Mechanistic Insights from the NTP Studies of Chromium

Abstract

Hexavalent chromium (Cr(VI)) is a contaminant of water and soil and is a human lung carcinogen. Trivalent chromium (Cr(III)), a proposed essential element, is ingested by humans in the diet and in dietary supplements such as chromium picolinate (CP). The National Toxicology Program (NTP) demonstrated that Cr(VI) is also carcinogenic in rodents when administered in drinking water as sodium dichromate dihydrate (SDD), inducing neoplasms of the oral cavity and small intestine in rats and mice, respectively. In contrast, there was no definitive evidence of toxicity or carcinogenicity following exposure to Cr(III) administered in feed as CP monohydrate (CPM). Cr(VI) readily enters cells via nonspecific anion channels, in contrast to Cr(III), which cannot easily pass through the cell membrane. Extracellular reduction of Cr(VI) to Cr(III), which occurs primarily in the stomach, is considered a mechanism of detoxification, while intracellular reduction is thought to be a mechanism of genotoxicity and carcinogenicity. Tissue distribution studies in additional groups of male rats and female mice demonstrated higher Cr concentrations in tissues following exposure to Cr(VI) compared to controls and Cr(III) exposure at a similar external dose, indicating that some of the Cr(VI) escaped gastric reduction and was distributed systemically. The multiple potential pathways of Cr-induced genotoxicity will be discussed.

Introduction

Chromium (Cr), a naturally occurring element, is abundant in the earth’s crust and occurs in a variety of valence states with trivalent (Cr(III)) and hexavalent (Cr(VI)) being the most stable and biologically relevant oxidation states. Cr(III) is a natural dietary constituent present in a variety of foods and has been proposed to be an essential trace element that may participate in carbohydrate and lipid metabolism. In addition, Cr(III) is present in dietary supplements, such as chromium picolinate (CP), that are marketed primarily for weight loss and antidiabetic effects.

Cr(VI) does not occur naturally but is a drinking water contaminant arising from certain industrial processes including electroplating, leather tanning, and textile manufacturing where it is used as an anticorrosive agent or as a dye/pigment. Cr(VI) has been detected in groundwater and soil samples in many states. The U.S. Environmental Protection Agency (US EPA) has set a maximum contaminant level (MCL) of 100 μg/L for total Cr in drinking water (US EPA 2003), although the limit in several states is 50 μg/L. These regulatory levels are not specific to the hexavalent form of Cr, however. Until recently, there was limited drinking water occurrence data for Cr(VI), specifically. In 2010, the Environmental Working Group (EWG 2010) reported that Cr(VI) was detected in 31 tap water samples from 35 U.S. cities that had previously measured only total Cr levels; the highest level of Cr(VI) detected was 12.9 μg/L, and the next highest levels ranged from 1.34 to 2.00 μg/L. In April 2012, Cr(VI) was added to the US EPA’s Third Unregulated Contaminant Monitoring Rule (UCMR), which is the new list consisting of no more than 30 unregulated contaminants that are required to be monitored by public water systems (US EPA 2012). The California Department of Health Services (CDPH) adopted its own UCMR regulation in 2001. Their most recent survey reported detectable levels of Cr(VI) in approximately 30% of drinking water sources monitored in California, which has a 1 μg/L detection limit for purposes of reporting; 86% of those sources had concentrations ranging from 1 to 10 μg/L (CDPH 2011). This required monitoring will provide much needed occurrence data to determine potential human exposure to Cr(VI) via drinking water sources.

In general, Cr(VI) compounds are more toxic than Cr(III) compounds. Cr(VI) has long been recognized as carcinogenic to humans following inhalation exposure, inducing lung tumors in Cr industry workers and in experimental animals exposed to these compounds by inhalation (International Agency for Research on Cancer [IARC] 1990; Cohen et al. 1993; National Toxicology Program [NTP] 2011). The potentially harmful health effects of Cr(VI) following oral exposure were brought to the public’s attention, most notably, in the movie Erin Brockovich that dealt with the alleged case of drinking water contamination in Hinkley, California, by Pacific Gas and Electric Company. In contrast, Cr(III) compounds display little or no toxicity or carcinogenic activity in rodents or in humans (MacKenzie et al. 1958; Schroeder, Balassa, and Vinton 1964, 1965; Ivankovic and Preussmann 1975; Anderson, Bryden, and Polansky 1997; NTP 2010).

Although the absorption of Cr(VI) and Cr(III) is low following oral exposure, Cr(VI) is absorbed more efficiently than Cr(III) (MacKenzie et al. 1959; Donaldson and Barreras 1966; Kerger et al. 1996; Febel, Szegedi, and Huszar 2001). This is thought to occur because Cr(VI) as chromate structurally resembles sulfate and phosphate and is taken up by all cells and organs throughout the body via sulfate transporters (Costa 1997). In contrast, Cr(III) is not a substrate for active transport (Proctor et al. 2002) but is thought to enter cells via diffusion or phagocytosis. Results of more recent in vitro studies conducted by the NTP in kidney membrane vesicles confirm that Cr(VI), but not Cr(III), is a competitive inhibitor of the sodium/sulfate cotransporter (Collins et al. 2010). Consistent with this mechanism of transport, higher Cr tissue concentrations were observed with Cr(VI) compared with Cr(III) following exposures to equivalent concentrations in drinking water (MacKenzie et al. 1958; Costa 1997; Costa and Klein 2006; Collins et al. 2010).

Both extracellular and intracellular reduction of Cr(VI) to Cr(III) occurs. As a result of the lower bioavailability of Cr(III), extracellular reduction, primarily in the stomach, has been suggested to be protective against the toxic and carcinogenic effects of Cr(VI) following oral exposure (De Flora et al. 1997; De Flora 2000; Proctor et al. 2002; Paustenbach et al. 2003). This hypothesis suggests that Cr(VI) would only cause harm at distal tissue sites if the endogenous reduction capacity of the stomach were exceeded. Alternatively, Stern (2010) has argued that kinetic competition exists among a number of processes affecting the disposition of Cr, including absorption across the stomach membrane, reduction of Cr(VI) to the relatively nontoxic trivalent form, and gastric emptying of stomach contents to the small intestine resulting in Cr(VI) escaping reduction. As discussed below, once Cr(VI) is absorbed into a cell, intracellular reduction is thought to be a mechanism of genotoxicity and carcinogenesis because of DNA damage that occurs when Cr(VI) is reduced through Cr(V) and Cr(IV) to Cr (III).

