TOXICOLOGICAL SCIENCES 118(2), 368–379 (2010) doi:10.1093/toxsci/kfq263 Advance Access publication September 15, 2010
Exposure to Hexavalent Chromium Resulted in Significantly Higher Tissue Chromium Burden Compared With Trivalent Chromium Following Similar Oral Doses to Male F344/N Rats and Female B6C3F1 Mice Bradley J. Collins,* Matthew D. Stout,* Keith E. Levine,† Grace E. Kissling,* Ronald L. Melnick,* Timothy R. Fennell,† Ramsey Walden,‡ Kamal Abdo,* John B. Pritchard,‡ Reshan A. Fernando,† Leo T. Burka,* and Michelle J. Hooth*,1 *National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; †Discovery and Analytical Sciences, RTI International, Research Triangle Park, North Carolina 27709; and ‡Laboratory of Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 1
To whom correspondence should be addressed at National Institute of Environmental Health Sciences, National Toxicology Program, 111 Alexander Drive, MD K2-13, Research Triangle Park, NC 27709-2233. Fax: (919) 541-4255. E-mail:
[email protected]. Received July 1, 2010; accepted August 26, 2010
In National Toxicology Program 2-year studies, hexavalent chromium [Cr(VI)] administered in drinking water was clearly carcinogenic in male and female rats and mice, resulting in small intestine epithelial neoplasms in mice at a dose equivalent to or within an order of magnitude of human doses that could result from consumption of chromium-contaminated drinking water, assuming that dose scales by body weight3/4 (body weight raised to the 3/4 power). In contrast, exposure to trivalent chromium [Cr(III)] at much higher concentrations may have been carcinogenic in male rats but was not carcinogenic in mice or female rats. As part of these studies, total chromium was measured in tissues and excreta of additional groups of male rats and female mice. These data were used to infer the uptake and distribution of Cr(VI) because Cr(VI) is reduced to Cr(III) in vivo, and no methods are available to speciate tissue chromium. Comparable external doses resulted in much higher tissue chromium concentrations following exposure to Cr(VI) compared with Cr(III), indicating that a portion of the Cr(VI) escaped gastric reduction and was distributed systemically. Linear or supralinear dose responses of total chromium in tissues were observed following exposure to Cr(VI), indicating that these exposures did not saturate gastric reduction capacity. When Cr(VI) exposure was normalized to ingested dose, chromium concentrations in the liver and glandular stomach were higher in mice, whereas kidney concentrations were higher in rats. In vitro studies demonstrated that Cr(VI), but not Cr(III), is a substrate of the sodium/sulfate cotransporter, providing a partial explanation for the greater absorption of Cr(VI). Key Words: National Toxicology Program; cancer; hexavalent chromium; trivalent chromium; chromium picolinate monohydrate; sodium dichromate dihydrate; disposition; rodent; inductively coupled plasma-mass spectrometry.
Chromium (Cr) exists in multiple oxidation states. The hexavalent chromium [Cr(VI)] and trivalent chromium [Cr(III)] states are most important from a biological and an industrial Published by Oxford University Press 2010.
standpoint. Cr(VI) is an industrial contaminant of water and soil and an established human lung carcinogen following inhalation exposure (Cohen et al., 1993; International Agency for Research on Cancer (IARC), 1990; National Toxicology Program [NTP] 1998). Sodium dichromate dihydrate (SDD) is the most watersoluble salt of Cr(VI). In contrast, Cr(III) is an essential element and a natural dietary constituent (Anderson, 1989; National Institutes of Health (NIH), 2007) and is also widely ingested in dietary supplements, most notably chromium picolinate monohydrate (CPM). CPM is an organic complex, with Cr(III) chelated to three molecules of picolinic acid to increase Cr(III) absorption. However, studies have shown that there is significant separation of the Cr(III) from the picolinic acid prior to absorption (Hepburn and Vincent, 2002; NTP, 2008b) and that the absorption of chromium(III) chloride, picolinate, and nicotinate is similar (out gradient (þNA). SDD: Na-driven sulfate uptake was inhibited by SDD to an extent equal to that produced by 4-acetamido-4#-isothiocyanostilbene2,2#-disulfonic acid (SITS), a well-established inhibitor of Na-sulfate co-transport. CPM: At equivalent doses, CPM was without effect. N ¼ 3 animals. **p < 0.05 versus þNA control uptake.
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 20fold 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; Fe´bel 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
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)
Exposure concentration (mg/l) Historical control rangea (drinking water; %) Historical control range (all routes; %) 0 14.3 28.6 57.3 85.7 172 257.4 516 a
Rats
Mice
Squamous cell papilloma or carcinoma of the oral mucosa or tongue
Adenoma or carcinoma of the duodenum, jejunum, or ileum
Males
Females
Males
Females
0–2 0–2 0/50b*** 1/50 (2.4)c — 0/49 — 0/50 — 7/49 (15.7)**
0–2 0–6 1/50 (2.2)*** 1/50 (2.3) — 0/50 — 2/50e (4.6) — 11/50 (23.9)**
0–10 0–10 1/50 (2.2)*** 3/50 (6.8) 2/50 (4.6) — 7/50 (15.1)* — 20/50 (43.8)*** —
0–4 0–4 1/50 (2.2)*** 1/50 (2.2) — 4/50d (8.3) — 17/50 (36.3)*** — 22/50 (45.9)***
The NTP historical database contains all studies that use the NTP-2000 diet with histopathology findings completed within the most recent 5-year period, including the present study. b Incidence/number examined. c Survival-adjusted percent incidence calculated using the poly-3 test. d The incidence exceeded the historical control range for both drinking water studies and all routes but was not significantly increased compared to the concurrent control. e The incidence exceeded the historical control range for drinking water studies but was not significantly increased compared to the concurrent control. *Significantly different than the control group by the poly-3 test or a significant trend if assigned to a control group (p 0.05). **p 0.05. ***p 0.001.
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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 (Table 4). This is consistent with previous studies in the literature (Coogan et al., 1991; Kargacin et al., 1993; Witmer et al., 1989, 1991). 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 (Fig. 1A) and incidences of small intestine neoplasms in male and female mice (Table 7; 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 lg/l total chromium in drinking water (U.S. EPA, 2003), although the limit in several states is 50 lg/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.
FUNDING
This research was supported (in part) by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences under Research Project Number ZO1 ES045004-11 BB and Z01 ES65554.
ACKNOWLEDGMENTS
The authors thank Drs Nigel Walker and Suramya Waidynatha for their critical review of this article.
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