259
Mutation Research, 63 (1979) 259--272
© Elsevier/North-Holland Biomedical Press
CRITICAL IMPORTANCE OF MICROSOME CONCENTRATION IN MUTAGENESIS ASSAY WITH V79 CHINESE HAMSTER CELLS
T. KUROKI .,a, C. MALAVEILLE a, C. DREVON a, C. PICCOLI a, M. MACLEOD b and J.K. SELKIRK b a Untt o f Chemical Carcmogenesls, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon Cedex 2 (France) and b Btology Divtsion, Oak Ridge National Laboratory, Oak Rtdge, TN 37830 (U.S.A.)
(Received 20 April 1979) Accepted 5 July 1979)
Summary For optimum mutagenesis in V79 Chinese hamster cells, the amount of liver postmitochondrial fraction in the assay was found to be of critical importance, depending on the chemicals being tested. Benzo[a]pyrene (BP) required lower (1--5%) concentrations of the liver 15 000 × g supernatant (S15) from methylcholanthrene pretreated rats for a maximum induction of cytotoxicity and mutagenicity, as determined by 8-azaguanine- and ouabain-resistance. A sharp peak of mutagenicity and cytotoxicity was induced by 7,8-dihydroxy-7,8d i h y d r o b e n z o [ a ] p y r e n e (7,8-diol BP) at a concentration of 1% of the S15 fraction. Little or no response was induced by these compounds with the $15 concentrations of more than 10%. Similarly, aflatoxin BI induced a sharp peak of mutagenicity and cytotoxicity at a concentration of 2% of the liver S15 fraction from Aroclor-pretreated rats. Under the same condition, non-carcinogenic ariatoxin G2 did not induce cytotoxicity and mutagenicity. Analysis of BP metabolites by high-pressure liquid chromatography indicates that with the 30% S15 fraction, more than 80% of BP was metabolized during the first 15 min, while with the 2% S15 fraction, 7,8-diol BP increased conTo w h o m correspondence and reprint requests should be addressed. Present address: D e p a r t m e n t of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shtrokanedai, Minatoku, T o k y o 108 (Japan). Abbrevtations: AZA r, 8-azaguanme-reslstance; BP, b e n z o [ a ] p y r e n e , 7,8-diol BP, trans-7,8-dthyd r o x y - 7 , 8 - d i h y d r o b e n z o [ a ] p y r e n e ; DEN, N-nitrosodiethylamine; DMN, N - n i t r o s o d i m e t h y l a m i n e ; DMSO, d l m e t h y l s u l f o x i d e ; FCS, fetal calf serum: HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HPLC, high-pressure liquid c h r o m a t o g r a p h y ; MCA, 3-methylcholanthrene, OUA r, ouaba~n-resistance; PB, phenobarbitone. TCPO, 1,2-epoxy-3,3,3otnchloropropane; $9, 9000 X g su peru atant of, unless otherwise specified, 25% w/v hver homogenate; $15, 15000 X g s upe rna t a nt of liver homogenate.
260 tinuously throughout the 120-min incubation period, suggesting a strong metabolic competition to rapidly remove BP and 7,8-diol BP with a high concentration of the $15. In contrast with these compounds, N-nitrosodimethylamlne induced mutagenicity and cytotoxicity which increased linearly in proportion to the increasing amount of the S15 fraction from phenobarbitone- and Aroclor-pretreated rats. Various nitrosamines with different lipophilicity were examined at a high (30%) and low (2%) concentration of the S15 fraction from Aroclor-pretreated rats, in which ratios of mutation frequencies at 30% and 2% correlated inversely with lipophilicity of the compound. This result suggests that the hpid solubility of test compounds may be one factor which determines the concentration of post-mitochondrial supernatant for optimum mutagenesis.
