Hexavalent chromium-induced apoptosis of granulosa cells involves selective sub-cellular translocation of Bcl-2 members, ERK1/2 and p53

Abstract

Hexavalent chromium (CrVI) has been widely used in industries throughout the world. Increased usage of CrVI and atmospheric emission of CrVI from catalytic converters of automobiles, and its improper disposal causes various health hazards including female infertility. Recently we have reported that lactational exposure to CrVI induced a delay/arrest in follicular development at the secondary follicular stage. In order to investigate the underlying mechanism, primary cultures of rat granulosa cells 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 intrinsic apoptotic pathway(s) were investigated. Our data indicated that CrVI: (i) induced DNA fragmentation and increased apoptosis, (ii) increased cytochrome c release from the mitochondria to cytosol, (iii) downregulated anti-apoptotic Bcl-2, Bcl-XL, HSP70 and HSP90; upregulated pro-apoptotic BAX and BAD, (iv) altered translocation of Bcl-2, Bcl-XL, BAX, BAD, HSP70 and HSP90 to the mitochondria, (v) upregulated p-ERK and p-JNK, and selectively translocated p-ERK to the mitochondria and nucleus, (vi) activated caspase-3 and PARP, and (vii) increased phosphorylation of p53 at ser-6, ser-9, ser-15, ser-20, ser-37, ser-46 and ser-392, increased p53 transcriptional activation, and downregulated MDM-2. Vitamin C pre-treatment mitigated CrVI effects on apoptosis and related pathways. Our study, for the first time provides a clear insight into the effect of CrVI on multiple pathways that lead to apoptosis of granulosa cells which could be mitigated by vitamin C.

Introduction

Chromium (Cr) exists in a series of oxidation states from −2 to + 6 valences; the most important stable states are elemental metal (0), trivalent (CrIII) and hexavalent (CrVI) compounds (Barceloux, 1999, Shi et al., 2004, Zhitkovich, 2005, Valko et al., 2006). CrVI is commonly used in numerous industrial processes and as emission or erosion byproducts of Cr-based catalytic converters, asbestos brake linings, cement dust, as well as in tobacco and food additives (Nriagu, 1988). Non-occupational sources of CrVI include contaminated soil, air and water (O'Brien et al., 2003). Occupational exposure to Cr is found among approximately half a million industrial workers in the United States and several millions worldwide. Significant contamination with CrVI has been found in approximately 30% of the drinking water in California (Salnikow and Zhitkovich, 2008). According to Environmental Working Group (EWG) water utility tests from 48,000 communities in 42 states (Sutton, 2010), at least 74 million people in nearly 7000 communities drink tap water polluted with CrVI. The USEPA has set a legal limit in tap water for total chromium of 100 ppb. However, chromium levels in the drinking water measured by EWG shows that total chromium is 1700 times higher than California's proposed public health goal for CrVI (Sutton, 2010). This disparity could indicate significant cancer risk and other health hazard for communities drinking chromium-containing tap water. The deposition of CrVI wastes in landfills and waterways by chromate industries affects millions of people residing in the vicinity of dangerously polluted sites who drink Cr containing water (BlacksmithInstitute, 2007). CrVI causes dermatitis, skin, lung and throat cancers, and infertility. Increased incidences of birth and developmental defects among children living around tanneries and chrome and leather industries are clearly evident in the developing world (BlacksmithInstitute, 2007).

Women working in Cr industries and living around Cr contaminated areas experience abnormal menses (Makarov and Shimtova, 1978), postnatal hemorrhage and birth complications with high levels of chromium in blood and urine (Shmitova, 1978, Shmitova, 1980). Cr is transported to offspring through milk in lactating women exposed to CrVI (Barceloux, 1999). CrVI can traverse the placental barrier in rodents (Tipton and Shafer, 1964, Barceloux, 1999). Within the pregnant uterus, CrVI alters early development and hatching of blastocysts (Jacquet and Draye, 1982), decreases the number of implantation sites and viable fetuses (Junaid et al., 1996, Kamath et al., 1997, Kanojia et al., 1998), produces embryotoxic and fetotoxic effects, and increases conceptus resorption in rodents (Junaid et al., 1996). Cr exposure through drinking water impairs ovarian follicular maturation and differentiation and promotes follicular atresia (Murthy et al., 1996), delays puberty, lengthens inter-estrus intervals and reduces number of ovulation (Kanojia et al., 1998) in rodents.

CrVI can escape from primary contact organs and blood erythrocytes and reach different organs (Barceloux, 1999, Dayan and Paine, 2001). In biological systems, after entry into cells, CrVI is rapidly detoxified/reduced to CrIII by an intracellular defensive reductant system that includes ascorbate (vitamin C), glutathione (GSH) and cysteine (Valko et al., 2005, Valko et al., 2006). CrIII is also a very popular nutritional supplement consumed by many people (Kirpnick-Sobol et al., 2006). Exposing yeast and mice via drinking water to CrVI and CrIII significantly increased the frequency of DNA deletions. Surprisingly, CrIII is a more potent inducer of DNA deletions than CrVI once CrIII is absorbed (Kirpnick-Sobol et al., 2006). Thus, both the environmental contaminant CrVI and the nutritional supplement CrIII increase DNA deletions in vitro and in vivo, when ingested via drinking water. Vitamin C accounts for ~ 80% of CrVI metabolism in target tissues such as lung, liver and kidney, being the fastest reducer of CrVI in vitro (Zhitkovich, 2005, Zhitkovich et al., 2005). Unlike rodents, human beings are unable to synthesize l-ascorbic acid because of their deficiency in t-gulono-g-lactone oxidase, the enzyme catalyzing the terminal step in l-ascorbic acid biosynthesis (Nishikimi et al., 1994). Therefore, the potential risk for CrVI exposure in humans might be more severe than what is reported in rodent models.

We have recently reported that lactational exposure to CrVI decreased primordial, primary, secondary, and antral follicles and thus delayed follicular development, decreased steroidogenesis, extended estrous cycle and pubertal onset in postnatal rat ovaries. Vitamin C supplementation protects ovary from these deleterious effects of CrVI (Banu et al., 2008a). However, the specific mechanism(s) responsible for CrVI-induced follicular arrest/atresia on follicular development are not yet understood. Follicular granulosa cell apoptosis or follicular atresia governs follicular growth and development in the ovary (Hirshfield, 1997, Hoyer, 2005). Metal toxins including CrVI and cadmium alter programmed granulosa cell death and follicular apoptosis (Blankenship et al., 1997, Matsuda-Minehata et al., 2006). In metal-induced apoptosis, mitochondria are reported to be the most pertinent target (Rana, 2008). Both mitochondrial damage and genotoxic effects determine the fate of CrVI-exposed cells to either growth arrest or apoptosis (Ye et al., 1999).

Therefore, we hypothesize that CrVI induces follicular atresia through apoptosis of granulosa cells by activating multiple cell signaling pathways. The objectives of the present study were to: (i) determine the effects of CrVI on activation of intrinsic apoptotic pathways and suppression of cell survival pathways in primary cultures of granulosa cells; (ii) understand the involvement of p53 and MAP-kinases in granulosa cell apoptosis; and (iii) evaluate the mitigative effects of vitamin C on CrVI-induced changes on the molecular end-points in granulosa cell apoptosis. Our results for the first time reveal that CrVI induces apoptosis of granulosa cells through activation of mitochondria-mediated intrinsic pathways, suppression of AKT pathways, and phosphorylation / activation of p53 through sustained and delayed activation of ERK1/2 pathways. Vitamin C partially mitigated these adverse effects of CrVI and protects granulosa cells from apoptosis.