A new structure-affinity relationship for TCDD receptor binding

We have investigated the capacity of various indoles to inhibit specific binding of [1,6-³H]2,3,7,8-tetrachlorodibenzo- -dioxin ([³H]TCDD) in rat liver cytosol, as analyzed by electrofocusing in polyacrylamide gels. Of these indoles, indolo[3,2- ]carbazole was the most active. The IC₅₀ value for TCDD receptor binding of indolo[3,2- ]carbazole as well as for 2,3,7,8-tetrachlorodibenzofuran was 3.6 nM. We have also studied the influence on binding exerted by introduction of some substituents on indolo[3,2- ]carbazole. Substitution with methyl groups at the 5 and 11 positions resulted in an increased affinity (IC₅₀ 1.2 nM) for the TCDD receptor as compared to the parent compound. Computer-supported molecular structure studies indicated that if the van der Waals radii of atoms are included, a rectangle of 6.8 × 13.7 Å may account for the binding of high-affinity ligands to the TCDD receptor.     

Gamma aminobutyric acid radioreceptor‐assay a possible biomarker for human exposure to certain agrochemicals

Abstract

Cyclodiene insecticides, hexachlorocyclohexanes, pyrethroids, bicyclophosphates, the bicycloorthocarboxylate insecticides and some of their metabolites and environmental degradation products are central nervous system toxicants with high specific binding affinity to the chloride channel of the γ‐ aminobutyric acid (GABA)_A receptor‐ionophore sites. [³⁵S] tertiary‐butylbicyclophosphorothionate (TBPS) with specific activity higher than 60 Ci/mmole has a high binding affinity to the same sites and is now commercially available and can be used to label the GABA_A receptor for the development of a radioreceptor assay technique. The GABA receptor was prepared by ultra centnfugation and dialysis of brain homogenates of either cow, goat, rat or catfish. The receptor was then labeled with [³⁵S] TBPS and the assay was conducted by measuring the displacement of radioactivity following incubation with samples containing the analytes. A radioreceptor assay protocol was developed to measure the amount of the α‐endosulfan in blood samples. The assay was extremely sensitive, and can detect 0.2 nM of endosulfan at a level equivalent to 0.08 ppb or 8x10^‐11 gm of endosulfan in each ml of the blood samples.     

Observations on overwintering juvenile chinook salmon ( ) exposed to bleached kraft mill effluent in the upper Fraser River, British Columbia

Chlorophenols and chloroguaiacols were quantified in juvenile chinook salmon captured near bleached kraft mills in the upper Fraser River in December 1987. Fish captured in April 1988 showed up to 55-fold induction of hepatic mixed function oxidase activity (EROD), and were contaminated with up to 370 ng·kg⁻¹ of 2,3,7,8-TCDF and 68 ng·kg⁻¹ of 2,3,7,8-TCDD. The implications for the survival of these salmon are uncertain.     

Properties of the 2,3,7,8-TCDD receptor- a QSAR approach

The Ah or 2,3,7,8-TCDD receptor protein plays an important role in mediating the biologic, toxic and genotoxic effects of aryl and halogenated aryl hydrocarbons. An assessment of the physicochemical parameters which facilitate ligand: receptor interactions was determined by multiple parameter linear regression analysis of the relative receptor binding affinities of a series of 7-substituted-2,3-dichlorodibenzo-p-dixoins (Eq 1), 8-substituted-2,3-di (Eq. 2) and 2,3,4-trichlorodibenzofurans (Eq 3). The results demonstrate that substituent lipophilicity (π) and molecular volumes were

(Eq.1)

log (I/EC₅₀) = 1.24 π + 6.10

(Eq.2)

log (I/EC₅₀) = 1.10 π + 5.19

(Eq.3)

log (I/EC₅₀) = 1.09 π + 5.77

the major determinant factors governing interactions between the rat hepatic cytosolic receptor protein and the diverse substituted ligands. It has been shown that there are marked differences in species sensitivity to 2,3,7,8-TCDD (i.e. guinea pig > rat maice > hamster) although hepatic receptor levels in these species are comparable. QSAR analysis of the receptor binding EC₅₀ data for the 7-substituted-2,3-dichlorodibenzo-p-dioxins using rat (Eq. 1), mouse (Eq. 4), guinea pig (Eq. 5) and hamster (Eq. 6) hepatic cytosol demonstrated that there were significant differences in these equations. However it was also noted that

(Eq.4)

log (I/EC₅₀) = 0.95 + 0.93 E_s + 5.28

(Eq.5)

log (I/EC₅₀) = 0.94 + 0.579 σ_p + 6.13

(Eq.6)

log (I/EC₅₀) = 0.70 + 1.227 σ_p + 6.38

physicochemical factors which are important for ligand-receptor interactions were identical for the most sensitive (guinea pig) and least sensitive (hamster) species. These results indicate that events which follow the initial ligand-receptor interaction must be the major factors which determine species sensitivity to 2,3,7,8-TCDD.     

