The VICC.ORG Directory of Doctors, Healthcare Providers & Researchers

Fritz F. Parl, M.D., Ph.D.

Prof Pathology Dir Clinical Chem
VICC Member
Researcher

Contact Information:

Vanderbilt University Medical Center
4605 The Vanderbilt Clinic
Nashville, TN 37232-2562
615-343-9117

Education

  • M.D. - University of Gottingen, 1968
  • Internship - Mercer Medical Center
  • Post Graduate Training - New York University Medical Center
  • Post Graduate Training - Rudolf Virchow Hospital
  • Residency - New York University Medical Center

 

Research Specialty

Breast cancer; molecular genetics; estrogens and estrogen receptor;

Research Description

Mammary estrogen metabolism leads to the formation of catechol estrogens, the 2-OH and 4-OH derivatives of 17b- estradiol (E2) and estrone (E1). Catechol estrogens have been implicated to play a causative role in breast cancer because they can directly or indirectly induce DNA damage. However, given the shared exposure of all women to estrogens and thereby to the potentially carcinogenic catechol estrogens, it is unclear how to explain differences in breast cancer risk between individuals. In normal breast tissue and breast cancer, catechol estrogens are produced by cytochromes P450 1A1 (CYP1A1) and 1B1 (CYP1B1) and inactivated by catechol-O-methyltransferase (COMT). Since the CYP1A1, CYP1B1, and COMT genes exist as wild type and polymorphic variants, we hypothesize that the inherited enzyme variants hold the potential to define differences in catechol estrogen/carcinogen exposure and thereby explain differences in breast cancer risk. In order to test this hypothesis, we will pursue the following specific aims. Aim 1. To determine whether the amino acid substitutions in variant CYP1B1 affect estrogen metabolism. To this end, we will perform a systematic analysis of each of the four common polymorphisms in exon 2 (codon 48:Arg & Gly; codon 119:Ala & Ser) and exon 3 (codon 432:Val & Leu; codon 453:Asn & Ser) individually and in combination. Each recombinant variant protein will be expressed in E. coli DH5aF''Iq and purified by Ni-chelate affinity chromatography using an N-terminal hexahistidine tag. We will carry out in vitro reconstitution assays combining recombinant purified CYP1B1 and NADPH-P450 reductase with E2 and E1 as substrates. Estrogen metabolites will be extracted and trimethylsilyl derivatives prepared for GC/MS analysis with quantitation by the stable isotope dilution method. These experiments will allow precise analysis of 4-OH and 2-OH catechol estrogen levels and 4-OH/2-OH ratios and define the catalytic effect (Km, Vmax) of each CYP1B1 variant on oxidative estrogen metabolism. Aim 2. To determine whether the amino acid substitutions in variant CYP1A1 and COMT affect estrogen metabolism. Variants of CYP1A1 (codons 171Leu & Ile, 461Thr & Asn, 462Ile & Val) and COMT (codon 158Val & Met) will be expressed, purified, and analyzed similar to CYP1B1 variants. Aim 3. To examine whether the in vitro interaction of CYP1A1, CYP1B1, and COMT variants affects estrogen metabolism. We will compare 4-OH and 2-OH catechol estrogen levels and 4-OH/2-OH ratios produced by wild type CYP1A1, CYP1B1, and COMT with those of variant CYP1A1, CYP1B1, and COMT genotype combinations identified in Aims 1 and 2 to deviate significantly from wild type activity. Aim 4. To determine whether the CYP1A1, CYP1B1, and COMT variants affect in vivo estrogen metabolism in human breast cancer cell lines, normal breast tissue and breast cancer biopsies. Guided by the results of the in vitro assays, we will quantitate 4-OH and 2-OH catechol estrogen levels and 4-OH/2-OH ratios in ten different human breast cancer cell lines, for which we already determined the CYP1B1 and COMT genotypes. Enzyme induction experiments with 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD), 12-O- tetradecanoylphorbol-13-acetate (TPA), and indolo[3,2-b] carbazole (ICZ) will answer the question whether the level of expression of variant forms of enzyme differs from that of wild type due to differences in inducibility. The final test of the hypothesis will consist of quantitation of 4-OH and 2-OH catechol estrogen levels and 4-OH/2-OH ratios in 25 - 30 breast tissues (benign and malignant) selected by CYP1A1, CYP1B1, and COMT genotyping. This project will define certain variant CYP1A1, CYP1B1, and COMT genotypes that, alone or in combination, are associated with higher life-long exposure to catechol estrogens and thereby potentially increased risk of breast cancer. .

Publications


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