Procarbazine genotoxicity in the MutaMouse; strong clastogenicity and organ-specific induction of lacZ mutations.

Procarbazine, a drug used for cancer chemotherapy, is carcinogenic in rodent bioassays. We analyzed the mutagenicity of procarbazine in various organs and the clastogenicity of the drug in hematopoietic cells of the lacZ transgenic MutaMouse. This was part of the second collaborative study of the Mammalian Mutagenesis Study Group of the Japanese Environmental Mutagen Society on the transgenic mouse mutation assay. At 50 mg kg(-1), procarbazine induced micronuclei in hematopoietic cells, but it did not increase the lacZ mutant frequency (MF) in bone marrow. It was also negative in liver, testis, spleen, kidney, and lung. Five daily administrations of 150 mg kg(-1) yielded highly positive responses in the drug's target organs for carcinogenesis (lung, bone marrow, and spleen). Lower positive responses were detected in kidney, which is a minor target organ. Liver showed only a slight increase in lacZ MF and brain showed no increase. The testis MF more than doubled which suggest that procarbazine is mutagenic to germ cells. Thus, we demonstrated that procarbazine has a strong clastogenic effect in hematopoietic cells and is mutagenic in a variety organs after high dose treatment. The induced MF was especially high in procarbazine's target organs for carcinogenesis, which supports the relevance of the transgenic mouse mutation assay for the assessment of potential genotoxins in vivo.     

Mutation induction by N-propyl-N-nitrosourea in eight MutaMouse organs.

As a part of the 2nd Collaborative Study for the Transgenic Mouse Mutation Assay, we studied the organ specificity and the temporal changes in mutant frequency (MF) of the lacZ gene following intraperitoneal injection of 250 mg/kg N-propyl-N-nitrosourea into male MutaMouse. We used a positive selection system and examined eight organs, i.e., bone marrow, liver, kidney, lung, spleen, brain, heart, and testis. The chemical caused a significant increase in MF in all organs except for brain, and the bone marrow was the most sensitive organ, exhibiting a MF on day 7 that was 10 times that of the control. The MF increased from day 7 to day 28 in liver, kidney, and testis, while it decreased in bone marrow. The relationship between the results of this study and the target organs of carcinogenesis, and the cause of the temporal changes in MF, are discussed.     

Multiple organ mutation in the lacZ transgenic mouse (Muta mouse) 6 months after oral treatment (5 days) with benzo[a]pyrene.

We have recently demonstrated that not all organs with high rates of mutation in the lacZ transgene develop tumors using the Muta Mouse. To better understand the role of in vivo mutation in carcinogenesis, we examined the mutant frequencies (MF) of the lacZ transgene in tumor-bearing and non tumor-bearing organs. MF, recovered after 2 weeks (the data taken from our previous study) and after 26 weeks following oral doses of 125 mg kg-1 day-1 benzo[a]pyrene (BP) for five days were compared. The organs examined included the target organs (forestomach, spleen, and lung) and non-target organs (colon, glandular stomach, and liver) for BP carcinogenesis. The data indicated that lacZ MF were markedly increased over spontaneous frequencies in the organs examined and that the organ which showed the highest MF was the colon, followed by the forestomach>spleen>glandular stomach, liver, and lung in that order. These findings indicate that the MF of the lacZ transgene in each organ, even 26 weeks after the start of the treatment does not fully correlate with the known target organs of BP. Furthermore, the lacZ MF in a non-papilloma region of a forestomach with a papilloma was equivalent to the two highest MF observed in the healthy colon (non-target organ) of mice at 26 weeks. These observations also indicate that the generation of tumors requires the induction of mutations as well as other factor(s) specific to the target organs. These results clearly suggest that highly mutated organs do not always progress to tumors in the transgenic mouse.     

N-Nitrosodi-n-propylamine induces organ specific mutagenesis with specific expression times in lacZ transgenic mice.

