Duodenal crypt health following exposure to Cr(VI): Micronucleus scoring, γ-H2AX immunostaining, and synchrotron X-ray fluorescence microscopy Academic research paper on "Biological sciences"

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Duodenal crypt health following exposure to Cr(VI): Micronucleus scoring, 7-H2AX immunostaining, and synchrotron X-ray fluorescence microscopy

Chad M. Thompson3 *.Jeffrey C. Wolfb, Reem H. Elbekaic, Madhav G. Paranjpec, Jennifer M. Seiterd, Mark A. Chappelld, Ryan V. Tapperoe, Mina Suhf, Deborah M. Proctorf , Anne Bichtelerg, Laurie C. Hawsg, Mark A. Harrisa

a ToxStrategies, Inc., Katy, TX 77494, USA b Experimental Pathology Laboratories, Sterling, VA 20166, USA c BioReliance, Rockville, MD, USA

d U.S. Army Engineer Research and Development Center, Vicksburg, MS 39180, USA e Photon Sciences Department, Brookhaven National Laboratory, Upton, NY 11973, USA f ToxStrategies, Inc., Mission Viejo, CA 92692, USA g ToxStrategies, Inc., Austin, TX 78731, USA

ABSTRACT

Lifetime exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water results in intestinal damage and an increase in duodenal tumors in B6C3F1 mice. To assess whether these tumors could be the result of a direct mutagenic or genotoxic mode of action, we conducted a GLP-compliant 7-day drinking water study to assess crypt health along the entire length of the duodenum. Mice were exposed to water (vehicle control), 1.4, 21, or 180ppm Cr(VI) via drinking water for 7 consecutive days. Crypt enterocytes in Swiss roll sections were scored as normal, mitotic, apoptotic, karyorrhectic, or as having micronuclei. A single oral gavage of 50mg/kg cyclophosphamide served as a positive control for micronucleus induction. Exposure to 21 and 180ppm Cr(VI) significantly increased the number of crypt enterocytes. Micronuclei and 7-H2AX immunostaining were not elevated in the crypts of Cr(VI)-treated mice. In contrast, treatment with cyclophosphamide significantly increased numbers of crypt micronuclei and qualitatively increased 7-H2AX immunostaining. Synchrotron-based X-ray fluorescence (XRF) microscopy revealed the presence of strong Cr fluorescence in duodenal villi, but negligible Cr fluorescence in the crypt compartment. Together, these data indicate that Cr(VI) does not adversely effect the crypt compartment where intestinal stem cells reside, and provide additional evidence that the mode of action for Cr(VI)-induced intestinal cancer in B6C3F1 mice involves chronic villous wounding resulting in compensatory crypt enterocyte hyperplasia.

© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/).

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Mutation Research/Genetic Toxicology and Environmental Mutagenesis

journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres

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ARTICLE INFO

Article history: Received 22 February 2015 Received in revised form 7 May 2015 Accepted 10 May 2015 Available online 14 May 2015

Keywords:

Hexavalent chromium Cr(VI)

Synchrotron Duodenum Carcinogenesis Mode of action 7-H2AX

* Corresponding author at: ToxStrategies, Inc. 23123, Cinco Ranch Blvd., Suite 220, Katy, TX 77494, USA. Tel.: +1 281 712 2062x2002; fax: +1 832 218 2756.

E-mail addresses: cthompson@toxstrategies.com (C.M. Thompson), JWolf@epl-inc.com (J.C. Wolf), reem.elbekai@bioreliance.com (R.H. Elbekai), madhav.paranjpe@bioreliance.com (M.G. Paranjpe), Jennifer.M.Seiter@erdc.dren.mil (J.M. Seiter), Mark.A.Chappell@usace.army.mil (M.A. Chappell), rtappero@bnl.gov (R.V. Tappero), msuh@toxstrategies.com (M. Suh), dproctor@toxstrategies.com (D.M. Proctor), abichteler@toxstrategies.com (A. Bichteler), lhaws@toxstrategies.com (L.C. Haws), mharris@toxstrategies.com (M.A. Harris).

1. Introduction

Chronic ingestion of high concentrations of hexavalent chromium [Cr(VI)] in drinking water has been associated with increased incidences of adenomas and carcinomas of the duodenum and jejunum in B6C3F1 mice [1,2]. When ingested, Cr(VI) is reduced to trivalent chromium [Cr(III)] by saliva and gastric fluid or taken into cells along the gastrointestinal tract through anion transporters [3-6]. Cr(III) absorption is minimal relative to Cr(VI), and Cr(III) has been shown to have minimal toxicity and no carcinogenicity in long term rodent bioassays [7,8]. Reduction of Cr(VI) to Cr(III) within cells can lead to oxidative stress and Cr(III)-ligands that can damage cellular constituents such as proteins and DNA

http://dx.doi.org/10.1016/j.mrgentox.2015.05.004

1383-5718/© 2015 The Authors. Published by Elsevier B.V. This is an open access article underthe CC BY license (http://creativecommons.org/licenses/by/4.0/).

