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 (<1% after 12 h) (Olin et al., 1994). Cr(III)
is not classifiable as to its human carcinogenicity (IARC, 1990).
The NTP conducted 2-year toxicity and carcinogenicity
studies of Cr(VI), as SDD administered in drinking water
(NTP, 2008a; Stout et al., 2009a), and Cr(III), as CPM
administered in feed (NTP, 2008b; Stout et al., 2009b). Cr(VI)
was carcinogenic in male and female rats and mice, inducing
squamous neoplasms of the oral cavity in rats and epithelial
neoplasms of the small intestine in mice. In addition,
microcytic hypochromic anemia in rats, erythrocyte microcytosis in mice, and increased incidences of histiocytic cellular
infiltration in various tissues of rats and mice were observed. In
contrast, at much higher doses, Cr(III) was not carcinogenic
in female rats or in mice. Increased preputial gland adenomas
in male rats may have been related to exposure. Cr(III) exposure
did not have any significant effect on survival, body weight, feed
consumption, or non-neoplastic lesions. This disparity in toxicity
and carcinogenicity is consistent with previous studies (Agency
for Toxic Substances and Disease Registry, 2000).
EXPOSURE TO HEXAVALENT CHROMIUM
Although the absorption of Cr(VI) and Cr(III) is low
following oral exposure, Cr(VI) is absorbed more efficiently
than Cr(III) (Donaldson and Barreras, 1966; Fébel et al., 2001;
Kerger et al., 1996; Mackenzie et al., 1959). This is thought to
occur because Cr(VI) as chromate (CrO2
4 ) structurally
resembles sulfate and phosphate and is taken up by all cells
and organs throughout the body through sulfate transporters
(Costa, 1997), whereas Cr(III) is not a substrate for transport
(Proctor et al., 2002) but is thought to enter cells via diffusion
or phagocytosis. Consistent with this mechanism of transport,
higher chromium tissue concentrations were observed with
Cr(VI) compared with Cr(III) following equivalent exposures
in drinking water (Costa, 1997; Costa and Klein, 2006;
Mackenzie et al., 1958).
Both extracellular and intracellular reduction of Cr(VI) to
Cr(III) occur. 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, 2000;
De Flora et al., 1997, 1989; Paustenbach et al., 2003; Proctor
et al., 2002). In contrast, intracellular reduction is thought to be
a mechanism of carcinogenesis because of DNA damage that
occurs when Cr(VI) is reduced through Cr(V) and Cr(IV) to
Cr(III) (reviewed in O#Brien et al., 2003). Because of its large
redox potential, Cr(III) is not expected to oxidize to Cr(VI)
in vivo. In summary, absorption and retention of chromium
depend on a number of factors: the rate of reduction of Cr(VI)
to Cr(III) outside the cells, the pH of the milieu, the rate of
transport of Cr(VI) into the cells, the rate of reduction of Cr(VI)
to Cr(III) inside the cells, and the rate of diffusion of Cr(III)
from the cells (NTP, 2008a).
As part of the NTP chronic oral studies of Cr(VI) (NTP,
2008a; Stout et al., 2009a) and Cr(III) (NTP, 2008b; Stout
et al., 2009b), total Cr was measured in selected tissues and
excreta of additional groups of male rats and female mice at
selected time points. The objective of these studies was to
determine the effect of administered species [Cr(VI) or Cr(III)]
on Cr uptake and tissue distribution. Because Cr(VI) is reduced
to Cr(III) in vivo, and current analytical procedures do not
allow for the speciation of Cr extracted from biological
samples, speciation of absorbed Cr(VI) was inferred by
comparing tissue uptake of Cr following exposure to Cr(VI)
or Cr(III). These are the first studies that provide a comprehensive comparison of chronic toxicity and carcinogenicity with
tissue distribution in rats and mice following exposure to
Cr(VI) and Cr(III) and may ultimately aid in the extrapolation
of the Cr(VI) carcinogenicity data to humans.
MATERIALS AND METHODS
Chemical and Dose Formulations
SDD (CAS 7789-12-0) was obtained from Aldrich Chemical Company
(Milwaukee, WI). The purity was determined using differential scanning
369
calorimetry; titration of the dichromate ion with sodium thiosulfate and
potassium ferrocyanide, speciation of the Cr ions using liquid chromatographyinductively coupled plasma-mass spectrometry (ICP-MS), and potentiometric
titrimetric analysis with sodium thiosulfate. Based on these analyses, the overall
purity was 99.7%. Dose formulations were prepared approximately every
2 weeks by mixing SDD with tap water. Periodic analysis using ultraviolet/
visible/near infrared spectroscopy (350–390 nm) confirmed that all dose
formulations varied by less than 10% of the target concentrations. CPM (CAS
No. 27882-76-4) used in the 2-year studies was a combination of chemical
obtained from TCI America (Portland, OR) and from Sigma-Aldrich (St Louis,
MO). Purity was determined by elemental analyses, proton-induced x-ray
emission spectroscopy, inductively coupled plasma-atomic emission spectroscopy, high-performance liquid chromatography (HPLC) with ultraviolet-visible
(UV) or mass spectrometric detection, or ICP-MS. The overall purity of the
chemical was 95%. The dose formulations were prepared monthly by mixing
CPM with feed. Homogeneity and stability of the dose formulations were
assessed by HPLC with UV detection. Periodic analysis confirmed that all dose
formulations were within 10% of the target concentrations. Concentrations of
total chromium in vehicles were 0.604 ± 0.253 ppm in feed [Cr(III) acetate
was added because Cr(III) is thought to be an essential element] and below
0.005 mg/l in tap water.
Animals and Animal Maintenance
The studies were conducted at Southern Research Institute (Birmingham,
AL). Male F344/N rats and female B6C3F1 mice were obtained from Taconic
Farms (Germantown, NY). Rats and mice were quarantined for 12 days (CPM)
or 14 days (SDD) and were 5–6 (CPM) or 6–7 (SDD) weeks old at the
beginning of the studies. Animals were distributed randomly into groups of
similar mean body weights and identified by tail tattoo. Rats were housed three
to a cage. Mice were housed five to a cage. Feed and tap water were available
ad libitum. For the SDD study, feed was irradiated NTP-2000 wafers. For
the CPM study, feed was irradiated NTP-2000 open formula meal diet. Both
diets were obtained from Zeigler Brothers, Inc. (Gardners, PA). Animals were
killed by asphyxiation with CO2.
