Chronic effects of the novel glucocorticosteroid RPR 106541 administered to beagle dogs by inhalation.

The preclinical safety of RPR 106541, a novel 17-thiosteroid, was evaluated in young adult and mature dogs by inhalation exposure for 26 weeks and 52 weeks, respectively. A dry powder formulation of RPR 106541 in lactose was administered to young adult dogs (approximately 6 months of age at initiation) at doses of 0 (air and placebo controls), 10, 100, or 1,000 microg/kg/d for 26 weeks. A solution-based aerosol formulation was administered to mature dogs (approximately 10 months at initiation) from a pressurized metered dose inhaler at 0 (air and placebo controls), 10, 50, and 150 microg/kg/d for 52 weeks. Clinical evidence of glucocorticosteroid-induced immunosuppression was observed by weeks 20-26 following relatively high dose exposures (100 microg/kg/d and 1,000 microg/kg/d) in young dogs receiving the dry powder formulation for 26 weeks. Classic glucocorticosteroid effects were observed, including adrenocortical atrophy, reduced bone mass with retention of epiphyseal growth plates in long bones, prominence of stromal adipose tissue in bone marrow, and atrophy of lymphoid tissues. Inhalation administration of RPR 106541 to sexually mature dogs facilitated more definitive characterization of endocrine affects of RPR 106541 as compared with administration in younger, sexually immature animals. Significant effects in female reproductive organs included absence of corpora lutea in association with atresia of vesicular follicles within the ovaries, endometrial hyperplasia, and lobular development of mammary tissue. Discordant development of mammary tissue, accumulation of secretory material within hyperplastic endometrial glands, and hypertrophy of uterine lining epithelium in absence of ovulation were consistent with a secondary progestin effect by a potent glucocorticosteroid.

Toxicologic and carcinogenic effects of the type IV phosphodiesterase inhibitor RP 73401 on the nasal olfactory tissue in rats.

RP 73401, a type IV phosphodiesterase inhibitor, caused toxic effects in the nasal olfactory region of Sprague-Dawley rats when administered by either oral or inhalation exposure. A single oral administration of RP 73401 (at a dose of > or = 50 mg/kg) or 5-day inhalation exposure (1 hr/day) at a dose of approximately 1.0 mg/kg per day caused degeneration and sloughing of the olfactory surface epithelium. Degeneration and loss of Bowman's glands were noted in the underlying lamina propria and submucosa. Electron microscopy of these lesions demonstrated that sustentacular cells and the epithelial cells lining Bowman's glands were the primary target cells in the olfactory mucosa. The earliest ultrastructural changes detected in these cells were dilatation and vesiculation of the endoplasmic reticulum, suggesting that metabolic activation is important for the toxic effects. In repeated-dose studies, 13 wk of oral dosing at 2.0 or 6.0 mg/kg per day resulted in subtle disorganization of the olfactory epithelium, whereas basal cell hyperplasia in the olfactory epithelium was identified in a 6-month inhalation study at a dose of 1.0 mg/kg per day. A 2-yr inhalation carcinogenicity study resulted in tumors of the nasal olfactory region in rats treated at 0.5 and 1.0 mg/kg per day. Most tumors were classified as olfactory neuroblastomas, and immunohistochemistry on selected tumors was consistent with their being of neuroectodermal origin. Of the species studied (rat, mouse, and dog), the olfactory toxicity of RP 73401 was confined to the rat, and the toxicity was likely related to metabolic activation by olfactory epithelial cells rather than the phosphodiesterase activity of the compound.

Ganglioneuromas of the thyroid gland in a colony of Sprague-Dawley rats.

Ganglioneuromas of the thyroid gland of Sprague-Dawley rats were found in 7.2% of 698 rats used in two, 2-year oncogenicity bioassays. The incidence of the tumor was unrelated to treatment or sex. Of the 52 ganglioneuromas of the thyroid gland identified in 50 animals, 40 (80%) had coincident C cell proliferations in the same or contralateral lobe. Ganglioneuromas were contiguous or commingled with C cell proliferations in 63.5% of the cases. The ganglioneuromas consisted of large ganglion cells and, in a few cases, cells thought to be less differentiated neuronal precursors, in a matrix of neurites and Schwann cells. They grew, infiltrating and expanding in the thyroid parenchyma, and did not metastasize. Immunohistochemical staining for calcitonin, S-100 protein, and neurofilaments, as well as electron microscopy, were used to further characterize the tumors. The close association of ganglioneuromas with C cell tumors supports the theory of neural crest origin of C cells and provides a parallel to the association of ganglioneuromas and pheochromocytomas in the adrenal medulla. This is the first report of ganglioneuromas occurring in the thyroid gland or occurring as a common entity in any species.

