Chem.-Biol. Interactions, 17 (1977) 129--136 © Elsevier/North-Holland Scientific Publishers, Ltd.
129
F A C T O R S F O R EFFICIENCY OF THE S A L M O N E L L A / M I C R O S O M E MUTAGENICITY ASSAY
C. MALAVEILLE, G. PLANCHE * and H. BARTSCH ** International Agency for Research on Cancer, Unit o f Chemical Carcinogenesis, 150 cours Albert Thomas, 69008 Lyon (France)
(Received October 5th, 1976) (Accepted January 21st, 1976)
SUMMARY
Factors were studied which m o d i f y the enzymatic capacity of mouse liver microsomal mixed-function oxidase to convert vinylidene chloride (1.1-dichloroethylene) (VDC) into mutagens in the S a l m o n e l l a / m i c r o s o m e mutagenicity test. A microsomal fraction incorporated in soft agar layer converted VDC into mutagens during 7 h at a constant rate; these were detected with S. t y p h i m u r i u m TA100. In absence of VDC the enzymatic activity declined gradually to nil after 14 h of incubation at 37°C. The presence o f EDTA greatly enhanced the microsome-mediated mutagenicity of VDC and led to prolonged enzymatic viability, b u t only when liver fractions from phenobarbitone (PB) pretreated mice were used. The efficiency o f the plate incorporation assay for the detection of mutagens is discussed in comparison with assays in liquid suspension.
INTRODUCTION It is n o w recognized that enzymatic conversion of many chemicals into ultimate reactive metabolites precedes carcinogenesis and mutagenesis [1]. Consequently, metabolic activation in vitro is a critical factor for the efficiency and reproducibility of mutagenicity testing procedures and, thus, must be rigorously controlled. In the plate incorporation assay described b y Ames et al. [2], whereby S. t y p h i m u r i u r n strains, a post-mitochondrial rat * Work in part fulfillment of a thesis at the University of Lyon. ** To whom correspondence and reprint requests should be addressed. Abbreviations: PB, phenobarbitone sodium; S-9, 9000 g tissue supernatant; VDC, vinylidene chloride (1,1-dichloroethylene).
130 liver supernatant and cofactors are combined in a soft agar layer, and the proposed modifications to this assay [3--7], the number of false negative results is relatively low in comparison with the number of carcinogens which can be detected as mutagens [8,9]. Previous studies have shown that N-nitrosodimethylamine and 3,3-dimethyl-l-phenyltriazene can be less efficiently detected in a plate incorporation assay than in a liquid incubation system [4,10]. In this assay, bacteria, liver microsomal enzymes and cofactors are incubated in a buffered solution [3,11]. On the other hand, c o m p o u n d s such as N-nitrosodibutylamine and N-nitrosodipentylamine exert rat liver microsome-mediated mutagenicity in the plate incorporation assay b u t cannot be detected in a liquid incubation assay [11]. Using an adapted form o f the plate incorporation assay [12] with S. typhimurium strains and liver microsomal enzymes to test gaseous compounds [5], it was noted that the microsomal-mediated mutagenicity of vinyl chloride increased linearly up to 9 h. Since it is primarily microsomal mixed-function oxidases which are responsible for the formation of mutagenic vinyl chloride metabolites [13] these data indicated that the viability of these enzymes is prolonged when they are incorporated in soft agar, whereas they undergo a relatively rapid inactivation after incubation for 1 h in a buffered vinyl chloride solution [14]. In order to explain these differences in mutagenicity in the 2 assay systems, we have investigated factors which modify the enzymic properties o f the liver microsomal mixed-function oxidase in vitro and, consequently, the enzyme-mediated mutagenicity o f chemicals in the Salmonella/microsome mutagenicity test. MATERIALS AND METHODS VDC (purity 99%, containing 0.3% 4-methoxyphenol as a n t i o x i d a n t ) w a s obtained from Merck-Schuchardt, Darmstadt, G.F.R. All other commercial products were o f the purest grades available.
