Pharmacokinetics of two major hydroxylated polychlorinated biphenyl metabolites with specific retention in rat blood.

1. Hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs) are, depending on their structure, strongly retained in mammalian, fish and bird blood. This is due to strong, though reversible, binding to the thyroxine binding and transporting protein transthyretin. 2,3,3',4',5-Pentachloro-4-biphenylol (4-OH-CB 107) and 2,2',3,4',5,5',6-heptachloro-4-biphenylol (4-OH-CB 187) are two of five major OH-PCB congeners in human plasma. 2. The relative amounts of OH-PCB congeners vary between species and also between human populations, in spite of similar PCB congener patterns, and may depend on different pharmacokinetic parameters of the OH-PCBs. In the present study, the pharmacokinetic parameters of 4-OH-CB 107 and 4-OH-CB 187 were determined in the rat after a single intravenous dose of 1 micromol kg(-1). Plasma samples were analysed by gas chromatography/mass spectrometry. 3. 4-OH-CB 107 had a half-life of 3.8 days; 4-OH-CB 187 had a half-life of 15 days. Volumes of distribution were 0.07 and 0.11 l kg(-1), respectively; clearances (ml h(-1)) were 0.67 and 0.22, respectively; and the areas under the curve were estimated as approximately 1500 and 4450 nmol h ml(-1). 4. The pharmacokinetic parameters thus determined help to explain the observed differences in the relative amounts of OH-PCBs in humans and other mammals exposed to environmental PCBs.

Tissue disposition, excretion and metabolism of 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) in the male Sprague-Dawley rat.

1. A disposition, metabolism and excretion study of orally administered 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) was conducted in the conventional and bile duct-cannulated male rat. 2. In the conventional rat, >50% of the radiolabelled dose was retained at 72 h, and lipophilic tissues were the preferred sites for disposition, i.e. adipose tissue, adrenals, gastrointestinal tract and skin. 3. Urinary excretion of BDE-99 was very low (<1% of dose), and glucuronidation of phenolic metabolites was suggested. 4. Biliary excretion of BDE-99 was slightly greater than observed in urine, i.e. 3.6% at 72 h. 5. Over 43% of the dose in the conventional male rat and 86% in the bile duct-cannulated rat was excreted in the faeces, mainly as the unmetabolized parent compound. 6. Metabolites in bile and faeces were not conjugated. Mono- and di-hydroxylated pentabromodiphenyl ether metabolites were characterized by mass spectrometry. Two thiol metabolites were characterized in the bile. Oxidative debromination was also observed in the faecal metabolites. 7. Tissue BDE-99 was readily extractable, except for in the liver. The tissue (14)C was not associated with lipids and was mainly the unmetabolized parent compound. 8. Total thyroxine (T4) plasma levels were elevated at 3 and 6 days, and returned to control levels by day 12.

Plasma concentrations of persistent organochlorines in relation to thyrotropin and thyroid hormone levels in women.

OBJECTIVES: There is a concern that persistent organohalogen toxicants, such as polychlorinated biphenyls (PCBs), might display endocrine-disrupting effects in exposed populations. In this study the correlations between PCBs and thyrotropin (TSH) and thyroid hormone concentrations in plasma were assessed in adult women. METHODS: The study group consisted of 182 fishermen's wives from the Swedish east coast, with a median age of 42 years (range 23-62) and a median current consumption of contaminated fatty fish from the Baltic Sea of two meals per month (range 0-12). TSH, free (FT3) and total (TT3) triiodothyronine and free (FT4) and total (TT4) thyroxin in plasma were analyzed by immunofluorometric assays, and 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) in plasma was analyzed by gas chromatography with electron capture detection. Twenty other PCB and two hydroxy-PCB congeners were analyzed in subgroups of the women. Plasma lipid analyses were performed with enzymatic techniques. RESULTS: The CB-153 concentration in plasma (range 16-776 ng/g lipid) was negatively correlated with the TT3 concentrations (range 1.0-3.0 nmol/l, rs = -0.29, P < 0.001). This association remained after age adjustment. CONCLUSIONS: The present study gives some support for the notion that dietary exposure to persistent organochlorine compounds (POCs) might weakly affect peripheral thyroid hormone concentrations in adult women.

