Transgenic rat gene-mutation assays can be used to assess genotoxicity of chemicals in target organs for carcinogenicity. Since gene mutations in transgenes are genetically neutral and thus accumulate along with treatment periods, the assays are suitable for genotoxicity risk assessment of chemicals using repeated-dose treatment methodologies. However, few studies have been conducted to examine the suitability of the assays in repeat-dose treatment protocols. In order to prove the utility of the transgenic rat assays, we treated gpt delta rats with aristolochic acid at 0.3 and 1 mg/kg by gavage daily for 28 days, and autopsied the rats 3 days after the final treatment, which is a protocol recommended by the International Workshop on Genotoxicity Testing (IWGT). Aristolochic acid exists in herbs and some other plants, and is carcinogenic in the kidney, bladder and stomach in rats. The mutant frequency (MF) in both the kidney and the liver increased significantly in a dose-dependent manner when the rats were treated with aristolochic acid. We concluded that the gpt delta rat assay is sensitive enough to detect gene mutations induced by aristolochic acid and also that the 28-day repeated-dose protocol is suitable for assessing genotoxicity of chemicals.

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A perceived shortcoming of transgenic rodent mutation assays is the relatively high spontaneous mutant frequencies (MFs) of the bacterial transgenes compared to endogenous genes. This background is dominated by G:C→A:T mutation, frequently in a CpG context, mammalian sites of cytosine methylation. The single A:T target site of the ΦX174 transgenic mouse reversion assay might avoid this background, yet in vivo mutagenic sensitivity at this site was poor because of background mutation in the recovery bacteria. In order to determine the actual spontaneous MF of the ΦX174 transgene in the mouse cell, several research tools have been developed: 1) single burst analysis, for distinguishing mouse from bacterial mutation; 2) a transgenic mouse embryonic cell line, PX-2; and 3) a forward mutational assay, which has few CpG sites among its target sites. In this study, single burst analysis was applied to the transgenic cell line for the first time, evaluating the response to UVB irradiation for potential phototoxicity studies. Under appropriate plating conditions, single burst analysis lowered the spontaneous MF10-fold from the original report to 0.17×10^-5. The MF 72 h after 70 J/m² UVB irradiation was 8.3×10^-5. The characteristic UVB-induced mutant spectrum included >80% G:C→A:T at dipyrimidine sites (primarily TpC dinucleotides) with 13% of these at a single CpG site, and 20% as multiple mutants, tandem and non-tandem. The spontaneous MF per nucleotide, 3×10^-8, was comparable to that of human disease genes in the germline. When normalized for target number and dose, single burst analysis of the ΦX174 forward mutational assay produced a UVB-induced MF that was equivalent to that of the cII transgene in mouse cell culture, but a spontaneous MF an order of magnitude lower. The results suggest this cell line is highly sensitive to UVB irradiation.

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This study was conducted to evaluate the effectiveness of a transgenic rat mutation assay using F344 gpt delta rats. We investigated the mutagenic potential in the lung of nickel subsulfide (Ni₃S₂), an insoluble fine-crystalline-metallic compound and a carcinogen in the rodent and human lung. Ni₃S₂ carcinogenicity has been proposed to act via both genotoxic and non-genotoxic mechanisms. Ni₃S₂ was intratracheally instilled into male gpt delta rats at doses of 0.5 and 1 mg/animal once a week for four weeks; these doses of Ni₃S₂ are high enough to induce inflammation in the lung. Following a period of 28 and 90 days after the first administration, the gpt mutant frequencies (MFs) in lung were determined in four independent laboratories, and Spi⁻ selection for larger deletion mutations was done in one laboratory. The gpt MFs of the rats treated with Ni₃S₂ were not increased: all four laboratories obtained similar results with no statistical differences. The Spi⁻ MFs were also not increased by exposure to Ni₃S₂. These results indicate that intratracheally instilled Ni₃S₂ is non-mutagenic in the lung of gpt delta transgenic rats; however, whether Ni₃S₂ is non-mutagenic in the lung or it induces mutations which are not detectable by transgenic rodent mutation assays requires further investigation.

