Exposure to 1-Bromopropane Causes Ovarian Dysfunction in Rats

Tetsuya Yamada*,1, Gaku Ichihara*, Hailan Wang*, Xiaozhong Yu*,2, Kei-ichiro Maeda{dagger}, Hiroko Tsukamura{dagger}, Michihiro Kamijima*, Tamie Nakajima* and Yasuhiro Takeuchi*,3

* Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; and {dagger} Department of Biological Resources and Environmental Sciences, Nagoya University Graduate School of Bioagricultural Sciences, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

Received August 16, 2002; accepted October 16, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although 1-bromopropane has been used in chemical and electronic industries as an alternative to ozone layer-depleting solvents, its toxicity on female reproductive organs has not been fully elucidated. The aim of this experiment was to determine the effect of 1-bromopropane on female reproductive function in rats. Forty female Wistar rats were divided into four equal groups. Each group was exposed daily to 0, 200, 400, or 800 ppm of 1-bromopropane for eight h a day. After exposure for 7 weeks, all rats in the 800-ppm group became seriously ill and were sacrificed during the 8th week. The other dose groups were exposed for 12 weeks. In the 800-ppm group, but not in the other two exposed groups, body weight was significantly less than the control at each time point from 2 to7 weeks after the beginning of exposure. Tests of vaginal smears showed a significant increase in the number of irregular estrous cycles with extended diestrus in the 400- and 800-ppm groups. Histopathological examination of the ovary showed a significant dose-dependent reduction of the number of normal antral follicles and a decrease in the number of normal growing follicles in the 400-ppm group. No significant change was found in plasma concentrations of LH or FSH in any group when compared with the control. Our results indicate that 1-bromopropane can induce a dose-dependent ovarian dysfunction in nonpregnant female rats associated with disruption in follicular growth process.

Key Words: 1-bromopropane; chlorofluorocarbon alternative; reproductive toxicity; female; estrous cycle; ovary; ovarian follicle; inhalation exposure; rat.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To preserve the ozone layer, certain chlorofluorocarbons and 1,1,1-trichloroethane have been banned from production in industrially developed countries since January 1996. Alternative compounds have been used as cleaning solvents in electronic industries. In Korea, amenorrhea, oligospermia, and anemia occurred in workers exposed to 2-bromopropane, which was used as an alternative to 1,1,1-trichloroethane (Kim et al., 1996Go). Subsequent studies revealed that 2-bromopropane is very toxic to reproductive and hematopoietic organs (Ichihara et al., 1997Go; Kamijima et al., 1997Go; Nakajima et al., 1997Go; Omura et al., 1997Go; Son et al., 1999Go; Takeuchi et al., 1997Go; Yu et al., 1999Go). Accordingly, the use of 2-bromopropane as a solvent was strictly limited in industry. Instead of 2-bromopropane, 1-bromopropane has recently come to be used as a new alternative. According to the report of the United Nations Environment Program (the Report of the Second Meeting of the Bureau of the Eleventh Meeting of the Parties to the Montreal Protocol on Substances that Deplete the Ozone Layer), the estimated sales of 1-bromopropane–based solvent for the period of January to December 1999 totaled 765 tons in Japan.

Our recent animal studies showed that 1-bromopropane could cause serious toxic effects on the central nervous system, peripheral nerves, and spermatogenesis (Ichihara et al., 2000aGo, 2000bGo; Wang et al., 2002Go; Yu et al., 1998Go). While 1-bromopropane is less toxic to spermatogonia when compared with 2-bromopropane, it is known to inhibit spermiation in the testis (Ichihara et al., 2000bGo). The underlying mechanism of the toxic effects of 1-bromopropane on the reproductive system is different from those of 2-bromopropane in male rats. On the other hand, previous study has revealed that a 10-week inhalation exposure to 1-bromopropane at 750 ppm reduced ovarian weight and numbers of corpora lutea, and also caused extended estrous cycle length (WIL Research Laboratories, 2001Go). The rats in the 500-ppm group displayed extended estrous cycle length without changing ovarian weight. Another study reported that exposure to 1-bromopropane increased relative weights of ovaries (Kim et al, 1999Go).

