In vitro genotoxicity studies using complex hydrophobic mixtures: efficient delivery of a petroleum sample to...

Petroleum fractions are a diverse group of extremely hydrophobic mixtures, some of which display strong carcinogenicity in animal skin painting experiments. Interpretation of in vitro genotoxicity experiments with these samples is complicated by inefficient delivery of these hydrophobic substances inside target cells. We therefore developed methods to assess and improve the efficiency of delivering a petroleum sample (Matrix, A.P.I. 81-17) to cultured C3H/10T1/2 cells for genotoxicity studies via lipid vesicle incorporation. Three radiolabeled compounds (14C-benzo(a)pyrene, 14C-decane, and 14C-naphthalene) of widely differing volatilities, broadly representative of the spectrum of compounds in petroleum samples, were separately added to Matrix. Lipid vesicles containing Matrix and radiolabeled compounds were prepared by the classical methods for preparing neutral, positive, and negatively charged multilamellar and unilamellar liposomes. Of these, the classical methods for preparing neutral unilamellar liposomes were the most successful for delivering radiolabeled compounds in Matrix to cells. Vesicles optimal for the delivery of tracers in Matrix were prepared with DSPC:cholesterol:lyso-PC (8.8:0.8:0.4, molar ratio) in a Matrix to lipid ratio of 31:69 (w/w). This new method of delivery resulted in proportional, dose-dependent, and reproducible uptake of all tracers. Further, cells treated with this preparation took up 2.5-fold more 14C-decane, 1.5-fold more 14C-BaP, and 18-fold more 14C-naphthalene added to Matrix than did cells treated with Matrix emulsified in tissue culture medium. In contrast, tracers were not taken up in a proportional or reproducible manner when emulsions were used, and in fact, uptake of 14C-naphthalene was consistently very small. Two petroleum fractions, C(2)029188 and C(3)029194, were 4- and 6-fold more cytotoxic, respectively, when delivered to C3H/10T1/2 cells by lipid vesicles than emulsions. The carcinogenic petroleum fraction C(5)0292202 induces type II transformed foci in C3H/10T1/2 cells when cells were treated with C(5)0292202 incorporated into lipid vesicles. The methods for lipid vesicle incorporation described here are effective in delivering hydrophobic petroleum fractions to cells and provide an alternative to the current inefficient and artifactual methods of emulsification currently used. With further validation and standardization, lipid vesicle incorporation of petroleum fractions and treatment of cells with these vesicles should be useful for studying the genotoxicity of complex hydrophobic mixtures in cell culture systems.