Animal use was in accordance with the United States Public Health Service
policy on humane care and use of laboratory animals and the Guide for the
Care and Use of Laboratory Animals. These studies were conducted in compliance with the Food and Drug Administration Good Laboratory Practice
Regulations (21CFR, Part 58).
Study Design
As part of the NTP 2-year bioassays of SDD (NTP, 2008a; Stout et al.,
2009a) and CPM (NTP, 2008b; Stout et al., 2009b), additional animals were
randomly assigned for measurement of total Cr in selected tissues and excreta;
these animals were treated the same as the core study animals used for
evaluation of toxicity and carcinogenicity with respect to exposure, housing,
and handling. SDD was administered in drinking water to groups of 40 male
rats and female mice at concentrations of 0, 14.3, 57.3, 172, and 516 mg/l.
CPM was administered in feed to groups of 30 male rats and female mice at
concentrations of 0, 2000, 10,000, and 50,000 ppm. On days 4, 11 and 180
(CPM and SDD), and on day 369 (SDD only), up to 10 rats and mice per
exposure group were removed from treatment and placed in individual
metabolism cages for separate collection of urine and feces. Animals were
provided undosed drinking water or feed ad libitum during this period to allow
unabsorbed chromium to be excreted. Two collections of urine and feces were
made to include the intervals from 0 to 24 and 24 to 48 h; measured values were
combined to yield the reported 48-h values. The 48-h washout period was based
on an elimination half-life of 8–21 h (Bragt and van Dura, 1983; Vanoirbeek
et al., 2003). On days 6, 13, and 182 (SDD and CPM), and on day 371 (SDD
only), at the end of the 48-h period, the animals were anesthetized with CO2/O2
and blood was taken from the retro-orbital sinus into heparinized tubes.
Erythrocytes and plasma were collected separately. Although the animals were
still under anesthesia, the abdominal wall was opened and the aorta was
severed. The liver, kidneys, and the stomach (separated into glandular stomach
370
COLLINS ET AL.
and forestomach) were removed, weighed, and frozen at 20C until further
use. Stainless steel was avoided during the tissue collection; only plastic,
Teflon, ceramic, or tungsten carbide instruments were used. Because a washout
period occurred prior to tissue collection, increased chromium concentrations in
plasma should represent the chromium entering plasma from the tissues. To
make comparisons of tissue concentrations between animal species and species
of chromium on day 182 (week 26), average time-weighted doses of Cr(VI) and
Cr(III) resulting from ingestion of SDD or CPM were calculated using the 1- to
25-week water and feed consumption data and body weight data (NTP, 2008b).
These doses are shown in Table 1.
Analysis of Total Chromium in Tissues and Excreta
The sample preparation and analysis procedures used to determine the concentration of chromium in tissues (Levine et al., 2010) and excreta (Levine
et al., 2009) in support of this investigation are presented in detail elsewhere
and are summarized here. Neither of these procedures could differentiate between
chromium oxidation states, so data reported are for total chromium, irrespective
of oxidation state administered.
Preparation of tissue samples. Each tissue sample was homogenized
using a Polytron tissue homogenizer (Brinkmann Instruments, Westbury, NY)
and returned to frozen storage. Prior to analysis, homogenized samples were
thawed and an aliquot of homogenate equivalent to 0.500 g of tissue was
transferred to a tared 50-ml centrifuge tube and the mass recorded. If
insufficient sample was available, the entire sample was used. Samples were
digested in 5 ml of concentrated nitric acid using a microwave digestion
procedure followed by a second microwave digestion step using hydrofluoric
acid. Following digestion, the contents of each tube were transferred to a 25-ml
volumetric flask to which was added 0.250 ml of internal standard solution
(ISS) and then diluted to volume with distilled water.
Preparation of plasma samples. A 0.500-g aliquot of plasma was
transferred to a tared 50-ml centrifuge tube, and the mass was recorded.
Plasma samples were digested as described above for tissue samples. Following
digestion, the contents of each tube were transferred to a 25-ml volumetric flask
to which 0.250 ml of ISS was added and then diluted to volume with distilled
water.
Preparation of excreta samples. Feces samples were transferred to a tared
50-ml centrifuge tube, and the wet mass was recorded. The tubes were
TABLE 1
Average Time Weighted Doses of Chromium following Exposure
of Male F344/N Rats and Female B6C3F1 Mice to Sodium
Dichromate Dihydrate (hexavalent chromium) or Chromium
Picolinate Monohydrate (trivalent chromium)
Chromium dose (mg chromium/kg
body weight)a
Exposure
Male rats
Female mice
0 mg/l SDD; 0 mg/l CPM
14.3 mg/l SDD
57.3 mg/l SDD
172 mg/l SDD
516 mg/l SDD
2000 ppm CPM
10,000 ppm CPM
50,000 ppm CPM
0
0.299
1.18
3.11
8.95
15.18
78.60
409.15
0
0.517
2.09
5.56
13.2
36.73
189.49
945.66
a
Calculated using food (CPM) or water (SDD) consumption data and body
weight data for weeks 1–25 of the studies, and the percent mass of Cr in CPM
(11.92) or SDD (34.90).
transferred to a freezer prior to lyophilization. All tubes were subjected to
freeze-drying for at least 24 h. After lyophilization, the dry mass and percent
loss on drying was recorded for each sample. Each sample was manually
homogenized with a clean glass rod and stored frozen until analysis. Each
freeze-dried feces sample was mixed with a Teflon-coated spatula prior to
transferring a target mass of freeze-dried feces homogenate, equivalent to
0.250 g wet mass, to a 50-ml centrifuge tube. Urine samples were vortexed
prior to transferring a 0.250-g aliquot to a 50-ml centrifuge tube. Excreta
samples were digested as described above for tissue samples. Following
digestion, the contents of each tube were transferred to a 25-ml volumetric
flask to which was added 0.150 ml of ISS and then diluted to volume with
distilled water.
