Genotoxicity of 255 chemicals in the Salmonella microsome test (Ames test) and 8-hydroxyguanine (8-OH-Gua) assay for the detection of carcinogens

A program of cancer prevention, which aimed at identifying and minimizing human exposure to hazardous chemicals, requires the development of a rapid inexpensive screening assay as a complement to expensive long-term animal tests. Among thousands of hazardous chemicals to which humans are exposed, very few have been tested for genotoxicity. This report presents 255 chemicals (mutagens, non-mutagens, etc.) of a wide variety of chemical types which have been tested for genotoxicity, both in the Salmonella microsome test (Ames test) and in 8-hydroxyguanine (8-OH-Gua) assay, in order to detect carcinogens. Of the 255 chemicals tested, 83 chemicals (32.5%) were mutagenic in S. Typhimurium YG1041, YG1042, YG3003 and/or YG7108 strain, and 21 chemicals (8.2%) formed 8-OH-Gua in the rat hepatocytes oxidized DNA. These results may demonstrate the utility and limitation of both the Ames test and 8-OH-Gua assay in detecting chemicals likely to be toxic

Genotoxicity of 255 chemicals in the Salmonella microsome test (Ames test)

and 8-hydroxyguanine (8-OH-Gua) assay for the detection of carcinogens

Nobuyuki Sera*, Yoshito Tanaka*, Hiroko Tsukatani*, Nobuhiro Shimizu*, Shigeji Kitamori* and Hideo Utsumi**

*Fukuoka Institute of Health and Environmental Sciences, 39 Dazaifu Fukuoka 818-0135, Japan

**Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku,

Fukuoka 812-8582, Japan

Abstract

A program of cancer prevention, which aimed at identifying and minimizing human exposure to hazardous

chemicals, requires the development of a rapid inexpensive screening assay as a complement to expensive

long-term animal tests Among thousands of hazardous chemicals to which humans are exposed, very few

have been tested for genotoxicity This report presents 255 chemicals (mutagens, non-mutagens, etc.) of a

wide variety of chemical types which have been tested for genotoxicity, both in the Salmonella microsome

test (Ames test) and in 8-hydroxyguanine (8-OH-Gua) assay, in order to detect carcinogens Of the 255

chemicals tested, 83 chemicals (32.5%) were mutagenic in S Typhimurium YG1041, YG1042, YG3003

and/or YG7108 strain, and 21 chemicals (8.2%) formed 8-OH-Gua in the rat hepatocytes oxidized DNA.

These results may demonstrate the utility and limitation of both the Ames test and 8-OH-Gua assay in

detecting chemicals likely to be toxic.

Keywords

Mutagenicity, Ames test, S Typhimurium YG1042, oxidative DNA damage, 8-OH-Gua

Introduction

Genotoxicity of carcinogens is considered one of the most important contributors to cancer and a marker of the cancer risk of various populations There are a number of short-term genotoxicity tests such as the Ames

test, chromosomal aberration test and micronuclei test, etc The Ames test is widely used as a simple rapid in vitro method for detecting the mutagenicity of a variety of genotoxic chemicals The Ames test, which is

also used for predicting possible carcinogenicity, has been sufficiently developed and seriously validated for widespread use There is a high correlation between mutagenicity in the Ames test and carcinogenicity in the animal tests (Sugimura 1986) 8-OH-Gua is one of the most popular markers for the evaluation of oxidative DNA damage and oxidative stress Exposure to a large number of genotoxic chemicals including -irradiation, 4-nitroquinoline-1-oxide and asbestos causes formation of 8-OH-Gua in their respective target organs (Floyd 1990) Herein we report the genotoxicity of 255 chemicals performed by two kinds of experiments; the Ames test using new strains and 8-OH-Gua assay using rat hepatocytes We suggest that the Ames test using YG1041 and/or YG1042 strain is quite useful for the efficient detection of genotoxic chemicals in the environment

