Activation of the Ah receptor by tryptophan and tryptophan metabolites.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of a variety of hydrophobic natural and synthetic chemicals, including the environmental contaminant 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). A variety of indole-containing chemicals, such as indole-3-carbinol, indolo[3, 2-b]carbazole, and UV photoproducts of tryptophan (TRP), have previously been identified as ligands for AhR. Here we have examined the ability of endogenous metabolites of tryptophan (TRP) to bind to and activate AhR in vitro and in cells in culture. Although hydroxylated TRP metabolites were inactive, two metabolites, namely tryptamine (TA) and indole acetic acid (IAA), were shown to be AhR agonists. Not only do TA and IAA bind competitively to AhR, but they also can stimulate AhR transformation and DNA binding and induce expression of an AhR-dependent reporter gene in cells. In addition to being an AhR ligand, TA is also a competitive substrate for cytochrome P4501A1, a well-characterized AhR- and TCDD-inducible gene product. Although these compounds are relatively weak ligands, compared to TCDD, they represent some of the first endogenous hydrophilic AhR agonists identified to date.

Brevetoxin-6 (PbTx-6), a nonaromatic marine neurotoxin, is a ligand of the aryl hydrocarbon receptor.

Brevetoxins (PbTx) are a family of marine polyether toxins that exert their toxic action by activating voltage-sensitive sodium channels. Two forms of brevetoxin, PbTx-2 and -3, induce hepatic cytochrome P4501A1, measured as ethoxyresorufin O-deethylase (EROD) activity, in redfish and striped bass. P4501A1 induction is transcriptionally regulated through the binding of a ligand, typically a planar aromatic compound, to the aryl hydrocarbon receptor (AhR) and subsequent complex formation with the dioxin response element (DRE), an upstream regulatory region of the CYP1A1 gene. To determine if PbTx, a nonaromatic compound, induced EROD by this mechanism, two sets of experiments were performed. Initially, saturation binding assays with PbTx-2, -3, and -6 were carried out to determine if PbTx-2, -3, or -6 was an AhR ligand. Results showed that PbTx-6 inhibited specific binding of dioxin to the AhR, whereas PbTx-2 and -3 had no effect. Subsequently, gel retardation assays showed that PbTx-6 caused a concentration-dependent increase in AhR-DRE complex formation. The most abundant and neurotoxic forms of brevetoxin, PbTx-2 and -3, did not appear to be involved in this process. However, PbTx-6, the epoxide which is a likely biotransformation product, is at least one of the forms of PbTx involved in EROD induction.

Ah-receptor-dependent modulation of gene expression by aged and diluted sidestream cigarette smoke.

Cigarette smoke is known to induce cytochrome P4501A1 expression and activity in a variety of species. Although the elevation of this isozyme is assumed to be associated with the activation of the CYP1A1 gene through a ligand-mediated mechanism involving the Ah-receptor (AhR), this has not been determined. In this study we have examined the mechanism by which an ambient level of aged and diluted sidestream cigarette smoke (ADSS) induces cytochrome P4501A1. Effects of ADSS on C57BL/6N and DBA/ 2N mice were examined. Induction of P4501A1-associated ethoxyresorufin-O-dealkylase (EROD) activity was observed in the lungs of C57BL/6N mice, while there was no induction in DBA/ 2N mice. ADSS also induced EROD in wild-type mouse hepatoma (Hepa1c1c7) cells (hepa1), but not in variant hepa1 cells defective in the AhR nuclear translocator (ARNT) protein. ADSS exposure of recombinant hepa1 cells, stably transfected with a reporter plasmid containing the luciferase gene under control of several dioxin responsive enhancers (DREs), resulted in a time- and exposure-dependent induction of luciferase activity. ADSS-mediated induction of luciferase activity was inhibited by alpha-naphthoflavone (alpha NF), an Ah-receptor antagonist. Gel retardation analysis demonstrated that exposure to ADSS induced transformation and DNA binding of the AhR complex. In summary, our results not only indicate a role for the AhR in mediating the induction of P4501A1 by ADSS, but also demonstrate that environmentally relevant levels of ADSS must contain AhR ligands at sufficient concentrations to activate gene expression in an AhR-dependent manner.

Identification of a novel cis-acting negative regulatory element affecting expression of the CYP1A1 gene in rat epidermal cells.

