Mutagenic and carcinogenic polyaromatic hydrocarbons (PAH) such as benzo[a]pyrene and 3-methylchoranthrene induce cellular cytochrome P450, and are metabolized by the P450. The metabolites bind to DNA bases, which lead to mutagenesis and carcinogenesis. On the other hand, some other toxic and carcinogenic hydrocarbons such as dioxins and polychlorinated biphenyls (PCB) induce the P450, but are not converted to mutagenic metabolites by the P450. The P450 induction is initiated by binding of these hydrocarbons to the cellular aryl hydrocarbon receptor (Ahr), followed by binding of the receptor to the xenobiotic responsive element that is located upstream the P450 gene. The level of activation of the Ahr pathway is roughly correlated with the toxicity of the aryl hydrocarbons. Here, we review the difference in primary structures of Ahr from vertebrates relative to their sensitivity to aryl hydrocarbons. We also describe our trial to establish recombinant yeasts for simply and rapidly measuring ligand-induced activation of Ahr from various vertebrates. Comparison in the activation of the Ahr pathway of different animals using the recombinant yeasts with the same genetic background is important for extrapolating animal data to humans.
The VitotoxTM test is a high-throughput bacterial genotoxicity test based on the SOS DNA-repair system induced by genotoxic compounds. Two genetically engineered Salmonella typhimurium strains are used in this system, TA104recN2-4 (Genox strain), that contains the bacterial luciferase (lux) operon (luxCDABE) under transcriptional control of recN promoter, and TA104 pr1 (Cytox strain), that constitutively expresses lux operon. The performance of the VitotoxTM test was evaluated with 33 known Ames positive chemicals, 26 known Ames negatives and 18 drug candidates developed at Mitsubishi Pharma Corporation. Ten compounds had inconclusive results because they caused SOS-independent enhancement of light emission. Among 49 known chemicals with conclusive results, 89% of the Ames positive compounds were detected as positive (genotoxic) with the VitotoxTM test, and all of the Ames negative compounds were detected as negative. There was a 94% concordance between the Ames test results and the VitotoxTM test results. In a practical validation study using 18 drug candidates developed at Mitsubishi Pharma Corporation, 7 of 8 Ames positive compounds were detected as genotoxic and all of the Ames negative compounds gave negative results with the VitotoxTM test. The concordance between the VitotoxTM test results and the Ames test results for 18 drug candidates was 94% (17/18). Moreover, the VitotoxTM test required a smaller sample quantity than the Ames test to detect genotoxicity. The present results indicate that the VitotoxTM test is useful for rapid screening of large numbers of chemicals when only a small quantity of a chemical is available.
We investigated improved methods for the preparation of metabolically activated forms of heterocyclic amines referred to as ‘activated heterocyclic amines’. We also described the influence of pH on the metabolic activation of Trp-P-2, and on the stability and mutagenicity of the activated form of Trp-P-2.
Some mutagens are inactivated rapidly by components included in culture media, especially by serum. Long incubation periods may not be appropriate for the comet assay because DNA lesions may be repaired during the time that mutagens are inactivated, leading to false negative results. We questioned how the exposure period of Chinese hamster ovary cells to 8 unstable mutagens affected outcome of the assay. Although the longest biological half-life of the test mutagens was 1.98 h, four were positive following 0.5—24 h incubations while other four were positive only when the incubation period was ≤ 4 h, suggesting that the DNA damage was repaired and the mutagens were inactivated. The rapid inactivation of mutagens in the medium did not affect whether the outcome of the comet assay was positive or negative when cells were exposed for 1—4 h. Based on these results, we concluded that long exposure should not be employed for the compounds that are unstable in culture media, and appropriate incubation time should be determined for them individually.
Gene disruption methods are useful to construct bacteria lacking a specific gene especially when a gene’s function is unknown. In the 1980s, complementation techniques were used as the first step in cloning a gene. With the advances made through genome projects, gene identification and cloning have allowed easier construction of deficient bacterial strains with cloned genes. In this report, I describe three methods of disrupting specific genes on chromosomes in a strain of interest, namely linear transformation, preligation and the ‘one-step’ method. Moreover, several genetic techniques which are necessary for conducting these methods are also reviewed.
