* Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya, Japan;
National Institute of Industrial Health, Kanagawa, Japan;
Institute for Laboratory Animal Experiments, Nagoya University School of Medicine, Nagoya, Japan;
§ Safety Assessment Laboratory, Sanwa Kagaku Kenkyusho Co. Ltd., Mie, Japan;
¶ Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Japan; and
|| Nagoya University Graduate School of Bioagricultural Sciences, Nagoya, Japan
Received August 31, 1999; accepted November 24, 1999
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: 1-bromopropane; reproductive toxicity; alternative to chlorofluorocarbons; spermiation failure; retention of elongated spermatid; spermiogenesis; Sertoli cell; testosterone; seminal vesicle; banana-like head.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present experiment aimed to clarify the dose-dependent reproductive toxicity of 1-bromopropane in male rats. Parts of this study were presented at the 71st and 72nd Annual Meetings of the Japan Society for Occupational Health (Ichihara et al., 1998, 1999b
).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The 4 groups were exposed to either 200, 400, or 800 ppm of 1-bromopropane or fresh air. The maximum concentration was 800 ppm because our previous study (Yu et al., 1998) showed that rats exposed to 1000 ppm of 1-bromopropane were debilitated after 4 weeks. The inhalation exposure was conducted from 1400 to 2200 h. The inhalation exposure system used in the present study has been described previously (Ichihara et al., 1997
; Takeuchi et al., 1989
). In brief, the regulated volume of 1-bromopropane was evaporated at room temperature and mixed with a larger volume of clean air to achieve the target concentrations. The vapor concentrations of 1-bromopropane in the chamber were measured every 10 s by gas chromatography and controlled to within ±5% of the target concentration by means of a personal computer. The mean concentration of levels measured every 10 s for 8 h was taken for the value on a given day. This was then averaged over 12 weeks in order to obtain the mean and standard deviations, and the daily gas concentrations in the 3 chambers were measured at 208 ± 15, 412 ± 24, and 821 ± 38 ppm, respectively. The 1-bromopropane (99.81% purity) was kindly supplied by Tosoh Co., Ltd., Japan. The Japanese law concerning protection and control of animals and the Guide of Animal Experimentation of the Nagoya University School of Medicine were followed throughout the experiments.
Epididymal sperm count, motility, tailless sperm ratio, and morphological abnormality of sperm head.
After 12 weeks' of exposure, the rats were weighed, and, under pentobarbital anesthesia, were killed by collecting all blood through the abdominal aorta. Epididymal sperm count and motility were evaluated according to our previous studies (Ichihara et al., 1996, 1997
). Sperm were collected as quickly as possible after the rat was killed. The sperm suspension was prepared by mincing the right cauda epididymidis in 2.5 ml of Hank's solution kept at 37°C, pipetting the suspension and filtering it through gauze. A part of the fraction was diluted with Hank's solution kept at 37°C. Progressive or non-progressive motile sperm were counted on an erythrocytometer (Neubauer type) under a light microscope. Another fraction was diluted with saline containing 0.5% formalin for detemination of the sperm count. A smear was made on a glass slide, and the sperm shape was examined under a phase-contrast microscope. Tailless sperm and sperm with morphologically abnormal heads were counted. A total of 300 sperm were examined to count tailless sperm, and 300 sperm with tails were examined to count sperm with morphologically abnormal heads, which were classified as straight, banana-like, teratic, (amorphous or pyknomorphous), and "other" (unclassified), according to Mori et al. (1991). An intermediate type with characteristics of both a banana-like (blunt) head and straight head was classified as banana-like, because most banana-like heads were also more or less straight.
Organ weights and histopathological examinations.
The epididymides, testes, prostate, seminal vesicle, liver, kidneys, spleen, lungs, heart, adrenal glands, thymus, pituitary gland and femur were dissected out of the rats at the end of the experiment, and were weighed, excluding the femur. The testes and the left epididymis were fixed in Bouin's solution and the other organs in 10% neutral buffered formalin for histopathological studies. The femur was decalcified after the fixation. The organs were embedded in paraffin and cut in 5-µm sections. Tissue sections of the testis were reacted with periodic acid-Schiff's reagent (PAS) and other organs were stained with hematoxylin and eosin (H-E).
Cell counts of spermatogenic cells and degenerating cells in seminiferous tubules.
