Congener Selectivity During Polychlorinated Biphenyls Degradation by Enterobacter sp. LY402

The relationship between the selectivity of a particular polychlorinated biphenyls (PCBs) congener and its biodegradability under the same concentration, especially by Enterobacter sp. LY402, is less well studied. To measure congener selectivity of Enterobacter sp. LY402, several influencing factors were studied. The results showed LY402 effectively degraded coplanar 3,4,3',4'-chlorobiphenyl (CB) at a concentration of 0.05 μM, but not 0.5 μM. The degradation rates of 2,4,5,2',3'-CB and 2,4,5,2',4',5'-CB were increased significantly when the sample constituents were changed from 12 to 5 congeners or to one congener. This indicated that bioremediation of individual congener was affected by other congeners present in the mixture. Moreover, for PCBs containing one chlorine on each phenyl ring, the reactivity preference of LY402 was 2,2'-CB ≥ 3,3'-CB ? 4,4'-CB. For two ortho chlorines congeners of PCBs, 2,2'-CB was degraded faster than 2,6-CB. Although 2,6-CB and 4,4'-CB were poorly degraded, the addition of one (i.e., 2,4,4'-CB and 2,6,3'-CB) or two more chlorines (i.e., 2,4,2',4'-CB) on the phenyl ring significantly increased their biodegradability. In addition, comparing the two congeners of ortho-meta-chlorinated biphenyl, 2,3,2',3'-CB with neighbor meta chlorines was degraded slower than 2,5,2',5'-CB with interval meta chlorines. All these indicated that the transformation rates of PCBs were not consistent with the number of chlorines, and PCBs containing the same numbers of chlorines but at different positions also resulted in different conversions. In principle, the extents of effect caused by the position of chlorine substituents on the degradation of PCBs by LY402 were ortho- > meta- > para-CB. In conclusion, the congener selectivity of LY402 was determined by many factors, including the composition of the congeners, their concentrations in the mixture and location and number of chlorine substituents on the phenyl rings.     

Transformation of polychlorinated biphenyls by a novel BphA variant through the meta-cleavage pathway

The transformation of 20 polychlorinated biphenyls (PCBs) through the meta-cleavage pathway by recombinant Escherichia coli cells expressing the bphEFGBC locus from Burkholderia cepacia LB400 and the bphA genes from different sources was compared. The analysis of PCB congeners for which hydroxylation was observed but no formation of the corresponding yellow meta-cleavage product demonstrated that only lightly chlorinated congeners including one tetrachlorobiphenyl (2,2',4,4'-CB) were transformed into their corresponding yellow meta-cleavage products. Although many other tetrachlorobiphenyls (2, 2',5,5'-CB, 2,2',3,5'-CB, 2,4,4',5-CB, 2,3',4',5-CB, 2,3',4,4'-CB) and one pentachlorobiphenyl (2,2',4,5,5'-CB) tested were depleted from resting cell suspensions, no yellow meta-cleavage products were observed. For most of these congeners, dihydrodiol compounds accumulated as the endproducts, indicating that the bphB-encoded biphenyl-2,3-dihydrodiol-2,3-dehydrogenase is a key limiting step for further degradation of highly chlorinated congeners. These results suggest that engineering the biphenyl dioxygenase alone is insufficient for an improved removal of PCB. Rather, improved degradation of PCBs is more likely to be achieved with recombinant strains containing metabolic pathways not only specifically engineered for expanding the initial dioxygenation but also for the mineralization of PCBs.     

Degradation of anaerobic reductive dechlorinationproducts of Aroclor 1242 by four aerobic bacteria

