Search

As demonstrated in Part I, cultured MutaMouse primary hepatocytes (PHs) are suitable cells for use in an in vitro gene mutation assay due to their metabolic competence, their "normal" phenotype, and the presence of the MutaMouse transgene for reliable mutation scoring. The performance of these cells in an in vitro gene mutation assay is evaluated in this study, Part II. A panel of 13 mutagenic and nonmutagenic compounds was selected to investigate the performance of the MutaMouse PH in vitro gene mutation assay. The nine mutagens represent a range of classes of chemicals and include mutagens that are both direct-acting and requiring metabolic activation. All the mutagens tested, except for ICR 191, elicited significant, concentration-dependent increases in mutant frequency (MF) ranging from 2.6- to 14.4-fold over the control. None of the four nonmutagens, including two misleading, or "false," positives (i.e., tertiary butylhydroquinone [TBHQ] and eugenol), yielded any significant increases in MF. The benchmark dose covariate approach facilitated ranking of the positive chemicals from most (i.e., 3-nitrobenzanthrone [3-NBA], benzo[a]pyrene [BaP], and aflatoxin B 1 [AFB1]) to least (i.e., N-ethyl-N-nitrosourea [ENU]) potent. Overall, the results of this preliminary validation study suggest that this assay may serve as a complimentary tool alongside the standard genotoxicity test battery. This study, alongside Part I, illustrates the promise of MutaMouse PHs for use in an in vitro gene mutation assay, particularly for chemicals requiring metabolic activation. Environ. Mol. Mutagen. 60:348-360, 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society., (© 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.)

Several studies suggest that MutaMouse is insensitive to clastogens, including the accompanying paper by Mahabir et al., which describes a study with bleomycin, camptothecin, m-AMSA (4'-(9-acridinylamino)-methanesulfon-m-anisidide) and its ortho-analogue, o-AMSA (4'-(9-acridinylamino)-methanesulfon-o-anisidide). Only camptothecin was clastogenic in MutaMouse and none of these four compounds induced mutations at the lacZ locus. However, to improve exposure, dose range-finding studies were performed in CD2F1 mice, the parental strain of MutaMouse. Male CD2F1 mice (n=3) were treated with bleomycin (25-100 mg/kg bw, p.o. and i.p.), camptothecin (1-10 mg/kg bw p.o.) and m-AMSA (10-50mg/kg bw p.o. and 1-5 mg/kg bw i.p.) for 5 days and blood was sampled on day 3 and/or day 6 for analysis by flow cytometry to determine % MN-RETs. Camptothecin (1 mg/kg bw, day 6) induced a 3.6-fold increase in % MN-RET (P<0.05) but was toxic at higher doses. All day-3 camptothecin samples were positive (P<0.05). Bleomycin was negative when administered p.o. but positive at all doses on both days when given i.p. (P<0.05) whereas m-AMSA was negative when given i.p. or orally. Based on these results, male MutaMouse mice (5 per group) were dosed daily with bleomycin (50 mg/kg bw) for 5 days or with camptothecin (5 mg/kg bw) for 2 days. Peripheral blood was sampled 24 h after the final dose in each group and tissues were sampled 37 days later. Both compounds induced significant increases in % MN-RET, but only bleomycin induced a significant increase in MF (6-fold in liver, 4.5-fold in kidney and 2-fold in lung) compared with the untreated control. These studies support the view that MutaMouse is insensitive to compounds where the genotoxic mechanism of action is predominantly clastogenesis, but demonstrates that the peripheral blood micronucleus test is a useful adjunct to the transgenic gene-mutation assay.

The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described. Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots. Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity. The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria. MucAB was cloned into the test plasmid to enhance error-prone DNA-repair. The conventional reversion principle has been combined with the luminometric measurement of an inducible reporter gene. The revertants are detected after induction of the beta-galactosidase-producing lacZ-gene either controlled by its natural lac-promotor or by the more stringently repressed (anhydrotetracyclin inducible) tetA promotor. The tester strains containing the tetA/lacZ reporter gene construct can grow in full medium over the complete assay. This test procedure enables screening for mutations within one working day. Incubation for 16 h reveals high sensitivity., (Copyright 2002 Elsevier Science B.V.)

