Muneyuki Miyagawa

Neurotoxicity and metabolism of methyl bromide in rats

Changes in locomotor activity, body temperature, and body weight gain, and the enhancement of thiopental-induced sleep were investigated in rats as indices of the functional changes in the CNS caused by methyl bromide (CH3Br) exposure. The correlations of these behavioral changes with CH3Br metabolism are discussed. The LC50 value and its 95% confidence limits for an 8-hr exposure of CH3Br was 302 ppm (267-340) ppm. Effects were examined following exposure to 63, 125, 188, or 250 ppm CH3Br for 8 hr. CH3Br concentrations as low as 63 ppm remarkably enhanced the sleep-inducing potency of thiopental, but CH3Br exerted no effect on thiopental metabolism. The body temperature and body weight gain were decreased at exposure to concentrations of 125 ppm or higher, and locomotor activity was reduced at 188 ppm or higher. These effects were reversible and, at 24 hr after the exposure, locomotor activity and body temperature were almost the same as in control rats. In a time-course study of CH3Br, bromine, and methyl alcohol, CH3Br was rapidly eliminated from rat tissues following the cessation of exposure, with a half-life of about 30 min in the early post-exposure period. In contrast, the elimination rate of bromine was very slow, with a half-life about 5 days. The methanol amount was below that reported to induce the changes in CNS functions. These results suggest that the CNS depression caused by CH3Br exposure may be due to the CH3Br molecule or the methyl moiety incorporated into tissues and may not be attributable to bromine or methanol. A linear relationship was obtained between bromine amounts in blood and the exposure concentration or duration. This result suggests the possibility that the extent of CH3Br exposure may be estimated from the bromine quantities in blood.

Dietary exposure to low doses of bisphenol A: effects on reproduction and development in two generations of C57BL/6J mice.

The present study was conducted to examine the effects of low‐dose exposure to bisphenol A on reproduction and development in two generations of mice. Pregnant female C57BL/6J mice (F0) were fed a diet containing low doses of bisphenol A (0, 0.33, 3.3, or 33 ppm) from gestational day 6 through postnatal day 22, and the weanlings (F1 and F2) from each F0 and F1 dam group, respectively, were also fed these same concentrations of bisphenol A ad libitum until sacrifice. There were no treatment‐related changes in body weight, body weight gain, food consumption, gestation length, or the number of live births on postnatal day 1 in F0 dams between the control group and bisphenol A groups. Sex ratio and viability were similar in all F1 pups. No treatment‐related changes were observed in body weight, food consumption, developmental parameters, anogenital distance, or weight of any of the organs (liver, kidney, heart, spleen, thymus, testis, ovary, or uterus) in F1 and F2 adults in either sex. The epididymis weight was slightly higher with 0.33 and 3.3 ppm in F1 males, but this slight increase was neither dose dependent nor seen across generations. There were no treatment‐related effects of bisphenol A on cauda epididymal sperm count or sperm motility in F1 or F2 males. These findings indicate that dietary exposure to bisphenol A between 0.33 and 33 ppm does not adversely affect reproduction or development as assessed in two generations of mice.

INHIBITION OF TYROSINE HYDROXYLASE ACTIVITY BY METHYL BROMIDE EXPOSURE

Rats were exposed to methyl bromide gas (16-250 ppm) for 8 hr, and tyrosine hydroxylase (TH) activity in the striatum, hypothalamus, frontal cortex, midbrain, and medulla oblongata was measured in brain homogenates from exposed rats, and in vivo following administration of decarboxylase inhibitor. Exposure to methyl bromide dose-dependently inhibited both in vitro and in vivo TH activity. Of the five brain areas, TH activity in the hypothalamus was most sensitive to methyl bromide. The time course of enzyme inhibition after exposure was similar to those of decreases in catecholamine concentrations, locomotor activity change, and body temperature reported previously. These results suggest methyl bromide reduces catecholaminergic neuronal activity in the brain via inhibition of TH activity.

Methyl bromide alters catecholamine and metabolite concentrations in rat brain.

