Fractionation, Chemical Analysis, and In Vitro Testing Identify Bioactive Components in MC252 Crude Oil

Abstract

Crude oil is a complex mixture that includes polycyclic aromatic hydrocarbons (PAHs) as one of its major components. The toxicity of some chemically substituted PAHs found in oil, such as the methylated species, are relatively understudied. A combination of chemical fractionation and analysis coupled with a bioassay was used to identify a subset of oil PAHs that activated aryl hydrocarbon receptor (AHR). Silica gel chromatography was used for primary and secondary oil fractionation, and standard and reverse phase high performance liquid chromatography (HPLC) were used for the final fractionation steps. Both gas chromatography (GC-) and HPLC-coupled with mass spectrometry (MS) were used to separate and identify compounds present in the petroleum fractions. Bioactivity of the individual fractions was identified and measured using a recombinant yeast strain that expressed the human aryl hydrocarbon receptor complex (AHRC) transcription factor that is composed of human AHR and the ARNT proteins. AHRC activation by oil components results in expression of β-galactosidase, and readout from this enzymatic activity is proportional to the amount and potency of the compounds that activated the system. Silica gel separations produced 25–29 fractions that were assessed for bioactivity using the AHRC reporter system. Bioactivity peaked with the fractions that contained larger PAHs that included four ring compounds such as the triphenylenes, benzanthracenes, and chrysenes (MW 228 + additional methyl groups). When tested as individual compounds, the triphenylenes and benzanthracenes were less potent than the chrysenes, so the latter constituted more of the AHRC signaling activity in the oil fractions. The chrysenes in bioactive fractions were mixtures of the parent compound along with mono-, di-, tri-, and tetra-methyl derivatives and other PAHs. The six possible mono-methylchrysenes were obtained and tested for AHRC activity and for their concentrations in oil. Chrysene, 1-, 2-, 3-, and 6-methylchrysene were present, but 4- and 5-methylchrysene were not detected in the bioactive fractions of oil that were resolved by HPLC. When tested individually in the AHRC bioassay, 4-methylchrysene was the most potent ligand, and 5-methylchrysene was the least potent. Synthetic mixtures of PAHs were reconstructed based upon the chemical composition of one fraction with the high AHRC activity. Collectively, these data show that: 1) the six methylchrysene isomers are within an order of magnitude of chrysene in their ability to activate the AHRC bioassay; 2) although they are a minor group, the chrysene compounds in oil potently activate AHRC signaling; 3) chrysenes diminish as oil weathers, while triphenylenes of identical molecular weight persist, 4) this methodology can be useful for identification and characterization of the bioactivity of sub-fractions and individual compounds found in oil.