Determining the best animal model for human cytochrome P450 activities: a comparison of mouse, rat, rabbit, dog, micropig, monkey and man.

1. In the present study, nine cytochrome P450 enzyme activities in seven species were characterized to allow a practical means of comparing this important metabolic step between various test animals and man. 2. Enzyme activities and kinetic parameters were first determined towards marker substrates for human cytochrome P450 enzymes. Inhibition profiles were then determined with both antibodies directed against various cytochrome P450 enzymes and with chemical inhibitors. 3. Both the enzyme kinetic parameters/enzyme activities, and the inhibition profiles obtained for the animal species were compared with those obtained for human liver microsomes in order to postulate the animal species most similar to man with regard to each individual cytochrome P450 enzyme activity. 4. It was found that, as expected, none of the tested species was similar to man for all the measured P450 enzyme activities, but that in each species only some of the P450 enzyme activities could be considered as similar to man. 5. When it is known which human cytochrome P450 enzymes are involved in the metabolism of a compound, the comparative data presented here can be used for selecting the most suitable species for in vitro and in it no experiments.

Glutathione-dependent biotransformation of the alkylating drug thiotepa and transport of its metabolite monoglutathionylthiotepa in human MCF-7 breast cancer cells.

In this study, the role of glutathione S-transferase (GST) P1-1, the cellular reduced glutathione (GSH) status, and ATP-dependent efflux pumps in the cellular glutathione-dependent biotransformation of thiotepa and transport of the main metabolite monoglutathionylthiotepa in relation to cytotoxicity was studied in control and GST-P1-1-transfected MCF-7 cell lines. It was demonstrated that an enhanced cellular level of GST-P1-1 leads to an enhanced formation of monoglutathionylthiotepa, which is transported out of the cell into the medium. Monoglutathionylthiotepa was able to reversibly inhibit the activity of purified GST-P1-1, but only at nonphysiological concentrations, indicating that feedback inhibition of GST by its metabolites is not a relevant process in vivo. The GST activity, cellular GSH level, and/or ATP-dependent efflux of monoglutathionylthiotepa were modulated using ethacrynic acid, D,L-buthionine-S,R-sulfoximine, probenecid, and verapamil to understand the interplay between GSTs, glutathione conjugation, and efflux of glutathione conjugates in more detail. Inhibition of the GSH biosynthesis by D,L-buthionine-R,S-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, significantly reduced the glutathione conjugation of thiotepa and potentiated the cytotoxicity of thiotepa. Pretreatment of cells with ethacrynic acid resulted in decreased formation of monoglutathionylthiotepa as a result of inhibition of GST in the GST-P1-1 transfectant. In addition, the intracellular amount of monoglutathionylthiotepa increased in both of the cell lines on exposure to ethacrynic acid, indicating that transport of the glutathione conjugate was partially inhibited by the glutathione conjugate of ethacrynic acid. Transport activity of monoglutathionylthiotepa could also be inhibited by probenecid and verapamil, inhibitors of organic anion transport, without influencing the biotransformation capacity of the cells. It was demonstrated that inhibition of glutathione conjugate efflux by probenecid and verapamil leads to enhanced cytotoxicity, which indicates that besides thiotepa, monoglutathionylthiotepa is also cytotoxic for the cells. Only enhanced biotransformation and subsequent transport of the glutathione conjugate into the medium (which occurs with the GST-P1-1 transfectant) results in enhanced viability. Therefore, it was concluded that only enhanced biotransformation of thiotepa represents a real detoxification pathway when the resulting conjugate is transported out of the cells. Altogether, the results indicate that it is not the overexpression of GST per se but the interplay between GSH/GST and glutathione conjugate efflux pumps that results in increased resistance to alkylating anticancer drugs such as thiotepa.

Species and strain differences in the hepatic cytochrome P450-mediated biotransformation of 1,4-dichlorobenzene.

