Induction of sister-chromatid exchanges by indirect mutagens/carcinogens in cultured rat hepatoma and esophageal tumor cells and in Chinese hamster Don cells co-cultivated with rat cells

2 rat cell lines originated from ascites hepatoma AH66-B and esophageal tumor R1 were examined for their inducibility of sister-chromatid exchanges (SCEs) after treatment with 14 kinds of indirect mutagens/carcinogens, including 6 amine derivatives, 4 azo compounds, 3 aromatic hydrocarbons and 1 steroid. Of the 14 chemicals tested, 2-acetylaminofluorene (AAF), butylbutanolnitrosamine (BBN), dimethylnitrosamine (DMN), cyclophosphamide (CP), urethane, 2-methyl-4-dimethylaminoazobenzene (2-MeDAB), 3′-methyl-4-dimethylaminoazobenzene (3′-MeDAB), 4-o-tolylazo-o-toluidine (4-TT), benzo[a]pyrene (BP), 7,12-dimethyl-benz[a]anthracene (DMBA) and diethylstilbestrol (DES) were estimated to be effective inducers of SCEs in AH66-B and/or R1 cells, without the use of exogenous activating systems. Cell-mediated SCE tests with 6 selected chemicals, CP, 2-MeDAB, 4-TT, BP, DMBA and DES, showed a significant increase of SCEs in Chinese hamster Don-6 cells co-cultivated with AH66-B or R1 cells, depending on the number and sensitivity of AH66-B or R1 cells, as well as on the dose of chemicals tested, whereas singly cultured Don-6 cells were much less sensitive or almost insensitive to these chemicals. The above findings suggest that AH66-B and R1 cells may retain metabolic activities to convert a wide range of indirect mutagens/carcinogens into their active forms to induce SCEs, and that these cell lines provide simple and reliable screening systems in vitro, including the cell-mediated SCE assay, for detection of genotoxic agents, without the use of exogenous activation systems.     

Evaluation of chemicals requiring metabolic activation in the EpiDerm™ 3D human reconstructed skin micronucleus (RSMN) assay

The in vitro human reconstructed skin micronucleus (RSMN) assay in EpiDerm? is a promising new assay for evaluating genotoxicity of dermally applied chemicals. A global pre-validation project sponsored by the European Cosmetics Association (Cosmetics Europe - formerly known as COLIPA), and the European Center for Validation of Alternative Methods (ECVAM), is underway. Results to date demonstrate international inter-laboratory and inter-experimental reproducibility of the assay for chemicals that do not require metabolism [Aardema et al., Mutat. Res. 701 (2010) 123–131]. We have expanded these studies to investigate chemicals that do require metabolic activation: 4-nitroquinoline-N-oxide (4NQO), cyclophosphamide (CP), dimethylbenzanthracene (DMBA), dimethylnitrosamine (DMN), dibenzanthracene (DBA) and benzo(a)pyrene (BaP). In this study, the standard protocol of two applications over 48 h was compared with an extended protocol involving three applications over 72 h. Extending the treatment period to 72 h changed the result significantly only for 4NQO, which was negative in the standard 48 h dosing regimen, but positive with the 72 h treatment. DMBA and CP were positive in the standard 48 h assay (CP induced a more reproducible response with the 72 h treatment) and BaP gave mixed results; DBA and DMN were negative in both the 48 h and the 72 h dosing regimens. While further work with chemicals that require metabolism is needed, it appears that the RMSN assay detects some chemicals that require metabolic activation (4 out of 6 chemicals were positive in one or both protocols). At this point in time, for general testing, the use of a longer treatment period in situations where the standard 48 h treatment is negative or questionable is recommended.     

The micronucleus assay as a test for the detection of aneugenic activity

The aim of this work was to determine the usefulness of the micronucleus assay for the detection of aneugenic potential. Chemicals affecting microtubule assembly, i.e., colchicine, vinblastine sulfate and tubulazole, and chemicals affecting targets other than microtubuli, i.e., mitomycin C, cyclophosphamide and miconazole, and the clastogens azathioprine and procarbazine were administered once orally or intraperitoneally to male and female mice. Bone marrow preparations were made at 24, 48 and 72 h after dosing. All the clastogens and aneugens, except miconazole, yielded positive results in the micronucleus test. Measurements of the area of the micronuclei and their distribution clearly showed that the chemicals affecting microtubule assembly produced larger micronuclei than did the clastogens. The pattern of area distribution of the micronuclei found with cyclophosphamide and mitomycin C was between those found for the tubulin inhibitors and the clastogens. These findings indicate that the micronucleus test not only detects chemicals affecting microtubule assembly, but also can discriminate them from clastogens by measurements of the area of the micronuclei.     

