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Relationship between aryl hydrocarbon receptor-affinity and the induction of EROD activity by 2,3,7,8-tetrachlorinated phenothiazine and derivatives
Abstract
Reported herein are semi-empirical calculations of the molecular geometry of TCDD, TCPT, TCPT-sulfoxide (TCPT-O), TCPT-sulfone (TCPT-O(2)), N-methyl-TCPT (Me-TCPT), N-methyl-TCPT-sulfoxide (Me-TCPT-O), and N-methyl-TCPT-sulfone (Me-TCPT-O(2)), the characterization of their AhR binding affinity in rat hepatic cytosol, and their ability to induce EROD activity in a rat hepatoma cell line in vitro. Semi-empirical calculations yielded detailed information about the stereochemistry and the preferred conformation of each of these compounds. These results in combination with observations reported in this paper were used to determine structure-activity relationships. In vitro displacement of (3)H-TCDD was measured by increasing concentrations of the respective ligands. This assay revealed a strong binding affinity of TCPT to the AhR with a K(i) value of 1.08 nM. TCDD had a K(i) value of 0.54 nM. The affinity of TCPT derivatives for the AhR decreased with increasing degree of oxidation. Moreover, N-methylation further lowered the affinity, so that the N-methyl sulfone derivative of TCPT displayed the highest K(i) at approximately 1200 nM (=460.4 ng/ml). A corresponding trend was observed regarding the potency of TCPT and derivatives to induce EROD activity in vitro. However, the potencies were considerably lower than that of TCDD. Enzyme induction was measured in a rat hepatoma cell line H4IIEC/T3 by quantification of ethoxyresorufin-O-deethylase (EROD) activity. Induction was measured at 12, 24, 48 and 72 h to determine time dependence. Sulfoxidated and N-methylated phenothiazines displayed a lower potency than their respective parent compounds. TCPT and all derivatives induced enzyme activity at an efficacy similar to TCDD at all time points measured. The reported findings clearly separate the induction of EROD activity by TCPT and derivatives from their binding affinities to the AhR. In contrast, a direct correlation between the two is generally assumed in drug development, leading to - in our view - unwarranted termination of drug candidates. Therefore, a lack of such a correlation for TCPT and derivatives in fact supports their further development as possible drug leads.
... The method of ligand-binding was adapted from but used TCAOB as a specific competitor because it binds with high affinity to the AhR (Poland et al., 1976). The method has been fully validated previously (Bazzi, 2008;Fan et al., 2009;Fried et al., 2007;Jiang et al., 2009). The cytosolic protein containing AhR was prepared from the liver of Charles River Wistar rats (a kind gift from Tim Smith, University of Nottingham, UK). ...
... The ability of the compound to bind to the AhR was measured using a [ 3 H]-TCDD competitive ligand-binding assay. The specific binding was determined for the [ 3 H]-TCDD by saturation binding analysis (Fig. 4A), as previously described (Bazzi et al., 2009;Fried et al., 2007;Jiang et al., 2009). The K d for [ 3 H]-TCDD was 1.24 nM (95% CI = 0.58-1.90 ...
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent ligands of the aryl hydrocarbon receptor (AhR). Here, we show that a novel fused mesoionic heterocyclic compound (AZ1) is ∼5-fold more potent than TCDD in both rat and human cell lines at inducing cytochrome P4501A1 RNA. In rat H4IIE cells, AZ1 gave an EC50 = 5.05 pM (95% CI = 2.81–9.09 pM) whereas TCDD had an EC50 = 25.5 pM (95% CI = 18.2–36.0 pM). AZ1 was also more potent than TCDD (5–10-fold) at inducing the AhR-related CYP1A2 and CYP1B1 genes, showing that AZ1 is more potent at inducing multiple genes. In human MCF-7 cells AZ1 gave an EC50 = 65.4 pM (95% CI = 45.6–93.7 pM) and TCDD an EC50 = 241 pM (95% CI = 161–362 pM), showing that AZ1 was more potent than TCDD at inducing CYP1A1 RNA in multiple species. Finally, the compound bound to rat cytosolic AhR with 6-fold higher affinity than TCDD, showing that the highly potent agonism of this substance is mediated via a high affinity for the receptor. This data shows that this novel compound, which shares structural similarities with various naphthoflavones, is a potent ligand of the AhR.
... It is not clear whether microsomal enzyme activity can be used to extrapolate to biotransformation kinetics due to the paucity of such data for birds. There have been human studies connecting in vitro biotransformation to compound structure (Bu 2006) and linking enzyme induction kinetics to structural functionality (Fried et al. 2007). A fish biotransformation study reported both microsomal enzyme activity and in vitro biotransformation kinetics without linking them quantitatively (Hou et al. 2018). ...
A literature review of bioaccumulation and biotransformation of organic chemicals in birds was undertaken, aiming to support scoping and prioritization of future research. The objectives were to characterize available bioaccumulation/biotransformation data, identify knowledge gaps, determine how extant data can be used, and explore the strategy and steps forward. An intermediate approach balanced between expediency and rigor was taken given the vastness of the literature. Following a critical review of > 500 peer-reviewed studies, > 25,000 data entries and 2 million information bytes were compiled on > 700 organic compounds for ~ 320 wild species and 60 domestic breeds of birds. These data were organized into themed databases on bioaccumulation and biotransformation , field survey , microsomal enzyme activity , metabolic pathway , and bird taxonomy and diet . Significant data gaps were identified in all databases at multiple levels. Biotransformation characterization was largely fragmented over metabolite/pathway identification and characterization of enzyme activity or biotransformation kinetics. Limited biotransformation kinetic data constrained development of an avian biotransformation model. A substantial shortage of in vivo biotransformation kinetics has been observed as most reported rate constants were derived in vitro. No metric comprehensively captured all key contaminant classes or chemical groups to support broad-scope modeling of bioaccumulation or biotransformation. However, metrics such as biota-feed accumulation factor, maximum transfer factor, and total elimination rate constant were more readily usable for modeling or benchmarking than other reviewed parameters. Analysis demonstrated the lack of bioaccumulation/biotransformation characterization of shorebirds, seabirds, and raptors. In the study of bioaccumulation and biotransformation of organic chemicals in birds, this review revealed the need for greater chemical and avian species diversity, chemical measurements in environmental media, basic biometrics and exposure conditions, multiple tissues/matrices sampling, and further exploration on biotransformation. Limitations of classical bioaccumulation metrics and current research strategies used in bird studies were also discussed. Forward-looking research strategies were proposed: adopting a chemical roadmap for future investigations, integrating existing biomonitoring data, gap-filling with non-testing approaches, improving data reporting practices, expanding field sampling scopes, bridging existing models and theories, exploring biotransformation via avian genomics, and establishing an online data repository.
... These include hepatotoxicity, immunotoxicity, teratogenicity, cancer, wasting and death (Pohjanvirta and Tuomisto 1994). It is firmly established that the essential first step in dioxin toxicity is the binding of TCDD and related dioxins to the aryl hydrocarbon receptor (AHR) (reviewed in Okey 2007) although not all compounds that bind the AHR with high affinity cause dioxin-like toxicity (Fried et al. 2007). The most conclusive evidence that the AHR mediates dioxin toxicity arises from experiments in mice whose AHR has been genetically knocked out; these Ahr-null mice are extraordinarily resistant to TCDD toxicities (Fernandez-Salguero et al. 1995Lin et al. 2001;Mimura et al. 1997;Thurmond et al. 1999). ...
