Comparison of hepatic in vitro metabolism of the pyrrolizidine alkaloid senecionine in sheep and cattle.
OBJECTIVE
To compare hepatic metabolism of pyrrolizidine alkaloids (PAs) between sheep and cattle and elucidate the protective mechanism of sheep.


METHODS
Liver microsomes and cytosol from 8 sheep and 8 cattle.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Duringer
Jennifer M
JM
Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA.
Buhler
Donald R
DR
Craig
A Morrie
AM
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D005978:Glutathione / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism*; D016686:Monocrotaline / Q000031:analogs & derivatives* / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism; D011763:Pyrrolizidine Alkaloids / Q000737:chemistry / Q000378:metabolism*; D012756:Sheep / Q000378:metabolism*; D013045:Species Specificity
D011763:Pyrrolizidine Alkaloids; D016686:Monocrotaline; C009237:senecionine; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D001189:Aryl Hydrocarbon Hydroxylases; D019362:Cytochrome P-450 CYP2B1; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D005978:Glutathione; C007241:dehydroretronecine