Acetaminophen bioactivation by human cytochrome P450 enzymes and animal microsomes.

Acetaminophen is a widely used analgesic antipyretic agent. When used at low doses, it is a safe drug, but at higher doses it can cause acute hepatic necrosis in humans and experimental animals. The key mechanism in the hepatotoxicity is cytochrome P450 (CYP)-catalysed formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) that is capable of binding to cellular macromolecules and in that way an LC/MS liquid chromatography/mass spectrometry (LC/MS) method was developed to measure NAPQI formation by trapping it to reduced glutathione. This method was used to determine the bioactivation of acetaminophen at two concentrations: 50 microM therapeutic and 1 mM toxic by using nine human recombinant CYP enzymes: CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4; and with different microsomes from experimental animals. At the toxic concentration the formation of NAPQI-glutathione was highest with CYP3A4 followed by CYP2E1, CYP1A2, and CYP2D6. At the therapeutic concentration, CYP3A4 had also the highest bioactivation capacity. In a comparison of the enzyme kinetics, CYP3A4 was the most efficient CYP with the lowest K(m) value 130 microM (95% confidence interval = 63-210 microM). Dexamethasone-induced rat liver microsomes had the most effective bioactivation capacity at therapeutic and toxic acetaminophen concentrations. This study suggests that CYP3A4 is the major CYP enzyme form catalysing acetaminophen oxidation to NAPQI in human liver.

Nicotine metabolism and urinary elimination in mouse: in vitro and in vivo.

This study aimed at elucidating the in vivo metabolism of nicotine both with and without inhibitors of nicotine metabolism. Second, the role of mouse CYP2A5 in nicotine oxidation in vitro was studied as such information is needed to assess whether the mouse is a suitable model for studying chemical inhibitors of the human CYP2A6. The oxidation of nicotine to cotinine was measured and the ability of various inhibitors to modify this reaction was determined. Nicotine and various inhibitors were co-administered to CD2F1 mice, and nicotine and urinary levels of nicotine and four metabolites were determined. In mouse liver microsomes anti-CYP2A5 antibody and known chemical inhibitors of the CYP2A5 enzyme blocked cotinine formation by 85-100%, depending on the pre-treatment of the mice. The amount of trans-3-hydroxycotine was five times higher than cotinine N-oxide, and ten times higher than nicotine N-1-oxide and cotinine. Methoxsalen, an irreversible inhibitor of CYP2A5, significantly reduced the metabolic elimination of nicotine in vivo, but the reversible inhibitors had no effect. It is concluded that the metabolism of nicotine in mouse is very similar to that in man and, therefore, that the mouse is a suitable model for testing novel chemical inhibitors of human CYP2A6.

Identification of inhibitors of the nicotine metabolising CYP2A6 enzyme--an in silico approach.

The compulsive nature of tobacco use is attributable to nicotine addiction. Nicotine is eliminated by metabolism through the cytochrome P450 2A6 (CYP2A6) enzyme in liver. Inhibition of CYP2A6 by chemical compounds may represent a potential supplement to anti-smoking therapy. The purpose of this study was to rationally design potent inhibitors of CYP2A6. 3D-QSAR models were constructed to find out which structural characteristics are important for inhibition potency. Specifically located hydrophobic and hydrogen donor features were found to affect inhibition potency. These features were used in virtual screening of over 60,000 compounds in the Maybridge chemical database. A total of 22 candidate molecules were selected and tested for inhibition potency. Four of these were potent and selective CYP2A6 inhibitors with IC(50) values lower than 1 muM. They represent novel structures of CYP2A6 inhibitors, especially N1-(4-fluorophenyl)cyclopropane-1-carboxamide. This compound can be used as a lead in the design of CYP2A6 inhibitor drugs to combat nicotine addiction.

Lactobacillus rhamnosus strain GG reduces aflatoxin B1 transport, metabolism, and toxicity in Caco-2 Cells.

