Background: Treatment with estrogen in early menopausal women protects against development of hepatic steatosis and nonalcoholic fatty liver disease but estrogen has undesirable side effects, which negate its beneficial effects in premenopausal and postmenopausal women. Targeted therapies require better understanding of the target sites and mechanisms by which estrogen signaling exerts its protective effects in women. Estrogen receptor α (ERα) is thought to be the primary mediator for estrogen signaling to protect against hepatic steatosis. ERα has several mechanisms for signal transduction: (1) inducing gene transcription by direct binding to specific DNA sequences, (2) inducing tethered transcription with other DNA-binding factors, and (3) stimulating nongenomic action through membrane-associated ERα. However, it is still unclear which mechanisms mediate ERα-dependent protection against hepatic steatosis. Methods: To understand the mechanisms of estrogen signaling for protection against hepatic steatosis in females, we analyzed the global ERα knockout mouse (αERKO), ERα DNA-binding domain mutant mouse (KIKO) and liver-specific ERα knockout mouse (LERKO) fed high-fat diets (HFD). The KIKO mouse disrupts the direct DNA-binding transcription activity but retains tethered transcription regulation and nongenomic action. Hepatic steatosis was evaluated by scoring the macrovesicular and microvesicular steatosis as well as serum alanine aminotransferase (ALT) levels. We analyzed serum testosterone to assess its correlation with hepatic steatosis. Results: Liver fat accumulation was far greater in HFD-fed αERKO and KIKO females than in HFD-fed wild-type (WT) controls. Conversely, HFD-fed LERKO females did not accumulate excess liver fat. HFD-fed αERKO and KIKO females showed higher microvesicular steatosis and ALT levels than WT controls that correlated with increased serum testosterone levels. Conclusions: ERα-mediated direct transcription in non-hepatic tissues is essential for estrogen-mediated protection against hepatic steatosis in HFD-fed females. The balance between non-hepatic estrogen signaling and hepatic or non-hepatic testosterone action may control hepatic steatosis.

Comparative analyses of canine and human soft tissue sarcomas (STSs) are lacking. This study compared the histological and immunohistochemical (labelling for desmin, smooth muscle actin [SMA], CD31, pancytokeratin, S100 and CD34) appearance of 32 archived, formalin-fixed, paraffin wax-embedded canine STS tumour specimens by board-certified veterinary and medical pathologists, both blinded to the other's interpretations. Comparison between the veterinary and human diagnoses revealed a generally consistent pattern of interpretation with few notable variations. Most tumours (13/32) were judged to display similar histomorphological appearance to human low-grade spindle cell sarcomas, appearing non-distinctive and morphologically of a fibroblastic/myofibroblastic type. Five canine cases resembled human liposarcoma, but with atypical desmin-positive epithelioid cells present. Five canine cases resembled human spindle cell sarcoma with myxoid features and two additional cases resembled human myxofibrosarcoma. Seven canine cases were noted to resemble human undifferentiated sarcoma. Findings in the present study demonstrate that canine STSs display histological and immunohistochemical features similar to their human equivalents. Because of these cross-species similarities, a particular opportunity exists to understand the biology and treatment of human STS by potentially including dogs as clinical models.

Cylindrospermopsin (CYN) is a toxin produced by a variety of fresh-water cyanobacterial species worldwide and induces significant adverse effects in both livestock and humans. This study investigated the course of CYN-induced toxicity in pregnant mice exposed daily during either the period of major organogenesis (gestation days [GD] 8–12) or fetal growth (GD13–17). Endpoints include clinical signs of toxicity, serum analyses to evaluate hepatic and renal function, histopathology of liver and kidney, and hematology. Study animals were administered 50 μg/kg CYN once daily by ip route and euthanized 24 h after 1, 2, 3, 4, or 5 consecutive doses, or 6 or 13 d after the dosing period. The course of the CYN-induced effects was determined at all euthanasia times for the endpoints just outlined. Results indicated that CYN is a toxin, producing lethality in dams during the early part of gestation, significant weight loss, and bleeding in the gastrointestinal tract, tail tip, and peri-orbital tissues. Effects also included alterations in serum markers for liver function, histopathological changes in liver and kidney tissues, electrolyte abnormalities, leukocytosis, and posttreatment thrombocytopenia and reticulocytosis. The onset of symptoms was rapid, producing reductions in weight gain in GD8–12 animals, bleeding in the vaginal area in GD13–17 animals, and significant increases in sorbitol dehydrogenase (SDH) in both groups after a single dose. Although the GD8–12 dams displayed a 50% lethality, in GD13–17 animals only a single death occurred. Alterations seen in hepatic and renal function or histopathology do not appear to be of sufficient severity to produce death. Evidence indicates that bleeding may play a critical role in the onset of symptoms and eventually, in the observed lethality.

