Fungal bioconversion of toxic polychlorinated biphenyls by white-rot fungus, Phlebia brevispora

Toxic coplanar polychlorinated biphenyls (Co-PCBs) were used as substrates for a degradation experiment with white-rot fungus, Phlebia brevispora TMIC33929, which is capable of degrading polychlorinated dibenzo-p-dioxins. Eleven PCB congener mixtures (7 mono-ortho- and 4 non-ortho-PCBs) were added to the cultures of P. brevispora and monitored by high resolution gas chromatography and mass spectrometry (HRGC/HRMS). Five PCB congeners, 3,3′,4,4′-tetrachlorobiphenyl, 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl were degraded by P. brevispora. To investigate the fungal metabolism of PCB, each Co-PCB was treated separately by P. brevispora and the metabolites were analyzed by gas chromatography and mass spectrometry (GC/MS) and identified on the basis of the GC/MS comparison with the authentic compound. Meta-methoxylated metabolite was detected from the culture containing each compound. Additionally, para-dechlorinated and -methoxylated metabolite was also detected from the culture with 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl, which are mono-ortho-PCBs. In this paper, we identified the congener specific degradation of coplanar PCBs by P. brevispora, and clearly proved for the first time by identifying the metabolites that the white-rot fungus, P. brevispora, transformed recalcitrant coplanar PCBs.     

Degradation of Polychlorinated Biphenyl Metabolites by Naphthalene-Catabolizing Enzymes

The ability of the dehydrogenase and ring cleavage dioxygenase of the naphthalene degradation pathway to transform 3,4-dihydroxylated biphenyl metabolites was investigated. 1,2-Dihydro-1,2-dihydroxynaphthalene dehydrogenase was expressed as a histidine-tagged protein. The purified enzyme transformed 2,3-dihydro-2,3-dihydroxybiphenyl, 3,4-dihydro-3,4-dihydroxybiphenyl, and 3,4-dihydro-3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl to 2,3-dihydroxybiphenyl, 3,4-dihydroxybiphenyl (3,4-DHB), and 3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl (3,4-DH-2,2′,5,5′-TCB), respectively. Our data also suggested that purified 1,2-dihydroxynaphthalene dioxygenase catalyzed the meta cleavage of 3,4-DHB in both the 2,3 and 4,5 positions. This enzyme cleaved 3,4-DH-2,2′,5,5′-TCB and 3,4-DHB at similar rates. These results demonstrate the utility of the naphthalene catabolic enzymes in expanding the ability of the bph pathway to degrade polychlorinated biphenyls.     

Functionality of biphenyl 2,3-dioxygenase components in naphthalene 1,2-dioxygenase

Naphthalene 1,2-dioxygenase (Nap dox) and biphenyl 2,3-dioxygenase (Bph dox) are related enzymes that have differentiated during evolution as their specificity has changed. Although their component arrangement is similar, the structure of each component has been modified quite extensively. The purpose of this work was to determine the catalytic capacity of purified Nap dox toward chlorobiphenyls and to investigate the functionality of Bph dox components in the Nap dox system. Both enzyme systems were purified by affinity chromatography as histidine-tagged fused proteins. Data show for the first time that Nap dox can catalyze the oxygenation of all three monochlorobiphenyl isomers, but it is unable to hydroxylate 2,5-, 2,2′-, 3,3′-, 4,4′-di- and 2,2′,5,5′-tetrachlorobiphenyl. The rates of cytochrome c reduction by the ferredoxin components of the two enzymes were identical when the Bph dox reductase component was used in the assay, showing an efficient electron transfer between the Bph dox reductase component and the Nap dox ferredoxin. However, when the Bph dox ferredoxin was used to reconstitute a hybrid Nap dox, the enzyme was only 22% as active as the parental enzyme. These data are discussed in terms of the potential use of Nap dox for the development of enhanced chlorobiphenyl-degrading dioxygenases. Received: 15 October 1998 / Received revision: 21 January 1999 / Accepted: 31 January 1999     

