Cytochrome P450 2J2, a new key enzyme in cyclophosphamide bioactivation and a potential biomarker for hematological malignancies.

The role of cytochrome P450 2J2 (CYP2J2) in cyclophosphamide (Cy) bioactivation was investigated in patients, cells and microsomes. Gene expression analysis showed that CYP2J2 mRNA expression was significantly (P<0.01) higher in 20 patients with hematological malignancies compared with healthy controls. CYP2J2 expression showed significant upregulation (P<0.05) during Cy treatment before stem cell transplantation. Cy bioactivation was significantly correlated to CYP2J2 expression. Studies in HL-60 cells expressing CYP2J2 showed reduced cell viability when incubated with Cy (half maximal inhibitory concentration=3.6 mM). Inhibition of CYP2J2 using telmisartan reduced Cy bioactivation by 50% and improved cell survival. Cy incubated with recombinant CYP2J2 microsomes has resulted in apparent Km and Vmax values of 3.7-6.6 mM and 2.9-10.3 pmol/(min·pmol) CYP, respectively. This is the first study demonstrating that CYP2J2 is equally important to CYP2B6 in Cy metabolism. The heart, intestine and urinary bladder express high levels of CYP2J2; local Cy bioactivation may explain Cy-treatment-related toxicities in these organs.

Cyclophosphamide induces mRNA, protein and enzyme activity of cytochrome P450 in rat.

The effects of cyclophosphamide (CPA) on CYP enzymes in vivo and its auto induction in rat were investigated in Wistar/Fu male rats at a single dose (40 or 200 mg kg(-1)) or as repeated dose of 200 mg kg(-1) CPA. After a single dose of CPA, mRNAs of CYPs 2B1, 2B2, 3A2, 2C11 were significantly induced up to 220-, 6.7-, 5.0- and 5.8-fold at the low dose CPA, and 4800-, 52-, 22- and 2.5-fold at the high dose. CYP2B1/2 and CYP3A proteins were increased by 4- and 2-fold (low dose) and by 28- and 1.7-fold (high dose). CYP2C11 protein levels were not altered. Microsomal activities of CYP2B, CYP3A and 2C11 were increased by 2-, 1.8- and 1.3-fold at low dose CPA, and 3.2-, 1.7- and 1.6-fold at high dose. A significant (p<0.05) decrease in CPA concentration and a significant (p<0.05) increase in 4-OH-CPA levels were observed with repeated administration of CPA. Acute induction effect on CYP2B1, 2B2, 2C11 and 3A2 and a substantial up regulation of CYP2B1 mRNA were observed after a single dose of CPA, auto induction was observed by repeated administration.

Metabolism of human cytochrome P450 marker substrates in mouse: a strain and gender comparison.

The aim was to characterize mouse gender and strain differences in the metabolism of commonly used human cytochrome (CYP) P450 probe substrates. Thirteen human CYP probe substrates (phenacetin, coumarin, 7-ethoxy-4-trifluoromethyl coumarin, amiodarone, paclitaxel, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, chlorzoxazone, p-nitrophenol, testosterone and lauric acid) were used in activity measurements. The metabolism of the probe substrates was compared in liver microsomes from male and female NMRI, CBA, C57bl/6, 129/SvJ and CD1 strains. The expression of proteins identified on Western blots with commonly available antibodies selective for specific human and rat CYP enzymes were compared in the different mouse strains. Males had higher metabolism than corresponding females for phenacetin O-deethylation (human marker for CYP1A2 activity), and a high correlation was found between phenacetin activity and immunoreactivity in Western blots produced with rat CYP1A2 antibodies. Protein detected by antibodies cross-reacting with human CYP2B6 and rat CYP2B1/2 antibodies was female specific except for the 129/SvJ strain, where it was absent in both genders. Females generally had a higher metabolism of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation (human markers for CYP2D activity). Bufuralol 1'-hydroxylation correlated with a female-dominant mouse CYP, which was detected with antibodies against rat CYP2D4. p-Nitrophenol 2-hydroxylation correlated better than chlorzoxazone 6-hydroxylation with the protein detected with antibodies against rat CYP2E1, indicating that p-nitrophenol is a more specific substrate for mouse CYP2E1.

