High Spontaneous Incidence Research Articles

Persistent Na + current couples spreading depolarization to seizures in Scn8a gain of function mice.

Spreading depolarization (SD) is a slowly propagating wave of massive cellular depolarization that transiently impairs the function of affected brain regions. While SD typically arises as an isolated hemispheric event, we previously reported that reducing M-type potassium current (I KM ) by ablation of Kcnq2 in forebrain excitatory neurons results in tightly coupled spontaneous bilateral seizure-SD complexes in the awake mouse cortex. Here we find that enhanced persistent Na + current due to gain-of-function (GOF) mutations in Scn8a (N1768D/+, hereafter D/+) produces a similar compound cortical excitability phenotype. Chronic DC-band EEG recording detected spontaneous bilateral seizure-SD complexes accompanied by seizures with a profound tonic component, which occurs predominantly during the light phase and were detected in the mutant mice across ages between P40-100. Laser speckle contrast imaging of cerebral blood flow dynamics resolved SD as bilateral wave of hypoperfusion and subsequent hour-lasting hypoperfusion in Scn8a D/+ cortex in awake head-restrained mice subjected to a subconvulsive PTZ. Subcortical recordings in freely moving mice revealed that approximately half of the spontaneous cortical seizure-SD complexes arose with concurrent SD-like depolarization in the thalamus and delayed depolarization in the striatum. In contrast, SD-like DC potential shifts were rarely detected in the hippocampus or upper pons. Consistent with the high spontaneous incidence in vivo , cortical slices from Scn8a D/+ mice showed a raised SD susceptibility, and pharmacological inhibition of persistent Na + current (I NaP ), which is enhanced in Scn8a D/+ neurons, inhibited SD generation in cortical slices ex vivo , indicating that I NaP contributes to SD susceptibility. Ex vivo Ca 2+ imaging studies using acute brain slices expressing genetic Ca 2+ sensor (Thy1-GCAMP6s) demonstrated that pharmacological activation of I KM suppressed Ca 2+ spikes and SD, whereas I KM inhibitor drastically increased the frequency of Ca 2+ spikes in the hippocampus of Scn8a D/+ mice, but not in WT, suggesting that I KM restrains the hyperexcitability resulting from Scn8a GOF mutation. Together, our study identifies a cortical SD phenotype in Scn8a GOF mice shared with the Kcnq2 - cKO model of developmental epileptic encephalopathy and reveals that an imbalance of non-inactivating inward and outward membrane currents bidirectionally modulates spatiotemporal SD susceptibility.

Heterozygous Tsc2 (Tsc2+/-) mouse model to study induced renal cancer in response to ionizing radiation at low doses.

Kidneys are one of the main dose-limiting organs in radiotherapeutic procedures of lower abdomen. Likewise, the threat of exposure of radiosensitive organs such as kidneys in warfare or radiation accidents among military personal or due to terrorist activities in general public is of increasing concern. These events warrant the need for appropriate animal models to study the acute and chronic effects of low- and high-dose rate radiation exposures. In this study, for the first time, we validated Tsc2+/- mouse model to study whether radiation accelerates carcinogenesis in kidneys. Tsc2+/- mice at increasing age groups at 8 and 10 months were exposed to repeated doses of gamma radiation (0.4 Gy × 5) and assessed for aggravated kidney tumor formation at 2 months post-irradiation. Animals from irradiated group showed a significant increase in numbers of bilateral, multifocal tumors compared with mock-irradiated animals. Intra-glomerular reactive oxygen species (ROS) levels measured by dihydroethidium florescence showed significant increases in ROS production in irradiated Tsc2+/- mice compared with non-irradiated animals. Similarly, selective hematological parameters and glomerular filtration rate were further reduced significantly in irradiated Tsc2+/- mice. Tsc2 protein, tuberin in irradiated mice, however, remains at the same reduced levels as that of the mock-irradiated heterozygous Tsc2 mice. The results indicate that radiation alters kidney homeostatic function and influences high spontaneous incidence of renal cell carcinoma in this rodent model. Repurposing of Tsc2+/- mice model will, therefore, provide a unique opportunity to study acute and delayed effects of radiation in the development of kidney cancers.