A subchronic inhalation toxicity study in rats exposed to silicon carbide whiskers.

To determine whether inhaled silicon carbide whiskers (SiC) cause lung damage in rats, four groups (50 males/50 females each) of rats were exposed to air only or to one of three concentrations of SiC 6 hr/day, 5 days/week for 13 weeks. Half (25 males/25 females/group) were euthanized at the end of exposure, the remainder 26 weeks later. Mean concentrations were 0, 630, 1746, and 7276 SiC whiskers/ml (0.09, 3.93, 10.7, and 60.5 mg/m3). Although there were no concentration-related changes in body weight, clinical chemistry, or hematological data attributable to SiC, lung weights were increased in the high concentration exposure group at both euthanization times. In all whisker-exposed groups, after 13 weeks of exposure, the incidence of the following lung and lymph node lesions was higher than in controls: inflammatory lesions; bronchiolar, alveolar, and pleural wall thickening; focal pleural fibrosis in lung; and reactive lymphoid hyperplasia in bronchial and mediastinal lymph nodes. After 26 weeks of recovery, lung inflammatory lesions had decreased and fewer rats had enlarged lymph nodes, but the incidence of alveolar wall thickening, focal pleural wall thickening, and adenomatous hyperplasia of lung had increased further. Incidence and severity appeared to be dose-related. Therefore, until longer term studies are undertaken and it is established whether the above observed lesions will progress to more severe pathological entities, it is prudent to adopt stringent handling procedures for silicon carbide whiskers.

Comparative aspects of contraceptive steroids: effects observed in the monkey.

The use of the non-human primate in long-term studies of contraceptive steroids has been questioned because of time, expense and apparent lack of results predictive for humans. Controversies have arisen primarily over the occurrence of mammary nodules in studies of different contraceptive steroids and the occurrence of uterine tumors in 2 high-dose group monkeys in the Depo-Provera study. The long-term studies have been criticized because of the experimental design and the small number of monkeys per dose group. Individual studies by themselves did not reveal lesions other than those expected from an exaggerated pharmacologic response of target tissues; however, a pattern may emerge from reviewing and combining results of different studies that indicate the results of these studies are in agreement with the clinical findings in man. Effects of contraceptive steroids on the mammary gland and genital organs will be discussed. Data from 17 contraceptive steroid studies involving 264 untreated control and 733 treated non-human primates were available.

Immunocytochemical evaluation of primary human esophageal carcinomas and their xenografts for keratin, beta-chorionic gonadotropin, placental lactogen, alpha-fetoprotein, carcinoembryonic antigen, and nonspecific cross-reacting antigen.

The unlabeled antibody peroxidase-antiperoxidase technique was used to examine esophageal neoplasms for the tumor markers beta-human chorionic gonadotropin, human placental lactogen (HPL), alpha-fetoprotein, carcinoembryonic antigen (CEA), and nonspecific cross-reacting antigen (NCA) before and after xenotransplantation to athymic nude mice. In addition, keratin was used as an epithelial cell marker. Immunoreactive beta-human chorionic gonadotropin was detected in four of seven primary tumors and in three of seven xenografts. Two of seven primary tumors contained HPL immunoreactive cells while four of seven tumor xenografts had HPL immunoreactivity. alpha-Fetoprotein was detected in two of seven primary tumors and in one of seven xenografts. NCA and CEA were detected in six of seven primary tumors and in all tumor xenografts. Five of seven primary neoplasms and six of seven tumor xenografts were found to contain both NCA and CEA, while one tumor and its xenografts displayed only NCA immunoreactivity. All seven primary carcinomas displayed keratin immunoreactivity, but only six of the seven xenograft tumors showed keratin positive cells. When a tumor marker was detected in a primary tumor, it was usually found in at least some of the xenografts arising from that tumor. However, marker loss did occur with repeated passage of tumors in some cases. On the other hand, markers were expressed in xenografts which were not present in the corresponding primary tumor. In three instances, HPL was detected in xenografts derived from HPL negative primary carcinomas. This was also true for CEA and NCA in one case. These results show that tumor markers are expressed to varying degrees by tumors growing as xenografts in nude mice. In primary tumors, HPL is associated with poorly differentiated squamous cell carcinomas and this marker was found to appear in HPL negative tumors as the tumor cells became less differentiated while growing as xenografts in nude mice.

Transformation of human bronchial epithelial cells transfected by Harvey ras oncogene.

