Polyhalogenated Aromatic Hydrocarbons Research Articles

Effects of CYP1A2 on Disposition of 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 2,3,4,7,8-Pentachlorodibenzofuran, and 2,2′,4,4′,5,5′-Hexachlorobiphenyl in CYP1A2 Knockout and Parental (C57BL/6N and 129/Sv) Strains of Mice

TCDD is the prototype and most potent member of the highly lipophilic polyhalogenated aromatic hydrocarbons (PHAHs), which are persistent and ubiquitous environmental contaminants. In both acute and subchronic animal studies, there is a specific accumulation of TCDD in liver greater than in adipose tissue. The inducible hepatic binding protein responsible for this hepatic sequestration of TCDD and its congeners has been shown by our laboratory to be CYP1A2 (J. J. Diliberto, D. Burgin, and L. S. Birnbaum, 1997, Biochem. Biophys. Res. Commun. 236, 431–433). The present study was conducted using knockout (KO) mice lacking expression of CYP1A2 (CYP1A2−/−) in order to investigate the role of CYP1A2 gene on the disposition of TCDD, 4-PeCDF (a dioxin-like PHAH), and PCB 153 (a nondioxin-like PCB) in KO (CYP1A2−/−) mice and age-matched parental mice strains (C57BL/6N: CYP1A2+/+, Ahb/b and 129/Sv: CYP1A2+/+, Ahd/d). Mice were dosed (25 μg [3H]TCDD/kg, 300 μg [14C]4-PeCDF/kg, or 35.8 mg [14C]PCB 153/kg bw in a corn oil vehicle) orally and terminated after 4 days. Residues of administered compounds in collected tissues and daily excreta were quantitated using 3H or 14C activity. Results demonstrated differential effects in disposition for the various treatments within the three genetically different groups of mice. In KO mice, TCDD, 4-PeCDF, and PCB 153 had very little hepatic localization of chemical, and the major depot was adipose tissue. In contrast, parental strains demonstrated hepatic sequestration of TCDD and 4-PeCDF, whereas disposition of PCB 153 in parental strains was similar to that in KO mice. Another difference between KO mice and parental strains was the enhanced urinary excretion of 4-PeCDF. This study demonstrates the importance of CYP1A2 in pharmacokinetic behavior and mechanistic issues for TCDD and related compounds.

Toxicity of mixtures of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls determined by dose-response curve analysis.

Polyhalogenated aromatic hydrocarbons (PHAH) such as polychlorinat ed dibenzop-dioxins (PCDD), dibenzofurans (PCDF), and biphenyls (PCB) are persistent environmental pollutants, that bioaccumulate throughout the food chain and may pose a health risk to humans and animals (Safe 1994). The most toxic and most studied PHAH is 2,3,7,8-TCDD. PCDD, PCDF, and PCB elicit a number of common biologic and toxic effects in laboratory animals and mammalian cells in culture. The induction of hepatoma microsomal EROD or the associated cytochrome P450 isoenzymes is one of the most characteristic and sensitive response. The toxic mechanism is mediated through combining with the arylhydrocarbon (Ah) receptor (Poland and Knutson 1982; Safe 1990; Whitlock 1993). Since biota in the environment is exposed to mixtures of these compounds rather than to a single congener, simultaneous or sequential exposure of organism to two or more of these compounds may alter quantitative and qualitative biological responses. Therefore, studies with mixtures could provide more information about the possible interactive effects between these compounds and the fundamental mechanisms behind these interactive effects. The concept of independent action, which is sometimes termed effect multiplication, is based on agents acting on different molecular target sites only. An evaluation of tendency and degree of over or under estimation of mixture toxicity can be done by comparative analysis of dose-additive and independent action. The new approach is the use of doseresponse curves (DRC) for evaluating observed combined effects with respect to additive as well as to independence (Poch 1993; Poch et al. 1995). This model and analysis method occasionally are applied in pharmacologic al studies. In general, the simplest cost and time saving approach is to do a dose-response study with a substance A alone and in the presence of a fixed dose of a “similarly” acting substance B. This method is proved can provide a rather simple procedure for obtaining evidence of magnitude and mechanism of action. The aim of the present study was to evaluate the combined effects of 2,3,7,8

Induction of Altered Hepatic Foci by a Mixture of Dioxin-like Compounds with and without 2,2′,4,4′,5,5′-Hexachlorobiphenyl in Female Sprague–Dawley Rats

