Nobuyuki Asaeda

Bisphenol A may cause testosterone reduction by adversely affecting both testis and pituitary systems similar to estradiol.

Bisphenol A (BPA) causes reproductive toxicities, but the mechanisms are still unclear. In the present study, we sought to clarify these mechanisms in comparison with those of 17beta-estradiol (E2). Prepubertal Wistar/ST male rats (4 weeks old) were subcutaneously administered BPA (0, 20, 100 and 200 mg/kg/day) or E2 (10 and 100 microg/kg/day) for 6 weeks. Both BPA and E2 treatments decreased plasma and testicular testosterone levels, and plasma luteinizing hormone (LH), but not E2 and follicle-stimulating hormone levels, though E2 treatment increased its plasma level. In relation to the decreased testosterone levels, BPA and E2 decreased expressions of steroidogenic enzymes and cholesterol carrier protein in Leydig cells. Thus, decreased testosterone levels in plasma might have resulted from decreased expressions of these enzymes and protein as well as from decreased plasma LH levels. Interestingly, the changes in steroidogenic enzymes and carrier protein were observed at lower levels of exposure to BPA or E2 than those inhibiting plasma LH levels. Microscopically, 200 mg/kg BPA and 100 microg/kg E2 significantly decreased Leydig cell numbers in the testis. In addition, BPA and E2 also decreased expression of estrogen receptor alpha-mRNA, which might be related to the decreased numbers of Leydig cells. Thus, BPA directly affects not only the Leydig cells but also the pituitary gland, but the former may be impaired at lower exposure concentrations than the latter.

Di(2-ethylhexyl)phthalate Induces Hepatic Tumorigenesis through a Peroxisome Proliferator-activated Receptor α-independent Pathway

Di(2‐ethylhexyl)phthalate Induces Hepatic Tumorigenesis through a Peroxisome Proliferator‐activated Receptor α‐independent Pathway: Yuki Ito, et al. Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine—Di(2ethylhexyl)phthalate (DEHP), a commonly used industrial plasticizer, causes liver tumorigenesis presumably via activation of peroxisome proliferator‐activated receptor alpha (PPARα). The mechanism of DEHP tumorigenesis has not been fully elucidated, and to clarify whether DEHP tumorigenesis is induced via PPARα, we compared DEHP‐induced tumorigenesis in wild‐type and Pparα‐null mice. Mice of each genotype were divided into three groups, and treated for 22 months with diets containing 0, 0.01 or 0.05% DEHP. Surprisingly, the incidence of liver tumors was higher in Pparα‐null mice exposed to 0.05% DEHP (25.8%) than in similarly exposed wild‐type mice (10.0%). These results suggest the existence of pathways for DEHP‐induced hepatic tumorigenesis that are independent of PPARα. The levels of 8‐OHdG increased dose‐dependently in mice of both genotypes, but the degree of increase was higher in Pparα‐null than in wild‐type mice. NFκB levels also significantly increased in a dose‐dependent manner in Pparα‐null mice. The protooncogene c‐jun‐mRNA was induced, and c‐fos‐mRNA tended to be induced only in Pparα‐null mice fed a 0.05% DEHP‐containing diet. These results suggest that increases in oxidative stress induced by DEHP exposure may lead to the induction of inflammation and/or the expression of protooncogenes, resulting in a high incidence of tumorigenesis in Pparα‐null mice.

Testicular and Hematopoietic Toxicity of 2-Bromopropane, a Substitute for Ozone Layer-Depleting Chlorofluorocarbons

Testicular and Hematopoietic Toxicity of 2‐Bromopropane, a Substitute for Ozone Layer‐Depleting Chlorofluorocarbons: Gaku Ichihara, et al. Department of Hygiene, Nagoya University School of Medicine—In 1995r unexpected amenorrhea, oligozoospermia and anemia were discovered in Korean workers exposed to solvents containing 2‐bromopropane which was a substitute for chlorofluorocarbon. We aimed to determine experimentally the testicular and hematopoietic toxicity of 2‐bromopropane in male rats. Thirty‐six Wistar male rats were divided into four groups of nine each. The rats were exposed to 300r 1,000 and 3,000 ppm 2‐bromopropane or only fresh air, respectively, 8 hr a day, 7 days per week. The 300 ppm and 1,000 ppm groups were exposed for 9 weeks, but the 3,000 ppm groups exposure was discontinued and three rats in this group were dissected after 9‐11 days’ exposure because of serious illness. The others were dissected at the end of the experiment. At 300 ppm or over, the testicular and epididymal weights per body weight, epididymal sperm count, motile sperm percentage and the number of erythrocytes and platelets had decreased compared to the control. Histopathologically, all types of germ cells decreased in the 300 ppm group. Germ cells were absent but Sertoli cells still remained in the 1,000 ppm and 3,000 ppm groups at the end of the experiment. Spermatogonia were absent and the number of spermatocytes decreased in the 3,000 ppm group rats sacrificed after 1 1 days’ exposure. Sertoli cell vacuolations were marked in two of these three rats. Bone marrow was hypocellular in the 1,000 ppm group and in all the rats in the 3,000 ppm group. These results clearly showed that 2‐bromopropane had a testicular and hematopoietic toxicity in male rats.