Genotoxic effects of PCB 52 and PCB 77 on cultured human peripheral lymphocytes

Polychlorinated biphenyls (PCBs) are known to be carcinogenic, but the mechanisms of this action are uncertain. Most, but not all, studies have concluded that PCBs are not directly mutagenic, and that much if not all of the carcinogenic activity resides in the fraction of the PCB mixture that contains congeners with dioxin-like activity. The present study was designed to determine genotoxic effects of an ortho-substituted, non-coplanar congener, 2,2′,5,5′-tetrachlorobiphenyl (PCB 52), and a non-ortho-substituted coplanar congener with dioxin-like activity, 3,3′,4,4′-tetrachlorobiphenyl (PCB 77) on cultured human peripheral lymphocytes. DNA damage was assessed by use of the comet assay (alkaline single-cell gel electrophoresis). After cell cultures were prepared, test groups were treated with different concentrations of PCB 52 (0.2 and 1 μM) and PCB 77 (1 and 10 μM) for 1 h at 37 °C in a humidified carbon dioxide incubator, and compared to a DMSO vehicle control group. The cells were visually classified into four categories on the basis of extent of migration such as undamaged (UD), low damage (LD), moderate damage (MD) and high damage (HD). The highest concentration of PCBs 52 and 77 significantly increased DNA breakage in human lymphocytes (p < 0.001). Our results indicate that both the non-coplanar PCB 52 and coplanar PCB 77 cause DNA damage, and that the ortho-substituted congener was significantly more potent than the dioxin-like coplanar congener.     

Effect of nutrient enrichment on the distribution and sedimentation of polychlorinated biphenyls (PCBs) in seawater

The effect of nutrient enrichment on the distribution of polychlorinated biphenyl's (PCBs) in the microbial food web and the residence time of PCBs in seawater was studied in an experimental mesocosm system. Two 5 m high temperature and light controlled mesocosm tubes (⊘ = 0,5 m) were filled with seawater from the northern Baltic Sea. Inorganic phosphorus and nitrogen were added daily to one mesocosm, while the other served as a control. Experiments were conducted at 5, 10 and 20°>C. Three ¹⁴C-labelled PCBs of different degree of chlorination were added to subsamples of the mesocosms: 4 chlorobiphenyl (MCB), IUPAC # 3; 2,2′,5,5′-tetrachlorobiphenyl (TCB), IUPAC # 52 and 2,2′, 4,4′,5,5′-hexachlorobiphenyl (HCB) IUPAC # 153. The biomasses and growth rates of the microorganisms as well as the sedimentation rate of particulate organic material increased with nutrient enrichment. The size distribution of the microorganisms changed with nutrient status, from dominance of picoplankton (< 2 μm) in the control towards increased importance of micro (> 10 μm) and nanoplankton (2– 10 μm) in nutrient enrichment. The specific growth rate of the bacterial community was found to be more temperature dependent than that of the phytoplankton community. The relative proportion of PCBs in the >2 μm fraction was observed to be in the order MCB < TCB < HCB, while the opposite distribution prevailed in the < 2 μm fraction. We hypothesize that this is due to the combined effect of the different Kow values of the PCBs and a different composition of the particulate organic carbon in the > 2 μm and < 2 μm fractions (e.g. different lipid composition). The residence time of the PCBs in the mesocosm generally decreased with nutrient enrichment, but was dependent on the degree of chlorination of the PCB. Our results indicate that the transport of organic pollutants up through the food web is more important in nutrient poor than in nutrient rich waters and that the importance of sedimentation is higher in eutrophic ecosystems. This revised version was published online in July 2006 with corrections to the Cover Date.     

Potential importance of protozoan grazing on the accumulation of polychlorinated biphenyls (PCBs) in the pelagic food web

Experiments were conducted to study the distribution of three selectedpolychlorinated biphenyl (PCB) congeners within the microbial food web attwo different nutrient levels; control and nutrient enriched. The objectivewas to quantify the uptake of PCBs through grazing by protozoa. The¹⁴C-PCBs tested were 4-chlorobiphenyl (IUPAC # 3),2,2,5,5-tetrachlorobiphenyl (IUPAC # 52), and2,2,4,4,5,5-hexachlorobiphenyl (IUPAC # 153). EachPCB was incubated in triplicate seawater samples at 20 idref;Cover one week. Daily, samples were separated into four fractions; <0.2µm (dissolved), 0.2-2 µm (bacteria), 2-10 µm(flagellate), and > 10 µm (microplankton; phytoplankton andprotozoa) by selective filtration. Of the PCB fraction that initiallyadsorbed to particles, 60–100% was associated to the bacterialfraction and 0–5% to the microplankton fraction. The totaluptake was highest in the nutrient enriched samples, but when normalized tothe carbon biomass the concentration was lower or equal to the control inall particle fractions. The recovery of the PCBs in the particulatefractions depended on the degree of chlorination, as the highest values wereobserved for the 2,2,4,4,5,5-hexachlorobiphenyl and thelowest for the 4-chlorobiphenyl. The concentrations in the bacterial andflagellate fractions decreased over the first 48–96 hours whilst theconcentration increased in the highest trophic level (>10 µmfraction). Approximately 75% of the increase in concentration of the2,2,4,4,5,5-hexachlorobiphenyl in the > 10 µmfraction was estimated to be the result of bacterivory. Our results indicatethe microbial food web can contribute to a rapid uptake of higherchlorinated PCBs, particularly in oligotrophic ecosystems where thebacterial biomass dominates.     

