Ingestion of Soil Contaminated with 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Alters Hepatic Enzyme Activities in Rats

Ingestion of Soil Contaminated with 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) Alters Hepatic Enzyme Activities in Rats. LUCIER, G.W., RUMBAUGH, R. C., MCCOY, Z., HASS, R., HARVAN, D., AND ALBRO,P. (1986). Fundam. Appl. Toxicol. 6, 36.4–371. Femalerats were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)in either corn oil or contaminated soil from the Minker sitein Missouri. Eight doses ranging from 0.015 to 5 µg TCDD/kgwere used in the corn oil group; the range was 0.015 to 5.5TCDD/kg in the TCDD-contaminated soil group. Rats in a thirdgroup were given equal amounts of soil uncontaminated with TCDD.No acute toxicity or effects on body weight gain were observedat these doses. In general, equivalent doses of TCDD in cornoil or TCDD in soil produced similar increases in hepatic arylhydrocarbon hydroxylase activity (AHH) and UDP glucuronyltransferaseactivity although effects were slightly greater in the TCDD–cornoil groups. In the corn oil groups, the induction of AHH rangedfrom about 30-fold at the highest dose to twofold at the lowestdose studied. TCDD also caused an increase in cytochrome P-450concentration and a shift in spectral peak from 450 to 448 nm.There was no effect of TCDD on ethylmorphine N-demethylase,consistent with previous reports. Liver concentrations of TCDD(mean ± SD) in the 5-µg/kg groups were 40.8 ±6.3 ppb in the TCDD- corn oil group and 20.3 ± 12.9 ppbin the TCDD-contaminated soil group. Our results suggest thatthe bioavailability of TCDD in soil in rats is approximately50%. Therefore, ingestional exposure to TCDD-contaminated soilmay constitute a significant health hazard in view of its extremelyhigh toxicity and relatively high bioavailability.     

Hepatic Aryl Hydrocarbon Hydroxylase and Cytochrome P450 Induction following the Transpulmonary Absorption of TCDD from Intratracheally Instilled Particles

Hepatic Aryl Hydrocarbon Hydroxylase and Cytochrome P450 Inductionfollowing the Transpulmonary Absorption of TCDD from IntratracheallyInstilled Particles. NESSEL, C. S., AMORUSO, M. A., UMBREIT,T. H., AND GALLO, M. A. (1990). Fundam. Appl. Toxicol. 15, 500-509.Inhalation of particles contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) will be an increasingly important route of human exposurein light of the increased utilization of municipal waste incinerationand the resultant emission of contaminated materials into theenvironment. The potential for pulmonary absorption of the compoundfrom respirable particles was assessed in the present studyfollowing the intratracheal instillation of TCDD (1) as a contaminantof gallium oxide particles and (2) in a corn oil vehicle. Groupsof five female Sprague-Dawley rats received 0, 0.005, 0.055,0.55, or 5.5 µg/kg TCDD in a single instillation and wereeuthanized 4 days later. Absorption was characterized by enzymeinduction [aryl hydrocarbon hydroxylase (AHH) activity and totalcytochrome P450] and histopathological examination of the liver.Induction of hepatic enzymes was dose-dependent with both treatmentregimes. Up to an 18-fold increase in AHH and an 80% increasein cytochrome P450 were observed in treated animals. Inductionwas slightly higher when animals received TCDD in com oil thanwhen animals received TCDD-contaminated particles and was relativelycomparable to induction following oral exposure. Similar resultswere obtained when animals were treated with particles contaminatedup to 4 weeks prior to instillation. Characteristics of TCDD-inducedhepatotoxicity, including enlarged hepatocytes and fatty infiltration,were apparent in treated rats, but were not present in vehicle-instilledanimals. These results indicate that systemic effects occurfollowing pulmonary exposure to TCDD and that inhalation maybe an important route Of exposure for TCDD.     