An oxygenated metabolite of benzo[a]pyrene increases hepatic β-oxidation of fatty acids in chick embryos

Polycyclic aromatic hydrocarbons (PAHs) are well-known carcinogens to humans and ecotoxicological effects have been shown in several studies. However, PAHs can also be oxidized into more water soluble-oxygenated metabolites (Oxy-PAHs). The first purpose of the present project was to (1) assess the effects of a mixture containing three parent PAHs: anthracene, benz[a]anthracene, and benzo[a]pyrene versus a mixture of their oxygenated metabolites, namely: anthracene-9,10-dione, benz[a]anthracene-7,12-dione, and 9,10-dihydrobenzo[a]pyrene-7-(8H)-one on the hepatic fatty acid β-oxidation in chicken embryos (Gallus gallus domesticus) exposed in ovo. The second and also main purpose of the project was to (2) assess the effects of the parent PAHs versus their oxy-PAHs analogues when injected individually, followed by (3) additional testing of the individual oxy-PAHs. The hepatic β-oxidation was measured using a tritium release assay with [9,10-³H]-palmitic acid (16:0) as substrate. The result from the first part (1) showed reduced hepatic β-oxidation after exposure in ovo to a mixture of three PAHs, however, increased after exposure to the mixture of three oxy-PAHs compared to control. The result from the second part (2) and also the follow-up experiment (3) showed that 9,10-dihydrobenzo[a]pyrene-7-(8H)-one was the causative oxy-PAH. The implication of this finding on the risk assessment of PAH metabolite exposure in avian wildlife remains to be determined. To the best of our knowledge, no similar studies have been reported.     

Levels and congener profiles of PCBs and PCDD/Fs in blue shark (Prionace glauca) liver from the South-Eastern Mediterranean Sea (Italy)

Liver of blue shark (Prionace glauca) specimens from the South-Eastern Mediterranean Sea were analyzed for the presence of polychlorinated biphenyls (PCBs), including coplanar congeners, polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). PCBs were the dominant chemicals, followed by PCDFs and PCDDs. The pattern of PCB congener concentrations in the hepatic tissue was dominated by higher chlorinated compounds. The specific profile of toxic PCDD/F congeners was characterized mainly by 2,3,7,8-TCDF and 2,3,7,8-TCDD, followed by 1,2,3,6,7,8-HxCDD and 2,3,4,6,7,8-HxCDF. The total 2,3,7,8-TCDD toxic equivalent (TEQs) was 149 pg g⁻¹ lipid wt. The profile of TEQ shows that PCDDs present the greatest risk to this species contributing to total toxicity with a percentage approximately of 60%, while the contribution of PCDFs and DL-PCBs is almost the same being 22.4% and 21.6%, respectively. Further investigations are urgently needed to characterize the PCDD/Fs contamination levels not only in elasmobranch fish but in all Mediterranean marine biota.     

The interaction of 1,3-diaryltriazenes with the receptor

Certain substituted 1,3-diaryltriazenes possessing anorectic activity were evaluated for their ability to compete with ³H-TCDD for specific binding to the receptor. The N-substituent had little effect while aromatic substitution was important in determining binding to the receptor.     

Determination of 2,3,7,8-TCDD toxic equivalent factors (TEFs): support for the use of the in vitro AHH induction assay

The in vitro induction of the cytochrome P₁-450-dependent monooxygenases, aryl hydrocarbon hydroxylase (AHH) or ethoxyresorufin O-deethylase (EROD) by 2,3,7,8-TCDD and related toxic halogenated aryl hydrocarbons in rat hepatoma H-4-II E cells has been developed as a short term quantitative bioassay for these toxic chemicals. There was a linear correlation between the -log EC₅₀ (in vitro) AHH induction vs the -log ED₅₀ (in vivo) for body weight loss, thymic atrophy, hepatic AHH and EROD induction in the rat for several polychlorinated biphenyl, dibenzo-p-dioxin and dibenzofuran congeners and mixtures. These data clearly support the utility of the in vitro AHH induction assay as a short term test system for quantitating the “toxic or 2,3,7,8-TCDD equivalents” in an extract containing toxic halogenated aromatics. The bioassay method is rapid, relatively accurate and much more cost effective than conventional analytical methods such as gas-chromatography-mass spectrometry from which it is difficult to determine the levels of 2,3,7,8-TCDD equivalents in specific analytes.     

