Translational Relevance of Rodent Models to Predict Human Liver Disease.

Animals models are essential to understand the complex pathobiology of human diseases. George Box's aphorism based on statistics "All models are wrong, but some are useful" certainly applies to animal models of disease. In this session, the translational relevance of various animal models applicable to human liver disease was explored starting with a historic overview of the rodent cancer bioassay with emphasis on hepatocarcinogenesis from early work at the National Cancer Institute, refinement by the National Toxicology Program and contemporary efforts to identify potential mechanisms and their relevance to human cancer risk. Subsequently, recently elucidated understanding of the molecular drivers and signaling mechanisms of liver pathophysiology and liver cancer, including factors associated with liver regeneration, metabolic hepatocellular zonation, and the role of macrophages and their crosstalk with stellate cells in understanding human liver disease was discussed. Next, our contemporary understanding of the role of nuclear receptors in hepatic homeostasis and drug response highlighting nuclear receptor activation and crosstalk in modulating biological responses associated with liver damage and neoplastic response were discussed. Finally, an overview and translational relevance of different drug-induced liver injury (DILI) rodent model systems focused on pathology and mechanisms with commentary on current relevant Food and Drug Administration (FDA) perspective were summarized with closing remarks.

Oral chronic toxicity and carcinogenicity study of alpha-glycosyl isoquercitrin (AGIQ) in Sprague Dawley rats.

alpha-Glycosyl isoquercitrin (AGIQ) is a flavonoid that possesses antioxidant and tumor suppressive capabilities and is marketed as a food additive in Japan. The aim of this study was to assess the potential for oral chronic toxicity and carcinogenicity of AGIQ in male and female Sprague Dawley rats following up to 5.0% dietary exposure. In the chronic toxicity study, rats were exposed to AGIQ or vehicle for one year with a 6-month interim termination point; for the carcinogenicity study, rats were treated for 24 months. No signs of AGIQ-related toxicity clinically or histologically were observed for up to one year except for yellow discoloration of bone. In the carcinogenicity study, a statistically significant increase in the incidence of malignant glioma of the brain or spinal cord was observed in female rats exposed to 5.0% AGIQ compared to those exposed to control feed. A Scientific Advisory Panel of experienced neuropathologists reviewed the gliomas (routine stains and glial cell markers) and concluded that the gliomas were a rare, spontaneous, rat-specific neoplasm: malignant microglial tumor. The lesions could not definitively be attributed to AGIQ exposure and have limited implications with respect to predicting human cancer risk.

Extended one-generation reproductive toxicity study evaluating gardenia blue in Sprague Dawley rats.

Gardenia blue powder was administered at 0.5%, 2.5%, or 5.0% in feed to male and female Sprague Dawley rats in an Extended One-Generation Reproductive Toxicity Study (OECD Test Guideline 443). The dosed diet began 14 days before mating and was continued at the same concentration level for the entire study for all parental animals (P0) and offspring (F1). At weaning, offspring were allocated into one of 5 cohorts for different endpoints. P0 and F1 animals had blue urine, blue or black feces, and blue discolorations in gastrointestinal organs, mesenteric lymph nodes, and kidneys. This treatment-related finding was not considered adverse as there were no histopathologic correlates. There was a dose-related increase in sperm concentration in P0 and F1 males. There were dose-related increases in heart weights of F1 postnatal day (PND) 21 males, male and female thyroid weights, and female TSH levels of PND 91 F1 offspring, with no histopathological correlate. There were no consistent treatment-related adverse effects on any other parameters evaluated for general toxicity, reproductive toxicity, developmental neurotoxicity, or developmental immunotoxicity. The highest dietary concentration (5.0%) of gardenia blue powder was the no observed adverse effect level (NOAEL) for male and female rats at all life stages evaluated.

Progressive disruption of neurodevelopment by mid-gestation exposure to lipopolysaccharides and the ameliorating effect of continuous alpha-glycosyl isoquercitrin treatment.