Potency of a Complex Mixture of Polychlorinated Dibenzo-p-dioxin, Dibenzofuran, and Biphenyl Congeners Compared to 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Causing Fish Early Life Stage Mortality

Use of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity equivalentsconcentration (TEC) assumes that polychlorinated di benzo-p-dioxins(PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs) act additivelyand via a common mechanism to cause toxicity. To test theseassumptions, 11 TCDD-like congeners and three non-TCDD-likecongeners were combined at ratios typically found in Lake Michiganlake trout. The potency of the mixture, expressed as TEC basedon fish-specific toxic equivalency factors, was compared toTCDD for producing lake trout and rainbow trout early life stagemortality. Signs of toxicity following exposure of newly fertilizedeggs to the mixture or to TCDD were indistinguishable; sac frymortality associated with blue-sac disease, and slopes of thedose-response curves for percentage sac fry mortality versusegg TEC or versus egg TCDD were parallel. However, the mixturedose-response curves were significantly shifted to the rightof the TCDD dose-response curves by 1.3- to 1.8-fold as illustratedby LD₅₀ values. Following exposure to the mixture or TCDD, LD₅₀sfor lake trout early life stage mortality were 97 (89–110)pg TE/g egg and 74 (70–80) pg TCDD/g (LD₅₀, 95% fiduciallimits) and for rainbow trout were 362 (312–406) pg TE/gegg and 200 (148–237) pg TCDD/g egg. These data suggestthat TCDD-like congeners act via a common mechanism to causetoxicity during trout early development, but may not act strictlyadditively when combined in a mixture of TCDD- and non-TCDD-likecongeners at ratios found in Great Lakes fish. The deviationfrom additivity, however, is less than current safety factorsof 10-fold commonly applied in ecological risk assessments,providing support for the continued use of a TE additivity modelfor assessing risk posed by complex mixtures of PCDDs, PCDFs,and PCBs to fish.     

In Vitro Bioassay for Dioxinlike Activity Based on Alterations in Epithelial Cell Proliferation and Morphology

In Vitro Bioassay for Dioxinlike Activity Based on Alterationsin Epithelial Cell Proliferation and Morphology. GIERTHY, J.F., AND CRANE, D. (1985). Fundam. Appl. Toxicol. 5, 754–759.2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown toinduce changes in morphology and proliferation characteristicsof a nonkeratinizing derivative (XBF) of a keratinizing epithelialcell line (XB), cloned from a mouse teratoma, when coculturedwith irradiated feeder cells. Polychlorinated dibenzodioxins(PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinatedbiphenyls (PCBs), polynuclear aromatic hydrocarbons (PAHs),and pesticides (24 compounds in total) were tested for theirability to induce these effects. The results indicated that,for the representative compounds tested, these changes are relativelyspecific for—and that the XBF cells are extremely sensitiveto—PCDDs and PCDFs. TCDD was the most potent congenertested, capable of inducing the effects at a concentration aslow as 10^–11 M. The activities of other tested PCDDs andPCDFs ranged from 10^–1 to 10^–3 of TCDD activity.The PCBs, PAHs, and pesticides had lower activities rangingfrom 10^–3 to 10^–6 that of TCDD. This assay systemusing XBF cells cocultured with irradiated 3T3 fibroblast feedercells was examined as a possible in vitro screening assay fordioxinlike activity by testing benzene extracts of soot froma fire involving a PCB-containing transformer. The results werecompared to a high-resolution gas chromatographic/mass spectrometricanalysis for total PCDFs in the same samples. This comparisonshowed a good correlation, suggesting that the XBF-3T3 systemhas potential for use as a semiquantitative assay for dioxinlikeactivity.     

Aryl Hydrocarbon Hydroxylase Induction in Adult Rat Hepatocytes in Primary Culture by Several Chlorinated Aromatic Hydrocarbons Including 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Aryl Hydrocarbon Hydroxylase Induction in Adult Rat Hepatocytesin Primary Culture by Several Chlorinated Aromatic HydrocarbonsIncluding 2,3,7,8-Tetrachlorodibenzo-p-dioxin. JANSING, R. L.,AND SHAIN, W. (1985). Fundam. Appl. Toxicol. 5, 713–720.The induction of aryl hydrocarbon hydroxylase (AHH) by Aroclor1254, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other polychlorinatedorganic compounds was examined in primary cultures of adultrat hepatocytes isolated by a collagenase perfusion technique.Following exposure of fresh hepatocyte cultures to 1 µg/mlAroclor 1254, AHH induction was undetected for 48 hr and thenincreased dramatically up to 96 hr. Cultures maintained in controlmedium for either 24, 48, or 72 hr prior to a 24-hr exposureto Aroclor 1254 displayed significant inducible AHH which wassustained to 96 hr. AHH induction was extremely sensitive totwo planar polyaromatic hydrocarbons, 2,3,7,8-TCDD and 2,3,7,8-tetrachlorodibenzofuran,and the PCB congener 3,4,3',4'-tetrachlorobiphenyl, but insensitiveto 2,6-dichlorodibenzofuran, 2,5,2',5'-tetrachlorobiphenyl,2,4,5,2',4',5'-hexachlorobiphenyl, and hexachlorobenzene. Theinduction of AHH activity in primary cultures of adult rat hepatocytesmay represent a useful bioassay for screening extracts of foodstuffs,biological fluids, or environmental samples for dioxin-likeactivity     