We studied the aerobic degradation of eight PCB congeners which comprise from 70 to 85% of the anaerobic dechlorination products from Aroclor 1242, including2-, 4-, 2,4-, 2,6-, 2,2'-, 2,4'-, 2,2',4-, and2,4,4'-chlorobiphenyl (CB), and the biodegradation of their mixtures designed to simulate anaerobic dechlorination profiles M and C. StrainsComamonas testosteroni VP44 and Rhodococcus erythreus NY05 preferentially oxidizeda para-substituted ring, while Rhodococcus sp. RHA1, similar to well known strain Burkholderia sp. LB400, preferably attackedan ortho-chlorinated ring. Strains with ortho-directed attack extensively degraded2,4'- and 2,4,4'-CB into 4-chlorobenzoate, while bacteria with para-directed attack transformed these congeners mostly into potentially problematicmeta-cleavage products. The strains that preferentiallyoxidized an ortho-substituted ring readily degradedseven of the eight congeners supplied individually; only 2,6-CB was poorly degraded. Degradationof 2,2'- and 2,4,4'-CB was reduced when present in mixtures M and C. Higher efficiencies of degradation of the individual congeners and defined PCB mixtures M and C and greater production of chlorobenzoates were observed with bacteria that preferentially attackan ortho-substituted ring. PCB congeners 2,4'-, 2,2',4-, and 2,4,4'-CB canbe used to easily identify bacteria with ortho-directed attack whichare advantageous for use in the aerobic stage of the two-phase (anaerobic/aerobic)PCB bioremediation scheme.     

Sequential reductive dechlorination of meta-chlorinated polychlorinated biphenyl congeners in sediment microcosms by two different Chloroflexi phylotypes

Three species within a deeply branching cluster of the Chloroflexi are the only microorganisms currently known to anaerobically transform polychlorinated biphenyls (PCBs) by the mechanism of reductive dechlorination. A selective PCR primer set was designed that amplifies the 16S rRNA genes of a monophyletic group within the Chloroflexi including Dehalococcoides spp. and the o-17/DF-1 group. Assays for both qualitative and quantitative analyses by denaturing gradient gel electrophoresis and most probable number-PCR, respectively, were developed to assess sediment microcosm enrichments that reductively dechlorinated PCBs 101 (2,2',4,5,5'-CB) and 132 (2,2',3,3',4,6'-CB). PCB 101 was reductively dechlorinated at the para-flanked meta position to PCB 49 (2,2',4,5'-CB) by phylotype DEH10, which belongs to the Dehalococcoides group. This same species reductively dechlorinated the para- and ortho-flanked meta-chlorine of PCB 132 to PCB 91 (2,2',3',4,6'-CB). However, another phylotype designated SF1, which is more closely related to the o-17/DF-1 group, was responsible for the subsequent dechlorination of PCB 91 to PCB 51 (2,2',4,6'-CB). Using the selective primer set, an increase in 16S rRNA gene copies was observed only with actively dechlorinating cultures, indicating that PCB-dechlorinating activities by both phylotype DEH10 and SF1 were linked to growth. The results suggest that individual species within the Chloroflexi exhibit a limited range of congener specificities and that a relatively diverse community of species within a deeply branching group of Chloroflexi with complementary congener specificities is likely required for the reductive dechlorination of different PCBs congeners in the environment.     

Destruction of mixture of tri-hexa-chlorinated biphenyls by Rhodococcus genus strains

Destruction of polychlorinated biphenyls (PCBs) by strain-destructors Rhodococcus sp. B7a and Rhodococcus sp. G12a has been studied. It was shown that these strains destruct 78-95% of PCB mixture containing tri-hexa-chlorinated biphenyls. Rhodococcus destruct all components of the mixture of tri-, tetra-, penta-, and hexa-chlorinated biphenyls without accumulation of toxic chlorinated metabolites. The studied bacteria destruct PCB that are the most stable for oxidation, such as 2,5,2',5'-CB; 3,4,3',4'-CB; and 2,4,5,2',4',5'-CB. The most perspective strains are R. rubber P25, Rhodococcus sp. B7a and Rhodococcus sp. G12a whose metabolic potential can be used for biotechnological refinement of the environment from highly toxic pollutants.     

Degradation of 4,4'-dichlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl by the white rot fungus Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium has demonstrated abilities to degrade many xenobiotic chemicals. In this study, the degradation of three model polychlorinated biphenyl (PCB) congeners (4,4'-dichlorobiphenyl [DCB], 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl) by P. chrysosporium in liquid culture was examined. After 28 days of incubation, 14C partitioning analysis indicated extensive degradation of DCB, including 11% mineralization. In contrast, there was negligible mineralization of the tetrachloro- or hexachlorobiphenyl and little evidence for any significant metabolism. With all of the model PCBs, a large fraction of the 14C was determined to be biomass bound. Results from a time course study done with 4,4'-[14C]DCB to examine 14C partitioning dynamics indicated that the biomass-bound 14C was likely attributable to nonspecific adsorption of the PCBs to the fungal hyphae. In a subsequent isotope trapping experiment, 4-chlorobenzoic acid and 4-chlorobenzyl alcohol were identified as metabolites produced from 4,4'-[14C]DCB. To the best of our knowledge, this the first report describing intermediates formed by P. chrysosporium during PCB degradation. Results from these experiments suggested similarities between P. chrysosporium and bacterial systems in terms of effects of congener chlorination degree and pattern on PCB metabolism and intermediates characteristic of the PCB degradation process.     