Use of a cocktail of probe substrates for drug-metabolizing enzymes for the assessment of the metabolic capacity of hepatocyte preparations.

A cocktail of the following probe substrates for human drug-metabolizing enzymes was used to characterize hepatocyte preparations: phenacetin (for CYP1A2), diclofenac (CYP2C9), diazepam (CYP2C19), bufuralol (CYP2D6), midazolam (CYP3A4/5) and 7-hydroxycoumarin (for glucuronidation and sulphation). The cocktail was incubated with cryopreserved human, dog or minipig hepatocytes or with freshly prepared rat hepatocytes. Sample analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an Open Access environment that allowed less experienced MS operators to login, submit and analyse sample sets using predefined settings without the immediate attendance of an experienced analyst. Intrinsic clearances (CLint) were calculated from the disappearance of the compounds from the incubations. Initially, the cocktail used for human, rat and dog hepatocyte incubations contained 7-ethoxycoumarin instead of 7-hydroxycoumarin. However, 7-ethoxycoumarin had an inhibitory effect on the metabolism of phenacetin. The highest CLint estimated with human and dog hepatocytes was observed for 7-hydroxycoumarin. For rat and minipig hepatocytes, the highest CLint was observed for bufuralol. In incubations with dog and minipig hepatocytes, the lowest CLint was seen with diclofenac, whereas for human and rat hepatocytes, the lowest value was observed with diazepam and phenacetin, respectively. When the cocktail was incubated together with human hepatocytes and 1 microM ketoconazole, the CLint of midazolam was decreased to about 7.5% of the control value, whereas the metabolism of the other cocktail compounds was virtually unaffected by this CYP3A inhibitor. It is suggested that a cocktail of specific human probe substrates for drug-metabolizing enzymes can be used routinely for the determination of the metabolic capacity of hepatocyte preparations in order to ensure the quality and reproducibility of experiments. Moreover, a cocktail of specific probe substrates can also be a useful tool for studies on enzyme inhibition.

Role of polymorphic human CYP2B6 in cyclophosphamide bioactivation.

The role of polymorphic CYP2B6 in cyclophosphamide (CPA) bioactivation was investigated in human liver microsomes. A total of 67 human liver specimens were first genotyped with respect to the CYP2B6*5 and CYP2B6*6 variant alleles. CYP2B6 apoprotein levels in 55 liver microsomal preparations were assessed by immunoblotting. 4-Hydroxy-CPA and hydroxy-bupropion were quantified by using HPLC and LC-MS, respectively. 7-Ethoxy-4-trifluoromethyl coumarin O-deethylase activity was measured fluorometrically. The frequencies of CYP2B6*5 and CYP2B6*6 mutant alleles were 9.0 and 16.4%, respectively. CYP2B6 protein expression was detected in 80% of the samples, with a large variation (0.003-2.234, arbitrary units). There was a high correlation between CYP2B6 apoprotein content and CPA 4-hydroxylation (n=55, r=0.81, P<0.0001). When based on the CYP2B6 apoprotein levels, the *6 carriers had significantly higher CPA 4-hydroxylation (P<0.05). CPA 4-hydroxylation also correlated significantly with other CYP2B6-specific reactions (n=20, P<0.0001). V(max) and K(m) for CPA 4-hydroxylation in recombinant CYP2B6 enzyme were 338 nmol/min/nmol enzyme and 1.4 mM, respectively. CYP2B6 showed much higher in vitro intrinsic clearance than previously observed in recombinant CYP2C19 and CYP2C9 variants in yeast expression system. Our results demonstrate that the polymorphic CYP2B6 is a major enzyme in the bioactivation of CPA. Moreover, we identified a strong impact of CYP2B6*6 on CPA 4-hydroxylation.

