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Mutation Research/Genetic Toxicology and Environmental Mutagenesis
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Assessment of K-Ras mutant frequency and micronucleus incidence in the mouse duodenum following 90-days of exposure to Cr(VI) in drinking water
Travis J. O'Brien3, Hao Dinga, Mina Suhb, Chad M. Thompsonc, Barbara L. Parsonsd, Mark A. Harrisc, William A. Winkelmane, Jeffrey C. Wolfe, J. Gregory Hixonf, Arnold M. Schwartzg, Meagan B. Myersd, Laurie C. Hawsf, Deborah M. Proctorb *
a George Washington University, Department of Pharmacology and Physiology, Washington, DC 20037, United States b ToxStrategies, Inc., Rancho Santa Margarita, CA 92688, United States c ToxStrategies, Inc., Katy, TX 77494, United States
d US Food and Drug Administration, National Center for Toxicological Research (NCTR), Jefferson, AR 72079, United States e Experimental Pathology Laboratories, Inc., Sterling, VA 20166, United States f ToxStrategies, Inc., Austin, TX 78759, United States
g George Washington University, Department of Pathology, Washington, DC 20037, United States
ABSTRACT
Chronic exposure to high concentrations of hexavalent chromium [Cr(VI)] as sodium dichromate dihy-drate (SDD) in drinking water induces duodenal tumors in mice, but the mode of action (MOA) for these tumors has been a subject of scientific debate. To evaluate the tumor-site-specific genotoxicity and cytotoxicity of SDD in the mouse small intestine, tissue pathology and cytogenetic damage were evaluated in duodenal crypt and villus enterocytes from B6C3F1 mice exposed to 0.3-520 mg/L SDD in drinking water for 7 and 90 days. Allele-competitive blocker PCR (ACB-PCR) was used to investigate the induction of a sensitive, tumor-relevant mutation, specifically in vivo K-Ras codon 12 GAT mutation, in scraped duodenal epithelium following 90 days of drinking water exposure. Cytotoxicity was evident in the villus as disruption of cellular arrangement, desquamation, nuclear atypia and blunting. Following 90 days of treatment, aberrant nuclei, occurring primarily at villi tips, were significantly increased at >60 mg/L SDD. However, in the crypt compartment, there were no dose-related effects on mitotic and apoptotic indices or the formation of aberrant nuclei indicating that Cr(VI)-induced cytotoxicity was limited to the villi. Cr(VI) caused a dose-dependent proliferative response in the duodenal crypt as evidenced by an increase in crypt area and increased number of crypt enterocytes. Spontaneous K-Ras codon 12 GAT mutations in untreated mice were higher than expected, in the range of 10-2 to 10-3; however no treatment-related trend in the K-Ras codon 12 GAT mutation was observed. The high spontaneous background K-Ras mutant frequency and Cr(VI) dose-related increases in crypt enterocyte proliferation, without dose-related increase in K-Ras mutant frequency, micronuclei formation, or change in mitotic or apoptotic indices, are consistent with a lack of genotoxicity in the crypt compartment, and a MOA involving accumulation of mutations late in carcinogenesis as a consequence of sustained regenerative proliferation.
Published by Elsevier B.V.
ARTICLE INFO
Article history:
Received 11 December 2012
Received in revised form 27 March 2013
Accepted 28 March 2013
Available online 9 April 2013
Keywords:
Hexavalent chromium
Mutation
Micronucleus
Cancer risk assessment
* Corresponding author at: ToxStrategies, Inc., 23142 Arroyo Vista, Rancho Santa Margarita, CA 92688, United States.
E-mail addresses: phmtjo@gwu.edu (T.J. O'Brien), dinghao@gwmail.gwu.edu (H. Ding), msuh@toxstrategies.com (M. Suh), cthompson@toxstrategies.com (C.M. Thompson), barbara.parsons@fda.hhs.gov (B.L. Parsons), mharris@toxstrategies.com (M.A. Harris), BWinkelman@epl-inc.com (W.A. Winkelman), JWolf@epl-inc.com (J.C. Wolf), ghixon@toxstrategies.com (J.G. Hixon), aschwartz@mfa.gwu.edu (A.M. Schwartz), meagan.myers@fda.hhs.gov (M.B. Myers), lhaws@toxstrategies.com (L.C. Haws), dproctor@toxstrategies.com (D.M. Proctor).
