BMS-189453, a novel retinoid receptor antagonist, is a potent testicular toxin.

BMS-189453 is a synthetic retinoid that acts as an antagonist at retinoic acid receptors alpha, beta, and gamma. In Sprague Dawley rats at daily oral doses of 15, 60, or 240 mg/kg for 1 month, BMS-189453 produced increases in leukocyte counts, alkaline phosphatase and alanine aminotransferase levels, and marked testicular degeneration and atrophy at all doses. Significant overt signs of toxicity and deaths occurred at 240 mg/kg, whereas body-weight and food-consumption decreases occurred at 60 and 240 mg/kg. When BMS-189453 was administered to male rats at daily doses ranging from 12.5 to 100 mg/kg for 1 week, only minimal testicular changes occurred at all doses, shortly after the dosing period. However, after a 1-month drug-free observation period, marked testicular atrophy was evident at all doses. BMS-189453 was then administered at doses of 2, 10, or 50 mg/kg to male rats for 1, 3, or 7 consecutive days. Dose- and duration-dependent testicular toxicity that occurred after a 1-month observation period did not recover, and, in some cases, was more severe 4 months after the last dose. In rabbits administered BMS-189453 at oral doses of 2, 10, or 50 mg/kg for 1 week, testicular degeneration and atrophy were evident in the high-dose group at 1 month following treatment. These studies indicate that retinoid antagonists can selectively produce progressive and prolonged testicular toxicity after single or repeated oral doses that are otherwise well tolerated.

BR96 sFv-PE40 immunotoxin: nonclinical safety assessment.

BR96 sFv-PE40, a recombinant DNA-derived fusion protein composed of the heavy- and light-chain variable region domains of the monoclonal antibody BR96 and the translocation and catalytic domains of Pseudomonas exotoxin A, is being developed for the treatment of solid tumors expressing cell surface Lewis(y)-related antigens. Single- and repeat-dose intravenous toxicity studies in rats and dogs and a comparative ex vivo tissue-binding study with rat, dog, and human tissues were conducted to assess the toxicity of BR96 sFv-PE40 and to estimate a safe starting dose in humans. Additional studies were performed to investigate the prevention of pulmonary vascular-leak syndrome, the dose-limiting toxicity of BR96 sFv-PE40 in rats, and the immunogenicity of BR96 sFv-PE40. In single-dose studies in rats, the vascular leak appeared to be primarily confined to the lungs; however, with a repeat-dose regimen (every other day for 5 doses) other organs including the brain and heart were involved at lethal doses (12-15 mg/m2 cumulative). Single doses of 1.8 mg/m2 and a cumulative 3.8 mg/m2 dose (0.75 mg/m2, every other day for 5 doses) were generally well tolerated in rats. These doses are significantly greater than doses required to cure rodents bearing human tumor xenografts. In dogs, the major target organ following single or repeated doses (every 3 days for 5 doses) was the pancreas. Morphologic changes in the exocrine pancreas ranged from atrophy with single-cell necrosis to diffuse acinar necrosis. After a 1-mo dose-free observation period, no residual pancreatic toxicity was observed in dogs given single doses up to 6.0 mg/m2 or 5 doses of 2.4 mg/m2 (12 mg/m2 cumulative). No significant pancreatic toxicity was observed at doses <0.6 mg/m2 in high Lewis(y)-expressing dogs. Assessment of trypsinlike immunoreactivity was useful in monitoring changes in pancreatic function. The immunogenicity of BR96 sFv-PE40 could be inhibited by combined treatment with an immunosuppressant in dogs, thus maintaining exposure to BR96 sFv-PE40.

In vivo efficacy and toxicity of a single-chain immunotoxin targeted to CD40.

G28-5 sFv-PE40 is a single-chain immunotoxin targeted to CD40, which is highly expressed on human hematologic malignancies, including non-Hodgkin's lymphoma, B-lineage leukemias, multiple myeloma, and Hodgkin's disease, as well as certain carcinomas. In vitro analysis showed that this monovalent immunotoxin had a binding affinity of 3 nmol/L, within 15-fold of the bivalent parental monoclonal antibody. G28-5 sFv-PE40 was stable when incubated in mouse serum at 37 degrees C for 6 hours and cleared from the circulation of mice with a half-life of 16.7 minutes. This immunotoxin was effective in treating human Burkitt's lymphoma xenografted SCID mice with complete responses, defined by an asymptomatic phenotype for greater than 120 days, obtained at doses of 0.13 to 0.26 mg/kg. The efficacy of treatment was dependent on the schedule used, with every three days for five injections being the most effective tested. The toxicity of G28-5 sFv-PE40 was examined in SCID mice, rats, and monkeys, with the maximum tolerated dose being 0.48, 1.0, and 1.67 mg/kg, respectively. Comparative immunohistology showed that the G28-5 specificity was qualitatively similar between human and monkey tissue. In summary, G28-5 sFv-PE40 was effective at inducing complete antitumor responses in lymphoma xenografted mice at doses that were well tolerated in mice, rats, and monkeys.

