Reproductive Toxicity Of Male Rats Exposed To Two Isomers Of Bromopropanes

ObjectiveTo study the reproductive toxicity of male rats exposed to 1-bromopropane and 2-bromopropane and to explore the toxic mechanism of two isomers. Therefore, it will provide useful baseline information on exploring the reproductive toxicity and the epigenetic mechanism of human exposed to bromopropanes and developing occupational health standards.Methods1. Eight SPF SD male rats were randomly divided into 3 groups of 6 each, respectively. The groups were exposed to corn oil, 1-BP (1g/kg) and 2-BP (1g/kg) for 7 days by intraperitoneally injection. Epididymal and testicular tissues were examined for biochemical and histopathological changes. Apoptotic cells in testis were detected by TUNEL staining and immunohistochemistry for active caspase-3. GSH, MDA, SOD, GST, GSH-RD, GSH-PX and CAT were assayed by biochemical methods. In addition, real-time quantitative PCR was used to analyze the mRNA levels of Bcl-2, Fas, Bax, P53 and FasL.2. Twenty-seven SPF SD male rats were randomly divided into 3 groups of 9 each, respectively. The groups were exposed to corn oil, 1-BP (1g/kg) and 2-BP (1g/kg) for 14 days by intraperitoneally injection. Three rats of each group were random selected for histological and apoptotic findings. Real-time quantitative PCR was used to analyze the levels of DNMT1, DNMT3a, and DNMT3b mRNA. In addition, ELISA-like were used to determine HDAC and HAT activity.3. SPSS 10.0 software and JUMP 7.0 were used for analysis. Homogeneity of variances was analyzed employing Levene's test. If variances were homogenous, ANOVA was used, followed by the LSD method to determine differences between the treated animals and the control ones. The Krusakal-Wallis test was used when variances were not homogenous. Differences between groups were analyzed using the Mann-Whitney U-test. A value of P<0.05 was considered significant.Results1. Seven-day exposure experimentSeven-day exposed to BPs resulted in reduction of body weight and the weights of testes and epididymis, although the change was significant for the body weight of 1-BP rats and the weights of testes of 2-BP rats only (P<0.05, P<0.05). There were no significant differences in the prostate and seminal vesicle between the 2-BP and control groups.Exposure to 1-BP and 2-BP decreased epididymal sperm count and morphological normal sperms, although the change was significant for 2-BP only (P<0.05). 1-BP induced an increase in sperm with an abnormal head shape (P<0.05). 2-BP induced an increase in tailless sperm and sperm with an abnormal head (P<0.01, P<0.05).Histopathological examination revealed atrophy of seminiferous tubules, reductions in the number of germ cells and vacuolation of Sertoli cells in 2-BP-treated group, compared with the control group. Retained, elongated spermatids were found near the lumen and the more basal region of stagesâ…¨-â…ªseminiferous tubules in the 1-BP exposed groups.No or only one TUNEL-positive cell was noted along the basement of the spermatogenic epithelium per tubule. Exposed to 1-BP increased the percentages of TUNEL-positive tubules, TUNEL-positive cells per tubule, and apoptotic cell index, but the changes was not significant. Exposed to 2-BP resulted in selective degeneration of germ cells at the periphery of the tubules. Most cells undergoing apoptosis were located along the basement of the seminiferous tubules, which were identified as pachytene spermatocytes. TUNEL-positive cells were usually observed in stagesâ… andâ…¦-â…§seminiferous epithelia. The percentages of TUNEL-positive tubules, TUNEL-positive cells per tubule, and apoptotic cell index increased significantly in 2-BP-treated animals (P<0.05, P<0.05, P<0.01). As compring with 1-BP group, exposed to 2-BP increased the percentages of TUNEL-positive tubules and apoptotic cell index (P<0.05, P<0.01). Active caspase-3 activity was both increased in 1-BP and 2-BP group. As compring with control group, 1-BP and 2-BP group showed a significant increase in the proportion of apoptotic germ cells and expressed a higher number of active caspase-3-positive cells per tubule (P<0.05, P<0.05, P<0.01, P<0.01). In addition, 2-BP group increased the proportion of apoptotic germ cells and the number of active caspase-3-positive cells per tubule, compared with 1-BP (P<0.01, P<0.01).1-BP increased GR activity in testis, and SOD activity and MDA level in epididymis. 2-BP decreased the GSH level and increased the MDA level in epididymis and testis, decreased GST and GR activity in epididymis.The mRNA expression of p53, Bax and Bcl-2 were decresed in 1-BP and 2-BP group, although the changes of those three genes in 2-BP group were significant (P<0.05, P<0.05, P<0.05), compared with control group. There are no significant changes of Fas and FasL genes in 1-BP and 2-BP.2. Two-week exopsure experimentAs comparing with control group, the mRNA expression of DNMT1, DNMT3a and DNMT3b were decresed in 2-BP group(P<0.001, P<0.05, P<0.001), while there were no significant changes of these genes in 1-BP group. Comparing with 1-BP group, the mRNA expression of DNMT1and DNMT3b were also decresed in 2-BP group(P<0.001, P<0.001)2-BP induced a high activity of HAT in testi(sP<0.05), while there was no significant different in HDAC activity. There are no significant differences in the HDAC and HAT activity between the 1-BP and control group.ConclusionThis study indicated that bromopropanes can cause reproductive disorder in male rats, the rats treatment with 2-BP had more toxicity than that with 1-BP, and it might have different toxic mechanism between two isomers. Exposure to 1-bromopropane inhibits spermiation in male rats, in contrast to 2-bomopropane, which targets spermatognia and spermatocytes. Our results also suggest that DNMTs, HAT and HDAC may have a significant effect for 2-BP reproductive toxicity.