1. Formation of Hydroxylated Compounds by Microsomal Metabolism of 2,2’,3,3’,6,6’-Hexachlorobiphenyl (PCB-136) Ananya Pramanik, Izabela Kania-Korwel, Jarline Encarnacion, Xianai Wu, Hans-Joachim Lehmler Department of Occupational and Environmental Health, The University of Iowa, Iowa City Background  Polychlorinated Biphenyls (PCB) are a type of organochlorine molecules that were previously used in industrial and commercial processes, including as dielectric fluids in transformers, coolants, plasticizers in paint, hydraulic fluids, and pesticide extenders.  PCB congeners are persistent organic pollutants and their production was banned in the U.S. in 1979.  Most PCB's are stored in the adipose tissue, of mammals, which makes their elimination more difficult.  Some PCB's can be metabolized by cytochrome P450 enzymes in the liver.  One group of potentially toxic metabolites produced by cytochrome P450 enzymes are hydroxylated PCBs, which may cause reduced blood plasma thyroid levels leading to possible fetal defects in pregnant women.Objective To investigate the correlation between the amount of hydroxylated PCB metabolites produced and the time of incubation in a series of time course experiments involving rat liver microsomes treated with PCB 136. Materials and Methods  Rat livers were harvested from male Sprague- Dawley rats and liver microsomes were prepared by differential centrifugation.  Microsomes were incubated with PCB 136 under the following conditions (at 37ºC): 0.5 mg microsomal protein per mL; 0.4 mg NADPH per mL; 10 mM PCB 136.  Reactions were stopped with 0.5 M NaOH after 5 minutes (carbon monoxide inhibition experiment) or 1 to 30 minutes (time course experiment).  PCB 136 and metabolites were extracted, derivatized and quantified by gas chromatograph. Results and Discussion  PCB 136 is metabolized by cytochrome P450 enzymes (CYPs) to 4- and 5-hydroxy PCB 136 (4- and 5-OH-PCB 136): Effect of Carbon Monoxide on the Formation of 4-OH PCB 136 Effect of Carbon Monoxide on the Formation of 5-OH PCB 136  Carbon monoxide irreversibly inactivates cytochrome P450 enzymes and, thus, prevents the formation of hydroxylated PCB 136 metabolites. Time-dependent Formation of Hydroxylated Metabolites of PCB 136  4- and 5-OH PCB 136 are detectable in the microsomal incubation after 1 minute, with 5-OH PCB 136 being the major metabolites.  4,5-OH PCB 136 was only detected after 20 minutes of incubation.Conclusions  The formation of OH-PCBs increased with time.  Rank order of metabolite formation: 5-OH-PCB 136 > 4-OH-PCB > 4,5-OH-PCB 136.  4,5-OH PCB is detected at later time points because it is formed from 4- and 5-OH PCB 136. Future Directions  Investigate the metabolism of PCB 136 by different cytochrome P450 isoforms (e.g., by using recombinant enzymes or microsomes prepared from rats treated with inducers, such as dexamethasone).  Study the toxicity of PCB 136 metabolites in vitro and in vivo. Acknowledgments This research was supported by grants ES05605, ES013661 and ES017425 from the National Institute of Environmental Health Sciences, NIH.