Ah Receptor Binding Affinities Research Articles

Effect Analysis of Substituent Characteristics of PBDEs on Its Ah Receptor Binding Affinities

Based on the known experimental Ah receptor binding affinities of 18 kinds of polybrominated diphenyl ethers (PBDEs), the quantitative structure-activity relationships (QSAR) model for PBDEs’ Ah receptor binding affinities was established via 13 substituent parameters (total number of substituent, substituent number in different position, substituent positional relationship parameters, substituent difference between two rings) to complement unknown binding affinities of other 191 PBDEs. Then, the full factorial experiment with 10 factors which correlated with each substituent position and 2 level (0,1) was applied to analyze the main effect and second-order interaction effect of each substituent position on PBDEs’ Ah receptor binding affinities. Meanwhile, different analysis methods were used for the views of the total number of substituent, the similarity of different phenyl ring in single congener and the distribution of substituents on single phenyl ring to expound the correlation between substituent characteristics and Ah receptor binding affinities of PBDEs comprehensively. The obtained results have shown that: PBDEs’ Ah receptor binding affinities are significantly affected by the main effect and second-order interaction effect of substitution positions, especially, the ortho-substituents can weaken the PBDEs’ Ah receptor binding affinities and para-substituents have the opposite effect. The order of the importance for different position is presented as: para>ortho>meta. The main effect of meta-substituent is small which often affects the Ah receptor binding affinities of PBDEs by representing the second-order interaction effects combined with ortho/para-substituent. For other substituent characteristics, the total number of substituent and the similarity of different phenyl ring in single congener cannot control the Ah receptor binding affinities of PBDEs effectively, but the more decentralized for substituents on single phenyl ring, the smaller Ah receptor binding affinities for PBDEs.

Analysis of Ah receptor binding affinities of polybrominated diphenyl ethers via in silico molecular docking and 3D-QSAR

Polybrominated diphenyl ethers (PBDEs) have become ubiquitous contaminations due to their use as flame retardants. The structural similarity of PBDE to some dioxin-like compounds suggested that they may share similar toxicological effects: they might activate the aryl hydrocarbon receptor (AhR) signal transduction pathway and thus might have adverse effects on wildlife and humans. In this study, in silico computational workflow combining molecular docking and three-dimensional quantitative structure–activity relationship (3D-QSAR) was performed to investigate the binding interactions between PBDEs and AhR and the structural features affecting the AhR binding affinity of PBDE. The molecular docking showed that hydrogen-bond and hydrophobic interactions were the major driving forces for the binding of ligands to AhR, and several key amino acid residues were also identified. The CoMSIA model was developed from the conformations obtained from molecular docking and exhibited satisfactory results as q 2 of 0.605 and r 2 of 0.996. Furthermore, the derived model had good robustness and statistical significance in both internal and external validations. The 3D contour maps generated from CoMSIA provided important structural features influence the binding affinity. The obtained results were beneficial to better understand the toxicological mechanism of PBDEs.

Docking and 3D-QSAR studies on the Ah receptor binding affinities of polychlorinated biphenyls (PCBs), dibenzo- p-dioxins (PCDDs) and dibenzofurans (PCDFs)

Polychlorinated biphenyls (PCBs), dibenzo- p-dioxins (PCDDs) and dibenzofurans (PCDFs) binding with the aryl hydrocarbon receptor (AhR) have been correlated with many toxic responses. Hence, it is very necessary to study the interactions between these ligands and AhR for further understanding of the mechanism of toxicity. In this study, an integrated molecular docking and 3D-QSAR approach was employed to investigate the binding interactions between PCBs, PCDDs, PCDFs and AhR. From molecular docking, hydrogen-bonding and hydrophobic interactions were observed to be characteristic interactions between compounds and AhR. Based on the mechanism of interactions, an optimum 3D-QSAR model with good robustness ( Q CUM 2 = 0.907 ) and predictability ( Q EXT 2 = 0.863 ) was developed by partial least squares. Additionally, the developed QSAR model indicated that the molecular size, shape profiles, polarizability and electropological states of compounds were related to the binding affinities to AhR.