1. Introduction
Chronic exposure to high concentrations of hexavalent chromium [Cr(Vl)], as sodium dihydrate dichromate (SDD), in drinking water was recently shown to cause small intestinal tumors in mice [1,2]. These tumors occurred at concentrations that are orders of magnitude greater than typical environmental exposures to Cr(Vl). Specifically, mouse duodenal tumors were observed at 57-516 mg/L SDD or ~20-180 mg/L Cr(Vl), whereas environmental exposures for humans are common at drinking water concentrations of 0.001-0.005 mg/L Cr(Vl) [3]. Ingestion of Cr(Vl) has generally not been considered carcinogenic at environmentally relevant exposures because most Cr(Vl) is reduced to trivalent
1383-5718/$ - see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.mrgentox.2013.03.008
chromium [Cr(III)] in the acidic conditions of the stomach thereby limiting uptake [4]. However, because Cr(VI) is genotoxic and thus may potentially act by a mutagenic mode of action (MOA) to cause mouse intestinal tumors [5,6], the potential for Cr(VI) to pose a cancer risk in the low dose range has been the subject of significant scientific debate and study [3,7]. Several MOAs have been proposed for Cr(VI)-induced small intestinal carcinogenesis [3,5,6]. If mutation is an early event for the MOA, then mutations in target tissues are expected to increase as a function of dose, and detected at lower doses and prior to the onset of pre-neoplastic and neoplas-tic lesions. Considering that environmental exposure to low levels of Cr(VI) in drinking is widespread [8-11], whether Cr(VI) acts by a mutagenic MOA and poses a potential cancer risk at low exposures are important questions for protecting public health.
The analysis of specific hotspot oncogene mutations is a powerful approach for understanding MOA. Further, in vivo mutational analysis in the tissues where tumors occur is the highest tier of evidence for determining whether a chemical operates via a mutagenic MOA as delineated in the U.S. EPA's draft Framework for Determining a Mutagenic Mode of Action for Carcinogenicity [12]. Studies using model mutagenic carcinogens (aristolochic acid, azoxymethane, benzo[a]pyrene, N-hydroxy-acetylaminofluorene, and simulated solar light) have shown that allele-specific competitive blocker-PCR (ACB-PCR) is sufficiently sensitive to detect the early, dose-related increases in mutations that lead to tumor formation. In addition, several studies show that K-Ras codon 12 GGT to GAT mutation, a mutation frequently detected in normal/control tissues (human/rodents), may be amplified during carcinogenesis, even when the mutational specificity of the mutagen is other than the G to A mutation being measured [13,14]. Both K-Ras codon 12 GGT to GAT and GGT to GTT mutations were recently shown to occur following 4 weeks of inhalation exposure to ethylene oxide in Big Blue B6C3F1 mouse lung tissue [15]. Because ethylene oxide induces oxidative stress, G to T mutation was hypothesized to occur, but the K-Ras codon 12 GAT mutation was the more sensitive endpoint, more than K-Ras codon 12 GTT or cII neutral reporter gene mutations [15]. These results are interpreted as meaning that amplification of spontaneous K-Ras codon 12 GAT mutation, potentially through cooperative interactions with other chemically induced mutations, can serve as a sensitive, generic reporter of the early effects of a carcinogen.
Although mutational specificity was not reported by the National Toxicology Program (NTP) for the mouse small intestinal tumors, K-Ras may be the mutation target for mouse small intestinal tumors induced by Cr(VI). It is among the most frequently activated oncogenes with mutations at codons 12, 13, and/or 61 found in mammalian tumors [16]. K-Ras-mediated signaling plays a key role in controlling cell cycle progression, growth, migration, cytoskeletal changes, proliferation, differentiation, and apoptosis [16,17]. Although very little is known about the potential role that K-Ras mutations play in the formation of relatively rare duodenal tumors in rodents or humans, codon 12 GGT to GAT mutation is one of the most commonly reported mutations in human intestinal cancers [18-22]. In addition, K-Ras mutation has been shown to contribute to small intestine tumorigenesis in mice and is mutated early in carcinogenesis [23,24]. Based on these facts, K-Ras is considered a potential target oncogene, in addition to being a nonspecific reporter gene, for intestinal carcinogenesis.
