| Our research group has previously tested water samples from the Chongqing section of the Jialing River and the Yangtze River, and found Phthalatic acid esters (PAEs) and Polycyclic aromatic hydrocarbon (PAHs) pollution in water was very marked. Di(n-butyl) phthalate (DBP) and Benzo(a)pyrene (BaP) were the typical representatives of PAEs and PAHs respectively.They commonly coexist in the water , known as POPs and endocrine disrupting chemicals (EDCs). Especially, exposure to DBP and BaP was always instantaneous and at low dose level. Though there were plenty of studies concerning the male reprodutive toxicity of DBP or BaP, reports about their combined exposure toxic effect had never been seen so far.In this sense, more studies should be performed to explore this issue.At the other hand,the available studies about toxicty of DBP or BaP alone were mainly focused on Leydig, Sertoli and spermatogenic cells in testis. In contrast, little is known about the role of testicular macrophages, especially after co-exposure to DBP and BaP. Because testicular macrophages, as a special immune cell population adjacent to Leydig cells, play an important role in regulating steroidogenesis of Leydig cells by paracrine and maintaining homeostasis within the testis .We hypothesized that testicular macrophages may be the target of DBP and BaP ,and thus disigned this study to observe the effect of DBP and BaP exposure on testicular macrophages function and testosterone biosynthesis, to probe the mechanism by which DBP and BaP affect steroidogenesis in a testicular macrophage-specific manner.These findings will form the basis of future studies.Methods:1. subchronic exposure to DBP and BaP at low dose level in vivo:(1) groups and dosageS.D Rats were randomly assigned into seven groups (24 rats/group) and treated with 50 or 250 mg/kg/day DBP alone (DBP-50, DBP-250), 1 or 5 mg/kg/day BaP alone (BaP-1, BaP-5), 50 mg/kg/day DBP plus 1 mg/kg/day BaP (D+B-50+1), 250 mg/kg/day DBP plus 5 mg/kg/day BaP (D+B-250+5), or vehicle alone (control).(2) experimental designRats were gastrically perfused with DBP and BaP in equal volumes every one day for 90 days. Tissues were collected from eight rats in each group at days 30 , 60 and 90 .The effect of DBP and BaP on the general toxicological parameters , testicular macrophages function and the intracelluar testosterone production were observed .2. low dosage exposure to DBP and BaP in vitro:At first we established the primary culture of testicular macrophages, after normal incubation for 24h, media was replaced with treated media. DMSO were used to as vehicle to dissolve the DBP and BaP.According to the dosage of DBP and BaP in media ,eight groups were randomly divided into two control groups(blank contro and DMSO control) and six exposure groups, which included DBP-0.1(final concentration 0.1μg/ml DBP alone), DBP-10(final concentration 10μg/ml DBP alone),BaP-0.01(final concentration 0.01μg/ml BaP alone), BaP-1(final concentration 1μg/ml BaP alone), D+B-0.1+0.01(final concentration 0.1μg/ml DBP plus 0.01μg/ml BaP ) and D+B-10+1(final concentration 10μg/ml DBP plus 1μg/ml BaP).After incubation for 24h with treated media,the cells were collected and apoptosis was measured by flow cytometry, and the concentration of cytokines in supernatant such as IL-1βand TNF-αwas tested by ELISA. At the same time, the neutral red phagocytosis of the testicular macrophage were also tested. Thus, the injury of testicular macrophage, as well as the change of secrection and phagocytic function were further examined in vitro, which would be helpful to undertand the mechanism of cytotoxic action of DBP and BaP on testicular macrophages.Results:1. Effect of DBP and BaP exposure on the general toxicological parameters in reprodutive system and other important organs.(1) There was no significant change in the body weight and neurobehavior of rats after exposure to DBP and BaP at low dose level.(2) The organ coefficient of epididymis and liver showed significant difference compared with the control group. (3) In testis, epididymis and liver, morphological changes induced by DBP and BaP exposure were seen easily. Changes were characterized by decrease of germinal epithelia layers and vacuolus degeneration of germinal epithelia in testis, decrease of sperm number in epididymis and broadening of hepatic sinusoids or punctiform necrosis in liver.(4) It was at days 60 and in groups of DBP-250 and D+B-250+5 that sperm number in epididymis decreased significantly. DBP seems to play the greatest role in the co-exposure effects observed.2. Effect of exposure to DBP and BaP on endocrinal function of testiculare macrophages.(1) IL-1βincreased significantly both at mRNA and protein level after oral administration of DBP and BaP. It should pointed out that the effect of mRNA level did not emerged until 90 days and did not occur in a dose-dependent manner. Co-exposure to DBP and BaP had additive effects on increased expression of IL-1β. In addition, DBP played the predominant role in this regulation, whereas BaP had relatively weak effects on IL-1βprotein expression.(2) Contrary to the IL-1β, TNF-αdecreased significantly both at mRNA and protein level.But co-exposure to DBP and BaP also had additive effects on decreased expression of TNF-α.