Environmental mutagens and carcinogens.

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ENVIRONMENTAL MUTAGENS

+3136

AND CARCINOGENS Minako Nagao and Takashi Sugimura National Cancer Center Research Institute, Chuo·ku, Tokyo,

104

Japan

Taijiro Matsushima Institute of Medical Science, University of Tokyo, Minato·ku, Tokyo, 108 Japan

CONTENTS INTRODUCTION METHODOLOGY Mutation Tests Using Microbes ................................................................................ Repair test and rec-assay ..........................................................................................

Mutation test ..........................................................................................................

Inductest ................................................................................................................

Metabolic activation

................................................................................................

In Vitro Trans/ormation Test Using Cultured Mammalian Cells .......................... Other Short-Term Test Systems lor Detection 0/ Environmental Mutagens and Carcinogens .............................................................................................. In Vivo Long-Term A nimal Tests lor Environmental Carcinogens ...................... .. NA TURALL Y OCCURRING AGENTS .................... . . . ....... . . . . . . . ............................. Fungi ......................................................................................................................... . Plants ....................................................................................................................... . Fatty Acid Peroxides in Foods ................................................................................ .. PYROLYSIS PRODUCTS .................................................................. ...................... ... . Cooking Products ..................................................................................................... . Tobacco Tar .............................................................................................................. Aromatic Hydrocarbons ........................................................................................... . NITROSA TION PRODUCTS ........................ . . .......................................... .................. SYNTHETIC CHEMICALS . . . . . . . . . . . . . . . . . .. . ........ . . . . . . . . . . .. . ..... . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . ........ .. Food Additives ................................................................................................... ...... . Cosmetics ..................... ............................................................................. ................ Drugs ........................................................................................................................ Pesticides ................................................................................................................... .

Household Materials ................................................................................................ Occupational Substances and Industrial Intermediates ......................................... .

118 119 119 119 120 121 121 123 123 123 124 124 125 127 127 127 129 130 130 131 131 131 133 134 13S 136 117

0066-4197/78/1215-0117$01.00

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NAGAO. SUGIMURA & MATSUSHIMA

POLLUTANTS ........................................ .............................................. ........ ................ Air ..............................................................................................................................

Water

........................................................................................................................

FACTORS MODIFYING MUTAGENESIS AND CARCINOGENESIS .............. EVALUATION AND QUANTITATION OF DATA .............................................. OVERLAPPING OF MUTAGENS AND CARCINOGENS ..................................

138 138 139 139 142 143


INTRODUCTION

Environmental mutagens and carcinogens may have two important effects in humans: They may cause mutation of germ cells resulting in the accumu lation of heritable abnormal genes in the population, and they may cause mutation of somatic cells resulting in the formation of malignant cells in individuals. In the past few years, much progress has been made in screening environ mental mutagens and carcinogens, using the Salmonella mutation test first developed by Ames and his group (1-3). The DNA repair test has also been used to detect environmental mutagens and carcinogens by Slater et al (4), by Kada et al (5), and by us (6). Other microbial systems using Escherichia coli (7), Bacillus subtilis (8), Neurospora ( 9), and Saccharomyces (10) have also been widely used. Meanwhile the overlapping of carcinogens and mutagens has been em phasized by McCann et al (11-13), Purchase et al (14), and us (15-17). Therefore, the problem of environmental mutagens directly reflects that of environmental carcinogens and vice versa. The literature on environmental mutagens is now enormous and cannot be fully covered in this review. Nowadays it is possible to obtain information from computerized sources on the results of mutagenic and carcinogenic tests on chemicals. In addition the survey of the literature on Chemical Mutagenesis (18 ) compiled by the Environmental Mutagen Information Center, Oak Ridge, is a useful source of information on environmental mutagens, and information on environ mental carcinogens can be obtained in Abstracts on Carcinogenesis (19) prepared by the National Cancer Institute. This review does not include all the available information on environmental mutagens and carcinogens, but reviews important recent progress in research, with emphasis on the rela tionship between mutagens and carcinogens. Modern industry has introduced many mutagens and carcinogens into our environment, but nature itself also created many mutagens and carcino gens, which can be found in molds and plants. In addition, environmental mutagens and carcinogens were recently discovered that have been pro duced ever since the first adoption of fire by our ancestors. This review attempts to give a balanced account of all these factors. Environmental mutagens and carcinogens are important to scientists, manufacturers. con-


ENVIRONMENTAL MUTAGENS AND CARCINOGENS

119

sumers, and the staff of regulatory agencies, and thus many facets of the importance of these compounds must be mentioned. For instance, the fact, which has not previously been taken into proper consideration, that the specific activities of environmental mutagens and carcinogens have a very wide range of 1 to 107 magnitude is discussed in this review. The terms, environmenta l mutagen and environmenta l ca rcinogen include compounds with very weak and very strong activities, which are difficult to include in the same category either scientifically or practically. Quantitative aspects of carcinogenic and mutagenic activities are especially important. METHODOLOGY

Many different types of biological systems have been used for detection of environmental mutagens. Each system has certain advantages and disad vantages. These systems can be classified into three categories: microbial systems, cultures of mammalian cells in vitro, and multicellular animals or plants. The microbial systems used have ranged from bacteriophages, to eucaryotic Neurospora cells, and much of the information reviewed in this article was obtained with microbial systems. In addition, some data on mutations, chromosome aberrations, and sister chromatide exchange in cultured cells and on Drosophila and silk worms are also cited. For detection of environmental carcinogens, the systems for in vitro cell transformation, which have been reported by many workers (20-25), are useful. To prove that an environmental substance is carcinogenic by long term animal experiments, defined experimental conditions are required, as reported in the guidelines of the National Cancer Institute (26) and of IARC-WHO (27). To obtain a definite conclusion about the activity of weak environmental carcinogens, very strict conditions must be maintained. Mutation Tests Using Microbes REPAIR TEST AND REC-ASSAY Slater et al (4) first reported the use of strains with a wild phenotype of DNA repair and with a DNA-polymerase 1 deficient mutant, poIA,. These two strains are spread out on agar plates with a central well, the test chemicals are placed in the wells, and the plates are kept overnight at 37°C to allow bacterial growth. Many directly acting carcinogens, such as N-alkyl-N-nitrosoureas, 2nitrosofluorene, and N-hydroxyl-2-aminofluorene cause much wider growth inhibition zones round the well with the polA, mutant than with the wild strain. Slater et al (4) reported that the DNA-modifying activities of N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN) could be detected by including a microsomal fraction in vitro. Nagao &


1 20

NAGAO, SUGIMURA & MATSUSHIMA

Sugimura (6) found a good correlation between carcinogenicity detected in in vivo tests and results of the repair test on many nitroquinoline and nitropyridine derivatives. Benzo(a)pyrene (BP), N-acetyl-2-aminoftuorene (AAF), cycloheximide, and melphalan gave positive results in the repair test (28). Tatsumi & Nishioka (29) tried to make the repair test more quantitative: They incubated E. eoli WP2, uvrA, and reeA and a double mutant with the test substance in liquid medium for 4 hr at 37°C and then measured the absorbance at 660 nm. This method is called the test-tube test and it is a sensitive, simple, and quantitative test (29). The ree-assay, developed by Kada et al (5), uses a B. subtilis recombina tion-deficient mutant, ree-. Cultures of wild and ree- strains are streaked on broth agar plates from one point, and filter paper soaked in a solution of the test chemical was placed on that point. Compounds causing DNA damage showed more inhibition along the streaked line of rec- strain than along that of the wild strain. Nishioka (30) tested 41 metal compounds using this ree-assay, finding that compounds, such as K2Cr04' K2Cr207, CH3HgCl, and (NH4) 2Mo04 inhibited the ree- mutant more than the wild strain. Mutation tests using Salmonella typhimurium or E. have proved the most practical for detecting environmental mutagens and carcinogens, the former becoming especially widely used since Ames and his collaborators introduced the UV-sensitive (uvrB) and deep rough (rfa) strains (2). The uvr gene makes the cells more sensitive to many environmental mutagens, and the deep rough gene, which results in a cell wall defective in lipopolysaccharide, enables many substances to penetrate into the cells. The mutants tested are TA1 535, a base-pair change mutant, and TA1 536-1538, frameshift mutants. The S. typhimurium series is generally thought to be able to detect most environmental mutagens and carcinogens. However, the typical mutagens nitrofuran derivatives were not mutagenic to the TA1535 series, although they were mutagenic to the WP2 mutant of E. eoli (31 ). S. typhimurium TAlOO and TA98, which were obtained by introducing R-factor plasmid pKM lOl into TA1535 and TA1538, respectively, are susceptible to nitrofu ran derivatives (32). Generally speaking, TA100 and TA98 can be recom mended for detecting most environmental mutagens and carcinogens ( 1 1 ). However, mutagens that produce interstrand cross-links, such as mitomy cin C, cannot be detected with the TA1 535 series or TA100 or TA98, although they can be detected with TA92 or TA94, which have the uvr+ gene (3, 33). It is interesting that some mutagens and carcinogens, such as N-methylnitrosourea (MNU) and methylazoxymethanol (MAM), MUTATION TEST

