Silver Nanoforms as a Therapeutic Agent for Killing Escherichia coli and Certain ESKAPE Pathogens.

The scope of this study included the preparation of silver nanoforms with high antimicrobial efficacy, low cost, and ease of application. The term 'silver nanoforms' refers to silver located on the amorphous or crystalline titanium dioxide (TiO2). Silver nanoforms may be used as an alternative to antibiotics in killing bacteria. Pure and silver-incorporated titanium (used as a carrier) was prepared using the sol-gel-modified method. Physical and chemical properties of the samples were described, and the antibacterial activity was indicated using the following strains of bacteria: Staphylococcus aureus, Klebsiella pneumoniae (ESKAPE pathogens), and Escherichia coli. The results have shown that the antibacterial activity of silver nanoforms with amorphous TiO2 is much better than that in the samples based on anatase (crystalline TiO2). The sensitivity of the tested bacteria to silver nanoforms depends on physical and chemical properties of the nanoforms and individual characteristics of the bacteria. For the first time, significant participation of amorphous TiO2 in antibacterial compounds has been described through this study.

Selection and electrophoretic characterization of Salmonella enterica subsp. enterica biocide variants resistant to antibiotics.

The proposed research outlines a serious common concern of Salmonella resistance to antimicrobials following prolonged exposure to the disinfectants (biocides). These phenotypes of bacteria could potentially result in hard to treat infections. Typical for avian sources, biocide sensitive S. enterica subsp. enterica serovars: Typhimurium, Enteritidis, Virchow and Zanzibar and their isogenic biocide-tolerant variants were studied in order to investigate bacteriostatic effect of two commercially available biocide formulations: potassium peroxymonosulfate (P) and dodecylamine based structure (triamine, D). We found that cultivating of the bacteria in the medium supplemented with a blend containing P did not influence their antibiotic susceptibility pattern. In contrast, tolerance of bacteria to D compound resulted in resistance to co-trimoxazole, cefotaxime and ciprofloxacin of which two cefotaxime and ciprofloxacin are used commonly for the treatment of invasive Salmonella infections in humans. The dependency between OMP patterns and the level of Salmonella survival in media containing the biocides was observed merely in serovar Typhimurium. In conclusion, these results suggest that Salmonella strains challenged by prolonged treatment with the disinfectants become resistant to antibiotics, however it depends on Salmonella serovar and the chemical used. This paper also highlights the loop-mediated isothermal amplification (LAMP) as a technique that offers great benefits to microbiological detecting of Salmonella species in the samples.

Influence of microflora composition on safety and colour parameters of "kumpia wieprzowa" during ripening.

The aim of this study was to define the influence of microbiological activity on the safety (microflora composition, biogenic amine amount) and colour of "kumpia wieprzowa" during the 3-month ripening period. The study included the amount of aerobic bacteria, yeast, lactobacilli rods, coagulase-negative cocci, pH and colour parameters as well as the content of nitrates (V) and (III), biogenic amines and amino acids. The lactobacilli and cocci constituted the predominant microflora of the ready-to-eat product (4.9-5.2 and 5.2-5.4 log cfu/g, respectively), although further mesophilic bacteria identification revealed the presence of numerous aerobic, aerotolerant and anaerobic species, mostly gram-positive, spore- and non-spore-forming. The absence of 2-phenylethylamine and putrescine and the low level of tryptamine (2.5 mg/kg) at the beginning of the ripening as well as the increase of tyramine and spermine amounts from 11.5 and 2.7 to 21.9 and 4.0 mg/kg, respectively during the treatment, denoted the good quality of raw meat used and dynamic growth of the desired acidifying and denitrifying microorganisms. The development of the coagulase-negative cocci population corresponded with the a* and C* colour parameters and the nitrate (III) content increase, the final result of which was 26.9, 27.5 as well as 19.4 mg/kg. The content of nitrates (V) and (III) was optimal to obtain a non-cured, safe and suitably coloured, long-term ripened meat product.

