Kazuya Fujishiro

Dose dependent effects of inhaled ethylene oxide on spermatogenesis in rats.

Male Wistar rats were exposed to ethylene oxide (EO) at concentrations of 50, 100, or 250 ppm for six hours a day, on five days a week for 13 weeks. Dose effect relations of inhaled EO on spermatogenesis were evaluated from testicular and epididymal weights, histopathological changes and lactate dehydrogenase X (LDH X) activity in the testis, and sperm counts and sperm head abnormalities in the epididymis. At 250 ppm, a decrease in epididymal weights, slight degenerations in the seminiferous tubules, decreased sperm counts, and increased numbers of abnormal sperm heads in the tail of the epididymis were found; these were not seen at lower doses. When the abnormal sperm heads were classified into immature types and teratic types, the number of immature heads increased only at 250 ppm. On the other hand, the teratic type had increased at doses of 50 and 100 ppm EO when compared with the control group. Hence, subchronic inhalation of EO at low concentrations affects spermatogenesis in rats.

Testicular toxicity and alterations of glutathione metabolism resulting from chronic inhalation of ethylene oxide in rats

Wistar male rats were exposed to ethylene oxide (EtO) at a concentration of 500 ppm, 6 hr a day, 3 days a week, for 2, 4, 6, or 13 weeks. Testicular toxicity and changes in glutathione metabolism in the testis were investigated. The relative weights of the testes and the epididymes of the EtO-exposed group decreased in a time-dependent manner. Light microscopic examination revealed degeneration and exfoliation of germ cells. Although the severity of damage became apparent over the course of exposure, some seminiferous tubules showed germ cell recovery at 13 weeks compared with 6 weeks. There was no alteration in plasma testosterone concentration. Glutathione reductase (GR) activity decreased during the entire examination period, and recovery from the decrease was not achieved by addition of flavin adenine dinucleotide (FAD). On the other hand, glutathione peroxidase (GPx) activity decreased at 2 weeks, and then increased at 6 and 13 weeks. In spite of alterations in the glutathione redox cycle, the level of reduced glutathione (GSH) in the testes was not affected. Glutathione S-transferase activity, measured with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, increased at 6 and 13 weeks and, measured with 1,2-epoxy-3-(p-nitrophenoxy)propane, increased at 4, 6, and 13 weeks. These data indicate that chronic inhalation of EtO induces testicular atrophy. Alterations in the glutathione redox cycle and glutathione S-transferase activity might play important roles in the toxicity and the detoxifying mechanism of the testis.

Preventive effects of methylcobalamin on the testicular damage induced by ethylene oxide

In this study, the effects of methylcobalamin on testicular damage induced by ethylene oxide (EtO) were studied. When Wistar male rats inhaled EtO at 500 ppm, 6 h a day, 3 days a week, for 6 weeks, testicular damage was observed histopathologically and by some other parameters. Subcutaneous injection of methylcobalamin at 500 μg/kg, 5 times/week was found to ameliorate the damage. However, the degree of the methylcobalamin effect differed among the parameters examined in this study. Decrease in testicular weight due to EtO exposure was completely prevented by methylcobalamin, and decrease in testicular mature spermatid count and LDH-X activity was fairly well prevented. The degree of prevention of alteration in the epididymis, such as epididymal weight, epididymal sperm count and sperm abnormality rate, was significant but not complete. EtO caused apparent alterations in glutathione metabolism in the testes, but methylcobalamin did not affect such alterations induced by EtO. From these results, it has been determined that methylcobalamin has definite preventive effects on testicular toxicity of EtO.

Effects of megadoses of pyridoxine on spermatogenesis and male reproductive organs in rats

Although it has been indicated that many neurotoxicants also cause reproductive toxicity, the reproductive toxicity of megadoses of pyridoxine, which is a neurotoxicant, has not been studied. In this paper, we studied the effects of megadoses of pyridoxine on male reproductive organs. Pyridoxine hydrochloride, 125 mg/kg, 250 mg/kg, 500 mg/kg or 1000 mg/kg, daily, was intraperitoneally injected into Wistar male rats 5 days a week for 2 or 6 weeks, and its effects on the male reproductive organs were investigated. After 2 weeks of administration, absolute weights of the testis in the 500 and 1000 mg/kg epididymis in all the exposed groups and prostate gland in the 1000 mg/kg group decreased, and mature spermatid counts in the testis decreased in the 1000 mg/kg group. After 6 weeks administration, the absolute and relative weights of the testis, epididymis, prostate gland and seminal vesicle decreased in the 500 mg/kg and 1000 mg/kg groups, and mature spermatid counts in the testis and sperm counts in the epididymis decreased in these groups. Among the marker enzymes of the testicular cells, LDH-X activity decreased, and β-glucuronidase activity, cytochrome P-450 content and cytochrome b5 content increased in the 1000 mg/kg group. Plasma testosterone concentration did not significantly alter in all the exposed groups. From these results, it was concluded that megadoses of pyridoxine affected the spermatogenesis and decreased reproductive organ weights in the rat.

