Aryl hydrocarbon receptor activity in intestinal epithelial cells in the formation of colonic tertiary lymphoid tissues.

After birth, the development of secondary lymphoid tissues (SLTs) in the colon is dependent on the expression of the aryl hydrocarbon receptor (AhR) in immune cells as a response to the availability of AhR ligands. However, little is known about how AhR activity from intestinal epithelial cells (IECs) may influence the development of tertiary lymphoid tissues (TLTs). As organized structures that develop at sites of inflammation or infection during adulthood, TLTs serve as localized centers of adaptive immune responses, and their presence has been associated with the resolution of inflammation and tumorigenesis in the colon. Here, we investigated the effect of the conditional loss of AhR activity in IECs in the formation and immune cell composition of TLTs in a model of acute inflammation. In females, loss of AhR activity in IECs reduced the formation of TLTs without significantly changing disease outcomes or immune cell composition within TLTs. In males lacking AhR expression in IECs, increased disease activity index, lower expression of functional-IEC genes, increased number of TLTs, increased T-cell density, and lower B- to T-cell ratio were observed. These findings may represent an unfavorable prognosis when exposed to dextran sodium sulfate (DSS)-induced epithelial damage compared with females. Sex and loss of IEC AhR also resulted in changes in microbial populations in the gut. Collectively, these data suggest that the formation of TLTs in the colon is influenced by sex and AhR expression in IECs.NEW & NOTEWORTHY This is the first research of its kind to demonstrate a clear connection between biological sex and the development of tertiary lymphoid tissues (TLT) in the colon. In addition, the research finds that in a preclinical model of inflammatory bowel disease, the expression of the aryl hydrocarbon receptor (AhR) influences the development of these structures in a sex-specific manner.

Lethal interaction of ubiquitous insecticide carriers with virus.

Large quantities of presumably nontoxic petroleum oil by-products are introduced into the environment as pesticide dispersal agents and emulsifiers. An increase in viral lethality with a concomitant influence on the liver and central nervous system occurs in young mice previously primed with such chemicals.

Indolo[3,2-b]carbazole: a dietary-derived factor that exhibits both antiestrogenic and estrogenic activity.

BACKGROUND: Indole-3-carbinol (I3C) and related compounds have been identified in vegetables of the Brassica genus. I3C and its acid-derived condensation product, indolo[3,2-b]carbazole (ICZ), bind to the aryl hydrocarbon (Ah) receptor and induce CYP1A1/1A2 gene expression in both in vivo and in vitro models. I3C also inhibits mammary tumor development in rodent models. PURPOSE: The major focus of this study was to investigate the induction of CYP1A1-dependent activity and antiestrogenic effects of ICZ in the MCF-7 human breast cancer cell line and determine if induction of CYP1A1 is required for observed antiestrogenic responses. METHODS: The induction of CYP1A1 in MCF-7 cells was determined by measuring time- and concentration-dependent changes in ethoxyresorufin O-deethylase (EROD) activity in response to ICZ treatment. The effects of ICZ on occupied nuclear estrogen receptor (ER) levels and inhibition of estrogen (17 beta-estradiol [E2])-induced cell proliferation, [3H]thymidine uptake, secretion of the 52-kd protein, and nuclear progesterone receptor (PR) levels were also measured. Chloramphenicol acetyl transferase (CAT) activity was assayed in MCF-7 cells transiently transfected with an estrogen-responsive vit-CAT plasmid. Competitive binding to rat cytosolic ER was also examined. RESULTS: ICZ (> or = 10 nM) induced CYP1A1 in MCF-7 human breast cancer cells. This compound also elicited a diverse spectrum of antiestrogenic responses, including inhibition of E2-induced cell proliferation, [3H]thymidine uptake, occupied nuclear PR binding, and CAT activity in cells transfected with the estrogen-responsive vit-CAT plasmid. In nuclear extracts from ICZ-treated cells, there was a decrease in ER levels and binding to an estrogen-responsive element in a gel shift assay. I3C also decreased nuclear ER binding in MCF-7 cells. ICZ bound with low affinity to the ER and exhibited weak estrogen-like activity. CONCLUSIONS: Like other Ah receptor agonists, ICZ is antiestrogenic in human breast cancer cells, and this activity is consistent with the inhibitory activity of I3C on mammary tumor formation in rodents. ICZ-induced antiestrogenic responses can be observed at times or concentrations in which EROD activity is unchanged, indicating an interaction between the Ah receptor and ER-mediated endocrine pathways that is independent of P450-induced hormone metabolism. ICZ also is a weak estrogen in MCF-7 cells and binds to the ER. IMPLICATIONS: The current focus on the role of dietary and environmental estrogens in human disease should take into account the possible contra-active effects of Ah receptor agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), ICZ, I3C, and related compounds that exhibit antiestrogenic activity.