Superantigen types in Staphylococcus aureus isolated from patients with cystic fibrosis.

The screening of 17 SAg genes of S. aureus isolated from the sputum of cystic fibrosis (CF) patients revealed that among 47 genetically different strains, 39 (83 %) carried SAg genes. Superantigens forming enterotoxin gene cluster were detected in 20 strains. The 2nd most common superantigen type was selk detected in 13 strains. In 9 strains, selk occurred together with the sea gene. Out of 74 strains recovered from nasal carriers, 56 (75 %) were found to carry SAg genes, 38 carried egc genes, while selk was detected in 5 strains. The predominant SAg types in both investigated S. aureus populations were egc and selk/sea, but selk gene frequency was significantly higher in the CF-derived strains.

Selective biotransformation of taxol to 6 alpha-hydroxytaxol by human cytochrome P450 2C8.

The principal taxol biotransformation reaction of humans and of human hepatic in vitro preparations is 6 alpha-hydroxylation of the taxane ring, but a separate, minor hydroxylation pathway (metabolite B formation) also exists. Taxol metabolism was studied using membrane fractions from Hep G2 cells infected with recombinant vaccinia viruses that contain complementary DNAs encoding several human cytochrome P450 enzymes. Only P450 2C8 formed detectable 6 alpha-hydroxytaxol. Metabolite B formation was catalyzed by complementary DNA-expressed 3A3 and 3A4, but not by 3A5. Each P450 3A preparation catalyzed felodipine oxidation. The apparent Km and Vmax values for taxol 6 alpha-hydroxylation were 5.4 +/- 1.0 microM and 30 +/- 1.5 nmol/min/nmol P450, respectively, for complementary DNA-expressed P450 2C8; the values were 4.0 +/- 1.0 microM and 0.87 +/- 0.06 nmol/min/mg protein, respectively, for human hepatic microsomes. The inhibition of 6 alpha-hydroxytaxol formation by quercetin was competitive with an apparent Ki of 1.3 or 1.1 microM with 2C8 or hepatic microsomes, respectively; retinoic acid was inhibitory, showing an apparent Ki of 27 microM with hepatic microsomes; inhibition by tolbutamide was complex, weak, and unlikely to be clinically relevant. The correlation between hepatic 2C8 protein content and 6 alpha-hydroxytaxol formation was high (r2 = 0.82), while the correlation with 2C9 content was low (r2 = 0.38). These data show that human biotransformation routes of taxol result from catalysis by specific enzymes of two P450 families and that taxol 6 alpha-hydroxylation is a useful indicator of P450 2C8 activity in human hepatic microsomes.

Metabolic activation of 4-ipomeanol by complementary DNA-expressed human cytochromes P-450: evidence for species-specific metabolism.

4-Ipomeanol is a pulmonary toxin in cattle and rodents that is metabolically activated by cytochromes P-450 (P-450s). P-450-mediated activation of 4-ipomeanol to DNA binding metabolites was evaluated using a vaccinia virus complementary DNA expression system and an in situ DNA-binding assay. Twelve human P-450s and two rodent P-450s were expressed in human hepatoma Hep G2 cells and examined for their abilities to metabolically activate this toxin. Three forms, designated CYP1A2, CYP3A3, and CYP3A4, were able to catalyze significant production of DNA-bound metabolites of 20-, 8-, and 5-fold, respectively, above binding catalyzed by Hep G2 cells infected with wild-type vaccinia virus. These enzymes, with highest activities, are not known to be expressed in human or rodent lung. CYP2F1 and CYP4B1, two enzymes that are expressed in lung, display only modest 3- and 2-fold respective increased abilities to metabolically activate 4-ipomeanol. Two human forms were inactive and seven other human forms showed activities ranging from 0.5- to 2-fold above control level. Surprisingly, rabbit complementary DNA-expressed CYP4B1 was the most active enzyme (180-fold above control) among all P-450s tested in producing DNA-binding metabolites from this mycotoxin. These studies demonstrate a species difference in 4-ipomeanol metabolism and suggest caution when attempting to extrapolate rodent data to humans.