This investigation tested the hypothesis that carriers of golden retriever muscular dystrophy (GRMD), a genetically homologous condition of Duchenne muscular dystrophy (DMD), have quantifiable abnormalities in myocardial function, structure, or cardiac rhythm. Eleven GRMD carriers and four matched controls had cardiac evaluations and postmortem examinations. 24-h ECG Holter monitoring disclosed ventricular ectopy in 10 of 11 carriers and 2 of 4 controls. Conventional echocardiography failed to demonstrate significant differences between carriers and controls in systolic function. All carriers had multifocal, minimal to marked myofiber necrosis, fibrosis, mineralization, inflammation, and/or fatty change in their hearts. Immunohistochemistry revealed a mosaic dystrophin deficiency in scattered cardiac myofibers in all carriers. No controls had cardiac histologic lesions; all had uniform dystrophin staining. Despite cardiac mosaic dystrophin expression and degenerative cardiac lesions, GRMD carriers at up to 3 years of age could not be distinguished statistically from normal controls by echocardiography or 24-h Holter monitoring.

Preclinical toxicity studies have demonstrated that exposure of laboratory animals to liver enzyme inducers during preclinical safety assessment results in a signature of toxicological changes characterized by an increase in liver weight, hepatocellular hypertrophy, cell proliferation, and, frequently in long-term (life-time) studies, hepatocarcinogenesis. Recent advances over the last decade have revealed that for many xenobiotics, these changes may be induced through a common mechanism of action involving activation of the nuclear hormone receptors CAR, PXR, or PPARα. The generation of genetically engineered mice that express altered versions of these nuclear hormone receptors, together with other avenues of investigation, have now demonstrated that sensitivity to many of these effects is rodent-specific. These data are consistent with the available epidemiological and empirical human evidence and lend support to the scientific opinion that these changes have little relevance to man. The ESTP therefore convened an international panel of experts to debate the evidence in order to more clearly define for toxicologic pathologists what is considered adverse in the context of hepatocellular hypertrophy. The results of this workshop concluded that hepatomegaly as a consequence of hepatocellular hypertrophy without histologic or clinical pathology alterations indicative of liver toxicity was considered an adaptive and a non-adverse reaction. This conclusion should normally be reached by an integrative weight of evidence approach.

Preclinical toxicity studies have demonstrated that exposure of laboratory animals to liver enzyme inducers during preclinical safety assessment results in a signature of toxicological changes characterized by an increase in liver weight, hepatocellular hypertrophy, cell proliferation, and, frequently in long-term (life-time) studies, hepatocarcinogenesis. Recent advances over the last decade have revealed that for many xenobiotics, these changes may be induced through a common mechanism of action involving activation of the nuclear hormone receptors CAR, PXR, or PPARα. The generation of genetically engineered mice that express altered versions of these nuclear hormone receptors, together with other avenues of investigation, have now demonstrated that sensitivity to many of these effects is rodent-specific. These data are consistent with the available epidemiological and empirical human evidence and lend support to the scientific opinion that these changes have little relevance to man. The ESTP therefore convened an international panel of experts to debate the evidence in order to more clearly define for toxicologic pathologists what is considered adverse in the context of hepatocellular hypertrophy. The results of this workshop concluded that hepatomegaly as a consequence of hepatocellular hypertrophy without histologic or clinical pathology alterations indicative of liver toxicity was considered an adaptive and a non-adverse reaction. This conclusion should normally be reached by an integrative weight of evidence approach.

