DEVELOPMENT OF AN IN-SITU TOXICITY ASSAY SYSTEM USING RECOMBINANT BACULOVIRUSES

A new method for experimentally analyzing the role of enzymes involved in metabolizing mutagenic, carcinogenic, or cytotoxic chemicals is de scribed. Spodoptera fugiperda (SF-21) cells infected with recombinant baculoviruses are used for high level expression of one or more cloned enzymes. The ability of these enzymes to prevent or enhance the toxic ity of drugs and xenobiotics is then measured in situ. Initial paramet ers for the system were developed and optimized using baculoviruses en gineered for expression of the mouse soluble epoxide hydrolase (msEH, EC 3.3.2.3) or the rat cytochrome P4501A1. SF-21 cells expressing msEH were resistant to trans-stilbene oxide toxicity as well as several ot her toxic epoxides including: cis-stilbene oxide, 1,2,7,8-diepoxyocran e, allylbenzene oxide, and estragole oxide. The msEH markedly reduced DNA and protein adduct formation in SF-21 cells exposed to [H-3]allylb enzene oxide or [H-3]estragole oxide. On the other hand, 9,10-epoxyoct adecanoic acid and methyl 9,10-epoxyoctadecanoate were toxic only to c ells expressing sEH, suggesting that the corresponding fatty acid diol s were cytotoxic. This was confirmed by showing that chemically synthe sized diols of these fatty acid epoxides were toxic to control SF-21 c ells at the same concentration as were the epoxides to cells expressin g sEH. A recombinant baculovirus containing a chimeric cDNA formed bet ween the rat P4501A1 and the yeast NADPH-P450 reductase was also const ructed and expressed in this system. A model compound, naphthalene, wa s toxic to SF-21 cells infected with the rat P4501A1/reductase chimeri c baculovirus, but was not toxic to cells infected with a control viru s. This susceptibility could be reversed by co-infecting SF-21 cells w ith either a human or a rat microsomal EH virus along with the P4501A1 /reductase virus. These results demonstrate the usefulness of this new system for experimentally analyzing the role of enzymes hypothesized to metabolize endogenous and exogenous chemicals of human health conce rn.