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Three new rat cell lines (designated as BP13, BP30 and BP36B), derived from rat basophilic-type renal cell carcinomas induced with N-ethyl-N-hydroxyethylnitrosamine, were established and characterized. Passaged up to 100 times in vitro for 3 years, each cell line forms epithelial monolayers with cell cycles for BP13, BP30 and BP36B of 29, 21 and 17 h, respectively. Positive glucose-6-phosphate dehydrogenase (G6PD) and gamma-glutamyltransferase (gamma-GT) activity in their cytoplasm, but negative succinate dehydrogenase (SD) and slightly positive carbonic anhydrase type II (CA) localization indicates an origin from proximal tubules. Ultrastructural examination showed the presence of variable numbers of mitochondria and many microvilli and intracellular junctions on the plasma membrane. BP13 and BP30 were found to be tetraploid and BP36B diploid. BP13 has one marker chromosome 15p+, and BP36B an isochromosome of 1q. Anchorage-independent growth and tumorigenicity in immunosuppressed nude mice of BP13 and BP36B, but not BP30, proved their neoplastic nature. These three cell lines should provide useful tools for studying the biological characteristics of renal cell tumors.

We conducted a collaborative validation study, under the auspices of the Japanese Ministry of Labour, on the in vitro micronucleus test to see if it could be used as an alternative to the in vitro chromosome aberration test for evaluation of chemical safety. We used the Chinese hamster lung cell line (CHL/IU), which is the most widely used system for the latter test in Japan, and evaluated 66 chemicals, including clastogens and polyploidy inducers. The cytochalasin B cytokinesis blocking method, which is commonly used in human lymphocyte culture, was applied to the established cell line, but did not improve the detection of chemically-induced micronuclei in continuously growing cells. The highest micronucleus frequencies were obtained at 48 or 72 h continuous treatments. In short treatments (6 h), a 42 h recovery time yielded the best responses. Concordance between the results of the micronucleus test and the chromosomal aberration test was satisfactorily high (88.7%), and we concluded that the in vitro micronucleus test could be used in place of the chromosomal aberration test as a simple and rapid method for detecting clastogens and aneugens in vitro. We also propose a protocol for the test.

BACKGROUND A recently developed laser scanning cytometry technique was applied to cytometric studies to detect rapidly stable chromosomal aberrations induced by a carcinogen in a Chinese hamster fibroblast cell line, CHO-WBLT. METHODS Individual chromosomes were collected from metaphase cells by a syringe technique and spread on slides. The DNA content of each chromosome stained with propidium iodide was measured with a laser scanning cytometer (LSC). A characteristic DNA histogram, designated as the "laser scanning karyotype (LSK)," was obtained from about 20,000 chromosomes of CHO-WBLT cells. Each chromosome was confirmed morphologically under the microscope by using a "re-location" system built into the LSC. RESULTS A total of 21 chromosomes, including marker chromosomes specific to the cell line, were assigned to 10 major peaks in the LSK, which was analogous to the karyotype demonstrated with the classical Q-banding technique. In contrast, clonal sublines isolated after exposure to the carcinogen benzo[a]pyrene showed LSKs different from those found in untreated control cells, and seven of 20 clones were found to be abnormal, with a small number of chromosomal translocations and/or deletions, which were confirmed by Q-banding. CONCLUSIONS The laser scanning cytometry technique was employed to detect stable chromosomal aberrations in CHO-WBLT cells after treatment with benzo[a]pyrene. The results obtained with this technique were comparable to those obtained by Q-banding; therefore, this method may be useful for rapid primary screening to detect stable, abnormal karyotypes induced by environmental chemicals and/or radiation.

