Kerry T. Blanchard

Methapyrilene Toxicity: Anchorage of Pathologic Observations to Gene Expression Alterations

Methapyrilene (MP) exposure of animals can result in an array of adverse pathological responses including hepatotoxicity. This study investigates gene expression and histopathological alterations in response to MP treatment in order to 1) utilize computational approaches to classify samples derived from livers of MP treated rats based on severity of toxicity incurred in the corresponding tissue, 2) to phenotypically anchor gene expression patterns, and 3) to gain insight into mechanism(s) of methapyrilene hepatotoxicity. Large-scale differential gene expression levels associated with the exposure of male Sprague—Dawley rats to the rodent hepatic carcinogen MP for 1, 3, or 7 days after daily dosage with 10 or 100 mg/kg/day were monitored. Hierarchical clustering and principal component analysis were successful in classifying samples in agreement with microscopic observations and revealed low-dose effects that were not observed histopathologically. Data from cDNA microarray analysis corroborated observed histopathological alterations such as hepatocellular necrosis, bile duct hyperplasia, microvesicular vacuolization, and portal inflammation observed in the livers of MP exposed rats and provided insight into the role of specific genes in the studied toxicological processes.

Mechanisms and Biomarkers of Cardiovascular Injury Induced by Phosphodiesterase Inhibitor III SK&F 95654 in the Spontaneously Hypertensive Rat

The cardiovascular injury of the type III selective PDE inhibitor SK&F 95654 was investigated in SHR. Twenty-four hours after a single sc injection of 100 or 200 mg/kg of the drug, rats exhibited cardiomyocyte necrosis and apoptosis, interstitial inflammation, hemorrhage and edema, as well as mesenteric arterial hemorrhage and necrosis, periarteritis, EC and VSMC apoptosis, EC activation, and MC activation and degranulation. Elevated serum levels of cTnT and decreased cTnT immunoperoxidase staining on cardiomyocytes were detected in the drug-treated rats. Serum levels of α 2-macroglobulin and IL-6 were significantly elevated following drug treatment. NMR spectral patterns of urine samples are significantly different between the drug-treated and control rats. These results indicate that measurement of serum cTnT, acute phase proteins, and cytokines as well as metabonomic urine profiles may serve as potential biomarkers for drug-induced cardiovascular injury in rats. Increased expression of CD63 on MC (tissue biomarker of MC), of nitrotyrosine on MC and EC (an indirect indicator of NO in vivo), and of iNOS on MC and EC (source of NO) suggest that NO produced by activated and degranulated MC as well as activated EC play an important role in SK&F 95654-induced mesenteric vascular injury.

Identification of genes implicated in methapyrilene-induced hepatotoxicity by comparing differential gene expression in target and nontarget tissue.

Background Toxicogenomics experiments often reveal thousands of transcript alterations that are related to multiple processes, making it difficult to identify key gene changes that are related to the toxicity of interest. Objectives The objective of this study was to compare gene expression changes in a nontarget tissue to the target tissue for toxicity to help identify toxicity-related genes. Methods Male rats were given the hepatotoxicant methapyrilene at two dose levels, with livers and kidneys removed 24 hr after one, three, and seven doses for gene expression analysis. To identify gene changes likely to be related to toxicity, we analyzed genes on the basis of their temporal pattern of change using a program developed at the National Institute of Environmental Health Sciences, termed “EPIG” (extracting gene expression patterns and identifying co-expressed genes). Results High-dose methapyrilene elicited hepatic damage that increased in severity with the number of doses, whereas no treatment-related lesions were observed in the kidney. High-dose methapyrilene elicited thousands of gene changes in the liver at each time point, whereas many fewer gene changes were observed in the kidney. EPIG analysis identified patterns of gene expression correlated to the observed toxicity, including genes associated with endoplasmic reticulum stress and the unfolded protein response. Conclusions By factoring in dose level, number of doses, and tissue into the analysis of gene expression elicited by methapyrilene, we were able to identify genes likely to not be implicated in toxicity, thereby allowing us to focus on a subset of genes to identify toxicity-related processes.