Anaerobic Dehalogenation of Hydroxylated Polychlorinated Biphenyls by Desulfitobacterium dehalogenans

Ten years after reports on the existence of anaerobic dehalogenation of polychlorinated biphenyls (PCBs) in sediment slurries, we report here on the rapid reductive dehalogenation of para-hydroxylated PCBs (HO-PCBs), the excreted main metabolites of PCB in mammals, which can exhibit estrogenic and antiestrogenic activities in humans. The anaerobic bacterium Desulfitobacterium dehalogenans completely dehalogenates all flanking chlorines (chlorines in ortho position to the para-hydroxyl group) from congeners such as 3,3′,5,5′-tetrachloro-4,4′-dihydroxybiphenyl.     

Cometabolic degradation of polychlorinated biphenyls (PCBs) by axenic cultures of Ralstonia sp. strain SA-5 and Pseudomonas sp. strain SA-6 obtained from Nigerian contaminated soils

Substantial metabolism of 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-tetraCB) and 2,3′,4′,5-tetraCB by axenic cultures of Ralstonia sp. SA-5 and Pseudomonas sp. SA-6 was observed in the presence of biphenyl supplementation, although, the strains were unable to utilize tetrachlorobiphenyls as growth substrate. The former was more amenable to aerobic degradation (∼70% degradation) than the latter (22–45% degradation). Recovery of 2,5-chlorobenzoic acid and chloride from 2,3′,4′,5-tetraCB assay is an indication of initial dioxygenase attack on the 3,4-dichlorophenyl ring. The PCB-degradative ability of both strains was also investigated by GC analysis of individual congeners in Aroclor 1242 (100 ppm) following 12-day incubation with washed benzoate-grown cells. Results revealed two different catabolic properties. Whereas strain SA-6 required biphenyl as inducer of the degradation activity, such induction was not required by strain SA-5. Nearly all the detectable congeners in the mixture were extensively degraded (% reduction in ECD area counts for individual congeners ranged from 50.0 to 100% and 14.2 to 100%, respectively, for SA-5 and SA-6). The two strains exhibited no noticeable specificity for congeners with varying numbers of chlorine substitution and positions. The degradative competence of these isolates most especially SA-5 makes them among the most versatile PCB-metabolizing organisms yet reported.     

Dechlorination of polychlorinated biphenyl congeners by an anaerobic microbial consortium

  An anaerobic methanogenic microbial consortium, developed in a granular form, exhibited extensive dechlorination of defined polychlorinated biphenyl (PCB) congeners. A 2,3,4,5,6-pentachlorobiphenyl was dechlorinated to biphenyl via 2,3,4,6-tetrachlorobiphenyl, 2,4,6-trichlorobiphenyl, 2,4-dichlorobi-phenyl and 2-chlorobiphenyl (CB). Removal of chlorine atoms from all three positions of the biphenyl ring, i.e., ortho, meta and para, was observed during this reductive dechlorination process. Biphenyl was identified as one of the end-products of the reductive dechlorination by GC-MS. After 20 weeks, the concentrations of the dechlorination products 2,4,6-CB, 2,4-CB, 2-CB and biphenyl were 8.1, 41.2, 3.0 and 47.8 μM respectively, from an initial 105 μM 2,3,4,5,6-CB. The extent and pattern of the dechlorination were further confirmed by the dechlorination of lightly chlorinated congeners including 2-CB, 3-CB, 4-CB, 2,4-CB and 2,6-CB individually. This study indicates that the dechlorination of 2,3,4,5,6-CB to biphenyl is due to ortho, meta and para dechlorination by this anaerobic microbial consortium. Received: 30 April 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996     

