Toru Yamauchi

Oxidative DNA Damage in Relation to Neurotoxicity in the Brain of Mice Exposed to Arsenic at Environmentally Relevant Levels

Oxidative DNA Damage in Relation to Neurotoxicity in the Brain of Mice Exposed to Arsenic at Environmentally Relevant Levels: Fengyuan Piao, et al. Department of Hygiene, Dalian Medical University, China—To clarify the association between oxidative DNA damage and the neurotoxicity of arsenic, the formation of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) as an index of oxidative DNA damage in the brain was examined in mice fed with drinking water containing 1 or 2 ppm arsenic, using an HPLC‐electrochemical detector and immunohistochemical method. 8‐OHdG levels were significantly increased in the brain of mice given arsenic and its immunoreactivity was distributed in the cerebral and cerebellar cortexes. Cerebral cortex neurons and Purkinje cells in the cerebellar cortex showed degenerative changes in accordance with the distribution of 8‐OHdG immunoreactivity. The levels of arsenic in this study were lower than those reported in epidemiological studies. Thus, we conclude that environmentally relevant levels of arsenic induce pathological changes through oxidative DNA damage in the brain tissues in vivo and that cerebral and cerebellar cortex neurons seem to be the major targets of arsenic neurotoxicity.

Effects of ALDH2 gene polymorphisms and alcohol-drinking behavior on micronuclei frequency in non-smokers

Alcohol abuse is a serious health problem, leading to life-threatening damage to most of the important organ systems. Genotoxic damage is used as an early effect indicator in the surveillance of human exposure to genotoxic substances. Intra- and inter-individual variations of baseline frequencies of micronuclei (MN) in peripheral blood lymphocytes of human populations have been reported previously. Polymorphisms in a few metabolic enzyme genes seem to account for a proportion of this variability, but the impact of specific genetic variants on MN frequencies has not yet been clarified. In 42 healthy Japanese non-smoking men, we investigated the relationship between the MN frequency levels and genetic polymorphisms in three different genes: aldehyde dehydrogenase 2 (ALDH2), X-ray repair cross-complementing group 1 (XRCC1) and excision repair cross-complementing group 2 (ERCC2). Genotyping was performed by PCR-RFLP analysis. The ALDH2 variant (deficient-type) was significantly associated with increased MN frequency levels in subjects with drinking more than three times per week, whereas the XRCC1 and ERCC2 variants seemed to be unrelated to the MN frequency. The ALDH2-deficient habitual drinkers had an average MN frequency of 5.88+/-0.58 (+/- S.E.) compared with 3.20 +/- 0.80 in the ALDH2-proficient habitual drinkers (P<0.05). The ALDH2-proficient non-habitual drinkers had the lowest MN frequency (1.56 +/- 0.41). Furthermore, subjects with highest levels of mean MN frequency, who consumed more than 100g of alcohol per week and more than three times per week, had A2 genotype of ALDH2. A significant odds ratio (12.25, P<0.05) for the MN frequency levels above the 50th percentile value was observed for the ALDH2-deficient individuals versus the ALDH2-proficient individuals after adjustment for several confounders. These results strongly suggest that human early genotoxic effect studies based on the cytogenetic markers of MN should take into account both the individual ALDH2 polymorphism and the potential confounding effect of the drinking behavior.

Influence of Gender, Age and Lifestyle Factors on Micronuclei Frequency in Healthy Japanese Populations

Cytogenetic biomarkers such as chromosomal aberrations (CAs), sister chromatid exchanges (SCEs) and micronuclei (MN) have been applied to the study of individuals exposed to known or potential genotoxic agents. Recent evidence also suggests the usefulness of MN tests for screening carriers of specific mutations that indicate cancer susceptibility 2, . Nevertheless, these biomarkers have not demonstrated as high values in exposed individuals as had been expected, perhaps because they had been influenced by dietary factors such as folate deficiency, the plasma levels of vitamin B12 and homocysteine, and habitual (exercising, drinking and smoking) and demographic factors (age and gender): it is suggested that age and gender are the most important variables affecting MN frequency . Our review of the literature found few reports about MN frequency in healthy Japanese populations as control groups 5, , but these reports did not refer to the contributions of either demographic or habitual factors in detail. MN assay is one of the most sensitive markers for detecting DNA damage, and has been used to investigate genotoxicity of a variety of chemicals. MN testing with interphase cells is more suited as a cytogenetic marker because it is not limited to metaphases, and has the advantage of allowing rapid screening of a larger numbers of cells than in studies with SCEs or CAs . MN is formed from acentric chromosomeor chromatid-type fragments and whole chromosomes that have lagged behind in cell division, being left outside both daughter nuclei. MN analysis therefore appears to be a good tool for investigating the effects of clastogens and aneuploidogens in occupational and environmental exposure in human epidemiological studies 7) and a lso in animal experiments 8, , but several factors could be associated with intraand inter-individual variations of MN. This study aimed to investigate the effects of demographic variables and several lifestyle factors on MN frequency in healthy Japanese subjects.

