The NTP characterized and compared the carcinogenicity and tissue distribution of SDD and CPM, two chromium compounds with widespread human exposure. The NTP chronic studies of Cr(VI), as SDD, demonstrated that it was carcinogenic in rats and mice after oral exposure (; NTP, 2008a
; Stout et al., 2009a
). In contrast, at much higher exposure concentrations, Cr(III), as CPM, may have been carcinogenic in male rats but was not carcinogenic in female rats or mice (NTP, 2008b
; Stout et al., 2009b
). As part of these studies, total Cr concentrations were measured in tissues and excreta of male rats and female mice at various exposure durations to provide internal dosimetry data to aid in the interpretation of the bioassay results. Because sex differences in chromium tissue accumulation were not expected based on studies conducted by Mackenzie et al. (1958)
and Sutherland et al. (2000)
, measurements were made in only one sex of each species. There were increases in total chromium in multiple tissues in both male rats and female mice, indicating that systemic exposure to chromium occurred following exposure to both Cr(VI) and Cr(III).
TABLE 7 Combined Incidences of Epithelial Neoplasms of the Alimentary Tract in F344/N Rats and B6C3F1 Mice following Exposure to Sodium Dichromate Dihydrate for 2 Years in Drinking Water (adapted from Stout et al., 2009a)
It had been hypothesized that oral exposure to Cr(VI) would not produce an increase in cancer, except perhaps in the stomach, because of the efficient capacity of the stomach to reduce Cr(VI) to Cr(III) (De Flora, 2000
; De Flora et al., 1997
; Proctor et al., 2002
). To determine if ingested Cr(VI) was systemically distributed, tissue Cr concentrations resulting from exposure to similar external doses of Cr(VI) and Cr(III) were compared. In all tissues examined, similar external doses of chromium resulted in much higher tissue Cr concentrations following exposure to Cr(VI) relative to Cr(III), indicating that at least a portion of the Cr(VI) was distributed to tissues prior to reduction. These data are consistent with reports in the literature in which the same concentration of Cr(VI) and Cr(III) was administered in drinking water for 11 months (Costa, 1997
; Costa and Klein, 2006
; Mackenzie et al., 1958
). The greater uptake of Cr following exposure to Cr(VI) is consistent with differences observed in toxicity and carcinogenicity, including the absence of small intestine tumors in mice following Cr(III) exposure, and the proposed mechanisms of transport from previous studies (Alexander and Aaseth, 1995
); reviewed by Costa (1997)
and Costa and Klein (2006)
and the present study.
Kerger et al. (1996
and Paustenbach et al. (1996)
have proposed that the pharmacokinetics of Cr(VI) following oral exposure can be explained largely by the reduction of ingested Cr(VI) in the gut, which results in the production of Cr(III) organic complexes. Although Cr(III) is relatively nondiffusible across cellular membranes, Kerger et al.
suggest that Cr(III) organic complexes are more easily absorbed into cells than inorganic Cr(III) and once absorbed are more rapidly eliminated than Cr(VI). These investigators point to similar Cr elimination profiles in red blood cells (RBCs) and plasma as evidence for the presence of Cr(III) organic complexes because Cr(VI) absorbed in blood is reduced to unstable intermediates, which form complexes with hemoglobin and other proteins resulting in retention of Cr for the lifetime of the RBC (Gray and Sterling, 1950
; Kerger et al., 1996
; Ottenwaelder et al., 1988
). Our data are not consistent with this hypothesis and suggest that Cr(VI) was taken up by RBCs and tissues following administration of SDD. Several lines of evidence support this conclusion. Cr concentrations were significantly increased in erythrocytes at all exposure durations following exposure to the two highest doses of SDD. Although we utilized a washout period of 48 h, the chromium concentrations in the erythrocytes were approximately sixfold higher than those in the plasma, indicating that the Cr taken up by RBCs was largely retained, rather than diffusing into the plasma from RBCs or other tissues. In contrast, with CPM, an organic complex, chromium concentrations were higher in plasma than in RBCs, indicating limited uptake or more extensive diffusion into the plasma from the RBCs or other tissues. The 15- to 20-fold higher Cr concentrations (on day 182) in the RBC following exposure to Cr(VI), relative to a comparable external dose of Cr(III), and the observed toxicity to RBCs with Cr(VI) but not Cr(III) provides additional evidence that Cr(VI) was preferentially taken up by and was toxic to erythrocytes. These data are consistent with previous reports in the literature demonstrating higher levels of chromium in blood following administration of Cr(VI) compared with Cr (III) (Gray and Sterling, 1950
; Mackenzie et al., 1959
Although chromium was not measured in the small intestine following exposure to Cr(VI) or Cr(III), previous reports suggest that Cr(VI) is also likely to be absorbed in this tissue to a greater extent than Cr(III) (Donaldson and Barreras, 1966
; Fébel et al., 2001
). The study by Davidson et al. (2004)
demonstrating increased susceptibility to skin cancer induction in hairless mice following co-exposure to ultraviolet light and Cr(VI) in the drinking water provides additional evidence that Cr(VI) can have systemic effects that are distant from the site of exposure. The data in the present report do not explain why neoplasms were not observed at sites distant from the alimentary tract.