Influence of the consumption of fatty Baltic Sea fish on plasma levels of halogenated environmental contaminants in Latvian and Swedish men.

We examined the influence of widely varied consumption of fatty fish from the Baltic Sea and of age on plasma concentrations of polychlorinated biphenyls (PCBs), polychlorobiphenylols (OH-PCBs), 2, 2-bis(4-chlorophenyl)-1,1,1-trichloroethane (4,4'-DDT), 2, 2-bis(4-chlorophenyl)-1,1-dichloroethane (4,4'-DDE), 2,2',4, 4'-tetrabromodiphenyl ether (BDE-47), hexachlorobenzene (HCB), and pentachlorophenol (PCP) in Latvian and Swedish men. Both age and fish consumption were significantly correlated with the concentrations of [sigman]PCB, [sigman]OH-PCB, 4,4'-DDE, 4,4'-DDT, and HCB. In the case of BDE-47, no significant relationship with age was observed, and fish consumption had the largest relative effect on plasma concentrations of this contaminant. This relationship may be a result of exposure to BDE-47 having been more recent than that of PCBs and DDE, or because the half-life of BDE-47 may be shorter than that of PCB and DDE. Latvian men demonstrated higher plasma levels of DDE and DDT but lower levels of [sigman]PCB and PCP than did Swedish men. The corresponding levels of HCB and BDE-47 were similar in both countries. The Spearman's rank correlation coefficient obtained by comparing the level of the metabolite 4-hydroxy-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107) to the combined levels of its parent compounds, 2,3,3',4, 4'-pentachlorobiphenyl (CB-105) and 2,3',4,4',5-pentachlorobiphenyl (CB-118), was higher than the median correlation coefficient obtained upon comparing the level of this metabolite to all other possible combinations of two PCB levels. No other increased correlation between metabolite and parent PCB concentration was observed.

Induction of aberrant mitosis with PCBs: particular efficiency of 2, 3,3',4,4'-pentachlorobiphenyl and synergism with triphenyltin.

The polychlorinated biphenyls 2,2',5,5'- and 3,3',4, 4'-tetrachlorobiphenyl, 2,3,3',4,4'- and 3,3',4,4', 5-pentachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl were tested for spindle-disturbing activity in V79 Chinese hamster cells. Clones lacking endogenous cytochrome P450 activity or expressing rat CYP1A1 or CYP2B1 were used. Induction of abnormal chromosomal arrangements in mitosis were found to be favoured by o-chlorine substitutions, but not by co-planarity giving affinity, for example, for the Ah receptor and CYP1A isoenzymes. Only 2,2',5, 5'-tetrachloro- and 2,3,3',4,4'-pentachlorobiphenyl gave dose-response curves similar to many other compounds tested in vitro, showing an increase from the background level of 10 to 100% disturbed mitoses with nominal concentrations >10(-6) M, i.e. concentrations far above the total PCB concentrations found in human blood. Cells transfected with rat CYP2B1 were more sensitive to the most active congener, 2,3,3',4,4'-pentachlorobiphenyl, than cells lacking P450 activity or expressing CYP1A1. Induction of abnormal mitosis by PCB metabolites formed by P450 enzymes cannot be excluded, but does not seem likely because of the short treatment time and the reportedly slow metabolism of PCBs. 2,3,3',4, 4'-Pentachlorobiphenyl showed synergistic activity with the potent spindle poison triphenyltin. Inactive concentrations of both agents (10 and 50 nM, respectively) caused abnormal configurations when combined. This is an important finding since exposure to mixtures of compounds is common and it motivates further studies of subthreshold activities of highly lipophilic environmental contaminants.

