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- Finnström, Niklas
(author)
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Variation and comparison of cytochromes P450 in human liver and blood : with special emphasis on gene expression
- 2001
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Doctoral thesis (other academic/artistic)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.
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| 2. |
- Lind, Anna-Britta, et al.
(author)
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Gene expression of cytochrome P450 1B1 and 2D6 in leukocytes in human pregnancy
- 2003
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In: Pharmacology and Toxicology. - : Wiley. - 0901-9928 .- 1600-0773. ; 92:6, s. 295-9
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Journal article (peer-reviewed)abstract
- We investigated the influence of human pregnancy on gene expression of two cytochrome P450 enzymes in white blood cells. Cytochrome P450 1B1 (CYP1B1) catalyses oestradiol 4-hydroxylation, and may participate in the endocrine regulation of oestrogens. Cytochrome P450 2D6 (CYP2D6) metabolises many commonly used drugs, and previous studies have suggested that it is induced during pregnancy. CYP1B1 and CYP2D6 were therefore considered to be of interest in human pregnancy. As it is not ethically possible to take liver biopsies from healthy mothers during pregnancy, easily accessible cells that express the genes were used as a surrogate tissue. White blood cells were collected from eighteen pregnant women, and were used to measure CYP1B1 and CYP2D6 ribonucleic acid (RNA). The analysis was repeated after pregnancy, the women, thus, serving as their own controls. Real-time reverse transcriptase - polymerase chain reaction methods were used with 18S ribosomal RNA as an internal control. A slight, but not significant, increase in gene activity of CYP1B1 was detected during pregnancy. Expression of CYP2D6 in blood was extremely low, and induction of CYP2D6 during pregnancy could not be confirmed. In conclusion, gene expression of CYP1B1 and CYP2D6 in leukocytes was not significantly up-regulated in the third trimester of pregnancy, but a trend indicating an altered metabolism during pregnancy was detected.
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| 3. |
- Thörn, Mari, et al.
(author)
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Cytochromes P450 and MDR1 mRNA expression along the human gastrointestinal tract
- 2005
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In: British Journal of Clinical Pharmacology. - : Wiley. - 0306-5251 .- 1365-2125. ; 60:1, s. 54-60
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Journal article (peer-reviewed)abstract
- AIM:The aim of this study was to quantify the mRNA expression of three cytochromes P450 (CYP) and P-glycoprotein (P-gp) in the human gastrointestinal (GI) tract.METHOD:Biopsies were obtained from gastric, duodenal, colonic and rectal mucosa during routine gastro-colonoscopy in 27 patients. The biopsies were snap-frozen in liquid nitrogen. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used for the quantitative analyses of mRNA expressed by the CYP2E1, CYP3A4 and CYP3A5 genes, and the MDR1 gene coding for P-gp protein. The mRNA expression of b-actin was used as an internal standard for comparisons between samples.RESULTS:All CYP genes were expressed at all locations throughout the GI tract, although all showed substantial interindividual variation. CYP2E1 had the highest expression at all locations (P < 0.05 to P < 0.0001), except in the right colon. CYP3A4 and CYP3A5 had their highest mRNA expression in the duodenum (P < 0.001 and P < 0.000 001, respectively) and CYP2E1 in the stomach (P < 0.01). MDR1 mRNA concentrations increased along the GI tract with the highest expression being in the left colon (P < 0.000001).CONCLUSION:Multiple sampling within the same individual enabled us to study the intraindividual variation in expression of CYP and MDR1 genes along the GI tract. We find that CYP2E1 mRNA expression is higher than that of the other CYPs. CYP3A expression is highest in the duodenum and that of MDR1 increases from stomach and duodenum to colon.
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| 4. |
- Thörn, Mari, et al.
(author)
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Expression of cytochrome P450 and MDR1 in patients with proctitis
- 2007
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In: Upsala Journal of Medical Sciences. - : Uppsala Medical Society. - 0300-9734 .- 2000-1967. ; 112:3, s. 303-312
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Journal article (peer-reviewed)abstract
- Background. The aim of this study was to investigate the effect of inflammation on the gene expression of three cytochrome P450's (CYP) and P-glycoprotein (P-gp) in the rectal and colonic mucosa in patients with proctitis. Methods. Biopsies were obtained from inflamed and normal mucosa in association with routine sigmoidoscopy in patients with proctitis. The biopsies were snap-frozen in liquid nitrogen. Real time PCR (polymerase chain reaction) was used for quantitative analyses of mRNA specific for the CYP2E1, CYP3A4 and CYP3A5 gene and the MDR1 genes. Values were normalised based on gene expression of beta-actin to enable comparisons between samples. Results. The gene expression of CYP2E1 and CYP3A4 was lower in mucosa with severe inflammation vs normal mucosa (p<0.05). For CYP3A5 and P-gp there was no significant difference when comparing normal and inflammatory changed mucosa. Conclusion. Our study suggests that at least for some of the CYP enzymes the expression decreases in response to the inflammatory process in the gastrointestinal tract.
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