These studies suggested that 1-bromopropane might be a reproductive toxicant to not only male rats but also female rats. However, the former studies on female rats provided limited information on histopathological alterations of ovaries. It would be of great interest to characterize histopathological changes of ovaries or ovarian follicles induced by 1-bromopropane in comparison to the effects on primordial follicles by 2-bromopropane (Yu et al., 1999Go), because previous studies on male rats demonstrated the difference of target in the testis between 1-bromopropane and 2-bromopropane (Ichihara et al., 2000bGo; Omura et al., 1997Go, 1999Go). The present study was designed to investigate the effects of 1-bromopropane on the female reproductive system, focusing on ovarian follicles, in rats.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemical.
1-Bromopropane was kindly supplied by Tosoh Co., Ltd., Japan. Analytical analysis by capillary gas chromatography confirmed that the supplied stock was more than 99.5% pure.

Animals and inhalation exposure.
Female Wistar rats, weighing 150–170 g at 10 weeks of age, were purchased from the Shizuoka Laboratory Animal Center, Japan. They were housed in stainless steel cages, provided food and water ad libitum, and kept on a 12-h/12-h light/dark cycle (lights on at 9 A.M.) at a constant temperature (23.0–25.0°C) and relative humidity (57–60%). The inhalation exposure system used in the present study has been described in detail previously (Huang et al., 1989Go, 1990Go; Takeuchi et al., 1989Go). The vapor concentration in the chamber was measured by gas chromatography and was digitally controlled within ±5% of the target concentration, using a personal computer system. Japanese laws concerning the protection and control of animals, standards related to care and management of experimental animals, and the Guide of Animal Experimentation of the Nagoya University Graduate School of Medicine were strictly followed throughout the study.

Experimental design.
Forty female rats were divided into four equal groups. After monitoring estrous cycles for three weeks, each group was exposed daily to 0, 200, 400, or 800 ppm of 1-bromopropane by inhalation for eight h a day (from 2 to 10 P.M.), seven days a week. After exposure for 7 weeks, all rats of the 800-ppm group became seriously ill and were sacrificed by decapitation during the 8th week. Other groups were exposed for 12 weeks and decapitated on the day of diestrus I, during the 13th week.

Monitoring of estrous cycle.
During the study period, daily vaginal smears were taken between 11:00 A.M. and 12:00 P.M. to monitor ovarian cycle pattern. The smears were stained with 0.5% methylene blue solution (Katayama Chemical, Japan) and examined under a light microscope. Cycle days were classified as proestrus, estrus, diestrus I, and diestrus II (Cooper et al., 1993Go). For statistical analysis, the 15-week study period was divided into five consecutive 3-week periods: one preexposure period and four exposure periods. The estrous cycles were defined as normal when they showed typical stages of proestrus, estrus, and diestrus, which were usually observed with a four- to six-day cycle. When a cycle overlapped two consecutive periods, it was counted as a 0.5 cycle in each period. Estrous cycles were defined as regular cycles (3.0–5.0 cycles/3 weeks), irregular cycles (0.5–2.5 cycles/3 weeks), and no cycle (0 cycle/3 weeks).

Histopathological examination.
Reproductive organs (right ovary, uterus, and vagina) and other organs (thymus, adrenal gland, kidney, spleen, liver, and brain) were dissected out carefully and weighed immediately. These organs, except the brain, were fixed in 10% neutral buffered formalin for light microscopic evaluation. Tissue blocks were embedded in paraffin and cut into 5-µm sections. They were mounted on glass slides, and stained with hematoxylin and eosin. Kidney tissue blocks were also stained by the PAS (Periodic Acid Schiff) method.

