Effects Of Di-n-butyl Phthalate And Benzo(a)pyrene On Hormone Synthesis Of Male Rat Leydig Cells And Its Mechanism

At present, organic contaminants are extensively distributed in the environment. Water surroundings were especially polluted by persistent organic pollutions(POPs) with increasing severity. Aquatic environment in China were contaminated by POPs at different degrees. Phthalatic acid esters (PAEs) and Polycyclic aromatic hydrocarbon (PAHs) are the most popular pollutants. As human beings could be exposed to PAEs and PAHs at a relatively low level for a long time, it's necessary to evaluate the toxic effects of PAEs and PAHs on human health. Di-n-butyl phthalate(DBP) and Benzo(a)pyrene (BaP) were typical pollutants of PAEs and PAHs, respectively. Although many researches have showed that PAEs and PAHs have male reproductive developmental toxicity, data of the combined toxicity and potential threats to male reproduction posed by DBP and BaP remain limited. Many studies have revealed that Leydig cell of testes is one of the main target cells of DBP or BaP, and testosterone synthesis and secretion could be impacted by DBP or BaP. In the present study we chose DBP and BaP as the model chemicals of organic pollutants, and tested the single or combined effect of them on morphology and function of testicle leydig cell in puberty SD male rats. In addition, some regulatory factors and synthetase were involved in testosterone synthesis pathway in Leydig cells, therefore, we observed the effect of DBP and BaP on the mRNA and protein expression of these factors, and further studied the relationship among these factors and steroidogenesis induced by DBP and BaP, which would provide some reference indicators for deep understanding of its mechanism.Methods1. AnimalsA total of 168 male Sprague-Dawley rats (4-5 weeks old) were equally randomized into 7 groups (24 per group), alt. dieb., and respectively received by gavage corn oil , 50 mg/ kg of DBP , 250 mg/ kg of DBP ,1 mg/ kg of BaP,5mg/ kg of BaP, 50 mg/ kg of DBP +1 mg/ kg of BaP , 250 mg/ kg of DBP +5 mg/ kg of BaP for 90days. At the end of experimental period, i.e. 30,60 and 90 day after gavage of DBP or BaP, 8 rats per group were sacrificed randomly.2. treatment and methodsThe rats were anesthetized with pentobarbital and sacrificed by decapitation. Arteria femoralis blood was immediately collected before decapitation. The serum testosterone was separated and was determined with commercially available radioimmunoassay kits, according to the manufacturer's instruction. Samples and standards were measured in duplicates in the same assay and the CV% were always <15%.Rats were immediately subjected to necropsy and the testes, epididymides and main organ samples were promptly removed, and weighed. Testes was used for gene expression of insulin-like factor 3(Insl3), luteinizing hormone receptor (LHR), peripheral benzodiazepine receptor (PBR) and protein expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 Side Chain Cleavage Enzyme (P450scc), 3 beta hydroxysteroid dehydrogenase (3β-HSD), 17 beta hydroxysteroid dehydrogenase (17β-HSD), transforming growth factor alpha (TGFα) and insulin-like growth factor 1 (IGF-Ⅰ)assay, and histopathological examination.3 rats randomly selected from each group were subjected to necropsy and the right testis was fixed in 4% Polyoxymethylene solution. One piece of tissues from each side of the testis were embedded in paraffin, sectioned at 4μm, stained with hematoxylin and eosin (H&E), and examined histologically. For electron microscope observation, testis of each group were selected randomly and fixed in 2.5% glutaraldehyde immediately after removal. After fixation, the specimens were processed through standard dehydration in graded ethanol before infiltration in Epon and embedded. The ultrathin sections were stained with uranyl acetate and lead citrate and viewed under transmission electron microscope (TECNAI-10, Philip, Netherlands).3. Statistical analysisStatistical significance was established using one-way analysis of variance (ANOVA) followed by the least significant difference test when multiple treatment groups were compared. The data were expressed as mean±standard deviation (SD) of 8 animals per group. The software package SPSS 13.0 was used for statistical analysis. For all statistical tests, significance was determined at a level of P<0.05. Results and discussion1. The effect of di-n-butyl phthalate and benzo(a)pyrene on rat growth and testis morphology.1.1 On the weight and organ coefficientAfter DBP individual exposure, BaP individual exposure and DBP/BaP coexposure at the dosage used in this study, no significant effect was found on rat growth or its weight increase, and no significant toxical effect was found in rat testis, heart and spleen. Compared with control, exposure to 1 mg/kg and 5 mg/kg BaP for 60 days could decrease liver coefficient (P<0.05); exposure to 250 mg/kg DBP for 90 days could increase liver coefficient (P <0.05) and exposure to 50 mg/kg BaP for 30 days could reduce liver coefficient (P <0.05); exposure to 5 mg/kg BaP for 30 days and coexposure to 50 mg/kg DBP and 1 mg/kg BaP for 30 days at low doses could reduce kidney coefficient (P <0.05). Coexposure to 250 mg/kg DBP and 5 mg/kg BaP for 60 days at hige doses could increase kidney coefficient (P <0.05). These results revealed that BaP had a greater effect on epididymis which might be the target organ of BaP. Liver and kidney might be two of the main target organs for DBP and BaP, which showed that they might have potential toxic effect on liver and kidney. (Table 1) 1.2 On the testis and Leydig cell morphologyNo significant pathological changes were observed by light microscope; After all groups exposed for 90 days, different levels of changes in ultramicrostructure of Leydig cells was found by electron microscope, which mainly demonstraed as the extension of mitochondria and smooth endoplasmic reticulum.2. The effect of di-n-butyl phthalate and benzo(a)pyrene on hormone synthesis in Leydig