To clarify the mechanism of gonadotropin imbalances and the differential response of rat and mouse testicular interstitial cells (Leydig cells) to procymidone, N-(3, 5-dichlorophenyl)-1, 2-dimethylcyclopropane-1, 2-dicarboximide, male Sprague-Dawley rats and ICR mice were fed procymidone in diet for 2 weeks at 0, 700, 2, 000 and 6, 000 ppm for rats, or 0, 1, 000, 5, 000 and 10, 000 ppm for mice. Testosterone and luteinizing hormone (LH) levels in serum, testis or pituitary and the in vitro binding affinities of procymidone, flutamide and related compounds to the androgen receptor in prostate cytosol of rats and mice were examined. Hypergonadotropism in rats and mice was clearly observed in the same order two weeks after the initiation of treatment with procymidone. Increased levels of testosterone and LH in serum at 6, 000 ppm and LH in pituitary at and above 700 ppm in rats were observed. In mice, testosterone levels in serum and testis elevated at 10, 000 ppm. LH levels in serum and pituitary elevated significantly as well at around 5, 000 to 10, 000 ppm. In the competitive binding assay, procymidone showed a significant but lower binding affinity comparing to that of cyproterone acetate, the steroidal androgen receptor antagonist, for the androgen receptor in both rats and mice under the condition that unlabeled dihydrotestosterone (DHT) effectively inhibited the binding of [3H]-DHT to the androgen receptor in both species. The relative binding affinity (RBA) of procymidone was of the same order as that of flutamide, a synthetic non-steroidal antiandrogen structurally similar to procymidone. These results indicate that procymidone is an active antiandrogen and the androgen receptor antagonism is the likely mechanism of action.
Ethanol, at 1.6 and 3.2 g/kg p. o., disrupted the discrete lever-press and shuttle avoidances in mice, eliciting decrease in the avoidance rate. The response rate was increased by 3.2 g/kg of ethanol in the shuttle avoidance. The single p. o. administration of caffeine 3-30 mg/kg, theophylline 3-100 mg/kg, or theobromine 3-100 mg/kg produced no significant change in both response rate and the avoidance rate in the lever-press avoidance. In the shuttle avoidance, up to 3 mg/kg of caffeine, and 10 and 30 mg/kg of theophylline or theobromine significantly increased the response rate, though there was no significant change in the avoidance rate at any doses of these methylxanthines. Intermediate doses of methylxanthines ameliorated the ethanol (1.6 g/kg)-induced decrease in the avoidance rate with an increase in the response rate, indicating non-specific amelioration of the avoidance rate. The ethanol (3.2 g/kg)-induced decrease in the avoidance rate was enhanced by methylxanthines in a dose-dependent manner. However, in the shuttle avoidance, the response rate was sometimes increased by the combined administration of methylxanthines and ethanol. The present results suggest that, although ethanol and methylxanthines possess CNS depressant and stimulant actions, respectively, methylxanthines do not specifically ameliorate, but rather may worsen, the ethanol-induced avoidance disruption, indicating an enhancement of the behavioral toxicity.
Rats were fed for 10 days a 10 or 20% casein diet, or the diets with 0.3% DL-methionine or L-cystine. Rats fed the 20% casein exceeded those fed, the 10% casein in growth, hepatic glutathione (GSH) levels, and microsomal drug-metabolizing activities including cytochrome P-450, aminopyrine N-demethylase, and aniline hydroxylase. Supplement with methionine or cystine had significantly elevated hepatic GSH, regardless of the casein content. After the feeding, rats were intraperitoneally injected with chlorobenzene (0.5 mmol/kg body weight), and the urinary metabolites (4-chlorophenylmercapturic acid (4-CPMA), 2-, 3- and 4-chlorophenol (CPs), 4-chlorocatechol (4-CC), and 2-, 3- and 4-chlorophenylmethylsulfide (CPMSs)) were measured for 24 hours post-injection. Rats fed the 20% casein exceeded those fed the 10% casein in 4-CPMA, CPs, and in total urinary metabolites. Supplement with methionine or cystine to the 10% casein significantly increased 4-CPMA and decreased 4-CC. Supplement with methionine or cystine to the 20% casein also significantly increased 4-CPMA excretion, but had no effect on urinary 4-CC. The highest urinary excretion of CPMSs was observed in rats fed the 10% casein. Both increase of dietary protein and addition of the sulfur-containing amino acids decreased urinary CPMSs. These results indicate that total urinary metabolites are strongly associated with the microsomal drug-metabolizing activity, formation of the mercapturic acid is dependent on the hepatic GSH level, and the urinary CPMS level is independent on the mercapturic acid formation.
To investigate the mechanism and toxicological significance of testicular interstitial cell tumors (ICT) observed in a long-term rat study with procymidone, N-(3, 5-dichlorophenyl)-1, 2-dimethylcyclopropane-1, 2-dicarboximide, male Sprague-Dawley rats were fed procymidone in diets for up to 6 months with a positive control group receiving a single subcutaneous injection of cadmium chloride. Examinations mainly for gonadal functions such as serum testosterone and luteinizing hormone (LH), reproductive organ weight and histopathology presented evidence of the indirect involvement of gonadotropins in the production of ICT in rats. A significant increase in both serum testosterone and LH was observed in the early stage at high dietary concentrations of procymidone without any lesion in gonadal systems in histopathology, whereas administration of cadmium chloride produced the expected substantial increase in serum LH and a concomitant decrease in serum testosterone with a marked damaging effect on gonadal systems. Increases in serum testosterone and LH levels in animals receiving procymidone were reversible. The no-effect level for procymidone on serum testosterone and LH was 300 ppm over six months of treatment. The possible mechanism of ICT production in rats by non-genotoxic procymidone, structurally similar to flutamide, a synthetic non-steroidal antiandrogen, is likely to be derived from its induction of a hypergonadotropism due to the competitive binding to the androgen receptor, preventing the normal effect of testosterone to control the circulating level of LH.
Glycerol has an action to enhance pulmonary tumorigenesis in mice treated with 4-nitroquinoline 1-oxide (4NQO). In order to evaluate factors contributing to enhancing effect of glycerol on 4NQO-induced pulmonary tumorigenesis, we selected alveolar macrophage (AM) as a source of active oxygen formation in the lung and investigated the effect of glycerol on active oxygen formation in AMs of mice treated with 4NQO in this study. AMs were stimulated with opsonized zymosan, and active oxygen formation in AMs after stimulation was examined. Continuous glycerol treatment within 4 weeks after 4NQO injection has no influence on the capacity of active oxygen generation in AMs (expressed as maximum count of chemiluminescence) and total amount of active oxygen generation in AMs (expressed as total count of chemiluminescence). These results suggest that active oxygen formation in AMs don't contribute to enhance 4NQO-induced pulmonary tumorigenesis in mice treated with glycerol.
The metabolic activation of aflatoxin B1 by human placental microsomes was studied. Aflatoxin B1 showed relatively high mutagenic activity in Ames test when incubated with human placental microsomes. Addition of α-naphthoflavone or aminoglutethimide, known inhibitors of cytochrome P450 1A and P450 19, respectively, into the test system partially inhibited the mutagen-producing activity. It was suggested that the activation of aflatoxin B1 in human placental microsomes is mediated by at least these two forms of cytochrome P450.