Abstract
To study cytochrome P-450 (CYP) 2B6 contribution to methoxychlor metabolism within human liver microsomes and to initiate an investigation of CYP2B6 protein expression, we developed a polyclonal antibody targeted to a 20-residue peptide within that protein. The antibody was found to be highly sensitive and monospecific for CYP2B6 on immunoblots. Although many immunological studies have described the absence or low expression of CYP2B6 in human livers, in the present investigation, we have found this not to be the case. We immunoquantified CYP2B6 apoprotein expression in a panel of 28 livers and found concentrations ranging from 2 to 82 pmol/mg protein, with a mean value of 25 pmol/mg protein. Five livers (∼18%) displayed relatively high levels of CYP2B6 (>40 pmol/mg protein). There were no sex-related differences, although the highest level was observed in a 1-week postpartum donor given several medications. A marked diminution in variability was found in individuals aged 56 or older (n = 12), but there were no age-related trends in mean CYP2B6 content. We suggest that CYP2B6 represents a significant portion of total CYP in human liver. The exquisite sensitivity of this antibody (fmol quantities are detected easily on immunoblots) may explain our detection of CYP2B6 in 100% of livers versus its detection in a limited number of livers by certain other investigators. The antibody also was found to immunoinhibit CYP2B6-catalyzed N-demethylation of (S)-mephenytoin in human liver microsomes by 68 to 79%. The utility of this antibody for determining human liver microsomal CYP2B6 contribution to theortho-hydroxylation of methoxychlor was demonstrated.
Footnotes
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Send reprint requests to: Dr. David Kupfer, Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical Center, 55 Lake Ave. North, Worcester, MA 01655. E-mail:David.Kupfer{at}ummed.edu
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↵1 Current address: Gentest Corporation, 6 Henshaw Street, Woburn, MA 01801.
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The project described was supported by Grants ES00834 and ES05737 from the National Institute of Environmental Health Sciences, National Institutes of Health. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of Environmental Health Sciences, National Institutes of Health.
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↵3 The conversion of buproprion to its major active metabolite, hydroxybuproprion, is catalyzed primarily by CYP2B6 in liver microsomes (E. P. Goodale and J. A. Ascher, personal communication).
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↵4 In another sample of human liver microsomes, R1-IgG was found to inhibit (S)-mephenytoin N-demethylation by 79% (data not shown).
- Abbreviations used are::
- CYP
- cytochrome P-450
- TBST
- Tris (10 mM, pH 7.5)-buffered 0.9% saline containing 0.05% Tween 20
- R1-IgG and R2-IgG
- IgG fraction obtained from rabbits 1 and 2, respectively
- Received October 28, 1998.
- Accepted January 4, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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