The objective of this study was to assess whether mutagene-sis is an early key event in the MOA for Cr(VI)-induced duodenal carcinogenesis. Using a multifaceted approach, we investigated genotoxicity and mutagenicity in mouse duodenal epithelium following oral exposures to SDD for 90 days under conditions similar to those employed in the NTP 2-year cancer drinking water bioassay (0,14, 60,170 and 520mg/L SDD) and at two lower exposure concentrations (0.3 and 4mg/L SDD) that are more representative of
possible human exposure. Considering the unique structure of the small intestine with non-proliferating villi and proliferating crypt compartments (Fig. 1A), duodenal tissues were examined for evidence of aberrant nuclei (e.g., micronuclei) in the crypt and villus regions. Taken together, assessment of the effects of Cr(VI) on the crypt compartment and on the frequency of K-Ras codon 12 GAT mutation greatly improves our understanding of the MOA for the small intestinal tumors observed in mice following lifetime exposure to Cr(VI) in drinking water.
2. Materials and methods
2.1. Animals and tissues
Test substance, animal husbandry, and study design have been described in detail elsewhere [7]. Briefly, female B6C3F1 mice (Charles Rivers Laboratories International, Inc., Raleigh, NC) were provided ad libitum access to SDD (Sigma-Aldrich, Inc. Milwaukee, WI) in drinking water at concentrations ranging from 0.3 to 520 mg/L for 7 or 90 days (effects are referred to herein as occurring on days 8 and 91). At the time of sacrifice, intestinal sections were flushed with ice-cold phosphate buffered saline. The duodena were cut longitudinally, and the epithelium was scraped, and stored at -80 °C.
For assessment of tissue histopathology, aberrant nuclei and mitotic figures, duodenal sections were fixed in 10% neutral buffered formalin, embedded in paraffin fortransverse sectioning and sectioned at approximately 5 |xm. All procedures were carried out with the approval of the Institutional Animal Care and Use Committee at Southern Research Institute.
2.2. Assessment of crypt area, aberrant nuclei and mitotic figures in mouse duodenum
Paraffin-embedded duodenal sections (3 sections per mouse) were stained for DNA using Feulgen's stain and analyzed by Experimental Pathology Laboratories, Inc. (EPL®; Sterling, VA). Unless otherwise stated, image analysis procedures were performed according to methods described in the EPL standard operating procedures. Using the 40x microscope objective and the Virtual Slice module ofthe Stereo Investigator software, a montage image (multiple high-resolution images stitched together) was obtained. Using Image-Pro® Plus (IPP; v7.0, Media Cybernetics, Silver Spring, MD) software, the total mucosal and villous surface areas were outlined manually and the internal borders of these areas were determined automatically by the software's "Count/Size" color segmentation tool and user-defined colorimetric criteria. The crypt area was calculated by subtracting the villous area from the total mucosal area.
Mitotic and apoptotic cells were counted in fully intact crypts in order to compute mitotic and apoptotic indices. Furthermore, karyorrhectic nuclei, micronuclei (MN) and apoptotic nuclei were counted in both the villus and crypt compartments ofthree slides obtained from each animal per dose group (4-5 mice in each of 7 dose groups). Digital images were randomized prior to the cell counting, and persons performing the counts were unaware of the treatment group status of individual animals. Using IPP software, aberrant nuclei in the digital image were counted by marking each nucleus manually. Identification criteria for aberrant nuclei (i.e., micronuclei, apoptotic nuclei, and karyorrhectic nuclei) were consistent with that of Goldberg et al. [25]. Specifically, apoptotic cells were characterized by nuclei that had a smudged, heterochromatic appearance, and a discrete rounding of the cell cytoplasm was often evident. Conversely, distinctly visible chromosomal components were apparent in cells with mitotic figures. Each 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 were fragmented into small, unequally sized, dense spherical bodies, and the cytoplasmic margins of such cells were often indistinct.
Duodenal tissue sections stained with hematoxylin and eosin (H&E) were previously prepared as described in Thompson et al. [7] for microscopic examination. Tissue sections were assessed and scored for stratification, desquamation, presence of lymphocytes in the lamina propria, nuclear atypia and the ratio of villous to crypt length (Appendix A for methods and scoring results).