Phenozan, a Synthetic Phenolic Antioxidant, Inhibits the Development of Spontaneous Tumors in Rats and Mice

Synthetic phenolic antioxidant β-(4-hydroxy-3,5-di-tert-butylphenyl) propionic acid, named phenozan, is a potential antiepileptic drug. In pre-clinical trials this substance did not manifest any toxicity, and also inhibited the development of some spontaneous tumors in animals. The purpose of this study was to evaluate inhibiting effect of phenozan on spontaneous carcinogenesis in rats and mice. In experiments with rats LIO and mice SHR of local breeding, with high spontaneous tumor incidence, phenozan was dissolved in sunflower oil and administered by gavage in therapeutic dose 5 mg/kg 3 times per week for 18 months. There were no any signs of toxicity and differences in weight of animals during the phenozan treatment compared with the control (sunflower oil). Phenozan significantly reduced the overall incidence and multiplicity of all tumors but only multiplicity of malignant tumors, compared with the control. Moreover a significant decrease of overall incidence and multiplicity was observed in pituitary and breast tumors in females and only overall multiplicity of tumors of pituitary and lymphoid tissue in males. In mice phenozan reduced overall incidence and multiplicity of lung tumors (in females) and also overall multiplicity of all tumors (in females) and only malignant tumors (in males). These findings allow us to classify phenozan as anticarcinogenic agent. Anticarcinogenic activity of phenozan is important because clinical study of this drug as the possible antiepileptic drug goes along and it is known that such drugs are designed for long-term use.

Hormone Receptor Expression in Spontaneous Uterine Adenocarcinoma in Fischer 344 Rats.

Most uterine cancers, the most common gynecological malignancies in women in developed countries, are hormone-dependent endometrial adenocarcinomas (EACs) that express estrogen and progesterone receptors. Although rat strains exist with a high spontaneous incidence of EAC, the Fischer 344 (F344) strain, previously one of the most commonly used strains in carcinogenicity testing, is not a high-incidence strain. To better understand the biology of this neoplasm, we assessed estrogen receptor α (ER), progesterone receptor (PR), and Ki-67 expression using immunohistochemistry in spontaneous EAC in 18 F344 rats used as control animals in 2-year National Toxicology Program bioassays. Of the 18 tumors, 9 were well-differentiated and 9 were poorly differentiated. Most tumors, 7/18, were ER+PR+, as observed in women. Of the remainder, 6/18 were ER+PR-, 2/18 were ER-PR+, and 3/18 were ER-PR-. Well-differentiated tumors were ER+ (8/9) more often than poorly differentiated tumors (5/9). The percentage of ER+ tumors (72%) in rats was similar to that seen in women, but rats less frequently had PR+ (50%) tumors than women. The heterogeneous estrogen and progesterone receptor immunophenotypes observed in F344 rats in this study highlight the importance of evaluating hormone receptor expression in animal models used for chemical evaluations.

The effect of sevoflurane on developing A/J strain mouse embryos using a whole-embryo culture system—the incidence of cleft lip in culture embryos

A/J strain mice have a high spontaneous incidence of cleft lip (ICL) and/or palate. The primary palate-related effects of sevoflurane on developing A/J strain mouse embryos (embryos) were studied using a whole-embryo culture (WEC) system. This system could separate the direct effects of sevoflurane from those that are maternally mediated. A total of 205 10.5-d embryos were cultured for 24 h in either a control group (control gas: 95% O2 and 5% CO2) or sevoflurane-administered groups (1/4, 1/2, and 1 minimum alveolar concentration (MAC) with control gas) for 8 h. After 16 h, 11.5-d culture embryos were examined in terms of crown-rump length, number of somites, and protein content. Crown-rump length in the 1 MAC was significantly shorter than in the control group (p < 0.05). Protein content in the 1/2 MAC (p < 0.05) and 1 MAC (p < 0.001) was significantly lower than in the control group. The ICL showed no significant differences between each group. (The ICL rose with an increase in the sevoflurane concentration, but this was not significant). The positive findings in this study indicate that a WEC system is useful for studying the mechanisms of ICL (teratogenicity) associated with sevoflurane.