Polychlorinated dibenzo-p-dioxins and dibenzofurans in marine mammals in the Canadian North

Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) were determined in pooled samples of ringed seal (Phoca hispida) blubber, beluga (Delphinapterus leucas) blubber and polar bear (Ursus maritimus) liver and fat from several areas throughout the Canadian north and compared to mean PCB and HCB levels in the same samples. All seal samples, and all but one polar bear sample, had detectable 2,3,7,8-TCDD at concentrations ranging from 2 to 37 ng/kg, but TCDD was not found in beluga blubber (< 2 ng/kg). All seal samples and one of three beluga samples contained 2,3,7,8-TCDF at levels of 2 to 7 ng/kg, but TCDF was not found in any bear sample. TCDF must therefore be cleared rapidly by polar bears. No other PCDF congeners were found at detection limits of 4 to 8 ng/kg. OCDD concentrations in seal blubber and polar bear samples ranged from not detected (< 8 ng/kg) to 43 ng/kg. No apparent biomagnification of TCDD, OCDD or TCDF occurred from seal to bear fat, similar to previous findings for DDT, and unlike SigmaPCBs and HCB which biomagnified 6- to 17-fold. Highest concentrations of 2,3,7,8-TCDD and OCDD in seals and bears were found in the central Canadian Arctic Archipelago, and lowest were found in Hudson Bay, the reverse of PCB concentration distribution. The reason for higher levels of TCDD and OCDD in the Arctic than in the sub-Arctic is suggested to be trans-polar movement of aerosols with combustion-related origins in Eurasia. Levels of 2,3,7,8-TCDF were more evenly distributed throughout the North, and were positively correlated with PCB, but not with HCB or 2,3,7,8-TCDD levels in seals.     

TCDD (2,3,7,8-tetrachlorodibenzo-P-dioxin) causes reduction in glucose uptake through glucose transporters on the plasma membrane of the guinea pig adipocyte.

A single dose of 2,3,7,8-TCDD (1 micrograms/kg, i.p. injection) resulted in a significant decrease in cellular 3-O-methyl-[3H]-glucose uptake by guinea pig adipose tissue and pancreas after 24 hours. An in situ tissue culture study in which pieces of adipose tissue were incubated with 10(-8)M TCDD showed a time-dependent decrease in glucose uptake. Reconstitution of adipocyte plasma membrane from tested or control animals into artificial liposomes also resulted in this difference in glucose uptake. Binding of [3H]-cytochalasin B, a specific inhibitor of glucose transporter proteins, was significantly lower in acetone-ether powder preparations of TCDD-treated adipose tissue than from controls, suggesting that the total titer of these proteins is decreased by TCDD. Finally, the relevance of these results to glucose or lipid metabolism was tested. Lipoprotein lipase (LPL) activity of guinea pig adipose tissue was decreased after 8 hours of in situ incubation with TCDD indicating that glucose uptake was depressed at an earlier time point. These findings may contribute to a better understanding of dioxin-induced "wasting syndrome".     

High affinity binding of 3H‐verapamil to rat brain synaptic membrane is antagonized by pyrethroid insecticides

Abstract

Deltamethrin is a powerful neuroactive agent causing increased neurotransmitter release at the synapse. To understand the cause for such an action of this pyrethroid insecticide, we have studied its binding characteristics to the calcium channel by utilizing ³H‐verapamil as a specific ligand. It was found that deltamethrin is capable of competing at the same binding site as ³H‐verapamil. Also, it was found that nonlabeled verapamil could reduce the binding of ³H‐deltamethrin (tested at 1 nM) to its binding site. The EC₅₀ value of verapamil to displace 50% of ³H‐deltamethrin was in the order of 0.3 μM, which coincides with the approximate K_D value for this ligand to bind with calcium channels in the rat brain synaptosomal plasma membranes.     