Most chemical carcinogens and/or mutagens are biologically inert, unless metabolically activated, mainly by microsomal mixed function monooxygenases, to electrophilic intermediates, which react with the nucleophilic groups in cellular macromolecules [12]. Since target cells in most in vitro mutagenesis assays are lacking such metabolic capacity, inclusion of a metabolic activation system is essential for the detection of the mutagenic activity of chemicals that require metabolic activation. Liver postmitochondrial fractions, coupled with an NADPH-generating system, have been used extensively for this purpose in microbial assays and, more recently, with mammalian cells [1,4--6,9,11,15,19, 23,25]. We have previously reported microsome-medlated mutagenesis in V79 Chinese hamster cells by various nitrosamines [6,11] and vinyl chloride [5]. In the present study, polycyclic aromatic hydrocarbons and aflatoxins have been tested with the aim of extending the use of this mammalian cell mutagenesis system to other groups of carcinogens. The results obtained here show that the optimum concentration of postmitochondrial fraction is of critical importance and dependent on the chemicals to be tested. Similar results were also obtained in parallel experiments using S. typhimurium strains as genetic indicator [Malaveille et al., submitted]. Possible mechanisms were discussed in relation to the lipophilicity of the compounds and the basis of HPLC-analyses of BP metabolites. Materials and methods
Chemicals. Aflatoxin B1 and G2 (Schuchardt, Munich, Federal Republic of Germany), 8-azaguanine (Pfaltz and Bauer, Flushing, NY, U.S.A.), BP (Schuchardt, Munich, Federal Republic of Germany and Aldrich, Milwaukee, WI, U.S.A.), DMN and DEN (Merck, Darmstadt, Federal Republic of Germany}, glucose-6-phosphate (Boehringer-Mannheim, Paris, France), MCA (Fluka, Buchs SG, Switzerland), N-nitrosomorpholine (Merck, Darmstadt, Federal Republic of Germany), ouabain (Sigma Chemicals, St. Louis, MO, U.S.A.), NADP and NADPH (Boehringer-Mannheim, Paris, France) and TCPO (Aldrich, Beerse, Belgium) were purchased. 3H-BP (24 Ci/mmol) was obtained from Amersham (Arlington Heights, IL, U.S.A.), of which radiochemical purity
261
was checked routinely b y HPLC. N-Nitrosodipropylamine was obtained from Dr. M. Castegnaro in our institute and 7,8-diol BP was provided by Dr. P.L. Grover, Chester Beatty Research Institute of Cancer Research (London, Great Britain). Autoclavable Eagle's MEM was obtained from Flow Laboratories {Irvine, Great Britain). FCS was purchased from Grand Island Biological Company (Grand Island, NY, U.S.A.). All other products were of the purest grades available. Cell culture. V79 Chinese hamster cells were generously provided by Dr. E. Huberman (Oak Ridge National Laboratory, Oak Ridge, TN, U.S.A.). The cells were grown in Eagle's MEM supplemented with 10% FCS at 37°C in an atmosphere of 5% CO2 in air. Preparation of postmitochondrial fraction ($15 fraction). Adult male BDVI rats (120--140 g), bred in our laboratory, were fed on a Charles River CRF diet (Charles River Breeding Laboratories, Wilmington, MA, U.S.A.). The animals were pretreated with inducers as follows: PB sodium at 1 mg/ml in drinking water for 7 days; MCA at 40 mg/kg b o d y weight, dissolved in olive oil and injected i.p. 48 h before; and Aroclor 1254 at 500 mg/kg b o d y weight, diluted in olive oil and injected i.p. 5 days before. The choice of $15 fraction in terms of inducers was made by referring the results in the microbial assay [2] as well as in the mammalian cell assay [11]. The pooled livers of 3 decapitated animals were washed, excised and homogenized with a Potter-Elvehjem-type teflon homogenizer in 3 vol. of 0.25 M sucrose at pH 7.4 containing 2 mM MgCI: and 20 mM HEPES (sucrose--HEPES buffer). The postmitochondrial fraction (S15 fraction) was prepared by two successive centrifugations at 9000 X g for 10 min and at 15 000 X g for 20 min. The microsome fraction was prepared by a further centrifugation at 105 000 X g for 60 min and the pellet was suspended in an equal volume of the sucrose--HEPES buffer. All procedures were carried o u t at 0--4 ° C. The resulting fractions were kept in liquid nitrogen until use. Mutagenic assays. Mutations in two genetic loci, comprising of resistances to 8-azaguanine, a purine analogue, and ouabain, a specific inhibitor of Na÷/K ÷ activated ATPase in cell membrane, were induced in V79 Chinese hamster cells in the presence of $15 fraction. Experimental procedures were reported in detail elsewhere [6,11]. In brief, the V79 cells were plated at a concentration of 1.0--1.5 X 106 cells/60-mm Petri dish and were cultured overnight. They were then incubated in 2.5 ml of the reaction mixture, consisting of 0.75 ml of S15 fraction, diluted with sucrose--HEPES buffer at a given concentration, 0.75 ml modified SCrensen--phosphate buffer (0.055 M, pH 7.4 containing 0.9% NaC1 and 1.6 mg MgCl2 • 6H20 per ml}, 0.25 ml glucose-6-phosphate solution (13 mg/ml in PBS), 0.25 ml NADP solution (6.3 mg/ml in PBS), 0.5 ml PBS and 20 pl of chemicals being tested. The final concentrations of the co-factors in the reaction mixture were 20 pmoles PO]- per ml, 3.1 pmoles Mg :÷ per ml, 5 pmoles glucose-6-phosphate per ml and 0.8 pmoles NADP per ml. A concentration of the $15 fraction is expressed as a percentage (v/v) in the reaction mixture, e.g., 10% $15 contains 0.25 ml $15 and 0.5 ml of the sucrose--HEPES buffer used in its preparation in 2.5 ml of the reaction micture, so that the final concentrations of the co-factors were not changes. Unless otherwise stated, incubation was at 37°C for 1 h for nitrosamines and 3 h for BP, 7,8-diol BP and aflatoxins.