Dioxins exert their major toxicologic effects by binding to the aryl hydrocarbon receptor (AHR) and altering gene transcription.
Numerous dioxin-responsive genes previously were identified both by conventional biochemical and molecular techniques and
by recent mRNA expression microarray studies. However, of the large set of dioxin-responsive genes the specific genes whose
dysregulation leads to death remain unknown. To identify specific genes that may be involved in dioxin lethality we compared
changes in liver mRNA levels following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in three strains/lines of dioxin-sensitive rats with changes in three dioxin-resistant rat strains/lines. The
three dioxin-resistant strains/lines all harbor a large deletion in the transactivation domain of the aryl hydrocarbon receptor
(AHR). Despite this deletion, many genes exhibited a “Type-I” response—that is, their responses were similar in dioxin-sensitive
and dioxin-resistant rats. Several genes that previously were well established as being dioxin-responsive or under AHR regulation
emerged as Type-I responses (e.g. CYP1A1, CYP1A2, CYP1B1 and Gsta3). In contrast, a relatively small number of genes exhibited
a Type-II response—defined as a difference in responsiveness between dioxin-sensitive and dioxin-resistant rat strains. Type-II
genes include: malic enzyme 1, ubiquitin C, cathepsin L, S-adenosylhomocysteine hydrolase and ferritin light chain 1. In silico searches revealed that AH response elements are conserved
in the 5′-flanking regions of several genes that respond to TCDD in both the Type-I and Type-II categories. The vast majority
of changes in mRNA levels in response to 100 μg/kg TCDD were strain-specific; over 75% of the dioxin-responsive clones were
affected in only one of the six strains/lines. Selected genes were assessed by quantitative RT-PCR in dose-response and time-course
experiments and responses of some genes were assessed in Ahr-null mice to determine if their response was AHR-dependent. Type-II genes may lie in pathways that are central to the difference
in susceptibility to TCDD lethality in this animal model.
... These include hepatotoxicity, immunotoxicity, teratogenicity , cancer, wasting and death (Pohjanvirta and Tuomisto 1994). It is firmly established that the essential first step in dioxin toxicity is the binding of TCDD and related dioxins to the aryl hydrocarbon receptor (AHR) (reviewed in Okey 2007) although not all compounds that bind the AHR with high affinity cause dioxin-like toxicity (Fried et al. 2007). The most conclusive evidence that the AHR mediates dioxin toxicity arises from experiments in mice whose AHR has been genetically knocked out; these Ahr-null mice are extraordinarily resistant to TCDD toxicities (Fernandez-Salguero et al. 1995, 1996; Lin et al. 2001; Mimura et al. 1997; Thurmond et al. 1999). ...
Dioxins exert their major toxicologic effects by binding to the aryl hydrocarbon receptor (AHR) and altering gene transcription. Numerous dioxin-responsive genes previously were identified both by conventional biochemical and molecular techniques and by recent mRNA expression microarray studies. However, of the large set of dioxin-responsive genes the specific genes whose dysregulation leads to death remain unknown. To identify specific genes that may be involved in dioxin lethality we compared changes in liver mRNA levels following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in three strains/lines of dioxin-sensitive rats with changes in three dioxin-resistant rat strains/lines. The three dioxin-resistant strains/lines all harbor a large deletion in the transactivation domain of the aryl hydrocarbon receptor (AHR). Despite this deletion, many genes exhibited a "Type-I" response-that is, their responses were similar in dioxin-sensitive and dioxin-resistant rats. Several genes that previously were well established as being dioxin-responsive or under AHR regulation emerged as Type-I responses (e.g. CYP1A1, CYP1A2, CYP1B1 and Gsta3). In contrast, a relatively small number of genes exhibited a Type-II response-defined as a difference in responsiveness between dioxin-sensitive and dioxin-resistant rat strains. Type-II genes include: malic enzyme 1, ubiquitin C, cathepsin L, S-adenosylhomocysteine hydrolase and ferritin light chain 1. In silico searches revealed that AH response elements are conserved in the 5'-flanking regions of several genes that respond to TCDD in both the Type-I and Type-II categories. The vast majority of changes in mRNA levels in response to 100 microg/kg TCDD were strain-specific; over 75% of the dioxin-responsive clones were affected in only one of the six strains/lines. Selected genes were assessed by quantitative RT-PCR in dose-response and time-course experiments and responses of some genes were assessed in Ahr-null mice to determine if their response was AHR-dependent. Type-II genes may lie in pathways that are central to the difference in susceptibility to TCDD lethality in this animal model.
The thermodynamic properties of phenothiazine and 135 polybrominated phenothiazine (PBPTH) in the ideal gas state at 298.15 K and 101.3 kPa have been calculated at the B3LYP/6-31G* level using Gaussian 03 program and their thermodynamic parameters were obtained. The isodesmic reactions were designed to calculate standard enthalpy of formation (ΔfHΘ) and standard free energy of formation (ΔfGΘ) of PBPTH congeners. The relationship of these thermodynamic parameters with the number and position of Br atom substitution (NPBS) was established, finding that there exists high correlation (r2 ≥ 0.998) of thermal correction to energy (Eth), heat capacity at constant volume (CVΘ), entropy (SΘ), enthalpy (HΘ), free energy (GΘ), standard enthalpy of formation (ΔfHΘ ), standard free energy of formation (ΔfGΘ) with NPBS. The number of Br atom substitution is the main factor, which has the greatest impact on the values of the thermodynamics properties. The stability of PBPTH congeners was theoretically proposed based on the relative magnitude of their ΔfGΘ.
The thermodynamic properties of 135 polychlorinated phenothiazines (PCPTZs) in the standard state are calculated using a combination of quantum mechanical computations performed with the Gaussian 03 program at the B3LYP/6-311G ** level, and their octanol-water partition coefficients (logKow) are calculated based on group contributions. The chlorine substitution pattern strongly influenced the thermodynamic properties and hydrophilicity of the compounds. The thermodynamic properties of congeners also depend on the chlorine substitution pattern. The effect of chlorine substitution pattern is quantitatively studied by considering the number and position of Cl atom substitution (NPCS). The results show that the NPCS model may be used to predict the thermodynamic properties and hydrophilicity for all 135 PCPTZ congeners.
Bosphorus and Dardanelles form the Turkish Straits System together with the Marmara Sea. The system provides a connection between the Mediterranean and the Black Sea. Istanbul and its coastal environment have been strongly affected by wastewater discharges, high population and heavy ship traffic. The aim of this study is to provide a comparison between the chemical and biological analysis of surface sediment samples, which were collected from 17 different coastal stations of the Istanbul Strait, to determine the pollutants and comparable effect related data. For the chemical analysis, PAH, PCDD/F and PCB isotope dilution techniques, and for the biological analysis, Micro EROD (24-h and 72-h) assay were employed. Highest values were found at the most polluted sites due to ship traffic and wastewater discharges. Most of the results of chemical analysis were lower or close to biological analysis, except for the samples from two stations where antagonistic modulators are indicated to be present in the Micro EROD assay. This study suggests that the WWTP discharges into Bosphorus and the heavy ship traffic are important sources of pollution in the sediment, as evidenced by high concentrations of toxic and persistent organic chemicals. Local discharges of the megacity should be further decreased.
