Kinetics of hydrolysis and reaction of aflatoxin B1 exo-8,9-epoxide and relevance to toxicity and detoxication.
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Aflatoxin B1↗
Biomedical subjects
W W Johnson
Publications and source records attributed to W 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.
Aflatoxin B1↗
Twenty years of biochemistry of human P450s: purification, expression, mechanism, and relevance to drugs.
Today cytochrome P450 (P450) research is accepted as an integral part of drug development and discovery. Work leading to this point included biochemical studies on P450 in experimental animal models and application to human systems. The development of recombinant expression systems has been an important part of the progress, and in this article we describe some recently developed bacterial systems that can be used for the production of metabolites, genotoxicity testing, and screening in random mutagenesis work. Rate-limiting aspects of P450 reactions vary with particular systems, and further investigations are in order. Non-ionic detergents have been utilized widely in P450 purification work; these compounds are now shown to be substrates for P450s. These oxidations are not only of fundamental interest in expanding the repertoire of P450 substrates but have significance in light of human exposure to these compounds.
Cytochrome P-450 Enzyme System↗
Kinetics of ferric cytochrome P450 reduction by NADPH-cytochrome P450 reductase: rapid reduction in the absence of substrate and variations among cytochrome P450 systems.
The reduction of ferric cytochrome P450 (P450) to ferrous is the first chemical step in almost all P450 reactions, and many characteristics of this step have been reported. Reduction kinetics of rabbit and human P450s were measured in a variety of systems. As reported earlier, P450 reduction is biphasic in microsomes and some purified P450 systems. However, this is not an inherent property of P450s, and some low- and high-spin iron P450s were reduced with single-exponential kinetics. Contrary to a generalized view, the presence of substrate is not necessary for rapid reduction of all P450s. Also, low-spin heme can be reduced as rapidly as high-spin in several P450s. P450s varied considerably in their reduction behavior, and even a single P450 showed remarkably different reduction kinetics when placed in various environments. P450 3A4 reduction was examined in liver microsomes, a reconstituted system, a fusion protein in which it was linked to NADPH-P450 reductase, and baculovirus and bacterial membranes in which P450 3A4 and NADPH-P450 reductase were coexpressed; the systems differed considerably in terms of the need for the substrate testosterone and cytochrome b5 (b5) for reduction and as to whether reduction was rate-limiting in the overall catalytic cycle. When b5 was included in reconstituted systems, it reduction kinetics were linked with those of some P450s. This behavior could be simulated in kinetic models in which electrons flowed from the ferrous P450.CO complex to oxidized b5. Overall, the kinetics of ferric P450 reduction cannot be generalized among different P450s in various systems, and concepts regarding influence of substrate, reaction sequence, and a rate-limiting step are not very universal.
Animals↗
Reconstitution of recombinant cytochrome P450 2C10(2C9) and comparison with cytochrome P450 3A4 and other forms: effects of cytochrome P450-P450 and cytochrome P450-b5 interactions.
Tolbutamide methyl hydroxylation and S-warfarin 7-hydroxylation activities were reconstituted in systems containing recombinant human cytochrome P450 (P450 or CYP) 2C10(2C9) and the optimal conditions for the systems were compared with those of bufuralol 1'-hydroxylation by CYP1A1, theophylline 8-hydroxylation by CYP1A2, bufuralol 1'-hydroxylation by CYP2D6, chlorzoxazone 6-hydroxylation by CYP2E1, and testosterone 6 beta-hydroxylation by CYP3A4. CYP2C10 required cytochrome b5 (b5) for optimal rates of tolbutamide and S-warfarin oxidations and b5 could be replaced by apo-b5; apo-b5 and b5 effects on the reconstituted systems have already been reported in systems containing CYP3A4 for the oxidation of testosterone and nifedipine and for the rapid reduction of CYP3A4 by NADPH-P450 reductase (H. Yamazaki et al., 1996, J. Biol. Chem. 271, 27438-27444). Stopped-flow studies, however, suggested that apo-b5 as well as b5 did not cause stimulation of the reduction of CYP2C10 by NADPH-P450 reductase, while the reduction rates were dependent on the substrates in reconstituted systems. Chlorzoxazone 6-hydroxylation by CYP2E1 was stimulated by b5, but not by apo-b5, in reconstituted systems. Neither apo- nor holo-b5 increased bufuralol 1'-hydroxylation activity by CYP1A1 or 2D6 or theophylline 8-hydroxylation by CYP1A2. Interestingly, we found that testosterone 6 beta-hydroxylation by CYP3A4 was stimulated by CYP1A2 (and also by a modified form in which the first 36 residues of the native human protein were removed) and CYP1A1 as well as by b5, and such stimulations were not seen when other P450 proteins (e.g., CYP2C10, 2D6, or 2E1) were added to the reconstituted systems. In contrast, substrate oxidations by CYP2C10 and CYP2E1 were not stimulated by other P450 proteins. The present results suggest that there are differences in optimal conditions for reconstitution of substrate oxidations by various forms of human P450 enzymes, and in some P450-catalyzed reactions protein-protein interactions between P450 and b5 and other P450 proteins are very important in some oxidations catalyzed by CYP2C10, 2E1, and 3A4.