Flame retardant exposure: polybrominated diphenyl ethers in blood from Swedish workers.

Polybrominated diphenyl ethers (PBDEs) are used as additives in polymers and textiles to prohibit the development of fires. Because of the production and use of PBDEs, their lipophilic characteristics, and persistence, these compounds have become ubiquitous environmental contaminants. The aim of the present study was to determine potential exposures of PBDEs to clerks working full-time at computer screens and personnel at an electronics-dismantling plant, with hospital cleaners as a control group. Five PBDE congeners--2,2',4,4'-tetraBDE; 2,2',4,4',5,5'-hexaBDE; 2,2',4,4',5, 6'-hexaBDE; 2,2',3,4,4',5',6-heptaBDE; and decaBDE--were quantified in blood serum from all three categories of workers. Subjects working at the dismantling plant showed significantly higher levels of all PBDE congeners in their serum as compared to the control group. Decabromodiphenyl ether is present in concentrations of 5 pmol/g lipid weight (lw) in the personnel dismantling electronics; these concentrations are comparable to the concentrations of 2,2',4, 4'-tetraBDE. The latter compound was the dominating PBDE congener in the clerks and cleaners. The major compound in personnel at the dismantling plant was 2,2',3,4,4',5',6-heptaBDE. Concentrations of this PBDE congener are almost twice as high as for 2,2',4, 4'-tetraBDE in these workers and seventy times the level of this heptaBDE in cleaners. The total median PBDE concentrations in the serum from workers at the electronics-dismantling plant, clerks, and cleaners were 37, 7.3, and 5.4 pmol/g lw, respectively. The results show that decabromodiphenyl ether is bioavailable and that occupational exposure to PBDEs occurs at the electronics-dismantling plant.

Metabolism of 2,2',4,4'-tetrabromodiphenyl ether in rat and mouse.

1. The distribution and excretion of orally administered 14C-labelled 2,2',4,4'-tetrabromodiphenyl ether (TBDE) have been studied in rat and mouse. 2. TBDE was efficiently absorbed and stored in adipose tissue where high concentrations were observed in both species. 3. In the rat, 86% of the dose was retained after 5 days, while 14% was excreted via the faeces and < 0.5% via the urine. 4. The mouse excreted 20% of the dose via the faeces and 33% via the urine, the latter as a hydrophilic and labile metabolite. 5. Metabolites covalently bound to macromolecules and lipids were noted in tissues and faeces from both species. 6. The major individual compound was parent TBDE in the faeces and tissues although small amounts of five hydroxylated metabolites were indicated by GC-MS. 7. In plasma from both rat and mouse only a few of the hydroxylated metabolites were present, indicating selective retention of these metabolites.

Hydroxylated polychlorinated biphenyls: distribution in the pregnant mouse.

1. In the pregnant C57BL mouse the disposition of a single, intravenous low dose of 14C-labelled 4-hydroxy-3,5,3',4'-tetrachlorobiphenyl (4-OH-TCB) or 4-hydroxy-3,5,2',3',4'-pentachloro-biphenyl (4-OH-PeCB1) was monitored by liquid scintillation counting and whole-body autoradiography. The compounds were placentally transferred and accumulated in the foetal tissues (e.g. plasma and liver). Also, maternal accumulation was observed in selected tissues, including liver, adrenal gland, adipose tissue and yolk sac placenta. 2. The foetal concentration of both hydroxy-PCBs increased with time up 24 h post-exposure and the foetal plasma concentration with at this time-point two-fold of that in maternal plasma. Chemical analysis of maternal plasma and liver showed no metabolism of the administered compounds. 3. In the pregnant C57BL mouse at late gestation, exposure to 4-OH-TCB generally resulted in a higher foetal and maternal tissue retention than did 4-OH-PeCB1. The estimated elimination half-lives (t 1/2) of 4-OH-TCB in maternal liver and plasma were 69 and 13 h respectively, and for 4-OH-PeCB1 were 17 and 13 h. 4. No differences in foetal tissue concentration of 4-OH-TCB were observed between the C57BL, and NMRI mouse. In contrast, earlier studies have shown that the PCB congener CB-77, the parent compound of 4-OH-TCB, resulted in a C57BL/NMRI foetal ratio of 1:5.