The aryl hydrocarbon receptor is a ligand-dependent transcription factor that induces expression of a number of genes encoding drug metabolizing enzymes, such as CYP1A1, CYP1A2 and CYP1B1. Recently, it was suggested that the AhR signaling pathway may be involved in mediating the anticancer activity of novel 2-(4-aminophenyl) benzothiazole drugs in MCF-7 breast cancer cells. There is no direct proof of direct binding between these drugs and AhR, and it is also unclear how AhR signaling per se plays a role in the activity of these drugs. This study investigates the role of AhR in the mechanism of action of the benzothiazole drugs by determining the ability of these drugs to bind to the rat hepatic AhR, to induce CYP1A1 mRNA and to inhibit cell growth in rat hepatoma H4-II-E cells.
The apparent binding kinetics of [3H]-TCDD to AhR in rat liver cytosol were, KD= 0.37nM and Bmax = 40 fmol/mg cytosolic protein. Using the standard assay conditions, 18 compounds competitively displaced [3H]-TCDD from specific sites, and are ligands for AhR. Induction of CYP1A1 mRNA by 5 compounds was determined in H4-II-E cells. The highest affinity ligand, IH445, was the most potent with an EC50 ~ 80-fold lower than that of TCDD (60 pM) with no detectable antagonistic activity in H4-II-E cells. The other high-affinity benzothiazoles tested were (30-100) x 103-fold less potent for inducing CYP1A1 mRNA than TCDD. The binding affinities of these compounds were 200-1000-fold higher than induction potency. For example, 5F 203 has a Ki value of 2.8 nM, induced CYP1A1 mRNA to similar maximal levels as seen with TCDD, and has an EC50 of 3 μM. The 1000-fold difference for 5F 203 between binding and CYP1A1 RNA induction was suggested to be a result of metabolism or that 5F 203 exhibits partial AhR antagonist activity. The time course effect on the CYP1A1/β-actin mRNA ratios by 5F 203 revealed that the response was increasing linearly in response to 5F 203 at 4 h treatment, indicating that the former possibilty is less likely to be a major factor. To address the second possibility, the antagonistic activity of 5F 203 on TCDD-induced CYP1A1 mRNA was investigated. H4-II-E cells were treated with increasing concentrations of TCDD ± 1μM 5F 203. The results demonstrated that 5F 203 shifted the EC50 of TCDD 100-fold to the right. Schild analysis on the antagonism of TCDD-induced CYP1A1 mRNA by different concentrations of 5F 203 provided a quantitative explanation for the 1000-fold difference between binding and induction for 5F 203. In contrast, the EC50 of 5F 203 in human MCF-7 cells was 2 nM, which is ~ 10-fold less potent than TCDD. Moreover, 5F 203 had no detectable antagonistic activity on TCDDinduced CYP1A1 mRNA. When 5F 203 was assessed for cell growth inhibition by MTT assay, it was found active in MCF-7 cells with a GI50 of 18 nM, but failed to elicit the same effect in H4-II-E cells.
These results prove that 5F 203 is a potent agonist in MCF-7 cells, but a partial agonist in H4-II-E cells. The partial agonism observed with 5F 203 is a compound-specific property given that another analogue, IH 445, was found potent inducer of CYP1A1 mRNA with no antagonistic activity. The results of this study reveal species-specific partial agonism of the AhR. The potency of the cytostatic effect of 5F 203 parallel potency for inducing CYP1A1 mRNA in both cells. Moreover, both, the cytostatic effect of 5F 203 and partial agonism of AhR for inducing CYP1A1 mRNA is species-specific. Whether agonism/antagonism for the induction of CYP1A1 mRNA is related to the anticancer activity of 5F 203 remains to be elucidated.
Nine groups of food items (freshwater fish, marine fish, pork, chicken, chicken eggs, leafy, non-leafy vegetables, rice and flour) and three types of human samples (human milk, maternal serum and cord serum) were collected for the analysis of PCDD/Fs. Results of chemical analysis revealed PCDD/Fs concentrations (pg g(-1) fat) in the following ascending order: pork (0.289 pg g(-1) fat), grass carp (Ctenopharyngodon idellus) (freshwater fish) (0.407), golden thread (Nemipterus virgatus) (marine fish) (0.511), chicken (0.529), mandarin fish (Siniperca kneri) (marine fish) (0.535), chicken egg (0.552), and snubnose pompano (Trachinotus blochii) (marine fish) (1.219). The results of micro-EROD assay showed relatively higher PCDD/Fs levels in fish (2.65 pg g(-1) fat) when compared with pork (0.47), eggs (0.33), chicken (0.13), flour (0.07), vegetables (0.05 pg g(-1) wet wt) and rice (0.05). The estimated average daily intake of PCDD/Fs of 3.51 pg EROD-TEQ/kg bw/day was within the range of WHO Tolerable Daily Intake (1-4 pg WHO-TEQ/kg bw/day) and was higher than the Provisional Tolerable Daily Intake (PMTL) (70 pg for dioxins and dioxin-like PCBs) recommended by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) [Joint FAO/WHO Expert Committee on Food Additives (JECFA), Summary and conclusions of the fifty-seventh meeting, JECFA, 2001.]. Nevertheless, the current findings were significantly lower than the TDI (14 pg WHO-TEQ/kg/bw/day) recommended by the Scientific Committee on Food of the Europe Commission [European Scientific Committee on Food (EU SCF), Opinions on the SCF on the risk assessment of dioxins and dioxin-like PCBs in food, 2000.]. However, it should be noted that micro-EROD assay overestimates the PCDD/Fs levels by 2 to 7 folds which may also amplify the PCDD/Fs levels accordingly. Although the levels of PCDD/Fs obtained from micro-EROD assay were much higher than those obtained by chemical analysis by 2 to 7 folds, it provides a cost-effective and rapid screening of dioxin levels in food and human samples.
New fluorinated 2-aryl-benzothiazoles, -benzoxazoles, and -chromen-4-ones have been synthesized and their activity against MCF-7 and MDA 468 breast cancer cell lines compared with the potent antitumor benzothiazole 5. Analogues such as 9a, b and 12a, d yielded submicromolar GI50 values in both cell lines; however, none of the new compounds approached 5 in terms of antitumor potency. For 5, binding to the aryl hydrocarbon receptor appeared to be necessary but not sufficient for growth inhibition.
A new parametric quantum mechanical molecular model, AM1 (Austin Model 1), based on the NDDO approximation, is described. In it the major weaknesses of MNDO, in particular failure to reproduce hydrogen bonds, have been overcome without any increase in computing time. Results for 167 molecules are reported. Parameters are currently available for C, H, O, and N.