262 The cells were then washed twice and incubated for 2--3 h in fresh culture medium, followed by replating for determination of the cytotoxicity and mutagenicity induced. Cytotoxicity was determined by plating 100 cells/60-mm dish (4 dishes at each point) and cultured for 7 days. For mutagenesis, 2 × 104 and 10 s cells/60-mm dish were plated for AZA ~ and OUA ~, respectively (8 or more dishes at each point). The expression period was for 48 h. When a high toxicity was expected, expression periods of 72 and 96 h were also examined, in order to ensure enough cell divisions for maximum expression and the highest value was chosen for the computation of induced mutation frequency. Selection drugs were added to give final concentrations of 20 pg/ml 8-azaguanine and 1 mM ouabain. The media containing the drug were changed once, 5-7 days later; for the 8-azaguanine medium, FCS was replaced by dialysed FCS and non-essential amino acids for Eagle's MEM were added at 0.1 mM. The cultures were fixed and stained with Giemsa at 12 days for AZA ~ and at 14 days for OUA ~. Mutation frequency was calculated per 10 s survivors, takmg into account the number of cells plated and the plating efficiency. Means and standard errors in the figures and the table refer to the variability in a single experiment. Reproducibility of the results was confirmed in most cases by repeating the experiments. Spontaneous mutation frequencies were 1.7 + 0.9 (mean and S.D. of 9 Expts.) for AZA ~ and 0.9 + 0.7 (mean and S.D. of 3 Expts.) for OUA ~. These values were not subtracted in calculating induced mutation frequencies. The stability of AZA r and OUA ~ colonies was demonstrated using 10 and 11 isolated colonies, respectively, which were cultured more than one m o n t h in the absence of the drug used for selection [6,11]. Analysis of metabolites of BP by HPLC. Incubations of the S15 fraction from MCA-pretreated rats was carried out under the conditions described above. Incubations were stopped by 1 ml of acetone and extracted 3 times with 2 vol. of ethyl acetate, dried over anhydrous magnesium sulfate, evaporated to dryness and redissolved in methanol for chromatographic analysis. 3H-BP was diluted with unlabeled BP in DMSO to a final concentration of 10 pM. Metabolite separation was performed with a Spectra Physics 3500 HPLC fitted with a DuPont 1-meter ODS-Permaphase column, as previously described [20]. Elution was done by reverse-phase using a 30--70% methanol--water gradient. Column pressure was 500 psi and oven temperature was 50°C. Effluent was monitored by a DuPont 835-multiwavelength fluorescence and ultraviolet spectrophotometer with a 250--390 nm transmission filter. Gradient sweep time was 30 min. Peaks were identified by coincident retention time and UVspectra. Fractions (0.2 ml) were collected and measured by radioactivity in a Searle Mark III scintillation counter using aquasol (New England Nuclear, Boston, MA, U.S.A.) at the counting solution. Raw scintillation data was automatically transmitted to a PDP-11 computer (Digital Equipment Corp., Maynard, MA, U.S.A.). Data reduction programs designed to adjust for specific activity converted the radioactivity into molar quantities. Simultaneously, control incubations without microsomes were extracted and chromatographed in an identical fashion and the background subtracted from the metabolism data by means of a control program. The reduced data for each label was plotted coincidentally by a Tektronix 4051 Graphics Terminal interfaced with the PDP-11 computer.
263 Results
Concentrations of S15 fraction required for optimum mutagenicity assays of BP, 7,8-diol BP and aflatoxin B~ Optimum conditions in detecting the mutagenicity of BP, 7,8-diol BP and aflatoxin B~ were investigated in the presence of various concentrations (1,2,5, 10,20 and 30%) of liver S15 fractions. As shown in Fig. 1, BP at 10 and 20 pM was cytotoxic and mutagenic, although weakly so, only when assayed in the reaction mixture containing the $15 fraction from MCA-pretreated rats at concentrations from 1--5%: 20 pM BP incubated with 2 or 5% of the $15 produced 15--17 AZA r colonies, mutation frequencies which were 6--7-fold higher than the solvent control. 7,8-Diol BP, a proximate metabolite of BP was much more cytotoxic and mutagenic than its parental BP under these conditions. A sharp peak of cytotoxicity and mutagenicity was observed at 1% of the S15 fraction from MCA-pretreated rats (Fig. 2): 208 AZA r and 28 OUA r colonies per l 0 s survivors were observed when treated with 5 #M 7,8-diol BP in the presence of the S15 fraction at a concentration of 1%, whereas little or no effects were observed with the S15 at concentrations of 5% or more. Fig. 3A shows the time courses of the induction of mutagenicity and cytotoxicity by 2 pM 7,8-diol BP. The number of AZA r mutants increased up to 3 h, while OUA r mutation reached a plateau at 1 h incubation. Consequently, an incubation time of 3 h was chosen for the following experiments. Dose--
O
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Concentration of $15 (%) Fig. 1. C y t o t o x i c l t y ( t o p ) a n d m u t a g e m c i t y ( b o t t o m : A Z A r) i n d u c e d b y BP a t 1 0 (o) a n d 2 0 ( e ) / ~ M m t h e p r e s e n c e o f t h e liver S 1 5 f r a c t i o n f r o m M C A - p r e t r e a t e d r a t s , a t c o n c e n t r a t i o n s o f 1, 2, 5, 100 2 0 a n d 30%. E x p r e s s i o n p e n o d s w e r e 4 8 h. C y t o t o x i c i t y w a s d e t e r m i n e d b y p l a t i n g e f f i c i e n c y a n d is e x p r e s s e d as percentages of the control containing no $15 fraction. Mutagenicity was determined by the frequency of d r u g - r e s i s t a n t c o l o m e s p e r 1 0 5 s u r v i v o r s , t a k i n g i n t o a c c o u n t t h e n u m b e r o f cells p l a t e d a n d p l a t i n g effic i e n c y . Bars" SE, w h e n n o t i n d i c a t e d SE are w i t h i n t h e s y m b o l s .