The effects of inhalation exposure of rats methyl bromide (MB) on dopamine (DA), homovanillic acid (HVA), norepinephrine (NE), 3-methoxy-4-hydroxyphenylglycol (MHPG), serotonin (5HT), and 5-hydroxyindoleacetic acid (5HIAA) concentrations of various brain regions (striatum, hypothalamus, frontal cortex, midbrain, and medulla oblongata) were investigated. Rats received a single 8 hr exposure to MB, and amines and metabolites were separated by a reverse-phase HPLC, and were quantified via native fluorescence. An exposure to 100 ppm MB decreased tissue levels of DA and NE in all brain areas at 0 or 2 hr following exposure. HVA and MHPG contents were significantly increased in almost all brain regions. In a second study, rats were exposed to four concentrations of MB ranging from 31-250 ppm, and monoamine and metabolite levels in brain regions measured immediately after the exposure. Again, there were dose-dependent decreases of DA and NE, and increases in HVA and MHPG. Less clear changes in 5 HT and 5HIAA contents were observed. These data suggest that alterations of catecholamine metabolism may be a factor in MB-induced neurotoxicity.

Effects of in utero exposure to 2,2′,4,4′,5,5′‐hexachlorobiphenyl (PCB 153) on somatic growth and endocrine status in rat offspring

ABSTRACT  Exposure to polychlorobiphenyl (PCB) mixtures at an early stage of development has been reported to affect endocrine glands; however, little is known about the precise toxicological properties of individual PCB. The present study was undertaken to determine whether prenatal exposure to 2,2′,4,4′,5,5′‐hexachlorobiphenyl (PCB 153), a di‐ortho‐substituted non‐coplanar congener, affects postnatal development in rat offspring. Pregnant Sprague‐Dawley rats (Crj: CD (SD) IGS) were given PCB 153 (0, 16, or 64 mg/kg/day) orally from gestational day (GD) 10 through GD 16, and developmental parameters in the male and female offspring were examined. We found no dose‐dependent changes in body weight, body length (nose–anus length), tail length, or the weights of kidneys, testes, ovaries and uterus in offspring at 1 or 3 weeks of age. Liver weights were increased in the PCB 153–treated groups, although we observed a significant difference only in males. Anogenital distance was unaffected in the PCB 153–treated groups. We observed a significant dose‐dependent decrease in the plasma concentrations of thyroxine and tri‐iodothyronine, whereas those of thyroid‐stimulating hormone were not significantly changed. In addition, there were no dose‐dependent changes in plasma concentrations of growth hormone and insulin‐like growth factor‐I in any dose group. These findings suggest that prenatal exposure to PCB 153 (GD 10–16, 16–64 mg/kg/day) may alter the thyroid status in rat offspring to some extent without affecting somatic growth or its related hormonal parameters.

Intratracheal Instillation Methods and the Distribution of Administered Material in the Lung of the Rat

Intratracheal instillation is widely used for respiratory toxicity tests in experimental animals. However, there are wide variations in the techniques used for instillation, and it is thus difficult to compare the results obtained using different techniques. To examine the effect of instillation methods, we compared the distribution of a test substance in the lungs of rats after intratracheal instillations under various conditions. Rats received an intratracheal instillation of 0.3 mL of india ink suspension under different conditions as follows: 3 different angles of body restraint, 0° (supine horizontal), 45° (supine head up) and 90° (vertical head up); 2 instillation speeds, high (40 mL/min) and low (4 mL/min); and 2 different devices, a standard bulb-tipped gavage needle and an aerosolizing microsprayer designed for intratracheal instillation. One hour after treatment under these various conditions, rats were sacrificed, and the local distribution of the suspension in the lungs was observed. No animal restrained in the supine head-up or vertical head-up position died from the treatment; however, fatalities were observed when rats were restrained in the supine horizontal position except under high-speed dosing conditions with a microsprayer. Better distribution of the suspension in the lungs was observed in the rats restrained in the supine head-up position after instillation at high speed when compared with other conditions. These results indicated that high-speed instillation to the subject restrained in the supine head-up position is an appropriate condition for performing intratracheal instillation.

Inhalation of 1-Bromopropane Causes Excitation in the Central Nervous System of Male F344 Rats

