Role of human cytochromes P450 in the metabolic activation of chemical carcinogens and toxins.
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Animals↗
Biomedical subjects
F J Gonzalez
Publications and source records attributed to F J Gonzalez.
1462V mutation in the human CYP1A1 gene: lack of correlation with either the Msp I 1.9 kb (M2) allele or CYP1A1 inducibility in a three-generation family of east Mediterranean descent.
A 15-member three-generation family of Eastern Mediterranean descent was previously studied, and an association between CYP1A1 (cytochrome P1450, benzo[a]pyrene hydroxylase) inducibility and a CYP1A1 3'-polymorphism (the Msp I 1.9 kb allele) was reported (Petersen et al., Am J Hum Genet 1991:48, 720-725). Here we have re-examined the original DNA (and in some cases, newly prepared DNA from freshly drawn blood) from these same individuals, in order to assess the association between CYP1A1 inducibility and both the CYP1A1 gene Msp I RFLP polymorphism and the CYP1A1 gene A-->G polymorphism at codon 462. This latter nucleotide change results in an altered amino acid (462Ile-->Val), which is purported to increase CYP1A1 enzyme activity and mutagenicity towards benzo[a]pyrene about two-fold among Japanese. Among the 15 members of this three-generation family examined, no absolute correlation was observed between the 1462V genotype and either the Msp I 1.9 kb allele or the CYP1A1 inducibility phenotype. We also found no absolute correlation between the Msp I 1.9 kb allele and the CYP1A1 inducibility phenotype.
Alleles↗
O-demethylation of epipodophyllotoxins is catalyzed by human cytochrome P450 3A4.
We previously demonstrated that O-demethylation of the pendant dimethoxyphenol ring of epipodophyllotoxins to produce their respective catechol metabolites is catalyzed by cytochrome(s) P450 in human liver microsomes. Our objective was to identify the specific human cytochrome(s) P450 responsible for catechol formation. Using a panel of prototypical substrates and inhibitors for specific cytochromes P450, we identified substrates for CYP3A4 (midazolam, erythromycin, cyclosporin, and dexamethasone) as inhibitors of catechol formation from both etoposide and teniposide. Dexamethasone inhibition was competitive, with Ki values of 60 and 45 microM for etoposide and teniposide, respectively. In 58 human livers, the correlation coefficients for teniposide catechol formation versus 1'- and 4-hydroxymidazolam formation were 80% and 85%, respectively; for etoposide catechol formation versus 1'- and 4-hydroxymidazolam formation r2 was 83% and 79%, respectively. Teniposide and etoposide catechol formation rates were also significantly correlated with immunodetectable CYP3A (r2 = 49% and 51%, respectively) and not with immunodetectable CYP1A2, 2E1, or 2C8. Finally, cDNAs for human CYP3A4, 3A5, 2A6, 2B6, 2C8, and 2C9 were functionally expressed in HepG2 cells, using a vaccinia viral vector. Teniposide and etoposide catechol formation was catalyzed primarily by 3A4 (15.4 and 40.9 pmol/pmol/hr, respectively) and to a lesser degree by 3A5 (1.94 and 11.3 pmol/pmol/hr, respectively), whereas there was no detectable O-demethylation of epipodophyllotoxins by 2A6, 2B6, 2C8, 2C9, or the control virus alone. Moreover, the relative activities of midazolam hydroxylation, compared with O-demethylation of epipodophyllotoxins, were similar for heterologously expressed 3A4 and for human liver microsomes. We conclude that catechol formation from teniposide and etoposide is primarily mediated by human CYP3A4, making these reactions susceptible to inhibition by prototypical 3A substrates and inhibitors.
Biotransformation↗
Validation of 4-nitrophenol as an in vitro substrate probe for human liver CYP2E1 using cDNA expression and microsomal kinetic techniques.
