PubMed Health⌕ Search

Biomedical subjects

F P Guengerich

Publications and source records attributed to F P Guengerich.

At least 19 recordsLinked to original sources
PubMed2001

Minipig cytochrome P450 3A, 2A and 2C enzymes have similar properties to human analogs.

BACKGROUND: The search for an optimal experimental model in pharmacology is recently focused on (mini)pigs as they seem not only to be an alternative source of cells and tissues for xenotherapy but also an alternative species for studies on drug metabolism in man due to similarities between (mini) pig and human drug metabolizing systems. The purpose of this work is to characterize minipig liver microsomal cytochromes P450 (CYPs) by comparing their N-terminal sequences with corresponding human orthologs. RESULTS: The microsomal CYPs exhibit similar activities to their human orthologous enzymes (CYP3A4, nifedipine oxidation; 2A6, coumarin 7-hydroxylation; 2D6, bufuralol 1'-hydroxylation; 2E1, p-nitrophenol hydroxylation; and 2C9, tolbutamide hydroxylation). Specific minipig CYP (2A, 2C and 3A) enzymes were partially purified and proteins identified by immunostaining (using antibodies against the respective human CYPs) were used for N-terminal amino acid sequencing. From comparisons, it can be concluded that the sequence of the first 20 amino acids at the N-terminus of minipig CYP2A is highly similar to human CYP2A6 (70% identity). The N-terminal sequence of CYP2C shared about 50% similarity with human 2C9. The results on the minipig liver microsomal CYP3A yielded identical data with those obtained for amino acid sequences of the pig CYP3A29 showing 60% identity with human CYP3A4. CONCLUSIONS: Thus, our results further support the view that minipigs may serve as model animals in pharmacological/toxicological studies with substrates of human CYP enzymes, namely, of the CYP3A and CYP2A forms.

Animals↗
PubMed2001

Oxidation of phenethylamine derivatives by cytochrome P450 2D6: the issue of substrate protonation in binding and catalysis.

Cytochrome P450 (P450) 2D6 oxidizes a wide variety of drugs typically at a distance of 5-7 A from a basic nitrogen on the substrate. To investigate the determinants of P450 2D6 catalysis, we analyzed the binding and oxidation of phenethylamine substrates. P450 2D6 discriminated between the various phenethylamines, as evidenced by binding and steady-state results. Whereas the spectral binding affinity for 3-methoxyphenethylamine and 4-methoxyphenethylamine was similar, the affinity for 4-hydroxyphenethylamine was 12-fold weaker than for 3-hydroxyphenethylamine at pH 7.4. The binding of 3,4-dihydroxyphenethylamine was equally poor. These equilibrium dissociation constants were based on the observation of both type I and type II perturbation difference spectra; the former involves displacement of the proximal H(2)O ligand, yielding an iron spin state change, and the latter requires nitrogen ligation to the heme iron. One explanation for the observed type II binding spectra is the presence of both protonated and unprotonated forms of these compounds. To address this possibility, the K(S) values for 3-methoxyphenethylamine and 4-methoxyphenethylamine were determined as a function of pH. Two apparent pK(a) values were determined, which corresponded to a P450 2D6 residue involved in binding and to a lowered pK(a) of a substrate amine group upon binding P450 2D6. The apparent pK(a) of the enzyme residue (6.6) is much higher than the expected pK(a) of Asp301, which has been hypothesized to play a role in binding. Interestingly, the apparent pK(a) for the methoxyphenethylamine derivatives decreased by as much as 2 pH units upon binding to P450 2D6. 3-Methoxyphenethylamine and 4-methoxyphenethylamine underwent sequential oxidations with O-demethylation and subsequent ring hydroxylation to form 3,4-dihydroxyphenethylamine (dopamine). At higher substrate concentrations, the second oxidation was inhibited. This result can be explained by the increasing concentration of the inhibitory unprotonated substrate. Nevertheless, the rates of methoxyphenethylamine oxidations are the highest reported for P450 2D6 substrates.