Different test results have been reported for the same chemicals in two in vitro chromosome aberration test systems, CHL cells tested by a Japanese protocol and CHO cells tested by the US National Toxicology Program [Sofuni et al., Mutat Res 241:173-213,1990]. Here, laboratories in Japan, the US and the UK tested 9 such chemicals in CHL and CHO cells using the same protocols and found all 9 positive in both cell types; differences in earlier conclusions with these chemicals were due mainly to test protocol, not to different sensitivities of the cells. The most important protocol difference is sampling time. Chemicals that were negative in the NTP series using a sampling time of 10 to 13 hours often produced positive results when retested here with a 20- to 24-hour sampling time. While positive results were obtained in both cell types, CHL cells sometimes had higher aberration levels and survived at higher doses than CHO cells would tolerate. This may reflect some intrinsic difference in sensitivity but may also be affected by factors such as cell cycle length and culture media (e.g., oxygen scavenging capacity). The collaboration reported here also contributed to a better understanding of scoring aberrations, especially "gaps"; there was good agreement on what types of aberrations should be included in the totals when scoring criteria were clearly defined, for example, many changes classified as "gaps" by the Japanese system were classified as "breaks" in the scoring systems used in the United States and the United Kingdom, and were appropriately included in total aberration counts.

In a collaborative study organized under the JEMS MMS, nine mouse lymphoma assay (MLA) "unique positive' NTP rodent carcinogens were re-evaluated by an in vitro chromosomal aberration assay using Chinese hamster lung fibroblast cells (CHL/IU). Six of nine chemicals induced chromosomal aberrations; bromodichloromethane, chlorendic acid and isophorone induced structural aberrations, and chlorodibromomethane, pentachloroethane and 1,1,1,2-tetrachloroethane induced numerical aberrations (polyploidy). These six chemicals, therefore, are not uniquely positive in the MLA. The difference between the NTP results and ours might be due to the use of different cell lines and protocols, and in some cases, to different interpretations of polyploidy. The remaining three chemicals, benzyl acetate, cinnamyl anthranilate and trichloroethylene, were negative in this study.

2-Amino-1-methyl-6-phenylimidazo[4,5-beta]pyridine (PhIP) induced structural chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) in human lymphocytes and human diploid fibroblasts (TIG-7) at concentrations above 12.5 microgram /ml in the presence of rat S9 mix. PhIP also elevated the frequencies of SCEs in human lymphocytes in the presence of rat S9 at concentrations above 2.0 microgram/ml with dose-dependency. A proximate form of metabolites of PhIP, 2-hydroxy-amino-1-methyl-6-phenylimidazo[4,5-beta]pyridine (N-OH-PhIP), caused CAs in human and Chinese hamster fibroblast cells in the absence of S9 mix at concentrations above 0.75 microgram/ml and 1.25 microgram/ml, respectively, which were 10 times lower than the effective concentration of PhIP. No marked differences were observed in the cytogenetic sensitivity to N-OH-PhIP between human and Chinese hamster cells, except between lymphocytes obtained from different donors.

Stevioside, a constituent of Stevia rebaudiana, is commonly used as a non-caloric sugar substitute in Japan. The genetic toxicities of stevioside and its aglycone, steviol, were examined with seven mutagenicity tests using bacteria (reverse mutation assay, forward mutation assay, umu test and rec assay), cultured mammalian cells (chromosomal aberration test and gene mutation assay) and mice (micronucleus test). Stevioside was not mutagenic in any of the assays examined. The aglycone, steviol, however, produced dose-related positive responses in some mutagenicity tests, i.e. the forward mutation assay using Salmonella typhimurium TM677, the chromosomal aberration test using Chinese hamster lung fibroblast cell line (CHL) and the gene mutation assay using CHL. Metabolic activation systems containing 9000 g supernatant fraction (S9) of liver homogenates prepared from polychlorinated biphenyl or phenobarbital plus 5,6-benzoflavone-pretreated rats were required for mutagenesis and clastogenesis. Steviol was weakly positive in the umu test using S.typhimurium TA1535/pSK1002 either with or without the metabolic activation system. Steviol, even in the presence of the S9 activation system, was negative in other assays, i.e. the reverse mutation assays using S.typhimurium TA97, TA98, TA100, TA102, TA104, TA1535, TA1537 and Escherichia coli WP2 uvrA/pKM101 and the rec-assay using Bacillus subtilis. Steviol was negative in the mouse micronucleus test. The genotoxic risk of steviol to humans is discussed.