Cylindrospermopsin (CYN) is a tricyclic alkaloid toxin produced by fresh water cyanobacterial species worldwide. CYN has been responsible for both livestock and human poisoning after oral exposure. This study investigated the toxicity of CYN to pregnant mice exposed during different segments of gestation. The course of recovery and individual responses to the toxin were evaluated. Adverse effects of CYN were monitored up to 7 weeks post-dosing by clinical examination, histopathology, biochemistry and gene expression. Exposure on gestational days (GD) 8-12 induced significantly more lethality than GD13-17 exposure. Periorbital, gastrointestinal and distal tail hemorrhages were seen in both groups. Serum markers indicative of hepatic injury (alanine amino transferase, aspartate amino transferase and sorbitol dehydrogenase) were increased in both groups; markers of renal dysfunction (blood urea nitrogen and creatinine) were elevated in the GD8-12 animals. Histopathology was observed in the liver (centrilobular necrosis) and kidney (interstitial inflammation) in groups exhibiting abnormal serum markers. The expression profiles of genes involved in ribosomal biogenesis, xenobiotic and lipid metabolism, inflammatory response and oxidative stress were altered 24 h after the final dose. One week after dosing, gross, histological and serum parameters had returned to normal, although increased liver/body weight ratio and one instance of gastrointestinal bleeding was found in the GD13-17 group. Gene expression changes persisted up to 2 weeks post-dosing and returned to normal by 4 weeks. Responses of individual animals to CYN exposure indicated highly significant inter-animal variability within the treated groups. Copyright (C) 2010 John Wiley & Sons, Ltd.

B6C3F1 mice chronically exposed to 3,3′,4,4′-tetrachloroazobenzene (TCAB), a contaminant of dichloroaniline-derived herbicides, developed a number of neoplastic and nonneoplastic lesions, including carcinoma of the urinary tract. Groups of fifty male and fifty female B6C3F1 mice were exposed by gavage to TCAB at dose levels of 0, 3, 10, and 30 mg/kg five days a week for two years. Control animals received corn oil:acetone (99:1) vehicle. Decreased survival of male mice in the mid-dose group and of male and female mice in the high-dose groups was related mainly to the occurrence of urethral transitional cell (urothelial) carcinoma and resulting urinary obstruction. Increased urethral transitional cell carcinomas were seen in all treated male groups in a dose-related manner as well as in the females treated with 30 mg/kg TCAB. Administration of TCAB was also associated with increased transitional cell hyperplasia of the urethra. Most nonneoplastic lesions of the urogenital tract were considered secondary to local invasion and urinary obstruction by the urethral transitional cell carcinomas. The mechanism of tumor induction is uncertain, but the high frequency of tumors in the proximal urethra of male mice suggests that the neoplasms result from the exposure of a susceptible population of urothelial cells to a carcinogenic metabolite of TCAB.

Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) signalling pathways play a role in carcinogenesis. Inhibition of EGF receptor (EGFR) and of VEGF is effective in increasing the radiation responsiveness of neoplastic cells both in vitro and in human trials. In this study, immunohistochemical evaluation was employed to determine and characterize the potential protein expression levels and patterns of EGFR and VEGF in a variety of canine malignant epithelial nasal tumours. Of 24 malignant canine nasal tumours, 13 (54.2%) were positive for EGFR staining and 22 (91.7%) were positive for VEGF staining. The intensity and percentage of immunohistochemically positive neoplastic cells for EGFR varied. These findings indicate that EGFR and VEGF proteins were present in some malignant epithelial nasal tumours in the dogs, and therefore, it may be beneficial to treat canine patients with tumours that overexpress EGFR and VEGF with specific inhibitors in conjunction with radiation.

Trivalent chromium (Cr(III)) has been proposed to be an essential element, which may increase sensitivity to insulin and thus participate in carbohydrate and lipid metabolism. Humans ingest Cr(III) both as a natural dietary constituent and in dietary supplements taken for weight loss and antidiabetic effects. Chromium picolinate (CP), a widely used supplement, contains Cr(III) chelated with three molecules of picolinic acid and was formulated in an attempt to improve the absorption of Cr(III). In order to examine the potential for CP to induce chronic toxicity and carcinogenicity, the NTP conducted studies of the monohydrate form (CPM) in groups of 50 male and female F344/N rats and B6C3F1 mice exposed in feed to concentrations of 0, 2000, 10,000 or 50,000ppm for 2 years; exposure concentrations were selected following review of the data from NTP 3-month toxicity studies. Exposure to CPM did not induce biologically significant changes in survival, body weight, feed consumption, or non-neoplastic lesions in rats or mice. In male rats, a statistically significant increase in the incidence of preputial gland adenoma at 10,000ppm was considered an equivocal finding. CPM was not carcinogenic to female rats or to male or female mice.

A 7-year-old captive female prothonotary warbler (Protonotaria citrea) died following chronic feather and weight loss. At necropsy, the right eye had a 2 X 2 X 1 mm corneal plaque of inspissated yellow-tan material and edema of the lower eyelid. Microscopically, both eyes exhibited diffuse, severe pyogr anulomatous endophthalmitis with retinal necrosis and detachment. Numerous intralesional branching, gram-positive, beaded, filamentous bacteria formed a thick mat attached to the retinal pigmented epithelium and extending into the pecten. Bacteria were strongly acid-fast positive by Fite's stain but only occasionally acid-fast positive by Ziehl-Neelsen staining, a characteristic consistent with a Nocardia spp. Infected regions demonstrated positive in situ hybridization reactivity with a probe complementary to the 16S rRNA gene of Nocardia spp. There was no evidence of primary bacterial infection in the other organs examined.