Polychlorinated biphenyls as inducers of hepatic microsomal enzymes: Structure-activity rules

A number of highly purified polychlorinated biphenyl (PCB) isomers and congeners were synthesized and administered to male Wistar rats at dosage levels of 30 and 150 μmol · kg⁻¹. The effects of this in vivo treatment on the drug-metabolizing enzymes were determined by measuring the microsomal benzo[a]pyrene (B[a]P) hydroxylase, dimethylaminoantipyrine (DMAP) N-demethylase and NADPH-cytochrome c reductase enzyme activities, the cytochrome b₅ content and the relative peak intensities and spectral shifts of the reduced microsomal cytochrome P-450: CO and ethylisocyanide (EIC) binding difference spectra. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC) and PB plus MC (coadministered) to the test animals. The synthetic PCB congeners used in this study included 3,4,4′,5-tetrachlorobiphenyl (TCBP-1), 2,3′,4,4′-tetrachlorobiphenyl (TCBP-2), 2,3′,4,4′,5′-pentachlorobiphenyl (PCBP-1), 2,3,4,4′,5-pentachlorobiphenyl (PCBP-2), 2,3,3′,4,4′,5-hexachlorobiphenyl (HCBP-1), 2,3,3′,4′,5,6-hexachlorobiphenyl (HCBP-2), 2,3,3′,5,5′,6-hexachlorobiphenyl (HCBP-3), 2,2′,3,5,5′,6-hexachlorobiphenyl (HCBP-4) and 2,3,3′,4,5,5′-hexachlorobiphenyl (HCBP-5) and were used to reappraise the structure-activity rules for PCBs as hepatic microsomal enzyme inducers. The results suggested that (a) PCBs which induce MC or mixed-type activity must be substituted at both para positions, at least two meta positions but not necessarily on the same phenyl ring and can also contain one ortho chloro substituent; (b) due to the considerable structural diversity of the PB-type inducers the rules for induction of this activity by PCB congeners are not readily defined.     

Active-site engineering of biphenyl dioxygenase: effect of substituted amino acids on substrate specificity and regiospecificity

Biphenyl dioxygenase (Bph Dox) catalyzes the initial dioxygenation step in the metabolism of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in the determination of the substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Previously, the substitution of Asn at Thr-376 near the active-site iron in the BphA1 of Pseudomonas pseudoalcaligenes KF707 expanded the oxidation range and altered the regiospecificity of Bph Dox for PCBs. In this study, we replaced Thr-376 with Gly, Ser, Gln, Tyr, Val, Phe, Asp, and Lys and expressed these enzymes in Escherichia coli. Bph Dox mutants of Thr376Asn, Thr376Val, Thr376Phe, and Thr376Lys showed novel degradation activity for dibenzofuran, which is a poor substrate for KF707 Bph Dox. All active Bph Dox mutants showed altered regiospecificity with 2,2′-dichlorobiphenyl and 2,5,4′-trichlorobiphenyl. The Thr376Gly, Thr376Val, Thr376Phe, and Thr376Asp Bph Dox mutants introduced molecular oxygen at the 2,3 position of 2,2′-dichlorobiphenyl, forming 2-chloro-2′,3′-dihydroxybiphenyl with concomitant dechlorination. The Bph Dox mutants of Thr376Gly, Thr376Ser, Thr376Asp, and Thr376Lys attacked 2,5,4′-trichlorobiphenyl via both 2′,3′- and 3,4-dioxygenation activities. In particular, the Thr376Phe Bph Dox mutant exhibited enhanced and expanded degradation activities toward all of the compounds tested. Further site-directed mutation was induced to change the oxidizing character of KF707 Bph Dox to that of the Bph Dox of Burkholderia xenovorans LB400 by the substitution of two amino acids, Ile335Phe and Thr376Asn, near the active-site.Electronic supplementary material Supplementary material is available in the online version of this article at .     