The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (473) for easy reference. Metabolic activation. S9 from animals induced either with Aroclor 1254 or with the combination of phenobarbital with beta-naphthoflavone is acceptable, and other systems could be used with suitable justification. Exposure concentrations. The upper limit of testing should be 10 mM (or 5 mg/ml where molecular weight is not known or mixtures are being tested), whichever is lower. Where this limit is inappropriate the investigator should give detailed justification of the choice of top concentration. Cytotoxicity should be measured not only in range-finding tests but also concurrently with the assay for chromosomal aberrations. Cytotoxicity should be assessed by measurements of cell growth such as cell counts or confluence estimation. Mitotic index data alone are not a sufficient measure of cytotoxicity, except in the case of blood cultures for which other methods are impractical. Cytotoxicity at the top dose should be greater than 50% of concurrent negative/solvent controls, if this can be achieved without exceeding a concentration limit of 10 mM or 5 mg/ml. There should be at least three concentrations scored for aberrations (each with and without S9), covering a toxicity range down to a concentration giving little or no cytotoxicity. This will usually mean that the concentrations scored will be quite closely spaced. It was not possible to reach a consensus on the issue of solubility limits. The group did not agree on whether (a) solubility rather than cytotoxicity should be the limiting factor, such that only one top dose with evident precipitate should be scored even if toxicity is not observed, or (b) several concentrations with evident precipitate should be scored for aberrations if this were necessary to obtain cytotoxicity. It was agreed that evidence of precipitation should be determined in the final culture medium. Controls. Concurrent positive controls are required but the working group thought it inappropriate to specify the control chemicals or the degree of response that should be obtained, leaving it up to the test laboratory to demonstrate that the system was working adequately based on historical data within the laboratory. It is not necessary to include both negative and solvent controls concurrently with the aberration test; solvent controls alone are acceptable provided that the laboratory has data to demonstrate that there is no effect of the solvent on baseline values. Preparation of cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

At present, the micronucleus test is the most popular short-term assay for the in vivo detection of clastogens or spindle poisons. As micronuclei are rare events, it has been argued that the conventional microscopical analysis based on 500-2000 cells per animal does not provide enough sensitivity. In addition, the manual scoring of micronuclei is time-consuming and tedious. As an attempt to solve these problems, an automated method for the analysis of micronuclei in mouse bone marrow erythrocytes was developed using flow cytometry. Femoral bone marrow cells, from mice treated with known in vivo clastogens [mitomycin C (MMC): 0.5, 1 or 2 mg/kg body weight intraperitoneally; benzene: 1000, 2000 or 4000 mg/kg body weight orally] or vehicles for the clastogens (physiological saline, olive oil; for control animals), were fixed with 1% glutaraldehyde, suspended in 70% ethanol for storing, stained with 4',6-diamidino-2-phenylindole (0.5 microgram/ml) and analyzed (50,000 cells per sample) in an EPICS V flow cytometer using a Coherent 5W UV laser at 150-200 mV. The frequency of micronucleated erythrocytes (MNEs) was calculated from the cytometer, by a computer program involving model fitting to normal Gaussian distribution. Correlations between MNE frequencies obtained by manual microscopic observations (1000-2000 cells per animal) and by the flow cytometric measurements were good for both group results (means of 4-5 mice; linear correlation coefficient r = 0.89-0.998) and individual data (r = 0.82-0.96). Repeated experiments with MMC showed good reproducibility for the flow cytometric scoring of micronuclei.

