Variation and comparison of cytochromes P450 in human liver and blood : with special emphasis on gene expression

Abstract

The liver is the major drug and xenobiotic metabolising organ, and is the target of many adverse reactions. The main hepatic metabolic enzymes belong to the mono oxygenase enzyme family of cytochromes P450 (CYP). Direct studies of these enzymes in small liver biopsy samples require ultrasensitive methodology.

In this thesis, two different methods for the study of CYP1A2, CYP1B1, CYP2E1, and CYP3A4 gene expression were developed. The methods were applied to small percutaneous needle biopsies of the liver.

The leukocyte fraction of blood was investigated as a potential "surrogate" organ, to determine whether blood mRNA levels mirrored hepatic mRNA expression. The mRNA levels in the two organs did not correlate for any of the four studied enzymes. Interestingly, the pattern of gene expression was reversed in the two tissues. CYP2E1 mRNA had the highest expression in liver tissue, followed by CYP3A4, CYP1A2 and CYP1B1. The opposite was shown in leukocytes where the CYP1B1 gene was expressed at the highest levels.

Gene expression in leukocytes was investigated in detail as repeated samples from the same individual displayed a spurious and inconsistent variation in mRNA levels. Systematic and regular measurements over a ten-week period revealed an intra-individual variation of between 40 and 347 %. The menstrual cycle did not have any significant effect on this variation. Exogenous factors, such as dietary constituents, may possibly influence this variation.

The consistently high levels of CYP1B1 mRNA in leukocytes may indicate important endogenous functions for this enzyme in the blood and/or bone marrow, probably in the metabolism and synthesis of hormones. This was suggested by the finding that women express higher levels than men of CYP1B1 mRNA in leukocyte fractions of blood.

In the liver there was no significant correlation between gene- and apoprotein expression as studied in twenty human livers for CYP1A2, CYP2E1 and CYP3A4. The liver samples used originated from two different sources. Ten of the livers were donor livers (time from excision to freezing less than 12 hours) while the other ten were snap-frozen surplus biopsy material obtained at surgery. Surprisingly, these two sources of liver tissue did not differ significantly.

In conclusion, sensitive methods for gene expression of CYP enzymes may not always be used for prediction of apoprotein levels in human tissues but may serve as a tool for mapping of CYP enzymes in tissues of limited accessibility, and for the study of mechanisms of regulation. Although blood is not a feasible substitute for liver biopsies in studies of hepatic CYP gene expression, the hematogeneous cytochrome P450 gene expression itself is of interest in e.g. studies of metabolic mechanisms for blood induced dyscrasias.