32P-postlabeling of DNA adducts arising from complex mixtures: HPLC versus TLC separation applied to adducts from petroleum products.

The carcinogenicity of petroleum products is mainly due to their content of polycyclic aromatic compounds (PACs). These compounds may be activated metabolically and react with DNA to form DNA adducts, which is a critical event in the initiation of cancer. One of the most common techniques for analyzing DNA adducts is (32)P-postlabeling. The chromatographic method often used has been (32)P-TLC (thin-layer chromatography), but the more recently developed (32)P-HPLC (high-performance liquid chromatography) method has shown advantages. The aim of this study was to test the hypothesis that the (32)P-HPLC method has a better ability of detecting DNA adducts derived from petroleum products than (32)P-TLC. It was found that some DNA adducts migrated from the application point in (32)P-TLC in such a way that it is doubtful if they could be detected and quantified properly. It was also found that, when using (32)P-HPLC, it is possible to use the same protocol for substances with a wide variety of DNA adduct forming potential, whereas (32)P-TLC needs to be optimized regarding time of exposure and/or the amount of DNA applied. Further, a pattern of recognition in (32)P-HPLC enables a selective assessment of DNA adducts derived from complex mixtures whereas (32)P-TLC is very limited when analyzing complex mixtures due to poor resolution. With more knowledge about the properties of the most mutagenic DNA adducts in HPLC, it could be possible to know also which pattern corresponds to a mutagenic or carcinogenic oil. Consequently, (32)P-HPLC is a good alternative when assessing the genotoxicity of petroleum products.

DNA adduct formation and physiological effects from crude oil distillate and its derived base oil in isolated, perfused rat liver.

A distillate, D431, originating from Venezuelan heavy crude oil and a severely hydro-treated base oil, BO100, derived from this distillate, were tested for DNA adduct formation capacities and overall impact on liver functions. D431 had earlier showed DNA adduct formation in vitro but not in vivo in the rat. In this study, isolated rat liver perfusions were performed to elucidate whether the lack of DNA adducts in vivo was because of lack of uptake or metabolism. The oils were extracted with dimethyl sulfoxide and the extracts added to the perfusion system. Bile production, lactate metabolism and perfusate flow through the organ, which are parameters that reveal the condition of the liver, were continuously monitored. Samples of liver were collected once every hour during perfusion for DNA adduct analysis with (32)P-HPLC. The results for the distillate D431 showed that the production of bile and metabolism of lactate decreased while DNA adduct formation increased with time. The DNA adduct pattern formed in the D431-treated livers was similar to that found earlier in in vitro studies performed on calf thymus DNA (CT-DNA). In the case of DNA adduct formation, virtually no difference with dose was seen, suggesting that perhaps a point of saturation of, for instance, enzymatic systems was reached. The results for base oil BO100 showed no significant difference regarding bile production, lactate metabolism and DNA adduct formation when compared with the control, indicating no toxic or genotoxic activity.