Transfection of normal human bronchial epithelial (NHBE) cells with a plasmid carrying the ras oncogene of Harvey murine sarcoma virus (v-Ha ras) changed the growth requirements, terminal differentiation, and tumorigenicity of the recipient cells. One of the cell lines isolated after transfection (TBE-1) was studied extensively and shown to contain v-Ha ras DNA. Total cellular RNA from TBE-1 cells hybridized to v-Ha ras structural gene fragment probes five to eight times more than RNA from parental NHBE cells. The TBE-1 cells expressed phosphorylated v-Ha ras polypeptide p21, showed a reduced requirement for growth-factor supplements, and became aneuploid as an early cellular response to v-Ha ras expression. As the transfectants acquire an indefinite life-span and anchorage independence they became transplantable tumor cells and showed many phenotypic changes suggesting a pleiotropic mechanism for the role of Ha ras in human carcinogenesis.

Chronic toxicity and carcinogenicity evaluation of fenvalerate in rats.

Groups of 93 male and 93 female Sprague-Dawley rats were fed diets containing 1, 5, 25, and 250 ppm fenvalerate for up to 2 yr. The control group consisted of 183 males and 183 females. Approximately 10 treatment and 20 control rats/sex . group were killed at intervals of 3, 6, 12, and 18 mo. When body weights, food consumption, hematology, clinical chemistry and organ weights did not reveal a treatment effect, two additional groups of 50 males and 50 female rats were placed on 0 or 1000 ppm fenvalerate diets and maintained for 2 yr. Body weight was decreased and organ/body weight ratios were increased in brain, liver, spleen, kidneys (females), heart (females), and testes (males) in the 1000 ppm group. Mammary and pituitary tumors were commonly observed, along with a variety of other tumors occurring randomly among all control and treatment groups. No statistically significant differences in the number and type of neoplasms were observed except for mammary tumors in females in the main study. These effects were judged not to be toxicologically significant, since mammary tumor incidences did not exceed expected incidences in aged female Sprague-Dawley rats, time to tumor appearance was unchanged, and no shift in percent benign versus malignant tumors occurred. Sarcomas identified in the subcutis and dermis in 5/51 1000-ppm-treated males were also identified in 2% (1/50), 2% (2/102), and 0-6% of concurrent, original, and historical controls, respectively. Microscopic examination did not reveal any treatment-related lesions. The no-observable-effect level was determined to be 250 ppm.

Long-term explant culture of normal mammary epithelium.

Mammary epithelium and surrounding stroma have been maintained in an explant system for 1 to 6 months and subsequently xenografted into athymic nude mice. The morphological characteristics of 26 cases of normal human mammary epithelium in long-term explant culture were described, using high-resolution light and electron microscopy. Normal human breast tissue specimens were obtained from immediate autopsy or surgical resection. The explants were cultured in Connaught Medical Research Laboratories Medium 1066 supplemented with serum, insulin, and hydrocortisone. The histotypic features of the mammary epithelium in both the central portion of the explant and the epithelial outgrowth onto the surface of the explant were described. In some cultures, the cells acquired more keratin and formed multicellular blister-like domes. Tissues from eight cases after 1 to 14 weeks in culture were xenografted in athymic nude mice and were maintained up to 924 days.

Long-term survival of normal adult human tissues as xenografts in congenitally athymic nude mice.

Bronchi, pancreatic ducts, and colons from adult human were maintained as xenografts in congenitally athymic nude N:NIH(S) mice for 715, 145, and 89 days, respectively. After an ischemic crisis and revascularization of the human tissue, the epithelium regenerated to a normal differentiated morphology. The long-term survival of normal adult human epithelial tissues as xenografts provides model systems for the study of the interactions of chemical and/or physical carcinogens with human tissues.

Spontaneous esophageal carcinoma and epithelial cell line of an adult rhesus monkey.

A continuous epithelial cell line, 816A, was established from a lymph node of an adult rhesus monkey with metastatic esophageal carcinoma. These cells are characterized by the presence of desmosomes and a markedly heteroploid karyotype. At a relatively early culture age, electron microscopy showed both budding and extracellular type C virus. Antigen reactive with antisera to Mason-Pfizer monkey virus was observed by complement-fixation. The level of this antigen decreased with increased culture age. To our knowledge, the 816A cells represent the only established simian or human cell line of esophageal carcinoma origin.

Disseminated lymphoma in a rhesus monkey: a case report.

A rhesus monkey was inoculated with rhesus cytomegalovirus. A leukocyte-associated herpes virus, unrelated to cytomegalovirus, was later isolated from the same monkey. Four years after the virus inoculation, the monkey developed a disseminated lymphoma.

Characterization of a spontaneous undifferentiated carcinoma from an African green monkey (Cercopithecus aethiops).

