Preliminary risk assessment of the wet landscape option for reclamation of oil sands mine tailings: bioassays with mature fine tailings pore water.

Chemical and biological assays have been carried out on the "pore water" that results from the settling of the tailings that accompany bitumen recovery from the Athabasca oil sands. Examination of the nonacidic extracts of pore water by gas chromatography-mass spectroscopy allowed the identification of numerous two- to three-ring polycyclic aromatic compounds (PACs), to a total concentration of 2.6 micrograms/L of pore water. The PACs were biodegraded by microflora naturally present in the pore water. Acute toxicity was associated principally with the acidic fraction (naphthenic acids) of pore water extracts according to the Microtox assay; other work has shown that acute toxicity dissipates fairly rapidly. Both individual PACs and concentrated pore water extracts showed minimal levels of binding to the rat Ah receptor and induced minimal ethoxyresorufin-O-deethylase activity in primary rat hepatocytes, showing an insignificant risk of inducing monooxygenase activity. Taken together with previous work showing negligible mutagenic activity of these extracts, we conclude that it should be possible to develop tailing slurries into biologically productive artificial lakes.

Application of the ethoxyresorufin-O-deethylase (EROD) assay to mixtures of halogenated aromatic compounds.

The ethoxyresorufin-O-deethylase (EROD) assay monitors the induction of the xenobiotic-metabolizing enzyme cytochrome P-450 (CYP) 1A1 and is a widely used biomarker for exposure of wildlife to substances that bind the aryl hydrocarbon (Ah) receptor. In this work the induction of EROD activity by single compounds and binary mixtures in primary rat hepatocytes was compared with the predictions of a kinetic model involving mixtures of inducers. The inducing agents were also examined for their ability to activate the Ah receptor to its DNA-binding form and for their ability to act as competitive inhibitors for CYP 1A1. Xenobiotics that failed to activate the Ah receptor did not induce EROD activity. Competitive inhibition for CYP 1A1 between the xenobiotic and 7-ethoxyresorufin caused EROD activity to fall at high xenobiotic concentrations. Competition for a limited number of Ah receptor sites depressed the EROD activity of a strong inducer such as 2,3,7,8-tetrachlorodibenzo-p-dioxin at high concentrations of a weak inducer. Application of the kinetic model to the example of a mixture of low concentrations of dibenzo-p-dioxins and much higher concentrations of polychlorinated biphenyls indicated that EROD assays often seriously underestimate the true potency of an environmental sample. Hence the EROD assay cannot be used for determining dioxin equivalent concentrations using the toxic equivalence factor approach.

Electrochemical treatment of 2,4,6-trinitrotoluene and related compounds.

This work involves electrolysis of nitrotoluene congeners, which are persistent pollutants that enter the environment as a consequence of their manufacture and use as explosives. Reduction to aminotoluenes occurred with high current efficiency at a variety of cathodes, at potentials -0.5 to -1 V vs SCE. The products were formed in high chemical yield and with excellent mass balance. Preliminary experiments were also carried out to find methods of removing the electrolysis products from solution by oxidative oligomerization. The most satisfactory method was partial reoxidation at a Ti/IrO2 anode, suggesting an overall remediation technology in which reduction is followed by reoxidation of the spent catholyte in the anode compartment of the same electrolytic cell.

Treatment methods for the remediation of nitroaromatic explosives.

The production and use of nitroaromatic explosives for military operations have resulted in their dissemination into the environment, where their presence in waterways and soil poses an ecological and health hazard. This paper reviews technologies that are available or under investigation to remediate areas contaminated with these compounds.

Synthesis of polybrominated diphenyl ethers and their capacity to induce CYP1A by the Ah receptor mediated pathway.

Polybrominated diphenyl ethers (PBDEs) have become widely distributed as environmental contaminants due to their use as flame retardants. Their structural similarity to other halogenated aromatic pollutants has led to speculation that they might share toxicological properties such as hepatic enzyme induction. In this work we synthesized a number of PBDE congeners, studied their affinity for rat hepatic Ah receptor through competitive binding assays, and determined their ability to induce hepatic cytochrome P-450 enzymes by means of EROD (ethoxyresorufin-O-deethylase) assays in human, rat, chick, and rainbow trout cells. Both pure PBDE congeners and commercial PBDE mixtures had Ah receptor binding affinities 10(-2)-10(-5) times that of 2,3,7,8-tetrachlorodibenzo-p-dioxin. In contrast with polychlorinated biphenyls, Ah receptor binding affinities of PBDEs could not be related to the planarity of the molecule, possibly because the large size of the bromine atoms expands the Ah receptor's binding site. EROD activities of the PBDE congeners followed a similar rank order in all cells. Some congeners, notably PBDE 85, did not follow the usual trend in which strength of Ah receptor binding affinity paralleled P-450 induction potency. Use of the gel retardation assay with a synthetic oligonucleotide indicated that in these cases the liganded Ah receptor failed to bind to the DNA recognition sequence.