Spermatogenic cells at stage VII were counted to evaluate the cellularity of spermatogenic cells in the testis, according to Omura et al. (1999). Twelve photomicrographs of stage VII seminiferous tubules were randomly taken under light microscope from one section of testis per rat with a digital camera (Fujix Digital Camera HC-300Z/CL, Olympus Japan Co., Ltd.). The digital pictures were printed out with a full-color, high-resolution digital printer (PICTROGRAPHY 3000, Fuji Film Japan Co., Ltd.). All of the spermatogonia, preleptotene spermatocytes, pachytene spermatocytes, round spermatids, and nuclei of Sertoli cells in the seminiferous tubule were counted. Spermatogonia, preleptotene spermatocytes, and nuclei of Sertoli cells were confirmed by direct observation under light microscope, because it was sometimes difficult to classify these cells on the print. We examined 12 seminiferous tubules per rat, because the digital camera could take only 12 pictures, making it easy to avoid counting of the same seminiferous tubules twice. We considered that 12 tubules was enough, because 12 was more than the 10 recommended in the review by Creasy (1997) and adopted in the original study by Omura et al. (1999). Degenerating pachytene spermatocytes and vacuoles or spaces in the seminiferous epithelium at stage VII were also counted. Elongated spermatid at stage VII were not counted because it was difficult to do so accurately. Retained, elongated spermatid and nuclei of Sertoli cells were counted in 12 round or ovoid seminiferous tubules at postspermiation stages (stages IX, X, and XI) in the same way. Retained, elongated spermatids near the lumen were not counted, because it was sometimes difficult to distinguish them from released spermatids in the lumen. Only retained and degenerating (probably phagocytized by Sertoli) elongated spermatids near the basement membrane were counted for quantitative evaluation. The criterion was that more than half of the profile should be in the basal one or two cell layers (layer of leptotene or pachytene spermatocytes). Stages of the cycle in rats were classified according to Russell et al. (1990). The numbers of the spermatogenic cells, degenerating cells, or vacuoles/spaces were expressed per tubule or per 100 Sertoli nuclei. A mean value of 12 tubules was treated as the representative value of each animal.
Hormone assay.
Plasma samples were stored at 80° until assayed for testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). Plasma testosterone levels were measured with a radioimmunoassay (RIA) kit for testosterone (Eiken Chemical, Tokyo). Ten µl of plasma was extracted with 2 ml of hexane-ethylether mixture (3:2). The detection limit was 1.2 ng/ml. LH and FSH concentrations in plasma were determined by a double antibody RIA with a rat LH RIA kit and FSH RIA kit provided by the National Hormone and Pituitary Program (Baltimore, MD), and were expressed in terms of NIDDK-rLH-RP-3 and NIDDK-rFSH-RP-2, respectively. The least detectable levels of LH and FSH were 0.16 ng/ml and 2.5 ng/ml for 50-µl samples, respectively.
Hematological examination.
The following hematologic parameters were determined: erythrocyte counts, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpusucular hemoglobin concentration (MCHC), total leukocyte count, and platelet count (with an F-800 Toa Microcell Counter, Toa Medical Electronics).
Statistical analysis.
Multiple comparisons were made between the exposure groups and the control using Dunnett's method following one-way analysis of variance (ANOVA). A probability (P) of <0.05 was accepted as statistically significant. The percentage values were converted by arcsine transformation before the above analysis. The log transformation was performed before ANOVA for the value of testosterone and the number of degenerating spermatids to equalize the variance, because the standard variation increased in proportion to the mean value. Root transformation was applied to the degenerating spermatocytes because the probability of degenerating spermatocytes was so low that it could be regarded as following the Poisson distribution.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Degenerating spermatocytes at stage VII at 800 ppm and retained spermatids at the postspermiation stages might be involved with hormonal changes, since hypophysectomy has been known to induce a degeneration of spermatocytes at stage VII and retention of step 19 spermatid (Russell et al., 1977). These changes were prevented by administration of LH and FSH (Russell et al., 1977). The present study showed a significant decrease in the plasma testosterone level at 800 ppm but not at 200 or 400 ppm. However, a dose-dependent decrease in the weight of the seminal vesicle might indicate a decrease in mean testosterone level at 200 ppm or higher, if the general presumption holds here that the weight of the seminal vesicle is specifically sensitive to the testosterone level (Hamilton 1990). We could not obtain data suggesting the involvement of change in gonadotropin secretion with a decrease in testosterone or testicular lesions. However, it should be noted that pentobarbital was used instead of decapitation, which sometimes causes hemolysis or contamination of blood with lymph, to evaluate hematotoxicity. Nazian (1988) showed that pentobarbital inhibited secretion of FSH, LH, or testosterone, while Morris and Knigge (1976) found it had no effect on LH. The present study also has the limitation of one-point blood sampling to evaluate the secretion of gonadotropins and testosterone, which are released in a pulsatile manner. Further studies are necessary to elucidate the involvement of gonadotropin secretion status with decreases in testosterone or Sertoli function.
