Ionic Liquid 1-Octyl-3-Methylimidazolium (M8OI) Is Mono-Oxygenated by CYP3A4 and CYP3A5 in Adult Human Liver

Abstract

Environmental sampling around a landfill site in the UK previously identified the methylimidazolium ionic liquid, 1-octyl-3-methylimidazolium (M8OI), in the soil. More recently, M8OI was shown to be detectable in sera from 5/20 PBC patients and 1/10 controls and to be oxidised on the alkyl chain in the human liver. The objective of this study was to examine the metabolism of M8OI in humans in more detail. In human hepatocytes, M8OI was mono-oxygenated to 1-(8-Hydroxyoctyl)-3-methyl-imidazolium (HO8IM) then further oxidised to 1-(7-carboxyheptyl)-3-methyl-1H-imidazol-3-ium (COOH7IM). The addition of ketoconazole-in contrast to a range of other cytochrome P450 inhibitors-blocked M8OI metabolism, suggesting primarily CYP3A-dependent mono-oxygenation of M8OI. Hepatocytes from one donor produced negligible and low levels of HO8IM and COOH7IM, respectively, on incubation with M8OI, when compared to hepatocytes from other donors. This donor had undetectable levels of CYP3A4 protein and low CYP3A enzyme activity. Transcript expression levels for other adult CYP3A isoforms-CYP3A5 and CYP3A43-suggest that a lack of CYP3A4 accounted primarily for this donor's low rate of M8OI oxidation. Insect cell (supersome) expression of various human CYPs identified CYP3A4 as the most active CYP mediating M8OI mono-oxygenation, followed by CYP3A5. HO8IM and COOH7IM were not toxic to human hepatocytes, in contrast to M8OI, and using a pooled preparation of human hepatocytes from five donors, ketoconazole potentiated M8OI toxicity. These data demonstrate that CYP3A initiates the mono-oxygenation and detoxification of M8OI in adult human livers and that CYP3A4 likely plays a major role in this process.

Keywords: C8mim; PBC; cytochrome P450; ionic solvent; methylimidazolium ionic liquids.

Figure 1

The metabolism of M8OI in the human liver. (A) The proposed metabolic pathway of M8OI metabolism. CYP, cytochrome P450 based on this inhibition by the CYP inhibitors SKF525a and metyrapone, as reported in [10]; ADH, alcohol dehydrogenase; AcDH, acetaldehyde dehydrogenase, based on inhibition by the ADH inhibitor 4-methyl pyrazole and AcDH inhibitor disulfiram, as reported in [10]. Note, R = -(CH₂)₇-. (B) A typical LC-MS chromatogram from human hepatocytes (NHL5) during incubation with 10 µM M8OI demonstrating the production of HO8IM and COOH7IM (with similar retention times but identified and quantified based on their different fragmentation patterns); MRM transitions for M8OI, HO8IM and COOH7IM were m/z 195.2 -> 83.1, m/z 211.2 -> 83.1 and m/z 225.2 -> 83.1. (C) The time course for the production of HO8IM and COOH7IM in human hepatocyte (NHL5) incubation with 10 µM M8OI with or without the co-incubation of the indicated CYP inhibitor (see Table 1 for details). Data are the mean and SD of 3 separate cultures from the same hepatocyte donor. * p > 0.95.

Figure 1

The metabolism of M8OI in the human liver. (A) The proposed metabolic pathway of M8OI metabolism. CYP, cytochrome P450 based on this inhibition by the CYP inhibitors SKF525a and metyrapone, as reported in [10]; ADH, alcohol dehydrogenase; AcDH, acetaldehyde dehydrogenase, based on inhibition by the ADH inhibitor 4-methyl pyrazole and AcDH inhibitor disulfiram, as reported in [10]. Note, R = -(CH₂)₇-. (B) A typical LC-MS chromatogram from human hepatocytes (NHL5) during incubation with 10 µM M8OI demonstrating the production of HO8IM and COOH7IM (with similar retention times but identified and quantified based on their different fragmentation patterns); MRM transitions for M8OI, HO8IM and COOH7IM were m/z 195.2 -> 83.1, m/z 211.2 -> 83.1 and m/z 225.2 -> 83.1. (C) The time course for the production of HO8IM and COOH7IM in human hepatocyte (NHL5) incubation with 10 µM M8OI with or without the co-incubation of the indicated CYP inhibitor (see Table 1 for details). Data are the mean and SD of 3 separate cultures from the same hepatocyte donor. * p > 0.95.

