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No Effect of CYP1B1 Val432Leu Polymorphism on CYP1B1 Messenger RNA Levels in an Organochlorine-Exposed Population in Slovakia

Majorie B.M. van Duursen¹, Rocío Fernández Cantón¹, Tony Koan², J. Thomas Sanderson¹, Krijn Kieviet¹ and Martin van den Berg¹

¹ Institute for Risk Assessment Sciences, Utrecht University, Utrecht, the Netherlands; and ² National Reference Center for Dioxins and Related Chemicals, Institute of Preventive and Clinical Medicine, Bratislava, Slovakia

Requests for reprints: Majorie B.M. van Duursen, Biochemical Toxicology, Institute for Risk Assessment Sciences, Yalelaan 2, P.O. Box 80176, 3508 TD, Utrecht, The Netherlands. Phone: 31-30-253-5400; Fax: 31-30-253-5077. E-mail: m.vanduursen{at}iras.uu.nl

	Introduction

Top Introduction Study Population and Methods Results and Discussion References

 
Cytochrome P450 1B1 (CYP1B1) is a phase I enzyme involved in the metabolic activation of many polycyclic aromatic hydrocarbons. It is also involved in the hydroxylation of estradiol to 4-hydroxyestradiol, a potentially genotoxic metabolite that is suggested to play a role in carcinogenesis. CYP1B1 is expressed in many tissues including mononuclear peripheral blood cells and is regulated through the aryl hydrocarbon receptor–mediated pathway, which can be induced by several environmental chemicals, including polycyclic aromatic hydrocarbons and persistent organochlorine pollutants such as polychlorinated biphenyls (PCB). A single nucleotide polymorphism of the CYP1B1 gene (1,249GC) leads to an amino acid substitution in codon 432 (Val to Leu). This substitution results in a lower catalytic activity of the enzyme (1), but the effect of this CYP1B1 Val432Leu polymorphism on CYP1B1 gene expression and its inducibility is not clear. Gene expression and genotype of CYP1B1 can easily be determined in blood lymphocytes, making it a potential candidate to be used as biomarker for exposure to aryl hydrocarbon receptor agonists. We studied the correlation between CYP1B1 gene expression level and exposure to environmental factors within the CYP1B1 Val432Leu genotype groups. This study was part of a European project (PCBRISK) that investigates a human population exposed to environmental pollution as a consequence of the 25-year-long production of PCBs in eastern Slovakia.

	Study Population and Methods

Top Introduction Study Population and Methods Results and Discussion References

 
Blood samples were taken from two populations in two different areas in Slovakia, the Michalovce District (polluted area) and the Stropkov District (less polluted, reference area). RNA was isolated from the lymphocytes and the samples (n = 334) were stored at –80°C until analysis. Methods for blood collection, isolation of human lymphocytes, and RNA isolation are described elsewhere (2). Blood concentrations of PCBs were measured as sum of all PCBs or as nanograms of toxic equivalent (TEQ) per kilogram of lipid, which was defined as the sum of PCBs normalized with the WHO toxic equivalent factors. CYP1B1 genotype was determined by a PCR-RFLP method using 750 ng RNA, adapted after Tang et al. (3). DNA (4.5 µg ; Fermentas, Inc., Hanover, MD) was used as positive control for endonuclease restriction.

CYP1B1 mRNA expression was normalized by a log₁₀ transformation. Deviations from the Hardy-Weinberg equilibrium were calculated using the two-sided ² test. One-way ANOVA was conducted and Pearson correlation coefficients were calculated to study associations between variables. Statistical calculations were done using GraphPad InStat 3.06 (GraphPad Software, Inc., San Diego, CA) and SAS (SAS Institute, Inc. Cary, NC).

	Results and Discussion

Top Introduction Study Population and Methods Results and Discussion References

 
Of the 334 RNA samples stored, we were able to determine the CYP1B1 Val432Leu genotype of 114 samples (34%). Of the other samples, RNA concentrations were too low for genotyping. Of one sample no information on gender was available. In the total population, the allele frequency for Leu was 0.44 (Table 1). This allele frequency is similar to other observed frequencies in various healthy Caucasian populations and did not statistically significantly deviate from the Hardy-Weinberg equilibrium (P = 0.1). Genotype frequencies were not different within the male or female subgroups and they did not deviate from the Hardy-Weinberg equilibrium (male P = 0.1 and female P = 0.5).