Counting of ovarian follicles.
Serial sections (8-µm) were prepared from the left ovaries for counting follicles. A modified method of Pedersen and Peters (1968)Go for differential follicle counts was used according to Plowchalk et al. (1993)Go, where types 1–3b, types 4–5b, and types 6–8 were grouped as primordial, growing, and antral follicles, respectively. Primordial follicles were included as oocytes with a complete one-layer ring of granulosa cells as well as oocytes devoid of such layers. Growing follicles were defined as oocytes, with multiple layers of surrounding granulosa cells without antrum formation. Antral follicles were defined as large oocytes with multiple layers of surrounding granulosa cells with fluid-filled antrum. No attempt was made to count atretic follicles or sum up total atresic follicle numbers. Follicles devoid of pyknotic nuclei, or that showed no chromatin redistribution, cytoplasmic condensation, or disarrangement of granulosa cells were considered as normal follicles. To avoid counting the same follicle that was large in diameter doubly, only follicles with explicit nucleoli on sections were counted. Follicles without visible nucleoli were not counted. Light microscopy was used for morphological characterization and maturation of follicles. Starting with the first serial section that contained the ovarian tissue, every fifth serial section was scored for differential follicle numbers. The numbers of each type of follicles in all sections were summed to determine the total primordial, growing, and antral ovarian follicle counts.

Hormonal assay.
Blood plasma for hormonal assays was collected at decapitation. Concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were determined by a double-antibody radioimmunoassay by using rat LH and FSH RIA kits supplied by the National Hormone and Pituitary Program (Baltimore, MD). The values were expressed in terms of NIDDK-rLH-RP-3 and NIDDK-rFSH-RP-2, respectively (Maeda et al., 1994Go; Tsukamura et al., 1994Go). The lowest detection limits of LH and FSH were 0.16 ng/ml and 2.5 ng/ml, respectively. The intra-assay coefficient of variation for LH and FSH assay were 5.6 % for 51.1 ng/ml and 10.9 % for 13.7 ng/ml, respectively.

Statistical methods.
Differences in body weight on every week, organ weight, and hormonal concentration between exposed groups and the control were analyzed by ANOVA followed by Dunnett’s multiple comparison method. The number of ovarian follicles in each group was compared to the control by Dunnett's multiple comparison method following root transformation for normalizing each distribution. The data of organ weight, hormonal concentration, and the number of ovarian follicles were obtained, also from the 800-ppm group, but these data in this group were excluded in the statistical analysis. This is because there was not an appropriate age-matched control group for comparison. The number of estrous cycles in each group was compared to the control in each pre-exposure or exposure period by the Kruskal-Wallis test, followed by Dunnett-type multiple comparison method. A single rat from the control group was excluded from the analysis because extreme splenomegaly, hepatomegaly, and polycythemia were identified at autopsy. Data are expressed as mean ± SD. A p value less than 0.05 denoted the presence of a statistically significant difference.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The actual vapor concentrations of the exposed groups were 813 ± 28, 415 ± 19, and 200 ± 8 ppm, respectively. In the 800-ppm group, body weight was significantly decreased at each time point from 2 to 7 weeks after the beginning of exposure compared to the control, but the weight of the other two exposure groups did not significantly differ from the control (Fig. 1Go).



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FIG. 1. Changes in body weight in rats exposed to 1-bromopropane. The rats in the 800-ppm group became seriously ill and were euthanized during the 8th week. Other groups were exposed to 1-bromopropane for 12 weeks. Body weight gain in the 800-ppm group was significantly less than the control, whereas the gain in rats of other groups did not significantly differ from the control. Differences in body weight were analyzed by ANOVA followed by Dunnett’s multiple comparison method; **p < 0.01.

