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

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

Analysis of difference in vivo effects of TCDD between c-src +/+ mice, c-src deficient, -/+ and -/- B6, 129-Src(tm 1 sor) mice and their wild-type littermates

Toxicities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were tested on two strains of c-src deficient B6, 129-Src(tm 1 sor) mice, c-src -/+ and c-src -/- and their matched wild-type littermates c-src +/+ mice along with another c-src +/+ mice, from the same genetic lineage, B6, 129-Fos(tm 1 Pa) mice. The most conspicuous effect of c-src deficiency on the toxicity of TCDD appears to be the reduced hepatotoxicity. TCDD-treated c-src deficient mice show only modest degrees of hepatomegaly and triglycerides accumulation as compared to treated wild-type mice.     

Method for evaluation of immunotoxicity of dioxin compounds using human T-lymphoblastic cell line, L-MAT

Caspase-3 was activated in apoptotic L-MAT cells by treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Treatment with tributyltin, which has been reported to induce apoptosis in rat thymocytes, also activated caspase-3 and led to cell death in L-MAT cells. Blocking caspase-3 activity with the peptide inhibitor, DEVE-CHO, prevented TCDD from inducing subsequent apoptotic changes. The potent Ah receptor ligand, 2,3,7,8-tetrachlorodibenzofuran (TCDF), the low acute toxicity compound, 1,2,3,4,6,7,9-heptachlorodibenzo-p-dioxin (HCDD), and one of the major contaminants in human milk, 3,3',4,4',5-pentachlorobiphenyl (PCBP), increased the activation level of caspase-3, each in a dose-dependent manner. Thus, we propose that measuring caspase-3 activation in the human T-lymphoblastic cell line, L-MAT, is a useful evaluation method for the immunotoxicity of dioxin compounds.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on serum insulin and glucose levels in the rabbit

Serum insulin and glucose were measured in young male rabbits after a single intraperitoneal dose of 1 or 50 micrograms/kg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Serum insulin levels in the high dosed rabbits were significantly decreased between 15 min and 8 h post treatment, equivalent after 24 h, significantly elevated 48 h post treatment, and they were not different at 10 days post-treatment when compared to weight matched and pair-fed controls. At the low dose, rabbits showed no differences in serum insulin from controls. In the high dose group, serum glucose levels were generally not different between treated and control animals, though there was a transient hyperglycemia 1 h after treatment, and both treated groups became hypoglycemic after ten days. The results indicate that TCDD altered serum insulin levels which were not coupled to changes in serum glucose.     

Effect of inducers of P-450 cytochrome isoenzymes on TCDD immunosuppressive activity

Drugs inducing different forms of P-450 cytochrome isoenzymes and binding the Ah receptor or not were investigated for their ability to modify 2,3,7,8, tetrachlorodibenzo-p-dioxin (TCDD) immunotoxicity in mice. 3-Methyl-cholanthrene (3MC) and β-naphthoflavone (BNF) administered to TCDD-treated mice caused additive inhibition of humoral antibody production and of responsiveness to concanavallin A (ConA) but not to phytohemagglutinin (PHA) and lipopolisaccharide (LPS) while phenobarbital (PB) never modified TCDD immunosuppression. Natural killer (NK) cell activity was not reduced by single drug treatment or by combined treatments. Hepatic aryl hydrocarbon hydroxylase (AHH) induction by TCDD was not significantly modified by PB, 3MC or BNF.     

The reliability of 2,3,7,8-tetrachlorodibenzo-p-dioxin and toxicity equivalence measurements in Missouri residents

This study evaluates the reliability of serum measurements of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and toxicity equivalence (TEQ) in individuals with no prior exposure to TCDD or dioxin-like compounds above background levels. Reliability was measured over three time-periods in the same individuals. TCDD and TEQ were measured in 127 and 128 participants. The values declined during the testing periods. Reliability coefficients for TCDD was 0.69 and for TEQ 0.91 but increased to 0.79 and 0.92, respectively, after removal of three outlying cases. Study results show good reliability over 22 months suggesting a consistency in the uptake and excretion of TCDD and other dioxin-like compounds.     