The computerized fact database for the toxicity data of chemicals was constructed at the National Institute of Hygienic Sciences, Tokyo, Japan (biological database, BL-DB). The BL-DB stores data on mutagenicity, teratogenicity, carcinogenicity, and other toxicological tests of chemicals that appeared in the scientific literature. The BL-DB includes information about chemical identification, test system, results of the assays, and a bibliography. The system consists of five modules: data collection, data maintenance, data search, data downloading, and backup. ADABAS is used as a core database management system. Many kinds of test data are stored with the same formats; therefore, users can retrieve data of different toxicological data by the same manner. A user of the BL-DB can use about 50 kinds of commands to interact with the system, and the majority of fields are defined as search fields, thereby facilitating retrieval of target data through many ways. Currently, there are mainly data for the mutagenicity, especially on the Salmonella/microsome assay and the rodent micronucleus assay. These data can be retrieved and used for structure-activity relationship studies.

Smoke condensates of woods used for food preservation and aromatization in Nigeria were tested for mutagenic activity using Salmonella typhimurium TA98 and TA100. The woods were: white mangrove ( A vicennia nitida ), red mangrove ( Rhizophora racemosa ), mahogany Khaya sp.), abura ( Mitragyna ciliata ), alstonia ( Alstonia boonei and black afara ( Terminalia ivorensis ). Cigarette tar was tested for comparison. The condensates induced dose-dependent increases in the number of His + revertants mainly with S9 mix. With the exception of mahogany and cigarette smoke condensate, the smoke condensates induced more revertants/μg condensate in TA100 than in TA98. The number of revertants/μg condensate ranged between 0.04 and 0.9 for the wood smoke condensates and was 0.12 for the cigarette smoke in TA100. The range was between 0.1 and 0.30 for the wood smoke condensates and 0.18 revertants/μg condensate for cigarette smoke condensate in TA98. Concentrations of 7 polycylic aromatic hydrocarbons (PAHs) in the condensates were determined namely, pyrene, benzo[ a ]pyrene, benzo[ a ]anthracene, benzo[ k ]fluoranthene, benzo[ b ]chrysene, benzo[ g , hi ]perylene and dibenzo[ a , e ]pyrene. The condensates contained varying concentrations of the individual PAHs and those with higher concentrations generally showed greater mutagenic activities. However, the order of mutagenic potency in the bacterial strains differed from the order of PAH concentrations, which were lower than the concentrations at which they are reported to induce mutations. When 6 of the PAHs were mixed in the concentrations in which they were found in the individual condensates, the mixtures did not induce mutation so that the contribution of the PAHs to the mutagenic activities of the condensates could hot be determined.

Two in vitro tests with different genetic end points, gene mutation and chromosomal damage, and an in vivo test, preferably the micronucleus test in mice, were recommended as a battery system for the primary assessment of genotoxic effects of chemicals. From our comparative studies on the mutagenic potency of chemicals, it was pointed out that results should be evaluated quantitatively rather than qualitatively, since the potency varies extensively, at range of 10(7) fold, among different chemicals, and the in vitro genotoxins relatively weak tend to be negative in in vivo mutagenicity tests as well as in carcinogenicity tests in rodents. New Salmonella tester strains, called YG-series, were established, which showed a high nitroreductase or acetyltransferase activity and specifically sensitive to nitroarens or aromatic anmines in the reverse mutation assays (Ames test). These strains could detect a small amount of mutagenic aromatic amines containing in the urine of cigarette smokers. A new technique in the micronucleus test using peripheral blood erythrocytes was introduced. A cumurative genotoxic effect of benzene, for an example, was detected in the peripheral blood even several weeks after treatment every week by gavage. A cyto-flowmetric analysis can be also applied to monitoring of such effects.

Ultra-vital staining with acridine orange (AO) is introduced into the micronucleus assay with mouse peripheral blood cells. Peripheral blood was stained vitally by dropping whole blood on an AO-coated slide and covering the sample with a coverslip. With this method, reticulocytes are identified easily by their red fluorescing reticulum structure. The distinction between young and mature erythrocytes was clearer and less subjective than the distinction between polychromatic and normochromatic erythrocytes by Giemsa staining or by conventional AO fluorescent staining. Although the induction of micronucleated peripheral reticulocytes (MNRETs) was delayed by about 12 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optimal sampling time. It is concluded that bone marrow cells can be replaced by peripheral blood as material for the micronucleus assay.