[3,9,10]. Cr(VI) is reported to induce Cr-DNA adducts, DNA-protein crosslinks, DNA-Cr-DNA crosslinks, DNA single strand and double strand breaks, and other forms of DNA modification [9,11,12]. Despite the potential for DNA damage, we previously showed that B6C3F1 mice exposed to <180 ppm Cr(VI) for 90 days did not exhibit increases in crypt micronuclei (MN), nor increases in kras codon 12 GAT mutant frequency [13]. Analyses from the same underlying 90-day study also demonstrated that B6C3F1 mice exposed to 180 ppm Cr(VI) did not exhibit increased DNA damage in the crypt compartment as measured by phosphorylated H2AX (7-H2AX) immunostaining [14]. Consistent with the absence of DNA damage, synchrotron-based X-ray fluorescence (XRF) microscopy revealed the presence of strong Cr fluorescence in duodenal villi, but negligible Cr fluorescence in the crypt compartment where intestinal stem cells reside [14]. Collectively, these data argue against a direct genotoxic mode of action in the development of Cr(VI)-induced intestinal tumors.

The aforementioned micronucleus and XRF microscopy analyses were conducted on standard 5 |im transverse tissue biopsies that provide ideal histological features for pathogenic diagnoses, but limited coverage along the anteroposterior axis. Because intestinal tumors are generally thought to arise from stem cells within the intestinal crypt compartment [15-21], we sought to expand upon the transverse analyses by employing the "Swiss roll" technique [22] - a histologic preparation method that allows the entire length of an intestinal segment to be examined microscopically. Herein, we describe the results of a GLP in vivo intestinal micronucleus study in B6C3F1 mice (with cyclophosphamide administered as a positive control). In addition to micronucleus scoring, immunostaining for 7-H2AX was conducted, and XRF microscopy was performed to correlate chromium localization with genotoxic effects in the crypt.

2. Materials and methods

2.1. Chemicals

Cr(VI), in the form of sodium dichromate dihydrate (SDD, 100.1% pure; CAS# 7789-12-0), cyclophosphamide (CPH; CAS# 6055-192), and dimethylhydrazine (DMH, 99.8% purity; CAS# 57-14-7) were obtained from Sigma-Aldrich Inc., (St. Louis, MO). Concentration of Cr(VI) in SDD formulations was confirmed by EPA Method 7196 at Eurofins Lancaster Laboratories (Lancaster, PA). All dose formulations, with the exception of the low dose, were within ±10% of the target concentration. The concentration of Cr(VI) at the low dose was determined to be ~0.91 ppm.

2.1.1. Animals and study design

The in-life portion of this study was conducted by BioReliance (Rockville, MD), and approved by the BioReliance Institutional Animal Care and Use Committee. Although the OECD has published several test guideline studies for examining toxicity of chemicals in animals, there is no specific guideline for the small intestine (or colon) at this time. Therefore, our study design was informed by the OECD Test Guideline 474 "Mammalian Erythrocyte Micronucleus Test" [23,24], as well as previously published in vivo intestinal micronucleus studies [25,26].

Male and female B6C3F1 responded similarly to Cr(VI) [1,2]; therefore, female B6C3F1 mice were used in the current study in an effort to further inform mechanistic studies conducted in female B6C3F1 mice [27,28]. Mice approximately 6 weeks of age were obtained from Charles River (Raleigh, NC), and were allowed to acclimate for approximately 1 week. Mice were allowed ad libitum access to the same food used in the National Toxicology Program

(NTP) cancer bioassay [1], specifically irradiated NTP-2000 chow (Zeigler Bros., Gardners, PA).

Mice (5 per group) were exposed 7 days to 4, 60, or 516mg/l SDD in tap water (equivalent to ~1.4,21, and 180 ppm Cr(VI)) using water bottles. Notably, 180 ppm was the highest concentration employed in the NTP cancer bioassay [1]. Vehicle control animals (n = 10) were exposed to tap water only (Washington Suburban Sanitary Commission Potomac Plant, Potomac, MD). The water is tested yearly and meets U.S. EPA drinking water standards (total chromium was not detected). Three positive controls were used: 65 mg/kg DMH by oral gavage, 65 mg/kg DMH by intraperitoneal injection, and 50 mg/kg CPH by oral gavage. All mice were sacrificed 24 h after the last exposure.

2.1.2. Histopathology

At study termination, mice were euthanized by CO2 asphyxiation and necropsied. Each duodenum was removed, prepared for longitudinal sectioning using the Swiss roll procedure [22], fixed in 10% neutral-buffered formalin and processed routinely for paraffin embedding. Blocks were processed and analyzed by Experimental Pathology Laboratories (Sterling, VA). Each paraffin block was microtomed to generate sections for DNA staining with Feulgen stain, 7-H2AX immunostaining, and XRF mapping. Sections for Feulgen staining were microtomed at approximately 4 |im thickness. Sections were mounted on glass slides with glass coverslips using an appropriate mounting medium as per routine.