Identification of a microorganism that links its growth to the reductive dechlorination of 2,3,5,6-chlorobiphenyl

Anaerobic bacteria reductively dechlorinate polychlorinated biphenyls (PCBs) in aquatic sediments, but these microorganisms remain uncultured and, until now, unidentified. Through denaturing gradient gel electrophoresis (DGGE) of 16S rDNA from a highly enriched ortho -PCB dechlorinating culture, the growth of a single microorganism was shown to be dependent upon the presence and dechlorination of 2,3,5,6-tetrachlorobiphenyl. This is the first identification of a microorganism that catalyses the reductive dechlorination of a PCB. The organism, bacterium o -17, has high sequence similarity with the green non-sulphur bacteria and with a group that includes Dehalococcoides ethenogenes . Bacterium o -17 required acetate for dechlorination and growth. H₂:CO₂ (80:20 at 101 kPa) did not support dechlorination or growth of the dechlorinator. Archaeal 16S rDNA was not detected in actively dechlorinating bromoethanesulphonate-treated non-methanogenic cultures, which indicated that methanogenic Archaea were not required for dechlorination. The consistent association with dechlorinating activity combined with high similarity to other known dechlorinating microorganisms indicates that bacterium o -17 catalyses the reductive ortho -dechlorination of 2,3,5,6-tetrachlorobiphenyl.     

Chemical and enzymatic synthesis of monomeric procyanidins (leucocyanidins or 3′,4′,5,7-tetrahydroxyflavan-3,4-diols) from (2R,3R)-dihydroquercetin

The major product from the reduction of (2R,3R)-dihydroquercetin with sodium borohydride is the 2,3-trans-3,4-trans isomer of leucocyanidin [(2R,3S,4R-3,3′,4,4′,5,7-hexahydroxyflavan] whereas the enzymatic reduction product is the 2,3-trans-3,4-cis isomer [(2R,3S,4S)-3,3′,4,4′,5,7-hexahydroxyflavan]. The 3,4-trans isomer may be partly converted to the 3,4-cis isomer under mild acid conditions. The 3,4-cis isomer is more acid-labile, and more reactive both chemically with thiols and enzymatically with a diol reductase, than the 3,4-trans isomer.     

Comparison of Gas Chromatography and Mineralization Experiments for Measuring Loss of Selected Polychlorinated Biphenyl Congeners in Cultures of White Rot Fungi

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than Phanerochaete chrysosporium ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4′-dichlorobiphenyl, 2,4′,5-trichlorobiphenyl (2,4′,5-TCB), 2,2′,4,4′-tetrachlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl. The congener tested for mineralization was 2,4′,5-[U-¹⁴C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of Bjerkandera adusta (UAMH 8258 and UAMH 7308), one strain of Pleurotus ostreatus (UAMH 7964), and Trametes versicolor UAMH 8272 gave the highest biodegradation and mineralization. P. chrysosporium ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of P. chrysosporium, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower, ≤11%, because it measures a complete oxidation of at least part of the congener molecule but the results were more consistent and therefore more reliable in assessment of PCB biodegradation.     

Embryolethality and induction of 7-ethoxyresorufin O-deethylase in chick embryos by polychlorinated biphenyls and polycyclic aromatic hydrocarbons having Ah receptor affinity.

The lethality and 7-ethoxyresorufin O-deethylase (EROD)-inducing potency of some individual polycyclic aromatic hydrocarbons (PAHs) and coplanar polychlorinated biphenyls (PCBs) in chick embryos were measured in order to compare the mechanisms of action of these compounds. In previous studies it was found that coplanar PCBs and certain PAHs have a high embryolethality in the chicken and that they induce embryonic EROD activity. Although the most potent PAHs were almost as embryolethal as the PCBs when injected into hens' eggs 72 h prior to measurement, they were considerably less potent EROD inducers. In the present study, three coplanar PCBs (3,3',4,4'-tetrachlorobiphenyl (TCB), 3,3',4,4',5-pentachlorobiphenyl (PeCB) and 3,3',4,4',5,5'-hexachlorobiphenyl (HCB)) and four of the most toxic PAHs (benzo[a]anthracene (BaA), benzo[k]fluoranthene (BkF), indeno[1,2,3-cd]pyrene (IP) and dibenzo[a, h]-anthracene (DBahA] were administered to chick embryos in different ways, including co-administration. Additive embryolethality was found when BkF and PeCB were co-administered as well as when BaA and DBahA were given simultaneously. The PAHs were more effective as EROD inducers when injected on day 9 (24 h prior to measurement) than when injected on day 7 (72 h prior to measurement). The opposite was found for PeCB and HCB, whereas no difference in potency was noted when comparing TCB injected 24 and 72 h before EROD determination. These substance-related differences were probably due, at least partly, to differences in biotransformation rates. EROD activities found after treatment with high doses of BkF, IP, or DBahA on day 9 were similar to those measured after treatment with PeCB in doses high enough to give maximal induction. Co-administration of high doses of BkF and PeCB did not further increase the activity, indicating that the PAHs and coplanar PCBs induce EROD to a common maximal value. To decrease the influence of metabolization of the PAHs on their EROD-inducing potency, EROD was determined early in development (day 8) and soon after treatment (24 h) in one experiment. In that experiment, the PAHs proved to be only a few times less potent EROD inducers in relation to their embryolethalities compared with the PCBs. The results of the present study, a previously observed similarity in pathology between chick embryos treated with PAHs and embryos treated with coplanar PCBs, and the fact that the most toxic PAHs also are the most avid Ah receptor binders suggest that the coplanar PCBs and the PAHs largely exert their toxicity in chick embryos via an Ah receptor-mediated mechanism. The differences between the compounds in their EROD-inducing potency/embryolethality ratios could probably be explained by their different rates of biotransformation.     

