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This article presents the historical control data of spontaneous tumors in Tg.rasH2 published in 2013 (2004–2012) and compares and contrasts it to more recent data collected from 2013 to 2018, reporting differences in the average percentage incidences or incidence ranges as well as the incidence of new tumors. In 2013, we published a comprehensive review of spontaneous tumors in Tg.rasH2 mice used in 26-week carcinogenicity studies, which included data from control dose groups from 26 studies and a total of 710 mice per sex. The total database, now including the more recent data, has nearly doubled the number of animals, completing to date a total of 52 studies in males and 51 studies in females for a total of 1,615 male mice and 1,560 female mice, respectively. In this article, we compare the data collected from 2004 to 2012 against the data collected from 2013 to 2018 and the overall tumor incidence change. [ABSTRACT FROM AUTHOR]

Three levels of nasal cavity sections (anterior, middle, and most posterior) are routinely examined as protocol required tissues in our 26-week carcinogenicity studies involving Tg.rasH2 mice. Exudative inflammation of the nasal cavity was noted in the most posterior section of both males and females that were administered vehicle and/or test article via oral gavage, particularly when the vehicle and/or test article had irritant properties, was in the form of a salt, had a low pH, and/or was viscous. The exudative inflammatory lesion was characterized by the presence of eosinophilic proteinaceous fluid, fibrin, mucin, sloughed cells, and degenerate neutrophils within the nasal cavities. In lesions of increased severity, there was often degeneration, necrosis, and erosion of the underlying mucosa. Often, there was hyperplasia as well as squamous metaplasia of the mucosa. Retrospective analysis of our data, involving thirty-two 26-week Tg.rasH2 carcinogenicity studies, revealed that despite the presence of these exudative inflammatory changes with degeneration, necrosis, and mucosal hyperplasia, progression to tumor formation in the nasal cavities was rare and the incidence of nasal tumors was comparable in animals with or without exudative inflammatory lesions. [ABSTRACT FROM AUTHOR]

Tg.rasH2 mice are predisposed to hemangiosarcomas. Following the spleen, the uterus is the second most commonly affected organ in the female mice. Female mice are also predisposed to spontaneous vascular proliferative lesions on the serosal surface of the uterus, in which there is proliferation of normal vessels that are lined by well-differentiated endothelial cells. The hemangiosarcomas and vascular proliferative lesions can occur independently. In our facility, we have recorded a total of 47 uterine hemangiosarcomas in 3,985 female Tg.rasH2 mice assigned to various groups in 38 studies. Of these 47 cases, we have seen 22 (46.8%) cases where there was a clear progression of the serosal uterine vascular proliferative lesion into a hemangiosarcoma. In the remaining 25 (53.2%) cases, the uterine hemangiosarcomas involved myometrium and endometrium, but there was no serosal vascular proliferation. Based on the retrospective analysis of our data, we demonstrate that the vascular proliferative lesions noted on the serosal surfaces can progress to hemangiosarcomas and therefore these vascular proliferative lesions should be considered as preneoplastic lesions. [ABSTRACT FROM AUTHOR]

We recently conducted a retrospective analysis of data collected from 29 Tg.rasH2 carcinogenicity studies conducted at our facility to determine how successful was the strategy of choosing the high dose of the 26-week studies based on an estimated maximum tolerated dose (MTD). As a result of our publication, 2 counterviews were expressed. Both counterviews illustrate very valid points in their interpretation of our data. In this article, we would like to highlight clarifications based on several points and issues they have raised in their papers, namely, the dose-level selection, determining if MTD was exceeded in 26-week studies, and a discussion on the number of dose groups to be used in the studies. [ABSTRACT FROM AUTHOR]

Exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water was associated with an increased incidence of oral tumors in F344 rats in a 2-year cancer bioassay conducted by the National Toxicology Program. These tumors primarily occurred at 180 ppm Cr(VI) and appeared to originate from the gingival mucosa surrounding the upper molar teeth. To investigate whether these tumors could have resulted from a mutagenic mode of action (MOA), a transgenic mutation assay based on OECD Test Guideline 488 was conducted in Big Blue® TgF344 rats. The mutagenic oral carcinogen 4-nitroquinoline-1-oxide (4-NQO) served as a positive control. Mutant frequency was measured in the inner gingiva with adjacent palate, and outer gingiva with adjacent buccal tissue. Exposure to 10 ppm 4-NQO in drinking water for 28 days increased mutant frequency in the cII transgene significantly, from 39.1 ± 7.5 × 10−6 to 688 ± 250 × 10−6 in the gingival/buccal region, and from 49.8 ± 17.8 × 10−6 to 1818 ± 362 × 10−6 in the gingival/palate region. Exposure to 180 ppm Cr(VI) in drinking water for 28 days did not significantly increase the mutant frequency in the gingival/buccal (44.4 ± 25.4 × 10−6) or the gingival/palate (57.8 ± 9.1 × 10−6) regions relative to controls. These data indicate that high (∼180,000 times expected human exposure), tumorigenic concentrations of Cr(VI) did not significantly increase mutations in the gingival epithelium, and suggest that Cr(VI) does not act by a mutagenic MOA in the rat oral cavity. Environ. Mol. Mutagen. 56:621-628, 2015. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced degradation of aryl hydrocarbon receptor (AhR) is inhibited by MG-132, a potent inhibitor of the 26S proteasome. Therefore, the current study aims to address the effect of MG-132 on the AhR-regulated gene, cytochrome P450 1a1 (Cyp1a1), using murine hepatoma Hepa 1c1c7 cells. Our results showed that MG-132 at the highest concentration tested, 10μM significantly increased the Cyp1a1 at mRNA, protein and catalytic activity levels through a transcriptional mechanism. On the other hand, MG-132 further potentiated the TCDD-mediated induction of Cyp1a1 at mRNA but not at protein level. In contrast, MG-132 significantly inhibited the TCDD-mediated induction of Cyp1a1 catalytic activity. In addition, we showed that the decrease in Cyp1a1 catalytic activity is not Cyp specific, as MG-132 significantly inhibited Cyp2b1 and total cytochrome P450 catalytic activities. These results prompted us to examine the effect of MG-132 on total cellular heme content and heme oxygenase-1 (HO-1) mRNA, a rate limiting enzyme of heme degradation. Our results showed that MG-132 significantly induced HO-1 mRNA in a concentration-dependent manner. Furthermore, MG-132 potentiated the induction of HO-1 mRNA by TCDD in a concentration-dependent manner. The induction of HO-1 mRNA level coincided with a decrease in total cellular heme content. In conclusion, the present study demonstrates for the first time that MG-132, despite of increasing Cyp1a1 mRNA expression, it decreases its activity probably through decreasing its heme content. [Copyright &y& Elsevier]

Abstract: Heavy metals alter the carcinogenicity of AhR ligands by modulating the induction of the Cyp1a1 enzyme, but the mechanism(s) remain unresolved. In this study, the effect of the heavy metals, As3+, Cd2+, and Cr6+, on the transcriptional and posttranscriptional regulation of the Cyp1a1 gene was investigated in Hepa 1c1c7 cells. A time-dependent study showed that all metals significantly induced the basal Cyp1a1 mRNA, but only As3+ and Cd2+ further potentiated the inducible Cyp1a1 mRNA level. Alternately, Cr6+ inhibited the TCDD-mediated induction of Cyp1a1 mRNA. As3+ potentiated the induction of Cyp1a1 mRNA by TCDD only at 3h and 6h after treatment. Cd2+ on the other hand further potentiated the induction up to 24h after treatment. The metal-mediated induction of Cyp1a1 mRNA was further potentiated by the protein synthesis inhibitor, cycloheximide and the 26S proteasome inhibitor, MG-132, but completely inhibited by the RNA transcription inhibitor, actinomycin-D, implying a transcriptional regulation of the Cyp1a1 gene by the heavy metals. Not surprisingly, Cd2+ and Cr6+ activated the DNA-binding capacity of the AhR for the xenobiotic responsive element, as measured by the electrophoretic-mobility shift assay while all three metals induced AhR-dependent luciferase reporter gene expression in transiently transfected Hepa 1c1c7 cells. On the other hand, only As3+ increased the Cyp1a1 mRNA half-life while Cd2+ and Cr6+ increased the Cyp1a1 protein half-life, suggesting the involvement of posttranslational modifications. A significant decrease in TCDD-mediated induction of Cyp1a1 activity was associated with an increase in HO-1 mRNA levels and a concomitant decrease in cellular heme content after all metal treatments. We clearly demonstrated that As3+, Cd2+, and Cr6+ increase Cyp1a1 mRNA levels at the transcriptional and posttranscriptional levels while decreasing Cyp1a1 activity at the posttranslational level. [Copyright &y& Elsevier]

