Anaemia, hypothyroidism and immune suppression associated with polychlorinated biphenyl exposure in bottlenose dolphins (Tursiops truncatus)

Polychlorinated biphenyls (PCBs), persistent chemicals widely used for industrial purposes, have been banned in most parts of the world for decades. Owing to their bioaccumulative nature, PCBs are still found in high concentrations in marine mammals, particularly those that occupy upper trophic positions. While PCB-related health effects have been well-documented in some mammals, studies among dolphins and whales are limited. We conducted health evaluations of bottlenose dolphins (Tursiops truncatus) near a site on the Georgia, United States coast heavily contaminated by Aroclor 1268, an uncommon PCB mixture primarily comprised of octa- through deca-chlorobiphenyl congeners. A high proportion (26%) of sampled dolphins suffered anaemia, a finding previously reported from primate laboratory studies using high doses of a more common PCB mixture, Aroclor 1254. In addition, the dolphins showed reduced thyroid hormone levels and total thyroxine, free thyroxine and triiodothyronine negatively correlated with PCB concentration measured in blubber (p = 0.039, < 0.001, 0.009, respectively). Similarly, T-lymphocyte proliferation and indices of innate immunity decreased with blubber PCB concentration, suggesting an increased susceptibility to infectious disease. Other persistent contaminants such as DDT which could potentially confound results were similar in the Georgia dolphins when compared with previously sampled reference sites, and therefore probably did not contribute to the observed correlations. Our results clearly demonstrate that dolphins are vulnerable to PCB-related toxic effects, at least partially mediated through the endocrine system. The severity of the effects suggests that the PCB mixture to which the Georgia dolphins were exposed has substantial toxic potential and further studies are warranted to elucidate mechanisms and potential impacts on other top-level predators, including humans, who regularly consume fish from the same marine waters.     

Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of PCB phytoremediation

The aim of this work is to increase the efficiency of the biodegradation of polychlorinated biphenyls (PCBs) by the introduction of bacterial genes into the plant genome. For this purpose, we selected the bphC gene encoding 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas testosteroni B-356 to be cloned into tobacco plants. The dihydroxybiphenyldioxygenase enzyme is the third enzyme in the biphenyl degradation pathway, and its unique function is the cleavage of biphenyl. Three different constructs were designed and prepared in E. coli: the bphC gene being fused with the beta-glucuronidase (GUS) gene, with the luciferase (LUC) gene, and with histidine tail in three separate plant cloning vectors. The GUS and LUC genes were chosen because they can be used as markers for the easy detection of transgenic plants, while histidine tail better enables the isolation of protein expressed in plant tissue. The prepared vectors were then introduced into cells of Agrobacterium tumefaciens. The transient expression of the prepared genes was first studied in cells of Nicotiana tabacum. Once this ability had been established, model tobacco plants were transformed by agrobacterial infection with the bphC/GUS, bphC/LUC, and bphC/His genes. The transformed regenerants were selected on media using a selective antibiotic, and the presence of transgenes and mRNA was determined by PCR and RT-PCR. The expression of the fused proteins BphC/GUS and BphC/LUC was confirmed histochemically by analysis of the expression of their detection markers. Western blot analysis was performed to detect the presence of the BphC/His protein immunochemically using a mouse anti-His antibody. Growth and viability of transgenic plants in the presence of PCBs was compared with control plants.     

Determinants of Serum PCBs in Adolescents and Adults: Regression Tree Analysis and Linear Regression Analysis

Regression tree analysis, a non-parametric method, was undertaken to identify predictors of the serum concentration of polychlorinated biphenyls (sum of marker PCB ¹ ABBREVIATIONS: BMI: body-mass index, CV: cross validation, ln: natural logarithm, ns: not significant, PCAHs: polychlorinated aromatic hydrocarbons, PCBs: polychlorinated biphenyls, R² _a: adjusted coefficient of determination, VIF: variance inflation factor. View all notes 138, 153, and 180) in humans. This method was applied on biomonitoring data of the Flemish Environment and Health study (2002–2006) and included 1679 adolescents and 1583 adults. Potential predictor variables were collected via a self-administered questionnaire, assessing information on lifestyle, food intake, use of tobacco and alcohol, residence history, health, education, hobbies, and occupation. Relevant predictors of human PCB exposure were identified with regression tree analysis using ln-transformed sum of PCBs, separately in adolescents and adults. The obtained results were compared with those from a standard linear regression approach. The results of the non-parametric analysis confirm the selection of the covariates in the multiple regression models. In both analyses, blood fat, gender, age, body-mass index (BMI) or change in bodyweight, former breast-feeding, and a number of nutritional factors were identified as statistically significant predictors in the serum PCB concentration, either in adolescents, in adults or in both. Regression trees can be used as an explorative analysis in combination with multiple linear regression models, where relationships between the determinants and the biomarkers can be quantified.     