The probiotic Lactobacillus rhamnosus GG is able to bind the potent hepatocarcinogen aflatoxin B1 (AFB1) and thus potentially restrict its rapid absorption from the intestine. In this study we investigated the potential of GG to reduce AFB1 availability in vitro in Caco-2 cells adapted to express cytochrome P-450 (CYP) 3A4, such that both transport and toxicity could be assessed. Caco-2 cells were grown as confluent monolayers on transmembrane filters for 21 days prior to all studies. AFB1 levels in culture medium were measured by high-performance liquid chromatography. In CYP 3A4-induced monolayers, AFB1 transport from the apical to the basolateral chamber was reduced from 11.1%+/-1.9% to 6.4%+/-2.5% (P=0.019) and to 3.3%+/-1.8% (P=0.002) within the first hour in monolayers coincubated with GG (1x10(10) and 5x10(10) CFU/ml, respectively). GG (1x10(10) and 5x10(10) CFU/ml) bound 40.1%+/-8.3% and 61.0%+/-6.0% of added AFB1 after 1 h, respectively. AFB1 caused significant reductions of 30.1% (P=0.01), 49.4% (P=0.004), and 64.4% (P<0.001) in transepithelial resistance after 24, 48, and 72 h, respectively. Coincubation with 1x10(10) CFU/ml GG after 24 h protected against AFB1-induced reductions in transepithelial resistance at both 24 h (P=0.002) and 48 h (P=0.04). DNA fragmentation was apparent in cells treated only with AFB1 cells but not in cells coincubated with either 1x10(10) or 5x10(10) CFU/ml GG. GG reduced AFB1 uptake and protected against both membrane and DNA damage in the Caco-2 model. These data are suggestive of a beneficial role of GG against dietary exposure to aflatoxin.

New potent and selective cytochrome P450 2B6 (CYP2B6) inhibitors based on three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis.

BACKGROUND AND PURPOSE: The cytochrome P450 2B6 (CYP2B6) enzyme metabolises a number of clinically important drugs. Drug-drug interactions resulting from inhibition or induction of CYP2B6 activity may cause serious adverse effects. The aims of this study were to construct a three-dimensional structure-activity relationship (3D-QSAR) model of the CYP2B6 protein and to identify novel potent and selective inhibitors of CYP2B6 for in vitro research purposes. EXPERIMENTAL APPROACH: The inhibition potencies (IC(50) values) of structurally diverse chemicals were determined with recombinant human CYP2B6 enzyme. Two successive models were constructed using Comparative Molecular Field Analysis (CoMFA). KEY RESULTS: Three compounds proved to be very potent and selective competitive inhibitors of CYP2B6 in vitro (IC(50)<1 microM): 4-(4-chlorobenzyl)pyridine (CBP), 4-(4-nitrobenzyl)pyridine (NBP), and 4-benzylpyridine (BP). A complete inhibition of CYP2B6 activity was achieved with 0.1 microM CBP, whereas other CYP-related activities were not affected. Forty-one compounds were selected for further testing and construction of the final CoMFA model. The created CoMFA model was of high quality and predicted accurately the inhibition potency of a test set (n=7) of structurally diverse compounds. CONCLUSIONS AND IMPLICATIONS: Two CoMFA models were created which revealed the key molecular characteristics of inhibitors of the CYP2B6 enzyme. The final model accurately predicted the inhibitory potencies of several structurally unrelated compounds. CBP, BP and NBP were identified as novel potent and selective inhibitors of CYP2B6 and CBP especially is a suitable inhibitor for in vitro screening studies.

Lactobacillus rhamnosus strain GG modulates intestinal absorption, fecal excretion, and toxicity of aflatoxin B(1) in rats.

In this study, the modulation of aflatoxin B(1) (AFB(1)) uptake in rats by administration of the probiotic Lactobacillus rhamnosus GG was demonstrated. Fecal AFB(1) excretion in GG-treated rats was increased via bacterial AFB(1) binding. Furthermore, AFB(1)-associated growth faltering and liver injury were alleviated with GG treatment.

More potent inhibition of human CYP2A6 than mouse CYP2A5 enzyme activities by derivatives of phenylethylamine and benzaldehyde.

