Intra- and extra-cellular formation of metabolites from chemically reactive species.

Many of the pharmacological and toxicological actions of foreign compounds are mediated through the formation of their metabolites, which are subsequently converted into other metabolites before they are eliminated from the body. Although many of the pharmacological and toxicological actions are caused by chemically stable metabolites, others are caused by chemically reactive metabolites having halflives in cells and in the body ranging from milliseconds to several days. Because the concentrations of reactive metabolites may be profoundly influenced by their halflives in different compartments of the body, it is useful to think of the chemically reactive metabolites as belonging to one or more functional categories. (1) Ultra-short-lived metabolites never leave the enzyme that catalyses their formation. They either immediately decompose to stable metabolites while reversibly bound to the enzyme or combine with the enzyme irreversibly and thereby inactivate it. The precursors of such metabolites have been called 'suicide enzyme inhibitors'. Because of the presumed specificity and irreversible action of such metabolites, their precursors are frequently used in therapy to obtain selective inhibition of specific enzymes. (2) Short-Iived metabolites leave the enzymes which catalyse their formation, but never leave the cells in which they are formed. After a metabolite in this category leaves the enzyme that catalyses its formation, it may be rapidly converted either enzymically or non-enzymically into stable metabolites or it may become covalently bound to various cell components, including the enzyme that catalyses its formation and inactivation. Nevertheless, it cannot react with components in other cells and therefore any toxicity caused by the metabolite is restricted to those cells in which it is formed. (3) Intermediate-lived metabolites leave the cells in which they are formed but never enter other organs. A metabolite in this category thus leaves the cells in which it is formed and enters blood and thus may be inactivated and combine with various components in blood and in cells located downstream from the cells in which the metabolite is formed. Any pharmacological or toxicological action, however, would be restricted to the organ in which the metabolite is formed. (4) Long-lived metabolites enter the blood and are distributed to virtually all cells in the body. Thus virtually any cell may be considered as a potential target for the action of metabolites in this category. (5 ) Ultra-long-lived metabolites not only enter the blood and are distributed throughout the body, but also are excreted into bile or urine. Because of the high concentrations of the chemically reactive metabolites attained in kidney, urinary bladder, and intestines, any toxicity caused by the metabolites in this category may be preferentially localized in these organs. The strategies used to detect chemically reactive metabolites in the different categories vary markedly. For example, it might be possible to detect metabolites in categories (3) and (4) and by measuring their covalent binding to haemoglobin or other substances in blood. But this approach would be useless in detecting metabolites in categories (1) and (2). As a first step in determining the category to which a metabolite belongs in a given organ, we have developed an approach based on the general equations of Gillette (1980), by which the portion of a chemically reactive metabolite that escapes isolated cells may be estimated. In this approach we assume that rate of formation of the metabolite within cells reaches a steady state almost instantaneously, and that a portion of the metabolite is converted into other metabolites, including covalently bound metabolites to macromolecules, and another portion of the chemically reactive metabolite diffuses out of cells and decomposes to other substances. The addition of a nucleophile, such as a protein, that cannot enter cells serves as a trap by reacting with the chemically reactive metabolite coming out of the cells at the expense of the decomposition products formed from it. At an infinite concentration of the nucleophile, all of the reactive metabolite that escapes the cells will react with the nucleophile and none of the decomposition products derived from the extracellular metabolite will be formed. Thus the objective of such studies is to predict the pattern of metabolites at an infinite concentration of the nucleop hile. If the number of potential binding sites on the nucleophile are not significantly decreased at low concentrations of the nucleophile during the time of incubation, the rate of reaction of the reactive metabolite with the nucleophile may be represented as a pseudo-first-order reaction with a rate constant of [N]k2, as illustrated in the model shown in Fig. 1. If the nucleophile also serves as an enzyme that converts the chemically reactive metabolite to second-generation metabolites, then the rate of formation of the secondgeneration metabolites may also be represented by apseudofirst-order-reaction with a rate constant of [Nlk,, in the model. If the second-generation metabolite is also formed intracellularly, the rate at which the metabolite is formed within the cells may be represented by a rate constant of k, 8 , and all other reactions occurring intracellularly may be represented as a sum of rate cpnstants, kl,, and all other reactions occurring extracellularly may % e represented by the sum of the rate constants kZ3 and k26. With this model, three extreme cases may be envisioned: In case 1, the chemically reactive metabolite is short-lived and thus it would not covalently bind to the added nucleophile, nor would the added nucleophile alter the pattern of metabolites formed from the metabolite. In case. 2, some of the reactive metabolite diffuses out of the cells and becomes covalently bound to the nucleophile, but the rate of decomposition of the extracellular metabolite is so rapid that little of it re-enters the cells; that is, k,, is negligible compared with k23 and kZ6. In this case, the amount of reactive metabolite that becomes covalently bound to the nucleophile will depend on the relative rates at which the reactive metabolite is inactivated within the cells and diffuses out of the cells, but the addition of the nucleophile will decrease only those metabolites that are formed from the extracellular reactive metabolite. In case 3, the reactive metabolite crosses the cellular membranes so rapidly that the steady-state intracellular and extracellular concentrations are virtually identical; that is, kzl is much greater than the sum of k23, [Nlk,,, [Nlk25 and k26. In this case, nearly all of the reactive metabolites will be inactivated extracellularly at an infinite concentration of the nucleophile. The proportion of the reactive metabolite that should be covalently bound to the extracellular nucleophile at an infinite concentration of the nucleophile will therefore depend solely on the relative rates at which the nucleophile irreversibly binds to the reactive metabolite and catalyses its conversion into other metabolites. All of the other metabolites formed either intracellularly or