537, TA 1538, and TA 98 (TA 1538 plus an R-factor plasmid) which detect frameshift mutagens and TA 1535 which detects base-pair substitution mutations, all strains kindly provided by Prof. B.N. Ames, University of California, Berkley, Calif. Polycyclic aromatic hydrocarbon samples were dissolved in dimethylsulfoxide
155 or in dimethylsulfoxide containing a small volume of acetone so as to give solutions between 0.2--9 mg/ml concentration. Measured volumes of these solutions (none more than 0.5 ml) were incorporated into liquid agar (2 ml) which was then thoroughly mixed. The agar containing the incorporated sample and test organism was poured over the base agar plate and allowed to set. Plates were incubated for 48 h at 37°C, after which time individual colonies of bacteria were counted. All tests of dimethylsulfoxide and of dimethylsulfoxide containing acetone (0.5 ml/plate) conducted regularly gave no signs of mutagenicity or of solvent toxicity. Samples were generally tested over a range of concentrations in order to discern indication,~ of dose response. A two-fold or greater increase in revertants, i.e., observed number of revertant colonies three-fold or greater than the spontaneous number, was considered a positive test. Toxic effects were checked by examination of the background lawn of bacterial growth and by the presence of at least the usual number of spontaneous revertants. Control counts of spontaneous revertants obtained for the four test strains averaged: TA 98, 48; TA 1535, 21; TA 1537, 12; TA 1538, 25. Thin-layer chromatography was conducted with 20 X 20 cm chromatoplates of Silica Gel HF2s4 (E. Merck GmbH., Darmstadt) 0.25 mm thick, irrigated in ascending fashion with benzene or with chloroform--methanol ( 4 : 1 , v/v). Individual components were visualized under 254 nm or 366 nm ultraviolet light. Results Irradiated and unirradiated control samples of 22 polycyclic aromatic hydrocarbons and their derivatives were tested with as m a n y as 4 S. typhimurium test strains for possible mutagenicity. Data of Table 1 establish that definite responses were obtained for benzo[a]pyrene subjected to air oxidations induced by various radiation. A similar pattern of mutagenicity responses was had whether visible light, 254 nm light, or ~°Co gamma radiation was used to initiate the oxidations. Linear or near-linear dose--response curves were obtained with the three strains TA 98, TA 1537, and TA 1538 for 6°Co gamma irradiated samples of crystalline benzo[a]pyrene and of benzo[a]pyrene adsorbed on silica gel and with TA 1537 and TA 1538 for samples of benzo[a]pyrene adsorbed on filter paper irradiated with visible fluorescent light. Unirradiated samples of benzo [a] pyrene subjected to the same manipulations as the irradiated samples were uniformly non-mutagenic as was benzo[a]pyrene irradiated with ~°Co gamma radiation under vacuum. Data of Table 2 confirm and extend our observations of the radiationinduced activation of benzo[a]pyrene. Here irradiated samples shown to be mutagenic were fractionated by thin-layer chromatography and material from several regions of the chromatoplate was tested for mutagenicity. In Expts. No. 1 and No. 2 {benzene as irrigation solvent) fractions l b and 2b containing recovered benzo[a]pyrene ( R f 0 . 6 ) showed negligible mutagenicity, b u t material from the more polar regions was mutagenic, as was material pooled from these several zones. Furthermore, retesting of pooled material recovered from agar test plates of the initial testing of samples l c - - l e suggested a marked