Animals and tissue preparation Male OF-1 mice (35 g bodyweight) were obtained from Iffa-Credo, St. Germain-sur-l'Arbresle, France, and were fed on Charles River C R F diet. Some mice recieved PB in the drinking water (1 mg/ml) for 7 days prior to the experiment. The 9000 g tissue supernatant (S-9) fractions were prepared from the pooled livers o f 4 animals (3 ml/g wet tissue) in 0.15 M KC1 as described previously [5]. The resulting tissue fraction was assayed immediately in some experiments; in others it was stored at --30°C for a m a x i m u m o f 15 h before it was used for mutagenicity assays. Storage under these conditions did not effect the tissue-mediated mutagenicity of VDC. All procedures were carried o u t at 0--4°C, using sterile glassware and solutions. Mutagenicity tests Tests for the mutagenicity of VDC were carried o u t according to an
131 adapted plate incorporation assay to test volatile compounds [5] using a histidine-auxotrophic strain o f S. typhimurium TA100, generously provided by Prof. B.N. Ames, Berkeley, Calif. U.S.A. Procedure A. Plates containing S-9 o f mouse liver, an NADPH generating system, 0.1/~mole of each histidine and biotine, the bacteria (1--2 • l 0 s) and all other ingredients as described [2,12] in a soft agar overlay (2.6 ml) were exposed to a gaseous mixture o f 2% VDC in air (v/v) in a dessicator at 37°C in the dark. After a specific exposure time, VDC was replaced by air; after further incubation for up to 48 h at 37°C the number of his÷-revertant colonies per plate was scored. The concentration of VDC dissolved in the incubation medium under these conditions was 3.3 • 10 -3 M [ 15]. Procedure B. Mouse liver S-9 and bacteria incorporated in the soft agar layer as described in procedure A were preincubated by keeping the plates at 37 °C for certain lengths o f time specified in the text. After preincubation, the plates were exposed to 2% VDC in air (v/v) for 3 h as described in procedure A. VDC was then removed under vacuum, and incubation was continued for a total of 48 h from the beginning of VDC exposure. Procedure C. On t o p of plates prepared as described in procedure A, but without histidine and biotine, 200 gl of a sterile aqueous solution containing 0.1 pmole o f histidine and biotine were spread as an even layer after preincubation b u t prior to VDC exposure. The plates were then exposed to 2% VDC in air (v/v) for 3 h as described in procedure B. Within the concentration range used, VDC in the presence of liver So9 and other ingredients as mentioned in procedures A, B and C showed no toxicity to the background lawn of bacteria which grows in the presence o f traces of histidine and biotine in the medium. The presence of an R factor in the TA100 strain was checked by seeding bacteria on ampicillin-containing agar [2]. Procedure D: Determination o f the bacterial growth curve. S. typhimurium strain TA100 (1--2 • l 0 s cells/plate) was incorporated in plates (9.2 cm in diameter) containing 0.1 #mole of histidine and biotine, as described in [12]. After various lengths of preincubation at 37°C in air, an aliquot o f the soft agar layer (1/11 o f the total surface area) was removed. This agar disc was then gently homogenized in 10 ml of 0.9% saline in a Potter--Elvejhem homogenizer. The cells were enumerated by seeding a suitable dilution on Vogel Bonner E nutrient agar plates [5] containing 1.5% agar, 2% glucose and 8 g/1 Difco nutrient broth. RESULTS
VDC requires metabolic activation b y microsomal mixed-function oxidase from rat or mouse liver to yield mutagenic reactants which can be detected with S. typhimurium stains TA1530 and TA100 [15]. A modification of the original Salmonella/microsome mutagenicity test [5] permits the measurement of time- and dose-dependent mutagenicity response curves (procedure A) and, therefore, the monitoring of microsomal enzyme activity
132 involved in metabolic activation o f VDC as a function o f incubation time under in vitro conditions. Exposure of S. t y p h i m u r i u m strain TA100 to 2% VDC in air in the presence o f a hepatic microsomal fraction from untreated or PB-pretreated mice caused a linear increase in the mutagenic response up to 4 h o f exposure (Fig. 1). This VDC concentration had previously been shown to be non-toxic to the bacteria [15]. Exposure to 2% VDC in air for 3 h was therefore used to study the viability of mouse liver microsomal mixed-function oxidase incorporated in a soft agar layer before VDC exposure. A maximal mutagenic response was noted after 2 h o f preincubation o f microsomal liver enzymes from both untreated or PB-pretreated mice prior to VDC exposure, the number o f his+-revertant colonies being about 30% higher than in plates which had not been preincubated (Fig. 2A). After preincubation for 4 h at 37 ° C, mouse liver S-9 showed the same enzymic capacity for converting VDC into mutagens, and the n u m b e r o f his ÷ revertants per plate remained as high as when the plates had not been preincubated. After preincubation for 14 h, the enzymes were inactive in converting VDC into mutagens, irrespective o f whether the S-9 was obtained from animals which had been pretreated with PB or not. In order to examine whether the peak activity seen in Fig. 2A is due to an increased mutagenicity caused by bacterial proliferation, histidine and biotine were omitted from plates during preincubation and only spread on the agar layer prior to VDC exposure (Fig. 2B). Under these conditions, the n u m b e r of his+ revertants per plate remained unchanged up to 4 h of preincubation,
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Fig. 1. T i s s u e - m e d i a t e d m u t a g e n i c r e s p o n s e o f S. typhimurium T A 1 0 0 a f t e r e x p o s u r e to V D C for various l e n g t h s o f time. M u t a g e n i c i t y assays ( p r o c e d u r e A) were p e r f o r m e d w i t h s t r a i n T A 1 0 0 a n d p o o l e d S-9 liver f r a c t i o n s f r o m 4 P B - p r e t r e a t e d m i c e (©) or f r o m 4 unt r e a t e d ( e ) f o l l o w i n g e x p o s u r e o f t h e b a c t e r i a to 2% V D C in air at 3 7 ° C (v/v). In t h e control (~), e o f a c t o r s ( N A D P ÷, glucose 6 - p h o s p h a t e ) were o m i t t e d . T h e n u m b e r o f s p o n t a n e o u s his*-revertant c o l o n i e s has n o t b e e n s u b t r a c t e d . M e a n values + S.E. were c o l l a t e d f r o m a series o f 2--5 e x p e r i m e n t s .
