Synthesis of Sterically Hindered Polychlorinated Biphenyl Derivatives.

A series of sterically hindered (methoxylated) polychlorinated biphenyl derivatives was synthesized using the Suzuki and the Ullmann coupling reaction. The Suzuki coupling with Pd(dba)(2)/2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (DPDB) gave better yields (65-98%) compared to the classic Ullmann coupling reaction (20-38%). Despite the reactive catalyst system, no significant coupling with aromatic chlorine substituents was observed. Crystal structure analysis of four PCB derivatives revealed solid state dihedral angles ranging from 69.7° to 81.0°, which indicates that these highly ortho substituted PCB derivatives have some conformational flexibility.

Synthesis and Solid State Structure of Fluorous Probe Molecules for Fluorous Separation Applications.

A series of colored hydrocarbon and fluorocarbon tagged 1-fluoro-4-alkylamino-anthraquinones and 1,4-bis-alkylamino-anthraquinone probe molecules were synthesized from a (fluorinated) alkyl amine and 1,4-difluoroanthraquinone to aid in the development of fluorous separation applications. The anthraquinones displayed stacking of the anthraquinone tricycle and interdigitation of the (fluorinated) alkyl chains in the solid state. Furthermore, intramolecular N-H···O hydrogen bonds forced the hydrocarbon and fluorocarbon tags into a conformation pointing away from the anthraquinone tricycle, with the angle of the tricycle plane normal and the main (fluorinated) alkyl vector ranging from 1 to 39°. Separation of the probe molecules on fluorous silica gel showed that the degree of fluorination of the probe molecules plays only a minor role with most eluents (e.g., hexane-ethyl acetate and methyl nonafluorobutyl ethers-ethyl acetate). However, toluene as eluent caused a pronounced separation by degree of fluorination for fluorocarbon, but not hydrocarbon tagged probe molecules on both silica gel and fluorous silica gel. These studies suggest that hydrocarbon and fluorocarbon tagged anthraquinones are useful probe molecules for the development of laboratory scale fluorous separation applications.

Development of a synthetic PCB mixture resembling the average polychlorinated biphenyl profile in Chicago air.

Studies of environmental and toxic effects of polychlorinated biphenyls (PCBs) are ideally performed with PCB mixtures reflecting the composition of environmental PCB profiles to mimic actual effects and to account for complex interactions among individual PCB congeners. Unfortunately, only a few laboratory studies employing synthetic PCB mixtures have been reported, in part because of the challenges associated with the preparation of complex PCB mixtures containing many individual PCB congeners. The objective of this study was to develop a PCB mixture that resembles the average PCB profile recorded from 1996 to 2002 at a satellite station of the Integrated Atmospheric Deposition Network located at the Illinois Institute of Technology (IIT) in Chicago, Illinois, using commercial PCB mixtures. Initial simulations, using published Aroclor profiles, showed that a mixture containing 65% Aroclor 1242 and 35% Aroclor 1254 was a good approximation of the target profile. A synthetic Chicago air mixture (CAM) was prepared by mixing the respective Aroclors in this ratio, followed by GC/MS/MS analysis. Comparison of the PCB profile of the synthetic mixture with the target profile suggests that the synthetic PCB mixture is a good approximation of the average IIT Chicago air profiles (similarity coefficient cos θ = 0.82; average relative percent difference = 84%). The synthetic CAM was also a reasonable approximation of the average of 184 PCB profiles analyzed in 2007 at 37 sites throughout Chicago as part of the University of Iowa Superfund Basic Research Program (isbrp), with a cos θ of 0.70 and an average relative percent difference of 118%. While the CAM and the two Chicago air profiles contained primarily di- to pentachlorobiphenyls, higher chlorinated congeners, including congeners with seven or eight chlorine atoms, were underrepresented in the synthetic CAM. The calculated TCDD toxic equivalency quotients of the synthetic CAM (2.7 ng/mg PCB) and the IIT Chicago air profile (1.6 ng/mg PCB) were comparable, but lower by two orders of magnitude than the isbrp Chicago air profile (865 ng/mg PCB) due to surprisingly high PCB 126 levels in Chicago air. In contrast, the calculated neurotoxic equivalency quotients of the CAM (0.33 mg/mg PCB) and the two Chicago air profiles (0.44 and 0.30 mg/mg PCB, respectively) were similar. This study demonstrates the challenges and methods of creating and characterizing synthetic, environmental mixtures of PCBs.

Influence of dietary fat on the enantioselective disposition of 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) in female mice.