The mutational specificities of DNA cross-linking compounds such as cisplatin, transplatin, carboplatin, mitomycin C, psoralen, and 8-methoxypsoralen were investigated in lacZ reversion assay systems of Escherichia coli. Tester strains were constructed by introducing the six kinds of F' plasmids (lacI-, lacZ461, and proAB+), each of which carries a different base-substitution mutation within the lacZ gene. Each of the six possible base-substitution mutations was assayed by Lac+ reversion. Cisplatin induced G.C-->A.T transitions and G.C-->T.A transversions, with the former predominating. Transplatin induced A.T-->G.C transitions in addition to G.C-->A.T transitions and G.C-->T.A. Carboplatin weakly induced G.C-->A.T transitions. On the other hand, mitomycin C induced only G.C-->T.A transversions, while psoralen and 8-methoxypsoralen reactivated with near-UV irradiation induced A.T-->G.C transitions preferentially. The Lac(+) reversion system was very convenient for rapidly determining mutational spectra., (Copyright 2001 Wiley-Liss, Inc.)

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.

We previously reported the development of mutation-specific Escherichia coli B tester strains WP3101 to WP3106 from strain WP2uvrA. In this study we constructed their pKM101-containing derivatives WP3101P to WP3106P, and further isolated their rfa derivatives WP4101-WP4106 and WP4101P-WP4106P. The six kinds of F' plasmids (lacI-, lacZ-, proAB+), each of which carries a different lacZ allele, contained in the above strains were originally derived from E. coli K-12 strains CC101-CC106. All the tester strains show Lac- and Trp- phenotype. Assays for transitions and transversions are based upon Lac+ reversion of a specific mutation located within the lacZ gene on an F' plasmid. The trpE65(ochre) allele in the same strains enables them to be used for Trp+ reversion assays as well. In the present paper, we evaluated the sensitivity, specificity, and usefulness of the newly developed tester strains. Strains WP3101P-WP3106P were highly sensitive to determine mutational profile of heterocyclic amines with S9 mix-mediated metabolic activation and most of the oxidative mutagens and free radical generators tested. Every type of base-pair substitutions induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) or 5-diazouracil were detected in strains WP3101P-WP3106P, while A:T-->C:G and G:C-->A:T mutations induced by MeIQ, and A:T-->C:G, G:C-->A:T, and G:C-->C:G by 5-diazouracil were not detected in pKM101-free tester strains. In pKM101-carrying strains, cumene hydroperoxide induced all types of base substitutions, while formaldehyde preferentially induced G:C-->T:A transversions. Phenazine methosulfate induced predominantly G:C-->A:T transitions and G:C-->T:A transversions, while H2O2 induced predominantly G:C-->T:A and A:T-->T:A transversions. Introduction of the rfa mutation considerably enhanced sensitivity to bulky mutagens such as polycyclic aromatic compounds. All six possible base substitutions induced by 9, 10-dimethyl-1,2-benzanthracene (DMBA) were detected in tester strains WP4101P-WP4106P. In conclusion, our tester strains WP3101P-WP3106P and WP4101P-WP4106P permitted rapid and simple detection of specific mutations induced by variety of mutagens., (Copyright 1999 Elsevier Science B.V.)

Benzo[a]pyrene (BP) has been investigated for the ability to induce mutation at the site of contact. Skin painting treatments with BP caused a time-dependent and statistically significant increase in mutation frequency (MF) in the treated areas of skin. The MF exceeded 500 x 10(-6) 21 days after either 1 x 25 or 5 x 5 micrograms treatments. Increases to > 700 x 10(-6) were seen when doses of 1 x 50 or 5 x 10 micrograms were used. Neither the liver nor the lung showed any increase in mutation frequency after 21 days in animals exposed to the 5 x 10 micrograms treatment regime. It is concluded that following topical administration, BP is able to induce mutation in the skin at the site of application, but not in either the lung or liver.

Transgenic mouse assays have provided an unprecedented opportunity to study mutagenesis in diverse rodent tissues. In this article data from MutaMouse mutagenicity assays based on the Escherichia coli gene lacZ were analyzed systematically using liver and bone marrow as potential target tissues. Sources of variation, including plates (within packaging reactions), packaging reactions (within animals) and animals, were evaluated for extra-binomial variation. Although hardly any evidence of overdispersion was detected at the plate level, limited evidence of extra-binomial variation was observed at the packaging reaction level. There was, however, much stronger evidence of overdispersion at the animal level. Statistical tests for increasing trend in mutant frequency with increasing dose were also performed at the animal level. A significant increasing trend following exposure to N-nitrosodibenzylamine was detected in liver but not in bone marrow. A logistical model was used to further describe the dose-response relationship observed in N-nitrosodibenzylamine-treated liver tissue.