Loss of palindromic symmetry in Tg.AC mice with a nonresponder phenotype

The Tg.AC transgenic mouse carries the v‐Ha‐ras oncogene under the control of the ζ‐globin promoter and is currently being used in a short‐term carcinogenesis assay for safety testing of pharmaceuticals. A subset of hemizygous Tg.AC mice was found to be nonresponsive to the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate, which characteristically induces skin papillomas in these mice with repeated dermal applications. We previously showed that responder and nonresponder hemizygous Tg.AC mice carry about 40 copies of transgene but that the nonresponders had lost a 2‐kb BamHI fragment containing the ζ‐globin promoter sequence. The present restriction enzyme and S1 nuclease digestion experiments strongly suggested that the 2‐kb BamHI fragment resulted from the orientation of two transgenes in an inverted repeat formation. Two subsets of nonresponder Tg.AC mice were identified. Restriction enzyme and S1 nuclease digestion experiments suggested that one nonresponder genotype was produced by a large deletion of one or more near complete copies of transgene sequence and the other genotype was produced by a small deletion near the apex of the “head‐to‐head” juncture of the inverted repeat. Polymerase chain reaction amplification, cloning, and sequencing results confirmed the palindromic orientation of transgene in Tg.AC mice. Our results indicated that, despite the presence of multiple copies of transgene in a direct repeat orientation, loss of symmetry in the palindromic array of transgene sequence results in the loss of the responder phenotype in Tg.AC mice. Mol. Carcinog. 30:99–110, 2001. Published 2001 Wiley‐Liss, Inc.

Dermal Carcinogenicity in Transgenic Mice: Relative Responsiveness of Male and Female Hemizygous and Homozygous Tg.AC Mice to 12-O-Tetradecanoylphorbol 13-Acetate (TPA) and Benzene

Assessment of the carcinogenic potential of chemical agents continues to rely primarily upon the chronic rodent bioassay, a resource-intensive exercise. Recent advances in transgenic technology offer a potential resource conserving approach to carcinogen detection. Incorporation of oncogenes with known roles in the development of neoplasms into the genomes of laboratory rodents may provide new models with the potential of quickly and accurately separating carcinogenic from noncarcinogenic chemicals. The insertion of the v-Ha-ras oncogene into the genome of FVB/N mice imparts the qualities of genetically initiated skin in the transgenic mouse line designated as Tg.AC. The skin of either hemizygous (animals carrying the transgene on 1 allele) or homozygous (transgene copies on both alleles) Tg.AC mice promptly responds to the application of nongenotoxic carcinogens, such as the classical tumor promoting phorbol esters, with the development of squamous papillomas. Tumor production generally begins after 8-10 applications of 2.5 μg/mouse (3 times/wk) of 12-O-tetradecanoylphorbol 13-acetate (TPA). Maximal tumor response is usually in evidence within 20 wk. If this transgenic mouse line is to be useful in the identification of carcinogenic chemicals, experimental protocols must be systematically optimized. Experiments were conducted to compare the relative responsiveness of male and female hemizygous and homozygous Tg.AC mice to the dermal application of TPA and the known human leukemogen, benzene. Results revealed shipment-related variabilities in the relative responsiveness of hemizygous male and female mice to the application of the proliferative agent. Homozygous mice of both sexes were more reliable and uniform in responsiveness to both TPA and benzene. Therefore, our standard protocol for the conduct of bioassays with the Tg.AC mouse line specifies the use of homozygous males and/or females.

Loss of critical palindromic transgene promoter sequence in chemically induced Tg.AC mouse skin papillomas expressing transgene-derived mRNA†