(3) Same with TNF-α, iNOS expression was downregulated after DBP and BaP exposure. Only at DBP 250mg/kg/d and BaP 5mg/kg/d level , Such effect be seen.Though no obvious change occurred when exposure DBP or BaP alone, the iNOS mRNA level decreased significantly when co-exposure to DBP and BaP, which indicated that DBP and BaP interact with each other and triggered the completely different effect from single exposure.3. Effect of exposure to DBP and BaP on subsets expression of testiculare macrophages.The balance in the subsets of testicular macrophages was altered.The ED1 protein levels showed a persistent increase during 90 days. The ED2 protein level in rats treated with low-dose DBP plus BaP (D+B-50+1) showed an initial, transient decrease at day 30, but returned to the normal level at day 90. Of note, the expression of ED2 remained very low in the D+B-250+5, suggesting that exposure to relatively high doses of DBP and BaP for 90 days irreversibly affected the composition of the macrophage subsets.4. Effect of exposure to DBP and BaP on phagocytic function-associated enzymes of testiculare macrophages. (1) The activity decrease of lysozyme after co-exposure to DBP and BaP was significant. Of note, the single exposure to DBP at 50mg/kg/d level showed marked increase at 30 and 60 days.(2) The acid phosphatase (ACP) activity decreased with the time increasing during 90 days, indicating there existed a time-dependent effect. At day 90 the ACP activity in all toxicant groups showed a significant decline.5. Effect of exposure to DBP and BaP on intratesticular testosterone production.(1) Ultrastructure Features of lydig cells mainly included the membrance change of mitochondria and ER, such as medullary sheath, dilatation and swelling. Besides, Co-exposure groups showed abnormity of nuclei,such as pyknosis and inclusion. No obvious difference in morphology had been seen at different dose level.(2) Testosterone levels in the testes were significantly lower in the most of toxicant groups compared with the control group in 90 days.The testosterone level in rats in both co-exposure groups was also much lower than that in the groups exposed to each toxicant alone.The suppressive effects of DBP and BaP were additive in both co-exposure groups relative to the individual groups.6. Initial exploration of Mechanism of testicular macrophages damage from DBP and BaP in vitro.(1) DBP and BaP could induce the apoptosis of testicular macrophages in vitro at extremely low dose level (DBP 0.1μg/ml; BaP 0.01μg/ml). Compared to BaP exposure, testicular macrophages was more sensitive to DBP exposure.There existed complicated interaction between DBP and BaP in inducing macrophages apoptosis.(2) DBP alone or DBP and BaP combined ,at high dose level,could dramatically enhance the IL-1βsecrection , decrease the TNF-αsecrection and repress nonspecific phagocytosis of testicular macrophages in vitro.These effects were not significant at low dose level.Conclusions:1. DBP and BaP exposure at low dose level caused morphological changes in testis, epididymis and liver, which indicate that these organs may be the target of DBP and BaP low-dose exposure.2. In co-exposure groups DBP played the predominant role in decreasing the sperm numbers in epididymis,3. DBP single exposure group at 50mg/kg/d showed some effects opposite to other toxicant groups in early period of exposure (effects on ED2 expression or lysozyme activity , for example).These indicate that there existed hormesis at such dose level.4. DBP and BaP decreased testosterone production additively and dose-dependently.5. Though little was know about the outline of the way DBP and BaP interact each other, addition joint action may be the main way they interact to affect the testicular macrophages function.6. Testicular macrophages produced a selective secretion (i.e. upregulation of IL-1βexpression and down-regulation of TNF-αand iNOS expression) after exposure to DBP and BaP at low dose level. As a key cytokine secrected by testicular macrophages affected by DBP and BaP, IL-1βmight play an important role in local regulation in testis .7. DBP and BaP may induce apoptosis of testicular macrophages (mainly ED2 subset) , change the balance in the subsets of testicular macrophages and enhance ability of resident testicular macrophages to secrete IL-1β, resulted in enhanced production of IL-1βas a potent steroidogenesis repressor. This may represent an important mechanism by which DBP and BaP repress steroidogenesis.In conclusion, this study in vivo and vitro has shown that testicular macrophages may constitute a target for the testicular steroidogenesis disrupting effects of DBP and BaP, low-dose DBP and BaP can change the balance in the subsets of testicular macrophages, decrease their ability of nonspecific phagocytosis ,especially alter secrection of some important cytokines and inhabit the steroidogenesis function of Leydig cells in a paracrine manner. It is clear that the'spectrum of toxic effects'of these two chemicals include massive upregulation of IL-1βsecrection, but how the enhanced IL-1βexerts its regulatory function locally, which needs more future researches in this area to elucidate this problem further.
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