We have developed a set of multipurpose tester strains (WP3101 to WP3106) derived from E. coli WP2uvrA for the detection and classification of mutagens. Six kinds of F' plasmid (lacI, lacZ, proAB+) in strains CC101-CC106, each of which carried a different lacZ allele, were transferred to a delta(lac-pro) derivative of WP2uvrA. Assays for transitions and transversions are based upon Lac+ reversion of a specific mutation located within the lacZ gene on an F' plasmid in strains WP3101-WP3106. In addition, the trpE65(ochre) allele in the same strains is available for Trp+ reversion assays. Using the new tester strains, we investigated the mutational specificities of various chemical mutagens. Base analog mutagens and alkylating mutagens induced specific types of base substitutions. G:C-->A:T transitions and G:C-->T:A transversions predominated in mutagenesis induced by 4-nitroquinoline 1-oxide. Only a slight increase in G:C-->T:A transversions was observed in cells treated with 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), although the potent mutagenicity of AF-2 was detected in a concurrent Trp+ reversion assay in the same strain. Sodium azide, on the other hand, was negative in the Trp+ reversion assay but specifically induced G:C-->A:T transitions. Present finding suggested that target sites for AF-2- and azide-induced lesions may largely depend on sequence context.

Quinoline is carcinogenic to the liver of rats and mice and mutagenic to bacterial tester strains in the presence of rat liver microsomal enzymes. The unscheduled DNA synthesis (UDS) study suggested that quinoline might be a non-genotoxic carcinogen because of the lack of UDS-inducing capacity. In order to determine whether or not cancer induction is initiated by mutagenic DNA lesions, the present study was undertaken to evaluate the mutagenicity of quinoline in an in vivo mutation assay system using the lac Z transgenic mouse (Muta Mouse). Mutation was only induced in the liver, the target organ of carcinogenesis by quinoline, but not in the other organs examined, i.e. lung, kidney and spleen. Mutant frequency in the liver was 4-fold higher than in the untreated control animals. Dimethylnitrosamine, used as a positive control, induced mutation at a frequency 5-fold higher in the liver and 3-fold higher in the spleen than in their respective control organs. It can be concluded that the genotoxicity of quinoline is responsible for its hepatocarcinogenesis, although UDS was not induced under the conditions previously reported.

The establishment in recent years of transgenic shuttle vector-based mutagenicity assays has provided improved systems for analysis of mutagenic and carcinogenic processes. Results in the mouse have stimulated the development of an alternate species suitable for mutation analysis and have increased our understanding of the existing models. A previously described shuttle vector (lambda LIZ), based on a lacI target gene, was constructed in this laboratory for the study of mutagenesis in transgenic mice and in cultured cell lines. The shuttle vector allows for several options in its recovery from the host genome and in mutant identification. Of the 9 transgenic lineages that were generated with the lambda LIZ vector, one was chosen for use in a standardized mutagenicity assay (Big Blue, mouse lineage A1). Characterization of this lineage included copy-number determination, chromosomal localization of transgene integration and analysis of copy-number stability. As part of the validation process, the standardized color-screening assay has been tested in the mouse, both for spontaneous mutant frequencies and with a variety of model mutagenic compounds, and has been shown to identify most major classes of mutations as evidenced by mutant spectra data. A discussion of the relative sensitivity of the shuttle vector to each of these classes of mutations is included. These studies have now been extended to the generation of transgenic rats containing the same shuttle vector for cross-species analysis. Spontaneous mutant frequencies in two transgenic rat lineages were measured in liver and in germ cells. Preliminary data suggest that spontaneous mutant frequencies in somatic tissue are lower in rats than in mice, a result consistent with historical observations of DNA damage and repair in these two species. Also under evaluation are alternative selectable systems for mutant identification, and hybrid animals obtained from mating lambda LIZ transgenics with genetically engineered mice possessing an inactivated tumor suppressor gene. It is expected that each of these widely varying endeavors will contribute, not only in furthering our understanding of the role transgenic systems should play in human risk assessment, but in illuminating the mechanisms of mutation in general.

