Sabine Rehm

The mammary pathology of genetically engineered mice: the consensus report and recommendations from the Annapolis meeting{

NIH sponsored a meeting of medical and veterinary pathologists with mammary gland expertise in Annapolis in March 1999. Rapid development of mouse mammary models has accentuated the need for definitions of the mammary lesions in genetically engineered mice (GEM) and to assess their usefulness as models of human breast disease. The panel of nine pathologists independently reviewed material representing over 90% of the published systems. The GEM tumors were found to have: (1) phenotypes similar to those of non-GEM; (2) signature phenotypes specific to the transgene; and (3) some morphological similarities to the human disease. The current mouse mammary and human breast tumor classifications describe the majority of GEM lesions but unique morphologic lesions are found in many GEM. Since little information is available on the natural history of GEM lesions, a simple morphologic nomenclature is proposed that allows direct comparisons between models. Future progress requires rigorous application of guidelines covering pathologic examination of the mammary gland and the whole animal. Since the phenotype of the lesions is an essential component of their molecular pathology, funding agencies should adopt policies ensuring careful morphological evaluation of any funded research involving animal models. A pathologist should be part of each research team.

Classification of Proliferative Pulmonary Lesions of the Mouse Recommendations of the Mouse Models of Human Cancers Consortium

Rapid advances in generating new mouse genetic models for lung neoplasia provide a continuous challenge for pathologists and investigators. Frequently, phenotypes of new models either have no precedents or are arbitrarily attributed according to incongruent human and mouse classifications. Thus, comparative characterization and validation of novel models can be difficult. To address these issues, a series of discussions was initiated by a panel of human, veterinary, and experimental pathologists during the Mouse Models of Human Cancers Consortium (NIH/National Cancer Institute) workshop on mouse models of lung cancer held in Boston on June 20–22, 2001. The panel performed a comparative evaluation of 78 cases of mouse and human lung proliferative lesions, and recommended development of a new practical classification scheme that would (a) allow easier comparison between human and mouse lung neoplasms, (b) accommodate newly emerging mouse neoplasms, and (c) address the interpretation of benign and preinvasive lesions of the mouse lung. Subsequent discussions with additional experts in pulmonary pathology resulted in the current proposal of a new classification. It is anticipated that this classification, as well as the complementary digital atlas of virtual histological slides, will help investigators and pathologists in their characterization of new mouse models, as well as stimulate further research aimed at a better understanding of proliferative lesions of the lung.

Cadmium and prostate cancer

Prostatic cancer is a common and frequently lethal malignant disease. In the United States and other countries the incidence and mortality rate of prostate cancer continue to rise. Cancer of the prostate has an extremely complex etiology and appears dependent on a variety of factors, making linkage to a single factor very difficult to detect. Cadmium is a metallic toxin of great environmental and occupational concern. Cadmium exposure has been associated with human prostatic cancer in some, but not all, epidemiologic studies. Some studies indicate that tissue levels of cadmium in the human prostate correlate with malignant disease. Any association between cadmium and prostatic cancer has been controversial, in large part because of a previous lack of relevant animal models. However, several chronic studies in rats revealing a correlation between cadmium exposure and prostatic tumors have been published over the last several years. These include a study of oral cadmium exposure, a route extremely relevant to human exposure. Several of these chronic studies indicate a hormonal dependence of cadmium-induced prostate cancer. Other supportive work continues to accumulate, such as studies showing in vitro malignant transformation of prostatic epithelial cells with cadmium exposure. In addition, there are indications that the primary biologic tolerance system for cadmium (i.e., the metallothionein gene) may be only poorly active in the specific lobes of the rat prostate in which cadmium induces tumors. The induction in rats of prostate cancer by cadmium treatment clearly supports, but does not definitively establish, a possible role for cadmium as an etiological agent in human prostate cancer. Further research, however, will be required to establish the precise role of cadmium in this important human malignancy.

Carcinogenicity of Oral Cadmium in the Male Wistar (WF/NCr) Rat: Effect of Chronic Dietary Zinc Deficiency