1. A rapid 96-well plate assay method was developed and validated to measure liver microsomal coumarin 7-hydroxylation in vitro. 2. The method was used to test inhibition of human and mouse CYP2A enzymes by three phenylethylamine derivatives 2-(p-tolyl)-ethylamine, amphetamine, 2-phenylethylamine and benzaldehyde, and two of its derivatives, 4-methylbenzaldehyde and 4-methoxybenzaldehyde. 3. The benzaldehyde derivatives were more potent inhibitors of CYP2A5 than the phenylethylamines. The K(ic) value of 4-methylbenzaldehyde was 3.4 micro M and for 4-methoxybenzaldehyde it was 0.86 micro M for CYP2A5. 4. Amphetamine is a weak inhibitor of CYP2A6, whereas benzaldehyde is a suicide inhibitor with K(inact) = 0.16 min(-1) and K(I) = 18 micro M. The K(ic) values of 2-phenylethylamine, 2-(p-tolyl)-ethylamine, 4-methylbenzaldehyde and 4-methoxybenzaldehyde were 1.13, 0.23, 0.36 and 0.73 micro M for CYP2A6, respectively. 5. Novel potent inhibitors were found for CYP2A6 and, except for 4-methoxybenzaldehyde, all the compounds inhibited CYP2A5 and CYP2A6 enzymes differentially. These data add to the refinement of CYP2A enzyme active sites and provide chemical leads for developing novel chemical inhibitors of the CYP2A6 enzyme.

Cytochromes P450 2A6, 2E1, and 3A and production of protein-aldehyde adducts in the liver of patients with alcoholic and non-alcoholic liver diseases.

BACKGROUND/AIMS: Interaction between CYP2E1, ethanol metabolites, and enhanced lipid peroxidation is linked to the pathogenesis of alcoholic liver disease. This study was conducted to compare the expression of various cytochrome enzymes and the appearance of aldehyde adducts in humans. METHODS: Acetaldehyde- and lipid peroxidation-derived protein adducts and CYP2A6, 2E1, and 3A4/5 were examined immunohistochemically from liver specimens of 12 alcohol abusers with either mild (n=7) or severe (n=5) liver disease, and from nine non-drinking patients with non-alcoholic steatosis (n=4), or hepatitis (n=5). RESULTS: Ethanol-inducible CYP2E1 was present in all alcoholic livers. While CYP2A6 in zone 3 hepatocytes was also abundant in the alcoholic patients with various degrees of liver disease, CYP3A415 was most prominent in alcoholic cirrhosis. The sites of CYP2E1 and CYP2A6 immunoreactivity co-localized with fatty deposits, and with the sites of acetaldehyde and lipid peroxidation-derived protein adducts. The CYP enzymes were also abundant in the centrilobular hepatocytes of patients with fatty liver due to obesity or diabetes. CONCLUSIONS: Alcohol-induced liver damage is associated with a generalized induction of CYP2A6, CYP2E1 and CYP3A4 and generation of acetaldehyde and lipid peroxidation-derived protein-aldehyde adducts. However, CYP induction also occurred in patients with non-alcoholic steatosis.

Pronounced differences in inhibition potency of lactone and non-lactone compounds for mouse and human coumarin 7-hydroxylases (CYP2A5 and CYP2A6).

1. The structural requirements for a compound to be a potent inhibitor for mouse CYP2A5 and human CYP2A6 enzymes catalysing coumarin 7-hydroxylase activity have been studied. 2. The IC50 of 28 compounds for the pyrazole-treated male DBA/2 mouse and human liver microsomal coumarin 7-hydroxylation were determined at 10 microm coumarin concentration 15 times over Km of coumarin. 3. The three most potent inhibitors for CYP2A5 were gamma-nonanoic lactone, gamma-decanolactone and gamma-phenyl-gamma-butyrolactone with an IC50 = 1.9+/-0.4, 2.1+/-0.2 and 2.4+/-0.3 microM and for CYP2A67-methylcoumarin, butylcyclohexane and indan with an IC50. = 30+/-3.2, 43+/-9 and 50+/-11 microM. 4. Among the 28 compounds studied, only 2-benzoxazolinone, 2-indanone and gamma-valerolactone showed similar inhibitory activity in both species. Indan had a lower IC50 for human than for mouse coumarin 7-hydroxylation, whereas the IC50 of 24 other compounds was higher for human than for mouse coumarin 7-hydroxylation. 5. The largest difference in IC50 between mouse and human activity was observed with 5-substituted phenyl, pentyl, hexyl, heptyl or octyl gamma-lactones or 6-substituted delta-lactones. IC50 of gamma-undecanolactone and gamma-decanolactone was 500 times lower for mouse than human coumarin 7-hydroxylation. 6. The difference in the IC50 between human and mouse coumarin 7-hydroxylation decreased substantially with the corresponding compounds without the lactone ring. 7. It is concluded that certain 5- or 6-position substituted gamma- and delta-lactones are potent inhibitors for mouse CYP2A5 but not for the orthologous human CYP2A6 and that the active site of CYP2A6 could be smaller than the active site of CYP2A5.