Systems used to collect and tabulate cell count data via image analysis included an Olympus® BX51 research microscope (Olympus America, Inc. Melville, NY), an Infinity 2-1R 1.4 megapixel color digital video camera (Lumenara® Corporation, Ottawa, Ontario, Canada), Image-Pro® Plus (IPP version 7.0, Media Cybernetics, Silver Spring, Maryland), computer running Microsoft® Windows® 7 Professional operating system, and Microsoft® Excel 2010 (Microsoft Corporation).

Blocks were randomized and coded, and the slides were masked so that the pathologist was unaware of the treatment group status of individual mice during photography and image scoring. Using the 20x microscope objective, images of duodenal crypts were acquired from Feulgen-stained slides; at least 15 full-length crypts were analyzed per animal. Five cell types were scored based on previously described criteria [26]: normal, apoptotic, mitotic, micronucleus, or karyorrhectic nucleus. Specifically, apoptotic cells were characterized by nuclei that had a smudged, heterochromatic appearance, and often a discrete rounding of the cell cytoplasm. Micronuclei (MN) consisted of a single dense, ovoid to spherical body that was located adjacent to a normal nucleus within the cytoplasm of the same cell. Karyorrhectic nuclei (KN) were fragmented into small, unequally sized, dense spherical bodies, and the cytoplasmic margins of such cells were often indistinct. A mitotic figure had distinctly evident chromosomal components. An American College of Veterinary Pathologists (ACVP) board-certified veterinary pathologist performed counts by differentially marking the various cell types in each of the arbitrarily selected crypts using the image analysis software, which also tabulated the results and exported them to a spreadsheet. Cell counts were peer-reviewed by an ACVP board-certified pathologist at BioReliance, and all cell scores represent consensus results.

Immunostaining for 7-H2AX was conducted as previously described [14]. Unstained 5 |im duodenal sections (1 per animal; 5 animals per group) were stained immunohistochemically for the 7-H2AX antigen with primary rabbit polyclonal c-H2AX antibody (Abcam, ab2893) and secondary goat anti-rabbit antibody (Vector, BA-6100) using routine avidin-biotin complex (ABC) methodology (Vector, PK-6100) and a diaminobenzidine (DAB) chromagen (Bio-care Medical, BDB2004H). Both negative and positive controls were prepared. The negative control consisted of a section of mouse

Table 1

Histological analyses of duodenal crypts.

Dose Enterocytes evaluated Crypts Enterocytes/crypt (mean ± sd) MN (total) KN (total) %A1 (mean ± sd) A/crypt (mean ± sd) M/Crypt (mean ± sd)

Water 6694 171 39.3 ± 3.5 4 0 0.39 ± 0.26 0.16 ± 0.10 0.60 ± 0.23

1.4ppmCr(Vl) 3159 77 41.0 ± 4.0 1 0 0.29 ± 0.32 0.11 ± 0.12 0.58 ± 0.23

21 ppmCr(Vl) 3946 76 51.9 ± 2.8* 1 1 0.64 ± 0.24 0.33 ± 0.11 0.57 ± 0.25

180ppmCr(Vl) 5161 77 67.1 ± 5.5* 0 0 0.40 ± 0.38 0.27 ± 0.26 0.48 ± 0.25

65 mg/kg DMHip 3697 94 39.4 ± 4.0 6 1 0.55 ± 0.37 0.22 ± 0.16 0.72 ± 0.18

65 mg/kg DMHpo 3153 77 41.0 ± 3.9 0 0 0.57 ± 0.15 0.24 ± 0.07 0.61 ± 0.22

50 mg/kg CPHpo 3447 87 39.3 ± 4.3 30** 5** 0.57 ± 0.48 0.23 ± 0.19 0.48 ± 0.07

MN: micronuclei; KN: karyorrhectic nuclei; %A1: apoptotic index; A/Crypt: apoptotic enterocytes per crypt; M/Crypt: mitotic figures per crypt; IP: intraperitoneal injection; PO: oral gavage. 'Significantly different from control via 1-way ANOVA and Dunnett's test (p <0.05). "Significantly different from control via Fisher's exact test (p <0.05) n = 10 in the control group, and n = 5 in treated groups.

duodenum in which normal rabbit serum was substituted for the primary antibody, and the positive control was a section of mouse testis. Appropriately, there was no immunoreactivity in the negative control, and the positive control exhibited very specific nuclear staining of spermatogonia and some stages of round to slightly elongated spermatids. An ACVP board-certified veterinary pathologist reviewed the immunostaining in the crypt and villus areas.