Influence of cell proliferation on initiating activity of pure polychlorinated biphenyls and complex mixtures in resistant hepatocyte in vivo assays for carcinogenicity.

The abilities of various pure polychlorinated biphenyls (PCB) and complex mixtures to generate resistant gamma-glutamyltransferase (GGT)-positive hepatocellular nodules was evaluated in F344 rats in which hepatocytes were proliferating. The PCB examined were 2,2',4,4',5,5'-hexachlorobiphenyl (CAS: 35065-27-1), 2,2',4,4'-tetrachlorobiphenyl, 2,2',5,5'-tetrachlorobiphenyl, Aroclor 1254 (CAS: 11097-69-1), and a prepared mixture of pure PCB isomers and congeners similar to those found in human breast milk. The PCB were administered either to male and female suckling rats (weekly for 3 wk) during liver growth or to adult male rats (150-160 g body wt) previously subjected to two-thirds partial hepatectomy (PH). Rats were subsequently given a selection regimen consisting of 3 daily doses (20 mg/kg) of 2-acetylamino-fluorene (2-FAA; CAS: 53-96-3) followed by either PH or necrotizing carbon tetrachloride (CAS: 56-23-5) in adult rats that previously underwent PH. None of the PCB exposures generated GGT-positive nodules after selection, whereas known initiators such as diethylnitrosamine (CAS: 55-18-5), 3-methylcholanthrene (CAS: 56-49-5), benzo[a]pyrene (CAS: 50-32-8), and 2-FAA were active initiators of nodules in suckling or hepatectomized rats. These findings indicate that short-term exposures to these PCB during liver cell proliferation do not show initiating action in an in vivo assay that detects both hepatic and nonhepatic initiating carcinogens.

Influences of different polychlorinated biphenyls on cytocidal, mitoinhibitory, and nodule-selecting activities of N-2-fluorenylacetamide in rat liver.

The influences of different polychlorinated biphenyl (PCB) isomers and congeners on distinct hepatotoxic responses to the hepatocarcinogen N-2-fluorenylacetamide [(2-FAA) CAS: 53-96-3] were examined in F344 rats. Cytocidal toxicity of 2-FAA (25-400 microM), determined by lactate dehydrogenase release during 20 hours in primary monolayer cultures of isolated rat hepatocytes, was reduced by in vivo pretreatment with either phenobarbitone [(PB) CAS: 50-06-6] or 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP), a PB-type PCB inducer. However, cytocidal toxicity of 2-FAA was substantially potentiated by either 3-methylcholanthrene [(MCA) CAS: 56-49-5] or 3,3',4,4'-tetrachlorobiphenyl [(TCBP) CAS: 32598-13-3], an MCA-type PCB. In the same cell culture assays, all four pretreatments similarly reduced cytocidal toxicity of N-hydroxy-N-2-fluorenylacetamide (0.32-32 microM; CAS: 53-95-2). By comparison, pretreatments with either the PB-type or MCA-type PCB's (50-200 mumol/kg) diminished mitoinhibitory toxicity of 2-FAA in vivo, as measured by hepatic regenerative growth and hepatocyte labeling indices 7 days after partial hepatectomy (PH) in rats given 3 consecutive daily doses of 2-FAA (20/mg/kg/day) before PH. This regimen of 2-FAA and PH promoted rapid selective growth of gamma-glutamyltranspeptidase-positive (gamma-GT+) nodules at 2 and 4 weeks after PH in rats previously given an initiating hepatocarcinogen, diethylnitrosamine [(DENA) CAS: 55-18-5]. However, various PCB's, including 2,2',4,4',5,5'-HCBP, 3,3',4,4'-TCBP, 2,2',4,4'-TCBP, 2,2',5,5'-TCBP, and the commercial mixture Aroclor 1254, each given as a single dose of 50 mumol/kg by gavage 10 days after DENA and 7 days before 2-FAA, all reduced the size of 2-FAA-selected gamma-GT+ nodules during the 4-week period after PH. These results indicate that, in spite of predictable inducer-specific opposite influences of different types of PCB's on cytocidal toxicity of 2-FAA, all PCB's similarly reduce nodule selection by 2-FAA in initiated livers. Reduced growth of 2-FAA-selected nodules correlated with the consistent ability of all PCB's to enhance regeneration of liver mass after 2-FAA and PH.