Combined effects of mercury and hexachlorobenzene in the rat

Abstract

The objective of the present study was to assess the potential interactive effects of two Great Lakes chemical contaminants, hexachlorobenzene (HCB) and mercury (HgCl₂). Groups of 10 female Sprague‐Dawley rats were administered by gavage single doses of HCB (400, 600 mg/kg b.w. in corn oil), HgCl₂ (10.0, 12.5 mg/kg b.w. aqueous) or combinations of both followed by observation for clinical signs of toxicity for 14 days. Five animals from treatment groups died before the termination of the study; one animal each in 600 mg HCB, 400 mg HCB + 10 mg HgCl₂, and 600 mg HCB + 10 mg HgCl₂, and two animals in 600 mg HCB + 12.5 mg HgCl₂. The surviving animals were necropsied at the termination of the study, and hematological, clinical chemistry, histopathological and tissue residue analyses were performed. Relative liver weights were increased in both low and high dose groups of HCB but not in animals treated with HgCl₂ alone. Co‐administration of HgCl₂ did not alter the HCB effects on the liver weight of the animals. Serum cholesterol levels were increased in all the groups receiving HCB but not HgCl₂. No interactive effects on other serum parameters were seen in animals administered with both chemicals. Mild to moderate morphological changes occurred in the liver, thyroid, thymus, ovary and bone marrow of rats exposed to HCB or HCB + HgCl₂, and in the kidney of HgCl₂ or HgCl₂ + HCB treated animals. More severe histological changes occurred in the groups receiving both chemicals. The histological effects appeared to be additive. It was concluded that co‐administration with HCB and HgCl₂ resulted in additive effects in some of the endpoints measured but no synergism or antagonism was observed.     

Human-derived cell lines to study xenobiotic metabolism

In vitro systems make for rapid identification of xenobiotic effects and can be used to study cellular and subcellular toxicity mechanisms. In this report the metabolic competence of two human-derived cell lines, a hepatic (Hep G2) and a pulmonary one (A549) was tested. In the two cell systems the capability to activate Benzo[a]Pyrene through the cytochrome P450 enzyme system and to form reactive metabolites was analysed. ³H-BaP and the scintillation counting analysis were used to show the differences of the metabolic activity in Hep G2 and A549. A similar time course of ³H-BaP uptake was observed in the cell systems. Nevertheless, in the two cell lines the distribution of radioactive metabolites seemed to reflect a specific tissue response to toxicity.     

Determination of PCDDs,PCDFs, PCBs and HCB emissions from the metallurgical sector in Poland

Background The aim of the project was to measure the actual emissions of PCDD/F, PCBs and HCB from 20 selected metallurgical installations in Poland, in order to update the national inventory of dioxin emission from metallurgical industry for developing a strategy for dioxins and furans emission abatement from the subject facilities (UNEP 2005). Methods Sampling methodology used in this work was developed at the Cracow University of Technology because of the complexity of simultaneous sampling and determining PCDFs, PCDDs, PCB and HCB. For the determination a GC-MS/MS system was used. Results and Discussion Results from the work indicate that the highest dioxins and PCB concentrations were recorded for iron ore sintering plants at 1.10–1.32 ng total¹ TEQ/Nm³ followed by aluminium scrap melting at 0.03–0.66 ng total TEQ/Nm³. The highest HCB concentrations at 613–1491 ng/Nm³ were also recorded for iron ore sintering plants, whereas at aluminium plants the HCB concentrations were in the range of only 10.1–22.7 ng/Nm³. Conclusions The above investigations indicate that secondary aluminium production is the most significant dioxins source, if calculated as emission factor values. However, iron ore sintering plants are operating at much higher production capacity, causing this process to become the major source of dioxins, PCB and HCB pollution to the atmosphere in Poland. Recommendations and Perspectives Based on the performed tests and the environmental reviews of selected plants several recommendations were formulated for the reduction of generation or of emission of these pollutants from iron ore sintering plants, electric arc furnace steel production processes, hot-blast furnace operations, secondary aluminium smelting and primary zinc production from zinc cathodes.     

Serum concentrations of chlorinated dibenzo-p-dioxins and dibenzofurans among former New Zealand trichlorophenol workers

This study examined serum levels of 2,3,7,8-substituted chlorinated dioxins and furans, and 15 PCBs for 346 New Zealand employees who worked at a site that manufactured 2,4,5-trichlorophenol (TCP) and 2,4,5-trichlorophenoxy acetic acid (2,4,5-T). Participants with potential TCP or 2,4,5-T exposures had mean lipid-adjusted 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) levels of 9.9 ng kg⁻¹ lipid compared to 4.9 ng kg⁻¹ for workers with no exposure at the site. Among exposed workers, we found evidence of differences in 2,3,7,8-TCDD levels by department and duties. Workers involved in an accidental release had the highest mean 2,3,7,8-TCDD levels, 37.9 ng kg⁻¹, followed by workers in the trichlorophenol plant, 23.4 ng kg⁻¹. Workers with potential intermittent exposures to 2,3,7,8-TCDD in construction, maintenance, mechanics, and transport had 2,3,7,8-TCDD levels above New Zealand background levels of 3.9 ng kg⁻¹, indicating workplace exposures. Among participants with work history indicating no 2,3,7,8-TCDD exposures, we observed some individuals with 2,3,7,8-TCDD levels above background levels. However, in most cases, these workers reported workplace exposures not recorded on their work histories or held other jobs with the potential for 2,3,7,8-TCDD exposures outside the plant. All other dioxin, furan, and PCB levels were similar among the exposed and unexposed workers.     