We investigated the effect of lipopolysaccharide (LPS)-induced maternal immune activation used as a model for producing neurodevelopmental disorders on hippocampal neurogenesis and behaviors in rat offspring by exploring the antioxidant effects of alpha-glycosyl isoquercitrin (AGIQ). Pregnant Sprague-Dawley rats were intraperitoneally injected with LPS (50 µg/kg body weight) at gestational days 15 and 16. AGIQ was administered in the diet to dams at 0.5% (w/w) from gestational day 10 until weaning at postnatal day 21 and then to offspring until adulthood at postnatal day 77. During postnatal life, offspring of LPS-injected animals did not show neuroinflammation or oxidative stress in the brain. At weaning, LPS decreased the numbers of type-2b neural progenitor cells (NPCs) and PCNA+ proliferating cells in the subgranular zone, FOS-expressing granule cells, and GAD67+ hilar interneurons in the dentate gyrus. In adulthood, LPS decreased type-1 neural stem cells, type-2a NPCs, and GAD67+ hilar interneurons, and downregulated Dpysl3, Sst, Fos, Mapk1, Mapk3, Grin2a, Grin2b, Bdnf, and Ntrk2. In adults, LPS suppressed locomotor activity in the open field test and suppressed fear memory acquisition and fear extinction learning in the contextual fear conditioning test. These results indicate that mid-gestation LPS injections disrupt programming of normal neurodevelopment resulting in progressive suppression of hippocampal neurogenesis and synaptic plasticity of newborn granule cells by suppressing GABAergic and glutamatergic neurotransmitter signals and BDNF/TrkB signaling to result in adult-stage behavioral deficits. AGIQ ameliorated most aberrations in hippocampal neurogenesis and synaptic plasticity, as well as behavioral deficits. Effective amelioration by continuous AGIQ treatment starting before LPS injections may reflect both anti-inflammatory and anti-oxidative stress effects during gestation and neuroprotective effects of continuous exposure through adulthood.

Twelve-month in utero safety assessment of gardenia blue, a natural food colorant.

Toxicity assessment of the food colorant Gardenia jasminoides Ellis at dietary exposures of 0.0%, 0.1%, 0.5%, 1.5%, 3.0% and 5.0% included measures of T-cell- dependent antibody response, neurotoxicity, and clinical and anatomic pathology in Sprague Dawley rats during mating, gestation, lactation, postnatal development, and following weaning for up to 12 months including 3- and 6-month interim evaluations. Blue coloration of the gastrointestinal tract, mesenteric lymph nodes and kidneys was present in treated rats only at necropsy with minimal blue coloration at the lowest dose and without histopathological correlates in any of the tissues. There was good survival with no consistent treatment-related changes in hematology, clinical chemistry, enhanced evaluation of lymphoid tissues, or tissue histopathology at interim and final time points. T-cell dependent antibody response and neurotoxicity screening were negative in treated rats. The no-observed-adverse-effect level (NOAEL) was determined to be 5.0% gardenia blue (2,854.5 and 3,465.4 mg/kg/day in parental males and females, respectively, prior to mating; 3,113.5 and 4,049.6 mg/kg/day in male and female offspring, respectively, following up to 12 months of exposure.

Contemporary Activities of Toxicologic Pathology Societies.

Educational activities and training opportunities in toxicologic pathology are major goals of 9 formally established Toxicologic Pathology Societies and the International Academy of Toxicologic Pathology. Some Toxicologic Pathology Societies have examination-based certification programs while others accept certification or registration by veterinary pathology organizations including the American College of Veterinary Pathologists, the European College of Veterinary Pathologists. We summarize here the membership numbers and current activities of formally established Toxicologic Pathology Socities.

Identification of compound causing yellow bone discoloration following alpha-glycosyl isoquercitrin exposure in Sprague-Dawley rats.

