Reactive metabolites generation

Identification of DNA-Reactive Metabolites Generated in a Target Tissue, Mouse Skin, In Vivo. Our initial studies focused on activation of DMBA in mouse embryo cells in culture because of the ease of isolation of sufficient DNA for adduct characterization. The cells were exposed to DMBA and the isolated DNA enzymatically hydrolyzed to deoxyribonucleosides. DMBA-deoxyribonucleoside adducts were characterized by fluorescence measurements (11,22), by photosensitivity studies (12) and by column chromatography (23,24). These studies provided evidence that the DNA-reactive metabolite generated in these cells is a bay region dihydrodiol epoxide. The enzymatic steps in this activation pathway (Figure 1) involve oxidation of DMBA by mixed function oxidases to a 3,4-epoxide which is converted by epoxide hydrase to a 3,4-dihydro-diol. This is, in turn, oxidized by mixed function oxidases to the dihydrodiol epoxide. 

Wulferink M, Gonzalez J, Goebel C, Gleichmann E (2001) T cells ignore aniline, a prohapten, but respond to its reactive metabolites generated by phagocytes possible implications for the pathogenesis of toxic oil syndrome. Chem Res Toxicol, 14(4) 389-397. 

Several enzyme systems exist as cellular defense (detoxification) pathways against the chemically reactive metabolites generated by GYP metabolism (91,92,102,103). These include GST, epoxide hydrolase, and quinone reductase, as well as catalase, glutathione peroxidase, and superoxide dismutase, which detoxify the peroxide and superoxide by-products of metabolism. The efficiency of the bioinactivation process is dependent on the inherent chemical reactivity of the electrophilic intermediate, its affinity and selectivity of the reactive metabolite for the bioinactivation enzymes, the tissue expression of these enzymes, and the rapid upregulation of these enzymes and cofactors mediated by the cellular sensors of chemical stress. The reactive metabolites that can evade these defense systems may damage target proteins and nucleic acids by either oxidation or covalent modification. 

Potential toxicity of metabolites may depend on activation reactions in resistant vs sensitive strains. Reactive metabolites generated in resistant plants are generally labile and would not likely be incorporated into animals in measurable levels. In animals, the resultant toxicity of a compound may depend on the proportion converted into active metabolites in sensitive vs resistant species of test animals. (See Gillette, Biochemical Aspects, herein). 

A relatively unique type of reactive metabolite is carbene, i.e., a divalent carbon, which is a proposed intermediate in the oxidation of methylene dioxy-containing compounds. A methylenedioxy group in aromatic compounds is subject to O-dealkylation, e.g., 3,4-methylenedioxyamphetamine, as shown in Figure 8.20. The process generates formic acid and the catechol metabolite as final products. However, in the course of the reaction, a... 

Cell-free systems, when supplemented with relevant cofactors, are remarkably proficient, despite their crudity in generating reactive electrophiles from most procarcinogens. However, they provide at best a broad approximation of in vivo metabolism and can fail to produce sufficient quantity of a particular reactive metabolite to be detectable by the indicator cells or they can produce inappropriate metabolites that do not play a role in vivo (see Gatehouse and Tweats, 1987, for discussion). 

Fig. 8.5 Schematic showing the generation of a reactive metabolite, its reaction with a protein target, and toxicity resulting from a direct mechanism (protein essential to cell function) or one involving the immune system.

Following incubation of a test compound in a system designed to generate trapped conjugates of reactive metabolites (e.g., supplemented microsomes or hepatocytes)... 

Electrochemical oxidation is a means of generating reactive metabolites either in the absence of biological nucleophiles or through the addition of nucleophiles under controlled conditions. Mass spectrometric, NMR and IR evaluations can all be performed as described for amodiaquine [41] but recapitulation of microsomal metabolism under electrochemical conditions is not always possible and hence this technique is probably of limited applicability. 

Liver injury is clinically defined as an increase of serum alanine amino transferase (ALT) levels of more than three times the upper limit of normal and a total bilirubin level of more than twice the upper limit of normal [4]. The clinical patterns of liver injury can be characterized as hepatocellular (with a predominant initial elevation of ALT), cholestatic (with an initial elevation of alkaline phosphatase) or mixed. The mechanisms of drug-induced hepatotoxicity include excessive generation of reactive metabolites, mitochondrial dysfunction, oxidative stress and inhibition of bile salt efflux protein [5]. Better understandings of these mechanisms in the past decades led to the development of assays and models suitable for studying such toxic mechanisms and for selecting better leads in the drug discovery stage. 

Haonzi D, Lekahal M, Moreau A et al (2000) Cytochrome P450-generated reactive metabolites cause mitochondrial permeability transition, cuspate activation and apoptosis in rat hepatocytes. Hepatology 32 303-311... 

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