coli



121

are most effectively detected with hisG46, which has the wild types of surface lipopolysaccharide and DNA repair. The sensitivities of various S. typhimurium strains to typical mutagens and carcinogens are listed in Table 1. Actinomycin D is mutagenic in a system of forward mutation using Neurospora (34), but it cannot be detected with the TA1535 series or TAlOO or T A98 (35, 36). A recent report on the production of 8-azaguanine resistance as a forward mutation in S. typhimurium (36) seems to be rele vant in detecting this kind of mutagen. The inductest was devised by Devoret and his associates (37, 38). The principle of the inductest was described by Lwoff (39). By combin ing lysogenic induction in E. coli K12 envA uvrB with a drug metabolic activation system, the inductest became very effective for detecting many environmental mutagens and carcinogens. The advantage of using a single bacterial strain to cover many different types of chemicals should be empha sized. The assay of galactokinase synthesized by induced phage is sensitive (38). Mutation in induced phage also could be counted (38). INDUCTEST

Many mutagens and carcinogens are meta bolized to active forms. Some mutagens, such as N-methyl-N'-nitro-N nitrosoguanidine (MNNG), do not require any metabolic activation, but react directly with DNA. Mutagens, such as AF-2, 2-(2-furyl)-3-(5-nitro-2furyl)acrylamide, can be activated by enzymes that are present in the bac terial cells. However, most mutagens and carcinogens are activated metabolically by enzymes present in mammalian cells. Mammalian cells METABOLIC ACTIVATION

Table 1

Detection of the mutagenicities of typical mutacarcinogens with various S. ty-

phimurium strains8 TA1535

TA1536-38

TAtOO

TA98

uvrB

uvrB

uvrB

rfa

rfa

-R

-R

rfa +R ++

+

AF-2

BP Aflatoxin B 1

+

hisG46

TA92

uvrB

uvr+

uvr+

rfa +R

wild

wild +R

+

++

+

+

++

+

-R

MNNG

+

+

++

++

DMN

+

+

++

++

+

+

++

++

MitomycinC

+

Methylnitrosourea 8+,

sensitive;

++,

strongly sensitive; -, insensitive.


122


have the capacities both to activate and inactivate mutagens. In view of the activating process, original mutagens may be called promutagens. Pro muta gens can be converted to proximate mutagens and finally to ultimate muta gens (40), which readily react with DNA. A typical example of this metabolic pathway is as follows: AAF (pro mutagen) is converted to N acetoxy-AAF (ultimate mutagen) through N-hydroxy-AAF (proximate mutagen) (40). Pro mutagens, proximate mutagens, and ultimate mutagens correspond to pro carcinogens, proximate carcinogens, and ultimate car cinogens, respectively. The most widely used system for metabolic activation is the rat liver S-9 fraction supplemented with the cofactor NADP and glucose-6-phosphate (G6P). The S-9 fraction is the supernatant fraction of tissue homogenate obtained by centrifugation at 9000 X g for 10 min. Before the S-9 fraction from rats was prepared, the animals were usually treated with poly chlorinated biphenyl (PCB), phenobarbital (PB), or 3-methylcholanthrene (MC). PCB can induce microsomal activating enzymes for many different types of environmental mutagens and carcinogens. However, it is a most undesirable compound even for laboratory use, and fortunately a combina tion of .B -naphthoftavone and PB has been found to be as effective as PCB (41). Rat liver has been the most widely used source of the S-9 fraction, but the livers from other species of mammals, including humans have also been used as sources. The results of experiments using human liver S-9 fraction seem to be important in evaluating the risk of environmental mutagens and carcinogens to humans (42-45). The liquid method of activation involved incubation of the bacteria with S-9 mix and the test-substance and then washing the bacterial cells and counting revertants and surviving bacteria (46). In the plate method de scribed by Ames et al (3), the mixture of bacteria, S-9 mix, and test sub stance is added directly to molten-soft agar and poured onto hard-agar. This method is very simple and quick. In the method reported by Nagao et al (47), the mixture of bacteria, test substance, and S-9 mix were incubated for 20 min at 37° before adding molten-soft agar. With this preincubation method, DMN, a typical mutagen and carcinogen, gave positive results (15). A method involving in vivo metabolic activation of a drug was reported by Legator & Malling (48). The test substance was administered to animals by gastric tube or subcutaneous injection and bacteria were injected in traperitoneally and withdrawn after some hours. This method is convenient for investigating the mutagenicity of crude material containing suspected mutagens and carcinogens or of material that is difficult to sterilize.


123


In Vitro Transformation Test Using Cultured Mammalian Cells

Since the demonstration of in vitro transformation of cultured cells by carcinogens (20-24), the in vitro transformation test using cultured mam malian cells has been considered very useful for studies on the mechanism of carcinogenesis and also for detecting many environmental mutagens and carcinogens (49). The test system developed by Pienta et al (25) using cryopreserved embryonal Syrian hamster cells can be used with more com pounds than other systems. In this test, cells of the same lot, which did not yield transformed colonies under defined control conditions, were first se lected, and since many vials of cells of the same lot were prepared at the beginning, this test could be used to screen many environmental mutagens and carcinogens using cells with identical cellular characters (25). For metabolic activation, x-irradiated feeder layers of embryonal Syrian ham ster cells were used. Other Short- Term Test Systems for Detection of Environmental Mutagens and Carcinogens

Systems for mutations in cultured mammalian cells were well reviewed by Chu (50), Arlett (51), and O'Neill et al (52). Chromosome aberrations (53, 54) and sister chromatid exchange systems (55) have been described in dc;tail. Cytotoxicity tests using fibroblasts from normal subjects and patients with xeroderma pigmentosum have also been useful for detecting environ mental mutagens and carcinogens, as described by Stich et al (56) and Maher et al (57). Mutation tests using Drosophila (58) and silk worm (Bombyx mori) (59) are also well documented. The unscheduled DNA synthesis is also a good method for detecting environmental mutagens (60); the system of cultures of hepatic cells de scribed by Williams seems especially promising, because these cells contain metabolic activation systems (6 1 ). In Vivo Long- Term Animal Tests for Environmental Carcinogens

Although there are a tremendous number of reports on carcinogenesis experiments using rats and mice, most of the experiments do not fulfill the conditions necessary for quantitative evaluations, or the conditions de scribed in the guideline of the National Cancer Institute of the United States (26). For the past few decades, the experiments on carcinogenicity have been carried out on moderate or very strong carcinogens. But recently experi-

124


ments on weak or very weak carcinogens have become more important. The principal experimental conditions required are as follows: use of more than two species of animals of both sexes and use of more than two dose levels of the test substance.


