Ventricular preexcitation sensitive to flecainide in late stage chick embryo ECGs: 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs inotropic but not chronotropic or dromotropic responses to isoproterenol and confers resistance to flecainide.

ECGs free of movement artefacts were obtained without anesthesia in 16- to 18-day-old chick embryos close to hatching and used to study the effect of the environmental toxin 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) on cardiac rhythm and conduction. The ECGs of normal late stage chick embryos exhibited short PR intervals, frequent nonisoelectric PR segments, delta waves, and inverted T waves. Those ECG characteristics are found in patients with the Wolff-Parkinson-White syndrome (WPW) in which they reflect ventricular preexcitation associated with the use of accessory conduction pathways and arrhythmias. Isoproterenol (30 microg/egg) did not alter the ECG preexcitation characteristics. Flecainide, a sodium channel blocker used clinically to suppress WPW accessory pathway activity, at 0.5 to 5 mg per egg diminished the preexcitation and caused atrioventricular (AV) block, supporting the use of accessory pathways together with AV-nodal conduction in normal late stage chick embryos. The findings challenge the dogma that accessory pathways are entirely replaced by AV conduction pathways in late fetal development. TCDD, at 1-2 nmol per egg for 48 h, did not affect heart rate, the increase in heart rate by isoproterenol, or the ECG characteristics, suggesting that short-term TCDD treatment did not affect sinus node function or cardiac conduction. The latter results taken together with prior findings indicate that TCDD differentially impairs the inotropic and lusitropic effects but not the chronotropic or dromotropic effects of isoproterenol. In TCDD-treated embryos, flecainide, tested at 5 mg per egg, caused much less inhibition of preexcitation or production of AV block than in the untreated or solvent-treated controls. The resistance to flecainide represents a new TCDD effect consistent with the reported increase of cardiac myocyte [Ca(2+)](i) by TCDD treatment.

TCDD induces CYP1A4 and CYP1A5 in chick liver and kidney and only CYP1A4, an enzyme lacking arachidonic acid epoxygenase activity, in myocardium and vascular endothelium.

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other Ah receptor ligands, species differences in sensitivity and the relationship of CYP1A induction to the toxicity, are poorly understood. Ah receptor ligands induce formation of CYP1A1 and 1A2 in mammals and of a different set of enzymes, CYP1A4 and 1A5, in chicks. We examined induction by TCDD of CYP1A4 and 1A5 mRNA and protein in chick embryo liver, heart, kidney, lung, intestine, bursa, spleen, thymus, brain, and muscle by in situ hybridization and immunohistochemistry and verified the histochemical findings by CYP-specific assays, 7-ethoxyresorufin deethylase for CYP1A4 and arachidonic acid epoxygenation for CYP1A5. CYP1A4 alone was extensively induced in the cardiovascular system, in cardiac myocytes, in perivascular cells having the same location as impulse-conducting Purkinje cells, and like CYP1A1, in vascular endothelium in every organ examined. Unlike mammalian CYP1A, CYP1A4 and 1A5 were both substantially induced in kidney proximal tubules as well as liver, and neither enzyme was induced in kidney glomeruli or lung or brain parenchymal cells. The findings demonstrate (a) a route for CYP1A4 to affect cardiac function, (b) that vascular endothelium is a major site of CYP1A induction across species, and (c) that CYP1A induced in heart or endothelial cells cannot affect cardiac or vascular function via generation of arachidonic acid epoxides because the CYP1A enzymes induced in those organs are not arachidonic acid epoxygenases. Further, the specificity of CYP1A induction sites and of the catalytically active enzymes induced at each site support a significant role for CYP1A induction in Ah receptor ligand toxicity and species differences in sensitivity.

Olfaction and symptoms in the multiple chemical sensitivities syndrome.

Whereas most idiosyncratic environmental sensitivity complaints do not fit known diagnoses, the multiple chemical sensitivities syndrome (MCS) is an extreme presentation that has defined diagnostic criteria. MCS symptomatics claim that they acquired a sensitized state as the result of a chemical exposure, usually to a solvent or pesticide, but not to a fragrance. Before this exposure, they did not experience symptoms. Following sensitization, symptoms increasing in number and severity with time are attributed by the MCS symptomatic to various exposures that are innocuous to most individuals. Although phenomenological studies have provided no evidence that particular odors elicit MCS symptoms, low levels of fragrances and perfumes are frequently associated with the reporting of MCS symptoms. This evaluation examines proposed mechanisms by which odorants and fragrances might cause either sensitization or elicitation of MCS symptoms, including altered odor sensitivity, primary irritancy or irritancy-induced upper airway reactivity, neurogenic switching of trigeminal irritancy signals, time-dependent sensitization and limbic kindling, CNS toxicity, and various psychiatric conditions. In no case was there persuasive evidence that any olfactory mechanism involving fragrance underlies either induction of a sensitized state or the triggering of MCS symptoms. Fragrances and other odorants could, however, be associated with symptoms as claimed by MCS symptomatics, because they are recognizable stimuli, but fragrance has not been demonstrated to be causal in the usual sense.