Estradiol metabolism by complementary deoxyribonucleic acid-expressed human cytochrome P450s.

Twelve forms of human cytochrome P450 were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. The cDNA-expressed enzymes were tested for their ability to oxidize estradiol. Incubation of [14C]estradiol with cell lysates containing P450 IA2 resulted in the production of 2-hydroxy and 4-hydroxy metabolites with substrate turnovers of 2.74 and 0.27 min-1, respectively. P450s IIIA3 and IIIA4 yielded the same metabolites at about one third the rate of P450 IA2. Low levels of estradiol hydroxylation were also catalyzed by P450s IIC9, IIIA5, and IVB1. Six other P450 forms yielded no detectable metabolism. The roles of P450s IA2, IIA3, and IIIA4 were further established by immunoinhibition using antirat P450 antibodies. Antibody that specifically binds to P450 IIIA3 and IIIA4 inhibited 60-70% of estradiol hydroxylation, and antibody against P450 IA2 inhibited 20-40% of the estradiol hydroxylase activity in microsomes from two human liver specimens, suggesting that these enzymes constitute the major forms catalyzing estradiol oxidation in human liver. Immunoinhibition results also suggest that 2-hydroxy- and/or 4-hydroxycatechol estrogens are further metabolized to other yet uncharacterized metabolites by P450s IIIA3 and IIIA4.

Predicting the cytochrome P450 mediated metabolism of xenobiotics.

The cytochrome P450s play a unique role in the metabolism of xenobiotics. Characteristics which allow a vast number of foreign compounds to be metabolized by a limited number of enzymes include broad substrate specificity and broad regioselectivity. Because of their importance in both the metabolism and toxicity of drugs and environmental contaminants, efforts are being made to use computational methods to predict these biotransformation pathways. This review describes the recent progress towards the prediction of the tertiary structures of the various P450s and the determination of the electronic characteristics of substrates which determine their tendency to be oxidized by the P450s.

Role of human cytochrome P450 3A4 and 3A5 in the metabolism of taxotere and its derivatives: enzyme specificity, interindividual distribution and metabolic contribution in human liver.

Taxotere, a promising anticancer agent, is metabolized almost exclusively in liver and excreted from bile in all species. To determine which cytochrome P450 is involved in taxotere biotransformation, 11 cDNA-expressed human cytochrome P450s were examined for their activity in the metabolism of taxotere and its derivatives. Of all P450s, cytochrome P450 3A4 and 3A5 were the most active for the oxidation of taxotere to the primary metabolite RPR104952 and for subsequent oxidation of RPR104952 to RPR111059 and RPR111026. RP70617, an epimer of taxotere was also metabolized by both P450 3A enzymes to form metabolite XII. The activity of 3A4/5 enzymes for these substrates was 4-50-fold greater than the other P450s examined. The Kms of 3A4 and 3A5 for taxotere were 0.91 and 9.28 microM, and Vmax for the formation of RPR104952 were 1.17 and 1.36 m(-1), respectively. The contribution of the 3A enzyme complex to the metabolism of taxotere in human livers from 21 individuals was assessed with the inhibitory monoclonal antibody and ranged from 64-93%. The primary oxidative metabolism of taxotere by human liver microsomes was well correlated with 3A4-dependent reactions for testosterone 6beta-hydroxylation (r2 = 0.84), taxol aromatic hydroxylation (r2 = 0.67) and aflatoxin B1 3alpha-hydroxylation (r2 = 0.63); whereas a poor correlation was found for reactions specifically catalysed by other P450s (all r2 < or =O.17). The extent of taxotere metabolism also closely correlated with levels of 3A4 enzyme in human livers quantified with immunoblot monoclonal antibody (r2 = 0.61). These results demonstrate that the P450 3A4 and 3A5 enzymes are major determinants in taxotere oxidation and suggest that care must be taken when administering this drug with other drugs that are also substrates for these enzymes.