NATURALLY OCCURRING AGENTS

Fungi Some fungi produce strong carcinogens or mutagens. Among these, afla toxin BI is the strongest mutagen (11, 62) and carcinogen (63, 64). Contami nation of foods, such as peanuts and grains, with aflatoxins has been reported (65, 66). Moreover fungi may grow and produce aflatoxins on corn damaged by the cornborer, while still in the field (67). Sterigmatocystin is also a very strong mutagen and carcinogen, and contamination of foods with sterigmatocystin has also been reported (68). Both compounds are bisfranoid derivatives, which are metabolically activated by a mammalian microsomal enzyme system to ultimate forms. DNA adducts of aflatoxin BI were prepared in the presence of a rat liver microsomal enzyme in vitro, and the structure of the main adduct was determined to be 2,3-dihydro-2(N7-guanyl)-3-hydroxyaflatoxin BI (69). The 2,3-epoxide of aflatoxin BI was deduced to be an ultimate form (70). The mutagenicity of aflatoxin BI was also observed with Saccharomyces cerevisiae (71), Neurospora (72, 73), Drosophila (58), and in mice in vivo (74). Aflatoxin BI induced 8azaguanine-resistant mutants and chromosome aberrations in mouse FM3A cells (75). Aflatoxin BI and B2 also induced cell transformation in Pienta's system (25). The specific mutagenic activities of aflatoxins de creased in order, BI > GI > B2, and this order coincided with the order of their carcinogenicities (63, 64). Versicolorin A, which was identified as an intermediate of aflatoxin bio synthesis in Aspergillus flavus and A. parasiticus, was also mutagenic and its specific activity on S. typhimurium TA98 was half that of sterigmatocys tin (76). Demethylsterigmatocystin, 5-methylsterigmatocystin, sterigmatin, and 6,8-dimethylversicolorin A were all strong, or fairly strong, mutagens on TAlOO and TA98 with metabolic activation (77). Demethylsterigmato cystin is a strong mutagen even without metabolic activation. The carcinogenicities of griseofulvin, patulin, penicillic acid, and ochratoxin A have been reported but no mutagenicity of these compounds was detected using S. typhimurium (71) or Saccharomyces cerevisiae (71). Veno & Kubota (78) reported that citrinin from Penicillium citrinum, luteoskyrin from P. islandicum, and zearalenone from Fusarium graminea-


1 25

rum gave positive results in the rec-assay using B. subtilis H17 (rec+) and M45 (rec-). Chaetoglobosin A obtained from Chaetomium globosum and PR-toxin from P. roqueforti showed weak but distinct mutagenicity with S. typhi murium TAIOO without metabolic activation (77).


Plants The plant kingdom is a major source of naturally occurring mutagens and carcinogens. Cycasin in cycad nuts is known to be a carcinogen (79). Cycasin, a glycoside of MAM was mutagenic to bacteria in the presence of glycosidase, inducing base-pair change mutation (80). Bracken fern, Pteris aquilina, eaten in Japan, also induced bladder tu mors in cows and tumors in the intestine and bladder of rats (81, 82). An acetone-ethanol extract of bracken was mutagenic to S. typhimurium with metabolic activation (83). Milk from cows fed bracken fern and urine from rats fed bracken fern were mutagenic to S. typhimurium TA100 without metabolic activation (84). Moreover, an ethanol or ethyl acetate extract of bracken fern was proved to damage the DNA in gastrointestinal epithelial cells in vitro (85). The active carcinogenic principle in bracken fern has been studied by many workers but has not yet been identified, and although it was reported to be shikimic acid (86), tumor formation with this compound was not reproducible (87) and its mutagenicity could not be demonstrated (16). Pyrrolizidine alkaloids are recognized as naturally occurring carcinogens and they have been found in some 50 species of the families Compositae, Boraginaceae, and Leguminosae (88), which were used as medicinal herbs. More than 1000 pyrrolozidine alkaloids have been isolated from plants and insects. The relationship between their structures and carcinogenicities has been reviewed by Clark (89), and Schoental (90), and in IARC Monographs (9 1 ). The following pyrrolizidine alkaloids were shown to be carcinogenic: jacobine, monocrotaline, riddelliine, and seneciphylline (9 1 ). The mutagen icities of pyrrolizidine alkaloids could not be detected by the S. typhi murium in vitro test, but could be detected in Drosophila (58, 92) and Aspergillus (93). Petasitenin, a pyrrolizidine alkaloid isolated from the flower stalks of coltsfoot, Petasitesjaponicus, used as a herbal remedy and food was recently proved to be hepatocarcinogenic to rats (94). The mutagenicity of coltsfoot was proved in S. typhimurium hisG46 by host-mediated assay by feeding coltsfoot-containing diet to mice (95). The DNA damaging activities of monocratalin and heliotrine were found in the presence of liver microsomal enzymes using a repair-deficient strain


1 26


of E coli (96). The active forms of this alkaloid were found to be pyrroles produced by the mixed function oxidases (97). Recently, many studies on the mutagenicities of flavonoids have been reported by Wang et al (98), Hardigree & Epler (99), Bieldanes & Chang (100), Brown et al (10 1 ), and Sugimura et al (102). Kaempferol, a flavonoid, is a constituent of bracken fern. Sugimura et al (102) demonstrated the mutagenicity of kaempferol while searching for the substance with carcino genic activity in bracken fern. Wang et al (98) also found that quercetin was mutagenic. There are many flavonoids in plants, and the mutagenicities of more than 70 naturally occurring flavonoids were tested (103). Of these, quercetin was the strongest mutagen, followed by kaempferol, rhamnetin, galangin, isorhamnetin, and fisetin (101-103). These mutagenic flavonoids have hydroxy groups at the 3 and 5 positions. All these compounds except quercetin required metabolic activation by rat liver enzymes, and the mutagenicity of quercetin was further enhanced by rat liver enzymes. S. typhimurium TA98 was more sensitive than TAl00 to all these flavonoids except woganin, which has no hydroxy group at the 3 position and which showed strong mutagenicity to TAloo with metabolic activation (103). Isoflavones and flavanones were either not mutagenic or only very weakly mutagenic (10 1 , 102). The highly mutagenic compounds quercetin and kaempferol are present in many edible plants, vegetables, and fruits (104106). Onion (Allium cepa) contains about 11-24 g of quercetin per kilogram fresh weight of outer epidermis (106). Astragalin and tiliroside, glycosides of kaempferol, and isoquercitrin, a glycoside of quercetin, were found in bracken fern (107-109). Flavonoids are mainly localized in the outer tis sues, skin, or peel of fruits, tubers, and roots. Rutin, a rutinoside of querce tin; astragalin; isaquercitrin; and tiliroside were all mutagenic to S. typhimurium (103) after treatment with a glycosidase, hesperidinase (110) obtained from Aspergill u s niger. A spice, sumac (genus Rhus), was recently found to be mutagenic and the active mutagenic substance in sumac was proved to be quercetin (11 1 ). Quercetin and quercitrin were not carcinogenic when fed to rats as 1% of the diet (112). Quercetin induced in vitro transformation in Pienta's system, but kaemp ferol did not induce transformation (1 1 3). Hydrazine was carcinogenic to mice on oral or intraperitoneal adminis tration and in rats on oral administration (114). Hydrazine was found in tobacco and tobacco smoke and one cigarette was reported to contain 30 ng of hydrazine (115). Hydrazine was mutagenic to S. typhimurium TA100 and TA1 535 (11). Edible mushrooms contain various hydrazine derivatives. Agaricus bis porus, the most commonly eaten commercial mushroom, contains agaritine