An environmentally-relevant mixture of organochlorines and its vehicle control, dimethylsulfoxide, induce ultrastructural alterations in porcine oocytes

Organochlorine chemicals accumulate in the environment, particularly in the Arctic, and constitute potential developmental hazards to wildlife and human health. Although some of their harmful effects are recognized, their mechanisms of action within the target cells need to be better understood. This study was designed to test the hypothesis that an environmentally-relevant organochlorine mixture alters oocyte ultrastructure in the porcine model. Immature cumulus-oocyte complexes (COCs), partially cultured (18 hr) COCs without treatment or exposed to the organochlorine mixture or its vehicle (0.1% dimethysulfoxide; DMSO) during culture were processed for light and transmission electronic microscopy (TEM). The organochlorines induced major ultrastructural changes in the COCs: decreased density of the lipid droplets, increased smooth endoplasmic reticulum (SER) volume and increased interactions among SER, mitochondria, lipid droplets and vesicles. We suggest that these ultrastructural changes facilitate energy formation necessary to produce metabolizing enzymes. Other ultrastructural changes may reflect some degree of organochlorine toxicity: fewer gap junctions and decreased electron density of the cortical granules. Unexpectedly, the DMSO control treatment also induced similar ultrastructural changes, but to a lesser degree than the organochlorine mixture. This study is the first to demonstrate the effect of environmental contaminants on mammalian oocyte ultrastructure.     

Direct evidence that an arene oxide is a metabolic intermediate of 2,2',5,5'-tetrachlorobiphenyl.

Radiolabeled arene oxide was recovered from incubations containing [³H]-2,2′,5,5′-tetrachlorobiphenyl (³H-TCB), unlabeled 2,2′,5,5′-tetrachlorobiphenyl-3,4-oxide (TCBAO), 3,3,3-trichloropropene-1,2-oxide (TCPO), NADPH, and liver microsomes from phenobarbital-induced rats. No labeled arene oxide was generated in the absence of NADPH, nor during the metabolism of unlabeled TCB in the presence of [³H]-H₂O. The recovered oxide (radiolabeled and carrier) was characterized by mobility on silica gel and by conversion to 3- and 4-hydroxy-TCB. Formation of a dihydrodiol metabolite was apparently blocked by inhibition of epoxide hydrase. These data provide the first direct evidence that arene oxides are intermediates of halogenated biphenyl metabolism.     

Association between exudates of brown algae and polychlorinated biphenyls

Exudates from the brown algaeCaepidium antarcticum andDesmarestia sp. were investigated for their ability to associate with hydrophobic pollutants such as polychlorinated biphenyls (PCB s). The percentage of PCB associated with algal exudates ranged from 79% for decachlorobiphenyl to 23% for the pentachlorobiphenyl congener No. 95. Exudates from the tested brown algae may therefore alter the bioavailability of PCBs in natural or artificial ecosystems.     

Molecular genetics and evolutionary relationship of PCB-degrading bacteria

Biphenyl-utilizing soil bacteria are ubiquitously distributed in the natural environment. They cometabolize a variety of polychlorinated biphenyl (PCB) congeners to chlorobenzoic acids through a 2,3-dioxygenase pathway, or alternatively through a 3,4-dioxygenase system. Thebph genes coding for the metabolism of biphenyl have been cloned from several pseudomonads. The biochemistry and molecular genetics of PCB degradation are reviewed and discussed from the viewpoint of an evolutionary relationship.Abbreviations BP biphenyl - bph BP/PCB-degradative gene - 23DHBP 2,3-dihydroxybiphenyl - HPDA 2-hydroxy-6-oxo-6-phenylhexa 2,4-dienoic acid - KF707 P. pseudoalcaligenes strain KF707 - LB400 Pseudomonas sp. strain LB400 - PCB polychlorinated biphenyls - Q1 P. paucimobilis strain Q1tod; toluene catabolic gene     

Transient decrease of liver cytosolic glutathione S-transferase activities in rats given 1,2-dibromoethane or CCl4

In vivo treatment of fasted male rats with 1,2-dibromoethane (DBE) (0.4 mmol/kg) or carbon tetrachloride (CCl4) (4 mmol/kg) was found to rapidly alter the activities of liver cytosolic and microsomal glutathione S-transferases. Microsomal activities towards chloro-2,4-dinitrobenzene (CDNB) were increased 2 h after either treatment. Cytosolic activities towards CDNB and 3,4-dichloronitrobenzene (DCNB), but not 1,2-epoxy-3-(p-nitrophenoxy)-propane (ENPP), were selectively and transiently decreased after either treatment. Time course studies in DBE animals indicated that the decrease in cytosolic activity was not evident until 2 h although liver glutathione (GSH) concentrations were diminished within 15 min. In contrast, in CCl4 animals the decrease in cytosolic activity was evident within 15 min and was not accompanied by diminished GSH concentrations. By 4 h, cytosolic activities had rebounded to control levels in both DBE and CCl4-treated animals. Kinetic studies of the enzyme in liver cytosol from animals 2 h after treatment with DBE or CCl4 indicated that both treatments decreased the apparent Vmax while neither treatment altered the apparent Km. This pattern of change allows exclusion of a simple competitive mechanism of enzyme inhibition, but cannot distinguish between reversible non-competitive inhibition and irreversible inhibition. It is possible that the observed decreases in the activities of the abundant cytosal enzyme are due to 'sacrificial' covalent linkages between the enzyme and reactive metabolites of DBE or CCl4.