Cytochrome P-450 CYP2D6↗
PubMed2001

A new selective and potent inhibitor of human cytochrome P450 1B1 and its application to antimutagenesis.

Human cytochrome P450 (P450) 1B1 is found mainly in extrahepatic tissues and is overexpressed in a variety of human tumors. Metabolic activation of 17beta-estradiol (E(2)) to 4-hydroxy E(2) by P450 1B1 has been postulated to be a factor in mammary carcinogenesis. The inhibition of recombinant human P450 1B1 by 2,4,3',5'-tetramethoxystilbene (TMS) was investigated using either bacterial membranes from a human P450/NADPH-P450 reductase bicistronic expression system or using purified enzymes. TMS showed potent and selective inhibition of the ethoxyresorufin O-deethylation (EROD) activity of P450 1B1 with an IC(50) value of 6 nM. TMS exhibited 50-fold selectivity for P450 1B1 over P450 1A1 (IC(50) = 300 nM) and 500-fold selectivity for P450 1B1 over P450 1A2 (IC(50) = 3 microM). The inhibitory effects of TMS on EROD activity of human liver microsomes were determined. TMS inhibited EROD activity of human liver microsomes at the same concentration as with recombinant human P450 1A2. TMS also strongly inhibited 4- and 2-hydroxylation of E(2) by P450 1B1-expressing membranes or purified P450 1B1. TMS was a competitive inhibitor of P450 1B1 with a K(i) of 3 nM. The inhibition by TMS was not mechanism-based, and the loss of activity was not blocked by the trapping agents glutathione, N-acetylcysteine, or dithiothreitol. Using purified histidine-tagged P450 1B1, the binding kinetic analysis was performed with TMS, yielding a K(d) of 3 microM. The activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline in an Escherichia coli lac-based mutagenicity tester system containing functional human P450 1B1 was strongly inhibited by TMS. Our results indicate that TMS is a very selective and potent competitive inhibitor of P450 1B1. TMS is selective for inhibiting P450 1B1 among other human P450s including 1A1, 1A2, and 3A4 and warrants consideration as a candidate for preventing mammary tumor formation by E(2) in humans.

Animals↗
PubMed2001

Random mutagenesis of human cytochrome p450 2A6 and screening with indole oxidation products.

Cytochrome P450 (P450) 2A6 mutants from randomized libraries generated in the substrate recognition sequence (SRS) regions were screened in Escherichia coli on the basis of indole metabolism. SRS 3 and 4 libraries yielded colonies that produced indigo at least as well as wild-type (WT) P450 2A6, and some colonies were consistently more blue upon replating. One mutant, F209T, showed indole 3-hydroxylation WT. The double mutant L240C/N297Q consistently produced very blue colonies. Five mutants yielded mixtures of pigments from indole different than WT, as judged by visible spectra and HPLC of products. When bacteria expressing the mutants were grown in the presence of each of 26 substituted indoles, a variety of patterns of formation of different dyes was seen with several of the mutants. This approach has potential value in understanding P450 2A6 function and generating new dyestuffs and other products.

Aryl Hydrocarbon Hydroxylases↗
PubMed2001

Metabolic activation of carcinogenic 1-nitropyrene by human cytochrome P450 1B1 in Salmonella typhimurium strain expressing an O-acetyltransferase in SOS/umu assay.