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Transgenic rodent mutation assays are useful models for investigating the genotoxicity of chemicals in vivo. Transgenic gpt delta mice contain multiple copies of chromosomally integrated lambda EG10 phage shuttle vector, which contains reporter genes that allow detection of mutations. This system can identify both point mutations by the gpt assay (6-thioguanine selection) and certain types of deletions using the Spi⁻ assay. Transgenic gpt delta rats, which have the same lambda EG10 DNA copies as gpt delta mice, have also been developed. The average spontaneous gpt mutant frequency (MF) in both gpt delta mice and rats is approximately 4.5×10^-6. In the Spi⁻ assay, the average spontaneous Spi⁻ MF is approximately 2.7×10^-6 in gpt delta mice, similar to that of gpt delta rats. More than 20 chemicals and irradiations have been analyzed with these systems, and this review summarizes the MFs and treatment conditions. The data demonstrate that these transgenic rodent models are useful for detection and analysis of point mutations and deletions in vivo.

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The emerging Pig-a gene mutation assay, a powerful and promising tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs), which are deficient in glycosylphosphatidylinositol anchored protein. Various approaches for measuring Pig-a mutant cells have been developed, particularly those focused on peripheral RBCs and reticulocytes (RETs). Previously, it had been reported that Pig-a and gpt mutant frequencies were relatively increased in N-ethyl-N-nitrosourea (ENU)- and benzo[a]pyrene (BP)-treated mice. The capacity and characteristics of the Pig-a assay relative to transgenic rodent (TGR) mutation assays, however, are unclear in rats. Here, using transgenic gpt delta rats, we compared the in vivo genotoxicity of single oral doses of ENU (40 mg/kg) in the gpt gene mutation assay in bone marrow and liver, and Pig-a gene mutation assays on RBCs and RETs in the same animals. The Pig-a gene mutation assays were conducted at 1, 2, and 4 weeks after treatment, whereas gpt assays were conducted on tissues collected at the 4-week terminal sacrifice. Consequently, we detected that Pig-a and gpt mutant frequencies were clearly increased in ENU-treated rats, indicating that both the Pig-a and TGR gene mutation assays can detect in vivo ENU genotoxicity equally.

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SMD-502, a new vitamin D₃ (VD₃) analog, is an antipsoriatic drug candidate. The compound exhibited fewer side effects than known VD₃ analogs in animal models, probably because the compound is rapidly converted into pharmacologically inactive form after permeating into systemic circulation from the application site on skin. This feature of the compound makes it difficult to assess genotoxic risk in vivo with the standard approach of bone marrow or peripheral blood micronucleus assays in rodents because of the low blood concentration level. To evaluate in vivo mutagenicity of the compound in the present study, mutant frequency (MF) in skin and liver of gpt delta transgenic mice was examined with percutaneous administration of SMD-502 for 28 days. In tissues collected 7 days after the end of administration, no significant increase in the MF was observed in either skin or liver. Additionally, when the compound was tested in a GDL1 cell line established from gpt delta mice, GDL1 cells exhibited no significant increase in MFs even under conditions in which they would be exposed to a much higher concentration of the compound than in the in vivo study. The results in this study further supported the consideration that SMD-502 has no mutagenic activity.

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The Pig-a gene is involved in the synthesis of glycosylphosphatidylinositol (GPI) anchors. Pig-a gene mutations can be detected by identifying the presence of CD59, the GPI anchor protein, on the surface of erythrocytes (RBC Pig-a assay) and reticulocytes (PIGRET assay) and can be identified using flow cytometry. The usefulness of these Pig-a gene mutation assays has been confirmed in multi-laboratory trials with referenced mutagens. Although 4,4′-methylenedianiline (MDA) is an aromatic amine and has been identified as a potent hepatic carcinogen, in vivo micronucleus tests for MDA in hematopoietic cells determined that it was negative to weakly positive for genotoxicity. In the present study, we examined the mutagenicity of MDA in the peripheral blood of rats after 1- and 28-day MDA dosing using the Pig-a gene mutation assays. We also examined the utility of the RBC Pig-a and PIGRET assays. No changes in mutation frequency were observed after one-day MDA administration. Repeated dosing caused a moderate increase in mutation frequency compared to vehicle control at days 14 and 28, as measured by the RBC Pig-a assay and at day 14 by the PIGRET assay. The highest mutation frequency was found on days 7 and 14 by the PIGRET and RBC Pig-a assays, respectively.
In this study, we detected the mutagenicity of MDA in peripheral blood samples using gene mutation assays and judged to be positive for the MDA mutagenicity since a significant increase in mutation frequency was observed at high dose. These assays are expected to be easily integrated into general toxicity tests and to be combined with existing genotoxicity studies.

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