Previous rat toxicity studies of alpha-glycosyl isoquercitrin (AGIQ), a water-soluble flavonol glycoside derived from rutin, revealed systemic yellow bone discoloration. This investigative study was conducted to determine the AGIQ metabolite(s) responsible for the discoloration. Female Sprague-Dawley rats were administered dietary AGIQ at doses of 0%, 1.5%, 3.0%, or 5.0% (0, 1735.0, 3480.8, and 5873.7 mg/kg/day, respectively) for 14 days, followed by a 14- or 28-day recovery period. Measurements of quercetin in urine and quercetin, quercetin 3-O-glucuronide, kaempferol, and 3-o-methylquercetin metabolites of AGIQ in bone (femur), white and brown fat, and cerebrum samples were conducted following the exposure period and each recovery period. Gross examination of the femur revealed yellow discoloration that increased in intensity with dose and was still present in a dose-related manner following both recovery periods. Quercetin, at levels correlating with AGIQ dose, was measured in the urine following the 14-day exposure period and, at lower concentrations, 14 or 28 days following cessation of AGIQ exposure. All four metabolites were present in a dose-dependent manner in the femur following 14 days of dietary exposure; only quercetin, quercetin 3-O-glucuronide, and 3-o-methylquercetin were present during the recovery periods. Quercetin, quercetin 3-O-glucuronide, and 3-o-methylquercetin were detected in white fat (along with kaempferol), brown fat (excluding quercetin due to analytical interference), and cerebrum samples, indicating systemic availability of the metabolites. Collectively, these data implicate quercetin, quercetin 3-O-glucuronide, or 3-o-methylquercetin (or a combination thereof) as the most likely metabolite of AGIQ causing the yellow discoloration of bone in rats administered dietary AGIQ.

Contemporary activities of Toxicologic Pathology Societies.

Educational activities and training opportunities in toxicologic pathology are major goals of 9 formally established Toxicologic Pathology Societies and the International Academy of Toxicologic Pathology. Some Toxicologic Pathology Societies have examination-based certification programs while others accept certification or registration by veterinary pathology organizations including the American College of Veterinary Pathologists, the European College of Veterinary Pathologists. We summarize here the membership numbers and current activities of formally established Toxicologic Pathology Socities.

Continuous exposure to α-glycosyl isoquercitrin from developmental stages to adulthood is necessary for facilitating fear extinction learning in rats.

We previously reported that exposure to α-glycosyl isoquercitrin (AGIQ) from the fetal stage to adulthood facilitated fear extinction learning in rats. The present study investigated the specific AGIQ exposure period sufficient for inducing this behavioral effect. Rats were dietarily exposed to 0.5% AGIQ from the postweaning stage to adulthood (PW-AGIQ), the fetal stage to postweaning stage (DEV-AGIQ), or the fetal stage to adulthood (WP-AGIQ). Fear memory, anxiety-like behavior, and object recognition memory were assessed during adulthood. Fear extinction learning was exclusively facilitated in the WP-AGIQ rats. Synaptic plasticity-related genes showed a similar pattern of constitutive expression changes in the hippocampal dentate gyrus and prelimbic medial prefrontal cortex (mPFC) between the DEV-AGIQ and WP-AGIQ rats. However, WP-AGIQ rats revealed more genes constitutively upregulated in the infralimbic mPFC and amygdala than DEV-AGIQ rats, as well as FOS-immunoreactive(+) neurons constitutively increased in the infralimbic cortex. Ninety minutes after the last fear extinction trial, many synaptic plasticity-related genes (encoding Ephs/Ephrins, glutamate receptors/transporters, and immediate-early gene proteins and their regulator, extracellular signal-regulated kinase 2 [ERK2]) were upregulated in the dentate gyrus and amygdala in WP-AGIQ rats. Additionally, WP-AGIQ rats exhibited increased phosphorylated ERK1/2+ neurons in both the prelimbic and infralimbic cortices. These results suggest that AGIQ exposure from the fetal stage to adulthood is necessary for facilitating fear extinction learning. Furthermore, constitutive and learning-dependent upregulation of synaptic plasticity-related genes/molecules may be differentially involved in brain regions that regulate fear memory. Thus, new learning-related neural circuits for facilitating fear extinction can be established in the mPFC.

Reevaluation of Hepatocellular Neoplasms in CD-1 Mice from a 2-year Oral Carcinogenicity Study with Permethrin.

