A Proposal for New Strategies in Dose Selection for 26-Week Tg.Rash2 Carcinogenicity Studies.

Our experience indicates that extrapolation of doses from the maximum tolerated doses (MTD) derived from 4-week dose range finding (DRF) studies conducted in CByB6F1 may overpredict tolerability and undermine utility of the high-dose groups in 26-week carcinogenicity studies conducted in Tg.rasH2. In the 26-week carcinogenicity studies conducted in Tg.rasH2 mice, we analyzed the initial body weights, food consumption (FC), terminal body weights, body weight gain (BWG), mortality, and tumor incidence for vehicle and test article-treated dose groups for 26 studies conducted from 2014 to 2018. Although not statistically significant compared to the control dose group, the % BWG decreased in male mice of mid- and high-dose groups by >10%, whereas in females there were no differences. The mortality increased in a statistically significant manner for medium and high doses of males. In female mice, the mortality increased in the high-dose group but not in a statistically significant manner. When the cause of death (COD) was analyzed in all dose groups of both sexes, the COD due to tumors was highest in the control groups, whereas it was lowest in high-dose groups of both sexes. At the same time, the COD due to undetermined causes, which is possible indication of test article-induced toxicity, was highest in high-dose groups of both sexes. These findings together indicate that MTD derived from earlier DRF studies was exceeded when applied to 26-week carcinogenicity studies and did not serve any purpose in the outcome of these studies.

A Comparison of Spontaneous Tumors in Tg.rasH2 Mice in 26-week Carcinogenicity Studies Conducted at a Single Test Facility during 2004 to 2012 and 2013 to 2018.

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.

Tg.rasH2 Mice and not CByB6F1 Mice Should Be Used for 28-Day Dose Range Finding Studies Prior to 26-Week Tg.rasH2 Carcinogenicity Studies.

Our recent retrospective analysis of data, collected from 29 Tg.rasH2 mouse carcinogenicity studies, determined how successful the strategy of choosing the high dose for the 26-week studies was based on the estimated maximum tolerated dose (EMTD) derived from earlier 28-day dose range finding (DRF) studies conducted in CByB6F1 mice. Our analysis demonstrated that the high doses applied at EMTD in the 26-week Tg.rasH2 studies failed to detect carcinogenic effects. To investigate why the dose selection process failed in the 26-week carcinogenicity studies, the initial body weights, terminal body weights, body weight gains, food consumption, and mortality from the first 4 weeks of 26-week studies with Tg.rasH2 mice were compared with 28-day DRF studies conducted with CByB6F1 mice. Both the 26-week and the earlier respective 28-day studies were conducted with the exact same vehicle, test article, and similar dose levels. The analysis of our results further emphasizes that the EMTD and subsequent lower doses, determined on the basis of the 28-day studies in CByB6F1 mice, may not be an accurate strategy for selecting appropriate dose levels for the 26-week carcinogenicity studies in Tg.rasH2 mice. Based on the analysis presented in this article, we propose that the Tg.rasH2 mice and not the CByB6F1 mice should be used in future DRF studies. The Tg.rasH2 mice demonstrate more toxicity than the CByB6F1 mice, possibly because of their smaller size compared to CByB6F1 mice. Also, the Tg.rasH2 males appear to be more sensitive than the female Tg.rasH2 mice.

Progression of Serosal Vascular Proliferative Lesions to Hemangiosarcomas in the Uterus of the 26-Week Tg.rasH2 Mice Carcinogenicity Studies.

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.

Renal Tumors in 26-Week Tg.Rash2 Mice Carcinogenicity Studies.