Role of estrogen receptor (ER) alpha in insulin-like growth factor (IGF)-I-induced responses in MCF-7 breast cancer cells.

Insulin-like growth factor-I (IGF-I) is a mitogenic polypeptide that induces proliferation of MCF-7 breast cancer cells, and cotreatment with the phosphoinositide 3-kinase (PI3-K) inhibitor LY294002 and the antiestrogen ICI 182780 inhibits IGF-I-induced growth. The role of estrogen receptor alpha (ERalpha) in mediating responses induced by IGF-I was investigated in cells transfected with small inhibitory RNA for ERalpha (iERalpha). The results showed that IGF-I-dependent phosphorylation of Akt and mitogen-activated protein kinase, induction of G(1)-S-phase progression and enhanced expression of cyclin D1 and cyclin E were dependent on ERalpha. Moreover, these same IGF-I-induced responses were also inhibited by the antiestrogen ICI 182780 and this was in contrast to a previous report suggesting that ICI 182780 did not affect IGF-I-dependent activation of PI3-K or induction of cyclin D1 expression. ICI 182780 exhibits antimitogenic activity and iERalpha inhibits G(1)-S-phase progression and proliferation of MCF-7 cells treated with IGF-I, suggesting that the effects of the antiestrogen are primarily related to downregulation of ERalpha.

Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds.

The aryl hydrocarbon (Ah) receptor binds several different structural classes of chemicals, including halogenated aromatics, typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polynuclear aromatic and heteropolynuclear aromatic hydrocarbons. TCDD induces expression of several genes including CYP1A1, and molecular biology studies show that the Ah receptor acts as a nuclear ligand-induced transcription factor that interacts with xenobiotic or dioxin responsive elements located in 5'-flanking regions of responsive genes. TCDD also elicits diverse toxic effects, modulates endocrine pathways and inhibits a broad spectrum of estrogen (17 beta-estradiol)-induced responses in rodents and human breast cancer cell lines. Molecular biology studies show that TCDD inhibited 17 beta-estradiol-induced cathepsin D gene expression by targeted interaction of the nuclear Ah receptor with imperfect dioxin responsive elements strategically located within the estrogen receptor-Sp1 enhancer sequence of this gene.

Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta.

The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ER alpha or ER beta protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ER alpha or ER beta complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ER alpha and ER beta protein revealed a single binding component for [3H]-17beta-estradiol (E2) with high affinity [dissociation constant (Kd) = 0.05 - 0.1 nM]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ER beta than to ER alpha. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and 2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity of ER alpha and ER beta at concentrations of 100-1000 nM. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 >> zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ER alpha and E2 >> genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ER beta. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ER alpha and a mixed agonist-antagonist for ER beta. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ER beta, and they may trigger many of the biological responses that are evoked by the physiological estrogens.

Interferon-tau activates multiple signal transducer and activator of transcription proteins and has complex effects on interferon-responsive gene transcription in ovine endometrial epithelial cells.