2.1.3. X-ray fluorescence microspectroscopy

XRF microscopy was conducted as previously described [14]. Swiss roll sections (~20 |im) were cut and placed on Mylar film. Synchrotron-based micro-X-ray fluorescence (||-XRF) was performed at beamline X27A at the National Synchrotron Light Source at Brookhaven National Laboratory in Upton, NY. The beam size on the sample was approximately 7 |im x 10 |im using Rh-coated Kirkpatrick-Baez focusing optics. X-rays were selected using a water-cooled channel-cut Si(111) monochromator. XRF data were collected using a Vortex ME4 Silicon Drift Detector Array. For XRF analysis, the monochromator was calibrated using a Chromium foil to the Cr K-edge (5989 eV), and XRF imaging was performed at 11 keV.

2.1.4. Statistical evaluations

Statistics for Cells/Crypt, apoptotic cells per crypt (A/Crypt), and mitotic figures per crypt (M/Crypt) were conducted using 1-way ANOVAs followed by Dunnett's test or Dunn's test, depending on whether the data were amenable to parametric or nonparamet-ric analyses. As the incidence of MN, KN, and apoptosis was very low (or zero) in the total number of cells observed, a small-sample method was required to test the significance of differences in outcomes between treated and control groups [29]. The Fisher's exact test was used on 2 x 2 contingency tables for each contrast to calculate odds ratios and confidence limits. Statistical packages used included R (http://www.R-project.org) and Prism 6 for Mac (Graph-Pad Software, San Diego, CA, www.graphpad.com)

3. Results and discussion

3.1. Assessment ofDNA damage in duodenal crypts

Using image analysis software, enterocytes were counted and scored in 15 or more crypts per animal (n = 5-10), resulting in analysis of approximately 3000-6000 crypt epithelial cells per treatment group (Table 1, Fig. 1A). Oral exposure to 50 mg/kg CPH significantly increased numbers of crypt MN and KN relative to duodena of vehicle control mice (Table 1; Fig. 1B). Exposure to <180ppm Cr(V1) did not increase numbers of crypt MN or KN (Table 1). Neither oral nor intraperitoneal exposure to DMH significantly increased MN or KN, although such aberrant nuclei were elevated in the latter group (Table 1). No treated animals exhibited increases in the number of apoptotic cells per crypt (A/crypt) or percentage of apoptotic cells in the crypt (apoptotic index; A1) (Table 1). The Cr(V1) findings in this study confirm those of earlier micronucleus results reported in B6C3F1 mice exposed to <180ppmCr(V1) for 90

days, in which scoring was performed on transverse duodenal sections but without a positive control group [13,27]. The absence of MN in intestinal crypts is consistent with Cr(Vl) in vivo peripheral blood cell micronucleus assay results, which are generally negative [30,31].

To further investigate potential for DNA damage in the crypt, immunohistochemical staining was conducted for ^-H2AX, which accumulates at sites of DNA double-strand breaks induced by chemicals and reactive oxygen species, as well as collapsed replication forks [32,33]. Cr(Vl) has been shown to increase ^-H2AX staining in Caco-2 cells at cytotoxic concentrations [34], and in other mammalian cell types pre-supplemented with dehy-droascorbic acid [35,36]. In vehicle control mice, immunoreactivity was observed in the chromatin material of mitotic figures and, to a lesser degree, in goblet cell mucus (Fig. 1C). Staining of mitotic figures was not unexpected, as DNA damage-independent H2AX phosphorylation has been shown to increase in mammalian cells as they progress from G1 phase through mitosis [37]. In mice exposed to CPH, discrete dense immunostaining was additionally observed in cells with KN and MN (Fig. 1D). Although ^-H2AX levels can increase within minutes of DNA double strand break formation and can disappear within several hours, ^-H2AX staining can remain elevated in micronuclei 24 h after exposure to insults such as irradiation [38]. In sharp contrast, the crypts of mice exposed to 180 ppm Cr(Vl) appeared healthy and exhibited no difference in 7-H2AX staining relative to those of untreated mice (Fig. 1E). Although the absence of a general increase in immunoreactivity could be attributed to the 24 h recovery period employed in this experiment, the findings herein are consistent with those of our earlier ^-H2AX studies in B6C3F1 mice that were allowed ad libitum access to 180 ppm Cr(Vl) for 90 days without a 24 h recovery [14].

3.2. Synchrotron-based imaging of chromium in the duodenum

XRF microscopy was used to map the distribution of chromium along the length of the duodenum. As an internal control, calcium (Ca) fluorescence was compared to chromium (Cr) fluorescence in the duodenal crypts and villi. ln mice exposed to vehicle control (water), Ca was detected throughout the crypt and villus regions, whereas, Cr signal was virtually nil (Fig. S1). In mice exposed to 180 ppm Cr(Vl) for 7 days, Ca was detected throughout the crypt and villus regions (Fig. 2A), whereas, Cr fluorescence was clearly visible in the intestinal villi and conspicuously absent in the crypt region (Fig. 2B). A similar pattern was observed in a duodenal section from another mouse exposed to 180 ppm Cr(Vl) (Fig. S2). These findings help explain the lack of aberrant nuclei in the crypts of mice exposed to Cr(Vl) (Table 1).