An adult male African green monkey (Cercopithecus aethiops) with an undifferentiated carcinoma, probably originating from the nasal mucosa, was received from the Akron, Ohio zoo. Cultivation of this tumor in vitro resulted in a mixture of fibroblastic and epithelial cells which was subsequently separated using differential trypsinization. The neoplastic nature of the cultured epithelial cells was verified by their ability to transplant into athymic nude, or antithymocyte serum-treated mice, where poorly differentiated carcinomas were produced, and cultures of the tumors that arose in nude mice were morphologically similar to pretransplantation cultures. Early cultures showed a normal male karyotype characteristic of the species; however, in long-term cultures, a clearly defined, small submetacentric Y chromosome was not observed. Electron microscopic examination of tumor tissue and cultured tumor cells revealed desmosomes and the presence of cytoplasmic (keratin-type) fibrils, which tended to be organized around the nucleus. In addition to the keratin-type fibrils, the cultured tumor cells also contained a large amount of cytoplasmic inclusion material that may represent keratohyalin granules. There was no evidence of a viral association with tumor material or cultured cells. The cultures were susceptible to infection by vesicular stomatitis virus, Herpesvirus hominis type 1, and H. saimiri, but were resistant to the Epstein-Barr virus.

Chronic Herpesvirus saimiri infection in an owl monkey.

An adult owl monkey (Aotus tricirgatus) used for immunologic studies of Herpesvirus saimiri (HVS) developed early, late, membrane, and neutralizing antibodies to HVS approximately 3 weeks after the beginning of the experiment. HVS was isolated by the cocultivation of peripheral blood for over 1 year. No clinical, gross, or histopathologic findings of malignancy were exhibited by the animal. The HVS isolate from the animal was indistinguishable biologically and serologically from the original HVS strain of Meléndez and from an isolate of an experimentally HVS-induced tumor. Inoculation of this isolate into 2 young white-lipped marmosets (Saguinus fuscicollis) produced typical malignant lymphoma and lymphocytic leukemia. Our findings suggested that the virus from the chronically infected animal was oncogenic and that host factors were primarily responsible for determining the disease manifestation of the virus infection. Another owl monkey chronically infected with HVS for over 2 years has remained asymptomatic.

Responses of infant rhesus monkeys to inoculation with Mason-Pfizer monkey virus materials.

Rhesus monkeys neonatally inoculated with Mason-Pfizer monkey virus (M-PMV) and virus-infected cells frequently developed viral and/or bacterial pneumonia and enteritis. Three characteristic hematologic patterns occurred among the inoculated animals and correlated well with the probability of survival. Postmortem examination of the animals revealed lymphadenopathy and thymic atrophy. M-PMV was present in lymph nodes, blood, brain, spleen, thymus, kidneys, and bone marrow. The disease induced in some animals had characteristics suggestive of a slow-virus-induced autoimmune response.

Spontaneous lymphoma associated with Herpesvirus saimiri in owl monkeys.

Two cases of lymphoma and one case of lymphoproliferative disease were found in a group of 7 owl monkeys imported into our colony as a single group. Herpesvirus saimiri (HVS) was isolated from the tumor cells of 1 lymphoma by cocultivation and from kidney cell cultures from the monkey with lymphoproliferative disease. Antibody to HVS was found in serum samples from 2 monkeys positive for HVS but not in the sera from the 4 clinically normal monkeys. Antibody to Epstein-Barr virus-infected cells was also found in the serum from the animal with lymphoma.

Fibrous plaques in the aortas of adult female rhesus monkeys (Macaca mulatta).

Lesions in the abdominal aorta were found in 36 of 40 mature female rhesus monkeys given various oral contraceptive steroids and in 8 of 10 monkeys of a non-treated control group. The lesions consisted of proliferation of subendothelial smooth muscle cells and collagen, often with fragmentation of the internal elastic lamina, forming a plaque. These occurred also, in order of descending frequency, in thoracic aorta, aortic arch, femoral and iliac arteries, and the carotid and pulmonary arteries. They appear statistically unrelated to steroid treatment and lack correlation with body weight and blood cholesterol levels.

PIP: The effects of oral contraceptive (OC) steroids on fibrous plaques in the aorta were studied in 50 adult female rhesus monkeys. Mestranol with or without norethindrone was administered in 1 or 10 times the human dose cyclically for 7 menstrual cycles. Blood was obtained throughout the study for cholesterol determinations and the animals were sacrificed at the end of 7 cycles for gross and microscopic tissue examinations. 36 of 40 monkeys given OCs and 8 of 10 controls revealed lesions in the abdominal aorta. Proliferation of subendothelial smooth muscle cells and collagen, often with fragmentation of internal elastic lamina, forming a plaque, was revealed in the lesions. The lesions also occurred in descending frequency in thoracic aorta, aortic arch, femoral and iliac arteries and the carotid and pulmonary arteries. There was a tendency for serum cholesterol to increase with body weight but the lesions appeared to be unrelated to steroid treatment and appeared to lack correlation with body weight and cholesterol levels.