Toxicologic Pathology

To study various polyhydroxy-alkaloid glycosidase inhibitors, 16 groups of 3 mice were dosed using osmotic minipumps with swainsonine (0, 0.1, 1, and 10 mg/kg/day), castanospermine, and calystegines A₃, B₂, and C₁ (1, 10, and 100 mg/kg/day). After 28 days, the mice were euthanized, necropsied, and examined using light and electron microscopy. The high-dose swainsonine–treated mice developed neurologic disease with neuro-visceral vacuolation typical of locoweed poisoning. Castanospermine- and calystegines-treated mice were clinically normal; however, high-dose castanospermine–treated mice had thyroid, renal, hepatic, and skeletal myocyte vacuolation. Histochemically, swainsonine- and castanospermine-induced vacuoles contained mannose-rich oligosaccharides. High-dose calystegine A₃–treated mice had increased numbers of granulated cells in the hepatic sinusoids. Electron microscopy, lectin histochemistry, and immunohistochemistry suggest these are pit cells (specialized NK cells). Histochemically, the granules contain glycoproteins or oligosaccharides with abundant terminal N-acetylglucosamine residues. Other calystegine-treated mice were histologically normal. These findings indicate that swainsonine produced lesions similar to locoweed, castanospermine caused vacuolar changes with minor changes in glycogen metabolism, and only calystegine A₃ produced minimal hepatic changes. These also suggest that in mice calystegines and castanospermine are less toxic than swainsonine, and as rodents are relatively resistant to disease, they are poor models to study such induced storage diseases.

A toxicogenomics study was performed in the mouse liver after treatment of a bile-duct–damaging chemical, 4,4′-methylene dianiline (MDA), across multiple doses and sampling times in an acute phase using the AB Expression Array System. Imprinting control region (ICR) mice were given a single oral administration of a low (10 mg/kg b.w.) or high (100 mg/kg b.w.) dose of MDA. Mice were sacrificed six, twenty-four, and seventy-two hours after treatment for serum chemistry, histopathology, and mRNA preparation from liver samples. Treatment with MDA increased liver-toxicity–related enzymes in blood and induced bile-duct cell injury, followed by regeneration. To explore potential biomarker gene profiles, the altered genes were categorized into four expression patterns depending on dose and time. Numerous functionally defined and unclassified genes in each category were up- or down-regulated throughout the period from cellular injury to the recovery phase, verified by RT-PCR. Many genes associated with liver toxicity and diseases belonged to one of these categories. The chemokine-mediated Th1 pathway was implicated in the inflammatory process. The genes associated with oxidative stress, apoptosis, and cell-cycle regulation were also dynamically responsive to MDA treatment. The Wnt/β-catenin signaling pathway was likely responsible for the reconstitution process of the MDA-injured liver.

Ovarian follicle counting is a method to assess ovarian toxicity in reproductive toxicity studies in rats. Although ovarian follicle counting has been traditionally performed manually on hematoxylin and eosin (H&E)-stained sections, the use of immunohistochemical methods, including human cytochrome P450 1B1 (CYP1B1) and proliferating cell nuclear antigen (PCNA), have been used to enhance the visibility of the primordial and primary follicles to facilitate manual counting. In this study, serial sections from both ovaries from ten 3-month-old female Sprague Dawley rats were stained using routine H&E and immunohistochemistry for PCNA. Counting of primordial and primary follicles was performed manually using these two stains and by semi-automated image analysis of PCNA-stained slides. Although manual counting of PCNA-stained slides is preferable to manual counting of H&E-stained slides, manual counting involves variability between individual counters. Semi-automated image analysis of PCNA-stained slides yields an accurate and consistent count of these primordial/primary follicles and eliminates variability between individual counters.

Exposure to particulate matter (PM) air pollution is associated with increased asthma morbidity. Residual oil flash ash (ROFA) is rich in water-soluble transition metals, which are involved in the pathological effects of PM. The objective of this study was to investigate the effects of intranasal administration of ROFA on pulmonary inflammation, pulmonary responsiveness, and excess mucus production in a mouse model of chronic pulmonary allergic inflammation. BALB/c mice received intraperitoneal injections of ovalbumin (OVA) solution (days 1 and 14). OVA challenges were performed on days 22, 24, 26, and 28. After the challenge, mice were intranasally instilled with ROFA. After forty-eight hours, pulmonary responsiveness was performed. Mice were sacrificed, and lungs were removed for morphometric analysis. OVA-exposed mice presented eosinophilia in the bronchovascular space (p < .001), increased pulmonary responsiveness (p < .001), and epithelial remodeling (p = .003). ROFA instillation increased pulmonary responsiveness (p = .004) and decreased the area of ciliated cells in the airway epithelium (p = .006). The combined ROFA instillation and OVA exposure induced a further increase in values of pulmonary responsiveness (p = .043) and a decrease in the number of ciliated cells in the airway epithelium (p = .017). PM exposure results in pulmonary effects that are more intense in mice with chronic allergic pulmonary inflammation.