Toxicity of diethanolamine. 1. Drinking water and topical application exposures in F344 rats.

Toxicology studies of diethanolamine were conducted in male and female F344 rats for 13 weeks' duration to characterize and compare effects of exposure in the drinking water with those caused by topical application. Doses of diethanolamine ranged from 160 to 5000 ppm in the drinking water study (equivalent to daily doses of 25-440 mg kg-1 in males and 15-240 mg kg-1 in females) and from 32 to 500 mg kg-1 in the topical application study. Dose-dependent toxic effects due to exposure to diethanolamine included hematological changes (a poorly regenerative, microcytic anemia), as well as toxic responses in the kidney (increased weight, tubular necrosis, decreased renal function, and/or tubular mineralization), brain and spinal cord (demyelination), testis (degeneration of the seminiferous tubules) and skin (site of application: ulceration, inflammation, hyperkeratosis and acanthosis). A no-observed-adverse-effect level was not achieved for hematological changes, nephropathy or hyperkeratosis of the skin. Differences in dose-response between the drinking water and topical application exposures were attributed largely to the limited dermal absorption of this chemical.

Transplantation of large granular lymphocyte leukemia in congenitally athymic rats.

Twenty-two congenitally athymic nude (rnu/rnu) rats were transplanted with large granular lymphocyte leukemia derived from F344 rats and then compared with ten similar rats inoculated with a suspension of normal F344 rat spleen cells. The normal spleen cells and tumor cells from a spontaneous, naturally occurring leukemia did not grow or cause clinical disease in any of the rats. All rats inoculated with a serially passaged leukemia cell inoculum had local growth at the inoculation site that spread widely and resulted in progressive tumor growth. Death occurred between 16 and 38 days after inoculation. The 22 rats that received passaged tumor cells developed leukemia and splenomegaly. Spleens were diffusely infiltrated by tumor cells and had severe depletion of lymphocytes in the white pulp. Leukemic rats were thrombocytopenic and had hemolytic anemia characterized by increased osmotic fragility, red cell width, and many nucleated erythrocytes. The disease syndrome appears similar to that of F344 rats transplanted with the same inoculum. Because the host rats lacked T cells, it is concluded that the hemolytic anemia and thrombocytopenia that develop in transplanted rats are independent of T cell function.

Reproductive effects in Long-Evans rats exposed to chlorine dioxide.

Long-Evans rats, 4-6 weeks of age, were dosed with 0.0, 2.5, 5.0, or 10.0 ml/kg chlorine dioxide (ClO2) in deionized water for up to 73 days. Males were exposed for 56 days and females for 14 days prior to breeding, and throughout the 10-day breeding period. Males were killed and evaluated for sperm parameters and reproductive tract histopathology following the breeding period. Females continued to be dosed throughout gestation and lactation until weaning on lactation day 21, when both dams and selected pups were necropsied. Neither clinical signs of toxicity nor adverse effects on any reproductive parameter examined were observed in the parental animals. Litter size, pup viability, and pup weight were unaltered by chlorine dioxide exposure. F0 reproductive tract organ weights and F1 organ weights for testis, epididymis, uterus, and ovaries were not different between groups, but vaginal weight was significantly decreased (P less than 0.03) for female weanlings in the high dose (10.0 mg/kg) group relative to controls. There were no changes in thyroid hormone parameters that appeared to be attributable to chlorine dioxide treatment.

Spleen cell population changes and hemolytic anemia in F344 rats with large granular lymphocyte leukemia.

A spontaneous large granular lymphocyte leukemia from a F344 rat was transplanted to 36 syngeneic recipients to study the interactions among leukemia, T lymphocytes, and the development of immunemediated hemolytic anemia. Six rats were euthanatized at biweekly intervals, and spleen weight, total spleen cellularity, and differential spleen cell counts were correlated with hemograms and osmotic fragility. Sequential changes in splenic architecture were correlated with hematologic parameters. Monoclonal antibodies defining all T lymphocytes (W3/13), T helper-inducer cells (W3/25), and T suppressor cells (OX-8) were used to identify T cells in immunocytochemical techniques on spleen sections, as well as in fluorescence activated cell sorter analysis of spleen cell suspensions. The onset of hemolytic anemic at 7 weeks after transplantation coincided with the first detection of tumor cells in the spleen and peripheral blood. Tumor cells first accumulated in the marginal zones, and then they infiltrated the red pulp sinusoids. Although the leukemia caused dispersion of the splenic lymphoid tissue, there was no significant lymphopenia, and the relative number of helper (W3/25+) and suppressor (OX-8+) lymphocytes did not change. Because the induction of anemia was a relatively early event in splenic involvement, we concluded that anemia was unrelated to disruption of lymphoid architecture; furthermore, it does not appear to be caused by changes in the numbers of regulatory T lymphocytes.