Cr(III) and Cr(VI) Cr compounds were nominated separately to the NTP for toxicity and carcinogenicity testing. The National Cancer Institute nominated CP for testing based on the potential for widespread consumer exposure from its use as a dietary supplement. The NTP selected CP monohydrate (CPM) for study because it is the commercially available form of CP. The California (CA) Congressional Delegation, the CA EPA, and the CA Department of Health Services nominated Cr(VI) to the NTP for study because of the lack of adequate experimental data on the toxicity and carcinogenicity of Cr(VI) ingested orally and because of the potential for exposure of large human populations via contaminated drinking water. The NTP selected sodium dichromate dihydrate (SDD) for study because it is the primary base material for the production of Cr compounds and is the most water-soluble chromate salt. While addressing each specific nomination, one of the strengths of these studies is that they allowed a direct comparison of the toxicity and carcinogenicity of trivalent and hexavalent Cr compounds. The major findings and conclusions from the NTP studies are presented here. In addition, the potential mechanisms involved in the genotoxicity and carcinogenicity of these compounds are discussed.

SDD and CPM Study Design

The designs of the 3-month and 2-year studies of SDD and CPM are presented in Table 1. Additional experimental details are available in the NTP technical reports for these compounds (NTP 2008, 2010). Animal use was in accordance with the U.S. Public Health Service Policy on humane care and use of laboratory animals and the Guide for the Care and Use of Laboratory Animals (National Research Council 1996). The 3-month study data were used to select exposure concentrations for the 2-year studies. The highest exposure concentration of 50,000 ppm in the 3-month and 2-year studies of CPM was considered to be the maximum feasible dose in feed studies that would not alter nutritional content of the diet. Average daily doses in the 2-year studies of CPM were approximately three to five orders of magnitude higher than those consumed by humans ingesting typical doses of supplements. For the 2-year studies of SDD, a wider spacing of doses was used to extend the dose–response curve, and an additional low-exposure concentration group was added to provide a dose that was closer to concentrations to which humans might be exposed through the drinking water. The lowest concentration of SDD (14.3 mg/L) used in the NTP 2-year studies is equivalent to 5 mg/L Cr and is less than 10 times the reported Cr(VI) concentration of 0.58 mg/L that was found in a groundwater monitoring well in Hinkley, California, (Pellerin and Booker 2000). Doses for the 2-year study of SDD in male mice were lower than female mice and rats due to decreased body weight and water consumption observed in the 3-month toxicity study.

Table 1.—

Design of the 3-month and 2-year studies of sodium dichromate dihydrate (SDD) and chromium picolinate monohydrate (CPM).

Cr compound	SDD	CPM
Species of Cr	Hexavalent (VI)	Trivalent (III)
Route (vehicle)	Oral (drinking water)	Oral (feed)
Animals	Male and Female F344/N rats and B6C3F1 mice
Group size
90-Day study	N = 10	N = 10
2-Year study	N = 50	N = 50
Exposure concentrations
90-Day	0, 62.5, 125, 250, 500, 1,000 mg SDD/L	0, 80, 240, 2,000, 10,000, 50,000 ppm CPM
2-Year	0, 14.3, 57.3, 172, 516 mg SDD/L (0, 14.3, 28.6, 85.7, 257.4 mg SDD/L in male mice)	0, 2000, 10,000, 50,000 ppm CPM
End points
90-Day	Survival, body weights, clinical observations, water (SDD)/food (CPM) consumption, hematology, clinical chemistry, sperm motility and vaginal cytology, micronucleus, histopathology
2-year	Survival, body weights, clinical observations, water (SDD)/food (CPM) consumption, hematology (SDD using tissue distribution animals), histopathology
Tissue distribution
Animals	Male rats and female mice as part of the 2-year studies
Exposure duration	6, 13, 182 days (SDD and CPM), 371 days (SDD only) including a 2 day washout; N = 10/species/duration
Tissues/excreta analyzed	Plasma (n = 6); RBC, glandular stomach, liver, kidney, urine, feces (n = 3)
End point measured	Total Cr by inductively coupled plasma mass spectrometry (ICP-MS)
Exposure for comparison of internal doses	516 mg SDD/L	2,000 ppm CPM
Time-weighted external dose at 182 days in rats	8.95 mg Cr/kg body weight	15.18 mg Cr/kg body weight
Time-weighted external dose at 182 days in mice	13.2 mg Cr/kg body weight	36.73 mg Cr/kg body weight

Major Results of the 3-month and 2-year Studies of CPM (Cr(III))

There were no biologically significant changes in survival, body weights, feed consumption, or nonneoplastic lesions in rats or mice in the 3-month or 2-year studies of CPM at doses up to 50,000 ppm in the diet (Stout, Nyska, et al. 2009; NTP 2010). The lack of an effect on body weight following exposure to CPM in these studies is notable because CP has been marketed as a dietary supplement for weight loss. In the 2-year studies, a statistically significant increase in the incidence of preputial gland adenoma in male rats at 10,000 ppm may have been related to exposure. CPM was not carcinogenic in female rats or in mice of either sex.

Major Results of the 3-month and 2-year Studies of SDD (Cr(VI))

In-life Effects in Rats and Mice

Survival, body weight, and water consumption data are presented in detail elsewhere (NTP 2007, 2008; Stout, Herbert, et al. 2009). In the 2-year study, survival of exposed groups of rats and mice was similar to that of the respective control groups. Mean body weights compared to controls were decreased in male and female rats and mice. In rats, mean body weights of 516 mg/L males and females were less than those of controls throughout the study and by the end of the study were 88 or 89% that of the respective controls. In male mice, mean body weights of the high-dose group were only slightly less (6%) than controls by the end of the study. In female mice, mean body weights were 8% less than controls in 172 mg/L females and 15% less in 516 mg/L females by the end of the study.

The lower body weights were partly attributed to poor palatability of the dosed water and consequent reductions in water consumption rather than direct toxic effects of SDD exposure. Water consumption by male and female rats and mice exposed to the two highest concentrations of SDD was less than that by the controls throughout the study. No clinical findings were attributed to SDD exposure in rats or mice.

Hematology in Rats and Mice

In the 3-month and 2-year studies of SDD, an exposure-related microcytic hypochromic anemia occurred in rats and was characterized by decreases in mean cell volumes, mean cell hemoglobin concentrations, hematocrits, hemoglobin concentrations, and erythrocyte counts, and an increase in reticulocyte counts; these data are presented in detail elsewhere (NTP 2007, 2008; Stout, Herbert, et al. 2009). The anemia was most prominent early in the study (22 days to 3 months) and ameliorated by the 12-month time point. The transient nature suggests an adaptive response by the exposed animals. The mice were less affected in the 3-month and 2-year studies compared to rats and had only a mild erythrocyte microcytosis. Similar hematological effects have been reported for rats and mice following oral exposure to Cr(VI) (Kumar and Barthwal 1991; NTP 1996a, 1996b, 1997; Agency for Toxic Substances and Disease Registry [ATSDR] 2000).