Transfer of Polychiorinated Dibenzo-p-dioxins and Dibenzofurans to Fetal and Neonatal Rats

A fly ash extract from a municipal incinerator, containing polychlorinateddibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), was orallyadministered to rats on Days 10 to 17 of the pregnancy and duringthe first 10 days of the lactation period. PCDD and PCDF congenersretained in the fetuses and livers of the offspring had a 2,3,7,8-chlorinesubstitution pattern. The only non-2,3,7,8-substituted congenerretained in the liver of the offspring and females was 2,3,4,6,7-pentachlorodibenzofuran(PnCDF). In the fetuses 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) had the highest retention (0.13% of total dose) and adecrease in retention was found with increasing number of chlorineatoms. In the livers of the offspring, the highest retentionwas found for 2,3,7,8-TCDD and the three 2,3,7,8-substitutedhexachlorodibenzo-p-dioxins (HxCDDs) (5.26–8.12%). Inthe livers of the pregnant and lactating females penta- andhexachlorinated congeners had the highest retention (53.91–80.20%). For both groups the liver retention of the tetra- tooctachlorinated congeners was similar, but the lactating femalesstored less PCDDs and PCDFs in their adipose tissue. A linearrelationship was found between the retention of congeners inthe livers of the females and offspring.     

Rapid bioassay for the determination of dioxins and dioxin-like PCDFs and PCBs in meat and animal feeds

Over the past several years, the numerous contamination incidents have raised concerns over the presence of halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and related chemicals in foods and feeds. Here we applied a sensitive recombinant mouse hepatoma cell (H1L1.1c2) bioassay for the determination of dioxins and dioxin-like polychlorinated dibenzofurans (PCDFs) and biphenyls (PCBs) in meat and animal feeds. These cells responded to TCDD-like chemicals with dose-dependent induction of firefly luciferase activity, and the minimal detection limit of TCDD in the cell was 16 fg. Induction equivalency factors determined for pure TCDD-like polychlorinated dibenzo-p-dioxins (PCDDs), PCDFs, and PCBs in the bioassay were well-correlated with the World Health Organization's toxic equivalency factors. To determine the applicability of the bioassay system to detect those compounds presence in meat and feed samples, cell bioassays for 17 TCDD-like PCDDs and PCDFs congeners-spiked lipid extracted from beef or animal feed were performed. Mean recoveries of TCDD-like chlorinated PCDDs and PCDFs congeners from spiked beef or feed fat ranged from 61.2 to 122.3%. Within-laboratory coefficients of variation for analysis as index of precision were lower than 5.2%, and the calculated limits of detection and quantitation were 0.33 and 1 pg toxicity equivalency quantity (TEQ)/0.5 g fat, respectively. Correlation between bioassay- and high-resolution gas chromatography-mass spectrometry (HR-GC-MS)-determined TEQs for 10 meat samples was 0.85, with 1.2 times higher in bioassay than HR-GC-MS. The correlation between bioassay- and HR-GC-MS-determined TEQs in 10 animal feed products was 0.81, with 2.1 times higher in bioassay than HR-GC-MS. Overall, these results demonstrated that the recombinant cell bioassay can be used for the rapid detection and quantitation of PCDDs and dioxin-like PCDFs and PCBs in meats and animal feeds.     

Health risk analysis of human exposures to soil amended with sewage sludge contaminated with polychlorinated dibenzodioxins and dibenzofurans

The risk of cancer to humans exposed to soil treated with wastewater/sewage treatment plant sludge, known to be contaminated with small amounts of polychlorinated dibenzodioxins and dibenzofurans (PCDDs and PCDFs), was evaluated. The particulate-bound PCDDs and PCDFs are found in trace amounts in the effluent from ground water pumping (dewatering) at an abandoned wood preservation facility. The water, which was sent to a water recovery plant, underwent primary and secondary treatment prior to discharge. The residual sludge was added to agricultural soil as a conditioner. The present analysis treats the extreme case of sludge applied near the home of a target individual, a lifetime resident, who is also a farm worker in the area of the application. The successive stages of infancy, childhood and adulthood are treated separately to assess the contributions of typical age-specific indoor and outdoor activities on exposure rates. Five toxicity rating schemes using so-called TCDD equivalents, and two unit risk slopes are applied to the chemical profile in sludge to determine the cancer potency of the soil contaminants. These risk estimates range from 1 X 10-8 to 3 X 10-7.     

