A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD) to generate TCDD: AhR: DNA complex which could protect receptor-binding DNA against exonuclease II (Exo III) digestion. With Exo III treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.
Binding Sites ; Cytochrome P-450 CYP1A1/*genetics ; Cytosol/metabolism ; DNA-Binding Proteins/chemistry ; Exodeoxyribonucleases/chemistry ; Liver/*metabolism ; Polymerase Chain Reaction ; Rats, Sprague-Dawley ; Receptors, Aryl Hydrocarbon/*chemistry ; Tetrachlorodibenzodioxin/*analogs & derivatives ; Tetrachlorodibenzodioxin/chemistry

There are substantial variations of relative risks (RR) in smoking-related mortality by country and time. We hypothesized the RRs in smoking-related mortality might differ depending on serum concentrations of persistent organic pollutants (POPs). We evaluated the associations of cigarette smoking with total mortality in 610 elderly (aged > or = 70 yr) (702 elderly for organochlorine pesticides [OCPs]) after stratification by serum concentration of POPs, in the National Health and Nutrition Examination Survey (NHANES) 1999-2004 followed through 2006. Summary measures of POPs subclasses showed significant or marginally significant interaction with cigarette smoking on the risk of total mortality. P values for interaction were 0.069 for polychlorinated dibenzo-p-dioxins (PCDDs), 0.008 for polychlorinated biphenyls (PCBs), and 0.024 for OCPs. The effect of smoking on total mortality showed different patterns according to the serum concentration of some POPs. Former or current smokers had 1.4 to 2.9 times higher mortality rates compared with never smokers among participants with higher serum concentrations of POPs (2nd or 3rd tertiles). However, when the level of PCBs or OCPs were low (1st tertile), there were little positive associations between smoking and mortality. Our study suggests that the background exposure to several POPs may be related to variability in smoking-related total mortality.
Aged ; Aged, 80 and over ; Environmental Exposure ; Environmental Pollutants/*blood ; Female ; Humans ; Hydrocarbons, Chlorinated/blood ; Male ; Nutrition Surveys ; Pesticides/blood ; Polychlorinated Biphenyls/blood ; Proportional Hazards Models ; Risk ; Smoking/*mortality ; Tetrachlorodibenzodioxin/analogs & derivatives/blood

There are substantial variations of relative risks (RR) in smoking-related mortality by country and time. We hypothesized the RRs in smoking-related mortality might differ depending on serum concentrations of persistent organic pollutants (POPs). We evaluated the associations of cigarette smoking with total mortality in 610 elderly (aged > or = 70 yr) (702 elderly for organochlorine pesticides [OCPs]) after stratification by serum concentration of POPs, in the National Health and Nutrition Examination Survey (NHANES) 1999-2004 followed through 2006. Summary measures of POPs subclasses showed significant or marginally significant interaction with cigarette smoking on the risk of total mortality. P values for interaction were 0.069 for polychlorinated dibenzo-p-dioxins (PCDDs), 0.008 for polychlorinated biphenyls (PCBs), and 0.024 for OCPs. The effect of smoking on total mortality showed different patterns according to the serum concentration of some POPs. Former or current smokers had 1.4 to 2.9 times higher mortality rates compared with never smokers among participants with higher serum concentrations of POPs (2nd or 3rd tertiles). However, when the level of PCBs or OCPs were low (1st tertile), there were little positive associations between smoking and mortality. Our study suggests that the background exposure to several POPs may be related to variability in smoking-related total mortality.
Aged ; Aged, 80 and over ; Environmental Exposure ; Environmental Pollutants/*blood ; Female ; Humans ; Hydrocarbons, Chlorinated/blood ; Male ; Nutrition Surveys ; Pesticides/blood ; Polychlorinated Biphenyls/blood ; Proportional Hazards Models ; Risk ; Smoking/*mortality ; Tetrachlorodibenzodioxin/analogs & derivatives/blood