Reduced food consumption and associated lower body weights may occur in subacute toxicity studies. The short-term effects of food restriction (FR) on body and reproductive organ weights, hormones, and testis histology were assessed in Sprague-Dawley rats fed 20% to 36% less (21 g feed/day) than rats fed ad libitum (AL) starting at six, eight, ten, or twelve weeks of age for two or six weeks. Body weight and relative seminal vesicle, ventral prostate, and/or epididymis weights were reduced in rats FR for two or six weeks. Degeneration of stage VII pachytene spermatocytes was seen in rats FR for six weeks when initiated at eight, ten, and twelve weeks of age. Plasma testosterone concentrations were lower in rats FR at ages six to eight weeks, eight to ten weeks, six to twelve weeks, and eight to fourteen weeks. Luteinizing hormone was not statistically different in FR rats compared with AL counterparts. Therefore, duration of lower food intake had a greater impact on spermatogenesis, whereas a younger initial age of lower food intake was more influential on testosterone levels. These interactions are important in the interpretation of subacute toxicology studies employing FR or when test articles lower food consumption relative to AL-fed rats.

Age- and gender-related lymphoid tissue variability in control male and female monkeys of various ages (under three years; three to six years; seven to fifteen years) was characterized. Spleen and thymus organ weights, organ-to-body and organ-to-brain ratios, morphology by light microscopy, and B- and T-cell immunohistochemistry (IHC) were evaluated. Splenic weights and ratios were not significantly different between various age groups or genders, except males and females in the three-to-six-years age group, who exhibited statistically significant changes from the under-three-years age group. No differences in the number of primary follicles, secondary follicles with germinal centers, B-cell follicles, and periarterial lymphoid sheath were seen between age groups or genders, and no trends were noted in the spleen. By IHC, no differences were observed in B- and T-cell splenic densities. Several age- and gender-related changes in weights and ratios were noted in the thymus. The thymus had a trend toward increased interlobular fat infiltration with increasing age in both males and females. Thymic delineation of the cortex and medulla was significantly decreased in the seven-to-fifteen-years age group for males only. The cortex-to-medulla ratio was significantly lower only in males in the seven-to-fifteen-years age group. B- and T-cell cellular density did not change across various ages.

Pregnant C57BL/6 mice were exposed to 5 μg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or vehicle by oral gavage between gestation days (GDs) 11 and 13. The thymus, spleen, and liver of the pups were examined histologically, and cell surface antigen expression was assessed on postnatal days (PNDs) 1, 14, 25, and 46. In addition to the expected decrease in thymic weight on PND 1, TCDD caused an increase in splenic weight on PND 14 and in hepatic weight on PNDs 14 and 25. The apoptotic index was increased and the corticomedullary border poorly defined in thymuses of TCDD-exposed mice on PND 1, but not at later endpoints. T lymphocytes were increased and B lymphocytes decreased in spleens of the TCDD-exposed mice on PND 46. TCDD-exposed mice had a nearly significant (p =.051) decrease in the number of splenic germinal centers on PND 46. Foci of extramedullary hematopoiesis (EMH) were increased in number in the livers of TCDD-exposed mice on PND 14, suggesting possible increased production of immune cells of unknown phenotype and function in this organ. These results suggest that late-gestation thymic architectural changes caused by TCDD resolve shortly after birth: however, abnormalities in other immunologically important areas may appear later in postnatal life.

We investigated the effects of erdosteine on acetaminophen (APAP)-induced hepatotoxicity in rats. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), AST (aspartate aminotransferase), and ALT (alanine transaminase) activities, and malonyldialdehyde (MDA) and nitric oxide levels as oxidant/antioxidant biochemical parameters were investigated with light microscopic evaluation in adult female Wistar Albino rats. APAP administration produced a decrease in hepatic SOD, CAT, and GSH-Px activities, and coadministration of erdosteine (150 and 300 mg/kg) resulted in increases in the activities. MDA and NO levels increased in the APAP group, and erdosteine treatments prevented these increases. Significant elevations in serum AST and ALT levels were observed in the APAP group, and when erdosteine and APAP were coadministered, their serum levels were close to those in the control group. Light microscopic evaluation of livers showed that there were remarkable centrilobular (zone III) hepatic necrosis and mild to moderate sinusoidal congestion in the APAP group, whereas in the erdosteine group, cellular necrosis was minimal and the hepatocytes maintained a better morphology when compared to the APAP group. Erdosteine prevented APAP-induced liver injury and toxic side effects probably through the antioxidant and radical scavenging effects of erdosteine.