Chromatophoroma in Nibea mitsukurii showed a unique geographic distribution in Japan. The epidemiological and laboratory studies demonstrated that the fish tumor was induced by several environmental mutagens/carcinogens which were discharged from industrial effluent. Especially, chlorinated acetones and dicarbonyl compounds were potential candidates as genotoxic compounds. After chlorine-disinfection of drinking raw water, many kinds of chlorinated organic compounds were formed. Among them, 3-chloro-4-dichloromethyl-5-hydroxy-2(5H)-furanone (MX) showed the highest mutagenic activity on S.typhimurium TA100 without metabolic activation. The distribution of MX in drinking water in Japan was determined. The precursor and DNA modification of MX were also studied. The mutagenic and carcinogenic potency of MX was observed by two-stage carcinogenicy test in rats. These experiments showed that environmental mutagens and carcinogens might be potential substances to induce certain tumors not only to aquatic animals but also human beings.
The mutational specificities of mutagens provide important information about the mechanisms of mutagenesis and DNA repair. For the purpose to investigate the mutational spectrum, the Lac+ reverse mutation system with a set of six specific tester strains have been employed. The mutational target is an F’ plasmid-encoded mutant lacZ461 allele (CC101 to CC106), by which each of the six possible base-pair substitution mutations can be independently detected by measuring Lac+ revertant colonies. Using this system, we investigated the mutational specificities of several carcinogenic heterocyclic amines and revealed that G:C→T:A transversions generated by heterocyclic amines such as Methyl-IQ and Trp-P-2 could be easily detected with clear dose-response relationships. We also demonstrated that a recA730lexA51 (Def) strain showed mutator activity generating A:T→T:A transversions most favorably. Compared with colony probe hybridization analysis or DNA sequencing analysis, one of the advantages of the Lac+ reverse mutation system is that very few transitions and transversions were quantitatively determined by this system. We introduced uvrA mutations to increase the sensitivity of strains CC101-CC106 to various mutagens. However, the sensitivity did not improved as we expected. We also found that cells carrying both F’ and pKM101 plasmids showed impaired growth in minimal medium. We, therefore, transferred the F’ plasmids from E. coli K-12 (CC101 to CC106) to a derivative of E. coli B/r WP2uvrA. To further increase the sensitivity to mutagens, we isolated their rfa derivatives. Since no obvious growth delay was observed in E. coli B cells containing F’ and pKM101, we could construct two sets of tester strains, WP3101P to WP3106P (uvrA, pKM101), and WP4101P to WP4106P (uvrA, rfa, pKM101). With these tester strains, more than 40 kinds of mutagens including photomutagenic compounds were analyzed for their mutational spectra. The strains we developed were highly sensitive to detect not only mutational specificity with a Lac+ reversion assay, but also mutagenicity with an ordinary Trp+ reversion assay. It is expected that the mutational spectra of chemical mutagens in bacterial cells with different genetic background for DNA repair would provide novel findings on mutagenesis and DNA repair.
The transgenic mouse mutation assay was developed as a striking new tool for mutation research in 1990. This assay enables the detection of mutations in a transgene in multiple organs including germinal tissues and thus reveals organ-specific genotoxicity of the mutagen. Following its introduction in MutaMouse and Big Blue mouse systems, modification of the methodology, mainly the introduction of the positive selection system and development of other transgenic animal models including rat, improved and assured the relevance of the assay. Accumulation of experimental data suggests the transgenic mouse mutation assay can be used as a standard in vivo test for mutagenesis. We have developed a multi-endpoint test, by combining the peripheral blood micronucleus assay with the transgenic mouse mutation assay. This test allows simultaneous detection of clastogenecity and mutagenecity in vivo. Since these two endpoints indicate different characteristics of the mutagen, data from many chemicals suggest the importance of detecting both endpoints. With this approach, the transgenic assay could detect the mutagenecity of diethylnitrosamine, which failed to be detected in micronucleus assay. Another important advantage of this assay is its suitability for sequence analysis. Sequencing of the transgene enables to draw mutagen-specific mutation spectrum, a molecular signature of the mutagen, and is very useful to deduce the mechanism of mutagenesis. In this regard, we have intensively used a positively selectable target gene ‘cII’. This gene is relatively short (300 bp) which made the sequencing process easier and less time consuming and enables us to generate data on mutagenesis of several mutagens. We hope the database will be useful for molecular epidemiology in future. A quantitative comparison of carcinogenic and mutagenic potency of chemicals revealed a good correlation with transgenic mutation assay and therefore suggesting a usefulness of this assay for the quantitative risk assessment.