127

amounting to 0.04% of the fresh tissue weight (116). y-Glutamyltrans ferase, which is present in mushrooms, catalyzes the breakdown of agari tine to L-glutamate and 4-hydroxymethylhydrazine. N'-Acetyl-4-(hydroxy methyl)-phenylhydrazine increased the incidence of tumors of the lung and blood vessels when given to Swiss mice in their drinking water at a concen tration of 0.0625% throughout life ( 1 1 7). The estimated consumption of A. bisporus in the United States from July 1 972 to June 1 973 was estimated to be about 300 million Ib (118). Gyromitra esculenta, a wild edible mush room, contains ethylidene gyromitrin (acetaldehyde formylmethylhydra zone), N-methyl-N-formylhydrazine, and methylhydrazine (11 8). The latter two compounds seem to be produced from ethylidene gyromitrin by hydrolysis during cooking (119). and fresh mushrooms obtained from Eu ropean sources were estimated to contain 1 . 2-1.6 g per kg of ethylidine gyromitrin (118). Ethylidene gyromitrin was not mutagenic to E. coli WP2 but N-methylhydrazine was mutagenic ( 1 20). N-Methylhydrazine was also mutagenic to S. typhimurium hisG46 and TA92 and was carcinogenic in mice and hamsters (121, 1 22). Safrole, isosafrole, and dihydrosafrole were weakly carcinogenic (123). The mutagenicity of safrole could be detected in S. typhimurium TA1535 using the S-9 fraction of the liver of rats treated with safrole or the 105,000 X g supernatant of liver homogenate of rats treated with PCB (124). 1 '-Acetoxysafrole and 2',3'-epoxysafrole were mutagenic in Ames test with out metabolic activation (32). Some flavonoids and safrole are also known to enhance drug metabolism (124, 1 25). Sassafras oil contains up to 93% safrole, and the essential oils from nutmeg, mace, ginger, star anise, cinna mon, and black pepper contain 0.01 % to 10% safrole ( 1 23).

Fatty Acid Peroxides in Foods A high incidence of cancer has been observed in men who have a diet with a high content of polyunsaturated fat ( 1 26). When polyunsaturated fatty acids were oxidized, malonaldehyde was produced as a peroxidized decom position product. Malonaldehyde was mutagenic to S. typhimurium TA1978, a frameshift mutant with normal excision repair ( 1 27). However, 1 2-hydroxystearic acid, 8-undec-2-enolactone, y-decano lactone, 4-keto decanoic acid, and 1 2-oxooleic acid, which were reported to be carcinogenic (128), did not show mutagenicity on TA1 978, TA98, or TA100 (129). PYROLYSIS PRODUCTS

Cooking Products Charred parts of broiled fish or beef were shown to be mutagenic in S. typhimurium TA98 after metabolic activation with the S-9 fraction of rat


128


liver (130). BP has been found in broiled fish (131), and broiled meat (132), but the mutagenicities of these charred materials are too great to be ex plained by their contents of B P. Pyrolysis of protein produced strong frameshift mutagens that required metabolic activation by the S-9 fraction (133). Pyrolysates of amino acids showed various mutagenicities (134-136). Among these, the pyrolysate of tryptophan was the most mutagenic, followed by those of serine, glutamic acid, ornithine, and lysine. The active principles from tryptophan tar were purified and their struc tures were reported by Sugimura et al (137) as shown in Figure 1. After metabolic activation with liver S-9, 3-amino- l ,4-dimethyl-5H-pyrido [4,3-b]indole (Trp- P- l ) and 3-amino- l -methyl-5H-pyrido[4,3-b]indole (Trp-P-2) were the strongest mutagens so far tested on TA98 (138). Human S-9 also could activate these compounds (139). Trp- P- l (140) and Trp- P-2 (140, 141) were synthesized chemically. Trp- P- l and Trp- P-2 could trans form in vitro cultured cells on Pienta's system (142). Trp- P-2 could bind with DNA in vitro in the presence of rat liver microsomes (143). Active principles in a glutamic acid pyrolysate were purified and two crystalline compounds were obtained, whose structures were determined to be 2amino-6-methyldipyrido[1,2-a : 3',2'-d]imidazole (Glu-P-I) and 2-amino dipyrido[1,2-a: 3',2'-d]imidazole (Glu-P-2) (Figure 1) (144). Pyrolysates of glucose, arabinose, fructose, and sorbitol were all muta genic to TAlOO without metabolic activation (134). A pyrolysate of glucosamine was mutagenic in TA98 with metabolic activation, like the pyrolysates of amino acids (134). A pyrolysate of glucose contained ace-

Trp-P-I

Trp-P-2

Glu-P-I

Glu-P-2

Figure I

O:N� N�NH2 , �.,� N

Structures of potent mutagens newly found in pyrolysates of tryptophan and

glutamic acid.



129

taldehyde and glyoxal, which were mutagenic to TA1 00 (145), but the mutagenicity of the glucose pyrolysate was too great to be explained by the contents of those two compounds. The mutagenicity of a sugar pyrolysate was also observed by Setliff & Mower ( 1 46). Caramel, which is widely used as a food coloring and flavoring, was slightly, but definitely, mutagenic to TAlOO ( 1 47), but recently it was reported that caramel had no carcinogenic effect when fed to rats as 6% of the diet for 2 years ( 1 48). Pyrolysates of garlic and onion showed strong mutagenicity in TA100 and T A98 with metabolic activation. Garlic contained alliin and allicin, and onion contained allicin, and pyrolysates of pure alliin and allicin were also mutagenic (149). Edible weeds, which are usually eaten broiled, also pro duced mutagenic pyrolysis products (150). The mutagenic potentials pro duced by pyrolysis depend very much on the dryness of the food: A low water content favors the production of mutagens when food is broiled (151). Charcoal-broiled beef was not only mutagenic, but also active in inducing the drug-metabolizing enzymes. A diet containing charcoal-broiled beef stimulated phenacetin metabolism in the intestine of rats (152). Other stud ies showed that a charcoal-broiled diet stimulated B P hydroxylase activity in the placenta and liver of pregnant rats (153).

Tobacco Tar Epidemiologic data have indicated that cigarette smoking is one of the most important causes of human cancer, followed by dietary factors (154). More than 1 200 components of tobacco tar have been identified ( 1 5 5). A promot ing action of tobacco tar was also reported (156). In attempts to improve tobacco, a short-term carcinogenicty test, namely the Salmonella mutation test was carried out by Hutton & Hackney (43), K ier et al (157), Sato et al ( 1 58), and Mizusaki et al ( 1 59). Results showed that 1 00 fJ.g of tobacco tar has the same (43), or similar (95) mutagenic activity as 0.6 fJ.g BP. The tar from one cigarette, about 20 mg, contained approximately 20 ng of B P. The potent mutagenic activity in this amount of tar was too great to be explained by this amount of BP, and thus other compounds besides BP must contribute to the mutagenicity, as pointed out by Hutton & Hackney (43) and Sugimura et al (95). Mutagenic activities were observed mainly in basic and weakly acidic fractions. However, tumor-initiating activity, detected by painting the tar on mouse skin, was found mainly in the neutral fraction (160). This may be because aromatic amines, such as 2-aminoanthracene, have strong mutagenicity but relatively low carcinogenicity. Another possible reason is that, since the basic fraction is toxic, animal experiments cannot be made with a sufficient amount of basic fraction to detect carcinogenicity. Sato et ,

130



al (158) found that the specific mutagenic activities of tobacco tars from low- and high-tar cigarettes were similar. The mutagenicities of tobacco tars were correlated with the contents of proteinaceous nitrogen and total nitro gen in the leaves (161). Therefore, it is likely that the mutagenic principles in tobacco tar were produced mainly by pyrolysis of amino acids and proteins, as in the cases of beef and fish (95). Yamasaki & Ames demon strated that the urine of smokers showed mutagenicity (162). Aromatic Hydrocarbons

There are many reports of the presence of carcinogenic polyaromatic hydro carbons in our environment. For instance, some barnacles and oysters contain polyaromatic hydrocarbons depending upon their habitat; some pyrogenic materials, Japanese foods, whiskey, and beefsteak were reported to contain polyaromatic hydrocarbons (163). Comprehensive reviews on mutagenic and carcinogenic activity of BP and benzo(a)anthrance and their derivatives were published by Jerina et al (164) and Levin et al (165). NITROSATION PRODUCTS