Animals↗
Reaction of aflatoxin B1 exo-8,9-epoxide with DNA: kinetic analysis of covalent binding and DNA-induced hydrolysis.
The exo isomer of aflatoxin B1 (AFB1) 8,9-epoxide appears to be the only product of AFB1 involved in reaction with DNA and reacts with the N7 atom of guanine via an SN2 reaction from an intercalated state. Although the epoxide hydrolyzes rapidly in H2O (0.6 s-1 at 25 degrees C), very high yields of DNA adduct result. Experimental binding data were fit to a model in which the epoxide forms a reversible complex with calf thymus DNA (Kd = 0.43 mg ml-1, or 1.4 mM monomer equivalents) and reacts with guanine with a rate of 35 s-1. Stopped-flow kinetic analysis revealed attenuation of fluorescence in the presence of DNA that was dependent on DNA concentration. Kinetic spectral analysis revealed that this process represents conjugation of epoxide with DNA, with an extrapolated rate maximum of 42 s-1 and half-maximal velocity at a DNA concentration of 1.8 mg ml-1 (5.8 mM monomer equivalents). The rate of hydrolysis of the epoxide was accelerated by calf thymus DNA in the range of pH 6-8, with a larger enhancement at the lower pH (increase of 0.23 s-1 at pH 6.2 with 0.17 mg DNA ml-1). The same rate enhancement effect was observed with poly[dA-dT].poly[dA-dT], in which the epoxide can intercalate but not form significant levels of N7 purine adducts, and with single-stranded DNA. The increased rate of hydrolysis by DNA resembles that reported earlier for epoxides of polycyclic hydrocarbons and is postulated to involve a previously suggested localized proton field on the periphery of DNA. The epoxide preferentially intercalates between base pairs, and the proton field is postulated to provide acid catalysis to the conjugation reaction.
Aflatoxin B1↗
Conjugation of highly reactive aflatoxin B1 exo-8,9-epoxide catalyzed by rat and human glutathione transferases: estimation of kinetic parameters.
Aflatoxin B1 (AFB1) exo-8,9-epoxide, the reactive product of the hepatocarcinogen AFB1, is stable in aprotic solvents but hydrolyzes rapidly in H2O at 25 degrees C and pH 7 (t1/2 = 1 s). However, it is also known that some glutathione (GSH) transferases can conjugate the epoxide with GSH to give the adduct in high yield. We developed an approach to estimating kinetic parameters for reactions involving this epoxide or other substrates that are unstable to H2O. Varying concentrations of the (anhydrous) epoxide and GSH transferase were mixed and the GSH conjugates were measured. The final concentrations of product were known for each set of the starting epoxide and enzyme concentrations in a modeling approach, where the competition with the hydrolysis reaction is considered with two variables, a K for binding of the enzyme and epoxide and a rate k2, which includes microscopic steps following complex formation and resulting in conjugate formation. The ratio k2/K, a measure of enzyme efficiency, varied among individual recombinant GSH transferases in the the order (rat) 10-10 >> 3-3 > (human) M1-1 > T1-1 > A1-1 > P1-1 > A2-2, from 3 x 10(6) to 10 M(-1) s(-1). The high ratio of M1-1 among the human GSH transferase enzymes tested is consistent with other work in which GSH-AFB1 conjugates were not detected in hepatocytes with an M1 null polymorphism. This general kinetic approach should be applicable to estimation of kinetic parameters involved in the interaction of other unstable substrates with enzymes.
Aflatoxin B1↗
Aflatoxin B1 8,9-epoxide hydrolysis in the presence of rat and human epoxide hydrolase.