cells2.1 On serum testosteroneExposure to 50 mg/kg DBP could increase serum testosterone levels (P <0.05), which indicated that exposure to low doses of DBP might led to hormesis effect of low doses; exposure to 1 mg/kg BaP for 60 days, exposure to 5mg/kg BaP for 30-60 days, and coexposure to 50 mg/kg DBP and 1 mg/kg BaP for 30 days could increase the level of surum testosterone (P <0.05). This indicated that exposure to≤5mg/kg BaP for 30-60 days could interrupt the synthesis of serum testosterone and could have hormesis effect of low doses (Table 2).2.2 On the expression of Insl3 RNA in the testisExposure and coexposure to high/low doses of DBP and BaP for 30, 60 and 90 days could reduce the expression of Insl3 mRNA in the testis (P < 0.01). This indicated that Insl3 at the transcriptional level was more sensitive than the low doses of DBP and BaP. In adolecent age, subchronical exposure to low doses of DBP and BaP might influence the function of Leydig cells (Table 2). 3. Discussion on the influence mechanisms of di-n-butyl phthalate and benzo(a)pyrene on testosterone synthesis in Leydig cells.Table 3 demonstrated the effect of individual exposure and coexposure to DBP and BaP (30 days, 60 days and 90 days) on cholesterol transmembrane transport regulatory factor, key enzymes in testosterone synthesis, hormone receptor and hormone-related adjusting factors and the related factors at the transcriptional level or translational level.3.1 The influence on cholesterol transmembrane transport regulatory factorIndividual exposure to DBP and BaP, and coexposure to low doses of DBP and BaP for 60 days mainly decreased PBR gene expression (P <0.01; P <0.05); coexposure to high doses of DBP and BaP for 60 days could increase PBR gene expression (P <0.01). This indicated individual exposure or coexposure to DBP and BaP had an effect on PBR gene expression, however, the increase of testosterone level on the 60th day might have little to do with the upregulation or downregulation of PBR.Exposure to DBP and BaP for 60 days could increase the expression of StAR protein (P >0.05), which is in accordance with the increase of testosterone level. This revealed that the increase of StAR protein expression might play a role in increasing the testosterone level, which might be one of the mechanisms for increasing the testosterone level.3.2 On key enzymes in testosterone synthesisThere is no statistical significance between individual exposure or coexposure to DBP and BaP for 60 days and P450scc protein expression,which demonstrated that the increase of testosterone level might be irrelevant to P450scc protein expression.Individual exposure to 5 mg/kg BaP for 30 days raised the expression of 3β-HSD protein (P >0.05). This suggested that 3β-HSD protein might be related to the increase of testosterone level after 30 days of 5 mg/kg BaP exposure.Exposure to 250 mg/kg DBP for 60 days could raise the expression of 17β-HSD protein (P <0.01) while exposure to 1 mg/kg BaP for 90 days could decrease its expression (P <0.01). This indicated that after exposed to BaP for 30 days 17β-HSD might not participate in the increase of testosterone level.3.3 On hormone receptor and hormone-related adjusting factorsBoth individual exposure and coexposure to DBP and BaP could reduce LHR mRNA expression (P <0.01). This revealed that LHR was sensitive to DBP and BaP at the transcriptional level, however the decrease of LHR mRNA expression might not be induced by the increase of serum testosterone after 60 days exposure, and the influence of DBP and BaP on the testosterone level was unrelated to the receptor adjusting pathway . This might relate to LH-independent pathway.Exposure to 50 mg/kg DBP and 1mg/kg BaP for 30 days and 60 days could raise the TGFαprotein expression, exposure to 5 mg/kg BaP and coexposure to high/low doses of DBP and BaP for 30 days could increase TGFαprotein expression (P <0.01). This demonstrated that the increase of testosterone level was in accord with the increase of TGFαprotein expression, which might prove BaP adjusting testosterone level by increasing TGFαprotein expression.The effect of these exposure group on IGF-I protein expression: exposure for 30 days and 60 days upregulated IGF-I protein expression (P <0.01; P <0.05) and 90 days exposure downregulated IGF-I protein expression (P <0.01; P <0.05). In this study, after exposure to BaP for 60 days, the upregulation of IGF-I protein expression is the same as the increase of testosterone level, which revealed that the upregulation of IGF-I protein expression was one of the mechanisms for DBP or BaP induced testosterone level increase.3.4 The combined effect of exposure to high/low doses of DBP and BaP showed in Table 4. ConclusionSubchoronic individual exposure or coexposure to DBP and BaP had negative effects on the adolecent SD male rat and different exposure time could induce different degrees of damage. Exposure to BaP could change epididymis coefficient,liver coefficient and kidney coefficient. Exposure to BaP for 60 days could increase testosterone level, which indicated that the upregulation of StAR protein, 3β-HSD protein, TGFαprotein and IGF-I protein was related to their functional mechanisms. After 60-day exposure to 50 ma/kg DBP, serum testosterone level was raised, which might be hormesis effect induced by low dose of DBP exposure. And TGFαmight play an important role in this process. Coexposure to DBP and BaP could have interaction effects, which demonstrated as antagonism and synergism with few showed non-interaction effect (addition). In this study, serum testosterone level was increased but not decreased, which might relate to the exposure doses and exposure age: the DBP exposure dosage adopted in this study was camparetively low, which might induce hormesis effect; and adolecent exposure were used instead of utero exposure which might not cause the rat be sensible to intrauterine exposure.Further studies is required to provide more evidence for the toxical effect of DBP and BaP coexposure and its mechanisms.