Electrochemical treatment of acidic aqueous ferrous sulfate and copper sulfate as models for acid mine drainage.

Acid mine drainage (AMD) is a serious environmental problem in the mining industry. The present work describes electrolytic reduction of solutions of synthetic AMD, comprising FeSO4/H2SO4 and CuSO4/H2SO4, in flow-through cells whose anode and cathode compartments were separated using ion exchange membranes. In the case of FeSO4/H2SO4 at constant flow rate, the pH of the effluent from the catholyte increased progressively with current at a variety of cathodes, due to electrolytic reduction of H+ ions to elemental hydrogen. Near-quantitative removal of iron was achieved by sparging air into the catholyte effluent, thereby precipitating iron outside the electrochemical cell, and avoiding fouling of the electrodes. The anode reaction was the oxidation of water to O2, a proton-releasing process. Using cation exchange membranes and sodium sulfate as the supporting electrolyte in the anode compartment, the efficiency of the process was compromised at high currents by transport of H+ competitively with Na+ from the anode to the cathode compartments. Higher efficiencies were obtained when anion exchange membranes were used, and in this case no additional supporting electrolyte other than dilute H2SO4 was needed, the net reaction being the electrochemically driven transfer of the elements of H2SO4 from the cathode to the anode compartments. Current efficiencies approximately 50% were achieved, the loss of efficiency being accounted for by ohmic heating of the solutions. In the case of CuSO4/H2SO4 and anion exchange membranes at high currents, reduction of Cu2+ and H+ ions and transport of SO4(2-) ions out of the catholyte caused unacceptably high potentials to be generated.

Competitive behavior in the interactive toxicology of halogenated aromatic compounds.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that binds and mediates responses to many halogenated aromatic compounds (HACs). Exposure to mixtures of HACs frequently results in nonadditive behavior in both in vivo and in vitro assays. One cause is antagonism, which results when two or more ligands compete for a limited supply of the AhR; one interacts agonistically to induce a strong response, and the other interacts unproductively, eliciting little or no response. This study involves the mechanism by which HACs induce CYP 1A1. Agonistic (e.g., TCDD) and unproductive (e.g., PCB 153) HACs behaved similarly through the stages of initial AhR binding and conversion of the initial AhR-ligand complex to the form that possesses increased affinity for the bound ligand. They diverged in the ability of the AhR-HAC complex to bind to a synthetic oligonucleotide containing the consensus dioxin response enhancer sequence, as studied by the gel retardation assay. Competition for the Ah receptor was used to explain antagonistic behavior between TCDD and other HACs in both the gel retardation assay and the downstream response of CYP 1A1 induction in primary rat hepatocytes.

Metabolism of polychlorinated dibenzo-p-dioxins by rat liver microsomes.

The in vitro metabolism of several chlorinated dibenzo-p-dioxin congeners (PCDDs) was studied using rat liver microsomes as a source of CYP 1 enzymes. The reactions were kinetically first order in both enzyme and substrate and showed a general trend toward decreasing reactivity with increasing chlorination. Michaelis-Menten kinetics were followed for 1-chlorodibenzo-p-dioxin (1-CDD); the reactivity of the enzyme preparation toward 1-CDD exactly paralleled its activity toward 7-ethoxyresorufin. The unreactive congeners 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) and 2,2'-dichlorobiphenyl (2,2'-DCB) acted as competitive inhibitors toward 1-CDD, with inhibition constants in the micromolar range, similar to the value of the Michaelis constant of 1-CDD. The inhibitory potency of furafylline, a mechanism-based inhibitor that is selective for CYP 1A2, declined in the order acetanilide (standard) > 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) > 1-CDD. We conclude that 1-CDD and 1,2,3,4-TCDD are oxidized almost exclusively by CYP 1A1, whereas 2,3,7,8-TCDD and 1,2,4,7,8-PeCDD are oxidized mainly by CYP 1A2. 1,2,3,7,8-PeCDD was oxidized too slowly for us to reach any conclusion about the P450 isozyme responsible.