Quantitative Structure–Activity Relationships Study on the Ah Receptor Binding Affinities of Polybrominated Diphenyl Ethers Using a Support Vector Machine

AbstractPolybrominated Diphenyl Ethers (PBDEs), which are widely distributed in the environment due to their use as flame retardant, may cause long‐term health problems in humans. Their structural similarity to Polychlorinated Biphenyls (PCBs) implies their possible dioxin‐like toxicity. By the use of Partial Least Square Regression (PLS ) with Leave‐One‐Out (LOO) Cross‐Validation (CV), seven net atomic charge descriptors have been extracted from more than 80 quantum descriptors for predicting the Aryl hydrocarbon Receptor Relative Binding Affinities (RBA) of PBDEs. Using Support Vector Machines (SVM) and Radial Basis Function Neural Networks (RBFNs), the RBAs of 15 PBDE congeners have been correlated with the extracted seven quantum chemical descriptors. The SVM models generalize better than the RBFN models. The CV correlation coefficients (q2) for the SVM models based on three‐ and five‐fold CVs are 0.889 and 0.896, respectively. The good performance of the QSAR models based on net atomic charges suggests that electrostatic interactions may play important roles in the Aryl hydrocarbon Receptor (AhR) binding of PBDEs.

QSPR/QSAR models for prediction of the physicochemical properties and biological activity of polybrominated diphenyl ethers

Polybrominated diphenyl ethers (PBDEs) are a group of important persistent organic pollutants. In the present study, geometrical optimization and electrostatic potential calculations have been performed for all 209 PBDE congeners at the HF/6-31G ∗ level of theory. A number of statistically-based parameters have been obtained. Linear relationships between gas-chromatographic relative retention time (RRT), n-octanol/air partition coefficient (lg K OA), 298 K supercooled liquid vapour pressures (lg p L), Henry’s law constant (lg H) and Ah receptor binding affinity (−lg RBA) of PBDEs and the structural descriptors have been established by multiple regression method. The result shows that the quantities derived from electrostatic potential V s, max , V s, min , Π , ∑ V s + , V s - ¯ , ν , σ tot 2 , and N v - , together with the molecular volume ( V mc) can be well used to express the quantitative structure–property relationships of PBDEs, which proves the general applicability of this parameter set to a great extent. Good predictive capabilities have also been demonstrated. Based on these equations, the predicted values have been presented for those PBDE congeners whose experimentally determined physicochemical properties are unavailable. The QSAR model for the Ah receptor binding affinity is relatively poor, which can be ascribed to the complexity of factors which affect biological activity and the limitations of the present parameter set in describing steric characters of the molecule.

QSAR study on the Ah receptor-binding affinities of polyhalogenated dibenzo-p-dioxins using net atomic-charge descriptors and a radial basis neural network

A radial basis function neural network (RBFN) has been used to correlate Ah receptor-binding affinities of polychlorinated, polybrominated, and polychlorinated-brominated dibenzo-p-dioxins with molecular weight and eight net atomic charge descriptors. Support vector machine (SVM) and partial least square (PLS) regression models based on the same data set have also been built. Leave-one-out cross-validation was used to train the RBFN, SVM, and PLS models. For predicting Ah receptor-binding affinities, the RBFN model with a squared cross-validation correlation coefficient (q2) of 0.8818 outperforms the SVM and PLS models and also compares favorably with any other reported quantitative structure-activity relationship model based on the same activity data set. The significance of the RBFN model with net atomic charges as descriptors suggests that electrostatic and dispersion-type interactions play important roles in governing the Ah receptor binding of polychlorinated, polybrominated, and polychlorinated-brominated dibenzo-p-dioxins.