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Fig. 2A, B. Tissue-mediated mutagenic response of S. typhimurium TA100 as a function of preincubation time of plates before exposure to VDC in air. Mutagenicity assays were performed with strain TA100 and S-9 mouse liver fraction following 3 h exposure of the plates to 2% VDC in air (v/v). A, Untreated, or o, PB-pretreated, mice (procedure B); e, PB-pretreated mice, but histidine and biotine omitted from the plates during preincubation (procedure C). In the control (dotted line), cofactors (NADP ÷, glucose 6-phosphate) were omitted. The number of spontaneous his÷-revertant colonies has not been subtracted. Mean values _+ S.E. were collated from 4 experiments.
reflecting a constant rate o f metabolism o f VDC. The enzyme activity declined gradually to nil after 14 h o f preincubation. By measuring bacterial growth (procedure D, Materials and Methods), it could be demonstrated that the maximal mutagenicity seen in Fig. 2A, after 2 h o f preincubation followed by 3 h o f VDC exposure, coincided with the exponential growth o f the TA100 strain, which occurred between approximately 3 and 7 h after plating. After this period, the number o f bacteria had increased 30--40-fold. EDTA is n o w known to have a protective effect against lipid peroxidation [16,17] which could destroy microsomal enzyme activity. We have therefore studied the effect of 0.1 mM EDTA (0.26 pmole/plate) in plates containing bacteria and a fortified S-9 liver fraction from PB-pretreated mice (Fig. 3). The presence of EDTA during preincubation greatly increased the liver microsome-mediated mutagenicity o f VDC; after 6 h of preincubation, the n u m b e r of his+-revertant colonies was 3 times higher than in the absence o f EDTA. No enzymic conversion of VDC into mutagens was detected in plates with or without EDTA after 14 h or preincubation. Using conditions identical to those described in Fig. 3, the experiments were repeated with liver S-9 from untreated mice. Neither the absence nor
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Fig. 3. Tissue-mediated m u t a g e n i c response o f S. typhimurium T A 1 0 0 as a f u n c t i o n o f p r e i n c u b a t i o n time o f plates c o n t a i n i n g E D T A before e x p o s u r e to V D C . M u t a g e n i c i t y assays ( p r o c e d u r e B) were p e r f o r m e d w i t h strain T A 1 0 0 and S-9 liver fraction from PBp r e t r e a t e d mice f o l l o w i n g 3 h e x p o s u r e o f the bacteria to 2% V D C in air (v/v) in the presence o f 0 . 2 6 p m o l e / p l a t e E D T A ( $ ) or in the absence o f E D T A (©). T h e n u m b e r o f s p o n t a n e o u s h/s+-revertant c o l o n i e s ( - - - ) has not b e e n subtracted. Mean values + S.E. were c o l l a t e d for 3 e x p e r i m e n t s .
the presence o f EDTA made any difference to the microsome-mediated mutagenicity o f VDC. DISCUSSION
VDC was used as substrate to measure the influence o f agar on the viability o f hepatic microsomal enzymes, since this compound has no direct mutagenic action but requires activation by mouse liver microsomal mixedfunction oxidase to yield mutagenic metabolites (Ref. 15 and Fig. 1). In this study, the number o f his÷-revertant colonies o f S. typhimurium TA100 was used as a measure o f the enzymic capacity of the S-9 mouse liver preparation. When bacteria are exposed to gaseous VDC, its removal in vacuo permits the establishment of linear time- and dose-dependent mutagenic response curves (Fig. 1 and Ref. 5). In plates containing mouse liver S-9 fractions, cofactors for mixed-function oxidase and bacteria, preincubated for various lengths of time before exposure to VDC for 3 h (procedure A), maximal mutagenicity was noted after 2 h o f preincubation (Fig. 2A). Liver S-9 mediated mutagenicity of VDC was 50% higher when PB-pretreated animals were used than in assays using tissues from untreated animals, confirming previous results [15]. En-
135 zymic ability to metabolize VDC into mutagens declined over a period o f 6 h and was nil after 14 h o f preincubation. The peak mutagenic activity observed after 2 h of incubation (Fig. 2A) appears to be due to an enhanced mutagenic effect during bacterial replication and/or increased bacterial numbers. This was shown by omitting growth factors from the agar during preincubation of the plates and by adding histidine and biotine just before VDC exposure (Fig. 2B); under these conditions, liver S-9 mediated mutagenicity o f VDC remained unchanged over 7 h (4 h preincubation plus 3 h VDC exposure), reflecting a constant rate of formation of VDC metabolites. This is also confirmed by the linearity of the time-dependent liver S-9 mediated mutagenicity o f VDC observed over 7 h (Fig. 1). Although the nature o f the stabilizing effect of agar on mouse liver microsomal enzymes remains to be investigated, alteration o f the structural organization of the multi-enzyme complex in the membrane [18] and/or reduction o f lipid peroxidation in the presence o f agar could be contributing factors. When EDTA, a known inhibitor of lipid peroxidation [16,17,19] was added to the plates, a marked enhancement of the liver S-9 mediated mutagenicity of VDC was noted but only when microsomal enzymes from PB-pretreated mice were used (Fig. 3). These data exclude the possibility that EDTA enhances the mutagenicity o f VDC, e.g. b y altering the permeability of the bacterial membrane, b u t are in agreement with a report that the liver of PB-pretreated rodents shows alterations of the lipid peroxidation [20]. A 2-fold enhancement by EDTA of microsomal-mediated mutagenicity in S. typhimurium was also shown with vinyl chloride using PB-pretreated mice or with 1,4-dichlorobutene-2 with S-9 liver fraction from polychlorinated biphenyl (Aroclor 1254)-pretreated mice (data not included). As a practical consequence, EDTA might be used to potentiate the microsomalmediated mutagenicity of certain chemicals. This study gives an explanation why the plate incorporation assay is effective in detecting chemicals whose metabolic conversion into mutagens occurs at a low rate or which require multi-step activation processes [11]. We have shown experimentally for the first time that incorporation o f liver microsomal enzymes in a soft agar layer prolongs their viability for up to several hours; this p h e n o m e n o n could explain w h y certain compounds, e.g. N-nitrosodi-n-butyl- and N-nitrosodi-n-pentylamine, were not detectable as mutagens in liquid suspension after an incubation up to 1 h [11]. ACKNOWLEDGEMENTS
The authors would like to thank Miss P. Stafford-Smith for secretarial assistance and Mrs. E. Ward for editorial aid. The authors are indebted to Mrs. G. Brun for technical help. Financial support for the authors' research activities in this area has been provided partly by Contract No. 1-CP-55630 from the National Cancer Institute, NIH, U.S.A. Note added in proof. Since completion of this work, VDC has been found to be carcinogenic in mice (C. Maltoni, personal communication).