The effect of chronic dietary zinc deficiency on the carcinogenic potential of dietary cadmium was assessed in male Wistar (WF/NCr) rats. Groups (n = 28) of rats were fed diets adequate (60 ppm) or marginally deficient (7 ppm) in zinc and containing cadmium at various levels (0, 25, 50, 100, or 200 ppm). Lesions were assessed over the following 77 weeks. Zinc deficiency alone had no effect on survival, growth, or food consumption. Cadmium treatment did not reduce survival or food consumption and only at the highest doses of cadmium (100 and 200 ppm) was body weight reduced (maximum 17%). The incidence of prostatic proliferative lesions, both hyperplasias and adenomas, was increased over that seen in controls (1.8%) in both zinc-adequate (20%) and zinc-deficient rats (14%) fed 50 ppm cadmium. The overall incidence for prostatic lesions for all cadmium treatment groups was, however, much lower in zinc-deficient rats, possibly because of a marked increase in prostatic atrophy that was associated with reduced zinc intake. Cadmium treatment resulted in an elevated leukemia incidence (maximum 4.8-fold over control) in both zinc-adequate and zinc-deficient groups, although zinc deficiency reduced the potency of cadmium in this respect. Testicular tumors were significantly elevated only in rats receiving 200 ppm cadmium and diets adequate in zinc. Both zinc-deficient and zinc-adequate groups showed significant positive trends for development of testicular neoplasia with increasing cadmium dosage. Thus, oral cadmium exposure is clearly associated with tumors of the prostate, testes, and hematopoietic system in rats, while dietary zinc deficiency has complex, apparently inhibitory, effects on cadmium carcinogenesis by this route.

Cytopathologic and Neurochemical Correlates of Progression to Motor/Cognitive Impairment in SIV-Infected Rhesus Monkeys

Neurochemical, pathologic, virologic, and histochemical correlates of simian immunodeficiency virus (SIV)- associated central nervous system (CNS) dysfunction were assessed serially or at necropsy in rhesus monkeys that exhibited motor and cognitive deficits after SIV infection. Some infected monkeys presented with signs of acquired immunodeficiency disease (AIDS) at the time of sacrifice. Seven of eight animals exhibited motor skill impairment which was associated with elevated quinolinic acid in cerebrospinal fluid (CSF). Examination of the brains revealed diffuse increases in glial fibrillary acidic protein immunoreacti vity in cerebral cortex in all animals, regardless of evidence of immunodeficiency disease. Reactive astrogliosis preceded or was coincident with the onset of neuropsychological impairments. Virus rescue from CSF of six of eight infected animals showed that one of three animals with AIDS and none of three animals without AIDS at necropsy had virus rescue-positive CSF. Multinucleated giant cells were seen in the brain of only one animal with end-stage AIDS and high systemic virus burden at death. Neither systemic nor CNS virus burden was associated with the onset of CNS dysfunction. SIV-associated motor/cognitive impairment is associated with subtle, widespread changes in CNS cytology and neurochemistry, rather than with large increases in brain virus burden or widespread virus-associated brain lesions.

Diagnostic Performance of Traditional Hepatobiliary Biomarkers of Drug-Induced Liver Injury in the Rat

Nonclinical studies provide the opportunity to anchor biochemical with morphologic findings; however, liver injury is often complex and heterogeneous, confounding the ability to relate biochemical changes with specific patterns of injury. The aim of the current study was to compare diagnostic performance of hepatobiliary markers for specific manifestations of drug-induced liver injury in rat using data collected in a recent hepatic toxicogenomics initiative in which rats (n = 3205) were given 182 different treatments for 4 or 14 days. Diagnostic accuracy of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (Tbili), serum bile acids (SBA), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), total cholesterol (Chol), and triglycerides (Trig) was evaluated for specific types of liver histopathology by Receiver Operating Characteristic (ROC) analysis. To assess the relationship between biochemical and morphologic changes in the absence of hepatocellular necrosis, a second ROC analysis was performed on a subset of rats (n = 2504) given treatments (n = 152) that did not cause hepatocellular necrosis. In the initial analysis, ALT, AST, Tbili, and SBA had the greatest diagnostic utility for manifestations of hepatocellular necrosis and biliary injury, with comparable magnitude of area under the ROC curve and serum hepatobiliary marker changes for both. In the absence of hepatocellular necrosis, ALT increases were observed with biochemical or morphologic evidence of cholestasis. In both analyses, diagnostic utility of ALP and GGT for biliary injury was limited; however, ALP had modest diagnostic value for peroxisome proliferation, and ALT, AST, and total Chol had moderate diagnostic utility for phospholipidosis. None of the eight markers evaluated had diagnostic value for manifestations of hypertrophy, cytoplasmic rarefaction, inflammation, or lipidosis.

Chronic Toxic and Carcinogenic Effects of Cadmium Chloride in Male DBA/2NCr and NFS/NCr Mice: Strain-Dependent Association with Tumors of the Hematopoietic System, Injection Site, Liver, and Lung