156 TABLE 1 MUTAGENIC ACTIVATION OF BENZO[a]PYRENE Expt. No.
1
Benzo[a]pyrene treatment a
Amount tested (pg/plate)
Crystals, 60Co gamma radiation, for 7 days ( 2 . 5 × 107 r a d )
10 20 50 100 200 3OO
Adsorbed, 60Co gamma r a d i a t i o n f o r 1.5 d a y s ( 0 . 5 X 107 r a d )
50 100 200
3
Adsorbed, 254 nm light for 2 days
500 1000 1200
4
A d s o r b e d , visible l i g h t f o r 18 d a y s
5
Control, unirradiated crystals
20~200
6
Control, unirtadiated material recovered f r o m silica gel
20--200
7
Control, unirradiated material recovered f r o m filter paper
50--100
8
Control, crystals 60Co gamma radiation for 7 d a y s ( 2 . 5 X 107 r a d ) in v a e UUlTI
2
BY RADIATION IN AIR M u t a g e n i c r e s p o n s e , i n c r e a s e in r e v e r t a n t s c
b
TA1535
0 0
0 0
50 100 2OO
TA1537
4 11 15 19
3.1 6.8 9.4
TA1538
TA98
4.3 10 20 26
1.5 3 10 15 21 35
1.9 3
1.5 2 2.6
4 2.5 0 d 3.4 3.5 6.7
6.3 9.6 13
4.4 13 16
0
0
0
0
0
0
100
a All c o n d i t i o n s c o n d u c t e d in t h e p r e s e n c e o f a i r e x c e p t as n o t e d . b W e i g h t o f s a m p l e a c t u a l l y t e s t e d p e r p l a t e , a d d e d as d i m e t h y l s u l f o x i d e s o l u t i o n i n c o r p o r a t e d i n t o t o p agar layer. c I n c r e a s e in r e v e r t a n t s = t o t a l n u m b e r o f r e v e r t a n t s - - n u m b e r o f s p o n t a n e o u s r e v e r t a n t s number of spontaneous revertants. d Toxic effects noted.
increase in revertant colonies of bacteria too high for proper counting. Therefore, the induced mutagenicity of irradiated benzo[a]pyrene was stable to chromatography and to manipulations. Moreover, dimethylsulfoxide solutions of mutagenic, irradiated benzo [a]pyrene samples retained their mutagenicity to the TA 1 5 3 8 strain for as long as 86 days storage at room temperature. In Expt. No. 3 using a more polar irrigation solvent polar fractions 3b and 3c (Rf 0.31--0.74} contained most of the mutagenic activity with negligible activity being found in the most mobile zone 3a which contained parent benzo[a]pyrene. As these chromatographic operations did not exclude with certainty components from one zone overlapping into adjacent zones, it is not possible to determine h o w many mutagenic components were formed, but it is clear that the activity is associated with components more polar than the parent hydrocarbon.
157 TABLE 2 PRELIMINARY CHROMATOGRAPHIC IRRADIATION PRODUCTS a Expt. No.
Sample No.
CHARACTERIZATION
Sample identity
OF MUTAGENIC BENZO[a]PYRENE
Mutagenic response, increase in r e v e r t a n t s TA1538
1
2
Crystals, 2 . 5 × 1 0 7 t a d la
Unfractionated products
lb lc ld le If lg
Rf 0.5--1.0 fraction b Rf 0.2--0.5 fraction b Rf 0.1--0.2 fraction b Rf 0--0.1 fraction b Pooled Nos. lb--le material Extracted agar from Nos. lc--le c
19/500 ~g 25/1000 #g 0 5.7 10 15 7.2 v e r y high
Adsorbed, 0 . 5 × 1 0 7 r a d 2a 2b 2c 2d 2e
3
TA1537
Unfractionated Rf 0.5--1.0 b Rf 0.2--0.5 b Rf 0.1--0.2 b Rf 0--0.1 b
6.2 0 2.7 1.5 8.3
1.6 3.4 2.3 5.5
Crystals, 2.5 X 1 0 7 r a d 3a 3b 3c 3d 3e
Rf Rf Rf Rf Rf
0.74--1.0 b 0.44--0.74 b 0.31--0.44 b 0.10--0.31 b 0--0.10 b
1.7 6.5 4.2 3.6 3.5
(0) d (4.8) d (5.0) d (1.0) d (0) d