136 REFERENCES 1 J.A. Miller and E.C. Miller, Chemical carcinogenesis, mechanisms and approaches to its control, J. Natl. Cancer Inst., 47 (1971) v. 2 B.N. Ames, J. McCann and E. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31 (1975) 347. 3 C.N. Frantz and H.V. Malling, The quantitative microsomal mutagenesis assay method, Mutation Res., 31 (1975) 365. 4 H. Bartsch, C. Malaveille and R. Montesano, In vitro metabolism and microsomemediated mutagenicity of dialkylnitrosanlines in rats, hamsters and mouse tissues, Cancer Res., 35 (1975) 644. 5 H. Bartsch, C. Malaveille and R. Montesano, Human, rat and mouse liver-mediated mutagenicity of vinyl chloride in S. typhimurium strains, Int. J. Cancer, 15 (1975) 429. 6 U. Rannug, A. Johansson, C. Ramel and C.A. Wachmeister, The mutagenicity of vinyl chloride after metabolic activation, Ambio, 3 (1974) 194. 7 T. Sugimura, T. Yahagi, M. Nagao, M. Takeuchi, T. Kawachi, K. Hara, E. Yamasaki, T. Matsushima, Y. Hashimoto and M. Okada, Validity of mutagenicity tests using microbes as a rapid screening method for environmental chemicals, in R. Montesano, H. Bartsch and L. Tomatis (Eds.), Screening Tests in Chemical Carcinogenesis, Scientific Publications No. 12, IARC, Lyon, France, 1976, pp. 81--101. 8 J. McCann, E. Choi, E. Yamasaki and B.N. Ames, Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. USA, 72 (1975) 5135. 9 J. McCann and B.N. Ames, Detection of carcinogens as mutagens in the Salmonella/ microsome test: assay of 300 chemicals. Part II, Proc. Natl. Acad. Sci. USA, 73 (1976) 950. 10 C. Malaveille, G.F. Kolar and H. Bartsch, Rat and mouse tissue-mediated mutagenicity of ring-substituted 3,3-dimethyl-l-phenyl-triazenesin Salmonella typhimurium, Mutation Res., 36 (1976) 1. 11 H. Bartsch, A. Camus and C. Malaveille, Comparative mutagenicity of N-nitrosamines in semi-solid and in a liquid incubation system in the presence of rat or human tissue fractions, Mutation Res., 37 (1976) 149. 12 B.N. Ames, N.E. Durston, E. Yamasaki and F.D. Lee, Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. USA, 70 (1973) 2281. 13 H. Bartsch and R. Montesano, Mutagenic and carcinogenic effects of vinyl chloride, Mutation Res., 32 (1975) 93. 14 N. Loprieno, R. Barale, S. Baroncelli, C. Bauer, G. Bronzetti, A. Cammellini, G. Cercignani, C. Corsi, G. Gervasi, C. Leporini, R. Nieri, A.M. Rossi, G. Stretti and G. Turchi, Evaluation of the genetic effects induced by vinyl chloride monomer (VCM) under mammalian metabolic activation: studies in vitro and in vivo, Mutation Res., 40 (1976) 85. 15 H. Bartsch, C. Malaveille, R. Montesano and L. Tomatis, Tissue-mediated mutagenicity of vinylidene chloride and 2-chlorobutadiene in Salmonella typhimurium, Nature (London), 255 (1975)641. 16 E.D. Wills, Lipid peroxide formation in microsomes. Relationship of hydroxylation to lipid peroxide formation, Biochem. J., 113 (1969) 333. 17 T. Kamataki, N. Ozawa, M. Kitada and H. Kitagawa, The occurrence of an inhibitor of lipid peroxidation in rat liver soluble fraction and its effect on microsomal drug oxidations, Biochem. Pharmacol., 23 (1974) 2485. 18 A. Stier, Lipid structure and drug metabolizing enzymes, Biochem. Pharmacol., 25 (1976) 109. 19 W. Levin, A.Y.H. Lu, M. Jacobson, R. Kuntzman, J.L. Poyer and P.B. McCay, Lipid peroxidation and the degradation of cytochrome P-450 heine, Arch. Biochem. Biophys., 158 (1973) 842. 20 H.K.J. Hahn, D.J. Tuma, A.J. Barak and M.F. Sonell, Effect of phenobarbital on lipid peroxidation in the liver, Biochem. Pharmacol., 25 (1976) 769.