Competitive inhibition by inducer as a confounding factor in the use of the ethoxyresorufin-O-deethylase (EROD) assay to estimate exposure to dioxin-like compounds.

The ethoxyresorufin-O-deethylase (EROD) assay has been extensively used in whole animals and in cell culture as a biomarker of exposure to environmental contaminants such as dioxin-like compounds (DLCs). This paper addresses two controversial phenomena that arise when DLCs are examined by the EROD assay. Firstly, the maximum level of induced EROD activity varies with the identity of the inducing compound; secondly, the induced EROD activity reaches a concentration-dependent maximum level that is followed by an apparent reduction in activity when the concentration of inducer is further increased. These phenomena are completely explained by competitive inhibition of the EROD enzyme-substrate reaction by the dioxin-like compound. A kinetic model explains the biphasic appearance of EROD induction curves as a function of a compound's binding affinity with the Ah receptor (Kd) and its binding affinity to CYP 1A1 (Ki) which results in inhibition of the EROD enzyme-substrate reaction. These results limit the reliability of the information obtained from calibration curves of EROD activity versus concentration of a standard DLC such as 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Metabolism of polychlorinated dibenzo-p-dioxins and related dioxin-like compounds.

Recent work on the metabolism of polychlorinated dibenzo-p-dioxins and related compounds such as polychlorinated dibenzofurans and coplanar polychlorinated biphenyls is reviewed. Most in vivo studies and epidemiological studies on humans have been concerned with rates of elimination of these compounds; in vitro work has shown that a wide variety of metabolites can be found, either in hydroxylated format (typically in feces) or as conjugates (in urine). Metabolism of this group of compounds seems always to represent a detoxification process rather than one of bioactivation.

Gel-filtration chromatographic method for determining relative binding affinities: rat hepatic estrogen receptor as an example system.

We report a gel-filtration-based chromatographic method for separation of specific, nonspecific, and free radioligand in a protein receptor-ligand binding assay for the example of the estrogen receptor ERalpha. This assay affords relative binding affinities (RBAs) without the need for a separate determination of nonspecific binding. The probit method is recommended as the most satisfactory method of evaluating the data. The assay responds to both estrogen agonists and antagonists, mixtures respond additively, and the slopes of the probit plots indicate that all ligands bind to the same site on the estrogen receptor. RBAs obtained with rat and rainbow trout ERalpha were in good agreement, and also with those from other reported assays, consistent with the interspecies conservation of key regions of the ligand binding domain among estrogen receptors.

Characterization of the Ah receptor from human placental tissue.

The rate of thermal inactivation of the unliganded human Ah receptor, studied by sucrose density gradient centrifugation, with respect to loss of ligand binding ability, was found to be greater than those of most rodents at 20 degrees C, but the temperature coefficient of the rate constant was much smaller than for the rodent species. This implies that the unliganded human Ah receptor would be thermally more stable than the rodent analogs at physiological temperatures. The liganded form of the human Ah receptor was found to be less stable with respect to ligand release than the rodent receptors. These differences in behavior between human and rodent Ah receptors underline the difficulties in using rodent data in the development of receptor-based models of dioxin toxicity. Attempts to develop an alternative to sucrose density gradient centrifugation, comparable with the hydroxylapatite adsorption method used to assay rodent hepatic Ah receptor, were unsuccessful.

Additive binding of polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin to the murine hepatic Ah receptor.

Fixed aliquots of both radiolabeled [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and hepatic Ah receptor from C57BL/6J mice were incubated competitively at 4, 23, and 30 degrees C with mixtures of 2,3,7,8-TCDD and several polychlorinated biphenyls (PCBs). The production of the radiolabeled receptor-ligand complex changed if the ligands were added sequentially, demonstrating that the competition between PCBs and TCDD for the Ah receptor in vitro is principally a kinetic rather than an equilibrium phenomenon and is irreversible on the time scale of our in vitro experiments. Examination of previous reports on the ability of TCDD, PCBs, and their mixtures to induce cleft palate in fetal mice suggests that the potency of receptor-ligand complexes is ligand-dependent. Receptor occupancy is not a sufficient condition for toxicity, and protection by one ligand against the toxic effect of a second, more potent one is only possible when a significant fraction of receptors is occupied.

