Environmental toxicants and effects on female reproductive function

Abstract

One of the most toxic substances known to humans, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin), is also highly pervasive in the environment. It is created naturally in volcanic eruptions and forest fires, and anthropogenically in waste incineration, chlorination processes and certain plastics manufacture. From reports of large industrial and other accidents, or from experimental studies, dioxin exposure has been correlated in animal models and/or humans with chloracne of the skin, organ cancers, hepatotoxicity, gonadal and immune changes, pulmonary and other diseases such as diabetes, skewing of the sex ratio, and infertility. We have demonstrated that the aromatic hydrocarbon receptor (AHR) that binds dioxin in tissues is localized in zebrafish, rat and rhesus monkey (Macaca mulatta) ovaries and in rat and human luteinizing granulosa cells (GC) (among other tissues), that labeled dioxin is specifically localized to granulosa cells of the ovarian follicle as observed by autoradiography, and that incubations of GC or ovarian fragments with environmentally relevant concentrations (fM to nM) of dioxin inhibit estradiol secretion significantly. Our experiments show that in human, non-human primate, rat, trout, and zebrafish ovarian tissues, dioxin inhibits estrogen synthesis at some level of the steroid biosynthetic pathway, most likely by inhibiting transcription of mRNAs for or activity of side-chain cleavage (Cyp11a1 gene) and/or aromatase (Cyp19a1 gene) enzymes, or conceivably other steroidogenic enzymes/factors. Such an untoward effect on estrogen synthesis in females exposed to dioxin environmentally may predispose them to defects in aspects of their fertility.

Figure 1. Simplified schematic of AHR activation by TCDD

TCDD binds the AHR in the cytoplasm, which displaces heat shock proteins and an AHR-interacting protein. TCDD-AHR translocates to the nucleus and is complexed with ARNT. This heterotrimeric complex binds AHREs or iAHREs and interacts with co-activators or co- represssors at the enhancer regions of DNA to achieve biologic effect (transcriptional activation or inhibition). Interactions with the ER may occur in multiple regions of the cell and this may represent a potential site of cross-talk between AHR- and ER-signaling pathways.

Figure 2. Brief schematic of the steroidogenic pathway to estrogens in ovarian GC

Cholesterol is transported to the inner mitochondrial membrane of ovarian follicle thecal cells by Star through the peripheral benzodiazepine receptor channel in the outer mitochondrial membrane, and cleaved to P₅. Thecal smooth endoplasmic reticulum is then the site for Hsd3b isozyme conversion of P₅ to P₄ and DHEA to A₄; and for the conversion of P₅ to DHEA under Cyp17a. A₄ ultimately diffuses across the basement membrane to layers of mural granulosa cells where it is aromatized by Cyp19a1 to estrone; and interconverted with E₂ via Hsd17b isozymes, as occurs between A₄ and testosterone.

Figure 3. Effects of TCDD on E₂ secretion by human LGC

TCDD at fM, pM and nM concentrations caused a significant decrease in E₂ secretion at 8, 12 and 24 h in all treatment concentrations versus control. Data are presented as mean ± S.E.M. Letters (a, b) denote significance among treatment groups, based on Tukey’s post-hoc test (P < 0.05). Treatments sharing the same letter did not differ significantly. N = 8 for each treatment group.

Figure 4. Comparison of average E₂ release noted in monkey ovarian fragments cultured with either control medium (CTL) or medium containing pM TCDD (TCDD)

TCDD exposure at this environmentally relevant concentration markedly suppressed E₂ release at 24 (*, p < 0.003) and 48 (*, p < 0.02) h. Although marked suppression in E ₂ release was also suggested at 72 and 96 h, differences between the CTL and TCDD cultures were not found to be statistically significant (p = 0.07 and p = 0.20, respectively). Rather, variability of response at these latter times increased dramatically. Data represent means ± SE. “n” = 3 at 24, 48, and 72 h; “n” = 2 at 96 h.