Transcriptional activation of avian CYP1A4 and CYP1A5 by 2,3,7, 8-tetrachlorodibenzo-p-dioxin: differences in gene expression and regulation compared to mammalian CYP1A1 and CYP1A2.

The toxicity, carcinogenicity, and biochemical effects of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) are dependent upon activation of the Ah receptor, a ligand-activated transcription factor. Ah receptor activation leads to the induction of cytochrome P450 (CYP) 1A enzymes, which include CYP1A1 and 1A2 in mammals and CYP1A4 and 1A5 in chickens. CYP1A induction is a major effect of TCDD exposure although its relationship to TCDD toxicity and carcinogenicity are not understood. In these studies we investigated by nuclear run-on transcription assays along with Northern and Western blotting in chick embryo liver, kidney, and heart whether avian CYP1A4 and 1A5, like mammalian CYP1A1 and 1A2, are transcriptionally induced by TCDD and whether the chick CYP1A enzymes exhibit differences analogous to mammalian CYP1A enzymes in organ expression. We report that CYP1A4 and 1A5, like CYP1A1 and 1A2, are transcriptionally induced by TCDD in liver. However, whereas CYP1A1 is not constitutively expressed in liver, CYP1A2 and both CYP1A4 and 1A5 are constitutively expressed. Further, whereas TCDD induces only CYP1A1 and not CYP1A2 in extrahepatic organs, TCDD induces both CYP1A4 and 1A5 in chick kidney. Also, TCDD induced CYP1A4 but not 1A5 in both myocardium and heart vessels whereas CYP1A1 induction has only been found in endocardium. Further, liver CYP1A4 and 1A5 mRNAs had the same half lives and were both superinduced by cycloheximide, whereas mRNA half lives differ for CYP1A1 and 1A2, and cycloheximide superinduces only CYP1A1. We suggest that there are species differences in the effects of TCDD on CYP1A gene expression, organ distribution, and regulation that are likely to be accompanied by differences in CYP1A function and that this diversity may contribute to the large differences in sensitivity to TCDD among species.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces hepatic cytochrome P450-dependent arachidonic acid epoxygenation in diverse avian orders: regioisomer selectivity and immunochemical comparison of the TCDD-induced P450s to CYP1A4 and 1A5.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) markedly induced cytochrome P450 (CYP)-dependent arachidonic acid metabolism in liver microsomes from hatchlings of four avian species belonging to four different orders: chick, pigeon, cormorant, and great blue heron, increasing formation of arachidonic acid epoxides (EETs), monohydroxyeicosatetraenoic acids (HETEs), omega-1, and omega-2 OH arachidonic acid products by fivefold or more. Microsomes from TCDD-induced hatchling chicks had the highest activity and the least restricted EET regioselectivity. omega-OH arachidonic acid, the principal constitutive metabolite in chick and pigeon liver microsomes and a major product for cormorant and great blue heron was not induced by TCDD. Constitutive EET formation in avian liver microsomes was very low except in cormorant microsomes where 8,9-EET was generated almost exclusively. Western blots of liver microsomes using polyclonal antisera to chick embryo-derived CYP1A4 and 1A5 recognized two TCDD-induced bands in each of the species. The chick bands had the same molecular weights as CYP1A4 and 1A5 (55 and 55.5 kDa, respectively) but those of the other species differed. Immunopurified antiserum monospecific for CYP1A5 recognized a band in microsomes from all of the avian species, and monospecific antiserum for CYP1A4 recognized a band in microsomes from chick, pigeon, and great blue heron. AntiCYP1A4 and 1A5 IgG immunoinhibited TCDD-induced mixed function oxidase activity completely in chick and chick embryo microsomes and only partially in the other avian microsomes. The results demonstrate that (1) TCDD causes much greater induction of CYP-dependent arachidonic acid metabolism, and of arachidonic acid epoxygenation in particular, in avian than in mammalian species; (2) TCDD induces two CYP1A-related enzymes in birds as in mammals; (3) CYP1A enzymes in the birds other than chicks are not identical to CYP1A4 and 1A5 but share some enzymatic and immunochemical characteristics with them; (4) constitutive omega-OH arachidonic acid in all of the avian species and 8,9-EET in cormorant are formed by CYP enzymes unrelated to CYP1A; and (5) two distinct characteristics of avian CYP1A enzymes are the acquisition by avian CYP1A4-related P450 of unique epitope(s) and by CYP1A5-related P450 of unusual catalytic effectiveness for arachidonic acid epoxygenation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induction of cytochrome P450-dependent arachidonic acid metabolism in mouse liver microsomes: evidence for species-specific differences in responses.