2.3. DNA isolation and amplification of K-Ras from mouse duodenum samples
For DNA isolation, previously reported methods [26] were followed. A region surrounding the K-Ras gene was amplified from 1 |xg of digested genomic DNA (~3 x 105 copies of a single-copy nuclear genome) per 200 |xl PCR reaction containing: 20|xl 10x Pfu reaction buffer, 16|xl of 2.5 mM dNTP, 4 ixl PfuUltra™ DNA polymerase and 4 |xl (10 |xM) each of primer (forward primer, 5'-TGGCTGCCGTCCTTTACAA-3' and reverse primer, 5'-GGCCTGCTGAAAATGACTGAGTATAAACTTGT-3'). Cycling conditions were: 2min at 94°C, 28 cycles of 1 min at 94°C, 2min at 58°C, 1 min at 72°C followed by a 7min final extension at 72°C. The PCR product (170bp) was concentrated by centrifugation under vacuum to ~50 |xl and resolved on a 1.5% TAE agarose gel,
Fig. 1. Effect of Cr(VI) treatment on intestinal tissues and structure. (A) Structure of the intestinal crypt and villi of the small intestine. Villi are projections into the lumen that are predominantly covered with mature, absorptive enterocytes, along with occasional mucus-secreting goblet cells. These cells live only for a few days, die and are shed into the lumen - approximately 1400 cells/villus/day are shed or ~2 x 106 cells per day in the small intestine of the mouse. Crypts, or glands of Lieberkuhn, are tubular invaginations of the epithelium around the villi, lined largely with younger epithelial cells. At the base of the crypts are stem cells, which divide continually and are the source of all the epithelial cells in the crypts and on the villi. (B) H&E stained image of a control mouse (0mg/L SDD) duodenum at day 91. Elongated villi and crypt structures are outlined in darker shade (blue in web version) and lighter shade (yellow in web version) boxes, respectively. (C) H&E stained duodenum of a mouse exposed to 520 mg/L SDD at day 91. Villi and crypt structures are outlined in darker shade (blue in web version) and lighter shade (yellow in web version) boxes, respectively. Arrows indicate damage to villus tips, blunting, and widening of villi. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Figure 1A was adapted from Potten and Loeffler [42].
excised and purified using the GENECLEAN® spin kit (MP Biomedicals, Solon, OH) to recover DNA in 0.5x TE buffer (5mM Tris, 0.05mM EDTA, pH 7.5). The gel-purified fragments were then divided into 2 |xl aliquots and stored at -80 °C. DNA concentrations of some aliquots were measured as previously described [27] and used to calculate the number of K-Ras codon 12 copies per |xl.
2.3.1. Allele-specific competitive-blocker PCR (ACB-PCR) standards
Synthesis of ACB-PCR standards for mouse K-Ras codon 12 GGT to GAT ACB-PCR was conducted as previously described [27]. Mutant standard (K-Ras codon 12 GAT) was serially diluted in reagent-grade water and mixed with wild-type standard (K-Ras codon 12 GGT) to generate mutant fraction (MF) standards with mutant to wild-type ratios of 10-1, 10-3, 10-4, 10-5, and 0 (wild-type alone, which defines the technical background of the assay). Five samples were analyzed per dose group, and the MF for each sample was measured in triplicate. Aliquots of the first-round PCR products synthesized from mouse duodenum samples were diluted to the same concentration as the standards, 5 x 107 K-Ras copies per |xl. Three primers (purified by polyacrylamide gel electrophoresis) were used in ACB-PCR, and an antibody-mediated hot-start ACB-PCR assay was implemented in two separate steps [27]. The PCR reaction mix was composed of 1.0 mg/mL Triton X-100, 0.1 mg/mL gelatin, 80|xM dNTP, 1.6 mM MgCl2, 1x buffer, 500 nM DP (5'-TCGTAGGGTCGTACTCATC-3'), 300 nM MSP (5'-fluorescein-ACTTGTGGTGGTTGGAGCCT-3'), 630 nM BP (5'-ACTTGTGGTGGTTGGAGCCddG-3'), 66.57 mU/^l Hotstart Stoffel and 0.4mU/|xl Perfect Match PCR enhancer (Aligent Technologies, Inc. Santa Clara, CA). The reaction mix was gently mixed and 40 |xl was added to each well of a 96-well plate containing 10 |xl of standards, a no DNA control, and samples in separate wells. The cycling conditions were as follows: 2 min at 94 °C, then 36 cycles of 30 sec at 94 °C, 45 sec at 45 °C, 1 min at 72 °C, and followed by a 4 "Csoak.