The hepatic tumor-promoting activity of a mixture of polyhalogenated aromatic hydrocarbons (PHAHs) was studied in a medium term two-stage initiation/promotion bioassay in female Sprague–Dawley rats. The PHAH mixture contained 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), 2,3′,4,4′,5-pentachlorobiphenyl (PCB 118), 2,3,3′,4,4′,5-hexachlorobiphenyl (PCB 156), 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153) and covered >90% of the total toxic equivalents (TEQ) present in Baltic herring. To determine possible interactive effects of di-ortho-substituted PCBs, the PHAH mixture was tested with (PHAH+) and without (PHAH−) PCB 153. Rats were initiated by a diethylnitrosamine injection (30 mg/kg body wt i.p.) 24 h after a partial23hepatectomy. Six weeks after initiation, the PHAH mixtures were administered once a week by subcutaneous injections for 20 weeks. Treatment with the PHAH mixtures caused liver enlargement and an increased activity of the hepatic cytochrome P4501A1/2 and P4502B1/2. All PHAH exposure groups exhibited an increased occurrence of hepatic foci positive for the placental form of glutathione-S-transferase. In the PHAH-group dosed 1 μg TEQ/kg body wt/week, the volume fraction of the liver occupied by foci was significantly lower compared to the TEQ equivalent dosed TCDD group (3.8 vs 8.7%). The volume fraction was significantly increased in the groups treated with 0.5, 1, or 2 μg TEQ/kg body wt/week of the PHAH+ mixture (4.5, 5.2, and 6.6%, respectively) compared to the corn oil group (2.0%), but to a lower extent than expected on basis of the TEQ doses. Overall, the TEQ-based administered dose overestimated the observed tumor-promoting effects of this PHAH mixture. The applicability of the toxic equivalency factor concept, the role of differences in toxicokinetic properties and interactive effects of PCB 153 on hepatic deposition of the dioxin-like congeners are discussed.

Induction of biotransformation enzymes by polyhalogenated aromatic hydrocarbons (PHAHs):potential impact on animal physiology and health

Biotransformation and its role in the elimination of polyhalogenated aromatic hydrocarbons (PHAHs) has been the subject of many studies from the late seventies onwards. The notion of specific, high affinity interactions of phenolic PHAH metabolites with the plasma transport proteins of thyroid hormone and vitamin A, both in vitro and in vivo, stimulated further research into the possible role of biotransformation in the toxicity of certain PHAHs such as PCBs. Currently, phenolic metabolites of PCBs and related compounds have been identified as major metabolites in blood plasma of e.g. grey seals (Halichoerus grypus) and humans with background environmental exposure to these chemicals. The concentrations of the hydroxy-PCBs were in the same range as the most persistent parent congeners, such as PCB 153, 138 and 180. These phenolic metabolites were found to possess a specific range of biological activities, which differed from the parent compounds. Another potential adverse effect associated with persistent induction of biotransformation enzymes, like UDP-glucuronyl transferases (UGTs) by PHAHs, is a long-term enhanced elimination of several important endogenous ligands such as vitamin A and thyroid hormones. Reduced levels of vitamin A and thyroid hormones have been reported in most experimental animal and wildlife species exposed to PHAHs. The recent observation of the accumulation of high levels of phenolic PCB metabolites in blood and brain of late gestational rat foetuses, in parallel with reductions in both vitamin A and thyroid hormone levels, suggests that these metabolites may play an important role in the observed developmental toxicity of PHAHs

CYP1A-catalyzed uroporphyrinogen oxidation in hepatic microsomes from non-mammalian vertebrates (chick and duck embryos, scup and alligator)

Uroporphyrin (URO) accumulation in the liver of animals treated with polyhalogenated aromatic hydrocarbons (PHAH) is associated with increased microsomal oxidation of uroporphyrinogen catalyzed by rodent CYP1A2 and by a similar form in chicken, CYP1A5. The planar biphenyl, 3,3′,4,4′-tetrachlorobiphenyl (TCB) stimulates uroporphyrinogen oxidation (UROX) in chick hepatic microsomes, but inhibits UROX activity in hepatic microsomes from mice and rats pre-induced for CYP1A2. Here we investigated whether TCB would stimulate or inhibit UROX in other non-mammalian species. UROX was stimulated 1.5–3-fold by TCB and 2–4-fold by 3,3′,4,4′,5,5′-hexachlorobiphenyl in hepatic microsomes from duck, alligator and scup treated with inducers of CYP1A. Hexachlorobenzene stimulated chick UROX, but was ineffective with microsomes from the other species. The stimulation of UROX by TCB was also observed in chick hepatocyte cultures. Pretreatment with up to 5 nM TCB induced CYP1A, but did not result in accumulation of URO. However, URO did accumulate if additional (post-induction) TCB was added along with 5-aminolevulinic acid. In this post-inductional TCB treatment, cycloheximide was included to prevent further induction of CYP1A. In duck hepatocytes, pretreatment with 25 nM TCB resulted in URO accumulation from 5-aminolevulinic acid. Post-induction TCB was not required and caused no further increase in URO accumulation. The differences in PHAH stimulation of UROX among the non-mammalian species have implications in the evolutionary changes in CYP1A, as well as the mechanism of development of PHAH-stimulated uroporphyria in different species.