The banana-like heads were similar to the heads of early-elongated spermatids. This morphological abnormality might be related to the immaturity of sperm. Mori et al. (1991) classified the straight heads or banana-like heads of sperm into an immature type in the rats exposed to ethylene oxide. In the rats administered gallium arsenide, spermiation failure was accompanied by an increase in sperm with straight heads (Omura et al., 1996). Such an increase would indicate the early release of elongated spermatids from Sertoli cells or a delay in the spermatid transformation process, which might also be related to a functional disorder of Sertoli cells.
The histopathological changes in the testis induced by 1-bromopropane are much different from those by 2-bromopropane, which causes severe testicular atrophy at 300 ppm for 9 weeks and an almost complete loss of germ cells at 1000 ppm for 9 weeks (Ichihara et al., 1996, 1997
). As shown in the count of spermatogonia or spermatocytes at VII stage, 1-bromopropane does not have the severe toxicity toward spermatogonia which 2-bromopropane has. In addition, the present study also showed that 1-bromopropane had less hematopoietic toxicity than 2-bromopropane.
Kim et al. (1999) administered 50, 300, and 1800 ppm of 1-bromopropane to rats, 6 h/day, 5 days/week, for 8 weeks, and did not observe any reproductive toxicity. In their study, the 1-bromopropane exposure increased the relative testicular weight per body weight, but they did not mention absolute testicular weight nor did they examine the epididymal sperm, seminal vesicle, or prostate. They found cytoplasmic vacuolation in the hepatocytes around the central veins without dose-dependency, but did not observe 1-bromopropane-related morphological changes in the other organs or any significant changes in feed consumption, urinalysis, hematology, or serum biochemistry. In the present study, fat droplets around the central vein were found in the control group and they tended to decrease in the 800-ppm group.
The effects of 1- and 2-bromopropane on reproductive organs differed, but the lowest adverse effect levels (LOAEL) were almost the same as for the 200-ppm group in our experiments. 1-Bromopropane is a newly introduced solvent, and its toxic effects on exposed workers have not been reported to date. However, as seen in the intoxication cases of 2-bromopropane in Korea (Kim et al., 1996; Park et al., 1997
), workers could be exposed to the solvent at higher concentrations when it is used in an open system in the workplace. Workers should thus be carefully protected from exposure to 1-bromopropane.
In conclusion, 1-bromopropane dose-dependently decreased the epidydimal sperm count and sperm motility, and increased tailless sperm and sperm with an immature head shape. It did not decrease the testicular weight, the number of spermatogonia, spermatocytes, or round spermatids at stage VII, but increased retained, elongated spermatids at the postspermiation stages IXXI. These changes show that the main effect on reproductive organs of 1-bromopropane is inhibition of spermiation activity, in contrast to 2-bromopropane, which impairs spermatogonia. However, the lowest adverse effect levels of both 1- and 2-bromopropane are similar. Thus, this agent should be very cautiously used in the workplace, from the viewpoint of its possible male reproductive toxicity.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Creasy, D. M. (1997). Evaluation of testicular toxicity in safety evaluation studies: The appropriate use of spermatogenic staging. Toxicol. Pathol. 25, 119131.[ISI][Medline]
Hamilton, D. W. (1990). Anatomy of mammalian male accessory reproductive organs. In Marshall's Physiology of Reproduction (G. E. Lamming, Ed.), pp. 691746. Churchill Livingstone, Edinburgh.
Hew K. W., Ericson W. A., and Welsh, M. J. (1993). A single low-cadmium dose causes failure of spermiation in the rat. Toxicol. Appl. Pharmacol. 121, 1521.[ISI][Medline]
Ichihara, G., Asaeda, N., Kumazawa, T., Tagawa, Y., Kamijima, M., Yu, X., Kondo, H., Nakajima, T., Kitoh, J., Yu, I. J., Moon, Y. H., Hisanaga, N., and Takeuchi, Y. (1996). Testicular toxicity of 2-bromopropane. J. Occup. Health 38, 205206.
