Michael A. Gallo

Ah Receptor and NF-κB Interactions, a Potential Mechanism for Dioxin Toxicity

The Ah receptor (AhR) mediates many of the toxic responses induced by polyhalogenated and polycyclic hydrocarbons (PAHs) which are ubiquitous environmental contaminants causing toxic responses in human and wildlife. NF-κB is a pleiotropic transcription factor controlling many physiological functions adversely affected by PAHs, including immune suppression, thymus involution, hyperkeratosis, and carcinogenesis. Here, we show physical interaction and mutual functional repression between AhR and NF-κB. This mutual repression may provide an underlying mechanism for many hitherto poorly understood PAH-induced toxic responses, and may also provide a mechanistic explanation for alteration of xenobiotic metabolism by cytokines and compounds that regulate NF-κB.

Role of NF-κB in Regulation of PXR-mediated Gene Expression A MECHANISM FOR THE SUPPRESSION OF CYTOCHROME P-450 3A4 BY PROINFLAMMATORY AGENTS

It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. Cytochrome P-450 3A4 cyp304 is responsible for the metabolism of over 50% of current prescription drugs, and cyp3a4 expression is transcriptionally regulated by pregnane X receptor (PXR), which is a ligand-dependent transcription factor. In this study, we report that NF-κB activation by lipopolysaccharide and tumor necrosis factor-α plays a pivotal role in the suppression of cyp3a4 through interactions of NF-κB with the PXR·retinoid X receptor (RXR) complex. Inhibition of NF-κB by NF-κB-specific suppressor SRIκBα reversed the suppressive effects of lipopolysaccharide and tumor necrosis factor-α. Furthermore, we showed that NF-κB p65 disrupted the association of the PXR·RXRα complex with DNA sequences as determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. NF-κB p65 directly interacted with the DNA-binding domain of RXRα and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by the PXR·RXRα complex. This mechanism of suppression by NF-κB activation may be extended to other nuclear receptor-regulated systems where RXRα is a dimerization partner.

Ah receptor and NF-κB interactions: mechanisms and physiological implications

Abstract The aryl hydrocarbon (Ah) receptor mediates most of the toxic effects induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds, which are ubiquitous environmental contaminants causing toxic responses in human and wildlife. Nuclear factor kappa B (NF-κB) is a pleiotropic transcription factor that plays a pivotal role in a wide array of physiological and pathological responses including immune modulation, inflammatory responses and apoptosis. Many physiological functions adversely affected by TCDD are also known to be regulated by NF-κB, such as immune activation, maintenance of skin differentiation, control of cell proliferation and survival, as well as induction of xenobiotic metabolizing enzymes. In the past few years, evidence has emerged to show that the Ah receptor and NF-κB interact and transcriptionally modulate each other. This review discusses Ah receptor–NF-κB interactions and examines potential mechanistic explanations for toxic responses as a result of TCDD exposure and the suppression of cytochrome P450 1A1/1A2 by stress stimuli such as inflammation and infection.

Antiestrogenic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin: Tissue-specific regulation of estrogen receptor in CD1 mice☆

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a polychlorinated aromatic hydrocarbon with teratogenic and carcinogenic properties. Previous studies in our and other laboratories have demonstrated that TCDD has antiestrogenic properties. In order to elucidate the mechanism of action of TCDD on estrogen sensitive tissues, we studied its effects on serum estradiol and estrogen receptor (ER) levels in liver and uteri of CD1 mice. Treatment with TCDD did not result in alterations of serum estradiol levels at any of the doses tested (1.0-30 micrograms/kg). In contrast, TCDD treatment induced a dose-dependent decrease in hepatic and uterine ER protein as determined by an enzyme immunoassay and equilibrium binding assays. A decrease in cytosolic and nuclear ER levels in uteri occurred as early as 24 hr after initial treatment with 30 micrograms/kg TCDD and recovery occurred by 14 days. Hepatic cytosolic and nuclear ER also decreased at a dose of 30 micrograms/kg TCDD at 24 hr after treatment, but recovery occurred only by 21 days. Studies in ovariectomized mice indicate that the regulation of hepatic ER by TCDD is independent of ovarian factors, but ovariectomy inhibited the downregulation of uterine ER by TCDD. Furthermore, determination of TCDD-induced cytochrome P-450 levels indicates that the downregulation of uterine ER is uncoupled from induction of hepatic cytochrome P-450. This study indicates that the antiestrogenic effects of low doses of TCDD are mediated through its ability to decrease hepatic and uterine ER and are not due to alterations in serum estradiol levels. Our results on ovariectomized mice indicate that TCDD-induced downregulation of ER is tissue specific and may involve different mechanisms at transcriptional or posttranscriptional levels.