Report of the who working group on the assessment of health risks for human infants from exposure to PCDDS, PCDFS and PCBS

On Nov. 20–22, 1995, a World Health Organization working group consisting of 12 scientific representatives from 6 different countries met to reassess the health risks to infants associated with perinatal exposure to polyhalogenated aromatic hydrocarbons (PHAHs). Following a review of previous WHO/EURO consultations, as part of their comprehensive programme on PCDDs, PCDFS and PCBs, current exposure information and recent experimental and epidemiologic data were discussed. Exposure assessments within the past decade have revealed that in the case of breast milk samples concentrations of PCDDs/DFs and PCBs have shown a continual decline, in certain countries by up to 50%. New experimental data has revealed that a variety of structural, functional and behavioural alterations can be induced in rodent species following exposure to PHAHs while a Dutch collaborative PCB/dioxin study has illustrated subtle clinical, endocrine and mental/psychomotor development effects can occur in breast fed infants. The provisional conclusions of the working group were: 1) current evidence does not warrant altering the previous WHO recommendation for promotion/support of breast feeding and 2) based on new clinical data which supports the biological plausibility of certain observed experimental observations, continued and enhanced effort should be directed towards identifying and controlling sources of environmental input for these contaminants.

Application of biomarkers for exposure and effect of polyhalogenated aromatic hydrocarbons in naturally exposed European otters ( Lutra lutra)

In the serious decline of European otters ( Lutra lutra) over the last decades, polychlorinated biphenyls (PCBs) are considered to be one of the major factors. As no experiments can be conducted with otters, an eco-epidemiological study was performed to derive no observed effect concentrations (NOECs) for PCBs in the otter. A strong negative correlation was found between hepatic vitamin A and polychlorinated biphenyl (PCB) concentrations expressed as TCDD-equivalents (TEQs), coinciding with a higher incidence of infectious diseases. The no-effect concentration for vitamin A reduction was 2 ng TEQ/g lipid, 10-fold reduction was already found in animals with 5 ng TEQ/g lipid. The TEQ-levels measured with a reporter gene assay based on chemical-activated luciferase expression (the CALUX assay) correlated well with the TEQ levels calculated based on non- and mono- ortho PCB concentrations. The TEQ levels in blood and liver correlated well when expressed on a lipid basis. In living captive otters blood plasma TEQ levels (either measured based on gas chromatography (GC) or CALUX measurement) were lower than in the feral otters, and positively correlated with plasma total and free thyroid hormone but not with plasma retinol levels. Hepatic vitamin A concentration was found to be a physiologically relevant effect parameter. The NOEC for hepatic vitamin A reduction was translated into TEQ levels in fish and sediment. The CALUX response in 50–500 μl blood plasma proved to be a sensitive non-destructive biomarker for quantification of internal TEQ levels.

In vitro inhibition of thyroid hormone sulfation by hydroxylated metabolites of halogenated aromatic hydrocarbons.

Earlier studies in our laboratory showed that hydroxylated metabolites of polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), and dibenzofurans (PCDFs) competitively inhibit thyroxine (T4) binding to transthyretin (TTR) and type I deiodinase (D1) activity. In this study, we investigated the possible inhibitory effects of hydroxylated metabolites of polyhalogenated aromatic hydrocarbons (PHAHs) on iodothyronine sulfotransferase activity. Rat liver cytosol was used as a source of sulfotransferase enzyme in an in vitro assay with 125I-labeled 3,3'-diiodothyronine (T2) as a model substrate. Increasing amounts of hydroxylated PCBs, PCDDs, or PCDFs or extracts from incubation mixtures of PHAHs and induced liver microsomes were added as potential inhibitors of T2 sulfotransferase activity. Hydroxylated metabolites of PCBs, PCDDs, and PCDFs were found to be potent inhibitors of T2 sulfotransferase activity in vitro with IC50 values in the low micromolar range (0.2-3.8 microM). The most potent inhibitor of T2 sulfotransferase activity in our experiments was the PCB metabolite 3-hydroxy-2,3',4, 4',5-pentachlorobiphenyl with an IC50 value of 0.2 microM. A hydroxyl group in the para or meta position appeared to be an important structural requirement for T2 sulfotransferase inhibition by PCB metabolites. Ortho hydroxy PCBs were much less potent, and none of the parent PHAHs was capable of inhibiting T2 sulfotransferase activity. In addition, the formation of T2 sulfotransferase-inhibiting metabolites of individual brominated diphenyl ethers and nitrofen as well as from some commercial PHAH mixtures (e.g., Bromkal, Clophen A50, and Aroclor 1254) was also demonstrated. These results indicate that hydroxylated PHAHs are potent inhibitors of thyroid hormone sulfation. Since thyroid hormone sulfation may play an important role in regulating free hormone levels in the fetus, and PCB metabolites are known to accumulate in fetal tissues after maternal exposure to PCBs, these observations may have implications for fetal thyroid hormone homeostasis and development.

