16R-HETE and 16S-HETE Alter Human Cytochrome P450 1B1 through Transcriptional and Allosteric Mechanism

• Author / Creator

  Hidayat, Rahmat

• Cardiac hypertrophy is a complex anomaly of the heart associated with increased muscle mass and thickening of the cell walls in response to accumulative stress. Although this condition occurs naturally, the prolonged state will progress into heart failure or even cause death. Recent studies have revealed the eccentricity of cytochrome P450 1B1 (CYP1B1) and its associated cardiotoxic mid-chain HETEs metabolites in developing cardiac hypertrophy and heart failure. Facilitated by CYP, AA can be metabolized into several bioactive compounds such as epoxyeicotrienoic acids (EETs) and hydoyeicosatetraenoic acids (HETEs). The latter is further categorized into mid-chain, subterminal, and terminal HETEs. Subterminal HETEs have been involved in various physiological and pathophysiological processes; however, their role in cardiac hypertrophy has not been fully defined. For that reason, the objectives of the current study are; 1) to determine the possible effect of subterminal HETEs, R and S enantiomers of 16 HETE, on CYP1B1 gene expression in vitro in RL-14 cells, 2) to investigate the modulatory effect of 16-HETE enantiomers in vitro on CYP1B1 enzyme activity medicated by human recombinant enzyme CYP1B1 and human liver microsomes, and 3) to examine the modulatory effect of 16R-HETE and 16S-HETE on different CYP450 enzyme. Our results showed that both enantiomers of 16-HETE significantly upregulated CYP1B1 at mRNA and protein levels in RL-14 cell lines. This modulation occurred through a transcriptional mechanism, as evident by transcriptional induction and luciferase assay. Furthermore, neither post-transcriptional nor post-transcriptional modification was involved in the phenomenon since there was no change in CYP1B1 mRNA and protein stability upon treatment of 16-HETE enantiomers. Surprisingly, 16-HETE enantiomers significantly increased CYP1B1 activity in RL-14 cells, recombinant human CYP1B1, and human liver microsomes. On the contrary, 16-HETE enantiomers significantly inhibited CYP1A2 catalytic activity mediated by the recombinant human CYP1A2 and human liver microsomes. The sigmoidal binding mode of these enzyme activities represents that CYP1B1 activation occurred through allosteric regulation. In conclusion, our study provides the first evidence that 16R-HETE and 16S-HETE increase CYP1B1 gene expression, protein through the transcriptional mechanism, and CYP1B1 at catalytic activity mediated by human recombinant CYP1B1 and liver microsome through an allosteric mechanism.

• Subjects / Keywords

  • CYP1B1
  • Cardiac Hypertrophy
  • Heart Failure
  • 16-HETE

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-cavq-yp14

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.