Physiological implications of estrogen receptor modulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin

The interactions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with hormones and hormone receptors have important implications for TCDD toxicity. Evidence suggests that TCDD modulates receptors for glucocorticoids, prolactin, thyroxine, low density lipids, epidermal growth factor, and estrogens. Estrogen receptor modulation and the animals physiological responses to this modulation appear to be particularly important effects and can explain much of the toxicity observed in TCDD-treated animals. Susceptibility of different species to TCDD correlates with their steroid glucuronidation capacity. Because of the close interactions and interdependent regulation of hormonal systems, other hormones may have a similar role in TCDD toxicity.

Establishing Data-Derived Adjustment Factors from Published Pharmaceutical Clinical Trial Data

In non-cancer risk assessment the goal traditionally has been to protect the majority of people by setting limits that account for interindividual variability in the human population. The Environmental Protection Agency (EPA) has assigned a default uncertainty factor (?UF) of 10 to account for interindividual variability in response to toxic agents in the general population. Previous studies have suggested that it is appropriate to equally divide this factor into sub-factors of 3.2 each for variability in human pharmacokinetics (PK) and pharmacodynamics (PD). As an extension of this model, one can envision using scientific data from the literature to modify the default sub-factors with compound-specific adjustment factors (AFs) and to create new and more scientifically based defaults. In this paper, data from published clinical trials on six pharmaceutical compounds were used to further illustrate how to calculate and interpret data-derived AFs. The clinical trial data were analyzed for content and the re...

Reproductive toxicity in female mice of dioxin-contaminated soils from a 2,4,5-trichlorophenoxyacetic acid manufacturing site

Reproduction in female C57B/6 mice treated with contaminated soils from a 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) manufacturing site and a metal scrap yard in Newark, New Jersey was studied. Soils contained a wide variety of contaminants, including halogenated dibenzodioxins and dibenzofurans, benzene, alkylbenzenes, chlorobenzenes, polyaromatic hydrocarbons, phenolics, phenoxy acids and other compounds. Acute and reproductive toxicity (primarily fewer live pups born and fewer pups surviving until weaning) was observed from the manufacturing site soil (soil A; with 2,050 μg 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD)/kg soil and 18 mg total dibenzodioxins and dibenzofurans/kg soil); the scrap yard soil (soil B; with 230 (μg 2,3,7,8-TCDD/kg soil) had no observable effect. Soil recontaminated with 2,3,7,8-TCDD, or 2,3,7,8-TCDD in corn oil interfered with estrus cycling and completely prevented reproduction. If 2,3,7,8-TCDD treatment was terminated, estrus cycling resumed within four weeks, but reproduction was still unsuccessful for these mice. The results suggest that 1) the manufacturing site soil (soil A) is toxic but the signs of toxicity cannot be solely attributed to the 2,3,7,8-TCDD content; 2) even though the bioavailability of 2,3,7,8-TCDD from this soil is low (Umbreitet al. 1986), other reproductive toxins are bioavailable or the low doses of 2,3,7,8-TCDD that are bioavailable may be sufficient to be reproductive toxins; 3) 2,3,7,8-TCDD blocks estrus in mice and (as previously reported) interferes with reproduction; and 4) after cessation of dosing, thein situ concentrations of 2,3,7,8-TCDD that block estrus are decreased below an effective level within four weeks.

Polyamine-mediated conformational perturbations in DNA alter the binding of estrogen receptor to poly(dG-m5dC).poly(dG-m5dC) and a plasmid containing the estrogen response element