Tissue distribution, excretion, and metabolism of 1,2,7,8-tetrachlorodibenzo-p-dioxin in the rat

A tissue distribution, excretion, and metabolism study was conducted using a relatively non-toxic dioxin congener, i.e., 1,2,7,8-tetrachlorodibenzo-p-dioxin (1278-TCDD), to gain a better understanding of mammalian metabolism of dioxins. Conventional, bile duct cannulated, and germ free male rats were administered mg/kg quantities as a single oral dose. Elimination of 1278-TCDD was largely complete by 72 h. Distribution of [¹⁴C]1278-TCDD was low in all tissues examined. Metabolites were identified in urine, bile, and feces by negative ion FAB-MS and ¹H-NMR, or GC/MS. The major fecal metabolite was a NIH-shifted hydroxylated TCDD. The bile contained a glucuronide conjugate of this hydroxy TCDD, and a diglucuronide conjugate of a dihydroxy-triCDD. The major metabolites in urine were glucuronide and sulfate conjugates of 4,5-dichlorocatechol.     

Dioxin formation in pulp and paper mills of India

Samples of effluents, sludge, pulp, final products (paper) and soil were collected from the identified pulp and paper mills in India. The samples were analysed for 2,3,7,8-tetrachloro-dibenzo-p-dioxin (2,3,7,8-TCDD) and other dioxin congeners and precursors. Pulp and paper mills using chlorine for the bleaching process showed the presence of 2,3,7,8-TCDD in effluent samples. In the effluent and pulp samples from mills where chlorine dioxide was used as a bleaching agent, the 2,3,7,8-TCDD congener ranged from below the detection limit 0.05 to 0.12 ngL⁻¹/ngg⁻¹. The relative standard deviation of reproducibility and the percent recovery of 2,3,7,8-TCDD were 2.07 and 82.4% in pulp and 2.8 and 92% in effluent, respectively. The 1,3,6,8-TCDD was the only other major dioxin congener found in the treated and untreated effluent and sludge samples. However, dichlorobenzene, trichlorophenyl, and hexachlorobiphenyl were detected in all samples. The formation of dioxins can be minimised by replacing chlorine with chlorine dioxide in bleaching processes in pulp and paper mills.     

Induction of cytochrome p‐450 and phosphatidylcholine synthesis by endosulfan in liver of rats : Effect of quality of dietary proteins

Abstract

Administration of endosulfan significantly increased microsomal protein, cytochrome P‐450 content and the activity of aminopyrine N‐demethylase. Effect of endosulfan and actinomycin D either alone or together on microsomal protein, cytochrome P‐450, NADPH cytochrome c reductase, aniline hydroxylase, aminopyrine N‐demethylase, phosphatidylcholine content, incorporation of ³H‐choline and ¹⁴C‐methionine were studied in rats given amino acid deficient and supplemented diets. Administration of endosulfan significantly increased the above parameters in both the dietary groups, whereas administration of actinomycin D did not have any effect in rats fed supplemented diets, however, significant decrease in the PC and the incorporation of choline and methionine into PC of rats fed deficient diet were observed. A positive correlation in the effect of endosulfan on hepatic mixed function oxidase activity and hepatic phosphatidylcholine is observed.     

Effect of phenobarbital, naphthoflavone and diethlmaleate on the sex dependent urinary pattern of hexachlorobenzene metabolites in the rat

The sex related difference in the urinary excretion of pentachlorothiophenol (PCThP) was investigated in rats after a single oral dose of hexachlorobenzene (HCB) and compared to that obtained after a single oral dose of pentachloronitrobenzene (PCNB). A ten fold difference was obtained in both cases in favour of females. The effect of phenobarbital (PB) and β-naphtoflavone (β–NF) and the effect of diethylmaleate (DM) on the urinary pattern of HCB metabolites was investigated in male and female rats. PB enhanced the urinary excretion of pentachlorophenol (PCP) and tetrachlorohydroquinone (TCHQ) but had no effect on the excretion of PCThP. β-NF did not have any effect on any of the three main metabolites while an hepatic glutathione depletion induced by DM significantly reduced the excretion of PCThP in females. The results suggest that, after HCB or PCNB intake, a common sex dependent biotransformation step leads to the formation of PCThP, after conjugation with glutathione, independently of the pathway leading to the formation of PCP which is under the control of a PB inducible form of cytochrome P-450.