Our data indicate that environmental exposure to PCBs in our study population had no statistically significant effect on mRNA expression of CYP1B1, regardless of the CYP1B1 Val432Leu genotype. This is inconsistent with the study described by Hanaoka et al. (4). They found a higher expression level of CYP1B1 in peripheral blood lymphocytes on exposure to polycyclic aromatic hydrocarbons in Chinese coke oven workers who had at least one CYP1B1 432Leu allele. However, the number of subjects in that study was small (37 cases and 13 control workers) and it concerned an Asian population. Two other studies reported a correlation between CYP1B1 expression levels in peripheral blood lymphocytes and exposure to aryl hydrocarbon receptor agonists. One found a correlation with dioxin exposure higher than 6.5 pg TEQs/g lipid in a Japanese population (5); the other found no effect of polycyclic aromatic hydrocarbon exposure in healthy volunteers of unknown ethnicity (6). Spencer et al. (7) have suggested using CYP1B1 expression as a biomarker for in vivo exposure to dioxin-like compounds. However, despite the high blood levels of PCBs and TEQs in our study population and the separation of the population into genetically more homogenous groups with respect to CYP1B1 Val432Leu genotype, no correlation was observed between PCB levels and CYP1B1 mRNA levels in human lymphocytes. In addition, the large interindividual variability in CYP1B1 expression and PCB blood levels in the population makes it unlikely that possible, subtle effects of PCB exposure on RNA expression can be detected. This makes the suitability to use CYP1B1 expression as biomarker for exposure questionable.

	Acknowledgments

 
We thank Tomas Trnovec and Jan Petrick who supplied the blood samples and Rob Beelen for his help with the statistical analyses.

	Footnotes

 
Grant support: European Union project (PCBRISK) no. QLK4-CT-2000-00488.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/21/04; accepted 10/12/04.

	References

Top Introduction Study Population and Methods Results and Discussion References

 

1. Aklillu E, Oscarson M, Hidestrand M, Leidvik B, Otter C, Ingelman-Sundberg M. Functional analysis of six different polymorphic CYP1B1 enzyme variants found in an Ethiopian population. Mol Pharmacol 2002;61:586–94.[Abstract/Free Full Text]
2. Cantón RF, Besselink HT, Sanderson JT, Botschuyver S, Brouwer B, van den Berg M. Expression of CYP1A1 and 1B1 mRNA in blood lymphocytes from two district populations in Slovakia compared to total TEQs in blood as measured by the DRE-CALUX® assay. Organohalogen Compounds 2003;64:215–8.
3. Tang YM, Green BL, Chen GF, et al. Human CYP1B1 Leu432Val gene polymorphism: ethnic distribution in African-Americans, Caucasians and Chinese; oestradiol hydroxylase activity; and distribution in prostate cancer cases and controls. Pharmacogenetics 2000;10:761–6.[CrossRef][Medline]
4. Hanaoka T, Yamano Y, Pan G, et al. Cytochrome P450 1B1 mRNA levels in peripheral blood cells and exposure to polycyclic aromatic hydrocarbons in Chines coke oven workers. Sci Total Environ 2002;296:27–33.[CrossRef][Medline]
5. Toide K, Yamazaki H, Nagashima R, et al. Aryl Hydrocarbon Hydroxylase Represents CYP1B1, and not CYP1A1, in Human Freshly Isolated White Cells: Trimodal Distribution of Japanese Population According to Induction of CYP1B1 mRNA by Environmental Dioxins. Cancer Epidemiol Biomarkers Prev 2003;12:219–22.[Abstract/Free Full Text]
6. Tuominen R, Warholm M, Moller L, Rannug A. Constitutive CYP1B1 mRNA expression in human blood mononuclear cells in relation to gender, genotype, and environmental factors. Environ Res 2003;93:138–48.[Medline]
7. Spencer DL, Masten SA, Lanier KM, et al. Quantitative analysis of constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1B1 expression in human lymphocytes. Cancer Epidemiol Biomarkers Prev 1999;8:139–46.[Abstract/Free Full Text]

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