Neoplastic and Nonneoplastic Lesions in Rats and Mice

Glandular Stomach

In the 3-month SDD study, the primary nonneoplastic lesions were observed in the glandular stomach of rats exposed to 1,000 mg/L and included focal ulceration, regenerative epithelial hyperplasia, and squamous epithelial metaplasia (NTP 2007). Notably, no concentration-related increases in neoplasms or nonneoplastic lesions were observed in the forestomach or glandular stomach of rats or mice in the 2-year study (NTP 2008).

Oral Cavity Neoplasms in Rats

In the 2-year SDD study of male and female rats, there were significantly increased incidences of neoplasms of the squamous epithelium that lines the oral cavity (oral mucosa and tongue) at 516 mg/L SDD (Table 2; NTP 2008; Stout, Herbert, et al. 2009) Specifically, these increases were observed for squamous cell carcinoma in the oral mucosa and for squamous cell papilloma or carcinoma (combined) of the oral mucosa or tongue of male and female rats at 516 mg/L. There were also squamous cell carcinomas in the oral mucosa of two 172 mg/L female rats; this incidence exceeded the historical control ranges for drinking water studies and for all routes of administration (Table 2).

Table 2.—

Squamous cell neoplastic lesions in the oral cavity (oral mucosa and tongue) of male and female F344/N rats in the 2-year study of sodium dichromate dihydrate (SDD).

	Male					Female
Lesion	0 mg/L	14.3 mg/L	57.3 mg/L	172 mg/L	516 mg/L	0 mg/L	14.3 mg/L	57.3 mg/L	172 mg/L	516 mg/L
Oral mucosa
Papilloma	0	0	0	0	1	0	0	0	0	0
Carcinoma	0***	0	0	0	6*	0***	0	0	2+	11***
Tongue
Papilloma	0	0	0	0	1	1	1	0	0	0
Carcinoma	0	1	0	0	0	0	0	0	1	0
Oral Mucosa or Tongue
Papilloma or carcinoma (combined)	0***	1	0	0	7**	1***	1	0	2++	11**

Note: N = 49–50; significant by the poly-3 test at *p ≤ .05, **p ≤ .01, ***p ≤ .001 (significant for trend if noted for controls); exceeded the historical control range for +all routes and drinking water or ++drinking water only (historical control data not collected for tongue because it is not a protocol required tissue)

Microscopically, the squamous cell carcinomas were highly invasive, irregular masses that appeared to originate from the epithelium of the palate adjacent to the upper molar teeth. The squamous cell carcinomas occurred as pleomorphic islands, cords, and clusters of dysplastic squamous epithelium surrounded by dense proliferative connective tissue stroma (Figure 1A and B) that invaded the adjacent submucosal tissue extending to the soft tissues surrounding the nasal structures. In some animals, they invaded the tongue and Harderian gland, and, in one case, penetrated the maxillary bone and invaded the brain. The squamous cell papillomas of the oral mucosa and tongue were exophytic masses that projected from the mucosa. They occurred as irregular papillary proliferations of well-differentiated keratinized squamous epithelium supported by a core of connective tissue stroma (Figure 1C and D; Figure 2A). In the tongue, squamous cell carcinoma was diagnosed when there was evidence of invasion in the form of irregular foci of dysplastic squamous epithelium that extended from the base of the epithelium into the stroma of these papillary masses (Figure 2B and C).

Figure 1.—

Squamous cell neoplasms of the oral mucosa in rats given sodium dichromate dihydrate (SDD) for 2 years. A, Squamous cell carcinoma (arrows) arising from the oral mucosa of the soft palate has invaded the adjacent submucosal tissue and surrounded a molar tooth; H&E,1.0×. B, Higher magnification of Figure 1A. Note pleomorphic islands, cords, and clusters of dysplastic squamous epithelium (arrows) surrounded by dense proliferative connective tissue stroma; hematoxylin and eosin (H&E), 6.5×. C, Squamous cell papilloma (arrows) projecting from the mucosal surface of the palate; H&E, 1.0×. D, Higher magnification of Figure 1C. The papilloma is composed of thick papillae of well-differentiated, mature, keratinized squamous epithelium (arrows); H&E, 4.0×.

Figure 2.—

Squamous cell neoplasms of the tongue in a rat given sodium dichromate dihydrate (SDD) for 2 years. A, Squamous cell papilloma arising from the surface of the tongue occurs as a broad-based, pedunculated mass composed of fronds of well-differentiated, keratinized squamous epithelium (arrows); H&E,1.0×. B, Squamous cell carcinoma projecting from the surface of the tongue (arrows) composed of thick papillae of well-differentiated, mature, keratinized squamous epithelium supported by connective tissue stroma; H&E, 1.0×. C, Higher magnification of Figure 2B showing dysplastic squamous epithelium (arrows) extending from the basal layer into the adjacent dense connective tissue stroma of the stalk; H&E, 15.0×.

Nonneoplastic lesions were not observed in the oral mucosa of rats or mice exposed to SDD for 2 years. Based on the neoplastic findings in the 2-year study, the NTP conducted a retrospective analysis of the oral cavity and tongue in the 3-month rat study for which doses up to 1,000 mg/L were used. No treatment-related lesions were observed. In addition, no histopathological lesions were observed in the oral cavity of rats or mice in 3-month drinking water studies of SDD conducted more recently using a similar design to the NTP studies (Thompson et al. 2011, 2012).

Histiocytic Infiltration in Rats and Mice

In the 3-month and 2-year studies of SDD, increased incidences of histiocytic cellular infiltration were observed in several tissues including the liver, duodenum, and mesenteric and pancreatic lymph nodes of rats and mice (NTP 2007, 2008; Stout, Herbert, et al. 2009); incidences for the 2-year studies are presented in Table 3. The severities of these lesions ranged from minimal to moderate. The infiltrating histiocytes were swollen with abundant, lightly eosinophilic cytoplasms and were morphologically similar in rats and mice. Histiocytic infiltrates were characterized by the presence of individual, small clusters, and sometimes syncytia of large histiocytes (macrophages) within the sinusoids of the liver and lymph nodes and the lamina propria at the tips of the duodenal and jejunal villi (Figure 3A–F). In the lymph nodes, the histiocytic infiltrates often occurred as expansive sheets that in some cases replaced much of the lymph node parenchyma (Figure 3E–F). In control rats and mice, very low numbers of histiocytes with similar morphology are not uncommon in the liver and lymph nodes. In general, such infiltrates occur individually or as small clusters and are randomly distributed and considered background changes. In these studies, no threshold was used when diagnosing the presence of histiocytic infiltrates in the tissues. However, in contrast to the controls, the histiocytic infiltrates in exposed rats and mice in general occurred with greater frequency and in clusters of greater numbers. The biological significance and cause of the histiocytic infiltrates is not clear but may be related to phagocytosis of the test material in the intestine and subsequent translocation to the liver and regional lymph nodes. This lesion was not observed in the 3-month or 2-year studies of CPM.