264
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Concentrat,on of $15 (%) Fig. 2. C y t o t o x l c l t y ( t o p ) a n d m u t a g e m c l t y ( m i d d l e . A Z A r a n d b o t t o m : BP m t h e p r e s e n c e o f t h e h v e r S 1 5 f r a c t i o n f r o m M C A - p r e t r e a t e d r a t s a t s i o n p e r m d s w e r e 72 h f o r A Z A r a n d O U A r at 1 a n d 2% S 1 5 a n d 48 h m u t a g e n i c i t y are e x p r e s s e d as d e s c r i b e d in t h e l e g e n d t o Fig. 1. Bars: within the symbols.
O U A r) i n d u c e d 1, 2, 5, 10 a n d for the others. SE, w h e n n o t
b y 5 pM 7,S-dlol 30%. T h e e x p r e s Cytotoxiclty and i n d i c a t e d SE are
response curves for 7,8-diol BP are shown in Fig. 4. At concentrations o f 1 and 2% of the $15 fraction, mutagenicity and c y t o t o x i c i t y were induced in proportion to dose of 7,8-diol BP: higher mutagenicity and c y t o t o x i c i t y were observed in the presence of 1% than in that of 2% 815 fraction. In the presence of 30% S15, however, no response was detected up to 10 pM 7,8-diol BP. Experiments were done in order to examine if the lack of mutagenicity at higher concentrations of S15 may be due to inactivation of active metabolites by enzymes, such as epoxide hydratase [18] and glutathione-S-transferase [7, 17]. However, when TCPO, an inhibitor o f microsomal epoxide hydratase, was added at 0.2 and 0.4 mM, no AZA r was induced by BP (data not included}. F u r t h e r m o r e , no mutagenicity was induced by BP and 7,8-diol BP when the S15 fraction and NADP were replaced by pure microsome fraction and
265
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Fig. 3. T i m e c o u r s e o f the r e d u c t i o n o f c y t o t o x i c l t y ( t o p ) a n d m u t a g e n i c l t y ( b o t t o m : e , A Z A r ; o, O U A r) b y 2 #M 7,8-diol BP (A: l e f t ) a n d 2 #M a f l a t o x m B 1 (B: right). V 7 9 Chinese h a m s t e r cells w e r e t r e a t e d w i t h t h e c h e m m a l s for 1, 3 a n d 5 h m t h e p r e s e n c e o f t h e liver S 1 5 f r a c t i o n e i t h e r f r o m M C A - p r e t r e a t e d rats at 1% ( f o r 7,8-dlol BP) o r f r o m A r o c l o r - p r e t r e a t e d r a t s at 2% ( f o r a f l a t o x m B 1). E x p r e s s i o n p e r i o d s w e r e 72 h for A Z A r b y 7,8-dlol BP at 1, 3 a n d 5 h a n d A Z A r b y a f l a t o x m B 1 at 3 h; 96 h f o r A Z A r b y a f l a t o x m B 1 at 5 h, 4 8 h f o r the o t h e r s . C y t o t o x l c i t y a n d m u t a g e n i c i t y are e x p r e s s e d as d e s c r i b e d m t h e l e g e n d t o Fig. 1. Bars" SE, w h e n n o t i n d i c a t e d SE are w i t h i n t h e s y m b o l s .
NADPH, which does not contain the soluble glutathione-S-transferase (data not included). Aflatoxin B1 also requires lower concentrations of S15 fraction for the induction of its mutagenicity in V79 Chinese hamster cells (Fig. 5): a sharp peak of mutagenicity and cytotoxicity was obtained at 2% of the liver S15 fraction from Aroclor-pretreated rats, b u t little or no response was induced at concentrations of higher than 10%. Time courses of the inductions are shown in Fig. 3B, in which mutagenicity and cytotoxicity increased linearly with time of incubation up to 5 h: for practical reasons, however, an incubation time of 3 h was chosen. Under these conditions, mutagenicity and cytotoxicity were proportional to the dose of aflatoxin BI (Fig. 6}. Aflatoxin G2, a structurally related, but non-carcinogenic compound [8], did not induce cytotoxicity and mutagenicity when tested up to a concentration of 10 #M under the conditions where aflatoxin B1 is mutagenic (Table 1).
