Archives of
Arch. Toxicol. 39, 159--169 (1977)
TOXICOLOGY 9 by Springer-Verlag 1977
Mutagenicity and Chromosomal Aberrations as an Analytical Tool for in vitro Detection of Mammalian Enzyme-Mediated Formation of Reactive Metabolites* H. Greim, D. Bimboes**, G. Egert***, Waltraud G6ggelmann, and Maria Kr/imer Abteilung Toxikologie, Gesellschaft ffir Strahlen- und Umweltforschung Miinchen, D-8042 Neuherberg, Institut ffir Toxikologie, Universit/it Tiibingen, WilhelmstraJ3e 56, D-7400 Tfibingen, Federal Republic of Germany
Abstract. 1. Incubation of trichloroethylene, 1,1-dichloroethylene, vinylchloride, tetra-chlorocyclopentadiene, the nitroso derivatives of the pesticides Carbaryl, Prometryn, and Dodin in the presence of metabolically active mouse liver microsomes and bacteria as target cells were mutagenic, whereas tetrachloroethylene, 1,2 cis- and transdichloroethylene, hexachlorocyclopentadiene, carbontetrachloride, chloroform, halothane, trichlorofluoromethane and styrene were not activated to mutagenic species. 2. In a similar in vitro test system using freshly isolated human lymphocytes as target cells dimethylnitrosamine induced chromosomal aberrations. 3. It is concluded from the experiments that submammalian or mammalian in vitro cell systems with metabolically active liver microsomes are not only suitable to screen for chemical mutagens but to demonstrate formation of reactive intermediates, which are short lived and cannot be detected by chemical procedures.
Key words: Submammalian and mammalian in vitro test systems - Mutagenicity - Chlorinated ethylenes - Chlorinated cyclopentadienes - Haloalkanes -Styrene -Styrene-oxide - Microsomal metabolism.
Zusammenfassung. 1. Trichlor~ithylen, 1,1-Dichlor~ithylen, Vinylchlorid, Tetrachlorzyklopentadien sowie die Nitrosoderivate der Pestizide Karbaryl, Prometryn und Dodin wurden von metabolisch aktiven M~iuselebermikrosomen zu reaktiven Zwischenprodukten umgesetzt, die f'dr zugesetzte Testbakterien mutagen waren. Tetrachlor~ithylen, 1,2 cis- und trans-Dichlor~ithylen, Hexachlorzyklopentadien, Tetrachlorkohlenstoff, Chloroform, Halothan, Trichlorfluorme* Presented at the Symposium "Influence of Metabolic Activations and Inactivations on Toxic Effects" held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977 ** Present address: Department of Organic Chemistry, University of Tfibingen, D-7400 Tiibingen *** Present address: Department of Chemistry, Max-Planck-Institut fiir Experimentelle Medizin, D-3400 Gfttingen
160
H. Greim et al. than und Styrol zeigten im Testsystem keine Mutagenitht, whhrend Styrolepoxid ohne metabolische Aktivierung mutagen war. 2. In einem metabolisch aktiven System mit frisch priiparierten menschlichen Lymphozyten als Testorganismen induzierte Dimethylnitrosamin Chromosomenaberrationen. 3. Die Untersuchungsergebnisse zeigen, dal3 sich metabolisierende in vitro Testsysteme mit Bakterien oder S~iugetierzeUen als Testorganismen nicht nur ffir Routineuntersuchungen zum Nachweis mutagener Umweltchemikalien eignen sondern auch zum Nachweis reaktiver Metaboliten, die aufgrund ihrer geringen Stabilitht mit chemischen Methoden nicht erfal3bar sind.