2.3.2. Analysis ofACB-PCR products
ACB-PCR products were analyzed by vertical electrophoresis through 8% polyacrylamide/Tris-acetate gels. The DNA bands were visualized using the Storm 865 scanning Fluor-Imager with an external blue laser, and fluorescence was quantified using ImageQuant 5.2. The pixels in each band of the standards were quantified, and the log10 of pixel counts of the 10-1 to 10-5 standards were plotted versus the log10 of MF. A power function was fit to the log10-log10 data to construct a standard curve.
2.4. Statistical analyses
Crypt area data were analyzed by ANOVA followed by Dunnett's or Dunn's post hoc tests. Aberrant nuclei counts were rarely observed events and were therefore analyzed via generalized linear models [28] and specifically via Poisson regression with the canonical log link, which is the natural choice for such data [29]. Statistical
packages used included R (http://www.R-project.org) and Prism 5 for Mac (Graph-Pad Software, San Diego California USA, www.graphpad.com). K-Ras codon 12 GAT mutation data were analyzed using Microsoft Excel and Prism 5.
3. Results
3.1. Assessment of cytogenetic damage
Following 90 days of exposure to Cr(VI), cytotoxicity was observed in the mouse duodenal villi, as evidenced by disruption of normal tissue architecture and desquamation of surface villus cells, nuclear injury and atypia, and a lamina propria inflammatory response involving lympho-histiocytic infiltration and multinu-cleate giant cells, and villus blunting (decreased ratio of crypt to villi length) (Appendix A Supplementary Figure 1). Compared to the elongated villi observed in the control mice (Fig. 1B), blunting of the villous structures was obvious in treated animals especially at the highest dose group (520 mg/L SDD) (Fig. 1C) (520 mg/L SDD).
A representative cross section of duodenum used in crypt area analysis is shown in Fig. 2A where crypt and villus compartments are differentiated. A representative image of cross section of duodenum that has been enhanced to dark blue pseudocolor by computer software and used in villus/crypt area ratio analysis is presented in Fig. 2B. A representative image of cross section of duodenum used in crypt enterocyte analyses is presented in Fig. 2C. Proliferation in the crypt was evident at day 8 with increase in crypt area of ~45% at >170 mg/L SDD (data not shown). At day 91, the crypt area was increased ~45% at 60 mg/L; crypt area was increased significantly at >170 mg/L, with a 2-fold or more increase compared to control (Fig. 2D). The ratio of the villus to crypt area was decreased at >170 mg/L SDD (Fig. 2E). If SDD were toxic to crypt enterocytes, then under a chronic exposure scenario, one would expect a dose-dependent decrease in crypt area accompanied by mucosal attenuation [30]. Consistent with the increase in crypt area, the number of enterocytes per crypt was also increased following exposure to >60 mg/L SDD (Fig. 2F). Bilinear dose-response modeling
Fig. 2. Quantitative assessment of intestinal structure in mice. (A) Representative cross section of duodenum used in crypt area analysis presented - results provided in (D) -green line differentiates crypt from villus compartments in web version. (B) Representative image (dark blue pseudocolor enhanced by computer software) of cross section of duodenum used in villus/crypt area ratio analysis - results presented in (E). (C) Representative image of crypt enterocyte analyses - results presented in (F) and Table 1. (D) Measures of crypt area in 3 contiguous tissue sections at day 91 (*p <0.05 by ANOVA/Dunn's). (E) Villus/crypt area ratios at days 8 and 91 (*p <0.05 by ANOVA/Dunnett's). (F) Dose-response of the number of enterocytes per crypt (mean ± s.d.). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 1
Mitotic and apoptotic indices in fully intact crypts of mice exposed to Cr(VI), as SDD, for 90 days of drinking water.