Analysis of BP metabolites by HPLC For understanding possible mechanism for lack of mutagenicity at high concentrations, metabolites of BP were separated b y HPLC under the same incubation condition for the mutagenesis induction. Incubation with 2% $15 from MCA-pretreated rats for 45 min (Fig. 7A) produced the whole series of metabolites; 9,10
Mutation Research, 63 (1979) 259--272
© Elsevier/North-Holland Biomedical Press
CRITICAL IMPORTANCE OF MICROSOME CONCENTRATION IN MUTAGENESIS ASSAY WITH V79 CHINESE HAMSTER CELLS
T. KUROKI .,a, C. MALAVEILLE a, C. DREVON a, C. PICCOLI a, M. MACLEOD b and J.K. SELKIRK b a Untt o f Chemical Carcmogenesls, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon Cedex 2 (France) and b Btology Divtsion, Oak Ridge National Laboratory, Oak Rtdge, TN 37830 (U.S.A.)
(Received 20 April 1979) Accepted 5 July 1979)
Summary For optimum mutagenesis in V79 Chinese hamster cells, the amount of liver postmitochondrial fraction in the assay was found to be of critical importance, depending on the chemicals being tested. Benzo[a]pyrene (BP) required lower (1--5%) concentrations of the liver 15 000 × g supernatant (S15) from methylcholanthrene pretreated rats for a maximum induction of cytotoxicity and mutagenicity, as determined by 8-azaguanine- and ouabain-resistance. A sharp peak of mutagenicity and cytotoxicity was induced by 7,8-dihydroxy-7,8d i h y d r o b e n z o [ a ] p y r e n e (7,8-diol BP) at a concentration of 1% of the S15 fraction. Little or no response was induced by these compounds with the $15 concentrations of more than 10%. Similarly, aflatoxin BI induced a sharp peak of mutagenicity and cytotoxicity at a concentration of 2% of the liver S15 fraction from Aroclor-pretreated rats. Under the same condition, non-carcinogenic ariatoxin G2 did not induce cytotoxicity and mutagenicity. Analysis of BP metabolites by high-pressure liquid chromatography indicates that with the 30% S15 fraction, more than 80% of BP was metabolized during the first 15 min, while with the 2% S15 fraction, 7,8-diol BP increased conTo w h o m correspondence and reprint requests should be addressed. Present address: D e p a r t m e n t of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shtrokanedai, Minatoku, T o k y o 108 (Japan). Abbrevtations: AZA r, 8-azaguanme-reslstance; BP, b e n z o [ a ] p y r e n e , 7,8-diol BP, trans-7,8-dthyd r o x y - 7 , 8 - d i h y d r o b e n z o [ a ] p y r e n e ; DEN, N-nitrosodiethylamine; DMN, N - n i t r o s o d i m e t h y l a m i n e ; DMSO, d l m e t h y l s u l f o x i d e ; FCS, fetal calf serum: HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HPLC, high-pressure liquid c h r o m a t o g r a p h y ; MCA, 3-methylcholanthrene, OUA r, ouaba~n-resistance; PB, phenobarbitone. TCPO, 1,2-epoxy-3,3,3otnchloropropane; $9, 9000 X g su peru atant of, unless otherwise specified, 25% w/v hver homogenate; $15, 15000 X g s upe rna t a nt of liver homogenate.
260 tinuously throughout the 120-min incubation period, suggesting a strong metabolic competition to rapidly remove BP and 7,8-diol BP with a high concentration of the $15. In contrast with these compounds, N-nitrosodimethylamlne induced mutagenicity and cytotoxicity which increased linearly in proportion to the increasing amount of the S15 fraction from phenobarbitone- and Aroclor-pretreated rats. Various nitrosamines with different lipophilicity were examined at a high (30%) and low (2%) concentration of the S15 fraction from Aroclor-pretreated rats, in which ratios of mutation frequencies at 30% and 2% correlated inversely with lipophilicity of the compound. This result suggests that the hpid solubility of test compounds may be one factor which determines the concentration of post-mitochondrial supernatant for optimum mutagenesis.