Although ingestion is the major route of exposure to polychlorinated biphenyls (PCBs), dietary factors altering their absorption and excretion are only poorly understood. In the present study, (+/-)-PCB 136 was administered orally to female C57BL/6 mice fed an unrefined (URD, 10% fat) or high fat (HFD, 40% fat) diet to investigate the effect of the dietary fat content on the disposition of PCBs. Three days after administration, PCB levels in the adipose tissue were significantly lower in HFD animals than URD animals, partly due to a higher excretion rate of PCB 136 in the HFD group. (+)-PCB 136 was enriched in all organs and in feces. In both groups, enantiomeric fractions in feces increased each day after administration. We hypothesize that low EF (enantiomeric fraction) values in feces excreted within 24h of exposure are due to the presence of undigested, racemic PCB. Higher EF values in feces excreted after two and three days are due to excretion of previously absorbed PCBs. Overall, our study suggests that the EF value may be a good tool to investigate the absorption and excretion of PCBs in vivo.

Effect of potassium perfluorooctanesulfonate, perfluorooctanoate and octanesulfonate on the phase transition of dipalmitoylphosphatidylcholine (DPPC) bilayers.

Perfluorooctanesulfonic acid (PFOS) is a persistent environmental pollutant that may cause adverse effects by inhibiting pulmonary surfactant. To gain further insights in this potential mechanism of toxicity, we investigated the interaction of PFOS potassium salt with dipalmitoylphosphatidylcholine (DPPC) - the major component of pulmonary surfactant - using steady-state fluorescence anisotropy spectroscopy and DSC (differential scanning calorimetry). In addition, we investigated the interactions of two structurally related compounds, perfluorooctanoic acid (PFOA) and octanesulfonic acid (OS) potassium salt, with DPPC. In the fluorescence experiments a linear depression of the main phase transition temperature of DPPC (T(m)) and an increased peak width was observed with increasing concentration of all three compounds, both using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) as fluorescent probes. PFOS caused an effect on T(m) and peak width at much lower concentrations because of its increased tendency to partition onto DPPC bilayers, i.e., the partition coefficients decrease in the K(PFOS)>K(PFOA)>>K(OS). Similar to the fluorescence anisotropy measurements, all three compounds caused a linear depression in the onset of the main phase transition temperature and a significant peak broadening in the DSC experiments, with PFOS having the most pronounced effect of the peak width. The effect of PFOS and other fluorinated surfactants on DPPC in both mono- and bilayers may be one mechanism by which these compounds cause adverse biological effects.

Mixing of perfluorooctanesulfonic acid (PFOS) potassium salt with dipalmitoyl phosphatidylcholine (DPPC).

Perfluorooctane-1-sulfonic acid (PFOS) is emerging as an important persistent environmental pollutant. To gain insight into the interaction of PFOS with biological systems, the mixing behavior of dipalmitoylphosphatidylcholine (DPPC) with PFOS was studied using differential scanning calorimetry (DSC) and fluorescence anisotropy measurements. In the DSC experiments the onset temperature of the DPPC pretransition (Tp) decreased with increasing PFOS concentration, disappearing at XDPPC < or = 0.97. The main DPPC phase transition temperature showed a depression and peak broadening with increasing mole fraction of PFOS in both the DSC and the fluorescence anisotropy studies. From the melting point depression in the fluorescence anisotropy studies, which was observed at a concentration as low as 10 mg/L, an apparent partition coefficient of K = 5.7 x 10(4) (mole fraction basis) was calculated. These results suggest that PFOS has a high tendency to partition into lipid bilayers. These direct PFOS-DPPC interactions are one possible mechanism by which PFOS may contribute to adverse effects, for example neonatal mortality, in laboratory studies and possibly in humans.

Mixing of perfluorinated carboxylic acids with dipalmitoylphosphatidylcholine.

Perfluorinated acids are emerging as an important class of persistent environmental pollutant, thus raising human health concerns. To understand the behavior of these compounds in biological systems, the mixing behavior of two perfluorinated acids, perfluorododecanoic and perfluorotetradecanoic acid, with dipalmitoylphosphatidylcholine (DPPC) was studied in monolayers at the air-water interface and in fully hydrated DPPC bilayers. The mixing behavior of both acids was indicative of an attractive interaction and partial miscibility with DPPC at the air-water interface. In the bilayer studies, the fluorinated acids cause peak broadening and elimination of the pretransition of DPPC. The onset temperature of the main phase transition remains constant in the presence of the fluorinated acids suggesting immiscibilities in the gel phase. Below X(DPPC) = 0.97 significant peak broadening of the main phase transition can be observed. These results suggest strong interaction between the respective acid and DPPC, and that both acids are able to partition into the lipid bilayer. However, their mixing behavior is far from ideal, thus suggesting the presence of domains or lipid aggregates with high acid concentrations which may (adversely) impact the function of biological mono- and bilayers.