Influence of chlorine substituents on rates of oxidation of chlorinated biphenyls by the biphenyl dioxygenase of Burkholderia sp. strain LB400

Biphenyl dioxygenase from Burkholderia (Pseudomonas) sp. strain LB400 catalyzes the first reaction of a pathway for the degradation of biphenyl and a broad range of chlorinated biphenyls (CBs). The effect of chlorine substituents on catalysis was determined by measuring the specific activity of the enzyme with biphenyl and 18 congeners. The catalytic oxygenase component was purified and incubated with individual CBs in the presence of electron transport proteins and cofactors that were required for enzyme activity. The rate of depletion of biphenyl from the assay mixture and the rate of formation of cis-biphenyl 2,3-dihydrodiol, the oxidation product, were almost equal, indicating that the assay accurately measured enzyme-specific activity. Four classes of CBs were defined based on their oxidation rates. Class I contained 3-CB and 2,5-CB, which gave rates that were approximately twice that of biphenyl. Class II contained 2,5,3',4'-CB, 2,3,2',5'-CB, 2,3,4,5-CB, 2,3,2',3'-CB, 2,4, 5,2',5'-CB, 2,5,3'-CB, 2,5,4'-CB, 2-CB, and 3,4,5-CB, which gave rates that ranged from 97 to 35% of the biphenyl rate. Class III contained only 2,3,4,2',5'-CB, which gave a rate that was 4% of the biphenyl rate. Class IV contained 2,4,4'-CB, 2,4,2',4'-CB, 3,4,5, 2'-CB, 3,4,5,3'-CB, 3,5,3',5'-CB, and 3,4,5,2',5'-CB, which showed no detectable depletion. Rates were not significantly correlated with the aqueous solubilities of the CBs or the number of chlorine substituents on the rings. Oxidation products were detected for all class I, II, and III congeners and were identified as chlorinated cis-dihydrodiols for classes I and II. The specificity of biphenyl dioxygenase for the CBs examined in this study was determined by the relative positions of the chlorine substituents on the aromatic rings rather than the number of chlorine substituents on the rings.     

Effects of polychlorinated biphenyls on cytochrome P450 induction in the chick embryo hepatocyte culture

The effects of pure synthetic polychlorinated biphenyl (PCB) congeners on the induction of cytochrome P450 and associated activities were examined in cultured chick embryo hepatocytes. Dose-response effects for the induction of total cytochrome P450 ethoxyresorufin-O-deethylase (EROD) activity, and benzphetamine demethylase (BPDM) activity were studied using 10 selected tetra- to hexachlorinated PCB congeners. These studies revealed that PCBs caused effects in the chick hepatocyte culture different from previously observed effects in rat liver. Based on their effects in chick hepatocytes, the PCBs could be categorized into two groups. The first group (consisting of 3,3',4,4'-PCB, 3,3',4,4',5-PCB, 3,3',4,4',5,5'-PCB, 2',3,3',4,5-PCB, 2,3,3',4,4',5'-PCB, and 2,3,4,4',5-PCB) induced total cytochrome P450 2.4- to 2.9-fold and EROD activity from 1-2 pmol/min/mg protein to 162-247. There was marked variation in potency, but all these congeners had a maximal inducing dose above which cytochrome P450 concentrations and EROD activities declined. BPDM activities were increased only slightly (1.2- to 1.6-fold) at the maximal cytochrome P450 inducing dose. The second group of congeners (consisting of 2,2',4,5,5'-PCB. 2,2',4,4',5,5'-PCB, and 2,2',3,4,4',6-PCB) induced total cytochrome P450 concentrations 4.0-fold and BPDM activities 2.2- to 2.6-fold with greatest activity occurring at the highest doses which could be added (10-50 microM). However, EROD activities were also increased by these congeners to 60-112 pmol/min/mg protein with declining activities seen at the highest PCB doses (i.e., resembling EROD induction patterns of the first group). The EROD induction patterns with these latter PCB congeners are noteworthy since these PCBs do not induce EROD activity in the rat. For both groups of PCB congeners, EROD induction was associated with increased accumulation of uroporphyrin in cultures exposed to exogenous 5-aminolevulinate. Studies investigating the reason for the depression of cytochrome P450 concentrations and/or EROD activities by high doses of the PCBs revealed that with the first group there was slightly decreased total protein synthesis, decreased total cell heme concentrations, and decreased accumulation of radiolabeled heme synthesized from 5-[14C]aminolevulinate. These changes might represent nonspecific toxic effects of the first group of PCBs. However, since these changes were not seen with the second group of PCBs, it is unlikely that either inhibition of heme synthesis or toxicity cause the depression of EROD activity with high PCB doses.     