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.

Hydroxylated polychlorinated biphenyl metabolites are anti-estrogenic in a stably transfected human breast adenocarcinoma (MCF7) cell line.

Hydroxylated metabolites of polychlorinated biphenyls (OHCBs) have been identified in blood of marine mammals, fish-eating birds, and humans at concentrations in some cases exceeding those of the unmetabolized polychlorinated biphenyls (PCBs). OHCBs have been associated with inhibition of vitamin A and thyroxin transport, estrogenicity in a mouse uterotrophic assay, and feminization of male turtle sexual development. OHCBs, representing both environmentally derived and laboratory exposure-derived metabolites, were tested in an in vitro bioassay utilizing an estrogen-responsive human breast adenocarcinoma cell line (MCF7-LUC) stably transfected with a luciferase reporter gene linked to estrogen responsive elements. OHCB activity was tested at three different media concentrations of 17beta-estradiol (E2), comparing the concentration-response curves using charcoal-stripped medium (0.0009 nM E2), and two physiologically relevant E2 concentrations (0.1 and 1.0 nM E2). Eleven of 13 OHCBs tested were anti-estrogenic. Evidence for an estrogen receptor mediated mechanism of action was apparent for only two OHCBs-4-OH-2',3,3',4',5,5'-Cl6-biphenyl and 4,4'-(OH)2-3,3',5,5'-Cl4-biphenyl. These two have not been identified in environmental samples. The remaining OHCBs exhibited "anti-estrogenicity" that was related to their effect on cell viability and, therefore, cannot be described as exhibiting "hormone disruption" solely by an estrogen receptor mediated mechanism. OHCB anti-estrogenic activity was eliminated in the presence of E2 concentrations normally found in humans, except for 4,4'(OH)2-3,3',5,5'-Cl4-biphenyl. 4-OH-2',3',4',5'-Cl4-biphenyl and 4-OH-2',4',6'-Cl3-biphenyl were partial estrogen agonists, exhibiting weak estrogenicity in the presence of 0.0009 nM E2 and weak anti-estrogenicity in the presence of 0.1 and 1 nM E2. Human metabolites of PCBs were not estrogenic in MCF7 cells.

Milk transfer and neonatal uptake of coplanar polychlorinated biphenyl (PCB) congeners in mice.

The selective accumulation of 3,3',4,4'-tetrachlorobiphenyl metabolites in late gestational foetal blood and soft tissues in mice as a result of administration of different coplanar polychlorinated biphenyl (PCB) congeners, is reported elsewhere. The situation in the nursing neonate after maternal exposure to the same congeners is now studied: The 14C-labelled congeners 3,3',4,4'-tetrachlorobiphenyl (IUPAC number CB-77),3,3',4,4'5-pentachlorobiphenyl (IUPAC number CB-126), 3,3',4,4',5,5'-hexachlorobiphenyl (IUPAC number CB-169) (all three non-ortho congeners) and 2,3,3',4,4'-pentachlorobiphenyl (IUPAC number CB-105) (mono-ortho congener) were injected intravenously in lactating mice at day 11 post partum. One day and four days later, milk and neonatal/maternal tissues and plasma radioactivity was monitored by liquid scintillation counting (dose: 2.0 mumol (20-50 microCi)/kg body weight). In milk, CB-126, -169 and -105 showed higher levels (1450-2520 pmol/ml; one day after administration) than did CB-77 (580 pmol/ml), and in neonates, the relative whole-body levels of radioactivity (CB-169 and -105 highest) were related to the levels seen in milk (probably the consequences of their metabolic persistence). The comparably high 14C-concentration found in neonatal liver (about 15,000 pmol/kg) after CB-126 administration and in plasma (880 pmol/ml) after CB-77 administration could be explained by binding to specific proteins. In general, neonatal mice had two to seven times higher plasma levels than those of their mothers. These results indicate that CB-126, -169 and -105 are transferred via milk to neonates in considerable quantity and are deposited mainly in neonatal liver, whereas CB-77 is transferred in a comparably lower amount and accumulated in neonatal plasma. The lower 14C-levels in the NMRI mothers and offspring (about half of C57BL values in maternal and neonatal plasma), could possibly be explained by a differentiated metabolism of CB-77 in these two strains.