Ichihara, G., Asaeda, N., Kumazawa, T., Tagawa, Y., Kamijima, M., Yu, X., Kondo, H., Nakajima, T., Kitoh, J., Yu, I. J., Moon, Y. H., Hisanaga, N., and Takeuchi, Y. (1997). Testicular and hematopoietic toxicity of 2-bromopropane, a substitute for ozone layer-depleting chlorofluorocarbons. J. Occup. Health 39, 5763.[ISI]
Ichihara, G., Ding, X., Yu, X., Wu, X., Kamijima, M., Peng, S., Jiang, X., and Takeuchi, Y. (1999a). Occupational health survey on workers exposed to 2-bromopropane at low concentrations. Am. J. Ind. Med. 35, 523531.[ISI][Medline]
Ichihara, G., Yu, X., Eiji, S., Kitoh, J., Asaeda, N., Xie, Z., Wang, H., and Takeuchi, Y. (1998). Neurotoxicity of 1-bromopropane. Sangyo Eiseigaku Zasshi 40, 414 (Japanese).
Ichihara, G., Yu, X., Kitoh, J., Shibata, E., Asaeda, N., Yamada, T., Wang, H., Xie, Z., Kumazawa, T., Iwai, K., Maeda, K., Tsukamura, H., and Takeuchi, Y. (1999b). Histopathological changes of nervous system and reproductive organ and bloodbiochemical findingsin rats exposed to 1-bromopropane. Sangyo Eiseigaku Zasshi 41, 513 (Japanese).
Kamijima, M., Ichihara, G., Yu, X., Xie, Z., Kitoh, J., Tsukamura, H., Maeda, K., Nakajima, T., Asaeda, N., Hisanaga, N., and Takeuchi, Y. (1997a). Ovarian toxicity of 2-bromopropane in the non-pregnant female rat. J. Occup. Health 39, 144149.[ISI]
Kamijima, M., Ichihara, G., Yu, X., Xie, Z., Kitoh, J., Tsukamura, H., Maeda, K., Nakajima, T., Asaeda, N., Hisanaga, N., and Takeuchi, Y. (1997b). Disruption in ovarian cyclicity due to 2-bromopropane in the rat. J. Occup. Health 39, 34.[ISI]
Kim, H. Y., Chung, Y. H., Jeong, J. H., Lee, Y. M., Sur, G. S., and Kang, J. K. (1999). Acute and repeated inhalation toxicity of 1-bromopropane in SD rats. J. Occup. Health 41, 121128.[ISI]
Kim, Y., Jung, K., Hwang, T., Jung, G., Kim, H., Park, J., Kim, J., Park, J., Park, D., Park, S., Choi, K., and Moon, Y. (1996). Hematopoietic and reproductive hazards of Korean electronic workers exposed to solvents containing 2-bromopropane. Scand. J. Work Environ. Health 22, 387391.[ISI][Medline]
Ku, W. W., Chapin, R. E., Wine, R. N., and Gladen, B. C. (1993). Testicular toxicity of boric acid (BA): Relationship of dose to lesion development and recovery in the F344 rat. Reprod. Toxicol. 7, 305319.[ISI][Medline]
Lim, C. H., Maeng, S. H., Lee, J. Y., Chung, Y. H., Kim, T. G., Park, J. H., Moon, Y. H., and Yu, I. J. (1997). Effects of 2-bromopropane on the female reproductive function in Sprague-Dawley rats. Ind. Health 35, 278284.[ISI][Medline]
Linder, R. E., Klinefelter, G. R., Strader, L. F., Suarez, J. D., and Dyer, C. J. (1994a). Acute spermatogenic effects of bromoacetic acids. Fundam. Appl. Toxicol. 22, 422430.[ISI][Medline]
Linder, R. E., Klinefelter, G. R., Strader, L. F., Suarez, J. D., and Roberts, N. L. (1997). Spermatotoxicity of dichloroacetic acid. Reprod. Toxicol. 11, 681688.[ISI][Medline]
Linder, R. E., Klinefelter, G. R., Strader, L. F., Suarez, J. D., Roberts, N. L., and Dyer, C. J. (1994b). Spermatotoxicity of dibromoacetic acid in rats after 14 daily exposures. Reprod. Toxicol. 8, 251259.[ISI][Medline]
Linder, R. E., Strader, L. F., and Rehnberg, G. L. (1990). Effect of acute exposure to boric acid on the male reproductive system of the rat. J. Toxicol. Environ. Health 31, 133146.[ISI][Medline]
Mori, K., Kaido, M., Fujishiro, K., Inoue, N., Koide, O., Hori, H., and Tanaka, I. (1991). Dose-dependent effects of inhaled ethylene oxide on spermatogenesis in rats. Br. J. Ind. Med. 48, 270274.[ISI][Medline]
Morris, M., and Knigge, K. M. (1976). The effects of pentobarbital and ether anesthesia on hypothalamic LH-RH in the rat. Neuroendocrinology 20, 193200.[ISI][Medline]
