Toxicologic Pathology

Reactive oxygen species (ROS) and other radicals are involved in a variety of biological phenomena, such as mutation, carcinogenesis, degenerative and other diseases, inflammation, aging, and development. ROS are well recognized for playing a dual role as deleterious and beneficial species. The objectives of this review are to describe oxidative stress phenomena, terminology, definitions, and basic chemical characteristics of the species involved; examine the biological targets susceptible to oxidation and the defense mechanisms of the organism against these reactive metabolites; and analyze methodologies, including immunohistochemical markers, used in toxicological pathology in the visualization of oxidative stress phenomena. Direct detection of ROS and other free radicals is difficult, because these molecules are short-lived and highly reactive in a nonspecific manner. Ongoing oxidative damage is, thus, generally analyzed by measurement of secondary products including derivatives of amino acids, nuclei acids, and lipid peroxidation. Attention has been focused on electrochemical methods based on voltammetry measurements for evaluating the total reducing power of biological fluids and tissues. This approach can function as a tool to assess the antioxidant-reducing profile of a biological site and follow changes in pathological situations. This review thus includes different topics essential for understanding oxidative stress phenomena and provides tools for those intending to conduct study and research in this field.

Diet-associated kidney diseases of rats includes nephropathy in both sexes and nephrocalcinosi s in females. High protein content of diets appears to be the major cause for severe nephropathy and changing the source of protein to one such as soy protein, restricting caloric intake, or modifying the diet to decrease protein consumption could decrease the severity of nephropathy. The NTP-2000 diet with lower protein content than most diets decreases the severity of nephropathy and increases the survival of Fischer 344 rats without substantial changes in growth patterns and body weights. Nephrocalcinosis, characterized by mineralization of renal tubules at the corticomedullary junction, has been reported in young and adult female rats of most strains and stocks suggesting a major contribution of female sex hormones to the development of this lesion. Calcium (Ca), phosphorou s (P), magnesium (Mg), and chloride (Cl) imbalances, especially a Ca:P ratio of less than 1.0 in diet, are considered to be associated with this lesion. Most commercial diets commonly used for toxicology studies have a Ca:P molar ratio of less than 1.0. Increasing the Ca:P molar ratio to more than 1.0 and closer to 1.3 in the AIN-93 purified diet and NTP-2000 nonpurified diet prevents the development of this lesion. Genetics will predispos e rats to some diseases and environmental factors will influence the severity of these diseases. Diet is one of the most important environmental factors. Diets balanced for nutrients without excesses could markedly improve the health of rats used in chronic studies leading to substantial increases in survival and thereby accomplish the objective of chronic toxicity and carcinogenicity studies.

Many rodent renal and bladder carcinogens rely upon epigenetic mechanisms of carcinogenesis; such mechanisms are likely to influence the spectrum of urinary tract tumors observed in control and treated animals. This is reflected in several features of chemically induced rodent urinary tract neoplasms, including a low overall tumor incidence, an increased prevalence of urinary tract tumors in rats compared to mice and males compared to females, the tendency for epithelial tumors to predominate over nonepithelial types, and demonstrated links to chronic progressive nephropathy and urolithiasis. Such tendencies are also characteristic of spontaneous urinary tract tumors in rodents. Data to support these observations can be derived from large historical databases such as the Toxicology Data Management System, maintained by National Toxicology Program.

Bladder neoplasia in humans consists of 2 diseases, a low-grade papillary tumor that does not invade or metastasize, and a high-grade lesion that usually invades and metastasizes. Bladder carcinogenesis in rats is most like the low-grade, papillary tumor, although it eventually does progress and invade. In the mouse, models are available that mimic each of these disease processes. Preneoplastic lesions in humans and rodents include various types of hyperplasia, proliferative cystitis, and dysplasia. These preneoplastic and neoplastic lesions arise throughout the urothelium, from the renal pelvis to the urethra, although most commonly in the bladder. Rarely, benign and malignant mesenchymal lesions occur in rats and mice, with a unique submucosal mesenchymal lesion present in some strains of mice. In addition, eosinophilic and clear inclusions in the superfi cial layer of urothelium in mice, which do not appear to be associated with toxicity or carcinogenesis, have been reported. An approach to evaluation of carcinogenic mechanisms involved in the urothelium is presented. It focuses on distinguishing between DNA reactive carcinogens vs those that act by increasing cell proliferation. Although rodent model s do not precisely mimic the human disease, they have provided useful model s for furthering our understanding of the carcinogenic process in the urothelium as it pertains to human diseases.