Estrogenic and antiestrogenic effect of in vitro treatment of follicular cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin

OBJECTIVE: Two types of follicular cells from preovulatory ovary were cultured in vitro separately and in co-culture to test difference in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) action on particular cell types. METHODS: The accumulation of TCDD in follicular wall was analysed using coupled capillary gas chromatography mass spectrometry. Whole preovulatory follicles were isolated from ovary and incubated with prolonged exposure to 0.1 nM TCDD or single exposure to 10 nM TCDD for four days. In the second part of experiments direct effects of TCDD on steroidogenesis were investigated in porcine theca cells (Tc) and granulosa cells (Gc) cultured alone and in co-culture (GT). The media were collected after four days for steroid analysis. RESULTS: 59.3% and 81.2% of TCDD added to the culture medium was accumulated after 0.1 and 10 nM, respectively. TCDD in a dose-dependent manner increased estradiol secretion with concomitant progesterone secretion by theca interna cells. On the other hand decrease of both progesterone and estradiol secretion by granulosa cells cultured alone and in co-culture with theca cells was noted. CONCLUSION: Different cell-specific estrogenic or antiestrogenic effect of TCDD were found in ovarian follicles.     

Mixture effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and polychlorinated biphenyl congeners in rats

Concern of the toxic effects and bioaccumulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls in the environment continues to be a focus of research in persistent organochlorine contaminants. Groups of five adult female S.D. rats were administered by gavage 0, 2.5, 25, 250 or 1000 ng TCDD/kg body weight/day or TCDD in combination with a mixture of PCB congeners (PCBs) at 2 or 20 microg/kg b.w./day for a period of 28 days. Growth suppression, increased absolute and relative liver weights, and decreased thymic weight were observed in either the 1000 ng TCDD group alone, or the groups receiving a mixture of 1000 ng TCDD + 2 microg PCBs. The TCDD induced increases in liver and thymic weights were not altered by co-administration with PCBs, however, growth suppression appeared to be more pronounced in the group receiving 1000 ng TCDD + 2 microg PCBs than with TCDD alone. Treatment with TCDD at 250 ng and 1000 ng/kg resulted in a significant increase in hepatic microsomal methoxy resorufin-O-demethylase and ethoxy resorufin-O-deethylase activities which were antagonized by co-administration with PCBs. Similarly, effects of 250 ng TCDD on serum cholesterol and liver UDP glucuronosyl transferase activity and ascorbic acid were significantly reduced by co-administration with 20 microg PCBs. Other biochemical effects elicited by treatment with 1000 ng TCDD, but not affected by co-administration with PCBs include the following: increased serum albumin, decreased liver vitamin A, and increased kidney vitamin A and liver microsomal glutathione-S-transferase activity. While decreased hemoglobin, platelet, packed cell volume and red cell indices were observed in TCDD treated rats, no interactive effects were seen. The above results indicate that the mixture effects of PCBs and TCDD may be additive or antagonistic depending on the dose level and endpoints measured. For the purpose of predicting mixture effects, knowledge of mechanisms of action and toxicokinetics is required.     

Carcinogenic and co-carcinogenic potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin in a host-mediated in vivo/in vitro assay

In an in vivo/in vitro assay system (Massa et al., 1990) we have detected the carcinogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The carcinogenic potential measured in this system is concentration-dependent. Experiments with other carcinogenic compounds have revealed that TCDD at low doses can act as co-carcinogen. At higher concentrations TCDD induces TNF-α production.     

TCDD alters PKC signaling pathways in developing neuronal cells in culture

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to induce neurodevelopmental deficits such as poor cognitive development and motor dysfunction. However, the mechanism of TCDD-mediated neurotoxicity remains unclear. Since PKC signaling is one of the most pivotal events involved in neuronal function and development, we analyzed the effects of TCDD on the PKC signaling pathway in cerebellar granule cells derived from PND-7 rat brain. Immunoblot analysis revealed the presence of PKC-alpha, betaII, delta, epsilon, lambda and iota in both cytosol and membrane fractions of cerebellar granule cells, but PKC-gamma was below the detectable level. TCDD induced a significant translocation of PKC-alpha, -betaII and -epsilon from cytosol to membrane fraction (p<0.05) and a marginal translocation of PKC-delta at high dose only (p<0.1). It also increased RACK-1, an adaptor protein for PKC, in a dose-dependent manner. Exposure to TCDD induced a dose-dependent increase of both [3H] PDBu binding and the intracellular calcium level. The results suggest that the selective PKC isozymes and RACK-1 are involved in TCDD-mediated signaling pathway and these proteins may be possible molecular targets in neuronal cells for TCDD exposure. Our study provides basic data to understand mechanism of TCDD-induced neurotoxicity with respect to PKC signaling pathway and a scientific basis for improving the health risk assessment of neurotoxicants by identifying intracellular target molecules in neuronal cells.     