Arachidonic acid is biotransformed to metabolites active in signal transduction by cytochrome P450 (CYP) as well as by cyclooxygenase and lipoxygenase enzymes. Inducers of CYP1 enzymes, including 2,3,7,8-tetrachlorodibenzo-p-dioxin and other Ah receptor ligands, markedly increase liver microsomal CYP-dependent arachidonic acid epoxygenation in chicks but depress epoxygenation in rat liver microsomes where they elicit about twofold increases in formation of other CYP products, omega-1 to omega-4-OH arachidonic acid. These studies examined the effect of TCDD on metabolism of [1-14C]-labeled arachidonic acid by mouse liver microsomes. Mouse liver microsomes metabolized arachidonic acid exclusively by a CYP-dependent mechanism as evidenced by lack of metabolism in the absence of NADPH and by formation of specific CYP-dependent metabolites. The major constitutive products were epoxygenase products (EETs and EET-diols) and omega-OH arachidonic acid. Treatment with TCDD increased formation of omega-2- to omega-4-OH arachidonic acid products 23-fold, formation of omega-1-OH arachidonic acid about 5-fold, and formation of epoxygenase products and HETEs each about twofold. In contrast, TCDD treatment decreased formation of omega-OH arachidonic acid by over 70%. EET-diols comprised a greater fraction of total epoxygenase products in mouse liver microsomes than has been found for liver microsomes of other species. The high EET-diol formation was attributable to a non-TCDD-inducible, EET epoxide hydrolase activity in mouse liver microsomes. For comparison, the effect of TCDD on [1-14C]-labeled arachidonic acid was examined in homogenates of spleen, an immune system target of TCDD. While levels of total [1-14C]-arachidonic acid metabolism were comparable in both tissues, virtually all of the metabolism by spleen was CYP-independent, and it was unaffected by TCDD. Western blotting experiments showed that TCDD-induced mouse Cyp1a1 and 1a2 share immunologic epitopes with chick CYP1A4 and 1A5. However, in immunoinhibition studies, an antibody to CYP1A5, the chick arachidonate epoxygenase, was ineffective against TCDD-induced arachidonic acid metabolism in mouse liver microsomes, suggesting that there are differences in the catalytic sites or tertiary structures of CYP1A5 and the CYP-enzyme catalyzing the TCDD-induced arachidonic acid metabolism in mouse liver. This study shows that the effects of TCDD of the profile of CYP-dependent arachidonic acid metabolities and the amounts produced in mouse liver microsomes differ from other species. The findings suggest that species differences in CYP1A catalytic activities including the metabolism of arachidonic acid may contribute to species differences in sensitivity to TCDD toxicity.

Identification of CYP1A5 as the CYP1A enzyme mainly responsible for uroporphyrinogen oxidation induced by AH receptor ligands in chicken liver and kidney.

Uroporphyrinogen is an intermediate of the heme biosynthetic pathway. The oxidation of uroporphyrinogen to uroporphyrin (UROX) has been demonstrated to be catalyzed by mammalian CYP1A2. This reaction has an important role in uroporphyria caused by halogenated aromatic compounds. Two CYP enzymes induced by Ah receptor ligands were purified recently from chick embryo liver. One, designated CYP1A5, was preferentially active in arachidonic acid epoxygenation and the other, designated CYP1A4, in 7-ethoxyresorufin deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH), reactions mainly catalyzed by CYP1A1 in rodents. The amino acid sequences of both CYP1A5 and CYP1A4 are more similar to CYP1A1 than to 1A2, and neither can be classified as an ortholog of mammalian CYP1A1 or 1A2. Here we report that reconstituted purified CYP1A5 was eight times more active than CYP1A4 in catalyzing UROX. The stimulation of UROX by 3,4,3',4'-tetrachlorobiphenyl that has been observed in microsomes was also observed with the reconstituted enzymes. Similar dose response relationships were found for induction of UROX and EROD in both chick embryo liver microsomes and in cultured chick hepatocytes, indicating coinduction of CYP1A5 and CYP1A4. UROX was induced by the Ah receptor ligand, 3-methylcholanthrene, in chicken kidney as well as liver. The findings reported here and other evidence that CYP1A4 and CYP1A5 tend to exhibit CYP1A1 and 1A2-like enzyme activites, respectively, indicate that the division of some enzyme activities among CYP1A enzymes applies to different vertebrate classes.