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL/6 mice

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and TCDD plus MCDF were administered to C57BL/6 mice and their effects on several aryl hydrocarbon (Ah) receptor-mediated responses including hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction, immunotoxicity and teratogenicity were determined. MCDF did not induce hepatic microsomal AHH and EROD at doses up to 500 mumol/kg, however, co-administration of MCDF (50 mumol/kg) with a dose of TCDD which elicited a submaximal induction response (i.e. ED80-100, 15 nmol/kg) resulted in some small but significant inhibition of the induction of hepatic microsomal AHH and EROD (14 and 17%, respectively) compared to that observed with TCDD alone. Co-administration of TCDD and other doses of MCDF (10, 100, 200 or 500 mumol/kg) did not effect the induction response. These results were in contrast to the effectiveness of MCDF as an antagonist of the induction of AHH and EROD by TCDD in the rat (up to 50% inhibition of monooxygenase induction). Administration of MCDF (4, 20 and 40 mumol/kg) to C57BL/6 mice caused some inhibition of the splenic plaque-forming cell response to sheep erythrocytes only at the highest dose (26% decrease); the interaction of MCDF (4, 20 and 40 mumol/kg) and an immunotoxic dose of TCDD (3.7 nmol/kg) resulted in significant protection from the immunotoxic effects of TCDD at the 2 higher dose levels of MCDF. Similarly, MCDF (400 mumol/kg) did not cause cleft palate in mice but at this dose level MCDF afforded some protection from TCDD (20 micrograms/kg)-mediated cleft palate in mice. However, studies utilizing [3H]TCDD suggested that the protective effects may be due to modulation of TCDD reaching the palate in the co-treated animals (MCDF plus TCDD). Although both MCDF and Aroclor 1254 were both weak Ah receptor agonists in C57BL/6 mice, the former compound was much less effective as a TCDD antagonist. The observed species-specific effects for these 2 TCDD antagonists may be related species-dependent differences in receptor structure and receptor-ligand (i.e. agonist or antagonist) interactions.     

Polychlorinated dibenzo-p-dioxins: quantitative in vitro and in vivo structure-activity relationships

There were marked effects of structure on the activities of 14 polychlorinated dibenzo-p-dioxins (PCDDs) as competitive ligands for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor and as inducers of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells in culture. 2,3,7,8-TCDD was the most active compound in both assays and several PCDD congeners which were fully substituted in the lateral 2, 3, 7 and 8 positions but also contained additional chlorosubstituents in non-lateral 1, 4, 6 and 9 positions were less active. It was also evident that there was a decrease in in vitro binding and induction activities with decreasing lateral chlorine substitution. Although comparable structure-activity relationships (SARs) for the PCDDs were observed for the induction and receptor binding assays, there was not a linear or rank order correlation between the 2 sets of data. Several in vivo biologic and toxic activities of 2,3,7-trichloro-, 2,3,7,8- and 1,3,7,8-tetrachloro-, 1,2,4,7,8- and 1,2,3,7,8-pentachloro- and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin were determined in a dose-response fashion in immature male Wistar rats. The ED50 values for hepatic microsomal AHH and EROD induction, body weight loss and thymic atrophy were obtained. There was an excellent linear correlation between the -log EC50 values for AHH or EROD induction in cell culture and the -log ED50 values for enzyme induction, body weight loss and thymic atrophy in the rat. The in vitro enzyme induction data could be used to quantitatively estimate the toxicity of the PCDD congeners in the rat: this latter correlation has previously been observed for a series of polychlorinated dibenzofurans.     