We have previously demonstrated that three potent iron chelators, hinokitiol, dithizone and deferoxamine, induce differentiation of F9 embryonal carcinoma cells, as do other well-known morphogens such as retinoic acid (RA) and sodium butyrate (NaB). In this study, we compared the patterns of cell proliferation, cell death and cell cycle arrest during the process of differentiation induced by these five agents. When F9 cells were cultured with the agents at their individual differentiation-inducing concentrations, cell proliferation was rapidly inhibited by treatment with the iron chelators and NaB. In contrast, RA did not influence the rate of increase of cell number at the concentration of 1 μ m . The three chelators also caused a marked reduction in cell viability, and the treated cells exhibited internucleosomal DNA fragmentation, whereas cells treated with NaB showed no apoptotic characteristics. RA induced apoptosis weakly at 1 μ m and strongly at higher concentrations. In addition, all the iron chelators hindered cell cycle progression, resulting in an arrest at the G1-S interface or S phase. The phenomena observed in chelator-treated cells were considerably different from those in RA- or NaB-treated cells. It is concluded that the three iron chelators cause both severe apoptotic cell death and cell cycle arrest of proliferating F9 cells via cellular iron deprivation, and that this apoptotic change may be independent of the process of differentiation.

At the Washington International Workshop on Genotoxicity Test Procedures (March 25-26, 1999), the current methodologies and data for the in vitro micronucleus test were reviewed. From this, guidelines for the conduct of specific aspects of the protocol were developed. Because there are a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at this time. Agreement was achieved on the following topics: Cells. The choice of cells is flexible, yet the choice of cell type should be justified and take into consideration doubling time, spontaneous frequency of micronuclei, and genetic background. Slide preparation. A fixation method that preserves the cytoplasm and cytoplasmic boundaries, and minimizes clumping should be used. Use of fluorescent DNA-specific dyes is encouraged for better detection of small micronuclei. Analysis. Micronuclei should have a diameter less than one-third of the main nucleus, and should be clearly distinguishable from the main nucleus. In the cytokinesis-block method, binucleated cells selected for analysis should have two clearly distinguishable main nuclei. Cells where the main nucleus(ei) is undergoing apoptosis should not be scored for micronuclei because the assumed micronuclei may have been the result of nuclear fragmentation during the apoptotic process. Toxicity. Cytotoxicity can be measured by various methods including cell growth, cell counts, nucleation (i.e., percent binucleated), division/proliferation index, confluence. A majority of the group recommended that the highest concentration should induce at least 50% cytotoxicity (by whatever measure is selected). Cytochalasin B. There is much debate regarding the use of cytochalasin B. For human lymphocytes, the use of cytochalasin B (6 microg/ml [lymphocytes cultured from whole blood cells] and 3-6 microg/ml [isolated lymphocyte cultures]) is recommended. For cell lines, because there were no definitive data showing a clear advantage or disadvantage of the use of cytochalasin B for a variety of chemicals, the majority opinion of the group was that at this time, the use of cytochalasin B for cell lines is considered optional. Further studies (many chemicals of a variety of potencies, tested both with and without cytochalasin B) are clearly needed to resolve this issue. Number of doses. At least three concentrations should be scored for micronuclei. Treatment/harvest times. At this time, there are not enough data to define the most appropriate treatment/harvest times. Following the principles of the in vitro metaphase assay (with or without metabolic activation), it was agreed that there was a need for a short treatment followed by a recovery time in the absence of test chemical, there was a need for a long treatment (maybe with and without recovery time), and ideally, treatment should cover cells in different cell cycle stages.