The mutagenic and clastogenic effects of N-nitrosodi-n-propylamine (NDPA) in lacZ transgenic mice (MutaMouse) were investigated as a part of the second collaborative study of the transgenic mouse mutation assay by a subgroup of the Mammalian Mutagenesis Study Group, a suborganization of the Environmental Mutagen Society of Japan. Male MutaMouse mice were administered NDPA intraperitoneally at a dose of 250 mg/kg, which is half of the LD(50) of the compound. The clastogenicity of NDPA was examined by the peripheral blood micronucleus test just before and at 24, 48 and 72 h after the treatment. The mutant frequencies in the bone marrow, liver, lung, kidney and urinary bladder were examined by the positive selection method for lacZ kidney. These findings demonstrate that NDPA induces organ-specific mutagenesis with specific expression times, and that the mutagenicity of NDPA in lacZ transgenic mice is consistent with its carcinogenicity.     

A method to distinguish between the de novo induction of thymidine kinase mutants and the selection of pre-existing thymidine kinase mutants in the mouse lymphoma assay

The mouse lymphoma assay (MLA) is the most widely used in vitro mammalian gene mutation assay. It detects various mutation events involving the thymidine kinase (Tk) gene in L5178Y/Tk+/- -3.7.2C mouse lymphoma cells. Mutants are detected using a thymidine analogue that arrests the growth of cells containing a functional Tk gene. However, there are a number of potential test chemicals that are thymidine analogues, and there is a problem when using the MLA to evaluate the mutagenicity of these chemicals. Thymidine analogues are activated by Tk before eliciting their toxicity. Therefore, any pre-existing Tk-/- mutants may avoid the toxicity of the test chemical and obtain a growth advantage over the Tk+/- cells, increasing the Tk mutant frequency (MF) in the culture via a selection mechanism. This potential mutant selection effect needs to be distinguished from de novo mutant induction in order to properly evaluate the mutagenicity of these chemicals. Here we describe a simple MLA study design that can differentiate between the selection of pre-existing mutants and de novo mutant induction. Trifluorothymidine (TFT), a thymidine analogue and the selection agent normally used in the MLA, and 4-nitroquinoline-1-oxide (4-NQO), a potent mutagen, were used to treat cells from two different Tk+/- mouse lymphoma cell cultures with different background MFs (approximately 112 and 305x10(-6)). Both agents significantly increased the Tk MFs in both the normal and high background cultures (p<0.01). In 4-NQO-treated cultures, the induced MFs (MF of treated culture-MF of control) for the cultures with different background MFs were about the same (p>0.1), while in TFT-treated cultures, they were significantly different (p<0.01). In TFT-treated cultures, the fold-increases of MF (MF of treated culture/MF of control) for the cultures with different background MFs were about the same (p>0.1), while in 4-NQO-treated cultures, they were significantly different (p<0.01). This study confirms that, when de novo mutations are induced, the induced MF is the same for cultures with normal and artificially high background MFs. In situations where the increase in MF is due solely to selection of pre-existing mutants, the "induced" MF will be a multiple of the background MF and the magnitude of the increase of the induced MF will depend upon the magnitude of the background MF. Our results demonstrate that it is possible, using this experimental design, to distinguish between chemicals acting primarily via the selection of pre-existing Tk mutants and those inducing de novo mutants in the MLA.     

The mouse spot test. Evaluation of its performance in identifying chemical mutagens and carcinogens