Comparison of substrate metabolism by wild type CYP2D6 protein and a variant containing methionine, not valine, at position 374.

We have analysed kinetic parameters of cDNA-derived CYP2D6 proteins derived from the original CYP2D6 cDNA isolate (Gonzalez FJ et al. Nature 1988: 331, 442-446) which contains methionine at position 374 (CYP2D6-Met) and a modified cDNA which contains valine at position 374 (CYP2D6-Val). This latter protein is predicted from the CYP2D6 genomic sequence. Several quantitative differences, but no qualitative differences in metabolism were observed. CYP2D6-Met was found to have a two-fold lower Km and a three-fold lower turnover rate for (R)(+)-bufuralol 1'-hydroxylation as compared to CYP2D6-Val. In contrast, CYP2D6-Met and CYP2D6-Val had a similar Km for debrisoquine 4-hydroxylation while CYP2D6-Val had an 18-fold higher turnover rate. CYP2D6-Val and CYP2D6-Met had similar Kms for metoprolol but CYP2D6-Val showed a three-fold higher capacity for the O-demethylation reaction compared to alpha-hydroxylation which is more similar to that seen in human liver. In the case of sparteine, CYP2D6-Val and CYP2D6-Met showed similar capacities for formation of the 2-dehydrosparteine metabolite but the Km value for CYP2D6-Met was six-fold higher than that for CYP2D6-Val. Kinetic differences between CYP2D6-Met and CYP2D6-Val were further probed by examination of apparent Ki for inhibition of (R,S)(+/-)-bufuralol 1'-hydroxylation. Similar Ki values (within a factor of three) were observed for perhexiline and (R,S)-propranolol while quinidine and dextromethorphan were 8.5-fold and 21-fold more effective inhibitors of CYP2D6-Val relative to CYP2D6-Met. An allele specific polymerase chain reaction assay was developed for the CYP2D6-Met allele. The CYP2D6-Met allele was not found among 83 individuals. In the aggregate, these data indicated that the CYP2D6-Val allele is the more common allele in human populations. The quantitative kinetic differences between these two enzymes appears most pronounced for substrates/inhibitors with rigid structures. CYP2D6-Val more often has a substantially lower Km and/or a substantially higher capacity to metabolize those substrates.

Inhibition of the contraction of the ductus arteriosus to oxygen by 1-aminobenzotriazole, a mechanism-based inactivator of cytochrome P450.

1. We have proposed that contractile tension of the ductus arteriosus is sustained by a cytochrome P450-linked mechanism acting as a limiting step in the synthesis of endothelin-1 (ET-1). In the present study, we have used the isolated ductus from near-term foetal lambs and guinea-pigs to investigate the effect on both muscle tone and ET-1 formation of 1-aminobenzotriazole (ABT), a suicide substrate for mono-oxygenase reactions. 2. ABT relaxed the lamb ductus at rest (2.5% O2) and during the oxygen contraction (15 to 95% O2). The effect was seen at 40 microM, and at 0.8 mM active tone was almost completely abolished. ABT (1 mM) also reversed the oxygen contraction in the guinea-pig ductus. 3. In the lamb ductus, the ABT response was not affected by removal of the endothelium or by treatment with 2.8 microM indomethacin (at 2.5% O2) and the ensuing contraction. 4. At both low and high concentration, ABT relaxed marginally, or not at all, the potassium-contracted (55 mM) ductus from either species. 5. ET-1 release from either the intact or endothelium-denuded lamb ductus tended to decrease in the presence of ABT (1 mM), whilst during the same treatment cyclic GMP content of the tissue remained unchanged. 6. We conclude that ABT relaxation is due to suppression of a contractile mechanism and not to activation of prostaglandin- and NO-mediated relaxing mechanisms. This contractile mechanism has a cytochrome P450-based mono-oxygenase reaction as a key component.