SDD (mg/L) Cells evaluated MN (total) KN (total) Mitotic index (%) Apoptotic index (%)
0 1921 0 0 1.43 ± 1.17 0.47 ± 0
0.3 1707 0 4 2.28 ± 1.07 1.0 ± 0
4 1825 0 0 2.36 ± 0.68 0.50 ± 0
14 1420 0 0 3.08 ± 0.46 0.70 ± 0
60 2386 0 0 2.43 ± 0.76 0.50 ± 0
170 2746 0 0 2.72 ± 0.97 0.84 ± 0
520 3194 0 0 2.11 ± 1.09 0.67 ± 0
Data represent counts in 10 fully intact crypts per animal (5 animals per group; 4 in 14mg/L group). MN=total number of micronuclei in crypts; KN = total number of karyorrhectic nuclei in crypts; mitotic index = cells/total cells x 100.
mitotic cells/total cells x 100; apoptotic index=apoptotic
using the 'segmented' R package of the number of enterocytes per crypt (not shown) indicated an apparent threshold in response at ~13 mg/L SDD.
To assess the potential for crypt damage, duodenal tissue sections were Feulgen-stained to visualize nuclear material so that cytogenetic analyses could be conducted. The number of mitotic and apoptotic cells were counted in fully intact crypts in order to compute mitotic index and apoptotic index. There were no significant or dose-dependent effects on either endpoint in the intact crypts (Table 1). The crypts were also evaluated for karyorrhectic nuclei and MN. The former occurs with destructive fragmentation of the nucleus upon cell death that can be either a result of programmed death (e.g., apoptosis) or necrosis, whereas MN can arise from DNA breakage or chromosomal disjunction. There were no treatment-related effects on either the number of karyorrhectic nuclei or MN in duodenal crypts (Table 1).
We also measured karyorrhectic nuclei and MN across entire sections, not just within fully intact crypts. There were no treatment-related increases in either form of nuclear damage in crypts at either day 8 or 91 (data not shown). In the villus
epithelium, there were dose-dependent increases in karyorrhectic nuclei, clustered at the tips of the villi (Table 2), consistent with the cytotoxicity reported in our 90-day study [7]. There was also an apparent dose-dependent increase in MN in the villi (Table 2).
Table 2
Total number of aberrant nuclei in duodenal villi.
SDD (mg/L) Day 8 Day91
KN MN KN MN
0 0 1 0 1
0.3 0 3 1 1
4 0 5 0 2
14 0 2 0 0
60 2 1 5' 2
170 3' 6 6' 9'
520 9' 11' 25' 9'
Values represent total number of aberrant nuclei in 15 sections (3 slides per animal; 5 animals per treatment group, except only 4 animals were examined for 14 mg/L SDD treatment group at day 91). KN = karyorrhectic nuclei; MN = micronuclei. * Significantly different from control group (p < 0.05) by Poisson regression.
Fig. 3. ACB-PCR assay of mouse K-Ras codon 12 GGT to GAT mutation in murine duodenum. (A) PAGE of ACB-PCR mutant fraction (MF) standards and duodenum DNA samples. A representative example of polyacrylamide gel images showing the 91 bp ACB-PCR product generated from MF standards including a no DNA control (NDC), and duodenal samples from Cr(Vl)-treated mice; (B) Standard curve relating pixel intensity of the 91 bp ACB-PCR product to K-Ras MF. The pixel intensity of each band of the duplicate standards was quantified, log10-transformed, and used to construct a standard curve. Both replicates are plotted. The regression equation derived from each standard curve was used to interpolate the MF of the samples run simultaneously with the standards. (C) Plot showing the average MF (based on triplicate analyses) from each animal. The dashed line is the geometric mean MF for each dose group (N=5).
3.2. Analysis of K-Ras codon 12 GAT mutations in mouse duodenum
Thirty-five samples (i.e., 7 dose groups/5 animals each) were evaluated for K-Ras codon 12 GAT mutations, and all except for one, which had a MF = 10-6, were within the ACB-PCR mutant standards range of detection. ACB-PCR products analyzed by vertical electrophoresis through 8% polyacrylamide/Tris-acetate gels are shown on Fig. 3A. The standard log curve is shown on Fig. 3B. Notably, Cr(Vl) treatment did not result in an increase in K-Ras codon 12 GGT to GAT mutations, and no dose-dependent trend was evident (Fig. 3C and Table 3).
ln an effort to determine the sensitivity of ACB-PCR relative to standard DNA sequencing (i.e., Sanger) techniques, we sequenced the region encompassing K-Ras codons 12 and 13 in select samples with MF ranging from 10-2 to 10-5. All samples were found to be wild-type for both codons 12 and 13. We further investigated the sensitivity of DNA sequencing for detecting K-Ras codon 12 GAT mutations using our mutant standards and found that this technique was insensitive even for detecting MFs as high as 10-1 (data not shown).