129
F A C T O R S F O R EFFICIENCY OF THE S A L M O N E L L A / M I C R O S O M E MUTAGENICITY ASSAY
C. MALAVEILLE, G. PLANCHE * and H. BARTSCH ** International Agency for Research on Cancer, Unit o f Chemical Carcinogenesis, 150 cours Albert Thomas, 69008 Lyon (France)
(Received October 5th, 1976) (Accepted January 21st, 1976)
SUMMARY
Factors were studied which m o d i f y the enzymatic capacity of mouse liver microsomal mixed-function oxidase to convert vinylidene chloride (1.1-dichloroethylene) (VDC) into mutagens in the S a l m o n e l l a / m i c r o s o m e mutagenicity test. A microsomal fraction incorporated in soft agar layer converted VDC into mutagens during 7 h at a constant rate; these were detected with S. t y p h i m u r i u m TA100. In absence of VDC the enzymatic activity declined gradually to nil after 14 h of incubation at 37°C. The presence o f EDTA greatly enhanced the microsome-mediated mutagenicity of VDC and led to prolonged enzymatic viability, b u t only when liver fractions from phenobarbitone (PB) pretreated mice were used. The efficiency o f the plate incorporation assay for the detection of mutagens is discussed in comparison with assays in liquid suspension.
INTRODUCTION It is n o w recognized that enzymatic conversion of many chemicals into ultimate reactive metabolites precedes carcinogenesis and mutagenesis [1]. Consequently, metabolic activation in vitro is a critical factor for the efficiency and reproducibility of mutagenicity testing procedures and, thus, must be rigorously controlled. In the plate incorporation assay described b y Ames et al. [2], whereby S. t y p h i m u r i u r n strains, a post-mitochondrial rat * Work in part fulfillment of a thesis at the University of Lyon. ** To whom correspondence and reprint requests should be addressed. Abbreviations: PB, phenobarbitone sodium; S-9, 9000 g tissue supernatant; VDC, vinylidene chloride (1,1-dichloroethylene).
130 liver supernatant and cofactors are combined in a soft agar layer, and the proposed modifications to this assay [3--7], the number of false negative results is relatively low in comparison with the number of carcinogens which can be detected as mutagens [8,9]. Previous studies have shown that N-nitrosodimethylamine and 3,3-dimethyl-l-phenyltriazene can be less efficiently detected in a plate incorporation assay than in a liquid incubation system [4,10]. In this assay, bacteria, liver microsomal enzymes and cofactors are incubated in a buffered solution [3,11]. On the other hand, c o m p o u n d s such as N-nitrosodibutylamine and N-nitrosodipentylamine exert rat liver microsome-mediated mutagenicity in the plate incorporation assay b u t cannot be detected in a liquid incubation assay [11]. Using an adapted form o f the plate incorporation assay [12] with S. typhimurium strains and liver microsomal enzymes to test gaseous compounds [5], it was noted that the microsomal-mediated mutagenicity of vinyl chloride increased linearly up to 9 h. Since it is primarily microsomal mixed-function oxidases which are responsible for the formation of mutagenic vinyl chloride metabolites [13] these data indicated that the viability of these enzymes is prolonged when they are incorporated in soft agar, whereas they undergo a relatively rapid inactivation after incubation for 1 h in a buffered vinyl chloride solution [14]. In order to explain these differences in mutagenicity in the 2 assay systems, we have investigated factors which modify the enzymic properties o f the liver microsomal mixed-function oxidase in vitro and, consequently, the enzyme-mediated mutagenicity o f chemicals in the Salmonella/microsome mutagenicity test. MATERIALS AND METHODS VDC (purity 99%, containing 0.3% 4-methoxyphenol as a n t i o x i d a n t ) w a s obtained from Merck-Schuchardt, Darmstadt, G.F.R. All other commercial products were o f the purest grades available.
