William W. Johnson

Activation and detoxication of aflatoxin B1

Aflatoxin B1 (AFB1) is a potent hepatocarcinogen in experimental animals and a hazard to human health in several parts of the world. Implementation of rational intervention plans requires understanding of aspects of the roles of individual chemical steps involved in its disposition. AFB1 is activated to AFB1 exo-8,9-epoxide primarily by cytochrome P450 (P450) enzymes, particularly P450 3A4. However, P450 3A4 and other P450s also oxidize AFB1 to less dangerous products. The exo-epoxide is unstable in H2O (t1/2 1 s at 25 degreesC, k=0.6 s-1) and the diol product undergoes base-catalyzed rearrangement to a dialdehyde that reacts with protein lysine residues. AFB1 exo-8, 9-epoxide reacts with DNA to give adducts in high yield (>98%). This interaction is characterized by a Kd of approximately 1.4 mM, intercalation between base pairs, and rapid reaction with the guanyl N7 atom (k approximately 40 s-1). A proton field on the periphery of DNA is postulated to catalyze hydrolysis and also conjugation. Rat and especially human epoxide hydrolase show very little rate acceleration of hydrolysis of AFB1 exo- or endo-8,9-epoxide. However, glutathione transferases (GSTs) can catalyze AFB1 exo-8,9-epoxide conjugation. Kinetic analysis indicates a range of ratios of kcat/Kd varying from 10 to 1700 s-1 M-1, with the polymorphic GST M1-1 having the highest activity of the human GSTs. Studies with human hepatocytes indicate a major role for GST M1-1 in AFB1 conjugation and that the model chemoprotective agent oltipraz can act by both inducing GSTs and inhibiting P450s.

Lack of Electron Transfer from Cytochrome b5 in Stimulation of Catalytic Activities of Cytochrome P450 3A4 CHARACTERIZATION OF A RECONSTITUTED CYTOCHROME P450 3A4/NADPH-CYTOCHROME P450 REDUCTASE SYSTEM AND STUDIES WITH APO-CYTOCHROME b5

Many catalytic activities of cytochrome P450 (P450) 3A4, the major human liver P450 enzyme, require cytochrome b5 (b5) for optimal rates. The stimulatory effect of b5 on P450 reactions has generally been thought to be due to transfer of electrons from ferrous b5 to the ferrous P450-O2-substrate complex. We found that apo-b5, devoid of heme, could substitute for b5 in stimulating two prototypic activities, testosterone 6β hydroxylation and nifedipine oxidation. The stimulatory effect was not seen with albumin, hemoglobin, catalase, or cytochrome c. Apo-b5 could not substitute for b5 in a testosterone 6β hydroxylation system composed of NADH-b5 reductase and P450 3A4. Rates of electron transfer from NADPH-P450 reductase to ferric P450 3A4 were too slow (<2 min−1) to support testosterone 6β hydroxylation (∼14 min−1) unless b5 or apo-b5 was present, when rates of ∼700 min−1 were measured. The oxidation-reduction potential (Em,7) of the ferric/ferrous couple of P450 3A4 was unchanged (∼−310 mV) under different conditions in which the kinetics of reduction were altered by the addition of substrate and/or apo-b5. Rapid reduction of P450 3A4 required substrate and a preformed complex of P450 3A4, NADPH-P450 reductase, and b5; the rates of binding of the proteins to each other were 2-3 orders of magnitude less than reduction rates. We conclude that b5 can facilitate some P450 3A4-catalyzed oxidations by complexing with P450 3A4 and enhancing its reduction by NADPH-P450 reductase, without directly transferring electrons to P450.

Reactive Intermediates in Biological Systems: What Have We Learned and Where Are We Going?

During the past 25 years and the six meetings in this series, there have been many advances in the study of reactive intermediates and their relevance to health. Many issues of risk assessment remain controversial. Nevertheless, the science has advanced considerably and has enabled real progress in many difficult areas. Two areas of major advancement in the field involve the availability of new technology and approaches—analytical chemistry and recombinant DNA technology (Guengerich, 2000). Since 1975 we have seen extensive development of HPLC, and both NMR and mass spectrometry are orders of magnitude more sensitive. Recombinant DNA technology has not only greatly facilitated studies of the involved enzymology but has also made possible the development of model cellular systems to deal with questions that could not have been addressed before.