Interaction of aflatoxin B1 with cytochrome P450 2A5 and its mutants: correlation with metabolic activation and toxicity.

Among members of the mouse cytochrome P450 2A family, P450 2A5 is the best catalyst of aflatoxin B1 (AFB1) oxidation to its 8,9-epoxide (Pelkonen, P., Lang, M., Wild, C. P., Negishi, M., and Juvonen, R. O. (1994) Eur. J. Pharmacol., Environ. Toxicol. Pharmacol. Sect. 292, 67-73). Here we studied the role of amino acid residues 209 and 365 of the P450 2A5 in the metabolism and toxicity of AFB1 using recombinant yeasts. The two sites have previously been shown to be essential in the interaction of coumarin and steroids with the P450 2A5. Reducing the size of the amino acid at position 209 or introducing a negatively charged residue at this site increased the 8,9-epoxidation of AFB1 compared to the wild type. In addition, replacing the hydrophobic amino acid at the 365 position with a positively charged lysine residue strongly decreased the metabolism of AFB1. These mutations changed the KM values generally less than the Vmax values. The changes in AFB1 metabolism contrast with the changes in coumarin 7-hydroxylation caused by these amino acid substitutions, since reducing the size of the 209 residue strongly reduced coumarin metabolism and increased the K(M) values. On the other hand, the results with AFB1 are similar to those obtained with steroid hydroxylation. This suggests that the size of the substrate is important when interacting with the residue 209 of the protein. The catalytic parameters of AFB1 correlated generally with its toxicity to the recombinant yeasts expressing the activating enzyme and with the binding of AFB1 to yeast DNA. Furthermore high affinity substrates and inhibitors (e.g., methoxsalen, metyrapone, coumarin 311, 7-methylcoumarin, coumarin, and pilocarpine) of P450 2A5 could efficiently block the toxicity of AFB1. It is suggested that the recombinant yeasts expressing engineered P450 enzymes are a useful model to understand the substrate protein interactions, to study the relationship of metabolic parameters to toxicity, and to test potential inhibitors of metabolism based toxicity.

CYP enzymes catalyze the formation of a terminal olefin from 2-ethylhexanoic acid in rat and human liver.

1. The metabolism of 2-ethylhexanoic acid (2-EHA) was studied in rat, mouse and human liver microsomes in vitro. The metabolites of 2-EHA were identified as methylated derivatives by gas chromatography-mass spectrometry. 2. 2-Ethyl-1,6-hexanedioic acid was the main metabolite produced in rat, mouse and human liver microsomes. Unsaturated 2-ethyl-5-hexenoic acid, a terminal olefin, was produced only in human liver microsomes and phenobarbital-induced rat liver microsomes. The cytochrome P450 (CYP) inhibitors metyrapone, SKF 525A, triacetyloleandomycin (TAO), quinidine and the cytochrome P450 reductase antibody abolished its formation both in rat and human microsomes. 3. The metabolites were analyzed also in vivo in urine of 2-EHA-exposed rats and in urine of sawmill workers exposed occupationally to 2-EHA. Both rat and human urine contained 2-ethyl-1,6-hexanedioic acid as the main metabolite and also 2-ethyl-5-hexenoic acid. Metyrapone, SKF 525A and TAO all decreased drastically the formation of 2-ethyl-5-hexenoic acid in the rat. 4. The data indicate that (1) several CYP families (CYP2A, CYP2B, CYP2D and CYP3A) could be responsible for the hepatic metabolism of 2-EHA, (2) the same metabolites were formed in rats and man and (3) an unsaturated terminal olefin, 2-ethyl-5-hexenoic acid is formed in the liver.

Structural flexibility and functional versatility of cytochrome P450 and rapid evolution.

P450 represents a large group of heme-thiolate enzymes that exhibit remarkably diverse activities for the metabolism of numerous endogenous and exogenous chemicals. Recent site-directed mutagenesis studies indicate that a single mutation at any of the key residues can be enough to alter the substrate and/or product specificities in the P450 activities. Molecular modeling predicts that these key residues are located within the substrate heme pocket. Structural elements involved in diversifying P450 activity appear to correspond to the B' helix, the F helix and the F/G interhelical loop in the bacterial P450s. Structures represented by these regions are extremely variable despite the fact that the core of the P450 substrate pocket is well conserved. A mutation within these regions may result in a significant geometrical alteration of the pocket and lead to diversify the P450 activity. Phylogenetical analysis shows a relatively high rate of nonsynonymous substitution within these substrate binding regions. The functional versatility of P450 can thus be largely accounted for in terms of pocket change brought about by rapid mutations.