The published results on 60 chemicals and X-rays investigated in the mouse spot test were compared with data on the same chemicals tested in the bacterial mutation assay (Ames test) and lifetime rodent bioassays. The performance of the spot test as an in vivo complementary assay to the in vitro bacterial mutagenesis test reveals that of 60 agents, 38 were positive in both systems, 6 were positive only in the spot test, 10 were positive only in the bacterial test and 6 were negative in both assays. The spot test was also considered as a predictor of carcinogenesis; 45 chemicals were carcinogenic of which 35 were detected as positive by the spot test and 3 out of 6 non-carcinogens were correctly identified as negative. If the results are regarded in sequence, i.e. that a positive result in a bacterial mutagenicity test reveals potential that may or may not be realized in vivo, then 48 chemicals were mutagenic in the bacterial mutation assay of which 38 were active in the spot test and 31 were confirmed as carcinogens in bioassays. 12 chemicals were non-mutagenic to bacteria of which 6 gave positive responses in the spot test and 5 were confirmed as carcinogens. These results provide strong evidence that the mouse coat spot test is an effective complementary test to the bacterial mutagenesis assay for the detection of genotoxic chemicals and as a confirmatory test for the identification of carcinogens. The main deficiency at present is the paucity of data from the testing of non-carcinogens. With further development and improvement of the test it is probable that the predictive performance of the assay in identifying carcinogens should improve, since many of the false negative responses may be due to inadequate testing.     

In vivo genotoxicity of 2-amino-3,8-dimethylimidazo[4, 5-f]quinoxaline in lacI transgenic (Big Blue) mice

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a heterocyclic amine found in cooked meat, is a strong mutagen in the Salmonella/microsome assay and was proven to be a hepatocarcinogen in rodents. We used the lacI transgenic (Big Blue(R)) mouse to investigate MeIQx genotoxicity in vivo. lacI mutant frequencies were examined in liver and colon after single intragastric administration of MeIQx (males) or 12 weeks of feeding in the diet (males and females). Micronucleus induction was monitored in the peripheral blood and cell proliferating activity was monitored by proliferating cell nuclear antigen (PCNA) immunostaining, but only after the intragastric administration. Intragastric treatment with MeIQx (100 mg/kg) did not increase mutant frequency (MF) in liver or colon but it did induce a slight but statistically significant increase in the incidence of micronucleated reticulocytes 48 h after the treatment. No apparent increase in PCNA-positive foci was observed in any of tissues analyzed 14 days after the treatment. Administration of MeIQx (300 ppm) in diet for 12 weeks, however, caused MF increases in liver and colon in male and female mice, with greater increases in the females. An increase was also obvious after 4 weeks, but only in females. The sex difference in MF is consistent with the fact that female mice are more susceptible to MeIQx carcinogenesis. These results demonstrated that in the transgenic mouse mutation assay, long-term feeding of MeIQx was more effective than single gastric exposures in revealing the compound's mutagenicity in the target organs of carcinogenicity and that sex differences in susceptibility can also be observed.     

Mutagenicity of 4-nitroquinoline 1-oxide in the MutaMouse.

As part of a collaborative study, the Mammalian Mutagenesis Study Group (MMS), a sub-organization of the Environmental Mutagen Society of Japan (JEMS) conducted mutagenicity tests in MutaMouse. Using a positive selection method, we studied the organ-specificity and time dependence of mutation induction by 4-nitroquinoline 1-oxide (4NQO). A single dose of 4NQO was administered intraperitoneally (7.5 or 15 mg/kg) or orally (200 mg/kg) to groups of male mice. On days 7, 14 and 28 after treatment, we isolated the liver, kidney, lung, spleen, bone marrow, testis and stomach in the intraperitoneal administration experiment and the liver, lung, bone marrow, testis and stomach in the oral administration experiment. In addition, we performed the peripheral blood micronucleus test to evaluate clastogenicity. In the mice treated intraperitoneally at 7.5 mg/kg, we found increased mutant frequency (MF) only in the lung, where the MF did not vary with expression time. In the mice treated at 15 mg/kg, we found increased MF in the liver, bone marrow and lung. In orally treated mice, the MF was high in the lung and liver and very high in the bone marrow and stomach while the increase in the testis was negligible. As the expression time was prolonged, the MF tended to increase in the liver, decrease in the bone marrow, and remain stable in the lung, testis and stomach. The incidence of micronucleus induction in peripheral blood cells was significantly increased (p<0.01) in the 4NQO groups when compared with the vehicle control group by intraperitoneal treatment. Thus, these assay systems appeared to be of use in detecting not only genetic mutation but also chromosomal aberration.     