Preparation of standards. Solvent standards were prepared from two
working stock solutions by adding appropriate aliquots of each working stock
to 100-ml volumetric flasks along with 10 ml of concentrated nitric acid and
diluting to volume with deionized water. Matrix standards were prepared in
a similar manner, except that aliquots of the working stocks were spiked into
0.500 g of control tissue or feces homogenate and were then carried through the
same digestion procedure as the samples.
Analysis of tissue, plasma, and excreta. Samples and standards were
analyzed by ICP-MS using a Thermo X7 instrument (ThermoElectron Corp.,
Winsford, Cheshire, U.K.) or a Plasma Quad XR Instrument (VG Elemental
Ltd., Winsford, Cheshire, U.K.) with a concentric nebulizer and a Peltier
impact-bead spray chamber cooled to 5C.
Sodium/Sulfate Co-transport Experiments
Preparation of rat kidney brush border membrane vesicles. Brush border
membrane vesicles (BBMV) were isolated from renal cortex of six male
Sprague-Dawley CD rats (~250 g) using a method previously described by
Beck and Sacktor (1978). Caþþ precipitation of a crude membrane homogenate
was followed by differential centrifugation to yield a purified vesicle
preparation. Marker enzyme analysis showed an enrichment of the brush
border membrane marker, alkaline phosphatase, of 10- to 20-fold. The
basolateral membrane marker, Naþ-Kþ-ATPase, showed enrichments of less
than onefold. The final membrane pellet was suspended in vesicle buffer
(100mM mannitol, 100mM KCl, 20mM N-2-hydroxyethylpiperazine-N#-2ethanesulfonic acid [HEPES]/tris (hydroxymethyl)aminoethane [Tris], 1mM
MgSO4) at pH 7.4 and stored in liquid nitrogen until use.
Effect of SDD and CPM on transport of [35S]-sodium sulfate in
BBMV. For transport experiments, BBMV were thawed, centrifuged,
resuspended, and allowed to equilibrate in fresh vesicle buffer for 60 min at
room temperature and then placed on ice. Ten microliters of vesicles were
incubated at room temperature for 2 min in 90 ll of media (100mM NaCl,
20mM Tris/HEPES, and 1 mM MgSO4, pH 7.4) containing 50lM [35S]sodium sulfate and 50, 250, and 500lM SDD or CPM. This suspension was
transferred to Millipore Glass Fiber Filters (0.7 lm) (Billerica, MA), presoaked
in distilled water, washed three times with buffer, and allowed to dry. The
filters were counted in a Packard 2900TR Tricarb Liquid Scintillation Analyzer
(Waltham, MA). Data were transformed and analyzed using GraphPad Prism
software (La Jolla, CA), and values represent analysis from triplicate
experiments. Internal controls include sodium-free uptake (KCl substituted
for NaCl in transport media) and SITS-sensitive uptake with sodium present but
no inhibitor (SITS, 1mM 4-acetamido-4#-isothiocyanostilbene-2,2#-disulfonic
acid).
Kinetics of SDD inhibition of the transport of [35S]-sodium sulfate in rat
kidney BBMV. For kinetic experiments, BBMV were thawed, centrifuged,
resuspended, and allowed to equilibrate in fresh vesicle buffer for 60 min at
room temperature and then placed on ice. Ten microliters of vesicles were
incubated for 30 s in 90 ll of media (100mM NaCl, 20mM Tris/HEPES, and
1mM MgSO4) containing 5–250 lM [35S]-sodium sulfate and 250lM SDD in
the presence or absence of 1mM SITS. This suspension was transferred to
Millipore Glass Fiber Filters (0.7 lm), presoaked in distilled water, washed
371
EXPOSURE TO HEXAVALENT CHROMIUM
TABLE 2
Concentrations of Chromium in Tissues and Excreta in Male F344/N Rats following Exposure to Sodium Dichromate Dihydrate
(hexavalent chromium) in Drinking Watera
0 mg/l
n
3
Erythrocytes (lg/g)
Day 6
0.044 ± 0.002
Day 13
0.051 ± 0.002
Day 182 0.050 ± 0.014
Day 371 0.055 ± 0.003
Plasma (lg/g)b
Day 6
0.052 ± 0.003
Day 13
0.054 ± 0.004
Day 182 0.063 ± 0.005
Day 371 0.054 ± 0.014
Liver (lg/g)
Day 6
0.072 ± 0.007
Day 13
0.080 ± 0.013
Day 182 0.081 ± 0.004
Day 371 0.092 ± 0.003
Kidney (lg/g)
Day 6
0.376 ± 0.070
Day 13
0.089 ± 0.006
Day 182 0.083 ± 0.003
Day 371 0.170 ± 0.011
Glandular stomach (lg/g)
Day 6
0.076 ± 0.003
Day 13
0.095 ± 0.008
Day 182 0.197 ± 0.031
Day 371 0.253 ± 0.066
Forestomach (lg/g)
Day 6
0.098 ± 0.024
Day 13
0.091 ± 0.015
Day 182 0.089 ± 0.018
Day 371 0.090 ± 0.015
Feces (lg)c
Day 6
11.56 ± 3.06
Day 13
9.056 ± 1.977
Day 182 10.04 ± 2.06
Day 371 6.869 ± 1.126
Urine (lg)c,d
Day 6
0.275 ± 0.073
Day 13
0.139 ± 0.028
Day 182 0.630 ± 0.131
Day 371 0.588 ± 0.087
14.3 mg/l
57.3 mg/l
172 mg/l
516 mg/l
3
3
3
3
Significant departure
from linearity?