a Data were obtained following irradiation of 4.0 mg benzo[a]pyrene and thin-layer chromatographic f r a c t i o n a t i o n o f t h e i r r a d i a t e d s a m p l e . F o o t n o t e s a a n d c o f T a b l e 1 p e r t a i n t o T a b l e 2 also. b C h r o m a t o g r a p h i c s o l v e n t f o r E x p t s . N o . 1 a n d 2: b e n z e n e ; f o r E x p t . N o . 3: c h l o r o f o r m - - m e t h a n o l (4 : 1, v/v). M o b i l i t y ( R f ) o f a u t h e n t i c r e f e r e n c e s a m p l e s i n t h e t w o s o l v e n t s y s t e m s w e r e : b e n z o [ a ] p y r e n e , 0.62, 1.0; 6-hydroxybenzo[a]pyrene, 0.26 --; 9-hydroxybenzo[a]pyrene, 0.15, 1.0: benzo[a]p~ene-l,6d i o n e , 0 . 2 0 , 0 . 8 ; b e n z o [ a ] p y r c n e - 6 , 1 2 - d i o n e , --, 0 . 8 ; benzo[a]pyrene-7,S.dihydro-trans-7,8-diol, 0, 0 . 6 7 ; benzo[a]pyrene-9,10-dihydro-trans-9,10-diol, Oo 0 . 6 3 . c Following mutagenicity testing of samples Nos. lc--le, agar plates were stored under refrigeration for 5 - - 7 d a y s , t h e n e x t r a c t e d w i t h b e n z e n e - - a c e t o n e , t h e s o l v e n t e x t r a c t s e v a p o r a t e d u n d e r v a c u u m , a n d the residue tested for mutagenicity. d D a t a in p a r e n t h e s i s w e r e o b t a i n e d u s i n g 8 . 0 m g b e n z o [ a ] p y r e n e f r a c t i o n a t e d b y t h i n - l a y e r c h r o m a t o g raphy twice.
Data of Table 2 taken with a dependency upon air for mutagenicity (Expt. 8, Table 1) suggest that the radiation-induced mutagenicity of b e n z o [ a ] p y r e n e is due to formation of oxidized products. Although the chemical nature of the mutagenic species formed in these experiments has not been discovered, we have preliminary evidence which identified the 1,6-,3,6-, and 6,12
Mutation Research, 49 (1978) 153--161 © Elsevier/North-Holland Biomedical Press
NON-ENZYMIC ACTIVATION OF POLYCYCLIC AROMATIC HYDROCARBONS AS MUTAGENS
THOMAS L. GIBSON, VERA B. SMART and LELAND L. SMITH Division of Biochemistry, The Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Tx. 77550 (U.S.A.) (Received 26 May 1977) (Revision received 20 September 1977) (Accepted 26 September 1977)
Summary Samples of 22 polycyclic aromatic hydrocarbons and related derivatives were subjected to ~°Co gamma radiation in air, and the irradiated samples were tested for mutagenicity with the Salmonella typhimurium strains TA 98, TA 1535, TA 1537, and TA 1538. Testing was conducted with the bacterial strains alone, thus not fortified with liver-microsomal enzymes or other metabolizing systems. Marked mutagen responses were obtained for several irradiated samples with the TA 98, TA 1537, and TA 1538 strains but not with the TA 1535 strain. Irradiated samples of benzo [a ] anthracene, benzanthrone, benzo[g,h,i]perylene, benzo[a]pyrene, chrysene, fluorene, 9-methylanthracene, 1-methylphenanthrene, 2-methylphenanthrene, and pyrene gave positive mutagenic tests and dose--responses, whereas unirradiated control samples of these were inactive. Acenaphthene, phenanthrene, and phenanthrenequinone exhibited toxicity which interfered with interpretation of mutagenicity testing. Samples of 2-methylanthracene and tetracene were mutagenic with or without irradiation. Alizarin, anthracene, anthraquinone, anthrone, dibenzo[a,h]anthracene, picene, and triphenylene gave negative results. Samples of benzo[a]pyrene adsorbed on silica gel irradiated in air by ~°Co gamma radiation or by 254 nm ultraviolet light and samples adsorbed on filter paper irradiated by visible light yielded preparations mutagenic towards the TA 98, TA 1537, and TA 1538 strains. These results suggest that parent polycyclic aromatic hydrocarbons not themselves mutagenic towards S. typhimurium may be oxidized in air by radiation-induced processes to products whose mutagenicity resembles that of liver-microsomal metabolites of the parent polycyclic aromatic hydrocarbon.