Molecular cloning and expression of two novel avian cytochrome P450 1A enzymes induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Transcriptional regulation by the aryl hydrocarbon receptor, for which the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent ligand, leads in mammalian liver to the induction of genes for two distinct cytochrome P450 (CYP)1A enzymes, CYP1A1 and -1A2. Fish seem to have only one CYP1A enzyme. CYP1A enzymes have been regarded as injurious largely because of their ability to activate chemical carcinogens. We report here the cloning and sequencing of cDNAs for two catalytically distinct TCDD-induced CYP enzymes in chick embryo liver. One mediates classic CYP1A1 activities. The other has some -1A2-like activities and is also responsible for TCDD-induced arachidonic acid epoxygenation, a much more conspicuous effect in liver of chicks than of mammalian species. Amino acid sequence analysis shows that although each chick enzyme can be classified in the CYP1A family, both are more like CYP1A1 than -1A2, and neither can be said to be directly orthologous to CYP1A1 or -1A2. Phylogenetic analysis shows that the two chick enzymes form a separate branch in the CYP1A family tree distinct from mammalian CYP1A1 and -1A2 and from fish CYP1A enzymes. The findings suggest that CYP1A progenitors split into two CYP enzymes with some parallel functions independently in two evolutionary lines, evidence for convergent evolution in the CYP1A family. Northern analysis shows that the chick enzymes have a different tissue distribution from CYP1A1 and -1A2. Polymerase chain reaction and in situ hybridization data show that both chick enzymes are expressed in response to TCDD even before organ morphogenesis. The findings further suggest that beyond their role in activating carcinogens, CYP1A enzymes have conferred evolutionary and developmental advantages, perhaps as defenses in maintaining homeostatic responses to toxic chemicals.

Arachidonic acid metabolism by human cytochrome P450s 2C8, 2C9, 2E1, and 1A2: regioselective oxygenation and evidence for a role for CYP2C enzymes in arachidonic acid epoxygenation in human liver microsomes.

The membrane-bound endogenous fatty acid arachidonic acid can be released from membranes by phospholipases and then metabolized to biologically active compounds by cyclooxygenases, lipoxygenases, and cytochrome P450 (CYP) enzymes. In the liver the CYP pathway is the most significant. Liver CYP arachidonate products include epoxyeicosatrienoic acids (EETs) and monohydroxylated products (HETEs). We examined metabolism of [1-14C]arachidonic acid by a panel of 10 human CYP enzymes expressed in HepG2 cells. In the absence of expressed CYP enzymes, control HepG2 cell microsomes generated only small amounts of omega- and omega--1-OH arachidonic acid (ratio 2:1). Microsomes from HepG2 cells expressing CYP2C8, 2C9, 1A2, and 2E1 were 7-21 times more active than microsomes from the HepG2 controls. CYP2C8, 2C9, and 1A2 principally generated epoxygenase products; 36 to 48% were in the form of EET-diols, reflecting host HepG2 microsomal epoxide hydrolase activity. CYP2C8 and 2C9 formed more 14,15- and 11,12-EET than did CYP1A2, while CYP1A2 formed more 8,9-EET. CYP2C9 also generated a peak with the retention time of 12-HETE. CYP2E1 generated omega--1-OH arachidonic acid and, to a lesser extent, omega-OH arachidonic acid (ratio 2:1). A small amount of epoxygenase activity was also detected for CYP2B6; its overall activity, however, was only about twice control levels. Activities of CYP2A6, 3A3, 3A4, and 3A5 were low and limited to the omega-/omega--1-OH arachidonic acid peak; CYP2D6 was inactive. Microsomes prepared from three individual human livers varied threefold in total arachidonic acid metabolism. For all three livers omega-OH arachidonic acid was the major product (up to 74% of total metabolites). Epoxygenase products constituted 14 to 28% of the total products; 60 to 83% of those were EET-diols, indicating that the human liver microsomes have substantial EET-epoxide hydrolase activity. 11,12-EET was the major EET for two livers and 14,15-EET for the third. The CYP2C inhibitor sulfaphenazole depressed human liver microsomal epoxygenase activity by 50% at 50 microM, while alpha-naphthoflavone inhibited arachidonic acid epoxygenase activity by 27% at 2 microM and by 32% at 10 microM. Collectively, these findings suggest that human liver microsomal arachidonic acid metabolism is catalyzed principally by CYP2C enzymes. CYP1A2, CYP2E1, and possibly CYP2B6 are likely to play more minor roles, though their contribution may be enhanced by exposure to inducers of those enzymes. CYP2A6, CYP2D6, and CYP3A enzymes are unlikely to make any significant contribution.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of hepatocytes as the major locus of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1-related P450s, TCDDAA and TCDDAHH, in chick embryo liver.

It was recently shown that the pleiotropic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in chick embryo liver includes the induction of cytochrome P450-mediated arachidonic acid epoxygenation, as well as 7-ethoxyresorufin deethylation (EROD) and aryl hydrocarbon hydroxylation (AHH). The TCDD-induced arachidonic acid metabolism in avian liver microsomes is catalyzed by a 55 kDa P450, TCDDAA, whereas the TCDD-induced AHH and EROD are catalyzed by a different 54.5 kDa P450, TCDDAHH. In this study, we investigated the distribution and inducibility of TCDDAA and TCDDAHH in hepatocytes and nonparenchymal cells. Sonicates of freshly isolated hepatocytes from embryos treated with solvent alone (control) metabolized [14C]arachidonic acid principally to a single metabolite, omega-OH arachidonic acid. Treatment with TCDD increased total arachidonic acid metabolism 2.9-fold and epoxygenase products [epoxyeicosatrienoic acids (EETs) and EET-diols] 36-fold. After treatment, EETs and EET-diols constituted 59% of the total metabolites. EROD in hepatocyte sonicates was increased 32-fold by TCDD treatment. The same pattern of arachidonate metabolites and degree of increase in arachidonate metabolism and EROD by TCDD treatment was observed in the hepatocyte sonicates and liver microsomes. TCDD treatment increased arachidonic acid metabolism and EROD activity 3.6- and 50-fold, respectively, in the nonparenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of tamoxifen-4-hydroxylation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (beta NF), and phenobarbital (PB) in avian liver: identification of P450 TCDDAA as catalyst of 4-hydroxylation induced by TCDD and beta NF.