We report renal tubular adenomas and a carcinoma in 26-week Tg.rasH2 mouse carcinogenicity studies, which have not been reported to date either at our facility or in other published data. However, during the year 2014, renal tubular tumors were present in 4 studies conducted at our facility. Because of their morphological similarity to the amphophilic-vacuolar (AV) phenotypic variant of renal tubule tumors noted in Sprague-Dawley and Fischer 344 rats, which are thought to be familial, as well as the genetic homogeneity of Tg.rasH2 mice, we tracked the parents of these mice with tumors in each study. The origin of these tumors could not be traced back to any of the parents or even an animal barrier, and these tumors were not attributed to the vehicle or test article. Although the exact mechanism of tumorigenesis was not known, based on the available information, the development of renal tumors in these mice was considered random and spontaneous.

Regulatory Forum Opinion Piece*: Retrospective Evaluation of Doses in the 26-week Tg.rasH2 Mice Carcinogenicity Studies: Recommendation to Eliminate High Doses at Maximum Tolerated Dose in Future Studies. A Response to the Counterpoints.

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.

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

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.

Assessment of the mutagenic potential of Cr(VI) in the oral mucosa of Big Blue® transgenic F344 rats.

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.

A robust method for assessing chemically induced mutagenic effects in the oral cavity of transgenic Big Blue® rats.

The Big Blue® (BB) in vivo mutation assay uses transgenic rodents to measure treatment-induced mutations in virtually any tissue. The BB assay can be conducted in rats or mice and is ideal for investigating tissue-specific mutagenic mode of action of tumor induction. Some tissues such as oral mucosa have not been thoroughly studied. Due to the small quantity and cartilaginous nature of oral cavity tissues, development of special prosection and DNA isolation methods was required to permit robust analysis of mutations in these tissues. Improved surgical methods permitted collection of adequate and reproducible quantities of tissue (∼45 mg gingiva/buccal and ∼30 mg gingiva/palate). Optimized DNA isolation methods included use of liquid nitrogen pulverization, homogenization, nuclei pelleting, digestion, and phenol/chloroform extraction, to yield sufficient quantities of DNA from these tissues. In preliminary optimization work, mutant frequency (MF) in tongue and gingiva was increased in rats exposed to the promutagen, benzo[a]pyrene, and the direct mutagen, N-ethyl-N-nitrosourea. The oral cavity carcinogen, 4-nitroquinoline-1-oxide (4-NQO; 10 ppm in drinking water; 28 days), was qualified as a positive control for mutagenesis in oral tissues since it caused significant increases in cII MFs in gingiva/palate (50.2-fold) and gingiva/buccal tissues (21.3-fold), but not in liver or bone marrow (0.9- and 1.4-fold, respectively). These results are consistent with the observation that 4-NQO primarily induces tumors in oral cavity. Results also demonstrate the utility of the BB rat mutation assay and optimized methods for investigation of oral cavity mutagenicity, and by extension, analysis of other small and cartilaginous tissues.

Regulatory Forum Opinion Piece*: Retrospective Evaluation of Doses in the 26-week Tg.rasH2 Mice Carcinogenicity Studies: Recommendation to Eliminate High Doses at Maximum Tolerated Dose (MTD) in Future Studies.

High doses in Tg.rasH2 carcinogenicity studies are usually set at the maximum tolerated dose (MTD), although this dose selection strategy has not been critically evaluated. We analyzed the body weight gains (BWGs), mortality, and tumor response in control and treated groups of 29 Tg.rasH2 studies conducted at BioReliance. Based on our analysis, it is evident that the MTD was exceeded at the high and/or mid-doses in several studies. The incidence of tumors in high doses was lower when compared to the low and mid-doses of both sexes. Thus, we recommend that the high dose in male mice should not exceed one-half of the estimated MTD (EMTD), as it is currently chosen, and the next dose should be one-fourth of the EMTD. Because females were less sensitive to decrements in BWG, the high dose in female mice should not exceed two-third of EMTD and the next dose group should be one-third of EMTD. If needed, a third dose group should be set at one-eighth EMTD in males and one-sixth EMTD in females. In addition, for compounds that do not show toxicity in the range finding studies, a limit dose should be applied for the 26-week carcinogenicity studies.

