Lack of in vivo mutagenicity of carbendazim in the liver and glandular stomach of MutaMice.

BACKGROUND: Carbendazim (methyl 2-benzimidazolecarbamate, CASRN: 10605-21-7) exhibits spindle poisoning effects and is widely used as a fungicide. With respect to genotoxicity, carbendazim is deemed to be non-mutagenic in vitro, but it causes indicative DNA damage in vivo and chromosome aberrations in vitro and in vivo. In this study, we examined the mutagenicity of carbendazim in vivo. RESULTS: MutaMice were treated with carbendazim orally at doses of 0 (corn oil), 250, 500, and 1,000 mg/kg/day once a day for 28 days. A lacZ assay was used to determine the mutant frequency (MF) in the liver and glandular stomach of mice. MutaMice were administered up to the maximum dose recommended by the Organization for Economic Co-operation and Development Test Guidelines for Chemicals No. 488 (OECD TG488). The lacZ MFs in the liver and glandular stomach of carbendazim-treated animals were not significantly different from those in the negative control animals. In contrast, positive control animals exhibited a significant increase in MFs in both the liver and glandular stomach. CONCLUSIONS: Carbendazim is non-mutagenic in the liver and glandular stomach of MutaMice following oral treatment.

In vivo mutagenicity assessment of orally treated tert-butyl hydroperoxide in the liver and glandular stomach of MutaMouse.

BACKGROUND: tert-Butyl hydroperoxide (TBHP; CAS 75-91-2), a hydroperoxide, is mainly used as a polymerization initiator to produce polyethylene, polyvinyl chloride, and unsaturated polyester. It is a high-production chemical, widely used in industrial countries, including Japan. TBHP is also used as an additive for the manufacturing of food utensils, containers, and packaging (UCP). Therefore, there could be consumer exposure through oral intake of TBHP eluted from UCPs. TBHP was investigated in various in vitro and in vivo genotoxicity assays. In Ames tests, some positive results were reported with and/or without metabolic activation. As for the mouse lymphoma assay, the positive result was reported, regardless of the presence or absence of metabolic activation enzymes. The results of some chromosomal aberrations test and comet assay in vitro also demonstrated the genotoxic positive results. On the other hand, in in vivo tests, there are negative results in the bone marrow micronucleus test of TBHP-administered mice by single intravenous injection and the bone marrow chromosomal aberration test using rats exposed to TBHP for 5 days by inhalation. Also, about dominant lethal tests, the genotoxic positive results appeared. In contrast, there is little information about in vivo mutagenicity and no information about carcinogenicity by oral exposure. RESULTS: We conducted in vivo gene mutation assay using MutaMice according to the OECD Guidelines for the Testing of Chemicals No. 488 to investigate in vivo mutagenicity of TBHP through oral exposure. After repeated dosing for 28 days, there were no significant differences in the mutant frequencies (MFs) of the liver and glandular stomach up to 300 mg/kg/day (close to the maximum tolerable dose (MTD)). The positive and negative controls produced the expected responses. CONCLUSIONS: These findings show that orally administrated TBHP is not mutagenic in the mouse liver and glandular stomach under these experimental conditions.

Derivation of subacute guidance values for chemical contaminants of drinking water quality standard in Japan.

The concentration of chemicals in drinking water may transiently and accidently exceed the Drinking Water Quality Standard (DWQS). If the level of a contaminant is not expected to cause adverse effects for a limited period of exposure, immediate suspension of the water supply may not be necessary. Assessments should be conducted using subacute guidance values (SGVs). In this study, we assessed 26 chemicals for the DWQS to establish the SGVs. Principally, a key study was selected from subacute studies to derive a Subacute Reference Dose (saRfD). The SGV was calculated from the saRfD for adults (drinking water intakes: 40 mL/kg/day) and children (drinking water intakes: 150 mL/kg/day). No allocation factor was applied to derive the SGV. We established the SGV for 20 chemicals, which were 2-38 times higher than the corresponding DWQS. However, SGVs for six chemicals were the same as the corresponding DWQS. Therefore, immediate action will be required for these six accidental contaminants. Our established SGVs are useful for assessing accidental contamination.

In vivo mutagenicity assessment of styrene in MutaMouse liver and lung.