Cytotoxic Response Profiles of Cultured Renal Epithelial and Mesenchymal Cells toSelected Aromatic Hydrocarbons

The cytotoxic responses of cultured rat renal cortico-tubular epithelial cells (TECs) and glomerular mesangial cells (GMCs) to selected polycyclic and polyhalogenated aromatic hydrocarbons (AHs) were evaluated to further define class specific differences in toxicity potential among these chemicals. Primary cultures of renal cells were exposed to benzo[ a]pyrene (BaP, 0.3–30 μ m); naphthalene (NAPH, 1–1000 μ m); 2-methylnaphthalene (2-MNAPH, 1–1000 μ m), 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD, 0.01–1n m); 2,3,7,8-tetrachlorodibenzofuran (TCDF, 0.05–5n m; and pentachlorophenol (PCP, 0.1–10 μ m for 4 or 24 hr. Measurements of mitochondrial membrane fragility in TECs revealed that the polycyclic aromatic hydrocarbons (PAHs) (BaP, NAPH and 2-MNAPH) preferentially injured these cells relative to halogenated aromatic hydrocarbons (HAHs). Extended exposures to all AHs tested were associated with moderate mitochondrial injury in TECs. Challenge of TECs with AHs for 4 hr did not modulate GSH levels, although modest increases in this tripeptide occurred on extended exposures. Similar response profiles were observed in GMCs, where PAHs elicited mitochondrial damage by 4 hr, while extended challenge was associated with injury in response to all AHs. Only aryl hydrocarbon receptor (AhR) ligands (BaP, TCDD and TCDF) depleted intracellular glutathione (GSH) in GMCs, while extended exposures to BaP and TCDD, as well as NAPH and 2-MNAPH, were associated with rebound increases in cellular GSH content. These data indicate that AHs can compromise renal cell function by interference with mitochondrial function and GSH homeostasis and implicate both epithelial and mesenchymal populations in the nephrotoxic response to this heterogeneous class of chemicals.

Changes in thyroid hormone economy following consumption of environmentally contaminated Great Lakes fish.

Epizootics of thyroid lesions in fish and piscivorous birds that are resident in the Great Lakes region of North America suggest that there are environmental factors present in the Great Lakes ecosystem that act as potent endocrine disruptors, and that they are transferred along the food chain. This paper examines the results of wildlife studies, as well as related studies on fish-eating human populations in the region. It also re-examines the results of experimental studies of the effects of Great Lakes fish diets on the thyroid physiology of rodents and shows that the thyroid responses of fish-fed rats and mice were essentially similar to those found in rats that had been administered specific polyhalogenated aromatic hydrocarbon (PHAH) congeners or commercial polychlorinated biphenyl mixtures. However, the responses to the Great Lakes fish diets were found at PHAH exposure levels that were commonly several orders of magnitude lower than those applied in the classical toxicology studies. These findings, together with the results of the Great Lakes piscivorous bird studies and one in which captive common seals were fed "environmentally contaminated" fish, suggest that the "environmental" PHAH mixtures accumulated in fish represent a significant threat to the thyroid hormone economy, and the effects are greater than could be predicted by virtue of the known levels of active congeners in this naturally bioaccumulated PHAH mix.

Interactions of persistent environmental organohalogens with the thyroid hormone system: mechanisms and possible consequences for animal and human health.