Our goal was to characterize possible species and strain differences in the hepatic microsomal biotransformation of 1,4-dichlorobenzene (1,4-DCB). Experiments compared extent of labeled 1,4-DCB conversion to oxidized metabolites, glutathione conjugates, and covalently bound metabolites by hepatic microsomes from humans, from male B6C3F1 mice, and from males of three rat strains (Fischer 344, Sprague-Dawley (SD), and Wistar). These rodents were selected for comparison because of their dissimilar responses to 1,4-DCB, notably, hepatocarcinogenicity in the B6C3F1 mouse but not the Wistar or Fischer rat, and nephrotoxicity and carcinogenicity in the Fischer rat. The species rank order for total in vitro conversion of 1,4-DCB was mouse > rat >> human. Conversion by microsomes from Fischer and Wistar rats was similar, whereas SD rats showed less biotransformation than the other two strains. Microsomes from the mouse produced most of the reactive metabolites as indicated by covalent binding to macromolecules (>20% of total metabolites formed). This covalent binding by mouse microsomes was extensively inhibited by ascorbic acid (AA), with a concomitant increase in hydroquinone formation, indicating an important role for benzoquinones as reactive metabolites. Phenobarbital pretreatment of rats enhanced the in vitro conversion of 1,4-DCB and the amount of covalent binding. Covalent binding for all rat microsomes was partly (33-79%) inhibited by AA. Addition of glutathione (GSH) plus AA further diminished the covalent binding with concomitant increased formation of the GSH-conjugated epoxide. Human microsomes produced the least reactive metabolites, with the majority (>70%) of this covalent binding prevented by GSH addition. The observed species differences, notably the more pronounced biotransformation of 1,4-DCB to reactive species including benzoquinones, could be factors in this compound's liver carcinogenicity in B6C3F1 mice but not other rodent species.

The use of human in vitro metabolic parameters to explore the risk assessment of hazardous compounds: the case of ethylene dibromide.

Ethylene dibromide (1,2-dibromoethane, EDB) is metabolized by two routes: a conjugative route catalyzed by glutathione S-transferases (GST) and an oxidative route catalyzed by cytochrome P450 (P450). The GST route is associated with carcinogenicity. An approach is presented to use human purified GST and P450 enzymes to explore the importance of these metabolic pathways for man in vivo. This strategy basically consists of four steps: (i) identification of the most important isoenzymes in vitro, (ii) scaling to rate per milligram cytosolic and microsomal protein, (iii) scaling to rate per gram liver, and (iv) incorporation of data in a physiologically based pharmacokinetic (PBPK) model. In the first step, several GST isoenzymes were shown to be active toward EDB and displayed pseudo-first-order kinetics, while the EDB oxidation was catalyzed by CYP2E1, 2A6, and 2B6, which all displayed saturable kinetics. In the second step, the predictions were in agreement with the measured activity in a batch of 21 human liver samples. In the third step, rat liver P450 and GST metabolism of EDB was predicted to be in the same range as human metabolism (expressed per gram). Interindividual differences in GST activity were modeled to determine "extreme cases." For the most active person, an approximately 1.5-fold increase of the amount of conjugative metabolites was predicted. Lastly, it was shown that the GST route, even at low concentrations, will always contribute significantly to total metabolism. In the fourth step, a PBPK model describing liver metabolism after inhalatory exposure to EDB was used. The saturation of the P450 route was predicted to occur faster in the rat than in man. The rat was predicted to have a higher turnover of EDB from both routes. Nevertheless, when all data are combined, it is crucial to recognize that the GST remains significantly active even at low EDB concentrations. The limitations and advantages of the presented strategy are discussed.

Differences in cytochrome P450-mediated biotransformation of 1,2-dichlorobenzene by rat and man: implications for human risk assessment.