Table 3.—

Tissues with histiocytic cellular infiltration in F344/N rats and B6C3F1 mice.

Exposure concentration	Male rats	0 mg/L	14.3 mg/L	57.3 mg/L	172 mg/L	516 mg/L
	Female rats	0 mg/L	14.3 mg/L	57.3 mg/L	172 mg/L	516 mg/L
	Male mice	0 mg/L	14.3 mg/L	28.6 mg/L	85.7 mg/L	257.4 mg/L
	Female mice	0 mg/L	14.3 mg/L	57.3 mg/L	172 mg/L	516 mg/L
Liver	Male rats (N = 49–50)	1	0	2	5	34**
	Female rats (N = 50)	1	5	21**	42**	47**
	Male mice (N = 50)	1	3	4	4	3
	Female mice (N = 49–50)	2	15**	23**	32**	45**
Duodenum	Male rats (N = 46–48)	0	0	6*	36**	47**
	Female rats (N = 46–50)	0	0	1	30**	47**
	Male mice (N = 50)	0	2	4	37**	35**
	Female mice (N = 50)	0	0	4	33**	40**
Mesenteric lymph node	Male rats (N = 49–50)	13	11	30**	39**	41**
	Female rats (N = 50)	21	18	27	36**	42**
	Male mice (N = 46–49)	14	38**	31**	32**	42**
	Female mice (N = 46–50)	3	29**	26**	40**	42**
Pancreatic lymph node	Male rats (N = 32–36)	17	22	17	17	25
	Female rats (N = 29–36)	17	20	23	32**	27
	Male mice (N = 5–16)	0	2	2	5*	12**
	Female mice (N = 12–15)	0	1	2	7**	8**

Note: Significant by the poly-3 test at *p ≤ .05 or **p ≤ .01.

Figure 3.—

Histiocytic infiltrates in the liver, duodenum, and mesenteric lymph node of rats given sodium dichromate dihydrate (SDD) for 2 years. A and B, Infiltrates (arrows) are multifocal and randomly distributed in the parenchyma and in the portal areas of the liver; H&E, 10.0× and 20.0×. C and D, Infiltrates (arrows) occur in the lamina propria at the tips of the duodenal villi. E and F, In the mesenteric lymph node, histiocytic infiltrates (arrows) are multifocal and have replaced large segments of the lymphoid tissue; H&E, 4.0× and 13.0×. The histiocytes are swollen with abundant lightly eosinophilic, faintly stippled cytoplasm.

Small Intestine Neoplasms and Hyperplasia in Mice

In contrast to rats, neoplasms in epithelial tissues of the lower alimentary system (small intestine) were observed in mice in the 2-year SDD study. In both males and females, there was a clear exposure concentration–response relationship when adenomas and carcinomas were combined at all sites of the small intestine (duodenum, jejunum, or ileum; Table 4). These increases were significant at the two highest exposure concentrations in each sex. In addition, the incidence in 57.3 mg/L females exceeded the historical control ranges for drinking water studies and for all routes of administration (Table 4); this increased incidence was also considered to be related to treatment. These increases were driven primarily by significant increases in the incidences of adenoma of the duodenum; the number of mice with multiple adenomas was also significantly increased at the high dose in both sexes (data not shown). In females, the incidence of carcinoma in the duodenum was significantly increased at 516 mg/L. In the jejunum, the incidence of adenoma was increased in 516 mg/L females.

Table 4.—

Epithelial neoplastic lesions in the small intestine of male and female B6C3F1 mice in the 2-year study of sodium dichromate dihydrate (SDD).

	Male					Female
Lesion	0 mg/L	14.3 mg/L	28.6 mg/L	85.7 mg/L	257.4 mg/L	0 mg/L	14.3 mg/L	57.3 mg/L	172 mg/L	516 mg/L
Duodenum
Adenoma	1***	0	1	5	15***	0***	0	2+	13***	12***
Carcinoma	0*	0	0	2++	3+	0	0	0	1++	6*
Jejunum
Adenoma	0**	0	0	0	3+	0**	1+	0	2+	5*
Carcinoma	0	2	0	1	2	1	0	2+	2+	1
Duodenum, jejunum, or ileum (combined)
Adenoma	1***	1	1	5+	17***	0***	1	2+	15***	16***
Carcinoma	0*	2	1	3++	5*	1***	0	2+	3+	7*
Adenoma or carcinoma (combined)	1***	3	2	7*	20***	1***	1	4+	17***	22***

Note: N = 50; Significant by the poly-3 test at *p ≤ .05, **p ≤ .01, ***p ≤ .001 (significant for trend if noted for controls); exceeded the historical control range for +all routes and drinking water or ++drinking water only.

Adenomas occurred as discrete, focally extensive, plaque-like areas of proliferating glandular epithelium that thickened and effaced the mucosa and protruded into the lumen (Figure 4A and B). Carcinomas were sessile, plaque-like neoplasms distinguished from adenomas by extensive invasion and effacement of the mucosa, underlying submucosa and muscle layers (Figure 4C). They were composed of atypical, pleomorphic and hyperchromatic cells that formed glandular structures and irregular clusters and cords variably interspersed with desmoplastic tissue (Figure 4D).

Figure 4.—

Epithelial neoplasms in the duodenum of mice given sodium dichromate dihydrate (SDD) for 2 years. A, An adenoma appears as a focally extensive, plaque-like lesion that has effaced a focally extensive segment of the mucosa and protruded into the lumen (arrows); H&E, 2.0×. Normal mucosa is seen at either side of and opposite the adenoma. B, Higher magnification of Figure 4A showing a portion of the adenoma. In contrast to the normal villi in the upper left, within the adenoma, plump, tall columnar, hyperchromatic epithelial cells line and thicken the villi and form irregular glandular structures; H&E, 12.0×. C, A carcinoma has completely invaded and effaced the duodenum; H&E, 3.5×. D, Higher magnification of Figure 4C. Note dysplastic pattern of growth with the large segments in which the neoplastic epithelium forms irregular glandular structures separated by desmoplastic tissue; H&E, 9.0×.

Within the gastrointestinal tract, epithelial neoplasms of the intestines are relatively uncommon. Of 290 carcinogenic agents identified by the NTP, approximately 19 (~7%) caused intestinal neoplasia, 14 in the rat and 5 in the mouse (Chandra, Nolan, and Malarkey 2010). Of these 19 agents, none were enteric carcinogens in both the rat and mouse. Neoplasms occurred in both males and females. In the mouse, neoplasms occurred almost exclusively in the small intestine (4 of 5), whereas, in the rat, neoplasms occurred in the large or both small and large intestine.

