The environmental toxin TCDD (2,3,7,8-tetrachlorodibenzop-dioxin, dioxin) produces diverse toxic effects including a lethal wasting syndrome whose hallmark is suppressed hepatic gluconeogenesis. All TCDD toxicities require activation of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor. Whereas the mechanism for AHR induction of target genes is well understood, it is not known how AHR activation produces any TCDD toxicity. This report identifies for the first time an AHR target gene, TiPARP (TCDD-inducible poly(ADPribose) polymerase, PARP7) that can mediate a TCDD toxicity, i.e. suppression of hepatic gluconeogenesis. TCDD suppressed hepatic glucose production, expression of key gluconeogenic genes, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase), and NAD ؉ levels, and increased PARP activity and

Photooxidized tryptophan (TRP) in tissue culture medium elicits a transient cytochrome P450 (CYP1) induction response in cultured cells. We show here that exposure of TRP to window sunlight (aTRP) greatly increased the potency, efficacy, and duration of CYP1A induction by TRP in primary chick embryo hepatocytes and in vivo. Aqueous TRP exposed to sunlight for 7 days exhibited a 100-fold or greater increase in potency over TRP in medium. The induction response was sustained for at least 48 h and was comparable in efficacy to 2,3,7,8-tetrachlorodibenzo-p-dioxin. In hepatocytes, increases in mRNAs for CYP1A4 and CYP1A5, chick orthologs of mammalian CYP1A1 and 1A2, preceded increases in CYP1A proteins and enzyme activities, 7-ethoxyresorufin deethylase (EROD) for CYP1A4 and arachidonic acid epoxygenation for CYP1A5, consistent with a transcriptional mechanism. Aryl hydrocarbon receptor (AhR) dependence was evidenced by aTRP induction of EROD in wild-type Hepa1c1c7 cells but not in AhR-defective (c35) mutants. Preparations of aTRP were stable for many months at 4 degrees C and were relatively resistant to metabolism by hepatocytes or liver microsomes. Fractionation of aTRP by HPLC analysis coupled with EROD assays showed that aTRP contained multiple photoproducts and CYP1A inducing components, which varied in sensitivity to metabolism by hepatocytes. The previously identified TRP photoproduct, 6-formylindolo[3,2-b]carbazole (FICZ), was one component, but FICZ was not required for CYP1A induction by the aTRP mixture. These findings identify the indoor environment, and window sunlight in particular, as a new source of CYP1A inducers. Further, the evidence that biologically active metabolites of an endogenous substrate, arachidonic acid, are formed by aTRP-induced CYP1A provides a pathway by which TRP photoproducts, like toxic xenobiotics, could have significant physiologic effects.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) is a carcinogenic and highly toxic industrial byproduct that persists in the environment and produces a pleiotropic toxicity syndrome across vertebrate species that includes wasting, hepatosteatosis, and thymus atrophy. Dioxin toxicities require binding and activation of the aryl hydrocarbon receptor (AhR), a ligand activated transcription factor. However, after nearly 50 years of study, it remains unknown how AhR activation by dioxin produces toxic effects. Here, using the chick embryo close to hatching, a well-accepted model for dioxin toxicity, we identify NAD+ loss through PARP activation as a novel unifying mechanism for diverse effects of dioxin in vivo. We show that NAD+ loss is attributable to increased PARP activity in thymus and liver, as cotreatment with dioxin and the PARP inhibitor PJ34 increased NAD+ levels and prevented both thymus atrophy and hepatosteatosis. Our findings additionally support a role for decreased NAD+ dependent Sirt6 activity in mediating dioxin toxicity following PARP activation. Strikingly, treatment in vivo with the NAD+ repleting agent nicotinamide, a form of vitamin B3, prevented thymus atrophy and hepatosteatosis by dioxin and increased sirtuin activity, providing a therapeutic approach for preventing dioxin toxicities in vivo.

ABSTRACT:Cytochrome P450 (P450) enzymes metabolize the membrane lipid arachidonic acid to stable biologically active epoxides [eicosatrienoic acids (EETs)] and 20-hydroxyeicosatetraenoic acid (20-HETE). These products have cardiovascular activity, primarily acting as vasodilators and vasoconstrictors, respectively. EET formation can be increased by the prototype CYP1A or CYP2 inducers, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or phenobarbital (PB), respectively. We report here that imidazole derivative drugs: the anthelminthics, albendazole and thiabendazole; the proton pump inhibitor, omeprazole; the thromboxane synthase inhibitor, benzylimidazole; and the aromatase (CYP19) inhibitor vorozole (R76713, racemate; and R83842, (؉) enantiomer) increased hepatic microsomal EET formation in a chick embryo model. Albendazole increased EETs by transcriptional induction of CYP1A5 and the others by combined induction of CYP1A5 and CYP2H, the avian orthologs of mammalian CYP1A2 and CYP2B, respectively. All inducers increased formation of the four EET regioisomers, but TCDD and albendazole had preference for 5,6-EET and PB and omeprazole for 14,15-EET. Vorozole, benzylimidazole, and TCDD also suppressed 20-HETE formation. Vorozole was a remarkably effective and potent inducer of multiple hepatic P450s at a dose range which overlapped its inhibition of ovarian aromatase. Increased CYP1A activity in mouse Hepa 1-6 and human HepG2 cells by vorozole and other imidazole derivatives demonstrated applicability of the findings to mammalian cells. The findings suggest that changes in P450-dependent arachidonic acid metabolism may be a new source of side effects for drugs that induce CYP1A or CYP2. They demonstrate further that in vivo induction of multiple hepatic P450s produces additive increases in arachidonic acid epoxygenase activity and can occur concurrently with inhibition of ovarian aromatase activity.