Off-Label Use of Bevacizumab for the Treatment of Age-Related Macular Degeneration

There is an active and controversial debate about the role of intravitreal bevacizumab versus approved drugs in the treatment of neovascular age-related macular degeneration (AMD). Because bevacizumab was available prior to the launch of ranibizumab, off-label use of the former became widespread and the cancer drug bevacizumab is the most commonly used medication in ophthalmology nowadays. This review considers every publication identified in MEDLINE using the keywords 'bevacizumab' and 'Avastin' between 1 June 2005 and 31 July 2008. The search identified 511 papers that were evaluated. In 33 studies, there was consistent and clear evidence for the efficacy of bevacizumab in neovascular AMD. However, the highest grade studies (three prospective, randomized, controlled trials) did not attain better than grade 2b level of evidence, and objective evaluation of the benefit of bevacizumab relative to representative controls was therefore not possible. Certainly, the available evidence is inferior to that obtained from the approval studies of ranibizumab and this should influence treatment selection and guidance of patients. These considerations indicate that important quality criteria need to be included in future studies to ensure more meaningful conclusions can be drawn. These include clearly defined inclusion criteria, information about the recruitment procedure (including data on withdrawals, excluded patients, concealed treatment allocation, use of intention-to-treat analyses and blinded assessment procedures). Although preclinical studies have almost exclusively found bevacizumab to be safe, the design utilized in clinical case series cannot rule out a possible increase in adverse events, which already show a high spontaneous incidence in elderly AMD patients. The superior evidence level for ranibizumab and the limited safety data for bevacizumab must be taken into consideration when evaluating the costs that a healthcare system is willing to spend. However, the superior grade of evidence for ranibizumab should not be confused with the (still missing) evidence for superior efficacy. The results of ongoing randomized, controlled, comparative trials will provide further data on the efficacy and cost effectiveness of bevacizumab and ranibizumab in the treatment of AMD. In the meantime, patients should be informed about the alternatives, the price differences and the restricted liability issue when off-label use of bevacizumab is offered.

Pulmonary Inflammation in Brown Norway Rats: Possible Association of Environmental Particles in the Animal Room Environment

Brown Norway (BN) rats have been found to develop granulomatous pneumonia with high spontaneous incidence under normal husbandry conditions. In our laboratory, ambient particles from the animal housing environment were suspected to be involved in the pathogenesis of these pulmonary lesions. In the present study, we correlated the histopathology of spontaneous granulomatous pneumonia with the cytology of bronchoalveolar lavage fluid (BALF) analysis and animal room particle counts. The results show that the sum of the macroscopic lesion score in the lung correlated with the number of eosinophils in the BALF. Microscopically, the severity of granulomatous lesions was significantly correlated with both pulmonary eosinophils and the number of eosinophils in the BALF, but not with those of neutrophils or lymphocytes. In addition, the histological features of the lesions were not influenced by housing conditions or sensitization with ovalbumin. Ambient particles (0.3-1.0 microm in diameter) in the animal room environment were measured and compared with the number of eosinophils in BALF. Interestingly, there was a significant correlation between them, suggesting a possible relationship between environmental particles and the pathogenesis of granulomatous pneumonia in BN rats. However, further study is needed to clarify the pathogenic mechanism.

Failure of Ureteric Bud Invasion: A New Model of Renal Agenesis in Mice

FUBI (failure of ureteric bud invasion) is a highly inbred strain of mouse with a high spontaneous incidence of uni- or bilateral renal agenesis (60%). Bilateral renal agenesis is lethal within 2 days after birth. The primary defect of FUBI is failure of the ureteric bud to penetrate into the metanephric mesenchyme at around embryonic day 11, resulting in apoptosis of metanephric cells and leading to renal agenesis on the affected side. The metanephros seemed to be normal because co-culturing of the FUBI metanephros with homologous spinal cord induced differentiation of the rudiment, but co-culturing with the homologous ureteric bud frequently did not. Genetic analysis revealed that more than two genes were involved in this malformation and we mapped one of the modifier loci, fubi1, on chromosome 2, at approximately 65 cM from the centromere. In this region, there are two possible candidate genes, Wilms' tumor 1 and formin, that play important roles in kidney development. Some of formin mutants shared a similar phenotype with FUBI; however, there was no difference in the expression of formin in embryonic kidneys between FUBI and control NFS/N mice. Studies of fubi1 congenic mice indicated that interaction of two or more loci is essential for the FUBI phenotype.

The Tg.AC (v-Ha-ras) Transgenic Mouse: Nature of the Model

The Tg.AC (v-Ha-ras) transgenic mouse model provides a reporter phenotype of skin papillomas in response to either genotoxic or nongenotoxic carcinogens. In common with the conventional bioassay, the Tg.AC model responds to known human carcinogens and does not respond to noncarcinogens. It also does not respond to most chemicals that are positive in conventional bioassays principally at sites of high spontaneous tumor incidence. The mechanism of response of the Tg.AC model is related to the structure and genomic position of the transgene and the induction of transgene expression through specific mediated interactions between the chemicals and target cells in the skin.