It is well established that mutagens induce recombination in cultured cells and experimental organisms. Presumably, this is a consequence of the DNA-damage-triggering cellular-repair mechanisms. The relationship between recombination and mutagenicity has been exploited in submammalian organisms, such as yeast, to assay the ability of chemical agents and radiation to induce a form of recombination called gene conversion--the non-reciprocal transfer of genetic information. This work has demonstrated the efficacy of predicting mutagenicity on the basis of recombination induction. Here, we describe the utilization of a transgenic mouse system for efficient detection of germ-line gene-conversion events as a mutagen-screening tool. These mice contain two mutually defective reporter (lacZ) genes under the regulatory control of a spermatogenesis-specific promoter. A particular intrachromosomal gene conversion event must occur for the generation of functional lacZ activity. Conversion events are visualized by histochemical staining or flow cytometric analysis of transgenic spermatids. The highly mutagenic compound chlorambucil induced a several fold percentage-wise increase of lacZ-positive spermatids, whereas acrylamide, a weak genotoxin, produced no marked increase in converted spermatids. The results indicate that recombination-based transgenic mouse models for genotoxin screening present a viable option for inexpensive and rapid whole-animal mutagen testing. The particular mice we describe may ultimately prove to be a useful tool for identifying agents which can cause heritable genetic mutations in humans.

Transgenic mice have introduced new possibilities in the field of mutation research and safety testing. Using lacZ transgenic mice (Muta Mouse), we have combined the peripheral blood micronucleus assay with the transgenic mouse mutation assay, enabling the concomitant detection of gene mutations and micronucleus induction in vivo in the same animals (Suzuki et al., 1993). Several mutagens, i.e., mitomycin C (MMC), ethyl nitrosourea (ENU), ethyl methanesulfonate (EMS) and diethyl nitrosamine (DEN), were tested in this combined assay. All of them increased the lacZ mutant frequency in bone marrow or liver, and all except DEN induced micronuclei in peripheral blood. These initial studies demonstrated that genotoxicity in vivo could be detected with these two endpoints and, more importantly, that some specificity exists among these tissues analyzed. Although transgenic mouse mutation assays have many potential applications in in vivo mutation research, several problems stand in the way of wider use. Paramount among these are cost and labor intensiveness. The color screening systems for lacZ or lacI mutation detection require large numbers of plates and tedious scoring processes. In order to make significant advances in this field, it will be necessary to use positive selection for induced mutants, such as has been described recently for the lacZ and lacI transgenic mouse models.

Streptozotocin has been reported to induce DNA damage in mouse liver although malignant tumors were not induced in this organ. DNA damage had not yet been monitored in the mouse kidney which was the tumor target organ in two mouse studies. In order to elucidate target organ specificity of genotoxicity and of tumorigenesis, we investigated the induction of DNA damage (microgel electrophoresis assay) and mutations (LacI transgenic mouse mutation assay) in the liver and kidney of male C57BL/6 mice. Our results show that the microgel electrophoresis assay was more sensitive and revealed the genotoxic potential of streptozotocin at lower doses than the mutation assay. It was, however, less specific in that DNA damage was induced both in target and non-target tissues of carcinogenesis at a similar potency. In contrast, the mutation analysis revealed the kidney to be more sensitive when the induced mutation frequencies are expressed as a multiple of the respective spontaneous rates. We conclude, therefore, that the carcinogenic organotropy of streptozotocin correlates better with its tissue-specific mutagenicity than with its pattern of inducing DNA damage when the two in vivo genotoxicity assays mentioned above are used. A combined use of the microgel electrophoresis assay and the transgenic mouse mutation assay is proposed for investigations of tissue-specific genotoxicity.

The use of transgenic animals for in vivo mutagenicity studies following rescue of the bacterial lacZ transgene has been hindered by the sheer scale of the experimental work involved. We describe here a new positive selection protocol which is based upon a modified E. coli bacterial host. This new system is potentially capable of generating mutation data much faster and more cheaply than previous methods.

Experimental features of a transgenic mouse mutation assay based on a lacI target transgene from Escherichia coli are considered in detail. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined with the goal of identifying sources of excess variation in the observed mutant fractions. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal (within study) variability. Data from two laboratories are evaluated, using various statistical methods to identify excess variability. Results suggest only scattered patterns of excess variability, except possibly in those cases where genomic DNA from test animals is stored for extended periods (e.g., > 90 days) after isolation from tissues. Further study is encouraged to examine the validity and implications of this time/storage-related effect.