Metabolic activation of 1-nitropyrene (1-NP) by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes was investigated. 1-NP induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of any P450 system, but the activities were influenced by the levels of bacterial O-acetyltransferase (OAT) and nitroreductase. Metabolic activation of 1-NP by human P450 1B1/NPR membranes was observed and was influenced by the levels of OAT levels in tester strains. Metabolic activation of 1-NP (0.3microM) by P450 1B1 was 750 umu units/min/nmol P450 1B1 in an OAT-overexpressing strain NM2009. The metabolic activation of 1-NP (3-30microM) was similar (approximately 300 umu units/min/nmol P450 1B1) using TA1535/pSK1002 or OAT-deficient strain NM2000. P450 1B1 had the highest catalytic activities among P450 family 1 enzymes for the activation of 1-aminopyrene (1-AP) in the OAT-overexpressing strain NM2009, suggesting nitrenium ion formation via N-hydroxylation/O-acetylation. High-performance liquid chromatography (HPLC) analyses revealed the formation of 1-nitropyrene-6-ol and also 1-nitropyrene-3-ol, 1-nitropyrene-8-ol, and trans-4,5-dihydroxy-4,5-diol-1-nitropyrene from 1-NP (10microM), catalyzed by P450 1B1. These results indicate that 1-NP can be activated by human P450 1B1 to a genotoxic agent by nitroreduction/O-acetylation at low substrate concentrations and probably by epoxidation (independent of OAT) at high concentrations.

Acetyltransferases↗
PubMed2001

Heterologous expression of cytochrome P450 2D6 mutants, electron transfer, and catalysis of bufuralol hydroxylation: the role of aspartate 301 in structural integrity.

Cytochrome P450 (P450) 2D6 is a polymorphic human enzyme involved in the oxidation of >50 drugs, most of which contain a basic nitrogen. In confirmation of previous work by others, substitutions at Asp301 decreased rates of substrate oxidation by P450 2D6. An anionic residue (Asp, Glu) at this position was found to be important in proper protein folding and heme incorporation, and positively charged residues were particularly disruptive in bacterial and also in baculovirus expression systems. Truncation of 20 N-terminal amino acids had no significant effect on catalytic activity except to attenuate P450 2D6 interaction with membranes and NADPH-P450 reductase. The truncation of the N-terminus increased the level of bacterial expression of wild-type P450 2D6 (Asp301) but markedly reduced expression of all codon 301 mutants, including Glu301. Reduction of ferric P450 2D6 by NADPH-P450 reductase was enhanced in the presence of the prototypic substrate bufuralol. Bacterial flavodoxin, an NADPH-P450 reductase homolog, binds tightly to P450 2D6 but is inefficient in electron transfer to the heme. These results collectively indicate that the acidic residue at position 301 in P450 2D6 has a structural role in addition to any in substrate binding and that the N-terminus of P450 2D6 is relatively unimportant to catalytic activity beyond a role in facilitating binding to NADPH-P450 reductase.

Adrenergic beta-Antagonists↗
PubMed2001

Diversity in mechanisms of substrate oxidation by cytochrome P450 2D6. Lack of an allosteric role of NADPH-cytochrome P450 reductase in catalytic regioselectivity.

Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Differences in the regioselectivity of oxidation products formed in systems containing NADPH-P450 reductase/NADPH and the model oxidant cumene hydroperoxide have been proposed by others to be due to an allosteric influence of the reductase on P450 2D6 (Modi, S., Gilham, D. E., Sutcliffe, M. J., Lian, L.-Y., Primrose, W. U., Wolf, C. R., and Roberts, G. C. K. (1997) Biochemistry 36, 4461-4470). We examined the differences in the formation of oxidation products of N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, metoprolol, and bufuralol between reductase-, cumene hydroperoxide-, and iodosylbenzene-supported systems. Catalytic regioselectivity was not influenced by the presence of the reductase in any of the systems supported by model oxidants, ruling out allosteric influences. The presence of the reductase had little effect on the affinity of P450 2D6 for any of these three substrates. The addition of the reaction remnants of the model oxidants (cumyl alcohol and iodobenzene) to the reductase-supported system did not affect reaction patterns, arguing against steric influences of these products on catalytic regioselectivity. Label from H(2)18O was quantitatively incorporated into 1'-hydroxybufuralol in the iodosylbenzene- but not in the reductase- or cumene hydroperoxide-supported reactions. We conclude that the P450 systems utilizing NADPH-P450 reductase, cumene hydroperoxide, and iodosylbenzene use similar but distinct chemical mechanisms. These differences are the basis for the variable product distributions, not an allosteric influence of the reductase.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2001

Binding and oxidation of alkyl 4-nitrophenyl ethers by rabbit cytochrome P450 1A2: evidence for two binding sites.