Regio- and stereo-selective metabolism of phenanthrene by twelve cDNA-expressed human, rodent, and rabbit cytochromes P-450.

The regio- and stereoselective metabolism of phenanthrene (PA) by seven cDNA-expressed human and five rodent and rabbit cytochromes P-450 has been examined using reverse-phase and chiral stationary phase high-pressure liquid chromatography (HPLC). Turnover numbers ranged from 0.2 to 55 nmol/min per nmol. Using vaccinia virus expression of P-450 enzymes in Hep G2 cells, m1A1 and m1A2 were found to be the most active P-450s. Of the human P-450s, 1A2 and 2B6 have the highest activity and 2C9 has moderate activity. Using cytochrome P-450s expressed in a lymphoblastoid cell line in presence of epoxide hydrolase (EH), human 1A1 had approximately twice the activity of human 1A2. Regioselectivities for PA metabolism were found to be both isozyme and species-dependent. Stereochemical analysis revealed that the P-450s 1A1, m1A1, m1A2, r2A1, r2B1, PB- and 3MC-treated rat liver microsomes preferentially formed 3R,4R-diol enantiomer (88-97%), whereas rabbit 4B formed the 3S,4S-diol enantiomer (72%). Eleven P-450s, 3MC and PB microsomes preferentially formed 1R,2R-diol enantiomer (80-96%). This is the same stereochemistry as the precursors to some diol epoxides that are potent carcinogens.

Studies of flurbiprofen 4'-hydroxylation. Additional evidence suggesting the sole involvement of cytochrome P450 2C9.

Flurbiprofen, a non-steroidal anti-inflammatory drug (NSAID), is metabolized by both oxidation via the cytochrome P450 system and by glucuronidation. The major oxidative pathway in flurbiprofen metabolism is to a 4'-hydroxy metabolite, and recently we demonstrated that cytochrome P450 2C9 and its R144C variant were involved in this process (Tracy et al., Biochem Pharmacol 49: 1269-1275, 1995). Using complementary DNA (cDNA)-expressed cell systems, it has been demonstrated that at physiological concentrations of flurbiprofen there is a lack of involvement of P450s 1A2, 2C8, 2E1, and 3A4. In evaluating flurbiprofen as a potential probe for cytochrome P450 2C9, it is important to assess the involvement of additional P450s in this process. To this end, further studies were undertaken using specific inhibitors of P450 2C9 and P450 cDNA-expressed microsomes for P450 1A1, 2A6, 2B6, 2C19, and 2D6 to assess their potential involvement. We observed the inhibition of (R)- and (S)-flurbiprofen 4'-hydroxylation by an inhibitor of P450 2C9, sulfaphenazole (Ki = 0.07 and 0.06 microM, respectively), and the NSAID piroxicam (Ki = 10 and 7 microM, respectively). Furthermore, using microsomes from a lymphoblastoid cell line, we found that P450s 1A1, 2A6, 2B6, 2C19, and 2D6 were not involved in flurbiprofen hydroxylation at physiological concentrations of flurbiprofen. This finding is particularly important due to the sequence homology and potential substrate overlap of P450 2C9 and 2C19. These studies then provide additional evidence to suggest that P450 2C9 may be the only isoform involved to any substantial degree in flurbiprofen 4'-hydroxylation, and thus this reaction is useful as an in vitro probe for this particularly cytochrome P450 isoform and may be useful as an in vivo probe.

Cross-reactivity of thirteen monoclonal antibodies with ten vaccinia cDNA expressed rat, mouse and human cytochrome P450s.