The present study investigates the effects of 1-bromopropane (1BP) on animal behavior to determine the extent of toxicity to the central nervous system (CNS). We measured the spontaneous locomotor activity (SLA) of rats before and after 3 weeks of exposure to 1BP for 8 h per day. In control and 10 ppm groups, the SLA values were similar to pre-exposure levels on post-exposure Day 1 and thereafter. However, the SLA values in the 50 and 200 ppm groups were higher than pre-exposure levels. Open-field behavior was evaluated after exposure and freezing time decreased with exposure to increasing concentrations of 1BP. Ambulation and rearing scores in the exposed groups were higher than control values, particularly in the 50 and 200 ppm groups. The frequency of defecation and urination decreased almost dose-dependently. Exposure to 50-1000 ppm of 1BP did not affect passive avoidance behavior examined using a step-through type apparatus. The amount of time swimming in the water maze test was not affected in the controls, or groups exposed to 50 and 200 ppm 1BP, but that in the 1000 ppm group was increased compared with control. Exposure at 50-1000 ppm dose-dependently decreased the traction performance of rats, indicating decreased muscle strength. We found that 10-200 ppm of 1BP exposure did not affect motor coordination determined by rota-rod performance. The increased SLA values and open-field activity support the notion that 1BP has excitatory effects on the CNS of F344 male rats. In addition, 1BP reduced the grip or muscle strength of the rats. Memory function was not disordered and the motor coordination of all four limbs remained normal.

Conditioned taste aversion induced by inhalation exposure to methyl bromide in rats

The toxic effect of methyl bromide vapor was assessed by a conditioned taste aversion regime. Rats kept under a water deprivation schedule for 7 days, were permitted access to 0.3% (w/v) sodium saccharin, and were exposed to methyl bromide at 0, 25, 50, and 100 ppm for 4 h. 3 days after the exposure, saccharin preference tests were carried out, revealing dose-dependent saccharin aversion in the exposure group. This result suggests that the conditioned taste aversion method is effective for assessing the toxicity of gaseous substances such as methyl bromide.

Genotoxicity assessment of intravenously injected titanium dioxide nanoparticles in gpt delta transgenic mice

Titanium dioxide (TiO2) nanoparticles are increasingly manufactured in large amounts for use in industrial applications such as cosmetics, pigments, foods, and as photo-catalysts. Many in vitro studies have examined the genotoxicity of TiO2 nanomaterials; some of these studies suggest that TiO2 nanoparticles (NPs) are genotoxic. Several in vivo studies have also been reported recently, but the results are inconsistent. In this study, we investigated, using several genotoxicity endpoints, the effects of dispersed TiO2 suspensions following multiple intravenous injections in mice. Male gpt Delta C57BL/6J mice were administered TiO2 NPs at doses of 2, 10 or 50mg/kg body weight per week for 4 consecutive weeks. Genotoxic effects were then analyzed by the Pig-a gene mutation assay and the micronucleus assay on peripheral blood, and by the alkaline comet, gpt mutation, and Spi(-) mutation assays on the liver. We also assessed the localization of TiO2 NPs in the liver, by transmission electron microscopy. Administration of TiO2 NPs did not significantly increase any of the following endpoints: frequency of Pig-a mutants (erythrocytes); frequency of micronuclei (reticulocytes); level of DNA damage (liver); frequencies of gpt and Spi(-) mutants (liver). Most TiO2 NPs in the liver were found in the sinuses and inside Kupffer cells, although some were occasionally observed in liver parenchymal cells. These results indicate that TiO2 NPs do not have genotoxic effects on mouse liver or bone marrow.

Significant association between decreased ALDH2 activity and increased sensitivity to genotoxic effects in workers occupationally exposed to styrene.

ALDH2 is involved in the metabolism of styrene, a widely used industrial material, but no data are available regarding the influence of this enzyme on the metabolic fate as well as toxic effects of this chemical. In this study, we recruited 329 workers occupationally exposed to styrene and 152 unexposed controls. DNA strand breaks, DNA-base oxidation in leukocytes and urinary 8-hydroxydeoxyguanosine (8-OH-dG) were assayed as biomarkers to measure genotoxic effects. Meanwhile, we examined the genetic polymorphisms, including ALDH2, EXPH1, GSTM1, GSTT1 and CYP2E1, and also analyzed the levels of styrene exposure through detecting urinary styrene metabolites and styrene concentration in air. In terms of DNA damage, the three genotoxic biomarkers were significantly increased in exposed workers as compared with controls. And the styrene-exposed workers with inactive ALDH2 *2 allele were subjected to genotoxicity in a higher degree than those with ALDH2 *1/*1 genotype. Also, lower levels of urinary styrene metabolites (MA + PGA) were observed in styrene-exposed workers carrying ALDH2 *2 allele, suggesting slower metabolism of styrene. The polymorphisms of other enzymes showed less effect. These results suggested that styrene metabolism and styrene-induced genotoxicity could be particularly modified by ALDH2 polymorphisms. The important role of ALDH2 indicated that the accumulation of styrene glycoaldehyde, a possible genotoxic intermediate of styrene, could account for the genotoxicity observed, and should be taken as an increased risk of cancer.