Trend analysis of body weight parameters, mortality, and incidence of spontaneous tumors in Tg.rasH2 mice.

Carcinogenicity studies have been performed in conventional 2-year rodent studies for at least 3 decades, whereas the short-term carcinogenicity studies in transgenic mice, such as Tg.rasH2, have only been performed over the last decade. In the 2-year conventional rodent studies, interlinked problems, such as increasing trends in the initial body weights, increased body weight gains, high incidence of spontaneous tumors, and low survival, that complicate the interpretation of findings have been well established. However, these end points have not been evaluated in the short-term carcinogenicity studies involving the Tg.rasH2 mice. In this article, we present retrospective analysis of data obtained from control groups in 26-week carcinogenicity studies conducted in Tg.rasH2 mice since 2004. Our analysis showed statistically significant decreasing trends in initial body weights of both sexes. Although the terminal body weights did not show any significant trends, there was a statistically significant increasing trend toward body weight gains, more so in males than in females, which correlated with increasing trends in the food consumption. There were no statistically significant alterations in mortality trends. In addition, the incidence of all common spontaneous tumors remained fairly constant with no statistically significant differences in trends.

Toxicity and biodistribution of the serotype 2 recombinant adeno-associated viral vector, encoding Aquaporin-1, after retroductal delivery to a single mouse parotid gland.

In preparation for testing the safety of using serotype 2 recombinant adeno-associated vector, encoding Aquaporin-1 to treat radiation-induced salivary gland damage in a phase 1 clinical trial, we conducted a 13 week GLP biodistribution and toxicology study using Balb/c mice. To best assess the safety of rAAV2hAQP1 as well as resemble clinical delivery, vector (10(8), 10(9), 10(10), or 4.4 × 10(10) vector particles/gland) or saline was delivered to the right parotid gland of mice via retroductal cannulation. Very mild surgically induced inflammation was caused by this procedure, seen in 3.6% of animals for the right parotid gland, and 5.3% for the left parotid gland. Long term distribution of vector appeared to be localized to the site of cannulation as well as the right and left draining submandibular lymph nodes at levels >50 copies/µg in some animals. As expected, there was a dose-related increase in neutralizing antibodies produced by day 29. Overall, animals appeared to thrive, with no differences in mean body weight, food or water consumption between groups. There were no significant adverse effects due to treatment noted by clinical chemistry and pathology evaluations. Hematology assessment of serum demonstrated very limited changes to the white blood cell, segmented neutrophils, and hematocrit levels and were concluded to not be vector-associated. Indicators for liver, kidney, cardiac functions and general tissue damage showed no changes due to treatment. All of these indicators suggest the treatment is clinically safe.

The 26-week Tg.rasH2 mice carcinogenicity studies: microscopic examination of only select tissues in low- and mid-dose groups.

A typical 26-week Tg.rasH2 mouse carcinogenicity study usually has 4 dose groups, composed of 25 mice/sex, which include 1 control and 3 test article-treated groups. In every study, there is a protocol required full tissue list of 49 tissues which is examined microscopically in all animals of these 4 dose groups. Based on retrospective analysis of the historical control data collected from studies conducted in Tg.rasH2 mice from 2004 to 2012, we propose that a full tissue list be examined as usual in the control and high-dose groups; however, in the low- and mid-dose groups, only select tissues should be examined. The select tissue list is generated after analyzing common tumors, uncommon tumors seen grossly, uncommon tumors not seen grossly, organ weight variations with accountable microscopic lesions, and target organs identified in the high-dose groups. The proposed changes to the International Conference on Harmonization S1 guidance may lead to an increased need for 26-week Tg.rasH2 mice studies. The time savings resulting from processing and evaluating a select tissue list rather than a full tissue list from low- and mid-dose groups of Tg.rasH2 mouse studies will further accelerate early completion of these studies without compromising the quality and integrity.