Animals and tissue preparation Male OF-1 mice (35 g bodyweight) were obtained from Iffa-Credo, St. Germain-sur-l'Arbresle, France, and were fed on Charles River C R F diet. Some mice recieved PB in the drinking water (1 mg/ml) for 7 days prior to the experiment. The 9000 g tissue supernatant (S-9) fractions were prepared from the pooled livers o f 4 animals (3 ml/g wet tissue) in 0.15 M KC1 as described previously [5]. The resulting tissue fraction was assayed immediately in some experiments; in others it was stored at --30°C for a m a x i m u m o f 15 h before it was used for mutagenicity assays. Storage under these conditions did not effect the tissue-mediated mutagenicity of VDC. All procedures were carried o u t at 0--4°C, using sterile glassware and solutions. Mutagenicity tests Tests for the mutagenicity of VDC were carried o u t according to an
131 adapted plate incorporation assay to test volatile compounds [5] using a histidine-auxotrophic strain o f S. typhimurium TA100, generously provided by Prof. B.N. Ames, Berkeley, Calif. U.S.A. Procedure A. Plates containing S-9 o f mouse liver, an NADPH generating system, 0.1/~mole of each histidine and biotine, the bacteria (1--2 • l 0 s) and all other ingredients as described [2,12] in a soft agar overlay (2.6 ml) were exposed to a gaseous mixture o f 2% VDC in air (v/v) in a dessicator at 37°C in the dark. After a specific exposure time, VDC was replaced by air; after further incubation for up to 48 h at 37°C the number of his÷-revertant colonies per plate was scored. The concentration of VDC dissolved in the incubation medium under these conditions was 3.3 • 10 -3 M [ 15]. Procedure B. Mouse liver S-9 and bacteria incorporated in the soft agar layer as described in procedure A were preincubated by keeping the plates at 37 °C for certain lengths o f time specified in the text. After preincubation, the plates were exposed to 2% VDC in air (v/v) for 3 h as described in procedure A. VDC was then removed under vacuum, and incubation was continued for a total of 48 h from the beginning of VDC exposure. Procedure C. On t o p of plates prepared as described in procedure A, but without histidine and biotine, 200 gl of a sterile aqueous solution containing 0.1 pmole o f histidine and biotine were spread as an even layer after preincubation b u t prior to VDC exposure. The plates were then exposed to 2% VDC in air (v/v) for 3 h as described in procedure B. Within the concentration range used, VDC in the presence of liver So9 and other ingredients as mentioned in procedures A, B and C showed no toxicity to the background lawn of bacteria which grows in the presence o f traces of histidine and biotine in the medium. The presence of an R factor in the TA100 strain was checked by seeding bacteria on ampicillin-containing agar [2]. Procedure D: Determination o f the bacterial growth curve. S. typhimurium strain TA100 (1--2 • l 0 s cells/plate) was incorporated in plates (9.2 cm in diameter) containing 0.1 #mole of histidine and biotine, as described in [12]. After various lengths of preincubation at 37°C in air, an aliquot o f the soft agar layer (1/11 o f the total surface area) was removed. This agar disc was then gently homogenized in 10 ml of 0.9% saline in a Potter--Elvejhem homogenizer. The cells were enumerated by seeding a suitable dilution on Vogel Bonner E nutrient agar plates [5] containing 1.5% agar, 2% glucose and 8 g/1 Difco nutrient broth. RESULTS
VDC requires metabolic activation b y microsomal mixed-function oxidase from rat or mouse liver to yield mutagenic reactants which can be detected with S. typhimurium stains TA1530 and TA100 [15]. A modification of the original Salmonella/microsome mutagenicity test [5] permits the measurement of time- and dose-dependent mutagenicity response curves (procedure A) and, therefore, the monitoring of microsomal enzyme activity
132 involved in metabolic activation o f VDC as a function o f incubation time under in vitro conditions. Exposure of S. t y p h i m u r i u m strain TA100 to 2% VDC in air in the presence o f a hepatic microsomal fraction from untreated or PB-pretreated mice caused a linear increase in the mutagenic response up to 4 h o f exposure (Fig. 1). This VDC concentration had previously been shown to be non-toxic to the bacteria [15]. Exposure to 2% VDC in air for 3 h was therefore used to study the viability of mouse liver microsomal mixed-function oxidase incorporated in a soft agar layer before VDC exposure. A maximal mutagenic response was noted after 2 h o f preincubation o f microsomal liver enzymes from both untreated or PB-pretreated mice prior to VDC exposure, the number o f his+-revertant colonies being about 30% higher than in plates which had not been preincubated (Fig. 2A). After preincubation for 4 h at 37 ° C, mouse liver S-9 showed the same enzymic capacity for converting VDC into mutagens, and the n u m b e r o f his ÷ revertants per plate remained as high as when the plates had not been preincubated. After preincubation for 14 h, the enzymes were inactive in converting VDC into mutagens, irrespective o f whether the S-9 was obtained from animals which had been pretreated with PB or not. In order to examine whether the peak activity seen in Fig. 2A is due to an increased mutagenicity caused by bacterial proliferation, histidine and biotine were omitted from plates during preincubation and only spread on the agar layer prior to VDC exposure (Fig. 2B). Under these conditions, the n u m b e r of his+ revertants per plate remained unchanged up to 4 h of preincubation,
0
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Fig. 1. T i s s u e - m e d i a t e d m u t a g e n i c r e s p o n s e o f S. typhimurium T A 1 0 0 a f t e r e x p o s u r e to V D C for various l e n g t h s o f time. M u t a g e n i c i t y assays ( p r o c e d u r e A) were p e r f o r m e d w i t h s t r a i n T A 1 0 0 a n d p o o l e d S-9 liver f r a c t i o n s f r o m 4 P B - p r e t r e a t e d m i c e (©) or f r o m 4 unt r e a t e d ( e ) f o l l o w i n g e x p o s u r e o f t h e b a c t e r i a to 2% V D C in air at 3 7 ° C (v/v). In t h e control (~), e o f a c t o r s ( N A D P ÷, glucose 6 - p h o s p h a t e ) were o m i t t e d . T h e n u m b e r o f s p o n t a n e o u s his*-revertant c o l o n i e s has n o t b e e n s u b t r a c t e d . M e a n values + S.E. were c o l l a t e d f r o m a series o f 2--5 e x p e r i m e n t s .