Materials and methods

255 chemicals of a wide variety of chemical types were purchased from Wako Pure Chemical Co Ltd (Osaka, Japan) and 10 complex mixtures such as condensates of river water were collected in Kyushu, Japan The 255 chemicals tested were 65 pesticides (26 insecticides, 16 herbicides and 14 fumigants, etc.), 168 industrial chemicals (104 aromatic hydrocarbons, 60 allyl hydrocarbons, etc.) and others (natural toxins, etc.) These samples were dissolved in dimethylsulfoxide (DMSO) or sterilized distilled water at sufficient concentration to react by dose-response curves

Mutagenicity was assayed by the standard Ames test (Maron et al 1983) with a modification of pre-incubation (37°C, 20min), in the presence or absence of a mammalian metabolic activation system (S9mix)

The newly constructed Salmonella tester strains used were YG1041 (hisD3052/pKM101/pYG233) and YG1042 (hisG46/pKM101/pYG233) strain (nitroreductase and O-acetyltransferase-overproducing) sensitive

to nitroarenes and/or aromatic amines, YG3003 (hisD(G)8476/rfa/pAQ1/pKM101/mutM ::Km) strain (deficient in 8-hydroxyguanine DNA glycosylase) sensitive to oxidative chemicals, and YG7108

(hisG46/rfa/d uvrB/ d adaST::Kmr/ d ogttST

::Cmr) strain (deficient in O6-methylguanine DNA methyltransferase) sensitive to alkylating chemicals These strains were kindly supplied by Dr T Nohmi of the National Institute of Health Sciences S9 fraction of phenobarbital/5,6-benzoflabone-pretreated male Sprague-Dawley (SD) rat liver and human liver were purchased from Oriental Yeast Co Ltd (Tokyo, Japan) and Funakoshi Co Ltd (Tokyo, Japan), respectively

Fresh rat hepatocytes were isolated from SD rats (8 weeks) and they were plated on a collagen substratum of

12 well plates with Dulbecco’s Modified Eagle Media (Gibco Co Ltd.) supplemented with fetal calf serum (5%), insulin (6.25g/ml), penicillin (50U/ml), streptomycin (50g/ml) and dexamethasone (1M) at a density

of 3x103 cells/viable cells/dish (Delclos et al 1987, Nakae et al 1995) 8-OH-Gua level in rat hepatocytes

DNA treated in an in vitro reaction with chemicals for 24hrs was measured by high performance liquid

chromatograph with an electrochemical detector (HPLC-ECD), and/or by a competitive ELISA system using

a monoclonal antibody specific for 8-OH-Gua

Results and discussion

Nitroreductase (NR) and O-acetyltransferase (OAT) are enzymes involved in the intracellular metabolic activation of aromatic hydrocarbons, etc in S Typhimurium TA strains YG1041 and YG1042 have 50-100 times respectively higher levels of both NR and OAT enzyme activity than their host strains TA98 and

TA100 (Hagiwara et al 1993) We compared the mutagenic activity (shown as the number of revertants per nanomolecule of chemicals, rev./nmol) of YG strains with that of the conventional TA strains as shown in Fig 1 YG1041 showed about 40-1,800 times higher mutagenic activity toward 2-nitrofuorene,

1-nitropyrene and 1,8-di1-nitropyrene than did TA98 YG1042 showed a 20-70 times stronger mutagenic response to 2-nitronaphthalene, 2,4-dinitroluene and 2-aminoanthracene than TA100 We tested the

1 10 100 1000 10000 100000 1000000 10000000

Chemicals

Fig 1 Comparison of mutagenicity in Ames test using S Typhimurium

YG starins (1041, 1042) and their host TA strains (98, 100)

TA strain YG strain

2-Nitrofluoranthene, 1-nitropyrene and 1,8-dinitropyrene are tested without S9mix, Glu-P-1 and Trp-P-2 are tested with S9mix using TA98 and YG1041.