Introduction
It is well known that many foreign compounds that are toxic to man are converted in the body to chemically reactive metabolites (Gillette, 1974a, b; McLean, 1973). Such reactants are capable to interact with tissue macromolecules thereby inducing cellular damage. Necrosis (Slater, 1966; Recknagel, 1967; Judah et al., 1970; Mitchell et al., 1973) or interaction with nuclear components with the possible consequence of mutagenic or carcinogenic effects (Miller and Miller, 1969; Miller, 1970; Heidelberger, 1975). To easily detect such effects, several in vitro test procedures have been developed. They are based on the evidence that many of the activated compounds do interact with target organelles. Providing targets to demonstrate such effects has been necessary because many of the reactants are short-lived and cannot be detected by analytical procedures. The method most frequently used has been to determine the amount of covalent binding of the reactants to the metabolically active system, the microsomes (Gelboin, 1969; Uehleko, 1973; Kappus et al., 1974; Kappus and Bolt, 1976; Maling et al., 1974; Uehleke et al., 1977). The advantage of the system is the relatively simple detection of reactive intermediates formed during metabolism of the parent compound. It is of disadvantage that the system does not provide a better defined biologic target than the nonspecific binding to the microsomal fraction. Consequently, it seems to be more significant for toxicologic evaluation to use systems with defined targets which permit evaluation of the compound investigated, such as mutagenesis. To evaluate mutagenic effects of reactants formed during metabolic conversion we preferentially use a bacterial in vitro test system using rat or mouse liver microsomes for metabolic activation (Mailing, 1971; Popper et al., 1973; Mailing and Frantz, 1974; Czygan et al., 1973a, b, 1974). This is a modification of the test developed by Ames (Ames et al., 1973, 1975) who performs metabolic activation by the 9000 x g supernatant of a rat liver homogenate (S-9 fraction) in the presence of bacteria in the top agar poured on top of the growth agar and incubated for 36-48 h. Preincubation in the absence of top agar will increase the rate of metabolic activation of certain compounds (Sugimura et al., 1976, Bartsch et al., 1976). Using
In vitro Mutagenicity Test Systems
161
microsomes instead of S-9 fraction again increases metabolic activation and reduces possible inactivation by cytoplasmic enzymes. However, several promutagens are activated into mutagens by soluble enzymes. These compounds will not be detected when pure microsomes are used. As target cells we use Salmonella typhimurium strains provided by Ames (Ames et al., 1973, 1975) and the E. eoli K 12 strain developed by Mohn (Mohn et al., 1974). In the following chapter the authors Purchase et ai. (1976, 1977) discuss the validity of such tests for screening procedures. I shall present evidence for the validity of the tests for research purposes to prove the formation of reactive compounds.
Materials and Methods a) Bacterial Systems. Mutagenic activity of the derivatives formed during microsomal activation was tested in a metabolizing in vitro system (Czygan et al., 1973b) with E. eoli K 12 or the Salmonella typhimurium strains TA 1535 or 1538, which require histidine for growth (Ames et al., 1973). The bioauxotrophic E. coli strain can be used in 4 mutation systems (Ellenberger and Mohn, 1975): In the 3 back mutation systems gal-, arg-, and had-, and in the MTR system where forward mutation leads to resistance to 5-methyl-DL-tryptophane. For the experiments 6--9 x 108 cells of an overnight culture were suspended in 1.5 ml incubate containing 5 mg microsomal protein, isolated from mouse livers, and the NADPH generating system of 5 mM MgC12, 16 mM DL-isocitrate-Na3, 0.66 mM NADP-Na3, 20 ~1 isocitrate-dehydrogenase (20 milliunits/p~l) in 0.1 M phosphate buffer pH 7.4, as well as different concentrations of the test compounds. These concentrations were selected from preliminary experiments so that they did not reduce cell survival by more than 20% (Table 1). After 1-2 h of incubation in a shaking water bath at 37 ~ the reaction was terminated in ice. The incubate was diluted in saline and plated on appropriate selective media (Czygan et ai., 1973a; Mohn et al., 1974), as described previously. Survival of the tester strains was determined by plating on the complete medium. Mutagenicity is expressed as colony-forming units (cfu) that were counted on the appropriate selective media per cfu counted on the complete medium. Liver mierosomes were isolated from male NMRI mice, 8 - 1 0 weeks of age, pretreated for 10 days with 0.1% phenobarbital (PB) in the drinking water or with a single intraperitoneal injection of 500 mg/kg Clophen A 50, 4 days before sacrifice to increase microsomal enzyme activity (Czygan et al., 1973b).
