Toxicologic Pathology

The ability of FRTL-5 rat thyroid follicular cells to engulf latex beads by phagocytosis was evaluated using flow cytometry and compared to iodide trapping in response to selected growth factors, second messengers, and chemicals. Cell suspensions were analyzed to determine the percentage of fluorescence-positive cells as well as the fluorescence intensity of positive cells. Phagocytosis was stimulated by forskolin, cholera toxin, 8-Br-cAMP, calcitriol, and transforming growth factor-β. In contrast, phagocytosis was inhibited by insulin, calcium, and aminotriazole, but not by sodium iodide. The results of this study showed that phagocytosis of latex beads was regulated in a manner similar to iodide trapping and could be altered by the addition of numerous compounds. Phagocytic activity was stimulated by both cAMP-dependent and cAMP-independent pathways. Flow cytometric evaluation of phagocytosis of fluorescent latex beads represents a simple, rapid, nonradioactive index of thyroid function in vitro.

Hepatic changes induced by phenethyl isothiocyanate (PEITC) in the liver of rats were determined by quantitative microscopy. Groups of male Fischer-344 rats were fed either a standard, cereal-based diet (Wayne rodent meal) or a purified diet (AIN-76A) containing PEITC at concentrations of 0.75 and 6.0 mmol/kg for 13 wk. Severe hepatic lipidosis was observed in control rats fed the purified diet. Addition of PEITC to the purified diet significantly reduced lipid content in hepatocytes. In contrast, lipid content in the liver of the rats fed the cereal-based diet containing PEITC was greater than in control rats maintained on the same diet. In addition, dose-related reductions in hepatocyte, lipid droplet, peroxisome, and mitochondrial volumes were observed in PEITC-treated rats fed the cereal-based diet. These results indicate that PEITC exerts differential effects on the liver of rats fed either the cereal-based or purified diet.

Histologic, electron microscopic, and immunohistochemical studies were made to analyze the structural features and the cellular composition of the pulmonary lesions produced in rats by the administration of interleukin-2 (IL-2). This agent induced pulmonary edema; thickening of alveolar septa; damage to endothelial cells in capillaries and venules, marked interstitial infiltration by cytotoxic T lymphocytes, lymphokine-activated killer (LAK) cells, macrophages, and dendritic cells (as demonstrated by cell counting in preparations stained immunohistochemically with peroxidase- and fluorochrome-labeled antibodies); and injury to bronchiolar and alveolar epithelial cells. Granular and agranular lymphocytes often were closely apposed to endothelial cells in capillaries and venules. Contacts between lymphocytes and type II alveolar epithelial cells also were observed. Damaged type II alveolar epithelial cells showed nuclear and cytoplasmic features that are considered indicative of apoptosis (confirmed by nick end labeling). Phagocytosis of apoptotic bodies by macrophages was occasionally found. These results support the concept that IL-2 induces cytotoxic vascular and parenchymal cell damage that is mediated by LAK cells and cytotoxic T lymphocytes, which make contacts with endothelial cells and type II alveolar epithelial cells. This damage appears to be exacerbated by the secondary release of a variety of vasoactive agents and inflammatory mediators.

The effects of coenzyme Q₁₀ (CoQ₁₀) on the hydrazine-induced changes in the structure of mitochondria and those in antioxidant systems of the liver were investigated using rats as experimental animals. Animals were placed on a powdered diet containing 1.0% hydrazine for 7-8 days in the presence or absence of the combined treatment with CoQ₁₀. Results obtained were as follows: (a) treatment of animals with CoQ₁₀ prevented the hydrazine-induced formation of megamitochondria in the liver; (b) changes observed in the liver of the hydrazine-treated animals in comparison to the control were increases in the contents of α-tocopherol and CoQ analogs, increases in the levels of lipid peroxidation, decreases in the level of reduced glutathione with increases in that of oxidized glutathione, and increases in the ratio of unsaturated to saturated fatty acids in phospholipid domains of mitochondrial membranes; and (c) administration of CoQ₁₀ to hydrazine-treated animals suppressed enhanced lipid peroxidation and improved lowered adenosine diphosphate/O ratios of mitochondria. The present data suggest that CoQ₁₀ suppresses the hydrazine-induced formation of megamitochondria by scavenging free radicals generated from hydrazine and its metabolites.