Inhibition of phosphoenolpyruvate carboxykinase activity appears to be the key biochemical lesion in the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exerts its acute toxicity by inducing a gradually increasing voluntary feed refusal. However, this seems not to be caused by a direct effect on the central nervous system, as far higher concentrations of TCDD were found in the brain after intracerebroventricular (i.c.v.) than after lethal intravenous (i.v.) injections, but were not accompanied by a wasting syndrome. TCDD causes inhibition of several key enzymes of gluconeogenesis, with phosphoenolpyruvate carboxykinase (PEPCK) responding earliest and strongest to the insult. Responses of pyruvate carboxylase (PC) and glucose-6-phosphatase (G-6-Pase) are less pronounced and begin at later time points. Blood and brain levels of tryptophan increase following TCDD treatment with a lag period of about three days, shortly after the decrease of PEPCK activity becomes apparent. Since thi samino acid is the precursor of the appetite suppressant neurotransmitter serotonin, and since it is normally degraded via gluconeogenesis, a series of events can be suggested to explain the TCDD-induced wasting syndrome. By an as yet unrevealed mechanism TCDD decreases the activity of PEPCK to about 40 percent of normal, leading to a back-up of gluconeogenic substrates, among them tryptophan, which in turn can further inhibit PEPCK activity in vivo. This causes an increase in serotonin turnover in brain and possibly in other tissues. Increased serotonergic activity in turn is likely to play an important role in the increasing feed refusal of TCDD-treated rats which eventually leads to death.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin or 2,2',4,4',5,5'-hexachlorobiphenyl on vitamin K-dependent blood coagulation in male and female WAG/Rij-rats

Newborns are susceptible to hemorrhages (hemorrhagic disease of the newborn or HDN) due to vitamin K deficiency. Induction of cytochrome P450 in the fetal liver by maternal anticonvulsant therapy such as phenobarbital or phenytoin is considered to be a major cause. An observed increase in late hemorrhagic disease (LHD) in breast fed neonates gave rise to the hypothesis that PCBs and dioxins, P450-inducing contaminants present in human milk, might effect vitamin K-dependent blood coagulation. This hypothesis was studied in rats. Administration of a single oral dose of 0.003, 0.03, 0.3, 3 or 30 nmol 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) per kg bodyweight or 0.75, 4, 20, 100 or 500 micromol 2,2',4,4',5,5'-hexachlorobiphenyl/kg bw (HxCB) to female and male rats resulted in dose-related reductions of the vitamin K-dependent coagulation factor VII. The highest factor VII reduction in female rats was 44%, observed after TCDD exposure. The Lowest Observed Adverse Effect Level (LOAEL) of TCDD on female factor VII levels was 0.3 nmol/kg bw (96 ng/kg). There was a significant inverse correlation between Factor VII levels and induction of hepatic ethoxyresorufin O-deethylating (EROD) activity, reflecting CYP1A1, and total P450 content. HxCB had no effect on female coagulation factors. In contrast, in male rats only exposure to HxCB, which induces mainly CYP2B1 and 2B2, decreased both coagulation factors dramatically up to 88%. The LOAEL of HxCB on factor VII in male rats was 100 micromol/kg bw (36 mg/kg). In general, effects on coagulation factors in male rats exceeded those in females. In addition, sex-dependent differences of TCDD and HxCB were observed on the hepatic vitamin K cycle enzyme activities in female and male rats. Vitamin K-dependent (gamma-glutamyl carboxylase activity was mainly induced in female rats; 2.3-fold in the highest dose group of TCDD. In male rats only vitamin K 2,3-epoxide reductase (KO-reductase) activity was induced 1.7-fold by the highest dose of HxCB. KO-reductase activity in female rats was also increased by TCDD, however, less pronounced than the carboxylase activity. Concluding, the hepatic vitamin K cycle still functions and is not blocked by TCDD or HxCB, thus explaining the observed reduction in factor VII. Finally, the possible role of P450 in vitamin K deficiency is discussed. Based on these results it is suggested to investigate the possible role of PCBs and dioxin-like compounds in LHD in more detail.     