We previously hypothesized that the genetic trait of aromatic hydrocarbon nonresponsiveness (the failure in certain inbred strains of mice of polycyclic hydrocarbons to induce aryl hydrocarbon hydroxylase activity, and the diminished sensitivity to the more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is due to mutation which results in an induction receptor with a diminished affinity for the inducing compound. Following the intraperitoneal administration of [14C]TCDD (6 nmol/kg), hepatic accumulation of the radiolabel was greatest in C57BL/6J mice, intermediate in the hybrid B6D2F1/J mice, and least in DBA/2J mice, a pattern which mirrors the strain sensitivity to hydroxylase induction by TCDD (C57BL/6J greater than B6D2F1/J greater than DBA/2J). These data are compatible with receptor mutation theory and suggested that the hepatic uptake of TCDD is determined by the affinity of the receptor. In vitro experiments on the binding of [3H]TCDD to hepatic cytosol from C57BL/6J mice revealed a small pool of high affinity sites which stereospecifically and reversibly bind TCDD. The specific binding of [3H]TCDD to hepatic cytosol had an equilibrium dissociation constant KD of 0.27 nM and a maximum binding capacity of 84 fmol/mg of cytosol protein. Much less high affinity specific binding of [3H]TCDD was observed in hepatic cytosol from DBA/2J mice, but the KD was not estimated because of the limited aqueous solubility of the ligand. The binding affinity of 23 halogenated dibenzo-p-dioxins and dibenzofurans for this hepatic cytosol-binding species closely correlated with the potencies of these compounds as inducers of hepatic aryl hydrocarbon hydroxylase activity. The polycyclic hydrocarbons that induce hepatic hydroxylase activity competed with [3H]TCDD for hepatic cytosol binding, but phenobarbital, pregnenolone-16alpha-carbonitrile, and the steroid hormones had no specific binding. The data suggest that the hepatic cytosol species which binds TCDD is the receptor for the induction of hepatic aryl hydrocarbon hydroxylase activity, and that the mutation in nonresponsive mice results in an altered receptor with a diminished affinity for inducing compounds.
Halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (MC) cause transcriptional activation of the CYP1A1 gene via their interaction with the aromatic hydrocarbon (Ah) receptor. Direct radioligand binding and competitive binding studies demonstrated that the cytosolic Ah receptor from the mouse hepatoma cell line Hepa-1 bound TCDD with an affinity approximately 3-4-fold greater than that for MC. However, TCDD was approximately 1,000-fold more potent than MC as an inducer of CYP1A1-mediated aryl hydrocarbon hydroxylase activity in cultured Hepa-1 cells as assessed at 14 h following exposure to inducer. To understand the basis for this quantitative discrepancy between Ah receptor binding affinity and CYP1A1 induction potency, we systematically compared TCDD and MC for their abilities to activate sequential events in the CYP1A1 induction mechanism that occur subsequent to initial binding to the cytosolic Ah receptor. Using a gel retardation assay, TCDD and MC were shown to be equipotent in causing in vitro transformation of the cytosolic Ah receptor to its DNA-binding form. In addition, the transformed Ah receptor bound to a specific dioxin-responsive enhancer sequence with the same apparent affinity when MC was the ligand as when TCDD was the ligand. At an early time point (i.e. 2 h) in the CYP1A1 induction process, TCDD was only approximately 4-25-fold more potent than MC in stimulating the nuclear uptake of the ligand-Ah receptor complex, and the two ligands displayed a relatively small difference (> or = 10-fold) in CYP1A1 mRNA induction potency. When assessed at 4 h following ligand treatment, TCDD was only approximately 10-fold more potent than MC as an aryl hydrocarbon hydroxylase inducer, suggesting a time-dependent reduction in the potency of MC in intact cells. Exposure of Hepa-1 cells to MC over a 16-h time course resulted in an increased ability of these cells to convert [3H]MC to alkali-extractable metabolites. Our data are consistent with the idea that TCDD and MC display relatively small differences in their intrinsic abilities to activate Ah receptor-mediated events. The reduced biological potency of MC observed in intact cells and whole animals is at least partially due to the more rapid metabolic inactivation of this ligand compared with the poorly metabolized TCDD. By extension, the extraordinary toxicity of TCDD may not be explained solely by its high affinity for the cytosolic Ah receptor.
Developmental toxicity to TCDD-like congeners in fish, birds, and mammals, and reproductive toxicity in mammals are reviewed. In fish and bird species, the developmental lesions observed are species dependent, but any given species responds similarly to different TCDD-like congeners. Developmental toxicity in fish resembles "blue sac disease," whereas structural malformations can occur in at least one bird species. In mammals, developmental toxicity includes decreased growth, structural malformations, functional alterations, and prenatal mortality. At relatively low exposure levels, structural malformations are not common in mammalian species. In contrast, functional alterations are the most sensitive signs of developmental toxicity. These include effects on the male reproductive system and male reproductive behavior in rats, and neurobehavioral effects in monkeys. Human infants exposed during the Yusho and Yu-Cheng episodes, and monkeys and mice exposed perinatally to TCDD developed an ectodermal dysplasia syndrome that includes toxicity to the skin and teeth. Toxicity to the central nervous system in monkey and human infants is a potential part of the ectodermal dysplasia syndrome. Decreases in spermatogenesis and the ability to conceive and carry a pregnancy to term are the most sensitive signs of reproductive toxicity in male and female mammals, respectively.
Severe toxicological responses have been attributed to certain chlorodibenzo p dioxins. Therefore, the acute and subacute toxicities of several of these compounds have been determined and these results provide information for evaluating potential health hazards. 2,3,7,8 Tetrachlorodibenzo p dioxin (2,3,7,8 TCDD) was the most toxic chlorodibenzo p dioxin studied, having an LD50 in the μg/kg range. Hexachlorodibenzo p dioxin (HCDD) was less toxic than 2,3,7,8 TCDD but more toxic than either 2,7 dichlorodibenzo p dioxin (2,7 DCDD) or octachlorodibenzo p dioxin (OCDD). Both 2,3,7,8 TCDD and HCDD were acnegenic, highly embryotoxic, and positive for the chick edema factor. 2,7 DCDD and OCDD were not chloracnegenic, caused little or no embryotoxicity, and were low in acute oral lethality; OCDD was negative for the chick edema factor.
In the present study we have utilized comparative molecular field analysis (CoMFA), a three-dimensional quantitative structure-activity relationship paradigm, to explore the physicochemical requirements for binding to the Ah (dioxin) receptor. Recent developments by Gillner et al. [(1993) Mol. Pharmacol. 44, 336-345] prompted us to review and revise our previous CoMFA/QSAR model [Waller, C. L., and McKinney, J. D. (1992) J. Med. Chem. 36, 3660-3666] to include a structurally-diverse training set of Ah receptor ligands ranging in size from naphthalene to indolo[3,2-b]carbazole nuclei. An exhaustive validation process utilizing external test sets and hierarchical cluster analysis routines was employed during model construction and is discussed herein. The limitations of the approach presented herein are discussed with respect to predictive ability of the CoMFA/QSAR models, which is demonstrated to be dependent on a balance between structural diversity and redundancy in the molecules comprising the training set. The results of our modified CoMFA/QSAR model are consistent with and unify all previously established structure-activity relationships established for less structurally-diverse training sets of Ah receptor ligands. As a result of the more complete nature of the series of molecules under examination in the present study, the CoMFA/QSAR steric and electrostatic field contour plots as well as the essential and excluded volume plots provide for a more detailed characterization of the molecular binding domain of the Ah receptor. The implications of the CoMFA/QSAR model presented herein are explored with respect to quantitative hazard identification of potential toxicants.