3.3. Assessment of crypt proliferation in the duodenum

Exposure to Cr(Vl) caused a dose-dependent increase in the number of enterocytes per crypt (Table 1, Fig. 1F). Previous studies have demonstrated that exposure to high concentrations of

Fig. 1. Micronucleus and ^-H2AX evaluation in mouse duodenum. (A) Example of computer-based coding of cell counts (Feulgen's stain). (B) Representative image of duodenal crypt from mice exposed to 50 mg/kg CPH (Feulgen's stain). Arrows and arrowheads indicate micronuclei and karyorrhectic nuclei, respectively. (C) Representative duodenal section (40x objective) from control B6C3F1 mouse stained with anti-^-H2AX. (D) Representative duodenal section (40x objective) stained with anti-^-H2AX from a B6C3F1 mouse treated with 50 mg/kg CPH. (E) Representative duodenal section (40x objective) stained with anti-^-H2AX from a B6C3F1 mouse exposed to 180 ppm Cr(Vl) for 7 days. Bars indicate 25 |xm. (F) The mean number of enterocytes counted per crypt n = 5 per treatment group, n =10 for controls.

Cr(Vl) can induce crypt hyperplasia after 7 days of exposure [28], 90 days of repeated exposure [27,28,30], and chronic lifetime exposure [1,2]. Mice exposed to 180 ppm Cr(Vl) for 90 days exhibited a significant increase in crypt length from 98.3 to 190.5 |im with a concomitant increase in the number enterocytes per crypt visible in cross-sections from 38.4 to 63.9 [14]. This increase in enterocytes

per crypt is almost identical to the increase observed herein after 7 days of exposure (39.3 ± 3.5 vs. 67.1 ± 5.5; Table 1 and Fig. 1F). An increase in crypt length is evident when comparing Figs. 1C and Fig. 1E; however, crypt length changes could not be accurately quantified in the Swiss roll preparations due to mild tissue compression in some instances.

Fig. 2. XRF (X-ray fluorescence) maps of Ca and Cr in the mouse duodenum. (A) Ca XRF maps of a duodenum from a mouse exposed to 180 ppm Cr(VI) in drinking water for 7 days. (B) Cr XRF maps of a duodenum from a mouse exposed to 180 ppm Cr(VI) in drinking water for 7 days. The white circles mark the crypt region. Note: the colors represent fluorescence signal intensity ranging from blue (low signal) to white (high signal).

We previously reported that a significant increase in crypt length and enterocyte number following Cr(V1) exposure occurred without a statistically significant increase in mitotic index [27] -despite increased expression of genes (e.g. Ki67) involved in cell proliferation [31,39].This apparent contradiction is likely explained by the overall increase in the number of crypt enterocytes, which begin to differentiate as they migrate from the stem cell zone through the transit amplifying region toward the crypt-villus junction [19]. In fact there are several cell positions near the top of the crypt that have been described as "post-mitotic" maturing regions [40]. When we instead characterize mitotic activity as the total number of mitotic figures per crypt (M/Crypt), there is a significant treatment-related increase after 90 days of exposure (Fig. S3). Thus, when there is obvious crypt hyperplasia, M/Crypt is a more appropriate measure of mitotic activity than mitotic index.

As shown in Table 1, M/Crypt did not change in the present study despite the increased cellularity. This could be due to the 24 h period without Cr(V1) exposure (see Section 2). To test this notion, we calculated the M/Crypt in transverse duodenal sections from B6C3F1 mice exposed to Cr(V1) for 7 days in our previous 90-day drinking water study [27]. 1n contrast to the present study, mice in the former study were allowed ad libitum access to Cr(V1) until study termination. Mice in the 180 ppm Cr(V1) group exhibited a statistically significant 1.4-fold increase in M/Crypt relative to control mice from that study (data not shown). This suggests that the lack of effect on M/Crypt observed in the current study could be due to a reduction in enterocyte demand after 24 h without Cr(V1) exposure. 1mportantly, none of the Cr(V1) drinking water studies published to date have reported shortened, damaged, or hypoplastic crypts that would suggest toxicity in the crypt cells. The data in the present study clearly indicate that Cr(V1) caused increased crypt cellularity without apparent cytogenetic damage (Table 1), increased ^-H2AX immunostaining (Fig. 1E), or evidence of chromium in the crypt region (Fig. 2).

4. Conclusion

1t was previously demonstrated that mice exposed to <180 ppm Cr(V1) for up to 90 days did not exhibit an increase in crypt micronuclei [27]. Strengths of the aforementioned study include an extended repeated-dose exposure, two time points of analysis (day 8 and day 91), six treatment groups in addition to a negative control, and evaluations on a "per fully intact crypt" and "per entire slide" basis. Because the previous study was not originally designed as an in vivo micronucleus study, it lacked a concurrent positive control for micronucleus induction in the crypt compartment. Herein, we have replicated the study findings of O'Brien et al. [13,27] with a 7-day GLP in vivo micronucleus assay using Swiss roll samples. Consistent with our previous studies, no increases in aberrant nuclei or 7-H2AX immunostaining were detected in the crypt compartment of mice exposed to Cr(V1). By contrast, exposure to CPH led to increased MN and KN in the crypts, as well as increased 7-H2AX immunoreactivity.

