Induction of benzo[a]pyrene Mono-oxygenase in liver cell culture by the photochemical generation of active oxygen species. Evidence for the involvement of singlet oxygen and the formation of a stable inducing intermediate
Abstract:1. The photochemical generation of excited states of oxygen in liver cell culture by the mild ilumination of culture medium containing riboflavin, results in stimulation of benzo[a]pyrene 3-mono-oxygenase, a cytochrome P-450-linked mono-oxygenase. 2. The same large increase in mono-oxygenase activity was found when medium containing riboflavin was illuminated in the absence of cells and then stored in the dark for 24h before contact with the cells. From this it may be inferred that stimulation is due to the fo… Show more
“…Furthermore, the present investigation offers a relatively straightforward explanation for the reported ability of numerous structurally diverse chemicals to activate the AHR; such reports have been used to argue that the AHR binds ligands promiscuously (37). Our results also may explain why the addition of fresh Trp-containing medium to cell cultures (42), oxidative stress [e.g., by hyperoxia (43)], and hydrodynamic shear that gives rise to oxidized LDL (44,45), as well as why the addition of various complex mixtures, such as extracts of paper and ink, can activate the AHR (46,47).…”
Altered systemic levels of 6-formylindolo [3,2-b]carbazole (FICZ), an enigmatic endogenous ligand for the aryl hydrocarbon receptor (AHR), may explain adverse physiological responses evoked by small natural and anthropogenic molecules as well as by oxidative stress and light. We demonstrate here that several different chemical compounds can inhibit the metabolism of FICZ, thereby disrupting the autoregulatory feedback control of cytochrome P4501 systems and other proteins whose expression is regulated by AHR. FICZ is both the most tightly bound endogenous agonist for the AHR and an ideal substrate for cytochrome CYP1A1/1A2 and 1B1, thereby also participating in an autoregulatory loop that keeps its own steady-state concentration low. At very low concentrations FICZ influences circadian rhythms, responses to UV light, homeostasis associated with pro-and anti-inflammatory processes, and genomic stability. Here, we demonstrate that, if its metabolic clearance is compromised, femtomolar background levels of this compound in cell-culture medium are sufficient to up-regulate CYP1A1 mRNA and enzyme activity. The oxidants UVB irradiation and hydrogen peroxide and the model AHR antagonist 3′-methoxy-4′-nitroflavone all inhibited induction of CYP1A1 enzyme activity by FICZ or 2,3,7,8-tetrachlorodibenzo-p-dioxin, thereby subsequently elevating intracellular levels of FICZ and activating AHR. Taken together, these findings support an indirect mechanism of AHR activation, indicating that AHR activation by molecules with low affinity actually may reflect inhibition of FICZ metabolism and raising questions about the reported promiscuity of the AHR. Accordingly, we propose that prolonged induction of AHR activity through inhibition of CYP1 disturbs feedback regulation of FICZ levels, with potential detrimental consequences.
“…Furthermore, the present investigation offers a relatively straightforward explanation for the reported ability of numerous structurally diverse chemicals to activate the AHR; such reports have been used to argue that the AHR binds ligands promiscuously (37). Our results also may explain why the addition of fresh Trp-containing medium to cell cultures (42), oxidative stress [e.g., by hyperoxia (43)], and hydrodynamic shear that gives rise to oxidized LDL (44,45), as well as why the addition of various complex mixtures, such as extracts of paper and ink, can activate the AHR (46,47).…”
Altered systemic levels of 6-formylindolo [3,2-b]carbazole (FICZ), an enigmatic endogenous ligand for the aryl hydrocarbon receptor (AHR), may explain adverse physiological responses evoked by small natural and anthropogenic molecules as well as by oxidative stress and light. We demonstrate here that several different chemical compounds can inhibit the metabolism of FICZ, thereby disrupting the autoregulatory feedback control of cytochrome P4501 systems and other proteins whose expression is regulated by AHR. FICZ is both the most tightly bound endogenous agonist for the AHR and an ideal substrate for cytochrome CYP1A1/1A2 and 1B1, thereby also participating in an autoregulatory loop that keeps its own steady-state concentration low. At very low concentrations FICZ influences circadian rhythms, responses to UV light, homeostasis associated with pro-and anti-inflammatory processes, and genomic stability. Here, we demonstrate that, if its metabolic clearance is compromised, femtomolar background levels of this compound in cell-culture medium are sufficient to up-regulate CYP1A1 mRNA and enzyme activity. The oxidants UVB irradiation and hydrogen peroxide and the model AHR antagonist 3′-methoxy-4′-nitroflavone all inhibited induction of CYP1A1 enzyme activity by FICZ or 2,3,7,8-tetrachlorodibenzo-p-dioxin, thereby subsequently elevating intracellular levels of FICZ and activating AHR. Taken together, these findings support an indirect mechanism of AHR activation, indicating that AHR activation by molecules with low affinity actually may reflect inhibition of FICZ metabolism and raising questions about the reported promiscuity of the AHR. Accordingly, we propose that prolonged induction of AHR activity through inhibition of CYP1 disturbs feedback regulation of FICZ levels, with potential detrimental consequences.