4. Discussion
This is the first in vivo study using ACB-PCR to quantify K-Ras codon 12 GAT mutations in mouse duodenal epithelium and the first to evaluate in vivo mutation formation in the small intestine following exposures to Cr(Vl) in drinking water. The lack of a concentration-dependent increase in K-Ras codon 12 GAT MF in proliferating tissues and at carcinogenic doses suggests that mutation is not an early key event of Cr(Vl)-induced small intestinal carcinogenesis. K-Ras codon 12 GAT was selected for analysis because it is frequently mutated in many different tissues and is shown to be involved in mouse small intestine tumorigenesis
[14,23]. Thus, K-Ras GAT mutation at codon 12 is a likely target of Cr(Vl)-induced mutagenesis [15].
Without knowing the predominant mutations in the NTP tumor tissues, other possible early mutational targets were also considered. For example, Cr(Vl) might preferentially cause a K-Ras codon 12 GTT mutation or another mutation in K-Ras; however had that occurred we would have expected to detect an accumulation of spontaneous GAT mutations occurring with the GTT mutation [15]. ln addition to K-Ras, mutations in p53 and adenomatous polyposis coli (APC) are frequently observed in intestinal tumors. Mutations in p53 appear to occur relatively late in tumorigenesis [20,32], and p53 mutations are not increased in Cr(Vl)-induced lung tumors [33]. Thus, p53 is not considered a likely somatic mutation target for Cr(Vl) in non-neoplastic tissue. lnactivation of APC results in uncontrolled cell proliferation and intestinal adenoma formation through activation of Wnt/P-catenin signaling pathways [34,35]. We have observed crypt hyperplasia in mice exposed to Cr(Vl) after only 7 days of exposure [7]. lf this proliferation were due to mutations in APC, one might expect signs of neoplasia earlier than observed in the NTP 2-year bioassay (first observed at 451 days). Moreover, toxicogenomic data collected in our mice [36] do not indicate changes in gene expression consistent with loss of APC or activation of P-catenin signaling pathways (unpublished data). Hence, the apparent absence of effects in K-Ras and APC are consistent with the lack of early tumors and metastases observed in the NTP (2008) bioassay as well as the absence of preneoplastic (e.g., focal hyperplasia) or neoplastic lesions in the 90-day Cr(Vl) drinking water study [7]. lmportantly, K-Ras codon 12 GAT mutation has been found to be a functional gene associated with tumor formation, and is expected to be amplified with mutational loading that is not necessarily specific to a chemically induced DNA lesion within K-Ras codon 12 [14,37,38].
lt is noteworthy that Meng et al. reported an accumulation ofK-Ras codon 12 GAT mutations at 28 days in the mouse lung tissues
Table 3
Summary of K-Ras codon 12 GGT to GAT mutant fraction (MF) in mouse duodenal tissue using ACB-PCR.
SDD (mg/L) Mean MF(log10) SEM(log10)
0 -2.61 0.444
0.3 -3.15 0.638
4 -3.01 0.514
14 -2.76 0.489
60 -3.79 0.486
170 -3.33 0.729
520 -2.93 0.403
MF = mutant fraction; SEM = standard error of mean.
following i.p. administration of benzo[a]pyrene which was positively correlated with benzo[a]pyrene DNA adducts [37]. To assess Cr-DNA adduct formation as a potential factor in the MOA, Cr bound to genomic DNA of scraped epithelial tissues from Cr(VI) treated mice was also measured (see Appendix B for methods and results). DNA isolation and measurement techniques were similar to those used in in vitro studies [39]. Although elevations in Cr-DNA binding were observed with increased SDD dose, no correlation with K-Ras codon 12 GAT MFs was observed, which suggests that Cr-DNA binding, as measured in our assay, is not representative of pre-mutagenic DNA damage. However, complicating the interpretations of our Cr-DNA binding data is the potential binding of Cr to DNA during the overnight digestion for DNA isolation. Indeed, chromium trichloride (Cr(III)), added to scraped intestinal tissue from control/untreated animals prior to DNA isolation, generated Cr(III)-DNA levels (Appendix B) that were comparable to those in the high dose treatment group. Hence, chromium present in (or on) scraped duodenal cells from treated animals was likely to bind to genomic DNA during DNA isolation. In addition, we examined Cr-DNA binding in (non-target) liver tissue from animals in the high dose group, and the amount of Cr associated with liver DNA was comparable to levels found in the mouse duodenum at carcinogenic doses (Appendix B). Cr-DNA binding data may be reflective of Cr uptake in the villous tissue but not indicative of Cr-DNA adducts and damage in the proliferative crypt cells based on the lack of nuclear aberrations, changes in apoptotic cell and mitotic indices in crypt enterocytes, or increase in K-Ras mutations.