The involvement of human cytochrome P450 (CYP) 2E1 in the hydroxylation of 4-nitrophenol (4NP) to 4-nitrocatechol (4NC) has been investigated using cDNA expression and liver microsomal kinetic and inhibitor techniques. 4NP hydroxylation by human liver microsomes and cDNA-expressed human CYP2E1 exhibited Michaelis-Menten kinetics; the respective apparent Km values were 30 +/- 7 and 21 microM. Mutual competitive inhibition was observed for 4NP and chlorzoxazone (CZ) (an alternative human CYP2E1 substrate) in liver microsomes, with close similarities between the calculated apparent Km and Ki values for each individual compound. 4NP and CZ hydroxylase activities in microsomes from 18 liver donors varied to a similar extent (3.3- and 3.0-fold, respectively) and 4NP hydroxylase activity correlated significantly (rs > or = 0.75, P < 0.005) with both CZ hydroxylation and immunoreactive CYP2E1 content. The prototypic CYP2E1 inhibitor, diethyldithiocarbamate, was a potent inhibitor of 4NC formation and decreased 4NP hydroxylation by cDNA-expressed CYP2E1 and human liver microsomes in parallel. Probes for other human CYP isoforms namely (alpha-naphthoflavone, coumarin, sulphaphenazole, quinidine, troleandomycin and mephenytoin) caused < 15% inhibition of liver microsomal 4NP hydroxylation. These data confirm that, as in animal species, 4NP hydroxylation is catalysed largely by CYP2E1 in human liver and 4NP may therefore be used as an in vitro substrate probe for the human enzyme.
Base Sequence↗
Glycosylation-dependent activity of baculovirus-expressed human liver carboxylesterases: cDNA cloning and characterization of two highly similar enzyme forms.
A cDNA, designated hCE, encoding the entire sequence of a carboxylesterase, was isolated from a human liver lambda gt11 library. The hCE-deduced protein sequence contained 568 amino acids, including an 18 amino acid signal peptide sequence, and had a calculated molecular mass of the mature protein of 60,609 Da. A second cDNA, designated hCEv, was isolated from the same lambda gt11 library and contained a 3-bp deletion resulting in the loss of the final amino acid in the signal peptide sequence (Ala-1) and a second 3-bp deletion leading to an in-frame loss of Gln345. Expression of mRNA corresponding to both hCE and hCEv was detected in eight adult human liver samples, with individual levels varying 5-fold (hCE) and 12-fold (hCEv). A single immunoreactive protein was detected in 13 adult human liver samples when probed with antibody directed against a rat carboxylesterase. Based on allele-specific oligonucleotide hybridizations, we believe that the hCE and hCEv cDNAs represent two distinct members of the carboxylesterase family. The carboxylesterase genes were localized to human chromosome 16 using a somatic cell hybrid mapping strategy. Baculovirus expression of hCE in Sf9 cells produced a protein with an estimated molecular mass of 59,000 Da. This enzyme was able to hydrolyze aromatic and aliphatic esters but possessed no catalytic activity toward amides or a fatty acyl CoA ester. Baculovirus-mediated expression of the hCEv cDNA yielded a second protein of 56,000 Da resulting from inefficient N-glycosylation of the hCEv protein. Although the substrate specificity for the hCEv protein was identical to that of expressed hCE for any given substrate, the specific activity for the hCE protein was always higher than that for the hCEv protein. Tunicamycin inhibition studies provided the first evidence that N-glycosylation of these luminal enzymes is essential for maximal catalytic activity.
Alleles↗
Molecular biology of human xenobiotic-metabolizing cytochromes P450: role of vaccinia virus cDNA expression in evaluating catalytic function.
Mammalian xenobiotic-metabolizing cytochromes P450s are membrane-bound enzymes that use O2 and electrons from NADPH to oxidize their substrates. For most chemical substrates, stable metabolites are produced that are destined for further metabolism and elimination from the cell. These enzymes are also capable of metabolically-converting promutagens and procarcinogens to their active proximate metabolites that can kill and transform cells. The xenobiotic-metabolizing P450s reside with three distinct families of the large P450 super-family. To study the catalytic activities of P450s, particularly human P450s that cannot be easily purified, a cDNA expression system was developed using vaccinia virus. P450 cDNAs incorporated into this lytic virus are efficiently expressed into catalytically-active enzymes that can be used to determine substrate specificities of specific human P450s forms. Activation of the hepatocarcinogen aflatoxin B1 was determined using a series of vaccinia virus-expressed P450s establishing that it is metabolically-activated to a DnA-binding derivative by several human P450 forms, albeit to differing extents.
Animals↗
cDNA cloning, chromosomal mapping, and functional characterization of the human peroxisome proliferator activated receptor.