The oxidative biotransformation of 1,2-dichlorobenzene (1,2-DCB) was investigated using hepatic microsomes from male Wistar, Fischer-344 and Sprague-Dawley (SD) rats, phenobarbital (PB)- and isoniazid (ISO) pretreated male Wistar rats and from man. In addition, microsomes from cell lines selectively expressing one cytochrome P450 (P4502E1, 1A1, 1A2, 2B6, 2C9, 2D6, /A6 and 3A4) were used. The rate of conversion was 0.09 nmol/min/mg. protein for both Wistar and Fischer-344 rat microsomes, 0.04 for SD-microsomes and 0.14 for human microsomes. Induction of Wistar rats with isoniazid (ISO, a P4502E1 inducer) or phenobarbital (PB, a P4502B1/2 inducer) resulted in an increased conversion rate of 0.20 and 0.42 nmol/min/mg. protein, respectively. Covalent binding of radioactivity to microsomal protein was similar for Wistar, Fischer and ISO-pretreated rats (16-17% of total metabolites), whereas induction with PB resulted in an increased covalent binding of 23% of total metabolites. Covalent binding was 31% for SD-microsomes and only 4.6% for human microsomes. Ascorbic acid notably reduced the amount of covalently bound metabolites for the SD-microsomes only, indicating that for these microsomes quinones were likely to be involved in this part of the covalent binding. Conjugation of epoxides with glutathione (GSH) inhibited most of the covalent binding for all microsomes. In the absence of GSH, the epoxides were hydrolyzed by epoxide hydrolase, resulting in the formation of dihydrodiols. Inhibition of epoxide hydrolase resulted in a decreased conversion and an increased covalent binding for all microsomes tested, indicating a role of epoxides in the covalent binding. Fischer-344 rat liver microsomes showed a lower epoxide hydrolase activity than microsomes from Wistar and Sprague-Dawley rats, which may explain the higher sensitivity to 1,2-DCB induced hepatotoxicity of Fischer rats in vivo. Conjugation of the epoxides with GSH was predominantly non-enzymatic for the rat, whereas for man, conjugation was nearly exclusively catalyzed by glutathione-S-transferases. This difference may be explained by the formation of a 'non-reactive' 3,4-epoxide by P4502E1 in human microsomes: incubations with microsomes selectively expressing human P4502E1 as well as human liver microsomes, resulted in the formation of similar amounts of 2,3- and 3,4-dichlorophenol (DCP), as well as two GSH-epoxide conjugates in equal amounts. For rat microsomes, one major GSH-epoxide conjugate was found, and a much higher covalent binding, particularly for the PB-microsomes. Therefore, we postulate that rat P4502B1/2 preferentially oxidizes the 4,5-site of 1,2-DCB, resulting in a reactive epoxide. Postulating these epoxides to be involved in the mechanism(s) of toxicity, human risk after exposure to 1,2-DCB will be overestimated when risk assessment is solely based on toxicity studies conducted in rat.

Glutathione conjugation of the cytostatic drug ifosfamide and the role of human glutathione S-transferases.

Development of drug resistance against alkylating cytostatic drugs has been associated with higher intracellular concentrations of glutathione (GSH) and increased expression of glutathione S-transferase (GST) enzymes. Therefore, enhanced detoxification by the glutathione/glutathione S-transferase pathway has been proposed as a major factor in the development of drug resistance toward alkylating agents. In this paper we describe 31P NMR and HPLC studies on the spontaneous and glutathione S-transferase catalyzed formation of glutathionyl conjugates of two metabolites of ifosfamide, i.e., 4-hydroxyifosfamide and ifosfamide mustard. At 25 degrees C activated ifosfamide (= 4-hydroxyifosfamide + aldoifosfamide) disappeared faster in the presence of a 10-fold excess of GSH (t1/2 = 107 min) compared to incubations without GSH (t1/2 = 266 min). No evidence for the formation of 4-glutathionyl ifosfamide was found. The ultimate alkylating species of ifosfamide is ifosfamide mustard (IM). In the absence of glutathione, the rate constant for the disappearance of the ifosfamide mustard signal at 25 degrees C (pH 7) was 1.98 x 10(-3) min-1 (t1/2 = 350 min). In the presence of a 10-fold molar excess of glutathione, this rate constant was 1.95 x 10(-3) min-1 (t1/2 = 355 min), indicating that the spontaneous formation of an aziridinium ion is the rate-limiting event in the reaction with glutathione. The aziridinium ion formed from IM can deprotonate upon formation, leading to the formation of a (noncharged) aziridine species. This intermediate (N-(2-chloroethyl)-N'-phosphoric acid diamide) was characterized by 31P, 1H, and 13C NMR spectra. When 2 mM ifosfamide mustard was incubated with 1 mM GSH in the presence of 40 microM GST P1-1, the formation of monoglutathionyl ifosfamide mustard was 2.3-fold increased above the spontaneous level. The other major human isoenzymes tested (A1-1, A2-2, and M1a-1a) did not influence the formation of monoglutathionyl ifosfamide mustard. The results of these studies demonstrate that increased levels of GST P1-1 can contribute to an enhanced detoxification of ifosfamide.