2-Nitronaphthalene, 2,4-dinitrotoluen and 4-nitroquinoline N -oxide (4-NQO)

are tested without S9mix, 2-aminoanthracene and benzo[a]pyrene are tested with S9mix using TA100 and YG1042.

mutagenicity of 255 chemicals and listed the top twenty chemicals as shown in Table1 It turned out that YG1041 and/or YG1042 were more sensitive to the mutagenicity of chemicals classified as nitroarenes, aromatic amines and heterocyclic amines In addition, free radicals may be the most important genotoxic agents contributing to age-related diseases including cancer, and produce various kinds of oxidative DNA damage; nearly 100 products (Floyd 1990) Among them, 8-OH-Gua is a major mutation-prone (G:C to T:A transversion) DNA base-modified product Alkylating chemicals, as well as free radicals, are regarded as endogenous genotoxic chemicals which might be involved in colon cancer because it is suggested that colon cells may be frequently exposed to them (Sugimura 1986) We have chosen two other sensitive strains (Suzuki et al 1997, Yamada et al 1995) in order to assess the mutagenicity of 255 chemicals YG3003 was used to aid the identification of a variety of mutagenic free radicals including quinones, nitro and chloro

derivatives, while YG7108 was used to aid the identification of mutagenic alkylating chemicals such as N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) Of the 255 chemicals tested, 8 chemicals (3.1%) were

mutagenic in YG3003 and 12 chemicals (4.7%) were mutagenic in YG7108 presented in Table 2

In order to predict possible carcinogenicity to humans, the mutagenicity of 10 chemicals, which are well recognized mutagens requiring a metabolic activation system (S9mix), were compared using rat liver S9 fraction and human liver S9 fraction The mutagenicity by each S9mix makes a small difference and is almost equivalent as shown in Fig 2 In particular, relatively higher mutagenicity was observed for 2-aminoanthracene, MeIQx and 3-methylcholathrene not only by rat liver S9 fraction but also by human liver S9 fraction These results suggest that most mutagens could be activated to the ultimate forms in the presence of a mammalian metabolic activation system, such as human liver S9 fraction as well as rat liver S9 fraction This result suggests that human liver S9 fraction in the Ames test is useful for evaluation of genotoxicity to human

Reactive oxygen species and free radicals, such as superoxide anion radical (O2-), hydrogen peroxide (H2O2), hydroxy radical (.OH) and singlet oxygen (1O2), have been well correlated with the genotoxicity of chemicals 8-OH-Gua is one of the most critical lesions generated from deoxyguanine (dG) in oxidized DNA which was

observed after reaction of DNA, protein and lipids both in vitro and in vivo with genotoxic chemicals that

generate free radicals 8-OH-Gua is related to the initiation, promotion and progression of many diseases

Table 1 The top 20 chemicals of mutagenicity in S Typhimurium YG1042 with or without S9mix

YG1042, +S9mix YG1042, -S9mix

5 3-Methylcholanthrene 47,000 5 4-Nitroquinoline-N-oxide 51,100

17 1,2,5,6-Dibenzanthracene 6,180 17 2-Aminoanthracene 7,020

20 1,2-Dibromo-3-chloropropane 3,910 20 N-Nitrosodiphenylamine 4,070

such as cancer, tissue injury and inflammation (Floyd 1990) 8-OH-Gua has been commonly detected and identified by one of the already-existing methods; HPLC-ECD method (Helbock er al 1998, Shigenaga et al 1994) Desp ite it s pop ulari ty, th ere h ave b een do ubts regar ding i ts ac c urac y (possibil ity o f

artefactual production during sample preparation processes), expensive running cost and discrepancy in 8-OH-Gua level detected using different methods (gas chromatograph-mass spectrometry, 32P postlabeling

method, etc.) Therefore, it is important to develop a simple rapid in vitro method for evaluating the

generation of reactive oxygen species from a variety of genotoxic chemicals We developed a competitive ELISA method (Musarrat et al 1994) and analyzed 8-OH-Gua level (expressed as the ratio of 8-OH-Gua per

YG1041 (Rat S9) YG1041 (Human S9) YG1042 (Rat S9) YG1042 (Human S9)

0 50000 100000 150000 200000 250000 300000 350000 400000 450000

Chemicals

Fig 2 Comparison of mutagenicity of chemicals

by rat and human liver S9mix

Table 2 Mutagenicity in Ames test using S Typhimurium YG strains

(YG3003, YG7108) and their host strains (TA1535, TA102)