Competitive inhibition of coumarin 7-hydroxylation by pilocarpine and its interaction with mouse CYP 2A5 and human CYP 2A6.

1. We have shown earlier that pilocarpine strongly inhibits mouse and human liver coumarin 7-hydroxylase activity of CYP 2A and pentoxyresorufin O-deethylase activity of CYP 2B in vitro. Since pilocarpine, like coumarin, contains a lactone structure we have studied in more detail its inhibitory potency on mouse and human liver coumarin 7-hydroxylation. 2. Pilocarpine was a competitive inhibitor of coumarin 7-hydroxylase in vitro both in mouse and human liver microsomes although it was not a substrate for CYP 2A5. Ki values were similar, 0.52 +/- 0.22 microM in mice and 1.21 +/- 0.51 microM in human liver microsomes. 3. Pilocarpine induced a type II difference spectrum in mouse, human and recombinant CYP 2A5 yeast cell microsomes, with Ka values of 3.7 +/- 1.6, 1.6 +/- 1.1 and 1.5 +/- 0.1 microM, respectively. 4. Increase in pH of the incubation medium from pH 6 to 7.5 increased the potency of inhibition of coumarin 7-hydroxylation by pilocarpine. 5. Superimposition of pilocarpine and coumarin in such a way that their carbonyls, ring oxygens and the H-7' of coumarin and N-3 of pilocarpine overlap yielded a common molecular volume of 82%. 6. The results indicate that pilocarpine is a competitive inhibitor and has a high affinity for mouse CYP 2A5 and human CYP 2A6. In addition the immunotype nitrogen of pilocarpine is coordinated towards the haem iron in these P450s.

A sensitive microassay reveals marked regional differences in the capacity of rat brain to generate carbon monoxide.

Heme oxygenase activity is the sole known physiological source for the production of carbon monoxide (CO), a gaseous messenger candidate. A sensitive radioenzymatic microassay was validated to study regional distribution of heme oxygenase activity within the rat brain. The assay utilized a 14,000 X g supernatant of brain homogenate and [14C]heme as the substrate. Thin layer chromatography revealed that incubation of cerebellar supernatant with (14C]heme yielded a single reaction product, indistinguishable from bilirubin, that was selectively extracted into toluene. Radioactivity in toluene increased linearly in respect to time and added protein, was totally dependent on NADPH and was not detected with boiled homogenate. The reaction was dose-dependently inhibited by Zn-protoporphyrin IX (IC50 0.3 microM) and by an antibody generated against rat NADPH-cytochrome P450 reductase indicating specific involvement of heme oxygenase. As little as 36 fmol [14C]bilirubin/min could be readily detected requiring only microgram-quantities of cerebellar homogenate. Heme oxygenase activity measurements from discrete brain regions revealed for the first time marked differences in enzyme activity with the increasing order: frontal cortex < cerebellum = caudate-putamen < hippocampus = hypothalamus = colliculi << trapezoid body. This activity pattern closely reflects the distribution of immunoreactivity and mRNA for heme oxygenase. The present microassay should offer a valuable tool for studies directly assessing a possible role for CO in neural signaling.

Molecular engineering of microsomal P450 2a-4 to a stable, water-soluble enzyme.

Peptitergented P450 2a-4 (Pepti-P450), a water-soluble form of the mouse microsomal P450 2a-4, was genetically engineered and expressed in Escherichia coli. The NH2-terminal hydrophobic sequence (positions 2 to 19) of Pepti-P450 was replaced by a peptitergent PD1, amphipathic peptide consisting of 24 residues (C. E. Schafmeister, L. J. Miercke, and R. M. Stroud (1993) Science 262, 734-738). The expression level of Pepti-P450 (90,000 molecules/cell) was at least four times greater than that of wild-type P450 2a-4. Since Pepti-P450 was quite stable and was expressed as a peripheral membrane protein, it can be easily purified from the membrane fraction treated with Na2CO3 without using any detergents during the chromatographic steps. The purified Pepti-P450 retained the spectral and catalytic properties of the unmodified enzyme with a similar Km value for steroid 15 alpha-hydroxylase activity (19.7 microM in comparison to 14.2 microM of the wild-type). Gel permeation chromatography showed that the purified Pepti-P450 in the detergent-free buffer was an oligomer with an approximate molecular mass of 450 kDa. The replacement of the hydrophobic anchor domain with an amphipathic helix such as peptitergent, therefore, may provide a general method for engineering membrane-bound P450s to soluble enzymes.