Differential enantioselective transformation of atropisomeric polychlorinated biphenyls by multiple bacterial strains with different inducing compounds

Five polychlorinated biphenyl (PCB)-degrading bacteria were tested for the ability to differentiate between the enantiomers of four atropisomeric PCB congeners (2,2',3,6-tetra-CB; 2,2',3,3',6-penta-CB; 2,2',3,4',6-penta-CB; and 2,2',3,5',6-penta-CB) after growth in the presence of tryptone-soytone, biphenyl, carvone, or cymene. Enantioselectivity was shown to vary with respect to strain, congener, and cosubstrate.     

Degradation of Polychlorinated Biphenyls by UV-Catalyzed Photolysis

Photolysis of five polychlorinated biphenyl (PCB) congeners [2,4,4′-trichlorobiphenyl (PCB 28), 2,2′,5,′5-tetrachlorobiphenyl (PCB 52), 2,2′,4,5,5′-pentachlorobiphenyl (PCB 101), 2,2′,4,4′,5,′5-hexachlorobiphenyl (PCB 153) and 2,2′,3,4,4′,5,′5-heptachlorobiphenyl (PCB 180)] individually and in combination were carried out in the solvents methanol, ethanol, and 2-propanol. The disappearance of parent congener generally increased with UV intensity. The solvents had significant or limited effect on the removal of PCBs depending on the congener used. Because 2-propanol was highly toxic and methoxylated products were formed when methanol was used, ethanol was selected as the optimum solvent. The results of photolysis of the PCB mixture showed that PCB 52 was formed and accumulated after 4 h of photolysis. The addition of sodium hydroxide increased the rate of photolysis of the PCB mixture. One hundred percent removal can be obtained of the PCB in mixture in 90 min under optimized conditions. Gas chromatography–mass spectrometry was used to determine the intermediates of the photolysis of PCBs under optimized conditions. For the PCB congeners and mixture studied, the major photolytic intermediates were less chlorinated congeners, and biphenyl was the major product with minor amounts of hydroxylated PCBs, ethylated, dimethylated, and methylated biphenyls. Biphenyl could be further degraded by a prolonged photolysis. Toxicity of the PCB mixture during photolysis was monitored by the Microtox^® test. It was found that the toxicity increased at the early stage of photolysis, and gradually decreased as the reaction proceeded. After 90 min, the EC₅₀ of the reaction mixture was similar to that of the untreated sample.     

Evidence for novel mechanisms of polychlorinated biphenyl metabolism in Alcaligenes eutrophus H850

Previous studies indicated that Alcaligenes eutrophus H850 attacks a different spectrum of polychlorinated biphenyl (PCB) congeners than do most PCB-degrading bacteria and that novel mechanisms of PCB degradation might be involved. To delineate this, we have investigated the differences in congener selectivity and metabolite production between H850 and Corynebacterium sp. strain MB1, an organism that apparently degrades PCBs via a 2,3-dioxygenase. H850 exhibited a superior ability to degrade congeners via attack on 2-, 2,4-, 2,5-, or 2,4,5-chlorophenyl rings in PCBs but an inferior ability to degrade congeners via attack on a 4-chlorophenyl ring. Reactivity preferences were also reflected in the products formed from unsymmetrical PCBs; thus MB1 attacked the 2,3-chlorophenyl ring of 2,3,2',5'-tetrachlorobiphenyl to yield 2,5-dichlorobenzoic acid, while H850 attacked the 2,5-chlorophenyl ring to yield 2,3-dichlorobenzoic acid and a novel metabolite, 2',3'-dichloroacetophenone. Furthermore, H850 oxidized 2,4,5,2',4',5'-hexachlorobiphenyl, a congener with no adjacent unsubstituted carbons, to 2',4',5'-trichloroacetophenone. The atypical congener selectivity pattern and novel metabolites produced suggest that A. eutrophus H850 may degrade certain PCB congeners by a new route beginning with attack by some enzyme other than the usual 2,3-dioxygenase.     