Since the discovery of the acute hepatotoxicity and hepatocarcinogenicity of DMN by Magee ( 1 66), many carcinogenic nitroso compounds have been found. The carcinogenicities and mutagenicities of nitrosamines and ni trosamides were reviewed by Magee & Barnes (167), and by Druckrey et al (168). The mutagenicities of several N-nitroso compounds and their metabolites in S. typhimurium have also been reported (169-1 75). Carcinogenic N-nitroso compounds are produced by the reaction of ni trite with other compounds containing nitrogen, such as amines and amides, in acidic conditions in the stomach or in vitro. Amines and amides are derived from foods, drugs, and pesticide residues in foods, while nitrite is present in foods and water and is produced from nitrate in water and vegetables, and also in vivo. Malignant tumors were induced in rats fed nitrite with secondary amines ( 1 76). N-Nitroso compounds are suspected to be closely related to the development of human cancers ( 1 77). Thermal energy analysis is a specific and sensitive method for detection of N-nitroso compounds, and thermal energy analysis by both gas chroma tography and high pressure liquid chromatography can detect N-nitroso compounds at ppb levels in our environment (178). In this way DMN has been detected in urban air; moreover, several N-nitroso compounds were found in drinking water (178). The formation and analysis of environmental N-nitroso compounds were discussed at a working conference of the IARe ( 1 7 9). Reviews of nitrosation compounds are given in references ( 1 80-1 82).


131

SYNTHETIC CHEMICALS


Food Additives AF-2,2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide was widely used as a food preservative in Japan from 1965. Its use was approved for preserving fish meat cakes, fish and meat sausages, soybean curd, and red bean paste, because it had low toxicity and was claimed to be noncarcinogenic ( 1 83). However, induction of chromosome aberrations in cultured human lym phocytes by AF-2 was observed in 1972 (184), and later its potent mutagenicity was demonstrated by mutation induction in E. coli ( 1 85, 1 86) and by repair tests in E. coli and S. typhimurium (31 ). Although AF-2 gave positive results in repair tests using S. typhimurium TA1978/TA 1 538, it gave negative results in the reverse mutation test using the S. typhimurium TA 1535 series (31). Similar negative responses of S. typhimurium were obtained with 26 other nitrofuran derivatives, including feed additives and medicines, although 24 of the 26 nitrofuran derivatives gave positive results in both the E coli mutation test and repair test (31 ). New tester strains S. typhimurium TAIOO and TA98 that gave positive results with AF-2 and other nitrofuran derivatives ( 1 1 , 187) were promptly developed by Ames and his associates in 1974. In 1 974, it was found that AF-2 produced cancer of the forestomach in mice ( 1 88) and the use of AF-2 as a food preservative was immediately banned. The carcinogenicity of AF-2 was shown in mice ( 1 89-1 91 ), rats ( 1 92), and hamsters ( 1 9 1 ). The mutagenicity of nitrofuran derivatives was reviewed by Tazima et al ( 1 93). Saccharin was synthesized in 1879 and since then it has been widely used as an artificial sweetener. It was synthesized commercially by several meth ods, but the final products might have contained several types of impurities at various concentrations. Reports on the m utagenicity and carcinogenicity of saccharin are conflicting and equivocal, and these activities may be due to impurities. The mutagenicity of saccharin was reviewed by Kramers (194). The relationship of the mutagenicity of saccharin to its impurities was reported recently (195-197). The mutagenicity of urine from mice fed saccharin ( 1 97) might be explained by the formation of a mutagenic metabolite of saccharin, by the accumulation and condensation of impuri ties of saccharin in the urine, or by excretion of other mutagens, due to the modulation of normal metabolic and detoxicating pathways by administra tion of large amounts of saccharin. Cosmetics

Plant dyes, metallic dyes, and especially synthetic dyes have been used as hair dyes. Synthetic hair dyes composed of a variety of aromatic amino- and nitro-compounds can be divided into three groups: temporary, semiperma-


132


nent, and permanent. Azo- and diazo-dyes, anthraquinones, and azinic dyes were used as temporary dyes, and nitrophenylenediamines, amino-nitro phenols and amino-anthraquionones as semipermanent dyes. Both tempo rary and semipermanent dyes are called direct dyes and contain colored compounds. On the contrary, permanent dyes, which are oxidative dyes, contain colorless primary intermediates, such as p-phenylenediamine, p toluenediamine, p-aminodiphenylamine, p-aminophenol, o-aminophenol, and o-phenylenediamine, and couplers or modifiers, such as 2,4-diamino anisole, m-phenylenediamine, m -aminophenol, and resorcinol. These small molecular compounds readily penetrate the hairs where they form larger colored molecules by oxidation, coupling, or condensation reactions (198). Ames et al (199) tested 169 permanent hair dyes commercially available in the United States for mutagenicity on S. typhimurium TA 1538 and found that 150 of these (89%) were mutagenic. This was confirmed by Sugimura et al (15) who found that 146 of 169 (82%) commercial oxidative hair dyes in Japan were mutagenic on S. typhimurium TA1538. Some of the ingredients used in oxidative hair dyes were not only muta genic but also carcinogenic. 2,4-Diaminotoluene was used in hair dyes in the United States until 1971, when its use was banned because its car cinogenicity in rats had been reported in 1969 (200). This compound was used for synthesizing a variety of dyes, and was also used as a direct dye for dyeing leather and furs. It was also used as an intermediate in synthesis of toluene diisocyanate, which was used in the manufacture of polyure thane. With a metabolic activation, 2,4-diaminotoluene was mutagenic to S. typhimurium TA1538 (199) and TA98 (201, 202). This compound also induced transformation of cultured embryonic cells of Syrian hamster in Pienta's system (203). 2,4-Diaminoanisole (p-methoxy-m-phenylenediamine) was the most po tent mutagen among the ingredients of hair dyes tested in S. typhimurium TA 1538, and TA98 and was recently reported to be carcinogenic in rats and mice (204). This compound was weakly mutagenic, causing sex-linked recessive lethals of Drosophila melanogaster when administered orally (205). 2,5-Diaminotoluene and 2,5-diaminoanisole induced mutation in S. typhimurium TA1538 (199) and TA98 (201) with metabolic activation. Other ingredients, 0-, m-, and p-phenylenediamines were mutagenic on S. typhimurium TA1538 and TA98 with the metabolic activation system (199, 201, 202, 206). 2-Nitro-p- phenylenediamine and 4-nitro-o-phenylenediamine were mutagenic on S. typhimurium TA1538 (199, 206, 207) and TA98 (201, 202) without the metabolic activation system, and the latter compound induced sex-linked recessive lethals in Drosophila by feeding (205). These com-


133

pounds induced chromosome aberrations in cultured human lymphocytes (207) and in hamster cell lines (208-210). 2-Amino-4-nitrophenol, 2-amino5-nitrophenol, and 4-amino-2-nitrophenol induced mutations in S. typhi murium TA1538 or TA98 without metabolic activation (199, 201, 206).