The higher than expected K-Ras codon 12 GAT MFs observed in the intestinal mucosa, at all doses including controls, suggest that long-term Cr(VI) exposure may facilitate tumorigenesis through accumulation of K-Ras mutations due to prolonged regenerative cellular proliferation. Spontaneous mutation frequency in the duodenal tissues of our control mice was approximately 100-fold greater than in lung tissues of mice exposed to benzo[a]pyrene [37]. According to Greaves, the enteric epithelium has the fastest turnover rate which is exceeded only by a few rapidly growing neoplasms [30]. Thus, it is expected that prolonged chemically-induced villous injury increased replication errors and the accumulation of spontaneous mutations within stem cells that reside in the base of crypts. Intestinal injury and the proliferative response were far less evident in rats [1,40,41] and preneoplastic and neoplastic lesions were not observed in rats [1,41]. These observations further support the involvement of villous tissue injury in the MOA for Cr(VI)-induced intestinal carcinogenicity.
We also considered whetherthe villus blunting observed in mice was due to Cr(VI)-induced toxicity within the crypt compartment; however, the findings of this study do not support such. Notably, cytoplasmic vacuolization was increased in villous but not crypt enterocytes, and at concentrations lower than those that caused crypt hyperplasia. Furthermore, the number of crypt enterocytes in treated mice was increased (not decreased) following Cr(VI) exposure (Fig. 2F), and MN were not detected in duodenal crypts at either day 8 or 91 (Table 1). The lack of a change in crypt apop-totic and mitotic indices indicates that Cr(VI) was not cytotoxic to
Geometric mean MF Number of animals
2.43 x 10-3 5
7.11 x10-4 5
9.86 x10-4 5
1.75 x 10-3 5
1.61 x 10-4 5
4.67 x 10-4 5
1.19 x 10-3 5
crypt enterocytes. A few MN were detected in villous enterocytes - especially at 170 and 520 mg/L SDD (Table 2); however, given the lack of MN within the proliferative crypt compartment, it seems highly implausible that MN present in the non-proliferating villus compartment originated within the crypts - especially under the chronic exposure scenario. Instead, these nuclear aberrations are likely the result of cytotoxic mechanisms occurring in the villi. It is plausible that, under the proliferative conditions induced at the highest doses, some transition cells might be present in the villi and directly exposed to high concentrations of Cr(VI) in the lumen which could result in MN formation. Because the villus enterocytes are destined to slough into the lumen, genetic damage to these cells is unlikely to result in tumor formation.
Taken together, the multifaceted data in this study support that Cr(VI)-induced intestinal carcinogenicity is unlikely to involve K-Ras mutations or cytogenetic damage to the proliferating crypt enterocytes. Instead these data support a MOA based on chronic wounding (i.e., cytotoxicity) of the intestinal villi that induce persistent proliferative pressure on intestinal crypt cells and result in the accumulation of spontaneous mutations and tumor formation late in life.
Conflict of interest statement
Mina Suh, Chad M. Thompson, Mark A. Harris, Laurie C. Haws, and Deborah M. Proctor are independent consultants, and continue to provide consulting services to the Cr(VI) Panel of the American Chemistry Council. William A. Winkelman and Jeffrey C. Wolf are independent contractors and have no competing interests. The other authors have no competing interests.
Funding
This work was supported by the Cr(VI) Panel of the American Chemistry Council.
Acknowledgement
The contents of the manuscript do not necessarily reflect the views or policies of the U.S. FDA, nor does the mention of trade names or commercial products constitute endorsement or recommendation for use.
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. 2013.03.008.
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