Polycyclic aromatic hydrocarbons such as 3-methylcholanthrene are toxic to rat epidermal cells in low passages (3 to 6), but cultures of high passage (>/=15) are resistant. Since such compounds can be metabolically activated by cytochrome P4501A1, we have examined the regulation of this gene in low and high passage cells. Consistent with this difference, little or no 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible P4501A1 mRNA or enzyme activity was observed in high passage as compared to low passage cultures. Similarly, transfection of a luciferase reporter construct containing -1317 to +256 base pairs of the 5'-flanking region of the murine CYP1A1 gene was TCDD-inducible in low but not high passage cells. Ligand binding and transfection experiments demonstrated the presence of functional Ah receptor complexes in both high and low passage cells. Deletion analysis identified a 26-base pair negative regulatory DNA (NeRD) element contained within the upstream regulatory region of the CYP1A1 gene responsible for this effect. Nuclear extracts from both low and high passage cells contain a protein which specifically binds to NeRD-containing DNA. Thus, the loss of polycyclic aromatic hydrocarbon sensitivity in high passage rat epidermal cells appears to be due to decreased expression of CYP1A1, and this effect may be mediated by an altered NeRD binding factor(s) present in these cells.

Dioxinlike properties of a trichloroethylene combustion-generated aerosol.

Conventional chemical analyses of incineration by-products identify compounds of known toxicity but often fail to indicate the presence of other chemicals that may pose health risks. In a previous report, extracts from soot aerosols formed during incomplete combustion of trichloroethylene (TCE) and pyrolysis of plastics exhibited a dioxinlike response when subjected to a keratinocyte assay. To verify this dioxinlike effect, the complete extract, its polar and nonpolar fractions, some containing primarily halogenated aromatic hydrocarbons, were evaluated for toxicity using an embryo assay, for antiestrogenicity using primary liver cell cultures, and for the ability to transform the aryl hydrocarbon receptor into its DNA binding form using liver cytosol in a gel retardation assay. Each of these assays detect dioxinlike effects. Medaka (Oryzias latipes) embryos and primary liver cell cultures of rainbow trout (Oncorhynchus mykiss) were exposed to concentrations of extract ranging from 0.05 to 45 micrograms/l. Cardiotoxicity with pericardial, yolk sac, and adjacent peritoneal edema occurred after exposure of embryos to concentrations of 7 micrograms/l or greater. These same exposure levels were associated with abnormal embryo development and, at the higher concentrations, death. Some of the fractions were toxic but none was as toxic as the whole extract. In liver cells, total cellular protein and cellular lactate dehydrogenase activity were not altered by in vitro exposure to whole extract (0.05-25 micrograms/l). However, induction of cytochrome P4501A1 protein and ethoxyresorufin O-deethylase activity occurred. In the presence of whole extract, estradiol-dependent vitellogenin synthesis was reduced. Of the fractions, only fraction 1 (nonpolar) showed a similar trend, although vitellogenin synthesis inhibition was not significant. The soot extract and fractions bound to the Ah receptor and showed a significantly positive result in the gel retardation/DNA binding test. Chemical analyses using GC-MS with detection limits for 2,3,7,8-tetrachlorodibenzo-p-dioxin and dibenzofuran in the picomole range did not show presence of these compounds. Our results indicate that other chemicals associated with TCE combustion and not originally targeted for analysis may also pose health risks through dioxinlike mechanisms.

Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals.

Exposure to specific polychlorinated diaromatic hydrocarbons (PCDH), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), produces a wide variety of species- and tissue-specific toxic and biological effects. Many of these responses are mediated by the Ah receptor (AhR) and are modulated by the interaction of the PCDH:AhR complex with its DNA recognition sequence (the dioxin-responsive element (DRE)). We have constructed a recombinant expression plasmid which contains the luciferase gene under TCDD-inducible control of several DREs and responds to TCDD-like chemicals with the induction of firefly luciferase. Stable transfection of this vector into various cell lines has produced a series of species-specific cell bioassay systems that respond to TCDD-like chemicals with the induction of luciferase in a time-, dose-, and AhR-dependent manner. In addition, these cell lines have been used to demonstrate that 2,2',5,5'-tetrachlorobiphenyl can act as a species-specific AhR antagonist. Overall, these recombinant cell lines can be used for the detection and relative quantitation of AhR agonists/antagonists in complex mixtures of environmental and biological samples, for identification and characterization of novel AhR agonists, and for examination of species differences in PCDH responsiveness.

Species-specific antagonism of Ah receptor action by 2,2',5,5'-tetrachloro- and 2,2',3,3'4,4'-hexachlorobiphenyl.