Twelve monoclonal antibodies (MAbs) to rat cytochrome P450s and one MAb to a scup (fish) P450 have been isolated, characterized, and are currently in common use. Expression of cDNAs for different P450s from a vaccinia vector offers a rapid and simple way toward the production of individual P450s. The thirteen MAbs were examined for their cross-reactivity with ten cDNA expressed human, rat, and mouse P450s. Three MAbs to rat 1A1 and fish 1A1 cross-reacted with cDNA expressed mouse 1A1. One of the latter MAbs, 1-7-1 but none of the others cross-reacted with mouse 1A2. Surprisingly, the fish MAb to 1A1 also cross-reacted with human 2E1. Two MAbs to rat 2B1/2B2 cross-reacted with rat 2A1. An MAb to rat 2C11 cross-reacted with human 2C9. Two MAbs to rat 2E1 cross-reacted with human 2E1. Finally, two MAbs to rat 3A1 cross-reacted strongly with human 3A4. These studies open the door to constructing a library of MAbs with defined binding activity to the P450s of human and other species.

Role of cytochrome P450 2C9 and an allelic variant in the 4'-hydroxylation of (R)- and (S)-flurbiprofen.

Flurbiprofen is a chiral non-steroidal anti-inflammatory drug used in the treatment of pain or inflammation. The primary routes of biotransformation for (R)- and (S)-flurbiprofen are oxidation (presumably cytochrome P450) and conjugation. To date, the specific cytochrome P450 (P450) involved in the oxidative metabolism of this compound (specifically 4'-hydroxylation) has not been elucidated. Experiments were conducted to characterize the kinetic parameters (Km and Vmax) for the 4'-hydroxylation of (R)- and (S)-flurbiprofen in human liver microsomes, to determine if enantiomeric interactions occur when both enantiomers are present, and to identify the specific P450 form(s) involved in this reaction. In human liver microsomes, the Km and Vmax (mean +/- SD) for (R)-4'-hydroxy-flurbiprofen formation were 3.1 +/- 0.8 microM and 305 +/- 168 pmol.min-1.mg protein)-1, respectively. In comparison, the Km and Vmax (mean +/- SD) for (S)-4'-hydroxy-flurbiprofen formation were 1.9 +/- 0.4 microM and 343 +/- 196 pmol.min-1.mg protein-1, respectively. Enantiomeric interaction studies revealed a decrease in Km and Vmax for both enantiomers and an apparent loss of stereoselectivity. Racemic-warfarin, tolbutamide, alpha-naphthoflavone and erythromycin were studied as potential inhibitors of this process. The estimated Ki values for the inhibition of (R)- and (S)-4'-hydroxy-flurbiprofen formation by racemic-warfarin were 2.2 and 4.7 microM. This reaction was also inhibited by tolbutamide. In contrast, erythromycin and alpha-naphthoflavone had no appreciable effect on 4'-hydroxy-flurbiprofen formation. cDNA-expression of individual forms was used to determine which P450 was involved in 4'-hydroxy-flurbiprofen formation. P450 2C9 and an allelic variant (R144C) readily catalyzed the formation of 4'-hydroxy-flurbiprofen. P450 1A2 was also active albeit with a turnover rate 1/140th that of P450 2C9R144C (P450s 2C8, 2E1 and 3A4 were not active toward either enantiomer). The results of these studies indicate that the enantiomers of flurbiprofen may exhibit stereoselectivity with respect to enzyme affinity but have roughly equal maximum formation velocities. Additionally, these two enantiomers may compete for the enzyme resulting in lower maximum velocities for both enantiomers. Finally, of those P450 forms examined, only P450 2C9 and an allelic variant catalyzed the 4'-hydroxylation of both (R)- and (S)-flurbiprofen.

Human liver oxidative metabolism of O6-benzylguanine.