Interferon-tau (IFNtau), a type I IFN produced by sheep conceptus trophectoderm, is the signal for maternal recognition of pregnancy. Although it is clear that IFNtau suppresses transcription of the estrogen receptor alpha and oxytocin receptor genes and induces expression of various IFN-stimulated genes within the endometrial epithelium, little is known of the signal transduction pathway activated by the hormone. This study determined the effects of IFNtau on signal transducer and activator of transcription (STAT) activation, expression, DNA binding, and transcriptional activation using an ovine endometrial epithelial cell line. IFNtau induced persistent tyrosine phosphorylation and nuclear translocation of STAT1 and -2 (10 min to 48 h), but transient phosphorylation and nuclear translocation of STAT3, -5a/b, and -6 (10 to <60 min). IFNtau increased expression of STAT1 and -2, but not STAT3, -5a/b, and -6. IFN-stimulated gene factor-3 and STAT1 homodimers formed and bound an IFN-stimulated response element (ISRE) and gamma-activated sequence (GAS) element, respectively. IFNtau increased transcription of GAS-driven promoters at 3 h, but suppressed their activity at 24 h. In contrast, the activity of an ISRE-driven promoter was increased at 3 and 24 h. These results indicate that IFNtau activates multiple STATs and has differential effects on ISRE- and GAS-driven gene transcription.

Potent inhibition of estrogen sulfotransferase by hydroxylated PCB metabolites: a novel pathway explaining the estrogenic activity of PCBs.

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants which exert a variety of toxic effects in animals, including disturbances of sexual development and reproductive function. The estrogenic effects of PCBs may be mediated in part by hydroxylated PCB metabolites (PCB-OHs), but the mechanisms by which they are brought about are not understood. PCBs as well as PCB-Hs show low affinities for both alpha and beta estrogen receptor isoforms. In the present study we demonstrate that various environmentally relevant PCB-OHs are extremely potent inhibitors of human estrogen sulfotransferase, strongly suggesting that they indirectly induce estrogenic activity by increasing estradiol bioavailability in target tissues.

Transcriptional activation of cathepsin D gene expression by growth factors.

Insulin-like growth factor-I (IGF-I), transforming growth factor alpha (TGFalpha) and epidermal growth factor (EGF) induced cathepsin D gene expression and reporter gene activity in MCF-7 human breast cancer cells transiently transfected with a construct (pCD1) containing a -2576 to -124 cathepsin D gene promoter insert. In contrast, IGF-I, but not TGFalpha or EGF, induced reporter gene activity in cells cotransfected with wild-type estrogen receptor (ER) expression plasmid and a construct (pCD2) containing estrogen-responsive downstream elements from -208 to -101. Promoter deletion and mutational analysis experiments identified four GC-rich sites and an imperfect palindromic estrogen responsive element required for IGF-I activation of the ER (ligand-independent). Subsequent studies with the mitogen-activated protein kinase (MAPK) inhibitor, PD98059, and a serine(118(-ER mutant confirmed the role of the MAPK pathway for IGF-I activation of the ER in MCF-7 cells. Thus, growth factor activation of ER can mediate transactivation vs ER/Sp1 binding to GC-rich sites and represents a novel pathway for ligand-independent ER action. The divergent pathways for IGF-I and TGFalpha/EGF activation of the ER observed in MCF-7 cells contrast with previous data indicating that pathways for growth factor activation of the ER are dependent on the gene and/or gene promoter and on cell context.

DNA damage induced by wood preserving waste extracts in vitro without metabolic activation, as assayed by 32P-postlabeling.

Aqueous wood preserving waste (WPW) extracts were tested for their ability to damage DNA in vitro without metabolic activation. Two extracts were prepared from a surface tar and a surface clay soil sample of a WPW site. As assayed by 32P-post-labelling incubation of DNA with these extracts gave rise to highly complex, extract-specific profiles of DNA adducts whose formation depended on the concentration of WPW material. Most of the adducts appeared to be derived from polycyclic aromatic hydrocarbons (PAHs). Three mg organic WPW residue gave rise to total adduct levels of 13.8 (extract 1) and 66.2 (extract 2) DNA modifications in 10(7) DNA nucleotides, corresponding to 13.9 and 26.9 modifications, respectively, per 10 mg of soil. Thus, extract 2 was more active, although the parent residue had a 1.4-times lower PAH content as determined by gas chromatography/mass spectrometry (GC/MS). DNA adduct formation presumably was a consequence of (i) free radical reactions, possibly involving semiquinones and oxygen free radicals, and (ii) reaction of direct-acting electrophiles, derived from metabolism of WPW toxicants by soil microorganisms. These reactions appeared to be more active in sample 2. The results suggest that ground water at WPW sites contains DNA-reactive compounds posing a cancer hazard to humans. The in vitro DNA adduct assay represents a novel tool to readily assess this type of hazard and the possible effects of remediation measures.