Tamoxifen has been found to be metabolized by liver primarily into three metabolites, tamoxifen-N-oxide, formed by the flavin-containing monooxygenase, and N-desmethyl- and 4-hydroxytamoxifen, formed by cytochrome P450. The N-demethylation was demonstrated to be catalyzed by P4503A in rat and human liver; however, the P450s catalyzing the 4-hydroxylation have not been identified. Although 4-hydroxytamoxifen exhibits more potent estrogen agonist/antagonist activity than tamoxifen, the relative contributions of the parent drug and its 4-hydroxy metabolite(s) to the activity of tamoxifen in vivo have not been established. We report here that the rate of tamoxifen 4-hydroxylation is higher in livers of adult chicken and chick embryos than in livers of mammalian species. Tamoxifen 4-hydroxylation was increased by treatment of chick embryos with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (beta NF), and to a lesser extent by phenobarbital (PB). The major effect of PB treatment was an increase in tamoxifen N-demethylation. Tamoxifen 4-hydroxylase activity of reconstituted purified chicken P450s was highest for TCDDAA, a P450 active in arachidonate epoxygenation and estradiol 2-hydroxylation, and one of the two major P450s induced by TCDD and beta NF in chick embryo liver. The second P450, TCDDAHH, which is active in aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase was inactive in tamoxifen 4-hydroxylation. Anti-TCDDAA IgG immunoinhibited tamoxifen 4-hydroxylation in microsomes from beta NF-treated embryos by over 80%, but was ineffective against this reaction in the controls. The immunochemical findings together with the reconstitution data identify TCDDAA as the P450 responsible for TCDD/beta NF-induced tamoxifen 4-hydroxylation in chick liver. In PB-treated livers, a P450 fraction containing CYP2H1/H2, the major PB-induced P450s, had the highest tamoxifen 4-hydroxylase and N-demethylase activities, a finding compatible with one or both of those P450s being responsible for the PB-induced tamoxifen 4-hydroxylation and N-demethylation. The findings reported here raise the possibility that exposure of women undergoing tamoxifen therapy to agents that induce human CYP1A2 or CYPB1/2 analogues may produce increased levels of 4-hydroxytamoxifen and that this may affect the therapeutic potency of tamoxifen.

Purification and biochemical characterization of two major cytochrome P-450 isoforms induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in chick embryo liver.

Two cytochrome P-450 isoforms induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in chick embryo liver microsomes were purified. The P-450s exhibit catalytic selectivity either for arachidonic acid metabolism, particularly epoxygenation (P-450 TCDDAA, 55 kDa), or for aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin deethylase (7-EROD) (P-450 TCDDAHH, 54.5 kDa). Turnover numbers for arachidonic acid epoxygenation, AHH, and 7-EROD, respectively, were 24.2, 0.23, and 0.45 for TCDDAA and 0.57, 9.7, and 35.5 for TCDDAHH. Both P-450s were low spin, with carbon monoxide-binding peaks at 448 nm. Their N-terminal amino acid sequences showed 80% homology and contained the sequence: PXXXSATEXL, common to CYP1A P-450s but not others. Polyclonal antibodies to TCDDAA and TCDDAHH cross-reacted with both P-450s on Western blots and immunoinhibited all TCDD-induced liver microsomal arachidonic acid metabolism, AHH, and 7-EROD. Immunoquantitation using antibodies made monospecific by immunoadsorption against the heterologous P-450 showed that TCDDAA and TCDDAHH were coinduced in liver in equal amounts and accounted for all of the TCDD-induced P-450. Enzyme assays and Western blots also showed expression of both TCDDAA and TCDDAHH in kidney but only of TCDDAHH in heart, indicating that these P-450s can be independently regulated and selectively induced. On Western blots non-immunopurified anti-TCDDAA and anti-TCDDAHH antisera recognized rat and guinea pig CYP1A1 and CYP1A2. Immunopurified TCDDAA antiserum only recognized rat CYP1A2, indicating that TCDDAA is more closely related immunochemically to CYP1A2 than to CYP1A1. The evidence that pleotropic responses to Ah receptor ligands can include induction of a P-450 that can form biologically active products from the endogenous membrane lipid, arachidonic acid, suggests that organ- and cell-specific expression of TCDD-induced P-450s could affect responses to TCDD.

Asbestos in the air of public buildings: a public health risk?

