Raymond E. Stoll

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.

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.

Five-month oral (diet) toxicity/infectivity study of Bacillus thuringiensis insecticides in sheep

Bacillus thuringiensis insecticides (Bt) [Dipel (test substance D or Thuricide-HP (test substance T)] were administered in the diet for 5 months to castrated mixed rambouillet/merino sheep (24-34 kg at the beginning of the study) at a dose of 500 mg/kg/day (approximately 10(12) spores per day). No treatment-related effect was seen on weight gain or clinical chemistry parameters nor were significant gross clinical changes observed. Several blood and tissue samples taken just prior to the time the animals were killed or at necropsy were found to be positive for Bt when cultured. Detailed gross and microscopic pathologic examination of the sheep revealed several incidental lesions. However, the only lesion that may have been associated with the treatment was lymphocytic hyperplasia in Peyers patches seen in the cecum of three sheep and it was not considered to be clinically significant.

Hemizygous Tg.AC transgenic mouse as a potential alternative to the two-year mouse carcinogenicity bioassay: evaluation of husbandry and housing factors

The dermal Tg.AC transgenic mouse model has been proposed as a potential alternative to the conventional (e.g. oral, dermal, parenteral, inhalation, etc.) 2‐year rodent bioassay for detecting chemical carcinogenicity. The present study was designed to address a number of technical aspects of this model as well as to augment the database being developed with the Tg.AC system at the NIEHS. Hemizygous Tg.AC mice were implanted s.c. with microchips for identification and housed individually in polycarbonate (i.e. ‘plastic’) or suspended stainless‐steel wire‐bottom (i.e. ‘metal’) cages. Treatment consisted of dermal application of the test or control material in treatment volumes of 200 μl of acetone. Groups of 10 males and 10 females were treated as follows: G1—shaved, no treatment; G2—acetone control seven times a week; G3—100 μl of benzene three times a week; G4—150 μl of benzene three times a week; G5—1.25 μg of phorbol ester (PMA) twice a week. The G1–G5 mice were housed in plastic caging with Alpha‐dri® bedding. Three additional groups were housed in stainless‐steel wire‐bottom caging: G6—shaved, no treatment; G7—acetone control seven times a week; G8—1.25 μg of PMA twice a week. The PMA‐treated mice (G5 and G8) served as the positive controls. Mice were treated for 20 weeks followed by a 6‐week recovery period prior to necropsy. The incidence of dermal papillomas in the shaved area was recorded weekly. There were no spontaneous papillomas in the target area of any of the untreated (G1) or vehicle control (G2) animals in the polycarbonate cages. One papilloma was observed in the untreated mice (G6) and one in the vehicle control group (G7) in the steel cages. This suggests that the type of caging, the shaving process, microchip implantation and daily acetone treatment for 20 weeks are all consistent with a very low background incidence of papillomas in this model. Papillomas were observed in the positive control groups as early as 4 weeks of treatment and increased both in number per mouse and number of mice affected up to a maximum average of 3.5 papillomas per mouse and 55% (11/20) mice with papillomas in G5 and 2.7 and 80% (16/20) in G8. A plateau was reached at about week 13 and the numbers of papillomas remained stable through the rest of the treatment and recovery phases. The low dose of benzene (100 μl) showed no significant effect, whereas the higher dose (150 μl) produced a moderate number of papillomas beginning at about week 11. The results of this study are comparable with earlier studies at the NIEHS and indicate reproducibility between laboratories and that the Tg.AC transgenic mouse model is suitable for use in an industrial pre‐clinical safety evaluation context.

Noninvasive Measurement of Systemic Arterial Blood Pressure in the Conscious Beagle Dog

The objectives of this study were to evaluate a technique for routine, noninvasive measurement of systemic arterial blood pressure and heart rate (HR) in conscious Beagle dogs for toxicologic research. HR, systolic, diastolic, and mean arterial (MAP) pressures were measured with a DINAMAP research monitor (Model 1255, Critikon, Inc.) as follows: Dogs were restrained in a Harvard dog sling, a neonatal cuff was wrapped around the base of the tail, and blood pressure and HR were determined once a minute. Initially, normal values were obtained, 5-10 trials/session, one to three sessions/day for 15 days in six dogs. The day to day, session to session, and trial variabilities were determined and found to be minimal. The day to day diastolic pressure ranged from 74 +/- 18 to 91 +/- 13 mm Hg, systolic pressure from 125 +/- 25 to 156 +/- 22 mm Hg, MAP from 94 +/- 20 to 113 +/- 15 mm Hg, and HR from 111 +/- 21 to 126 +/- 24 beats/minute (bpm). The effects of various drugs on these parameters were determined. Norepinephrine increased diastolic, systolic, and MAP by 75 to 110 mm Hg and decreased HR by half. Epinephrine increased HR by 20 bpm. Phentolamine decreased diastolic, systolic, and MAP by up to 25 mm Hg. Isoproterenol increased HR by up to 130 bpm and decreased diastolic, systolic, and MAP by 20 mm Hg. In addition, the effect of a classic drug interaction on these parameters was determined. When dogs pretreated with the monoamine oxidase inhibitor tranylcypromine were challenged with tyramine, diastolic, systolic, and MAP pressures were increased, whereas HR was decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Dermal Carcinogenicity in Transgenic Mice: Effect of Vehicle on Responsiveness of Hemizygous Tg.AC Mice to Phorbol 12-Myristate 13-Acetate (TPA):

The Tg.AC mouse is being evaluated for use in short-term carcinogenicity bioassays. Because the dermal test protocol necessitates dissolving test agents we determined the effects of several solvents on responsivenes s of hemizygous mice to dermal applications of the classical skin tumor promoter, phorbol 12-myristate 13-acetate (TPA). Mice of both sexes received dermal applications of either acetone (negative control) or TPA in various vehicles [acetone, 100% methanol, 70% and 100% ethanol, DMSO and mixtures of acetone and ethanol (1:1), acetone and DMSO (4:1 and 1:1), and acetone and olive oil (4:1)]. Negative control animals did not exhibit papillomas. When administered in acetone, ethanolic or methanolic vehicles TPA caused prompt and robust papillomatous responses. TPA was also tumorigenic in all nonalcoholic vehicles, except the acetone-olive oil mixture. Papilloma responses were generally delayed when TPA was applied in the nonalcoholic solvents but the distinction between TPA-dosed and negative control groups was unequivocal. These results show that choice of vehicle may affect the quantitative and qualitative nature of the response of Tg.AC mice to TPA, but 8 of 9 vehicles proved satisfactory for delivery of TPA.

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.