Chromatographic fractionation and analysis by mass spectrometry of conjugated metabolites of bis(2-ethylhexyl)phthalate in urine.

Mono(2-ethylhexyl)phthalate (MEHP), the primary metabolite of the plasticizer bis(2-ethylhexyl)phthalate (DEHP), was given to guinea pigs and mice and the methods for the isolation, separation and analysis of its metabolites in urine were developed. Following solid-phase extraction with octadecylsilane-bonded silica, individual metabolites were purified and separated using a combination of ion-exchange chromatography on lipophilic gels and reversed-phase high-performance liquid chromatography. Analysis of intact conjugates, as well as nonconjugated metabolites, was performed by fast atom bombardment mass spectrometry (FAB-MS) and, after derivatization, by gas chromatography-mass spectrometry. Enzymatic methods were used for further characterization. The study confirms glucuronidation as the major conjugation pathway for MEHP in the investigated species. Although less important quantitatively, glucosidation is shown to be an alternative conjugation pathway in mice. The methods developed were applied to a sample of urine from a hyperbilirubinemic newborn infant subjected to DEHP-exposure in conjunction with an exchange transfusion. It was demonstrated that metabolites of DEHP were excreted in amounts which could be analyzed by FAB-MS.

Binding of a 3,3', 4,4'-tetrachlorobiphenyl (CB-77) metabolite to fetal transthyretin and effects on fetal thyroid hormone levels in mice.

The present study was conducted in order to study the effect of the PCB congener 3,3', 4,4'-tetrachlorobiphenyl (CB-77) on fetal thyroxin homeostasis in the mouse, and to examine a possible underlying mechanism behind the effect. C57BL mice were treated with 14C-labelled or unlabelled CB-77 (1 or 10 mg/kg body wt.) on day 13 of gestation, and control animals were treated with corn oil. The experiment was terminated at 4 days after exposure. Maternal and fetal plasma and livers, and whole fetuses for homogenate preparation, were collected and analysed for total radioactivity, in vitro binding of 125I-thyroxin to plasma transthyretin (TTR; a thyroxin-transporting protein), and free and total thyroxin (FT4, TT4) levels. Maternal plasma, fetal plasma and homogenates were also analyzed for presence of CB-77 and metabolites. Results showed a dose-dependent uptake of radioactivity in plasma and liver, fetal plasma 14C-levels being about five-times higher in 10 mg/kg dosed animals as after 1 mg/kg. Fetal; plasma levels of total radioactivity were four- to nine-times above maternal levels and corresponded to only one compound, the metabolite 4-OH-3,3', 4',5-tetrachlorobiphenyl (4-OH-tCB). 4-OH-tCB was the major metabolite also in whole fetuses, with only small amounts of the parent compound (approximately 15% of the 4-OH-tCB) and traces (approximately 6%) of two other metabolites, 2-OH-3,3, 4,4'-tetrachlorobiphenyl and 5-OH-3,3', 4,4'-tetrachlorobiphenyl. Polyacrylamide gel electrophoresis confirmed that the 14C-radioactivity in fetal plasma was bound to TTR, and revealed that in vitro binding of 125I-T4 to fetal TTR was reduced to 50% of control values in treated animals (10 mg/kg body wt.). Fetal plasma FT4 and TT4 levels were significantly decreased (64 and 55% of control fetuses) after 10 mg/kg treatment. In conclusion, exposure of pregnant mice to CB-77 results in the accumulation of the metabolite 4-OH-tCB in fetal mouse plasma. The metabolite binds to TTR and is accompanied by a significant decrease in fetal plasma T4 levels. A causative correlation between TTR binding and effects on T4 levels is suggested.

