Effect Of Diphenytriazol On Rat CYP1A1/2 And UGT1As Enzymes

Aim:To investigate the modulation of cytochrome P4501A1/2(CYP1A1/2)and Family 1 UDP-Glucuronosyltransferases(UGT1As)expression by giving diphenytriazoi to adult rats.Methods:To investigate the mechanism involved in the induction effort of diphenytriazol on Rat Hepatic CYP1A1/1A2 Enzymes:Sprague-Dawley rats were pretreated intraperitoneally with the same daily dose of diphenytriazol for 1,2 and 4 days.The rat pretreated byβ-naphthoflavone is positive control group;the rat pretreated by tea oil is negative control group.Liver CYP1A1/2 mRNA was measured using reverse transcription-polymerase chain reaction(RT-PCR).CYP1A2 protein was detected by using immunoblotting.CYP1A1 activity was determined by using spectrofluorometric method with ethoxyresorufin as probe substrate.CYP1A2 activity was assayed by using HPLC with phenacetin as probe substrate.To study the effects of diphenytriazol on the mRNA expression and enzymatic activity of UGT1As in liver and intestine:The mRNA levels of the expression of the UGT1A1,UGT1A3,UGT1A6 and UGT1A9 gene of the rat liver and intestine were assayed with RT-PCR.UGT1A6 and UGT1A9 activity was determined by using HPLC with 4-nitrophenol and propofol as probe substrates.Results:Both of the expression level of CYP1A1 and CYP1A2 mRNA was significantly improved.A_{CYP1A1 mRNA}/A_β-actinwas 0.270±0.040 for control group,0.343±0.055,0.417±0.045 and 0.603±0.083 for 1,2 and 4days diphenytriazol treated groups.A_{CYP1A2 mRNA}/A_β-actinwas 0.613±0.189 for control group,1.510±0.226,3.0574±0.518 and 4.1204±0.458 for 1,2 and 4days diphenytriazol treated groups.The correlation coefficient between expression level of CYP1A1/2 mRNA and treatment day of DL111 was 0.9984 and 0.9563 respectively.The activity of ethoxyresorufin-O-deethylase(EROD)and the metabolic degree of phenacetin in the mierosomal incubates pretreated by diphenytriazol were increased obviously contrast to blank control.There was a trend of induction day-dependent increase for activity of CYP1A1 and CYP1A2.Both of the expression level of UGT1A1 and UGT1A3 mRNA in liver and intestine was no significant different comparing with control group(p＞0.05).Both of the expression level of UGT1A6 and UGT1A9 mRNA was significantly improved.A ₍UGT1A6 mRNA/A_β-actinin liver was 1.31±0.28 for control group,2.894±1.05 and 4.86±1.21 for 2 and 4 days diphenytriazol treated groups.A_{UGT1A6 mRNA}/A_β-actinin intestine was 0.724±0.21 for control group,1.384±0.28 and 2.48±0.11 for 2 and 4 days diphenytriazol treated groups.A_{UGT1A9 mRNA}/A_β-actinin liver was 0.64±0.24 for control group,1.49±0.29 and 1.934±0.29 for 2 and 4 days diphenytriazol treated groups.A_{UGT1A9 mRNA}/A_β-actinin intestine was 0.40±0.11 for control group,0.94±0.23 and 2.23±0.49 for 2 and 4 days diphenytriazol treated groups.There was a trend of induction day-dependent increase for mRNA levels of UGT1A6 and UGT1A9.The metabolic degree of 4-nitrophenol and propofol in the liver and intestine microsomal incubates pretreated by diphenytriazol were increased obviously contrast to blank control.But there was no correlation between the metabolic degree and the induction dosage of diphenytriazol.Conclusion:Diphenytriazol has inductive effects on mRNA expression level,protein level and activity of rat hepatic CYP1A1/1A2.Diphenytriazol has inductive effects on mRNA expression level and activity of rat hepatic and intestinal UGT1A6/1A9.