Ovarian toxicity of 2-bromopropane in the non-pregnant female rat

Ovarian Toxicity of 2‐Bromopropane in the Non‐Pregnant Female Rat: Michihiro Kamijima, et al. Department of Hygiene, Nagoya University School of Medicine—A cluster of patients with amenorrhea, oligospermia and anemia were found among workers in an electronics factory in the Republic of Korea. 2‐Bromopropane was suspected to cause the disorders. This study aimed to clarify its ovarian toxicity in the virgin rat. Female Wistar rats were daily exposed to 0, 100, 300, or 1,000 ppm 2‐bromopropane for eight hours a day for 9 weeks. During the experimental period, vaginal smears were taken everyday to monitor ovarian cyclicity. Tissues were histopathologically examined and plasma concentrations of luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) were determined at the end of experiment. The vaginal smear test showed that the number of normal cycles decreased significantly both in the 300 and 1,000 ppm groups. The histopathological examination revealed dose‐dependent ovarian follicle atresia accompanied by a decreased number of normal antral and growing follicles in the 300 and 1,000 ppm groups. Especially, in the ovaries of rats with persistent estrous smears in the 1,000 ppm group, most of the follicles were atretic and there were no newly formed corpora lutea. There still remained normal antral follicles and corpora lutea in the ovaries of the remaining rats of the group and of the 300 ppm group with constant diestrous and occasional estrous smears. Hormonal examination revealed no statistically significant change in LH or FSH concentrations between any groups. The results showed that 2‐bromopropane has ovarian toxicity in rats, indicating that the secondary amenorrhea in human cases was due to 2‐bromopropane exposure.

Dose dependent effects of chronic exposure to toluene on neuronal and glial cell marker proteins in the central nervous system of rats.

The dose dependent effects of chronic exposure to toluene on the neuronal marker proteins (gamma-enolase, calbindin-D28k) and glial cell marker proteins (alpha-enolase, creatine kinase-B, and beta-S100 protein) were investigated in the central nervous system (CNS) of rats. Three groups of animals were exposed to 100 ppm, 300 ppm, or 1000 ppm toluene vapour eight hours a day, six days a week for 16 weeks. The contents of the marker substances were determined with enzyme immunoassays. A significant increase in the three glial cell marker proteins was noted in the cerebellum after exposure to 100 ppm toluene; a more pronounced increase occurred at the higher toluene concentrations. beta-S100 protein also exhibited a dose dependent increase in the brainstem and spinal cord. On the other hand, the two neuronal cell markers did not show a quantitative decrease in the CNS. This means that the development of gliosis, rather than neurone death, is induced by chronic exposure to toluene. The significant biochemical changes induced around the threshold limit value and the concentration dependent alterations suggest that these nerve specific marker proteins may be used to evaluate solvent related damage to the CNS.

Effect of inhalation exposure to 2-bromopropane on the nervous system in rats

Exposure to 2-bromopropane (2-BP) is suspected to have adverse effects on the nervous system. The aim of this study was to investigate whether the exposure of rats to 2-BP had neurotoxic effects using histological and electrophysiological studies. Wistar strain male rats were exposed daily to either 100 or 1000 ppm 2-BP or to fresh air for 8 h a day for 12 weeks. Body weight was measured before exposure and every 2 weeks. Motor nerve conduction velocity (MCV) and distal latency (DL) were measured before exposure and every 4 weeks during exposure. Histological examination of the nervous system was also performed. Exposure of rats (n = 9) to 1000 ppm resulted in suppression of body weight gain and a significant decrease in brain weight compared to the control (n = 9). Electrophysiological measurements showed a significant decrease in MCV in 1000 ppm exposed rats at 8 weeks and significant prolongation of DL at 8 and 12 weeks. Abnormalities of the myelin sheath were detected in the common peroneal nerves. In 100-ppm exposed rats (n = 9), no significant changes were noted in body weight and the peripheral nerve. In conclusions, long-term exposure to 1000 ppm of 2-BP may result in peripheral neuropathy in rats.