Although the acute toxic effects of cadmium in mice vary greatly with strain, relatively little is known about strain differences in cadmium carcinogenesis. Therefore, this work was performed to assess the chronic toxic and carcinogenic effects of cadmium in two strains of mice generally thought to be susceptible to the acute effects of cadmium. Male DBA/2NCr (DBA) and NFS/NCr (NFS) mice were given CdCl2 (40 mumol/kg, sc) either as a single dose (1 x 40) or as weekly doses for 16 weeks (16 x 40) starting at 8 weeks of age. Controls received saline. The animals were observed for the next 104 weeks and mice at risk were defined as those surviving to the time of appearance of a particular tumor. Cadmium-induced dose-related increases in lymphoma (primarily follicular center cell) incidence (1 x 40, 11 cases/23 mice at risk; 16 x 40, 16/28) over control (7/27) in DBA mice but not in NFS mice. Only NFS mice receiving repeated cadmium injections (16 x 40) showed sarcoma development at the injection site (9/35), as no sarcomas occurred in control NFS mice or any group of DBA mice. On the other hand, cadmium-treated (16 x 40) NFS mice, but not DBA mice, had more hepatocellular adenomas and carcinomas (9/27) than control (1/15) but only at the high dose (16 x 40). More cadmium-treated NFS mice had pulmonary tumors than controls, but only at the lower dose (1 x 40). Although testicular tumors were rare, nonneoplastic lesions (fibrosis and mineralization) were induced by cadmium to a similar extent in both strains. Clearly cadmium carcinogenicity varies widely with strain, indicating a genetic basis to susceptibility. The basis of these strain differences deserves further study.

Cadmium-induced ovarian toxicity in hamsters, mice, and rats

The effects of cadmium on the female genital tract of Syrian hamsters (Cr:RGH), of four mouse strains (BALB/cAnNCr, DBA/2NCr, C57BL/6NCr, NFS/NCr), and two rat strains (F344/NCr and WF/NCr) were studied by light microscopy after a single sc injection of cadmium chloride (CdCl2). Experiments involved animals prior to and after sexual maturity, and employed CdCl2 doses ranging from 20 to 47.5 mumol/kg. Animals were examined at intervals from 24 hr to 8 weeks following treatment. Syrian hamsters were most susceptible to CdCl2-induced ovarian hemorrhagic necrosis at all ages tested. Most severe ovarian lesions occurred in immature hamsters, in mature hamsters at high doses, and shortly before ovulation at all doses. The small arteries of the developing follicles and interstitial stroma seemed selectively susceptible to CdCl2 toxicity while the corpora lutea, mesothelium, primordial oocytes, and the rete ovarii appeared resistant. Pretreatment with zinc acetate markedly reduced the extent of ovarian lesions in hamsters. Although reduced in weight by 45%, the hamster ovaries recovered morphologically within 2 months after severe acute hemorrhagic necrosis. Uterine and cervical stromal hemorrhages were seen only in immature hamsters at doses of greater than or equal to 30 mumol CdCl2/kg. Of the mice, only the DBA/2NCr strain showed significant CdCl2-induced ovarian hemorrhages, and these hemorrhages occurred at doses also producing lethal liver toxicity. Lesions of the uterus were rare. Rats showed dose- and age-dependent toxicity in the ovaries, uterus, cervix, and liver. CdCl2 exposure in mature rats induced uterine lesions only in F344 rats, while acute ovarian and hepatic toxicity was less severe in mature animals of both strains. No lesions were noted after 7 days in mature WF rats. In both rats and mice, no cycle dependency of the ovarian lesions was evident.

Effects of Food Restriction on Testis and Accessory Sex Glands in Maturing Rats

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.

Effect of chronic dietary zinc deficiency on cadmium toxicity and carcinogenesis in the male Wistar [Hsd: (WI)BR] rat☆

Though it is known that excess zinc will prevent cadmium carcinogenesis, the impact of zinc deficiency on cadmium carcinogenesis has not been defined. This study assessed the effect of dietary zinc deficiency on the carcinogenic potential of cadmium in rats. Groups (n = 28 each) of male Wistar [Hsd: (WI)BR] rats were fed diets adequate (60 ppm) or deficient (7 ppm) in zinc and received a single sc dose of cadmium (5, 10, or 30 mumol Cd/kg). Lesions were assessed over the next 92 weeks. All cadmium doses increased the incidence of testicular interstitial cell tumors. The incidence of cadmium-induced testicular tumors was unaffected by dietary zinc status. However, when multiplicity of testicular lesions was considered, zinc-deficient diets markedly increased the number of testicular interstitial cell adenomas generated by cadmium exposure while significantly reducing the number of preneoplastic lesions (interstitial cell hyperplasias). The combined total number of neoplastic and preneoplastic lesions of the testes was independent of zinc status clearly indicating a shift from hyperplasia to neoplasia within the testes of zinc-deficient rats. The highest cadmium dose (30 mumol/kg) increased injection site sarcomas in zinc-deficient rats (7 tumors/27 rats at risk) but not zinc-adequate rats (3/26) when compared to control (0/49). Chronic progressive renal nephropathy was accelerated by cadmium in zinc-deficient rats. Results indicate that dietary zinc deficiency enhances carcinogenic response at the injection site of cadmium, promotes the neoplastic progression of cadmium-induced testicular lesions, and enhances chronic progressive nephropathy. Thus, dietary zinc deficiency appears to cause a generalized increase in the chronic toxic effects of cadmium.