Table 1. Mutagenic effects of N-nitroso-pesticides (NO-derivatives) on E. coil K 12 in the presence of mouse liver microsomes and NADPH (:~ + sx, n = 4). Unsubstituted Carbaryl, Dodin and Prometryn did not alter spontaneous mutation frequency (Egert and Greim, 1976)
Concentration Survival (~M) rate
(%)
Spontaneous mutation frequency
Mutation frequency (cfu/10s) gal-
nad-
arg-
mtr +
2.02 _+ 1.12
0.02 + 0.01
0.29 _+ 0.14
1.79 _+ 0.42
91 + 4
6.73 + 2.12
0.67 + 0.14
2.58 + 0.21
8.29 + 1.46
100
94 + 6
3.97 + 1.43
0.03 + 0.02
1.14 + 0.34
3.83 _+ 1.12
Dodin-NO
30
84 + 5
2.73 + 1.28
0.02 + 0.02
1.82 + 0.52
3.33 + 1.64
Prometryn-NO
30
90 + 6
4.50 + 2.14
0.55 + 0.32
1.49 + 0.38
7.82 + 9.38
DMN Carbaryl-NO
50 x 103
162
H. Greim et al.
b) Human Lymphocytes as Target Cells. Lymphocytes have been isolated from 10 ml of freshly drawn human blood and centrifuged on Ficoll-Ronpacon. The lymphocytes containing layer was removed, washed in TC 199 medium and resuspended in RPMI 1640 medium. Approximately 106 cells were incubated in 5 ml of the complete culture medium consisting of RPMI 1640 medium, fetal bovine serum, phytohemeagglutinine-M, heparin, Streptomycin and Penicillin. After 48 h incubation at 37~ the lymphocytes were sedimented by centrifugation at 280 x g for 10 min. After removal of the supernatant the cells were exposed to the metabolizing system (Bimboes and Greim, 1976). The metabolizing test system consisted of 106 lymphocytes in 1 ml RPMI 1640 medium containing 50 mM Hepes buffer pH 7.3, 2 mg of microsomal protein from livers of mice, pretreated with PB, 0.5 mM NADPH and 50 mM dimethyinitrosamine (DMN). After 30 min incubation at 37~ in a shaking water bath, the lymphocytes were sedimented by centrifuging 5 min at 220 x g. After removal of the supernatant, the cells were washed in TC 199 and resedimented by centrifugation. Thymidine incorporation studies were performed as described previously (Bimboes and Greim, 1976). They have been done immediately after exposure of the lymphocytes to the test system to investigate cytotoxic effects of the different components of the incubation system.
Results and Discussion
Bacterial in vitro Test Systems a) Chlorinated Ethylenes. The attempts to correlate the stability of the epoxides of chlorinated ethylenes formed during microsomal activation with mutagenicity are presented in a preceding chapter of this book. The epoxides of symmetrically chlorinated ethylenes are suggested to be stable and have not been mutagenic, whereas the non-symmetrically chlorinated ethylenes are more reactive and have been found to be mutagenic (Greim et al., 1975), correlating with the carcinogenicity of vinylchloride in man (Creech and Johnson, 1974; Heath et al., 1975) or the capability of dichloroethylene and trichloroethylene to induce tumors in experimental animals (Memorandum, 1975; Viola et al., 1976).