Comparison of acute toxicities of indolo[3,2- b]carbazole (ICZ) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in TCDD-sensitive rats

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) and related halogenated aromatic hydrocarbons are environmental toxicants that act via the AH receptor (AHR). In vitro studies have demonstrated that some indole derivatives present in cruciferous vegetables also bind to the AHR. One of the highest AHR binding affinities is exhibited by indolo[3,2- b]carbazole (ICZ). Since exposure to these dietary indoles is quantitatively far larger than that to halogenated aromatic compounds, their potential toxic risks have raised concern. In the present study, we compared the effects of ICZ with those of a single dose of 20 μg/kg TCDD in the most TCDD-sensitive rat strain (Long–Evans [ Turku AB]) (L-E). Whereas TCDD elicited the expected toxicity syndrome, ICZ, either as a single subcutaneous dose (63.5, 127 or 508 μg/kg) or with repeated sc dosing (508 μg/kg for 5 days) failed to reproduce any toxic impacts of TCDD. Furthermore, a simultaneous ICZ treatment (63.5 or 127 μg/kg for 10 days) did not interfere with TCDD (20 μg/kg; single exposure) action. A moderate hepatic induction of CYP1A1 could be triggered by repeated intragastric administration of ICZ (127 μg/kg for 4 days, the last treatment 2.5 h prior to termination). In control experiments in a reconstituted yeast system, ICZ potently and dose-dependently activated L-E rat AHR function demonstrating that it represents a bona fide high-affinity ligand for the rat receptor in vivo. Thus, the present study does not support the view that dietary exposure to ICZ would present a hazard of AHR-mediated adverse health effects to humans.

Synthesis of polybrominated diphenyl ethers and their capacity to induce CYP1A by the Ah receptor mediated pathway.

Polybrominated diphenyl ethers (PBDEs) have become widely distributed as environmental contaminants due to their use as flame retardants. Their structural similarity to other halogenated aromatic pollutants has led to speculation that they might share toxicological properties such as hepatic enzyme induction. In this work we synthesized a number of PBDE congeners, studied their affinity for rat hepatic Ah receptor through competitive binding assays, and determined their ability to induce hepatic cytochrome P-450 enzymes by means of EROD (ethoxyresorufin-O-deethylase) assays in human, rat, chick, and rainbow trout cells. Both pure PBDE congeners and commercial PBDE mixtures had Ah receptor binding affinities 10(-2)-10(-5) times that of 2,3,7,8-tetrachlorodibenzo-p-dioxin. In contrast with polychlorinated biphenyls, Ah receptor binding affinities of PBDEs could not be related to the planarity of the molecule, possibly because the large size of the bromine atoms expands the Ah receptor's binding site. EROD activities of the PBDE congeners followed a similar rank order in all cells. Some congeners, notably PBDE 85, did not follow the usual trend in which strength of Ah receptor binding affinity paralleled P-450 induction potency. Use of the gel retardation assay with a synthetic oligonucleotide indicated that in these cases the liganded Ah receptor failed to bind to the DNA recognition sequence.

Electronic eigenvalue (EEVA): a new QSAR/QSPR descriptor for electronic substituent effects based on molecular orbital energies. A QSAR approach to the Ah receptor binding affinity of polychlorinated biphenyls (PCBs), dibenzo- p-dioxins (PCDDs) and dibenzofurans (PCDFs)

A new descriptor of molecular structure for use in the derivation of predictive QSAR and QSPR models, electronic eigenvalue (EEVA), is described. This is a modification of the recently proposed EVA approach, but is based on computationally-derived molecular orbital energies instead of vibrational frequencies. Like EVA, it is also invariant as to the alignment of the structures concerned. Its performance has been tested with respect to the Ah receptor binding of PCBs, PCDDs and PCDFs, and its predictive ability has been clearly demonstrated. In particular, it seems to be suitable for `pure' electronic substituent effects, i.e., for cases in which both hydrophobic and steric factors are of minor importance.