Several classes of environmental contaminants have been claimed or suggested to possess endocrine-disrupting potency, which may result in reproductive problems and developmental disorders. In this paper the focus is on the multiple and interactive mechanisms of interference of persistent polyhalogenated aromatic hydrocarbons (PHAHs) and their metabolites with the thyroid hormone system. Evidence suggests that pure congeners or mixtures of PHAHs directly interfere with the thyroid gland; with thyroid hormone metabolizing enzymes, such as uridine-diphosphate-glucuronyl transferases (UGTs), iodothyronine deiodinases (IDs), and sulfotransferases (SULTs) in liver and brain; and with the plasma transport system of thyroid hormones in experimental animals and their offspring. Changes in thyroid hormone levels in conjunction with high PHAH exposure was also observed in captive as well as free ranging wildlife species and in humans. Maternal exposure to PHAHs during pregnancy resulted in a considerable fetal transfer of hydroxylated PHAHs, which are known to compete with thyroxine (T4) for plasma transthyretin (TTR) binding sites, and thus may be transported to the fetus with those carrier proteins that normally mediate the delivery of T4 to the fetus. Concomitant changes in thyroid hormone concentrations in plasma and in brain tissue were observed in fetal and neonatal stages of development, when sufficient thyroid hormone levels are essential for normal brain development. Alterations in structural and functional neurochemical parameters, such as glial fibrillary acidic protein (GFAP), synaptophysin, calcineurin, and serotonergic neurotransmitters, were observed in the same offspring up to postnatal day 90. In addition, some changes in locomotor and cognitive indices of behavior were observed in rat offspring, following in utero and lactational exposure to PHAHs. Alterations in thyroid hormone levels and subtle changes in neurobehavioral performance were also observed in human infants exposed in utero and through lactation to relatively high levels of PHAHs. Overall these studies indicate that persistent PHAHs can disrupt the thyroid hormone system at a multitude of interaction sites, which may have a profound impact on normal brain development in experimental animals, wildlife species, and human infants.

Multiple Roles of Polyhalogenated Biphenyls in Causing Increases in Cytochrome P450 and Uroporphyrin Accumulation in Cultured Hepatocytes

Uroporphyrin (URO) accumulation occurs in chick embryo hepatocytes treated with a number of polyhalogenated aromatic hydrocarbons (PHAHs) that are known inducers of cytochrome P4501As (CYP1A). Previous dose response studies had shown that URO accumulation does not begin until CYP1A, as indicated by ethoxyresorufin O-deethylase (EROD) activity, is maximally induced. The reason why the concentrations of PHAHs required for URO accumulation were higher than those required to induce EROD had not been explained. PHAHs, such as 3,3′,4,4′-tetrachlorobiphenyl (PCB77, IUPAC nomenclature, TCB) stimulate uroporphyrinogen (UROGEN) oxidation by microsomes from 3-methylcholanthrene (MC)-treated chick embryos. Here we used a new protocol to investigate whether the requirement for more TCB to stimulate in vitromicrosomal UROGEN oxidation extended to TCB-induced URO accumulation in intact cultured hepatocytes. Cultures were treated with increasing concentrations of TCB or other PHAHs to induce CYP1As, then with cycloheximide (CX) to prevent further P450 synthesis. The CX treatment was shown to block any further increases in CYP1A as determined by immunoblots. 5-Aminolevulinic acid and a high concentration of TCB (“postinduction TCB”) were then added to stimulate intracellular UROGEN oxidation. Using the protocol with postinduction TCB, the inducing concentrations of TCB which caused URO to begin to accumulate were now much lower than in the absence of postinduction TCB. Increases in CYP1A proteins, measured immunochemically, were detected at about the same inducing TCB concentrations that began to increase URO accumulation. The new protocol, with postinduction TCB, using URO accumulation as the end point, greatly increased the sensitivity of the culture system for detection of PHAHs with EC50s (n m) for 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), TCB, 3,3′,4,4′,5,5′-hexachlorobiphenyl, MC, and hexachlorobenzene being about 0.003, 0.11, 0.75, 3.5, and 30, respectively. As little as 2–4 fmol TCDD per culture dish caused detectible increases in URO accumulation. We conclude that URO accumulation in chick hepatocyte cultures is limited not only by the induction of CYP1A, but also by the stimulation of intracellular UROGEN oxidation.

Extrathyroidal effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on thyroid hormone turnover in male Sprague-Dawley rats.