Drugs

Iatrogenic cancers (211) have gradually increased and have become a major problem, as summarized in Table 2. Several antitumor agents are carcino genic and mutagenic. Alkylating agents, such as nitrogen mustard, uracil mustard, cyclophosphamide (Endoxan®), busulfan, melphalan, and thio TEPA are carcinogenic and mutagenic (33, 3 5). Antitumor antibiotics, such as adriamycin, daunomycin, mitomycin C, and actinomycin 0, are carcino genic and mutagenic (33, 35, 212), although bleomycin was found to be carcinogenic but not mutagenic in Salmonella (33, 35). Although daunomycin and adriamycin, anthracycline glycosides contain ing L-daunosamine, were highly mutagenic in S. typhimurium TA98, aclacinomycin A was nonmutagenic in S. typhimurium TA98 (2 l 3). Aclacinomycin A contained L-rhodosamine (dimethyl-L-daunosamine) and an additional disaccharide. The free amino group in the sugar moiety of anthracycline glycosides plays an important role in their mutagenicity (2 1 3). Intercalation of the anthracycline moiety between DNA base-pairs and the electrostatic interaction of the amino group with the phosphate group of DNA strands may be essential for the induction of mutation. Hycanthone, widely used for treatment of schistosomiasis in Africa, Brazil, and the Middle East, was carcinogenic and mutagenic in S. ty phimurium (214). Modification of the side-chain structure of hycanthone Table 2 Iatrogenic human carcinogens Cancer

Drugs Arsenic

skin cancer

Chloramphenicol

leukemia

Chlornaphazine Ibis (2-Chloroethyl)2-naphthylamine 1

bladder cancer

Cyclophosphamide

leukemia

Diphenylhydantoin

lymphoma

Imuran

reticulosarcoma

Liquid paraffin

stomach, colon, rectum

Melphalan Phenacetin

myeloid leukemia

Steroid contraceptives

hepatic hamartoma

Stilbestrol

breast, vaginal, and

Thorotrast

cervical adenocarcinomas liver hemangioendothelioma

cancers renal pelvis carcinoma


134


resulted in a marked reduction of mutagenic potency, while retaining anti schistosomal activity (215). These findings show that it is important and useful to use short-term mutagenicity tests during development of new drugs in order to obtain compounds with low mutagenicity and probably low carcinogenicity. Although phenacetin was carcinogenic, it was not mutagenic in S. ty phimurium TA100 or TA98, even in the presence of the 8-9 fraction of liver from rats treated with PCB. However, phenacetin was mutagenic in S. typhimurium TA100 in the presence of the S- 9 fraction from liver of ham sters treated with PCB (216). N-Hydroxyphenacetin was mutagenic to S. typhimurium TAloo in the presence of the S- 9 fraction of liver from rats or hamsters treated with PCB (216, 217). Imuran (azathioprine) was widely used as an immunodepressant in organ transplant patients, and an increased incidence of lymphomas among pa tients receiving this drug has been reported (218). Demonstration of the mutagenic activity of this drug in S. typhimurium TAloo required anaero bic incubation of the tester strain with this agent for 14 hr and then aerobic incubation for an additional 34 hr (219). Pesticides

Pesticides have potential carcinogenicity, teratogenicity, and mutagenicity. Continuous and extensive use of pesticides increases the chance of exposure of the general human population to pesticides as contaminants in foods, and the possibility of deleterious effects of pesticides on human health has been discussed. Evaluation of their safety is an urgent problem and microbial mutagenicity screening tests have also been carried out to predict their carcinogenicity. The mutagenicities of 193 pesticides, including 64 fungi cides, 60 insecticides, 67 herbicides, and 2 plant-growth regulators, were studied by rec-assay using B. subtilis rec+ and rec- (220) and by the mutation test using E coli WP2 and S. typhimurium TA1535, TA1537, TA1538, TA100, and TA98 (221, 222). On screening by rec-assay, 23 pesticides gave positive results. Among these, 12 fungicides, namely, Captafol, captan, Dexon® (DAPA), dichlo fluanid, N,N'-bis(dimethyldithiocarbamoyl) ethylenediamine (EMSC), ferbam, folpet, 2,4-dinitrophenyl thiocyanate (NBT), 5-nitro-I-naphtho nitrorile (NNN), bis(dimethylthiocarbamoyl)disulfide (TMTD), arsenic dimethyldithiocarbamate (TTCA) and ziram; 2 insecticides, namely di chlorovos (DDVP, vapona) and vamidothion; and 1 herbicide, N-(2-hy droxyethyl)hydrazine (HEH) were mutagenic in E. coli and S. typhi murium (222). Ethylenethiourea, a decomposition product and a metabo lite in animals of ethylene bis-dithiocarbamate fungicides, such as zineb, maneb, and nabam, was also mutagenic (222).



135

In screening tests using E. coli and S. typhimurium, carcinogenic pesti cides containing chloride, such as DDT, benzene hexachloride (BHC), aldrin, and dieldrin gave negative results (222), but some of these com pounds were mutagenic in Drosophila (223). The mutagenicities of I I organophosphorus insecticides, including di chlorovos, were reviewed by Wild (224). Among them, five insecticides, namely, Bidrin® (dicrotophos), dichlorovos, dimethoate, methyl parath ion, and oxydemetonmethyl (Meta-Systox R®) were mutagenic in bacteria and yeast; but three insecticides, diazinon, malathion, and parathion gave negative results in all tests on bacteria and yeast (224). Several pesticides were examined in our laboratory by the preincubation method using S. typhimurium TAIOO and TA98 with and without meta bolic activation (17, 147); captan, dichlorovos, TMTD, dimethoate, tri chlorfon (dipterex), and allethrin were mutagenic and diazinon, maneb, and tetrachloroisophthalonitrile were not. Kepone® (chlordecone) was an insecticide used against leaf-eating in sects, ants, and cockroaches. In 1975, the Environmental Protection Agency of the United States banned sales and use of the compound and prohibited further manufacture. This compound produced liver tumors in both rats and mice (225), but its mutagenicity has not yet been proved. Household Materials

tris(2,3'-Dibromopropyl) phosphate (Tris-BP) was widely used as a flame retardant additive for textiles, especially for children's sleepwear in the United States. This compound was mutagenic in S. typhimurium TA1535 and TAIOO without metabolic activation, and addition of the S-9 fraction from rat liver enhanced its mutagenic potency (226, 227). Dimethyl sulfox ide extracts of fabrics treated with this compound showed mutagenicity in S. typhimurium TA1535 (226). Although 2,3-dibromopropanol, 1,2,3-tri bromo propane, and 1,2-dibromo-3-chloropropane, which are impurities present in Tris-BP, were also mutagenic, they had lower mutagenic activi ties than that of Tris-BP and the impurities were not sufficient to account for the mutagenicity of Tris-BP (226, 227). tris( l -Chloromethyl-2-chloroethyl)phosphate and tris(2-chlorethyl) phosphate, which were used as flaine retardants in certain plastics, were not mutagenic in this assay system (226). tris(1,3-Dichloro-2-propyl)phosphate (Fyrol-FR2®), which is another flame retardant used as a replacement for Tris-BP and in children's sleep wear, was mutagenic in S. typhimurium TAloo with a metabolic activation (228). 1,3-Dichloropropanone, a possible metabolite of Fyrol-FR2, was a potent mutagen without metabolic activation. 1,3-Dichloro-2-propanol, an other expected metabolite, was mutagenic with a metabolic activation sys-

1 36



tern from the liver of mice and hamsters, but not rats, treated with PCB (228). N-Hydroxymethyl dimethyl phosphonopropionamide ( Pyrovatex Cp®), a flame retardant used in Japan, was also mutagenic in S typhi murium TAloo, but tetrakis(hydroxymethyl)phosphonium chloride was not ( 1 7). Tris-BP was recently shown to be carcinogenic (228). Sodium hypochlorite, NaOCl, widely used in homes for cleaning and disinfecting, was mutagenic in S typhimurium TA1 530 (229). Occupational Substances and Industrial Intermediates Chemicals producing cancer in man are summarized in Table 3 . The human carcinogens were mainly detected by their effects on man through occupa tional exposure or accidental contact (211, 230). Benzidine and 2-naphthylamine produced urinary bladder tumors in workers who were exposed to them, and their manufacture has been prohib ited in several countries. Benzidine was mutagenic in S typhimurium TA1538 and TA98, and 2-naphthylamine was m utagenic in TA1535 and TA 1 00, both in the presence of the S-9 fraction of rat liver (11). A variety of azo- and diazo-dyes were synthesized from benzidine, 2-naphthylamine, and their derivatives, and widely used in dyeing textiles, leathers, and Table 3 Human chemical cllIcinogensa Chemicals

Type of exposure

Acrylonitrile Aflatoxins 4-Aminobiphenyl Arsenic compound Asbestos (crocidolite, amosite, crysotile) Auramine Benzene Benzidine Cadmium oxide bis (2-Chloroethyl) sulfide (mustard gas) bis (Chloromethyl) ether Chloromethyl methyl ether Chromium compound Cigarette smoke Hematite 2 -Naphthylamine Nickel compounds Soots and tars Vinyl chloride

0 E

0 0 O, E

0 0 0 0 0 0 0 0 E

0 0 0 E, O 0

aType of exposure : E. environmental and others; O. occupational. gens to humans are listed in Table 2 .