Shape, if
not linear
0.051
0.036
0.054
0.064
±
±
±
±
0.002
0.005
0.003
0.006*
0.126
0.203
0.208
0.160
±
±
±
±
0.012*
0.019
0.002
0.059**
0.252
0.504
0.591
0.526
±
±
±
±
0.022*
0.066*
0.014*
0.031**
0.391
0.899
0.997
0.693
±
±
±
±
0.012**
0.134**
0.051**
0.029**
Y
N
Y
Y
Supralinear
—
Supralinear
Supralinear
0.068
0.048
0.064
0.062
±
±
±
±
0.007*
0.006
0.003
0.005
0.079
0.079
0.081
0.071
±
±
±
±
0.003**
0.005*
0.004*
0.011
0.087
0.103
0.099
0.110
±
±
±
±
0.008**
0.008**
0.007**
0.010**
0.109
0.146
0.146
0.146
±
±
±
±
0.007**
0.008**
0.009**
0.003**
N
Y
N
Y
—
Supralinear
—
Supralinear
0.097
0.080
0.249
0.289
±
±
±
±
0.015
0.005
0.007*
0.029*
0.326
0.700
1.568
1.145
±
±
±
±
0.027*
0.065
0.040**
0.334**
1.116
1.899
4.101
4.383
±
±
±
±
0.200**
0.347*
0.110**
0.419**
1.589
3.239
6.650
7.735
±
±
±
±
0.125**
0.385**
0.249**
0.155**
Y
Y
Y
Y
Supralinear
Supralinear
Supralinear
Supralinear
0.252
0.225
1.153
1.564
±
±
±
±
0.049
0.009*
0.036*
0.055*
0.591
1.125
5.464
4.879
±
±
±
±
0.043
0.041**
0.054**
1.259**
1.122
2.556
10.847
13.423
±
±
±
±
0.087*
0.390**
0.195**
0.307**
1.570
4.409
15.263
18.530
±
±
±
±
0.024**
0.596**
0.280**
0.430**
Y
Y
Y
Y
Supralinear
Supralinear
Supralinear
Supralinear
0.143
0.254
0.414
0.334
±
±
±
±
0.008*
0.073*
0.012*
0.029
0.333
0.310
1.043
1.038
±
±
±
±
0.040**
0.049*
0.081**
0.115*
0.773
1.331
4.300
4.801
±
±
±
±
0.105**
0.060**
0.367**
0.345**
1.967
1.762
9.886
14.643
±
±
±
±
0.109**
0.042**
0.354**
0.121**
N
Y
N
N
—
Supralinear
—
—
0.076
0.102
0.099
0.118
±
±
±
±
0.004
0.034
0.003
0.008
0.122
0.171
0.338
0.328
±
±
±
±
0.008
0.050
0.022*
0.081*
0.294
0.221
0.574
1.338
±
±
±
±
0.029
0.055*
0.171*
0.444**
0.285
0.593
1.654
2.849
±
±
±
±
0.123
0.159**
0.244**
0.975**
N
N
N
N
—
—
—
—
26.87
52.335
63.64
36.299
±
±
±
±
5.56*
6.275*
6.07*
4.185*
122.29
171.734
229.71
139.488
±
±
±
±
5.49**
9.232**
17.38**
11.446*e
246.86
362.994
467.84
311.849
±
±
±
±
84.73**
74.118**
33.30**
10.293**
1,030.2
972.867
1,947.6
1,146.27
±
±
±
±
117.71**
54.316**
287.25**
114.235**
N
N
N
N
—
—
—
—
0.480
0.396
1.145
2.023
±
±
±
±
0.053*
0.114*
0.101*
0.267*
2.520
1.100
7.076
7.406
±
±
±
±
0.732**
0.253**
0.075**
1.020**
4.594
1.943
12.580
13.702
±
±
±
±
1.118**
0.390**
0.898**
2.107**
6.967
4.561
20.137
23.969
±
±
±
±
0.717**
0.486**
0.579**
3.875**
N
N
Y
N
—
—
Supralinear
—
a
Mean ± SE. Statistical tests were performed on unrounded data. Data reflects 2-day washout period after dosing.
n ¼ 6 for all plasma values.
c
Cumulative concentration for 48 h ending on the day indicated.
d
Measured values less than the ELOQ (0.025 lg Cr with a nominal sample mass of 0.250 g) were included in the analysis as ½ ELOQ.
e
n ¼ 2.
*Significantly different (p < 0.05) from the control group by Shirley’s test.
**p < 0.01.
b
three times with buffer, and allowed to dry. The filters were counted in
a Packard 2900TR Tricarb Liquid Scintillation Analyzer. All transport
experiments were carried out at room temperature. Data were transformed
and analyzed using Microsoft Excel graphics software. The Michaelis-Menten
constant (Km) and the maximum rate of uptake (Vmax) were estimated from
x- and y-intercepts of the Lineweaver-Burk plots, and values represent
SITS-sensitive sodium sulfate transport from three experiments.
Statistical Methods
Feces, urine, and tissue concentrations typically have skewed distributions
and were analyzed using the nonparametric multiple comparison methods of
Shirley (1977) and Dunn (1964). The Jonckheere (1954) test was used to assess
the significance of dose-response trends and to determine whether a trendsensitive test (Shirley’s test) was more appropriate for pairwise comparisons
372
COLLINS ET AL.
TABLE 3
Concentrations of Chromium in Tissues and Excreta in Female B6C3F1 Mice following Exposure to Sodium Dichromate Dihydrate
(Hexavalent Chromium) in Drinking Watera,d
0 mg/l
n
3
Erythrocytes (lg/g)
Day 6
0.040 ± 0.003
Day 13
0.043 ± 0.005
Day 182 0.058 ± 0.004
Day 371 0.036 ± 0.004
Plasma (lg/g)b
Day 6
0.064 ± 0.006
Day 13
0.034 ± 0.016
Day 182 0.051 ± 0.006
Day 371 0.065 ± 0.004
Liver (lg/g)
Day 6
0.098 ± 0.039
Day 13
0.126 ± 0.003
Day 182 0.147 ± 0.015
Day 371 0.069 ± 0.007
Kidney (lg/g)
Day 6
0.095 ± 0.011
Day 13
0.127 ± 0.032
Day 182 0.067 ± 0.006
Day 371 0.070 ± 0.002
Glandular stomach (lg/g)
Day 6
0.306 ± 0.056
Day 13
0.207 ± 0.053
Day 182 0.305 ± 0.078
Day 371 0.731 ± 0.306
Forestomach (lg/g)
Day 6
0.328 ± 0.132
Day 13
0.201 ± 0.094
Day 182 0.173 ± 0.064
Day 371 0.320 ± 0.049
Feces (lg)c
Day 6
2.599 ± 0.899
Day 13
2.348 ± 1.173
Day 182 1.275 ± 0.569
Day 371 7.485 ± 2.863
14.3 mg/l
57.3 mg/l
172 mg/l
516 mg/l
3
3
3
3
Significant departure
from linearity?