154
Introduction Although much attention is currently being given to the metabolic activation by mammalian tissues of polycyclic aromatic hydrocarbons as mutagenic agents, little interest in such activation by non-enzymic means has developed. In that polycyclic aromatic hydrocarbons found quite broadly in nature, including soils, drinking water, polluted air, cigarette smoke, etc. are exposed to the oxygen of the air and to radiation from many sources the question of radiation-induced oxidations of this class of compounds to active mutagens attracted our interest. We have pursued the matter using as convenient means the Salmonella typhimurium bacterial mutagen testing procedure of Ames et al. [1--3,9]. We report herein our results using this approach, results which demonstrate that several polycyclic aromatic hydrocarbons are indeed transformed into mutagenic agents following irradiation in air with 6°Co gamma radiation. Materials and Methods Samples of anthracene (99.9% gold label), anthrone, anthraquinone, 2methylanthracene, 9-methylanthracene (99%), alizarin (97%), benzanthrone, phenanthrene (98+%), triphenylene (98%), and benzo[g,h,i]perylene were purchased from Aldrich Chemical Co., Milwaukee, WI. Chrysene, fluorene, 1-methylphenanthrene, 2-methylphenanthrene, picene, tetracene, acenaphthene, benzo[a]pyrene, and benzo[a]anthracene were from Analabs, North Haven, CT, and pyrene from Eastman Organic Chemicals, Rochester, NY. The purity of the compounds was checked by ultraviolet spectroscopy, thin-layer or high-pressure liquid chromatography, mass spectrometry or other methods without finding impurities. Benzo[a]pyrene was irradiated under four different conditions: (1) crystals irradiated by 6°Co gamma radiation from a Gammacell 200 (Atomic Energy of Canada Ltd., Ottawa) for 7 days, providing 2.5 X 107 rad, (2) adsorbed (1% by weight) on silica gel (60--200 mesh) irradiated by 6°Co gamma radiation (0.5 X 107 tad), (3) adsorbed (1% by weight) on Silica Gel HF2s4 (Merck, Darmstadt) and irradiated by 254 nm ultraviolet light from two 15-W quartz lamps for 2 days, and (4) adsorbed on Whatman No. 1 filter paper (2.5 mg per 50 sq.cm.), irradiated for 18 days by room light from overhead 40-W fluorescent tubes. Irradiation of the other polycyclic aromatic hydrocarbons was conducted only with 6°Co gamma radiation (2.5 X 107 rad). Irradiated samples were stored under dry nitrogen until tested for mutagenicity. Mutagenicity was determined with the Salmonella typhimurium test of Ames et al., [1--3,9] in which mutant strains of the bacterium nutritionally dependent upon histidine were used. No tissue homogenate, enzyme, or cell preparation was included for intermediate metabolic transformation of irradiated samples. The test strains used included TA 1537, TA 1538, and TA 98 (TA 1538 plus an R-factor plasmid) which detect frameshift mutagens and TA 1535 which detects base-pair substitution mutations, all strains kindly provided by Prof. B.N. Ames, University of California, Berkley, Calif. Polycyclic aromatic hydrocarbon samples were dissolved in dimethylsulfoxide
155 or in dimethylsulfoxide containing a small volume of acetone so as to give solutions between 0.2--9 mg/ml concentration. Measured volumes of these solutions (none more than 0.5 ml) were incorporated into liquid agar (2 ml) which was then thoroughly mixed. The agar containing the incorporated sample and test organism was poured over the base agar plate and allowed to set. Plates were incubated for 48 h at 37°C, after which time individual colonies of bacteria were counted. All tests of dimethylsulfoxide and of dimethylsulfoxide containing acetone (0.5 ml/plate) conducted regularly gave no signs of mutagenicity or of solvent toxicity. Samples were generally tested over a range of concentrations in order to discern indication,~ of dose response. A two-fold or greater increase in revertants, i.e., observed number of revertant colonies three-fold or greater than the spontaneous number, was considered a positive test. Toxic effects were checked by examination of the background lawn of bacterial growth and by the presence of at least the usual number of spontaneous revertants. Control counts of spontaneous revertants obtained for the four test strains averaged: TA 98, 48; TA 1535, 21; TA 1537, 12; TA 1538, 25. Thin-layer chromatography was conducted