A direct relationship between the 13C nmr chemical shifts of the quaternary carbon atoms of the central ring of phenothiazine and the dihedral angle of the respective derivatives has been observed. This correlation allows for the useful and quick estimation of the dihedral angles of novel phenothiazines from readily obtainable 13C nmr solution measurements. In addition, the change in the dihedral angle appears to be directly related to the S-C4a-C10a bond angle; however, the C4a-S-C5a bond angle is not affected by changes in the dihedral angle. This indicates that the “flattening” of the phenothiazine tricyclic ring system is compensated for by the vertical displacement of the sulfur atom, by changes in hybridization of N10, and by other angular distortions of the middle ring.
A recent intervention study demonstrated a powerful method for helping children with difficulty in sound categorization to learn to read and to spell. The aim of this project is to explain the underlying reasons for its success. The project is divided into three stages, Pretests, Training and Post-tests, which cover a period of 3 years. The subjects were young beginning readers, who were pre-tested on the strategies used in the successful teaching method, and then followed in school for 3 years to see how their skill in these strategies affected their progress in reading and spelling. They were divided into four matched groups in the second term of the project for a short training study. Phonological awareness when the child started school proved to be critical for success in early reading, and in spelling 2 years later. Memory for letter strings becomes increasingly important for spelling. Beginning readers who were taught the connection between the two strategies made early gains in reading text.
Concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat liver and adipose tissue, and hepatic ethoxyresorufin O-deethylase (EROD) activity were studied subsequent to a single subcutaneous injection of TCDD. Two types of experiments were performed to study: (a) time-dependent changes following a single injection of 300 ng TCDD/kg body wt (points 1–4), and (b) dose-dependent changes measurable after 7 days following a single injection (points 5–7).1.
Absorption of TCDD following a single subcutaneous injection was about 90% after 3 days and 98% after 5 days.
2.
Following a single dose of 300 ng TCDD/kg body wt peak concentrations were: liver (after 3 days): 4.70.9 ng/g wet wt, and adipose tissue (after 7 days): 0.820.07 ng/g wet wt.
3.
T1/2 of TCDD in liver was 13.6 days over the total experimental period (from day 10 to 91 of the study), apparently with an initial faster phase: 11.5 days (from day 10 to 49), and a slower period at the end of the experiment: 16.9 days (from day 49 to 91); in adipose tissue the t1/2 was 24.5 days (from day 14 to 91 of the study).
4.
Maximum induction of EROD in the liver was observed (14-fold at 300 ng TCDD/kg body wt) 3–7 days following the injection; the activity was decreased to about one third of the maximum 3 weeks after the injection; increase in total cytochrome P-450 at this dose was only about 1.4-fold at the induction maximum.
5.
The ratio of the TCDD concentrations in liver and adipose tissue increased considerably between doses of 3 ng TCDD/kg body wt (ratioabout 0.74) and 3000 ng TCDD/kg body wt (ratioabout 7.7).
6.
The extent of EROD induction in the liver increased dose dependently. A significant effect was first observed with a dose of 3 ng TCDD/kg body wt (activity about +32% above control activity). The corresponding tissue concentration was about 10 pg TCDD/g liver wet wt.
7.
An almost perfect linear relationship exists (when using a double-log plot) between the hepatic TCDD concentration and the EROD activity for tissue concentrations ranging from 40 to 30 000 pg TCDD/g wet wt.
Previous studies have shown that in two inbred strains of mice, straightforward correlations exist among the number of hepatic Ah-receptors, enzyme inducibility by TCDD, and lethality of TCDD. Here, studies were conducted in two strains of rats (Han/Wistar and Long-Evans) which differ widely in susceptibility to the lethal effects of TCDD, to determine if these are general phenomenona in TCDD toxicity. The total number of specific binding sites (Ah-receptors) for [3H]TCDD proved to be approximately equal in the livers of both rat strains. Likewise, no notable difference was detected in the effect of TCDD on the activities of 7-ethoxyresorufin O-deethylase, 7-ethoxycoumarin O-deethylase, and ethylmorphine N-demethylase or on the amount of cytochrome P-450 in hepatic microsomal fractions. Immunoblot analysis was carried out with monoclonal antibodies (Mabs). Mab 1-7-1 directed against rat liver 3-methylcholanthrene (MC)-inducible P-450 recognized forms P-450c and P-450d in TCDD-treated rats in a dose-dependent fashion and to a similar extent in both strains. In contrast, Mab 2-66-3 (against phenobarbital-inducible P-450) did not recognize any proteins in either strain, confirming the conclusion that TCDD elicites a MC-type induction of hepatic cytochrome P-450 in both strains of rats. Thus, it seems that the correlations observed in mice do not hold in rats and therefore should not be generalized. The parameters measured in the present study are causally unrelated to the mechanism of lethal action of TCDD in these rat strains.
Polychlorinated dibenzo-p-dioxins are persistent environmental pollutants. The most potent congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes a wasting syndrome and is a potent carcinogen and immunosuppressant in the rat at high doses. However, low doses cause opposite effects to some of those observed at higher doses, resulting in chemoprevention, stimulation of the immune system, and longevity in experimental animals. The new TCDD analogue, 2,3,7,8-tetrachlorophenothiazine (TCPT), was developed to take advantage of the low-dose effects of dioxins that have potential application as therapeutics. Its development marked a deviation from the traditional scope of phenothiazine drug design by deriving biological effects from aryl substituents. TCPT was synthesized in three steps. The key ring-closing step was performed utilizing a Buchwald-Hartwig amination to provide TCPT in 37% yield. Its potency to induce CYP1A1 activity over 24 h was 370 times lower than that of TCDD in vitro. The elimination half-life of the parent compound in serum was 5.4 h in the rat and 2.7 h in the guinea pig, compared to 11 and 30 days, respectively, for TCDD. These initial findings clearly differentiate TCPT from TCDD and provide the basis for further studies of its potential as a drug lead.