Most chemical carcinogens and/or mutagens are biologically inert, unless metabolically activated, mainly by microsomal mixed function monooxygenases, to electrophilic intermediates, which react with the nucleophilic groups in cellular macromolecules [12]. Since target cells in most in vitro mutagenesis assays are lacking such metabolic capacity, inclusion of a metabolic activation system is essential for the detection of the mutagenic activity of chemicals that require metabolic activation. Liver postmitochondrial fractions, coupled with an NADPH-generating system, have been used extensively for this purpose in microbial assays and, more recently, with mammalian cells [1,4--6,9,11,15,19, 23,25]. We have previously reported microsome-medlated mutagenesis in V79 Chinese hamster cells by various nitrosamines [6,11] and vinyl chloride [5]. In the present study, polycyclic aromatic hydrocarbons and aflatoxins have been tested with the aim of extending the use of this mammalian cell mutagenesis system to other groups of carcinogens. The results obtained here show that the optimum concentration of postmitochondrial fraction is of critical importance and dependent on the chemicals to be tested. Similar results were also obtained in parallel experiments using S. typhimurium strains as genetic indicator [Malaveille et al., submitted]. Possible mechanisms were discussed in relation to the lipophilicity of the compounds and the basis of HPLC-analyses of BP metabolites. Materials and methods
Chemicals. Aflatoxin B1 and G2 (Schuchardt, Munich, Federal Republic of Germany), 8-azaguanine (Pfaltz and Bauer, Flushing, NY, U.S.A.), BP (Schuchardt, Munich, Federal Republic of Germany and Aldrich, Milwaukee, WI, U.S.A.), DMN and DEN (Merck, Darmstadt, Federal Republic of Germany}, glucose-6-phosphate (Boehringer-Mannheim, Paris, France), MCA (Fluka, Buchs SG, Switzerland), N-nitrosomorpholine (Merck, Darmstadt, Federal Republic of Germany), ouabain (Sigma Chemicals, St. Louis, MO, U.S.A.), NADP and NADPH (Boehringer-Mannheim, Paris, France) and TCPO (Aldrich, Beerse, Belgium) were purchased. 3H-BP (24 Ci/mmol) was obtained from Amersham (Arlington Heights, IL, U.S.A.), of which radiochemical purity
261
was checked routinely b y HPLC. N-Nitrosodipropylamine was obtained from Dr. M. Castegnaro in our institute and 7,8-diol BP was provided by Dr. P.L. Grover, Chester Beatty Research Institute of Cancer Research (London, Great Britain). Autoclavable Eagle's MEM was obtained from Flow Laboratories {Irvine, Great Britain). FCS was purchased from Grand Island Biological Company (Grand Island, NY, U.S.A.). All other products were of the purest grades available. Cell culture. V79 Chinese hamster cells were generously provided by Dr. E. Huberman (Oak Ridge National Laboratory, Oak Ridge, TN, U.S.A.). The cells were grown in Eagle's MEM supplemented with 10% FCS at 37°C in an atmosphere of 5% CO2 in air. Preparation of postmitochondrial fraction ($15 fraction). Adult male BDVI rats (120--140 g), bred in our laboratory, were fed on a Charles River CRF diet (Charles River Breeding Laboratories, Wilmington, MA, U.S.A.). The animals were pretreated with inducers as follows: PB sodium at 1 mg/ml in drinking water for 7 days; MCA at 40 mg/kg b o d y weight, dissolved in olive oil and injected i.p. 48 h before; and Aroclor 1254 at 500 mg/kg b o d y weight, diluted in olive oil and injected i.p. 5 days before. The choice of $15 fraction in terms of inducers was made by referring the results in the microbial assay [2] as well as in the mammalian cell assay [11]. The pooled livers of 3 decapitated animals were washed, excised and homogenized with a Potter-Elvehjem-type teflon homogenizer in 3 vol. of 0.25 M sucrose at pH 7.4 containing 2 mM MgCI: and 20 mM HEPES (sucrose--HEPES buffer). The postmitochondrial fraction (S15 fraction) was prepared by two successive centrifugations at 9000 X g for 10 min and at 15 000 X g for 20 min. The microsome fraction was prepared by a further centrifugation at 105 000 X g for 60 min and the pellet was suspended in an equal volume of the sucrose--HEPES buffer. All procedures were carried o u t at 0--4 ° C. The resulting fractions were kept in liquid nitrogen until use. Mutagenic assays. Mutations in two genetic loci, comprising of resistances to 8-azaguanine, a purine analogue, and ouabain, a specific inhibitor of Na÷/K ÷ activated ATPase in cell membrane, were induced in V79 Chinese hamster cells in the presence of $15 fraction. Experimental procedures were reported in detail elsewhere [6,11]. In brief, the V79 cells were plated at a concentration of 1.0--1.5 X 106 cells/60-mm Petri dish and were cultured overnight. They were then incubated in 2.5 ml of the reaction mixture, consisting of 0.75 ml of S15 fraction, diluted with sucrose--HEPES buffer at a given concentration, 0.75 ml modified SCrensen--phosphate buffer (0.055 M, pH 7.4 containing 0.9% NaC1 and 1.6 mg MgCl2 • 6H20 per ml}, 0.25 ml glucose-6-phosphate solution (13 mg/ml in PBS), 0.25 ml NADP solution (6.3 mg/ml in PBS), 0.5 ml PBS and 20 pl of chemicals being tested. The final concentrations of the co-factors in the reaction mixture were 20 pmoles PO]- per ml, 3.1 pmoles Mg :÷ per ml, 5 pmoles glucose-6-phosphate per ml and 0.8 pmoles NADP per ml. A concentration of the $15 fraction is expressed as a percentage (v/v) in the reaction mixture, e.g., 10% $15 contains 0.25 ml $15 and 0.5 ml of the sucrose--HEPES buffer used in its preparation in 2.5 ml of the reaction micture, so that the final concentrations of the co-factors were not changes. Unless otherwise stated, incubation was at 37°C for 1 h for nitrosamines and 3 h for BP, 7,8-diol BP and aflatoxins.