b) Styrene. Styrene is widely used in the manufacture of plastics and synthetic resins and as an organic solvent (Loprieno et al., 1976). The reports on the mutagenic and carcinogenic effects of vinylchloride induced Milvy and Garro (1976) to investigate whether styrene oxide, a proposed metabolite of styrene (Leibman and Ortiz, 1969, 1970; Ohtsuji and Ikeda, 1971; Leibman, 1975)is mutagenic. F r o m the positive results they concluded that inhaled styrene m a y be activated to a potential carcinogen in man. We have been able to confirm the mutagenic effects of styrene oxide. However, when styrene has been investigated in an in vitro metabolizing test system, no mutagenic effects could be observed using S. typhimurium T A 1535 or E. coli K 12 strains. Consequently, it is suggested that microsomal activation to the oxide is not the obligatory pathway in the microsomal fraction and that other reactions such as hydroxylation to the vinylphenol or to 1-phenylethanol and 2-phenylethanol m a y be the major route of metabolism (Leibman and Ortiz, 1970; Leibman, 1975; Watabe and Maynert, 1968). However, rapid conversion of styrene oxide to the glycol by microsomal epoxide hydrase activity cannot be excluded although Clophen A-50induced mouse liver microsomes have been used. In such microsomes the rate of styrene oxide hydration would not be expected to exceed that of styrene oxide formation (Oesch, personal communication).
In vitro Mutagenicity Test Systems
163
c) Chlorinated Cyclopentadienes. Studying the metabolism of chlorinated cyclic hydrocarbons, it has been suggested that chlorinated cyclopentadienes are metabolically converted to the cyclopentadienone and that the rate of conversion may be due to the degree of chlorination at C 1 (Bonse and G/Sggelmann, 1977). In analogy to other vinyl-acid-chloride compounds, tetrachlorocyclopentadienone has been suggested to have acylating properties. It has been obvious to use the bacterial mutagenicity test system to detect the formation of this reactant (Fig. 1). It could be demonstrated that tetrachlorocyclopentadiene became highly mutagenie in the test system, whereas the hexachlorinated compound did not, confirming the suggestion that chlorination at C 1 hinders metabolic insertion of oxygen forming the dienone. Unfortunately, the pentachlorinated eyclopentadiene is unstable so that all attempts to use this compound failed until now.
d) Nitrosoderivatives of Pesticides. The test system has also been of value to study formation of mutagenic nitroso compounds from environmental chemicals such as pesticides occurring in the presence of nitrate at low pH values which resemble conditions of the human stomach (Sander and Bfirkle, 1969; Elespuru and Lijinsky, 1973; Eisenbrand et al., 1975). Secondary amino groups being present in pesticides such as carbaryl, the isopropylaminotriazine Prometryn and the N-dodecyl guanidino compound Dodin form N-nitroso derivatives (Egert and Greim, 1976). After synthesis of the nitroso derivatives and analysis by GC-mass-spectroscopy, infrared spectroscopy and quantitative analysis, the purified compounds have been tested for mutagenicity. Unsubstituted Carbaryl, Dodin and Prometryn were not mutagenic either in the absence or in the presence of metabolically active microsomes (Table 1). The nitroso compounds showed mutagenic activity only after metabolic activation, the
Ct
H
H
Ct
Tetrachloro- " ~ H... /CI CI
Nucteophil
CI
CI
-Ct-
CI" "Ct PentachloroCI\ /CI
CL t
Ct
Nucleophil
Tetrachtorocyclopentadienone S
Hexachtorocyclopentadiene Fig. 1. Proposed metabolic conversion of halogenated cyclopentadienes forming acylating tetrachlorocyclopentadienone (Bonse and Gfggelmann, 1977)
