Aryl hydrocarbon receptor response to indigoids in vitro and in vivo.

Indigo and indirubin have been reported to be present at low levels in human urine. The possibility that indigoids are physiological ligands of the aryl hydrocarbon receptor (AhR) has been suggested by initial studies in yeast, where indirubin was found to be 50 times more potent than 2,3,7,8-tetrachlorodibenzo[p]dioxin (TCDD), and indigo was found to be equipotent. To demonstrate that these indigoids are bona fide agonists in mammalian systems, we employed a number of in vitro and in vivo measures of AhR agonist potency. In a hepatoma cell reporter system, indigo yielded an EC50 of approximately 5x10(-6)M (indirubin 3' -oxime EC50 approximately 5x10(-7)M, indirubin EC50 approximately 1x10(-7)M). A comparison of these EC50 values with that of 2,3,7,8-tetrachlorodibenzofuran (TCDBF) ( approximately 3x10(-9)M) indicated that these compounds are less potent than classic halogenated-dibenzofurans or -dibenzo-p-dioxins. Competitive binding assays for AhR occupancy showed similar IC50 values for indirubin and TCDBF ( approximately 2x10(-9) and 5x10(-9)M), with the IC50 values of indigo and indirubin 3' -oxime being approximately 10-fold higher. When rats were treated with these indigoids in the range of 1.5-50mg/kg, induction of hepatic cytochrome P450 1A1 was detected. Differences in the rank-order of potency observed in vivo and in vitro could, in part, be explained by metabolism. Although their biological potencies are not as high as has been previously suggested, collectively the results show that these indole-derived pigments are agonists of AhR in vivo. The in vivo results suggest that solubility, distribution, and metabolism influence the response to the compounds.

Formation and mass spectrometric analysis of DNA and nucleoside adducts by S-(1-acetoxymethyl)glutathione and by glutathione S-transferase-mediated activation of dihalomethanes.

The dihalomethane CH(2)Cl(2) is an industrial solvent of potential concern to humans because of its potential genotoxicity and carcinogenicity. To characterize DNA damage by dihalomethanes, a rapid DNA digestion under acidic conditions was developed to identify alkali labile DNA-dihalomethane nucleoside adducts using HPLC-electrospray mass spectrometry. DNA digestion worked best using pH 5.0 sodium acetate buffer, a 30 min incubation with DNase II and phosphodiesterase II, and a 2 h acid phosphatase digest. DNA was modified with S-(1-acetoxymethyl)glutathione (GSCH(2)OAc), a reagent modeling activated dihalomethanes. Adducts to G, A, and T were detected at high ratios of GSCH(2)OAc/DNA following digestion of the DNA with the procedure used here. The relative efficacy of adduct formation was G > T > A >> C. The four DNA nucleosides were also reacted with the dihalomethanes CH(2)Cl(2) and CH(2)Br(2) in the presence of glutathione (GSH) and GSH S-transferases from bacteria (DM11), rat (GST 5-5), and human (GST T1-1) under conditions that produce mutations in bacteria. All enzymes formed adducts to all four nucleosides, with dGuo being the most readily modified nucleoside. Thus, the pattern paralleled the results obtained with the model compounds GSCH(2)OAc and DNA. CH(2)Cl(2) and CH(2)Br(2) yielded similar amounts of adducts under these conditions. The relative efficiency of adduct formation by GSH transferases was rat 5-5 > human T1-1 > bacterial DM11, showing that human GSH transferase T1-1 can form dihalomethane adducts under the conditions used. Although the lability of DNA adducts has precluded more sophisticated experiments and in vivo studies have not yet been possible, the work collectively demonstrates the ability of several GSH transferases to generate DNA adducts from dihalomethanes, with G being the preferred site of adduction in both this and the GSCH(2)OAc model system.

Analysis of the kinetic mechanism of haloalkane conjugation by mammalian theta-class glutathione transferases.

Glutathione (GSH) transferases (GSTs) catalyze the conjugation of small haloalkanes with GSH. In the case of dihalomethanes and vic-1,2-dihaloalkanes, the reaction leads to the formation of genotoxic GSH conjugates. A generally established feature of the reaction of the mammalian theta-class GSTs, which preferentially catalyze these reactions, is the lack of saturability of the rate with regard to the substrate concentration. However, the bacterial GST DM11 catalyzes the same reactions with a relatively low K(m). Recently, DM11 has been shown to exhibit burst kinetics, with a rate-determining k(off) rate for product (Stourman et al. (2003) Biochemistry 42, 11048-11056). We examined rat GST 5-5 and human GST T1-1 and did not detect any burst kinetics in the conjugation of C(2)H(5)Cl, CH(2)Br(2), or CH(2)Cl(2), distinguishing these enzymes from GST DM11. The kinetic results were fit to a minimal mechanism in which the rate-limiting step is halide displacement. The differences in the steady state kinetics of conjugations catalyzed by bacterial GST DM11 and the mammalian GSTs 5-5 and T1-1 are concluded to be the result of differences in the rate-limiting steps and not to inherent enzyme affinity for the haloalkanes. The results may be interpreted in the context of a model in which the halide order affects the rate of carbon-halogen bond cleavage of all such reactions catalyzed by the GSTs. With GST DM11, the halide order is manifested in the K(m) parameter but not k(cat). With mammalian GSTs, the high K(m) is difficult to estimate. With all of the GSTs, the halide order is seen in the enzyme efficiency, k(cat)/K(m), with C-Br cleavage approximately 10-fold faster than C-Cl cleavage. The ratio k(cat)/K(m) is the most relevant parameter for issues of risk assessment.