Association of brominated proteins and changes in protein expression in the rat kidney with subcarcinogenic to carcinogenic doses of bromate.

The water disinfection byproduct bromate (BrO3(-)) produces cytotoxic and carcinogenic effects in rat kidneys. Our previous studies demonstrated that BrO3(-) caused sex-dependent differences in renal gene and protein expression in rats and the elimination of brominated organic carbon in their urine. The present study examined changes in renal cell apoptosis and protein expression in male and female F344 rats treated with BrO3(-) and associated these changes with accumulation of 3-bromotyrosine (3-BT)-modified proteins. Rats were treated with 0, 11.5, 46 and 308 mg/L BrO3(-) in drinking water for 28 days and renal sections were prepared and examined for apoptosis (TUNEL-staining), 8-oxo-deoxyguanosine (8-oxoG), 3-BT, osteopontin, Kim-1, clusterin, and p-21 expression. TUNEL-staining in renal proximal tubules increased in a dose-related manner beginning at 11.5mg BrO3(-)/L in female rats and 46 mg/L in males. Increased 8-oxoG staining was observed at doses as low as 46 mg/L. Osteopontin expression also increased in a dose-related manner after treatment with 46 mg/L, in males only. In contrast, Kim-1 expression increased in a dose-related manner in both sexes, although to a greater extent in females at the highest dose. Clusterin and p21 expression also increased in a dose-related manner in both sexes. The expression of 3-BT-modified proteins only increased in male rats, following a pattern previously reported for accumulation of α-2u-globulin. Increases in apoptosis in renal proximal tubules of male and female rats at the lowest doses suggest a common mode of action for renal carcinogenesis for the two sexes that is independent of α-2u-globulin nephropathy.

Changes in mRNA and protein expression in the renal cortex of male and female F344 rats treated with bromate.

Bromate (BrO3(-)), a by-product of ozonation of drinking water, induces nephrotoxicity in male rats at much lower doses than in female rats. This difference appears to be related to the development of α-2u-globulin nephropathy in males. To determine sex-dependent changes in mRNA and protein expression in the renal cortex attributable to α-2u-globulin nephropathy, we performed microarray and immunohistochemical analyses in proximal renal tubules of male and female F344 rats treated with KBrO3 for 28 days. Particular attention was paid to molecular biomarkers of renal tubular injury. Microarray analysis of male and female rats treated with BrO3(-) at low doses (125 mg/L KBrO3) displayed marked sex-dependent changes in renal gene expression. The greatest differences were seen in genes encoding for cellular differentiation, apoptosis, ion transport, and cell proliferation. Differences by sex were especially prominent for the cell cycle checkpoint gene p21, the renal injury protein Kim-1, and the kidney injury and cancer biomarker protein osteopontin. Dose-related nephrotoxicity, assessed by hematoxylin and eosin staining, was greater in males compared to female rats, as was cellular proliferation, assessed by bromodeoxyuridine staining. The fraction of proximal renal cells with elevated 8-oxodeoxyguanosine (8-OH-dG) was only increased at the high dose and did not differ by sex. Dose-dependent increases in the expression of osteopontin were detected immunohistochemically only in male rats and were localized in proximal tubule cells. Similarly, BrO3(-) treatment increased clusterin and Kim-1 staining in the proximal tubules; however, staining for these proteins did not differ appreciably between males and females. These data demonstrate both qualitative and quantitative differences in the response of male versus female kidneys to BrO3(-)-treatment. These sex-dependent effects likely contribute to renal carcinogenesis of BrO3(-) in the male rat.

Absorption and disposition of bromate in F344 rats.

Bromate (BrO(3)(-)) is a ubiquitous by-product of using ozone to disinfect water containing bromide (Br(-)). The reactivity of BrO(3)(-) with biological reductants suggests that its systemic absorption and distribution to target tissues may display non-linear behavior as doses increase. The intent of this study is to determine the extent to which BrO(3)(-) is systemically bioavailable via oral exposure and broadly identify its pathways of degradation. In vitro experiments of BrO(3)(-) degradation in rat blood indicate a rapid initial degradation immediately upon addition that is >98% complete at concentrations up to 66µM in blood. As initial concentrations are increased, progressively lower fractions are lost prior to the first measurement. Secondary to this initial loss, a slower and predictable first order degradation rate was observed (10%/min). Losses during both phases were accompanied by increases in Br(-) concentrations indicating that the loss of BrO(3)(-) was due to its reduction. In vivo experiments were conducted using doses of BrO(3)(-) ranging from 0.077 to 15.3mg/kg, administered intravenously (IV) or orally (gavage) to female F344 rats. The variable nature and uncertain source of background concentrations of BrO(3)(-) limited derivation of terminal half-lives, but the initial half-life was approximately 10min for all dose groups. The area under the curve (AUC) and peak concentrations (C(t=5')) were linearly related to IV dose up to 0.77mg/kg; however, disproportionate increases in the AUC and C(t=5') and a large decrease in the volume of distribution was observed when IV doses of 1.9 and 3.8mg/kg were administered. The average terminal half-life of BrO(3)(-) from oral administration was 37min, but this was influenced by background levels of BrO(3)(-) at lower doses. With oral doses, the AUC and C(max) increased linearly with dose up to 15.3mgBrO(3)(-)/kg. BrO(3)(-) appeared to be 19-25% bioavailable without an obvious dose-dependency between 0.077 and 1.9mg/kg. The urinary elimination of BrO(3)(-) and Br(-) was measured from female F344 rats for four days following administration of single doses of 8.1mgKBrO(3)/kg and for 15 days after a single dose of 5.0mgKBr/kg. BrO(3)(-) elimination was detected over the first 12h, but Br(-) elimination from BrO(3)(-) over the first 48h was 18% lower than expected based on that eliminated from an equimolar dose of Br(-) (15.5±1.6 vs. 18.8±1.2µmol/kg, respectively). The cumulative excretion of Br(-) from KBr vs. KBrO(3) was equivalent 72h after administration. The recovery of unchanged administered BrO(3)(-) in the urine ranged between 6.0 and 11.3% (creatinine corrected) on the 27th day of treatment with concentrations of KBrO(3) of 15, 60, and 400mg/L of drinking water. The recovery of total urinary bromine as Br(-)+BrO(3)(-) ranged between 61 and 88%. An increase in the fraction of the daily BrO(3)(-) dose recovered in the urine was observed at the high dose to both sexes. The deficit in total bromine recovery raises the possibility that some brominated biochemicals may be produced in vivo and more slowly metabolized and eliminated. This was supported by measurements of dose-dependent increases of total organic bromine (TOBr) that was eliminated in the urine. The role these organic by-products play in BrO(3)(-)-induced cancer remains to be established.