Differences in Enantioselective Hydroxylation of 2,2',3,6-Tetrachlorobiphenyl (CB45) and 2,2',3,4',6-Pentachlorobiphenyl (CB91) by Human and Rat CYP2B Subfamilies.

Although polychlorinated biphenyls (PCBs) were commercially banned half a century ago, contamination of the environment and organisms by PCBs is still observed. PCBs show high persistence and bioaccumulation, resulting in toxicity. Among PCBs, chiral PCBs with more than three chlorine atoms at the ortho-position exhibit developmental and neurodevelopmental toxicity. Because toxicity is dependent on the atropisomer, atropisomer-specific metabolism is vital in determining toxicity. However, structural information on enantioselective metabolism remains elusive. Cytochrome P450 (CYP, P450) monooxygenases, particularly human CYP2B6 and rat CYP2B1, metabolize separated atropisomers of 2,2',3,6-tetrachlorobiphenyl (CB45) and 2,2',3,4',6-pentachlorobiphenyl (CB91) to dechlorinated and hydroxylated metabolites. Docking studies using human CYP2B6 predict 4'-hydroxy (OH)-CB45 from (aR)-CB45 as a major metabolite of CB45. Di-OH- and dechlorinated OH-metabolites from human CYP2B6 and rat CYP2B1 are also detected. Several hydroxylated metabolites are derived from CB91 by both P450s; 5-OH-CB91 is predicted as a major metabolite. CB91 dechlorination is also detected by identifying 3-OH-CB51. A stable conformation of PCBs in the substrate-binding cavity and close distance to P450 heme are responsible for high metabolizing activities. As hydroxylation and dechlorination change PCB toxicity, this approach helps understand the possible toxicity of chiral PCBs in mammals.

Enhanced metabolism of 2,3',4,4',5-pentachlorobiphenyl (CB118) by bacterial cytochrome P450 monooxygenase mutants of Bacillus megaterium.

Bacterial cytochrome P450 monooxygenase P450BM3 is a promising enzyme to provide novel substrate specificity and enhanced enzymatic activity. The wild type (WT) has been shown to metabolize the widely distributed polychlorinated biphenyl (PCB) 2,3',4,4',5-pentachlorobiphenyl (CB118) to hydroxylated metabolites. However, this reaction requires the coexistence of perfluoroalkyl carboxylic acids (PFCAs). To locate P450BM3 mutants metabolizing CB118 without PFCAs, mutations were selected from amino acids comprising the substrate-binding cavity and the substrate entrance. The mutant A264G showed enhanced hydroxylation activities compared to the WT for the production of five hydroxylated metabolites. Perfluorooctanoic acid addition provided the highest activity, as found in the WT. The docking model of A264G and CB118 indicated that the enlargement of the space above the heme brought CB118 close to the heme, resulting in high activity. In contrast, the mutants L188Q, QG, LVQ, and GVQ, which contain the L188Q mutation, showed higher activity than WT even without PFCAs. Docking models revealed that the closed form found in substrate binding was induced by the L188Q mutation in the substrate non-binding state of the mutants. These mutants are promising for bioremediation of PCBs using enhanced metabolizing activities.

Hydroxylation and dechlorination of 3,3',4,4'-tetrachlorobiphenyl (CB77) by rat and human CYP1A1s and critical roles of amino acids composing their substrate-binding cavity.

Cytochrome P450 (CYP) monooxygenases play critical roles in determining the toxicity of polychlorinated biphenyls (PCBs) in mammals. Hydroxylation of PCBs by these enzymes leads to increased water solubility, promoting the elimination of PCBs from the body. The CYP1 family is mainly responsible for metabolizing PCBs that exhibit a dioxin-like toxicity. Although the dioxin-like PCB 3,3',4,4'-tetrachlorobiphenyl (CB77) is abundant in the environment and accumulates in organisms, information on CB77 metabolism by CYP1A1s is limited. In this study, recombinant rat CYP1A1 metabolized CB77 to 4'-hydroxy (OH)-3,3',4,5'-tetrachlorobiphenyl (CB79) and 4'-OH-3,3',4-trichlorobiphenyl (CB35), whereas human CYP1A1 produced only 4'-OH-CB79. Rat CYP1A1 exhibited much higher metabolizing activity than human CYP1A1 because CB77 was stably accommodated in the substrate-binding cavity of rat CYP1A1 and was close to its heme. In a rat CYP1A1 mutant with two human-type amino acids, the production of 4'-OH-CB79 decreased, whereas that of the dechlorinated metabolite 4'-OH-CB35 increased. These results are explained by a shift in the CB77 positions toward the heme. This study provides insight into the development of enzymes with high metabolizing activity and clarifies the structural basis of PCB metabolism, as dechlorination contributes to a drastic decrease in dioxin-like toxicity.

Enantioselective metabolism of chiral polychlorinated biphenyl 2,2',3,4,4',5',6-Heptachlorobiphenyl (CB183) by human and rat CYP2B subfamilies.

Chiral polychlorinated biphenyls (PCBs) have atropisomers that have different axial chiralities and exist as racemic mixtures. However, biochemical processes often result in the unequal accumulation of these atropisomers in organisms. This phenomenon leads to enantiospecific toxicity enhancement or reduction because either of the atropisomers mainly affects toxicity expression. Enantioselective accumulation is caused by cytochrome P450 (CYP, P450) monooxygenases, especially the CYP2B subfamilies. Therefore, this study investigates the metabolism of a chiral PCB in vitro. Both atropisomers isolated from racemic 2,2',3,4,4',5',6-heptachlorobiphenyl (CB183) were metabolized by human CYP2B6, but not rat CYP2B1. This may be due to the difference in the size of the substrate-binding cavities of CYP2B6 and CYP2B1. The stable accommodation of (-)-CB183 in the cavity without any steric hindrance explained the preferential metabolism of (-)-CB183 compared to (+)-CB183. Two hydroxylated metabolites, 3'-OH-CB183 and 5-OH-CB183, were identified. The docking study showed that the 3'-position of the trichlorophenyl ring closely approaches the heme of CYP2B6. To our knowledge, this is the first study to elucidate the structural basis of chiral PCB metabolism by P450 isozymes. These results will help promote the precise toxicity evaluation of chiral PCBs and provide an explanation of the structural basis of chiral PCB metabolism.