The negative genotoxicity in the crypts of mice exposed to Cr(V1) is supported by the XRF mapping that indicated a near absence of Cr signal from the crypt region. 1t is unlikely that the Cr signal detected in villi 24 h after the last exposure to Cr(V1) originated in the crypt compartment because tritiated thymidine labeling studies indicate that cells in the lower half of the crypt would only reach the upper crypt and lower villus by 24 h [41]. Considering further that the crypt compartment expanded, it might take longer for labeled crypt enterocytes to reach the villi. The abundant presence of micronuclei in the crypts of mice 24 h after bolus exposure to CPH further indicates that crypt enterocytes did not have sufficient time to migrate out of the crypts and onto the villi. Furthermore, very little Cr was

detected in the crypts of transverse sections from mice exposed to 180 ppm Cr(V1) for 90 days without a 24 h water-only exposure period prior to study termination [14]. 1mportantly, chromium in intestinal villi poses little carcinogenic risk because villus entero-cytes are differentiated cells that soon slough into the intestinal lumen [42]. To date, none of the Cr(V1) drinking water studies have reported aberrant foci, a potential preneoplastic lesion, in intestinal villi [1,2,14,30].

The data herein indicate that Cr(V1) concentrations as high as 180ppmdo not adversely effect the crypt compartment. This study and others reviewed elsewhere [31] support that the mode of action for Cr(V1)-induced intestinal cancer in B6C3F1 mice does not involve direct genotoxicity, but rather chronic villous wounding and compensatory crypt enterocyte hyperplasia. 1ncreases in the stem cell population and/or increases in lifetime stem cell divisions are known risk factors for carcinogenesis [17,43]. Considering that Cr(V1) concentrations in U.S. drinking water supplies average 0.001 ppm (range: 0.03-97 ppb) [44,45], which are many log-fold lower than concentrations expected to cause villous damage, it is unlikely that environmental levels of Cr(V1) pose a carcinogenic risk to the small intestine.

Competing interest

The authors' employment affiliation is as shown on the cover page. ToxStrategies is a private consulting firm providing services to private and public organizations on toxicology and risk assessment issues. Some authors [C.T., M.H., D.P., L.H., M.S.,J.W.] have presented study findings in meetings with regulators including public meetings on behalf of the Cr(V1) Panel of the American Chemistry Council (ACC). The contents of this article reflect solely the views of the authors.

Sources of funding

This work was supported by the Cr(V1) Panel of the American Chemistry Council. Beamline X27A is supported in part by the U.S. Department of Energy (DOE) - Geosciences (DE-FG02-92ER14244 to The University of Chicago - CARS). Use of the NSLS was supported by the DOE, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886.

Acknowledgments

We thank Dr. J. Gregory Hixon (ToxStrategies) for statistical consultation, Shelley Gruntz (EPL) for assistance in preparation of tissue samples, Nancy Harris (EPL) for immunostaining, and Eric Choi (EPL) for slide photography. The views expressed in this article are solely those of the authors and do not reflect the official policies or positions of the ACC, Department of the Army, the Department of Defense, or any other department or agency of the US government.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mrgentox.2015. 05.004

References

[1] NTP, National Toxicology Program technical report on the toxicology and carcinogenesis studies ofsodium dichromate dihydrate (CAS No. 7789-12-0) in F344/N rats and B6C3F1 mice (drinking water studies), NTP TR 546, NIH Publication No. 08-5887, (2008).

[2] M.D. Stout, R.A. Herbert, G.E. Kissling, B.J. Collins, G.S. Travlos, K.L. Witt, R.L. Melnick, K.M. Abdo, D.E. Malarkey, M.J. Hooth, Hexavalent chromium is

[9 [10

[12 [13

[15 [16

[20 [21

[22 [23 [24

carcinogenic to F344/N rats and B6C3F1 mice after chronic oral exposure, [25

Environ. Health Perspect. 117 (2009) 716-722.

S. De Flora, Metabolic deactivation of mutagens in the Salmonella-microsome [26 test, Nature 271 (1978) 455-456.

S. De Flora, A. Camoirano, M. Bagnasco, C. Bennicelli, G.E. Corbett, B.D. Kerger, Estimates of the chromium(Vl) reducing capacity in human body [27

compartments as a mechanism for attenuating its potential toxicity and carcinogenicity, Carcinogenesis 18 (1997) 531-537.

C.R. Kirman, L.L. Aylward, M. Suh, M.A. Harris, C.M. Thompson, L.C. Haws, D.M. Proctor, S.S. Lin, W. Parker, S.M. Hays, Physiologically based pharmacokinetic

model for humans orally exposed to chromium, Chem. Biol. Interact. 204 [28

(2013) 13-27.