Aflatoxin B1 (AFB1) must be activated to the electrophilic AFB1 exo-8,9-epoxide to be genotoxic and carcinogenic. A role for epoxide hydrolase in detoxication has been suggested but never directly addressed. In light of recent studies determining the instability of AFB1 exo-8,9-epoxide in H2O, a role for epoxide hydrolase appears dubious. Rat liver or recombinant rat epoxide hydrolase provided an enhancement to the already fast hydrolysis rate of up to 22%. Purified human epoxide hydrolase provided no detectable enhancement to the rate of chemical hydrolysis. Some reduction in the genotoxicity of AFB1 was observed when the ratio of rat epoxide hydrolase to cytochrome P450 was high (approximately 50-fold). An 80-fold excess of human epoxide hydrolase over cytochrome P450 only produced an effect of approximately 25% inhibition. It appears, therefore, that there is little evidence to support a role for epoxide hydrolase in the detoxication of AFB1.
Aflatoxin B1↗
Reduction of postoperative pain: a double-blind, randomized clinical trial.
The authors investigated the capability of two commonly used basing techniques to reduce postoperative sensitivity. The authors measured the time it took the subjects to respond to a standardized stimulus of cold water (cold response measure, or CRM) at baseline and one week after treatment. The authors found no significant reduction in the mean CRM for the group receiving Amalgambond Plus (Parkell), but they did find a significant decrease in the mean CRM for the group receiving Copalite (Harry J. Bosworth Co.) with or without Vitrebond (3M). The authors concluded that the subjects in the Amalgambond Plus group experienced no more sensitivity to cold at one week postoperative than they did at baseline, while the subjects in the Copalite/Vitrebond group did.
Adolescent↗
Sealant leakage with and without isolation.
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Acid Etching, Dental↗
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 6beta 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 6beta 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 6beta hydroxylation ( approximately 14 min-1) unless b5 or apo-b5 was present, when rates of approximately 700 min-1 were measured. The oxidation-reduction potential (Em,7) of the ferric/ferrous couple of P450 3A4 was unchanged ( approximately -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.
Animals↗
Location of a potential transport binding site in a sigma class glutathione transferase by x-ray crystallography.
The crystal structure of the sigma class glutathione transferase from squid digestive gland in complex with S-(3-iodobenzyl)glutathione reveals a third binding site for the glutathione conjugate besides the two in the active sites of the dimer. The additional binding site is near the crystallographic two-fold axis between the two alpha 4-turn-alpha 5 motifs. The principal binding interactions with the conjugate include specific electrostatic interactions between the peptide and the two subunits and a hydrophobic cavity found across the two-fold axis that accommodates the 3-iodobenzyl group. Thus, two identical, symmetry-related but mutually exclusive binding modes for the third conjugate are observed. The hydrophobic pocket is about 14 A from the hydroxyl group of Tyr-7 in the active site. This site is a potential transport binding site for hydrophobic molecules or their glutathione conjugates.
Animals↗
First-sphere and second-sphere electrostatic effects in the active site of a class mu gluthathione transferase.
The activation of the thiol of glutathione (GSH) bound in the active site of the class mu glutathione transferase M1-1 from rat involves a hydrogen-bonding network that includes a direct (first-sphere) interaction between the hydroxyl group of Y6 and the sulfur of GSH and second-sphere interactions involving a hydrogen bond between the main-chain amide N-H of L12 and the hydroxyl group of Y6 and an on-face hydrogen bond between the hydroxyl group of T13 and the pi-electron cloud of Y6 (i.e., T13-OH---pi-Y6-OH--- -SG). The functions of these hydrogen bonds have been examined with a combination of site-specific mutagenesis and X-ray crystallography. The hydroxyl group of Y6 has a normal pKa of about 10 even though it is shielded from solvent and is in a largely hydrophobic environment. The apparent pKa of GSH in the binary Y6F.GSH complex is increased by 1.6 log units, and the reactivity of the enzyme-bound nucleophile is reduced. The catalytic properties of the Y6L mutant are identical to those of Y6F, suggesting that the weakly polar on-edge interaction between the aromatic ring and sulfur has no influence on catalysis. The refined three-dimensional structure of the Y6F mutant in complex with GSH shows no major structural perturbation of the protein other than a change in the coordination environment of the sulfur. Removal of the second-sphere influence of the on-face hydrogen bond between the hydroxyl groups T13 as in the T13V and T13A mutants elevates the pKa of enzyme-bound GSH by about 0.7 pKa units. Crystal structures of these mutants show that structural changes in the active site are minor and suggest that the changes in pKa of E.GSH are due to the presence or absence of the on-face hydrogen bond. The T13S mutant has a completely different side-chain hydrogen-bonding geometry than T13 in the native enzyme and catalytic properties similar to the T13A and T13V mutants consistent with the absence of an on-face hydrogen bond. The gamma-methyl group of T13 is essential in enforcing the on-face hydrogen bond geometry and preventing the hydroxyl group from forming more favorable conventional hydrogen bonds.
Animals↗
Scientific data-exchange in periodontology via Internet.