[Preliminary studies on the retention of hydroxylated PCDF metabolites in rat blood].

We recently reported that certain hydroxylated PCB (OH-CB) metabolites were selectively retained in blood of rats experimentally dosed with PCB (Aroclor 1254), and also in blood of seals and humans environmentally exposed to PCBs. We also showed that metabolism in vivo of polychlorinated dibenzofurans (PCDFs) gave rise to a large number of hydroxylated PCDF (OH-CDF) metabolites, excreted in faeces, after oral administration to rats of PCDFs mixture (1, 2, 7, 8-tetraCDF 14%, 2, 3, 7, 8-tetraCDF 35%, 1, 2, 3, 7, 8-pentaCDF 48%). These results suggest that OH-CDFs could be present in blood. In the present study, potential retention of OH-CDF metabolites in blood (serum) was investigated in a female Wistar rat exposed to the PCDFs mixture. Serum was analyzed for the methylated OH-CDFs by gas chromatography (GC) and GC-mass spectrometry. Two major metabolites were determined in serum 1 day after oral administration. These were identified to be 3-OH-2, 4, 7, 8-tetraCDF (A) and 3-OH-2, 4, 7, 8, 9-pentaCDF (B) by comparison with synthesized reference compounds. 3-OH-2, 4, 7, 8-tetraCDF and 1, 2, 3, 7, 8-pentaCDF, respectively, via 3, 4(6, 7)-epoxide intermediate and subsequent NIH-shift of the 3(7)-chlorine to the 4(6)-position. The amounts of 3-OH-2, 4, 7, 8-tetraCDF and 3-OH-2, 4, 7, 8, 9-pentaCDF in the serum (5.72 g) analyzed accounted for 0.018% of 2, 3, 7, 8-tetraCDF and 0.003% of 1, 2, 3, 7, 8-pentaCDF dosed, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

beta-Naphthoflavone- and self-induced metabolism of 3,3',4,4'-tetrachlorobiphenyl in hepatic microsomes of the male, pregnant female and foetal rat.

1. The in vitro metabolism of 3,3',4,4'-tetrachloro-[14C]-biphenyl ([14C]-TCB) by hepatic microsomes from the Wistar rat was investigated with liver microsomes from the male, pregnant female and foetus. 2. Three hydroxylated metabolites (4-OH-3,3',4,5'-tetrachlorobiphenyl, 5-OH-3,3',4,4'-tetrachlorobiphenyl, and 6-OH-3,3',4,4'-tetrachlorobiphenyl) were identified by hplc and gc-ms after incubations of liver microsomes from the beta-naphthoflavone-pretreated male rat and TCB-treated pregnant rat. No metabolites of [14C]-TCB were found after incubation with foetal liver microsomes from dams pretreated with [14C]-TCB. The results indicate that the in vivo accumulation of 4-OH-tetraCB in the foetal compartment is probably due to transplacental transport rather than the formation of this metabolite in the foetus. 3. Pretreatment of the male rat with beta-naphthoflavone substantially induced the formation of hydroxylated metabolites, but pretreatment with phenobarbital and dexamethasone was without effect. Based on in vitro incubations of liver microsomes from the beta-naphthoflavone pretreated male rat, an apparent Km and Vmax of 4.5 microM and 240 pmol/mg protein/min respectively was determined for the metabolism of [14C]-TCB. The formation of phenolic metabolites of [14C]-TCB was most likely dependent on P4501A induction.