The binding estrogen receptor (ER) to the upstream regions of estrogen-responsive genes, the estrogen response elements (ERE), is of fundamental importance in the regulation of gene expression by estradiol. Multiple cell-specific factors affect ER-ERE binding and modulate the responses of estradiol. We studied the role of polyamines in the recognition of ER, a ligand-activated transcription factor, with a left-handed Z-DNA forming polynucleotide as well as with a plasmid containing ERE. Polyamines are cellular organic cations with multiple functions in cell growth and differentiation. Polyamines induce Z-DNA conformation in alternating purine-pyrimidine sequences. To understand the role of polyamine-induced DNA conformational transition in ER-DNA interaction, we studied the binding of partially purified rabbit uterine ER to poly(dG-m5dC).poly(dG-m5dC). The induction of Z-DNA in the polynucleotide was monitored by circular dichroism and ultraviolet spectroscopic measurements. Binding of ER to poly(dG-m5dC).poly(dG-m5dC) increased from 15% to approx. 50-60% in the presence of 7.5 mM putrescine, 0.5 mM spermidine or 0.25 mM spermine. Maximal binding of ER to the polynucleotide was observed near the midpoint of the B-DNA to Z-DNA transition of the polynucleotide. N1-acetyl spermidine and N1-acetyl spermine facilitated the B-DNA to Z-DNA transition and the binding of ER although they were less effective than the unacetylated analog. Co(NH3)6(3+), a trivalent inorganic cation, also provoked the B-DNA to Z-DNA transition of the polynucleotide and increased its binding to ER. At higher polyamine concentrations, there was an inhibition of ER binding to the polynucleotide. In the presence of polyamines, the binding of ER to a plasmid containing ERE was 2-3-fold higher than that to a control plasmid devoid of ERE. Polyamine-induced facilitation of ER-ERE binding was also confirmed by gel mobility shift assay. Our data indicate that conformational perturbations, similar to that of the early stages of B-DNA to Z-DNA transition, are important in the recognition of ER and ERE.

Alteration of the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by estradiol and tamoxifen

We have hypothesized that part of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is mediated by interaction with the estrogen receptor complex. The experiments reported here investigate the interactions of TCDD with agonists and antagonists of the estrogen receptor. CD-1 female mice were observed for 2 months after treatment with various combinations of corn oil, estradiol, or tamoxifen, and/or TCDD in corn oil on 3 consecutive days. Estradiol had little effect on acute TCDD lethality but increased severity of TCDD-induced ascites and antagonized TCDD-induced uterine suppression. Severe liver damage did occur in TCDD and estradiol:TCDD treatment groups. Tamoxifen, a competitive inhibitor and a mixed agonist of the mouse estrogen receptor, antagonized the estrogenic effects of estradiol and estradiol:TCDD. Tamoxifen or tamoxifen:TCDD treatment greatly slowed body weight gain in comparison to controls and estrogen-treated animals. While the dose of tamoxifen used was otherwise non-toxic, tamoxifen greatly increased toxicity of TCDD as measured by time to death and percent lethality while having no effect on relative liver weight or relative uterine weight changes induced by TCDD. These findings are consistent with the hypothesis that a portion of the toxicity of TCDD is manifest through activity of the estrogen receptor complex.

Pulmonary bioavailability and fine particle enrichment of 2,3,7,8-tetrachlorodibenzo-p-dioxin in respirable soil particles

The pulmonary bioavailability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the enrichment of polychlorinated dioxins (PCDDs) and furans (PCDFs) in fine particles were evaluated to assess the implications that these factors have on risk and exposure assessments. Respirable subfractions of PCDD-contaminated soil from a former 2,4,5-trichlorophenoxyacetic acid manufacturing site were isolated by chemical dispersion and gravity sedimentation. Analysis of the subfractions revealed that there was a size-dependent enrichment of PCDDs and PCDFs, with smaller particles more highly contaminated. TCDD was enriched up to 33-fold as compared to unfractionated soil. Soil and laboratory-recontaminated gallium oxide, which served as the positive control, were administered by intratracheal instillation to female Sprague-Dawley rats. Animals were terminated up to 28 days following treatment and pulmonary bioavailability of TCDD was assessed by hepatic enzyme induction and TCDD concentration. Enzyme induction was dependent on the duration of exposure with up to 56 and 918% increases in cytochrome P450 and aryl hydrocarbon hydroxylase (AHH) activity, respectively, following exposure to PCDD-contaminated soil. There was no significant difference in AHH induction between animals which received contaminated soil and those treated with the positive control. Hepatic concentration of TCDD in soil-exposed rats was 115, 101, and 179% of positive controls at 1, 7, and 28 days post-treatment, suggesting that the soil or cocontaminants influenced retention of TCDD in the liver. These data indicate that the relative pulmonary bioavailability of TCDD on respirable soil particles is 100% as compared to laboratory-recontaminated gallium oxide and that PCDDs and PCDFs are highly enriched on respirable particles.(ABSTRACT TRUNCATED AT 250 WORDS)