Oxidative chemicals

Alkylating chemicals

(N -methyl-N '-nitro-N -nitrosoguanidine)

(N -ethyl-N '-nitro-N -nitrosoguanidine)

(N -propyl-N '-nitro-N -nitrosoguanidine)

(N -butyl-N '-nitro-N -nitrosoguanidine)

105 dG per nanomolecule (nmol) of chemicals) induced by in vitro incubation of rat hepatocytes with 255

chemicals The results suggest that 8-OH-Gua level was relatively correlated with mutagenicity for YG3003,

as shown in Fig 3 In particular, OH-Gua level by incubation of rat hepatocytes with 4-NQO (0.86–2.8 8-OH-Gua/105 dG/nmol) was drastically increased in a dose-dependent curve using log transformation at the dose of 0.001-0.1mM (HPLC-ECD method) and 0.01-10mM (ELISA method)

Fig 3 Relationship between mutagenicity in YG3003 and 8-OH-Gua level in rat hepatocytes induced by chemicals

0 1 2 3

Mutagenicity (rev./nmol)

Chemicals tested are Aflatoxin B1, 2-aminoanthracene, 2-aminoanthraquinone, 3-nitrofluoranthene, 1-nitropyrene, 4-nitroquinoline N-oxide, Cumene, dibenzyl ether, diethyl benzene mixture, formaldehyde, MEP, methoxychlor, paraquat, pentachlorophenol, potassium dichromate, simetryne, thiobencarb, TPN, 1,2,4-trichlorobenzene, 2,4,5-trichlorophenol and triphenyltin (IV) chloride.

4-NQO

Fig 4 Summary of 255 chemicals for mutagenicity in Ames test and oxidative DNA damage in 8-OH-Gua assay

Ames test

(83 chemicals)

8-OH-Gua assay

(21 chemicals)

Not detected

(151 chemicals )

YG1041

(21 chemicals)

YG1042

(51 chemicals)

YG3003

(8 chemicals)

YG7108

(12 chemicals)

Fig 3 Relationship between mutagenicity in YG3003 and 8-OH-Gua level in rat hepatocytes induced by chemicals

0 1 2 3

Mutagenicity (rev./nmol)

Chemicals tested are Aflatoxin B1, 2-aminoanthracene, 2-aminoanthraquinone, 3-nitrofluoranthene, 1-nitropyrene, 4-nitroquinoline N-oxide, Cumene, dibenzyl ether, diethyl benzene mixture, formaldehyde, MEP, methoxychlor, paraquat, pentachlorophenol, potassium dichromate, simetryne, thiobencarb, TPN, 1,2,4-trichlorobenzene, 2,4,5-trichlorophenol and triphenyltin (IV) chloride.

4-NQO

Of 255 chemicals tested, 83 chemicals (32.5%) were mutagenic in the Ames test using YG1041, YG1042, YG3003 and/or YG7108 strain, and 21 chemicals (8.2%) formed 8-OH-Gua in the rat hepatocytes oxidized DNA as shown in Fig 4 These sensitive strains could detect a variety of mutagens that were not detected in the standard tester strains They are suitable for mutagenicity of aromatic hydrocarbons, allyl hydrocarbons and insecticides, but not suitable for that of herbicides, fumigants, heavy metals, inorganic chemicals and natural toxins, etc These results may suggest that the utility and limitations of both the Ames test and 8-OH-Gua assay in detecting chemicals likely to be environmental mutagens We recommend the Ames test using these sensitive strains (especially YG1041 and/or YG1042) in combination with the standard tester strains (TA98 and TA100 series), in order to detect the mutagenicity of environmental chemicals efficiently, in extracts of river water, exhaust gases and urban atmosphere

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Acknowledgements

This research was supported by the research project ‘The research on the development of the total evaluation technique for the hazardous impacts by the chemical substances towards human and ecology (the project leader is Prof Hideo Utsumi, Graduate School of Pharmaceutical Sciences, Kyushu University)’ This research project was supported by Fundamental Research Fund for the Environmental Future from Environmental Agency, Government of Japan