When metabolically activated, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine isolated from cooked food, is clastogenic in cultured Chinese hamster and human cells. Secondary metabolites of PhIP are formed via acetyltransferase (AT) and sulfotransferase (ST) activity; however, which is responsible for its clastogenic effect is unknown. We addressed this question. We used a parental Chinese hamster lung cell line and three sublines transfected with different AT genes to test the clastogenic (i.e., micronucleus-inducing) effects of metabolically activated PhIP and 7,12-dimethylbenz[a]anthracene (DMBA) in the presence and absence of pentachlorophenol (PCP), a ST inhibitor. PhIP was significantly more clastogenic in the three AT-enriched sublines than in the parental line (p < 0.001). DMBA (a ST-activated mutagen), on the other hand, equally induced MNs in all the cell lines. When PCP was added to the test system, the MN-induction ability of DMBA, but not of PhIP, decreased significantly (p < 0.001). These findings strongly suggest that PhIP clastogenicity is due to AT activity and not to ST activity.

The ability of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to induce chromosomal aberrations (CAs) in Chinese hamster lung fibroblast (CHL/IU) cells in vitro was examined. On incubation with rat S9 (2.5-10%, v/v) for 3 h, followed by a recovery culture period of 21 h, IQ caused significant induction of CAs at a concentration 20 micrograms/ml, but had less effect at 40 micrograms/ml. With longer recovery culture times such as 27-33 h, however, IQ was much more effective at 40 micrograms/ml. No significant induction was observed with 1 or 6 h treatments followed by 23 or 18 h recovery cultures, respectively. On incubation without S9, only weak CA induction by IQ was observed. These results show that IQ is a clastogen and that its clastogenic effect varied with the experimental conditions, such as the time of exposure and the time of recovery culture. The cell cycle perturbation effect is suggested to be one of the critical factors for the detection of the clastogenic potential of IQ.

We attempted to establish the optimum conditions for the calcium phosphate (CaPO4) precipitation protocol by counting G418 resistant (G418r) colonies after transfection of pSV2-neo DNA into BALB 3T3 cells. The amount and molecular size of carrier DNA, number of plating cells, treatment period of DNA-CaPO4 precipitates and expression time of G418 selection were found to be important factors in the induction of G418r colonies. Six G418r clones were derived from BALB 3T3, NIH 3T3 and FRSK cells, and cocultured with G418 sensitive (G418s) parent cells in G418 medium. The colony formation capacity of all G418r cell clones decreased with the increasing number of plated G418s cells. Cell-cell contact appeared to be necessary to reduce the colony formation of G418r cells, and contact-dependent G418r cell killing was probably not related to gap junction formation. Contact-mediated cell killing is a likely explanation for the observation that induction of G418r colonies is often reduced under conditions of high-density plating, long treatment of DNA-CaPO4 precipitates, and long expression time of G418 selection. These results suggest that in some instances transfection efficiency using pSV2-neo DNA should be carefully evaluated because culture conditions can mask the induction of G418r colonies.

A sensitive umu test system for the detection of mutagenic nitroarenes has been developed using a new tester strain Salmonella typhimurium NM1011 having a high nitroreductase activity. The new strain was constructed by subcloning the bacterial nitroreductase gene into a plasmid pACYC184 and introducing the plasmid into the original strain S. typhimurium TA1535/pSK1002 harboring a fusion gene umuC′-′lacZ (pSK1002). Thus, the tester strain enabled us to monitor the genotoxic activities of various nitroarene compounds by measuring the β-galactosidase activity in the cells. The sensitivity of strain NM1011 was compared with that of the parent tester strain S. typhimurium TA1535/pSK1002 or a nitroreductase-deficient strain S. typhimurium NM1000 with respect to the induction of umuC gene expression by 17 mutagenic nitroarenes. The newly developed strain with high nitroreductase activity had about 3 times higher nitrofurazone-reductase activvity than the parent strain and was highly sensitive to the compounds 2-nitrofluorene, 1-nitronaptthalene, 2-nitronaphthalene, 1-nitropyrene, m-dinitrobenzene, 4,4′-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 3,9-dinitrofluoranthene, 5-nitroacenaphthene and 2,4-dinitrotoluene. By contrast, the enzyme-deficient strain did not show any considerable response to 2-nitrofluorene, m-dinitrobenzene,, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, 4,4′-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 2,4-dinitrotoluene and 5-nitroacenaphthene. These results suggest that the newly developed tester strain with high nitroreductase activity is very useful for the detection of potent mutagenic nitroarene compounds.

