Short Communication 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

The expression level of mRNAs for cytochrome P450 (CYP) 1A1 and 1B1 in freshly prepared white cells from 72 subjects exposed to dioxins at waste incinerators was investigated. The amounts of CYP1B1 mRNA ranged from 0.16 to 671 molecules/10 molecules of 18S rRNA, whereas the amounts of CYP1A1 mRNA were <6 molecules/10 ng total RNA, indicating that CYP1A1 was not induced to a detectable level by environmentally exposed dioxins. The inducibility of CYP1B1 mRNA in leukocytes, defined as the ratio of CYP1B1 mRNA to the plasma concentration of dioxins, varied among the subjects. It was found that the subjects showed trimodal distribution according to inducibility: 39 (54.2%), 25 (34.7%), and 8 (11.1%) of 72 subjects were judged as poor, intermediate, and high responders to environmental dioxins, respectively. The amounts of CYP1B1 mRNA in leukocytes of the intermediate and high responders were highly correlated with the plasma concentrations of dioxins (P < 0.05 and <0.01). These results suggest that CYP1B1 with polymorphic inducibility by dioxins is involved in aromatic hydrocarbon hydroxylase activities in human lymphocytes. Introduction Carcinogenic PAHs are metabolically activated by enzymes to generate reactive intermediates capable of binding to DNA to cause the mutation of cancer-related genes (1, 2). Thus, the capacity of enzymes responsible for the activation of the carcinogenic PAHs has been regarded as one of the factors affecting the risk to chemical carcinogenesis. Kellermann et al. (3) first reported that the capacity of individuals to induce AHH in lymphocytes in response to PAH was apparently associated with the individual differences in the risk of bronchogenic carcinoma, suggesting that the amount of AHH was one of the key determinants of the cancer risk. After this result, Guirgis et al. (4) and Trell et al. (5) reported that AHH activity in individuals correlated well with the risk of lung cancer and laryngeal carcinoma. The original assay of Kellermann et al. (3) was to measure PAH-inducible AHH activity in mitogen-activated lymphoblasts cultured for 96 h. CYP1A1 had been thought to be a sole enzyme acting as AHH until the role of CYP1B1 in the metabolic activation of PAHs was discovered (6, 7). The role of CYP1B1 in the metabolic activation of chemical carcinogens was found during the course of studies on the responsiveness to 2,3,7,8-tetrachlorodibenzo-p-dioxin (8). Thus, it seemed interesting to determine whether the AHH activity in the lymphoblast cultures prepared by the method of Kellermann et al. (3) was attributable to CYP1A1 or CYP1B1. We initiated this study to determine whether either one or both mRNAs for CYP1A1 and CYP1B1 were induced in human leukocytes in response to environmental dioxins in Japanese incinerator workers. We found that CYP1B1 mRNA, but not CYP1A1 mRNA, was detectable in essentially all subjects and that there were individual variations in the responsiveness to environmental dioxins to induce CYP1B1 mRNA. Materials and Methods Human Blood Samples. Blood samples used in this study were taken from Japanese subjects who were occupationally exposed to dioxins at waste incinerators. The concentrations of dioxins (pg TEQ/g lipid), which were defined as the sum of polychlorinated dibenzo-p-dioxins normalized with a WHO toxic equivalent factor, were determined by gas chromatography high-resolution mass spectrometry as described previously (9). This study was approved by the ethics committee of Hokkaido University. First-strand cDNA Synthesis. Total RNAs in human leukocytes were isolated by a TRIzol LS reagent system (Invitrogen, Carlsbad, CA). Nothing was done to separate the different white cell types. First-strand cDNA was then synthesized. Reverse transcriptase reaction was performed under the following conditions: a reaction mixture contained 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each deoxynucleotide triphosphate, 10 ng/ l total RNA, 4.5 ng/ l random primer pd(N)6 (Promega, Madison, WI), 2 units/ l a Received 8/8/02; revised 1/3/03; accepted 1/9/03. 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. 1 Supported in part by Grant-in-Aid 99-2 from The Organization for Pharmaceutical Safety and Research; Ministry of Education, Culture, Sports, Science and Technology of Japan; Ministry of Health, Labour and Welfare of Japan and Core Research for Evolutional Science and Technology. 2 To whom requests for reprints should be addressed, at Laboratory of Drug Metabolism, Graduate School of Pharmaceutical Sciences, Hokkaido University N12W6, Kita-ku, Sapporo 060-0812, Japan. Fax: 81-11-706-4978; E-mail: [email protected]. 3 The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; AHH, aromatic hydrocarbon hydroxylase; CYP, cytochrome P450; TEQ, toxic equivalents to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 219 Vol. 12, 219–222, March 2003 Cancer Epidemiology, Biomarkers & Prevention Research. on September 20, 2017. © 2003 American Association for Cancer cebp.aacrjournals.org Downloaded from Maloney murine leukemia virus reverse transcriptase (Promega), and 0.4 units/ l RNase inhibitor (Toyobo, Osaka, Japan). The mixtures were treated at 25°C for 10 min and then 37°C for 60 min. PCR Condition. Primers used for the amplification of cDNAs for CYP1A1 and CYP1B1 were CYP1A1-RTF, 5 -ccttcatcctggagaccttcc-3 ; CYP1A1-RTR, 5 -atggttgatctgccactggttt-3 ; CYP1B1-RTF, 5 -agaacgtaccggccactatc-3 ; and CYP1B1RTR, 5 -cacgacctgatccaattctg-3 . A quantitative PCR to determine the amount of CYP1B1 mRNA was performed in a real-time PCR machine, LightCycler (Roche Diagnostics, Mannheim, Germany), using a LightCycler-FastStart DNA Master SYBR Green I (Roche Diagnostics) according to the manufacturer’s instructions. PCR amplifications were performed in 40 cycles with melting at 95°C for 15 s, annealing at 60°C for 5 s, and extension at 72°C for 7 s. The amounts of 18S rRNA used as an internal standard were determined by a TaqMan Ribosomal RNA Control Reagents VIC Probe (Applied Biosystems, Foster City, CA). PCR conditions for amplification of -actin mRNA were as described previously (10).