Because of the observed species differences in sites of induced neoplasms following exposure to Cr(VI), tissue Cr concentrations normalized to external dose were compared between rats and mice. Cr uptake was found to be significantly higher in the kidney of rats and the liver and glandular stomach of mice (). This is consistent with previous studies in the literature (Coogan et al., 1991
; Kargacin et al., 1993
; Witmer et al., 1989
). In addition, higher tissue concentrations were achieved in rats than occurred in tissues of mice exposed to the lower concentrations of SDD that also resulted in small intestine neoplasms (57.3 in female mice and 172 mg/l in mice of both sexes). Based on these lines of evidence, the tissue concentration data do not explain the species differences in target sites of carcinogenicity.
It has been previously hypothesized that the small intestine neoplasms observed in the NTP 2-year bioassay of SDD would occur only at doses that exceeded the gastric reduction capacity (De Flora et al., 2008
). If the gastric reduction capacity had been exceeded, the dose that resulted in saturation would likely represent an inflection point for a sublinear exposure-response, with doses above this point demonstrating a greater rate of response than lower doses. Following exposure to SDD, the shapes of the exposure-response curves for both tissue concentration data in male rats and female mice () and incidences of small intestine neoplasms in male and female mice (; Stout et al., 2009a
) were either linear or supralinear. In addition, Cr(VI) doses from the NTP 2-year mouse study were compared with gastric reductive capacity estimates originally reported for humans (De Flora et al., 1997
) and allometrically scaled to mice and compared with average daily doses of Cr(VI) following exposure to SDD (Stout et al., 2009a
). This comparison revealed that the calculated dose that might saturate gastric reduction is higher than all the doses in male mice and is nearly equivalent to the highest dose in female mice (Stout et al., 2009a
). Collectively, these data indicate that the gastric reduction capacity was not saturated in rats or mice exposed to Cr(VI) in drinking water.
The lowest concentration of Cr(VI) in this study that produced an increase in tumor incidence in the small intestine of female mice was 20 mg/l (57.3 mg SDD/l). Using the time weighted average daily dose of Cr(VI) for the entire 2-year study and assuming mouse and human external exposure concentrations scale by body weight3/4
(body weight raised to the 3/4 power), exposure of mice to 20 mg Cr(VI)/l (1.016 mg/kg) for 2 years would result in a human equivalent daily dose of 0.166 mg/kg. This calculated dose is equivalent or within an order of magnitude to the doses estimated for a 70 kg person drinking 2 l of water per day at the highest concentrations reported in a survey of drinking water sources collected in Texas (5.41 mg/l; Texas Department of State Health Services, 2009
) or California (0.603 mg/l; California Department of Public Health, 2007a
). The U.S. EPA has set a maximum contaminant level of 100 μg/l total chromium in drinking water (U.S. EPA, 2003), although the limit in several states is 50 μg/l.
In conclusion, the results of these studies support the hypothesis that Cr(VI) is the species of chromium responsible for the induction of carcinogenesis in the NTP chronic toxicity and carcinogenicity studies of SDD. In addition, these results indicate that gastric reduction was not saturated following exposure to Cr(VI) and that differences in tissue uptake cannot account for the species differences in sites of Cr(VI)-induced carcinogenicity. The transport studies confirm previous reports that Cr(VI) is taken up by cells via the sodium/sulfate co-transporter, whereas Cr(III) is not, providing at least a partial explanation for the observed differences in tissue uptake.