Abnormal offspring syndrome can occur in fetuses and calves resulting from embryos produced in vitro or by nuclear transfer procedures. This study was conducted to determine the effects of in vitro embryo culture on fetal biochemistry profiles and histology of the liver and kidneys during late gestation. Embryos were produced in vivo by using superovulated cows (In Vivo) or in vitro by using a serum-containing culture system (In Vitro) as previously described (Miles et al. 2004 Biol. Reprod. 71, 1919–1926). Single blastocysts from each embryo production system were transferred nonsurgically into heifers. On Day 222 of gestation, fetuses from the In Vivo group (n = 12) and the In Vitro group (n = 12) were recovered in utero. Samples of fetal serum were collected for biochemical analysis. Samples of liver and kidney were prepared for histological evaluation. Stereological methods were used to determine the volume density of hepatocytes as well as kidney glomeruli and kidney tubules. Fetuses from the In Vitro group were heavier (P = 0.03) than those from the InVivo group (17.3 1.0 kg and 20.7 1.0 kg for InVivo and InVitro, respectively; least squares means SEM). Liver and paired kidney weights per kilogram of body weight did not differ (P ≥ 0.10) with treatment (26.4 0.6 g kg–1 v. 27.6 0.6 g kg–1 and 7.8 0.5 g kg–1 v. 9.1 0.5 g kg–1 for liver and kidney, respectively). In addition, there was no effect of treatment on the volume densities of hepatocytes, kidney glomeruli, and kidney tubules. However, compared with the In Vivo group, fetuses from the In Vitro group had increased (P ≤ 0.02) concentrations of blood urea nitrogen (BUN; 13.8 1.8 mg dL–1 v. 19.8 1.8 mg dL–1) and BUN:creatinine ratio (4.6 0.8 v. 7.9 0.8). No differences were observed between the In Vivo and In Vitro groups for serum levels of creatinine, total bilirubin, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, glucose, insulin, and insulin-like growth factor-I. In summary, compared with bovine fetuses from in vivo-produced embryos, fetuses from in vitro-produced embryos had increased body weight, normal liver and kidney morphology, and increased concentrations of BUN during late gestation. Supported by the State of North Carolina.

To respond to potential adverse exposures properly, health care providers need accurate indicators of exposure levels. The indicators are particularly important in the case of acetaminophen (APAP) intoxication, the leading cause of liver failure in the U.S. We hypothesized that gene expression patterns derived from blood cells would provide useful indicators of acute exposure levels. To test this hypothesis, we used a blood gene expression data set from rats exposed to APAP to train classifiers in two prediction algorithms and to extract patterns for prediction using a profiling algorithm. Prediction accuracy was tested on a blinded, independent rat blood test data set and ranged from 88.9% to 95.8%. Genomic markers outperformed predictions based on traditional clinical parameters. The expression profiles of the predictor genes from the patterns extracted from the blood exhibited remarkable (97% accuracy) transtissue APAP exposure prediction when liver gene expression data were used as a test set. Analysis of human samples revealed separation of APAP-intoxicated patients from control individuals based on blood expression levels of human orthologs of the rat discriminatory genes. The major biological signal in the discriminating genes was activation of an inflammatory response after exposure to toxic doses of APAP. These results support the hypothesis that gene expression data from peripheral blood cells can provide valuable information about exposure levels, well before liver damage is detected by classical parameters. It also supports the potential use of genomic markers in the blood as surrogates for clinical markers of potential acute liver damage.

Feline oral squamous cell carcinomas (SCC) have a poor prognosis despite aggressive treatment with surgery, radiation and anticancer drugs. Overexpression of the epidermal growth factor receptor (EGFR), a membrane-bound tyrosine kinase receptor, has been found in many human epithelial neoplasms, including oral SCC. EGFR overexpression has been associated with advanced disease and a poor prognosis. The purpose of this study was to determine whether feline oral SCC express EGFR. Thirteen formalin-fixed paraffin wax-embedded biopsy samples from feline oral SCC were analysed for EGFR expression using immunohistochemistry. Nine of 13 tumours (69%) were positive for EGFR expression, suggesting that altered EGFR expression plays a role in feline oral SCC and provides a rationale for a potential clinical benefit using EGFR inhibitors in combination with conventional treatments.

