Naomi Hisanaga

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.

A comparative study on the neurotoxicity of n-pentane, n-hexane, and n-heptane in the rat.

The neurotoxicity of n-pentane, n-hexane, and n-heptane have been studied in Wistar strain male rats after exposure to 3000 ppm of n-pentane, n-hexane, or n-heptane for 12 hours a day for 16 weeks. The nerve conduction velocity and the distal latency were measured before the beginning of the exposure and after exposure for four, eight, 12, and 16 weeks. The experiment showed that n-hexane disturbed the conduction velocity of the motor nerve and the mixed nerve and prolonged the distal latency in the rats tail, but that n-pentane and n-heptane did not. The light and electron microscopic examination showed that the peripheral nerve, the neuromuscular junction, and the muscle fibre of the rats exposed to n-hexane were severely impaired, but those of the rats exposed to n-pentane or n-heptane showed no particular changes even after 16 weeks of exposure. These results show that n-hexane is far more toxic to the peripheral nerve of the rat than n-pentane or n-heptane. It is necessary to study the neurotoxicity of other petroleum hydrocarbons, since some reports suggest that petroleum solvents might possibly contain neurotoxic hydrocarbons other than n-hexane.

A study on biological monitoring of n-hexane exposure.

Summaryn-Hexane is one of the solvents widely used in industry and well known to be neurotoxic. Recently it was clearly revealed that n-hexane is metabolized in vivo and its metabolites are excreted in the urine. However, the relationship between the exposed dose of n-hexane and the metabolites in the urine has not yet been substantially determined. Therefore, in this investigation we intended to clarify the above relationship in order to establish its usefulness for biological monitoring of n-hexane exposure.The exposed dose was measured by means of a personal monitoring badge worn by workers in seven factories manufacturing vinyl sandals. The time-weighted average (TWA) concentration of n-hexane was 0.2–47.4 ppm.The n-hexane metabolites in the urine of 22 workers were measured with modified Perbellinis method [12] in the early morning (6:00–7:00 hrs) and at 17:00 hrs. 2,5-Dimethylfuran, 2,5-hexanedione and γ-valerolactone were identified by gas chromatography and mass spectrometory. At 17:00 hrs the means ± SD of the metabolites were 0.21 ± 0.11 mg/l for 2,5-dimethylfuran, 1.13 ± 0.71 mg/1 for 2,5-hexanedione, and 2.04±2.31 mg/l for γ-valerolactone. The metabolites were also found in the urine in the early morning. 2-Hexanol was not detected in the urine of any worker examined. A strong correlation between TWA concentration of n-hexane and 2,5-hexanedione in the urine was found at 17:00 hrs (r = 0.895, P < 0.001).The results suggest that the urinary metabolites of n-hexane, especially 2,5-hexanedione, could be useful indicators for biological monitoring of n-hexane exposure. Furthermore the present study offers the advantage of a better estimate of n-hexane TWA.

An experimental study on the combined effects of n-hexane and toluene on the peripheral nerve of the rat.

An electrophysiological study was undertaken to determine whether toluene affected the neurotoxicity of n-hexane. Separate groups of eight rats were exposed to 1000 ppm n-hexane, 1000 ppm toulene, 1000 ppm n-hexane plus 1000 ppm toluene, of fresh air in an exposure chamber for 12 hours a day for 16 weeks. The body weight, MCV, DL, MNCVs were measured before exposure, after four, eight 12, and 16 weeks exposure; and four weeks after exposure was discontinued. Exposure to 1000 ppm n-hexane considerably impaired the function of the peripheral nerve, but exposure to a mixture of 1000 ppm n-hexane plus 1000 ppm toluene resulted in only slight impairment; 1000 ppm toluene had little effect. These results strongly suggest that toluene decreases the toxic effects of n-hexane on the peripheral nerve.

Changes of n-hexane metabolites in urine of rats exposed to various concentrations of n-hexane and to its mixture with toluene or MEK