Malformations of the maxillary incisors, diagnosed as dental dysplasia, were observed as a spontaneous background lesion in 3% (females) to 9% (males) of CD-1® mice and 14.5% (females) to 10.5% (males) of CD@ (Sprague-Dawley) rats in a chronic inhalation study. Lesions were reported grossly as overgrown, maloccluded, or malformed incisors. Microscopic findings included tooth pulp and periodontal abscesses, fractured and necrotic teeth, periodontal cysts, malformations of the incisor roots, and expansile masses, including odontomas, of the incisor roots. Development of lesions followed a pattern of tooth pulp necrosis and/or traumatic disruption of the epithelial root sheath at the base of the tooth. Feeding a powdered ration, which reduced the normal wearing of the incisors, and repeated clipping of overgrown incisors were believed to contribute to the incidence of disease.

Reactive oxygen species, especially iron-catalyzed hydroxyl radicals (·OH), are implicated in the pathogenesis of asbestos-induced pulmonary toxicity. We previously demonstrated that phytic acid, an iron chelator, reduces amosite asbestos-induced OH generation, DNA strand break formation, and injury to cultured pulmonary epithelial cells (268 [1995, Am. J. Physiol. (Lung Cell. Mol. Physiol.) 12: L471-480]). To determine whether phytic acid diminishes pulmonary inflammation and fibrosis in rats after a single intratracheal (it) instillation of amosite asbestos, Sprague-Dawley rats were given either saline (1 ml), amosite asbestos (5 mg; 1 ml saline), or amosite treated with phytic acid (500 μM) for 24 hr and then instilled. At various times after asbestos exposure, the rats were euthanized and the lungs were lavaged and examined histologically. A fibrosis score was determined from trichrome-stained specimens. As compared to controls, asbestos elicited a significant pulmonary inflammatory response, as evidenced by an increase (∼ 2-fold) in bronchoalveolar lavage (BAL) cell counts at 1 wk and the percentage of BAL neutrophils (PMNs) and giant cells at 2 wk (0.1 vs 6.5% and 1.3 vs 6.1%, respectively; p < 0.05). Asbestos significantly increased the fibrosis score at 2 wk (0 ± 0 vs 5 ± 1; p < 0.05). The inflammatory and fibrotic changes were, as expected, observed in the respiratory bronchioles and terminal alveolar duct bifurcations. The increased percentage of BAL PMNs and giant cells persisted at 4 wk, as did the fibrotic changes. Compared to asbestos alone, phytic acid-treated asbestos elicited significantly less BAL PMNs (6.5 vs 1.0%; p < 0.05) and giant cells (6.1 vs 0.2%; p < 0.05) and caused significantly less fibrosis (5 vs 0.8; p < 0.05) 2 wk after exposure. We conclude that asbestos causes pulmonary inflammation and fibrosis in rats after it instillation and that phytic acid reduces these effects. These data support the role of iron-catalyzed free radicals in causing pulmonary toxicity from asbestos in vivo.

A spontaneous mammary adenocarcinoma was detected in a 10-wk-old female virgin Sprague-Dawley rat in a subacute toxicity study for safety assessment. The rat was sacrificed according to the study schedule at 23 wk of age and was subjected to complete necropsy. Gross pathological examination revealed that the tumor had reached 20 x 35 x 45 mm. It was settled in the right posterior abdominal subcutaneous tissue and had not invaded the surrounding tissues. Histopathologically, it was diagnosed as a typical mammary adenocarcinoma of rats. In our literature search, we found no previous reports of spontaneous adenocarcinomas of the mammary gland in such a young rat. Therefore, attention should be paid to the fact that mammary adenocarcinomas can occur spontaneously at an early age in rats.