Ethanol-inducible cytochrome P4502E1: genetic polymorphism, regulation, and possible role in the etiology of alcohol-induced liver disease.

In the Tsukamoto-French model, ethanol causes an important 10-20-fold induction of ethanol-inducible cytochrome P4502E1 (CYP2E1), mediated through enzyme stabilization and increased rate of gene transcription. The CYP2E1 induction results in a pronounced increase in the rate of NADPH-dependent microsomal lipid peroxidation, an elevation which is not seen after simultaneous administration of the CYP2E1 inhibitor diallylsulfide. Increased amounts of lipid peroxides are seen in plasma and red blood cells of both rats and humans during high ethanol intake. A mechanism for ethanol-dependent liver damage is proposed which involves the CYP2E1-dependent lipid peroxide formation, either directly by its capability to induce NADPH-dependent peroxidation in the microsomal membranes or indirectly by a hypoxia-mediated transformation of xanthine dehydrogenase to xanthine oxidase, in activation of Ito cells and Kupffer cells to yield cytokine and collagen production. The CYP2E1 gene is polymorphic among Caucasians. Four different unrelated or partially linked polymorphisms have been observed. One polymorphism in the 5'-flanking region has been described to be associated with altered enzyme expression in vitro, and the rare allele was found to be less frequent among Swedish patients having lung cancer when compared to two different control groups. Another polymorphism, detectable with Dra I restriction endonuclease fragment length polymorphism (RFLP), was localized to intron 6, and the rare allele was less common among Italian alcoholics with clinical signs of liver cirrhosis, as compared to controls. Several other mutations in the CYP2E1 gene were found to be associated with this allele. However, further research is needed to relate the CYP2E1 gene polymorphism with incidence of liver cirrhosis.

Exposure to various benzene derivatives differently induces cytochromes P450 2B1 and P450 2E1 in rat liver.

Benzene (B), toluene (T), ethylbenzene (EB), styrene (S) and xylene isomers (oX, mX, pX) are important environmental pollutants and B is a proved human carcinogen. Their inhalation by male Wistar rats (4 mg/l, 20 h/day, 4 days) caused cytochrome P450 (P450) induction. The degree of P450 2B1 induction increased and that of 2E1 decreased in the series B, T, EB, S, oX, mX and pX, as estimated by Western blots, while neither solvent was as effective for 2B1 induction as phenobarbital and B was more effective for 2E1 than ethanol. The levels of several other P450s decreased after exposure to these solvents, B being most effective. Exposure to these solvents increased in vitro hepatic microsomal oxidation of B and aniline (AN) (2E1 substrates) 3 to 6-fold, indicating induction of this P450. T oxidation was increased 2 to 4-fold and chlorobenzene (ClB) oxidation 3-fold. Sodium phenobarbital (PB, 80 mg/kg/day, 4 days, i.p.) did not increase ethylmorphine (EM) and benzphetamine (BZP) demethylation (2B1 substrates), neither of the B derivatives did so, and oX decreased it; however, pentoxyresorufin O-dealkylation was well related to the immunochemically detected 2B1 levels in control, PB and B microsomes. PB did not increase B, but increased T and ClB oxidation 2-4 and 3-fold, respectively, indicating possible 2B1 role in their oxidation. B oxidation after various inducers was related to immunochemical 2E1 levels, T and ClB oxidation to both 2B1 and 2E1 and AN oxidation to 2E1 and 1A2 levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde as a substrate for ethanol-inducible cytochrome P450 (CYP2E1).

