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

The detrimental effects of environmental pollutants on the health of the individual are generally accepted, although the mechanisms of these effects remain to be incompletely understood. In the present study, we examined the effects of B[a]P, 2-BP, phenol and TCDD on proinflammatory cytokine gene expression in mice spleen cells which were stimulated with anti-CD3. 10-9M TCDD increased IFN gammar and TNF alpha gene expression, but suppressed IL-1 gene expression. 10-6M phenol inhibited IL-1, IL-6 and TNF alpha gene expression, and 10-6M of 2-BP downregulated TNF alpha gene expression. However, 10-6M of B[a]P did not influence on IL-1, IL-6, IFN gammar and TNF alpha gene expression. These findings suggest that TCDD may impair the immune functions of mice by enhancing proinflammatory cytokines production, whereas phenol and 2-BP may impair the functions by inhibiting the production of these cytokines.
Animals ; Antigens, CD3/immunology ; Apoptosis/drug effects ; Benzo(a)pyrene/*toxicity ; Cells, Cultured ; Cytokines/*biosynthesis/genetics ; Environmental Pollutants/*toxicity ; Gene Expression/drug effects ; Hydrocarbons, Brominated/*toxicity ; Male ; Mice ; Mice, Inbred C3H ; Phenol/*toxicity ; RNA/chemistry/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Spleen/*drug effects/metabolism ; Tetrachlorodibenzodioxin/*toxicity