Acute Alpha-Naphthylisothiocyanate-induced Liver Toxicity in Germfree and Conventional Male Rats.

Differences in the responses of conventional and germfree male Sprague-Dawley rats to acute injury induced by alpha-naphthylisothiocyanate (ANIT), a well-characterized biliary epithelial toxicant, were evaluated. Conventional and germfree rats were dosed once orally with 50 mg/kg of ANIT or corn oil alone and serially sacrificed daily for the next 3 days. Germfree rats treated with ANIT tended to have greater increases in virtually all liver and biliary-related analytes compared with conventional rats treated with ANIT; however, significant differences were found only in a few of these analytes including increased bile acids on day 3, total bilirubin on day 4, glutamate dehydrogenase (GLDH) on day 3, and reduced paraoxonase 1 (PON1) on days 2 and 3. Histologic differences between the conventional and germfree rats were modest, but most pronounced on day 2 (24-hr post dosing). Based on subjective scoring, biliary necrosis, neutrophilic cholangitis, and portal tract edema were more severe in germfree rats at 24 hr post dosing compared with conventional rats. Biliary epithelial replication did not differ between treated groups, however. Overall, germfree rats had a modestly greater level of biliary tract injury based on subjective histologic scoring and clinical chemistry measurements following an acute exposure to the well-characterized biliary toxin, ANIT; however, the difference between the ANIT-treated germfree and conventional groups was modest and most evident only within the first day following exposure. These findings suggest that the microbiome did not significantly affect ANIT-induced acute biliary tract injury in the conditions of this study.

Integrated approach to early detection of cardiovascular toxicity induced by a ghrelin receptor agonist.

Cardiovascular (CV) safety concerns are among the leading causes of compound attrition in drug development. This work describes a strategy of applying novel end points to a 7-day rodent study to increase the opportunity to detect and characterize CV injury observed in a longer term (ie, 28 days) study. Using a ghrelin receptor agonist (GSK894281), a compound that produces myocardial degeneration/necrosis in rats after 28 days at doses of 0.3, 1, 10, or 60 mg/kg/d, we dosed rats across a range of similar doses (0, 0.3, 60, or 150 mg/kg/d) for 7 days to determine whether CV toxicity could be detected in a shorter study. End points included light and electron microscopies of the heart; heart weight; serum concentrations of fatty acid-binding protein 3 (FABP3), cardiac troponin I (cTnI), cardiac troponin T (cTnT), and N-terminal proatrial natriuretic peptide (NT-proANP); and a targeted transcriptional assessment of heart tissue. Histologic evaluation revealed a minimal increase in the incidence and/or severity of cardiac necrosis in animals administered 150 mg/kg/d. Ultrastructurally, mitochondrial membrane whorls and mitochondrial degeneration were observed in rats given 60 or 150 mg/kg/d. The FABP3 was elevated in rats given 150 mg/kg/d. Cardiac transcriptomics revealed evidence of mitochondrial dysfunction coincident with histologic lesions in the heart, and along with the ultrastructural results support a mechanism of mitochondrial injury. There were no changes in cTnI, cTnT, NT-proANP, or heart weight. In summary, enhancing a study design with novel end points provides a more integrated evaluation in short-term repeat dose studies, potentially leading to earlier nonclinical detection of structural CV toxicity.

Nontraditional applications in clinical pathology.

Most published reviews of preclinical toxicological clinical pathology focus on the fundamental aspects of hematology, clinical chemistry, coagulation, and urinalysis in routine toxicology animal species, for example, rats, mice, dogs, and nonhuman primates. The objective of this continuing education course was to present and discuss contemporary examples of nonroutine applications of clinical pathology endpoints used in the drug development setting. Area experts discussed bone turnover markers of laboratory animal species, clinical pathology of pregnant and growing laboratory animals, clinical pathology of nonroutine laboratory animal species, and unique applications of the Siemens Advia(®) hematology analyzer. This article is a summary based on a presentation given at the 31st Annual Symposium of the Society of Toxicologic Pathology, during the Continuing Education Course titled "Nontraditional Applications of Clinical Pathology in Drug Discovery and Preclinical Toxicology."

Biliary proliferative lesions in the Sprague-Dawley rat: adverse/non-adverse.

Whether biliary proliferative lesions in nonclinical species are predictive of potential hepatotoxicity in humans depends, at least in part, on the nature and severity of such changes in the nonclinical species. We reviewed published literature (clinical and nonclinical) and experimental data from rat toxicology studies conducted by GlaxoSmithKline and the National Institute of Environmental Health Sciences' National Toxicology Program in an effort to better characterize the relative risk of hepatobiliary effects in humans. Available evidence supports the interpretation that minimal "typical" appearing bile duct hyperplasia limited to the portal triads may be considered non-adverse in the rat and is of little to no concern to humans. The toxicological relevance of mild to moderate "typical" hyperplasia is less certain, and may be considered adverse in the rat and potentially pose a risk for humans, particularly if accompanied by evidence of hepatobiliary injury or functional compromise. In addition, any proliferative lesion that includes atypical or dysplastic epithelial changes, oval cell proliferation, and/or significant extension beyond the portal tracts is considered more ominous and may be considered adverse in the rat.