Strain difference in the micronucleus test

The Collaborative Study Group for the Micronucleus Test, a task group of the Environmental Mutagen Society of Japan, has earlier addressed the question of sex difference as a source of variation in the micronucleus test. Strain difference, another issue in test protocols requiring urgent clarification, was selected as the subject of the second study. Male mice of strains Slc: ddY (ddY), CRJ: CD-1(ICR) (CD-1), Slc: BDF₁ (BDF₁), and ms: Hal (ms) were treated with 6 different chemicals chosen from various classes of micronucleus inducers: colchicine, 7,12-dimethylbenz[a]anthracene, ethyl methanesulfonate, N-ethyl-N-nitrosourea, 6-mercaptopurine, and potassium chromate. All 4 strains gave positive results with all 6 chemicals, although ms tended to show the highest responses. ddY and CD-1 were low responders, while BDF₁ was intermediate between ms and the other two. Although ms seemed superior to the other strains, its high responses became manifest mostly at high dose levels. ms was not always the most sensitive strain; it responded moderately to ethyl methanesulfonate. Also the background level of micronucleated polychromatic erythrocytes was the highest in ms, but this did not explain the apparent high sensitivity of this strain. Despite the strain differences, it can be concluded that any of the other strains used seems to suffice as a tester for the micronucleus test.     

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells.UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle.Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine.The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.     

Short-term tests for transplacentally active carcinogens: I. Micronucleus formation in fetal and maternal mouse erythroblasts

A cell-kinetic model for the application of the micronucleus test to polychromatic erythrocytes in mouse fetal liver, fetal blood, and maternal bone marrow after exposure to clastogenic agents is described. The time of expression and dose-response relationships obtained with γ-radiation, methyl methanesulphonate, procarbazine, mitomycin C and benzo[a]pyrene are analysed in terms of this model. The numbers of target cells damaged per unit dose has been calculated and the dose equivalents obtained. Maternal and fetal cells show similar sensitivity to γ-radiation, but fetal cells are markedly more sensitive to MMS and procarbazine. This probably due to differences in tissue distribution and metabolism. Maternal and fetal erythroid tissues can show linear and exponential dose-response relationships, which may not coincide (e.g. with MMS). It is concluded that risks from fetal exposure to genotoxic agents cannot be reliably predicted from in vivo tests restricted to adult animals. However, the micronucleus technique appled to fetal erythroid cells proveds a rapid and reliable short-term test, appropriate to minimising risks of genome damage during prenatal development.     

A combined testing protocol approach for mutagenicity testing.

The antischistosomal agent, hycanthone methanesulfonate (HMS), was employed to illustrate the utility of carrying out several mutagenicity tests in a single concurrent animal experiment. Several commonly used procedures that were successfully integrated into a multiple testing protocol included (1) metaphase analysis in bone marrow, (2) micronucleus test in bone marrow, (3) analysis of the urine for mutagenic constituents, and (4) the host-mediated assay using Salmonella typhimurium. In addition to these animal studies, in vitro mutagenicity testing with and without activation was carried out using S. typhimurium. HMS produced positive, dose--response effects in in vitro tests, metaphase analysis, micronucleus test, and urine analysis, but not in the host-mediated assay. The results of these integrated techniques suggest that such a protocol may be a benefit to those concerned with mutagenicity testing of chemicals.     

Short-term tests for transplacentally active carcinogens. A comparison of sister-chromatid exchange and the micronucleus test in mouse foetal liver erythroblasts

