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Oxidation of 2,6-dimethylaniline by recombinant human cytochrome P450s and human liver microsomes
J Gan1, P L Skipper, S R Tannenbaum
1Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Abstract:
2,6-Dimethylaniline (2,6-DMA) is classified as a rodent nasal cavity carcinogen and a possible human carcinogen. The major metabolite of 2,6-DMA in rats and dogs is 4-amino-3,5-dimethylphenol (DMAP) but oxidization of the amino group to produce metabolites such as N-(2,6-dimethylphenyl)hydroxylamine (DMHA) is also indicated by the occurrence of hemoglobin adducts of 2,6-DMA in human and rats. Previous studies have shown a large interindividual variability in human 2,6-DMA hemoglobin adduct levels. In the present study, 2,6-DMA oxidation in vitro by human liver microsomes and recombinant human P450 enzymes was investigated to assess whether the hemoglobin adduct variability could be attributed to metabolic differences. At micromolar concentrations, the only product detectable (UV) was DMAP, while at 10 nM, DMHA was a substantial product. 2E1 and 2A6 were identified as the major P450s in human liver microsomes responsible for the production of DMAP by using P450-specific chemical inhibitors and mouse monoclonal antibodies that selectively inhibit human P450 2E1 and 2A6. 2A6 was identified as the major P450 responsible for the N-hydroxylation. Native P450 2E1 and human liver microsomes catalyzed the rearrangement of DMHA to DMAP independent of NADPH. Consistent with a mechanism involving oxygen rebound to the heme iron center, labeled oxygen was not incorporated into DMAP from either 18O2 gas or H2 18O in this rearrangement. Results presented here suggest much of the observed interindividual variability of 2,6-DMA hemoglobin adduct levels could be due to differences in the relative amounts of hepatic 2E1 and 2A6.
Insights
Interindividual variability in human 2,6-dimethylaniline (2,6-DMA) hemoglobin adducts may stem from differing levels of hepatic P450 2E1 and 2A6 enzymes, which are key in 2,6-DMA metabolism.
Area of Science:
- Toxicology
- Metabolism
- Carcinogenesis
Background:
- 2,6-Dimethylaniline (2,6-DMA) is a potential human carcinogen.
- Human exposure to 2,6-DMA can result in hemoglobin adducts, with significant interindividual variability observed.
- The metabolism of 2,6-DMA, particularly N-hydroxylation, is crucial for understanding its toxicological effects.
Purpose of the Study:
- To investigate the in vitro metabolism of 2,6-DMA by human liver microsomes and recombinant P450 enzymes.
- To identify the specific cytochrome P450 (CYP) enzymes involved in 2,6-DMA oxidation and N-hydroxylation.
- To explore the potential contribution of metabolic differences to the observed variability in 2,6-DMA hemoglobin adduct levels.
Main Methods:
- In vitro incubation of 2,6-DMA with human liver microsomes and recombinant human P450 enzymes.
- Use of P450-specific chemical inhibitors and monoclonal antibodies to identify key enzymes.
- Analysis of metabolites, including 4-amino-3,5-dimethylphenol (DMAP) and N-(2,6-dimethylphenyl)hydroxylamine (DMHA).
- Investigation of DMHA rearrangement to DMAP using labeled oxygen and NADPH dependency.
Main Results:
- At micromolar concentrations, DMAP was the primary detectable metabolite, while at nanomolar concentrations, DMHA was substantial.
- Cytochrome P450 2E1 (2E1) and 2A6 (2A6) were identified as the major enzymes responsible for DMAP production in human liver microsomes.
- CYP2A6 was identified as the primary enzyme for 2,6-DMA N-hydroxylation.
- DMHA rearrangement to DMAP occurred independently of NADPH, suggesting a mechanism not involving direct reduction.
Conclusions:
- Variability in hepatic CYP2E1 and CYP2A6 levels likely contributes significantly to the interindividual differences in 2,6-DMA hemoglobin adduct formation.
- Understanding the specific roles of CYP2E1 and CYP2A6 in 2,6-DMA metabolism is critical for assessing human health risks.
- The non-reductive rearrangement of DMHA to DMAP warrants further mechanistic investigation.
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