Characterization of cytochrome P450 and glutathione S-transferase activity and expression in male and female ob/ob mice.
OBJECTIVE
To characterize the effect(s) of gender, age (glycemic status) and obese state, on hepatic biotransformation activities, expression of cytochrome P450 (CYP450) mRNAs and glutathione transferase activity in the ob/ob mouse.


METHODS
Male and female, ob/ob or ob/+ mice were killed at 3-4 months or 7-8 months of age. Hepatic microsomes, cytosol and RNA were prepared from each animal.


METHODS
Male and female ob/ob and ob/+ mice, 3-4 or 7-8 months of age.


METHODS
CYP450 form-specific activities of CYP1A1/1A2, CYP3A and CYP2B were estimated by determining the 0-dealkylation of alkoxyresorufin substrates (ethoxy-EROD, benzoxy-BROD and pentoxy-resorufin, PROD, respectively). CYP2E1-dependent, 4-nitrophenol hydroxylase (PNP-OH) and CYP3A-dependent erythromycin N-demethylase (ERY-DM) were also measured in hepatic microsomes. CYP1A2, CYP2E1 and CYP3A protein in microsomal fractions was determined by ELISA. Glutathione transferase activity (GST) was determined in hepatic cytosol and CYP1A2 and CYP2E1 mRNA was estimated by Northern blot analysis.


RESULTS
Female mice, regardless of glycemic status, showed an obesity enhanced level of CYP2E1-dependent PNP-OH activity and CYP2E1 protein as shown by ELISA. These increases were observed to be independent of the diabetic state, since 7-8 month-old mice had blood glucose levels identical to lean mice. The mRNA level of CYP2E1 in female mice also exhibited age-and obesity-influenced decreases in expression. No significant differences in CYP2E1 activity or expression were observed in male mice. CYP3A-dependent ERY-DM activity was significantly higher in young males, regardless of phenotype. CYP3A and CYP2B activities did not differ among any animals; however, CYP1A activity, while depressed in obese animals of both genders, was significantly different in old animals. Glutathione S-transferase activity was lower in obese male mice, whereas no difference was observed between lean and obese females


CONCLUSIONS
This study supports earlier observations in man and rats that the obese state produces alterations in the expression of important oxidation and conjugation pathways. In addition, this report more thoroughly examines the role of gender and glycemic status on biotransformation activities in the ob/ob mouse as demonstrated by increased CYP2E1 protein and CYP2E1-dependent activity in obese females, decreased CYP1A2 protein and CYP1A2-dependent activity in obese animals, and obesity had no effect of glutathione transferase in female mice, in contrast with the previously reported obesity-dependent decrease of this activity in male mice.
Roe
A L
AL
Graduate Center for Toxicology, Division of Pharmacology and Experimental Therapeutics, University of Kentucky, Lexington 40536-0082, USA.
Howard
G
G
Blouin
R
R
Snawder
J E
JE
D000375:Aging / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001711:Biotransformation; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D003920:Diabetes Mellitus / Q000378:metabolism; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008297:Male; D051379:Mice; D008820:Mice, Obese; D009765:Obesity / Q000201:enzymology*; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism
D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D005982:Glutathione Transferase