The effectiveness of 6 chemicals (benzo[a]pyrene, (BaP), cyclophosphamide (CP), diethylnitrosamine (DEN), methyl methanesulphonate (MMS), mitomycin C (MC) and procarbazine (PC) ) as inducers of micronuclei in foetal liver and maternal bone marrow erythroblasts has been determined, and related to that of gamma-radiation. CP, DEN, MMS and PC were all more effective in the foetal liver. The induction of micronuclei and SCEs by each chemical in foetal erythroblasts after in vivo exposure was measured. When expressed as induction of sister-chromatid exchanges (SCEs) per erythroblast/induction of micronuclei per erythroblast (/microM/kg), the ratios obtained were MC 580, BaP 470, DEN 430, CP 258, MMS 140 and PC 13. The lowest doses detected as potentially genotoxic by each test in foetal liver erythroblasts are (with the exception of PC which is a relatively ineffective inducer of SCEs) similar. When isolated foetal livers were exposed in vitro, SCE dose responses to BaP, MC, MMS and PC could be directly related to those from in vivo exposure, indicating the role of the foetal liver in metabolic activation, but CP was considerably more cytotoxic. The transplacental micronucleus test, and in vivo/in vitro method for SCEs in foetal liver erythroblasts, provide sensitive, complementary assays for genotoxic effects of chemicals during prenatal life. Since foetal liver possesses greater metabolic potential than adult bone marrow, the transplacental tests respond to genotoxic agents not detected by bone-marrow systems.     

Induction of sister chromatid exchanges in Chinese hamster cells by carcinogenic mutagens requiring metabolic activation

The induction of SCEs has proven to be the most sensitive mammalian system for detecting the effects of mutagenic carcinogens. Several chemicals that are mutagenic in the exquisitely sensitive Salmonella mutagenesis test have now been tested in Chinese hamster ovary (CHO) cells in culture. Cells were grown for 24 h (two rounds of DNA replication) in the presence of bromodeoxyuridine (Brd Urd) to form harlequin chromosomes in which it is possible to see the SCEs. To test whether the chemicals increase SCEs without metabolic activation, they were added at various concentrations for the entire culture period. To test if they induce SCEs after activation they were added for 30 min along with microsomes from rat liver (S-9 Mix of Ames). After this treatment the cells were cultured with Brd Urd. N-hydrosy-2-acetylamino-fluorene (10^?6?10^?4 M), N-acetoxy-2-acetylaminofluorenee (10)^?9?10^?7 M), and aflatoxin B₁ (10^?6?10^?4 M) all increased the yield of SCEs with increasing concentration. Further, aflatoxin B₁ was dramatically activated by the addition of rat liver microsomes. Benzo(a)pyrene (10^?6?10^?4 M), however, gave an increase only when activated. 2-aminofluorene (10^?6?10^?4 M) gave a slight increase only after long treatments without activation. In no case did 2-acetylamino-fluorene (10^?6?10^?4 M) increase SCE's. It thus appears that some of the chemicals that are positive in the Salmonella system are negative in the mammalian SCE system. Whether this reflects a difference in sensitivity between the two tests or the ability of the SCE test to discriminate between those chemicals that are active in bacteria, but not in mammals, is as yet unknown.     

Induction of in vivo DNA adducts by 4 industrial by-products in the rat-lung-cell system

Benz[a]anthracene (BA), dibenz[a,h]anthracene (DBA), dibenzo[a,i]pyrene (DBP), and dibenz[a,h]acridine (DBAC) are by-products found in many industrial wastes and emissions. Workers in the related occupational settings are potentially exposed to these substances through inhalation. In the present study, induction of DNA adducts in vivo by these chemicals was investigated using ³²P-postlabeling analysis in the rat-lung-cell system. The potency of DNA-adduct inducing activity was also compared to that of two cytogenetic endpoints i.e., sister-chromatid exchange (SCE) and micronucleus formation. Via intratracheal instillation, male CD rats (6/group) were dosed 3 times with BA, DBA, DBP or DBAC in a 24-h interval. Lung cells were enzymatically separated and used to determine the frequency of DNA adducts, SCE and micronuclei. Results show that all 4 test compounds induced DNA adducts, SCEs, and micronuclei in the rat-lung cell in vivo and that the postlabeling DNA adduct assay detected genotoxic activity at lower dose levels than the two cytogenetic assays. These findings suggest that BA, DBA, DBP or DBAC are rat pulmonary genetoxicants and the DNA-adduct assay is more sensitive than SCE or micronucleus assays for detecting the pulmonary genotoxicity of these industrial PAHs in the in vivo rat-lung-cell system.     

Modified glucose-oxidase/peroxidase residual glucose assay for use with anaerobic bacteria

Because Lactobacillus species compounds that interfere with the o-toluidine assay for glucose, use of the glucose-oxidase/peroxidase method was investigated. Although this method gave satisfactory results for Lactobacillus acidophilus, L. plantarum and L. lactis cultured in media with ≤ 40 mM glucose, at higher initial glucose concentrations, residual glucose values were anomalously high. This anomaly caused by hydrogen peroxide produced by the lactobacilli. Preincubating samples with catalase removed the endogenous hydrogen peroxide and resulted in accurate residual glucose values. The presence of catalase did not interfere with the subsequent peroxidase portion of the assay.     