Diarrheal and Environmental Isolates of Aeromonas spp. Produce a Toxin Similar to Shiga-Like Toxin 1

Abstract. Diarrheal and environmental isolates of 39 strains of Aeromonas spp. were studied for detection of virulence factors. Although these 39 strains did not produce either heat-labile or heat-stable enterotoxins, culture filtrates of 31 strains produced cytopathic effects on Vero cells. Among these, culture filtrates of three strains of Aeromonas hydrophila and one strain of Aeromonas caviae could be neutralized by Escherichia coli O157:H7 Shiga-like toxin 1 antiserum. A single band of plasmid DNA of 2.14 kbp was isolated from these strains of Aeromonas spp. and E. coli O157:H7, which could be amplified by the polymerase chain reaction (PCR), employing oligonucleotide primers from the Shiga-like toxin 1 (SLT1) gene of E. coli O157:H7. E. coli HB 101 cells when transformed with the same plasmid showed cytopathic effects on Vero cells, which indicates that the SLT 1 homolog gene(s) of Aeromonas spp. is plasmid encoded. These results suggest that Aeromonas spp. may also produce Shiga-like toxin 1, or at least a cytotoxin with some homology with the Shiga-like toxin 1 of E. coli O157:H7.

Seasonal mood variation among Japanese residents of Stockholm

Depressive symptoms estimated by the Beck Depression Inventory (BDI) were examined in winter and summer in a total of 242 Japanese adults staying less than 2 years or longer than 10 years in Stockholm, where the length of daylight changes dramatically throughout the winter and summer seasons. In spite of the difference in the period of residency, both groups of subjects showed more mental and somatic depressive symptoms in the winter than in the summer. Moreover, the winter BDI score of long stayers was significantly higher than that of short stayers. Accordingly, our results suggest that, although seasonal mood variation is essentially produced by a chronobiological factor, Swedish lifestyle to which long stayers have been accustomed also influences the seasonal mood variation.

Induction of micronuclei formation in preimplantation mouse embryos after maternal treatment with 2-bromopropane

We examined effects of 2-bromopropane (2-BP), a chlorofluorocarbon replacement, on mouse embryonic mutagenicity. 2-BP was administered to pregnant mice intraperitoneally (i.p.) (300, 600, 900, and 1800 mg/kg) during the early preimplantation period. On day 3 of gestation, micronuclei (MN) frequency and embryo cell number were determined. 2-BP induced a dose-related significant increase in MN frequency and a treatment-related decrease in embryo cell number. Furthermore, the cell numbers were significantly smaller in the MN-positive embryos by two-way ANOVA taking it into account an interactive effect between 2-BP dose and the presence or absence of MN. A simultaneous decrease in cell number and increase in MN frequency may reflect an embryonic developmental disadvantage resulting from maternal treatment with 2-BP. Further study is needed to establish how 2-BP contributes to postimplantation embryonic development.

Subacute toxic effects of zinc on various tissues and organs of rats

In order to expand our knowledge of zinc toxicity and to assess further the toxicities of zinc systematically, we observed the toxic effects of zinc on the functions of various tissues and organs in rats. The rats were randomly divided into four groups (14 in each group), viz. one normal control group (received saline), two zinc groups (Znlow: 4 mg/kg of zinc acetate; Znhigh: 8 mg/kg of zinc acetate), and one cyclophosphamide group (50 mg/kg, as positive control of micronucleated polychromatic erythrocytes (MPCEs)). Saline and zinc acetate were administered intraperitoneally to the rats once every 2 days, seven times in total. Cyclophosphamide was given intraperitoneally to the rats once. The concentration of blood zinc was determined and accumulation of zinc was not observed in the experimental groups. The frequencies of basophilic stippled erythrocyte (BSE) and MPCEs in the Znhigh group were significantly higher than those in the control group (P<0.05). The levels of serum glutamic oxalacetic transaminase (GOT) and serum triiodothyronine (T3) in the Znhigh groups decreased significantly, compared with the control group (P<0.01 or 0.05). Moreover, we also observed that the level of serum cortisol, another adrenal corticoid hormone in rats, was increased by zinc acetate in a dose-dependent manner. According to the literature and our findings, exposure to zinc, especially at higher doses, may produce toxic effects on various tissues and organs including the hematopoietic system, cytogenetics, biochemistry and endocrine system function. Therefore, it is suggested that zinc should be used carefully, especially by high risk groups such as children and pregnant women despite its use as a food additive or in self-medication. At the same time, it is necessary to investigate and research further these toxicities of zinc with long-term administration of low dosage.

Analysis of cytogenetic and developmental effects on pre-implantation, mid-gestation and near-term mouse embryos after treatment with trichlorfon during zygote stage.