The incidences of alveolar/bronchiolar adenomas and carcinomas in cumene-treated B6C3F1 mice were significantly greater than those of the control animals. We evaluated these lung neoplasms for point mutations in the K-ras and p53 genes that are often mutated in humans. K-ras and p53 mutations were detected by cycle sequencing of PCR-amplified DNA isolated from paraffin-embedded neoplasms. K-ras mutations were detected in 87% of cumene-induced lung neoplasms, and the predominant mutations were exon 1 codon 12 G to T transversions and exon 2 codon 61 A to G transitions. P53 protein expression was detected by immunohistochemistry in 56% of cumene-induced neoplasms, and mutations were detected in 52% of neoplasms. The predominant mutations were exon 5, codon 155 G to A transitions, and codon 133 C to T transitions. No p53 mutations and one of seven (14%) K-ras mutations were detected in spontaneous neoplasms. Cumene-induced lung carcinomas showed loss of heterozygosity (LOH) on chromosome 4 near the p16 gene (13%) and on chromosome 6 near the K-ras gene (12%). No LOH was observed in spontaneous carcinomas or normal lung tissues examined. The pattern of mutations identified in the lung tumors suggests that DNA damage and genomic instability may be contributing factors to the mutation profile and development of lung cancer in mice exposed to cumene.

Dogs treated with AR-H047108, an imidazopyridine potassium competitive acid blocker (P-CAB), developed clinical signs of hepatic dysfunction as well as morphologically manifest hepatotoxicity in repeat-dose toxicity studies. An investigative one-month study was performed, with interim euthanasia after one and two weeks. A detailed histopathological and immunohistochemical characterization of the liver lesions was conducted, including markers for fibrosis, Kupffer cell activation, apoptosis, and endothelial injury. In addition, hepatic retinoid and procollagen 1α2 mRNA levels in livers of dogs treated with AR-H047108 were analyzed. The results showed an early inflammatory process in central veins and centrilobular areas, present after one week of treatment. This inflammatory reaction was paralleled by activation of stellate/Ito cells to myofibroblasts and was associated with sinusoidal and centrivenular fibrosis. The early activation of stellate cells coincided with a significant decrease in retinyl ester levels, and a significant increase in procollagen 1α2 mRNA levels, in the liver. At later time points (three and six months), there was marked sinusoidal fibrosis in centrilobular areas, as well as occlusion of central veins resulting from a combination of fibrosis and increased thickness of smooth muscle bundles in the vessel wall. The pattern of lesions suggests a veno-occlusive-disease (VOD)–like scenario, possibly linked to the imidazopyridine chemical structure of the compound facilitated by specific morphological features of the dog liver.

Myopericytoma is a perivascular tumor that has been recently described in humans, but not in laboratory rodents. The authors encountered an intra-abdominal tumor resembling human malignant myopericytoma in a Fischer rat. Grossly, the tumor was found as two brown-colored masses located in the mesentery of rectum. Microscopically, the tumor was composed of oval to spindle-shaped cells, which were arranged in sheets around numerous thin-walled branching vessels and partly showed a concentric perivascular growth pattern. Mitoses were frequently seen, and the tumor cells showed a local invasion. Immunohistochemically, the tumor cells were strongly positive for alpha-smooth muscle actin and weakly positive for vimentin and desmin. Ultrastructurally, the tumor cells had dendritic processes, actin-like thin filaments with dense bodies, basement membranes, hemidesmosomes, and micropinocytotic vesicles. These findings suggest that the most appropriate term for diagnosis of the present case could be a malignant myopericytoma.

National Toxicology Program (NTP) inhalation studies demonstrated that cumene significantly increased the incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. Cumene or isopropylbenzene is a component of crude oil used primarily in the production of phenol and acetone. The authors performed global gene expression analysis to distinguish patterns of gene regulation between cumene-induced tumors and normal lung tissue and to look for patterns based on the presence or absence of K-ras and p53 mutations in the tumors. Principal component analysis segregated the carcinomas into groups with and without K-ras mutations, but failed to separate the tumors based on p53 mutation status. Expression of genes associated with the Erk MAP kinase signaling pathway was significantly altered in carcinomas with K-ras mutations compared to tumors without K-ras mutations or normal lung. Gene expression analysis also suggested that cumene-induced carcinomas with K-ras mutations have greater malignant potential than those without mutations. In addition, significance analysis of function and expression (SAFE) demonstrated expression changes of genes regulated by histone modification in carcinomas with K-ras mutations. The gene expression analysis suggested the formation of alveolar/bronchiolar carcinomas in cumene-exposed mice typically involves mutation of K-ras, which results in increased Erk MAP kinase signaling and modification of histones.

Although there are a few national schemes for accreditation/certification of toxicologic pathologists (e.g., in Japan and the United Kingdom), a global recognition system for bench toxicologic pathologists is missing, as are universal standards defining their core competencies. This paper summarizes basic means regarding how proficiency in toxicologic pathology is acquired, provides an overview over examinations of interest to toxicologic pathologists, and emphasizes the value of practical experience in the field. The paper then discusses basic approaches to evaluate the proficiency of toxicologic pathologists and examines potential means to recognize qualified toxicologic pathologists. With progressive globalization, it is important that the toxicologic pathology community deepens the discussion regarding a global recognition mechanism for their discipline.