P53 DECREASED CYP3A4 ACTIVITY AND PROTEIN IN NONALCOHOLIC FATTY LIVER (NAFL) AND NONALCOHOLIC STEATOHEPATITIS (NASH)

Rohitash Jamwal , University of Rhode Island, Kingston, RI
Benjamin J Barlock , University of Rhode Island, Kingston, RI
Sravani Adusumalli , University of Rhode Island, Kingston, RI
Ken Ogasawara , University of Rhode Island, Kingston, RI
Suzanne M De La Monte , Warren Alpert Medical School of Brown University, Providence, RI
Fatemeh Akhlaghi , Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI
Statement of hypothesis: Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease in Western populations. The objective of this work was to determine the impact of NAFLD on the CYP3A4 mRNA and protein levels, and midazolam hydroxylation in human liver.

Methodology: The effect of NAFLD on CYP3A4 activity was studied in liver microsomes (HLM, N=74) prepared from a repository of well-characterized human liver tissue. All the samples included in this study were CYP3A5 poor metabolizers (CYP3A5*3/*3). Human liver samples were graded by a certified pathologist and divided into three groups, normal (n=24), nonalcoholic liver (NAFL; n=26) and nonalcoholic steatohepatitis (NASH; n=24). The rate of formation of midazolam 1-hydroxylation was used to assess in vitro CYP3A4 activity in human liver microsomes. Protein levels of CYP3A4, CPR and Cyb5 was determined using a LC-MS/MS based label-free quantification method. Liver samples were also analyzed for mRNA expression of CYP3A4, CAR, PXR, and HNF4α.

Results: CYP3A4 activity in HLM with NAFL and NASH was 2 and 3.1-fold (p<0.05) lower than normal donors, respectively. Michaelis-constant (Km) of midazolam 1-hydroxylation among study groups ranged from 1.58 to 2.32 µM. Compared to normal, intrinsic clearance (CLint, whole liver) was 2.7 (p<0.05) and 4.1 (p<0.05) fold lower in donors with NAFL and NASH, respectively. Furthermore, CYP3A4 protein abundance in HLM from NAFL and NASH donors was significantly lower than that of normal (p<0.05). Multiple linear regression analysis showed that CYP3A4 protein expression significantly accounted for variability in CYP3A activity. Samples with NAFLD also had lower CYP3A4 mRNA, but the difference was statistically insignificant. Hepatic steatosis was significantly associated with a decrease in midazolam CLint and CYP3A4 protein. Age, sex, and gender did not exhibit any significant association with the observed alterations.

Conclusion: Overall, the evidence from different in vitro studies in HLM suggest that NAFLD is associated with decreased hepatic enzyme activity of CYP3A4 substrates in human, which worsens as the disease progresses from NAFL to NASH. Moreover, the expression of CYP3A4 protein can partially explain the reduction in CYP3A4 activity by disease state.