The Environmental Health and Safety Council of the American Health Foundation has examined current estimates of cancer risks associated with the presence of asbestos-containing materials (ACM) in public buildings. The Council finds that even complete removal of asbestos from all of these buildings will provide no measurable benefit to public health. The removal of nonfriable ACM only can be postulated to protect the public against a small hypothetical risk that cannot be measured epidemiologically. Moreover, examination of the assumptions used in the risk assessment calculations leads to the conclusion that these small calculated risks are likely to represent overestimates. In recent surveys, the measured asbestos levels in indoor air cast some doubt on whether occupant exposure to asbestos levels are contributed to significantly by ACM even when some of the material is friable or in bad condition. Furthermore, the models used for cancer risk estimates assume no threshold level for cancer and conclude that any exposure is carcinogenic. This may be unjustified in light of information on the mechanisms for some asbestos-caused disease. Based on the best available data, it is very unlikely that cancer will result from indoor asbestos exposure, especially where ACM is well maintained.

2,3,7,8-tetrachlorodibenzo-p-dioxin increases cardiac myocyte intracellular calcium and progressively impairs ventricular contractile responses to isoproterenol and to calcium in chick embryo hearts.

Binding by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the Ah receptor leads to transcriptional activation of several genes and a toxicity syndrome that includes tumor promotion, wasting, hormonal and immune system dysfunction, and death. Recent findings indicate that TCDD may also affect cardiac function. Here, we used the chick embryo, a TCDD-sensitive species, to further characterize the effects of TCDD on ventricular muscle contraction and on cardiac myocyte [Ca2+]i assessed with fura 2. The results show that TCDD causes an evolving sequence of contractile defects, independent of changes in diet, first impairing cAMP-modulated contraction (after 48 hr) and later (by seven days) decreasing responses to [Ca2+]o. Phenobarbital, even at high doses, failed to affect the inotropic response to isoproterenol, supporting the specificity of the ventricular contractile effects of TCDD. TCDD treatment also depressed inotropic responses to theophylline and forskolin, indicating that it has a post-beta-adrenergic receptor effect on cAMP action. In contrast to its depression of responses to beta-adrenergic stimuli and to [Ca2+]o, TCDD did not affect initial tensions of ventricular muscle stimulated at 1 Hz or the force-frequency response up to 1 Hz, indicating that TCDD-treated ventricles can respond normally at slow rates of stimulation. TCDD treatment depressed lusitropic (relaxation) responses to isoproterenol and to increasing [Ca2+]o indicating that it impairs the ability of the sarcoplasmic reticulum to sequester Ca2+. Fura 2-based measurements showed that [Ca2+]i was nearly doubled after TCDD treatment. The increase in [Ca2+]i is consistent with the decrease in the contractile response to [Ca2+]o, amelioration of the response to isoproterenol by subphysiologic concentrations of [Ca2+]o, and intermittent lack of response to electrical stimulation in high K+ observed in ventricles from TCDD-treated embryos. TCDD treatment also depressed the initial increase in [Ca2+]i by isoproterenol, consistent with the decreased contractile response to isoproterenol. The findings show that TCDD causes well defined, progressive impairment of avian ventricular responses to inotropic stimuli, providing new evidence that the heart is a target of TCDD action and that TCDD disturbs intracellular calcium processing.

Nitric oxide is a mediator of the decrease in cytochrome P450-dependent metabolism caused by immunostimulants.

Bacterial lipopolysaccharide (LPS) and a diverse array of other immunostimulants and cytokines suppress the metabolism of endogenous and exogenous substances by reducing activity of the hepatic cytochrome P450 mixed-function oxidase system. Although this effect of immunostimulants was first described almost 40 yr ago, the mechanism is obscure. Immunostimulants are now known to cause NO overproduction by cells via induction of nitric oxide synthase. We have investigated whether NO overproduction is involved in suppressing hepatic metabolism by LPS. In vitro treatment of hepatic microsomes with NO, produced by chemical decomposition of 3-morpholinosydnonimine or by nitric oxide synthase, substantially suppressed cytochrome P450-dependent oxygenation reactions. This effect of NO was seen with hepatic microsomes prepared from two species (rat and chicken) and after exposure to chemicals that induce distinct molecular isoforms of cytochromes P450 (beta-naphthoflavone, 3-methylcholanthrene, and phenobarbital). Spectral studies indicate that NO reacts in vitro with both Fe(2+)- and Fe(3+)-hemes in microsomal cytochromes P450. In vivo, LPS diminished the phenobarbital-induced dealkylation of 7-pentoxyresorufin by rat liver microsomes and reduced the apparent P450 content as measured by CO binding. These LPS effects were associated with induction of NO synthesis; LPS-induced NO synthesis showed a strong positive correlation with the severity of cytochrome P450 inhibition. The decrease in both hepatic microsomal P450 activity and CO binding caused by LPS was largely prevented by the selective NO synthase inhibitor N omega-nitro-L-arginine methyl ester. Our findings implicate NO over-production as a major factor mediating the suppression of hepatic metabolism by immunostimulants such as LPS.