Synergistic interactions of 2,3,7,8-TCDD and 2,2',4,4',5,5'-hexachlorobiphenyl in C57BL/6J and DBA/2J mice: role of the Ah receptor

Treatment of C57BL/6J mice with 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP, 500 mumol/kg) elevated hepatic cytosolic Ah receptor levels 82-107% for up to 14 days. Scatchard analysis of the [3H]2,3,7,8-TCDD (TCDD)-Ah receptor saturation binding curves from corn oil and HCBP treated rats gave KD values of 0.80 and 0.90 nM, respectively and confirmed that treatment with HCBP did not significantly alter receptor-radioligand affinities. Administration of HCBP to DBA/2J mice did not result in detectable hepatic cytosolic Ah receptor levels. Cotreatment of C57BL/6J mice with HCBP (500 mumol/kg) at a dose level of TCDD (1 nmol/kg) which elicited less than 10% of the maximum induction response resulted in significant synergistic induction of hepatic EROD and AHH [compared to animals treated only with TCDD (1 nmol/kg)]. In contrast, cotreatment of C57BL/6J mice with HCBP (500 mumol/kg) and maximally inducing dose levels of TCDD (100 or 500 nmol/kg) resulted in either a slight or no difference in the induction of AHH or EROD compared to the induction responses observed in mice treated only with TCDD. In contrast, cotreatment of DBA/2J mice with TCDD and HCBP (500 mumol/kg) resulted in significant synergistic induction of AHH and EROD at both submaximal (10-500 nmol/kg) and maximal (5000 nmol/kg) induction levels of TCDD. The only significant interactive effect of HCBP (500 mumol/kg) on the toxicity of TCDD in C57BL/6J and DBA/2J was protection from body weight loss observed after cotreatment of HCBP and TCDD in DBA/2J mice.     

Polychlorinated aromatic hydrocarbon lethality, mixed-function oxidase induction, and uroporphyrinogen decarboxylase inhibition in the chick embryo: dissociation of dose-response relationships

Effects on survival of chick embryos and on the activity of hepatic enzymes involved in heme biosynthesis and hemoprotein function were compared as a function of dose of 3,4,3',4'-tetrachlorobiphenyl (TCB), 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), 2,3,6,2',3',6'-HCB, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at 24 hr and at 9 days after polyhalogenated aromatic hydrocarbon (PAH) administration. 2,3,6,2',3',6'-HCB did not alter enzyme activities or survival. The other PAH increased delta-aminolevulinic acid synthetase up to 10- to 20-fold after 24 hr or 9 days of exposure. Hepatic porphyrins and uroporphyrinogen decarboxylase (Uro-D) activity were unaffected except by 3,4,3',4'-TCB which after 9 days of exposure increased porphyrins slightly (less than 2-fold) at 500 and 1000 nmol/egg and decreased Uro-D by 20% at 1000 nmol/egg. 3,4,3',4'-TCB, 3,4,5,3',4',5'-HCB, and TCDD preferentially induced cytochrome P-448-mediated mixed-function oxidases. The degree of induction was the same or greater after 9 days of exposure than after 24 hr. The three PAH increased aminopyrine demethylase and 7-ethoxycoumarin deethylase up to 2-fold and aryl hydrocarbon hydroxylase (AHH) 10- to 12-fold. 7-Ethoxyresorufin deethylase (7-ER) was increased 45-fold by 3,4,3',4'-TCB, 28-fold by 3,4,5,3',4',5'-HCB, and 55-fold by TCDD. The three PAH decreased survival after 9 days but not after 24 hr. Decreases in survival were accompanied by decreased thymus weights and increased incidences of pericardial and subcutaneous edema in surviving embryos. 3,4,3',4'-TCB caused dose-related decreases in survival at 100 to 1000 nmol/egg. 3,4,5,3',4',5'-HCB decreased survival at 500 and 1000 nmol/egg and TCDD at 6 nmol/egg. The effects on survival were greatest for 3,4,3',4',-TCB and least for TCDD, notwithstanding that all three PAH induced AHH to the same degree and that TCDD caused the greatest induction of 7-ER. The results demonstrate that the dose-response relationships for hepatic induction and lethality are dissociated and that the maximal induction levels are not correlated with the incidence of lethality. The findings indicate that the induction per se does not lead directly to toxicity and that other factors must intervene. The results further show that PAH lethality occurs independently of effects on hepatic Uro-D.     

