Sunlight Generates Multiple Tryptophan Photoproducts Eliciting High Efficacy CYP1A Induction in Chick Hepatocytes and In Vivo

Abstract

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.

Inhibition of cytochrome P4501-dependent clearance of the endogenous agonist FICZ as a mechanism for activation of the aryl hydrocarbon receptor

“…S4C and S5 A-C). The known formation of FICZ from Trp (3,4,33,34) suggested to us that, if its metabolic degradation is suppressed, background levels of FICZ in cell-culture media (even in media that had been protected from light) might be sufficient to activate such AHR-dependent responses. Indeed, liquid chromatography (LC)-MS analysis of DMEM revealed the presence of FICZ, even when the medium had been protected from light.…”

“…FICZ is formed from Trp in aqueous solutions, including cellculture media, upon exposure to UV and visible light (3,4,33,34) as well as in human skin cells exposed in vitro to UVB light (4). Moreover, FICZ is conjugated metabolically with sulfate, and such conjugates have been detected in human urine (3), further verifying the presence of the parent compound in vivo.…”

Mitochondrial P450-dependent arachidonic acid metabolism by TCDD-induced hepatic CYP1A5; conversion of EETs to DHETs by mitochondrial soluble epoxide hydrolase

“…Several factors should serve to increase attention to CYP1A2/1A5 mediated aa metabolism: (1) CYP1A2 is induced not only in liver, but in kidney, a well recognized site of action for P450-dependent aa metabolites [19,58], (2) CYP1A enzymes are induced not only by toxic chemicals but also by many aryl hydrocarbon receptor ligands such as tryptophan derivatives and other substances in food, as well as some endogenous CYP1A substrates, i.e. bilirubin and melatonin [1,59,60], and (3) CYP1A2, and to a lesser extent CYP1A1, catalyzes metabolism of those and other endogenous substrates (reviewed in Rifkind [1]). Thus, CYP1A induction and associated effects may be expected to have much wider implications than has been appreciated.…”

Identification of the Aryl Hydrocarbon Receptor Target Gene TiPARP as a Mediator of Suppression of Hepatic Gluconeogenesis by 2,3,7,8-Tetrachlorodibenzo-p-dioxin and of Nicotinamide as a Corrective Agent for This Effect

“…Treatment and processing of livers for hepatocyte cultures was as previously described (17). For primary cultures, chick embryo hepatocytes (CEH) were plated in 24-well Cell Culture Cluster plates (Corning Inc., Corning, NY) at 0.5 ϫ 10 6 cells per well or in 6-well Cell Culture Cluster plates at 3 ϫ 10 6 cells per well and maintained in culture in the medium described (17) for 48 h before treatment. 16 -18-day-old CE were used for in ovo treatment and 15-or 16-day-old CE for hepatocyte cultures.…”

“…We investigated this hypothesis using the chick embryo (CE) close to hatching, a well-established model for studying TCDD toxicity (4,17). This model exhibits the metabolic effects of TCDD seen in mammalian liver and allows liver metabolism to be studied independently of confounding effects of changes in food intake (18).…”