Immunochemical identity of the 2,3,7,8-tetrachlorodibenzo-p-dioxin- and beta-naphthoflavone-induced cytochrome P-450 arachidonic acid epoxygenases in chick embryo liver: distinction from the omega-hydroxylase and the phenobarbital-induced epoxygenase.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (beta NF), and phenobarbital (PB) cause marked induction of cytochrome P-450 (P-450)-mediated arachidonic acid metabolism in chick embryo liver. We show here that the P-450 arachidonic acid epoxygenases induced by TCDD and beta NF are immunochemically indistinguishable from each other and unrelated to the arachidonic acid epoxygenase induced by PB. On Western blots, IgG from an antiserum against beta NFAA, a 55-kDa P-450 arachidonic acid epoxygenase purified from beta NF-treated chick embryo liver, immunoreacted selectively and to the same extent with a 55-kDa band in liver microsomes from chick embryos treated with TCDD or beta NF. It failed to react with proteins from untreated, solvent-treated, or PB-treated embryos on immunoblots or to immunoinhibit PB-induced arachidonic acid metabolism. Anti-beta NFAA IgG immunoinhibited all arachidonic acid metabolism by reconstituted beta NFAA and formation of arachidonic epoxides (EETs) and monohydroxylated derivatives (HETEs) by microsomes from TCDD- and beta NF-treated livers; it did not inhibit omega-hydroxylation. In contrast, IgG from an antiserum against the major PB-induced chicken P-450s, 2H1 and 2H2, immunoreacted with two major PB-induced P-450s, of 48 and 49 kDa, on Western blots. It also immunoinhibited formation of EETs and HETEs by PB-treated microsomes entirely and omega-hydroxylation by 50%. It failed to react with TCDD- or beta NF-induced P-450s on Western blots or to immunoinhibit TCDD- or beta NF-induced arachidonic acid metabolism. Because other P-450s with which anti-beta NFAA and anti-PB IgG cross-reacted were inactive in arachidonic acid epoxygenation, the findings are consistent with beta NFAA being principally responsible for the epoxygenation induced by TCDD and beta NF and 2H1 and/or 2H2 being responsible for epoxygenation induced by PB. Further, the P-450 arachidonate omega-hydroxylase and the epoxygenase in livers of TCDD- or beta NF-treated embryos are immunochemically unrelated, whereas those in livers of PB-treated embryos may be partly related.

Beta-naphthoflavone induction of a cytochrome P-450 arachidonic acid epoxygenase in chick embryo liver distinct from the aryl hydrocarbon hydroxylase and from phenobarbital-induced arachidonate epoxygenase.

Cytochrome P-450-mediated arachidonic acid metabolism in chick embryo liver microsomes was increased by both Ah receptor-dependent (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphthoflavone) and independent (phenobarbital) P-450 inducers. Arachidonic acid epoxides and monohydroxyeicosatetraenoic acids were increased 9-12-fold. omega-1-OH arachidonic acid was also significantly increased by TCDD and beta-naphthoflavone while omega-OH arachidonic acid, the main metabolite in uninduced livers, was decreased by all three agents. The P-450s catalyzing the enhanced arachidonate metabolism in beta-naphthoflavone- and phenobarbital-treated liver were investigated in reconstituted systems containing wholly or partially purified P-450s. beta-Naphthoflavone induced formation of a 55-kDa P-450 selective for arachidonate metabolism and for epoxygenation in particular. This P-450 was purified (beta NFAA). It was found to be distinct from a 54.5-kDa beta-naphthoflavone-induced P-450 catalyzing aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase (designated NF1). Mean turnover numbers for arachidonate epoxygenase, aryl hydrocarbon hydroxylase, and 7-ethoxyresorufin deethylase were 11.2, 0.56, and 0.04, respectively, for reconstituted beta NFAA and 0.33, 11.8, and 2.4 for NF1. beta NFAA and NF1 also differed in chromatography elution characteristics and N-terminal amino acid sequences. Both were low spin, with carbon monoxide binding peaks at 448 nm. The phenobarbital-induced arachidonate epoxygenation was catalyzed by P-450 fractions containing the main 48- and 49-kDa phenobarbital-induced P-450s; fractions in which the 49-kDa P-450 predominated were the most active. Turnover numbers for arachidonic acid epoxygenation were not correlated with those for aminopyrine demethylation or 7-ethoxycoumarin deethylation for P-450s from phenobarbital-treated livers or with aryl hydrocarbon hydroxylase, 7-ethoxyresorufin deethylase, or 7-ethoxycoumarin deethylase for P-450s from beta-naphthoflavone-treated livers. Also, different P-450s catalyzed the epoxygenation and the omega-hydroxylation of arachidonic acid in both beta-naphthoflavone- and phenobarbital-treated livers. The findings support a physiologic role for P-450-induced arachidonate metabolism and provide a basis for a possible link between TCDD's induction of P-450 and alterations of cellular homeostasis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin increases reliance on fats as a fuel source independently of diet: evidence that diminished carbohydrate supply contributes to dioxin lethality.