Bioavailability of Soil-Bound TCDD: Oral Bioavailability in the Rat

Bioavailability of Soil-Bound TCDD: Oral Bioavailability inthe Rat. SHU, H., PAUSTENBACH, D., MURRAY, F. J., MARPLE, L.,BRUNCK, B., DEI ROSSI, D., TEITELBAUM, P. (1988). Fundam ApplToxicol. 10, 648–654. The implications to the public healthof trace amounts of 2,3,7,8-TCDD in the environment are underevaluation by regulatory agencies in the United States and WesternEurope. One major consideration in such evaluations is the contributionto human exposure via ingestion of TCDD-contaminated soil. An80% figure is under consideration by some regulators for estimatedhuman exposure. A contractor for one agency has, in fact, useda value of 1007% bioavailability for estimating human bioavailability.Several studies have investigated the oral bioavailability ofTCDD from contaminated soil in animals. Most have reported estimatesof 25–50%, although one has reported <0.5 and 85%,depending on the source of the contaminated soil. This paperreports an oral bioavailability of approximately 43% in therat dosed with three environmentally contaminated soil samplesfrom Times Beach, Missouri. This figure did not change significantlyover a 500-fold dose range of 2 to 1450 ng TCDD/kg of body weightfor soil contaminated with approximately 2, 30, or 600 ppb ofTCDD. The relevance of animal oral bioavailability data forthe human remains to be evaluated. However, since regulatoryagencies use animal data for extrapolating to humans, the 43%or 25–50% figure would be more accurate than the 80 or100% estimates.     

In vitro/in vivo effects of 7,8-benzoflavone on aryl hydrocarbon hydroxylase activity of mice.

The activity of aryl hydrocarbon hydroxylase (AHH) in mouse liver microsomes was assayed in vitro in the presence of 7,8-benzoflavone (7,8-BF). The mice had been previously treated with the AHH inducer 3-methylcholanthrene (MC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or vehicle (olive oil) alone. The effects of 7,8-BF on the AHH activity were markedly different according to the genetic responsiveness of the mice towards aromatic hydrocarbons (Ah). 7,8-BF had either an inhibitory or an enhancing effect on AHH, depending on the Ah responsiveness and previous treatment with MC or TCDD. In contrast, a single administration of 7,8-BF induced microsomal AHH, but the effect was much lower than MC or TCDD in both strains of mice. Simultaneous treatment or pre-treatment with 7,8-BF produced an inhibitory effect on AHH induction by MC or TCDD. Post-treatment with 7,8-BF inclined to promote the induction of AHH by MC or TCDD. These results suggest that the inhibitory effect of 7,8-BF is more exaggerated in vitro than in vivo. Inhibitory effects would be limited to AHH induction from MC or TCDD in in vitro studies, while they would depend on the time of application or Ah responsiveness in studies in vitro.     

Comparative Potencies of Aroclors 1232, 1242, 1248, 1254, and 1260 in Male Wistar Rats--Assessment of the Toxic Equivalency Factor (TEF) Approach for Polychlorinated Biphenyls (PCBs)

Immature male Wistar rats were treated with several differentdoses of the commercial polychlorinated biphenyls (PCBs) Aroclors1232, 1242, 1248, 1254, and 1260 (10, 40, 160, 480, and 2000mg/kg) and the effects on body weight gain, thymic atrophy,and the induction of hepatic microsomal aryl hydrocarbon hydroxylase(AHH), ethoxyresorufin O-deethylase (EROD), and pentoxyresorufin(O-deethylase (PROD) activities were measured 14 days aftertreatment. A significant inhibition in body weight gain wasobserved only in rats treated with high doses of Aroclors 1232and 1248 and thymic atrophy was not observed for any of theAroclors. All the Aroclors caused a dose-dependent increasein hepatic microsomal AHH, EROD, and PROD activities. The correspondingED50 values for the induction of AHH-EROD activities variedfrom 51 to 678 mg/kg. Aroclor 1260 was the least active inducerof the P4501A1-mediated enzyme activities. In contrast, Aroclor1260 was a potent inducer of PROD activity (ED50=37 mg/kg),but Aroclors 1232, 1242,1248, and 1254 did not induce 50% ofthe maximal response at the highest dose used in this experiment(2000 mg/ kg). Previous studies have quantitated the levelsof those PCB congeners which induce AHH or EROD activities inAroclors 1232, 1242, 1254, and 1260 and their potencies or toxicequivalency factors (TEFs) relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) have also been estimated or experimentally determined.Using highly conservative TEF values it was demonstrated thatthe calculated ED50s for the Aroclors as inducers of AHH andEROD activity were significantly lower than the observed ED50values. However, if the TEFs were derived directly from therelative potencies of the PCBs (compared to 2,3,7,8-TCDD) asinducers of AHH and EROD activity, then there was a good correspondencebetween the observed and calculated ED50 Values.     