Background: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-activated aryl hydrocarbon receptor (AHR) decreases gluconeogenesis by suppressing phosphoenolpyruvate carboxykinase (PEPCK) transcription via TCDD-inducible poly(ADPribose)-polymerase (TiPARP). Results: AHR enhances PEPCK ADP-ribosylation through TiPARP, and AHR suppression increases ADP-ribosylation PARP-independently. Conclusion: ADP-ribosylation, a new PEPCK posttranslational modification, is subject to complex regulation by the AHR. Significance: AHR gene activation leads to ADP-ribosylation of PEPCK with implications for energy metabolism beyond TCDD toxicity.

Activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is required for AHR dependent transcriptional activation and TCDD toxicity. We previously reported that aqueous tryptophan exposed to sunlight through window glass (aTRP) contains multiple photoproducts, including the well characterized 6-formylindolo[3,2-b]carbazole (FICZ), capable of activating the AHR and inducing CYP1A and CYP1A-mediated enzyme activities. We report here the isolation from aTRP and chemical characterization and synthesis of 1-(1H-indol-3-yl)-9H-pyrido[3,4-b]indole (IPI), a compound previously identified as a natural product of marine ascidia and now shown to be a TRP photoproduct with AHR-inducing properties. IPI, FICZ and TCDD produced equieffective induction of CYP1A-mediated 7-ethoxyresorufin deethylase (EROD) activity in chick embryo primary hepatocytes and mammalian Hepa1c1c7 cells. EROD induction by IPI was markedly curtailed in AHR-defective c35 cells, supporting the AHR dependence of the IPI response. Although IPI had a higher EC50 for EROD induction than FICZ, the much larger amount of IPI than FICZ in aTRP makes IPI a prominent contributor to EROD induction in aTRP. IPI was detected in TRP-containing culture medium under ambient laboratory conditions but not in TRP-free medium, consistent with its production from TRP. Cotreatment of hepatocytes with submaximal EROD-inducing doses of IPI and FICZ or TCDD produced additive increases in EROD without synergistic or inhibitory interactions. IPI and FICZ were readily metabolized by cultured hepatocytes. In addition to increasing CYP1A4 mRNA and EROD, IPI and FICZ decreased hepatocyte phosphoenolpyruvate carboxykinase mRNA expression and glucose output, biological effects associated with TCDD metabolic dysregulation. The findings underscore a role for sunlight in generating AHR-activating bioactive molecules.

The environmental toxin and carcinogen 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD, dioxin) binds and activates the transcription factor aryl hydrocarbon receptor (AHR), inducing CYP1 family cytochrome P450 enzymes. CYP1A2 and its avian ortholog CYP1A5 are highly active arachidonic acid epoxygenases. Epoxygenases metabolize arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs) and selected monohydroxyeicosatetraenoic acids (HETEs). EETs can be further metabolized by epoxide hydrolases to dihydroxyeicosatrienoic acids (DHETs). As P450-arachidonic acid metabolites affect vasoregulation, responses to ischemia, inflammation, and metabolic disorders, identification of their production in vivo is needed to understand their contribution to biologic effects of TCDD and other AHR activators. Here we report use of an acetonitrile-based extraction procedure that markedly increased the yield of arachidonic acid products by lipidomic analysis over a standard solid-phase extraction protocol. We show that TCDD increased all four EETs (5,6-, 8,9-, 11,12-, and 14,15-), their corresponding DHETs, and 18-and 20-HETE in liver in vivo and increased 5,6-EET, the four DHETs, and 18-HETE in heart, in a chick embryo model. As the chick embryo heart lacks arachidonic acid-metabolizing activity, the latter findings suggest that arachidonic acid metabolites may travel from their site of production to a distal organ, i.e., heart. To determine if the TCDDarachidonic acid-metabolite profile could be altered pharmacologically, chick embryos were treated with TCDD and the soluble epoxide hydrolase inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA). Cotreatment with AUDA increased hepatic EET-to-DHET ratios, indicating that the in vivo profile of P450-arachidonic acid metabolites can be modified for potential therapeutic intervention.

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