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Fig. 2A, B. Tissue-mediated mutagenic response of S. typhimurium TA100 as a function of preincubation time of plates before exposure to VDC in air. Mutagenicity assays were performed with strain TA100 and S-9 mouse liver fraction following 3 h exposure of the plates to 2% VDC in air (v/v). A, Untreated, or o, PB-pretreated, mice (procedure B); e, PB-pretreated mice, but histidine and biotine omitted from the plates during preincubation (procedure C). In the control (dotted line), cofactors (NADP ÷, glucose 6-phosphate) were omitted. The number of spontaneous his÷-revertant colonies has not been subtracted. Mean values _+ S.E. were collated from 4 experiments.
reflecting a constant rate o f metabolism o f VDC. The enzyme activity declined gradually to nil after 14 h o f preincubation. By measuring bacterial growth (procedure D, Materials and Methods), it could be demonstrated that the maximal mutagenicity seen in Fig. 2A, after 2 h o f preincubation followed by 3 h o f VDC exposure, coincided with the exponential growth o f the TA100 strain, which occurred between approximately 3 and 7 h after plating. After this period, the number o f bacteria had increased 30--40-fold. EDTA is n o w known to have a protective effect against lipid peroxidation [16,17] which could destroy microsomal enzyme activity. We have therefore studied the effect of 0.1 mM EDTA (0.26 pmole/plate) in plates containing bacteria and a fortified S-9 liver fraction from PB-pretreated mice (Fig. 3). The presence of EDTA during preincubation greatly increased the liver microsome-mediated mutagenicity o f VDC; after 6 h of preincubation, the n u m b e r of his+-revertant colonies was 3 times higher than in the absence o f EDTA. No enzymic conversion of VDC into mutagens was detected in plates with or without EDTA after 14 h or preincubation. Using conditions identical to those described in Fig. 3, the experiments were repeated with liver S-9 from untreated mice. Neither the absence nor
134 /.r
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Fig. 3. Tissue-mediated m u t a g e n i c response o f S. typhimurium T A 1 0 0 as a f u n c t i o n o f p r e i n c u b a t i o n time o f plates c o n t a i n i n g E D T A before e x p o s u r e to V D C . M u t a g e n i c i t y assays ( p r o c e d u r e B) were p e r f o r m e d w i t h strain T A 1 0 0 and S-9 liver fraction from PBp r e t r e a t e d mice f o l l o w i n g 3 h e x p o s u r e o f the bacteria to 2% V D C in air (v/v) in the presence o f 0 . 2 6 p m o l e / p l a t e E D T A ( $ ) or in the absence o f E D T A (©). T h e n u m b e r o f s p o n t a n e o u s h/s+-revertant c o l o n i e s ( - - - ) has not b e e n subtracted. Mean values + S.E. were c o l l a t e d for 3 e x p e r i m e n t s .
the presence o f EDTA made any difference to the microsome-mediated mutagenicity o f VDC. DISCUSSION
VDC was used as substrate to measure the influence o f agar on the viability o f hepatic microsomal enzymes, since this compound has no direct mutagenic action but requires activation by mouse liver microsomal mixedfunction oxidase to yield mutagenic metabolites (Ref. 15 and Fig. 1). In this study, the number o f his÷-revertant colonies o f S. typhimurium TA100 was used as a measure o f the enzymic capacity of the S-9 mouse liver preparation. When bacteria are exposed to gaseous VDC, its removal in vacuo permits the establishment of linear time- and dose-dependent mutagenic response curves (Fig. 1 and Ref. 5). In plates containing mouse liver S-9 fractions, cofactors for mixed-function oxidase and bacteria, preincubated for various lengths of time before exposure to VDC for 3 h (procedure A), maximal mutagenicity was noted after 2 h o f preincubation (Fig. 2A). Liver S-9 mediated mutagenicity of VDC was 50% higher when PB-pretreated animals were used than in assays using tissues from untreated animals, confirming previous results [15]. En-
135 zymic ability to metabolize VDC into mutagens declined over a period o f 6 h and was nil after 14 h o f preincubation. The peak mutagenic activity observed after 2 h of incubation (Fig. 2A) appears to be due to an enhanced mutagenic effect during bacterial replication and/or increased bacterial numbers. This was shown by omitting growth factors from the agar during preincubation of the plates and by adding histidine and biotine just before VDC exposure (Fig. 2B); under these conditions, liver S-9 mediated mutagenicity o f VDC remained unchanged over 7 h (4 h preincubation plus 3 h VDC exposure), reflecting a constant rate of formation of VDC metabolites. This is also confirmed by the linearity of the time-dependent liver S-9 mediated mutagenicity o f VDC observed over 7 h (Fig. 1). Although the nature o f the stabilizing effect of agar on mouse liver microsomal enzymes remains to be investigated, alteration o f the structural organization of the multi-enzyme complex in the membrane [18] and/or reduction o f lipid peroxidation in the presence o f agar could be contributing factors. When EDTA, a known inhibitor of lipid peroxidation [16,17,19] was added to the plates, a marked enhancement of the liver S-9 mediated mutagenicity of VDC was noted but only when microsomal enzymes from PB-pretreated mice were used (Fig. 3). These data exclude the possibility that EDTA enhances the mutagenicity o f VDC, e.g. b y altering the permeability of the bacterial membrane, b u t are in agreement with a report that the liver of PB-pretreated rodents shows alterations of the lipid peroxidation [20]. A 2-fold enhancement by EDTA of microsomal-mediated mutagenicity in S. typhimurium was also shown with vinyl chloride using PB-pretreated mice or with 1,4-dichlorobutene-2 with S-9 liver fraction from polychlorinated biphenyl (Aroclor 1254)-pretreated mice (data not included). As a practical consequence, EDTA might be used to potentiate the microsomalmediated mutagenicity of certain chemicals. This study gives an explanation why the plate incorporation assay is effective in detecting chemicals whose metabolic conversion into mutagens occurs at a low rate or which require multi-step activation processes [11]. We have shown experimentally for the first time that incorporation o f liver microsomal enzymes in a soft agar layer prolongs their viability for up to several hours; this p h e n o m e n o n could explain w h y certain compounds, e.g. N-nitrosodi-n-butyl- and N-nitrosodi-n-pentylamine, were not detectable as mutagens in liquid suspension after an incubation up to 1 h [11]. ACKNOWLEDGEMENTS
The authors would like to thank Miss P. Stafford-Smith for secretarial assistance and Mrs. E. Ward for editorial aid. The authors are indebted to Mrs. G. Brun for technical help. Financial support for the authors' research activities in this area has been provided partly by Contract No. 1-CP-55630 from the National Cancer Institute, NIH, U.S.A. Note added in proof. Since completion of this work, VDC has been found to be carcinogenic in mice (C. Maltoni, personal communication).
