Exposure to Iodoacetic Acid, a Water Disinfection Byproduct, Leads to Abnormal Expression of Key Reproductive Axis Genes in the Hypothalamus and Pituitary

Abstract

Iodoacetic acid (IAA) – a water disinfection byproduct (DBP) formed from the reaction between an oxidizing disinfectant, i.e. chlorine, and iodide – is an understudied, yet potentially dangerous environmental toxicant. DBPs have been epidemiologically associated with reproductive dysfunction. In vitro studies have indicated that IAA is one of the most cyto- and genotoxic DBPs. Further, murine ovarian research has shown that IAA exposure significantly inhibits antral follicle growth and reduces estradiol levels. Despite this evidence, little is known about the other components of the reproductive axis: the hypothalamus and pituitary. To address this, we tested the hypothesis that IAA exposure would lead to disrupted expression of key hypothalamic and pituitary genes related to reproductive function. We exposed adult female CD1 mice to 0.5, 10, 100, or 500 mg/L IAA in their drinking water from postnatal day 40 (P40) to their first day in diestrus after P75. From this experiment, we collected whole pituitaries and hypothalamic punches containing the arcuate nucleus (ARC), anteroventral periventricular zone (AVPV), and medial preoptic nucleus (mPOA), and processed them for mRNA analysis. We also exposed pituitary explant cultures to IAA to observe direct effects on gene expression. In vivo, we found that mRNA levels of kisspeptin (Kiss1) are significantly increased in the ARC, the region that controls pulsatile GnRH release, at 0.5 and 10 mg/L IAA concentrations. Kiss1 is unchanged in the AVPV, the neuron population responsible for generating the LH surge. We also measured ARC expression of neurokinin B (Tac2) and dynorphin (Pdyn), neuropeptides secreted by kisspeptin co-expressing neurons to autosynaptically stimulate Kiss1 release. We saw no difference in either. GnRH (Gnrh1) expression was also unchanged. Both in vivo at 10 mg/L IAA and in culture, we found IAA exposure significantly reduced Fshb mRNA. Preliminary immunohistochemistry (IHC) data suggests it also leads to an apparent reduction in FSH-positive cells in vitro (N=2). Lhb and the α-subunit (Cga) were unaltered in vivo, though were significantly reduced with in vitro exposure. In neither context was mRNA expression of the GnRH receptor (Gnrhr) changed. Noting apparent direct effects of IAA on the pituitary, we assessed expression of the cell-cycle inhibitor p21 (Cdkn1a), which has been shown to increase with toxicant exposure. We found Cdkn1a increased in vivo at 500 mg/L IAA, trending at 100 mg/L (p=.070), and in vitro. IHC data in vitro suggests a marked increase in P21-positivity following IAA exposure. These data, together with prior ovarian findings, implicate IAA as a potential reproductive axis disruptor at each major level – through ARC Kiss1 expression, Fshb expression in vivo and in vitro, FSH expression in vitro, and Lhb and Cga in vitro. Further, Cdkn1a/P21 induction indicates IAA toxicity at the level of the pituitary.??