Combined Effects Of Dibutyl Phthalate And Benzo [a] Pyrene On Spermatozoa In Male Rats

POPs, short for Persistent Organic Pollutants, refers to the organic chemicals that could persistently exist in the environment, have a long half-life, accumulate through the food chain, and negatives affects human health and the environment. The harm POPs has on human beings and the environment has become a global environmental problem. As there are thousands of POPs substances worldwide, it is difficult to conduct a comprehensive study due to limited financial, material, and human resources. Therefore, it is important to choose to study the kind of POPs that needs concerns and monitoring, as well as people's priority POPs. This is vital for a high efficiency in environmental monitoring, pollution control and health protection.Our previous studies show that polycyclic aromatic hydrocarbon (PAHs) and Phthalic Acid Esters (PAEs) are the most popular pollutants in the Three Gorges area. This experiment intended to study and use the DBP as a representative for PAEs, the Bap as a representative for PAHs. Previous studies have reported that both DBP and Bap have certain male reproductive toxicity, but the male reproductive toxicity under the exposure to Combination of Bap and DBP, especially for the toxicity of spermatogenic cell and its mechanism. In the present study, we test the single or combined reproductive toxicity of DBP and Bap on male rats, observe the toxic affection and discusses the effect of the combination of two toxic substances from the toxic effects level and mechanisms level. This will lay a solid foundation for objectively evaluating and predicting the possible damage POPs pollution will do to human beings and establishing an early-warning system for environmental health effects.Methods1. A total 245 male Sprague- Dawley rats (4-5 weeks old) were equally randomized into 7 groups (24 per group) and respectively received by gavage corn oil, 1 mg/ kg of Bap, 5 mg/ kg of Bap, 50mg/ kg of DBP, 250mg/ kg of DBP, 1 mg/ kg of Bap+ 50mg/ kg of DBP, 5 mg/ kg of Bap+ 250mg/ kg of DBP alt. dieb. For 90 days. At the 30, 60 and 90 days after gavage, 8 rats per group were sacrificed randomly. 4 rats per group were left for reproduction experiments.2. treatment and methordsThe testes, epididymis, liver and other visceral organs were harvested, weighed and the organ coefficients were counted. Blood samples were collected to assess the effect of Bap and DBP on plasma testosterone (T) and luteinizing hormone (LH) concentrations with Chemiluminescence immumo- assay. Cauda epididymides were isolated for the determination of progressive motility and density of stored spermatozoa.Testes were prepared for histologic and morphometric analysis. Stained testis sections were evaluated using an OLYMPUS Photomicroscope equipped which coupled with a computerized morphometric planimetry system (DP72- BSW) to facilitate the measurement of tubule area and area percentages (volume percentages) occupied by seminiferous tubules.The testes were surgically removed and germ cells were isolated. Flow cytometry (FCM) was used to detect changes of cell cycle and Two- dimensional electrophoresis (2-DE) was used to separate the proteins. The proteins which has different expression were analyzed using the Image master 5.0 and were identified by mass spectrometric analysis.Results and Discussion1. The effect of di-n-butyl phthalate and benzo(a)pyrene on rat growthThe exposure concentration of Bap and DBP used in this study did not affect weekly weight gains in exposed versus UNC rats (P>0.05). These data suggest that inhaled BaP, at the exposure concentration administered, the daily duration of exposures and the manner in which animals were restrained, did not impose undue stress on the rats, hence the identical weight gains between exposed and UNC rats.The organ coefficients of heart, spleen, kidney and testis also had no changes between exposed versus UNC rats (P>0.05), which means the exposing dosage didn't trigger a significant toxical effect in exposed rats.Compared with other groups, the co- exposure to 1 mg/kg/d Bap and 50 mg/kg/d DBP for 30 days could reduce liver coefficient (P<0.05), and the difference disappeared at 60 days and 90 days. It suggested that the liver is not only the organ where the Bap and DBP metabolize, but also the target organ of Bap and DBP. And the injuries to liver by Bap and DBP may be acute and provisional. The body may tolerated to them gradually. The epididymis might be sensitive to the toxic effect of Bap and DBP. In our study, the epididymis coefficient were significant decreased at the co-exposure to 1 mg/kg/d Bap and 50 mg/kg/d DBP for 90 days compared with the UNC rats. Suggested that the long time co- exposure to Bap and DBP might have the potential toxic effext to epididymis and might cause the atrophy of it.2. The effect of di-n-butyl phthalate and benzo(a)pyrene on rat fertility.In our study, the stored sperm density, sperm viability and movement parameters, which often be used in semen analyses to appraise the male reproductive ability, were not changed both in mono- and co- exposure to B[a]p and DBP. Showed that these indicator may not sensitive to Bap and DBP exposure and routine clinical check to the male reproductive ability by semen analyses may not identify problems in the people exposed to Bap and DBP.Spermatozoa with abnormal morphologies were disproportionately represented in samples of stored spermatozoa from exposed to DBP 250 mg/kg/d and co- exposed 1 mg/kg/d Bap and 50 mg/kg/d DBP for 90 days to rats than their control counterparts (P<0.05).Compared with the significant decreased of epididymis coefficient at co-exposure to 1 mg/kg/d Bap and 50 mg/kg/d DBP for 90 days. We thought that the decreased of epididymis coefficient may not arise from the decreased of sperm density, but from the regression in the epididymal epithelium, which construct the microenvironment in the epididymis for further