164
H. Greim et al.
Table 2. Mutagenic effects of polycyclichydrocarbons, Carbaryl and N-nitroso-carbarylon S. typhimuriurn TA 1538 in the absence (-) and presence (+) of metaboficallyactive mouse liver microsomes (Egert and Greim, 1976) Concentration (~M) Spontaneous mutation frequency Aminofluorene Methyleholanthrene Benzo(a)pyrene Carbaryl Carbaryl N-nitroso-Carbaryl N-nitroso-Carbaryl
40 25 25 100 100 100 100
Microsomal activity
+ + + -+ +
Survivalrate (%)
his+ revertants (ofu/10s)
100
Arch. Toxicol. 39, 159--169 (1977)
TOXICOLOGY 9 by Springer-Verlag 1977
Mutagenicity and Chromosomal Aberrations as an Analytical Tool for in vitro Detection of Mammalian Enzyme-Mediated Formation of Reactive Metabolites* H. Greim, D. Bimboes**, G. Egert***, Waltraud G6ggelmann, and Maria Kr/imer Abteilung Toxikologie, Gesellschaft ffir Strahlen- und Umweltforschung Miinchen, D-8042 Neuherberg, Institut ffir Toxikologie, Universit/it Tiibingen, WilhelmstraJ3e 56, D-7400 Tfibingen, Federal Republic of Germany
Abstract. 1. Incubation of trichloroethylene, 1,1-dichloroethylene, vinylchloride, tetra-chlorocyclopentadiene, the nitroso derivatives of the pesticides Carbaryl, Prometryn, and Dodin in the presence of metabolically active mouse liver microsomes and bacteria as target cells were mutagenic, whereas tetrachloroethylene, 1,2 cis- and transdichloroethylene, hexachlorocyclopentadiene, carbontetrachloride, chloroform, halothane, trichlorofluoromethane and styrene were not activated to mutagenic species. 2. In a similar in vitro test system using freshly isolated human lymphocytes as target cells dimethylnitrosamine induced chromosomal aberrations. 3. It is concluded from the experiments that submammalian or mammalian in vitro cell systems with metabolically active liver microsomes are not only suitable to screen for chemical mutagens but to demonstrate formation of reactive intermediates, which are short lived and cannot be detected by chemical procedures.
Key words: Submammalian and mammalian in vitro test systems - Mutagenicity - Chlorinated ethylenes - Chlorinated cyclopentadienes - Haloalkanes -Styrene -Styrene-oxide - Microsomal metabolism.
Zusammenfassung. 1. Trichlor~ithylen, 1,1-Dichlor~ithylen, Vinylchlorid, Tetrachlorzyklopentadien sowie die Nitrosoderivate der Pestizide Karbaryl, Prometryn und Dodin wurden von metabolisch aktiven M~iuselebermikrosomen zu reaktiven Zwischenprodukten umgesetzt, die f'dr zugesetzte Testbakterien mutagen waren. Tetrachlor~ithylen, 1,2 cis- und trans-Dichlor~ithylen, Hexachlorzyklopentadien, Tetrachlorkohlenstoff, Chloroform, Halothan, Trichlorfluorme* Presented at the Symposium "Influence of Metabolic Activations and Inactivations on Toxic Effects" held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977 ** Present address: Department of Organic Chemistry, University of Tfibingen, D-7400 Tiibingen *** Present address: Department of Chemistry, Max-Planck-Institut fiir Experimentelle Medizin, D-3400 Gfttingen
160
H. Greim et al. than und Styrol zeigten im Testsystem keine Mutagenitht, whhrend Styrolepoxid ohne metabolische Aktivierung mutagen war. 2. In einem metabolisch aktiven System mit frisch priiparierten menschlichen Lymphozyten als Testorganismen induzierte Dimethylnitrosamin Chromosomenaberrationen. 3. Die Untersuchungsergebnisse zeigen, dal3 sich metabolisierende in vitro Testsysteme mit Bakterien oder S~iugetierzeUen als Testorganismen nicht nur ffir Routineuntersuchungen zum Nachweis mutagener Umweltchemikalien eignen sondern auch zum Nachweis reaktiver Metaboliten, die aufgrund ihrer geringen Stabilitht mit chemischen Methoden nicht erfal3bar sind.