Renal papillary necrosis (RPN) is a signifi cant problem in human beings, especially in England and in Australia where it has been reported to account for 15% to 20% of patients needing renal transplants. Many compounds, including aspirin, phenacetin, phenylbutazone , indomethacin, mefenamic acid, flufenamic acid, fenoprofin, naproxen, and ibuprofen have been linked to renal papillary necrosis in human beings. Although the exact mechanism of RPN is unknown, there are several theories that have good scientifi c evidence behind them. Study of RPN in animals as models for the disease in human beings is limited by several factors, including anatomical differences between human beings and most animal species as well as technical diffi culties in studying the renal papilla.

The kidney is a frequent site for chemically induced cancers in rodents and among the 10 most frequent sites for cancer in human patients. Renal cell carcinoma (RCC) is the most frequent upper urinary tract cancer in humans and accounts for 80—85% of malignant renal tumors. Hereditary RCC occurs in Eker rats that are heterozygou s for an insertion mutation in the Tsc2 tumor suppressor gene. The germline mutation renders heterozygous mutants highly susceptible to renal carcinogens. The utility of this model in studying potential renal carcinogens is due to an ordered progression of proliferative renal lesions that can be identified and counted microscopically. The quantitative nature of the model allows for the production of statistically powerful data to understand the relative degree and potency of chemical effects and allow analysis of genetic alterations that may be chemical specific.

Chronic progressive nephropathy (CPN) is the most frequently diagnosed lesion in the rat kidney. It has many component s including degeneration and regeneration of renal tubule (RT) epithelium, glomerular lesions and interstitial inflammation and fi brosis. The incidence and severity of CPN is strain, age, and sex dependent and may be altered by a number of factors including exposure to xenobiotics. In National Toxicology Program (NTP) 2-year bioassays, xenobiotic-associated increased severity (exacerbation) of CPN often occurs in association with a marginal increased incidence of renal tubule cell neoplasms (RTCN). The relationship between CPN and RTCN development has not been defi nitively determined. The present study evaluated the association between severity of CPN and the occurrence of RTCN in control male F344 rats. A slight but statistically signifi cant increase in CPN severity was present in those animals with RTCN compared to aged-matched controls without RTCN. Although these data suggest there is a positive correlation between CPN and RTCN, cause and effect were not determined. This marginal association suggests that the number of RTCNs that may develop secondary to chemically exacerbated nephropathy would be few.

The nonnucleoside inhibitor (NNI) of HIV-1 reverse transcriptase, PHI-346 (N -[2-(1-cyclohexenyl)ethyl]- N'-[2-(5-bromopyridyl)]-thiourea), is a dual-function spermicidal agent with potent anti-HIV activity against drug-sensitive as well as drug-resistant HIV-1 strains with genotypic and phenotypic NNI resistance. PHI-346 was formulated via a lipophilic gel-microemulsion for intravaginal use as a potential dual-function microbicide. To evaluate the toxicity potential of short-term intravaginal exposure to PHI-346, groups of 15 female B₆C₃F ₁ and CD-1 mice were exposed intravaginally to a gel-microemulsion containing 0, 0.5, 1.0, or 2.0% PHI-346, 5 days per week for 13 consecutive weeks. On a molar basis, these concentrations of PHI-346 are 350 to 1,400-times higher than its spermicidal EC₅₀ and nearly 5 × 10⁶ to 2 × 10⁷ times higher than its in vitro anti-HIV IC₅₀. After 13 weeks of intravaginal treatment, B₆C₃F ₁ mice were evaluated for survival, body weight gain, absolute and relative organ weights. Blood was analyzed for hematology and clinical chemistry profiles. Microscopic examination was performed on hematoxylin and eosin-stained tissue sections from each study animal. Placebo control and PHI-346 dosed female CD-1 mice were mated with untreated males in order to evaluate if PHI-346 has any deleterious effects on the reproductive performance. There were no treatment-related mortalities. Mean body weight gain during the dosing period was not reduced by PHI-346 treatment. The hemogram or blood chemistry profiles revealed lack of systemic toxicity following daily intravaginal instillation of PHI-346 for 13 weeks. No clinically significant changes in absolute and relative organ weights were noted in PHI-346 dose groups. Extensive histopathological examination of tissues revealed no treatment-related abnormalities in any of the three PHI-346 dose groups. Repeated intravaginal exposure of CD-1 mice to increasing concentrations of PHI-346 for 13 weeks had no adverse effect on their subsequent reproductive capability, perinatal outcome, growth, and development of offspring. Collectively, these findings demonstrate that repetitive intravaginal administration of PHI-346 at concentrations as high as 1,400-times its spermicidal EC₅₀ and 2 ×10⁷ times its in vitro anti-HIV IC₅₀ was not associated with local or systemic toxicity and did not adversely affect the reproductive performance in mice. PHI-346 may be useful as an active ingredient of a safe vaginal microbicide for prevention of the sexual transmission of multidrug-resistant HIV-1.