Effects of Solutol (Kolliphor) and cremophor in polyethylene glycol 400 vehicle formulations in Sprague-Dawley rats and beagle dogs.

When conventional vehicles (eg, methylcellulose and water) impart inadequate physical, chemical, and/or biological properties for proper toxicological assessment of test article formulations, nonconventional vehicles may be considered. Often toxicity data for nonconventional vehicle formulations are limited. Studies were conducted to collect toxicity data from a rodent and a non-rodent species given 2 nonconventional vehicles, Solutol HS15/polyethylene glycol (PEG) 400 and Cremophor RH40/PEG 400, with differing formulations and dose volumes (10 mL/kg for rats; 2 or 5 mL/kg for dogs). In rats, both vehicles caused increase in kidney weights (males only) and decrease in thymic weights (males only) without concurrent microscopic findings; altered urine electrolytes, minimally decreased serum electrolytes (males only), and increased serum total cholesterol (females only) were also present. The Cremophor formulation was also associated with increased serum urea (males only) and urine phosphorus: creatinine. For rats given the Solutol formulation, both genders had decreased urine glucose parameters and males had increased urine volume. In dogs, loose/watery feces and emesis were present given either vehicle, and mucus-cell hyperplasia of the ileum was present given the Solutol formulation. Increased red blood cell mass and decreased urine volume in dogs given 30% Solutol/70% PEG 400 (5 mL/kg/d) were likely due to subclinical dehydration and hemoconcentration. For the Cremophor formulations, dose volume-dependent increased incidence of minimal subepithelial gastric hemorrhage was noted in dogs, and dogs given 5 mL/kg/d showed increased serum urea nitrogen. Overall, regardless of the formulation or dose volume, neither vehicle produced overt toxicity in either species, but the Solutol formulation produced fewer effects in rats. Generally, lower dose volumes minimized the severity and/or incidence of findings.

Effects of Kupffer cell depletion on acute alpha-naphthylisothiocyanate-induced liver toxicity in male mice.

Depletion of Kupffer cells, known to modulate chemical-induced hepatocellular injury, has not been studied with regard to biliary epithelial injury. Here, the authors investigated the effect of Kupffer cell depletion by clodronate on the toxicity of alpha-naphthylisothiocyanate (ANIT), known to injure biliary epithelium as well as hepatocytes. Up to 99% depletion of Kupffer cells occurred in ANIT and liposome-encapsulated clodronate-treated mice. The effect of Kupffer cell depletion was most evident one day following ANIT treatment. Histologically, there was a modest increase in neutrophil infiltration of the bile ducts, hepatocytic necrosis, and microvesicular vacuolization in the ANIT and clodronate-treated mice, but differences between other groups did not persist. Clinical pathology analytes related to the biliary or hepatocellular injury were significantly elevated in ANIT and clodronate-treated mice compared to mice given clodronate only. This was also true for mice given ANIT and empty liposomes in the case of the biliary analytes. However, group means were typically higher for the ANIT and clodronate-treated group than others on the first 2 days following ANIT injection. These findings suggest that Kupffer cell reduction increases hepatobiliary damage due to ANIT treatment.

Effect of estrous cycle phase on clinical pathology values in beagle dogs.

BACKGROUND: In dogs, the diestrus phase is considerably longer than in most domestic animals, and is characterized by high circulating progesterone concentrations that may influence clinical pathology values. OBJECTIVE: The objective of this retrospective study was to investigate differences in clinical pathology data in dogs in diestrus compared with data from dogs in all other phases of the estrous cycle. METHODS: Phase of the estrous cycle was determined by histologic evaluation of reproductive tissues from 86 control female Beagles that had participated in 23 toxicity studies. Serum biochemical, hematologic, and urinalysis values from dogs in diestrus were compared with data from dogs in all other estrous cycle phases using a 2-tailed t-test. RESULTS: In Beagles in diestrus (n = 38), serum cholesterol concentrations and eosinophil counts were 35% (P < .0001) and 45.8% (P = .0035) higher, respectively, than for Beagles in all other phases of the estrous cycle (n = 48). Furthermore, Beagles in diestrus had 14% lower AST activity (P = .0011), 1% lower chloride concentration (P = .0224), 7.8% lower hemoglobin concentration (P < .0001), 7.8% lower RBC count (P < .0001), and 7.6% lower hematocrit (P < .0001) compared with female dogs in all other phases of the estrous cycle. Urine values did not differ significantly between groups. CONCLUSIONS: Differences in clinical pathology values between dogs in different phases of the estrous cycle could potentially confound interpretation of data in toxicity studies, which often have small group sizes. Interpretation of clinical pathology data in female dogs should be performed with due consideration given to the phase of the estrous cycle.

An initial characterization of N-terminal-proatrial natriuretic peptide in serum of Sprague Dawley rats.