Menadione-resistant subclones were selected from cultured Chinese hamster lung (CHL) cells which had been mutagenized with MNNG or ENU. The frequency of surviving colonies and the level of resistance were higher in mutagenized cells than in non-mutagenized cells. A subclone (designated MM1) was isolated from MNNG-treated cells and showed the highest level of resistance, 3 times higher than the parental CHL cells. The level of resistance was stable in non-selective medium over 3 months. The MM1 cells were also 2–3 times more resistant to other naphthoquinones. The activity of NADPH-cytochrome P-450 reductase, which is thought to play an important role in activation of menadione, was reduced in the MM1 cells to half that in the parental CHL cells. On the other hand, no differences between MM1 and CHL cells were found in the activity of superoxide dismutase and catalase which are assumed to defend against the cytotoxicity of menadione. Karyotype analyses indicated that one small chromosome was lost in the MM1 cells. The MM1 cells showed a 3-fold resistance to menadione in the chromosomal aberration test. The frequencies of chromosomal aberrations induced by adriamycin and mitomycin C which could be activated by NADPH-cytochrome P-450 reductase were almost the same in the MM1 and CHL cells, suggesting that the reductive activation of these compounds by this enzyme in microsomes may not be involved in the induction of chromosomal aberrations.

Salmonella typhimurium YG1021, YG1024, YG1026 and YG1029 are new derivatives of the Ames tester strains TA98 and TA100, with elevated ‘classical’ nitroreductase or acetyl-CoA: N-hydroxyarylamine O-acetyltransferase level. Thirty mutagens with different structures were tested using these strains and the sensitivities were compared with those of the conventional strains and of the enzyme-deficient strains. Elevated O-acetyltransferase activity of the indicator strains specifically increased their ability to detect the mutagenicity of aromatic nitro, amino and hydroxylamino compounds, whereas the strains with high nitroreductase activity were very sensitive to some nitroaromatics. The combined use of the isogenic tester strains with different metabolic capacities was quite useful to assess the intracellular metabolic activation and detoxification mechanisms of chemical mutagens.

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on 1-nitropyrene (NP), 3 dinitropyrenes (DNPs), fluorene and 4 mononitrofluorenes with and without metabolic activation (rat S9 mix). The 3 DNPs (1,3-, 1,6- and 1,8-DNP) induced chromosomal aberrations in the absence of S9 mix. The frequencies of cells with aberrations after treatment for 48 h were 43% at 2 micrograms/ml of 1,3-DNP, 55% at 0.1 microgram/ml of 1,6-DNP and 45% at 0.025 microgram/ml of 1,8-DNP, indicating the order of clastogenic potency as 1,8- greater than 1,6- greater than 1,3-DNP. On the other hand, 1-NP, which is known to be a direct-acting mutagen in bacteria, was negative in the chromosomal aberration test without S9 mix, but clearly positive with S9 mix. This effect was dependent on the concentration of the S9 fraction in the reaction mixture. High-pressure liquid chromatography analysis showed that 1-NP was converted by S9 mix to several metabolites, including 1-aminopyrene (AP). The clastogenic activity of 1-AP, however, was equivocal without S9 mix, suggesting that active clastogens other than 1-AP exist. Fluorene induced chromosomal aberrations only in the presence of S9 mix (61.8% at 25 micrograms/ml). 1-, 2-, 3- and 4-nitrofluorene (NF) were more clastogenic in the presence of S9 mix than in the absence of S9 mix, suggesting that NFs were converted to more active clastogens by S9 mix.