The carcinogenic potential of chlorpromazine hydrochloride, a psychotropic agent, was assessed in the p53 heterozygou s mouse assay. In a 4-week dose range fi nding study in p53 wild-type mice, doses of 20, 40, 60, and 80 mg/kg were poorly tolerated becaus e of mortality secondary to the severe sedative and hypotensive effects of chlorpromazine. Based on 40% mortality at a dose of 20 mg/kg in the dose-range fi nding study, a high dose of 10 mg/kg was chosen for the 26-week carcinogenicity study in p53 heterozygous mice. Doses of 2.5, 5, and 10 mg/kg chlorpromazin e hydrochloride were well tolerated in the 26-week study. The administration of chlorpromazine hydrochloride at dose levels up to and including 10 mg/kg to p53 heterozygous and wild-type mice did not result in a dose-related increase in tumor incidence or in the type of tumors seen in comparison to controls. Findings related to the administration of chlorpromazine in the 26-week study were limited to minimal uterine and ovarian atrophy in p53 wild-type mice dosed with 10 mg/kg chlorpromazine hydrochloride. However, p53 heterozygous mice administered 400 mg/kg p-cresidine, a genotoxic carcinogen commonly used as a positive control for this model, developed urinary bladder tumors. Administration of p-cresidine also resulted in a regenerative anemia, splenic and hepatic hemosiderosis, renal findings, and ovarian and uterine atrophy. This study demonstrated that chlorpromazine hydrochloride, at the doses tolerated, was not carcinogenic in the p53 heterozygou s mouse assay.

In the multistep process of tumor development, several events occur to transform cells from normal to malignant. Although p53 is one of the most commonly mutated genes in a wide variety of tumors, how other genes interact with p53 to transform cells is only just beginning to be understood. To study the effects of the interaction of the Fas system with p53 in tumor progression and development, mice with a targeted disruption of the p53 tumor supressor gene and a mutation in Fas ligand were bred. Organ weights, life expectancy, and tumor and tissue histology were assessed. Although spleen weights were drastically increased in FasL^-/- p53^{- /-} mice, the FasL deficiency had no effect on life expectancy or the tumor spectrum of homozygous p53-deficient mice. The FasL deficiency reduced the median time to death from 12.1 months in FasL^+/+ p53^{+ /-} mice to 9.6 months in FasL^-/- p53 ^{+ /-} mice, and led to a shift in tumor spectrum from predominantly sarcomas (63%) when FasL was present to a large number of lymphomas (76%) in FasL^-/- p53^{+ /-} mice. Given the reduced life span and increased incidence of lymphoma in FasL ^-/- p53^{+ /-} mice, these mice could be useful in carcinogenicity testing, particularly for understanding mechanisms of compounds that are nongenotoxic.

The outbreak of methylmercury poisoning in the geographic areas around Minamata Bay, Kumamoto, Japan in the 1950s has become known as Minamata disease. Based on earlier reports and extensive pathological studies on autopsied cases at the Kumamoto University School of Medicine, destructive lesions in the anterior portion of the calcarine cortex and depletion predominantly of granular cells in the cerebellar cortex came to be recognized as the hallmark and diagnostic yardstick of methylmercury poisoning in humans. As the number of autopsy cases of Minamata disease increased, it became apparent that the cerebral lesion was not restricted to the calcarine cortex but was relatively widespread. Less severe lesions, believed to be responsible for the motor symptoms of Minamata patients, were often found in the precentral, postcentral, and lateral temporal cortices. These patients also frequently presented with signs of sensory neuropathy affecting the distal extremities. Because of few sufficiently comprehensive studies, peripheral nerve degeneration has not been universally accepted as a cause of the sensory disturbances in Minamata patients. The present paper describes both biopsy and autopsy findings of the peripheral nerves in a male fisherman who died at the age of 64 years and showed the characteristic central nervous system lesions of Minamata disease at autopsy. A sural nerve biopsy with electron microscopy performed 1 month prior to his death showed endoneurial fibrosis and regenerated myelin sheaths. At autopsy the dorsal roots and sural nerve showed endoneurial fibrosis, loss of nerve fibers, and presence of Büngner's bands. The spinal cord showed Wallerian degeneration of thefasciculus gracilis (Goll's tract) with relative preservation of neurons in sensory ganglia. These findings support the contention that there is peripheral nerve degeneration in Minamata patients due to toxic injury from methylmercury.