D.M. Proctor, M. Suh, L.L. Aylward, C.R. Kirman, M.A. Harris, C.M. Thompson, H. Gurleyuk, R. Gerads, L.C. Haws, S.M. Hays, Hexavalent chromium reduction kinetics in rodent stomach contents, Chemosphere 89 (2012) 487-493. [29 NTP, National Toxicology Program technical report on the toxicology and carcinogenesis studies of chromium picolinate monohydrate (CAS NO. [30 27,882-76-4) in F344/N rats and B6C3F1 mice (feed studies), NlH Publication

No. 08-5897, (2008).

M.D. Stout, A. Nyska, B.J. Collins, K.L. Witt, G.E. Kissling, D.E. Malarkey, M.J. Hooth, Chronic toxicity and carcinogenicity studies of chromium picolinate monohydrate administered in feed to F344/N rats and B6C3F1 mice for 2 [31

years, Food Chem. Toxicol. 47 (2009) 729-733.

K.P. Nickens, S.R. Patierno, S. Ceryak, Chromium genotoxicity: a double-edged sword, Chem. Biol. Interact. 188(2010) 276-288.

T.J. O'Brien, S. Ceryak, S.R. Patierno, Complexities of chromium [32

carcinogenesis: role of cellular response repair and recovery mechanisms,

Mutat. Res. 533 (2003) 3-36. [33

N. McCarroll, N. Keshava, J. Chen, G. Akerman, A. Kligerman, E. Rinde, An

evaluation of the mode of action framework for mutagenic carcinogens case

study ll: chromium (VI), Environ. Mol. Mutagen. 51 (2010) 89-111. [34

A. Zhitkovich, Chromium in drinking water: sources, metabolism, and cancer

risks, Chem. Res. Toxicol. 24 (2011) 1617-1629.

T.J. O'Brien, H. Ding, M. Suh, C.M. Thompson, M.A. Harris, J.G. Hixon, B.L. [35

Parsons, M.B. Myers, D.M. Proctor, K-Ras codon 12 GGT to GAT mutation is not elevated in the duodenum of mice subchronically exposed to hexavalent chromium in drinking water, in: 51st Annual Meeting of the Society of Toxicology, San Francisco, CA, 2012. [36

C.M. Thompson, J. Seiter, M.A. Chappell, R.V. Tappero, D.M. Proctor, M. Suh, J.C. Wolf, L.C. Haws, R. Vitale, L. Mittal, C.R. Kirman, S.M. Hays, M.A. Harris, Synchrotron-based imaging of chromium and gamma-H2AX immunostaining [37 in the duodenum following repeated exposure to Cr(Vl) in drinking water, Toxicol. Sci. 143 (2015) 16-25.

C.S. Potten, C. Booth, D.M. Pritchard, The intestinal epithelial stem cell: the [38

mucosal governor, Int. J. Exp. Pathol. 78 (1997) 219-243.

S.M. Cohen, E.B. Gordon, P. Singh, G.T. Arce, A. Nyska, Carcinogenic mode of

action of folpet in mice and evaluation of its relevance to humans, Crit. Rev. [39

Toxicol. 40 (2010) 531-545.

C. Tomasetti, B. Vogelstein, Cancer etiology. Variation in cancer risk among

tissues can be explained by the number of stem cell divisions, Science 347

(2015)78-81. [40

N. Barker, R.A. Ridgway, J.H. van Es, M. van de Wetering, H. Begthel, M. van

den Born, E. Danenberg, A.R. Clarke, O.J. Sansom, H. Clevers, Crypt stem cells

as the cells-of-origin of intestinal cancer, Nature 457 (2009) 608-611. [41

P. Rizk, N. Barker, Gut stem cells in tissue renewal and disease: methods,

markers, and myths Wiley interdisciplinary reviews, Syst. biol. Med. 4 (2012)

475-496. [42

L.G. van der Flier, H. Clevers, Stem cells, self-renewal, and differentiation in

the intestinal epithelium, Annu. Rev. Physiol. 71 (2009) 241-260.

C. Blanpain, E. Fuchs, Stem cell plasticity. Plasticity of epithelial stem cells in [43

tissue regeneration, Science 344 (2014) 1242281.

C. Moolenbeek, E.J. Ruitenberg, The Swiss roll: a simple technique for [44

histological studies of the rodent intestine, Lab. Anim. 15 (1981) 57-59.

OECD, Guideline for Testing of Chemicals. 2014. Test Guideline 474, -

Mammalian Erythrocyte Micronucleus Test, OECD, Paris, France, (2014). [45

OECD, Guideline for Testing of Chemicals. 1997. Test Guideline 474, -

Mammalian Erythrocyte Micronucleus Test, OECD, Paris, France, (1997).