Cryoprotective effect of low-molecular-weight hyaluronan on human dermal fibroblast monolayers.

The purpose of this study was to assess the availability of low-molecular-weight (low-MW) hyaluronan (HA) as a cryoprotectant for cellular cryopreservation. To clarify whether low-MW HA is cryoprotective, we evaluated the effect of HA concentration (0-5% w/w) in a cryoprotectant solution on cell membrane integrity after freeze-thaw. A test sample was created using human dermal fibroblast monolayers incubated in a culture dish for 24 h (37 degrees C, 5% CO2). Sodium hyaluronate (MW 3 x 10(4)-5 x 10(4)) dissolved in medium served as the cryoprotectant solution. Samples were immersed in the solution for 2 h at 0-4 degrees C. They were frozen at a cooling rate of 3 degrees C/min from 4 to -80 degrees C, cooled further to below -185 degrees C, and then thawed. Cell membrane integrity after thawing was evaluated using a trypan blue exclusion assay. The sample and freezing procedures were repeated in subsequent experiments, while the conditions of the solution immersion with respect to the sample varied. Next, to clarify whether the cryoprotective action of HA is intra- or extracellular, we performed three experiments. The first studied the dependence of membrane integrity after freeze-thaw on preliminary incubation time (0.75-24 h at 37 degrees C) with a sample immersed in the solution (5% w/w HA). In the second, membrane integrity of thawed samples that were initially frozen in a medium instead of solution, by removing extracellular HA following a preliminary 6-h incubation period, were evaluated. Thirdly, we investigated cellular uptake of fluorescein isothiocyanate-labeled HA (MW 10(5), 1% w/w) after a preliminary 6-h incubation period under fluorescent microscopy (without freeze-thaw). The results show that HA had a cryoprotective effect, and that this cryoprotective action was intracellular. Therefore, low- MW HA proves to be a promising cellular cryoprotectant.

Metabolic enhancement of 2,3',4,4',5-pentachlorobiphenyl (CB118) using cytochrome P450 monooxygenase isolated from soil bacterium under the presence of perfluorocarboxylic acids (PFCAs) and the structural basis of its metabolism.

2,3',4,4',5-Pentachlorobiphenyl (CB118) is one of the most abundant polychlorinated biphenyl (PCB) congeners in the environment, and perfluoroalkyl acids, including perfluorocarboxylic acids (PFCAs), are widely distributed in the environment. Although CB118 and perfluoroalkyl acids are present in all humans and biota, effects in the metabolic fate of CB118 leading to toxicity change are unclear. P450BM3, which is isolated from the soil bacterium Bacillus megaterium, metabolized CB118 to three different hydroxylated pentachlorobiphenyls (M1-M3). M2 was identified as 4'-OH-2,3',4,5,5'-pentachlorobiphenyl. These reactions were promoted by the presence of PFCAs, and perfluorooctanoic acid (PFCA-C8) was the most effective for accelerating these reactions among PFCAs with different carbon chain length. The production rate of M2 was accelerated by 25-times using PFCA-C8. Furthermore, the docking models of P450BM3 with CB118 and PFCAs revealed that the conformational changes of the substrate-binding cavity of P450BM3 after binding of PFCAs to P450BM3 were important for selective production of CB118 metabolites. This study leads to the clarification of the different metabolic fates of PCBs under complex contamination with PFCAs.

From the Cover: Structural Determinants of the Position of 2,3',4,4',5-Pentachlorobiphenyl (CB118) Hydroxylation by Mammalian Cytochrome P450 Monooxygenases.

Polychlorinated biphenyls (PCBs) accumulate in mammals via the food chain because of their characteristics such as slow degradation and high hydrophobicity. One of the 209 PCB congeners, 2,3',4,4',5-pentachlorobiphenyl (CB118), is abundantly found in the environment and in mammals. Understanding the metabolic fate of CB118 can provide important information toward evaluating its toxicity. In vitro studies on the metabolism of CB118 by cytochrome P450 enzymes (P450 or CYP) revealed that human CYP2B6 and rat CYP2B1 primarily metabolized it to 3-hydroxy (OH)-CB118, whereas rat CYP1A1 metabolized CB118 to 4-hydroxy-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107). Docking models of CYP2Bs with CB118 revealed a short distance between the 3-position of CB118 and the heme iron caused by polarization of the substrate-binding cavity, and maintenance of this pose through interaction with the peripheral amino acids determines the activity and position of hydroxylation. 4-Hydroxylation by rat CYP1A1 occurs owing to the longitudinal shape of the substrate-binding cavity toward the heme of CYP1A1. The metabolites 3-OH-CB118 and 4-OH-CB107 decreased potential for activating the aryl hydrocarbon receptor compared with that of CB118, thereby leading to a decrease in dioxin-like toxicity; however, the neurodevelopmental toxicity of 4-OH-CB107 has been previously reported. The results suggest that these 3 P450 isoforms play an important role in determining the extent of CB118 toxicity. This study will contribute to understanding of the metabolic fates and toxicity of CB118 in vivo.