Comparative kinetic study of the binding between 2,3,7,8-tetrachlorodibenzo-p-dioxin and related ligands with the hepatic Ah receptors from several rodent species.

Kinetic analysis of the time course of association of [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin with hepatic cytosol from five rodent species gave additional evidence for differences in the properties of the Ah receptor ligand binding subunit between species. A parallel study of the association of six tritiated polychlorinated dibenzo-p-dioxins and dibenzofurans with hepatic Ah receptor from Wistar rat and C57BL/6 mouse showed that their rank order for kinetic affinity did not correlate with the rank ordering of their toxic potency and may vary according to the source of the Ah receptor.

Characterization of a phenobarbital-inducible Ah receptor-like protein in the Sprague-Dawley rat.

Immature male Sprague-Dawley rats were treated with phenobarbital (PB) at a dose of 50 mg/kg for 3 successive days and then sacrificed 1, 6, or 9 days after the last treatment. An increase in the concentration of hepatic cytosolic proteins able to bind specifically to [3h]-2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H])TCDD) was observed. The level of the induced protein fell in parallel with the elimination of PB in vivo. Readministration of PB 9 days after the last treatment, by which time only the constitutive receptor was present, induced additional protein at levels similar to those obtained upon the original administration of the drug. There was considerable variability within a given treatment group in both the ratio of constitutive to induced protein and the total amounts of these proteins. The kinetic properties of the induced protein were significantly different from those of the constitutive Ah receptor. In particular, [3H]TCDD was released faster from the liganded induced protein than from the constitutive Ah receptor. The protein induced by PB had properties very similar to those of the protein induced by a combination of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,2',4,4',5,5'-hexachlorobiphenyl.

Factors affecting the toxicity of dioxin-like toxicants: a molecular approach to risk assessment of dioxins.

The numerous toxic responses of dioxin-like compounds are mediated by the intracellular Ah (aryl hydrocarbon) receptor. It has been suggested that the regulation of dioxins and similar substances could be placed on a molecular foundation by considering the proportion of Ah-receptor sites occupied by toxicant molecules. The present work has shown that the following formation not yet available would be needed in order to develop this approach: correlation between dioxin exposure and human tissue levels; accurate determination of the association constants for human Ah-receptor with toxicant, and for human receptor-ligand complex with DNA; and knowledge of the intracellular concentrations of both receptor binding sites and DNA binding sites. Furthermore, since not all dioxin-like substances behave identically, this information would need to be gathered for a wide variety of substances.

Kinetics of the association of several tritiated polychlorinated dibenzo-p-dioxin and dibenzofuran congeners with hepatic cytosolic Ah receptor from the Wistar rat.

Several tritiated chlorinated dibenzo-p-dioxins and dibenzofurans have been prepared with specific activities in the range of 30-50 Ci/mmol in order to investigate the effects of structure on the kinetics of their association with the rat cytosolic Ah receptor. The compounds were 2,3,7-trichlorodibenzo-p-dioxin (TrCDD), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-pentchlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,7,8-TCDF, and 1,2,3,7,8-pentachlorodibenzofuran (PeCDF). Although these congeners differed by up to 2 orders of magnitude in their biochemical and toxic potencies, their affinities for the Ah receptor as determined by conventional Scatchard analysis varied by less than 2-fold (range of KD values 5.0-9.3 nM). The rate of association of these ligands with the Ah receptor was studied at several temperatures, taking into account the competing thermal inactivation of the unbound receptor. The equilibrium constants (KD) were also obtained as the ratio of the rate constants for dissociation and formation, respectively, of the receptor-ligand complexes. The following conclusions were derived from the kinetic studies: (1) 2,3,7-TrCDD and 1,2,7,8-TCDF bound significantly more slowly to the Ah receptor than the other radioligands at all temperatures (13.5-37 degrees C), and this paralleled the lower biochemical potencies of the congeners; (2) the KD values obtained kinetically were in the subnanomolar range, with the smallest KD values observed for those ligands which bound most rapidly to the receptor; and (3) the temperature dependence of the KD values indicated that receptor-ligand association was favorable both enthalpically and entropically.