In the clinical setting, natriuretic peptides (NPs) have proven to be reliable noninvasive markers for diagnostic, prognostic, and therapeutic monitoring of heart failure. Given their proven utility in humans, NPs are potential candidates for translational biomarkers during drug development to detect drug-induced hemodynamic stress resulting in cardiac hypertrophy in preclinical species. We evaluated the intra- and interassay precision and the stability of serum N-terminal-proatrial natriuretic peptide (NT-proANP) using a commercially available enzyme-linked immunoassay (EIA). We then measured NT-proANP concentrations in 532 serum samples from 337 male Crl:CD(SD) rats with or without pressure-induced cardiac hypertrophy. Additionally, we established a reference range using samples from control animals across multiple studies. The data demonstrate that the NT-proANP EIA is a robust and reproducible assay for the measurement of NT-proANP. The noninvasive translational utility, minimal sample volume requirement, and the lack of existing hypertrophic biomarkers in the male rat make NT-proANP an excellent candidate for further interrogation as a biomarker of cardiac hypertrophy in preclinical toxicology investigations.

Acute ANIT toxicity in male IL-10 knockout and wild-type mice.

The innate immune response is known to modify hepatocellular injury induced by toxicants. To assess the role of IL-10, a component of the innate immune response, in toxicant-induced injury of biliary epithelium, wild-type (WT) and IL-10 knockout mice (KO) were given a single toxic dose (50 mg/kg) of alpha-napthylisothiocyanate (ANIT) and assessed at twenty-four-hour intervals for four days following treatment. Clinical signs of toxicity were greater in WT mice. Unexpectedly, over the course of the study, there was a consistent tendency for ANIT-treated IL-10 KO mice to have less hepatocellular injury than WT mice. However, changes in the biliary epithelium differed in that there was more histologic evidence of inflammation and necrosis on days 2 and 3, respectively, in ANIT-treated IL-10 KO mice compared with WT mice. Proliferation of biliary epithelium and hepatocytes was greater and/or occurred earlier in the ANIT-treated IL-10 KO mice compared with the ANIT-treated WT mice, suggesting a greater reparative response was needed for recovery after toxicant injury in the IL-10 KO mice. Overall, our data suggest that IL-10 KO mice have less hepatocellular injury than WT mice following a toxic dose of ANIT and that biliary epithelial injury is accentuated in the KO mice.

Toxicity and toxicokinetics of metformin in rats.

Metformin is a first-line drug for the treatment of type 2 diabetes (T2D) and is often prescribed in combination with other drugs to control a patient's blood glucose level and achieve their HbA1c goal. New treatment options for T2D will likely include fixed dose combinations with metformin, which may require preclinical combination toxicology studies. To date, there are few published reports evaluating the toxicity of metformin alone to aid in the design of these studies. Therefore, to understand the toxicity of metformin alone, Crl:CD(SD) rats were administered metformin at 0, 200, 600, 900 or 1200 mg/kg/day by oral gavage for 13 weeks. Administration of > or =900 mg/kg/day resulted in moribundity/mortality and clinical signs of toxicity. Other adverse findings included increased incidence of minimal necrosis with minimal to slight inflammation of the parotid salivary gland for males given 1200 mg/kg/day, body weight loss and clinical signs in rats given > or =600 mg/kg/day. Metformin was also associated with evidence of minimal metabolic acidosis (increased serum lactate and beta-hydroxybutyric acid and decreased serum bicarbonate and urine pH) at doses > or =600 mg/kg/day. There were no significant sex differences in mean AUC(0-24) or C(max) nor were there significant differences in mean AUC(0-24) or C(max) following repeated dosing compared to a single dose. The no observable adverse effect level (NOAEL) was 200 mg/kg/day (mean AUC(0-24)=41.1 microg h/mL; mean C(max)=10.3 microg/mL based on gender average week 13 values). These effects should be taken into consideration when assessing potential toxicities of metformin in fixed dose combinations.

Detection of feline immunodeficiency virus RNA by two nucleic acid sequence based amplification (NASBA) formats.

Feline immunodeficiency virus (FIV) is an AIDS-inducing lentivirus that infects domestic cats worldwide. Because of its clinicopathologic similarities to human immunodeficiency virus type 1 (HIV-1) infection, the FIV/cat infection system is a valuable animal model for investigating comparative aspects of HIV-1 biology. An assay that detects quickly and efficiently FIV RNA in relatively small volume samples of feline blood or other body fluids would be of benefit in studies of viral transmission and antiviral interventions. Nucleic acid sequence based amplification (NASBA) technology is particularly suited for the detection of RNA in a variety of body fluids. In this report, the development of two rapid, sensitive and versatile NASBA formats is described for the detection of FIV gag RNA in plasma from infected cats. RNA detection by either format was unaffected by the presence of feline plasma. The limits of detection were at least 200 copies of input RNA for both formats. Results from seropositive and seronegative feline plasma samples were clearly distinguishable. These results demonstrate that NASBA provides a rapid and sensitive alternative to RT-PCR and culture isolation for detecting FIV RNA in infected feline plasma.