SummaryIt is well known that n-hexane produces peripheral neuropathy, and 2,5-hexanedione, one of the metabolites of n-hexane, is thought to be the main causative agent. Recently, the metabolites of n-hexane in urine have been measured by gas chromatography, and 2,5-hexanedione was proved to be useful for the biological monitoring of n-hexane exposure. In the present experiment, we intended to clarify the change of n-hexane metabolites in the urine of rats exposed to various concentrations of n-hexane and to its mixture with toluene or MEK. In the first experiment, five separate groups of five rats each were exposed to 100, 500, 1000, or 3000 ppm of n-hexane, or fresh air respectively in an exposure chamber for 8 h a day. Urinary samples were gathered during exposure, 16, 24, and 40 h after exposure. Half of each sample was analyzed by gas chromatography after hydrolysis with acid and enzymes, and the other half was analyzed without hydrolysis. 2,5-Dimethylfuran, MBK, 2-hexanol, 2,5-hexanedione, and γ-valerolactone could be identified as n-hexane metabolites in the urine. The main metabolites were 2-hexanol and 2,5-hexanedione. 2-Hexanol was mostly excreted during exposure, while most of the 2,5-hexanedione was excreted after the end of exposure. The amount of metabolites in the urine correlatively increased with the concentration of n-hexane from 100 to 1000 ppm, but the amount of metabolites scarcely increased when the concentration of n-hexane increased from 1000 to 3000 ppm. The maximum concentration of the excreted metabolites in urine was delayed when the concentration of n-hexane increased. These findings may indicate that the capacity of hydroxylation in rat liver microsomes was saturated when exposed to high concentrations of n-hexane. Free (not conjugated) metabolites were also excreted in the urine in all concentrations during exposure and 16 h after exposure. In the second experiment, four separate groups of five rats each were exposed to 1000 ppm of n-hexane, 1000 ppm of n-hexane plus 1000 ppm of toluene, 1000 ppm of n-hexane plus 1000 ppm of MEK, or fresh air. The analyzing procedure was the same as that in the first experiment. Distributions of n-hexane metabolites in the mixed exposure group were almost similar to that of the n-hexane-alone group. The amount of metabolites decreased to about one-sixth of that in the n-hexane group by co-exposure with toluene and to about one-fourth by co-exposure with MEK. From these results, it can be considered that toluene decreases the neurotoxicity of n-hexane by the inhibition of n-hexane metabolism. However, the reason the neurotoxicity of n-hexane is increased by co-exposure with MEK cannot be clearly explained.

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.

Absence of blue-yellow color vision loss among workers exposed to toluene or tetrachloroethylene, mostly at levels below occupational exposure limits

SummaryPossible color vision loss was examined with Lanthonys new color test and Ishiharas color vision test in 261 solvent workers and 120 controls (48 men and 72 women). The solvent workers were exposed to either predominantly toluene [46 ppm as geometric mean (GM); 63 men and 111 women], tetrachloroethylene alone (13 ppm; 30 men and 34 women), or a mixture (14 men and 9 women) of tetrachloroethylene (12 ppm) and trichloroethylene (7 ppm). The only instances of color vision loss that were detected in either the exposed workers or the controls were six cases of red-green loss (all in men). These six cases of red-green loss showed an unbiased distribution between the exposed workers and the nonexposed controls.

Toluene induces behavioral activation without affecting striatal dopamine metabolism in the rat: behavioral and microdialysis studies.

We examined the effects of toluene on the release of dopamine (DA) and its metabolites in rat striatum using microdialysis. Intraperitoneal injection of 800 mg/kg toluene significantly increased motor activity in rats, as did methamphetamine (MAP) (1 mg/kg). However, 800 mg/kg toluene did not affect the extracellular levels of DA, 3,4-dihydroxyphenylacetic acid, homovanillic acid, or 5-hydroxyindoleacetic acid. This is in contrast to MAP, which significantly increased extracellular DA and decreased the extracellular levels of its metabolites. These results suggest that toluene-induced behavioral augmentation may not be associated with alterations in DA or serotonin neurochemistry such as are associated with MAP-induced behavioral augmentation.

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.

DETERMINATION OF URINARY 2,5-HEXANEDIONE CONCENTRATION BY AN IMPROVED ANALYTICAL METHOD AS AN INDEX OF EXPOSURE TO N-HEXANE

2,5-Hexanedione is a main metabolite of n-hexane and is considered as the cause of n-hexane polyneuropathy. Therefore, it is useful to measure 2,5-hexanedione for biological monitoring of exposure to n-hexane. The analytical methods existing for n-hexane metabolites, however, were controversial and not established enough. Hence, a simple and precise method for determination of urinary 2,5-hexanedione has been developed. Five ml of urine was acidified to pH 0.5 with concentrated hydrochloric acid and heated for 30 minutes at 90-100 degrees C. After cooling in water, sodium chloride and dichloromethane containing internal standard were added. The sample was shaken and centrifuged. 2,5-Hexanedione concentration in an aliquot of dichloromethane extract was quantified by gas chromatography using a widebore column (DB-1701). Urinary concentration of 2,5-hexanedione showed a good correlation with exposure to n-hexane (n = 50, r = 0.973, p less than 0.001). This method is simple and precise for analysis of urinary 2,5-hexanedione as an index of exposure to n-hexane.