Evidence for cytochrome P450 3A expression and catalytic activity in rat blood lymphocytes.
Freshly isolated peripheral blood lymphocytes from control rats were found to catalyze the N-demethylation of erythromycin, known to be mediated by cytochrome P450 3A (CYP3A) isoenzymes in rat liver. Pretreatment of rats with dexamethasone (100 mg/kgx3 days, i.p.), a CYP3A inducer, resulted in 3-4-fold increase in the activity of erythromycin demethylase (EMD) in freshly isolated peripheral blood lymphocytes. This increase in the enzyme activity was found to be associated with an increase in the rate of the reaction and affinity of the substrate towards the enzyme. Significant inhibition of the EMD activity on in vitro addition of ketoconazole, a specific CYP3A inhibitor in liver and polyclonal antibody raised against rat liver CYP3A have suggested that EMD activity in blood lymphocytes is catalyzed primarily by CYP3A isoenzymes. Further, immunoblot analysis with polyclonal antibody raised against rat liver CYP3A revealed significant immunoreactivity, co-migrating with the liver isoenzyme, indicating constitutive expression of CYP3A in blood lymphocytes. Pretreatment with dexamethasone was found to significantly increase the expression of CYP3A protein in freshly isolated rat blood lymphocytes, as observed with liver. Likewise, significant CYP3A mRNA detected in control rat blood lymphocytes has further demonstrated constitutive expression of CYP3A isoenzymes in blood lymphocytes. Furthermore, several fold increase in CYP3A mRNA expression following pretreatment with dexamethasone showed similarities in the regulation of CYP3A isoenzymes in rat blood lymphocytes with the liver enzyme. The data suggest that the blood lymphocytes can be used to monitor tissue expression of CYP3A isoenzymes and validate the suitability of lymphocytes as surrogates of CYP status in less accessible target tissues.
Dey
Aparajita
A
Developmental Toxicology Division, Industrial Toxicology Research Centre, M.G. Marg, P.O. Box 80, Lucknow-226001, India.
Yadav
Sanjay
S
Dhawan
Alok
A
Seth
Prahlad K
PK
Parmar
Devendra
D
D000818:Animals; D002384:Catalysis / Q000187:drug effects; D051544:Cytochrome P-450 CYP3A / Q000097:blood / Q000378:metabolism*; D065692:Cytochrome P-450 CYP3A Inhibitors; D003907:Dexamethasone / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D007654:Ketoconazole / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D008214:Lymphocytes / Q000187:drug effects / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats
D065692:Cytochrome P-450 CYP3A Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D012333:RNA, Messenger; D003907:Dexamethasone; D051544:Cytochrome P-450 CYP3A; D007654:Ketoconazole