Increased Circulating Colony-Stimulating Factor-1 (CSF-1) in SJL/J Mice With Radiation-Induced Acute Myeloid Leukemia (AML) Is Associated With Autocrine Regulation of AML Cells by CSF-1

AbstractThe SJL/J mouse strain has a high spontaneous incidence of a B-cell neoplasm, reticulum cell neoplasm type B (RCN B). In addition, following irradiation, 10% to 30% of these mice develop acute myelomonocytic leukemia (radiation-induced acute myeloid leukemia [RI-AML]), an incidence that can be increased to 50% by treatment of the mice with corticosteroids after irradiation. The role played by the mononuclear phagocyte growth factor, colony-stimulating factor-1 (CSF-1), in the development of RI-AML in SJL/J mice was investigated. Mice dying of RI-AML, but not those dying of RCN B or without disease, possessed elevated concentrations of circulating CSF-1. In addition, in mice developing RI-AML with a more prolonged latency, circulating CSF-1 concentrations were increased before overt expression of RI-AML. First-passage tumors from 14 different RI-AMLs all contained high concentrations of CSF-1, and six of six different first- or second-passage tumors expressed the CSF-1 receptor (CSF-1R). Furthermore, in vitro colony formation by first- or second-passage tumor cells from 20 of 20 different RI-AMLs was blocked by neutralizing anti–CSF-1 antibody, and four of four of these tumors were inhibited by anti–CSF-1R antibody. The results of these antibody neutralization studies, coupled with the observation of elevated circulating CSF-1 in mice developing RI-AML, show an autocrine role for CSF-1 in RI-AML development in SJL/J mice. Southern blot analysis of tumor DNA from six of six of these tumors failed to reveal any rearrangements in the genes for CSF-1 or the CSF-1R. Studies in humans have shown that patients with AML possess elevated levels of circulating CSF-1 and that AML cells can express CSF-1 and the CSF-1R. Thus, RI-AML in the SJL/J mouse appears to be a useful model for human AML.

Analysis of National Toxicology Program rodent bioassay data for anticarcinogenic effects

We reanalyzed data from 218 two-year rodent carcinogenicity studies carried out by the National Toxicology Program (NTP). These data were originally collected for the purpose of identifying potential human carcinogens. However, the objective of our analysis was to investigate the frequency of possible anticarcinogenic effects in these data, since recurring cases of chemical-associated tumor reductions have been noted in the course of these studies over time. Our analysis reveals that most (> 90%) NTP-tested chemicals show at least one statistically significant ( p < 0.05) decrease in site-specific tumor incidence. Because of the large number of statistical comparisons made in a long-term bioassay, random variability can account for many of these tumor decreases. However, we found that certain tumors (predominantly those with a high spontaneous incidence) show chemically related decreases far more frequently than chance expectation. Many of these decreases, particularly those for pituitary and mammary gland tumors, adrenal pheochromocytoma and uterine polyps in rats and liver and lung tumors in mice, are associated with the reduced body weights frequently observed in the dosed groups. The chemically related decreased incidences of leukemia in rats appear to be related to spleen damage, i.e., chemically related splenic toxicity is evident for most chemicals showing decreased incidences of leukemia. While random variability, associations with body weight and splenic toxicity can account for most of the decreased tumor incidences observed in NTP studies, there are other tumor decreases that could not be totally explained by these factors. Further investigations of possible mechanisms of action are underway. These data are relevant to the concept of chemoprevention as well as to the task of using long-term laboratory animal studies to predict enhanced human environmental-cancer risk for regulatory purposes.

Proliferating cell nuclear antigen staining of Fischer-344/N rat spleens affected by large granular lymphocyte leukemia.

Large granular lymphocyte (LGL) leukemia commonly occurs in the Fischer-344/N rat. The high spontaneous incidence complicates the interpretation of results from chronic carcinogenicity studies that use this rat strain. As a result, a comprehensive characterization of LGL leukemia is necessary to help understand the leukemogenic process and the applicability of staging for assessing the progression of this disease. In the current study, the proliferation rate of LGL leukemia cells from untreated control Fischer-344 (F-344) rats in 3 stages of leukemia compared to nonleukemic age-matched rats was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA). In histologic sections of spleen from aged F-344/N rats affected by LGL leukemia, there was a significant increase of both PCNA labeling and mitotic indices that was most advanced in the spleen of rats with more severe LGL leukemia. These results support biological significance for the morphologic staging system currently in use.