Although most cytochrome P450 (P450) reactions demonstrate saturation kinetics that fit to the standard Michaelis-Menten equation, there are important exceptions where sigmoidal or nonhyperbolic behavior is observed and have been fit instead to kinetic models involving two binding sites. To assess these models, we demonstrate the consistency of a two binding site model to interpret both steady-state kinetics and binding events. Rates of 4-nitrophenol and formaldehyde production from the O-demethylation of 1-methoxy-4-nitrobenzene by P450 1A2 isolated from rabbit liver produced biphasic plots, when plotted against substrate concentration. Experiments confirmed the absence of the further oxidation of the products. Recombinant rabbit P450 1A2 yielded the same maximal velocity and more marked biphasicity. Overall, these steady-state data fit well to kinetic models involving two binding sites. Steady-state studies of substrates with bulkier O-ethyl or O-isopropoxy groups indicated decreased affinity for the second site. Based on binding studies, the affinity of P450 1A2 for these substrates increased 200-fold with the larger alkyl groups. To analyze the single binding site model, competition studies were conducted with 1,4-phenyldiisocyanide and the alkyl 4-nitrophenyl ethers. Although the observed dissociation constants and the competing titrant demonstrated a linear dependence, the affinity for the competing titrant depended on the presence of the other titrant, which violates the single binding site model. Alternatively, we applied a two binding site model to these data to obtain dissociation constants for the binary and ternary complexes. The agreement between the dissociation constants for the heterogeneous complexes supports the appropriateness of the two binding site model. This novel finding for P450 1A2 may be more common than originally perceived for P450s.

Animals↗
PubMed2001

Stimulation of cytochrome P450 reactions by apo-cytochrome b5: evidence against transfer of heme from cytochrome P450 3A4 to apo-cytochrome b5 or heme oxygenase.

Many cytochrome P450 (P450)-dependent reactions have been shown to be stimulated by another microsomal protein, cytochrome b(5) (b(5)). Two major explanations are (i) direct electron transfer from b(5) and (ii) a conformational effect in the absence of electron transfer. Some P450s (e.g. 3A4, 2C9, 17A, and 4A7) are stimulated by either b(5) or b(5) devoid of heme (apo-b(5)), indicating a lack of electron transfer, whereas other P450s (e.g. 2E1) are stimulated by b(5) but not by apo-b(5). Recently, a proposal has been made by Guryev et al. (Biochemistry 40, 5018-5031, 2001) that the stimulation by apo-b(5) can be explained only by transfer of heme from P450 preparations to apo-b(5), enabling electron transfer. We have repeated earlier findings of stimulation of catalytic activity of testosterone 6beta-hydroxylation activities with four P450 preparations, in which nearly all of the heme was accounted for as P450. Spectral analysis of mixtures indicated that only approximately 5% of the heme can be transferred to apo-b(5), which cannot account for the observed stimulation. The presence of the heme scavenger apomyoglobin did not inhibit the stimulation of P450 3A4-dependent testosterone or nifedipine oxidation activity. Further evidence against the presence of loosely bound P450 3A4 heme was provided in experiments with apo-heme oxygenase, in which only 3% of the P450 heme was converted to biliverdin. Finally, b(5) supported NADH-b(5) reductase/P450 3A4-dependent testosterone 6beta-hydroxylation, but apo-b(5) did not. Thus, apo-b(5) can stimulate P450 3A4 reactions as well as b(5) in the absence of electron transfer, and heme transfer from P450 3A4 to apo-b(5) cannot be used to explain the catalytic stimulation.

Apoproteins↗
PubMed2001

Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase.