Food antioxidants or reductones, ascorbic acid and its derivatives, monophenolics including α-tocopherol, polyphenolics in tea leaves and red wine, SH compounds including cysteine, carotenoids including β-carotene and other reductones, can scavenge reactive oxygen species, reactive nitrogen oxide species and lipid peroxy radicals. By contrast, these antioxidants or reductones can be converted into prooxidants in the presence of oxygen or act to produce harmful compounds. Formation of heterocyclic amine mutagens in the model system is inhibited by scavenging pyrazine cation radical with monophenolics, polyphenolics, ascorbate and SH compounds, and the mutagenicity in cooked hamburger is effectively inhibited by ascorbate. Formation of nitrosamines is effectively inhibited by ascorbate, but inhibited or stimulated by polyphenolics and monophenolics. Monophenolics are converted into possibly harmful nitroso-, nitro- and diazo-compounds by reaction with N2O3. Hydroxyfuranones found in the Maillard reactions and hydroxyhydroquinone found in coffee cause DNA single strand breaks by generation of reactive oxygen species, and cause lung lipid peroxidation and increased type IV and I allergy responses by oral administration to experimental animals. On the other hand, food polyunsaturated fatty acids, which has been so far considered to give deteriorate effect, are found to be protective against oxidative stress-induced DNA damage.
Because of the recent advances in the acquisition of human materials for research in addition to the value in the evaluation of the mutagenicity in humans, the use of human S9 fractions in the Ames test is starting to attract attention. However, until recently, available data on the mutagenicity with human S9 fractions has been limited. We have thus accumulated a large and extensive body of data on the Ames test with human S9 fractions during the last 5 years. In this report, these data are reviewed, and the utility of the human S9 fractions in mutagenicity testing systems is discussed.
Substances causing persistent inflammation (asbestos, viruses and pathogens) are often carcinogenic even if they are not directly mutagenic. Reactive oxygen species from inflammatory cells injure DNA and are cell-proliferative leading to accelerated carcinogenesis. Lipid mediators in the linoleic acid (LA) cascade through arachidonic acid (n-6) and some cytokines form amplification cascades to stimulate these processes, whereas the fatty acids of n-3 type competitively suppress the LA cascade and carcinogenesis. This interpretation is consistent with the observations that (1) dietary oils with low n-6/n-3 ratios are suppressive compared with high-LA oils, (2) inhibitors of the LA cascade are suppressive, and (3) manipulations to knockout genes related to the LA cascade are suppressive for carcinogenesis. On the other hand, many kinds of genes are affected differently by the chain length and unsaturation of fatty acids regardless of the n-6 or n-3 type. Saturates, monounsaturates and LA up-regulate cholesterol synthesis leading to enhanced prenylation of oncogene products, cell-proliferation and carcinogenesis. Dietary cholesterol and high tissue cholesterol levels feedback suppress cholesterol synthesis and cell-proliferative stimuli, which partly accounts for epidemiological observations that cancer mortality is lower in the group with higher cholesterol level. For the cancers, the incidence of which is high in the US and has been increasing rapidly in Japan, reducing the intake of LA to half while maintaining those of n-3 fatty acids and animal fats at the levels of average Japanese is recommended.