Evidence for novel mechanisms of polychlorinated biphenyl metabolism in Alcaligenes eutrophus H850.

Previous studies indicated that Alcaligenes eutrophus H850 attacks a different spectrum of polychlorinated biphenyl (PCB) congeners than do most PCB-degrading bacteria and that novel mechanisms of PCB degradation might be involved. To delineate this, we have investigated the differences in congener selectivity and metabolite production between H850 and Corynebacterium sp. strain MB1, an organism that apparently degrades PCBs via a 2,3-dioxygenase. H850 exhibited a superior ability to degrade congeners via attack on 2-, 2,4-, 2,5-, or 2,4,5-chlorophenyl rings in PCBs but an inferior ability to degrade congeners via attack on a 4-chlorophenyl ring. Reactivity preferences were also reflected in the products formed from unsymmetrical PCBs; thus MB1 attacked the 2,3-chlorophenyl ring of 2,3,2',5'-tetrachlorobiphenyl to yield 2,5-dichlorobenzoic acid, while H850 attacked the 2,5-chlorophenyl ring to yield 2,3-dichlorobenzoic acid and a novel metabolite, 2',3'-dichloroacetophenone. Furthermore, H850 oxidized 2,4,5,2',4',5'-hexachlorobiphenyl, a congener with no adjacent unsubstituted carbons, to 2',4',5'-trichloroacetophenone. The atypical congener selectivity pattern and novel metabolites produced suggest that A. eutrophus H850 may degrade certain PCB congeners by a new route beginning with attack by some enzyme other than the usual 2,3-dioxygenase.     

Isolation and characterization of a novel polychlorinated biphenyl-degrading bacterium, Paenibacillus sp. KBC101

The biphenyl-utilizing bacterial strain KBC101 has been newly isolated from soil. Biphenyl-grown cells of KBC101 efficiently degraded di- to nonachlorobiphenyls. The isolate was identified as Paenibacillus sp. with respect to its 16S rDNA sequence and fatty acid profiles, as well as various biological and physiological characteristics. In the case of highly chlorinated biphenyl (polychlorinated biphenyl; PCB) congeners, the degradation activities of this strain were superior to those of the previously reported strong PCB degrader, Rhodococcus sp. RHA1. Recalcitrant coplanar PCBs, such as 3,4,3,4-CB, were also efficiently degraded by strain KBC101 cells. This is the first report of a representative of the genus Paenibacillus capable of degrading PCBs. In addition to growth on biphenyl, strain KBC101 could grow on dibenzofuran, xanthene, benzophenone, anthrone, phenanthrene, naphthalene, fluorene, fluoranthene, and chrysene as sole sources of carbon and energy. Paenibacillus sp. strain KBC101 presented heterogeneous degradation profiles toward various aromatic compounds.     

Dechlorination of individual congeners in aroclor 1248 as enhanced by chlorobenzoates, chlorophenols, and chlorobenzenes

Previous investigations showed that three classes of haloaromatic compounds (HACs; chlorobenzoates, chlorophenols, and chlorobenzenes) enhanced the reductive dechlorination of Aroclor 1248, judging from the overall extent of reduction in Cl atoms on the biphenyl. In the present study, we further investigated the kind of polychlorinated biphenyl (PCB) congeners involved in the enhanced dechlorination by four isomers belonging to each class (2,3-, 2,5-, 2,3,5-, and 2,4,6-chlorobenzoates; 2,3-, 3,4-, 2,5-, and 2,3,6-chlorophenols; and 1,2-, 1,2,3-, 1,2,4-, and penta-chlorobenzenes). Although the PCB congeners involved in the enhanced dechlorination varied with the HACs, the enhancement primarily involved paradechlorination of the same congeners (2,3,4'-, 2,3,4,2'- plus 2,3,6,4'-, 2,5,3',4'- plus 2,4,5,2',6'-, and 2,3,6,2',4'- chlorobiphenyls), regardless of the HACs. These congeners are known to have low threshold concentrations for dechlorination. To a lesser extent, the enhancement also involved meta dechlorination of certain congeners with high threshold concentrations. There was no or less accumulation of 2,4,4'- and 2,5,4'-chlorobiphenyls as final products under HAC amendment. Although the dechlorination products varied, the accumulation of orthosubstituted congeners, 2-, 2,2'-, and 2,6-chlorobiphenyls, was significantly higher with the HACs, indicating a more complete dechlorination of the highly chlorinated congeners. Therefore, the present results suggest that the enhanced dechlorination under HAC enrichment is carried out through multiple pathways, some of which may be universal, regardless of the kind of HACs, whereas others may be HAC-specific.     

