Cytochrome P450 1B1 is critical in the development of TNF-α, IL-6, and LPS-induced cellular hypertrophy.

Heart failure (HF) is preceded by cellular hypertrophy (CeH) which alters expression of cytochrome P450 enzymes (CYPs) and arachidonic acid (AA) metabolism. Inflammation is involved in CeH pathophysiology, but mechanisms remain elusive. This study investigates the impacts of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and lipopolysaccharides (LPS) on the development of CeH and the role of CYP1B1. AC16 cells were treated with TNF-α, IL-6, and LPS in the presence and absence of CYP1B1-siRNA or resveratrol. mRNA and protein expression levels of CYP1B1 and hypertrophic markers were determined using PCR and Western blot analysis, respectively. CYP1B1 enzyme activity was determined, and AA metabolites were analyzed using liquid chromatography-tandem mass spectrometry. Our results show that TNF-α, IL-6, and LPS induce expression of hypertrophic markers, induce CYP1B1 expression, and enantioselectively modulate CYP1B1-mediated AA metabolism in favor of mid-chain HETEs. CYP1B1-siRNA or resveratrol ameliorated these effects. In conclusion, our results demonstrate the crucial role of CYP1B1 in TNF-α, IL-6, and LPS-induced CeH.

Alteration of Hepatic Cytochrome P450 Expression and Arachidonic Acid Metabolism by Arsenic Trioxide (ATO) in C57BL/6 Mice.

The success of arsenic trioxide (ATO) in acute promyelocytic leukemia has driven a plethora studies to investigate its efficacy in other malignancies. However, the inherent toxicity of ATO limits the expansion of its clinical applications. Such toxicity may be linked to ATO-induced metabolic derangements of endogenous substrates. Therefore, the primary objective of this study was to investigate the effect of ATO on the hepatic formation of arachidonic acid (AA) metabolites, hydroxyeicosatetraenoic acids (HETEs), as well as their most notable producing machinery, cytochrome P450 (CYP) enzymes. For this purpose, C57BL/6 mice were intraperitoneally injected with 8 mg/kg ATO for 6 and 24 h. Total RNA was extracted from harvested liver tissues for qPCR analysis of target genes. Hepatic microsomal proteins underwent incubation with AA, followed by identification/quantification of the produced HETEs. ATO downregulated Cyp2e1, while induced Cyp2j9 and most of Cyp4a and Cyp4f, and this has resulted in a significant increase in 17(S)-HETE and 18(R)-HETE, while significantly decreased 18(S)-HETE. Additionally, ATO induced Cyp4a10, Cyp4a14, Cyp4f13, Cyp4f16, and Cyp4f18, resulting in a significant elevation in 20-HETE formation. In conclusion, ATO altered hepatic AA metabolites formation through modulating the underlying network of CYP enzymes. Modifying the homeostatic production of bioactive AA metabolites, such as HETEs, may entail toxic events that can, at least partly, explain ATO-induced hepatotoxicity. Such modification can also compromise the overall body tolerability to ATO treatment in cancer patients.

16R-HETE and 16S-HETE alter human cytochrome P450 1B1 enzyme activity probably through an allosteric mechanism.