Figure 2

Concentrations of M8OI and its metabolites in human hepatocyte cultures. (A) Hepatocytes were isolated from Liver A or Liver B and cultured as outlined in the Materials and Methods Section. Hepatocytes were incubated with 100 μM M8OI and the concentrations of M8OI, HO8IM and COOH7IM were determined in triplicate cultures directly (within 5 min) after addition and after 24 h. Concentrations were determined by reference to authentic standards. * p > 0.95. (B) H & E or immunohistochemical staining of liver sections from Liver A and Liver B for the indicated protein. For CYPs, a centrilobular region of the lobule is shown. POR, cytochrome P450 oxidoreductase; CPSI, carbamylphosphate synthase I (a liver-specific protein). Bar = 100 μm. (C) qRT-PCR for the indicated transcripts in hepatocytes isolated from Liver A and Liver B. RNA was isolated from 3 aliquots of cells and transcript levels were determined after normalisation to 18S rRNA levels. Normalised expression for each transcript was then expressed relative to the normalised level present in Liver A. * p > 0.95. (D) Western blot for the indicated protein from hepatocytes isolated from the indicated liver. Note: liver extracts, 20 µg protein/well; supersomes, 2 µg protein/well. (E) Luciferin PPXE activities. Data are the mean and SD of 3 determinations from each extract and are normalised to total protein levels. * p > 0.95.

Figure 3

M8OI metabolism with individual CYPs in membranes (supersomes) isolated from insect cells infected with recombinant CYP baculovirus. (A) A typical LC-MS chromatogram from the indicated supersome incubations with M8OI demonstrating the production of HO8IM; MRM transition m/z 211.2 -> 83.1. The identity of HO8IM was determined using an authentic standard (also seen with hepatocyte incubations) and is indicated by arrows. (B) The time course for the production of HO8IM with the indicated recombinant CYP. HO8IM concentrations were determined using authentic HO8IM as the standard. Note that in all cases, supersomes contained additionally both CYP reductase and cytochrome B5. The results are typical of at least 3 separate determinations of the same batch of supersome CYP.

Figure 3

M8OI metabolism with individual CYPs in membranes (supersomes) isolated from insect cells infected with recombinant CYP baculovirus. (A) A typical LC-MS chromatogram from the indicated supersome incubations with M8OI demonstrating the production of HO8IM; MRM transition m/z 211.2 -> 83.1. The identity of HO8IM was determined using an authentic standard (also seen with hepatocyte incubations) and is indicated by arrows. (B) The time course for the production of HO8IM with the indicated recombinant CYP. HO8IM concentrations were determined using authentic HO8IM as the standard. Note that in all cases, supersomes contained additionally both CYP reductase and cytochrome B5. The results are typical of at least 3 separate determinations of the same batch of supersome CYP.

Figure 4

The toxicity of M8OI in human hepatocytes is potentiated by the inhibition of its metabolism. (A) Left panel, human hepatocytes (NHL5) were treated with increasing concentrations of either M8OI, HO8IM or COOH7IM for 24 h, or right panel, 200 μM chlorpromazine (CPZ) for 1 h; control hepatocytes were treated with a 0.1% v/v DMSO vehicle. For MTT reduction, the data are the mean and SD of 4 separate determinations from the same donor. * p > 0.95 using Student’s t-test. (B) Photomicrographs of hepatocytes treated for 24 h as indicated, unless otherwise indicated. Bar = 100 μm. (C) Cryopreserved cultured hepatocytes treated for 24 h as indicated (except CPZ: 1 h). For MTT reduction, the data are the mean and SD of 4 separate determinations. Shown is the statistically significantly different % MTT activity versus the control (2-tailed, p < 0.05 level) using Student’s t-test. (D) Cryopreserved cultured hepatocytes treated for 24 h with or without 50 μM M8OI and increasing concentrations of ketoconazole. For MTT reduction, the data are the mean and SD of 4 separate determinations. * Statistically significantly different % MTT activity versus equivalent ketoconazole-free cultures (2-tailed, p < 0.05 level) using Student’s t-test. Note: chlorpromazine leads to hepatocyte death via necrosis [30].