Introduction
It is well known that many foreign compounds that are toxic to man are converted in the body to chemically reactive metabolites (Gillette, 1974a, b; McLean, 1973). Such reactants are capable to interact with tissue macromolecules thereby inducing cellular damage. Necrosis (Slater, 1966; Recknagel, 1967; Judah et al., 1970; Mitchell et al., 1973) or interaction with nuclear components with the possible consequence of mutagenic or carcinogenic effects (Miller and Miller, 1969; Miller, 1970; Heidelberger, 1975). To easily detect such effects, several in vitro test procedures have been developed. They are based on the evidence that many of the activated compounds do interact with target organelles. Providing targets to demonstrate such effects has been necessary because many of the reactants are short-lived and cannot be detected by analytical procedures. The method most frequently used has been to determine the amount of covalent binding of the reactants to the metabolically active system, the microsomes (Gelboin, 1969; Uehleko, 1973; Kappus et al., 1974; Kappus and Bolt, 1976; Maling et al., 1974; Uehleke et al., 1977). The advantage of the system is the relatively simple detection of reactive intermediates formed during metabolism of the parent compound. It is of disadvantage that the system does not provide a better defined biologic target than the nonspecific binding to the microsomal fraction. Consequently, it seems to be more significant for toxicologic evaluation to use systems with defined targets which permit evaluation of the compound investigated, such as mutagenesis. To evaluate mutagenic effects of reactants formed during metabolic conversion we preferentially use a bacterial in vitro test system using rat or mouse liver microsomes for metabolic activation (Mailing, 1971; Popper et al., 1973; Mailing and Frantz, 1974; Czygan et al., 1973a, b, 1974). This is a modification of the test developed by Ames (Ames et al., 1973, 1975) who performs metabolic activation by the 9000 x g supernatant of a rat liver homogenate (S-9 fraction) in the presence of bacteria in the top agar poured on top of the growth agar and incubated for 36-48 h. Preincubation in the absence of top agar will increase the rate of metabolic activation of certain compounds (Sugimura et al., 1976, Bartsch et al., 1976). Using
In vitro Mutagenicity Test Systems
161
microsomes instead of S-9 fraction again increases metabolic activation and reduces possible inactivation by cytoplasmic enzymes. However, several promutagens are activated into mutagens by soluble enzymes. These compounds will not be detected when pure microsomes are used. As target cells we use Salmonella typhimurium strains provided by Ames (Ames et al., 1973, 1975) and the E. eoli K 12 strain developed by Mohn (Mohn et al., 1974). In the following chapter the authors Purchase et ai. (1976, 1977) discuss the validity of such tests for screening procedures. I shall present evidence for the validity of the tests for research purposes to prove the formation of reactive compounds.
Materials and Methods a) Bacterial Systems. Mutagenic activity of the derivatives formed during microsomal activation was tested in a metabolizing in vitro system (Czygan et al., 1973b) with E. eoli K 12 or the Salmonella typhimurium strains TA 1535 or 1538, which require histidine for growth (Ames et al., 1973). The bioauxotrophic E. coli strain can be used in 4 mutation systems (Ellenberger and Mohn, 1975): In the 3 back mutation systems gal-, arg-, and had-, and in the MTR system where forward mutation leads to resistance to 5-methyl-DL-tryptophane. For the experiments 6--9 x 108 cells of an overnight culture were suspended in 1.5 ml incubate containing 5 mg microsomal protein, isolated from mouse livers, and the NADPH generating system of 5 mM MgC12, 16 mM DL-isocitrate-Na3, 0.66 mM NADP-Na3, 20 ~1 isocitrate-dehydrogenase (20 milliunits/p~l) in 0.1 M phosphate buffer pH 7.4, as well as different concentrations of the test compounds. These concentrations were selected from preliminary experiments so that they did not reduce cell survival by more than 20% (Table 1). After 1-2 h of incubation in a shaking water bath at 37 ~ the reaction was terminated in ice. The incubate was diluted in saline and plated on appropriate selective media (Czygan et ai., 1973a; Mohn et al., 1974), as described previously. Survival of the tester strains was determined by plating on the complete medium. Mutagenicity is expressed as colony-forming units (cfu) that were counted on the appropriate selective media per cfu counted on the complete medium. Liver mierosomes were isolated from male NMRI mice, 8 - 1 0 weeks of age, pretreated for 10 days with 0.1% phenobarbital (PB) in the drinking water or with a single intraperitoneal injection of 500 mg/kg Clophen A 50, 4 days before sacrifice to increase microsomal enzyme activity (Czygan et al., 1973b).