Nitrite and Nitrous Oxide Accumulation During Denitrification in the Presence of Pesticide Derivatives

Temporary accumulation of nitrite and nitrous oxide was observed in soil incubated under anaerobic conditions when derivatives of the insecticide chlordimeform [(N-4-chloro-o-tolyl)-N′,N′ -dimethylformamidine] were added. Chlordimeform did not affect the denitrification process, but N-formyl-4-chloro-o-toluidine and 4-chloro-o-toluidine caused an inhibition as determined by the accumulation of nitrite and nitrous oxide. A simultaneous application of the insecticide and its derivatives resulted in a stronger inhibitory effect than the application of each compound separately. Aniline intermediates of other pesticides also inhibited denitrification in soil, and they proved to be more effective than their parent compound.     

Studies on the oxidation products from 2,4-diaminotoluene by hydrogen peroxide and their mutagenicities. II.

2,4-Diaminotoluene (DAT) was reacted with hydrogen peroxide at room temperature for 2 days, and the resulting red precipitates were separated into 5 fractions on silica gel column chromatography. On the gas chromatographic (GC) study, the first fraction (Fr. 1), which is mutagenic (1425 and 1391 revertants/μg in the absence and presence of S9 respectively) in Salmonella typhimurium TA98, contained several peaks. Fr. 1 was further separated into 4 subfractions (Fr. 1-I-Fr. 1-IV) by silica gel column chromatography. The red crystals were separated from Fr. 1-III and the structure of the compound was determined to be 1,8-diamino-2,7-dimethylphenazine from physicochemical and chemical evidence.Further, o-nitro-p-toluidine, p-nitro-o-toluidine, 3,3′-diamino-4,4′-dimethylazobenzene and 3,3′ diamino-4,4′-dimethylazoxybenzene were identified with authentic and synthesized samples by gas chromatography/mass spectrometry. These compounds without nitrotoluidines were mutagenic, and phenazine, azo and azoxy compounds induced 49, 301 and 245 revertants/nmole in Salmonella typhimurium TA98 with 25 μl S9 per plate, respectively.     

Review of the current status of the mei-9a test for chromosome loss in Drosophila melanogaster: An assay with radically improved detection capacity for chromosome lesions induced by methyl methanesulfonate (MMS), dimenthylnitrosamine (DMN), and especially diethylnitrosamine (DEN) and procarbazine

A review of previous findings as well as new data are included in the present paper on recent invetigations by Zimmering and co-workers regarding a radical improvement in the detection capacity of the conventional test for chromosome loss to assay for induced chromosome lesions/breakage. The improvement has been achieved through the use of mei-9^a repair-deficient P1 females to which treated males are mated. 4 compounds have been tested including MMS, DMN, DEN and procarbazine. Not only the mei-9^a test yielded significantly higher frequencies of induced chromosome loss with MMS and DMN than the conventional test, preliminary data, in fact, providing evidence of a positive response in the mei-9^a test at a concentration one order of magnitude below that producing no effect in the conventional test, but, more critically, it has permitted detection of highly significant increases in induced chromosome loss with DEN and procarbazine, compounds proving negative in the conventional tests for chromosome loss and heritable traslocations at all concentrations employed including those producing substantial to high frequencies of recessive lethal.     

Pharmacological properties of dibenzo[a,c]cyclooctene derivatives isolated from Fructus Schizandrae chinensis I. Interaction with rat liver cytochrome P-450 and inhibition of xenobiotic metabolism and mutagenicity

Seven compounds isolated from Fructus Schizandrae chinensis, a traditional Chinese tonic, which is also able to decrease liver lesions by hepatoxic chemicals, are named Schizandrin (Sin) A, B and C, Schizandrol (Sol) A and B and Schizandrer (Ser) A and B. They are dibenzo[a,c]cyclooctene derivatives. Dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylenedioxy-biphenyl-2,2′-dicarboxylate (DDB) is an intermediate for synthesizing Sin C. The interactions of these compounds with rat liver microsomes in vitro have been investigated. Sol A and Sol B gave type I difference spectrum, the other six compounds gave ‘reverse type I’ difference spectrum. When Schizandrins or DDB were incubated with NADPH-reduced microsomes, Sin B, Sin C, Sol B, Ser A and Ser B generated dual Soret peaks at 455–460 nm and 425–430 nm, the other three compounds caused a difference spectrum without 455 nm peak. All these compounds more or less inhibit liver microsomal hydroxylation of benzo[a]pyrene (BP) demethylation of aminopyrine. Sin B, Sol B and DDB decreased mutagenicity of BP in Ames test.     