A 24-month oral carcinogenicity study of permethrin was conducted by feeding male and female CD-1 mice diets containing concentrations of 0, 20, 500, and 2,000 ppm of permethrin (males) or 0, 20, 2,500, and 5,000 ppm of permethrin (females). After approximately two years on study, surviving mice were sacrificed for the evaluation of chronic toxicity and/or carcinogenicity. An expert panel of pathologists was convened as a Pathology Working Group (PWG) to review coded liver histology sections from male and female mice and to classify all liver neoplasms according to current nomenclature and diagnostic criteria guidelines. The PWG results indicate that permethrin induced a significant dose-dependent increase in the incidence of hepatocellular neoplasms in treated female mice ( p < .01) as well as a nonstatistically significant increase in the incidence of hepatocellular tumors in treated male mice. Given the continuum of the diagnoses of adenoma and carcinoma, and the difficulty in distinguishing some of the lesions, it is appropriate to consider only the combined incidences of hepatocellular tumors (adenoma and/or carcinoma) for biological significance and risk assessment.

Lack of preventive effect of maternal exposure to α-glycosyl isoquercitrin and α-lipoic acid on developmental hypothyroidism-induced aberrations of hippocampal neurogenesis in rat offspring.

Hypothyroidism during the developmental stage induces disruption of hippocampal neurogenesis in later life, as well as inducing oxidative stress in the brain. The present study investigated the preventive effect of co-exposure to an antioxidant on disruptive neurogenesis induced by developmental exposure to anti-thyroid agent in rats. For this purpose, we used two antioxidants, α-glycosyl isoquercitrin (AGIQ) and α-lipoic acid (ALA). Mated female Sprague Dawley rats were either untreated (control) or treated with 12 ppm 6-propyl-2-thiouracil (PTU), an anti-thyroid agent, in drinking water from gestational day 6 to postnatal day (PND) 21, the latter group being subjected to feeding basal diet alone or diet containing AGIQ at 5,000 ppm or ALA at 2,000 ppm during PTU exposure. On PND 21, PTU-exposed offspring showed reductions in a broad range of granule cell lineage subpopulations and a change in the number of GABAergic interneuron subpopulations. Co-exposure of AGIQ or ALA with PTU altered the transcript levels of many genes across multiple functions, suggestive of enhancement of synaptic plasticity and neurogenesis. Nevertheless, immunohistochemical results did not support these changes. PTU exposure and co-exposure of AGIQ or ALA with PTU did not alter the hippocampal lipid peroxidation level. The obtained results suggest a possibility that thyroid hormone depletion itself primarily disrupts neurogenesis and that oxidative stress may not be involved in the disruption during development. Transcript expression changes of many genes caused by antioxidants may be the result of neuroprotective actions of antioxidants rather than their antioxidant activity. However, no preventive effect on neurogenesis suggested impairment of protein synthesis via an effect on mRNA translation due to hypothyroidism.

Spironolactone in Combination with α-glycosyl Isoquercitrin Prevents Steatosis-related Early Hepatocarcinogenesis in Rats through the Observed NADPH Oxidase Modulation.

Administration of the diuretic, spironolactone (SR), can inhibit chronic liver diseases. We determined the effects of SR alone or in combination with the antioxidant α-glycosyl isoquercitrin (AGIQ) on hyperlipidemia- and steatosis-related precancerous lesions in high-fat diet (HFD)-fed rats subjected to a two-stage hepatocarcinogenesis model. Rats were fed with control basal diet or HFD, which was administered with SR alone or in combination with an antioxidant AGIQ in drinking water. An HFD increased body weight, intra-abdominal fat (adipose) tissue weight, and plasma lipids, which were reduced by coadministration of SR and AGIQ. SR and AGIQ coadministration also reduced hepatic steatosis and preneoplastic glutathione S-transferase placental form-positive foci, in association with decrease in NADPH oxidase (NOX) subunit p22phox-positive cells and an increase in active-caspase-3-positive cells in the foci. Hepatic gene expression analysis revealed that the coadministration of SR and AGIQ altered mRNA levels of lipogenic enzymes ( Scd1 and Fasn), antioxidant-related enzymes ( Catalase), NOX component ( P67phox), and anti-inflammatory transcriptional factor ( Pparg). Our results indicated that SR in combination with AGIQ had the potential of suppressing hyperlipidemia- and steatosis-related early hepatocarcinogenesis through the reduced expression of NOX subunits.

Role of pathology peer review in interpretation of the comet assay.