Treatment of rats with different polyhalogenated aromatic hydrocarbons strongly decreases plasma T4, with little or no decrease in plasma T3. The extrathyroidal effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thyroid hormone turnover were studied by i.p. administration of a single dose of 10 microg TCDD/kg BW or vehicle (corn oil) to euthyroid (Eu) rats, thyroidectomized (Tx) rats, and Tx rats infused with 1 microg T4 (Tx+T4) or 0.4 microg T3 (Tx+T3)/100 g BW x day by osmotic minipumps. Tx rats showed decreased plasma T4 and T3 and increased plasma TSH levels, decreased hepatic type I deiodinase (D1) and malic enzyme activities, and increased brain type II deiodinase (D2) activities. All parameters were largely restored to Eu levels in Tx+T4 rats and, except for plasma T4 and brain D2 activity, in Tx+T3 rats, validating the thyroid hormone-replaced Tx rats as models to study the peripheral effects of TCDD. Three days after TCDD administration, plasma T4 and free T4 levels were significantly reduced in Eu and Tx+T4 rats, and plasma T3 was significantly reduced in Tx+T3, but not in Eu or Tx+T4 rats. Plasma TSH was not affected by TCDD in any group. Hepatic T4 UDP-glucuronyltransferase (UGT) activity was induced approximately 5-fold by TCDD, whereas T3 UGT activity was only increased by about 20% (P = NS) in the different groups. TCDD produced an insignificant decrease in liver D1 activity in Tx rats and an insignificant increase in brain D2 activity in Tx rats and hormone-replaced Tx rats. Hepatic malic enzyme activity was significantly increased by TCDD in all groups, except Tx rats. These results strongly suggest that the thyroid hormone-decreasing effects of TCDD are predominantly extrathyroidal and mediated by the marked induction of hepatic T4 UGT activity.

Impaired cellular immune response in rats exposed perinatally to Baltic Sea herring oil or 2,3,7,8-TCDD.

While the immunotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been well established, the effects of complex environmental mixtures of polyhalogenated aromatic hydrocarbons (PHAHs) are poorly understood. Many PHAHs, including the polychlorinated-biphenyls (PCBs), -dibenzofurans (PCDFs), and dibenzo-p-dioxins (PCDDs), possess 'dioxin-like' activities, and accumulate in the aquatic food chain. Organisms occupying high trophic levels may therefore be exposed to concentrations which may present an immunotoxic risk. In this study, pregnant PVG rats were administered a daily oral dose of 1 ml of the following during pregnancy and lactation: (1) oil extracted from herring caught in the relatively uncontaminated Atlantic Ocean; (2) oil extracted from herring caught in the contaminated Blastic Sea; or (3) the Atlantic herring oil extract spiked with 2,3,7,8-TCDD. The daily intakes of aryl hydrocarbon (Ah)-receptor dependent toxic equivalents (TEQ) for mothers were 0.3 in the Atlantic group, 2.1 in the Baltic group, and 134 ng/kg body wt. in the 2,3,7,8-TCDD positive control group. Immune function and host resistance to rat cytomegalovirus (RCMV) were assessed in offspring aged 11, 25, 46 or 59 days. Rat pups in the positive control TCDD-spiked group exhibited immunosuppression characterized by reduced thymus weight and cellularity, reduced thymocyte and splenocyte proliferative responses to T-dependent mitogens in vitro, reduced virus-associated natural killer (NK) cell and specific antibody responses. While less pronounced, a similar pattern of effects was observed in the rat pups exposed only to the Baltic Sea herring oil. These immunotoxic effects were transient in both exposure groups, with a time-related recovery in immune function possibly due to the half-life of TCDD in rats and the waning exposure levels in the rapidly growing pups. We previously demonstrated that the same Baltic Sea herring led to impaired natural killer cell and T-lymphocyte function in harbour seals during the course of a long-term captive feeding study. The collective results of these studies in rats and seals indicate the immunotoxic potential of environmental mixtures at current levels in the aquatic environment, and suggest that the developing immune system of young mammals may be at particular risk.

Promotion of Endometriosis in Mice by Polychlorinated Dibenzo-p-Dioxins, Dibenzofurans, and Biphenyls