Low incidences of focal epithelial hyperplasia occurred in the duodenum of exposed male and female mice in the 2-year study (Table 5). Although the increased incidences were not statistically significant, this lesion was considered a preneoplastic lesion in the continuum of benign to malignant neoplasia of the intestines because of its morphologic similarities to adenoma; it was, therefore, considered to be related to SDD exposure. Focal epithelial hyperplasia occurred as small areas of hypercellularity and increased basophilia in the superficial epithelium of the duodenum (Figure 5A and B). They were distinguished from adenomas by small size, common location in the superficial mucosa, and less discrete margins that tended to blend with the normal surrounding mucosal epithelium.

Table 5.—

Hyperplasia of the small intestine of male and female B6C3F1 mice in the 2-year study of sodium dichromate dihydrate (SDD).

Males	0 mg/L	14.3 mg/L	28.6 mg/L	85.7 mg/L	257.4 mg/L
Duodenum
Focal epithelial hyperplasia	0	0	0	1	2
Diffuse epithelial hyperplasia	0	11**	18**	42**	32**
Females	0 mg/L	14.3 mg/L	57.3 mg/L	172 mg/L	516 mg/L
Duodenum
Focal epithelial hyperplasia	0	0	1	2	0
Diffuse epithelial hyperplasia	0	16**	35**	31**	42**
Jejunum
Diffuse epithelial hyperplasia	0	2	1	0	8**

Note: N = 50; significant by the poly-3 test at *p ≤ .05, **p ≤ .01.

Figure 5.—

Focal hyperplasia in the duodenum of a mouse given sodium dichromate dihydrate (SDD) for 2 years. A, Note focal area of hyperplastic epithelium in the superficial mucosa (arrows); H&E, 3.0×. B, Higher magnification of Figure 5A; H&E, 15.0×.

The incidences of diffuse epithelial hyperplasia were significantly increased in the duodenum of all exposed groups of male and female mice (Table 5). In the jejunum, the incidence of diffuse epithelial hyperplasia was significantly increased in 516 mg/L females. Diffuse epithelial hyperplasia generally involved the entire mucosa. Compared to the mucosa of the controls (Figure 6A and C), the mucosa of the exposed mice appeared hypercellular and more basophilic (Figure 6B and D). The duodenal villi of exposed mice were short, broad, and blunt and there were increased numbers and disorganization of the mucosal epithelial cells that was particularly prominent in the epithelium lining the villi. Epithelial hyperplasia was also observed in male and female mice at all exposure concentrations in the 3-month studies (NTP 2007).

Figure 6.—

Diffuse hyperplasia in a mouse given sodium dichromate dihydrate (SDD) for 2 years. A, Low magnification of a section of duodenum from a control mouse showing long slender villi (arrows); H&E, 2.5×. B, In contrast to the normal duodenum, the duodenal mucosa in the exposed mouse is hypercellular, hyperbasophilic, and villi are short and blunt; H&E, 13.0×. C, Higher magnification of Figure 6A. Duodenal villi are tall and slender and lined by a single layer of tall columnar epithelial cells; H&E, 13.5×. D, Higher magnification of Figure 6B. Note increased mucosal cellularity and disorganization of the mucosal epithelial cells; H&E, 13.5×.

Tissue Distribution Studies

As part of the NTP 2-year studies of SDD and CPM, total Cr concentrations were measured in excreta and selected tissues of additional groups of male rats and female mice at selected time points (NTP 2008, 2010; Collins et al. 2010). These animals were treated the same as the animals in the core study groups with respect to exposure, housing, and handling. The measurement of total Cr in both 2-year studies allowed for tissue Cr concentrations to be compared in animals exposed to Cr(III) or Cr(VI). 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 Cr. There were increases in total Cr relative to controls in multiple tissues in both male rats and female mice after exposure to Cr(VI) and Cr(III) (Figure 7), indicating that there was systemic exposure to Cr following exposure to either form of Cr. In general, tissue concentrations at each time point increased with increasing exposure concentration. In all tissues examined, similar external doses of Cr resulted in much higher tissue Cr concentrations following exposure to Cr(VI) compared with Cr(III), indicating that at least a portion of the Cr(VI) was distributed to tissues prior to reduction. When tissue concentrations are normalized to ingested Cr dose, Cr concentrations were highest in the kidney of rats and in the liver and glandular stomach of mice (Collins et al. 2010).

Figure 7.—

Comparison of total chromium (Cr; μg Cr/g tissue) in red blood cells (RBCs), liver, kidney, and glandular stomach (GS) of male rats (left) and female mice (right) after 182 days of ingestion of similar time-weighted average daily doses of Cr(III) (resulting from exposure in feed to 2,000 ppm chromium picolinate monohydrate [CPM]) and Cr(VI) (resulting from exposure in drinking water to 516 mg sodium dichromate dihydrate (SDD)/L). In rats, ingested doses were 15.18 mg Cr(III)/kg body weight (CPM) or 8.95 mg Cr(VI)/kg body weight (SDD). In mice, ingested doses were 36.73 mg Cr(III)/kg body weight (CPM) or 13.2 mg Cr(VI)/kg body weight (SDD).

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 sublinear dose–response, with doses above this point demonstrating a greater rate of response than lower doses. Statistical analysis of the shapes of the exposure–concentration curves revealed either linear or supra-linear dose responses, indicating that these exposure concentrations did not saturate gastric reduction (Collins et al. 2010). A similar analysis of the data from the NTP 21-day comparative absorption study of SDD in rats, mice, and guinea pigs (NTP 2007), which utilized a wider range of exposure concentrations including doses as low as 2.87 mg/L SDD (1 mg Cr(VI)/L), yielded similar findings (data not shown).

In the NTP 2-year studies, the oral cavity and small intestine were not collected for total Cr analysis. However, Thompson et al. (2011, 2012) reported significant increases in total Cr concentrations in the oral cavity, glandular stomach, duodenum, jejunum, and ileum of rats and mice following 90 days of exposure to SDD in the drinking water. Tissue concentrations were highest in the duodenum of rats and mice. In the NTP 2-year studies, higher tissue concentrations were achieved in male rats than those that occurred in the tissues of female mice exposed to an external dose of SDD (57.3 mg/L) that also resulted in small intestine neoplasms. Thompson et al. (2012) demonstrated higher Cr concentrations in the duodenum relative to the oral cavity of rats after 90 days of exposure. These data indicate that tissue uptake does not explain the species differences in target sites of carcinogenicity.