When a comet assay, an increasingly popular in vivo genotoxicity test, shows a positive test result, interpretation of that response requires ruling out any confounding tissue site toxicity. Since the comet assay typically uses only two or three daily doses of test agent, precursor tissue changes indicative of toxicity may be easily overlooked. Using case examples for two flavoring agents, perillaldehyde and 4,5-epoxydec-2(trans)-enal, we highlight the role of pathology peer review in verifying precursor tissue changes indicative of tissue site toxicity, thereby increasing confidence in final interpretation of comet assay results. Given global deliberation regarding safety assessment of compounds entering the marketplace, we recommend consideration of pathology peer review for equivocal and positive comet assays so that interpretations are universally consistent.

Utilizing novel telepathology system in preclinical studies and peer review.

The demand for board certified toxicologic pathologists and expansion of IT capabilities has resulted in an increase of "virtual" and "distance" pathology (telepathology), and a need for greater flexibility in real time consultations. Newly developed microscope-integrated telepathology systems enable geographically remote stakeholders to view the live histopathology slide as seen by the microscope user. Simultaneous online viewing, annotation, and dialog between pathologists and study experts facilitates good science and economic benefits by enabling more timely and informed clinical decisions.

New Technologies: Real-time Telepathology Systems-Novel Cost-effective Tools for Real-time Consultation and Data Sharing.

Real-time telepathology for use in investigative and regulated preclinical toxicology studies is now feasible. Newly developed microscope-integrated telepathology systems enable geographically remote stakeholders to view the live histopathology slide as seen by the study pathologist within the microscope. Simultaneous online viewing and dialog between study pathologist and remote colleagues is an efficient and cost-effective means for consultation, pathology working groups, and peer review, facilitating good science and economic benefits by enabling more timely and informed clinical decisions.

Ninety-day Local Tolerability and Toxicity Study of ND0612, a Novel Formulation of Levodopa/Carbidopa, Administered by Subcutaneous Continuous Infusion in Minipigs.

A 90-day study in Göttingen minipigs was conducted to test the local tolerability and systemic toxicity of ND0612, a novel aqueous solution of carbidopa (CD)/levodopa (LD) intended for the treatment of Parkinson's disease by continuous subcutaneous administration using a discrete infusion pump. To evaluate tissue site reactions, we used a unique study design involving multiple infusion sites to evaluate the effect of dose per site (270/63, 360/45, and 360/84 mg LD/CD), volume of infusion per site (4.5 and 6 ml per site), formulation concentration (60/14 and 60/7.5 mg/ml LD/CD), daily rate of infusion per site (240 µl/hr for16 hr and 80 µl/hr for 8 hr, 320 µl/hr for 16 hr and 100 µl/hr for 8 hr, or 750 µl/hr for 8 hr), frequency (once every 5, 10, 15, or 20 days), and number of infusions (4, 6, or 9) to the same infusion site. No systemic adverse effects were observed. Histopathological changes at infusion sites started with localized minimal necrosis and acute inflammation that progressed to subacute and chronic inflammatory and reparative changes with evidence of progressive recovery following the final infusion. None of the infusion site effects were judged to be adverse, and clinical exposures to ND0612 are not expected to result in adverse responses.

Regulatory Forum Opinion Piece*: Imaging Applications in Toxicologic Pathology-Recommendations for Use in Regulated Nonclinical Toxicity Studies.

Available imaging systems for use in preclinical toxicology studies increasingly show utility as important tools in the toxicologic pathologist's armamentarium, permit longitudinal evaluation of functional and morphological changes in tissues, and provide important information such as organ and lesion volume not obtained by conventional toxicology study parameters. Representative examples of practical imaging applications in toxicology research and preclinical studies are presented for ultrasound, positron emission tomography/single-photon emission computed tomography, optical, magnetic resonance imaging, and matrix-assisted laser desorption ionization-imaging mass spectrometry imaging. Some of the challenges for making imaging systems good laboratory practice-compliant for regulatory submission are presented. Use of imaging data on a case-by-case basis as part of safety evaluation in regulatory submissions is encouraged.

The legacy of the F344 rat as a cancer bioassay model (a retrospective summary of three common F344 rat neoplasms).