Rats were maintained for 2 years on diets supplying 0.1, 0.01, and 0.001 μg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg/day. Analysis of these diets indicated 2200, 210, and 22 parts per trillion (ppt) of TCDD. Ingestion of 0.1 μg/kg/day caused an increased incidence of hepatocellular carcinomas and squamous cell carcinomas of the lung, hard palate/nasal turbinates, or tongue, whereas a reduced incidence of tumors of the pituitary, uterus, mammary glands, pancreas, and adrenal gland was noted. Other indications of toxicity at this dose level included increased mortality, decreased weight gain, slight depression of erythroid parameters, increased urinary excretion of porphyrins and δ-aminolevulinic acid, along with increased serum activities of alkaline phosphatase, γ-glutamyl transferase and glutamic-pyruvic transaminase. Gross and histopathologic changes were noted in the hepatic, lymphoid, respiratory, and vascular tissues. The primary hepatic ultrastructural change at this high dose level was proliferation of the rough endoplasmic reticulum. Terminal liver and fat samples from rats at this high dose level contained 24,000 and 8100 ppt of TCDD, respectively. Rats given 0.01 μg/kg/day for 2 years had a lesser degree of toxicity than that seen at the highest dose level. This included increased urinary excretion of porphyrins in females, liver lesions (including hepatocellular nodules), and lung lesions (including focal alveolar hyperplasia). Terminal liver and fat samples from rats of this dose level contained 5100 and 1700 ppt of TCDD, respectively. Ingestion of 0.001 μg of TCDD/kg/day (∼22 ppt in the diet) caused no effects considered to be of any toxicologic significance. At this lower dose level, terminal liver and fat samples each contained 540 ppt of TCDD. These data indicate that continuous doses of TCDD sufficient to induce severe toxicity increased the incidence of some types of tumors, while reducing other types. During the 2-year study in rats, no increase in tumors occurred in those rats receiving sufficient TCDD to induce slight or no manifestations of toxicity.
The development of modern toxicology was strongly influenced by parallel evolutions of sister disciplines as well as regulatory requirements. This led to an overemphasis of some areas of toxicology while other aspects of it experienced at least a temporary benign neglect. The author of this paper believes that the few and far between successes in elucidating the mechanism of toxicity of chemicals are related to this uneven approach to a very complex problem. Based on one of the most important and controversial contemporary issues in toxicology, viz. that of dioxins, it is argued that by a systematic step-wise use of methodologies borrowed from sister disciplines, our capabilities of elucidating the mechanism of action of toxicants can be considerably improved.
To investigate the relationship between vitamin A status and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) lethality, the influence of TCDD on tissue and serum vitamin A levels was determined in the most TCDD-susceptible (Long-Evans) and the most TCDD-resistant (Han/Wistar) rat strains. The TCDD LD50 values of these two strains differ by a factor of more than 300. Groups of three rats per strain were used in a dose-response study (given single ip doses of 0, 4, 40, 400, 800 or 1600 micrograms TCDD/kg body weight and killed on day 11) and in a time-course experiment (given single ip doses of 0, 4 and, in the case of Han/Wistar rats only, 1600 micrograms TCDD/kg body weight, and killed on days 4, 11, 23, 50 and 76). The strains showed similar response over the 76-day study with respect to vitamin A levels in the liver, kidneys, testicles and serum after exposure to a sublethal dose of TCDD (4 micrograms/kg body weight). In contrast, TCDD doses lethal to the Long-Evans strain only (40-1600 micrograms/kg, day 11) markedly increased kidney and serum vitamin A levels in Han/Wistar rats, while they were practically without effect in Long-Evans rats. Hepatic cytochrome P-450 concentration, and the activities of 7-ethoxyresorufin O-deethylase, ethylmorphine N-demethylase, and uridine diphosphate glucuronosyltransferase (towards p-nitrophenol) were affected by the TCDD doses in much the same manner in both strains. These findings show that the correlations between TCDD lethality and changes in vitamin A status found among species of laboratory animals do not hold for Long-Evans and Han/Wistar strains of rat.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been an important issue in occupational and environmental health for nearly two decades. During this period scientists have studied its possible impacts on exposed human populations. At the same time enormous efforts were made to elucidate the mechanism of TCDD action in various biological models. This paper provides a critical view of the advances made towards understanding the mechanism of TCDD action. Major topics discussed include the Ah-receptor hypothesis, TCDD as a thyroid hormone agonist, TCDD and vitamin A deficiency, TCDD's effect on receptor regulation, and its effect on intermediary metabolism including related hormonal responses. Although the exact mechanism of TCDD action is not yet known, more information is available on the toxicity of this compound than perhaps on that of any other substance. This wealth of information allows important conclusions regarding the assessment of acute, as well as of chronic, toxicities of TCDD for humans. There is no documented case of human death as a result of exposure to TCDD. It appears that humans are acutely less sensitive to TCDD than some animal species. The cause of TCDD-induced lethality in rats is a progressive lethal hypoglycemia due to inhibition of gluconeogenesis. Regulation of this metabolic pathway is quite different amongst species, although primates share great similarities. The assumption that the cause of TCDD-induced death in primates, in analogy to rats, is inhibition of gluconeogenesis would suggest that the acute toxicity of TCDD in humans would be in the range seen in rhesus monkeys (70-300 micrograms/kg). These values are about midway between the most (guinea pig) and least (hamster) sensitive species. TCDD is not a genotoxic agent and not an initiator, but promoter of tumor formation. There is considerable evidence that promotion of cancer, like any other chronic end point of toxicity, is a threshold-type biological process. Therefore, a linear extrapolation of the dose-response is an unnecessarily conservative approach in the safety assessment of TCDD. This paper, based on several studies with different end points of toxicity, supports the notion that 10 pg/kg/day of TCDD represent a safe lifetime exposure level for humans with regard to promotion of cancer, porphyria and chloracne.
In this report, we have used the radioligand [125I]2-iodo-7,8-dibromo-dibenzo-p-dioxin to describe the kinetics of ligand binding to the Ah receptor prepared from C57BL/6J mouse liver. The higher specific activity of this radioligand (2176 Ci/mmol), compared with the usual tritiated ligand [1,6-3H]2,3,7,8-tetrachloro-dibenzo-p-dioxin (58 Ci/mmol) permitted the study of ligand-receptor interactions at much lower component concentrations. For this radioiodinated ligand, Scatchard analysis of saturation binding curves, determined at six different protein concentrations, indicated that the apparent equilibrium dissociation constant, KD, was directly related to the dilution of the receptor preparation; for example, at 1160 micrograms of protein/ml, KD = 1.6 x 10(-10) M; at 36 micrograms of protein/ml, KD = 1.2 x 10(-11) M. Extrapolation of this function to infinite receptor dilution yielded KD = 6 x 10(-12) M. The addition of 70 micrograms/ml of bovine serum albumin to a receptor preparation of 30 micrograms of protein/ml produced a 10-fold decrease in the slope of the Scatchard plot (i.e., 10-fold increase in the apparent KD). Conversely, enrichment of the receptor by high performance liquid chromatography led to an increased slope and thus decreased estimate of KD. The association rate constant (k1), calculated from the integrated second-order rate equation, was 2.8 x 10(10) M-1 hr-1 and, from the initial velocity equation, had a value of 5.25 x 10(10) M-1 hr-1. The dissociation rate constant was biphasic, consisting of a predominant fast component with a rate constant of 0.36 hr-1 (k-1) and a slower component with a rate constant of 4.2-9.4 x 10(-3) hr-1 (k-2). Higher protein concentrations produced a decrease in estimates of k1 but not k-1 or k-2. The KD determined from the ratio of the kinetic rate constant, k-1/k1 = 6.9 x 10(-12) M, is in excellent agreement with that derived from the results of equilibrium binding experiments extrapolated to infinite dilution, KD = 6 x 10(-12) M. The decrease in KD, observed in equilibrium binding studies upon dilution of the receptor preparation, is best explained by a more accurate classification of "free" radioligand at lower protein concentrations. Finally, ligand binding to the Ah receptor is best described by a two-step process, the formation of an initial complex, characterized by rapid ligand dissociation, which undergoes transformation to a second distinct complex displaying a much slower ligand dissociation rate.