The oxidation of O6-benzylguanine, an inactivator of O6-alkylguanine-DNA alkyltransferase, was examined using human liver cytosol, microsomes, and several P450 isoforms. Incubation of O6-benzylguanine with human liver cytosol resulted in the formation of O6-benzyl-8-oxoguanine, which was inhibited by menadione, a potent inhibitor of aldehyde oxidase. Inhibition by allopurinol, a xanthine oxidase inhibitor, was less dramatic. Oxidation of O6-benzylguanine also occurred with pooled human liver microsomes and was inhibited by both furafylline and troleandomycin, selective inhibitors of CYP1A2 and CYP3A4, respectively. Human P450s CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2E1, and CYP3A4 expressed in Hep G2 hepatoma cells using vaccinia virus vectors were incubated with 10 or 200 microM O6-benzylguanine. At 10 microM, O6-benzylguanine was oxidized primarily by CYP1A2 and to a lesser extent by CYP3A4. However, an appreciable increase in CYP3A4 contribution was noted at 200 microM. CYP1A2 exhibited a more than 200-fold higher relative catalytic activity (Vmax/Km) compared with CYP3A4. Therefore, at therapeutically relevant concentrations of O6-benzylguanine, CYP1A2 could be primarily involved in its oxidation since it shows a much lower Km value (1.3 microM) than CYP3A4 (52.2 microM) and cytosol (81.5 microM). However, one would expect interindividual variation in the extent of oxidation of O6-benzylguanine depending on the levels of aldehyde oxidase, CYP1A2, and CYP3A4.

Role of cDNA-expressed human cytochromes P450 in the metabolism of diazepam.

The metabolic conversion of diazepam (DZ) to temazepam (TMZ, a C3-hydroxylation product of DZ) and N-desmethyldiazepam (NDZ, an N1-demethylation product of DZ) was studied using cDNA-expressed human cytochrome P450 (CYP) isozymes 1A2, 2B6, 2C8, 2C9, 2C9R144C, 2E1, 3A4, and 3A5 and human liver microsomes from five organ donors. Of the CYPs examined, 3A5, 3A4, and 2B6 exhibited the highest enzymatic activities with turnovers ranging from 7.5 to 12.5 nmol of product formed/min/nmol for the total metabolism of DZ, while 2C8, 2C9, and 2C9R144C showed lesser and moderate activities. 1A2 and 2E1 produced insignificant amounts of metabolites of DZ. The regioselectivity of CYPs was determined, and 2B6 was found to catalyze exclusively and 2C8, 2C9, and 2C9R144C preferentially the N1-demethylation of DZ to form NDZ. 3A4 and 3A5 catalyzed primarily the C3-hydroxylation of DZ, which was more extensive than the N1-demethylation. The ratios of TMZ to NDZ formed in the metabolism of DZ by 3A4 and 3A5 were approximately 4:1. Enzyme kinetic studies indicated that 2B6- and 2C9-catalyzed DZ metabolism followed Michaelis-Menten kinetics, whereas 3A4 and 3A5 displayed atypical and non-linear curves in Lineweaver-Burk plots. Human liver microsomes converted DZ to both TMZ and NDZ at a ratio of 2:1. Our results suggest that hepatic CYP3A, 2C, and 2B6 enzymes have an important role in the metabolism of DZ by human liver.

Inhibitory and non-inhibitory monoclonal antibodies to human cytochrome P450 3A3/4.