BACKGROUND: Styrene (CAS 100-42-5) is widely used as polystyrene and acrylonitrile-butadiene-styrene resin such as plastic, rubber, and paint. One of the primary uses of styrene is food utensils and containers, but a small amount of styrene transferred into food can be ingested by eating. Styrene is metabolized into styrene 7,8-oxide (SO). SO is mutagenic in bacteria and mouse lymphoma assays. It is clastogenic in cultured mammalian cells. However, styrene and SO are not clastogenic/aneugenic in rodents, and no rodent in vivo gene mutation studies were identified. METHODS: To investigate the mutagenicity of orally administered styrene, we used the transgenic rodent gene mutation assay to perform an in vivo mutagenicity test (OECD TG488). The transgenic MutaMouse was given styrene orally at doses of 0 (corn oil; negative control), 75, 150, and 300 mg/kg/day for 28 days, and mutant frequencies (MFs) were determined using the lacZ assay in the liver and lung (five male mice/group). RESULTS: There were no significant differences in the MFs of the liver and lung up to 300 mg/kg/day (close to maximum tolerable dose (MTD)), when one animal with extremely high MFs that were attributed to an incidental clonal mutation was omitted. Positive and negative controls produced the expected results. CONCLUSIONS: These findings show that styrene is not mutagenic in the liver and lung of MutaMouse under this experimental condition.

Initial hazard assessment of ethyl(dimethyl)(tetradecyl)ammonium ethyl sulfate: Genotoxicity tests and combined repeated-dose and reproductive/developmental toxicity screening in rats.

Ethyl(dimethyl)(tetradecyl)ammonium ethyl sulfate, used in laundry detergents, shampoos, and body soaps, is classified by the Japanese Chemical Substances Control Law as a priority assessment chemical substance for environmental effects. However, its toxicity data for human health are insufficient. This study evaluated this chemical under the Safety Examination of Existing Chemicals and Safety Programmes of the Ministry of Health, Labour and Welfare (MHLW). The MHLW conducted bacterial reverse mutation (Ames test), in vitro chromosomal aberration, and combined repeated-dose and reproductive/developmental toxicity screening tests. We performed a screening assessment of ethyl(dimethyl)(tetradecyl)ammonium ethyl sulfate for human health. The chemical showed a negative reaction in the Ames test and a positive reaction in the in vitro chromosomal aberration test with metabolic activation in rats. The combined repeated-dose and reproductive/developmental toxicity screening test showed significantly decreased food consumption at 50 mg/kg body weight/day, but no reproductive and developmental toxicity was observed. The no-observed-effect level of 15 mg/kg/day was obtained as a screening value. Therefore, this chemical was classified as hazard class 3, with a derived-no-effect level of 0.025 mg/kg/day. The results of this study will be useful for risk assessment of groups of structurally similar alkyl quaternary ammonium surfactants.

Effects of Low Temperature on Spider Mite Control by Intermittent Ultraviolet-B Irradiation for Practical Use in Greenhouse Strawberries.

The application of ultraviolet-B (UVB) radiation to control spider mites is challenging as a key technology for integrated pest management (IPM) in greenhouse strawberries in Japan. To address this, concurrent use of phytoseiid mites and reduced UVB irradiance is desirable to ensure control effects in areas shaded from UVB radiation and to minimize the sunscald in winter, respectively. We designed experiments reproducing the UVB dose on the lower leaf surfaces in strawberry and evaluated the effects of intermittent UVB irradiation at midnight for practical application in the greenhouse and low temperature on the survival of the spider mite Tetranychus urticae Koch (Acari: Tetranychidae) and damage to the phytoseiid mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae). The midnight intermittent UVB irradiation effectively suppressed egg hatching and development of larvae of T. urticae, and the control effect was reinforced at 20°C (no eggs hatched at 0.13 kJ m-2 d-1) rather than, at 25°C (70.8% eggs hatched). In contrast, the hatchability of N. californicus eggs was unaffected by intermittent UVB irradiation at 0.27 kJ m-2 d-1 at 25°C and 20°C. However, residual effects of UVB irradiation to N. californicus eggs on survival of hatched larvae were seen, so that reducing the UVB dose is also advantageous for this phytoseiid mite. N. californicus showed a photoreactivation capacity, whereas their UVB tolerance was improved by prey species, suggesting the possibility of the improvement of phytoseiid mites by diet. The reduction of UVB dose and concurrent use of phytoseiid mites increase reliability of the UVB method in IPM strategies in strawberry greenhouse.

Low level of polyandry constrains phenotypic plasticity of male body size in mites.