Inhibition of carcinogen-induced rat mammary tumor growth and other estrogen-dependent responses by symmetrical dihalo-substituted analogs of diindolylmethane.

90%) by the haloDIMs at concentrations of 5 or 10 microM, and only 4, 4'-dichloroDIM alone increased cell proliferation. With the exception of 5,5'-difluoroDIM, the remaining compounds also inhibited E2-induced growth of MCF-7 human breast cancer cells. DihaloDIMs (100 mg/kg/dayx3) were not estrogenic in the immature female B6C3F1 mouse uterus; however, in animals co-treated with E2 (0.02 microg/mouse), 5,5'-dichloro- and 6,6'-dichloroDIM inhibited uterine progesterone receptor (PR) binding and uterine peroxidase activity, whereas 5,5'-dichloro- and 5,5'-dichloro-2,2'-dimethylDIM inhibited only the latter response. The antitumorigenic activities of the dihaloDIMs were determined by their inhibition of carcinogen-induced mammary tumor growth in female Sprague-Dawley rats. 4,4'-Dichloro-, 5,5'-dibromo- and 6,6'-dichloroDIM, significantly inhibited mammary tumor growth at doses of 1 mg/kg every second day, and no significant changes in organ weights or liver and kidney histopathology were observed. These three compounds were more active than DIM in the same in vivo assay.

Induction of rat hepatic microsomal cytochrome P-450 by 2,3',4,4',5,5'-hexachlorobiphenyl.

The following evidence suggests that 2,3',4,4',5,5'-hexachlorobiphenyl resembles isosafrole as an inducer of hepatic microsomal cytochrome P-450d in the immature male Wistar rat. First, the major hepatic microsomal polypeptide (Mr = 52,000), intensified after treatment of rats with 2,3',4,4',5,5'-hexachlorobiphenyl, comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with cytochrome P-450d (i.e. the major isosafrole-inducible polypeptide) but had an electrophoretic mobility intermediate between cytochrome P-450b (Mr approximately equal to 51,500) and cytochrome P-450c (Mr = 56,000) (i.e. the major phenobarbital- and 3-methylcholanthrene-inducible polypeptides respectively). Second, when pairs of various xenobiotics were coadministered to rats at doses effecting maximal induction of hepatic microsomal cytochrome P-450, the inductive effects of 2,3',4,4',5,5'-hexachlorobiphenyl were additive with those of phenobarbital, 3-methylcholanthrene and pregnenolone-16 alpha-carbonitrile but not with those of isosafrole. The inductive effects of phenobarbital, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile and isosafrole were all expressed additively with each other. Third, in contrast to phenobarbital and pregnenolone-16 alpha-carbonitrile treatment, treatment of rats with 2,3',4,4',5,5'-hexachlorobiphenyl, isosafrole or 3-methylcholanthrene failed to increase markedly the proportion of total cytochrome P-450 capable of forming a 446 nm-absorbing complex with metyrapone. Fourth, the in vitro metabolism of isosafrole, catalyzed by hepatic microsomes from rats treated with 2,3',4,4',5,5'-hexachlorobiphenyl, isosafrole or 3-methylcholanthrene, produced complexes between ferrous cytochrome P-450 and a methylenedioxyphenyl metabolite, the spectra of which were between 400 and 500 nm and were similar to each other but which were readily distinguishable from the spectra of the product adducts formed during the metabolism of isosafrole by hepatic microsomes from rats treated with corn oil (control), phenobarbital, or pregnenolone-16 alpha-carbonitrile.

Inhibition of CYP1A1-dependent activity by the polynuclear aromatic hydrocarbon (PAH) fluoranthene.