New violet 3,3′-bipyridyl pigment purified from deep-sea microorganism <Emphasis Type="Italic">Shewanella violacea</Emphasis> DSS12

We have purified a new violet pigment derived from Shewanella violacea DSS12 to determine its chemical structure. The pigment colored blue in tetrahydrofuran (THF) or chloroform and showed a broad absorption spectrum from 500 to 700 nm. X-ray diffraction analysis of single crystals showed that the chemical structure of this pigment was 5,5′-didodecylamino-4,4′-dihydroxy-3,3′-diazodiphenoquinone-(2,2′), containing the same chromophore as an indigoidine known as microbial blue pigment. The violet color of this pigment was due to hypsochromic shift (blue shift) caused by the side-by-side orientation of this pigment molecule, revealed by X-ray structural analyses of a single crystal. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Structural basis of species differences between human and experimental animal CYP1A1s in metabolism of 3,3',4,4',5-pentachlorobiphenyl

Coplanar polychlorinated biphenyls included in dioxin-like compounds are bio-accumulated and adversely affect wildlife and human health. Although many researchers have studied the metabolism of PCBs, there have been few reports of the in vitro metabolism of 3,3',4,4',5-pentachlorobiphenyl (PCB126), despite the fact that it has the highest toxicity among PCB congeners. Cytochrome P450 (CYP) 1A1 proteins can metabolize some dioxins and PCBs by hydroxylation, but the activities of human and rat CYP1A1 proteins are very different. The mechanism remains unclear. From our results, rat CYP1A1 metabolized PCB126 into 4-OH-3,3',4',5-tetrachlorobiphenyl and 4-OH-3,3',4',5,5'-pentachlorobiphenyl, but human CYP1A1 did not metabolize. Homology models of the two CYP proteins, and docking studies, showed that differences in the amino acid residues forming their substrate-binding cavities led to differences in the size and shape of the cavities; only the cavity of rat CYP1A1 allowed PCB126 close enough to the haem to be metabolized. Comparison of the amino acid residues of other mammalian CYP1A1 proteins suggested that rats have a unique metabolism of xenobiotics. Our results suggest that it is necessary to be careful in human extrapolation of toxicity data estimated by using the rat as an experimental animal, especially in the case of compounds metabolized by CYP1A1.     