Table 1. Mutagenic effects of N-nitroso-pesticides (NO-derivatives) on E. coil K 12 in the presence of mouse liver microsomes and NADPH (:~ + sx, n = 4). Unsubstituted Carbaryl, Dodin and Prometryn did not alter spontaneous mutation frequency (Egert and Greim, 1976)
Concentration Survival (~M) rate
(%)
Spontaneous mutation frequency
Mutation frequency (cfu/10s) gal-
nad-
arg-
mtr +
2.02 _+ 1.12
0.02 + 0.01
0.29 _+ 0.14
1.79 _+ 0.42
91 + 4
6.73 + 2.12
0.67 + 0.14
2.58 + 0.21
8.29 + 1.46
100
94 + 6
3.97 + 1.43
0.03 + 0.02
1.14 + 0.34
3.83 _+ 1.12
Dodin-NO
30
84 + 5
2.73 + 1.28
0.02 + 0.02
1.82 + 0.52
3.33 + 1.64
Prometryn-NO
30
90 + 6
4.50 + 2.14
0.55 + 0.32
1.49 + 0.38
7.82 + 9.38
DMN Carbaryl-NO
50 x 103
162
H. Greim et al.
b) Human Lymphocytes as Target Cells. Lymphocytes have been isolated from 10 ml of freshly drawn human blood and centrifuged on Ficoll-Ronpacon. The lymphocytes containing layer was removed, washed in TC 199 medium and resuspended in RPMI 1640 medium. Approximately 106 cells were incubated in 5 ml of the complete culture medium consisting of RPMI 1640 medium, fetal bovine serum, phytohemeagglutinine-M, heparin, Streptomycin and Penicillin. After 48 h incubation at 37~ the lymphocytes were sedimented by centrifugation at 280 x g for 10 min. After removal of the supernatant the cells were exposed to the metabolizing system (Bimboes and Greim, 1976). The metabolizing test system consisted of 106 lymphocytes in 1 ml RPMI 1640 medium containing 50 mM Hepes buffer pH 7.3, 2 mg of microsomal protein from livers of mice, pretreated with PB, 0.5 mM NADPH and 50 mM dimethyinitrosamine (DMN). After 30 min incubation at 37~ in a shaking water bath, the lymphocytes were sedimented by centrifuging 5 min at 220 x g. After removal of the supernatant, the cells were washed in TC 199 and resedimented by centrifugation. Thymidine incorporation studies were performed as described previously (Bimboes and Greim, 1976). They have been done immediately after exposure of the lymphocytes to the test system to investigate cytotoxic effects of the different components of the incubation system.
Results and Discussion
Bacterial in vitro Test Systems a) Chlorinated Ethylenes. The attempts to correlate the stability of the epoxides of chlorinated ethylenes formed during microsomal activation with mutagenicity are presented in a preceding chapter of this book. The epoxides of symmetrically chlorinated ethylenes are suggested to be stable and have not been mutagenic, whereas the non-symmetrically chlorinated ethylenes are more reactive and have been found to be mutagenic (Greim et al., 1975), correlating with the carcinogenicity of vinylchloride in man (Creech and Johnson, 1974; Heath et al., 1975) or the capability of dichloroethylene and trichloroethylene to induce tumors in experimental animals (Memorandum, 1975; Viola et al., 1976).