Nakajima, T., Shimodaira, S., Ichihara, G., Asaeda, N., Kumazawa, T., Iwai, H., Ichikawa, I., Kamijima, M., Yu, X., Xie, Z., Kondo, H., and Takeuchi, Y. (1997a). 2-Bromopropane-induced hypoplasia of bone marrow in male rats. J. Occup. Health 39, 228233.[ISI]
Nakajima, T, Shimodaira, S., Ichihara, G., Asaeda, N., Kumazawa, T., Iwai, H., Ichikawa, I., Kamijima, M., Yu, X., Xie, Z., Kondo, H., and Takeuchi, Y. (1997b). Histopathological findings of bone marrow induced by 2-bromopropane in male rats. J. Occup. Health 39, 8182.[ISI]
Nazian, S. J. (1988). Serum concentrations of reproductive hormones after administration of various anesthetics to immature and young adult male rats. Proc. Soc. Exp. Biol. Med. 187, 482487.[Abstract]
Omura, M., Romero, Y., Zhao, M., and Inoue, N. (1997a). Histopathological changes of the testis in rats caused by subcutaneous injection of 2-bromopropane. J. Occup. Health 39, 234239.[ISI]
Omura, M., Romero, Y., Zhao, M., and Inoue, N. (1999). Histopathological evidence that spermatogonia are the target cells of 2-bromopropane. Toxicol. Lett. 104, 1926.[ISI][Medline]
Omura, M., Tanaka, A., Hirata, M., Zhao, M., Makita, Y., Inoue, N., Gotoh, K., and Ishinishi, N. (1996). Testicular toxicity of gallium arsenide, indium arsenide, and arsenic oxide in rats by repetitive intratracheal instillation. Fundam. Appl. Toxicol. 32, 7278.[ISI][Medline]
Omura, M., Zhao, M., Romero, Y., and Inoue, N. (1997b). Toxicity of 2-bromopropane on spermatogonia and spermatocytes. J. Occup. Health 39, 12.[ISI]
Park, J. S., Kim, Y., Park, D. W., Choi, K. S., Park, S. H., and Moon, Y. H. (1997). An outbreak of hematopoietic and reproductive disorders due to solvents containing 2-bromopropane in an electronic factory: South Korea: Epidemiological Survey. J. Occup. Health 39, 138143.[ISI]
Russell, L. D., and Clermont, Y. (1977). Degeneration of germ cells in normal, hypophysectomized, and hormone-treated hypophysectomized rats. Anat. Rec. 187, 347366.[ISI][Medline]
Russell, L., Ettlin, R., Sinha-Hikim, A., and Clegg, E. (1990). Histological and Histopathological Evaluation of theTestis, pp. 62118. Cache River Press, United States.
Swierstra, E. E., Whitefield, J. W., and Foote, R. H. (1964). Action of amphotericin B (fungizone) on spermatogenesis in the rabbit. J. Reprod. Fertil. 29, 1319.[Medline]
Takeuchi, Y., Huang, J., Shibata, E., Hisanaga, N., and Ono, Y. (1989). A trial to automate an organic solvent exposure system for small animals. Jpn. J. Ind. Health 31, 722 (Japanese).
Toth, G. P., Kelty, K. C., George, E. L., Read, E. J., and Smith, M. K. (1992). Adverse male reproductive effects following subchronic exposure of rats to sodium dichloroacetate. Fundam. Appl. Toxicol. 19, 5763.[ISI][Medline]
Treinen, K. A., and Chapin, R. E. (1991). Development of testicular lesions in F344 rats after treatment with boric acid. Toxicol. Appl. Pharmacol. 107, 325335.[ISI][Medline]
Yu, I. J., Chung, Y. H., Lim, C. H., Maeng, S. S., Lee, J. Y., Kim, H. Y., Lee, S. J., Kim, C. H., Kim, T. G., Lim, C. H., Park, J. S., and Moon, Y. H. (1997). Reproductive toxicity of 2-bromopropane in Sprague Dawley rats. Scand. J. Work Environ. Health 23, 281288.[ISI][Medline]
Yu, X., Ichihara, G., Kitoh, J., Xie, Z., Shibata, E., Kamijima, M., Asaeda, N., and Takeuchi, Y. (1998). Preliminary report on the neurotoxicity of 1-bromopropane, an alternative solvent for chlorofluorocarbons. J. Occup. Health 40, 234235.[ISI]
Yu, X., Kamijima, M., Ichihara, G., Li, W., Shibata, E., Hisanaga, N., and Takeuchi, Y. (1999). 2-Bromopropane causes ovarian dysfunction by damaging primordial follicles and their oocytes in female rats. Toxicol. Appl. Pharmacol. 159, 185193.[ISI][Medline]