Exposure to polychlorinated biphenyls causes endothelial cell dysfunction

Environmental chemicals, such as polychlorinated biphenyls (PCBs), may be atherogenic by disrupting normal functions of the vascular endothelium. To investigate this hypothesis, porcine pulmonary artery-derived endothelial cells were exposed to 3,3′,4,4′-tetrachlorobiphenyl (PCB 77), 2,3,4,4′,5-pentachlorobiphenyl (PCB 114), or 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153) for up to 24 hours. These PCBs were selected for their varying binding avidities with the aryl hydrocarbon (Ah) receptor and differences in their induction of cytochrome P450. PCB 77 and PCB 114 significantly disrupted, in a dose-dependent manner, endothelial barrier function by allowing an increase in albumin transfer across endothelial monolayers. These PCBs also contributed markedly to cellular oxidative stress, as measured by 2,7-dichlorofluorescin (DCF) fluorescence and lipid hydroperoxides, and caused a significant increase in intracellular calcium ([Ca²⁺]_i) levels. Enhanced oxidative stress and [Ca²⁺]_i in PCB 77- and PCB 114-treated cells were accompanied by increased activity and content of cytochrome P450 1A and by a decrease in the vitamin E content in the culture medium. In contrast to the effects of PCB 77 and PCB 114, cell exposure to PCB 153 had no effect on cellular oxidation, [Ca²⁺]_i, or endothelial barrier function. These results suggest that certain PCBs may play a role in the development of atherosclerosis by causing endothelial cell dysfunction and a decrease in the barrier function of the vascular endothelium. It is possible that interaction of PCBs with the Ah receptor and activation of the cytochrome P450 1A subfamily are involved in this pathology. © 1995 John Wiley & Sons, Inc.     

Isolation and potential cancer chemopreventive activities of phenolic compounds of beer

Beer contains a variety of phenolic compounds. During the brewing process, some of these compounds are removed by polyvinylpolypyrrolidone (PVPP) to prevent haze formation. We have analyzed the phytochemical composition of a PVPP residue as well as of unstabilized beer and isolated a total of 51 compounds. Eight structures were identified as novel, i.e., 2-(4′-hydroxyphenyl)-3,5-dihydroxybenzoic acid (6), 2′-(4″-hydroxyphenyl)isoferulic acid ester (12), 1,2,5,7-tetrahydroxyanthraquinone (23) and 4,7-dihydroxy-5-(2′,4′,6′-trihydroxyphenyl)-indan-1,2-dione (24) from the PVPP residue, and catechin-7-O-β-(6″-O-nicotinoyl)-β-D-glucopyranoside (41), ent-epigallo-catechin-(4αto8, 2αtoOto7)catechin (44), ent-epigallocatechin (4αto6, 2αtoOto7)catechin (45) and 2,3-cis-3,4-trans-2-[2,3-trans-3,3′,4′,5,7-pentahydroxyflavan-8-yl]-4-(3,4-dihydroxyphenyl)3,5,7-trihydroxybenzopyran (46) from the unstabilized beer. Most of the compounds were tested for potential cancer chemopreventive activities in in vitro test systems detecting a modulation of carcinogen metabolism (inhibition of phase 1 cytochrome P450 1A (Cyp1A) activity, induction of NAD(P)H:quinone oxidoreductase (QR) activity) and anti-inflammatory mechanisms (inhibition of lipopolysaccharide (LPS)-mediated induction of inducible nitric oxide synthase (iNOS), inhibition of cyclooxygenase 1 (Cox-1) activity). 1,2,5,7-Tetrahydroxyanthraquinone (23) and xanthohumol (25), a prenylated chalcone derived from hop, were identified as the most potent compounds and were additionally tested for inhibition of chemically-induced preneoplastic lesions in an ex vivo mouse mammary gland organ culture model (MMOC). Importantly, both agents inhibited lesion formation with halfmaximal inhibitory concentrations (IC₅₀) of 0.1 and 0.02 μM, respectively. Our results demonstrate that beer is an interesting source of potential cancer chemopreventive agents and should be further investigated with this respect. This revised version was published online in June 2006 with corrections to the Cover Date.     

Different molecular capacity in the induction of apoptosis by polychlorinated biphenyl congeners in rat renal tubular cell cultures

The effects of a commercial polychlorinated biphenyl (PCB) mixture (Aroclor 1248) and two individual PCB congeners were evaluated on rat renal proximal tubule culture cell viability and internucleosomal DNA fragmentation (DNA ladder) characteristic of apoptosis. Treatment with Aroclor 1248 caused the loss of cell viability and promoted apoptosis in a concentration- and time-dependent manner. The two PCB congeners assessed can also induce apoptosis. However, the extent of apoptosis generated was greater for the non-ortho-substituted planar congener (3,3,4,4-tetrachlorobiphenyl) than for the di-ortho-substituted nonplanar congener (2,2,4,4,5,5-hexachlorobiphenyl). This correlated with the loss of cell viability since the planar compound is much more cytotoxic. The results suggest a different molecular mechanism in the induction of apoptosis by planar or nonplanar PCB congeners.