Polynuclear aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, and recently bioassay-based induction studies have been used to determine exposures to complex mixtures of PAHs. Induction of CYP1A1-dependent activity in H4IIE rat hepatoma cells has been used extensively as a bioassay for halogenated aromatic hydrocarbons and more recently for PAHs. Fluoranthene (FL) is a prevalent PAH contaminant in diverse environmental samples, and FL did not induce CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity significantly in H4IIE cells. However, in cells cotreated with 2 x 10(-5) M FL plus the potent inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[k]fluoranthene (BkF) (2 x 10(-8) M), there was a significant decrease in EROD activities. Furthermore, treatment of TCDD-induced rat microsomes with FL caused an 80% decrease in EROD activity. Studies showed that FL did not affect induction of CYP1A1 protein or mRNA levels in H4IIE cells, and analysis of enzyme inhibition data using microsomal CYP1A1 indicated that FL noncompetitively inhibited CYP1A1-dependent activity. 32P-Postlabeling revealed no significant FL-DNA adduct formation in H4IIE cells treated with FL. However, in cells cotreated with FL plus BkF or benzo[a]pyrene (BaP), certain PAH-DNA adducts were induced 2-fold. This study demonstrated that FL is an inhibitor of CYP1A1-dependent enzyme activity in rat hepatoma H4IIE cells and that the genotoxic potency of some carcinogenic PAHs may be modulated by FL in mixtures containing relatively high levels of this compound.

Atypical cytochrome P450 induction profiles in glomerular mesangial cells at the mRNA and enzyme level. Evidence for CYP1A1 and CYP1B1 expression and their involvement in benzo[a]pyrene metabolism.

Recent studies in this laboratory have shown that benzo[a]pyrene (BaP) modulates growth factor-related gene expression and proliferation of renal glomerular mesangial cells (GMCs) in vitro. Because many of the toxic and biochemical effects of this polycyclic aromatic hydrocarbon are mediated through oxidative metabolism, the present studies were conducted to examine the patterns of cytochrome P450IA1 (CYP1A1) and P4501B1 (CYP1B1) inducibility in mesangial cells and the molecular consequences of this response. Exposure of cultured GMCs to BaP (30 microM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 10 nM) for 24 hr induced CYP1A1 mRNA levels, a response abolished by cotreatment with 10 microM cycloheximide. The pattern of hydrocarbon inducibility was atypical in that BaP was a more effective inducer of CYP1A1 gene expression than TCDD, and both hydrocarbons induced aryl hydrocarbon hydroxylase (AHH) activity, but not ethoxyresorufin-O-deethylase activity. Cotreatment with alpha-naphthoflavone (alpha NF, 1 microM) or ellipticine (ELLIP, 0.1 nM) only partially inhibited the induction of AHH activity by BaP (30 microM). BaP and TCDD also induced expression of the CYP1B1 protein and the pattern of induction was comparable to that observed for CYP1A1. Treatment of GMCs with 30 microM BaP was associated with the formation of eight DNA adducts, and their occurrence could be inhibited by pretreatment with alpha NF (1 microM), but not ELLIP (0.1 nM). These results demonstrate that CYP1A1 and CYP1B1-related activities are induced in GMCs by BaP and TCDD and this induction is associated with metabolism of BaP to reactive intermediates that bind covalently to DNA.

Is there an association between exposure to environmental estrogens and breast cancer?

It was initially reported that levels of polychlorinated biphenyls (PCBs) or p,p'-DDE were elevated in breast cancer patients (serum or tissue) versus controls. These results, coupled with reports that selected environmental estrogens decreased 17beta-estradiol (E2) 2-hydroxylase activity and increased the ratio of 16alpha-hydroxyestrone/2-hydroxyestrone metabolites in MCF-7 human breast cancer cells, have led to the hypothesis that xenoestrogens are a preventable cause of breast cancer. More recent studies and analysis of organochlorine levels in breast cancer patients versus controls show that these contaminants are not elevated in the latter group. Moreover, occupational exposure to relatively high levels of PCBs and DDT/DDE are not associated with an increased incidence of breast cancer. A reexamination of the radiometric E2 2-hydroxylase assay in MCF-7 cells with diverse estrogens, antiestrogens, and carcinogens showed that the mammary carcinogen benzo[a]pyrene induced this response and the antiestrogen ICI 164,384 decreased E2 2-hydroxylase activity. Thus, E2 2-hydroxylase activity and the 16alpha-hydroxyestrone/2-hydroxyestrone metabolite ratio in MCF-7 cells does not predict xenoestrogens or mammary carcinogens.