Polyandry, i.e. females mating with multiple males, is more common than previously anticipated and potentially provides both direct and indirect fitness benefits to females. The level of polyandry (defined by the lifetime number of male mates of a female) is an important determinant of the occurrence and intensity of sexual selection acting on male phenotypes. While the forces of sexual selection acting on phenotypic male traits such as body size are relatively well understood, sexual selection acting on phenotypic plasticity of these traits is unexplored. We tackled this issue by scrutinizing the link between polyandry and phenotypic plasticity of male body size in two sympatric plant-inhabiting predatory mite species, Phytoseiulus persimilis and Neoseiulus californicus. These two species are similar in life history, ecological niche requirements, mating behavior, polygyny and female body size plasticity but strikingly differ in the level of both polyandry and phenotypic plasticity of male body size (both lower in P. persimilis). We hypothesized that deviations from standard body size, i.e. the size achieved under favorable conditions, incur higher costs for males in the less polyandrous P. persimilis. To test our hypotheses, we conducted two experiments on (i) the effects of male body size on spermatophore transfer in singly mating females and (ii) the effects of mate sequence (switching the order of standard-sized and small males) on mating behavior and paternity success in doubly mating females. In P. persimilis but not N. californicus, small males transferred fewer but larger spermatophores to the females; in both species, females re-mated more likely with standard-sized following small than small following standard-sized males; in P. persimilis, first standard-sized males sired a higher proportion of offspring produced after re-mating by the female than first small males, whereas in N. californicus the paternity success of small and standard-sized males was independent of the mating sequence. Based on our results and pertinent previous studies, which showed that females of P. persimilis, but not N. californicus, prefer mating with standard-sized over small males and allow them fertilizing more eggs, the lack of interspecific difference in female body size plasticity, and the absence of any clue pointing at a role of natural selection, we suggest that the interspecific difference in male body size plasticity is sexually selected. Our study provides an indication of sexual selection constraining plasticity of male phenotypes, suggesting that the level of polyandry may be an important co-determinant of the level of phenotypic plasticity of male body size.

Developmental Phase-Specific Mortality After Ultraviolet-B Radiation Exposure in the Two-Spotted Spider Mite.

Exposure to ambient ultraviolet-B (UVB) radiation generates DNA lesions, such as cyclobutane pyrimidine dimers and 6-4 pyrimidine-pyrimidine photoproducts in Tetranychus urticae Koch (Acari: Tetranychidae). Larvae appeared normal and healthy after UVB irradiation. Conversely, many mites were trapped in their old epidermis or experienced retarded development and shrunk, thus failing to molt from protochrysalises to protonymphs and died. This suggested that DNA lesions per se were not causing lethality in mites unless damaged genes were expressed. UVB-induced DNA lesions may have interfered with DNA replication and gene expression during the physiological changes of morphogenesis in the chrysalis stage. Comprehensive gene expression analysis by RNA sequencing revealed that gene expression involving epidermal tissue (characteristically cuticular protein genes) and myosin heavy chain muscle-like genes were downregulated in protochrysalises irradiated with UVB at the larval stage. We conclude that the success of protochrysalis molting is determined by whether the DNA lesions of genes, particularly those connected with morphogenesis, are repaired before expression at the protochrysalis stage.

Photo-enzymatic repair of UVB-induced DNA damage in the two-spotted spider mite Tetranychus urticae.

Ambient ultraviolet-B (UVB) radiation induces lethal effects in the two-spotted spider mite Tetranychus urticae, whereas photoreactivation by irradiation with ultraviolet-A and visible light (VIS) plays an important role to increase survival of mites irradiated by UVB. The physiological mechanisms and ecological significance of photoreactivation in terrestrial arthropods have not been shown clearly. We verified the biological impact and accumulation of DNA lesions by UVB irradiation and the repair of them by photoreactivation in T. urticae larvae. Survival of UVB-irradiated larvae decreased with increasing UVB dose, but recovered remarkably with VIS exposure after UVB irradiation (photoreactivation). The DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidine photoproducts (6-4PPs) linearly increased with the UVB dose. The CPDs were repaired after exposure to VIS, whereas the frequency of 6-4PPs was unaffected by VIS; CPD photolyase genes, but not (6-4) photolyase genes, have been found in the T. urticae genome. Therefore, DNA damage and CPD photo enzymatic repair (PER) is significant for survival in this mite under ambient UVB radiation. Unexpectedly, gene expression of CPD photolyase was unaffected by irradiation with UVB and VIS. Instead, expression of xeroderma pigmentosum A (XPA) was increased by irradiation. XPA is a core factor in nucleotide excision repair (NER), which is a repair system unrelated to photo energy. The relationship between gene expression and enzymatic repair remains unclear. To elucidate the PER process in T. urticae, further study will be necessary on the gene expression patterns and molecular functions of CPD photolyase in PER and of XPA in NER.