Target organ(s) lung, large

intestine liver b ladder skin, lung lung, pleural cavity, gastrointestinal bladder bone marrow bladder

prostate lung lung lung

lung lung lung

bladder lung, nasal cavity skin, lUng liver, brain Iatrogenic carcino



1 37

papers. When benzidine diazo-dyes were administered to monkeys, carcino genic benzidine was liberated metabolically and excreted in the urine (23 1 ). The incidence of bladder tumors has increased among workers painting silk kimonos who accidentally ingest these azo- and diazo-dyes by licking their brush to moisten or point it (232). Several of these azo- and diazo-dyes were mutagenic in S. typhimurium TA loo and TA9S with metabolic reduction by azo-reductase, and by metabolic activation with the S-9 fraction of rat liver (95). The mutagenicities of several azo-dyes were also examined by the Salmonella mutation test (233, 234). Vinyl chloride was used in production of vinyl chloride polymer. It was also used as an aerosol propellant. Its use was banned in 1 974. Angiosar coma of the liver, a rare type of liver cancer in humans, and lung and brain cancer were induced in workers in vinyl chloride-polyvinyl chloride produc tion plants (235, 236). Vinyl chloride was carcinogenic in rats and mice (237, 238). Residual contamination of polymer plastics with vinyl chloride monomer would result in exposure of the general population. The mutagenicity of vinyl chloride was reviewed by Bartsch & Mon tesano (239) and Fishbein (240). Vinyl cholride induced mutation in S. typhimurium hisG46, TA 1 530, TA 1 535, and TA loo when the tester strains were exposed to its vapor with metabolic activation, but it was not muta genic when tested in aqueous solution (239, 241 ). It induced mutation in Salmonella without a metabolic activation system, but the presence of a metabolic activation system enhanced the mutagenic response. Vinyl chloride was mutagenic in E. coli K 1 2 (242), in Schizosac charomyces pombe, and in Saccharomyces cerevisiae (243). It was not muta genic in Neurospora crassa in the presence or absence of a metabolic activation system (244). The rates of chromosome aberrations were in creased in cultured blood lymphocytes from workers occupationally ex posed to vinyl chloride (245, 246). The fetal death rate was increased in women whose husbands were exposed to vinyl chloride (247). Acrylonitrile (cyanoethylene) was used in the manufacture of acrylic fibers, acrylonitrile-butadiene-styrene and styrene-acrylonitrile copolymers, and as a chemical intermediate in the synthesis of antioxidants, phar maceutical compounds, dyes, and surface-active agents. Preliminary epidemiological studies showed an increased incidence of cancers of the lung and large intestine of workers exposed to acrylonitrile in a textile fibers plant (248). Acrylonitrile was mutagenic to S. typhimurium TA 1 535 and TA 1 538 (249, 250) and in E. coli WP2, WP2 uvrA, and WP2uvrApoiA (25 1). The carcinogenicity of asbestos has been reviewed in IARe Monographs (252, 253) and by Selikoff (254). The incidence of lung cancer and pleural and peritoneal mesotheliomas increased among workers exposed occupa-


13 8

NAGAO. SUGIMURA & MATSUSHIMA

tionally to asbestos. and abnormally high incidences of cancers of the gastrointestinal tract and larynx were also observed in these workers. Meso thelioma were found amoung people living in the neighborhood of asbestos factories and mines and among families of asbestos workers. The general population may be exposed to asbestos in the air, drinking water, beverages, foods, and cosmetics. Asbestos was carcinogenic in mice, rats, hamsters, and rabbits (253). Asbestos and glass fibers were not mutagenic to E. coli and S. ty phimurium (255) but induced chromosome aberrations in cultured Chinese hamster cells (256, 257). Chromosome aberrations by asbestos were proba bly caused by physical factors. 2.3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was a by-product pro duced during the synthesis of 2,4,5-trichlorophenol (TCP), which was an intermediate in the manufacture of hexachlorophene and a herbicide, 2,4,5T (2,4, 5-trichloro phenoxyacetic acid). An explosion of a TCP-producing factory in northern Italy resulted in the TCDD-contamination of a large populated area of Seveso in 1 976 (258). TCDD was very toxic, mutagenic, and teratogenic, but its carcinogenicity was not adequately tested. It was reviewed in the IARC Monograph (259). Trichloroethylene, widely used as an industrial solvent, was reported to be carcinogenic in mice (260). Information on carcinogenicity of trichloro ethylene is also available (261). Mutagenicity of dichloroethylene and tri chloroethylene was reported (242). Dichloroethylene was used also as an industrial solvent. Chloroform, used in the manufacture of fluorocarbons for refrigerators, aerosol propellants, and plastics produced hepatocar cinoma in mice (262). POLLUTANTS

Air Particulate air pollutants collected on a glass fiber filter and extracted with an organic solvent were mutagenic and carcinogenic. Airborne particulate materials collected at six different points in industrial areas of Ohmuta and residential areas of Fukuoka in Japan were all mutagenic in S. typhimurium TA 98 and TA1538 with metabolic activation (263). Airborne particulate pollutants collected in Buffalo, New York, contained both direct acting mutagens and mutagens that required metabolic activation, whereas those collected in Berkeley, California, contained only direct acting mutagens on S. typhimurium (264). Airborne pollutants collected in Kobe. Japan (265), and in Duisburg, an industrial city in West Germany (266), were also mutagenic in S. typhimurium. The carcinogenicities of organic fractions of particulate pollutants collected in New York City were tested by subcutane ous injection into infant mice (267).


1 39


Water

A wide variety of organic contaminants, including polycyclic hydrocarbons and organochlorine pesticides, have been identified in municipal drinking water. Drinking water is usually treated with chlorine to free it from mi crobial contamination. However, chlorine treatment produces organochlo rine compounds (268), such as chloroform, which is a carcinogen. The relationship between drinking water supply from the Mississippi River and the cancer mortality rate in Louisiana was statistically significant, suggest ing that some organic contaminants in the water may be causative agents of cancer (269, 270). The carcinogenicities of 1 6 organic contaminants identified in samples of drinking water in the United States were tested by the pulmonary tumor induction test in strain A mice (271). Bromoform (tribromomethane) was carcinogenic and dichloromethane was a possible carcinogen, which requires further investigation (271). Sodium hypochlor ite, present in chlorinated water, was mutagenic to S. typhimurium TA1530 (229). The geographic distribution of gastric cancers in Colombia suggested their relation with the water supply, but arsenic, sulfur, and nitrosamines in the wat�r were not responsible for the gastric cancer. The nitrate contents of water samples from dug wells in a high risk area were higher than those of samples from a low-risk area ( 1 82, 272). Nitrosamines synthesized in the stomach from dietary amines and nitrite, formed by reduction of nitrate, may be a cause of gastric cancers. FACTORS MODIFYING MUTAGENESIS AND CARCINOGENESIS

A comutagenic effect was recently found on a bacterial mutagenesis system. On pyrolysis of tryptophan, large amounts of the ,a -carboline compounds norharman and harman are produced, and these compounds are also present in tobacco tar at 1000 times the concentration of B P ( 1 59). These compounds were not themselves mutagenic to S. typhimurium, but they enhanced the mutagenicities of the carcinogenic mutagens, B P (273), AAF ( 1 34, 273, 274), and N-dimethylaminoazobenzene (DAB) (273). DAB is a fairly strong hepatocarcinogen but is only weakly mutagenic (275). How ever, in the presence of norharman and the S-9 fraction from rat liver it was strongly mutagenic in S. typhimurium TA98 (273, 276). The mutagenicities of the carcinogenic azo-dyes, N-methylaminoazobenzene, 3'-methyl-DAB, and aminoazobenzene were also enhanced by norharman (276). However, 2-methyl-DAB, which was not carcinogenic on administration to normal animals but was carcinogenic on administration to hepatectomized animals, did not show enhanced mutagenicity with norharman (276).