Hepatic Ah receptor binding affinity for 2,3,7,8-tetrachlorodibenzo- p-dioxin: similarity between beagle dog and cynomolgus monkey

Hepatic AhR binding affinity for [ 3H]-2,3,7,8-tetrachlorodibenzo- p-dioxin ([ 3H]TCDD) was compared between two species widely used as laboratory animals: beagle dog and cynomolgus monkey ( Macaca fascicularis). The enriched 9S fractions from both species were obtained by sucrose gradient sedimentation. After incubation with [ 3H]TCDD, dextran-coat charcoal treatment (10 mg/ml) revealed that dog and monkey possess an AhR with a low binding affinity for [ 3H]TCDD. Saturation experiments were then achieved according to the method developed in experiments on human samples. The binding characteristics were determined after analysis of the data by Scatchard and Woolf plots. Receptor concentrations were quite similar in dog and monkey liver (26.6 and 14.4 pmol/mg, respectively) as well as the affinity ( K d) for [ 3H]TCDD (17.1 and 16.5 nM, respectively). The low binding affinity of dog and monkey AhRs appeared to be similar to those observed in human.

Characterization of polycyclic aromatic compounds in diesel exhaust particulate extract responsible for aryl hydrocarbon receptor activity

Chemical fractions of a model diesel exhaust particulate extract, notably the fraction containing polycyclic aromatic hydrocarbons (PAH) (Fraction II), mono-nitro PAH (Fraction III), and dinitro-PAH (Fraction IV) have been shown to displace binding of 2,3,7,8-tetrachloro[1,6-[ 3H]]dibenzo- p-dioxin (TCDD) from the rat liver cytosol aryl hydrocarbon (Ah) receptor. The Ah receptor binding activity of twenty-seven PAH, identified in Fraction II and recombined in the same proportions as those observed in the original fraction, was not significantly different to that of Fraction II, suggesting that the binding activity of this, fraction can be explained by the identified components. In addition, these two samples had the ability to induce expression of an Ah receptor target gene, CYP450lA1, as indicated by solution hybridisation assay of mRNA levels in mouse Hepa 1c1c7 cells. On screening of the individual reference PAH, the following compounds displayed IC 50 values for the Ah receptor binding < l μM: benzo(a)-fluorene, benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(a)pyrene and indeno(1,2,3-cd)pyrene. These six compounds also induced the expression of CYP4501A1 in the range 2–6-fold above baseline levels. Further studies demonstrated that the Ah receptor binding activity of Fraction II could be explained by the presence of these six PAR The chromatographic behaviour and Ah receptor binding affinity of reference mono-nitro PAH, indicate that 3-nitroperylene, 6-nitrobenzo(a)pyrene and 7-nitrobenz(a)-anthracene are candidate compounds responsible for the high Ah binding activity observed in Fraction III.

Substituted flavones as aryl hydrocarbon (Ah) receptor agonists and antagonists

The structure-dependent aryl hydrocarbon (Ah) receptor agonist and antagonist activities of the following substituted flavones were investigated: flavone, 4′-methoxy-, 4′-amino-, 4′-chloro-, 4′-bromo-, 4′-nitro-, 4′-chloro-3′-nitro-, 3′-amino-4′-hydroxy-, 3′,4′-dichloro-, and 4′-iodoflavone. The halogenated flavones exhibited competitive Ah receptor binding affinities ( IC 50 = 0.79 to 2.28 nM) that were comparable to that observed for 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) (1.78 nM). The compounds also induced transformation of the rat cytosolic Ah receptor and induced CYP1A1 gene expression in MCF-7 human breast cancer cells. However, despite the high Ah receptor binding affinities for these responses, the halogenated flavones were > 1000 times less active than TCDD for the other responses. Moreover, for other substituted flavones, there was no correlation between Ah receptor binding affinities and their activities as Ah receptor agonists. For example, 4′-aminoflavone induced CYP1A1 mRNA levels in MCF-7 cells but exhibited relatively low Ah receptor binding affinity ( IC 50 = 362 nM) and did not induce transformation of the rat cytosolic Ah receptor. All of the substituted flavones inhibited TCDD-induced transformation of the Ah receptor, and 4′-iodoflavone, an Ah receptor agonist at high concentrations (1–50 μM), inhibited the transformation at concentrations as low as 0.05 and 0.5 μM. Subsequent interaction studies with TCDD and 4′-iodoflavone confirmed that the latter compound inhibits induction of CYP1A1 gene expression by TCDD in MCF-7 cells. The results obtained for the substituted flavones suggest that within this structural class of compounds, various substituent groups can affect markedly the activity of each individual congener as an Ah receptor agonist or antagonist. These substituent-dependent differences in activity may be related to ligand-induced conformational changes in the Ah receptor complex and/or support the proposed existence of more than one form of the Ah receptor.