Using recombinant cell lines showing Ah receptor-controlled expression of a luciferase reporter gene, the interaction of di-ortho-substitute polychlorinated biphenyls (PCBs) with Ah receptor agonists was studied. In the recombinant Hepa1c1c7 mouse hepatoma (H1L1.1c7) cells strong antagonistic interaction of 2,2',5,5'-tetrachlorobiphenyl (PCB52) with luciferase expression induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3,3',4,4'-tetrachlorobiphenyl (PCB77) was observed, and similarly, between 2,2',3,3',4,4'-hexachlorobiphenyl (PCB128) and PCB77. Accordingly, PCB52 was found to inhibit ethoxyresorufin-O-deethylase (EROD) induction by PCB77 in wild-type Hepa1c1c7 cells. In contrast, the antagonistic effect of PCB52 on TCDD-induced luciferase expression was only minor in recombinant guinea pig GPC16 colon adenocarcinoma (G16L1.1c8) and human HepG2 hepatoma (HG2L1.1c3) cells, and intermediate in recombinant H4IIE rat hepatoma (H4L1.1c4) cells. Gel retardation studies using a 32 P-labelled dioxin responsive element (DRE)-containing oligonucleotide, and ligand binding studies using [3H]TCDD, demonstrated that the species-specific antagonistic activity of PCB52 on Ah receptor-controlled luciferase expression is due to inhibition of Ah receptor ligand and DNA binding. We conclude, that Ah-mediated luciferase expression provides a useful tool to study the species specificity of Ah receptor (ant)agonists.

DNA binding of the transformed guinea pig hepatic Ah receptor complex: identification and partial characterization of two high-affinity DNA-binding forms.

We have examined and characterized the binding of transformed guinea pig hepatic Ah receptor to its specific DNA recognition site, the dioxin-responsive element (DRE), using gel retardation analysis. Saturation binding analysis of transformed TCDD:AhR complexes were indicative of a single high-affinity binding site (Kd = 2.5 +/- 0.8 nM); however, DNA-binding analysis revealed the presence of two distinct TCDD-inducible protein-DRE complexes. Sucrose gradient centrifugation and subsequent gel retardation analysis of the fractions demonstrated a similarity in the distribution of [3H]TCDD-specific binding and TCDD-inducible protein-DNA complex formation, supporting the presence of the AhR in both complexes. In addition, the formation of both DNA-binding complexes exhibited the same nucleotide specificity previously determined for the AhR complex. Since labeling studies using a radio-iodinated photoaffinity dioxin agonist demonstrated that guinea pig cytosol contains a single ligand binding subunit of 105 kDa, the difference in migration of the complexes is due to other proteins associated with each complex. Overall, our results demonstrate the presence of two distinct high affinity DNA-binding forms of transformed guinea pig AhR complex which exhibit similar DNA-binding affinity and nucleotide specificity.

Protein kinase C is not involved in Ah receptor transformation and DNA binding.

Induction of cytochrome P4501A1 by 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) is mediated by the Ah receptor (AhR) complex, a ligand-dependent DNA-binding transactivator. Recently a role for protein kinase C (PKC) in the induction response has been reported in which PKC or a related kinase positively modulates AhR activity. We have examined the role of PKC by determining the effect of two nonspecific PKC inhibitors, H7 and staurosporine, and one specific PKC inhibitor, calphostin c, on AhR functionality. Although no kinase activity was detectable in cytosol, under the conditions used for our assays, AhR transformation and DNA binding still occurred. Addition of relatively high concentrations of the kinase inhibitors also had no significant effect on TCDD:AhR:DRE complex formation. Thus, our results indicate that protein kinase activity does not appear to be necessary for TCDD-dependent AhR transformation and DNA binding and they imply that protein kinases must play a role in another step(s) in the AhR-dependent mechanism of P4501A1 induction.

Binding of transformed Ah receptor complex to a dioxin responsive transcriptional enhancer: evidence for two distinct heteromeric DNA-binding forms.

Guinea pig hepatic Ah receptor (AhR) complex was transformed in vitro to its DNA-binding form by incubation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Transformed TCDD-AhR was covalently cross-linked by UV-irradiation to a bromodeoxyuridine-substituted oligonucleotide containing its specific DNA recognition site, the dioxin responsive element (DRE). Denaturing gel electrophoresis and autoradiography identified four TCDD-inducible protein-DNA complexes, with molecular masses of approximately 97, 105, and 115 kDa and a somewhat broader complex at 247 kDa. The 247-kDa complex appears to contain two distinct protein-DNA complexes of approximately 232 and 256 kDa and represents two proteins covalently cross-linked to a single DRE oligonucleotide, while the 97, 105, and 115-kDa complexes represent single protein-DRE cross-links. UV cross-linking to DRE oligonucleotides containing variable numbers of BrdU residues revealed that the 105-kDa protein, identified as the AhR ligand-binding subunit by photoaffinity labeling with a radioiodinated AhR agonist, cross-links to the DRE core consensus (5'-GCGTG-3'); the 97- and 115-kDa non-ligand-binding proteins differentially cross-link immediately 5'-ward of the core. Overall, our results not only demonstrate that the critical protein-DNA contacts which occur between the AhR complex and the DRE are made primarily by the ligand-binding subunit but also indicate that the AhR complex exists as two distinct heteromeric DNA-binding forms, containing one 105-kDa ligand-binding subunit and either one 115- or one 97-kDa non-ligand-binding subunit.