A sensitive competitive binding assay for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other ligands of the Ah receptor was developed using a stable preparation of the Ah receptor, the 40-55% ammonium sulfate fraction of liver cytosol from C57BL/6J mice, and the radioligand [125I]2-iodo-7,8-dibromodibenzo-p-dioxin (specific radioactivity, 2176 Ci/mmol, and binding affinity, KD = 6.5 pM). Conditions are described which maximize assay precision and sensitivity, while minimizing sample counting time, ensuring ligand solubility, and permitting attainment of binding equilibrium for competing ligands. Assay conditions were developed to allow calculation of the binding affinity for competing ligands and to ensure that an unknown competitor could be quantified in terms of "TCDD binding equivalents." Standard assay conditions consisted of incubation of 8 pM radioligand and 18-20 pM Ah receptor with 5-1000 pM TCDD, in a 1-ml volume, for 16 hr at 4 degrees. Statistical analysis of the standard curve of bound radioligand versus the log of the concentration of competing TCDD indicated the minimal detectable concentration of TCDD to be 10 pM (3.2 pg in a 1-ml assay alpha less than or equal to 0.01). The simplicity, sensitivity, and reproducibility of this competitive binding assay should prove useful as a screen to detect planar halogenated aromatic hydrocarbons and other ligands of the Ah receptor. The availability of this 125I-labeled dioxin congener also permitted the characterization of Ah receptor-ligand binding over a range of ligand and receptor concentrations not possible with currently available 3H-ligands.
TCDD and thyroxine have common molecular reactivity properties which enable them to present a planar face and lateral halogens in interactions with proteins. These molecular properties are consistent with the structure-toxicity relationship for TCDD and related compounds. Biological evidence is discussed including preliminary studies on the effects of TCDD exposure on tadpole growth and development which is consistent with the possible thyroxine-like activity of TCDD. The work suggests the possibility that toxicity is at least in part the expression of potent and persistent thyroid hormone activity (responses induced by TCDD which qualitatively correspond to those mediated by thyroid hormones). A mechanism for toxicity is proposed which involves receptor proteins; the planar aromatic system controls binding to cytosolic proteins and halogen substituents regulate binding to nuclear proteins. This simple model based on molecular reactivity sheds light on the diversified effects of TCDD and related compound toxicity and on certain thyroid hormone action. The model also permits predictions to be made with regard to the toxicity and thyroid hormone activity of untested compounds. In addition, the model suggests a general mechanism for hormone action based on metabolically regulated differential and cooperative protein receptor binding events in cellular compartments which can explain agonism, antagonism and potentiation within the framework of receptor occupancy theory.
Concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat liver and adipose tissue, and hepatic ethoxyresorufin O-deethylase (EROD) activity were studied subsequent to a single subcutaneous injection of TCDD. Two types of experiments were performed to study: (a) time-dependent changes following a single injection of 300 ng TCDD/kg body wt (points 1-4), and (b) dose-dependent changes measurable after 7 days following a single injection (points 5-7). 1. Absorption of TCDD following a single subcutaneous injection was about 90% after 3 days and 98% after 5 days. 2. Following a single dose of 300 ng TCDD/kg body wt peak concentrations were: liver (after 3 days): 4.7 +/- 0.9 ng/g wet wt, and adipose tissue (after 7 days): 0.82 +/- 0.07 ng/g wet wt. 3. T1/2 of TCDD in liver was 13.6 days over the total experimental period (from day 10 to 91 of the study), apparently with an initial faster phase: 11.5 days (from day 10 to 49), and a slower period at the end of the experiment: 16.9 days (from day 49 to 91); in adipose tissue the t1/2 was 24.5 days (from day 14 to 91 of the study). 4. Maximum induction of EROD in the liver was observed (14-fold at 300 ng TCDD/kg body wt) 3-7 days following the injection; the activity was decreased to about one third of the maximum 3 weeks after the injection; increase in total cytochrome P-450 at this dose was only about 1.4-fold at the induction maximum. 5. The ratio of the TCDD concentrations in liver and adipose tissue increased considerably between doses of 3 ng TCDD/kg body wt (ratio: about 0.74) and 3000 ng TCDD/kg body wt (ratio: about 7.7). 6. The extent of EROD induction in the liver increased dose dependently. A significant effect was first observed with a dose of 3 ng TCDD/kg body wt (activity about +32% above control activity). The corresponding tissue concentration was about 10 pg TCDD/g liver wet wt. 7. An almost perfect linear relationship exists (when using a double-log plot) between the hepatic TCDD concentration and the EROD activity for tissue concentrations ranging from 40 to 30,000 pg TCDD/g wet wt.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) caused a depletion of serum thyroxine, but paradoxically did not change L-3,5,3'-triiodothyronine (T3) levels in serum of rats. The activities of the thyroid-regulated enzymes alpha-glycerol phosphate dehydrogenase (GPD) and malic enzyme (ME) were determined in livers of normal and thyroidectomized (THX) rats treated with 0.1 to 100 nmol TCDD/kg body weight. Mitochondrial GPD activity did not change significantly as a function of TCDD dose in either normal or THX rats. ME activity was induced by TCDD in a dose-dependent fashion, but only in non-THX animals. The absence of ME induction in THX rats treated with TCDD indicates that TCDD is not intrinsically thyromimetic. The dependence of ME induction on thyroid hormones is much like the thyroid-hormone-dependent, multihormonal induction of ME by insulin and glucocorticoids. However, TCDD had no additive or synergistic effects on induction of ME activity in THX rats fed T3. A 30% decrease in steady-state plasma T3 levels of T3-fed animals treated with TCDD relative to T3-fed controls suggested that T3 catabolism was more rapid in TCDD-treated rats than controls. Thus a thyroid-hormone-dependent, multihormonal interaction is suggested as the basis for induction of ME by TCDD, but a strictly T3-dependent process has not been ruled out.
Variation in toxic parameters as single oral LD50 dose, range of time interval from dosing until death, organ weights and other toxic manifestations were studied in different animal species. Guinea pigs are most sensitive to the lethal effects of TCDD with 90% dying from a single 3 μg/kg dose. In contrast, a 100 μg/kg dose was lethal to 6 of 14 rats. In both species the time interval until death is similar and a large weight loss over a period of days preceded death. Female rats appear to be more sensitive to TCDD than males. There is an overall tendency for feed consumption to be reduced following TCDD exposure, but this depression is not sufficient magnitude to account for the body weight changes which occurred. Administration of daily or weekly sublethal doses does not seem to raise the threshold level of TCDD toxicity. For example decreased weight gain at the 1 μg/kg daily dose occurred in rats once the total dose administered exceeded approximately 20 μg/kg; decreased weight gain first occurred at the single 25 μg/kg level. A comparison of the body weight gains of 5 μg/kg weekly dose and the 1 μg/kg daily dose also reveals a similar weight pattern change. Parallel growth rates after 1 or 2 wk in the single dose rat experiments and weight gains exceeding that of controls subsequent to 30 day exposure at 1 μg/kg/rat suggest that toxicity of TCDD, at least in some circumstances, are reversible. The thymus appears to be a most sensitive indicator of TCDD exposure. Decreases in thymus weight consistently occurred in all species at a dose level below which body weight effects occurred.