Genotoxicity of o-aminoazotoluene (AAT) determined by the Ames test, the in vitro chromosomal aberration test, and the transgenic mouse gene mutation assay

o-Aminoazotoluene (AAT) has been evaluated as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). The Ames test found it to be mutagenic in the presence of a metabolic activation system, whereas it has little clastogenicity either in vitro or in vivo in the chromosomal aberration assay. AAT is also carcinogenic in the lung or liver of mice and rats given long-term administrations. Therefore, metabolites generated in the liver etc. may have gene mutation activity, and carcinogenesis would occur. We examined the mutagenicity of AAT in a gene mutation assay, using lacZ transgenic mice (MutaMice) and a positive selection method. AAT showed positive results for organs with metabolic functions, such as liver and colon and other organs. Positive results were also seen in an Ames test in the presence of metabolic activation and negative results seen in a chromosomal aberration test. Therefore, AAT had the potential to cause gene mutation in the presence of metabolic activation systems in vitro and the same reaction was confirmed in vivo with organs with metabolic function, such as liver and colon, but little clastogenicity in vitro or in vivo. Thus, metabolites with gene mutation activity may be responsible for the carcinogenicity of AAT. The transgenic mouse mutation assay proved to be useful for concurrent assessment of in vivo mutagenicity in multiple organs and to supplement the standard in vivo genotoxicity tests, such as the micronucleus assay which is limited to bone marrow as the only target organ.     

Mutagenic properties of a nitrofuran, 7-methoxy-2-nitronaphtho[2, 1-b]furan (R7000), in lacI transgenic mice

The in vivo mutagenic properties of a 5-nitrofuran, the 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), already well known in bacteria, was evaluated in lacI transgenic mice (Big Blue). The mutation frequency was determined in various organs of i.p. - treated mice and the nature of induced mutations was determined for the target organs in which mutation induction was significant. It was found that R7000 is mutagenic in mice, although, on the basis of the number of induced mutants per unit mass in comparison with other known mutagenic chemicals, R7000 appears to be considerably less mutagenic in mice than in bacteria. The most affected organs, small intestine, caecum and colon organs belong to the digestive apparatus. The distribution of R7000-induced mutations in the lacI gene recovered from small intestine of transgenic mice was very similar to that which had been found in E. coli. The difference between mouse and E. coli in the R7000 induced mutational spectra are mainly in the proportion of single base frameshifts versus base substitutions. Since R7000 induced mutations seemed to arise in the population of stem cells and that the stem cells are important for carcinogenesis, our results are compatible with a possible carcinogenic effect of R7000 and other nitrofurans.     

Comparison of the mutational spectra of the lacZ transgene in four organs of the MutaMouse treated with benzo[a]pyrene: target organ specificity

We recently demonstrated that not all organs with a high rate of induction of mutation in the lacZ transgene develop tumors in the lambdalacZ transgenic mice (MutaMouse) used for a long-term carcinogenicity study with benzo[a]pyrene (BP). To better understand the role of chemical-induced in vivo mutations in carcinogenesis, we compared the mutational spectra of the lacZ transgene in four organs of the MutaMouse obtained 2 weeks after five daily consecutive oral treatments with 125 mg/kg/day BP. lacZ transgenes were analyzed in two target organs (forestomach and spleen) and two non-target organs (colon and glandular stomach) for BP-induced carcinogenesis in MutaMouse, and all of these organs were highly mutated in the lacZ transgene. The sequence data showed similar mutational spectra of the lacZ transgene between the two target organs; the predominant mutations were G:C-->T:A transversions (55% and 50% for forestomach and spleen, respectively), followed by deletions (20% and 21% for forestomach and spleen, respectively) mainly at G:C site. The frequent G:C-->T:A transversions are consistent with reports of the mutational spectra produced in the p53 gene in tumors generated in rats and mice exposed to BP. In contrast, the mutational spectra of the lacZ transgene in the two non-target organs are different from those in the target organs, and are also suggested to differ from one another. These findings suggest an organ/tissue-specific mechanism of mutagenesis.     