Evidence for the Genotoxicity of Cr Compounds

Cr(VI) is genotoxic in a wide variety of in vitro and in vivo test systems, although responses may vary depending on protocol details and the type of Cr salt that is assayed. Overall, the data clearly indicate that in appropriate test systems and protocols, exposure to Cr(VI) results in increased frequencies of gene mutations, chromosomal damage, and DNA damage. The extensive literature on the mutagenicity of Cr compounds has been reviewed previously by a number of authors (e.g., IARC 1990; De Flora et al.1990; McCarroll et al. 2010; Zhitkovich 2011). To briefly summarize the findings, positive results were obtained in vitro with Cr(VI) compounds in gene mutation tests using a number of different Salmonella typhimurium and Escherichia coli tester strains that revert by frame shifting and base substitution; forward mutation and mitotic gene conversion assays in yeast; in vitro mammalian cell (including human cell lines) chromosomal damage assays that measured increases in sister chromatid exchanges, chromosomal aberrations, or micronuclei; assays measuring mutation induction at the tk locus in L5178Y mouse lymphoma cells; and tests for induction of DNA strand breaks and adduct formation or DNA synthesis inhibition in a variety of mammalian cell test systems. In vivo, positive results were reported in short-term assays measuring induction of chromosomal damage and micronuclei in laboratory rodents, and mutagenicity in transgenic animals. Results of comparative micronucleus assays in various strains of mice (ms, BDF1, CD-11, ddY) indicated differential strain and route susceptibilities to the chromosome-damaging effects of Cr(VI) (CSGMT 1986, 1988; Shindo et al. 1989), and these may underlie some of the variation observed in in vivo studies.

Cr(III) has been shown to be genotoxic in acellular test systems that allow direct contact with DNA, and it is this valence state that is hypothesized to be the active genotoxicant following exposure to Cr(VI) (Snow 1991; Bridgewater et al. 1994; Snow 1994). However, Cr(III) compounds give negative or conflicting results in standard genetic toxicity assays, likely a consequence of the low level of absorption of most Cr(III) salts, preventing access to DNA in cell-based systems. A comprehensive review of the genotoxicity literature for Cr(III) compounds was provided in NTP Technical Report 556 (NTP 2010) and the available information is summarized briefly here. Cr(III) compounds are not active in bacterial mutagenicity assays (Zeiger et al. 1992; Whittaker et al. 2005), but mixed results have been seen with Cr(III) compounds in a variety of mammalian cell mutagenicity assays (Stearns et al. 2002; Slesinski et al. 2005; Whittaker et al. 2005; Coryell and Stearns 2006). Both positive and negative results have also been reported for Cr(III) compounds in chromosomal aberration and DNA damage assays in cultured mammalian cells (Stearns et al. 1995; Seoane and Dulout 2001; Gudi et al. 2005; Andersson et al. 2007). In vivo, salivary gland chromosomal aberrations were reported to be increased in Drosophila melanogaster larvae whose male parents were reared on medium containing 260 mg/L CP (Stallings et al. 2006). In contrast to the observations in Drosophila, no significant increases in micronucleus frequencies in peripheral blood erythrocytes or DNA damage in lymphocytes or hepatocytes, measured by the comet assay, were seen in male CBA/Ca mice administered a single intraperitoneal injection of 3 mg/kg CP (Andersson et al. 2007). There is one report of DNA deletions identified in mouse pups transplacentally exposed to chromium(III) chloride (1,875 or 3,750 mg/L, calculated to yield an average daily dose of 375 or 750 mg/kg) in drinking water during gestational days 10 to 20 (Kirpnick-Sobol, Reliene, and Schiestl 2006). Additional studies conducted by these authors in yeast also revealed increases in DNA deletions following exposure to Cr(III).

Results of NTP Genetic Toxicology Studies

In studies conducted by the NTP, Cr(VI), in the form of SDD (concentration range 5–300 μg/plate), was shown to be mutagenic in S. typhimurium strains TA100 and TA98 and in E. coli strain WP2 uvrA pKM101 with and without induced rat liver S9 enzymes (NTP 2007). Mutagenic responses were stronger in the strains that mutate via base substitution (TA100, E. coli WP2), even though their genetic target sequences are different. TA100 has a G-G-G-G- target and the E. coli strain has an –A-T- target sequence. In mammalian cells, the target sequence is believed to be almost exclusively –G-C-G-C-G- (Salnikow and Zhitkovich 2008).

The results of four peripheral blood erythrocyte micronucleus tests from 2 independent toxicity studies conducted by the NTP in 3 strains of mice were mixed (NTP 2007). In the first study, no significant increases in the frequencies of micronucleated erythrocytes were measured in male or female B6C3F1 mice administered SDD in drinking water (concentration range of 62.5–1,000 mg/L) for 3 months. In the second study conducted by NTP, micronucleus frequencies were evaluated in B6C3F1, BALB/c, and transgenic am3(C57BL/6) male mice administered SDD in drinking water over an exposure concentration range of 62.5 to 250 mg/L for 3 months. An increase in micronucleated erythrocytes, judged to be equivocal, was noted in male B6C3F1 mice, and no increases in micronucleated erythrocytes were observed in male BALB/c mice. In contrast, a significant exposure concentration-related increase (p < .001) in micronucleated erythrocytes was seen in male am3(C57BL/6) mice. The differences in responses among these strains of mice are consistent with observations from the earlier comparative micronucleus assays that revealed differential strain and route susceptibilities to the chromosome-damaging effects of Cr(VI) in various strains of mice, perhaps related to differential extracellular reduction capability, DNA repair capability, or concentration of intracellular reductants (CSGMT 1986, 1988; Shindo et al. 1989).

The NTP also examined the genotoxic potential of Cr(III) in the form of CP and CPM; neither form of Cr(III) showed evidence of genotoxicity in standard assays (NTP 2010). Over a concentration range of 100 to 10,000 μg/plate, no evidence of mutagenicity was observed in S. typhimurium strains TA100 and TA98 or E. coli strain WP2 uvrA pKM101 following exposure to CPM, with or without exogenous metabolic activation. In addition, no increase in the frequency of micronucleated erythrocytes was observed in male B6C3F1 mice administered CPM (80–50,000 ppm) in feed for 3 months (Stout, Nyska, et al. 2009). In female mice, however, a small increase in micronucleated erythrocytes was judged to be equivocal. Similar results were obtained in an earlier investigation conducted by NTP, in which no increases in micronucleated erythrocytes were seen in male B6C3F1 mice administered chromium(III) carbonyl (0.51–255 μg) once daily by intraperitoneal injection for 4 weeks (Witt et al. 2000). Additional genotoxicity testing conducted with CP (not the monohydrate form of the compound) revealed no induction of gene mutations in two independent studies in several strains of S. typhimurium, with and without hamster or rat liver S9, and no induction of micronucleated erythrocytes in bone marrow of male F344 rats administered 3 daily doses of CP (156–2,500 mg/kg) by oral gavage.