Environmental fate and bioavailability of agent orange and its associated dioxin during the vietnam war

BACKGROUND: In 1996, the Committee on the Assessment of Wartime Exposure to Herbicides in Vietnam of the National Academy of Sciences' Institute of Medicine (IOM) issued a report on an exposure model for use in epidemiological studies of Vietnam veterans. This exposure model would consider troop locations based on military records; aerial spray mission data; estimated ground spraying activity; estimated exposure opportunity factors; military indications for herbicide use; and considerations of the composition and environmental fate of herbicides, including changes in the TCDD content of the herbicides over time, the persistence of TCDD and herbicides in the environment, and the degree of likely penetration of the herbicides into the ground. When the final report of the IOM Committee was released in October 2003, several components of the exposure model envisioned by the Committee were not addressed. These components included the environmental fate of the herbicides, including changes in the TCDD content over time, the persistence of TCDD and herbicides in the environment, and the degree of likely penetration of herbicides into the ground. This paper is intended to help investigators understand better the fate and transport of herbicides and TCDD from spray missions, particularly in performing epidemiological studies. METHODS: This paper reviews the published scientific literature related to the environmental fate of Agent Orange and the contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and discusses how this affected the potential exposure to TCDD of ground troops in Vietnam. Specifically, the mechanisms of dissipation and degradation as they relate to environmental distribution and bioavailability are addressed. RESULTS: The evaluation of the spray systems used to disseminate herbicides in Vietnam showed that they were capable of highly precise applications both in terms of concentrations sprayed and area treated. Research on tropical forest canopies with leaf area indices (a measure of foliage density) from 2 to 5 indicated that the amount of herbicide and associated TCDD reaching the forest floor would have been between 1 and 6% of the total aerial spray. Studies of the properties of plant surface waxes of the cuticle layer suggested that Agent Orange, including the TCDD, would have dried (i.e., be absorbed into the wax layer of the plant cuticle) upon spraying within minutes and could not be physically dislodged. Studies of Agent Orange and the associated TCDD on both leaf and soil surface have demonstrated that photolysis by sunlight would have rapidly decreased the concentration of TCDD, and this process continued in shade. Studies of 'dislodgeable foliar residues' (DFR, the fraction of a substance that is available for cutaneous uptake from the plant leaves) showed that only 8% of the DFR was present 1 hr after application. This dropped to 1% of the total 24 hrs after application. Studies with human volunteers confirmed that after 2 hrs of saturated contact with bare skin, only 0.15-0.46% of 2,4,5-T, one of the phenoxy acetic acid compounds that was an active ingredient of Agent Orange, entered the body and was eliminated in the urine. CONCLUSIONS: The prospect of exposure to TCDD from Agent Orange in ground troops in Vietnam seems unlikely in light of the environmental dissipation of TCDD, little bioavailability, and the properties of the herbicides and circumstances of application that occurred. Photochemical degradation of TCDD and limited bioavailability of any residual TCDD present in soil or on vegetation suggest that dioxin concentrations in ground troops who served in Vietnam would have been small and indistinguishable from background levels even if they had been in recently treated areas. Laboratory and field data reported in the literature provide compelling evidence on the fate and dislodgeability of herbicide and TCDD in the environment. This evidence of the environmental fate and poor bioavailability of TCDD from Agent Orange is consistent with the observation of little or no exposure in the veterans who served in Vietnam. Appreciable accumulation of TCDD in veterans would have required repeated long-term direct skin contact of the type experienced by United States (US) Air Force RANCH HAND and US Army Chemical Corps personnel who handled or otherwise had direct contact with liquid herbicide, not from incidental exposure under field conditions where Agent Orange had been sprayed.     