Toxic equivalency factors of polychlorinated dibenzo- p-dioxin, dibenzofuran and biphenyl congeners based on early life stage mortality in rainbow trout ( Oncorhynchus mykiss)

Toxic equivalency factors (TEFs), which relate the potency of individual polychlorinated dibenzo- p-dioxin (PCDD), dibenzofuran (PCDF), and biphenyl (PCB) congeners to 2,3,7,8- tetrachlorodibenzo- p-dioxin (TCDD), are used to assess the risk to fish early life stage development posed by complex mixtures of these congeners in eggs. Newly fertilized rainbow trout ( Oncorhynchus mykiss) eggs were injected with graded doses of 5 PCDD, 4 PCDF or 4 nonortho substituted PCB congeners, and sac fry mortality was used to determine TEFs. Potencies of non-ortho substituted PCBs in causing early life stage mortality were much lower than expected based on Ah receptor binding affinities in mammals and 4 mono-ortho and 5 di-ortho substituted PCB congeners did not cause rainbow trout early life stage mortality at doses of 24 300–130 000 ng/g egg. To determine if increased elimination of PCBs, relative to PCDDs and PCDFs, was responsible for the lower toxic potency of PCBs in fish compared to mammals, PCDD, PCDF and PCB congeners were injected into newly fertilized rainbow trout eggs and the percent dose remaining 35 days later at the sac fry stage of development was determined. The percent dose of various PCB congeners remaining in sac fry (62–88%) was similar to that of PCDD and PCDF congeners (78–91%), indicating that increased elimination is not responsible for the extremely low potency of the non-ortho substituted PCB congeners or for the failure of the mono- and di-ortho substituted PCB congeners to cause rainbow trout early life stage mortality.

2-Phenylphenanthridinone and Related Compounds: Aryl Hydrocarbon Receptor Agonists and Suicide Inactivators of P4501A1

The effects of several 2-substituted phenanthridinones (2-nitro-, 2-t-butyl-, 2-bromo-, 2-phenyl-, 2-ethyl-, 2-methoxy-, 2-iodo-, 2-n-butyl-, 2-chloro-, 2-trifluoromethyl-, 2-fluoro-, 2-isopropyl-, and 2-methyl) and phenanthridinone as ligands for the rat liver cytosolic aryl hydrocarbon (Ah) receptor were determined using a competitive binding assay and 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the radioligand. The competitive binding IC50 values varied from 317 (2-trifluoromethyl-) to 5870 nM (2-methoxyphenanthridinone); the relative low Ah receptor binding affinities for these compounds were paralleled by their weak activity as inducers of ethoxyresorufin O-deethylase (EROD) activity in rat hepatoma H4IIE cells; however, there was not a correlation between their structure-binding and structure-induction relationship. In cells cotreated with 1 nM TCDD plus different concentrations (0.01-10 μM) of the 2-substituted phenanthridinones, several of these compounds inhibited induction of EROD activity by TCDD; 2-t-butyl- and 2-phenylphenanthridinone (2-PP) were the most active compounds, causing a >80% reduction in the induced response. 2-PP was selected as a model to further investigate the mechanism of this inhibitory response. The results of interactive studies in rat hepatoma H4IIE cells cotreated with 2-PP plus TCDD or [3H]TCDD showed that 2-PP did not inhibit formation of the nuclear Ah receptor complex or induction of CYP1A1 mRNA levels or CYP1A1 protein. In contrast, incubation of 2-PP with either rat hepatoma H4IIE cells treated with TCDD or hepatic microsomes from TCDD-treated rats resulted in a rapid loss of EROD activity. In parallel experiments, [3H]2-PP was incubated with hepatic microsomes from TCDD-treated rats and analysis by denaturing electrophoresis showed that [3H]2-PP formed a covalent adduct with a 50- to 55-kDa protein and thus acted as a suicide inactivator of CYP1A1.