Cytochrome P450 1B1 (CYP1B1) has been widely associated with the development of cardiac pathologies due to its ability to produce cardiotoxic metabolites like midchain hydroxyeicosatetraenoic acids (HETEs) from arachidonic acid (AA) through an allylic oxidation reaction. 16-HETE is a subterminal HETE that is also produced by CYP-mediated AA metabolism. 19-HETE is another subterminal HETE that was found to inhibit CYP1B1 activity, lower midchain HETEs, and have cardioprotective effects. However, the effect of 16-HETE enantiomers on CYP1B1 has not yet been investigated. We hypothesized that 16(R/S)-HETE could alter the activity of CYP1B1 and other CYP enzymes. Therefore, this study was carried out to investigate the modulatory effect of 16-HETE enantiomers on CYP1B1 enzyme activity, and to examine the mechanisms by which they exert these modulatory effects. To investigate whether these effects are specific to CYP1B1, we also investigated 16-HETE modulatory effects on CYP1A2. Our results showed that 16-HETE enantiomers significantly increased CYP1B1 activity in RL-14 cells, recombinant human CYP1B1, and human liver microsomes, as seen by the significant increase in 7-ethoxyresorufin deethylation rate. On the contrary, 16-HETE enantiomers significantly inhibited CYP1A2 catalytic activity mediated by the recombinant human CYP1A2 and human liver microsomes. 16R-HETE showed stronger effects than 16S-HETE. The sigmoidal binding mode of the enzyme kinetics data demonstrated that CYP1B1 activation and CYP1A2 inhibition occurred through allosteric regulation. In conclusion, our study provides the first evidence that 16R-HETE and 16S-HETE increase CYP1B1 catalytic activity through an allosteric mechanism.

Sex- and enantiospecific differences in the formation rate of hydroxyeicosatetraenoic acids in rat organs.

Hydroxyeicosatetraenoic acids (HETEs) are hydroxylated arachidonic acid (AA) metabolites that are classified into midchain, subterminal, and terminal HETEs. Hydroxylation results in the formation of R and S enantiomers for each HETE, except for 20-HETE. HETEs have multiple physiological and pathological effects. Several studies have demonstrated sex-specific differences in AA metabolism in different organs. In this study, microsomes from the heart, liver, kidney, lung, intestine, and brain of adult male and female Sprague-Dawley rats were isolated and incubated with AA. Thereafter, the enantiomers of all HETEs were analyzed by liquid chromatography-tandem mass spectrometry. We found significant sex- and enantiospecific differences in the formation levels of different HETEs in all organs. The majority of HETEs, especially midchain HETEs and 20-HETE, showed significantly higher formation rates in male organs. In the liver, the R enantiomer of several HETEs showed a higher formation rate than the corresponding S enantiomer (e.g., 8-, 9-, and 16-HETE). On the other hand, the brain and small intestine demonstrated a higher abundance of the S enantiomer. 19(S)-HETE was more abundant than 19(R)-HETE in all organs except the kidney. Elucidating sex-specific differences in HETE levels provides interesting insights into their physiological and pathophysiological roles and their possible implications for different diseases.

Identifying simultaneous matrix metalloproteinases/soluble epoxide hydrolase inhibitors.

Matrix metalloproteinase (MMP) and soluble epoxide hydrolase (sEH) have completely unrelated biological functions; however, their dysregulation produce similar effects on biological systems. Based on the similarity in the reported structural requirements for their inhibition, the current study aimed to identify a simultaneous inhibitor for MMP and sEH. Six compounds were identified as potential simultaneous MMP/sEH inhibitors and tested for their capacity to inhibit MMP and sEH. Inhibition of MMP and sEH activity using their endogenous and exogenous substrates was measured by liquid chromatography/mass spectrometry, spectrophotometry, and zymography. Two compounds, CTK8G1143 and ONO-4817, were identified to inhibit both MMP and sEH activity. CTK8G1143 and ONO-4817 inhibited the recombinant human sEH activity by an average of 67.4% and 55.2%, respectively. The IC50 values for CTK8G1143 and ONO-4817 to inhibit recombinant human sEH were 5.2 and 3.5 µM, respectively, whereas their maximal inhibition values were 71.4% and 42.8%, respectively. Also, MMP and sEH activity of human cardiomyocytes were simultaneously inhibited by CTK8G1143 and ONO-4817. Regarding other compounds, they showed either MMP or sEH inhibitory activity but not both. In conclusion, these two simultaneous inhibitors of MMP and sEH could provide a promising intervention for the prevention and control of several diseases, especially cardiovascular diseases.