Relationship of body weight parameters with the incidence of common spontaneous tumors in Tg.rasH2 mice.

The mechanistic relationship between increased food consumption, increased body weights, and increased incidence of tumors has been well established in 2-year rodent models. Body weight parameters such as initial body weights, terminal body weights, food consumption, and the body weight gains in grams and percentages were analyzed to determine whether such relationship exists between these parameters with the incidence of common spontaneous tumors in Tg.rasH2 mice. None of these body weight parameters had any statistically significant relationship with the incidence of common spontaneous tumors in Tg.rasH2 males, namely lung tumors, splenic hemangiosarcomas, nonsplenic hemangiosarcomas, combined incidence of all hemangiosarcomas, and Harderian gland tumors. These parameters also did not have any statistically significant relationship with the incidence of lung and Harderian gland tumors in females. However, in females, increased initial body weights did have a statistically significant relationship with the nonsplenic hemangiosarcomas, and increased terminal body weights did have a statistically significant relationship with the incidence of splenic hemangiosarcomas, nonsplenic hemangiosarcomas, and the combined incidence of all hemangiosarcomas. In addition, increased body weight gains in grams and percentages had a statistically significant relationship with the combined incidence of all hemangiosarcomas in females, but not separately with splenic and nonsplenic hemangiosarcomas.

Incidence of spontaneous non-neoplastic lesions in transgenic CBYB6F1-Tg(HRAS)2Jic mice.

Since 2003, the Tg.rasH2 model has been accepted by regulatory agencies worldwide for 26-week short-term carcinogenicity assays as an alternative to the standard 2-year assays in conventional mice. However, over the decade, the number of actual studies conducted with alternative mouse models has remained low. The primary cause for low acceptance of this model has been lack of a historical database for the incidence of spontaneous lesions. Recently, we published the historical control database on spontaneous tumors in the Tg.rasH2 mice. The purpose of this publication is to present a large database pertaining to the non-neoplastic spontaneous lesions noted in Tg.rasH2 mice from studies conducted at our facility. Lesions that are considered unique in Tg.rasH2 mice are skeletal muscle myopathy, vascular anomalies involving various organs, and mesenteric arterial thrombosis. Other notable lesions are extramedullary hematopoiesis of spleen, subacute inflammatory foci in the liver, and infiltration of histiocytes in the lungs.

Regulation of CYP1A1 by heavy metals and consequences for drug metabolism.

Cytochrome P450 1A1 (CYP1A1) is a hepatic and extrahepatic enzyme that is regulated by the aryl hydrocarbon receptor signaling pathway. With the growing human exposure to heavy metals, emerging evidence suggests that heavy metals exposure alter CYP1A1 enzyme activity. Heavy metals regulate CYP1A1 at different levels of its aryl hydrocarbon receptor signaling pathway in a metal- and species-dependent manner. The importance of CYP1A1 emerges from the fact that it has been always associated with the metabolism of pro-carcinogenic compounds to highly carcinogenic metabolites. However, recently CYP1A1 has gained status along with other cytochrome P450 enzymes in the metabolism of drugs and mediating drug-drug interactions. In addition, CYP1A1 has become a therapeutic tool for the bioactivation of prodrugs, particularly cytotoxic agents. In this review, we shed light on the effect of seven heavy metals, namely arsenic, mercury, lead, cadmium, chromium, copper and vanadium, on CYP1A1 and the consequences on drug metabolism.

Selective inhibitors of CYP2J2 related to terfenadine exhibit strong activity against human cancers in vitro and in vivo.