The Fischer 344 (F344) rat was used by the National Toxicology Program (NTP) for over 5 decades for toxicity and carcinogenicity studies. However, in 2006, the NTP decided to switch to a different rat stock due largely to high background control incidences of Leydig cell tumors (LCTs) and mononuclear cell leukemia (MNCL), also known as large granular lymphocytic (LGL) leukemia. In the current review, we aim (1) to provide a summary of NTP bioassays with treatment-associated effects involving MNCL and LCTs in addition to male F344-specific tunica vaginalis mesothelioma (TVM); (2) to describe important pathobiological differences between these F344 rat tumor responses and similar target tissue-tumor response in humans; and (3) to present the NTP reasons for switching away from the F344 rat. We show that due to the highly variable background incidence of F344 MNCL, more reliance on historical control data than is usual for most tumor responses is warranted to evaluate potential effect of any chemical treatment in this rat strain. The high spontaneous incidence of LCTs in the testes of male F344 rats has made this tumor endpoint of little practical use in identifying potential testicular carcinogenic responses. TVM responses in F344 rats have a biological plausible relationship to LCTs unlike TVM in humans. Given their high spontaneous background incidence and species-specific biology, we contend that MNCL and LCT, along with TVM responses, in F344 rat carcinogenicity studies are inappropriate tumor types for human health risk assessment and lack relevance in predicting human carcinogenicity.

Recommendations from the INHAND Apoptosis/Necrosis Working Group.

Historically, there has been confusion relating to the diagnostic nomenclature for individual cell death. Toxicologic pathologists have generally used the terms "single cell necrosis" and "apoptosis" interchangeably. Increased research on the mechanisms of cell death in recent years has led to the understanding that apoptosis and necrosis involve different cellular pathways and that these differences can have important implications when considering overall mechanisms of toxicity, and, for these reasons, the separate terms of apoptosis and necrosis should be used whenever differentiation is possible. However, it is also recognized that differentiation of the precise pathway of cell death may not be important, necessary, or possible in routine toxicity studies and so a more general term to indicate cell death is warranted in these situations. Morphological distinction between these two forms of cell death can sometimes be straightforward but can also be challenging. This article provides a brief discussion of the cellular mechanisms and morphological features of apoptosis and necrosis as well as guidance on when the pathologist should use these terms. It provides recommended nomenclature along with diagnostic criteria (in hematoxylin and eosin [H&E]-stained sections) for the most common forms of cell death (apoptosis and necrosis). This document is intended to serve as current guidance for the nomenclature of cell death for the International Harmonization of Nomenclature and Diagnostic Criteria Organ Working Groups and the toxicologic pathology community at large. The specific recommendations are:Use necrosis and apoptosis as separate diagnostic terms.Use modifiers to denote the distribution of necrosis (e.g., necrosis, single cell; necrosis, focal; necrosis, diffuse; etc.).Use the combined term apoptosis/single cell necrosis whenThere is no requirement or need to split the processes, orWhen the nature of cell death cannot be determined with certainty, orWhen both processes are present together. The diagnosis should be based primarily on the morphological features in H&E-stained sections. When needed, additional, special techniques to identify and characterize apoptosis can also be used.

Practical Applications of in Vivo and ex Vivo MRI in Toxicologic Pathology Using a Novel High-performance Compact MRI System.

Magnetic resonance imaging (MRI) is widely used in preclinical research and drug development and is a powerful noninvasive method for assessment of phenotypes and therapeutic efficacy in murine models of disease. In vivo MRI provides an opportunity for longitudinal evaluation of tissue changes and phenotypic expression in experimental animal models. Ex vivo MRI of fixed samples permits a thorough examination of multiple digital slices while leaving the specimen intact for subsequent conventional hematoxylin and eosin (H&E) histology. With the advent of new compact MRI systems that are designed to operate in most conventional labs without the cost, complexity, and infrastructure needs of conventional MRI systems, the possibility of MRI becoming a practical modality is now viable. The purpose of this study was to investigate the capabilities of a new compact, high-performance MRI platform (M2™; Aspect Imaging, Israel) as it relates to preclinical toxicology studies. This overview will provide examples of major organ system pathologies with an emphasis on how compact MRI can serve as an important adjunct to conventional pathology by nondestructively providing 3-dimensional (3-D) digital data sets, detailed morphological insights, and quantitative information. Comparative data using compact MRI for both in vivo and ex vivo are provided as well as validation using conventional H&E.