2,3,7,8-Tetrachlorodibenzo-p-dioxin versus 3-methylcholanthrene: comparative studies of Ah receptor binding, transformation, and induction of CYP1A1.

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.

Effects of polychlorinated dibenzofurans on compounds in hepatic DNA of female Sprague-Dawley rats: Structure dependence and mechanistic considerations

Previous work indicated that covalent age-dependent DNA modifications of endogenous origin termed I-compounds may represent useful biomarkers for tumor promotion/carcinogenesis, as various tumor promoters/carcinogens, including 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and phenobarbital, reduce rat I-compound levels in liver, the target organ. The present study addressed the question as to whether polychlorinated dibenzofurans (PCDFs), which are related to TCDD and its congeners with regard to their toxic and biochemical properties, would also affect hepatic I-compound patterns and levels, and whether such effects would be chemical structure-dependent. Female Sprague-Dawley rats were treated once a week with a single dose (100 μg/kg) of 1,2,3,7,8-pentachlorodibenzofuran (1,2,3,7,8-PeCDF), 1,2,4,7,8-PeCDF, 2,3,4,7,8-PeCDF, or 2,3,4,6,7,8-hexachlorodibenzofuran (2,3,4,6,7,8-HeCDF) for 4 weeks and liver DNA was analyzed at the end of the last week by 32P-postlabeling assay. No carcinogen-DNA adducts were detected; however, levels of both non-polar and polar I-compounds were reduced in a structure-dependent manner. Potencies increased in the order, control (100%, 122 modifications in 10 9 DNA nucleotides = 1,2,4,7,8-PeCDF (104%) <1,2,3,7,8-PeCDF (80%) <2,3,4,7,8-PeCDF (61%) and 2,3,4,6,7,8-HeCDF (61%). Structure-activity relationships for total I-compounds, therefore, paralleled those reported for Ah receptor agonist activity, i.e., compounds that exhibit high cytosolic Ah receptor binding affinities and are also potent inducers of aryl hydrocarbon hydroxylase activity (1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF, and 2,3,4,6,7,8-HeCDF) were active, while 1,2,4,7,8-PeCDF, which is a less potent Ah receptor agonist, was inactive. Polar I-compounds responded to a greater extent than did non-polar ones and, in general, individual I-compounds were affected differentially, thus decreased formation or increased removal of I-compounds played a role in the observed effects of the toxins on DNA. It is proposed that Ah receptor-mediated enzyme induction, particularly of cytochrome P450, is involved in reduced hepatic I-compound formation and that subnormal I-compound levels may contribute to tumor promotion.

Binding of some dioxins and dibenzofurans to the Ah receptor. A QSAR model based on comparative molecular field analysis (CoMFA)

Based on comparative molecular field analysis (CoMFA), a quantitative structure-activity relationship, model for the cytosolic aryl hydrocarbon (Ah) receptor binding affinity of 50 chlorinated dibenzo- para-dioxin and dibenzofuran analogues is presented. The electronic structures of these compounds were calculated using the semiempirical AM1 method. Their extreme stabilities were rationalized using the frontier molecular orbital theory. The results indicate that their binding affinity was mainly influenced by steric and electrostatic interactions, whereas charge-transfer and hydrogen bonding mechanisms are probably of minor importance.

Structure-dependent reduction in DNA I-compound adducts by polychlorinated dibenzofurans in female Sprague-Dawley rat liver