Endocrine disruptors and human health--is there a problem? An update.

It has been hypothesized that environmental exposure to synthetic estrogenic chemicals and related endocrine-active compounds may be responsible for a global decrease in sperm counts, decreased male reproductive capacity, and breast cancer in women. Results of recent studies show that there are large demographic variations in sperm counts within countries or regions, and analyses of North American data show that sperm counts have not decreased over the last 60 years. Analyses of records for hypospadias and cryptorchidism also show demographic differences in these disorders before 1985; however, since 1985 rates of hypospadias have not changed and cryptorchidism has actually declined. Temporal changes in sex ratios and fertility are minimal, whereas testicular cancer is increasing in most countries; however, in Scandinavia, the difference between high (Denmark) and low (Finland) incidence areas are not well understood and are unlikely to be correlated with differences in exposure to synthetic industrial chemicals. Results from studies on organochlorine contaminants (DDE/PCB) show that levels were not significantly different in breast cancer patients versus controls. Thus, many of the male and female reproductive tract problems linked to the endocrine-disruptor hypothesis have not increased and are not correlated with synthetic industrial contaminants. This does not exclude an endocrine-etiology for some adverse environmental effects or human problems associated with high exposures to some chemicals.

Environmental and dietary estrogens and human health: is there a problem?

It has been hypothesized that organochlorine pesticides and other environmental and dietary estrogens may be associated with the increased incidence of breast cancer in women and decreased sperm concentrations and reproductive problems in men. However, elevation of organochlorine compounds such as dichlorodipehenyldichloroethylene (DDE) and polychlorinated biphenyls (PCBs) in breast cancer patients is not consistently observed. Reanalysis of the data showing that male sperm counts decreased by over 40% during 1940 to 1990 indicated that inadequate statistical methods were used and that the data did not support a significant decline in sperm count. Humans are exposed to both natural and industrial chemicals which exhibit estrogenic and antiestrogenic activities. For example, bioflavonoids, which are widely distributed in foods, and several industrial compounds, including organochlorine pesticides and various phenolic chemicals, exhibit estrogenic activity. Humans are also exposed to chemicals which inhibit estrogen-induced responses such as the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin and related chlorinated aromatics, polynuclear aromatic hydrocarbon combustion products, and indole-3-carbinol, which is found in cruciferous vegetables. Many of the weak estrogenic compounds, including bioflavonoids, are also antiestrogenic at some concentrations. A mass balance of dietary levels of industrial and natural estrogens, coupled with their estimated estrogenic potencies, indicates that the dietary contribution of estrogenic industrial compounds is 0.0000025% of the daily intake of estrogenic flavonoids in the diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Hazard and risk assessment of chemical mixtures using the toxic equivalency factor approach.

There is considerable public, regulatory, and scientific concern regarding human exposure to endocrine-disrupting chemicals, which include compounds that directly modulate steroid hormone receptor pathways (estrogens, antiestrogens, androgens, antiandrogens) and aryl hydrocarbon receptor (AhR) agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Based on quantitative structure-activity relationships for both AhR and estrogen receptor (ER) agonists, the relative potency (RP) of individual compounds relative to a standard (e.g. TCDD and 17-beta-estradiol) have been determined for several receptor-mediated responses. Therefore, the TCDD or estrogenic equivalent (TEQ or EQ, respectively) of a mixture is defined as TEQ = sigma[T(i)]xRP(i)or EQ=sigma[E(i)]xRP(i), where T(i) and E(i) are concentrations of individual AhR or ER agonists in any mixture. This approach for risk assessment of endocrine-disrupting mixtures assumes that for each endocrine response pathway, the effects of individual compounds are essentially additive. This paper will critically examine the utility of the TEQ/EQ approach for risk assessment, the validity of the assumptions used for this approach, and the problems associated with comparing low dose exposures to xeno and natural (dietary) endocrine disruptors.