TCDD causes stimulation of c-ras expression in the hepatic plasma membranes in vivo and in vitro.

A series of in vivo and in vitro experiments were conducted to determine the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) administered on the expression of c-ras. Differences in c-ras expression between control and TCDD treated groups were determined by immunoassay of p21ras protein, or indirectly measured by the specific binding of 3H-GTP to hepatic plasma membrane preparations. Intraperitoneal injection of sublethal doses of TCDD significantly elevated (P less than 0.05, Student t test) levels of hepatic p21ras protein in Sprague-Dawley rats and TCDD sensitive C57BL/6J mice. Such an increase occurred at an early stage of poisoning in the C57BL/6J mice. The earliest increase was detectable 6 hr after dosing, and the difference became statistically significant by 12 and 24 hr after dosing. In contrast, TCDD tolerant DBA/2J mice had only a marginal increase in hepatic p21ras protein which did not become statistically significant even at 24 hr host-dosing. TCDD evoked increases in hepatic p21ras protein of C57BL/6J mice were accompanied by the increase in the specific binding of GTP to hepatic plasma membranes. Column chromatography of solubilized rat hepatic membrane proteins on sephadex G-50 showed TCDD administration increased levels of a 3H-GTP binding protein with MW of approximately 21 Kd. 3H-GTP binding in total hepatic membranes was also elevated (P less than 0.05, Fisher PLSD multiple comparison test) 6 hr and 24 hr after dosing of C57BL/6J mice, but as expected the effect of TCDD was not as conspicuous as that found in the plasma membrane. TCDD treatment increased levels of a 21 Kd protein found in the in vitro translation products of RNA purified from guinea pig liver. This protein was identified as a c-ras protein based upon its ability to bind GTP, precipitation by a polyclonal antibody against the rasHa and Ki proteins and subsequent SDS-PAGE which showed a single protein band of approximately 21 Kd.

The relationship between labor and route of delivery in the preterm infant.

The purpose of this prospective study was to evaluate the effects of the active phase of labor and route of delivery on the frequency of germinal layer/intraventricular hemorrhage in 89 infants with ultrasound-estimated fetal weights less than or equal to 1750 gm. Twenty-eight infants (31.5%) had germinal layer/intraventricular hemorrhage within 1 hour after birth and an additional 15 infants (17%) had germinal layer/intraventricular hemorrhage beyond 1 hour after birth. Infants with germinal layer/intraventricular hemorrhage had a significantly lower gestational age (p less than 0.003) and birth weight (p less than 0.007). Germinal layer/intraventricular hemorrhage within 1 hour after delivery was increased in the infants of women who experienced the active phase of labor regardless of the route of delivery. However, the incidence of germinal layer/intraventricular hemorrhage beyond 1 hour after delivery and the overall incidence were similar in the vaginal delivery and cesarean delivery groups. In addition, there was an increased incidence of progression to grades III and IV hemorrhage regardless of route of delivery in the infants whose mothers experienced the active phase of labor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes increases in expression of c-erb-A and levels of protein-tyrosine kinases in selected tissues of responsive mouse strains.

2,3,7,8,-Tetrachlorodibenzo-p-dioxin (TCDD) administered in vivo causes drastic reduction in the weight of the mouse thymus at low doses (e.g., 30 micrograms/kg single i.p. injection), the reduction becoming statistically significant after 2 days. To understand the cause for such thymic involution TCDD-evoked changes in various biochemical parameters in this tissue were examined. The most noticeable change was observed in the increased activity of specific protein-tyrosine kinases and protein kinase C and an increased level of p21ras-associated binding of [3H]GTP. Since no significant change was observed with cAMP-stimulated protein kinases and cAMP levels, the above changes appear to be a selective effect on these special classes of proteins. As a result of a time sequence study it has become apparent that the rise in protein-tyrosine kinase activities becomes significant within 24 hr, whereas the rise in protein kinase C does not become significant until 48 hr. Among protein-tyrosine kinases, pp60c-src and probably pp561skT were found to be significantly elevated by TCDD treatment. In view of similarities between TCDD and thyroid hormones in causing thymic involution, the levels of c-erb-A expression were assessed in the liver by using avian 32P-labeled v-erb-A probe and RNA transfer blot hybridization technique. The results clearly indicate that TCDD has the property to elevate levels of mRNA bearing homology to v-erb-A. Such changes in c-erb-A expression and protein-tyrosine kinase occurred only in TCDD-susceptible (responsive) strains but not in tolerant (nonresponsive) strains of mice at the dose tested. Based on such observations a hypothesis has been proposed that TCDD owes its potency to its ability to stimulate the expression of one of a family of DNAs bearing homology to v-erb-A and that one of the major consequences of such an action is stimulation of various tyrosine kinases.