Revisiting the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase toward 2,2'-dichlorobiphenyl and 2,3,2',3'-tetrachlorobiphenyl

2,2'-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2'-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2',3'-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2'-CB and 2,3,2',3'-CB, thus providing new insights on the mechanism by which 2,2'-CB is dehalogenated to generate 2,3-dihydroxy-2'-chlorobiphenyl. We reacted 2,2'-CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2'-CB-d(8) was determined to be a dihydroxychlorobiphenyl-d(7) by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2',3'-CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2'-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2'-CB and 2,3,2,',3'-CB disfavors the dioxygenation of the chlorine-free ortho-meta carbons 5 and 6 for both congeners.     

Polychlorinated biphenyls (PCBs): mutagenicity and carcinogenicity

The potential mutagenicity and carcinogenicity of commercial PCBs has been investigated in both in vivo and in vitro systems and several conclusions can be drawn from these studies. (1) PCBs can covalently adduct DNA both in vivo and in vitro (using a source of metabolic activation); the more highly chlorinated biphenyls are poorly metabolized and these compounds tend to exhibit very low binding to DNA. Based on the structure-activity relationships for PCBs (Safe, 1984) it is unlikely that the more toxic compounds such as 3,3',4,4',5-penta- and 3,3',4,4',5,5'-hexachlorobiphenyl, would form covalent adducts with DNA. (2) PCB mixtures and individual compounds exhibit minimal mutagenic activity in most assay systems. (3) The more highly chlorinated PCB mixtures (i.e. greater than 50% Cl by weight) are hepatocarcinogens in rodents whereas data from a limited number of studies suggest that the lower chlorinated mixtures are not carcinogenic. (4) In some model systems, the higher chlorinated PCB mixtures act as promoters of preneoplastic lesions and hepatocellular carcinomas in rodents treated with a variety of initiators. (5) Aroclor 1254 acts as a promoter of skin papilloma formation in HRS/J hairless mice and structure-activity and genetic studies suggest that the Ah receptor is necessary but not sufficient for the activity of halogenated aryl hydrocarbons as promoters in hairless mice. (6) Individual PCB congeners and higher chlorinated commercial mixtures also exhibit anti-carcinogenic activity in the CD-1 mouse skin cancer model. (7) Results from occupational studies suggest that individuals exposed to PCBs may have an excess of cancer at some sites, however, the most comprehensive study (Brown, 1987) suggests that there are no significant increases in the overall cancer rate in workers exposed to PCBs. Follow-up and continuing epidemiological studies on the PCB-exposed workers are required to further clarify the potential carcinogenic effects of PCBs on humans. In several strains of rats and mice, there is a high incidence of hepatic preneoplastic lesions and carcinomas and these lesions can be induced by diverse promoting agents (Schulte-Hermann et al., 1983; Weinstein, 1984). Since PCBs are not mutagenic and do not readily form covalent adducts with cellular DNA, it is likely that the higher chlorinated biphenyls are not genotoxic and act as promoters of carcinogenesis in rodents. A comparable mechanism has been suggested for 2,3,7,8-TCDD (Shu et al., 1987; Weinstein, 1984). For PCBs, the role of the Ah receptor in mediating their activity as promoters has not been delineated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid assay for screening and characterizing microorganisms for the ability to degrade polychlorinated biphenyls

