In order to investigate the role of Fibroblast Growth Factors in hematopoietic cells, we studied the expression of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5 and FGF-6 mRNAs both in murine myelomonocytic leukemia WEHI-3B and in a murine stromal cell line SR-4987. Secretion of FGF-2 in the cell culture supernatant was also studied. Expression of mRNA encoding for the above-mentioned FGFs was analyzed by RT-PCR. The production of FGF-2 in the conditioned media of WEHI-3B and SR-4987 cell cultures was evaluated by techniques of affinity chromatography, chromatofocusing and immunoblotting. The biological activity of FGF-2 was checked on SR-4987 cells by a agar clonogenic assay. In both cell lines mRNA was found encoding for FGF-1, FGF-2 and FGF-6 and WEHI-3B cells express also mRNA for FGF-3 (int-2) and FGF-4 (K-FGF/hst). Furthermore, supernatant from WEHI-3B cells was found to stimulate dramatically the agar clonogenicity of SR-4987 cells which have a very poor basal capacity for growth in agar. The clonogenic activity of WEHI-3B conditioned medium is due to FGF-2 secreted into cell culture supernatant whereas SR-4987 cells, although express FGF-2 mRNA, do not seem able to secrete this factor. The expression in myeloid leukemia cells of oncogene-related factors such as FGF-3, FGF-4 and FGF-6 together with the secretion of FGF-2 able to support a positive regulation of bone marrow stromal cells function suggest that FGFs may have an important role in sustaining the leukemogenic process and related disorders.

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 …

The toxicity in humans of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a man-made compound and environmental pollutant, is still debated. The industrial accident at Seveso, Italy, in 1976 exposed a large population of both sexes and of all ages to a massive concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Monitoring of soil and measurement of blood samples allowed classification of the exposed population into three categories: A, B and R (high, medium and low exposure, respectively). This article presents data from longitudinal health monitoring of the population, including liver function, immune function, neurological impairment, dermatological effects, reproductive pathology, and mortality.

Cytochrome P450 (CYP) 1B1 activates polycyclic aromatic hydrocarbons and aryl aromatic hydrocarbons to carcinogens. We describe a competitive reverse transcription-PCR (RT-PCR) assay for the quantification of CYP1B1 mRNA in blood mononuclear cells (BMCs) by simultaneous RT and PCR amplification of cellular RNA with decreasing amounts of an internal standard. The concentration of CYP1B1 mRNA is derived from the ratio between the intensities of the bands corresponding to the amplified products. To reduce the variability of mRNA extraction efficiency, the measured amount of CYP1B1 has been calculated in relation to the beta-actin gene products. We measured CYP1B1 expression in the BMCs of 75 human subjects; no significant differences in the CYP1B1:beta-actin ratio were detected between women (range, 0.47-4.35; median, 2.0) and men (range, 0.72-3.85; median, 2.09). The analytical imprecision (CV) of duplicates was 14% (n = 25 pairs), and the intraindividual CV for two samples, 1 month apart, was 22% (n = 20). No significant differences were detected in smokers (n = 25; range, 0.77-3.55; median, 2.14) compared with nonsmokers (n = 50; range, 0.47-4.35; median, 2.0). The method has a wide range of linearity, good sensitivity and precision, and is suitable for studies of individual susceptibility as indicated by CYP1B1 expression in BMCs.