Titolo della tesi: Toxicogenomic effects of direct Benzo[a]pyrene-exposure on adult testicular organotypic culture
RATIONAL: BaP is a polycyclic aromatic hydrocarbon generated by the incomplete combustion of organic compounds. It is a ubiquitous air pollutant, and it is absorbed by the body mainly through the respiratory tract and orally. BaP is an inert molecule, however once inside cells it is bioactivated into a series of toxic metabolites that can damage cellular DNA, causing cell apoptosis and cancer. BaP bioactivation is triggered by the binding of BaP to the aryl hydrocarbon receptor (AhR). When AhR is activated by BaP, it induces the expression of several target genes containing the xenobiotic response element (XRE) in the promoter region. Among these are the genes coding for phase I drug metabolizing enzymes (DMEs). CYP1A1 and CYP1B1 are the main phase I DMEs responsible for the transformation of BaP into toxic products. Another enzyme that is not under the control of AhR, EPHX1, also plays an important role in the bioactivation of BaP. In the presence of cellular damage, the transcription factor NRF2 is activated, which induces phase II DMEs (eg SOD1, CATALASE, GST, UGT). These enzymes eliminate the toxic products of BaP, converting them into more hydrophilic molecules. It is known that BaP has negative effects on male reproductivity. In humans, it has been shown that in vitro exposure to BaP causes alteration of sperm parameters. In rodents, long exposures to BaP in vivo induce an apoptotic effect on germ and somatic cells of the testis and can dysregulate steroidogenesis by altering the gene expression of enzymes such as STARD1, CYP11A1 and CYP19A1. Moreover, it has been reported that the liver and lung are the main responsible for the biotransformation of BaP and that the toxic metabolites produced can reach peripheral organs such as the testis through the circulation and can cause damage there.
GENERAL OBJECTIVES: The effect of direct exposure to BaP on the adult testis has not been studied, therefore it is not known whether the testis is a BaP-resistant organ, damaged by the prevalent production of toxic metabolites produced in the liver and the lung. For this purpose, we investigated the toxicogenomic effect of direct Benzo[a]pyrene exposure on human and mouse adult testicular organotypic cultures.
EXPERIMENTAL DESIGN AND METHODS: For this study, we used fresh and cryopreserved human testicular biopsies and testes from adult C57/BL6 mice (2-5 months). 2-4mm testicular fragments were cultured in the absence or in presence of BaP 0,1-10 μM for 12h and 24h, respectively for humans and mice. In each experiment, an uncultured sample was made (time zero, T0). At the end of the treatment, for each condition RNA was extracted from part of the testis fragments while histological analysis was performed on the remaining testicular fragments. The analysis of genes involved in the metabolism of BaP (phase I in humans and mice, phase II only in mice), in steroidogenesis (in humans and mice) and in apoptosis (only in mice) was evaluated by RT-PCR and ddPCR. TUNEL assay was performed to evaluate the apoptotic effect of BaP.
RESULTS: In fresh and cryopreserved human testis fragments, BaP did not generally induce CYP1A1, which increased its expression only in a few cases. On the other hand, CYP1B1 and EPHX1 expression levels were increased in BaP-treated fresh tissue, but mostly unaffected in cryopreserved tissue. The bio-activation of BaP would therefore appear stronger in fresh tissue than in cryopreserved tissue. To confirm this, the TUNEL assay demonstrated an increase in the number of apoptotic cells in the BaP-treated fresh testicular tissue, while in most cases the BaP-mediated apoptotic effect in cryopreserved tissues is not observed. As for the modulation of steroidogenic genes, the expression of STARD1 and CYP11A1 tended to increase in fresh tissue and to remain mostly unchanged in cryopreserved tissue. The expression of CYP19A1, instead, is mostly unaffected in both fresh and cryopreserved tissue. These results suggest that testis directly exposed to BaP could undergo steroidogenic impairment, a conclusion deserving further studies. It is worth noting that, despite the general BaP-mediated effect just described, a certain variability of response was observed between the different donors, which can be explained by the presence of genetic polymorphisms of target genes. Although BaP in vitro exposure was longer in mouse testis cultures, no alteration of any of the genes studied was observed, with the only exception of CYP1B1 which significantly increased its expression. However, as for human testis, TUNEL analysis also shows a strong apoptotic effect of BaP in mouse testis fragments.
CONCLUSIONS: Our results show for the first time that the testis of mammals such as humans and mice is susceptible to direct exposure to BaP. This study increases our knowledge on the impact of environmental pollution on fertility, a topic of great interest today.