The beneficial effects derived from the use of chemicals in agriculture, energy production, transportation, pharmaceuticals, and other products that improve the quality of life are clearly established. However, continued exposure to these chemicals is only advantageous in conditions where the benefit far outweighs toxic manifestations. By law, determination of risk of toxicity necessitates the use of laboratory animals to establish whether chemical exposure is safe for humans. To simulate the human condition, it is incumbent upon investigators to choose a species in which pharmacokinetic and toxicokinetic principles are established and resemble those of humans. Some of the advantages to the use of rat in chemical toxicity testing include (a) similarities in metabolism, anatomy, and physiological parameters to humans; (b) the short life span, especially for carcinogenesis study; (c) the availability, ease of breeding, and maintenance at a relatively low cost; and (d) the existence of a large database to enable comparison of present to reported literature findings. However, the choice of rat can be complicated by several factors such as sex, age, and nutrition, but especially strain, where currently there are over 200 different strains of rat known to exist. The aim of this review is to demonstrate that there are differences in the responsiveness of rat strains to chemicals and that the susceptibility observed is dependent on the tissue examined. It is evident that the genotype differs among strains, and this may be responsible for differences in sensitivities to chemicals. Awareness of strain as a factor in susceptibility to toxicant action needs to be taken into account in interpretation of relevance of risk of toxicity for humans.

Since 1985, pathologists at Cornell University have investigated the causes of lesions in freshwater fish throughout New York waters in order to clarify possible impairment of fish health by environmental contaminants. Fishermen and biologists alerted us to several relatively protected reservoirs and ponds in which we have found no evidence of elevated levels of anthropogenic environmental contaminants but in which up to 100% of brown bullheads exhibited skin neoplasia. Complete necropsies and histologic study revealed that over 30% of mature brown bullheads from some of these sites had benign or malignant hepatocellular or biliary liver neoplasia. Up to 50% of brown bullheads had benign or malignant liver neoplasia in other relatively unpolluted waters with no evidence of skin neoplasia in bullheads. Multiple samplings of brown bullheads from several of these sites have revealed puzzling variability in the prevalence of skin, liver, and other neoplasia in these fish populations. The cause of these striking epizootics of neoplasia in brown bullheads in unpolluted waters in New York State remains unclear. We hypothesize that natural carcinogens such as N-nitroso compounds formed in aquatic sediments or radon from geologic formations may contribute to epizootic fish neoplasia in New York waters.

Since 1985, fish pathologists at Cornell University have studied lesions in freshwater fish inhabiting natural waters throughout New York State in order to clarify possible adverse effects of contaminants on fish health. In the course of these studies, we have conducted complete necropsies on over 400 brown bullheads and histologic examinations of the major organs of over 370 of these fish. In the course of our histologic studies, we observed islands of well-differentiated hepatocytes in the spleens of neoplasm-free brown bullheads from 2 of the 37 diverse lakes, rivers, and ponds that we sampled. The splenic hepatocyte islands occurred in 11-100% of fish from the 2 affected sites, with 1-50 hepatocyte clusters per section of spleen. Hepatocyte islands measured up to 1 mm in diameter. Unlike the metastases of hepatocellular carcinomas, which we have described elsewhere in this issue (9), these islands of hepatocytes in the spleens of neoplasm-free fish were not evident grossly. These hepatocyte clusters in the spleen of certain populations of brown bullheads may lead to confusion in studies of neoplasia in brown bullheads. Caution is advised in interpretation of metastasis of liver neoplasms to the spleen of brown bullheads.

Spermatic granulomas are inflammatory lesions that occur in the efferent ductule and epididymis of humans, goats, rats, and bulls. Three clinically normal dogs from a control group had histologic lesions of the initial segment of the caput epididymis consisting of intratubular spermatic granulomas. The granulomas were located within the efferent ductules, and the inflammatory response consisted primarily of aggregates of spermiophagic macrophages in the ductules. Sperm stasis of the affected ductules was evident by the dilation and accumulation of large numbers of spermatozoa in adjacent cross-sections of efferent ductules. Blind-ending efferent ductules were demonstrated by serially sectioning the initial segment of the epididymis. The intratubular granulomas did not completely occlude the outflow tract because spermatozoa were present in the cauda epididymis. While spermatic granulomas can be induced by trauma, infection, or toxins, spontaneous granulomas due to blind-ending ductules should be considered as a differential. Clinical history, location of the granuloma, and serial sectioning can help determine the most likely etiology in early cases.