 
Vaginal smear examination showed that the number of normal estrous cycles in the 800-ppm group was significantly lower in the first exposure period compared with the control. Moreover, none of these rats had normal estrous cycles in the next exposure period. All anestrus rats displayed constant diestrus stage. In the 400-ppm group, the estrous cycles gradually prolonged around the 7–9th week of exposure, and all rats showing irregular estrous cycles displayed extended diestrus. The number of normal estrous cycles was significantly lower in the last two periods. All anestrus rats also displayed constant diestrus smears in this group. In the 200-ppm group, there was no statistically significant change in estrous cycle compared to the control (Table 1Go).


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TABLE 1 Changes in Estrous Cycle in Rats Exposed to 1-Bromopropane by Inhalation
 
The weight of reproductive organs in the 200- and 400-ppm groups was not significantly different from the control. The absolute weights of the liver and kidney were significantly higher in the 200- and 400-ppm groups compared with the control. The brain weights of the 400-ppm group were significantly lower than those of the control (Table 2 and Table 3GoGo).


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TABLE 2 Body and Absolute Organ Weights at the End of the Experiment in Rats Exposed to 1-Bromopropane
 

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TABLE 3 Relative Organ Weights at the End of the Experiment in Rats Exposed to 1-Bromopropane
 
Histopathological examination revealed abnormal findings in the examined organs only in the ovaries, kidneys, and liver. Normal growing and antral ovarian follicles in the 800-ppm group were observed less frequently than the control. In contrast, normal primordial follicles in the 800-ppm group were observed more frequently than in the control. No newly formed corpus luteum could be identified. The 400-ppm group showed reduced numbers of normal growing and antral ovarian follicles compared to the control, but the histopathological changes were milder than in the 800-ppm group. No significant change was found in the number of primordial follicles. In the 200-ppm group, histological examination of the ovary was almost normal, but antral follicle count was significantly less than in the control (Table 4Go, Fig. 2Go). Histopathological examination of the kidneys revealed mild dilatation of the proximal tubules in the 800-ppm group but no abnormal findings in the other exposure groups. In the 800-ppm group, scattered cytoplasmic degeneration was detected in hepatocytes located around the central veins in the liver, accompanied by nuclear pyknosis, but no serious changes, such as diffuse or focal necrosis, were noted. No abnormal findings were found in the livers of the other exposed groups.


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TABLE 4 Number of Normal Ovarian Follicles in Rats Exposed to 1-Bromopropane
 


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FIG. 2. Histopathological examination of ovaries of control rats and those exposed to 1-bromopropane. Small arrows: normal primordial follicles. Large arrows: normal growing follicles. (A) Control group. Most of the ovarian follicles and corpora lutea are normal; (B) 400-ppm group. Note the decrease in normally growing and antral ovarian follicles. However, these changes are milder than those of the 800-ppm group; (C) 800-ppm group. Note that most of the ovarian follicles are atretic. Note also the lack of fresh corpus luteum. Cl: corpus luteum, Af: atretic follicle. Bar, 200 µm. (Hematoxylin-eosin staining; original magnification x100).

 
Hormonal assays revealed no significant changes in plasma LH and FSH concentrations in the 200- and 400-ppm groups compared with the control (Table 5Go).


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TABLE 5 Plasma Concentrations of Luteinizing Hormone (LH) and Follicle-Stimulating Hormone (FSH) in Rats Exposed to 1-Bromopropane
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The major finding of the present study was that the disruption of estrous cycle in rats exposed to 400 ppm of 1-bromopropane was accompanied by decreases in numbers of growing and antral follicles. Exposure of the 200-ppm group also resulted in a significant decrease in the number of antral follicles, although such exposure did not cause disruption of the estrous cycle. These findings indicate that the number of antral follicles is the most sensitive indicator in evaluating female reproductive toxicity to 1-bromopropane in rats. Interestingly, the number of primordial follicles appeared to be larger in the 800-ppm group. Given the tendency for an increase in primordial follicles, along with the decrease in growing and antral follicles at higher exposure levels, the result might reflect at least part disruption of the follicular growth process. However, that might also reflect the difference of age at necropsy. The rats in the 800-ppm group were sacrificed at 21 weeks of age although other groups were in necropsy at 26 weeks of age. Younger rats would have larger numbers of primordial follicles than older rats, since primordial follicles could not be created in adult rats.