Comparison of the effects of Wyeth-14,643 in Crl:CD BR and Fisher-344 rats

Wyeth-14,643 (WY) belongs to a diverse class of compounds which have been shown to produce hepatic peroxisome proliferation and hepatocellular carcinoma in rodents. Based on a review of bioassay data, a relationship appears to exist between peroxisome proliferating compounds and Leydig cell adenoma formation. Most rat bioassays with peroxisome proliferators have been conducted in the Fisher-344 (F344) rat, which has a high spontaneous incidence of Leydig cell adenomas. Thus, it was necessary to use an alternative animal model to investigate this relationship further. Therefore the Crl:CD BR (CD) rat, which has a low spontaneous Leydig cell adenoma incidence, was chosen for a 2-year feeding study with WY. Before initiating this 2-year feeding study, it was necessary to investigate whether any strain differences existed between CD and F344 rats with respect to WY-induced peroxisome and cell proliferation. In this study, male CD and F344 rats were fed diets containing 0, 50, or 1000 ppm WY for 21 days. Peroxisome proliferation in the liver and testis was determined biochemically by measuring β-oxidation activity and was confirmed ultrastructurally. Serum hormone levels and cell proliferation rates in the liver and testis were also measured. In addition, basal β-oxidation activity and cell proliferation rates were compared between the CD and F344 rats. A significant decrease in final body weight was observed in the 1000 ppm WY groups for both CD and F344 rats. Similar increases in liver weights were observed in both strains of rat at 50 and 1000 ppm WY (1.96- and 2.15-fold increase in CD rats and 2.29- and 2.38-fold increase in F344 rats, respectively). Peroxisome proliferation, assessed by measuring hepatic β-oxidation activity, was also increased to a similar extent in both strains of rats at 50 and 1000 ppm WY (21.9- and 22.4-fold increase for CD rats and 33.7- and 27.6-fold increase for F344 rats, respectively). The increase in hepatic β-oxidation activity was confirmed by electron micrographs which demonstrated increased numbers of peroxisomes in the livers of WY-treated rats. Hepatic cell proliferation rate was similar in CD rats at 50 and 1000 ppm WY (3.7-fold increase above control at each concentration). However, hepatic cell proliferation rate increased with increasing dose in F344 rats (2.7- and 5.4-fold increase above control at 50 and 1000 ppm WY, respectively). Wy did not induce significant increases in the rate of Leydig cell β-oxidation activity or Leydig cell proliferation rate in either strain of rat. Treatment with 1000 ppm WY did not appear to affect the number of peroxisomes observed by electron microscopy in the Leydig cells of either strain of rat. Moreover, the rate of β-oxidation in Leydig cells of the control rats was approximately 20 times less than the rate of hepatic β-oxidation, irrespective of strain. Serum testosterone, estradiol, prolactin, and LH levels were unchanged in CD and F344 rats. There were, however, significant differences between the strains of rat with respect to β-oxidation activity and cell proliferation. The CD rats had basal and WY-induced rates of hepatic β-oxidation activity and hepatic cell proliferation that were significantly higher than those of the F344 rats. Rates of basal and WY-induced Leydig cell proliferation were also greater in the CD rats than in the F344 rats. These data demonstrate that there are subtle differences in the basal β-oxidation activity and cell proliferation rates between the two strains and that 50 ppm WY produces a maximal response in the CD rats for the parameters measured in this study.