A “spiral test” applied to bacterial mutagenesis assays

A new procedure (the spiral test) has been set up and validated for the distribution of chemicals in bacterial multagenesis asssays. This metzhod involves the use of a special instrument (spiral plater), which dispenses, along a spiral track, decreasing volumes of liquid samples, from the near centre to the periphery of a rotating agar plate. A gradient of concentration of a compound up to about 1500:1 is thus formed on a single plate. The activity of 18 mutagens of various potencies and chemical classes was checked in the Salmonella/microsome test by dispensing their solutions either on the surface of top agar (method A) or of the minimal-glucose agar medium, before the addition of molten top agar incorporating bacteria and eventually S9 mix (method B).Compared with the spot test, the gradient of concentration of a compound produced by the spiral diluter was much wider and more gradual. Even non-diffusible chemicals (e.g. benzo[a]pyrene and benz[a]anthracene) were efficiently detected in the spiral test, as well as very weak (e.g. mebanazine and trimethylphosphate) or borderline (e.g. perylene, 1,1-dimethylhydrazine and procarbazine) mutagens, which were negative in the spot test. Method B was at least as sensitive as the plate-incorporation test, such a goal being achieved in a single plate instead of in serial plates. Technical problems made method A less sensitive, but it was more efficient in detecting unstable mutagens (e.g. β-propiolactone). Like the plate test, the spiral test appeared to be suitable for a semi-quantitative assessment of mutagenicity data, and was efficient in demonstrating both the activation of promutagens and the deactivation of some directly acting mutagens. Preliminary assays were also carried out with repair-proficient (WP2) or -deficient (TM1080: lex A^?/polA^?/R391 and CM871: lexA^?/uvrA^?/recA^?) trp^? strains of E. coli.     

Evaluation of the genotoxicity of cis-bis(3-aminoflavone)dichloroplatinum(II) in comparison with cis-DDP

Short-term tests that detect genetic damage have provided information needed for evaluating carcinogenic risks of chemicals to man. The mutagenicity of cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-[Pt(AF)2Cl2]) in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) was evaluated in the standard plate-incorporation assay in four strains of Salmonella typhimurium: TA97a, TA98, TA100 and TA102, in experiments with and without metabolic activation. It was shown that cis-[Pt(AF)2Cl2] acts directly and is mutagenic for three strains of S. typhimurium: TA97a, TA98 and TA100. In comparison with cis-DDP this compound showed a weaker genotoxicity. Contrary to cis-DDP it has not shown toxic properties in the tester bacteria. The genotoxicity of both tested compounds was evaluated using chromosomal aberration, sister chromatid exchange and micronucleus assays, without and with metabolic activation, in human lymphocytes in vitro. The inhibitory effects of both compounds on mitotic activity, cell proliferation kinetics and nuclear division index were also compared. In all test systems applied, cis-[Pt(AF)2Cl2] was a less effective clastogen and a weaker inducer of both sister chromatid exchanges and micronuclei in comparison with cis-DDP, with and without metabolic activation. cis-[Pt(AF)2Cl2] has a direct mechanism of action and is less cytostatic and cytotoxic than the other compound. These results provide important data on the genotoxicity of cis-[Pt(AF)2Cl2] and indicate its beneficial properties as a potential anticancer drug, especially in comparison with cis-DDP.     

Cytogenetic effects in the mouse of 17 chemical mutagens and carcinogens evaluated by the micronucleus test.

2 dialkylnitrosamines, 4 oxazaphosphorines, 6 aryldialkyltriazenes, urethane, N-hydroxyurethane, 4-nitroquinoline-1-oxide, procarbazine (natulan) and the inorganic carcinogen potassium chromate were studied for cytogenetic activity in the micronucleus test on mouse bone marrow. Except diethylnitrosamine, all chemicals were active. The results are compared with those known from studies in other mammalian and sub-mammalian test systems. The results of the micro nucleus test correlate well with results from other mutagenicity tests and with the carcinogenicity of the chemicals. The lack of an effect on N-nitrosodiethylamine (DENA) is discussed with regard to the short life-time of the ultimate mutagen.