Magnus Zeisig

Formation of DNA adducts from oil-derived products analyzed by 32P-HPLC

Abstract. The aim of this study was to investigate the genotoxic potential of DNA adducts and to compare DNA adduct levels and patterns in petroleum vacuum distillates, coal tar distillate, bitumen fume condensates, and related substances that have a wide range of boiling temperatures. An in vitro assay was used for DNA adduct analysis with human and rat S-9 liver extract metabolic activation followed by 32P-postlabeling and 32P-high-performance liquid chromatography (32P-HPLC). For petroleum distillates originating from one crude oil there was a correlation between in vitro DNA adduct formation and mutagenic index, which showed an increase with a distillation temperature of 250°C and a peak around a distillation point of approximately 400°C. At higher temperatures, the genotoxicity (DNA adducts and mutagenicity) rapidly declined to very low levels. Different petroleum products showed a more than 100-fold range in DNA adduct formation, with severely hydrotreated base oil and bitumen fume condensates being lowest. Coal tar distillates showed ten times higher levels of DNA adduct formation than the most potent petroleum distillate. A clustered DNA adduct pattern was seen over a wide distillation range after metabolic activation with liver extracts of rat or human origin. These clusters were eluted in a region where alkylated aromatic hydrocarbons could be expected. The DNA adduct patterns were similar for base oil and bitumen fume condensates, whereas coal tar distillates had a wider retention time range of the DNA adducts formed. Reference substances were tested in the same in vitro assay. Two- and three-ringed nonalkylated aromatics were rather low in genotoxicity, but some of the three- to four-ringed alkylated aromatics were very potent inducers of DNA adducts. Compounds with an amino functional group showed a 270-fold higher level of DNA adduct formation than the same structures with a nitro functional group. The most potent DNA adduct inducers of the 16 substances tested were, in increasing order, 9,10-dimethylanthracene, 7,12-dimethylbenz[a]anthracene and 9-vinylanthracene. Metabolic activation with human and rat liver extracts gave rise to the same DNA adduct clusters. When bioactivation with material from different human individuals was used, there was a significant correlation between the CYP 1A1 activity and the capacity to form DNA adducts. This pattern was also confirmed using the CYP 1A1 inhibitor ellipticine. The 32P-HPLC method was shown to be sensitive and reproducible, and it had the capacity to separate DNA adduct-forming substances when applied to a great variety of petroleum products.

32P-Postlabeling high-performance liquid chromatographic improvements to characterize DNA adduct stereoisomers from benzo[a]pyrene and benzo[c]phenanthrene, and to separate DNA adducts from 7,12-dimethylbenz[a]anthracene.

Single compounds can generate complex DNA adduct patterns by reactions through different pathways, with different target nucleotides and through different configurations of the products. DNA adduct analysis by 32P-HPLC was improved by adding an isocratic plateau in an otherwise linear gradient, thereby enhancing resolution of predictable retention time intervals. This enhanced 32P-HPLC technique was used to analyze and at least partly resolve 14 out of 16 available benzo[c]phenanthrene deoxyadenosine and deoxyguanosine adduct standards, 8 out of 8 available benzo[a]pyrene deoxyadenosine and deoxyguanosine adduct standards, and 51 peaks from 7,12-dimethylbenz[a]anthracene-calf thymus DNA reaction products. The same type of gradient modifications could be used to enhance resolution in analyses of other complex DNA adduct mixtures, e.g., in vivo in humans.

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 32P-postlabeling. The chromatographic method often used has been 32P-TLC (thin-layer chromatography), but the more recently developed 32P-HPLC (high-performance liquid chromatography) method has shown advantages. The aim of this study was to test the hypothesis that the 32P-HPLC method has a better ability of detecting DNA adducts derived from petroleum products than 32P-TLC. It was found that some DNA adducts migrated from the application point in 32P-TLC in such a way that it is doubtful if they could be detected and quantified properly. It was also found that, when using 32P-HPLC, it is possible to use the same protocol for substances with a wide variety of DNA adduct forming potential, whereas 32P-TLC needs to be optimized regarding time of exposure and/or the amount of DNA applied. Further, a pattern of recognition in 32P-HPLC enables a selective assessment of DNA adducts derived from complex mixtures whereas 32P-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, 32P-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 32P-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.