The inhibition of CYP enzymes in mouse and human liver by pilocarpine.

1. Pilocarpine is a cholinomimetic natural alkaloid. Its interactions with testosterone hydroxylations, coumarin 7-hydroxylase (COH), dimethylnitrosamine N-demethylase (DMNA), pentoxyresorufin O-dealkylase (PROD) and 7-ethoxyresorufin O-deethylase (EROD), which are indicative of the activities of cytochrome P4502A5 (CYP2A5) or 6, 2E1, 2B, 1A, were examined in mouse and human liver microsomes. 2. In mouse liver microsomes the IC50 values of pilocarpine were 6 microM for COH and testosterone 15 alpha-hydroxylase (T15 alpha OH) activities, 4 microM for PROD, approximately 100 microM for DMNA and testosterone 6 beta-hydroxylase (T6 beta OH) activities and > 1 mM for EROD activity. 3. In human liver microsomes, the IC50 value for COH was 6 microM and for DMNA 10 microM; T15 alpha OH and PROD activities were not detectable but T6 beta OH and testosterone 16 beta/2 beta-hydroxylase activities were moderately inhibited (IC50 70 microM). 4. These results suggest that pilocarpine has (i) a high affinity towards phenobarbitone-inducible CYP2A4/5 and CYP2B activities in mouse liver, (ii) a high affinity towards CYP2A6 in human liver microsomes and (iii) a moderate affinity towards CYP3A enzyme(s) in both microsomal preparations. 5. The low IC50 concentrations in vitro indicate potential metabolic interactions between pilocarpine and several P450 enzymes.

The role of CYP enzymes in cocaine-induced liver damage.

Cocaine is hepatotoxic in several species, including man. A high dose of cocaine produces metabolism-dependent, mainly pericentral, liver damage. At 24 h after a single dose of cocaine, mouse hepatic P450 content decreases but CYP2A activities; coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylase increase concomitant with prominent diffuse cell necrosis. Repeated administration of cocaine for up to 5 days decreases CYP1A1/2, 2A4/5, 2Cx, and 2E1 related enzymatic activities. However, after five doses of cocaine, CYP2B10 increases in conjunction with the healing process. In the acute phase, the increased CYP2A activities do not participate in cocaine bioactivation. CYP3A enzymes are principally responsible for the cocaine N-demethylation in human and mouse liver microsomes. The hepatic metabolic CYP enzyme profile will change during prolonged cocaine intake, this being accompanied by altered cell morphology. Possible connections to cocaine toxicity in man are discussed.

Activation of aflatoxin B1 by mouse CYP2A enzymes and cytotoxicity in recombinant yeast cells.

The ability of three highly homologous mouse liver CYP2A enzymes to activate aflatoxin B1 was studied by expressing them in recombinant AH22 Saccharomyces cerevisiae yeast cells. The reconstituted monooxygenase complex with CYP2A5 purified from yeast cell microsomes produced epoxide at a rate of 17.2 nmol/min per nmol P450 in the presence of 50 microM aflatoxin B1 while CYP2A4 had about 10% and P4507 alpha only 1.5% of this activity. However, Km values were 530 and 10 microM and Vmax values 12.5 and 14.3 nmol/min per nmol P450 for CYP2A4 and CYP2A5, respectively. When recombinant yeast cells were exposed to aflatoxin B1 LC50 concentrations were 7.5 +/- 5.5 microM for CYP2A4, 0.45 +/- 0.10 microM for CYP2A5 and > 320 microM for P4507 alpha expressing yeast cells. Aflatoxin B1-DNA adduct levels in the same yeast cells were 50, 890 pmol/mg DNA and below detection limit when 3.0 microM aflatoxin B1 was used in the incubation mixture. Coumarin an inhibitor for CYP2A4 and a substrate for CYP2A5 diminished the toxicity of aflatoxin B1 in a dose-dependent manner for these recombinant yeast cells. These data demonstrate that (1) highly homologous mouse CYP2A enzymes activate aflatoxin B1 in a different manner and (2) that recombinant yeast cells expressing mammalian CYP enzymes are a useful and inexpensive system to test the role of different enzymes in aflatoxin B1 toxicity. The data also indicate that mouse CYP2A5 and its counterpart in other species could have a significant role in aflatoxin B1 toxicity in organs where it is expressed at high levels.