262 The cells were then washed twice and incubated for 2--3 h in fresh culture medium, followed by replating for determination of the cytotoxicity and mutagenicity induced. Cytotoxicity was determined by plating 100 cells/60-mm dish (4 dishes at each point) and cultured for 7 days. For mutagenesis, 2 × 104 and 10 s cells/60-mm dish were plated for AZA ~ and OUA ~, respectively (8 or more dishes at each point). The expression period was for 48 h. When a high toxicity was expected, expression periods of 72 and 96 h were also examined, in order to ensure enough cell divisions for maximum expression and the highest value was chosen for the computation of induced mutation frequency. Selection drugs were added to give final concentrations of 20 pg/ml 8-azaguanine and 1 mM ouabain. The media containing the drug were changed once, 5-7 days later; for the 8-azaguanine medium, FCS was replaced by dialysed FCS and non-essential amino acids for Eagle's MEM were added at 0.1 mM. The cultures were fixed and stained with Giemsa at 12 days for AZA ~ and at 14 days for OUA ~. Mutation frequency was calculated per 10 s survivors, takmg into account the number of cells plated and the plating efficiency. Means and standard errors in the figures and the table refer to the variability in a single experiment. Reproducibility of the results was confirmed in most cases by repeating the experiments. Spontaneous mutation frequencies were 1.7 + 0.9 (mean and S.D. of 9 Expts.) for AZA ~ and 0.9 + 0.7 (mean and S.D. of 3 Expts.) for OUA ~. These values were not subtracted in calculating induced mutation frequencies. The stability of AZA r and OUA ~ colonies was demonstrated using 10 and 11 isolated colonies, respectively, which were cultured more than one m o n t h in the absence of the drug used for selection [6,11]. Analysis of metabolites of BP by HPLC. Incubations of the S15 fraction from MCA-pretreated rats was carried out under the conditions described above. Incubations were stopped by 1 ml of acetone and extracted 3 times with 2 vol. of ethyl acetate, dried over anhydrous magnesium sulfate, evaporated to dryness and redissolved in methanol for chromatographic analysis. 3H-BP was diluted with unlabeled BP in DMSO to a final concentration of 10 pM. Metabolite separation was performed with a Spectra Physics 3500 HPLC fitted with a DuPont 1-meter ODS-Permaphase column, as previously described [20]. Elution was done by reverse-phase using a 30--70% methanol--water gradient. Column pressure was 500 psi and oven temperature was 50°C. Effluent was monitored by a DuPont 835-multiwavelength fluorescence and ultraviolet spectrophotometer with a 250--390 nm transmission filter. Gradient sweep time was 30 min. Peaks were identified by coincident retention time and UVspectra. Fractions (0.2 ml) were collected and measured by radioactivity in a Searle Mark III scintillation counter using aquasol (New England Nuclear, Boston, MA, U.S.A.) at the counting solution. Raw scintillation data was automatically transmitted to a PDP-11 computer (Digital Equipment Corp., Maynard, MA, U.S.A.). Data reduction programs designed to adjust for specific activity converted the radioactivity into molar quantities. Simultaneously, control incubations without microsomes were extracted and chromatographed in an identical fashion and the background subtracted from the metabolism data by means of a control program. The reduced data for each label was plotted coincidentally by a Tektronix 4051 Graphics Terminal interfaced with the PDP-11 computer.
263 Results
Concentrations of S15 fraction required for optimum mutagenicity assays of BP, 7,8-diol BP and aflatoxin B~ Optimum conditions in detecting the mutagenicity of BP, 7,8-diol BP and aflatoxin B~ were investigated in the presence of various concentrations (1,2,5, 10,20 and 30%) of liver S15 fractions. As shown in Fig. 1, BP at 10 and 20 pM was cytotoxic and mutagenic, although weakly so, only when assayed in the reaction mixture containing the $15 fraction from MCA-pretreated rats at concentrations from 1--5%: 20 pM BP incubated with 2 or 5% of the $15 produced 15--17 AZA r colonies, mutation frequencies which were 6--7-fold higher than the solvent control. 7,8-Diol BP, a proximate metabolite of BP was much more cytotoxic and mutagenic than its parental BP under these conditions. A sharp peak of cytotoxicity and mutagenicity was observed at 1% of the S15 fraction from MCA-pretreated rats (Fig. 2): 208 AZA r and 28 OUA r colonies per l 0 s survivors were observed when treated with 5 #M 7,8-diol BP in the presence of the S15 fraction at a concentration of 1%, whereas little or no effects were observed with the S15 at concentrations of 5% or more. Fig. 3A shows the time courses of the induction of mutagenicity and cytotoxicity by 2 pM 7,8-diol BP. The number of AZA r mutants increased up to 3 h, while OUA r mutation reached a plateau at 1 h incubation. Consequently, an incubation time of 3 h was chosen for the following experiments. Dose--
O
60 *
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5
10
20
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10
Concentration of $15 (%) Fig. 1. C y t o t o x i c l t y ( t o p ) a n d m u t a g e m c i t y ( b o t t o m : A Z A r) i n d u c e d b y BP a t 1 0 (o) a n d 2 0 ( e ) / ~ M m t h e p r e s e n c e o f t h e liver S 1 5 f r a c t i o n f r o m M C A - p r e t r e a t e d r a t s , a t c o n c e n t r a t i o n s o f 1, 2, 5, 100 2 0 a n d 30%. E x p r e s s i o n p e n o d s w e r e 4 8 h. C y t o t o x i c i t y w a s d e t e r m i n e d b y p l a t i n g e f f i c i e n c y a n d is e x p r e s s e d as percentages of the control containing no $15 fraction. Mutagenicity was determined by the frequency of d r u g - r e s i s t a n t c o l o m e s p e r 1 0 5 s u r v i v o r s , t a k i n g i n t o a c c o u n t t h e n u m b e r o f cells p l a t e d a n d p l a t i n g effic i e n c y . Bars" SE, w h e n n o t i n d i c a t e d SE are w i t h i n t h e s y m b o l s .
