LETTER TO EDITOR Assessing the Threshold for the Duodenal GSH/GSSG Ratio in the Mouse Associated With Sodium Dichromate Dihydrate Ingestion Reported by Thompson et al. (2011)

To the Editor The U.S. Environmental Protection Agency (EPA) is currently considering its characterization of the human carcinogenicity potential of ingested hexavalent chromium (Crþ6), the outcome of which can have a major impact on risk-based environmental regulation. The key study in the U.S. EPA’s assessment is the 2year chronic bioassay in mice conducted by the National Toxicology Program (NTP, 2008) that found a dose-response relationship between Crþ6 ingested as sodium dichromate dihydrate (SDD) in drinking water and tumors of the small intestine. A critical issue in the U.S. EPA assessment is whether, with low-dose ingestion, toxicologically significant amounts of Crþ6 can escape the endogenous capacity of the stomach to reduce Crþ6 to the relatively nontoxic Crþ3. If Crþ6 can only pass out of the stomach to the duodenum after the stomach’s reductive capacity has been saturated by high doses of Crþ6, there may be a threshold for the production of small intestine tumors that precludes extrapolation of cancer potency from the NTP study doses to environmentally relevant levels of human exposure. If, on the other hand, as described in Stern (2010), the equilibrium redox process in the stomach competes with the kinetics of gastric emptying and absorption across the stomach membrane, even low doses of Crþ6 can pose a cancer risk. In their recent paper in Toxicological Sciences, Thompson et al. (2011) exposed mice for 90 days to Crþ6 in drinking as SDD. The water concentrations of SDD were designed to be similar to those in the 2-year NTP (2008) chronic bioassay but also included two concentrations below those employed by NTP. Thompson et al. (2011) posit that the ratio of reduced to oxidized glutathione (GSH/GSSG) reflects the overall redox status of the tissues in which it is measured and that glutathione potentially plays a direct role in the reduction of Crþ6 in those tissues. They therefore view a change in this ratio in the mouse duodenum in response to ingestion of SDD as a measure of the unreduced Crþ6 that escapes the stomach. At and above the dose at which the reduction capacity of the mouse stomach becomes saturated, they hypothesize that there would be a marked decrease in the GSH/GSSG ratio in the duodenum. On the other hand, they hypothesize that at doses where unreduced Crþ6 does not escape the stomach, there should be no change in the duodenal GSH/GSSG ratio. Using a statistical approach for analysis of trend and difference from control values employed in a different context in the NTP study, Thompson et al. (2011), in their 90-day exposure, claim to detect a significant decrease in the duodenal GSH/GSSG ratio at SDD concentrations 14 mg SDD/l. This is the lowest SDD drinking water concentration in the NTP study. At the two lower concentrations they employ, however, they claim to find no significant change in this ratio. Taken at face value, one might conclude from this report that 14 mg/l is a threshold for saturation of the reductive capacity of the mouse stomach. In other words, at SDD water concentrations 14 mg/l, there is ‘‘breakthrough’’ of unreduced Crþ6 into the duodenum, but that at lower more environmentally relevant concentrations, ingested Crþ6 is quantitatively reduced in the stomach and is unavailable to the duodenum. However, NTP applied this statistical approach to a data set that contained 50 animals per concentration group. Thompson et al. (2011) exposed only five animals per concentration group. Clearly, with such a small number of animals per category, the statistical power to detect a meaningful change is low, and the identification of a threshold is driven more by sample size than by the actual extent of changes in biological markers. Even cursory observation of Figure 2a in Thompson et al. (2011) shows that there is a dramatic and monotonic decrease in the duodenal GSH/GSSG ratio starting with the lowest SDD concentration employed by Thompson et al. (2011). Given the limitations on statistical hypothesis testing imposed by their small sample sizes, a more appropriate approach to the analysis