Evidence of Genotoxicity in Rodents Following Oral Exposure

Other in vivo chromosomal damage assays of Cr(VI) in animals using drinking water as the route of administration have given mixed results, consistent with what was observed in the NTP studies in mice. In F344 rats exposed to potassium chromate in drinking water (100 or 200 ppm for 3 weeks), increases in DNA-protein cross-links, an end point of Cr-induced genotoxicity, were detected in liver but not splenic lymphocytes (Coogan et al. 1991). However, DNA-protein cross-links were detected in isolated splenic lymphocytes exposed for 2 hr in vitro to 100 μM potassium chromate. Based on the levels of Cr required to induce measurable DNA damage in splenic lymphocytes in vitro, the authors suggested that the amount of Cr(VI) absorbed in vivo following oral exposure was insufficient to induce DNA-protein cross-links in splenic lymphocytes. De Flora, Iltcheva, and Balansky (2006) reported no increases in micronucleated erythrocytes in peripheral blood of BDF1 or Swiss mice exposed to SDD or potassium dichromate in drinking water (up to 500 mg Cr(VI)/L for 210 days). In contrast, Cr(VI) administered by intraperitoneal injection (50 mg/kg potassium dichromate or SDD) did produce significant increases in micronucleated erythrocytes (De Flora et al. 2006). In studies designed to measure genotoxicity of Cr following transplacental exposure, Kirpnick-Sobol, Reliene, and Schiestl (2006) reported that treatment of pregnant mice (C57BL/6J p^un/p^un) with potassium dichromate (Cr(VI); 62.5 or 125.0 mg/L) or chromium(III) chloride (1,875 or 3,750 mg/L) via drinking water during gestational days 10 to 20 resulted in significant increases in the frequencies of DNA deletions in retinal pigment epithelium of their pups examined at 20 days of age; these deletions were expressed as black spots in the retinal epithelial cells following somatic deletion of the p^un locus duplication resulting in reversion to wild-type phenotype. Furthermore, Kirpnick-Sobol, Reliene, and Schiestl (2006) reported that in mouse fetuses transplacentally exposed to Cr(III) and examined on gestational day 17.5, significant increases in DNA deletions were seen at threefold lower tissue concentrations of Cr than in fetuses transplacentally exposed to Cr(VI). These authors conducted additional studies in yeast, comparing intracellular Cr concentrations with DNA deletion frequencies, and they demonstrated that Cr(III) was a more potent inducer of DNA deletions than was Cr(VI). The authors concluded that the data from both the mouse and the yeast studies indicated that, although only small amounts of Cr(III) were absorbed, Cr(III) was highly effective at inducing DNA damage.

Although a number of studies have been conducted in animals using drinking water or dietary routes of administration similar to the protocols used by the NTP, studies in rodents evaluating the genotoxicity of Cr(VI) in the gastrointestinal (GI) tract after oral exposure are limited. De Flora et al. (2008) reported no evidence of oxidative damage or DNA-protein cross-links, both of which are associated with genotoxicity, in forestomach, glandular stomach, or duodenum of female SKH-1 mice administered 5 or 20 mg Cr(VI)/L in drinking water for 9 months.

Evidence of Genotoxicity in Humans Following Inhalation Exposure

Studies of Cr-induced genetic damage in humans have focused on inhalation exposure exclusively; no studies were located in the literature regarding genotoxic effects in humans after oral exposure to Cr(VI). Most studies measuring genetic damage end points in humans have involved occupational exposure to atmospheric Cr(VI), and results were consistent with Cr-induced chromosomal damage (IARC 1990). In several studies, elevated frequencies of sister chromatid exchanges, chromosomal aberrations, and/or micronuclei were observed in lymphocytes or buccal cells of Cr(VI)-exposed workers (IARC 1990; Vaglenov et al. 1999; Benova et al. 2002; Danadevi et al. 2004). In addition, DNA damage, measured by the comet assay (single-cell gel electrophoresis), was reported to be increased in lymphocytes of workers exposed to Cr and nickel in a stainless steel welding facility (Danadevi et al. 2004). Furthermore, direct correlations have been reported between levels of workplace Cr or the duration of Cr exposure, and the amount of genetic damage detected (IARC 1990).

Mechanisms of Genotoxicity

Despite the abundance of evidence for genotoxicity, particularly in in vitro tests, the mechanisms whereby Cr(VI) induces genetic damage are still a matter of some debate. It seems clear, however, that intracellular reduction of Cr(VI) to Cr(III), a process that generates intermediate Cr valences, as well as Cr(III) access to DNA are critical factors in the amount and type of damage induced. It is likely that multiple mechanisms are involved in Cr-induced genotoxicity, depending on exposure conditions, identity and concentration of reductants, and intracellular concentrations of Cr(III).

Cr(VI) absorption is facilitated by active sulfhydryl transporters, in contrast to Cr(III), which is not a substrate of the transmembrane transport system and, thus, cannot easily pass through the cell membrane (IARC 1990). Because it has been shown to be relatively nonreactive toward DNA itself (i.e., in cell free systems), Cr(VI) has been postulated to exert its genotoxic effects, at least in part, through the generation of oxygen radicals during metabolic transformation from the hexavalent form through the more reactive Cr(V) and Cr(IV) valences to the final product, Cr(III) (Sugden, Burris, and Rogers 1990; Sugiyama 1992; Kasprzak 1995; Shi et al. 1999; Vaglenov et al. 1999; Benova et al. 2002; O’Brien, Ceryak, and Patierno 2003; Jomova and Valko 2011). A variety of reactive carbon-based radical species are also formed. These, along with the final product of reduction, Cr(III), have been postulated to produce DNA single-strand breaks, DNA adducts, protein-DNA and Cr-reductant-DNA cross-links, and DNA interstrand cross-links, among other types of damage (see reviews by O’Brien, Ceryak, and Patierno 2003; McCarroll et al. 2010; Jomova and Valko 2011). Although, both enzymatic and nonenzymatic pathways may be involved in this process, nonenzymatic reduction is believed to dominate at normal physiological conditions (De Flora et al. 1990; Standeven and Wetterhahn, 1991). The three principal reductants involved in transformation of Cr(VI) are ascorbic acid, glutathione, and cysteine; additional compounds that may serve as reductants include lipoic acid, hydrogen peroxide, NAD(P)H, fructose, ribose, riboflavin, and glutathione reductase (Jomova and Valko 2011). The relative concentrations of Cr species and available reductants determine the rate and pathways involved in the reduction process, and, hence, the type and extent of DNA damage that may be produced. Ascorbate is the principal reductant of Cr(VI) in vivo; but in cell cultures, reduction of Cr(VI) is mainly facilitated by glutathione, which has been shown to produce a much higher concentration of oxidants than ascorbate (Wong, Armknecht, and Zhitkovich 2012), and this difference in reduction processes may underlie the different types and amounts of DNA damage seen with Cr(VI) in vivo compared with in vitro exposure situations.