Synthesis of hydroxylated PCB metabolites with the Suzuki-coupling.

An improved synthesis of hydroxylated polychlorinated biphenyls (PCBs) which are structurally related to the major hydroxy PCB congeners identified in human plasma is described. The coupling of (chlorinated) aryl boronic acids with bromochloro anisoles using the standard conditions of the Suzuki coupling gave the desired hydroxylated PCB metabolites in good to excellent yields. The approach offers the advantage of high selectivity and good yields compared to conventional methods such as the Cadogan reaction and allows the use of less toxic starting materials.

Synthesis of polychlorinated biphenyls (PCBs) using the Suzuki-coupling.

An improved synthesis of polychlorinated biphenyls (PCBs) utilizing a palladium-catalyzed cross-coupling reaction (Suzuki-coupling) is described. The coupling of (chlorinated) aryl boronic acids 1-3 with bromochlorobenzenes 4 using the standard conditions of the Suzuki-coupling gave the desired PCB congeners 5-7 in good to excellent yields. The self-coupling product of the aryl boronic acids is the major impurity of this reaction. 3,4,5-trichlorophenyl derivatives such as 10 can be synthesized by coupling of an aryl boronic acid with the corresponding bromochloroaniline 8. The approach offers the advantage of high selectivity and good yields compared to conventional methods such as the Cadogan reaction and allows the use of less toxic starting materials.

2,4,4'-trichlorobiphenyl increases STAT5 transcriptional activity.

The promoting effects of polychlorinated biphenyls (PCBs) have been studied extensively in a variety of two-stage carcinogenesis models. However, the molecular mechanisms responsible for the promotion effects of PCBs have not been elucidated. We measured the effect of PCBs on DNA-binding proteins involved in cell proliferation and transformation. Male Sprague-Dawley rats were injected intraperitoneally with mono-, di-, tri-, tetra-, or hexachlorobiphenyls (300 micromol/kg/d) each day for 4 d and killed 4 h after the last injection. To detect alterations in nuclear proteins that could explain the tumor-promoter activity of PCBs, liver nuclear extracts were analyzed by electrophoretic mobility shift assays. Electrophoretic mobility shift assay analysis of signal transducers and activators of transcription (STAT)-binding activity to a consensus gamma-interferon-activated sequence (GAS) element was compared in liver nuclear extracts from treated rats. STAT-binding activity was eightfold to tenfold higher in nuclear extracts from animals treated with 2,4,4'-trichloro- (PCB 28) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153). Analysis of the protein complex binding to the GAS element, with antibodies specific for STAT3, STAT5, and STAT6, indicated that the protein complex was made up of STAT5 and STAT6 proteins. HepG2 cells transiently transfected with a luciferase reporter gene construct containing many STAT5 binding sites were treated with PCB 28 and PCB 153. PCB 28 stimulated a greater than 25-fold increase in luciferase activity at the highest concentration tested, 1.0 microg/mL. However, enhanced luciferase activity did not occur with PCB 153 treatment. 4-Chlorobiphenyl (PCB 3), PCB 28, and PCB 153 treatment of Sprague-Dawley rats resulted in a large increase in protein binding to a consensus activated protein-1 (AP-1) element. However, 3,4-dichlorobiphenyl (PCB 12) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77) treatments did not increase AP-1 transcription activity. Further analysis of the proteins binding to the AP-1 consensus sequence with antibodies specific for c-fos, junD, and junB indicated that the protein composition consists of junD proteins. These data showed functional differences between noncoplanar and coplanar PCBs with respect to STAT activation and AP-1-DNA binding.

Production of DNA strand breaks in vitro and reactive oxygen species in vitro and in HL-60 cells by PCB metabolites.