Comparative dermal absorption of 2,3,7,8-tetrachlorodibenzo-p-dioxin and three polychlorinated dibenzofurans

Polychlorinated dibenzodioxins (PCDDs) and dibenzofurans (PCDFs) are toxic environmental contaminants which have the potential to accumulate in human tissues. In order to examine the potential for systemic exposure following dermal exposure, the absorption, distribution, and elimination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (1PeCDF), and 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF) were evaluated in male F344 rats. TCDD (0.00015, 0.001, 0.01, 0.1, 0.5, and 1.0 mumol/kg) and the three PCDFs (0.1, 0.5, and 1.0 mumol/kg) were applied to a preclipped region on the back of the rat and covered with a perforated cap. The rats were held in individual metabolism cages for 3 days. In animals administered 0.1 mumol/kg, the absorption of TCDF was greater than that of 4PeCDF, 1PeCDF, and TCDD. Relative absorption (percentage of administered dose) declined with increasing dose while the absolute absorption (microgram/kg) increased nonlinearly with dose. Absorption of TCDF at 0.1 mumol/kg was 48% of the administered dose which was significantly greater than that of the other compounds. At this dose, absorption of 4PeCDF was greater than that of TCDD. Absorption at the higher doses was similar for all four compounds. Maximum relative absorption of TCDD (approximately 40% of the administered dose) was obtained at 0.001 and 0.00015 mumol/kg. Major tissue depots for these four chemicals included liver, adipose, skin, and muscle tissue; however, the liver:fat ratio for 4PeCDF was approximately fourfold higher than that for the other three compounds. When normalized to 100% of dose absorbed, the distribution of 4PeCDF-derived radioactivity in liver and adipose tissue was similar to that previously observed after oral and iv administration. In animals administered 0.1 mumol TCDF or 1PeCDF/kg, 56 and 32% of the respective absorbed dose was excreted as polar metabolites within 3 days. Very little of the absorbed dose of either TCDD (approximately 10%) or 4PeCDF (approximately 2%) was eliminated. Results indicate that the dermal absorption of these compounds is incomplete and that systemic toxicity following acute dermal exposure to levels found in the environment is unlikely.     

6-Methyl-1,3,8-trichlorodibenzofuran as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: inhibition of the induction of rat cytochrome P-450 isozymes and related monooxygenase activities

In addition to being one of the most toxic chemicals known, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent inducer of rat liver microsomal cytochrome P-4501A1 (P-450c). Previous studies have demonstrated that a high affinity, low capacity cytosolic receptor (the Ah receptor) mediates the activity of TCDD to induce cytochrome P-4501A1, which catalyzes benzo[a]pyrene hydroxylation [aryl hydrocarbon hydroxylase (AHH]) and 7-ethoxyresorufin O-dealkylation (EROD). The results of the present study indicate that 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) effectively competes with [3H]TCDD for binding to the Ah receptor in rat liver cytosol. The concentration of MCDF effecting 50% displacement of [3H]TCDD was 4.9 X 10(-8) M, which is approximately 50 times greater than the EC50 for unlabeled TCDD (approximately 1 X 10(-9) M). However, in contrast to TCDD, MCDF was only a weak inducer of AHH and EROD activity in rat hepatoma H-4-II cells in culture. When co-incubated, MCDF diminished in a concentration-dependent manner the ability of TCDD to induce AHH and EROD activity in vitro. Treatment of rats with 20-200 mumol/kg MCDF in vivo had little or no effect on liver microsomal AHH and EROD activity, whereas treatment of rats with 16 nmol/kg TCDD caused a 6- and a 70-fold induction of AHH and EROD activity, respectively. When co-administered, MCDF diminished by approximately 50% the ability of TCDD to induce AHH and EROD activity in vivo. The partial antagonism produced by 50 mumol/kg MCDF could be partially overcome by doubling the dosage of TCDD from 16 to 32 nmol/kg. Immunochemical analysis of rat liver microsomes revealed that treatment of rats with 20-200 mumol/kg MCDF caused little or no induction of cytochromes P-4501A1 and P-4501A2 (P-450d), whereas these isozymes were induced 33- and 5-fold, respectively, in rats treated with 16 nmol/kg TCDD. When co-administered, MCDF diminished by approximately 50% the ability of TCDD to induce cytochrome P-4501A1 in vivo, which established that MCDF was not simply acting as an inhibitor of AHH and EROD activity. MCDF also antagonized the ability of TCDD to induce cytochrome P-4501A2, which suggests that the induction of both cytochromes P-4501A1 and P-4501A2 is regulated by the Ah receptor. These results indicate that MCDF binds with high affinity to the Ah receptor in rat liver cytosol and competitively blocks the binding of TCDD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of Mycoplasma arginini infection on the induction of aryl hydrocarbon hydroxylase by TCDD in rat hepatoma cell cultures