Previous studies showed exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) enhances the development of endometriotic lesions. In this study we examined the effects of other polyhalogenated aromatic hydrocarbons on endometriotic proliferation. B6C3F1 female mice were treated via oral gavage a total of five times, with 3 weeks between each dosing, with 0, 1, 3, or 10 micrograms 2,3,7,8,-TCDD/kg body weight (bw); 3 or 30 mg 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153)/kg bw; 100, 300, or 1000 micrograms 3,3',4,4',5-pentachlorobiphenyl (PCB 126)/kg bw; 10, 30, or 100 micrograms 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF)/kg bw; or 2 or 20 mg 1,3,6,8-TCDD/kg at 10 ml/kg bw. Endometriosis was surgically induced during the week of the second dosing. Three weeks following the final dose, the mice were euthanized and endometriotic lesions, whole body, liver, ovaries, uterine horn, and thymus were weighted, and lesion diameters were measured. Lesions, uterine horns, and ovaries were fixed for histopathology and livers were processed for measurement of ethoxyresorufin O-deethylase (EROD) activity. Both 2,3,7,8-TCDD (1 and 3 micrograms/kg bw) and 4-PeCDF (100 micrograms/kg bw) significantly enhanced the growth of endometrial lesions. No statistically significant increase in endometriotic lesion size was detected in animals treated with either PCB 126 or with the highest dose of 2,3,7,8-TCDD, possibly due to the effects of histologically observed ovarian toxicity. The nondioxin-like compounds, PCB 153 and 1,3,6,8-TCDD, produced no observable effects on endometriosis. Hepatic EROD activity was significantly induced by 2,3,7,8-TCDD, 4-PeCDF, and PCB 126, but not by PCB 153 or 1,3,6,8-TCDD. The results of this study provide preliminary support for the hypothesis that halogenated aromatic hydrocarbon-promoted endometriosis may be Ah receptor mediated.

Promotion of endometriosis in mice by polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls.

Previous studies showed exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) enhances the development of endometriotic lesions. In this study we examined the effects of other polyhalogenated aromatic hydrocarbons on endometriotic proliferation. B6C3F1 female mice were treated via oral gavage a total of five times, with 3 weeks between each dosing, with 0, 1, 3, or 10 micrograms 2,3,7,8,-TCDD/kg body weight (bw); 3 or 30 mg 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153)/kg bw; 100, 300, or 1000 micrograms 3,3',4,4',5-pentachlorobiphenyl (PCB 126)/kg bw; 10, 30, or 100 micrograms 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF)/kg bw; or 2 or 20 mg 1,3,6,8-TCDD/kg at 10 ml/kg bw. Endometriosis was surgically induced during the week of the second dosing. Three weeks following the final dose, the mice were euthanized and endometriotic lesions, whole body, liver, ovaries, uterine horn, and thymus were weighted, and lesion diameters were measured. Lesions, uterine horns, and ovaries were fixed for histopathology and livers were processed for measurement of ethoxyresorufin O-deethylase (EROD) activity. Both 2,3,7,8-TCDD (1 and 3 micrograms/kg bw) and 4-PeCDF (100 micrograms/kg bw) significantly enhanced the growth of endometrial lesions. No statistically significant increase in endometriotic lesion size was detected in animals treated with either PCB 126 or with the highest dose of 2,3,7,8-TCDD, possibly due to the effects of histologically observed ovarian toxicity. The nondioxin-like compounds, PCB 153 and 1,3,6,8-TCDD, produced no observable effects on endometriosis. Hepatic EROD activity was significantly induced by 2,3,7,8-TCDD, 4-PeCDF, and PCB 126, but not by PCB 153 or 1,3,6,8-TCDD. The results of this study provide preliminary support for the hypothesis that halogenated aromatic hydrocarbon-promoted endometriosis may be Ah receptor mediated.

Role of CYP1A2 in Hepatic Sequestration of Dioxin: Studies Using CYP1A2 Knock-Out Mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin), most potent of the polyhalogenated aromatic hydrocarbons, has been studied in a variety of genetically normal species. Transgenic mice lacking a cytochrome P450 1A2 gene were used to study the influence of the CYP1A2 gene on the hepatic sequestration and distribution of TCDD, 4-PeCDF (2,3,4,7,8-pentachlorodibenzofuran; dioxin-like compound), and PCB 153 (2,2′,4,4′,5,5′-hexachlorobiphenyl; non-dioxin-like PCB (polychlorinated biphenyl)). The knock-out mice were compared to their age-matched lineage strains of C57BL/6N (1A2+/+;Ahb) and 129/Sv (1A2+/+;Ahd) for each compound. As demonstrated by the liver-to-adipose tissue (L/F) concentration ratios, there was no hepatic sequestering of TCDD and 4-PeCDF in the transgenic knock-out mice.

