Hexavalent Chromium Through Lactational Exposure Delayed Follicular Development in Pre-Pubertal F1 Offspring: Mechanistic Insights into Cell Cycle Regulation of Granulosa Cells.

Abstract

The United States is one of the world’s leading producers of chromium compounds. In California, 30% of drinking water sources are contaminated with significant levels of CrVI. More than two million tons of CrVI processing wastes contaminate 150 sites in Northern New Jersey alone, as it is the chromate capital of the world. According to the National Tap Water quality data base and Environmental Working Group (EWG) report (2008), 1.7 million people from 42 communities living in the New Jersey area drink chromium contaminated water. Moreover, in California, ~74 million people in nearly 7,000 communities drink tap water polluted with hexavalent and other forms of Cr (3), and these people are predisposed to various health problems, including increased menstrual bleeding, ovulatory dysfunctions and infertility. Recently we have reported that lactational exposure to CrVI induced a delay/arrest in follicular development at the secondary follicular stage, delayed puberty, prolonged inter-estrous intervals, and increased FSH, decreased estradiol. CrVI also down-regulated steroidogenic/and steroid signaling machineries such as FSHR, LHR, StAR, SF1, ERα, ERβ, 17bHSD-1, and -2. In order to investigate the underlying mechanism the following studies were undertaken: In Vivo studies: Pregnant rats were divided into the following groups and given the following treatments from postpartum day 1-21: (i) Control (n=5); (iia-d) CrVI (potassium dichromate, 5, 25, 50 and 100 ppm); and (iii) CrVI plus Vitamin C supplementation (CrVI through drinking water with vitamin C supplementation, 20 mg/kg b.wt; through gavage). During this period the F1 female offspring (4 pups/mother) received CrVI and/or vitamin C through the mother's milk from postnatal days (PND) 1-21. At the end of the treatment, F1 offspring were euthanized, ovaries collected, and follicle numbers were counted. Results: CrVI decreased primordial, primary, secondary and antral follicle numbers in F1 rats in a dose-dependent manner. In vitro studies: Primary cultures of rat granulosa cells (GC) from normal PND 21 F1 female rats, and spontaneously-immortalized rat granulosa cells (SIGC) were treated with 10 µM potassium dichromate (CrVI) for 12 and 24 h, with or without vitamin C pre-treatment for 24 h. The effects of CrVI on cell proliferation, cell cycle, and expression and regulation of cell cycle regulatory proteins were investigated in GC and SIGC. Our data indicated that CrVI: (i) decreased cell proliferation, (ii) arrested cell cycle at the G1-S, and G2-M phase check-points, (iii) down regulated G1-S phase-specific cyclins D2 and D3, and cyclin E; and cyclin-dependent kinases (CDKs)-4, -6, and -2; (iv) down regulated G2-S phase-specific cyclins B1, A; and CDK-1; (v) CrVI up regulated CDK-inhibitors p15, p16, and p27 in GC and SIGC; (vi) CrVI also decreased cyclin D2-associated transcriptional factors c-myc, c-fos and c-jun; (vii) down regulated P13K/Akt. (viii) Vitamin C pre-treatment mitigated CrVI effects on the deregulation of cell cycle, decrease in cell proliferation, expression/regulation of cyclins, CDKs and CDKIs, and associated mitogenic pathways. Our study, for the first time provides a clear mechanistic insight into the effect of CrVI on granulosa cells which could be mitigated by vitamin C. (poster)