b) Styrene. Styrene is widely used in the manufacture of plastics and synthetic resins and as an organic solvent (Loprieno et al., 1976). The reports on the mutagenic and carcinogenic effects of vinylchloride induced Milvy and Garro (1976) to investigate whether styrene oxide, a proposed metabolite of styrene (Leibman and Ortiz, 1969, 1970; Ohtsuji and Ikeda, 1971; Leibman, 1975)is mutagenic. F r o m the positive results they concluded that inhaled styrene m a y be activated to a potential carcinogen in man. We have been able to confirm the mutagenic effects of styrene oxide. However, when styrene has been investigated in an in vitro metabolizing test system, no mutagenic effects could be observed using S. typhimurium T A 1535 or E. coli K 12 strains. Consequently, it is suggested that microsomal activation to the oxide is not the obligatory pathway in the microsomal fraction and that other reactions such as hydroxylation to the vinylphenol or to 1-phenylethanol and 2-phenylethanol m a y be the major route of metabolism (Leibman and Ortiz, 1970; Leibman, 1975; Watabe and Maynert, 1968). However, rapid conversion of styrene oxide to the glycol by microsomal epoxide hydrase activity cannot be excluded although Clophen A-50induced mouse liver microsomes have been used. In such microsomes the rate of styrene oxide hydration would not be expected to exceed that of styrene oxide formation (Oesch, personal communication).
In vitro Mutagenicity Test Systems
163
c) Chlorinated Cyclopentadienes. Studying the metabolism of chlorinated cyclic hydrocarbons, it has been suggested that chlorinated cyclopentadienes are metabolically converted to the cyclopentadienone and that the rate of conversion may be due to the degree of chlorination at C 1 (Bonse and G/Sggelmann, 1977). In analogy to other vinyl-acid-chloride compounds, tetrachlorocyclopentadienone has been suggested to have acylating properties. It has been obvious to use the bacterial mutagenicity test system to detect the formation of this reactant (Fig. 1). It could be demonstrated that tetrachlorocyclopentadiene became highly mutagenie in the test system, whereas the hexachlorinated compound did not, confirming the suggestion that chlorination at C 1 hinders metabolic insertion of oxygen forming the dienone. Unfortunately, the pentachlorinated eyclopentadiene is unstable so that all attempts to use this compound failed until now.
d) Nitrosoderivatives of Pesticides. The test system has also been of value to study formation of mutagenic nitroso compounds from environmental chemicals such as pesticides occurring in the presence of nitrate at low pH values which resemble conditions of the human stomach (Sander and Bfirkle, 1969; Elespuru and Lijinsky, 1973; Eisenbrand et al., 1975). Secondary amino groups being present in pesticides such as carbaryl, the isopropylaminotriazine Prometryn and the N-dodecyl guanidino compound Dodin form N-nitroso derivatives (Egert and Greim, 1976). After synthesis of the nitroso derivatives and analysis by GC-mass-spectroscopy, infrared spectroscopy and quantitative analysis, the purified compounds have been tested for mutagenicity. Unsubstituted Carbaryl, Dodin and Prometryn were not mutagenic either in the absence or in the presence of metabolically active microsomes (Table 1). The nitroso compounds showed mutagenic activity only after metabolic activation, the
Ct
H
H
Ct
Tetrachloro- " ~ H... /CI CI
Nucteophil
CI
CI
-Ct-
CI" "Ct PentachloroCI\ /CI
CL t
Ct
Nucleophil
Tetrachtorocyclopentadienone S
Hexachtorocyclopentadiene Fig. 1. Proposed metabolic conversion of halogenated cyclopentadienes forming acylating tetrachlorocyclopentadienone (Bonse and Gfggelmann, 1977)
164
H. Greim et al.
Table 2. Mutagenic effects of polycyclichydrocarbons, Carbaryl and N-nitroso-carbarylon S. typhimuriurn TA 1538 in the absence (-) and presence (+) of metaboficallyactive mouse liver microsomes (Egert and Greim, 1976) Concentration (~M) Spontaneous mutation frequency Aminofluorene Methyleholanthrene Benzo(a)pyrene Carbaryl Carbaryl N-nitroso-Carbaryl N-nitroso-Carbaryl
40 25 25 100 100 100 100
Microsomal activity
+ + + -+ +
Survivalrate (%)
his+ revertants (ofu/10s)
100