with 20 X 20 cm chromatoplates of Silica Gel HF2s4 (E. Merck GmbH., Darmstadt) 0.25 mm thick, irrigated in ascending fashion with benzene or with chloroform--methanol ( 4 : 1 , v/v). Individual components were visualized under 254 nm or 366 nm ultraviolet light. Results Irradiated and unirradiated control samples of 22 polycyclic aromatic hydrocarbons and their derivatives were tested with as m a n y as 4 S. typhimurium test strains for possible mutagenicity. Data of Table 1 establish that definite responses were obtained for benzo[a]pyrene subjected to air oxidations induced by various radiation. A similar pattern of mutagenicity responses was had whether visible light, 254 nm light, or ~°Co gamma radiation was used to initiate the oxidations. Linear or near-linear dose--response curves were obtained with the three strains TA 98, TA 1537, and TA 1538 for 6°Co gamma irradiated samples of crystalline benzo[a]pyrene and of benzo[a]pyrene adsorbed on silica gel and with TA 1537 and TA 1538 for samples of benzo[a]pyrene adsorbed on filter paper irradiated with visible fluorescent light. Unirradiated samples of benzo [a] pyrene subjected to the same manipulations as the irradiated samples were uniformly non-mutagenic as was benzo[a]pyrene irradiated with ~°Co gamma radiation under vacuum. Data of Table 2 confirm and extend our observations of the radiationinduced activation of benzo[a]pyrene. Here irradiated samples shown to be mutagenic were fractionated by thin-layer chromatography and material from several regions of the chromatoplate was tested for mutagenicity. In Expts. No. 1 and No. 2 {benzene as irrigation solvent) fractions l b and 2b containing recovered benzo[a]pyrene ( R f 0 . 6 ) showed negligible mutagenicity, b u t material from the more polar regions was mutagenic, as was material pooled from these several zones. Furthermore, retesting of pooled material recovered from agar test plates of the initial testing of samples l c - - l e suggested a marked
156 TABLE 1 MUTAGENIC ACTIVATION OF BENZO[a]PYRENE Expt. No.
1
Benzo[a]pyrene treatment a
Amount tested (pg/plate)
Crystals, 60Co gamma radiation, for 7 days ( 2 . 5 × 107 r a d )
10 20 50 100 200 3OO
Adsorbed, 60Co gamma r a d i a t i o n f o r 1.5 d a y s ( 0 . 5 X 107 r a d )
50 100 200
3
Adsorbed, 254 nm light for 2 days
500 1000 1200
4
A d s o r b e d , visible l i g h t f o r 18 d a y s
5
Control, unirradiated crystals
20~200
6
Control, unirtadiated material recovered f r o m silica gel
20--200
7
Control, unirradiated material recovered f r o m filter paper
50--100
8
Control, crystals 60Co gamma radiation for 7 d a y s ( 2 . 5 X 107 r a d ) in v a e UUlTI
2
BY RADIATION IN AIR M u t a g e n i c r e s p o n s e , i n c r e a s e in r e v e r t a n t s c
b
TA1535
0 0
0 0
50 100 2OO
TA1537
4 11 15 19
3.1 6.8 9.4
TA1538
TA98
4.3 10 20 26
1.5 3 10 15 21 35
1.9 3
1.5 2 2.6
4 2.5 0 d 3.4 3.5 6.7
6.3 9.6 13
4.4 13 16
0
0
0
0
0
0
100
a All c o n d i t i o n s c o n d u c t e d in t h e p r e s e n c e o f a i r e x c e p t as n o t e d . b W e i g h t o f s a m p l e a c t u a l l y t e s t e d p e r p l a t e , a d d e d as d i m e t h y l s u l f o x i d e s o l u t i o n i n c o r p o r a t e d i n t o t o p agar layer. c I n c r e a s e in r e v e r t a n t s = t o t a l n u m b e r o f r e v e r t a n t s - - n u m b e r o f s p o n t a n e o u s r e v e r t a n t s number of spontaneous revertants. d Toxic effects noted.
increase in revertant colonies of bacteria too high for proper counting. Therefore, the induced mutagenicity of irradiated benzo[a]pyrene was stable to chromatography and to manipulations. Moreover, dimethylsulfoxide solutions of mutagenic, irradiated benzo [a]pyrene samples retained their mutagenicity to the TA 1 5 3 8 strain for as long as 86 days storage at room temperature. In Expt. No. 3 using a more polar irrigation solvent polar fractions 3b and 3c (Rf 0.31--0.74} contained most of the mutagenic activity with negligible activity being found in the most mobile zone 3a which contained parent benzo[a]pyrene. As these chromatographic operations did not exclude with certainty components from one zone overlapping into adjacent zones, it is not possible to determine h o w many mutagenic components were formed, but it is clear that the activity is associated with components more polar than the parent hydrocarbon.