Selective retention of hydroxylated PCB metabolites in blood.

Polychlorinated biphenyls (PCBs) are important environmental contaminants, and their toxicity to wildlife and humans are of major concern. PCBs form persistent and abundant metabolites, PCB methyl sulfones, that accumulate in biota. We now report that certain hydroxylated PCB metabolites show a strong and selective accumulation in mammalian blood. Plasma from experimentally PCB-dosed rats and blood from environmentally exposed grey seals (Halichoerus grypus) and humans were analyzed. Among all possible hydroxylated metabolites of PCB that may be formed, only a few, dominated by 4-OH-2,3,5,3',4'-pentachlorobiphenyl and 4-OH-2,3,5,6,2',4',5'-heptachlorobiphenyl, were found in the blood samples. All identified compounds have a structure with the hydroxy group in a para or meta position, with chlorine atoms on vicinal carbon atoms. The concentrations of hydroxylated PCB in the blood were almost in the same range as the most persistent PCB congeners both for seals and humans.

Structure-dependent, competitive interaction of hydroxy-polychlorobiphenyls, -dibenzo-p-dioxins and -dibenzofurans with human transthyretin.

Previous results from our laboratory indicated specific and competitive interactions of hydroxylated metabolites of 3,3', 4,4'-tetrachlorobiphenyl with the plasma thyroid hormone transport protein, transthyretin (TTR), in rats in vivo and with human TTR in vitro. In the present study the structural requirements for competition with thyroxine (T4) for TTR-binding were investigated in more detail. Several hydroxylated polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) were tested in an in vitro competitive binding assay, using purified human TTR and [125I]T4 as a displaceable radioligand. All hydroxylated PCBs, but not the single PCB tested, competitively displaced [125I]T4 from TTR with differential potency. The highest competitive binding potency was observed for hydroxylated PCB congeners with the hydroxygroup substituted on meta or para positions and one or more chlorine atoms substituted adjacent to the hydroxy group on either or both aromatic rings (IC50 range 6.5-25 nM; Ka range: 0.78-3.95 x 10(8) M-1). The relative potency of all meta or para hydroxylated PCBs was higher than that of the physiological ligand, T4 (relative potency range: 3.5-13.6 compared to T4). There were no marked distinctions in TTR-T4 competitive binding potencies between the ortho- and non-ortho-chlorine substituted hydroxy-PCB congeners tested. Marked differences in TTR-T4 binding competition potency were observed between the limited number of hydroxylated PCDDs and PCDFs tested. The hydroxy-PCDD/Fs, with chlorine substitution adjacent to the hydroxy-group, i.e. 7-OH-2,3,8-trichlorodibenzo-p-dioxin, 2-OH-1,3,7,8-tetrachlorodibenzo-p-dioxin and 3-OH-2,6,7,8-tetrachlorodibenzofuran, all showed a similar or higher relative binding potency, i.e. 1, 4.4 and 4.5 times higher, respectively, than T4. No detectable [125I]T4 displacement was observed with 2-OH-7,8-dichlorodibenzofuran, 8-OH-2,3,4-trichlorodibenzofuran and 8-OH-2,3-dichlorodibenzo-p-dioxin, which did not contain chlorine substitution adjacent to the OH-group. These results indicate a profound similarity in structural requirements for TTR binding between hydroxy-PCB, -PCDD and -PCDF metabolites and the physiological ligand, T4, e.g. halogen substitution adjacent to the para hydroxy group, while planarity does not seem to influence the ligand-binding potency.

Enrichment of metabolites in the cerebrospinal fluid of cod (Gadus morhua) following oral administration of hexachlorobenzene and 2,4',5-trichlorobiphenyl.