The human peroxisome proliferator activated receptor (hPPAR) was cloned from a human liver cDNA library. The cDNA exhibited 85% and 91% DNA and deduced amino acid sequence identity with mouse PPAR (mPPAR), respectively. The hPPAR gene was mapped on human chromosome 22 slightly telomeric to a linkage group of six genes and genetic markers that are located in the general region 22q12-q13.1. Cotransfection assays of mouse Hepa 1 cells were used to roughly compare the ability of hPPAR- and mPPAR-expressed cDNAs to trans-activate the acyl CoA oxidase (ACO) PPAR response element located 5' upstream to the minimal thymidine kinase promoter driving the expression of the chloramphenicol acetyl transferase (CAT) reporter gene. Both receptors elicited a response with the prototypical peroxisome proliferators nafenopin, clofibrate, and WY-14,643. Moreover, using cotransfection assays in which the CAT reporter plasmid contained the CYP4 A6 gene response element rather than the ACO element, it was shown that hPPAR is capable of very efficiently trans-activating a second PPAR response element. These results indicate that the PPAR is present in humans in a form that is functional and can trans-activate response elements derived from two different genes, the rat ACO and the rabbit CYP4A6.
Acyl-CoA Oxidase↗
Utilization of ketone bodies by chick brain and spinal cord during embryonic and postnatal development.
Lipid synthesis from acetoacetate and 3-hydroxybutyrate was studied in chick embryo from 15 to 21 days and in chick neonate from 1 to 21 days. Embryonic spinal cord showed higher ability than brain to incorporate acetoacetate into total lipids, although a sharp decrease was found at hatching. 3-Hydroxybutyrate incorporation into total lipids was also higher in spinal cord than in brain, especially during the embryonic period. Phospholipids were the main lipids formed in both tissues from both precursors. An appreciable percentage of radioactivity was also recovered as free cholesterol, especially during the embryonic phase. The developmental patterns of amino acid synthesis from acetoacetate and 3-hydroxybutyrate were similar in both tissues: a clear increase after hatching was followed by a decrease at day 4 of neonatal life. Acetoacetate was a better substrate for amino acid synthesis than 3-hydroxybutyrate during the embryonic development in both tissues. Oxidation of both precursors to CO2 strongly decreased between 15 and 21 days of embryonic development both in brain and spinal cord.
3-Hydroxybutyric Acid↗
Metabolic differences in colon mucosal cells.
The colonic expression of cytochromes P450 from the CYP1A, CYP3A and CYP4B subfamilies has been characterized in rabbit and human tissues using RNA blotting, immunoblotting, immunohistochemistry and hybridization histochemistry. These studies demonstrate negligible expression of the CYP1A subfamily in either rabbit or human colon. The CYP3A6 gene is expressed in rabbit colon although at markedly reduced levels relative to liver and small intestine. Whilst at least two CYP3A genes are expressed at the mRNA level in human colon tissue from some individuals, no expression was demonstrated in others. Where expression was observed, this expression was continuous throughout the length of the colon. In rabbits, CYP4B1 represents a major colon P450 enzyme, expressed at levels in colon comparable to liver and small intestine. In contrast, the human CYP4B1 gene is expressed at low levels in some individuals. These studies highlight individual differences in the expression of cytochrome P450 enzymes of importance in procarcinogen metabolism.
Animals↗
Changes in ketone body utilization by chick liver, duodenal mucosa and kidney during embryonic and postnatal development.
1. Lipid synthesis from acetoacetate and 3-hydroxybutyrate in chick kidney and duodenal mucosa showed a clear decrease between 15 and 19 days of the embryonic phase, followed by an increase at hatching and a new decrease during the first neonatal period. The hepatic synthesis of lipids presented a different profile: a peak at 19-day-old embryo and a new increase during postnatal development. 2. These changes would be related to those in 3-hydroxybutyrate concentration in chick plasma throughout the perinatal period. 3. Phospholipids were the main kind of lipid formed in the three tissues. An appreciable percentage of radioactivity was also recovered as free cholesterol, especially during the embryonic phase. Triglycerides were also formed from acetoacetate in a high proportion in liver from neonatal animals. 4. Chick kidney showed the maximal ability to incorporate both precursors into amino acids. The peak obtained around hatching time would be related to the availability of the substrates. 5. Ketone body oxidation to CO2 was also maximal in kidney. In this tissue, a drastic decrease was observed during the final embryonic period, followed by a strong increase at day 1 after hatching and a new decrease at 4 days.
3-Hydroxybutyric Acid↗
Genetic toxicology using human cell lines expressing human P-450.
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Aflatoxin B1↗
Complete cDNA sequence and cDNA-directed expression of CYP4A11, a fatty acid omega-hydroxylase expressed in human kidney.