140


Aniline, which is a reduction product of DAB, also exhibited mutagen icity only in the presence of norharman and rat liver S-9 fraction (278). o-Toluidine, which induced benign papillary tumors in the bladder of rats (279), was not mutagenic alone, but exhibited mutagenicity in the presence of norharman, an aniline did. However, m -and p -toluidines were not muta genic at all even with norharman (278). N-Hydroxy-AAF, N-acetoxy-MAB, and 4'-methyoxycarbonyl-N-hy droxy-MAB were mutagenic to S. typhim urium TAloo and TA98 without metabolic activation. Their mutagenicities were not enhanced by norhar man alone, but were enhanced by norharman with rat liver S-9 fraction (274, 276). However, the mutagenicity of the ultimate form of AAF, N acetoxy-AAF, was enhanced by norharman and harman even without rat liver S-9 fraction (274). The mechanism of the comutagenic action of norharman has not yet been elucidated, but two interesting effects of norharman have been found that are probably related to its action. One is the intercalation of norharman and harman into DNA (280). The comutagenic effects of norharman with vari ous azo dyes, aniline, and a-toluidine were observed only in T A98, a frame shift mutant. Therefore the intercalation of norharman and harman into DNA may be partly responsible for their comutagenic action. The other interesting effect of norharman is on metabolism. With small amounts of rat liver enzymes, norharman inhibited the mutagenicity of B P, whereas with large amounts, norharman enhanced it. It was found that norharman inhibited the metabolism of B P. B P is known to be metabolized to water insoluble metabilites and then to water-soluble ones. Norharman was found to inhibit both steps, but especially the second one; that is, it caused accu mulation of water-insoluble metabolites, such as 7,8-dihydrodiol and 3hydroxy B Ps. Thus the accumulations of 7,8-dihydrodiol B P may be a reason for the comutagenic action of norharman with B P (281). Levitt et al (282) reported that norharman inhibited B P metabolism and suppressed the mutagenicity of B P observed in the presence of MC-induced mouse liver S-9. The comutagenic effect of norharman with aniline was as strong in TA1 538 (uvr-) as in TA98 (uvc -,R+), but was weaker in TA1978 (uvr+). Therefore, the damage induced by aniline plus norharman could be repaired by the uvr gene product, and the presence of the R factor was not required. Since the mutagenic activities of aniline and o -toluidine were newly demon strated with norharman, it is important to elucidate the cocarcinogenic action of norharman as an environmental factor. Cocarcinogenic phenom ena, which were discovered before comutagenic phenomena, were reviewed recently by Van Duuren (283). Pyrene, benzo(e)pyrene, catechol, pyrogal lol, fluoranthene, and linolyl oleate were reported to be cocarcinogens.



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12-0-Tetradecanoyl-phorbol-13-acetate (T PA) is an active tumor-pro moting principle in croton oil. T PA caused a two-to-three-fold increase in the mutagenicity of UV light in Salmonella (284). Anthralin, phenol, n dodecane, lauric acid, methyl-12-oxo- trans - lO-octadecanoate, and methyl hydroxy-octadecadienoate also had tumor-promoting activities (283). The tumor-promoting activities of cigarette smoke condensate in mouse skin and lime oil in mouse forestomach have been reported (283). T PA was recently found to enhance the spontaneous mutations and mutagenicity induced by UV-light in cultured Chinese hamster cells (285). Protease plays an important role in the process of carcinogenesis (286), and a protease inhibitor blocked tumor formation (287). Actinomycetes produce several protease inhibitors, such as leupeptin, antipain, chymosta tin pepstatin, and elastatinal (288). The leupeptin was found to inhibit carcinogenesis by dimethyl benz(a)anthracene (DMBA) and croton oil in mouse skin (289). In E. coli, mutation, phage induction, and filamentous growth depend on an inducible system, SOS function (290). Because protease is concerned with induction of SOS function (291), protease inhibitors affect the SOS function in bacteria. Antipain inhibited the mutagenesis induced by UV light or tif (292) in E. coli. Phage induction and filamentous growth in duced by tif were also inhibited by antipain (292) and elastatinal (293). Elastatinal inhibited the mutagenic actions of MNNG and N-ethyl-N' nitro-N-nitrosoguanidine (ENNG) in S. typhimurium (294). It was recently reported that extracts of some vegetables could inactivate the mutagens produced by pyrolysis of amino acids. Extracts of cabbage, turnip, and ginger decreased the mutagenicity of trytophan pyrolysis prod ucts. The inactivating factor in cabbage was purified and identified as a heat-labile high molecular weight protein (295, 2 96). Use of the antioxidants, butylated hydroxytoluene (BHT), and butylated hydroxyanisole (BHA) as food additives is permissible. BHT was reported to inhibit induction of hepatomas by 2-AAF (297). BHT also decreased chromosome aberrations induced by sodium cyclamate or DMBA in hu man leukocytes in vitro (298). BHT and BRA were not mutagenic in S. typhimurium but gave potitive results in B. subtilis rec-assay (147). Fur thermore, BHT has been reported to behave as a promoter of squamous cell carcinoma of the forestomach induced by DEN (299) and liver tumors induced by AAF (300). The antitumor activities of vitamin A and its synthetic analogs (retinoids) were reported. The mutagenicity of retinol acetate on S. typhimurium TAloo with or without S-9 fraction was found recently (147). Retinoids were reported to prevent mammary carcinomas induced by DMBA and bladder carcinomas induced by NMU (302, 303), but at high doses natural ,

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retinoids were toxic. Synthetic retinyl methyl ether was superior in many respects to vitamin A or vitamin A acetate (301), and l 3-cis-retinoic acid effectively inhibited induction of bladder cancer by NMU without any evident toxicity (303).


EVALUATION AND QUANTITATION OF DATA

The expression of a mutagenic potential under certain defined conditions seems to be very reproducible and quantitative. The mutagenic potential can thus be calculated from the number of revertants measured in the linear region of the dose-response curve and can be expressed as the number of revertants per nmole or p.g per plate (11). However, many factors affect expression of the mutagenic potential. The amount of S-9 added has a marked effect on the number of revertants: For instance, with 10, 50, and 150 p. l of S-9, the mutagenic potentials of BP in TA I00 were 812, 1440, and 760, respectively, when experiments were carried out with 5 p.g of BP. Moreover, the source of the S-9 fraction affects the results, values being different with S-9 fraction from different species organs, sexes, and inducers. Provided that mutagenesis is measured in vitro with S-9 fraction from the liver of rats treated with some inducers, it should be possible eventually to standardize the experimental conditions and to express the mutagenic po tentials of chemicals quantitatively. However, before the procedure is finally standardized, more studies are required on factors that modify mutagenesis. However, it should be mentioned that the mutagenic potential, expressed as revertants per nmole, varies widely in the range of 10-3 to 105: Aflatoxin B1 can produce 27,500 revertants per microgram, whereas isonicotinic acid hydrazide produces only 0.0039 revertants per microgram. Because the specific mutagenic potential varies so widely, the specific activity should be expressed in a logarithmic scale. The variations in the mutagenic potentials of mutagens under different experimental conditions are not nearly so great as this, and thus are not really so serious. Figure 2 shows a histogram of the specific activities of various environmental mutagens. The samples were selected at random. The peak of the distribution frequency of mutagens was observed with moderately mutagenic compounds, such as naturally occur ring flavonoids. It should be pointed out that this mutagenic potential is based entirely on results obtained in vitro with S. typhimurium and a microsomal activation system, and it is hard to extrapolate these data to effects on germ cells, especially of human. There is still no direct evidence of the roles of environmental mutagens in producing an increase in the . mutation of human germ cells. With respect to the growing number of substances with mutagenic activ ity found in the past few years, Sobels pointed out the importance of four phases in the evaluation of the effects of mutagens: (a) primary identifica-


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