Recently, the use of selected genetically altered mouse models in the detection of carcinogens after shea-term chemical exposures has been evaluated. Studies of several chemicals conducted by the National Toxicology Program in Tg.AC transgenic and heterozygous p53-deficient mice have been completed recently and represent a major contribution to this effort, as well as the largest accumulation to date of toxicologic pathology data in these 2 lines of mice. The purpose of this report is to describe the proliferative target organ effects observed in this set of studies, as well as to present the tumor profile in the control groups of this data set. These findings provide a comprehensive toxicologic assessment of these 2 genetically altered mouse strains, which are of emerging importance in toxicologic pathology.

Polymerase chain reaction (PCR) and in situ hybridization (ISH) have revolutionized the study of genes and gene expression, and many of these molecular biology advances will greatly impact research in toxicological pathology. PCR is one of the most powerful tools in molecular biology and involves primer-mediated enzymatic in vitro amplification of specific target DNA sequences. Recent innovative methods utilizing PCR technology have been developed to detect mutations in neoplastic and small subpopulations of cells, to study biomarkers of genetic susceptibility and genes involved with carcinogen metabolism, to estimate mutation frequencies, to find novel genes induced by chemical exposure, and to characterize gene expression. LSH provides data on individual cells rather than an average of total cellular populations and allows analysis for heterogeneity. When combined with PCR, the sensitivity of ISH is elevated, and single-copy DNA sequences, single-base mutations, or low copies of messenger RNA (mRNA) can potentially be detected within individual cells. Herein are reviewed ISH- and PCR-based techniques such as single-strand conformation polymorphism analysis to detect point mutations, allelotypic analysis for loss of heterozygosity, differential display of mRNA to characterize gene expression, quantitative reverse transcriptase polymerase chain reaction, and in situ polymerase chain reaction with emphasis on current or potential applications in toxicological pathology. These new and evolving techniques offer tremendous potential in providing new insights into the molecular basis of toxicity and carcinogenesis.

The mouse liver is a frequent target organ for chemical carcinogenesis (Huff et al., 1988, 1991; Gold et al., 1989) and tumor development exhibits preferential strain sensitivity (Dragani et al., 1992; Drinkwater and Bennett, 1991). In some reports a positive correlation has been observed between the degree of spontaneous liver tumor incidence and the propensity to develop liver tumors after treatment with chemical carcinogens (Della Porta et al., 1967; Flaks, 1968; Dragani et al., 1984, 1987; Diwan et al., 1986; Drinkwater and Ginsler, 1986), but this is not always the case (Grasso and Hardy, 1975; Hanigan et al., 1988; Dragani et al., 1992). Thus, the interpretation of this endpoint in assessing potential health hazards to humans continues to be the subject of active debate. Studies of molecular and genetic factors that modulate the genesis of mouse liver tumors should enhance our understanding of the relevance of this response following exposure to genotoxic as well as nongenotoxic chemicals. To utilize intelligently animal models as surrogates for human carcinogenesis, the validity of rodent tumor endpoints in assessing potential human health hazards from chemical exposure remains an important issue. One approach has been to understand the animal system itself and the mechanisms by which chemicals induce tumors in the animal model. Information regarding the molecular events associated with tumor induction should make the relevance of results from rodent carcinogenicity studies to human risk easier to assess. Results to date have identified activation of ras proto-oncogenes as one early event and an important factor associated with chemical induction of mouse liver neoplasia (Reynolds et al., 1986, 1987; Wiseman et al., 1986), although ras-independent pathways appear to account for an appreciable proportion of some chemically induced mouse liver tumors (Fox et al., 1990; Buchmann et al., 1991). Available data emphasize the complexity of H-ras activation in murine hepatocarcinogenesis. Not only the genetic background of the mouse but also the dose of the carcinogen may influence significantly the number of tumors containing activated H-ras. Both high sensitivity and low sensitivity strains of mice can develop liver tumors which contain activated H-ras oncogenes, showing that the ability to activate this gene does not in itself determine susceptibility to hepatocarcinogenesis. Ras gene mutational profiles in chemically induced liver tumors may be different and distinguishable from those in spontaneous tumors. Since multiple genetic as well as nongenetic events are associated with tumor development, defining a precise role for ras gene mutations when they occur in mouse liver tumors is often difficult.(ABSTRACT TRUNCATED AT 400 WORDS)