We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY1002/3A4, which express respective human P450 enzymes and NADPH-cytochrome P450 reductase (reductase) and bacterial O-acetyltransferase (O-AT). These strains were established by introducing two plasmids into S. typhimurium TA1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double tac promoter and the other, pOA102, carrying O-AT and umuC"lacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 125 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5-f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole and 3-amino-1-methyl-5H-pyrido[4,3-b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B(1) exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. beta-Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrome P450 enzyme involved in bioactivation of HCAs.

Acetyltransferases↗
PubMed2001

Coumarin substrates for cytochrome P450 2D6 fluorescence assays.

A set of nine 4-aminomethyl-7-alkoxycoumarin derivatives was synthesized and characterized as substrates for O-dealkylation by recombinant cytochrome P450 2D6, a major human enzyme involved in drug metabolism. Enzymatic O-dealkylation yields 7-hydroxycoumarins, which have useful fluorescence properties. The substrates, which differed in substitution at the amino and 7-hydroxy positions, varied in terms of catalytic efficiency of O-dealkylation and in their selectivity as substrates for cytochrome P450 2D6 in human liver microsomes. Several of the compounds are useful as cytochrome P450 2D6 substrates in single-phase, rapid-throughput assays.

Amino Acid Sequence↗
PubMed2001

Oxidations of p-alkoxyacylanilides catalyzed by human cytochrome P450 1A2: structure-activity relationships and simulation of rate constants of individual steps in catalysis.

Human cytochrome P450 (P450) 1A2 is involved in the oxidation of many important drugs and carcinogens. The prototype substrate phenacetin is oxidized to an acetol as well as the O-dealkylation product [Yun, C.-H., Miller, G. P., and Guengerich, F. P. (2000) Biochemistry 39, 11319-11329]. In an effort to improve rates of catalysis of P450 1A2 enzymes, we considered a set of p-alkoxyacylanilide analogues of phenacetin and found that variations in the O-alkyl and N-acyl substituents altered the rates of the two oxidation reactions and the ratio of acetol/phenol products. Moving one methylene group of phenacetin from the O-alkyl group to the N-acyl moiety increased rates of both oxidations approximately 5-fold and improved the coupling efficiency (oxidation products formed/NADPH consumed) from 6% to 38%. Noncompetitive kinetic deuterium isotope effects of 2-3 were measured for all O-dealkylation reactions examined with wild-type P450 1A2 and the E225I mutant, which has 6-fold higher activity. A trend of decreasing kinetic deuterium isotope effect for E225I > wild-type > mutant D320A was observed for O-demethylation of p-methoxyacetanilide, which follows the trend for k(cat). The set of O-dealkylation and acetol formation results for wild-type P450 1A2 and the E225I mutant with several of the protiated and deuterated substrates were fit to a model developed for the basic catalytic cycle and a set of microscopic rate constants in which the only variable was the rate of product formation (substrate oxygenation, including hydrogen abstraction). In this model, k(cat) is considerably less than any of the microscopic rate constants and is affected by several individual rate constants, including the rate of formation of the oxygenating species, the rate of substrate oxidation by the oxygenating species, and the rates of generation of reduced oxygen species (H(2)O(2), H(2)O). This analysis of the effects of the individual rate constants provides a framework for consideration of other P450 reactions and rate-limiting steps.

Alanine↗
PubMed2001

Forging the links between metabolism and carcinogenesis.

Metabolism plays important roles in chemical carcinogenesis, both good and bad. The process of carcinogen metabolism was first recognized in the first half of the twentieth century and developed extensively in the latter half. The activation of chemicals to reactive electrophiles that become covalently bound to DNA and protein was demonstrated by Miller and Miller [Cancer 47 (1981) 2327]. Today many of the DNA adducts formed by chemical carcinogens are known, and extensive information is available about pathways leading to the electrophilic intermediates. Some concepts about the stability and reactivity of electrophiles derived from carcinogens have changed over the years. Early work in the field demonstrated the ability of chemicals to modulate the metabolism of carcinogens, a phenomenon now described as enzyme induction. The cytochrome P450 enzymes play a prominent role in the metabolism of carcinogens, both in bioactivation and detoxication. The conjugating enzymes can also play both beneficial and detrimental roles. As an example of a case in which several enzymes affect the metabolism and carcinogenicity of a chemical, aflatoxin B1 (AFB1) research has revealed insight into the myriad of reaction chemistry that can occur even with a 1s half-life for a reactive electrophile. Further areas of investigation involve the consequences of enzyme variability in humans and include areas such as genomics, epidemiology, and chemoprevention.