PCBs are industrial chemicals that continue to contaminate our environment. They cause various toxic effects in animals and in exposed human populations. The mechanisms of toxicity, however, are not completely understood. PCBs are metabolized by cytochromes P450 to mono- and dihydroxylated compounds. Dihydroxy-PCBs can potentially be oxidized to the corresponding quinones. We hypothesized that reactive oxygen species (ROS) are produced by redox reactions of PCB metabolites. We tested several synthetic dihydroxy- and quinoid-PCBs with 1-3 chlorines for their potential to produce ROS in vitro and in HL-60 human leukemia cells, and DNA strand breaks in vitro. All dihydroxy-PCBs tested produced superoxide. The quinones generated superoxide only in the presence of GSH, probably during the autoxidation of the glutathione conjugates. We observed increased superoxide production with decreasing halogenation. Incubation of dihydroxy-PCBs or PCB quinones + GSH with plasmid DNA resulted in DNA strand break induction in the presence of Cu(II). Tests with various ROS scavengers indicated that hydroxyl radicals and singlet oxygen are likely involved in this strand break induction. Finally, dihydroxy- and quinoid PCBs also produced ROS in HL-60 cells in a dose- and time-dependent manner. We conclude that dihydroxylated PCBs, and PCB quinones after reaction with GSH, produce superoxide and other ROS both in vitro and in HL-60 cells, and oxidative DNA damage in the form of DNA strand breaks in vitro. The reactions seen in vitro and in cells may well be a predictor of the toxicity of PCBs in animals.

Cellular glutathione status modulates polychlorinated biphenyl-induced stress response and apoptosis in vascular endothelial cells.

Exposure to environmental contaminants, such as polychlorinated biphenyls (PCBs), may severely compromise normal function of vascular endothelial cells (EC). We have previously shown that PCB 77 (3,3',4,4'-tetrachlorobiphenyl), an arylhydrocarbon receptor (AhR) agonist, can induce oxidative stress in cultured EC. We now show that PCB 77 can activate EC and induce a cellular stress response that is reflected by the activation of c-Jun N-terminal/stress-activated protein kinases (JNK/SAPK). Our data also suggest that this PCB 77-mediated stress response can be modulated by the intracellular glutathione content. EC treated with buthionine-sulphoximine (BSO), an inhibitor of glutathione synthesis, further enhanced PCB-induced JNK/SAPK activity. This stress response was sustained only in the presence of BSO plus PCB 77. Media supplementation with the glutathione precursor N-acetyl-cysteine (NAC) reduced PCB 77-induced JNK/SAPK. Intracellular glutathione also may be implicated in PCB-induced EC apoptosis. Individual treatment with PCB, BSO, or linoleic acid induced activation of caspase 3. Compared to PCB 77 alone, annexin V activity was further amplified during combined treatment with BSO and PCB 77. DNA fragmentation was mostly observed when cells were treated with both BSO and PCB 77. The caspase 3-specific inhibitor DEVD-CHO protected cells against PCB 77/BSO-mediated apoptosis and inhibited the caspase activity without affecting JNK/SAPK activation or cellular glutathione levels. These results suggest that AhR ligands, such as PCB 77, cause vascular EC dysfunction by modulating intracellular glutathione, which subsequently leads to activation of stress-specific kinases. Furthermore, inhibition of glutathione synthesis by BSO can further potentiate the PCB 77-induced stress response and ultimately lead to apoptotic cell death.

Solubility enhancement of phenol and phenol derivatives in perfluorooctyl bromide.

Perfluorinated solvents are gaining popularity as pulmonary ventilation fluids, but they suffer from poor solvent quality in concurrent drug delivery applications. The present study examines the use of a hydrophobic solubilizing agent capable of interacting with model drug solutes by hydrogen bonding with the purpose of enhancing solubility in perfluorooctyl bromide (PFOB). A series of solubilizing agents containing a ketone carbonyl to act as a hydrogen bond acceptor and a perfluoroalkyl chain to maintain the solubility of the putative complex in PFOB are investigated. The solubility of phenol in PFOB is enhanced to the greatest extent by 1-(4-perfluorobutyl phenyl)-1-hexanone (III) where the ketone carbonyl is protected from the electron withdrawing effects of the perfluorobutyl chain by a phenyl ring. Experiments with solubilizers lacking the ketone group suggest that pi-pi bond interactions of III with phenol do not significantly enhance solubility. For a series of phenol derivatives, a rank-order correlation exists between the magnitude of solubility enhancement by III, as reflected by the calculated association constants, and the Hammett sigma parameter of the phenols. Because the O-methyl-substituted phenols do not have the ability to hydrogen bond, their solubility is not enhanced by the presence of III. The results of the present study indicate that solubility of model drug hydrogen bond donating compounds can be enhanced in PFOB by the presence of fluorocarbon-soluble hydrogen bond acceptors.