Hexavalent chromium genomic instability and aging effects in female reproductive tissues
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Background and purpose: Over the last 60 years infertility has increased globally at an alarming rate. While strong evidence supports aging as a major contributor to female infertility, environmental contaminants may exacerbate infertility issues by inducing toxicity and accelerating reproductive aging. However, the mechanistic connection between chemical exposure, toxicity, aging, and infertility has not been widely addressed. This study investigated female reproductive toxicity focusing on hexavalent chromium [Cr(VI)], which is a widespread environmental pollutant in air and drinking water. Epidemiological studies identify Cr(VI) as a reproductive toxicant, but the mechanisms are poorly understood. Cr(VI) is well known to induce DNA damage and disrupt repair pathways resulting in genomic instability, which is a hallmark of aging and a recognized mechanism of infertility in gametes. However, whether Cr(VI) induces these effects in somatic endocrine cells, which regulate reproductive function, is unknown. In this study, we hypothesized Cr(VI) drives infertility by inducing genomic instability and accelerating aging in somatic endocrine cells of the female reproductive system.
Methods: We used an in vitro cell culture model and an in vivo rat model to assess Cr(VI) toxicity and aging across the female reproductive health span. Human granulosa cells (HGL5) were exposed to sodium chromate for 24 h and analyzed for cytotoxicity, chromium uptake, and senescence. Female Sprague-Dawley rats of three age groups (3-, 7-, and 18-month old) were exposed to environmentally relevant concentrations (0, 0.05, or 0.1 mg/L, the World Health Organization and U.S. EPA maximum contaminant levels, respectively) of Cr(VI) in drinking water for 90 days, and ovaries were collected for metals analyses (whole tissue ICP-MS and laser ablation ICP-MS) and histology. We utilized immunohistochemistry to assess ovarian DNA damage (γH2AX), DNA repair (RAD51), and aging markers (p21 and p16). We performed lipid analyses using TimsTOF-MALDI spatial mass spectrometry.
Results: Exposure to Cr(VI) resulted in a concentration-dependent increase in intracellular chromium, cytotoxicity, and senescence in HGL5 cells at low Cr(VI) concentrations. In rats, chromium did not significantly accumulate in ovarian tissues when analyzed by whole-tissue ICP-MS, but spatial analyses with LA-ICP-MS revealed mild changes in essential metal (iron, magnesium, copper, zinc) distribution in ovarian tissue at the 0.1 mg/L concentration. Tissue staining intensity for γH2AX, RAD51, p16, or p21 in rat ovaries increased approximately 3-30% with increasing age within exposure groups. Cr(VI) exposure increased yH2AX-positive cells and RAD51-positive cells an additional 5%, but not p16 expressing cells. Cr(VI) slightly increased p21-positive cells in young and middle-aged rats (6 and 12% increases at the 0.1 mg/L concentration, respectively), but not geriatric rats. Finally, TimsTOF-MALDI imaging mass spectrometry revealed Cr(VI) altered the spatial distribution and relative levels of lipid species in ovaries potentially impacting reproductive function.
Conclusions: These results indicate levels of Cr(VI) at and below the U.S. EPA maximum contaminant level (MCL) may result in toxicity and accelerate aging in female reproductive organs. Despite no apparent accumulation in reproductive tissues, chromium exposure resulted in observable changes in essential metal homeostasis and genotoxicity that may impact reproductive function. This study fills key gaps in the understanding of Cr(VI)-induced toxicity and aging outcomes that may drive infertility. These results are significant because they suggest the current MCLs for chromium exposure may need to be re-evaluated to protect environmental and human health. This work does not reflect the policies or views of the U.S. EPA.