Comparison of the Effects of Wyeth-14,643 in Crl:CD BR and Fisher-344 Rats

Wyeth-14,643 (WY) belongs to a diverse class of compounds which have been shown to produce hepatic peroxisome proliferation and hepatocellular carcinoma in rodents. Based on a review of bioassay data, a relationship appears to exist between peroxisome proliferating compounds and Leydig cell adenoma formation. Most rat bioassays with peroxisome proliferators have been conducted in the Fisher-344 (F344) rat, which has a high spontaneous incidence of Leydig cell adenomas. Thus, it was necessary to use an alternative animal model to investigate this relationship further. Therefore the Crl:CD BR (CD) rat, which has a low spontaneous Leydig cell adenoma incidence, was chosen for a 2-year feeding study with WY. Before initiating this 2-year feeding study, it was necessary to investigate whether any strain differences existed between CD and F344 rats with respect to WY-induced peroxisome and cell proliferation. In this study, male CD and F344 rats were fed diets containing 0, 50, or 1000 ppm WY for 21 days. Peroxisome proliferation in the liver and testis was determined biochemically by measuring β-oxidation activity and was confirmed ultrastructurally. Serum hormone levels and cell proliferation rates in the liver and testis were also measured. In addition, basal β-oxidation activity and cell proliferation rates were compared between the CD and F344 rats. A significant decrease in final body weight was observed in the 1000 ppm WY groups for both CD and F344 rats. Similar increases in liver weights were observed in both strains of rat at 50 and 1000 ppm WY (1.96-and 2.15-fold increase in CD rats and 2.29-and 2.38-fold increase in F344 rats, respectively). Peroxisome proliferation, assessed by measuring hepatic β-oxidation activity, was also increased to a similar extent in both strains of rats at 50 and 1000 ppm WY (21.9-and 22.4-fold increase for CD rats and 33.7-and 27.6-fold increase for F344 rats, respectively). The increase in hepatic β-oxidation activity was confirmed by electron micrographs which demonstrated increased numbers of peroxisomes in the livers of WY-treated rats. Hepatic cell proliferation rate was similar in CD rats at 50 and 1000 ppm WY (3.7-fold increase above control at each concentration). However, hepatic cell proliferation rate increased with increasing dose in F344 rats (2.7-and 5.4-fold increase above control at 50 and 1000 ppm WY, respectively). Wy did not induce significant increases in the rate of Leydig cell β-oxidation activity or Leydig cell proliferation rate in either strain of rat. Treatment with 1000 ppm WY did not appear to affect the number of peroxisomes observed by electron microscopy in the Leydig cells of either strain of rat. Moreover, the rate of β-oxidation in Leydig cells of the control rats was approximately 20 times less than the rate of hepatic β-oxidation, irrespective of strain. Serum testosterone, estradiol, prolactin, and LH levels were unchanged in CD and F344 rats. There were, however, significant differences between the strains of rat with respect to β-oxidation activity and cell proliferation. The CD rats had basal and WY-induced rates of hepatic β-oxidation activity and hepatic cell proliferation that were significantly higher than those of the F344 rats. Rates of basal and WY-induced Leydig cell proliferation were also greater in the CD rats than in the F344 rats. These data demonstrate that there are subtle differences in the basal β-oxidation activity and cell proliferation rates between the two strains and that 50 ppm WY produces a maximal response in the CD rats for the parameters measured in this study.

Chronic carcinogenic and toxic effects of a single subcutaneous dose of cadmium in the male fischer rat

The carcinogenic effects of cadmium have not been thoroughly assessed in the commonly used Fischer (F344) rat. This study determined tumor incidence in various tissues of male F344/NCr rats after a single dose of cadmium. Cadmium (as CdCl2) was given sc in the dorsal thoracic midline at 30 mumole/kg to 70 8-week old male F344 rats while controls (n = 50) received saline. Rats were observed during the next 90 weeks. Early deaths (less than or equal to 32 weeks), due mostly to acute cadmium-induced hepatotoxicity, accounted for 37 of the cadmium-treated rats while no control rats died in the same period. A high incidence of injection site sarcomas (ISS) occurred in the cadmium-treated group (21 ISS/32 rats at risk; 66%) while only 1/50 occurred in controls (2%). In fact, ISS were the major cause of morbidity after 35 weeks in cadmium-treated rats. These tumors were mostly fibrosarcomas, although histiocytic and osteogenic sarcomas also occurred. Testicular interstitial cell tumors, which show a very high spontaneous incidence in this strain (41/49; 84%), were not markedly affected by cadmium (30/31; 97%). This is in sharp contrast to other strains, such as the Wistar, in which cadmium treatment is reported to cause as much as an eightfold increase in interstitial cell (Leydig cell) tumor incidence. The incidence of large granular lymphocyte (LGL) leukemia, which also occurred frequently in control F344 rats (12/47; 26%) was markedly decreased (2/31; 7%) by cadmium. Our recent studies indicate acute lymphonecrotic effects occur with cadmium in lymphoid tissues of rats, and this may be related to the suppression of the leukemia. These results indicate that cadmium is very effective in inducing ISS in F344 rats, as is the case with other strains thus far tested, and also markedly reduces spontaneous leukemia incidence.