A cDNA was isolated from a human kidney lambda gt10 library using the rat CYP4A3 cDNA as a probe. The cDNA-deduced amino acid sequence encoded a protein of 519 amino acids that was designated CYP4A11 (Nelson et al., 1993) and exhibited 76%, 72%, 80%, and 53% similarities to rat CYP4A1, rat CYP4A3, rabbit CYP4A6, and human CYP4B1, respectively. The deduced amino-terminal amino acid sequence of this cDNA agreed with the amino-terminal amino acid sequence of a major P450 protein purified from human renal microsomes. A second variant form of CYP4A11 cDNA, designated CYP4A11v, was isolated from the same library and had a deletion of a single adenine residue, thereby extending the reading frame and resulting in a protein of 591 amino acids. CYP4A11v is probably encoded by a rare allelic variant of CYP4A11, since no mutant alleles were uncovered in 15 normal individuals, as determined by a polymerase chain reaction (PCR) diagnostic test. Baculovirus-mediated cDNA expression of CYP4A11 yielded a P450 protein having a lambda max of 452 nm when reduced and complexed with carbon monoxide. The expressed enzyme efficiently catalyzed omega-hydroxylation of lauric acid. No detectable activity was uncovered toward arachidonic acid and prostaglandin E1. The cDNA-expressed variant, CYP4A11v, was found to be unstable and not to efficiently metabolize lauric acid, as assessed by both baculovirus and monkey kidney COS cell cDNA expression systems. These studies indicate that CYP4A11 is a major fatty acid-metabolizing P450 that is expressed in human kidney.
Amino Acid Sequence↗
Retroviral mediated expression of human cytochrome P450 2A6 in C3H/10T1/2 cells confers transformability by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).
In order to develop more efficient in vitro systems for the study of pro-mutagenic or pro-carcinogenic chemicals, we have produced transgenic C3H/10T1/2 cell lines expressing human cytochrome P450 (CYP) 2A6. A retroviral vector containing the cDNA was packaged in psi-2 cells, and used to infect C3H/10T1/2 cells. From 100 G418-resistant clones initially isolated, three cell lines were chosen for further study based upon their morphologies, growth rates and CYP2A6-dependent coumarin 7-hydroxylase activities. Infected clone 10T1/2-04, like the 10T1/2 cells, had no detectable CYP2A6 enzyme activity, while clones 10T1/2-10 and 10T1/2-29 had microsomal CYP2A6 enzyme activities within the range found in human liver microsomes. CYP2A6 protein levels were in agreement with the observed enzyme activities. Southern blots revealed that cells from clone 10T1/2-04 contained a vector lacking the CYP2A6 cDNA, while cells from clones 10T1/2-10 and 10T1/2-29 contained multiple full-length inserts. Southern analysis also indicated the presence of an endogenous CYP2A6 ortholog in the four cell lines. All cell lines exhibited about equal sensitivity to induction of cytotoxicity and conversion to ouabain resistance by the direct acting mutagen N-methyl-N'-nitro-N-nitrosoguanidine. The four lines were also about equally sensitive to transformation by benzo[a]pyrene, a chemical requiring metabolic activation. However, only clones 10T1/2-10 and 10T1/2-29, which express CYP2A6 activity, were mutated and morphologically transformed by the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.
Animals↗
Characterization of a human cell line expressing high levels of cDNA-derived CYP2D6.
We have developed a human B-lymphoblastoid cell, designated h2D6v2, which expresses high levels of CYP2D6 cDNA. Microsomal P450 contents of 160 pmol mg-1 protein were observed. NADPH-fortified microsomes exhibited a substantial capacity to hydroxylate the prototype CYP2D6 substrates bufuralol and debrisoquine. Kinetic parameters, apparent Km, turnover number, Ki for quinidine inhibition and stereospecificity of bufuralol hydroxylation, observed with the human lymphoblast expressed enzyme were similar to those observed in human liver microsomes or purified liver CYP2D6 proteins. Therefore, the human lymphoblast expressed material appears to faithfully reflect the authentic protein. Relative to control cells, h2D6v2 cells were more sensitive to the cytotoxicity and mutagenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), supporting our previous observation with a cell line expressing lower levels of CYP2D6. h2D6v2 microsomes were capable of metabolizing NNK and NNK metabolism and mutagenicity were markedly inhibited by the addition of quinidine, a CYP2D6 inhibitor. h2D6v2 cells coupled with control cells, represent a useful in vitro system for studying xenobiotic metabolism by the clinically important, polymorphic CYP2D6. The human lymphoblast system offers the desirable ability to couple metabolic transformation studies with toxicological endpoints such as cytotoxicity and mutagenicity.