T cell-derived IL-5 production is a sensitive target of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The immune system is one of the organs most vulnerable to the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Among the various immunotoxic effects of TCDD, the thymus involution and suppression of IgM antibody production are well known sensitive reactions of the thymocytes and B cells affected by TCDD. Recently, we reported that TCDD greatly inhibits the production of type-2 helper T (Th2) cell-derived cytokines, especially IL-5, by the splenocytes in mice immunized with ovalbumin (OVA). In the present study, we investigated the dose-dependency of these TCDD immunotoxic effects in OVA-immunized mice to identify the most sensitive target. Mice of two age groups, 6 weeks old and 3 weeks old, were dosed with 0.3, 1.0, or 3.0 microg TCDD/kg and immunized with OVA using alum as an adjuvant. Seven days later, the thymus weight, thymocyte population, antigen-specific IgM in the plasma, and IL-5 production by the splenocytes were examined. Among them, IL-5 production was significantly suppressed by all three doses of TCDD and reduced to about 30% by even a small dose of 0.3 microg TCDD/kg in both age groups. The thymus weight was significantly reduced by 1.0 microg or 3.0 microg TCDD/kg, but IgM production was not affected by up to 3.0 microg/kg of TCDD in both age groups. Taken together, the Th2 cell-derived IL-5 production was the most sensitive endpoint detecting TCDD toxicity among those examined. Our results also suggest that effector T cells are targets more vulnerable to TCDD toxicity than thymocytes or antibody-producing B cells in the OVA-immunized mice.     

The potential inhalation hazard posed by dioxin contaminated soil.

Mathematical models and field data were used to estimate the airborne concentrations of 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) vapor and particulates which could originate from soil containing 100 ppb TCDD. The model of Jury et al. (1983) and the box approach were used to predict the concentration of TCDD vapor from soil. The daily soil temperature was assumed to vary between 20 degrees C and 40 degrees C for six months of the year to account for diurnal warming and cooling of the soil. The depth of contamination was 50 mm. The model predicted average vapor flux rate for TCDD from soil for this temperature profile was 1.5 x 10(-14) mg/sec-cm2. The upper-bound estimates of the TCDD vapor concentration on-site at 40 degrees C and 20 degrees C were 2.5 pg/m3 and 1.8 pg/m3, respectively. Using a recently proposed unit risk value (URV) of 2.9 x 10(-6) (pg/m3)-1 [slope factor = 1.0 x 10(-14) (mg/kg-day)-1], the maximum plausible cancer risk is about 1 x 10(-5). If one accepts the EPA URV of 3.3 x 10(-5) (pg/m3)-1 (slope factor = 1.2 x 10(-13) (mg/kg-day)-1), then the risk is no greater than 1 x 10(4). A maximum TCDD vapor concentration of 0.21 pg/m3 was predicted 100 meters downwind (for summer days). The on-site concentration of TCDD in suspended particulate was estimated to be 1.4 pg/m3 (based on a TSP level of 0.07 mg/m3 from site soil). For persons exposed to vapors and particulates about 100 meters off-site, the exposure was about 10-fold less.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of binary mixtures of six xenobiotics on hormone concentrations and morphometric endpoints of northern bobwhite quail (Colinus virginianus)

This study investigated the effects of six endocrine disrupters in five different doses (0.1, 0.3, 1, 3, 10 mg/kg or microg/kg) in ethanol administered by oral gavage to bobwhite quail eggs. Six eggs each were in each dose group of coumestrol, ethynyl estradiol, indole-3-carbinol, o,p'-DDE, p,p'-DDE, or TCDD. Eggs were also dosed in two sets. One set was ethynyl estradiol (0, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0 microg/kg) and TCDD (0, 0.003, 0.01, 0.03, 0.1, 0.3 microg/kg). This set was dosed below the air cell with corn oil as vehicle. Also, northern bobwhite quail eggs were injected in ovo with nine binary mixtures of six xenobiotics prior to incubation (coumestrol (0.3 mg/kg), ethynyl estradiol (3.0 microg/kg), indole-3-carbinol (3.0 mg/kg), o,p'-DDE (1.0 mg/kg), p,p'-DDE (1.0 mg/kg), TCDD (0.1 microg/kg)). The mixtures injected were p,p'-DDE+indole-3-carbinol, coumestrol+indole-3-carbinol, TCDD+indole-3-carbinol, p,p'-DDE+o,p'-DDE, p,p'-DDE+ethynyl estradiol, coumestrol+ethynyl estradiol, coumestrol+TCDD, o,p'-DDE+ethynyl estradiol, TCDD+ethynyl estradiol. Eggs were dosed once prior to initiating incubation. Quail were allowed to hatch and were sacrificed at 21 days of age. Blood, measurements, and tissues were collected. Survival was significantly affected by increasing concentrations of TCDD in ethanol as revealed by trend analysis. Survival was also affected significantly by o,p'-DDE in ethanol but not by trend. Survival results of mixtures indicate significant differences among mixture, mixture components, and controls for coumestrol+TCDD, ethynyl estradiol+TCDD, and indole-3-carbinol+TCDD. Some trends from doses of single compounds that are supported by results in the literature were observed for hatchling weight of ethynyl estradiol dosed females, weight gain of indole-3-carbinol dosed males, weight gain and liver somatic index of o,p'-DDE dosed males, spleen somatic index of TCDD dosed males, and weight gain, gonad somatic index and egg gland somatic index of TCDD dosed females. In conclusion, the dose response treatments appeared to have effects beyond effects on survival of in ovo dosed quail. For mixtures, plasma estradiol concentrations were significantly different among coumestrol+ethynyl estradiol, ethynyl estradiol, coumestrol, and vehicle treatments. Liver somatic index among the same treatments was also significantly different. Kidney somatic index among ethynyl estradiol+p,p'-DDE, ethynyl estradiol, p,p'-DDE, and vehicle treatments was significantly different. Plasma estradiol and plasma testosterone ratios were very different among o,p'-DDE+p,p'-DDE, o,p'-DDE, p,p'-DDE, and vehicle treatments. Coumestrol and ethynyl estradiol appear antagonistic for plasma estradiol concentrations and liver somatic index when both chemicals are present together. Ethynyl estradiol and p,p'-DDE appear to act additively on kidney somatic index when combined together. Mixtures of compounds, used in this study indicate effects very different from either or both mixture components, indicating the lack of predictability of chemicals when combined in mixtures.     