2-bromopropane-induced hypoplasia of bone marrow in male rats

2‐Bromopropane‐lnduced Hypoplasia of Bone Marrow in Male Rats: Tamie Nakajima, et al. Department of Hygiene, Shinshu University School of Medicine—The hematotoxicity of 2 ‐bromopropane was investigated in thirty‐six male Wistar rats. The rats were put into four groups: three groups were exposed to 0, 300, and 1,000 ppm 2‐bromopropane for 8 hr per day, for 9 weeks, respectively, and the remaining group was exposed to 3,000 ppm only for 9‐11 days. Hematotoxicity was assessed by measuring peripheral blood cells as well as cellularity, the number of megakaryocytes and morphological findings in the bone marrow. Exposure to 2‐bromopropane decreased the numbers of erythrocytes in the peripheral blood at 300 ppm or higher, leukocytes at 1,000 ppm, and platelets at 300 and 1,000 ppm. Exposure to 300 ppm 2‐bromopropane did not influence the indices of bone marrow toxicity. Exposure to 1,000 ppm 2‐bromopropane or a higher dose‐dependently induced a hypoplastic profile with replacement of fatty spaces in the bone marrow, though the durations of exposure to 3,000 ppm were under one sixth. These exposures also induced dose‐dependent decreases in the number of megakaryocytes, with the maintained ratio of granulocytes to erythrocytes in the bone marrow. Residual progenitor cells showed some dysplastic or megaloblastic changes. These results suggest that exposure to 2‐bromopropane leads to a reduction in the numbers of hematopoietic cells in the bone marrow, the result being a persistent pancytopenia in male rats.

Chronic exposure to n-hexane induces changes in nerve-specific marker proteins in the distal peripheral nerve of the rat

1 After long-term n-hexane exposure (2000 ppm, 12 h d-1, 6 d week-1, for 24 weeks), the content of neuron-specific enolase (gamma-enolase), creatine kinase-B and beta-S100 protein in the cortex, cerebellum, spinal cord and proximal and distal sciatic nerves of rats was determined by enzyme immunoassay. 2 The amounts of the three proteins decreased significantly in the distal segment of sciatic nerve, whereas they remained unchanged in the brain and proximal sciatic nerve. The quantitative decline in these marker proteins in the distal sciatic nerve could be related to neurophysiological deficits in the peripheral nerves. 3 This study indicates that the biochemical changes observed are consistent with the clinical and pathological findings of n-hexane neuropathy. These nerve-specific marker proteins can be used to assess solvent-related peripheral neurotoxicity.

Nerve-Specific Marker Proteins as Indicators of Organic Solvent Neurotoxicity

Effects of chronic exposure to n-hexane and toluene on some nerve-specific marker proteins in rat central nervous system (CNS) and peripheral nervous system (PNS) were assessed and compared. The rats were exposed to 2000 ppm n-hexane, 12 hr/day, 6 days/ week, for 24 weeks, and to 1000 ppm toluene, 8 hr/day, 6 days/week, for 16 weeks. The level of neuron-specific enolase (NSE), creatine kinase-B (CK-B), and s-S100 protein in cortex, cerebellum, spinal cord, and proximal and distal sciatic nerve was determined by enzyme immunoassay method. In n-hexane-exposed rats, the level of NSE, CK-B, and s-S100 decreased significantly in the distal segment of the sciatic nerve, while the marker proteins in CNS and proximal sciatic nerve remained unchanged. In contrast, chronic exposure to toluene mostly affected these marker proteins in CNS tissues, displaying the increase of NSE, CK-B, s-S100 in cerebellum, as well as the increase of s-S100 in spinal cord. No quantitative changes of the three proteins in distal sciatic nerve were observed after exposure to toluene. n-Hexane-induced peripheral distal neuropathy and toluene-induced brain gliosis appeared to be responsible for this different pattern of biochemical changes. The present study suggests the usefulness of using these nerve-specific marker proteins to assess the solvent-related CNS and PNS neurotoxicity.