Effects of 2-[1-(ethoxyimino)propyl]-3-hydroxy-5-(2,4,6-trimethylphenyl) cyclohex-2-enone on hepatic haem biosynthesis: species differences in hepatic porphyria.

2-[1-(Ethoxyimino)propyl]-3-hydroxy-5-(2,4,6-trimethylphenyl) cyclohex-2-enone (ETC) is a novel alkyl ketone herbicide. Continuous administration of ETC to mice for 28 days resulted in marked liver enlargement and severe intrahepatic cholestasis. These effects have been shown to result directly from a rapid and marked accumulation of porphyrin in the liver. The porphyrin which accumulates in the liver has been identified as protoporphyrin IX and dose response and time course studies confirm prior inhibition of mitochondrial ferrochelatase as the causal lesion. ETC was a very potent porphyrinogenic compound in mice, with a no-effect level for a single oral dose of 1 mg/kg. Rats and hamsters were insensitive to this type of hepatotoxicity following single oral doses of up to 750 mg/kg or following repeated, and indeed prolonged administration. The sensitivity of different species to ETC-induced porphyria correlated with the effect of ETC on hepatic ferrochelatase activity. The inhibition of ferrochelatase activity and the hepatic porphyria in mice were both found to be readily reversible upon withdrawal of ETC.

Comparison of the effects of griseofulvin and dihydropyridines on ferrochelatase activity and porphyrin accumulation in primary cultures of mouse and rat hepatocytes.

The ability of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), 3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethyl pyridine (EDDC) and griseofulvin to induce porphyria in primary cultures of mouse and rat hepatocytes was determined. Exposure of mouse hepatocytes to DDC, EDDC or griseofulvin (5-100 mum) for 4 days resulted in a marked inhibition of ferrochelatase activity (up to 95%). However, whereas exposure of rat hepatocytes to DDC or EDDC (5-100 mum) for 4 days also resulted in marked inhibition of ferrochelatase activity (up to 96%), exposure to griseofulvin (5-100 mum) had no effect. DDC, EDDC and griseofulvin induced porphyrin accumulation in both mouse and rat hepatocyte cultures. In mouse hepatocyte cultures exposed to each xenobiotic the porphyrin that accumulated was predominantly protoporphyrin. In rat hepatocyte cultures exposed to DDC or EDDC the porphyrin that accumulated was also predominantly protoporphyrin, whereas following exposure to griseofulvin it was coproporphyrin. Time course studies confirmed that in rat hepatocyte cultures exposed to griseofulvin (25 or 100 mum) over a 4-day exposure period, ferrochelatase activity was not inhibited and coproporphyrin was always the predominant porphyrin accumulating (45-72% of total). Addition of 5-aminolaevulinic acid to mouse or rat hepatocyte cultures (10-1000 mum) also resulted in marked accumulation of porphyrin but whereas uroporphyrin accumulated in mouse hepatocyte cultures, coproporphyrin accumulated in rat hepatocyte cultures. These studies demonstrated that the hepatic porphyrias produced by the dihydropyridines and griseofulvin can be modelled in vitro in primary cultures of hepatocytes. Furthermore, the species differences in sensitivity of mouse and rat hepatocyte cultures in vitro to inhibition of ferrochelatase activity by griseofulvin mirrors, and therefore probably explains, the species differences in porphyria observed in vivo.

Reduced estradiol production by a substituted triazole results in delayed ovulation in rats.

Ovulation in the rat is delayed by a single administration of the substituted triazole R151885 (1,1-di(4-fluorophenyl)-2-(1,2,4-triazol-1-yl)-ethanol). This delay results from a 24-hr shift in the preovulatory luteinizing hormone (LH) surge since administration of chorionic gonadotrophin on proestrus restores ovulation. Plasma levels of estradiol are markedly reduced (42-45%) 6-12 hr after administration of R151885. The restoration of ovulation in R151885-pretreated rats, by administration of exogenous estradiol benzoate, indicates that the reduced estradiol levels play a pivotal role in the delay of ovulation. Granulosa cells isolated from rat ovaries produce estradiol and progesterone in vitro in the presence of both follicle-stimulating hormone and testosterone. The addition of R151885 to such cultures results in a dose-dependent inhibition of estradiol production (69% by 1 microM) without a significant effect on progesterone production. This inhibition occurs at concentrations of R151885 similar to those measured in vivo. R151885 is a competitive inhibitor of human placental aromatase (apparent Ki with androstenedione substrate of 410 nM) and produces a type II spectral perturbation of cytochrome P-450 from placental microsomes. Pituitaries isolated from R151885-treated rats have reduced LH output in response to gonadotrophin-releasing hormone stimulation compared with those of controls. It is proposed that R151885 competitively inhibits aromatase activity in developing ovarian follicles. The resultant temporary reduction of plasma estradiol levels at a critical time in the estrous cycle, and consequent inadequate pituitary sensitization, produces a 24-hr delay in the preovulatory LH surge and hence ovulation is delayed by 24 hr.

Ovulation in rats is delayed by a substituted triazole.

When a single oral dose of 5 or 25 mg/kg of the substituted triazole R151885 [1,1-di(4-fluorophenyl)-2-(1,2,4-triazol-1-yl)-ethanol] was administered at midday on diestrus-2 to rats with regular 4-day estrous cycles, the subsequent ovulation was delayed by 24 or 48 hr, respectively. There was no evidence of toxicity at the doses used. The only morphological changes detected in the reproductive tract were a delay in accumulation of uterine fluid and prolonged but normal follicular maturation prior to the delayed ovulation. The delayed follicles were slightly larger than normal follicles at the time of ovulation. The preovulatory peak plasma concentrations of progesterone, follicle stimulating hormone (FSH), and luteinizing hormone (LH) were delayed by 24 hr in rats treated with 5 mg/kg of R151885 on diestrus-2. Although there was a normal preovulatory peak plasma concentration of estradiol, values were reduced by between 30 to 50% late on diestrus-2 and early on proestrus. Additionally, in ovariectomized rats, 3 daily doses of 25 mg/kg of R151885 antagonized the action of estradiol on the uterus by 45%. We suggest that the reductions in plasma estradiol concentrations during diestrus-2 and proestrus, combined with some antagonism of estradiol's action, may prevent adequate priming of the pituitary thereby suppressing the preovulatory LH surge required for ovulation. The suppression of this LH surge is of a temporary nature indicating a reversible effect of R151885 on the hormonal control system.