Nitration of benzo[a]pyrene (BaP) with HNO3 (d=1.38) produced a mixture of dinitroBaPs (1, 6- and 3, 6-isomers) and mononitroBaPs (1-, 3- and 6-isomers). Pure 1, 6-dinitroBaP and 3, 6-dinitroBaP were obtained by the reduction of the dinitroBaPs mixture with NaSH to yield the separable products 1-amino-6-nitroBaP and 3-amino-6-nitroBaP, followed by conversion to dinitroBaPs via the the diazonium salts. The half-wave potentials (E1/2) corresponding to the one-electron reduction of dinitroBaPs were measured and the relationship of these values to the mutagenicity is discussed.

Acetyl-CoA: N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the intracellular metabolic activation of arylhydroxylamines derived from mutagenic nitroarenes and aromatic amines. The acetyltransferase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1024 and YG1029) had about 100 times higher 2-hydroxyamino-6-methyldipyrido[1,2-a: 3′, 2′-d]-imidazole (N-hydroxy-Glu-P-1) O-acetyltransferase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic actions of 2-nitrofluorene, 1-nitropyrene, 1,8-dinitropyrene, 2-amino-6-methyldipyrido[1,2-a: 3′, 2-d]-imidazole (Glu-P-1), 2-aminofluorene and 2-aminoanthracene. These results indicate that the new strains permit the efficient detection of the mutagenicity of environmental nitroarenes and aromatic amines.

Salmonella typhimurium YG1024, an O-acetyltransferase-overproducing derivative of TA98, was found to be more sensitive in detecting mutagenicity in human urine caused by cigarette smoking, than the conventional strain TA98, in Ames test in the presence of S9 mix. YG1021, another derivative of TA98 with elevated nitroreductase levels, did not show increased sensitivity. These results suggest that the mutagenic compounds concentrated by Blue Rayon from smokers' urine are aromatic amino compounds.

Multiple-dosing effects of benzo[ a ]pyrene in the micronucleus test were studied using CD-1 male mice. Mice were treated orally once, twice or 3 times with 250, 5000, 1000 or 1000 mg/kg, at 24-h intervals. Bone marrow cells were sampled 24 h after the last administration. The present study indicated that the incidence of polychromatic erythrocytes with micronuclei significantly increased more in the group of animals that received B[a]P twice than in those receiving it one or 3 times. The dose of 500 mg/kg B[a]P yielded the greated response of any dose regimen.

'Classical nitroreductase' is an enzyme involved in theintracellular metabolic activation ofmutagenic nitroarenes (1).Wehave already cloned the nitroreductase gene ofSalmonellatyphimuriumTA1538(2) anddeterminedthenucleotide sequenceby dideoxy sequence analysis ofboth strands. Below is shownthe nucleotide sequence of 1690base fragment which containsanopenreadingframeof651 nucleotides withpotential to encodethe nitroreductase. Themaxicell technique wasusedto identifythe nitroreductase and its molecular weight was estimated as28KDa, which is close to the calculated molecular weight of23,955. Possible sequence of -35, -10, S.D. and rho-independent transcriptional termination signal are indicated.

In the detection of cancer-inducing agents, it is important to establish appropriate screening systems, not only for initiators but also for promoters. Almost all the initiators can be detected by short-term tests using bacteria, such as the Ames test, or cultured mammalian cells, such as cytogenetic tests and drug-resistant mutation tests (4). In recent years, some studies indicate that active ras genes act as initiators in two-stage carcinogenesis in vivo as well as in vitro (1, 2, 3, 7). We cloned v-Ha-ras-transfected BALB 3T3 cells (Bhas 42) by co-transfection with pSV2-neo genes. Bhas 42 cells were found to be sensitive to contact inhibition, but also to undergo a drastic transformation by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) (7). These results suggest that Bhas 42 cells are initiated cells in two-stage transformation. Therefore, we tried to apply Bhas 42 cells to screening of promoters by using the two-stage transformation assay.