Trichlorfon has been widely used in agriculture as a broad spectrum insecticide. We examined cytogenetic and developmental effects on early mouse zygotes exposed to trichlorfon in vivo. Pregnant female mice were intraperitoneally administered a single dose of trichlorfon (100 or 200mg/kg) at 6h post presumed conception and either sacrificed on day of gestation (dg) 3, 9 or 17 to assess the developmental toxicity and mutagenic effects on embryos. Mean cell number (dg 3) and somite number (dg 9) of embryos in the two trichlorfon-treated groups were significantly fewer than in the control group and the mean micronucleus (MN) number (dg 3) and the frequency of mosaic aneuploidies including monosomic or trisomic cell lines (dg 9) was significantly increased in both trichlorfon-treated groups compared with the control group. However, there was no difference in fetal body weight (dg 17) between the control and trichlorfon-treated groups and no increased incidence of external malformations was observed in the trichlorfon-treated groups. These findings suggest that acute exposure of trichlorfon around fertilization induces a high frequency of MN, mosaic aneuploidies and developmental retardation in pre-implantation and mid-gestation embryos, and thereafter these embryos with MN or chromosome damage appear to develop past mid-gestation and catch up with normal embryos by near-term.

The effect of Calcicol® as calcium tonic on delayed neurotoxicity induced by organophosphorus compounds

To examine whether delayed neuropathy is prevented or alleviated when Ca is administered to experimental animals before or after organophosphorus compounds (OPs) dosing, we observed the effects of Calcicol administration as a calcium tonic on delayed neurotoxicity by OPs in hens. The hens (n=28) were randomly divided into seven groups (four in each group). One group received glycerol formal as vehicle group, two groups received 30 mg/kg leptophos or 40 mg/kg triortho-cresyl phosphate (TOCP) (L group and T group), two groups received 2.4 mg/kg Ca(2+) (0.3 ml/kg Calcicol) 24 h before leptophos or TOCP administration, and the last two groups received 2.4 mg/kg Ca after leptophos or TOCP administration, respectively. Although delayed polyneuropathy induced by OPs could not be prevented completely by Calcicol, the clinical signs of organophosphorus-induced delayed neuropathy (OPIDN) in hens that received Calcicol soon before or after OPs administration were less severe than those in hens that received only OPs and there were significant differences in OPIDN score between groups (P<0.05). This shows that polyneuropathy and the recovery function of nerves and muscles suffering from polyneuropathy can be alleviated, as long as calcium tonic is administered before the clinical signs develop. This study offers hope of recovery to humans who are exposed to these OPs because of work, attempted suicide, accidental ingestion or other accidents, etc. Meanwhile, our results indicate further that there is a relationship between a decrease in Ca(2+) concentration in tissues and induction of delayed neuropathy.

Delayed neurotoxicity of triphenyl phosphite in hens: Pharmacokinetic and biochemical studies

The organophosphorus compound, triphenyl phosphite (TPP), caused ataxia in chickens 8-14 days after single po or iv administration. The po and iv ED50 values were 1414 and 35.4 mg/kg, respectively. Chickens which developed ataxia lost 14.4 +/- 2.5% (mean +/- SEM, n = 14) of their initial weight at 28 days and the paralyzed birds showed a severe reduction of 29.3 +/- 2.9% (n = 13) of their initial weight at death or at 28 days after dosing. For the first 4-hr interval after iv injection of 50 mg/kg, the elimination of TPP from plasma consisted of at least two exponential phases; the half-lives of the first and second phases were approximately 30 and 60 min, respectively. When the birds received 100 mg/kg (iv) fatty tissue showed the highest TPP concentration, e.g., 215 micrograms/g fresh wt at 6 hr postdosing. The half-life was approximately 24 hr. Among neural tissues, the sciatic nerve had the highest concentration, followed by the spinal cord, the cerebellum, and the cerebrum. The red muscles, such as adductor magnus, contained about 4-30 times as much TPP as did the white muscles, such as biceps brachii, 6 hr after treatment. Time course effects of TPP treatment on mitochondrial enzymes in leg skeletal muscles were examined by treating hens with 50 mg/kg (iv) and euthanizing the birds at 6 hr to 8 days postdosing. The creatine kinase (CK) activities of the adductor and the soleus were significantly decreased at 2 (48 hr), 4, and 8 days, and at 4 and 8 days postdosing, respectively. Adductor magnus and soleus succinate dehydrogenase (SDH) activities were decreased markedly at 24 and 48 hr, and at 2 (48 hr), 4, and 8 days, respectively. Cytochrome oxidase (COD) activity in adductor magnus and soleus did not decrease during the time course. Biceps femoris CK, SDH, and COD activities were not affected by TPP treatment at this dosage. These results suggest that TPP administration affects the mitochondrial metabolism in skeletal muscle, especially red muscle of chickens.