B-Lymphocytes↗
Regulation of cytochrome P450 genes: molecular mechanisms.
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Animals↗
Evidence for a new variant CYP2D6 allele CYP2D6J in a Japanese population associated with lower in vivo rates of sparteine metabolism.
A group of Japanese subjects were phenotyped for CYP2D6 activity by administration of sparteine and determination of urine metabolic ratios (MR). The CYP2D6 alleles from two subjects having a high MR, characteristic of slower rates of sparteine metabolism, were cloned in lambda EMBL3 and subjected to sequence analysis. One individual possessed a CYP2D6B allele, typically found in Caucasians, that is inactive due to an altered 3' splice recognition site and other potentially disruptive mutations. The second allele from this individual was identical to the wild type normal Caucasian CYP2D6 allele except for C188T and G4268C base differences in exons 1 and 9, respectively, that result in P34S and S486T amino acid substitutions. This allele was designated CYP2D6J. The second individual possessed two CYP2D6J alleles. PCR assays were performed to detect this allele and other alleles from a group of subjects exhibiting low rates of sparteine metabolism, i.e. with MRs > 1.5. Eleven CYP2D6J alleles were detected in 14 subjects exhibiting low rates of metabolism and including four individuals who were homozygous for this variant and had very low rates of sparteine metabolism (MRs > 2.5). In contrast, only two CYP2D6J alleles were found in 14 subjects having MRs of < 1.0. These data suggest that CYP2D6J encodes an enzyme having lower rates of sparteine metabolism.
Alleles↗
Characterization of the human cytochrome P450 isozymes responsible for styrene metabolism.
The rate of formation of styrene glycol from styrene was compared in human, rat and mouse liver microsomes. At a low styrene concentration (0.085 mM), the rates decreased in the order, mouse > rat > human; at a high concentration (1.85 mM), the order was rat > mouse > human. The forms of cytochrome P450 that are responsible for transforming styrene to styrene glycol were determined by vaccinia virus-mediated cDNA expression of individual P450 forms in cultured cells. Of the 10 human P450 forms studied, CYP2B6 was the most effective in forming of styrene glycol, followed by CYP1A2, CYP2E1 and CYP2C8; the human P450s CYP3A3, CYP3A4 and CYP3A5 also catalysed metabolism, but were much less active; and CYP2A6, CYP2C9 and CYP2D6 had little detectable activity. CYP1A1 from mouse liver was more active in forming styrene glycol than mouse CYP1A2; the latter was less active than human CYP1A2. CYP2B1 from rat liver was more active than rat CYP2B2 or CYP2B6 from human liver. The rate of styrene glycol formation was higher in lung microsomes from smokers than in those from current nonsmokers.
Animals↗
Identification of CYP2C23 expressed in rat kidney as an arachidonic acid epoxygenase.
A cDNA was isolated from a rat kidney cDNA library using mixed probes of CYP (cytochrome P450) 2C6, 2C7, 2C8, 2C9 and 2C18 cDNAs. The 3'-terminal and 5'-terminal regions of the cDNA were sequenced and were identical with those of cytochrome P450 2C23 (CYP2C23) except for a one-base deletion and a one-base addition in coding region. These changes caused a frame shift and changed the deduced amino acid sequence relative to the previously published sequence. This cDNA was expressed using a baculovirus expression system, and the resultant P450 had a lambda max of 450 nm when reduced and complexed with carbon monoxide. Specific content of the expressed P450 ranged from 0.27 to 0.43 nmol/mg of cell lysate protein. Arachidonic acid metabolism catalyzed by expressed CYP2C23 indicated that CYP2C23 efficiently produced epoxyeicosatrienoic acids (EETs). These EETs were characterized further by gas-liquid chromatography/negative ion chemical ionization mass spectrometry (GC/NCIMS) and were found to include 8,9-EET, 11,12-EET and 14,15-EET in a ratio of 1:2:1. No 5,6-EET was detected. A low rate of lauric acid hydroxylation at the (omega-1)-position was found, but the enzyme was unable to metabolize prostaglandin E1. These studies suggest that CYP2C23 is responsible, in part, for the production of EETs in rat kidney.
Alprostadil↗