Cytochrome P4501A induction and testosterone hydroxylation in cultured hepatocytes of four fish species

Primary hepatocytes of the rainbow trout (Oncorhynchus mykiss), flounder (Platychthis flesus), dab (Limanda limanda) and lemon sole (Microstomus kitt) were exposed to 3,3'4,4'5 pentachlorobiphenyl (PCB 126) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for two days. This resulted in a dose-dependent induction of cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O-deethylase (EROD), or methoxyresorufin O-deethylase (MROD) activity. In all species, a linear relationship was observed between EROD and MROD activities, suggesting that the same CYP1A enzyme metabolizes the two alkoxy-resorufin substrates. Exposures of hepatocytes of flounder or dab to TCDD, resulted in a 59-fold and 8.2-fold induction of EROD activity, respectively. This did not concur with a change in the in vitro testosterone hydroxylation profiles of both species. These and other in vitro data indicate that TCDD exposure does not influence monooxygenase activities involved in testosterone hydroxylation. Furthermore, CYP1A is of minor importance for testosterone hydroxylation in these fish species.     

Evaluation of TCDD biodegradability under different redox conditions

Polychlorinated dibenzo-p-dioxins have been generated as unwanted by-products in many industrial processes. Although their widespread distribution in different environmental compartments has been recognized, little is known about their fate in the ultimate environment sinks. The highly stable dioxin isomer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been called the most toxic compound known to man. In this laboratory microcosm study, TCDD bioavailability was evaluated under five reduction/oxidation (redox) conditions including aerobic biodegradation, aerobic cometabolism, methanogenesis, iron reduction, and reductive dechlorination. Activated sludge and aquifer sediments from a TCDD and a pentachlorophenol (PCP) contaminated site were used as the inocula. Acetate, sludge cake, and cane molasses were used as the primary substrates (carbon sources) in cometabolism and reductive dechlorination microcosms. After a 90-day incubation period, microcosms constructed under reductive dechlorination conditions were the only treatment showing promising remediation results. The highest TCDD degradation rate [up to 86% of TCDD removal (with an initial concentration of 96 microg/kg of soil)] was observed in the microcosms with anaerobic activated sludge as the microbial inocula and sludge cakes as the primary substrates. Except for reductive dechlorination microcosms, no significant TCDD removal was observed in the microcosms prepared under other conditions. Thus, application of an effective primary substrate to enhance the reductive dechlorination process is a feasible method for TCDD bioremediation. Bioremediation expense can be significantly reduced by the supplement of some less expensive alternative substrates (e.g., sludge cakes, cane molasses). Results would be useful in designing a scale-up in situ or on-site bioremediation system such as bioslurry reactor for field application.     

A new structure-affinity relationship for TCDD receptor binding

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