Ultimate Drivers and Proximate Correlates of Polyandry in Predatory Mites.

Polyandry is more widespread than anticipated from Bateman's principle but its ultimate (evolutionary) causes and proximate (mechanistic) correlates are more difficult to pinpoint than those of polygyny. Here, we combined mating experiments, quantification of reproductive traits and microsatellite genotyping to determine the fitness implications of polyandry in two predatory mite species, where males are highly polygynous (up to 45 fertilized females during life), whereas females range from monandry to various polyandry levels. The medium-level polyandrous (up to eight male mates possible) Neoseiulus californicus received clear direct and indirect benefits: multiply mated females produced more offspring with higher survival chances over longer times than singly mated females. In contrast, singly and multiply mated females of the low-level polyandrous (commonly two male mates at maximum) Phytoseiulus persimilis produced similar numbers of offspring having similar survival chances. In both species, multiple mating resulted in mixed offspring paternities, opening the chance for indirect fitness benefits such as enhanced genetic compatibility, complementarity and/or variability. However, the female re-mating likelihood and the paternity chance of non-first male mates were lower in P. persimilis than in N. californicus. Regarding proximate factors, in both species first mating duration and female re-mating likelihood were negatively correlated. Based on occasional fertilization failure of first male mates in P. persimilis, and mixed offspring paternities in both species, we argue that fertilization assurance and the chance to gain indirect fitness benefits are the ultimate drivers of polyandry in P. persimilis, whereas those of N. californicus are higher offspring numbers coupled with enhanced offspring viability and possibly other indirect fitness benefits. Overall, the adaptive significance and proximate events well reflected the polyandry levels. Our study provides a key example for linking behavioral experiments, quantification of reproductive traits and paternity analysis via offspring genotyping to explain the evolution of differing levels of polyandry.

Factors affecting photoreactivation in UVB-irradiated herbivorous spider mite (Tetranychus urticae).

Ambient ultraviolet-B (UVB) radiation causes lethal damage to spider mites, and the extent of photochemical effects is determined by cumulative irradiance in the two-spotted spider mite, Tetranychus urticae. However, the LD50 values obtained using a UVB lamp were much lower than those elicited by solar UVB radiation. As solar radiation includes intense visible light and UVA, we assumed that a photoreactivation mechanism would play a role to survive under solar radiation. We assessed the capacity for photoreactivation in T. urticae eggs and larvae, and found that the efficacy of photoreactivation was determined by the cumulative irradiance of visible light (VIS) after exposure to UVB radiation. The wavelength range effective for photoreactivation went from UVA to green. Next, we found that an increased time lag between UVB and VIS radiation reduced the photoreactivation efficacy in eggs. In contrast, a time lag ≤4 h did not affect that in larvae. We discussed the possibility that the timing of photoreactivation occurs related with phase-specific UVB vulnerability and outbreak symptoms due to UVB-induced DNA damage. Our results suggest that T. urticae depends on a photoreactivation mechanism, and that the photoreactivation efficiency probably caused the divergence in UVB impact between UV lamp and solar radiation.

The Bunsen-Roscoe reciprocity law in ultraviolet-B-induced mortality of the two-spotted spider mite Tetranychus urticae.

To determine whether the Bunsen-Roscoe reciprocity law (i.e., the extent of photochemical effects is determined by cumulative irradiance) is applicable to ultraviolet-B (UVB) damage in the twospotted spider mite Tetranychus urticae, egg hatchability and survival of individuals were assessed after irradiation with a UVB lamp using various combinations of intensity and time length. A positive linear correlation between probit mortality and cumulative UVB irradiance was detected in eggs, larvae, teleiochrysalis females, and adult females, regardless of UVB intensity (0.19-0.58 Wm(-2)). LD50 values were clearly higher in adult females, followed by teleiochrysalis females, larvae, and eggs. In eggs, reciprocity was obeyed not only at the UVB intensities listed above, but also at very low UVB intensity (0.014-0.023 Wm(-2)). Such reciprocity in the negative effects of UVB radiation was also observed for the developmental rate of juveniles and egg production of adult females. However, the LD50 value of eggs obtained using the UVB lamp (0.58 kJm(-2)) was lower than that elicited by solar UVB radiation in a previous outdoor experiment (about 50 kJm(-2)). These results suggest that a photoreactivation mechanism plays an important role in the survival of this mite under solar radiation.