Serial syngeneic transplantation of large granular lymphocyte leukemia in F344 rats.

A spontaneous large granular lymphocyte (LGL) leukemia was serially transplanted in 92 male F344 rats kept under standard laboratory conditions. Serial transplantation into groups of four rats each resulted in a rapid reduction in the latent period of the disease. After 23 serial transplantations, F344 rats in groups that were injected intraperitoneally with 10(7) cells died between 12 and 16 days after transplantation. At necropsy, "transplanted" rats had enlarged mesenteric lymph nodes, thymus, and spleen. Neoplastic cells were detected in the spleen on day 3 and in peripheral blood on day 6. Extreme leukocytosis with leukemia was present on day 9. Severe hemolytic anemia coincided with a sharp increase in osmotic fragility on day 12. Splenic lymphoid depletion was observed histologically and confirmed by differential cell counts of isolated spleen cells. Analysis for surface markers of splenic lymphocytes by monoclonal antibodies and flow cytometry indicated that cells with T helper/inducer phenotypes were disproportionately decreased, while the number of T suppressor cells did not significantly change. The T helper/T suppressor lymphocyte ratio (normal = 2.09 +/- 0.35) was decreased on day 9 (0.76 +/- 0.10) and day 12 (0.25 +/- 0.04). Hemolytic anemia was not related to a decrease in the number of T suppressor cells. The passaged leukemia cell model should provide investigators with an easily maintained neoplasm of short latency with which to study pathogenesis of leukemia-related disorders.

Inhibition of in vitro plaque formation by large granular lymphocyte leukemia cells from F344 rats.

The effect of large granular lymphocyte leukemia on B lymphocyte function was studied by determining the number of plaques formed in an in vitro hemolytic plaque assay. Leukemia cells inhibited plaque formation by normal splenic lymphocytes in a logarithmic, dose-dependent manner. At the highest leukemia cell concentrations, spleen cell suspensions made 50% fewer plaques. Plaque forming responses were very sensitive to duration of preincubation time in all assays. The number of plaques formed decreased markedly if incubated 2 hr before the assay was performed. Incubation of the cells at 56 degrees C for 8 min did not alter the inhibitory activity but pretreatment with 0.01% trypsin did. Supernatant fluids from leukemia cell suspensions did not inhibit plaque formation. These data suggest that diffuse infiltration of lymphoid tissues by leukemia cells may interfere with some normal lymphocyte functions. Although leukemia cells inhibited splenic B lymphocyte function, leukemic rats did not have hypogammaglobulinemia.

Pulmonary endothelial and alveolar epithelial lesions induced by O,O,S-trimethyl phosphorothioate in rats.

The morphogenesis of pulmonary injury induced by an impurity present in a commercially important organophosphorus insecticide, O,O,S-trimethyl phosphorothioate (OOS-TMP), was studied by combined light and transmission electron microscopy. Weanling female WAG/Rij rats received OOS-TMP dissolved in corn oil by gavage and were studied at intervals from 6 to 168 h after treatment. Sequestration of neutrophils was initially observed at 12 h after treatment and was accompanied by interstitial oedema. Plasmalemma alterations in endothelium lining capillaries and small arteries and veins were observed from 12 to 120 h after treatment and were accompanied by endothelial cell detachment and separation from the basal lamina. Abundant aggregates of fibrin were sequentially observed in intravascular, interstitial, and alveolar spaces. Platelet aggregation and degranulation were occasionally observed in capillaries as early as 12 h after treatment, and frequently observed in capillaries and small vessels from 24 to 96 h after treatment. Significant increases in wet lung weight and lung water content occurred at the same time that morphologic changes were observed in pulmonary endothelium. Alterations in type I alveolar epithelial cells were initially observed at 24 h after treatment. Cell swelling, fragmentation, and necrosis were observed in both type I and type II cells and resulted in a bare basal lamina. Marked attenuation, hypertrophy, and proliferation of type II epithelial cells followed alveolar epithelial cell injury and loss. Minimal changes were observed in non-ciliated bronchiolar epithelial (Clara) cells; predominant changes included the loss of surface microvilli and apical cytoplasmic bulge. The results of this study indicate that the endothelium and alveolar epithelium are the predominant cell types in the rat lung injured following OOS-TMP administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Examination of the reproductive effects of tricresyl phosphate administered to Long-Evans rats.

