Phosphatidylinositol-4-kinase III␣ (PI4KIII␣) is an essential host cell factor for hepatitis C virus (HCV)replication. An N-terminally truncated 130-kDa form was used to reconstitute an in vitro biochemical lipid kinase assay that was optimized for smallmolecule compound screening and identified potent and specific inhibitors. Cell culture studies with PI4KIII␣ inhibitors demonstrated that the kinase activity was essential for HCV RNA replication. Two PI4KIII␣ inhibitors were used to select cell lines harboring HCV replicon mutants with a 20-fold loss in sensitivity to the compounds. Reverse genetic mapping isolated an NS4B-NS5A segment that rescued HCV RNA replication in PIK4III␣-deficient cells. HCV RNA replication occurs on specialized membranous webs, and this study with PIK4III␣ inhibitor-resistant mutants provides a genetic link between NS4B/NS5A functions and PI4-phosphate lipid metabolism. A comprehensive assessment of PI4KIII␣ as a drug target included its evaluation for pharmacologic intervention in vivo through conditional transgenic murine lines that mimic target-specific inhibition in adult mice. Homozygotes that induce a knockout of the kinase domain or knock in a single amino acid substitution, kinase-defective PI4KIII␣, displayed a lethal phenotype with a fairly widespread mucosal epithelial degeneration of the gastrointestinal tract. This essential host physiologic role raises doubt about the pursuit of PI4KIII␣ inhibitors for treatment of chronic HCV infection.

Chlorine dioxide (ClO 2 ) is an effective drinking water disinfectant, but sodium chlorate (NaClO 3 ) has been identi ed as a potentially harmful disinfection by-product . Studies were performed to describe the developmen t of thyroid lesions in animals exposed to NaClO 3 in the drinking water. Male and female F344 rats and B6C3F 1 mice were exposed to 0, 0.125, 0.25, 0.5, 1.0, or 2.0 g/L NaClO 3 for 21 days. Additional male F344 rats were exposed to 0, 0.001, 0.01, 0.1, 1.0, or 2.0 g/L NaClO 3 for 90 days. Female F344 rats were exposed to 0, 0.5, 1.0, 2.0, 4.0, or 6.0 g/L of NaClO 3 for 105 days. Thyroid tissues were processed by routine methods for light microscopi c examination, and follicular cell hyperplasia was diagnosed using a novel method. Thyroid hormone levels were altered signi cantly after 4 and 21 days. NaClO 3 treatment induced a concentration-dependen t increase in the incidence and severity of thyroid follicular cell hyperplasia. Male rats are more sensitive to the effects of NaClO 3 treatment than females. Follicular cell hyperplasi a was not present in male or female B6C3F 1 mice. These data can be used to estimate the human health risk that would be associated with using ClO 2 , rather than chlorine, to disinfect drinking water.

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Environmental exposures occurring early in life may have an important influence on cancer risk later in life. Here, we investigated carryover effects of dichloroacetic acid (DCA), a small molecule analog of pyruvate with metabolic programming properties, on age-related incidence of liver cancer. The study followed a stop-exposure/promotion design in which 4-week-old male and female B6C3F1 mice received the following treatments: deionized water alone (dH2O, control); dH2O with 0.06% phenobarbital (PB), a mouse liver tumor promoter; or DCA (1.0, 2.0 or 3.5g/l) for 10 weeks followed by dH2O or PB (n = 20-30/group/sex). Pathology and molecular assessments were performed at 98 weeks of age. In the absence of PB, early-life exposure to DCA increased the incidence and number of hepatocellular tumors in male and female mice compared with controls. Significant dose trends were observed in both sexes. At the high dose level, 10 weeks of prior DCA treatment induced comparable effects (≥85% tumor incidence and number) to those seen after continuous lifetime exposure. Prior DCA treatment did not enhance or inhibit the carcinogenic effects of PB, induce persistent liver cytotoxicity or preneoplastic changes on histopathology or alter DNA sequence variant profiles within liver tumors compared with controls. Distinct changes in liver messenger RNA and micro RNA profiles associated with prior DCA treatment were not apparent at 98 weeks. Our findings demonstrate that early-life exposure to DCA may be as carcinogenic as life-long exposures, potentially via epigenetic-mediated effects related to cellular metabolism.