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The mechanisms explaining arsenic toxicity are not well understood, but physiological consequences of stimulated aryl hydrocarbon receptor (AHR) signaling both directly and through cross-talk with other pathways have been indicated. The aim of this study was to establish how arsenic interacts with AHR-mediated transcription. The human hepatoma cell line (HepG2-XRE-Luc) carrying a luciferase reporter under the control of two AHR response elements (AHREs) and immortalized human keratinocytes (HaCaT) were exposed to sodium arsenite (NaAsO2; As(3+)), alone or in combination with the endogenous high affinity AHR ligand 6-formylindolo[3,2-b]carbazole (FICZ). Luciferase activity, cytochrome P4501A1 (CYP1A1) activity, oxidative stress-related responses, metabolic clearance of FICZ, and NADPH oxidase (NOX) activity as well as nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent gene expression were measured. Arsenic inhibited CYP1A1 enzyme activity and reduced the metabolic clearance of FICZ. Arsenic also led to activated CYP1A1 transcription but only in cells grown in medium containing trace amounts of the endogenous ligand FICZ, pointing to an indirect mechanism of activation. Initially, arsenic caused dose-dependent inhibition of FICZ-activated AHR signaling, disturbed intracellular GSH status, and increased expression of oxidative stress-related genes. Silencing of NOX4, addition of N-acetylcystein, or pretreatment with arsenic itself attenuated the initial dose-dependent inhibition of AHR signaling. Arsenic pretreatment led to elevated GSH levels and sensitized the cells to ligand-dependent AHR signaling, while silencing of Nrf2 significantly reduced arsenic-mediated activation of the AHR. In addition, influence of NOX on AHR activation was also observed in cells treated with the SH-reactive metals cadmium, mercury, and nickel. Together, the results suggest that SH-reactive agents via a new and possibly general NOX/H2O2-dependent mechanism can interfere with the endogenous regulation of the AHR.

Activation of the aryl hydrocarbon receptor (AhR), a conserved transcription factor best known as a target for highly toxic halogenated substances such as dioxin, under normal xenobiotic-free conditions is of considerable scientific interest. We have demonstrated previously that a photoproduct of tryptophan, 6-formylindolo[3,2-b]carbazole (FICZ), fulfills the criteria for an endogenous ligand for this receptor and proposed that this compound is the enigmatic mediator of the physiological functions of AhR. Here, we describe novel light-independent pathways by which FICZ can be formed. The oxidant H2O2 was shown to convert tryptophan to FICZ on its own in the absence of light. The enzymatic deamination of tryptamine yielded indole-3-acetaldehyde (I3A), which then rearranged to FICZ and its oxidation product, indolo[3,2-b]carbazole-6-carboxylic acid (CICZ). Indole-3-pyruvate (I3P) also produced I3A, FICZ, and CICZ. Malassezia yeast species, which constitute a part of the normal skin microbiota, produce a number of AhR activators from tryptophan. We identified both FICZ and CICZ among those products. Formation of FICZ from tryptophan or I3P produces a complex mixture of indole derivatives, some of which are CYP1A1 inhibitors. These can hinder the cellular clearance of FICZ and thereby increase its power as an AhR agonist. We present a general molecular mechanism involving dehydrogenations and oxidative coupling for the formation of FICZ in which I3A is the important precursor. In conclusion, our results suggest that FICZ is likely to be formed systemically.

Several polyphenols have been shown to activate the aryl hydrocarbon receptor (AHR) in spite of the fact that they bind to the receptor with low affinity. The aim of this study was to investigate whether quercetin (QUE), resveratrol (RES), and curcumin (CUR) interfere with the metabolic degradation of the suggested endogenous AHR ligand 6-formylindolo[3,2-b]carbazole (FICZ) and thereby indirectly activate the AHR. Using recombinant human enzyme, we confirmed earlier reported inhibitory effects of the polyphenols on cytochrome P4501A1 (CYP1A1) activity, and inhibition of metabolic clearance of FICZ was documented in FICZ-treated immortalized human keratinocytes (HaCaT). CYP1A1 activity was induced in HaCaT cells by all three compounds, and when they were added together with FICZ, a prolonged activation was observed after a dose-dependent inhibition period. The same pattern of responses was seen at the transcriptional level as determined with a CYP1A1 reporter assay in human liver hepatoma (HepG2) cells. To test the ability of the polyphenols to activate the AHR in the absence of FICZ, the cells were treated in medium, which in contrast to commercial batches of medium did not contain background levels of FICZ. Importantly, AHR activation was only observed in the commercial medium. Taken together, these findings suggest that QUE, RES, and CUR induce CYP1A1 in an indirect manner by inhibiting the metabolic turnover of FICZ. Humans are exposed to these compounds through the diet and nutritional supplements, and we propose that altered systemic levels of FICZ caused by such compounds may have physiological consequences.

The emission of diesel exhaust particulates is associated with potentially severe biological effects, e.g., cancer. The aim of the present study was to apply multivariate statistical methods to identify factors that affect the biological potency of these exhausts. Ten diesel fuels were analyzed regarding physical and chemical characteristics. Particulate exhaust emissions were sampled after combustion of these fuels on two makes of heavy duty diesel engines. Particle extracts were chemically analyzed and tested for mutagenicity in the Ames test. Also, the potency of the extracts to competitively inhibit the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the Ah receptor was assessed. Relationships between fuel characteristics and biological effects of the extracts were studied, using partial least squares regression (PLS). The most influential chemical fuel parameters included the contents of sulfur, certain polycyclic aromatic compounds (PAC), and naphthenes. Density and flash point were positively correlated with genotoxic potency. Cetane number and upper distillation curve points were negatively correlated with both mutagenicity and Ah receptor affinity. Between 61% and 70% of the biological response data could be explained by the measured chemical and physical factors of the fuels. By PLS modeling of extract data versus the biological response data, 66% of the genotoxicity could be explained, by 41% of the chemical variation. The most important variables, associated with both mutagenicity and Ah receptor affinity, included 1-nitropyrene, particle bound nitrate, indeno[1,2,3-cd]pyrene, and emitted mass of particles. S9-requiring mutagenicity was highly correlated with certain PAC, whereas S9-independent mutagenicity was better correlated with nitrates and 1-nitropyrene. The emission of sulfates also showed a correlation both with the emission of particles and with the biological effects. The results indicate that fuels with biologically less hazardous potentials should have high cetane number and contain less PAC and sulfur. The results also indicate that engine factors affect the formation and emission of nitrated PAC.