P. Chidiac, M.T. Goldberg, Lack of induction of nuclear aberrations by captan in mouse duodenum, Environ. Mutagen. 9 (1987) 297-306. M.T. Goldberg, D.H. Blakey, W.R. Bruce, Comparison of the effects of 1 2-dimethylhydrazine and cyclophosphamide on micronucleus incidence in bone marrow and colon, Mutat. Res. 109 (1983) 91-98. T.J. O'Brien, H. Ding, M. Suh, C.M. Thompson, B.L. Parsons, M.A. Harris, W.A. Winkelman, J.C. Wolf, J.G. Hixon, A.M. Schwartz, M.B. Myers, L.C. Haws, D.M. Proctor, Assessment of K-Ras mutant frequency and micronucleus incidence in the mouse duodenum following 90-days of exposure to Cr(Vl) in drinking water, Mutat. Res. 754 (2013) 15-21.

C.M. Thompson, D.M. Proctor, L.C. Haws, C.D. Hebert, S.D. Grimes, H.G. Shertzer, A.K. Kopec, J.G. Hixon, T.R. Zacharewski, M.A. Harris, lnvestigation of the mode of action underlying the tumorigenic response induced in B6C3F1 mice exposed orally to hexavalent chromium, Toxicol. Sci. 123 (2011) 58-70. A. Agresti, A survey of exact inference for contingency tables, Stat. Sci. 7 (1992)131-153.

NTP, National Toxicology Program technical report on the toxicity studies of sodium dichromate dihydrate (CAS No. 7789-12-0) administered in drinking water to male and female F344/N rats and B6C3F1 mice and male BALB/c and am3-C57BL/6 mice, NTP Toxicity Report Series Number 72, NlH Publication No. 07-5964, (2007).

C.M. Thompson, D.M. Proctor, M. Suh, L.C. Haws, C.R. Kirman, M.A. Harris, Assessment of the mode of action underlying development of rodent small intestinal tumors following oral exposure to hexavalent chromium and relevance to humans, Crit. Rev. Toxicol. 43 (2013) 244-274. W.M. Bonner, C.E. Redon, J.S. Dickey, A.J. Nakamura, O.A. Sedelnikova, S. Solier, Y. Pommier, GammaH2AX and cancer, Nat. Rev. Cancer 8 (2008) 957-967. K. Rothkamm, S. Barnard, J. Moquet, M. Ellender, Z. Rana, S. Burdak-Rothkamm, DNA damage foci: meaning and significance, Environ. Mol. Mutagen. (2015).

C.M. Thompson, Y. Fedorov, D.D. Brown, M. Suh, D.M. Proctor, L. Kuriakose, L.C. Haws, M.A. Harris, Assessment of Cr(Vl)-lnduced cytotoxicity and genotoxicity using high content analysis, PLoS One 7 (2012) e42720. Z. DeLoughery, M.W. Luczak, S. Ortega-Atienza, A. Zhitkovich, DNA double-strand breaks by Cr(Vl) are targeted to euchromatin and cause ATR-dependent phosphorylation of histone H2AX and its ubiquitination, Toxicol. Sci. 143 (2015) 54-63.

M. Reynolds, L. Stoddard, l. Bespalov, A. Zhitkovich, Ascorbate acts as a highly potent inducer of chromate mutagenesis and clastogenesis: linkage to DNA breaks in G2 phase by mismatch repair, Nucleic Acids Res. 35 (2007) 465-476. K.J. McManus, M.J. Hendzel, ATM-dependent DNA damage-independent mitotic phosphorylation of H2AX in normally growing mammalian cells, Mol. Biol. Cell 16 (2005) 5013-5025.

N.G. Medvedeva, l.V. Panyutin, l.G. Panyutin, R.D. Neumann, Phosphorylation of histone H2AX in radiation-induced micronuclei, Radiat. Res. 168 (2007) 493-498.

A.K. Kopec, S. Kim, A.L. Forgacs, T.R. Zacharewski, D.M. Proctor, M.A. Harris, L.C. Haws, C.M. Thompson, Genome-wide gene expression effects in B6C3F1 mouse intestinal epithelia following 7 and 90days of exposure to hexavalent chromium in drinking water, Toxicol. Applied Pharmacol. 259 (2012) 13-26. K. ljiri, C.S. Potten, Response of intestinal cells of differing topographical and hierarchical status to ten cytotoxic drugs and five sources of radiation, Br. J. Cancer 47 (1983) 175-185.

C.S. Potten, Stem cells in gastrointestinal epithelium: numbers characteristics and death philosophical transactions of the Royal Society of London, Series B Biol. sci. 353 (1998) 821-830.

C.S. Potten, M. Loeffler, Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt, Development 110 (1990) 1001-1020.

S.M. Cohen, L.B. Ellwein, Cell proliferation in carcinogenesis, Science 249 (1990) 1007-1011.

L.S. McNeill, J.E. Mclean, J.L. Parks, M.A. Edwards, Hexavalent chromium review, part 2: chemistry, occurrence, and treatment, J. Am. Waterworks Assoc. 104 (2012) E395-E405.

U.S. EPA, The Third Unregulated Contaminant Monitoring Rule (UCMR3): Occurrence Data., (2014).