Common marmosets were used as model animals for methylmercury (MeHg) poisoning. Six marmosets were given MeHg of 5 ppm Hg in drinking water. The animals were divided into 3 groups of 2 each. The first group was examined for acute symptomatic MeHg poisoning. They were given MeHg for 70 and 90 days, respectively, to manifest severe symptoms. The second group was sacrificed after 38 days of MeHg exposure, when they had acute-subclinical MeHg poisoning. The third group of animals was exposed for 21 days, and then observed for 2.5 years without MeHg exposure. One of them showed typical symptoms of MeHg poisoning after MeHg exposure had ended, but the other one showed only slight symptoms without ataxia. This experiment demonstrated that MeHg causes pathological changes in neural tissues including the peripheral nerves in common marmosets. Furthermore, common marmosets were found to show MeHg-induced pathological changes similar to those in humans in the cerebrum and cerebellum.

We provide a set of Standard Operating Procedures (SOPs) for preparing samples for electron microscopic evaluation that allow storage of samples in the primary fixative for at least 17 years without noticeable degradation, do not compromise the ability to prepare the same samples for standard light microscopic evaluation, and provide tips for orientation of samples that may be necessary for evaluation. Guidelines for proper sample size, buffer composition, and fluid concentration s during processing are given. The impact of these procedures on specimen quality, ability to produce truly comparable samples for drug development studies, and ways to minimize time spent by technicians preparing these samples during necropsies is evaluated. Although many laboratories routinely employ most of these techniques, this compilation will facilitate the simultaneou s light and electron microscopic preparation by the pathologist of comparable specimens that can be stored long-term at 4°C in McDowell's and Trump's 4F:1G fixative (4F:1G).

Multiple intrapulpal denticles were observed in maxillary incisors of control and treated B6C3F1 mice used in a chronic inhalation study. Histologically, the denticles originated from multiple small budlike projections emanating from the epithelial sheath, immediately adjacent to the pulp chamber. The denticles were round to ovoid in shape with a central cavity surrounded by tubular dentin. Immature denticles contained epithelial cells within the central cavity, whereas mature denticles were either devoid of cells or contained cell fragments. A layer of columnar odontoblasts surrounded the outer surface of each denticle. Denticles advanced in a coronal direction as the incisors grew. With continued incisor growth, some denticles impacted the tooth wall and were associated with defects in dentinogenesis, altered tooth shape, and microfractures. Some denticles became partly or entirely incorporated into the dentin of the tooth. Intrapulpal denticle formation may represent a previously unidentified alteration that could contribute to the development of dental dysplasia in mice by interfering with normal tooth development and predisposing affected teeth to malformation and biomechanical failure with fracture.

Different models of isolated and perfused livers and precision cut liver slices have been developed for studies on liver toxicology the past years. As most of these models were limited by nonphysiologi c settings, a new model of normothermic hemoperfused isolated porcine slaughterhous e livers to examine hepatotoxicity was established encompassing the advantages of slaughterhouse organs to reduce animal experiments and autologous blood as an optimal perfusate. As model compound, the analgesic substance diclofenac was used and the effects of this drug on organ function parameters were compared to an untreated control group. Using an amount of 2,000 ml, the organs were perfused over 180 minutes, metabolically controlled via a dialysis and oxygenation system and various hematological and hepatic parameters were examined. In contrast to the untreated control organs, significant differences were found in the diclofenac group for parameters such as lactate, creatinine, ALT, bicarbonate, or bile flow. In summary, the presently established model of isolated hemoperfused slaughterhouse livers displays a useful new approach to assess hepatotoxicity of different substances on the organ level. As a major economic advantage in comparison to setups using laboratory animals, the new model can be run with blood and organs obtained from slaughterhouse animals.