Tricresyl phosphate (TCP) is used commercially as a plasticizer and a flame retardant. The reproductive effects of TCP (less than 9.0% TOCP) were examined. Male Long-Evans rats received 0, 100, or 200 mg/kg and females received 0, 200, or 400 mg/gk TCP in corn oil by gavage. The 100 mg/kg TCP males were mated with 200 mg/kg TCP females, and 200 mg/kg TCP males were bred with 400 mg/kg TCP females. Males were dosed for 56 days and females for 14 days prior to breeding and throughout the 10-day breeding period. Following breeding, the males were necropsied and evaluated for sperm parameters and reproductive tract histopathology. Females were dosed throughout gestation and lactation. Pups and adult females were necropsied on postnatal day 21. Sperm concentration, motility, and progressive movement were decreased for 200 mg/kg dose group males. A dose-dependent increase in abnormal sperm morphology was observed for males in both TCP dose groups. The percent of sperm-positive females per group was unchanged, but the number of females delivering live young was severely decreased by TCP exposure. Litter size and pup viability were decreased in the 400 mg/kg dose group. Pup body weight and developmental landmarks were unaffected by TCP exposure. Histopathologic changes were observed in the testes and epididymides of male rats and in the ovaries of female rats exposed to TCP.

Echocardiography, electrocardiography, and radiography of cats with dilatation cardiomyopathy, hypertrophic cardiomyopathy, and hyperthyroidism.

The echocardiographic, ECG, and radiographic findings of sequentially examined cats with dilatation cardiomyopathy (DCM, n = 7), hypertrophic cardiomyopathy (HCM, n = 8), and hyperthyroidism (HT, n = 20) were compared with those of healthy control cats (n = 11). Cats with DCM were easily differentiated from healthy cats by echocardiography and from cats with HCM and HT by a dilated left ventricle at end-diastole with a mean +/- SD of 2.20 +/- 0.36 cm, reduced fractional shortening (2.9% +/- 3.7%), reduced aortic amplitude (0.07 +/- 0.05 cm), reduced left ventricular wall amplitude (0.09 +/- 0.09 cm), and increased E-point septal separation (0.83 +/- 0.29 cm). The cats with HCM were most consistently recognized echocardiographically by increased left ventricular wall thickness at end-diastole (0.75 +/- 0.12 cm). Some cats with HT had abnormal echocardiograms with left ventricular wall hypertrophy. These cats could usually be differentiated from the cats with HCM because of normal or increased ventricular wall amplitude, aortic amplitude, or percentage of thickening of the left ventricular wall and interventricular septum. Left atrial enlargement (left atrial diameter greater than 1.57 cm or left atrium/aorta greater than 1.75) was commonly detected by the echocardiogram in cats with DCM, HCM, or HT. The echocardiogram was helpful in differentiating the type of cardiomyopathy (DCM, HCM, or HT) when plain thoracic radiographs indicated that cardiomegaly existed. The ECG may have indicated incorrectly that there was left ventricular enlargement in some cats with HT, and it did not indicate consistently that left ventricular enlargement existed when present in cats with DCM or HCM. The ECG was a poor indicator of left atrial enlargement in all cats.

Adenocarcinoma of the jejunum in two cows.

Two cows from separate farms were examined for gastrointestinal disturbances. The first animal had an acute gastrointestinal disturbance with colic and the second animal had chronic weight loss and anemia. Both cows had jejunal adenocarcinoma with metastasis. They were euthanized and necropsied. Adenocarcinomas of each cow were similar histologically. Adenocarcinoma of the intestine is a rare disorder of cattle, but may present as either an acute or chronic gastrointestinal disturbance.

Characterization of spontaneous amyloidosis of Syrian hamsters using the potassium permanganate method.

Amyloid from 16 Syrian hamsters with spontaneous age-related systemic amyloidosis retained affinity for Congo red dye after treatment with potassium permanganate. Serum electrophoresis showed a slight rise in gamma globulins without a monoclonal spike. Bence-Jones proteins were not present in the urine. There was no histological evidence of plasmacytosis. Amyloidosis could not be associated with chronic inflammation. The lack of apparent etiology of this amyloid and its resistance to potassium permanganate treatment suggests that it is a primary non-AA amyloid. This finding varies with a report suggesting that the spontaneous amyloidosis of aged Syrian hamsters is associated with the AA fibril.

Antitubular basement membrane autoantibody in a dog with chronic tubulointerstitial nephritis.

Linear deposition of immunoglobulin G was seen by direct immunofluorescence along the tubular basement membranes in the kidney of a dog with chronic tubulointerstitial nephritis. Autoantibody eluted from the affected kidney bound to the tubular basement membrane of normal canine kidney, but not to several other normal canine basement membranes. The pathogenic significance of the autoantibody in this dog was not determined.