The cytochrome P450 epoxygenase, CYP2J2, converts arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs). We found recently that this enzyme is dramatically up-regulated in a variety of established human carcinoma cell lines and in human cancerous tissue and promotes the neoplastic phenotype. In the present study, we tested the hypothesis that specific inhibitors of CYP2J2 related to the drug terfenadine are effective antitumor agents. Four of these inhibitors (compounds 4, 5, 11, and 26) were tested for effectiveness in vitro and in vivo. In Tca-8113 cells, the CYP2J2 inhibitors decreased EET production by approximately 60%, whereas they had no effect on CYP2J2 mRNA or protein expression. Compound 26 inhibited the proliferation of human tumor cells, reduced their ability to adhere, invade, and migrate, and attenuated activation of epithelial growth factor receptor signal and kinases and phosphatidylinositol 3 kinase/Akt pathways. Inhibition of CYP2J2 also significantly potentiated human tumor cell apoptosis and caused a corresponding increase in caspase-3 activity and change in expression of apoptosis-related proteins Bax and Bcl-2. In murine xenograft models using MDA-MB-435 cells, treatment with compound 26 significantly repressed tumor growth, decreased lung metastasis, and was associated with increased expression of the anticancer genes CD82 and nm23, without causing toxicity. These data suggest that CYP2J2 inhibitors hold significant promise for use in treatment of neoplastic diseases.

MG-132 inhibits the TCDD-mediated induction of Cyp1a1 at the catalytic activity but not the mRNA or protein levels in Hepa 1c1c7 cells.

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, 10 microM 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.

Arsenite and cadmium, but not chromium, induce NAD(P)H:quinone oxidoreductase 1 through transcriptional mechanisms, in spite of post-transcriptional modifications.

NAD(P)H:quinone oxidoreductase (Nqo1)-mediated detoxification of quinones plays a critical role in cancer prevention. Metals alter the carcinogenicity of AhR ligands, such as TCDD, by modulating the induction of Nqo1, but the mechanism(s) remain unresolved. To decipher the molecular mechanisms involved in the alteration of Nqo1, we analyzed the effect of the metals As3+ (5 microM), Cd2+ (5 microM), and Cr6+ (25 microM) on the transcriptional activation of the Nqo1 gene and post-transcriptional modifications, in Hepa 1c1c7 cells. Both As3+ and Cd2+ induced Nqo1 mRNA in a time-dependent manner and potentiated TCDD-induced Nqo1 mRNA. Cr6+ on the other hand, completely inhibited the induction of Nqo1 mRNA by TCDD. The induction of Nqo1 mRNA by the metals was completely inhibited with the DNA transcription inhibitor actinomycin-D, indicating a requirement for de novo mRNA synthesis for the induction. Furthermore, the protein synthesis inhibitor cycloheximide decreased Nqo1 mRNA induction, suggesting a role for a labile protein in the transcriptional induction of Nqo1 mRNA by metals. Surprisingly, all three metals decreased Nqo1 mRNA stability while having no effect on Nqo1 protein half-life. Meanwhile, As3+ and Cd2+ induced constitutive Nqo1 activity and potentiated the induction by increasing concentrations of TCDD. On the other hand, Cr6+ inhibited inducible Nqo1 activity. It is apparent that metals alter Nqo1 expression at the transcriptional level, through a labile protein-mediated pathway.

Transcriptional activation and posttranscriptional modification of Cyp1a1 by arsenite, cadmium, and chromium.

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, As(3+), Cd(2+), and Cr(6+), 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 As(3+) and Cd(2+) further potentiated the inducible Cyp1a1 mRNA level. Alternately, Cr(6+) inhibited the TCDD-mediated induction of Cyp1a1 mRNA. As(3+) potentiated the induction of Cyp1a1 mRNA by TCDD only at 3h and 6h after treatment. Cd(2+) 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, Cd(2+) and Cr(6+) 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 As(3+) increased the Cyp1a1 mRNA half-life while Cd(2+) and Cr(6+) 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 As(3+), Cd(2+), and Cr(6+) increase Cyp1a1 mRNA levels at the transcriptional and posttranscriptional levels while decreasing Cyp1a1 activity at the posttranslational level.