P. L. Appel

Relationship of the single-electron reduction potential of quinones to their reduction by flavoproteins

Abstract The aerobic and anaerobic metabolism of a series of quinones of known single-electron reduction potential has been studied using flavoenzymes catalyzing single-electron reduction. Metabolism was more closely related to single-electron reduction potential than to structural features or lipid solubility of the quinones studied. The pattern of quinone reduction with purified NADPH-cytochrome P-450 reductase was similar to that seen with NADH: ubiquinone oxidoreductase with NADPH as the cofactor; the lower limit for reduction was a quinone single-electron reduction potential of −240 mV. The lower limit for quinone reduction with purified NADH-cytochrome b 5 , reductase and NADH: ubiquinone oxidoreductase with NADH as the cofactor was a single-electron reduction potential of −170 mV. With all three enzymes there was a decreased quinone metabolism at higher single-electron reduction potentials. The same pattern of quinone metabolism was seen using purified or microsomal NADPH-cytochrome P-450 reductase and purified or microsomal NADH-cytochrome b 5 , reductase respectively. Microsomal quinone metabolism under aerobic conditions showed an increased V max and an unchanged K m , compared to metabolism under anaerobic conditions.

Metabolic stability of experimental chemotherapeutic agents in hepatocyte: tumor cell co-cultures

SummaryA U.S. National Cancer Institute screening program for new anticancer drugs, based on the growth of primary human tumor cells in an in vitro soft agar colony formation assay, has resulted in the identification of a number of compounds that have cytotoxic activity against primary human tumor cells in vitro but are inactive in the conventional in vivo murine P388 leukemia animal model pre-screen. To investigate whether metabolic inactivation ov the compounds might be a factor in the lack of in vivo cytotoxicity we have co-cultured rat hepatocytes with A204 rhabdomyosarcoma and murine P388 leukemia cell lines in the soft agarose colony formation assay for 24 h during exposure to the compounds. Twenty compounds with a range of in vitro activities were studied. Thirteen compounds exhibited cytotoxicity against A204 cells in culture; nine of them were less active when co-cultured with hepatocytes, two were activated by hepatocyte co-culture, and two showed no effect of hepatocyte co-culture. P388 cells were more sensitive to the antiproliferative effects of the compounds than A204 cells. Two compounds that were not active against A204 cells exhibited cytotoxicity against P388 cells. One compound was inactivated by hepatocyte co-culture and one showed no effect. Five compounds showed no cytotoxicity toward either A204 cells or P388 cells. Two of the compounds showing hepatocyte inactivation in vitro possess activity in one or more in vivo tumor models. Thus, evidence for metabolic inactivation in hepatocyte co-culture is not always an indication for lack of in vivo antitumor activity. Hepatocyte co-culture methodology provides a simple and objective means, amenable to large-scale screening, of distringuishing metabolic activation or inactivation of a given compound from other pharmacokinetic and pharmacodynamic factors with a minimum of material.

Carbon tetrachloride-induced increase in the antitumor activity of cyclophosphamide in mice: A pharmacokinetic study

SummaryCarbon tetrachloride is an hepatotoxin that depresses hepatic microsomal cytochrome P-450 and other enzyme activities. Cyclophosphamide is an anticancer drug that is activated by hepatic microsomal cytochrome P-450, while the products of cyclophosphamide metabolism by cytochrome P-450 can be metabolized by other hepatic enzymes. Carbon tetrachloride pretreatment has been found to increase the in vivo antitumor activity of cyclophosphamide against murine leukemia P-388. Carbon tetrachloride did not, however, affect the direct cytotoxicity of cyclophosphamide or 4-hydroxycyclo-phosphamide to cells in culture. Pharmacokinetic studies in mice revealed a delayed plasma disappearance of cyclophosphamide after carbon-tetrachloride pretreatment with an apparent initial half-time of 20.4 min compared to 9.0 min in non carbon-tetrachloride-pretreated mice. Plasma levels of total alkylating activity and plasma 4-hydroxycyclophosphamide increased more slowly and reached a lower peak, but were maintained for a longer time period in mice pretreated with carbon-tetrachloride than in untreated mice. The half-life for plasma elimination of 4-hydroxycyclophosphamide in untreated mice was 12 min and in carbon-tetrachloride-pretreated mice 27 min. There was, however, no difference in the area under the curve for either plasma total alkylating activity or plasma 4-hydroxycyclophosphamide between the two groups. It is suggested that prolonged exposure of tumor cells to 4-hydroxycyclophosphamide might be responsible for the increased antitumor activity of cyclophosphamide following carbon-tetrachloride pretreatment.

Factors Affecting the Intracellular Generation of Free Radicals from Quinones

Isolated hepatocytes do not liberate appreciable amounts of superoxide into the external medium. Simple quinones stimulate the release of superoxide up to 15 nmol/min/10(6) hepatocytes. Superoxide release stimulated by a variety of simple quinones and more complex antitumor quinones was maximal at a quinone one-electron reduction potential of -70 mV. This was qualitatively similar to the pattern of superoxide formation seen with NADH-cytochrome b5 reductase and NADH: ubiquinone oxidoreductase. Superoxide production by NADPH-cytochrome P-450 reductase was maximal at a quinone single-electron reduction potential at -200 mV. Phenobarbital pretreatment had no effect on superoxide formation by hepatocytes suggesting that NADPH-cytochrome P-450 reductase activity is not rate limiting for quinone stimulated superoxide formation. Sulfonated stilbenes, specific inhibitors of anion exchange, had no effect on the release of superoxide by hepatocytes suggesting that superoxide is not transported through anion channels in the plasma membrane. Pretreatment of hepatocytes with 10(-5) M diethyldithiocarbamate produced over a two fold increase in the release of superoxide.