The Tg.AC transgenic mouse carries a v‐Ha‐ras transgene. Skin papillomas develop in Tg.AC mice upon repeated dermal application of tumor promoters and carcinogens. The transgene is inserted at a single site on chromosome 11 in a multiple‐copy array. Although most of the ≥ 40 copies are arranged in a direct‐repeat orientation, two copies of the transgene are inserted in a palindromic, inverted‐repeat orientation. Deletion of the palindromic transgene promoter sequence is associated strongly with and diagnostic of loss of phenotypic responsiveness to Tg.AC papillomagens, such as 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Unexpectedly, a loss of palindromic transgene sequence, in the absence of an observable reduction in copy number of the direct‐repeat‐oriented transgene sequence, is seen in DNA from papillomas when compared to genomic DNA from tail clips or skin samples away from the application site. Transgene‐derived transcripts were detectable in all Tg.AC papillomas sampled. The transgene locus was hypomethylated in papillomas but not in samples from tail clips from the same animal or from skin samples away from the application site in responder Tg.AC mice, as shown by loss of resistance to digestion by HpaII. A cell line derived from a Tg.AC squamous cell carcinoma showed complete loss of the palindromic transgene sequence, hypomethylation of the transgene locus, and strong expression of v‐Ha‐ras mRNA. These data indicate that the palindromic transgene sequence, which appears to be necessary for initial responsiveness to tumorigens, may be susceptible to deletion during rapid cellular proliferation and is not required for transgene expression in later phases of papilloma growth. Published 2001 Wiley‐Liss, Inc.

Oxymetholone: III. Evaluation in the p53+/- Transgenic Mouse Model

Oxymetholone has been identified as a suspected nongenotoxic carcinogen and has recently completed testing in a conventional National Toxicology Program (NTP) 2-yr rodent bioassay program. As a synthetic androgen with a limited historical database in toxicology, oxymetholone is an ideal candidate for prospective examination of the performance of short-term transgenic mouse models in the detection of carcinogenic activity. In the present series of 3 articles, studies are described where oxymetholone was evaluated prior to disclosure of the results of the NTP 2-yr bioassay. The accompanying articles provide evidence showing that oxymetholone is devoid of mutagenic activity yet elicits a positive carcinogenic response in the Tg.AC transgenic mouse model. In the present study, oxymetholone was administered by oral gavage to p53 heterozygous male and female mice for 26 wk at doses of 125, 625, and 1,250 mg/kg/day. The vehicle was 0.5% aqueous methylcellulose. Positive controls consisted of mice treated daily by oral gavage with 200 or 400 mg/kg/day of p-cresidine in corn oil. The oxymetholone-treated females showed significantly increased body weight gain and clitoral enlargement attributable to drug treatment. In addition, significant alterations in kidney, liver, and testis weights were attributable to oxymetholone. However, there were no neoplastic lesions that were attributable to oxymetholone in either sex. p-Cresidine produced unequivocal bladder neoplasms in both sexes at the high dose and in males at the lower dose. The absence of a neoplastic response with oxymetholone is consistent with the selectivity of the p53+/- mouse model for detecting carcinogens that act by genotoxic mechanisms.

Nonclinical Safety of the Sodium-Glucose Cotransporter 2 Inhibitor Empagliflozin

Empagliflozin, a selective inhibitor of the renal tubular sodium-glucose cotransporter 2, was developed for treatment of type 2 diabetes mellitus. Nonclinical safety of empagliflozin was studied in a battery of tests to support global market authorization. Safety pharmacology studies indicated no effect of empagliflozin on measures of respiratory or central nervous system function in rats or cardiovascular safety in telemeterized dogs. In CD-1 mouse, Wistar Han rat, or beagle dogs up to 13, 26, or 52 weeks of treatment, respectively, empagliflozin exhibited a toxicity profile consistent with secondary supratherapeutic pharmacology related to glucose loss and included decreased body weight and body fat, increased food consumption, diarrhea, dehydration, decreased serum glucose and increases in other serum parameters reflective of increased protein catabolism, gluconeogenesis, and electrolyte imbalances, and urinary changes such as polyuria and glucosuria. Microscopic changes were consistently observed in kidney and included tubular nephropathy and interstitial nephritis (dog), renal mineralization (rat) and tubular epithelial cell karyomegaly, single cell necrosis, cystic hyperplasia, and hypertrophy (mouse). Empagliflozin was not genotoxic. Empagliflozin was not carcinogenic in female mice or female rats. Renal adenoma and carcinoma were induced in male mice only at exposures 45 times the maximum clinical dose. These tumors were associated with a spectrum of nonneoplastic changes suggestive of a nongenotoxic, cytotoxic, and cellular proliferation-driven mechanism. In male rats, testicular interstitial cell tumors and hemangiomas of the mesenteric lymph node were observed; both tumors are common in rats and are unlikely to be relevant to humans. These studies demonstrate the nonclinical safety of empagliflozin.