136 REFERENCES 1 J.A. Miller and E.C. Miller, Chemical carcinogenesis, mechanisms and approaches to its control, J. Natl. Cancer Inst., 47 (1971) v. 2 B.N. Ames, J. McCann and E. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31 (1975) 347. 3 C.N. Frantz and H.V. Malling, The quantitative microsomal mutagenesis assay method, Mutation Res., 31 (1975) 365. 4 H. Bartsch, C. Malaveille and R. Montesano, In vitro metabolism and microsomemediated mutagenicity of dialkylnitrosanlines in rats, hamsters and mouse tissues, Cancer Res., 35 (1975) 644. 5 H. Bartsch, C. Malaveille and R. Montesano, Human, rat and mouse liver-mediated mutagenicity of vinyl chloride in S. typhimurium strains, Int. J. Cancer, 15 (1975) 429. 6 U. Rannug, A. Johansson, C. Ramel and C.A. Wachmeister, The mutagenicity of vinyl chloride after metabolic activation, Ambio, 3 (1974) 194. 7 T. Sugimura, T. Yahagi, M. Nagao, M. Takeuchi, T. Kawachi, K. Hara, E. Yamasaki, T. Matsushima, Y. Hashimoto and M. Okada, Validity of mutagenicity tests using microbes as a rapid screening method for environmental chemicals, in R. Montesano, H. Bartsch and L. Tomatis (Eds.), Screening Tests in Chemical Carcinogenesis, Scientific Publications No. 12, IARC, Lyon, France, 1976, pp. 81--101. 8 J. McCann, E. Choi, E. Yamasaki and B.N. Ames, Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. USA, 72 (1975) 5135. 9 J. McCann and B.N. Ames, Detection of carcinogens as mutagens in the Salmonella/ microsome test: assay of 300 chemicals. Part II, Proc. Natl. Acad. Sci. USA, 73 (1976) 950. 10 C. Malaveille, G.F. Kolar and H. Bartsch, Rat and mouse tissue-mediated mutagenicity of ring-substituted 3,3-dimethyl-l-phenyl-triazenesin Salmonella typhimurium, Mutation Res., 36 (1976) 1. 11 H. Bartsch, A. Camus and C. Malaveille, Comparative mutagenicity of N-nitrosamines in semi-solid and in a liquid incubation system in the presence of rat or human tissue fractions, Mutation Res., 37 (1976) 149. 12 B.N. Ames, N.E. Durston, E. Yamasaki and F.D. Lee, Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. USA, 70 (1973) 2281. 13 H. Bartsch and R. Montesano, Mutagenic and carcinogenic effects of vinyl chloride, Mutation Res., 32 (1975) 93. 14 N. Loprieno, R. Barale, S. Baroncelli, C. Bauer, G. Bronzetti, A. Cammellini, G. Cercignani, C. Corsi, G. Gervasi, C. Leporini, R. Nieri, A.M. Rossi, G. Stretti and G. Turchi, Evaluation of the genetic effects induced by vinyl chloride monomer (VCM) under mammalian metabolic activation: studies in vitro and in vivo, Mutation Res., 40 (1976) 85. 15 H. Bartsch, C. Malaveille, R. Montesano and L. Tomatis, Tissue-mediated mutagenicity of vinylidene chloride and 2-chlorobutadiene in Salmonella typhimurium, Nature (London), 255 (1975)641. 16 E.D. Wills, Lipid peroxide formation in microsomes. Relationship of hydroxylation to lipid peroxide formation, Biochem. J., 113 (1969) 333. 17 T. Kamataki, N. Ozawa, M. Kitada and H. Kitagawa, The occurrence of an inhibitor of lipid peroxidation in rat liver soluble fraction and its effect on microsomal drug oxidations, Biochem. Pharmacol., 23 (1974) 2485. 18 A. Stier, Lipid structure and drug metabolizing enzymes, Biochem. Pharmacol., 25 (1976) 109. 19 W. Levin, A.Y.H. Lu, M. Jacobson, R. Kuntzman, J.L. Poyer and P.B. McCay, Lipid peroxidation and the degradation of cytochrome P-450 heine, Arch. Biochem. Biophys., 158 (1973) 842. 20 H.K.J. Hahn, D.J. Tuma, A.J. Barak and M.F. Sonell, Effect of phenobarbital on lipid peroxidation in the liver, Biochem. Pharmacol., 25 (1976) 769.