Monooxygenase activities in carp as biochemical markers of pollution by polycyclic and polyhalogenated aromatic hydrocarbons: choice of substrates and effects of temperature, gender and capture stress

Cytochrome P450-dependent monooxygenase activities (7-ethoxyresorufin, 7-pentoxyresorufin and 7-ethoxycoumarin O-dealkylases and aminopyrine N-demethylase) were measured in hepatopancreatic microsomes of carp collected in one control and one polychlorinated biphenyl- and polycyclic aromatic hydrocarbon-polluted ponds in the Czech Republic. The magnitudes of responses of monooxygenase activities to the exposure to polycyclic and polyhalogenated aromatic compounds (PAC), namely polycyclic aromatic hydrocarbons and polychlorinated biphenyls, and effects of other factors including ambient temperature and stress associated with reduction of water volume at capture were assessed. An increase of the level of 7-ethoxyresorufin O-deethylase (EROD) i.e. the specific monooxygenase activity mediated by the cytochrome P4501A (CYP1A) isoenzymes, was the most sensitive biochemical marker of environmental pollution and exposure to PAC. Carp hepatic 7-pentoxyresorufin O-depentylase (PROD) was unsuitable as a specific indicator of elevation of cytochrome P4502B-like isoenzymes (and thus as a biomarker of presence of CYP2B-type of inducers e.g. organochlorine pesticides) due to a probable inducibility by CYP1A inducers. PROD, unlike aminopyrine N-demethylase, another mammalian CYP2B-dependent activity which was not increased by exposure to PAC, was strongly inhibited by CYP1A inhibitors, α-naphthoflavone and 9-hydroxyellipticine. The respective dose-response curves were related to the EROD inhibition. Chloramphenicol, an inhibitor of CYP2B enzymes. inhibited both activities only weakly or not at all. Adaptation of carp to lower ambient temperatures and to the stress associated with reduction of water volume and subsequent capture by netting proved to highly significantly inhibit levels of EROD and PROD activities. Summer appeared to be the most suitable season for the biochemical monitoring of contamination. Activity of 7-ethoxycoumarin O-deethylase (ECOD) was also markedly induced by PAC, but the enzyme was less responsive than the EROD activity. The ECOD activity was not significantly influenced by ambient temperature or by stress associated with capture and removal of fish. This probably reflected a modulation of partly different CYP enzymes other than EROD. Unlike in ECOD, the results confirmed the high specificity and sensitivity of induction of CYP1A-dependent EROD activity in carp (PAC in environmental exposure levels affected EROD highly significantly).

Interactive effects on the erod‐inducing potency of polyhalogenated aromatic hydrocarbons in the chicken embryo hepatocyte assay

AbstractThe chicken embryo hepatocyte‐7‐ethoxyresorufin O‐deethylase (EROD) assay is used as a method to measure the amount of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (2,3,7,8‐TCDD) equivalents (TEQs) in environmental samples. A common feature of EROD‐induction assays in vertebrates is that they generate biphasic dose‐response relationships that show dose‐related increases of the EROD induction to a maximum activity, followed by a dose‐related decrease at higher concentrations. In general, the maximum achievable enzyme activity decreases with increasing median effective concentration (EC50). This suggests that aryl hydrocarbon (Ah)‐receptor binding affinity is not the only factor determining the enzyme activity. An additional factor can obscure the maximum EROD activity (Ymax) and EC50 of the enzyme activity. Cytotoxicity and competitive inhibition are ruled out as possible influencing factors. Coadministration of 2, 2′, 4, 4′, 5, 5′‐hexachlorobiphenyl (PCB 153) and 2, 3, 7, 8‐TCDD significantly reduces the EC50 value compared to administration of TCDD alone. The dose‐related decrease at higher concentrations has been suggested to be induced by mechanisms other than the Ah‐receptor‐related mechanism responsible for the observed increases at low concentrations. These interactive effects have serious consequences for risk assessment based on bioassay‐derived TEQs.

Lack of effects of some individual polybrominated diphenyl ether (PBDE) and polychlorinated biphenyl (PCB) congeners on human lymphocyte functions in vitro

The structural similarities between polybrominated diphenyl ethers and immunotoxic halogenated aromatic compounds suggest that the polybrominated diphenyl ethers might affect the immune system. The present study was undertaken to investigate the immunological effects of some purified PBDE-congeners on human lymphocyte function in vitro. Polychlorinated biphenyl congeners were also included in the study. Mitogen-induced DNA synthesis and immunoglobulin synthesis by lymphocytes from blood donors were examined following polybrominated diphenyl ether or polychlorinated biphenyl exposure in vitro in order to determine the immunotoxic potential of these substances. No effects on mitogen-induced proliferation or immunoglobulin synthesis were observed after exposure of cells to concentrations up to 10 −5 M. The negative findings in this study indicate that certain functions of human peripheral lymphocytes, i.e. proliferation and immunoglobulin synthesis, are insensitive to the direct action of polybrominated diphenyl ethers and polychlorinated biphenyls. Our results are in accordance with other recent studies in which no effects on immunological parameters were demonstrated by exposure of lymphocytes to polyhalogenated aromatic hydrocarbons in vitro.