Genomic-Derived Markers for Early Detection of Calcineurin Inhibitor Immunosuppressant–Mediated Nephrotoxicity

Calcineurin inhibitor (CI) therapy has been associated with chronic nephrotoxicity, which limits its long-term utility for suppression of allograft rejection. In order to understand the mechanisms of the toxicity, we analyzed gene expression changes that underlie the development of CI immunosuppressant-mediated nephrotoxicity in male Sprague-Dawley rats dosed daily with cyclosporine (CsA; 2.5 or 25 mg/kg/day), FK506 (0.6 or 6 mg/kg/day), or rapamycin (1 or 10 mg/kg/day) for 1, 7, 14, or 28 days. A significant increase in blood urea nitrogen was observed in animals treated with CsA (high) or FK506 (high) for 14 and 28 days. Histopathological examination revealed tubular basophilia and mineralization in animals given CsA (high) or FK506 (low and high). We identified a group of genes whose expression in rat kidney is correlated with CI-induced kidney injury. Among these genes are two genes, Slc12a3 and kidney-specific Wnk1 (KS-Wnk1), that are known to be involved in sodium transport in the distal nephrons and could potentially be involved in the mechanism of CI-induced nephrotoxicity. The downregulation of NCC (the Na-Cl cotransporter coded by Slc12a3) in rat kidney following CI treatment was confirmed by immunohistochemical staining, and the downregulation of KS-Wnk1 was confirmed by quantitative real-time-polymerase chain reaction (qRT-PCR). We hypothesize that decreased expression of Slc12a3 and KS-Wnk1 could alter the sodium chloride reabsorption in the distal tubules and contribute to the prolonged activation of the renin-angiotensin system, a demonstrated contributor to the development of CI-induced nephrotoxicity in both animal models and clinical settings. Therefore, if validated as biomarkers in humans, SLC12A3 and KS-WNK1 could potentially be useful in the early detection and reduction of CI-related nephrotoxicity in immunosuppressed transplant patients when monitoring the health of kidney xenographs in clinical practice.

Oxymetholone: II. Evaluation in the Tg-AC transgenic mouse model for detection of carcinogens.

Several rodent models are under examination as possible alternatives to the classical 2-yr carcinogenicity bioassay. The Tg.AC transgenic mouse has been proposed as a shorter term model offering the possibility of detecting nongenotoxic and genotoxic carcinogenic agents. Retrospective studies of chemicals with established carcinogenic potential have revealed a close correlation between classical bioassay results and the production of skin tumors in the Tg.AC mouse model. Oxymetholone is a synthetic testosterone derivative that is a suspected carcinogen but has shown no evidence of genotoxic activity in a comprehensive battery of genetic toxicity assays. It currently is being tested by the National Toxicology Program (NTP) in a 2-yr rat carcinogenicity bioassay. Because of its nongenotoxicity and the ongoing chronic bioassay, oxymetholone was considered an ideal candidate for a prospective evaluation of the predictive validity of the Tg.AC dermal carcinogenicity model. Consequently, a 6-mo dermal study with oxymetholone in the Tg.AC mouse model was initiated and completed prior to disclosure of the NTP rat bioassay results. In this study, male and female hemizygous Tg.AC mice, 7-8 wk old, were housed individually in suspended plastic cages. An area of dorsal skin was shaved to accommodate dermal applications of 200-μl doses of vehicle control (acetone), drug (1.2, 6.0, or 12 mg oxymetholone in dimethylsulfoxide: acetone, 20:80), or positive control (1.25 μg 12- o-tetradecanoyl-phorbol-13-acetate [TPA]) solutions. Mice received oxymetholone or acetone daily or TPA twice weekly for 20 wk followed by a 6-wk recovery period. The acetone control groups exhibited low spontaneous incidences of papillomas, whereas dermal application of oxymetholone produced dose-related increases in the numbers of papilloma-bearing mice and the numbers of papillomas per animal. Females showed a somewhat greater response to the androgen than did the males. TPA caused an unequivocal increase in papillomas, with males exhibiting a greater response than females. The results of this study indicate that this nongenotoxic androgenic compound possesses proliferative properties. The results predict that chronic systemic administration of oxymetholone will most likely be associated with increased incidences of neoplasms.