In studies with laboratory rodents, administration of radical scavengers simultaneously with or prior to administration of Cr(VI) salts reduced clastogenic potency, consistent with the oxygen radical mechanism of action (Chorvatovicova et al. 1991, 1993; Sarkar, Sharma, and Talukder 1993). Results of in vitro mammalian cell studies showing reduction of Cr(VI)-induced DNA damage in the presence of a variety of oxygen radical scavengers, reducing agents, and metal chelators provide additional support for this mechanism (Pattison et al. 2001; Cemeli et al. 2003; O’Brien, Ceryak, and Patierno 2003). In mouse epidermal JB6 Cl41 cells exposed in vitro to Cr(VI), dose-dependent increases in intracellular levels of reactive oxygen species such as hydrogen peroxide and super-oxide anion radicals were detected using electron spin resonance (Son et al. 2010).

Recently, studies in female F344/N rats and B6C3F1 mice dosed with Cr(VI) in the drinking water (0.3–520 mg SDD/L) for 90 days revealed no significant increases in 8-hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, in the oral mucosa or duodenum of either species (Thompson et al. 2011, 2012). Significant decreases in the ratio of reduced/oxidized glutathione were reported in the oral mucosa and jejunum of the rats and the duodenum and jejunum of the mice, suggesting upregulation of a response to Cr(VI)-induced oxidative stress in these cells. As part of these studies, whole genome microarrays were used to identify gene expression profiles in duodenum and jejunum of female B6C3F1 mice (Kopec, Kim, et al. 2012) and F344/N rats (Kopec, Thompson, et al. 2012). In female mice, 16 genes with dose-dependent differential expression in duodenal epithelial samples were identified. Functions identified for these genes included activities associated with oxidative stress, cell cycle regulation, or lipid metabolism. Of the 16 genes, 3 were determined to be regulated by Nrf2, which is active in cellular pathways upregulated in response to oxidative stress. Comparative analysis with data from the female F344/N rats showed differences between the two species both in the number and functionality of upregulated genes (Kopec, Thompson, et al. 2012).

Cr(III), the final product of intracellular reduction of Cr(VI) and the most DNA-reactive valence state for Cr, accumulates within the cell as a result of its inability to pass through the cell membrane (Nickens, Patierno, and Ceryak 2010). Cr(III) has been shown to interact directly with DNA and other macromolecules to induce chromosomal alterations and mutational changes in DNA as a consequence of DNA adduct formation (Zhitkovich et al. 2001, 2002; Quievryn et al. 2003; Reynolds et al. 2007). Cr(III) forms both binary and ternary DNA adducts (O’Brien, Ceryak, and Patierno 2003; Zhitkovich 2005), with ternary adducts (Cr-reductant-DNA) being the most predominant and biologically relevant. Ascorbate-DNA adducts, which are substrates for nucleotide excision repair, are the most mutagenic (Zhitkovich 2011). In addition to DNA adducts, Cr(III) also forms ternary DNA cross-links with glutathione, ascorbate, cysteine, and histidine (Zhitkovich 2011).

The availability of intracellular ascorbate for Cr(VI) reduction may be key to the amount of Cr-induced DNA damage observed. Reynolds et al. (2007) have proposed that high intracellular levels of ascorbate are associated with marked increases in mutagenic DNA lesions in the presence of Cr(VI), possibly resulting from DNA mismatch repair as well as nonoxidative mechanisms of genotoxicity such as direct Cr(III)-DNA interaction (Wong, Armknecht, and Zhitkovich 2012). DNA adducts, DNA-protein cross-links, and DNA-reductive agent cross-links have all been identified as products of Cr(III)-DNA interactions. Detailed discussions of the types of Cr-DNA adducts that are formed in vivo and in vitro, along with the biological consequences of these adducts, including mutagenicity, DNA replication inhibition, and cytotoxicity, have been provided (O’Brien, Ceryak, and Patierno 2003; Zhitkovich 2005; McCarroll et al. 2010).

In summary, there are many potential mechanisms involved in the genotoxicity of Cr(VI). The hexavalent form of Cr does not itself react with DNA, but intracellular reduction of Cr(VI) produces reactive intermediate Cr species and the final DNA-reactive species, Cr(III). Reduction also produces intracellular reactive oxygen species and other reactive molecules, all of which have the potential to damage DNA and induce mutagenic events. There is competition among extracellular reduction of Cr(VI) and absorption of Cr(VI) into cells, and the balance between these conflicting processes and the availability of intracellular and extracellular reducing agents determines the extent of Cr(VI)-associated genotoxicity. The relative contributions of the multiple, complex pathways of Cr-induced genotoxicity are not yet fully understood and they continue to be the subject of intense investigation.

Major Conclusions and Significance of the NTP Studies of Chromium

Taken together, these NTP studies demonstrate that SDD was clearly carcinogenic in male and female rats and mice following exposure in the drinking water. SDD induced toxicity to the erythron and nonneoplastic lesions in multiple tissues in rats and mice. The toxicity and carcinogenicity observed with SDD was not observed with CPM. SDD was mutagenic in S. typhimurium and in E. coli and produced mixed results in micronucleus tests conducted in three mouse strains. CPM was not genotoxic in standard assays. Increased tissue concentrations of total Cr in both male rats and female mice indicated that systemic exposure to Cr occurred following exposure to both Cr(VI) and Cr(III). Higher total Cr concentrations following SDD exposure relative to CPM exposure suggests that at least a portion of the ingested Cr(VI) escaped gastric reduction and entered systemic circulation; the exposure concentrations used in the 2-year study of SDD did not appear to overwhelm the gastric reduction capacity. Differences in the oral toxicities of Cr(VI) and Cr(III) in animal studies provide additional evidence that Cr(VI) is not completely converted into Cr(III) in the stomach.

The NTP data were used as the basis to develop the California Public Health Goal for Cr(VI) in drinking water of 0.02 ppb that was established in 2011. Public Health Goals are nonmandatory goals developed for use by the CDPH in establishing primary drinking water standards (state MCLs) (CA EPA 2011). The NTP data were also used in a risk assessment conducted by the New Jersey Department of Environmental Protection in 2009 to derive a human cancer potency estimate for Cr(VI) by ingestion and an associated Soil Cleanup Standard of 1 ppm (NJ DEP 2009). Currently, there is no national US EPA cleanup criterion or standard for Cr(VI) in soil.

Abbreviations:

CA

California

CP

chromium picolinate

CPM

chromium picolinate monohydrate

Cr

chromium

Cr(III)

trivalent chromium

Cr(IV)

tetravalent chromium

Cr(V)

pentavalent chromium

Cr(VI)

hexavalent chromium

MCL

Maximum Contaminant Level

NTP

National Toxicology Program

SDD

sodium dichromate dihydrate

UCMR

Unregulated Contaminant Monitoring Rule

US EPA

United States Environmental Protection Agency

8-OhdG

8-hydroxy-2′-deoxyguanosine