Enumeration of 6-thioguanine-resistant tumour cells using flow cytometry and comparison with a microtitration cloning assay

Measurement of mutant frequency in tumour specimens has been hampered by low cloning efficiency in soft agar. A method was developed to detect cell proliferation using the thymidine analogue 5-bromo-2'-deoxyuridine (BrUdR). BrUdR incorporation was monitored by immunofluorescent staining of fixed cells using a monoclonal antibody highly specific for this nucleoside analogue. The 6-thioguanine (6TG) exposure conditions which inhibited DNA synthesis, as measured by BrUdR incorporation, in wild-type cells while allowing proliferation of spontaneous hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutants were investigated using tumour cell lines. It was shown that exposure to 10(-5) M BrUdR for the equivalent of 1 cell cycle time did not affect growth of wild-type cells, nor did it affect the growth of HPRT- mutants in the presence of 6TG. Methods for rapid flow cytometric enumeration of BrUdR-labelled 6TG-resistant cells were developed using fluorescent microspheres as an internal standard. To validate the BrUdR mutation assay, the 6TG mutant frequency (MF) was measured in L1210 R/S, a mouse leukaemic cell line (BrUdR 6TG MF = 7.0 X 10(-5] and the results directly compared with those from a microtitration cloning assay (MF = 4.6 X 10(-5]. The results were similar and within the range reported for HPRT MF in mammalian cells.     

Nitrosamine-induced mutagenesis in Escherichia coli K12 (343/113). 1. Mutagenic properties of certain aliphatic nitrosamines

The Escherichia coli K12 (343/113) test system developed by G. Mohn was used to detect the mutagenic activity induced by a group of aliphatic nitrosamines. Metabolic activation was incorporated into the assay by the addition of liver homogenates induced in either Sprague-Dawley rats or C3H mice with the addition of 0.1% phenobarbital to the drinking water. Nitrosodiethylamine (NDEA) was mutagenic upon metabolic activation and exhibited a preference to revert the missense mutation at the arginine locus. NDEA was also capable of inducing the forward mutation, selected as an ability to utilize galactose. NDEA was converted effectively into a mutagen in a time period of 30 min to 2 h. Metabolic activation with the mouse and rat liver preparations did not result in quantitative differences. Aliphatic nitrosamines that gave unexpected results with the Salmonella assay [4-10] were examined in the E. coli system. Nitrosodipropylamine (NDPA) and nitrosodiallylamine (NDAA) were mutagenic in both E. coli and Salmonella. Nitrosomethylethylamine (NMEA) was not mutagenic in Salmonella but was mutagenic in E. coli, and a strong carcinogen, nitrosomethylneopentylamine (NMNA), was not mutagenic in either assay. These results indicate the use of multiple genetic assays for the detection of genotoxic chemicals in our environment.     

Comparative investigation of multiple organs of mice and rats in the comet assay

Mice and/or rats are usually used to detect chemical carcinogenicity and it has been known that there are species differences in carcinogenicity. To know whether there are species difference in genotoxicity, we conducted comparative investigation of multiple organs of mice and rats in the comet assay. Since the sensitivity to xenobiotics is different for different species, we queried species difference in the genotoxic sensitivity at one equitoxic level but not at one equidose. Therefore, groups of four mice or rats were treated once intraperitoneally or orally with a chemical at highest dose without death and distinct toxic manifestation. When the death was not observed at 2000 mg/kg of a chemical, 2000 mg/kg was used for the comet study. The stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were sampled 3, 8, and 24h after treatment. Among chemicals tested, benzyl acetate, chlorodibromomethane and p-chloro-o-toluidine are carcinogenic to mice but not rats, and aniline, azobenzene, o-phenylphenol Na, and D-limonene are carcinogenic to rats but not mice. Although the two species differed in genotoxicity target organs and migration values, the judgement of a positive or negative response was the same for all chemicals studied except for 2,4-dimethoxyaniline, 2,5-diaminotoluene, and p,p'-DDT when chemicals with positive responses in at least one organ are judged to be comet assay-positive. 2,4-Dimethoxyaniline and 2,5-diaminotoluene that are Ames test-positive non-carcinogens in both species were positive in one organ (urinary bladder for 2,4-dimethoxyaniline and stomach for 2,5-diaminotoluene) in rats, but negative in all mouse organs. p,p'-DDT, which is an Ames test-negative but in vitro cytogenetic test-positive hepatic carcinogen in mice and rats, was positive in multiple rat organs, but not in any mouse organ. These results suggest that species differences in genotoxicity at one equitoxic level are not consistent with species difference in carcinogenicity and that the use of both species is appropriate to indicate a carcinogenic potential in the comet assay with multiple organs, when chemicals being positive in at least one organ are judged to be comet assay-positive.     