157 TABLE 2 PRELIMINARY CHROMATOGRAPHIC IRRADIATION PRODUCTS a Expt. No.
Sample No.
CHARACTERIZATION
Sample identity
OF MUTAGENIC BENZO[a]PYRENE
Mutagenic response, increase in r e v e r t a n t s TA1538
1
2
Crystals, 2 . 5 × 1 0 7 t a d la
Unfractionated products
lb lc ld le If lg
Rf 0.5--1.0 fraction b Rf 0.2--0.5 fraction b Rf 0.1--0.2 fraction b Rf 0--0.1 fraction b Pooled Nos. lb--le material Extracted agar from Nos. lc--le c
19/500 ~g 25/1000 #g 0 5.7 10 15 7.2 v e r y high
Adsorbed, 0 . 5 × 1 0 7 r a d 2a 2b 2c 2d 2e
3
TA1537
Unfractionated Rf 0.5--1.0 b Rf 0.2--0.5 b Rf 0.1--0.2 b Rf 0--0.1 b
6.2 0 2.7 1.5 8.3
1.6 3.4 2.3 5.5
Crystals, 2.5 X 1 0 7 r a d 3a 3b 3c 3d 3e
Rf Rf Rf Rf Rf
0.74--1.0 b 0.44--0.74 b 0.31--0.44 b 0.10--0.31 b 0--0.10 b
1.7 6.5 4.2 3.6 3.5
(0) d (4.8) d (5.0) d (1.0) d (0) d
a Data were obtained following irradiation of 4.0 mg benzo[a]pyrene and thin-layer chromatographic f r a c t i o n a t i o n o f t h e i r r a d i a t e d s a m p l e . F o o t n o t e s a a n d c o f T a b l e 1 p e r t a i n t o T a b l e 2 also. b C h r o m a t o g r a p h i c s o l v e n t f o r E x p t s . N o . 1 a n d 2: b e n z e n e ; f o r E x p t . N o . 3: c h l o r o f o r m - - m e t h a n o l (4 : 1, v/v). M o b i l i t y ( R f ) o f a u t h e n t i c r e f e r e n c e s a m p l e s i n t h e t w o s o l v e n t s y s t e m s w e r e : b e n z o [ a ] p y r e n e , 0.62, 1.0; 6-hydroxybenzo[a]pyrene, 0.26 --; 9-hydroxybenzo[a]pyrene, 0.15, 1.0: benzo[a]p~ene-l,6d i o n e , 0 . 2 0 , 0 . 8 ; b e n z o [ a ] p y r c n e - 6 , 1 2 - d i o n e , --, 0 . 8 ; benzo[a]pyrene-7,S.dihydro-trans-7,8-diol, 0, 0 . 6 7 ; benzo[a]pyrene-9,10-dihydro-trans-9,10-diol, Oo 0 . 6 3 . c Following mutagenicity testing of samples Nos. lc--le, agar plates were stored under refrigeration for 5 - - 7 d a y s , t h e n e x t r a c t e d w i t h b e n z e n e - - a c e t o n e , t h e s o l v e n t e x t r a c t s e v a p o r a t e d u n d e r v a c u u m , a n d the residue tested for mutagenicity. d D a t a in p a r e n t h e s i s w e r e o b t a i n e d u s i n g 8 . 0 m g b e n z o [ a ] p y r e n e f r a c t i o n a t e d b y t h i n - l a y e r c h r o m a t o g raphy twice.
Data of Table 2 taken with a dependency upon air for mutagenicity (Expt. 8, Table 1) suggest that the radiation-induced mutagenicity of b e n z o [ a ] p y r e n e is due to formation of oxidized products. Although the chemical nature of the mutagenic species formed in these experiments has not been discovered, we have preliminary evidence which identified the 1,6-,3,6-, and 6,12