We designed a rapid assay that assesses the polychlorinated biphenyl (PCB)-degradative competence and congener specificity of aerobic microorganisms, identifies strains capable of degrading highly chlorinated biphenyls, and distinguishes among those that degrade PCBs by alternative pathways. Prior attempts to assay PCB-degradative competence by measuring disappearance of Aroclors (commercial PCB mixtures) have frequently produced false-positive findings because of volatilization, adsorption, or absorption losses. Furthermore, these assays have generally left the chemical nature of the competence obscure because of incomplete gas chromatographic resolution and uncertain identification of Aroclor peaks. We avoided these problems by using defined mixtures of PCB congeners and by adopting incubation and extraction methods that prevent physical loss of PCBs. Our assay mixtures include PCB congeners ranging from dichloro- to hexachlorobiphenyls and representing various structural classes, e.g., congeners chlorinated on a single ring (2,3-dichlorobiphenyl), blocked at 2,3 sites (2,5,2'5'-tetrachlorobiphenyl), blocked at 3,4 sites (4,4'-dichlorobiphenyl), and lacking adjacent unchlorinated sites (2,4,5,2',4',5'-hexachlorobiphenyl). The PCB-degrative ability of microorganisms is assessed by packed-column gas chromatographic analysis of these defined congener mixtures following 24-h incubation with resting cells. When tested with 25 environmental isolates, this assay revealed a broad range of PCB-degradative competence, highlighted differences in congener specificity and in the extent of degradation of individual congeners, predicted degradative competence on commercial PCBs, and (iv) identified strains with superior PCB-degradative ability.     

Steady-state kinetic characterization and crystallization of a polychlorinated biphenyl-transforming dioxygenase

The oxygenase component of biphenyl dioxygenase (BPDO) from Comamonas testosteroni B-356 dihydroxylates biphenyl and some polychlorinated biphenyls (PCBs), thereby initiating their degradation. Overexpressed, anaerobically purified BPDO had a specific activity of 4.9 units/mg, and its oxygenase component appeared to contain a full complement of Fe(2)S(2) center and catalytic iron. Oxygenase crystals in space group R3 were obtained under anaerobic conditions using polyethylene glycol as the precipitant. X-ray diffraction was measured to 1.6 A. Steady-state kinetics assays demonstrated that BPDO had an apparent k(cat)/K(m) for biphenyl of (1.2 +/- 0.1) x 10(6) M(-1) s(-1) in air-saturated buffer. Moreover, BPDO transformed dichlorobiphenyls (diClBs) in the following order of apparent specificities: 3,3'- > 2,2'- > 4, 4'-diClB. Strikingly, the ability of BPDO to utilize O(2) depended strongly on the biphenyl substrate: k(cat)/K(m(O(2))) = (3.6 +/- 0. 3), (0.06 +/- 0.02), and (0.4 +/- 0.07) x 10(5) M(-1) s(-1) in the presence of biphenyl and 2,2'- and 3,3'-diClBs, respectively. Moreover, biphenyl/O(2) consumed was 0.97, 0.44, 0.63, and 0.48 in the presence of biphenyl and 2,2'-, 3,3'-, and 4,4'-diClBs, respectively. Within experimental error, the balance of consumed O(2) was detected as H(2)O(2). Thus, PCB congeners such as 2, 2'-diClB exact a high energetic cost, produce a cytotoxic compound (H(2)O(2)), and can inhibit degradation of other congeners. Each of these effects would be predicted to inhibit the aerobic microbial catabolism of PCBs.     

Isolation and Characterization of a Mixed Culture That Degrades Polychlorinated Biphenyls

A mixed culture composed of two Pseudomonas strains, designated as KKL101 and KKS102, was isolated from soil. This mixed culture had an enhanced ability to degrade various polychlorinated biphenyls (PCBs) which include highly chlorinated components. They did not grow individually on the mineral salts medium supplemented with a highly chlorinated PCB (PCB48, a mixture of mainly tetrachlorobiphenyl) and biphenyl. When the spent medium of KKL101 was added to the washed cell preparation of KKS102, however, the latter grew on these carbon sources, producing yellow compounds which were identified as metabolic intermediates of the carbon sources, biphenyl and PCBs. These results suggest that KKL101 produces a growth factor(s) essential for KKS102 to grow on PCBs and that the growth of KKL101 is supported by the metabolic intermediates produced by KKS102. It appears that these two bacterial strains have a symbiotic relationship. From the analysis of the degradation products of various PCB congeners, it was found that strain KKS102 degrades a wide range of PCBs which have been considered to be refractory to biological degradation.