sperm maturation and consequently. And the change in circulating testosterone concentrations may sharp the effect because the level of testosterone reaching the epididymides from general circulation and testicular fluid may not be sufficient to optimally regulate the epididymides. The increased percentage of decapitated spermatozoa in the cauda epididymides of co- exposed to 1 mg/kg/d Bap and 50 mg/kg/d DBP for 90 days compared with UNC could be likened to the degenerating conditions of stored spermatozoa due to damaged microenvironment in the epididymis and chronic deprivation of the epididymides of adequate testosterone.Coeoposure to low dose of Bap and DBP for 30, 90 days could reduce the circulating testosterone concentrations (P<0.05). Decreased plasma concentrations of testosterone in coexposed rats to low dose of Bap and DBP were accompanied by concomitant increases in plasma LH concentrations throughout the time periods studied, indicating that coeoposure to low dose of Bap and DBP did not have a direct effect on this pituitary gonadotropin, but rather a reflection of Leydig cell failure to respond to LH. It is also very likely that the reduction in the circulating testosterone concentrations by coexposed to low dose of Bap and DBP is secondarily contributed to by the heightened induction of the liver cytochrome P450s that are necessary for detoxification of Bap and DBP. The observed elevated plasma concentrations of LH among could result from the negative feedback on GnRH- induced LH synthesis and release.Compared with the control group, the stillbirth rate and absorption rate are increased at the groups exposed to high dosage of Bap and co- exposed to high dose of Bap and DBP, which had not significant changes in the determination of progressive motility and density of stored spermatozoa and plasma testosterone and luteinizing hormone concentrations.3. The effect of di-n-butyl phthalate and benzo(a)pyrene on histologic and morphometric analysis.The testicular ultra structure observed by electronic microscope showed that both of DBP and Bap caused mitochondrion dropsy and endoplasmic reticulum expanding in testicular cells. The adverse effect of combined groups were similar with Bap groups. The toxicity in combined groups were not stronger than that of DBP or Bap groups alone.The changes of histomorphology in the groups co- exposed to Bap and DBP were insignificant, but the reductions in the number of spermatogenic cell, sloughing of immature sperm atogenic cels and vacuoles degeneration of seminiferous epithelium were observed in the groups treated with Bap or DBP at both dosages.We analysis the morphometric characteristics of rat testis exposed to Bap and DBP versus UNC. The atrophy seminiferous tubules were observed in the groups treated with Bap and DBP at both dosages. It occurred earliest at the testes from co- exposed to Bap and DBP at low dosage at 30 days. Secondary was the testes from expososed to DBP at low dosage at 60 days. Other group exposed to Bap and DBP occurred at 90 days.Volume percentages occupied by seminiferous tubules in testis was increased in the groups exposed to DBP at 30 days. Compared with the results of tubule area, we ascribed the increasing of Volume percentages to the shrinkage of interstitium.4. The change in cell cycle and total proteic expression. On the basis of DNA content, three main germ cell peaks could be identified flow cytometrically: 2C (spermatogonia), 4C (primary spermatocytes), 1C (round and elongated spermatids). The region between the 2C and 4C peaks is comprised of cells actively synthesizing DNA and is termed as S-phase (synthetic phase). We compared the mean relative percentage of the different germ cell populations and germ cell ratios of 1C:2C, 1C:4C, 2C:4C. The effect of Bap and DBP in spermatogenesis focus on meiosis. The coexposure to Bap and DBP at low dosage had the early hormesis at 30d and 60d and became decompensation and inhibition at 90d. the mono- exposure to Bap and DBP at low dosage had the same effect, but the hormesis came later than co- exposure (60d ). No significant difference in the population of germ cell types was detected in the group treated with Bap at high dosage. The exposure to DBP at high dosage had the same effect with the exposure to it at low dosage and the co-exposure at high dosage could have interaction effects which demonstrated as antagonism.Two- dimensional electrophoresis (2-DE) was used to separate the proteins and ImageMaster 2D Platinum 5.0 analysis revealed 35 different proteins in UNC and treated group. The 35 proteins expressed were by mass spectrometric analysis. The expression of hemopexin and mitochondrial aldehyde dehydrogenase precursor had enhanced at treated group versus UNC. Compared with UNC, the expression of chromobox homolog 2 disappeared and the expression of albumin, tubulin beta and transferring enhanced at Bap treated group. Compared with the co- exposed group, the expression of pyruvate kinase 3 isoform 2 disappeared at the group exposed to DBP, and the expression of Heterogeneous nuclear ribonucleoprotein D-like enhanced and the expression of beta actin, tumor rejection antigen gp96, albumin and trichoplein decreased.ConclusionBoth DBP and Bap can cause certain adverse effect on the male reproductive system at the dosages of our experiment, including a reduction in mean tubular area and the epididymis coefficients, affect in spermatogenesis and histopathology observation, the increasing of the sperm deformation, and the different at proteins expression. But the semen analyses including stored sperm density, sperm viability and movement parameters had not changed. Suggesting that when we evaluate the reproductive risk associated with exposure to organic pollution in water, we should considered the combined effects of the toxicant and the impact in sub-clinical conditions, and can not only rely on the toxicological results of the single toxicant and the epidemiological investigations.