Urinary crystal formation and urothelial effects of pyroxasulfone administered to male rats.

Pyroxasulfone induced a low incidence of urinary bladder tumors in male rats in a 2-year bioassay at 1000 and 2000 ppm, with occasional urinary calculi. No increased incidence of tumors of any tissue occurred in female rats or in mice of either gender. We performed three short-term studies to evaluate early development of pyroxasulfone-induced urinary crystals and urothelial cytotoxicity with consequent regenerative proliferation. First, male rats were treated with dietary 50, 1000 or 2000 ppm pyroxasulfone for 1, 3 or 7 days. The urothelium was examined by light and scanning electron microscopy (LM, SEM) and bromodeoxyuridine labeling index (BrdU LI). In two other studies, male rats were treated with dietary 20 000 ppm pyroxasulfone for 1 week. Urine collected at various times of day was examined by SEM and energy dispersive spectroscopy (EDS) or by LM, SEM, EDS, and infrared spectroscopy (IFS). Urinary crystals were present at various time points. EDS and IFS showed some contained calcium; others contained organic matter. Cytotoxicity was detected by SEM as cellular swelling, craters, and necrosis and by LM as cellular hypertrophy. Increased cell proliferation was detected by LM (hyperplasia), SEM (piling up of round cells), and by increased BrdU LI. There was no evidence of increased apoptosis. These findings support a mode of action for pyroxasulfone-associated bladder tumors in male rats involving formation of urinary crystals leading to urothelial cytotoxicity and regenerative proliferation. This is a high dose phenomenon, therefore, pyroxasulfone is not likely to be carcinogenic to humans at exposure levels that do not cause crystals with subsequent calculi formation in the urinary tract.

The effect of different methods and image analyzers on the results of the in vivo comet assay.

INTRODUCTION: The in vivo comet assay is a widely used genotoxicity test that can detect DNA damage in a range of organs. It is included in the Organisation for Economic Co-operation and Development Guidelines for the Testing of Chemicals. However, various protocols are still used for this assay, and several different image analyzers are used routinely to evaluate the results. Here, we verified a protocol that largely contributes to the equivalence of results, and we assessed the effect on the results when slides made from the same sample were analyzed using two different image analyzers (Comet Assay IV vs Comet Analyzer). FINDINGS: Standardizing the agarose concentrations and DNA unwinding and electrophoresis times had a large impact on the equivalence of the results between the different methods used for the in vivo comet assay. In addition, there was some variation in the sensitivity of the two different image analyzers tested; however this variation was considered to be minor and became negligible when the test conditions were standardized between the two different methods. CONCLUSION: By standardizing the concentrations of low melting agarose and DNA unwinding and electrophoresis times between both methods used in the current study, the sensitivity to detect the genotoxicity of a positive control substance in the in vivo comet assay became generally comparable, independently of the image analyzer used. However, there may still be the possibility that other conditions, except for the three described here, could affect the reproducibility of the in vivo comet assay.

The PIGRET assay, a method for measuring Pig-a gene mutation in reticulocytes, is reliable as a short-term in vivo genotoxicity test: Summary of the MMS/JEMS-collaborative study across 16 laboratories using 24 chemicals.

The in vivo mutation assay using the X-linked phosphatidylinositol glycan class A gene (Pig-a in rodents, PIG-A in humans) is a promising tool for evaluating the mutagenicity of chemicals. Approaches for measuring Pig-a mutant cells have focused on peripheral red blood cells (RBCs) and reticulocytes (RETs) from rodents. The recently developed PIGRET assay is capable of screening >1×106 RETs for Pig-a mutants by concentrating RETs in whole blood prior to flow cytometric analysis. Additionally, due to the characteristics of erythropoiesis, the PIGRET assay can potentially detect increases in Pig-a mutant frequency (MF) sooner after exposure compared with a Pig-a assay targeting total RBCs (RBC Pig-a assay). In order to test the merits and limitations of the PIGRET assay as a short-term genotoxicity test, an interlaboratory trial involving 16 laboratories was organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagenicity Society (MMS/JEMS). First, the technical proficiency of the laboratories and transferability of the assay were confirmed by performing both the PIGRET and RBC Pig-a assays on rats treated with single doses of N-nitroso-N-ethylurea. Next, the collaborating laboratories used the PIGRET and RBC Pig-a assays to assess the mutagenicity of a total of 24 chemicals in rats, using a single treatment design and mutant analysis at 1, 2, and 4 weeks after the treatment. Thirteen chemicals produced positive responses in the PIGRET assay; three of these chemicals were not detected in the RBC Pig-a assay. Twelve chemicals induced an increase in RET Pig-a MF beginning 1 week after dosing, while only 3 chemicals positive for RBC Pig-a MF produced positive responses 1 week after dosing. Based on these results, we conclude that the PIGRET assay is useful as a short-term test for in vivo mutation using a single-dose protocol.

Evaluation of the in vivo mutagenicity of melamine by the RBC Pig-a assay and PIGRET assay.