Aflatoxin B1↗
PubMed2001

Metabolism of ochratoxin A: absence of formation of genotoxic derivatives by human and rat enzymes.

Ochratoxin A (OTA) is a potent renal carcinogen in male rats, although its mode of carcinogenicity is not known. The metabolism and covalent binding of OTA to DNA were investigated in vitro with cytochromes P450, glutathione S-transferases, prostaglandin H-synthase, and horseradish peroxidase. Incubation of OTA with rat or human liver microsomes fortified with NADPH resulted in formation of 4-(R)-hydroxyochratoxin A at low rates [10-25 pmol min(-1) (mg of protein)(-1)]. There was no evidence of OTA metabolism and glutathione conjugate formation with rat, mouse, or human kidney microsomes or postmitochondrial supernatants (S-9) [<5 pmol min(-1) (mg of protein)(-1)]. Recombinant human cytochromes P450 (P450) 1A1 and 3A4 formed 4-(R)-hydroxyochratoxin A at low rates [0.08 and 0.06 pmol min(-1) (pmol of P450)(-1), respectively]; no oxidation products of OTA were detected with recombinant human P450 1A2 or 2E1 or rat P450 1A2 or 2C11 [<0.02 pmol min(-1) (pmol of P450)(-1)]. Prostaglandin H-synthase produced small amounts of an apolar product [33 pmol min(-1) (mg of protein)(-1)], and OTA products were not formed with horseradish peroxidase. There was no evidence of DNA adduct formation when [(3)H]OTA was incubated with these enzyme systems in the presence of calf thymus DNA (<20 adducts/10(9) DNA bases); however, these enzymes catalyzed DNA adduct formation with the genotoxins aflatoxin B(1), 2-amino-3-methylimidazo[4,5-f]quinoline, benzo[a]pyrene, and pentachlorophenol. There was also no detectable [(3)H]OTA bound in vivo to kidney DNA of male Fischer-344 rats treated orally with [(3)H]OTA (1 mg/kg, 100 mCi/mmol, 24 h exposure, <2.7 adducts/10(9) DNA bases), based upon liquid scintillation counting. However, (32)P-postlabeling experiments did show evidence of DNA lesions with total adduct levels ranging from 31 to 71 adducts/10(9) DNA bases, while adducts in untreated rat kidney ranged from 6 to 24 adducts/10(9) DNA bases. These results do not support the premise that OTA or metabolically activated species covalently bind to DNA and suggest that the (32)P-postlabeled lesions are due to products derived from OTA-mediated cytotoxicity.

Animals↗
PubMed2001

Metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in human hepatocytes: 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid is a major detoxification pathway catalyzed by cytochrome P450 1A2.