BIOCHEMICAL CHANGES IN RAT ERYTHROCYTES CAUSED BY ETHYLENE OXIDE EXPOSURE

When Wistar male rats were exposed to ethylene oxide (EtO) at a concentration of about 500 ppm, 6 hr a day, 3 days a week for 2, 6, or 13 weeks, hematological examination showed macrocytic, normochromic anemia with a high reticulocyte count. This result raised the possibility that the hemolytic process was responsible for the anemia. Thus, the following possible causes of hemolysis were investigated with erythrocytes obtained from control and EtO-exposed rats. (1) Metabolism in erythrocytes; (a) Hexose monophosphate cycle: The activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, or glutathione peroxidase was not affected, but the activity of glutathione reductase (GR) significantly decreased and did not recover by the addition of flavin adenine dinucleotide. Reduced glutathione content also decreased and the glutathione stability test was positive. (b) Embden-Meyerhof pathway: Adenosine triphosphate content did not decrease. (c) Lapoport-Luebering cycle: 2,3-Diphosphoglycerate content was not affected. (2) Membrane alterations: Osmotic fragility was not affected and the activity of acetylcholine esterase in the ghost membranes of the exposed group increased. (3) Hemoglobin stability: The heat test and the isopropanol test were negative. GR has an important function in maintaining the reducing power in erythrocytes, and the decrease in the activity caused by EtO induced an alteration of the glutathione stability. Although the mechanism of EtO-induced anemia could not be clearly explained, the inhibition of GR activity might be related to the anemia.

Effects of dimethylformamide on hepatic microsomal monooxygenase system and glutathione metabolism in rats

The effects of repeated exposure to N,N-dimethylformamide (DMF) on hepatic microsomal monooxygenase system and glutathione metabolism were investigated. DMF was administered to Wistar male rats by subcutaneous (s.c.) injection at 0.5 ml/kg body weight daily for 1 week. Macroscopically, mild liver swelling was observed and liver weights significantly increased after 1 week of exposure to DMF. Hematological changes were not detected. In exposed rats, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, cholinesterase and total cholesterol significantly increased. Hepatic microsomal cytochrome P-450 and protoheme decreased by 34% and 24%, respectively, while microsomal protein and cytochrome b5 were not affected. NADH-ferricyanide reductase activity decreased by 24% while NADPH-cytochrome c reductase activity showed no change. Glutathione reductase (GR) activity showed a significant decrease after the first injection and remained depressed throughout the study, with no change in glutathione peroxidase (GPx) activity. Glutathione S-transferase (GST) activity showed a significant increase at 3 days after DMF treatment and gradually increased by 66% at 1 week. In a subsequent experiment with a single administration of DMF (4 ml/kg), reduced glutathione (GSH) in the liver was decreased by 28% at 8 h, but recovered to control levels by 24 h. These results indicate that DMF alters the hepatic microsomal monooxygenase system and glutathione metabolism. These findings may greatly contribute to the elucidation of the pathogenesis of DMF hepatotoxicity.

Cumulative toxicity potential of methomyl aerosol by repeated inhalation.

There are few investigations concerning the cumulative toxicity of agricultural chemicals by repeated inhalation. In this study, Wistar male rats were exposed to methomyl powder (mass median aerodynamic diameter, 4.4 microns) for a single 4-hr exposure, or for 4 hr/day, 5 days/week for 3 months. The average exposure concentrations were controlled at 9.9 mg/m3 for the single exposure and at 14.8 mg/m3 for repeated exposures by a dust generator consisting of a continuous fluidized bed with an overflow pipe and a screw feeder. After the repeated exposures, plasma and red cell cholinesterase activities, and lipid concentrations of the rat lungs were measured and histopathological examinations were performed. There was no evidence of cumulative effects on the red cell cholinesterase activity, histopathological changes and lipid concentration in 3-month repeated inhalation.

Chronic inhalation effects of ethylene oxide on porphyrin-heme metabolism

The effects of chronic ethylene oxide (EtO) inhalation on porphyrin-heme metabolism were investigated. When Wistar male rats were exposed to 500 ppm EtO for 6 h a day, 3 times a week for 13 weeks, hemoglobin content significantly decreased, and a normocytic and normochromic anemia was found. In the liver, cytochrome P-450 and protoheme significantly decreased but wet weight, microsomal protein and cytochrome b5 were not affected. The activity of delta-aminolevulinic acid (ALA) synthase increased while ALA dehydratase did not change. The activity of hepatic ferrochelatase decreased time-dependently. Uroporphyrin increased 37% and coproporphyrin tended to increase in the liver. The concentration of protoporphyrin in the liver and erythrocytes tended to increase. Coproporphyrin excretion in the urine showed a 5-6-fold increase while there was no significant increase in urinary ALA excretion. These results indicate that chronic inhalation of EtO causes alterations of hepatic porphyrin-heme metabolism as well as anemia and may affect mechanisms of adaptation to xenobiotics.

An Investigation of Symptoms in Ethylene Oxide Sterilization Workers in Hospitals

An Investigation of Symptoms In Ethylene Oxide Sterilization Workers in Hospitals: Katsuya Yahata,et al. The Institute of Industrial Ecological Sciences, The University of Occupational and Environmental Health, Japan—We investigated the frequency of particular symptoms reported during daily sterilization work with ethylene oxide (EO). By means of a self‐administered questionnaire we received 287 replies from 66 institutions where sterilization work was performed, (148 used EO, and 139 used other substances). In these two groups, complaints such as stiff shoulders (19.2%), low back pain (13.9%), and skin burning (13.2%) were frequently reported. When we compared the EO sterilization workers with other workers by means of Fishers exact test, significant differences were observed among the cases of diarrhea, headache, dullness, sore throat and eye irritation. We also investigated some safety and health management factors that suggested correlations with the manifestation of these symptoms.

Effects of inhaled ethylene oxide on the lens glutathione redox cycle in rats

The effects of chronic ethylene oxide (EtO) inhalation on the lens glutathione redox cycle were investigated. When Wistar male rats were exposed to 500 ppm EtO for 6 h a day, 3 times a week for 13 weeks, glutathione reductase decreased significantly in the lens while glutathione peroxidase did not. Glutathione reductase activity decreased time dependently, by as much as 81% after 13 weeks. In spite of changes in the glutathione redox cycle, reduced and oxidized glutathione levels were not affected. Our results raise the possibility that EtO inhalation may produce a cataract via changes in the glutathione redox cycle.