Structural alteration of mouse P450coh by mutation of glycine-207 to proline: spin equilibrium, enzyme kinetics, and heat sensitivity.

Mouse cytochrome P450coh is a high-spin haem protein which specifically catalyses coumarin 7-hydroxylase activity. A mutation of Gly-207 to Pro shifts the P450coh completely to the low-spin form, indicating that the sixth axial position of the haem is hexaco-ordinated with a water molecule in the mutant G207P. Moreover, the G207P mutation increases the Km value for coumarin 7-hydroxylase activity 100-fold and the Kd value for coumarin binding 200-fold. Conversely, the mutation decreases the Ki and Kd values 10- and 20-fold respectively when testosterone, a larger molecule, is used as a substrate. The results, therefore, are consistent with an idea that the substrate pocket may be larger in the mutant G207P than in the wild-type cytochrome P-450. A Gly-207 to Ala mutation (G207A) of P450coh (G207A), on the other hand, affects neither the spectral nor the enzymic properties of P450coh. Pro-207, through cis/trans isomerization or formation of a kink, may confer on the G207P a structural alteration of its substrate-haem pocket. Our previous studies [Iwasaki, Juvonen, Lindberg and Negishi (1991) J. Biol. Chem. 266, 3380-3382; Juvonen, Iwasaki and Negishi (1991) J. Biol. Chem. 266, 16431-16435] show that the residue at position 209 in P450coh resides close to the sixth axial position of the haem, and the spin equilibrium of the cytochrome P-450 shifts toward the high-spin state as residue 209 becomes more hydrophobic and larger. A Gly-207 to Pro mutation, therefore, results in the creation of a larger substrate pocket in the mutant cytochrome P-450 by altering the protein structure around residue 209 so that a water molecule and testosterone can be accommodated.

Site-directed mutagenesis of mouse steroid 7 alpha-hydroxylase (cytochrome P-450(7) alpha): role of residue-209 in determining steroid-cytochrome P-450 interaction.

We have cloned a cDNA encoding mouse steroid 7 alpha-hydroxylase P450(7) alpha (cytochrome P-450(7) alpha) and expressed it in Saccharomyces cerevisiae. Mouse P450(7) alpha is 70% identical in its amino acid sequence with the mouse steroid 15 alpha-hydroxylase P450(15) alpha (2A4). The Leu at position 209 of P450(15) alpha is the most important residue to determine the steroid hydroxylase activity of the P450 [Lindberg and Negishi (1989) Nature (London) 339, 632-634]. The P450(7) alpha contains Asn at the position corresponding to the Leu-209 of P450(15) alpha, although both P450s hydroxylate testosterone. The CO-reduced P450(7) alpha complex is unstable, so that it is quickly converted into the inactive P420, whereas the P450(15) alpha is very stable. The P450(7) alpha, however, is stabilized either by addition of testosterone or by a mutation of Asn-209 to Leu. The mutant P450(7) alpha displays a 17-fold lower Vmax. value than the wild-type enzyme. Unexpectedly, it also has 3-fold lower Km and Kd values. Residue 209 in P450(7) alpha, therefore, appears to be located at a critical site of the haem-substrate-binding pocket. Corticosterone inhibits the testosterone 7 alpha-hydroxylase activity of the wild-type P450(7) alpha, whereas it does not inhibit the mutant P450(7) alpha. Conversely, the P450(15) alpha activity becomes inhibited by corticosterone upon the replacement of Leu-209 by Asn. In addition, this mutation increases the corticosterone 15 alpha-hydroxylase activity of P450(15) alpha at least 20-fold. Whereas the inhibition by corticosterone depends on the presence of Asn at position 209, deoxycorticosterone inhibits the activities of the P450s regardless of the type of residue at 209. The results indicate, therefore, that the identity of residue 209 determines the affinity as well as specificity of steroid binding to both P450(7) alpha and P450(15) alpha.