Cytochromes P450 3A3/4 are inordinately important P450 enzymes catalyzing the metabolism of a large variety of clinically useful drugs, steroids, and carcinogens. Two monoclonal antibodies, MAb 3-29-9 and MAb 275-1-2, were prepared to human P450 3A4 from mice immunized with baculovirus-expressed human P450 3A4. MAb 3-29-9 was a powerful inhibitor of the enzymatic activity of P450 3A3/4/5. MAb 3-29-9 inhibited the P450 3A3, 3A4, and 3A5 catalyzed metabolism of substrates of divergent molecular weights, e.g., p-nitroanisole, phenanthrene, diazepam, testosterone, taxol, and cyclosporin. However, MAb 3-29-9 did not give a western blot with P450 3A3 or 3A4. MAb 275-1-2 was non-inhibitory but yielded a strong western blot with P450 3A3 and 3A4 but not with 3A5, and thus distinguished between 3A3/4 and 3A5. The two MAbs did not cross-react with human 2E1, 1A2, 2B6, 2C8, and 2C9; rat 2A1, 3A1/2, 4A1, 4A3, and 2B1; and mouse 1A1 and 1A2. MAb 3-29-9 has been used successfully to measure the quantitative contribution of P450 3A3 and 3A4 to the metabolism of the above-designated substrates in human adult liver. MAb 3-29-9 and MAb 275-1-2 are precise and sensitive reagents for P450 3A studies.

Cytochrome P450 2A1, 2E1, and 2C9 cDNA-expression by insect cells and partial purification using hydrophobic chromatography.

High-level expression of three cloned cytochrome P450 enzymes was accomplished using the baculovirus-insect cell expression system. The amount of enzyme expression was enhanced by cell infections in the presence of medium-supplements containing hemin and by growth in suspension cultures. Human cytochromes P450 2E1 and 2C9 and rat cytochrome P450 2A1 were partially purified from cell extracts using hydrophobic interaction and hydroxyapatite chromatography. The resulting enzymes were of estimated molecular masses similar to those reported previously and analyzed by PAGE. Reconstitution of enzyme activity resulted when the enzymes were incubated together with NADPH-cytochrome P450 reductase, phospholipid, NADPH, and appropriate substrates. The cytochrome P450 activity of the partially purified enzymes was comparable to that of the corresponding enzymes expressed in the vaccinia virus-Hep G2 system. These results provide evidence for a general means of obtaining cytochrome P450 enzymes for mechanistic, immunochemical, and biophysical investigations.

Studies on the mechanism of aromatase and other cytochrome P450 mediated deformylation reactions.

Aromatase is a microsomal cytochrome P450 that converts androgens to estrogens by three sequential oxidations. The isolation of the 19-hydroxy and 19-oxo androgens suggests that the first two oxidations occur at the C19 carbon. However, the mechanism of the third oxidation, which results in C10--C19 bond cleavage, has not been determined. Two proposed mechanisms which remain viable involve either initial 1 beta-hydrogen atom abstraction or addition of the ferric peroxy anion from aromatase to the C19 aldehyde. Semiempirical molecular orbital calculations (AM1) were used to study potential reaction mechanisms initiated by initial 1 beta-hydrogen atom abstraction. Initially, the energetics of carbon--carbon bond cleavage of the keto and enol forms of C1-radicals were studied and were found to be energetically similar. A mechanism was proposed in which the 19-oxo intermediate is subject to initial nucleophilic attack by the protein. The geometry of the A-ring in the androgens is between that for the 1-radicals and estrogen, suggesting that some transition state stabilization for the homolytic cleavage reaction can occur. More recently, studies on liver microsomal cytochrome P450 mediated deformylation of xenobiotic aldehydes supports mechanisms involving an alkyl peroxy intermediate formed by addition of the ferric peroxy anion from aromatase to the C19 aldehyde. Although this intermediate could proceed through several different concerted or non-concerted pathways, one non-concerted pathway involves the heterolytic cleavage of the dioxygen bond resulting in an active oxygenating species (iron-oxene) and a diol. The diol could then undergo hydrogen atom abstraction followed by homolytic carbon--carbon bond cleavage as in the mechanisms modeled previously. When this cleavage was modeled for seven aldehydes, a good correlation with reported experimental aldehyde turnover numbers was obtained. However, when dialkoxy derivatives of the aldehydes are subject to microsomal metabolism, the rates of carbon-carbon cleavage products do not approach the rates of deformylation of the aldehyde analog.