In this review, we have examined the biochemical and toxic responses produced by halogenated aromatic hydrocarbons and have tried to develop a model for their mechanism of action. These compounds bind to a cellular receptor and evoke a sustained pleiotropic response. In many tissues this response consists of the expression of a battery of enzymes which are, for the most part, involved in drug metabolism, but in other tissues, those which develop toxicity, an additional set of genes is expressed which effects cellular involution, division, and/or differentiation. The toxicity of these compounds appears to be due to the sustained expression of a normal cellular regulatory system, of which we were previously unaware. In future investigations it is hoped that we will learn the nature and physiologic role of this regulatory system. Only then can we hope to understand the mechanism of toxicity of these compounds.
The in vivo turnover of several rat liver microsomal proteins was studied using techniques designed to maximize antibody recognition specificity and minimize reutilization of radioactive labels. The kinetics of degradation of seven cytochrome P-450 isozymes, NADPH-cytochrome P-450 reductase, and epoxide hydrolase were determined in untreated rats and rats treated with phenobarbital or beta-naphthoflavone. In the cases where induction of these enzymes occurred with the above chemicals, rates of synthesis of the proteins were also estimated. In general, the degradation rates of the different proteins were rather similar to each other, and the effects of phenobarbital and beta-naphthoflavone on these rates were not very great. However, in the case of cytochromes P-450, a general trend was observed in which the heme moiety was degraded more rapidly than the apoprotein. Changes in the rates of synthesis of the individual proteins appear to contribute more to the altered steady-state levels which are expressed than do the rates of degradation, and profiles of steady-state enzyme concentrations predicted by the kinetic constants approximate those observed in vivo.
This paper shows that the relative toxic potency of four chlorinated dibenzo-p-dioxins (CDDs) is similar in two species with different sensitivities (guinea pig, Sprague-Dawley rat). More importantly, it also demonstrates that the relative toxic potencies of these homologues are very similar for acute, subchronic and chronic dosing in the same species (rat). Furthermore, examination of different endpoints of toxicity (mortality, porphyria, carcinogenicity) suggests that the dose-responses for these diverse end-points after acute, subchronic, and chronic administration are very similar if not identical for tetra-CDD. Based on toxicokinetic and toxicodynamic considerations, a new, possibly generalizable rule (average tissue concentration x time = toxicity) is derived for CDDs. Implicit in the relative potency arguments of CDDs is the requirement of a practical threshold dose for all endpoints of toxicity including cancer.
Long-Evans rats (strain Turku AB; L-E) are at least 1000-fold more sensitive (LD50 about 10 microg/kg) to the acute lethal effects of 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) than are Han/Wistar (Kuopio; H/W) rats (LD50 > 9600 microg/kg). The AH receptor (AHR) is believed to mediate the toxic effects of TCDD and related halogenated aromatic hydrocarbons. We compared the AHRs of L-E and H/W rats to determine if there were any structural or functional receptor differences that might be related to the dramatic difference in the sensitivity of these two strains to the lethal effects of TCDD. Cytosols from liver and lung of the sensitive L-E rats contained about twofold higher levels of specific binding sites for [3H]TCDD than occurred in H/W rats; the Kd for binding of [3H]TCDD to AHR in hepatic cytosols was similar between the two strains. Addition of the oxyanions, molybdate or tungstate (20 mM), had little effect upon ligand binding to AHR in hepatic cytosols from L-E rats whereas in cytosols from H/W rats these agents substantially diminished or totally abolished TCDD binding. The AHR in H/W cytosols also lost ligand-binding function when NaCl (20 to 400 mM) was added to the buffer whereas, in cytosols from L-E rats, the addition of 400 mM NaCl caused the receptor complex to shift from 9S to 6S during velocity sedimentation but did not destroy ligand binding function. AHR from hepatic cytosol of both the L-E and H/W rats could be transformed to the DNA-binding state in the presence of TCDD or other dioxin congeners as assessed by gel mobility shift assays. The most dramatic difference in AHR properties between L-E and H/W rats is molecular mass. Immunoblotting of cytosolic proteins revealed that the AHR in L-E rats has an apparent mass of approximately 106 kDa, similar to the mass of the receptor previously reported in several other common laboratory rat strains. In contrast, the mass of the AHR in H/W rats is approximately 98 kDa, significantly smaller than the mass of receptor reported in any other rat strains. F1 offspring of a cross between L-E and H/W rats expressed both the 106- and the 98-kDa protein. There was no apparent difference in the mass of the AHR nuclear translocator protein (ARNT) between the two strains, but the hepatic concentration of ARNT was about three times as high in L-E as in H/W rats. It will be interesting to find out how the altered structure of the AHR in H/W rats is related to their remarkable resistance to the lethal effects of TCDD.
The involvement of protein tyrosine kinases (PTKs) in aryl hydrocarbon receptor (AhR)-mediated signalling by omeprazole and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in hepatoma cells. Both omeprazole- and TCDD-dependent AhR signalling was attenuated by inhibition of c-src kinase, either by using pyrazolopyrimidine 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4 ]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibitors or by expression of dominant-negative c-src. These results indicate that the overall AhR function is modulated by c-src kinase activity. In contrast, a selective inhibition of omeprazole-mediated AhR signalling was revealed by tyrosine kinase inhibitors, tyrphostins AG17 and AG879. Furthermore, omeprazole-dependent AhR activation was abolished by mutation of Tyr320 to Phe, suggesting that this residue is a putative phosphorylation site. TCDD-dependent AhR signalling was neither affected by tyrphostins nor by this mutation. Our results are consistent with activation of the AhR by omeprazole in a ligand-independent manner, via a signal transduction pathway that involves protein tyrosine kinases, and are different from the mechanism exerted by high-affinity ligands.
Chronic toxicity of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) including its carcinogenicity was studied in female Sprague-Dawley rats in lifetime experiments. Six single dose and three multiple dose rate experiments were conducted with a single dose corn oil control group and a multiple dose rate corn oil control group, respectively. The lowest dose (1.0 mg/kg) of HpCDD and multiple dose rates of corn oil (4.0 ml/kg every other week) both prolonged the life of rats by about 2 months over that of single dose corn oil controls. Higher doses resulted in a predictable shortening of the life of rats after single dose administrations as well as after multiple dose rate administrations. The c x t = k paradigm previously validated for acute toxicity [Toxicol. Sci. 49 (1999) 102] was confirmed for chronic toxicity including carcinogenicity of HpCDD. The c x t = k product was independent of dosing regimen. Anemia and squamous cell carcinoma of the lungs were the earliest and most prevalent endpoints of toxicity. A dose of 2.1 mg/kg and 3.1 mg/kg of HpCDD caused 16.6% and 73.3% lung cancer, respectively. Liver cancer had a low prevalence and was a very late effect occurring only at doses lethal acutely for most rats in the three highest dosage groups. There was no correlation in the dose-dependence of non-malignant hepatic lesions and liver cancer.
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