The disposition of 14C-labelled hexachlorobenzene (HCB) and 2,4',5-trichlorobiphenyl (triCB) was studied in cod (Gadus morhua) and rainbow trout (Oncorhynchus mykiss). For both compounds tape section autoradiography revealed substantial amounts of radiolabelled material in the central nervous system (CNS) of cod, whereas only traces of radioactivity were observed in the CNS of rainbow trout. Furthermore, an enrichment of radiolabelled compound in the cerebrospinal fluid (CSF) was observed in the cod, whereas no radioactivity could be detected in the CSF of rainbow trout. According to autoradiography, the CNS of cod dosed with HCB contained the parent compound, whereas the major part of radioactivity in CSF was due to HCB metabolites. Thin-layer chromatography of extracts from cod dosed with triCB showed the presence of parent compound in the CNS, whereas part of the radioactivity in the CSF was due to triCB metabolites. The activities of cytochrome P-450 and UDP-glucuronosyltransferase in the CNS of cod and rainbow trout were determined in microsomal and mitochondrial fractions. Both species expressed activities which were in the same order of magnitude as those reported for the corresponding fractions from rat brain. Incubation of triCB with cod brain mitochondria and microsomes resulted in the formation of two polar metabolites. It is suggested that cod may be more vulnerable than rainbow trout regarding neurotoxicological effects of HCB, triCB and related environmental pollutants.

Glucuronidation of mono(2-ethylhexyl)phthalate. Some enzyme characteristics and inhibition by bilirubin.

A method for assaying mono(2-ethylhexyl)phthalate (MEHP) uridine diphosphate (UDP) glucuronyl transferase activity in microsomal preparations from guinea pig liver is described. The quantitation of the MEHP-glucuronide was performed by HPLC after direct injection of a sample of deproteinized incubation mixture. Solubilization of microsomal UDP-glucuronyltransferase activity was achieved by use of Lubrol, and optimal conditions for glucuronidation of MEHP were established. To investigate whether there is competition between MEHP and bilirubin for glucuronidation, inhibition experiments were performed with solubilized enzyme preparations. In these incubations addition of bilirubin decreased the formation of MEHP-glucuronide. No change in the maximal conversion rate (Vmax) was observed, indicating the occurrence of competitive inhibition. This observation may have implications in clinical situations where patients with hyperbilirubinemia are exposed to MEHP, e.g. in exchange transfusions in newborn infants.

A methylsulphonyl metabolite of a polychlorinated biphenyl can serve as a ligand for alpha 2mu-globulin in rat and major-urinary-protein in mice.

1. Kidneys from rats given an intraperitoneal dose of 4,4'-bis[( 3H]methylsulphonyl)-2,2',5,5'- tetrachlorobiphenyl[(CT3SO2)2TCB)] contained (CT3SO2)2TCB associated with a protein which has been isolated and characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblot and immunodiffusion analysis to be alpha 2 mu-globulin (alpha 2 mu). 2. The same radioactive alpha 2 mu-globulin complex was isolated from urine of rats given an i.p. dose of (CT3SO2)2TCB. This complex represented 12.3% (0.27% dose) and 9.3% (0.06% dose) of the radioactivity excreted in 0-24 h and 24-48 h urine, respectively. 3. The radioactivity was extractable from alpha 2 mu and characterized by t.l.c. (co-chromatography in two solvent systems) to be (CT3SO2)2TCB. 4. A radioactive-protein complex was isolated from urine of mice given an i.p. dose of (CT3SO2)2 TCB. The radioactive-protein complex was characterized to be mouse major urinary protein (MUP) by SDS-PAGE, immunoblot and immunodiffusion analysis. This complex was not detected in mouse kidney. 5. Urinary excretion of radioactive-MUP complex represented 51.7% (2.3% dose) and 28% (1.3% dose) of the radioactivity excreted in 0-24 h and 24-48 h urine, respectively.