Shape, if
not linear
0.056
0.042
0.079
0.042
±
±
±
±
0.010
0.002
0.008
0.011
0.108
0.341
0.194
0.094
±
±
±
±
0.009*
0.100
0.016*
0.002
0.260
0.747
0.719
0.340
±
±
±
±
0.047**
0.087*
0.052**
0.020*
0.374
1.190
1.561
0.795
±
±
±
±
0.078**
0.137**
0.165**
0.089**
N
Y
N
N
—
Supralinear
—
—
0.075
0.038
0.070
0.086
±
±
±
±
0.016
0.018
0.010
0.010
0.111
0.133
0.116
0.118
±
±
±
±
0.026
0.024**
0.005**
0.014**
0.150
0.204
0.167
0.150
±
±
±
±
0.039**
0.045**
0.009**
0.012**
0.213
0.311
0.253
0.209
±
±
±
±
0.059**
0.077**
0.016**
0.009**
N
N
Y
Y
—
—
Supralinear
Supralinear
0.340
0.591
0.746
0.792
±
±
±
±
0.010*
0.027*
0.068*
0.019*
1.421
2.659
4.575
3.782
±
±
±
±
0.146**
0.094**
0.559**
0.143**
4.638
9.374
17.270
13.777
±
±
±
±
0.950**
0.892**
2.746**
0.951**
5.622
14.830
52.047
39.450
±
±
±
±
0.795**
2.347**
2.890**
1.655**
Y
Y
N
N
Supralinear
Supralinear
—
—
0.166
0.245
0.361
0.340
±
±
±
±
0.011*
0.003*
0.061*
0.014*
0.415
1.259
2.062
1.291
±
±
±
±
0.020**
0.051**
0.191**
0.064**
0.892
4.061
10.187
7.868
±
±
±
±
0.106**
0.718**
1.078**
0.510**
1.338
4.027
17.487
21.827
±
±
±
±
0.157**
0.286**
0.500**
2.446**
Y
Y
Y
N
Supralinear
Supralinear
Supralinear
—
0.645
0.324
0.644
0.676
±
±
±
±
0.253
0.030
0.035*
0.104
1.258
2.614
3.659
2.807
±
±
±
±
0.290*
0.190*
0.547**
0.330*
2.450
7.048
11.520
9.994
±
±
±
±
0.266**
1.751**
3.017**
1.079*
5.785
13.130
52.673
49.867
±
±
±
±
0.131**
2.604**
12.310**
12.251**
N
N
N
N
—
—
—
—
0.683
0.288
0.444
0.381
±
±
±
±
0.262
0.056
0.099
0.077
1.308
0.400
1.033
1.271
±
±
±
±
0.553
0.044
0.102*
0.300*
1.102
2.030
2.141
1.812
±
±
±
±
0.373
0.532*
0.643**
0.208*
1.286
3.849
9.624
7.442
±
±
±
±
0.116
1.811*
3.638**
0.764**
N
N
N
N
—
—
—
—
9.382
12.103
5.217
9.505
±
±
±
±
1.405*
3.355*
0.471*
1.569
13.032
85.804
17.606
18.719
±
±
±
±
2.779*
21.392**
3.100**
2.172*
43.741
46.934
24.624
41.017
±
±
±
±
10.325**
4.884*
5.736**
3.214**
162.632
109.687
122.928
72.197
±
±
±
±
31.032**
29.885**
21.563**
19.151**
N
Y
N
N
—
Linear with outliers
—
—
a
Mean ± SE. Statistical tests were performed on unrounded data. Data reflects 2-day washout period after dosing.
n ¼ 6 for all plasma values.
c
Cumulative concentration for 48 h ending on the day indicated.
d
Measured values less than the ELOQ (0.025 lg Cr with a nominal sample mass of 0.250 g) were included in the analysis as ½ ELOQ.
*Significantly different (p < 0.05) from the control group by Shirley’s test.
**p < 0.01.
b
with the controls than a test that does not assume a monotonic dose response
(Dunn’s test). Measurements less than the experimental limit of quantitation
(ELOQ) were included in the statistical analyses as ½ ELOQ. Trend-sensitive
tests were used when Jonckheere’s test was significant at p < 0.01. Departures
from a linear dose-response relationship for feces, urine, and tissue
concentrations were tested using the lack-of-fit test for simple linear regression
(Neter et al., 1996). When a significant departure was found, the shape of the
dose-response curve was determined by visual examination. For similar
administered doses of Cr that resulted from exposure to 516 mg/l SDD and
2000 ppm CPM, concentrations were compared using Mann-Whitney tests
(Hollander and Wolfe, 1973). Concentrations were compared between mice
and rats using Mann-Whitney tests at each dose level. For sulfate transport
experiments, control and treated groups were compared with the two-tailed
Student’s t-test (p < 0.05).
RESULTS
Concentrations of Total Chromium in Tissue and Excreta
After Exposure to Sodium Dichromate Dihydrate
Chromium concentration in tissues and excreta are given in
Tables 2 and 3 for male rats and female mice, respectively,
following exposure to SDD. The 48-h feces collection
EXPOSURE TO HEXAVALENT CHROMIUM
contained up to 1.9 mg of chromium for rats and 160 lg for
mice. In rats, 49.2%, and in mice, 48.8%, of the ingested
chromium was excreted in the feces, indicating that the
absorption of chromium was low. The amount of chromium
consumed was estimated by calculating the mg SDD consumed
from the average daily drinking water consumption data for
weeks 1–25 for each dose and converting that amount to mg
Cr. The 48-h urine collection contained up to 24 lg of
chromium for rats exposed to SDD at 516 mg/l at day 371. Rat
urine chromium values ranged from 0.58–2.4% of dose for the
lowest two exposure concentrations, to 0.19–0.95% for the
highest exposure concentration, with the lower values in each
range seen at the earlier time points. Insufficient mouse urine
was collected for analysis at most time points.