The environmental toxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a wasting syndrome marked by hypophagia, loss of body fat, changes in intermediary metabolism and death. Use of conventional laboratory animals has not resolved whether or not TCDD affects intermediary metabolism independently of hypophagia. We used the chick embryo, which does not require an exogenous food supply for energy, to answer this question. Our results show that TCDD treatment increases dependence on fats as a fuel source independently of changes in food intake and therefore can affect intermediary metabolism independently of hypophagia. Results of experiments using aminocarnitine to inhibit fatty acid oxidation suggest that TCDD treatment impairs carbohydrate production rather than its utilization and that the former effect contributes to TCDD lethality.

Arachidonic acid metabolism by dioxin-induced cytochrome P-450: a new hypothesis on the role of P-450 in dioxin toxicity.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) is a highly potent inducer of cytochrome P-450. The role of the induced P-450 in TCDD toxicity has been obscure as P-450 neither detoxifies TCDD nor activates it to genotoxic or cytotoxic metabolites. We show, using a chick embryo model, that TCDD causes major increases in the NADPH dependent metabolism of arachidonic acid (AA), a predominant cell membrane fatty acid, that it does so with extremely high potency (ED50, 6.3 pmol per egg) and that this metabolism is catalyzed by TCDD-induced cytochrome P-450 species. Thus, TCDD treatment increased by six to ten fold the P-450 mediated hepatic microsomal metabolism of AA to epoxides and monohydroxyeicosatetraenoic acids, products whose diverse biological activities suggest links to TCDD's toxic effects. In contrast only x and x-1 hydroxy AA, inactive products, were significantly formed by the controls. These findings open a new perspective on how P-450 induction could be related to the diverse toxic effects of TCDD. They lead to the novel hypothesis that TCDD-induced cytochrome P-450 metabolizes an endogenous fatty acid to reactive products that in turn mediate or modulate varied manifestations of TCDD toxicity.

NAD(P)H: quinone oxidoreductase (DT-diaphorase) in chick embryo liver. Comparison to activity in rat and guinea pig liver and differences in co-induction with 7-ethoxyresorufin deethylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

NAD(P)H:quinone oxidoreductase (EC 1.6.99.2; DT-diaphorase) was present in the liver of 18- and 19-day-old chick embryos as assayed both by reduction of resorufin and by the more traditional assay, reduction of 2,6-dichlorophenolindophenol (DCPIP). Both reductions had the classic characteristics of DT-diaphorase: they were equally supported by NADPH and NADH and almost entirely inhibited by dicumarol. Chick embryo liver DT-diaphorase was entirely cytosolic. It was undetectable in the microsomal and mitochondrial fractions. Chick embryo liver cytosol and mitochondrial fractions contained an enzyme oxidizer of resorufin but not of DCPIP. The Km for NADPH for resorufin reductase was an order of magnitude higher in chick embryo than in rat or guinea pig cytosol (1 mM vs 0.1 mM). Resorufin reductase activity was higher for chick embryo than for rat or guinea pig cytosols: Vmax (nmol resorufin reduced per mg cytosolic protein per min +/- SEM) 355 +/- 28 for chick embryo, 159 +/- 10 for guinea pig and 68 +/- 28 for rat. The Vmax for DCPIP reduction was also twice as high in chick embryo as rat liver cytosol. In the chick embryo, 7 days after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at 6.4 micrograms/kg egg (1 nmol/egg) mortality was increased 2.4-fold, hepatic DT-diaphorase 1.3-fold, and 7-ethoxyresorufin deethylase (7-EROD) 72-fold over control levels. At 32 micrograms/kg, mortality was increased 4.2-fold, DT-diaphorase 2.3-fold and 7-EROD 100-fold. In the guinea pig, 5 days after treatment with TCDD at 10 micrograms/kg, TCDD toxicity was also evident (loss of body weight and thymus weight); there was no change in DT-diaphorase as measured by resorufin reduction, confirming by a different assay the observation of Beatty and Neal (Biochem Pharmacol 27: 505-510, 1978) that TCDD does not induce DT-diaphorase in guinea pig liver, and 7-EROD was increased 8-fold. In contrast, in the rat, 7 days after exposure to TCDD at 10 micrograms/kg, there was no evidence of toxicity, DT-diaphorase was increased close to 7-fold and 7-EROD, 100-fold. The results demonstrate that avian liver contains DT-diaphorase and show that the extent to which DT-diaphorase is part of the pleiotypic response of the liver to an Ah (aryl hydrocarbon) receptor ligand is species dependent. They also suggest that DT-diaphorase induction and TCDD toxicity may be inversely related. The possibility that DT-diaphorase protects against TCDD toxicity and participates in species differences in sensitivity to TCDD toxicity warrants further investigation.