Carcinogenesis in Relation to the Stem-Cell-Mutation Hypothesis

Abstract. From reports on fish, mice, rats, and humans, it can be concluded that at early developmental stages, especially stages before organogenesis, vertebrates are resistant to the induction of tumors by carcinogens. This conclusion and results on the molecular biology of chemical carcinogenesis in mice support the hypothesis that carcinogenesis of an organ is initiated by mutation of its stem cells formed during organogenesis. Convincing support for the existence of mutations that cause development of tumors is that heritable tumors are induced in mice and Drosophila by exposure of germ cells to radiation and chemicals. Various lines of evidence support the notion that tumor genes, which increase the predisposition of their carriers to develop tumors, are at least partly regulatory mutations. In this paper, the interrelation of tumorigenesis and teratogenesis, the high susceptibility of growing or regenerating organs to induction of tumors by carcinogens, and the latent period of induced neoplasms are discussed in relation to the stem-cell-mutation hypothesis.     

Age-dependent sensitivity of Big Blue transgenic mice to the mutagenicity of N-ethyl-N-nitrosourea (ENU) in liver

The incidence of childhood cancer is increasing and recent evidence suggests an association between childhood cancer and environmental exposure to genotoxins. In the present study, the Big Blue transgenic mouse model was used to determine whether specific periods in early life represent windows of vulnerability to mutation induction by genotoxins in mouse liver. Groups of mice were treated with single doses of 120 mg N-ethyl-N-nitrosourea (ENU)/kg body weight or the vehicle either transplacentally to the 18-day-old fetus or at postnatal days (PNDs) 1, 8, 15, 42 or 126; the animals were sacrificed 6 weeks after their treatment. The cII mutation assay was performed to determine the mutant frequencies (MFs) in the livers of the mice. Liver cII MFs for both sexes were dependent on the age at which the animals were treated. Perinatal treatment with ENU (either transplacental treatment to the 18-day-old fetus or i.p. injection at PND 1) induced relatively high MFs. However, ENU treatment at PNDs 8 and 15 resulted in the highest mutation induction. The lowest mutation induction occurred in those animals treated as adults (PND 126). For instance, the cII MF for the PND 8 female group was 646 x 10(-6) while the MF for female adults was only 145 x 10(-6), a more than 4-fold difference. Molecular analysis of the mutants found that A:T-->T:A transversions and A:T-->G:C transitions characterized the pattern of mutations induced by ENU in both the neonate and adult mice, while the predominate type of mutation in the controls was G:C-->A:T. The results indicate that mouse liver is most sensitive to ENU-induced mutation during infancy. This period correlates well with the age-dependent sensitivity to carcinogenicity in mouse liver, suggesting that mutation is an important rate-limiting factor for age-related carcinogenesis.     