The aim of the present paper was to study the current state of electronic scientific data-exchange in the field of dentistry and particularly in the specialty of periodontology. The use of electronic communication is unquestionably growing in all areas of life and has established itself in almost all areas of sciences and research. The potential use of Internet resources in the area of periodontology is the main topic of this paper. An overview explains the Internet system, its history, its structure, and the necessary hardware and software to use it. Text-orientated software and graphic interfaces like the World Wide Web will be described. Examples will be included to describing the basic services of Internet. The pros and cons of Internet use will be discussed and a preview of possible future developments in the field of communication for scientifically-oriented information will be included. This paper indicates that modern periodontal therapy and research may be enhanced by the use of Internet resources.
Communication↗
Involvement of cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer.
In recent years there has been considerable interest in the effect of variations in activities of xenobiotic-metabolizing enzymes on cancer incidence. This interest has accelerated with the development of methods for analyzing genetic polymorphisms. However, progress in epidemiology has been slow and the contributions of polymorphisms to risks from individual chemicals and mixtures are often controversial. A series of studies is presented to show the complexities encountered with a single chemical, aflatoxin B1 (AFB1). AFB1 is oxidized by human cytochrome P450 enzymes to several products. Only one of these, the 8,9-exo-epoxide, appears to be mutagenic and the others are detoxication products. P450 3A4, which can both activate and detoxicate AFB1, is found in the liver and the small intestine. In the small intestine, the first contact after oral exposure, epoxidation would not lead to liver cancer. The (nonenzymatic) half-life of the epoxide has been determined to be approximately 1 sec at 23 degrees C and neutral pH. Although the half-life is short, AFB1-8,9-exo-epoxide does react with DNA and glutathione S-transferase. Levels of these conjugates have been measured and combined with the rate of hydrolysis in a kinetic model to predict constants for binding of the epoxide with DNA and glutathione S-transferase. A role for epoxide hydrolase in alteration of AFB1 hepatocarcinogenesis has been proposed, although experimental evidence is lacking. Some inhibition of microsome-generated genotoxicity was observed with rat epoxide hydrolase; further information on the extent of contribution of this enzyme to AFB1 metabolism is not yet available.
Aflatoxin B1↗
Enamel microleakage of in-vivo Class V composite resin restorations using phosphoric acid versus maleic acid.
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Acid Etching, Dental↗
Three-dimensional structure, catalytic properties, and evolution of a sigma class glutathione transferase from squid, a progenitor of the lens S-crystallins of cephalopods.
The glutathione transferase from squid digestive gland is unique in its very high catalytic activity toward 1-chloro-2,4-dinitrobenzene and in its ancestral relationship to the genes encoding the S-crystallins of the lens of cephalopod eye. The three-dimensional structure of this glutathione transferase in complex with the product 1-(S-glutathionyl)-2,4-dinitrobenzene (GSDNB) has been solved by multiple isomorphous replacement techniques at a resolution of 2.4 A. Like the cytosolic enzymes from vertebrates, the squid protein is a dimer. The structure is similar in overall topology to the vertebrate enzymes but has a dimer interface that is unique when compared to all of the vertebrate and invertebrate structures thus far reported. The active site of the enzyme is very open, a fact that appears to correlate with the high turnover number (800 s-1 at pH 6.5) toward 1-chloro-2,4-dinitrobenzene. Both kcat and kcat/KmCDNB exhibit pH dependencies consistent with a pKa for the thiol of enzyme-bound GSH of 6.3. The enzyme is not very efficient at catalyzing the addition of GSH to enones and epoxides. This particular characteristic appears to be due to the lack of an electrophilic residue at position 106, which is often found in other GSH transferases. The F106Y mutant enzyme is much improved in catalyzing these reactions. Comparisons of the primary structure, gene structure, and three-dimensional structure with class alpha, mu, and pi enzymes support placing the squid protein in a separate enzyme class, sigma. The unique dimer interface suggests that the class sigma enzyme diverged from the ancestral precursor prior to the divergence of the precursor gene for the alpha, mu, and pi classes.
Amino Acid Sequence↗
Microleakage of light-cured glass-ionomer restorative materials.
The microleakage of three glass-ionomer restorative materials, one chemically cured and two light cured, was evaluated. Ten restorations of each material were placed, according to manufacturer's instructions, in Class V cavities in bovine incisors. All cavities were prepared with 90-degree cavosurface margins and were located at the cementoenamel junction. Sectioning of the teeth after thermocycling and immersion in methylene blue dye revealed only occasional slight leakage and no statistically significant difference among the three materials.
Analysis of Variance↗