Metabolic pathways of the mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) remain incompletely characterized in humans. In this study, the metabolism of MeIQx was investigated in primary human hepatocytes. Six metabolites were characterized by UV and mass spectroscopy. Novel metabolites were additionally characterized by 1H NMR spectroscopy. The carcinogenic metabolite, 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline, which is formed by cytochrome P450 1A2 (P450 1A2), was found to be transformed into the N(2)-glucuronide conjugate, N(2)-(beta-1-glucosiduronyl)-2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline. The phase II conjugates N(2)-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)sulfamic acid and N(2)-(beta-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, as well as the 7-oxo derivatives of MeIQx and N-desmethyl-MeIQx, 2-amino-3,8-dimethyl-6-hydro-7H-imidazo[4,5-f]quinoxalin-7-one (7-oxo-MeIQx), and 2-amino-6-hydro-8-methyl-7H-imidazo[4,5-f]quinoxalin-7-one (N-desmethyl-7-oxo-MeIQx), thought to be formed exclusively by the intestinal flora, were also identified. A novel metabolite was characterized as 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH), and it was the predominant metabolite formed in hepatocytes exposed to MeIQx at levels approaching human exposure. IQx-8-COOH formation is catalyzed by P450 1A2. This metabolite is a detoxication product and does not induce umuC gene expression in Salmonella typhimurium strain NM2009. IQx-8-COOH is also the principal oxidation product of MeIQx excreted in human urine [Turesky, R., et al. (1998) Chem. Res. Toxicol. 11, 217-225]. Thus, P450 1A2 is involved in both the metabolic activation and detoxication of this procarcinogen in humans. Analogous metabolism experiments were conducted with hepatocytes of untreated rats and rats pretreated with the P450 inducer 3-methylcholanthrene. Unlike human hepatocytes, the rat cell preparations did not produce IQx-8-COOH but catalyzed the formation of 2-amino-3,8-dimethyl-5-hydroxyimidazo[4,5-f]quinoxaline as a major P450-mediated detoxication product. In conclusion, our results provide evidence of a novel MeIQx metabolism pathway in humans through P450 1A2-mediated C(8)-oxidation of MeIQx to form IQx-8-COOH. This biotransformation pathway has not been detected in experimental animal species. Considerable interspecies differences exist in the metabolism of MeIQx by P450s, which may affect the biological activity of this mutagen and must be considered when assessing human health risk.

Animals↗
PubMed2001

Reaction of trichloroethylene oxide with proteins and dna: instability of adducts and modulation of functions.

Trichloroethylene (TCE) shows several types of toxicities, some of which may be the result of bioactivation. Oxidation by P450s yields the electrophile TCE oxide. We previously analyzed N(6)-acyllysine adducts formed from the reaction of TCE oxide with proteins [Cai, H., and Guengerich, F. P. (2000) Chem. Res. Toxicol. 13, 327-335]; however, we had been unable to measure ester adducts under the prolonged conditions of proteolysis and derivatization. Protein amino acid adducts were directly observed by mass spectrometry during the reaction of TCE oxide with the model polypeptides insulin and adrenocorticotropic hormone (ACTH, residues 1-24). The majority (80%) of the protein adducts were unstable under physiological conditions and had a collective t(1/2) of approximately 1 h, suggesting that they are ester type adducts formed from reactions of Cys, Ser, Tyr, or Thr residues with intermediates formed in TCE oxide hydrolysis. Synthetic O-acetyl-L-Ser and O-acetyl-L-Tyr had half-lives of 1 h and 10 min at pH 8.0, respectively, similar to the stabilities of the protein adducts. The effects of TCE oxide adduct formation on catalytic activities were examined with five model enzymes. No recovery of catalytic activity was observed during the reaction of TCE oxide with two model enzymes for which the literature suggests roles of a Lys, rabbit muscle aldolase and glucose-6-phosphate dehydrogenase. However, in the cases of papain (essential Cys residue in the active site), alpha-chymotrypsin (critical Ser residue), and D-amino acid oxidase (essential Cys and Tyr residues), time-dependent recoveries of enzyme activity were observed following reaction with TCE oxide or either of two model nucleophiles (dichloroacetyl chloride and acetic formic anhydride), paralleling the kinetics of removal of adducts from insulin and ACTH. Formation of adducts ( approximately 2%) was detected in the direct reaction of TCE oxide with 2'-deoxyguanosine, but not with the other three nucleosides found in DNA. During the reaction of TCE oxide with a synthetic 8-mer oligonucleotide, formation of adducts was observed by mass spectrometry. However, the adducts had a t(1/2) of 30 min at pH 8.5. These results indicate the transient nature of the adducts formed from the reaction of TCE oxide with macromolecules and their biological effects.

Adrenocorticotropic Hormone↗