Abstract: Heavy metals alter the capacity of AhR ligands to induce the bioactivating phase I and the detoxifying phase II xenobiotic-metabolizing enzymes but the mechanism(s) remain unknown. In the present study, we evaluated the role of As3+-, Cd2+-, and Cr6+-induced oxidative stress on the expression of Cyp1a1, Nqo1, and Gst ya in Hepa 1c1c7 cells. Both Cd2+ and Cr6+, but not As3+, increased the production of ROS. However, all metals increased cellular GSH content and heme oxygenase-1 mRNA levels. Although all three metals inhibited the induction of Cyp1a1 activity by TCDD, Cyp1a1 mRNA levels were potentiated. When cellular GSH was depleted with buthionine-(S,R)-sulfoximine (BSO), Cyp1a1 mRNA expression was further potentiated whereas Cyp1a1 activity was further inhibited. In parallel, pretreatment with the antioxidant N-acetylcysteine (NAC) did not alter Cyp1a1 mRNA expression but completely abrogated the inhibition of Cyp1a1 activity induction by all three metals. On the other hand, all three metals, alone or in the presence of TCDD, enhanced Nqo1 and Gst ya mRNA levels and Nqo1 activity. These effects were potentiated in the presence of BSO and abrogated with NAC. Our data clearly show that As3+-, Cd2+-, and Cr6+-induced oxidative stress modulates Cyp1a1 at transcriptional and posttranscriptional levels but induces Nqo1 and Gst ya at the transcriptional level. [Copyright &y& Elsevier]

Polycyclic aromatic hydrocarbons have been shown to cause oxidative stress in vitro and in vivo in various animal models but the mechanisms by which these compounds produce oxidative stress are unknown. In the current study we have investigated the role of the aryl hydrocarbon receptor (AHR) in the production of reactive oxygen species (ROS) by its cognate ligands and the consequent effect on cyp1a1 activity, mRNA and protein expressions. For this purpose, Hepa 1c1c7 cells wild-type (WT) and C12 mutant cells, which are AHR-deficient, were incubated with increasing concentrations of the AHR-ligands, benzo[a]pyrene (B[a]P, 0.25-25 μM), 3-methylcholanthrene (3MC, 0.1-10 μM) and β-naphthoflavone (βNF, 1-50 μM). The studied AHR-ligands dose-dependently increased lipid peroxidation in WT but not in C12 cells. However, only B[a]P and βNF, at the highest concentrations tested, significantly increased H 2 O 2 production in WT but not C12 cells. The increase in lipid peroxidation and H 2 O 2 production by AHR-ligands were accompanied by a decrease in the cyp1a1 catalytic activity but not mRNA or protein expressions, which were significantly induced in a dose-dependent manner by all AHR-ligands, suggesting a post-translational mechanism is involved in the decrease of cyp1a1 activity. The AHR-ligand-mediated decrease in cyp1a1 activity was reversed by the antioxidant N -acetylcysteine. Our results show that the AHR-ligands induce oxidative stress by an AHR-dependent pathway. [ABSTRACT FROM AUTHOR]

Aryl hydrocarbon receptor (AhR) ligands and heavy metals are environmental co-contaminants and their molecular interaction may disrupt the coordinated regulation of AhR-dependent phase I and II drug metabolizing enzymes. To determine the effect of heavy metals on the AhR-regulated genes: cytochrome P4501A1 (Cyp1a1), NAD(P)H: quinone oxidoreductase (QOR) and glutathione S-transferase Ya (GST Ya), murine hepatoma Hepa 1c1c7 cells were treated with increasing concentrations of As3+ (1–10 μM), Cd2+ (1–25 μM) and Cr6+ (1–25 μM) with or without the AhR ligands: 2,3,7,8-tetrachlorodibenzo-p-dioxin (0.1 nM), 3-methylcholanthrene (0.25 μM), β-naphthoflavone (10 uM), or benzo[a]pyrene (1 μM). Our results show that AhR ligands alone and As3+ or Cd2+ alone increased the catalytic activities and mRNA levels of all AhR-regulated genes. When metals were co-administered with an AhR ligand, all three metals inhibited the induction of Cyp1a1 activity by the AhR ligands but potentiated its mRNA and protein expression. In addition, all metals enhanced QOR and GST Ya at the activity and mRNA levels but modulated their induction by AhR ligands in a concentration, metal, and AhR ligand-dependent manner. Generally, Cr6+ inhibited while As3+ and Cd2+ potentiated the induction of QOR and GST Ya activities and mRNA levels. The three metals enhanced the expression of heme oxygenase-1, which coincided with the changes in the phase I and phase II enzyme activities. These results show that the ability of metals to alter the capacity of AhR ligands to induce the bioactivating phase I and the detoxifying phase II enzymes will influence the carcinogenicity and mutagenicity of the AhR ligands. [Copyright &y& Elsevier]

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 180 ppm 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 180 ppm Cr(VI) significantly increased the number of crypt enterocytes. Micronuclei and γ-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 γ-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., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)