Differential DNase I hypersensitivity ofrasoncogenes in B6C3F1, C3H/He, and C57BL/6 mouse liver

The male hybrid B6C3F1 mouse exhibits a 30% spontaneous hepatoma incidence, whereas the paternal C3H/He strain and the maternal C57BL/6 strain exhibit a 60% and a negligible incidence, respectively. In addition, both male and female B6C3F1 mice are extremely sensitive to chemical induction of hepatocarcinogenesis. The Ha-ras, Ki-ras, and myc oncogenes have been implicated in a variety of solid tumors. Specifically, Ha- and, less frequently, Ki-ras have been reported to be activated in B6C3F1 mouse liver tumors. The objective of this study was to examine a possible point of transcriptional control of Ha-ras, Ki-ras, and myc in all three mouse strains, our hypothesis being that these oncogenes may be primed for expression in the nascent liver of those strains exhibiting a high spontaneous hepatoma incidence. A positive correlation has been established between gene expression and the presence of DNase I hypersensitive sites. DNase I hypersensitive sites were observed in the Ha-ras and myc oncogenes in the three mouse strains. However, Ha-ras appears to possess an additional site in B6C3F1 and C3H/He as compared to C57BL/6. Similarly, the Ki-ras oncogene exhibited a DNase I hypersensitive site only in B6C3F1 and C3H/He mouse liver. These results indicate that the hepatoma-prone strains (B6C3F1 and C3H/He) may have a greater potential for Ha- and Ki-ras expression than does the non-hepatoma-prone strain (C57BL/6).

Comparative gastrointestinal enzyme activity and activation of the promutagen 2,6-dinitrotoluene in male CD-1 mice and male Fischer 344 rats

Comparative intestinal nitroreductase, azo reductase, β-glucuronidase, dechlorinase and dehydrochlorinase activities in young male Fischer 344 rats and young male CD-1 mice were measured in vitro while the comparative biotransformation of 2,6-dinitrotoluene to mutagenic metabolites was determined in vivo. The mice, which exhibit a high spontaneous incidence of hepatomas, had markedly greater nitroreductase activity and metabolized significantly more 2,6-dinitrotoluene to mutagenic metabolites than did Fischer 344 rats, which show a low incidence of liver tumors. Results of this study indicate that species differences in the incidence of hepatomas may be influenced by microbial flora and/or the biotransformation of xenobiotics in the G.I. tract.

Chronic toxicity and carcinogenicity of bis(tri- n-butyltin)oxide (TBTO) in the rat

In a 106-wk toxicity and carcinogenicity study, groups of 60 male and 60 female weanling Wistar rats were fed 0, 0.5, 5 or 50 mg bis(tri- n-butyltin)oxide (TBTO)/kg diet. In males, feed consumption was increased in all treated groups and increased water consumption occurred at 5 and 50 mg/kg. During the second year, body weight decreased in the 50-mg/kg males, while the females in that group showed no weight gain. Excess mortality was confined to the 50-mg/kg group towards the end of the study. Haematological changes, comprising anaemia, lymphocytopenia and thrombocytosis were noted mainly at the high-dose level. Also, signs of decreased kidney function and increased plasma enzyme activities (alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase) were noted. No effects on serum hormone concentrations (thyrotropin, follicle stimulating hormone, luteinizing hormone or insulin) were observed, except for a decrease in the free thyroxin:thyroxin ratio in both sexes at the high-dose level. Higher serum IgM and IgA levels were present at 50 mg/kg, while, in females, IgG was decreased. At 50 mg/kg, the ovaries, adrenals, spleen (females), heart (males), pituitary, liver and kidneys were increased in weight, but the thyroid weight was decreased in females. The total tin concentrations in liver and kidneys showed a dose relationship and, in general, the concentrations were similar after 1 and 2 yr. Non-neoplastic histological alterations after 1 yr consisted of a decrease in the cell height of the thyroid follicles in all dose groups, with a reduced number of psammoma bodies at 50 mg/kg, a decrease in splenic iron content at 5 (females only) and 50 mg/kg, and a slight bile-duct activation. After 2 yr, only the thyroid changes were still present. In addition, at 2 yr, vacuolation and pigmentation of the proximal tubular epithelium and nephrosis were enhanced at 50 mg/kg. The incidence of benign tumours of the pituitary was significantly elevated and enhanced at 0.5 and 50 mg/kg. At 50 mg/kg increases in pheochromocytomas in the adrenal medulla and in parathyroid adenomas (males) were noted, while adrenal cortical tumours were decreased (males). There was a low, non-dose-related incidence of pancreatic carcinoma. Other tumour rates were in line with control data. It is concluded that lifetime feeding of 50 mg TBTO/kg diet induces toxicity in various organ systems. An increase in some common tumours was found at the high dose, probably due to hormonal or immunological changes. Effects on the incidence of some of these tumours were observed also in the low-dose groups, but not at the intermediate dose. Because of the high spontaneous incidence of the tumours in this strain of rats, the variable incidence in the treated groups, and the absence of a dose-effect relationship, the significance of the increased incidence of these tumours is questionable. On the basis of the marginal effects at 5 mg/kg, a no-adverse-effect level of 0.5 mg TBTO/kg can be established.