In rats and mice, the highest mean chromium concentrations
were observed in the glandular stomach, kidney, and liver.
Mean concentrations of chromium in the kidney were greater
than those in the liver at most time points for rats, whereas the
reverse was generally true for mice. In general, tissue
concentrations at each time point increased with increasing
exposure concentration. Statistical analysis of the shapes of the
exposure-concentration curves revealed either linear or supralinear responses (Tables 2 and 3; Fig. 1A); a supralinear dose
response is defined as a decreasing rate of response with
increasing exposure concentration.
When tissue concentrations are normalized to external Cr
dose (in milligram/kilogram body weight), at 516 mg/l on Day
182 (Table 1) and compared between rats and mice, female
mice show statistically higher tissue chromium concentrations
in liver (~53) and glandular stomach, (~43), whereas male
rats had a higher concentration of chromium in the kidney
(~1.33) (Table 4).
In some tissues, such as kidney, liver, forestomach, and
glandular stomach, tissue concentrations increased with
duration of exposure at each exposure concentration, but the
rate of accumulation of total chromium in tissues decreased
with longer exposure duration (Fig. 1). This pattern of tissue
accumulation was consistent for all tissues when normalized to
ingested Cr dose per unit body weight. Concentrations of
chromium in erythrocytes and plasma did not increase with
increasing duration of exposure at any exposure concentration.
Concentrations of Total Chromium in Tissue and Excreta
after Exposure to CPM
Chromium concentration in tissues and excreta are given in
Tables 5 and 6 for male rats and female mice, respectively,
following exposure to CPM. The 48-h feces collection contained
up to 59.3 mg of chromium for rats and 5.6 mg for mice. In rats,
42.2% of the ingested chromium was excreted in the feces,
whereas 20.2% was found in the feces of mice. The total amount
of chromium consumed was determined by calculating the mg
CPM consumed from the average daily food consumption data
for weeks 1–25 for each dose and converting that amount to mg
373
FIG. 1. Uptake of chromium in female mouse liver following exposure to
SDD (Cr(VI)) or CPM (Cr(III)). Exposure response curves for uptake of
chromium in the liver of female B6C3F1 mice following exposure to (A) SDD
or (B) CPM for various exposure durations. (C) Comparison of liver chromium
concentrations in female mice following exposure to control feed (CPM-Con),
control drinking water (SDD-Con), 2000 ppm (36.73 mg/kg/day) CPM (CPMExp), or 516 mg/l (13.2 mg/kg/day) SDD (SDD-Exp) for 182 days.
Cr. The 48-h urine collection for CPM-exposed animals
contained up to 31 lg of chromium for rats. Insufficient mouse
urine was collected for analysis at most time points.
As with Cr(VI), the tissues with the highest mean chromium
concentrations after exposure were the glandular stomach,
kidney, and liver in rats and mice. Tissue Cr concentrations
were higher than would be expected based only on the presence
of erythrocytes in the blood of these tissues. Mean chromium
concentrations in the kidney were greater than those in the liver
at most time points for rats, whereas the reverse was true for
mice. However, the observed chromium concentrations were
much lower following exposure to CPM than following exposure
to SDD, even though exposures to chromium were much higher
with CPM. Chromium tissue concentrations generally increased
374
COLLINS ET AL.
TABLE 4
Comparison of Chromium Uptake Normalized to Time-Weighted Average Dose in Tissues and Excreta of Male F344/N Rats and
Female B6C3F1 Mice Exposed to 516 mg/l Sodium Dichromate Dihydrate (Hexavalent Chromium) in Drinking Water on Day 182a
Male rats
n
Erythrocytes ((lg/g)/(mg/kg))
Plasma ((lg/g)/(mg/kg))b
Liver ((lg/g)/(mg/kg))
Kidney ((lg/g)/(mg/kg))
Glandular stomach ((lg/g)/(mg/kg))
Forestomach ((lg/g)/(mg/kg))
Feces (lg/(mg/kg))c
0.111
0.016
0.743
1.705
1.104
0.185
217.564
3
±
±
±
±
±
±
±
0.006
0.001
0.028
0.031
0.040
0.027
32.088
Female mice
Significant difference?
3
±
±
±
±
±
±
±
N
Y
Y
Y
Y
N
Y
0.118
0.019
3.956
1.327
3.997
0.730
9.313
0.013
0.001
0.220
0.038
0.934
0.276
1.640
a
Mean ± SE. Statistical tests were performed on unrounded data. Data reflects 2-day washout period after dosing.
n ¼ 6 for all plasma values.
c
Cumulative concentration for 48 h ending on day 182.
b
with exposure up to 10,000 ppm; however, levels did not
increase above this dose. The exposure-total Cr concentration
curves were supralinear or flat (Tables 5 and 6; Fig. 1B).
In kidney and liver, chromium concentrations increased with
increasing exposure duration, at all exposure concentrations, in
both rats and mice; however, the rate of tissue chromium
uptake decreased with longer exposure duration. In rats, mean
concentrations in the kidney increased dramatically between
day 13 and day 182. Tissue chromium concentrations did not
increase with exposure duration in erythrocytes, forestomach,
or glandular stomach for all doses in both rats and mice. These
observations can be interpreted as the tissues approaching
equilibrium between Cr(III) uptake and Cr(III) loss via
diffusion from the tissues and from cell turnover.
Comparison of Total Chromium Concentrations following
Exposure to Similar Doses of Cr(III) and Cr(VI)
Calculation of Cr dose in milligram/kilogram body weight
revealed that exposure to 516 mg SDD/l, which resulted in
increased squamous neoplasms of the oral cavity in male and
female rats and increased epithelial neoplasms of the small
intestine in male and female mice (NTP, 2008a; Stout et al.,
2009a), and 2000 ppm CPM resulted in ingestion of
comparable doses of Cr per unit body weight. Cr doses from
CPM exposure averaged 1.7 and 2.8 times higher, respectively,
in rats and mice than those of SDD. All other CPM dose groups
resulted in appreciably higher Cr exposures than the 516 mg
SDD/l dose group (Table 1).