The Pig-a assay is a new in vivo genotoxicity test for detecting mutagens in the bodies of animals, using the endogenous Pig-a gene as the target. There are two types of Pig-a assays: the red blood cell (RBC) Pig-a assay, which uses RBCs, and the PIGRET assay, which uses reticulocytes. The Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group collaborative study of the Pig-a assay was carried out to investigate the usefulness of the PIGRET assay. The mutagenicity of melamine was evaluated as part of this study. Eight-week-old male Crl:CD (SD) rats were administered a single gavage dose of melamine as a non-genotoxic bladder carcinogen. Blood samples were collected at the first, second and fourth weeks after administration, and the RBC Pig-a assay and PIGRET assays were conducted using these samples. Three dose levels were used in the study: the highest dose was 2000mg/kg, which is generally used as the maximum dose in in vivo genotoxicity testing, and 1000 and 500mg/kg were also used. As a positive control, a group of rats was administered a single dose of N-nitroso-N-ethylurea (ENU) by gavage at 40mg/kg. The Pig-a mutant frequencies (Pig-a MFs) did not increase in any of the melamine groups throughout the experimental period in either the RBC Pig-a assay or the PIGRET assay. Both the RBC Pig-a and PIGRET assays revealed significant increases in the Pig-a MFs in the ENU group, starting at day 7 after a single administration. Therefore, these two assays, when evaluated after a single administration, can be used to determine that melamine is non-mutagenic.

Measuring reproducibility of dose response data for the Pig-a assay using covariate benchmark dose analysis.

The reproducibility of the in vivo Pig-a gene mutation test system was assessed across 13 different Japanese laboratories. In each laboratory rats were exposed to the same dosing regimen of N-nitroso-N-ethylurea (ENU), and red blood cells (RBCs) and reticulocytes (RETs) were collected for mutant phenotypic analysis using flow cytometry. Mutant frequency dose response data were analysed using the PROAST benchmark dose (BMD) statistical package. Laboratory was used as a covariate during the analysis to allow all dose responses to be analysed at the same time, with conserved shape parameters. This approach has recently been shown to increase the precision of the BMD analysis, as well as providing a measure of equipotency. This measure of equipotency was used here to demonstrate a reasonable level of interlaboratory reproducibility. Increased reproducibility could have been achieved by increasing the number of cells scored, as this would reduce the number of zero values within the mutant frequency data. Overall, the interlaboratory trial was successful, and these findings support the transferability of the in vivo Pig-a gene mutation assay.

Genotoxic assessment of small mammals at an illegal dumpsite at the Aomori-Iwate prefectural boundary.

In 2003, we examined the chromosomes of grass voles at an illegal dumpsite at the Aomori-lwate prefectural boundary. In subsequent years, from 2003-2006, we surveyed the chromosomes of four species of small mammals, namely, the Japanese grass vole (Microtus montebelli), the large Japanese field mouse (Apodemus speciosus), the small Japanese field mouse (A. argenteus), and the greater Japanese shrew mole (Urotrichus talpoides). Each annual survey revealed, both on a yearly basis and during the entire period in question, that the frequencies of breaks and gaps in chromosomes of M. montebelli were significantly higher at the dumpsite than on the outskirts and in controls, suggesting that grass voles at the dumpsite have been subject to continuous genotoxic effects since the establishment of the dumpsite. We also ascertained that grass voles are much more susceptible to chromosomal damage than field mice and shrew moles, which had very low levels of chromosomal aberrations at the dumpsite, on the outskirts of the dumpsite, and in controls. Our four-year survey revealed two variants of M. montebelli from the dumpsite with M6 fission (2n=31), two variants of A. speciosus from the outskirts with XO monosomy (2n=47, XO), and a variant of A. speciosus from the dumpsite with situs inversus. Our analysis confirms our previously proposed hypothesis that M. montebelli might be useful as an indicator species for genotoxic assessment of below-ground pollution by industrial waste at illegal dumpsites.

Chromosomal aberrations in Japanese grass voles in and around an illegal dumpsite at the Aomori-Iwate prefectural boundary.

Bone marrow chromosomes of thirty specimens of the Japanese grass vole, Microtus montebelli (2n=30), which had been caught on and near an illegal dumpsite at the Aomori-Iwate prefectural boundary, were analyzed and compared with those of fifteen grass voles from non-polluted areas as part of an effort to assess genotoxic influences on grass voles in the dumpsite area. Fifty metaphases per specimen were examined with particular attention to numerical and structural aberrations. Two specimens from the dumpsite had 2n=31, which was confirmed by G-banding analysis to have been caused by centric fission of M6 homologs, while control specimens had no such abnormality. In specimens from the polluted area, the mean number of chromosomal aberrations (breaks and/or gaps) per 50 metaphases per specimen was 2.57+/-0.41, which was significantly higher than that (0.80+/-0.14; P<0.01) in control specimens. Chromosomal aberrations were randomly distributed on chromosomes, with frequencies being proportional to the relative lengths of chromosomes. Our findings suggest that grass voles at and around the dumpsite have been seriously damaged at the chromosomal level and, moreover, that M. montebelli might be useful as an indicator species for genotoxic assessment of below-ground pollution by industrial waste at illegal dumpsites.