Altered methylation of ras oncogenes in benzidine-induced B6C3F1 mouse liver tumors

The B6C3F1 mouse is a hybrid strain which exhibits a high (30%) spontaneous hepatoma incidence and sensitivity to chemical induction of liver tumors. The spontaneous hepatoma incidence of the paternal C3H/He strain is approximately 60%, while that of the maternal C57BL/6 strain is very low. The presence of activated oncogenes, primarily Ha- ras, and to a lesser extent, Ki- ras, has been reported in B6C3F1 mouse liver tumors. Because alterations in a gene's capacity for expression, as well as mutation, may be involved in oncogene activation, this investigation was directed toward an examination of a putative control point for transcription, i.e., the methylation state of a gene. Hypomethylation is believed to be necessary, but not sufficient, for transcription. It was therefore hypothesized that alterations in the methylation state of the Ha- ras and Ki- ras oncogenes may facilitate the aberrant expression of these genes in B6C3F1 mouse liver. Restriction enzyme analysis ( MspI, HpaII, and HhaI) was used to assess the extent of DNA methylation. MspI digestion of B6C3F1 and C3H/He DNA revealed the absence of a 15-kb Ha- ras band presen in MspI-digested C57BL/6 DNA, suggesting that the Ha- ras oncogene of B6C3F1 and C3H/He mouse liver lacks a methylated site. In other respects, the Ha- ras and Ki- ras oncogenes are methylated to a degree which suggests that these oncogenes have a low potential for expression in normal mouse liver. The methylation state of the Ha- ras and Ki- ras oncogenes was also assessed in benzidine-induced hepatomas and adjacent nontumor tissue from B6C3F1 mice. In four out of four cases, the Ha- ras oncogene was hypomethylated in tumor as compared to nontumor tissue and increased expression of the gene was detected in three out of four hepatomas; the Ki- ras oncogene was hypomethylated in two out of four cases. These results suggest that hypomethylation of oncogenes may provide an epigenetic mechanism for facilitating their aberrant expression. The lack of a methylated site observed in the Ha- ras oncogene in B6C3F1 and C3H/He mouse liver may indicate an increased potential for its expression which could, in part, account for the high propensity for hepatoma development in these two strains.

A transgenic mouse model for human neurofibromatosis.

Human T-lymphotropic virus type 1 (HTLV-1) has been associated with the neurologic disorder tropical spastic paraparesis and possibly with multiple sclerosis. The tat gene of HTLV-1 under control of its own long terminal repeat is capable of inducing tumors in transgenic mice. The morphologic and biologic properties of these tumors indicate their close resemblance to human neurofibromatosis (von Recklinghausen's disease), the most common single gene disorder to affect the nervous system. The high spontaneous incidence of this disease, together with the diverse clinical and pathologic features associated with it, suggests that environmental factors may account for some of the observed cases. Multiple tumors developed simultaneously in the transgenic tat mice at approximately 3 months of age, and the phenotype was successfully passed through three generations. The tumors arise from the nerve sheaths of peripheral nerves and are composed of perineural cells and fibroblasts. Tumor cells from these mice adapt easily to propagation in culture and continue to express the tat protein in significant amounts. When transplanted into nude mice, these cultured cells efficiently induce tumors. Evidence of HTLV-1 infection in patients with neural and other soft tissue tumors is needed in order to establish a link between infection by this human retrovirus and von Recklinghausen's disease and other nonlymphoid tumors.