“…In vitro and in vivo studies have shown that the byproducts of tryptophan photooxidation possess a high AhR-binding capacity and are able to induce the expression of Cyp1a1 and other AhR target genes. [61][62][63][64] A prime example of a tryptophan photoproduct is a 6formylindolo[3,2-b]carbazole (FICZ), which displays a significant structural similarity and potency to ICZ and can agonistically stimulate AhR activity in as low as picomolar ranges [65][66][67] (Fig. 2).…”
Section: Indole and Tryptophan Metabolitesmentioning
Aryl hydrocarbon receptor (AhR) is a member of the basic helix-loop-helix-(bHLH) superfamily of transcription factors, which are associated with cellular responses to environmental stimuli, such as xenobiotics and oxygen levels. Unlike other members of bHLH, AhR is the only bHLH transcription factor that is known to be ligand activated. Early AhR studies focused on understanding the role of AhR in mediating the toxicity and carcinogenesis properties of the prototypic ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In recent years, however, it has become apparent that, in addition to its toxicological involvement, AhR is highly receptive to a wide array of endogenous and exogenous ligands, and that its activation leads to a myriad of key host physiological functions. In this study, we review the current understanding of the functions of AhR in the mucosal immune system with a focus on its role in intestinal barrier function and intestinal immune cells, as well as in intestinal homeostasis.
“…S1C,D), indicating that there was not a general effect on nucleocytoplasmic shuttling arising from UV exposure. Light-activated AHR signaling has been observed in a variety of cultured cells since the first report by Paine in 1976 24 , and the functional relevance of tryptophan as an UVB chromophore for formation of an endogenous AHR-activating photoproduct that explains the effect of light on cells has been repeatedly documented 13,25–28 .Figure 1UVB induces AHR translocation to the nucleus and the expression of its target genes, in vitro . ( A ) Western Blot analyses of AHR from nuclear and cytoplasmic fractions from SCC25 cells 1 hr following irradiation with 15 min UVB.…”
Links between solar UV exposure and immunity date back to the ancient Greeks with the development of heliotherapy. Skin contains several UV-sensitive chromophores and exposure to sunlight can produce molecules, such as vitamin D3, that act in an endocrine manner. We investigated the role of the aryl hydrocarbon receptor (AHR), an environmental sensor and ligand-regulated transcription factor activated by numerous planar compounds of endogenous, dietary or environmental origin. 15- to 30-minute exposure of cells to a minimal erythemal dose of UVB irradiation
in vitro
induced translocation of the AHR to the nucleus, rapidly inducing site-specific DNA binding and target gene regulation. Importantly,
ex vivo
studies with
Ahr
wild-type or null fibroblasts showed that serum from mice whose skin was exposed to a 15 min UVB dose, but not control serum, contained agonist activity within 30 min of UV irradiation, inducing AHR-dependent gene expression. Moreover, a 15-min cutaneous UVB exposure induced AHR site-specific DNA binding and target gene regulation
in vivo
within 3–6 hr post-irradiation in blood and in peripheral tissues, including intestine. These results show that cutaneous exposure of mice to a single minimal erythemic dose of UVB induces rapid AHR signaling in multiple peripheral organs, providing compelling evidence that moderate sun exposure can exert endocrine control of immunity through the AHR.
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