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.

The toxicity of repeated exposures to rolipram, a type IV phosphodiesterase inhibitor, in rats.

Rolipram is a selective inhibitor of Type IV phosphodiesterase isozymes (PDE IV) which is often used as a baseline comparator for compounds in this class. To document the toxicological effects of rolipram, it was administered to female rats at 0, 10, 30 or 100 mg/kg/day orally for up to 2 weeks. One treatment-related death in the 100 mg/kg/day dose group was observed on day 3, and all rats at this dose level were considered moribund and euthanatized on day 5. Several clinical signs were observed in treated rats, including increased salivation, slight distention of the abdomen, emaciated appearance, and ataxia. After 14 days of treatment, the rats were necropsied and tissues examined microscopically. A number of compound-related histopathological changes were observed in rats receiving 30 or 100 mg/kg/day. Myocardial degeneration and necrosis, endocardial fibrosis, epicarditis, and arteritis/periarteritis of intramural and extramural coronary arteries were observed in the heart. A necrotizing vasculitis and inflammation were observed in the mesentery and interstitial areas of the liver, affecting medium-sized portal arteries and veins. Focal necrosis was also observed in the glandular mucosa of the stomach at these 2 dose levels. Other treatment-related effects included squamous hyperplasia and hyperkeratosis with or without ulceration in the nonglandular stomach of at least one animal from all treatment groups. Enlarged salivary glands were noted at necropsy in animals treated with 100 mg/kg/day, and this finding correlated microscopically with dilatation and degeneration of ducts and acini in the sublingual gland with secondary inflammation and edema. The results of this study demonstrate that rolipram, a selective inhibitor of the type IV class of PDE, can cause effects on the heart and vasculature of rats which heretofore have been ascribed only to selective inhibitors of the PDE III class of isozymes. Therefore, these organs should be closely examined in studies with other PDE IV inhibitors. In addition, the gastrointestinal tract and salivary glands were sites for rolipram-induced toxicity and may be targets of other PDE IV inhibitors.

Distribution, excretion, and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin in C57BL/6J, DBA/2J, and B6D2F1/J mice.

The strains of mice, C57BL/6J, DBA/2J, and B6D2F1/J, have been used as models to study the mechanism of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The distribution, excretion, and metabolism of this compound was studied in male C57BL/6J, DBA/2J, and B6D2F1/J mice following the intraperitoneal administration of radiolabeled TCDD at a dose of 10 micrograms/kg. In all strains, the liver and adipose tissue were the major sites for the accumulation of 3H-TCDD, with more 3H-TCDD being distributed to the livers of the C57BL/6J and B6D2F1/J strains as compared to the DBA/2J strain. While in all strains the feces were the major route of elimination, the total amount of 3H-TCDD-derived radioactivity excreted in the feces amounted to approximately 72% of the original dose in the C57BL/6J and B6D2F1/J strains whereas this was only 54% in the DBA/2J strain. The half-lives for the cumulative excretion of radioactivity in the feces were similar in all strains. The half-life for the excretion of radioactivity in the urine was considerably greater in the DBA/2J strain as compared to the C57BL/6J and B6D2F1/J strains. The estimated half-lives for the total cumulative excretion of 3H-TCDD-derived radioactivity by all routes was 11.0, 24.4, and 12.6 days for the C57BL/6J, DBA/2J, and B6D2F1/J strains, respectively. Greater than 85% of the total radioactivity excreted in urine, bile, and feces from all three mouse strains was present as metabolites of TCDD.

Metabolism of acrylonitrile by isolated rat hepatocytes.

Several of the pathways of metabolism of the suspected carcinogen acrylonitrile (AN) were identified previously in this laboratory with the use of subcellular fractions and purified enzymes (Guengerich, F.P., Geiger, L.E., Hogy, L.L., and Wright, P.L., Cancer Res., 41: 4925-4933, 1981). In order to establish the relative contributions of the various pathways leading to activated and detoxicated products, we examined AN metabolism in isolated Fischer 344 rat hepatocytes as a model. Reduced glutathione (GSH) was depleted, and cell viability was lost in an AN concentration-dependent manner. The major GSH adduct formed at all AN concentrations was identified as S-(2-cyanoethyl)GSH using thin-layer and high-performance liquid chromatography. Acid hydrolysis and amino acid analysis of labeled hepatocellular, protein revealed S-(2-carboxyethyl)-cysteine as the major adduct formed, indicating direct alkylation of cysteinyl residues by AN. 2-Cyanoethylene oxide accumulated in the hepatocyte incubations but did not appear to contribute extensively to alkylation of GSH or protein. Cyanide, resulting from hydrolysis of 2-cyanoethylene oxide, appeared to be completely converted to thiocyanate, which was identified by gel exclusion chromatography and mass spectrometry of the methyl derivative. The concentration of thiocyanate formed was directly proportional to the concentration of AN used. Cyanide does not appear to play a role in AN-mediated cell death. Alkylation of hepatocellular DNA and RNA and extracellular DNA was not observed to an extent greater than one base in 3.5 X 10(5). The relative rates of the various pathways were compared, and more than 97% of the metabolites can be accounted for by the described reactions. These results are of use in evaluating the contribution of the various pathways and modes of binding of AN to toxicity and carcinogenicity in liver and extrahepatic target tissues.

Specificity of the spermidine requirement for the replication of phi X174 DNA be cell-free extracts of Escherichia coli.

A new experimental approach for assessing the biological significance of spermidine interactions in isolated systems is applied to the stimulation by spermidine of the conversion of phi X174 virion DNA to its replicative form by cell-free extracts of Escherichia coli. At 2.5 mM Mg2+, spermidine activated the reaction 20-fold. Varying the spermidine concentration affected both the rate and extent of this DNA synthetic reaction without altering the nature of the reaction products. We evaluated the biological significance of the spermidine requirement by measuring reaction rates in the presence of a homologous series of spermidine analogs of known activity in vivo. There was a lack of specificity, in that all of these analogs were capable of efficiently substituting for spermidine in stimulating the reaction rate. The relevance of this in vitro spermidine stimulation to Escherichia coli chromosome replication in vivo is discussed in light of the results obtained with the spermidine analogs.

Stimulation of deoxyribonucleic acid replication fork movement by spermidine analogs in polyamine-deficient Escherichia coli.

We examined the rate of deoxyribonucleic acid (DNA) replication fork movement in polyamine-deficient cells of Escherichia coli by two independent techniques. DNA autoradiography was used to directly visualize the length of DNA produced during a given time interval, and replication rates were calculated. The amount of DNA synthesized after blocking protein synthesis also allowed calculation of replication rates. We found that the DNA chain elongation rate in polyamine-deficient cells was about half that of putrescine- or spermidine-supplemented cells. We also found that spermidine homologs of increasing chain length, when present at equal intracellular concentrations, exhibited a decreasing ability to support growth and the rate of DNA replication fork movement. The kinetics of recovery of DNA synthesis from the polyamine-deficient state were also investigated. A new rate of DNA synthesis was reached about 20 min after addition of spermidine to polyamine-limited cells. The rise in the rate of DNA synthesis was preceded by a rise in the intracellular concentration of spermidine.

Etiologies of Parkinsonism.

A case report is presented of a patient with Parkinsonism. Three contributory etiological factors are considered with the conclusion that craniocerebral trauma may have been the most significant cause of Parkinsonism in this individual.