3-Methylindole

Journal of Pharmacology and Experimental Therapeutics, 1996, 276(1):21-29.

Metabolism of 3-methylindole by vaccinia-expressed P450 enzymes: correlation of 3-methyleneindolenine formation and protein-binding.[Reference: WebLink]

The toxicity of 3-Methylindole (3 MI), a selective pneumotoxin, is dependent upon cytochrome P450-mediated bioactivation 3. Using vaccinia-expressed P450 enzymes, the metabolites of radiolabeled 3 MI produced by 14 individual P450s were identified and quantified by high performance liquid chromatography. Indole-3-carbinol was produced from incubations of 3 MI with only four enzymes. Although 3-methyloxindole was a product of several P450s, human 1A2 was most efficient in producing this metabolite. The toxic intermediate of 3 MI is believed to be a reactive methylene imine, 3-methyleneindolenine.
METHODS AND RESULTS:
In this study, this intermediate was detected as its mercapturate adduct, when N-acetylcysteine was added to the incubations. 3-Methyleneindolenine was produced by CYP2A6 at a rate of 50.9 +/- 8.9 pmol/mg protein/hr and by CYP2F1 at a rate of 205.7 +/- 12.5 pmol/mg/hr. The mouse 1a-2 and rabbit 4B1 enzymes produced the reactive intermediate in amounts that exceeded that of the human 2F1 enzyme by 1.4-fold and 1.9-fold, respectively. The toxicity of 3 MI is believed to be due to covalent binding of a P450-generated intermediate to critical pulmonary proteins. Comparison of covalent binding studies to the formation of the metabolites revealed a strong correlation between the amount of the 3 MI adduct detected and covalent binding.
CONCLUSIONS:
This study showed that the methylene imine electrophile is produced by only a few P450 enzymes and is the metabolite responsible for the covalent binding and presumably, the toxicity of 3 MI. Remarkable product preferences between the desaturation pathway to form the methyleneindolenine by CYP2F1 and the ring epoxidation pathway to form the oxindole by CYP1A2, were observed.

Toxicological Sciences,2000,52(2):284–292.

Metabolism of 3-Methylindole by Porcine Liver Microsomes: Responsible Cytochrome P450 Enzymes[Reference: WebLink]

The role of different cytochrome P450 enzymes on the metabolism of 3-Methylindole (3MI) was investigated using selective chemical inhibitors.
METHODS AND RESULTS:
Eight chemical inhibitors of P450 enzymes were screened for their inhibitory specificity towards 3MI metabolism in porcine microsomes: alpha-naphthoflavone (CYP1A1/2), 8-methoxypsoralen (CYP2A6), menthofuran (CYP2A6), diethyldithiocarbamate (CYP2A6), 4-methylpyrazole (CYP2E1), sulphaphenazole (CYP2C9), quinidine (CYP2D6), and troleandomycin (CYP3A4). The production of 3MI metabolites was only affected by the presence of inhibitors of CYP2A6 and CYP2E1 in the microsomal incubations. In a second experiment, a set of porcine microsomes (n = 30) was analyzed for CYP2A6 content by protein immunoblot analysis and for their coumarin 7-hydroxylation activity (CYP2A6 activity). Both CYP2A6 content and enzymatic activity were found to be highly and negatively correlated with 3MI fat content.
CONCLUSIONS:
The results of the present study indicate that the CYP2A6 porcine ortholog plays an important role in the metabolism of 3MI and that measurement of CYP2A6 levels and/or activity could be a useful marker for 3MI-induced boar taint.

Free radic biol med, 1994, 17(1):19-25.

Identification of 3-MI-derived N-centerred radicals obtained from incubation of 3-MI with microsomal-NADPH system by EPR-HPLC spin trapping.[Reference: WebLink]

3-Methylindole (3-MI) is a metabolite of tryptophan that causes acute pulmonary edema and emphysema in ruminants when administered orally or intravenously. Electron paramagnetic resonance (EPR) spin-trapping techniques have been used to investigate the in vitro and in vivo formation of free radicals during 3-MI metabolism by goat lung. Utilizing C-phenyl-N-tertbutyl nitrone (PBN), a nitrogen-centered free radical has been detected from 3-MI in goat lung muicrosomal incubation. The EPR spectrum of the spin adduct is identical to the observed when 3-MI is irradiated with ultraviolet light. The formation of a nitrogen-centered 3-MI free radical is followed by the appearance of a carbon-centered radical in microsomal preparations.
METHODS AND RESULTS:
The objective of the present study is to prove that the nitrogen-centered radical generated from the 3-MI incubation systems is a 3-MI radical utilizing [14C]-3 MI and the EPR-HPLC technique. The HPLC chromatogram includes three peaks that give EPR signals. These peaks are assigned to nitrogen-, oxygen- and carbon-centered radical adducts. The polarity of the three peaks follows the order: carbon-centered radical adduct > oxygen-centered radical adduct > nitrogen-centered radical adduct. The last has a polarity that is weaker tha 3-MI. Only the nitrogen-centered peak and the 3-MI peak possessef radioactivity. The retention time of the nitrogen centered radical is the same as the spin adduct generated by 3-MI irradiation with ultraviolet light.
CONCLUSIONS:
These results demonstrate that the nitrogen-centered radical is a 3-MI-PBN spin adduct, and supports the hypothesis that 3-MI-induced lung damage results from activation of 3-MI to a free radical. Also, in this study the stability of the radical spin adducts and the best conditions to produce the radicals in the incubation system was investigated.