Effect of Alcohol on Drug Efflux Protein and Drug Metabolic Enzymes in U937 Macrophages

Abstract

Background ATP-binding cassette (ABC) proteins and cytochrome P450 (CYP) enzymes regulate the bioavailability of HIV-1 antiretroviral therapeutic (ART) drugs, non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs). They are also involved in regulating, and responding to, oxidative stress in various tissues and organs including liver. The present study is designed to assess the effect of alcohol on the ABCC1 and CYP enzymes involved in the metabolism of NNRTIs and PIs (CYP2B6, CYP2D6, CYP3A4) and oxidative stress (CYP1A1, CYP2A6, CYP2E1) in U937 macrophages. The U937 cell line has been utilized as an in vitro model of human macrophages. Methods The expression levels of the ABCC1 and CYP enzymes in U937 macrophages were characterized in terms of mRNA quantification, protein analysis, and assays for functional activity. In addition, oxidative stress was monitored by measuring the activities of oxidative stress marker enzymes and production of reactive oxygen species (ROS). Results The order of mRNA expression in U937 macrophages was ABCC1 ~ CYP2A6 > CYP3A4 ~ CYP2E1 ~ CYP1A1 > CYP2D6 > CYP2B6. Alcohol (100 mM) increased the mRNA levels of ABCC1 and CYP2A6 (200%), CYP2B6 and CYP3A4 (150%), and CYP2E1 (400%) compared with the control. Alcohol caused significant upregulation of ABCC1, CYP2A6, CYP2E1, and CYP3A4 proteins (50-85%) and showed >50% increase in the specific activity of CYP2A6 and CYP3A4 in U937 macrophages. Furthermore, alcohol increased the production of ROS and significantly enhanced the activity of oxidative stress marker enzymes, superoxide dismutase and catalase in U937 macrophages. Conclusions Our study showed that alcohol causes increases in genetic and functional expressions of ABCC1 and CYP enzymes in U937 macrophages. This study has clinical implications in alcoholic HIV-1 individuals, because alcohol consumption is reported to reduce the therapeutic efficacy of NNRTIs and PIs and increases oxidative stress.

Regulation of Cytochrome P450 2E1 expression by ethanol: Role of oxidative stressmediated PKC/JNK/SP1 pathway

“…Our observation that acute ethanol treatment induces CYP2E1 expression by approximately 1.5-fold in SVGA astrocytes is significant and consistent with our earlier observation in U937 cells, 15 as well as with observations from other studies. 31,32 However, in primary monocytes of chronic alcohol users, CYP2E1 mRNA expression showed B10-fold induction (unpublished observations) compared with healthy individuals, which is consistent with hepatic CYP2E1 induction in chronic alcohol users.…”

“…As previously shown in U937 monocytic cells, 15 we examined whether ethanol also induces ROS in SVGA astrocytes at 100 mM ethanol (near physiological concentration in binge drinkers) at 12-36 h. Single treatment of 100 mM ethanol induced ROS production by 420% at 24 and 36 h (Figure 1a). Further, to examine whether CYP2E1 is responsible for the generation of ROS, we knocked down CYP2E1 expression through transfection using 10 nM predesigned CYP2E1 siRNA and 10 nM scrambled siRNA as control.…”

“…However, its relative level in SVGA astrocytes compared with other CYPs is similar to that in the liver. 22 Further, as previously shown in U937 monocytic cells, 15 we investigated whether ethanol induces CYP2E1 in SVGA astrocytes. Initial results showed that 50 mM ethanol is optimum to induce CYP2E1 for up to 24 h (data not shown).…”

Transtorno Por Uso De Álcool E Hiv/Aids: Uma Revisão Da Literatura

“…A isoenzima CYP3A4, a qual pertence ao complexo citocromal P450, é responsável em grande parte pela metabolização dos ARVs 159,160 . Nesse aspecto, o consumo crônico de álcool tem sido relacionado com a capacidade de induzir os níveis de expressão de CYP3A4, o que, por sua vez, pode afetar o metabolismo e, consequentemente, reduzir a eficácia do tratamento e potencializar a toxicidade 45,161 . Além disso, a presença de TUA em indivíduos HIV+ tem demonstrado afetar significativamente as concentrações terapêuticas de ARVs plasmáticos, incluindo estavudina, lamivudina e nevirapina 45 .…”

“…A isoenzima CYP3A4 é responsável por parte do metabolismo do álcool, sendo ativada de forma importante em consumos crônicos. O metabolismo do álcool resulta na produção de espécies reativas do oxigênio, as quais causam toxicidade em monócitos / macrófagos e astrócitos 161,162 . Indivíduos HIV+ com TUA em tratamento com ARVs podem apresentar aumento de intoxicação, pois a isoenzima CYP3A4 atua no metabolismo de moléculas ARVs e também pode ser influenciada pelo álcool 163 .…”