2-Bromopropane, an isomer of 1-bromopropane, also causes disruption of the estrous cycles and the disappearance of such cycles following long-term exposure in nonpregnant female rats exposed to 1000 ppm (Kamijima et al., 1997Go). In another study, the same dose was found to decrease the number of ovarian follicles of all stages including primordial follicles (Yu et al., 1999Go). These results suggest that 2-bromopropane may target primordial follicles. Several chemicals exhibit toxic effects on primordial follicles such as 9,10-dimethylbenzanthracene, 3-methylcholanthrene, benzo[a]pyrene, 4-vinylcyclohexene, and its diepoxide metabolite (Borman et al., 2000Go). In this regard, a decrease in the number of primordial follicles could cause early menopause.

In contrast to the above findings on 2-bromopropane, 1-bromopropane seems to mainly alter the maturation of follicles and is less toxic to follicles at early stages. A number of chemical compounds are known to impair ovarian follicle maturation. For example, at high dose, ethinylestradiol increased the number of apoptotic corpora lutea and early-stage ovarian follicles (Andrews et al., 2002). Methoxychlor induced atrophy of the ovary, accompanied by degeneration of small and antral follicles and complete atrophy of the corpora lutea (Chapin et al., 1997Go). These effects may be due to inhibition of the hypothalamic-pituitary-ovarian axis (Sarkar et al., 1989Go).

The above differences between the toxic effects of 1-bromopropane and 2-bromopropane on female reproductive organs seem to parallel the differences in the actions of the two isomers on the male reproductive system. Previous studies showed that long-term exposure to 2-bromopropane resulted in reductions in the numbers of all types of spermatogenic cells (Ichihara et al., 1997Go) and that acute or subacute exposure impaired spermatogenesis (Omura et al., 1999Go), followed by apoptosis of spermatocytes (Yu et al., 2001Go). On the other hand, 1-bromopropane had little effect on spermatogonia, spermatocytes, and round spermatid (Ichihara et al., 2000bGo), and only a mild effect on weight gain of seminal vesicles and a failure of spermiation in seminiferous tubules in male rats (Ichihara et al., 2000bGo). Considered together, these results indicate that 1-bromopropane has adverse effects on both male and female reproductive organs, although the mechanisms of these effects are different from those of 2-bromopropane.

It is well known that 1-bromopropane also exhibits severe neurotoxicity (Ichihara et al., 2000; Yu et al., 1998Go). Sclar (1999)Go reported a case of an intoxicated male worker who developed encephalomyeloradiculoneuropathy following exposure to 1-bromopropane. Recently, Ichihara et al. (2002)Go reported three female cases of 1-bromopropane toxicity. Because one of these cases was a 50-year-old female, she had menopause before exposure to 1-bromopropane. However, the remaining two females showed temporal disruption of the estrous cycle. Thus, 1-bromopropane may cause severe toxicity in humans, including reproductive and neurological dysfunction, and may impair the central nervous system as well as the peripheral nerves.

The adverse effects of 1-bromopropane on the ovaries may be indirect through the disruption of hormonal regulation following central nervous system impairment. It is known that suppression of pulsatile luteinizing hormone secretion impairs ovarian follicle maturation (Maeda et al., 1994Go; Tsukamura et al., 1994Go). However, our results in sex hormonal assays showed no significant changes. LH and FSH concentrations may not be good indicators of endocrine regulation since the levels of these hormones are to a large extent influenced by estrous cycle and pulsatile secretion. In addition, the present study did not investigate the effect on the surging level of hormones. Studies are currently underway in our laboratories to examine the effect of 1-bromopropane on pulsatile gonadotropin secretion and surging level.