The effects of polychlorinated dibenzofurans (PCDFs) on hepatic I-compound DNA adducts in female Sprague-Dawley rats were determined at 2 dose levels. In the first experiments, rats (4 per group) were treated with each of 3 polychlorinated dibenzofuran (PCDF) congeners at 20 μg/kg/week or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) at 10 μg/kg/week for 4 weeks. Effects on total I-compound levels when compared with age-matched control animals (corn oil) were as follows: control (116) > 1,2,4,7,8-pentachlorodibenzofuran (PeCDF) (112) > 1,2,3,7,8-PeCDF (93) > 2,3,4,7,8-PeCDF (91) > TCDD (72) where the numbers in parentheses were the number of I-compounds in 10 9 DNA nucleotides. In the second experiment, rats (5 per group) were treated with each of the congeners at a higher dose of 100 μg/kg/week for 4 weeks. The effects were as follows: control (123) ≈ 1,2,4,7,8-PeCDF (125) > 1,2,3,7,8-PeCDF (98) > 2,3,4,7,8-PeCDF (75) ≈ 2,3,4,6,7,8-hexachlorodibenzofuran (HCDF) (75). The active compounds (1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF and 2,3,4,6,7,8-HCDF) were those that exhibited high cytosolic Ah receptor binding affinities (1.5 – 7.5 × 10 −8 M) and were also potent AHH inducers. The inactive compound (1,2,4,7,8-PeCDF) was less active in the receptor binding and enzyme induction assays. The results showed that there was a structure-dependent correlation between the Ah receptor agonist activity of a congener and its potency to reduce I-compounds in female Sprague-Dawley rat liver.

Inhibition of insulin-like growth factor-I responses in MCF-7 cells by 2,3,7,8-tetrachlorodibenzo- p-dioxin and related compounds

Insulin-like growth factor-I (IGF-I) stimulated the growth and [ 3H]thymidine uptake in MCF-7 human breast cancer cells grown in serum- and growth factor-inactivated serum-containing media. Cotreatment of the cells with IGF-I plus 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) resulted in a significant decrease in mitogen-induced cell proliferation and [ 3H]thymidine uptake. Similar effects were observed for cells treated with 2,3,7,8-TCDD and IGF-I plus 17β-estradiol. The relative antimitogenic activities of 2,3,7,8-TCDD and related compounds followed the order 2,3,7,8-TCDD > 2,3,7,8-tetrachlorodi benzofuran (TCDF) > 1,2,7,8-TCDF > 1,3,7,8-TCDD which was similar to their aryl hydrocarbon (Ah) receptor binding affinities. The results showed that 2,3,7,8-TCDD did not alter the IGF-I receptor mRNA levels or the K D values for binding of [ 125I]IGF-I to the IGF-I receptor in MCF-7 cells. However, 2,3,7,8-TCDD significantly decreased the number of IGF-I-induced IGF-I receptor binding sites and this may play a role in the growth-inhibitory properties of 2,3,7,8-TCDD and related compounds and in the ‘cross-talk’ between the two endocrine-response pathways.

Certain tryptophan photoproducts are inhibitors of cytochrome P450-dependent mutagenicity.

Two photoproducts, derived from UV-irradiation of the amino acid L-tryptophan and with high Ah (TCDD) receptor binding affinity, were tested for genotoxic and antimutagenic effects. The two indolo[3,2-b]carbazole derivatives, with the molecular weights of 284 and 312, respectively, were tested in Saccharomyces cerevisiae strain D7 for mitotic gene conversion and reverse mutation and in strain RS112 for sister chromatid conversion and gene conversion. No significant (P > 0.05) genotoxic effects were found in strain D7, while strain RS112 showed a small but significant increase in the frequency of sister chromatid conversions. In Chinese hamster ovary (CHO) cells the two compounds induced a statistically significant but less than twofold increase in the frequency of sister chromatid exchanges (SCE). No mutations were detected when the compounds were tested in Salmonella typhimurium strains TA98 and TA100. However, both 284 and 312 acted as antimutagens on strain TA100 + S9 in the presence of benzo(a)pyrene. The decrease in mutagenicity by the most potent compound 284 was 20 revertants/nmol. This effect could be explained by an inhibitory effect on the cytochrome P450-dependent ethoxyresorufin O-deethylase (EROD) activity as seen in rat hepatocytes. The two compounds were also tested with hamster cells expressing rat cytochrome P-450IA1. The results support the conclusion that this cytochrome P-450 isozyme is inhibited by the tryptophan photoproducts. Similar results were also seen with two other high affinity Ah receptor ligands the quinazolinocarboline alkaloids rutaecarpine and dehydrorutaecarpine.