To elucidate the enzymatic excision-repair process operative on cyclobutane-type pyrimidine photodimers in human dermal fibroblasts, we have examined excised dimer-containing material recovered in the trichloroacetic acid soluble fraction from far-ultraviolet-irradiated (254 nm, 40 J m-2) and incubated (24 h) cell cultures. The excised DNA photoproducts were found in oligonucleotide fragments with an estimated mean chain length of approximately 3.7 bases. Exposure of these isolated excision fragments, labeled with [3H]thymidine (dT), to a secondary, dimer-photoreversing fluence of far-UV (5.5 kJ m-2) resulted in the release of free dT and thymidine monophosphate (TMP). Photorelease of these two radioactive species was measured by high-performance liquid chromatography, with TMP being detected as the increase in dT following bacterial alkaline phosphatase treatment. These data imply that the photoliberated dT and TMP moieties were attached to the excision fragments solely by the cyclobutane ring of the dimer. No evidence was obtained for the photoliberation of free thymine, thus corroborating a conclusion reached by others that the excision of dimers in human cells is not initiated by scission of an intradimer N-glycosyl bond. The sum of the tritium label recovered in dT plus TMP corresponded to approximately 40% of that disappearing from thymine-containing dimers on photoreversal, suggesting that in about 80% of the isolated excision fragments the dimer is located at one end of the oligonucleotide and contains a break in its internal phosphodiester bond.(ABSTRACT TRUNCATED AT 250 WORDS)
Applications of an improved gel-solvent system for cleanup of pesticide residues by gel permeation chromatography (GPC) were investigated. Elution characteristics using Bio-Beads SX-3 gel and a toluene-ethyl acetate (1+3) elution solvent were determined for 16 nonicnic chlorinated pesticides, 3 polychlorinated biphenyls (PCBs), 14 chlorophenoxy herbicide esters, and 7 organophosphate insecticides. Elution patterns for vegetable and animal lipids were also studied. Quantitative recoveries of the pesticides were achieved. No liquid-liquid partitioning cleanup steps were required with any type of nonionic chlorinated pesticide or sample matrix. Only GPC cleanup was required for the nonionic chlorinated pesticides, PCBs, and organophosphate pesticide residues in chicken and turkey fat samples. Electron capture and flame photometric detectors were used in the gas chromatographic method for the respective pesticides. Samples containing up to 0.5 g lipid each were processed at the rate of one every 30–40 min with the automated system. Results were in excellent agreement with those from accepted manual partitioning methods and were achieved with significant savings of both labor and chemicals.
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