Mycoplasma arginini was eliminated from a rat hepatoma cell line (H-4-II-E) by plating at low cell density and treatment with chlortetracycline (250 micrograms/ml), kanamycin (250 micrograms/ml), tylosin (100 micrograms/ml), 3% M. arginini antiserum and 5% fresh guinea-pig serum. The induction of AHH activity in the cell culture was measured in response to increasing concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The ED50 values (estimated doses that produce 50% maximum enzyme induction) were calculated to be 0.256, 0.452 and 0.344 pmol TCDD/plate for original, mycoplasma-free and reinfected cells, respectively. Although the absence of M. arginini in the rat hepatoma cell line makes the cells slightly less responsive to AHH induction by TCDD, this decrease does not detract from the use of the method to screen food extracts and environmental samples for the presence of certain toxic planar organic compounds.     

Effects of postnatal exposure to mixtures of non-ortho-PCBs, PCDDs, and PCDFs in prepubertal female rats.

There are concerns that postnatal exposure to organochlorines present in breast milk could lead to adverse health effects. We reconstituted four mixtures of aryl-hydrocarbon receptor (AhR) agonists (3 non-ortho polychlorinated biphenyls [PCBs], 6 polychlorinated dibenzodioxins [PCDDs], 7 polychlorinated dibenzofurans [PCDFs], or all 16 chemicals together [referred to as AhRM]) based on their concentrations in breast milk, and examined their effects following exposure by gavage from day 1 until day 20 of age. Female neonates received dosages of AhRM equivalent to 1, 10, 100, or 1000 times the amount consumed by an infant over the first 24 days of life. Other groups received the PCBs, the PCDDs, or the PCDFs at the 1000x level. All rats were sacrificed at 21 days of age. Changes in ethoxyresorufin-o-deethylase hepatic activity, thymus and body weights, and serum thyroxin were linked to the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalents (TEQ) of the four mixtures (1000x-AhRM > PCDDs > PCBs > PCDFs). To test for AhRM antiestrogenicity, two additional groups received 1.5 microg/kg of 17alpha-ethynyl estradiol (EE) with or without the 1000x-AhRM. The AhRM had no effect on uterine weight or EE-stimulated uterine growth. The actions of the combined EE and AhRM treatments suggest additive effects in decreasing pentoxyresorufin-o-deethylase activity and spleen weight, but nonadditive/antagonistic effects on adrenal weight and serum thyroxin. In conclusion, (1) 10x-AhRM had no detectable effects, (2) TEQ values relate to observed toxicities, even when testing complex mixtures of AhR agonists, and (3) indications of tissue-specific additive and nonadditive/antagonistic effects, but no synergism, were observed when doses of AhRM were increased, or combined with EE.     

A comparative study of polychlorinated dibenzofurans,polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin on aryl hydrocarbon hydroxylase inducing potency in rats

The aryl hydrocarbon hydroxylase (AHH) inducing potency of toxic chlorinated aromatic hydrocarbons such as polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in the young male Wistar rats. Alternatively, a technical PCDF mixture, 15 individual PCDF isomers or TCDD were administered i.p. in doses of 5 g/kg; a PCB mixture was given in a dose of 50 mg/kg. The order of AHH inducing ability was TCDD > PCDFs PCBs in kidney, lung, and liver. In the prostate, thymus, and spleen, only TCDD enhanced the AHH activity. The AHH inducibility in the lung and liver, induced by 15 pure PCDF isomers with varying chlorine substitutions was also examined. Only 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-tetra-CDF) and 2,3,4,7,8-pentachlorodibenzofurans (2,3,4,7,8-penta-CDF) significantly induced the hepatic AHH activity (4- and 2-fold, respectively), while eight PCDF isomers, including these two, significantly enhanced the pulmonary AHH activity (6- to 30-fold). Taking into account both the potent AHH inducibility and the high bioaccumulation of these compounds, 2,3,7,8-tetra- and 2,3,4,7,8-penta-CDF should be given due attention with regard to environmental-related factors and the possibility of involvement in the etiology of Yusho disease.A part of this work was presented at the 51st annual meeting of the Japanese Society for Hygiene, May 1–3, 1981, Sapporo, Japan and the 52nd annual meeting of the Japanese Society for Hygiene, March 29–31, 1982, Tokyo, Japan