Exposure to 516 mg/l SDD for 182 days, which was the
longest common exposure duration, resulted in significantly
higher chromium tissue concentrations compared with those
resulting from exposure to 2000 ppm CPM for 182 days. For
example, rats exposed to SDD had 13 times more chromium in
the liver and 5 times more chromium in the kidney than rats
exposed to CPM, and mice exposed to SDD had 39 times more
chromium in the liver (Fig. 1C) and 22 times more chromium
in the kidney than mice exposed to CPM.
Sodium/Sulfate Co-transport Experiments
To confirm that interactions with the sodium-sulfate co-transporter might underlie differences in Cr(VI) versus Cr (III) uptake
observed in these studies, experiments were conducted in
membrane vesicles from kidney, a preparation that has high
levels of sodium-sulfate transport. In these experiments, SDD
and CPM were incubated with rat kidney BBMV in the presence
of 35S to determine if the chromium compounds interacted with
the sulfate transporter. Exposure to 50, 200, or 500lM SDD
resulted in significant exposure-dependent decreases in sulfate
uptake by the vesicles, indicating that SDD inhibited sulfate
uptake (Fig. 2). In contrast, exposure of up to 500lM CPM had
no effect on sulfate uptake.
Because SDD inhibited the uptake of sulfate, kinetic experiments were conducted to determine the type of interaction with
the transporter. The Km for binding of sulfate was significantly
increased (p 0.05) from 12.6 ± 0.69lM in the control
experiment to 40.5 ± 3.18lM in the presence of 250lM SDD,
whereas the Vmax values were similar between control (65.9 ±
25.3 pmol/30 s/mg protein) and SDD (50.1 ± 15.8 pmol/30 s/
mg protein) samples. An increased Km (decreased affinity)
without a change in Vmax indicates that the inhibition of sulfate
transport was competitive and provides additional evidence that
Cr(VI) is taken up by sulfate transporters.
DISCUSSION
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 (Table 7;
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
375
EXPOSURE TO HEXAVALENT CHROMIUM
TABLE 5
Concentrations of Chromium in Tissues and Excreta in Male F344/N Rats following Exposure to Chromium Picolinate Monohydrate
(trivalent chromium) in Feeda
0 ppm
n
3
Erythrocytes (lg/g)
Day 6
0.0343 ±
Day 13
0.0405 ±
Day 182
0.0462 ±
Plasma (lg/g)b
Day 6
0.0609 ±
Day 13
0.0782 ±
Day 182
0.0796 ±
Liver (lg/g)
Day 6
0.0610 ±
Day 13
0.0779 ±
Day 182
0.1110 ±
Kidney (lg/g)
Day 6
0.1247 ±
Day 13
0.1019 ±
Day 182
0.1534 ±
Glandular stomach (lg/g)
Day 6
0.0721 ±
Day 13
0.4378 ±
Day 182
1.0366 ±
Forestomach (lg/g)
Day 6
0.0709 ±
Day 13
0.1183 ±
Day 182
0.0863 ±
Feces (lg)c
Day 6
15.5 ±
Day 13
10.5 ±
Day 182
23.3 ±
Urine (lg)c
Day 6
0.313 ±
Day 13
0.539 ±
Day 182
0.430 ±
2000 ppm
10,000 ppm
50,000 ppm
3
3
3
Significant departure
from linearity?
Shape, if
not linear
0.0100
0.0114
0.0028
0.0446 ± 0.0023
0.0517 ± 0.0089
0.0616 ± 0.0104
0.0600 ± 0.0057
0.0725 ± 0.0095
0.0956 ± 0.0078*
0.0392 ± 0.0109
0.0676 ± 0.0058
0.1154 ± 0.0064**
N
N
Y
—
—
Supralinear
0.0068
0.0057
0.0034
0.0919 ± 0.0048**
0.0940 ± 0.0030*
0.1118 ± 0.0100**
0.1050 ± 0.0044**
0.1141 ± 0.0061**
0.1638 ± 0.0042**
0.1259 ± 0.0064**
0.1351 ± 0.0060**
0.1760 ± 0.0087**
Y
Y
Y
Supralinear
Supralinear
Supralinear
0.0072
0.0093
0.0120
0.1423 ± 0.0056*
0.2333 ± 0.0116*
0.5130 ± 0.0328*
0.2151 ± 0.0038**
0.3384 ± 0.0181**
1.1857 ± 0.0622**
0.2357 ± 0.0164**
0.3954 ± 0.0305**
1.2843 ± 0.0171**
Y
Y
Y
Supralinear
Supralinear
Supralinear
0.0172
0.0098
0.0080
0.3757 ± 0.0228*
0.7173 ± 0.0336*
2.8677 ± 0.0159*
0.4946 ± 0.0210**
1.2823 ± 0.0900**
6.0673 ± 0.3618**
0.5778 ± 0.0285**
1.2500 ± 0.0896*
6.7137 ± 0.0489**
Y
Y
Y
Supralinear
Supralinear
Supralinear
0.0005
0.3547
0.8837
0.1671 ± 0.0639
0.1450 ± 0.0298
0.6991 ± 0.1218
0.1061 ± 0.0101
0.1679 ± 0.0288
1.4057 ± 0.6062
0.1604 ± 0.0463
0.2299 ± 0.0745
0.6560 ± 0.1255
N
N
N
Flat
Flat
Flat
0.0068
0.0133
0.0155
0.1222 ± 0.0092*
0.1354 ± 0.0222
0.1592 ± 0.0100*
0.1991 ± 0.0385*
0.2400 ± 0.1112
0.3615 ± 0.0589**
0.1701 ± 0.0202*
0.1390 ± 0.0109
0.3479 ± 0.0332*
Y
N
Y
Supralinear
Flat
Supralinear
5.4
0.3
9.3
981.5 ± 133.5*
1,974.1 ± 97.1*
2,182.5 ± 308.7*
2,670.5 ± 761.1**
8,619.4 ± 839.5**
11,451.2 ± 1,417.2**
32,055.2 ± 3,383.3**
34,786.4 ± 10,954.5**
59,338.5 ± 6,272.3**
N
N
N
—
—
—
10.323 ± 1.727*
14.653 ± 2.218**
23.106 ± 1.102**
23.549 ± 4.426**
31.366 ± 5.451**
30.026 ± 9.869**
N
N
N
—
—
—
0.056
0.068
0.101
4.755 ± 1.183*
6.728 ± 0.427*
8.169 ± 1.195*
a
Mean ± SE. Statistical tests were performed on unrounded data. Measured values less than the ELOQ were included in the analysis as ½ ELOQ. Data reflects
2-day washout period after dosing.
b
n ¼ 6 for all plasma values.
c
Cumulative chromium content for 48 h ending on the day indicated.