264
100 80 60 40
.
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Concentrat,on of $15 (%) Fig. 2. C y t o t o x l c l t y ( t o p ) a n d m u t a g e m c l t y ( m i d d l e . A Z A r a n d b o t t o m : BP m t h e p r e s e n c e o f t h e h v e r S 1 5 f r a c t i o n f r o m M C A - p r e t r e a t e d r a t s a t s i o n p e r m d s w e r e 72 h f o r A Z A r a n d O U A r at 1 a n d 2% S 1 5 a n d 48 h m u t a g e n i c i t y are e x p r e s s e d as d e s c r i b e d in t h e l e g e n d t o Fig. 1. Bars: within the symbols.
O U A r) i n d u c e d 1, 2, 5, 10 a n d for the others. SE, w h e n n o t
b y 5 pM 7,S-dlol 30%. T h e e x p r e s Cytotoxiclty and i n d i c a t e d SE are
response curves for 7,8-diol BP are shown in Fig. 4. At concentrations o f 1 and 2% of the $15 fraction, mutagenicity and c y t o t o x i c i t y were induced in proportion to dose of 7,8-diol BP: higher mutagenicity and c y t o t o x i c i t y were observed in the presence of 1% than in that of 2% 815 fraction. In the presence of 30% S15, however, no response was detected up to 10 pM 7,8-diol BP. Experiments were done in order to examine if the lack of mutagenicity at higher concentrations of S15 may be due to inactivation of active metabolites by enzymes, such as epoxide hydratase [18] and glutathione-S-transferase [7, 17]. However, when TCPO, an inhibitor o f microsomal epoxide hydratase, was added at 0.2 and 0.4 mM, no AZA r was induced by BP (data not included}. F u r t h e r m o r e , no mutagenicity was induced by BP and 7,8-diol BP when the S15 fraction and NADP were replaced by pure microsome fraction and
265
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Aflatoxm B1
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Incubation
Time
(hours)
Fig. 3. T i m e c o u r s e o f the r e d u c t i o n o f c y t o t o x i c l t y ( t o p ) a n d m u t a g e n i c l t y ( b o t t o m : e , A Z A r ; o, O U A r) b y 2 #M 7,8-diol BP (A: l e f t ) a n d 2 #M a f l a t o x m B 1 (B: right). V 7 9 Chinese h a m s t e r cells w e r e t r e a t e d w i t h t h e c h e m m a l s for 1, 3 a n d 5 h m t h e p r e s e n c e o f t h e liver S 1 5 f r a c t i o n e i t h e r f r o m M C A - p r e t r e a t e d rats at 1% ( f o r 7,8-dlol BP) o r f r o m A r o c l o r - p r e t r e a t e d r a t s at 2% ( f o r a f l a t o x m B 1). E x p r e s s i o n p e r i o d s w e r e 72 h for A Z A r b y 7,8-dlol BP at 1, 3 a n d 5 h a n d A Z A r b y a f l a t o x m B 1 at 3 h; 96 h f o r A Z A r b y a f l a t o x m B 1 at 5 h, 4 8 h f o r the o t h e r s . C y t o t o x l c i t y a n d m u t a g e n i c i t y are e x p r e s s e d as d e s c r i b e d m t h e l e g e n d t o Fig. 1. Bars" SE, w h e n n o t i n d i c a t e d SE are w i t h i n t h e s y m b o l s .
NADPH, which does not contain the soluble glutathione-S-transferase (data not included). Aflatoxin B1 also requires lower concentrations of S15 fraction for the induction of its mutagenicity in V79 Chinese hamster cells (Fig. 5): a sharp peak of mutagenicity and cytotoxicity was obtained at 2% of the liver S15 fraction from Aroclor-pretreated rats, b u t little or no response was induced at concentrations of higher than 10%. Time courses of the inductions are shown in Fig. 3B, in which mutagenicity and cytotoxicity increased linearly with time of incubation up to 5 h: for practical reasons, however, an incubation time of 3 h was chosen. Under these conditions, mutagenicity and cytotoxicity were proportional to the dose of aflatoxin BI (Fig. 6}. Aflatoxin G2, a structurally related, but non-carcinogenic compound [8], did not induce cytotoxicity and mutagenicity when tested up to a concentration of 10 #M under the conditions where aflatoxin B1 is mutagenic (Table 1).
Analysis of BP metabolites by HPLC For understanding possible mechanism for lack of mutagenicity at high concentrations, metabolites of BP were separated b y HPLC under the same incubation condition for the mutagenesis induction. Incubation with 2% $15 from MCA-pretreated rats for 45 min (Fig. 7A) produced the whole series of metabolites; 9,10