Mutagenic properties of a nitrofuran, 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), in lacI transgenic mice

The in vivo mutagenic properties of a 5-nitrofuran, the 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), already well known in bacteria, was evaluated in lacI transgenic mice (Big Blue). The mutation frequency was determined in various organs of i.p. — treated mice and the nature of induced mutations was determined for the target organs in which mutation induction was significant. It was found that R7000 is mutagenic in mice, although, on the basis of the number of induced mutants per unit mass in comparison with other known mutagenic chemicals, R7000 appears to be considerably less mutagenic in mice than in bacteria. The most affected organs, small intestine, caecum and colon organs belong to the digestive apparatus. The distribution of R7000-induced mutations in the lacI gene recovered from small intestine of transgenic mice was very similar to that which had been found in E. coli. The difference between mouse and E. coli in the R7000 induced mutational spectra are mainly in the proportion of single base frameshifts versus base substitutions. Since R7000 induced mutations seemed to arise in the population of stem cells and that the stem cells are important for carcinogenesis, our results are compatible with a possible carcinogenic effect of R7000 and other nitrofurans.     

Evidence for NQO2-mediated reduction of the carcinogenic estrogen ortho-quinones

The physiological function of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase) is to detoxify potentially reactive quinones by direct transfer of two electrons. A similar detoxification role has not been established for its homologue NRH:quinone oxidoreductase 2 (NQO2). Estrogen quinones, including estradiol(E(2))-3,4-Q, generated by estrogen metabolism, are thought to be responsible for estrogen-initiated carcinogenesis. In this investigation, we have shown for the first time that NQO2 catalyzes the reduction of electrophilic estrogen quinones and thereby may act as a detoxification enzyme. ESI and MALDI mass spectrometric binding studies involving E(2)-3,4-Q with NQO2 clearly support the formation of an enzyme-substrate physical complex. The problem of spontaneous reduction of substrate by cofactor, benzyldihydronicotinamide riboside (BNAH), was successfully overcome by taking advantage of the ping-pong mechanism of NQO2 catalysis. The involvement of the enzyme in the reduction of E(2)-3,4-Q was further supported by addition of the inhibitor quercetin to the assay mixture. NQO2 is a newly discovered binding site (MT3) of melatonin. However, addition of melatonin to the assay mixture did not affect the catalytic activity of NQO2. Preliminary kinetic studies show that NQO2 is faster in reducing estrogen quinones than its homologue NQO1. Both UV and liquid chromatography-tandem mass spectrometry assays unequivocally corroborate the reduction of estrogen ortho-quinones by NQO2, indicating that it could be a novel target for prevention of breast cancer initiation.     

In vitro testing of an indirect mutagen (cyclophosphamide) with human leukocyte cultures: activation with liver microsomes and use of a dialysis bag.

A system of cell-mediated mutagenesis is described for the study of compounds which require metabolic activation to exert their cytotoxic and mutagenic effects. This system combines BHK21 cells for metabolism of the compounds and V79 cells as targets for mutagenesis. Using the two polycyclic hydrocarbon carcinogenesis benzo(a)pyrene and 7-methylbenz(a)anthracene we have shown that the hydrocarbon-DNA reaction which accompanies mutagenesis in the target cell is indistinguishable from that reported to occur in vivo and in primary cell cultures. Our results also support the view that a diol epoxide metabolite is responsible for the biological activity of benzo(a)-pyrene.The application of cell-mediated mutagenesis to the routine testing of suspect environmental chemicals for biological activity is discussed.     

Collaborative validation study of the in vivo micronucleus test using mouse colonic epithelial cells

The in vivo micronucleus test using mouse colonic epithelial cells was evaluated as the 11th collaborative study organized by the Collaborative Study Group on the micronucleus test (CSGMT) with three model chemicals that were known to induce chromosome damage in mouse colonic cells. Five laboratories participated in this validation study. All three model chemicals, i.e. 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), N-methyl-N-nitrosourea (MNU), and mitomycin C (MMC), induced micronucleated colonic epithelial cells in a 4-day exposure protocol in all participating laboratories. We confirmed that the present single cell suspension method could be used to detect the model chemicals as micronucleus inducers in mouse colonic epithelial cells. Advantages of this method are that experiments are easy to perform and that intact cells can be analyzed. The present study suggested that the colon micronucleus assay proposed here is useful for mechanistic studies of colon carcinogenesis.