senecionine and dehydroretronecine

To compare hepatic metabolism of pyrrolizidine alkaloids (PAs) between sheep and cattle and elucidate the protective mechanism of sheep.. Liver microsomes and cytosol from 8 sheep and 8 cattle.. The PA senecionine, senecionine N-oxide (nontoxic metabolite) and 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP; toxic metabolite) were measured in microsomal incubations. The kcat (turnover number) was determined for DHP and N-oxide formation. Chemical and immunochemical inhibitors were used to assess the role of cytochrome P450s, flavin-containing monooxygenases (FMOs), and carboxylesterases in senecionine metabolism. The CYP3A, CYP2B, and FMO concentrations and activities were determined, in addition to the role of glutathione (GSH) in senecionine metabolism.. DHP concentration did not differ between species. Sheep formed more N-oxide, had higher N-oxide kcat, and metabolized senecionine faster than cattle. The P450 concentrations and isoforms had a large influence on DHP formation, whereas FMOs had a large influence on N-oxide formation. In cattle, CYP3A played a larger role in DHP formation than in sheep. FMO activity was greater in sheep than in cattle. Addition of GSH to in vitro microsomal incubations decreased DHP formation; addition of cytosol decreased N-oxide formation.. Hepatic metabolism differences alone do not account for the variation in susceptibility seen between these species. Rather, increased ruminal metabolism in sheep appears to be an important protective mechanism, with hepatic enzymes providing a secondary means to degrade any PAs that are absorbed from the rumen.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Blotting, Western; Cattle; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2B1; Cytochrome P-450 CYP3A; Glutathione; Microsomes, Liver; Monocrotaline; Oxidoreductases, N-Demethylating; Oxygenases; Pyrrolizidine Alkaloids; Sheep; Species Specificity

Spironolactone (SPL), a known inducer of cytochrome P4503A, was injected into rats and guinea pigs in order to investigate species differences in pyrrolizidine alkaloid (PA) metabolism. Liver microsomes from treated male rats showed an increased (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) formation of 47% from the PA senecionine, coincident with the induction of P4503A1, whereas senecionine N-oxidation was decreased by 49%, probably due to a reduction in hepatic P4502C11 concentrations. By contrast, liver microsomes from SPL-treated female rats exhibited almost a 500% increase in both DHP and senecionine N-oxide production, coincident with the marked induction of P4503A1. In guinea pigs of both sexes, oral treatment with SPL caused an approximately 50% increased formation of both DHP and senecionine N-oxide by liver microsomes. Only a slight increase in hepatic concentrations of P4503A1 occurred in the treated guinea pigs. SPL treatment increased testosterone 16 beta-hydroxylase activity by 100% in both sexes of guinea pigs. Use of the P4503A1 specific inhibitor triacetyloleandomycin showed that the P4503A sub-family played an important role in senecionine bioactivation in untreated or SPL-treated rats but not in both untreated and SPL-treated guinea pigs. This study demonstrated that P4503A was not the major enzyme for senecionine metabolism in guinea pigs.

Topics: Animals; Antineoplastic Agents, Phytogenic; Aryl Hydrocarbon Hydroxylases; Biotransformation; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Female; Guinea Pigs; Isoenzymes; Male; Microsomes, Liver; Monocrotaline; Oxidation-Reduction; Oxidoreductases, N-Demethylating; Pyrrolizidine Alkaloids; Rats; Rats, Sprague-Dawley; Species Specificity; Spironolactone

Studies were carried out to investigate the metabolism of senecionine by human liver microsomes and the role of human cytochrome P450IIIA4 in this process. Human liver microsomes metabolized senecionine to two major products, (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) and senecionine N-oxide. The rates of product formation (DHP and senecionine N-oxide) varied widely with the microsomal samples tested. There was a 30-fold difference in DHP formation and a 25-fold difference in N-oxidation between the poorest metabolizer and the highest metabolizer of senecionine. The conversion of senecionine to DHP and senecionine N-oxide in human liver microsomes was markedly inhibited by the mechanism-based inactivators of P450IIIA4, gestodene and triacetyloleandomycin. Anti-P450IIIA4 IgG, at a concentration of 1 mg/nmol of P450, was found to inhibit completely the formation of DHP and senecionine N-oxide in human liver microsomes (HL101) having low activity toward senecionine. At 5 mg IgG/nmol P450, anti-P450IIIA4 inhibited 90 and 84% respectively of the formation of DHP and senecionine N-oxide in liver microsomes (HL110) with the highest activity toward senecionine. The formation of DHP or senecionine N-oxide was highly correlated with the amount of P450IIIA4 measured in the microsomes using polyclonal anti-P450IIIA4 IgG. The rate of DHP production also had a strong correlation with the rate of senecionine N-oxide formation (r = 0.999) and with the rate of nifedipine oxidation (r = 0.998). Our present studies provide evidence that P450IIIA4 is the major enzyme catalyzing the bioactivation (DHP formation) and detoxication (senecionine N-oxide formation) of senecionine in human liver.

Topics: Antineoplastic Agents, Phytogenic; Carcinogens; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Humans; Immunoglobulin G; Kinetics; Liver; Microsomes, Liver; Mixed Function Oxygenases; Monocrotaline; Norpregnenes; Oxidation-Reduction; Pyrrolizidine Alkaloids; Troleandomycin

Topics: Animals; Biotransformation; Glutathione; In Vitro Techniques; Inactivation, Metabolic; Male; Microsomes, Liver; Monocrotaline; Pyrrolizidine Alkaloids; Rats; Rats, Inbred Strains

In vitro metabolism of senecionine with rat hepatic microsomes was studied. The main pyrrolic metabolite, 6,7-dihydro-7-hydroxy-1-hydroxy-methyl-5H-pyrrolizine was isolated by preparative high performance liquid chromatography (PRP-1 column) and examined by mass spectrometry. Results of the incubation performed in the presence of H218O indicated that both hydroxyl groups of the metabolite came from the solvent. Thus, formation of the pyrrolic metabolite is accomplished by alkyl-oxygen fission of both bonds C7-O and C9-O of the dehydroalkaloid, an intermediate supposedly formed during metabolic transformation of pyrrolizidine alkaloids (PAs).

Topics: Animals; Chromatography, High Pressure Liquid; Circular Dichroism; In Vitro Techniques; Male; Mass Spectrometry; Microsomes, Liver; Monocrotaline; Pyrrolizidine Alkaloids; Rats