Compared to monitoring of the estrous cycle or fertility, counts of ovarian follicle differentiation may be the most sensitive quantitative indicator of female reproductive toxicity and could predict the type of reproductive disruption that may be caused by exposure to chemicals (Bolon et al., 1997GoYu et al., 1999Go). However, only limited work is available in which follicle counting was used in serial sections (Bolon et al., 1997GoBorman et al., 2000GoYu et al., 1999Go). The present study showed a significant decrease in antral follicles in the 200-ppm group, though there was no significant change in the estrous cycle in this group. This finding supports the notion that ovarian follicle count may be the most sensitive indicator of female reproductive dysfunction.

Our results demonstrated significant increases in the absolute weights of kidney and liver in the 200- and 400-ppm groups without any changes in body weight. Mild dilation of proximal tubules observed at 800 ppm might relate to increase in kidney weight, but such histopathological change is not clear at lower levels. Dilation of proximal tubules may suggest tubular dysfunction (Hanley, 1980Go; Olsen et al., 1993Go), but the present study lacks the data of biological markers of tubular function, such as N-acetylglutamate. There are also no qualitative histopathological alterations explaining weight gain in liver. However, a possibility of microsomal induction might be considered, as liver weight gain usually accompanied hepatic microsomal induction in the rats administered many kind of organic solvents (Nakajima et al., 1991Go). Weight gain in adrenal glands at 400-ppm dosage might suggest stress reaction involved with the central nervous system. On the other hand, male rats did not show such a weight change in kidney or adrenal gland in any exposed groups, although the absolute weight of liver increased at 800-ppm doses (Ichihara et al., 2000bGo). These results might reflect a sex difference in the sensitivity of kidneys to 1-bromopropane. Increase in liver or kidney weights was not observed in male (Ichihara et al., 1997Go) or female rats (Kamijima et al., 1997Go) exposed to 2-bromopropane; rather, decrease in kidney and liver weights were observed in male rats exposed to 2-bromopropane (Ichihara et al., 1997Go). These phenomena also might suggest a difference of toxic mechanism between 1-bromopropane and 2-bromopropane.

A significant absolute brain weight loss, which was detected in our females rats exposed to 400 ppm of 1-bromopropane, was also observed in male rats exposed to 800 ppm (Ichihara et al., 2000aGo). In this regard, toluene is a well-known neurotoxic agent but it never results in a decrease in brain weight in rats, even after exposure to 1000 ppm (Huang et al., 1990Go). These findings highlight the severity of neurotoxicity of 1-bromopropane.

In conclusion, we have demonstrated in the present study that 1-bromopropane impaired female reproductive functions in rats in a manner different from that of 2-bromopropane. We emphasize the need for careful handling of this chemical compound in the industry.


    ACKNOWLEDGMENTS
 
We thank Nobuo Nakashima (Department of Pathology, Nagoya University Hospital) for advice on histopathological findings of ovaries. Thanks are also due to Junzoh Kitoh (Professor Emeritus, Institute for Laboratory Animal Research, Nagoya University Graduate School of Medicine) for advice about the method for evaluating ovarian follicles, and Yoshiki Morita (Department of Nephrology, Nagoya University Hospital) for advice about the histopathological findings of the kidney. This study was supported in part by grants No. 11670367, 13470088, and 14770165 from the Ministry of Education, Science, Sports and Culture, Japan, and the Aichi Health Promotion Foundation, Japan.


    NOTES
 
Portions of this data were presented at the 39th annual meeting of the Society of Toxicology, March 2000, Philadelphia, PA.

1 To whom correspondence should be addressed. Fax: +81-52-744-2126. E-mail: tetsu{at}med.nagoya-u.ac.jp. Back

2 Present address: Institute for Risk Analysis and Risk Communication, Department of Environmental Health, University of Washington School of Public Health and Community Medicine, 4225 Roosevelt Way NE #100, Seattle, WA 98105–6099. Back

3 Present address: Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan. Back


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