*Significantly different (p < 0.05) from the control group by Shirley’s test.
**p < 0.01.
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).
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
376
COLLINS ET AL.
TABLE 6
Concentrations of Chromium in Tissues and Excreta in Female B6C3F1 Mice following Exposure to Chromium Picolinate
Monohydrate (trivalent chromium) in Feeda
0 ppm
n
3
Erythrocytes (lg/g)
Day 6
0.0688 ±
Day 13
0.0672 ±
Day 182
0.0361 ±
Plasma (lg/g)b
Day 6
0.0703 ±
Day 13
0.0792 ±
Day 182
0.0644 ±
Liver (lg/g)
Day 6
0.1732 ±
Day 13
0.1860 ±
Day 182
0.2118 ±
Kidney (lg/g)
Day 6
0.0970 ±
Day 13
0.0813 ±
Day 182
0.0799 ±
Glandular stomach (lg/g)
Day 6
0.3810 ±
Day 13
0.4913 ±
Day 182
0.2594 ±
Forestomach (lg/g)
Day 6
0.2618 ±
Day 13
0.3582 ±
Day 182
0.2941 ±
Feces (lg)d
Day 6
5.0 ±
Day 13
4.0 ±
Day 182
2.7 ±
Significant departure
from linearity?
Shape, if
not linear
2000 ppm
10,000 ppm
50,000 ppm
3
3
3
0.0171
0.0110
0.0040
0.0543 ± 0.0110
0.0609 ± 0.0052
0.0773 ± 0.0084*
0.0749 ± 0.0182
0.1635 ± 0.0065
0.1198 ± 0.0132**
0.0813 ± 0.0073
0.7116 ± 0.5998
0.1292 ± 0.0204*
N
N
Y
Flat
Flat
Supralinear
0.0080
0.0024
0.0055
0.1283 ± 0.0097**
0.1130 ± 0.0147*
0.1162 ± 0.0066**
0.1389 ± 0.0145**
0.1404 ± 0.0293*
0.1262 ± 0.0081**
0.1749 ± 0.0354**
0.1960 ± 0.0453**
0.2762 ± 0.0606**
N
N
N
—
—
—
0.0093
0.0107
0.0421
0.3566 ± 0.0026*
0.5705 ± 0.0273*
1.3467 ± 0.0552*
0.5619 ± 0.0885**
0.8671 ± 0.0591**
2.6553 ± 0.3195**
0.4904 ± 0.0075*
1.0055 ± 0.1068**
2.8937 ± 0.4221**
Y
Y
Y
Supralinear
Supralinear
Supralinear
0.0092
0.0021
0.0013
0.2165 ± 0.0220*
0.3533 ± 0.0129*
0.7907 ± 0.0309*
0.3253 ± 0.0309**
0.7171 ± 0.1738**
1.0035 ± 0.0357**
0.3242 ± 0.0543*
0.6933 ± 0.1200*
1.1981 ± 0.1849**
Y
Y
Y
Supralinear
Supralinear
Supralinear
0.1045
0.1635
0.0951
0.7671 ± 0.2943
0.4520 ± 0.1541
1.9187 ± 0.5852*
0.4174 ± 0.1225
6.4730 ± 3.3601*
1.6635 ± 0.5503
0.9349 ± 0.3279
1.3614 ± 0.5943
1.2947 ± 0.0846
N
Y
N
Flat
Flat with outlier
Flat
0.0558
0.0214
0.1099
1.0015 ± 0.3925
0.3746 ± 0.2022
1.4537 ± 0.3915*
0.3211 ± 0.0420
17.1577 ± 14.2041c
0.7424 ± 0.0737
0.6222 ± 0.2099
1.3203 ± 0.8859
1.1491 ± 0.2961
N
N
Y
Flat
Flat
Linear with outlier
129.2 ± 22.9*
156.6 ± 8.1*
129.9 ± 37.5*
632.5 ± 149.1**
586.9 ± 81.9**
1,022.8 ± 230.0**
2,831.8 ± 522.2**
3,360.1 ± 77.3**
5,562.8 ± 258.9**
N
N
N
—
—
—
2.5
0.9
1.2
a
Mean ± SE. Statistical tests were performed on unrounded data. Urine data are not presented due to inadequate sample sizes. Measured values less than the
ELOQ were included in the analysis as ½ ELOQ. Data reflects 2-day washout period after dosing.
b
n ¼ 6 for all plasma values.
c
Measured chromium concentrations for the three samples were 5.692, 45.4 and 0.4007 lg/g forestomach. Because reanalysis of the first two samples resulted in
similar chromium concentrations (measured at 5.518 and 44.05 lg/g forestomach) and there were three samples analyzed from the group, outliers could not be
excluded.
d
Cumulative chromium content for 48 h ending on the day indicated.
*Significantly different (p < 0.05) from the control group by Dunn’s or Shirley’s test.
**p < 0.01.
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, 1997) 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
377
EXPOSURE TO HEXAVALENT CHROMIUM
FIG. 2. Effect of SDD and CPM on transport of [35S]-sodium sulfate in rat
kidney BBMV. Control: The extent of sodium-driven sulfate transport is
demonstrated by the increase in sulfate uptake seen in the presence of a Na
in>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; 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
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.
378
COLLINS ET AL.
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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