Quantification of Aromatic Hydrocarbon Receptor (AHR) and Related Genes by Calibrated Reverse Transcription-PCR in Blood Mononuclear Cells

The aromatic hydrocarbon ( Ah ) gene battery (1) comprises Ah receptor ( AHR ), its nuclear translocator ( ARNT ), and the genes that encode the detoxifying enzymes of phase I (e.g., cytochrome P4501A1, -1A2, and -1B1) and phase II. These enzymes and growth regulatory factors such as interleukin 1-β (IL-1β) are coordinately induced by polychlorinated dibenzo- p -dioxins and polycyclic aromatic hydrocarbons, toxic chemicals that are ubiquitous in ambient air and in cigarette smoke, via AHR (1)(2)(3)(4). Quantification of the regulation of AHR and related genes could be useful to monitor the degree of polycyclic aromatic hydrocarbon exposure and to understand the pathways to chemical carcinogenesis and atherosclerosis (5). The expression of AHR and related genes has been studied using Northern blot analysis (6) or quantitative reverse transcription-PCR (RT-PCR) using labeled primers (7), which are low-sensitivity techniques; competitive RT-PCR (8), which is more sensitive but quite laborious; and real-time PCR (9), which is expensive. An interesting technique, proposed for the first time by Tsai and Wiltbank (10) and considered later by Bor et al. (11), is called “calibrated ” RT-PCR; it is a competitive RT-PCR based on the addition of a fixed amount of internal standard (IS) to the sample and to a set of calibrators. The labor, time, and costs for this method are lower than those for classic competitive RT-PCR. We present calibrated RT-PCR assays to study the expression of AHR , ARNT , cytochrome P4501B1 ( CYP1B1 ), and IL-1β for the AHR gene battery and of β-actin ( β-ACT ) as housekeeping gene in peripheral blood mononuclear cells from 32 healthy subjects. Total RNA was extracted according to the method of Chomczynski and Sacchi (12) from 1 × 107 peripheral blood mononuclear cells in suspension isolated by step-gradient centrifugation on Histopaque. RNA was resuspended in 50 μL …