Liver microsomes from starved and acetone-treated rats catalyzed NADPH-supported metabolism of acetaldehyde at a rate 8-fold higher than corresponding control microsomes; the Vmax was about 6 nmol/mg microsomal protein/min and the apparent Km 30 microM. The reaction was efficiently inhibited by anti-CYP2E1 IgG, but not by control IgG. Reconstituted membranes containing rat CYP2E1 and cytochrome b5 metabolized acetaldehyde with a Vmax of 20 nmol/nmol/min and an apparent Km of 30 microM, whereas CYP2B4 containing vesicles or vesicles without b5 were ineffective. Gas chromatographic/mass spectrometric analysis of products formed from [2H4]-acetaldehyde with CYP2E1-containing reconstituted membrane vesicles revealed the formation of acetate as the only detectable product, although other water soluble products were also formed as evidenced from incubations with [1,2-14C]acetaldehyde. The results indicate that CYP2E1 is an aldehyde oxidase and thus metabolizes both ethanol and its primary oxidation product. This might have implications in vivo for acetaldehyde metabolism in liver and brain.

Role of ethanol-inducible cytochrome P450 (P450IIE1) in catalysing the free radical activation of aliphatic alcohols.

Incubation of rat liver microsomes with 1-propanol and 1-butanol in the presence of NADPH and of the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) allowed the detection of free radical intermediates tentatively identified as 1-hydroxypropyl and 1-hydroxybutyl radical, respectively. Microsomes isolated from rats treated chronically with ethanol (EtOH) or with the combination of starvation and acetone treatment (SA), exhibited a two-fold increase in the ESR signal intensity as compared to untreated controls, whereas no increase was observed in phenobarbital-induced (PB) microsomes. Consistently, in reconstituted membrane vesicles, ethanol-inducible cytochrome P450IIE1 was twice as active as phenobarbital-inducible P450IIB1 in producing 1-butanol free radicals. In the microsomal preparations from EtOH and SA pretreated rats the addition of antibodies against cytochrome P450IIE1, but not of preimmune IgGs, lowered the ESR signal of 1-butanol radicals by more than 50%. The same antibodies decreased the free radical production by untreated microsomes by 35-40%, but were ineffective on microsomes from PB-treated animals. This indicated that cytochrome P450IIE1 is the major enzyme responsible for the free radical activation of alcohols in control and ethanol-fed rats. The generation of 1-hydroxybutyl radicals by EtOH microsomes was inhibited by 40, 48 and 68%, respectively, by the addition of isoniazid, tryptamine and octylamine, compounds known to specifically affect the NADPH oxidase activity of this isoenzyme. This effect was not due to the scavenging of the alcohol radical since none of these compounds affected the ESR signals originated from 1-butanol in a xanthine-xanthine oxidase system. When added to reconstituted membrane vesicles isoniazid, tryptamine and octylamine also decreased 1-butanol radical formation by P450IIE1 by 54, 38 and 66%, respectively. Such an inhibition corresponded to the effect exerted by the same compounds on O2- release from P450IIE1 containing vesicles. These results indicate that the capacity of cytochrome P450IIE1 to reduce oxygen is related to its ability to generate alcohol free radicals and suggest that ferric cytochrome P450-oxygen complex might act as oxidizing species toward alcohols.

Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Lack of production of 2,3-dihydroxybenzoate unless hydroxyl radical formation is permitted.

Attack by hydroxyl radicals (.OH) upon salicylate (2-hydroxybenzoate) leads to formation of both 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (gentisate, 2,5-DHB). It has been suggested that formation of 2,3-DHB from salicylate is a means of monitoring .OH formation. Production of 2,3-DHB and 2,5-DHB by liver microsomal fractions and isoforms of cytochrome P-450 was investigated. Liver microsomes prepared from variously treated rats and rabbits catalysed the formation of 2,5-DHB but not 2,3-DHB. Formation of 2,5-DHB was inhibited by CO, metyrapone and SKF-525A, but not by the .OH scavengers mannitol and formate or by the iron chelator desferrioxamine. Purified P-450s IIE1, IIB4 or IA2 from rabbit liver microsomes, reconstituted together with NADPH-cytochrome P-450 reductase, led to formation of equal amounts of 2,3-DHB and 2,5-DHB in reactions that were almost completely inhibited by mannitol or formate. Addition of Fe3+/EDTA either to microsomes or to membranes containing reconstituted P-450 caused formation of approximately equal amounts of 2,3-DHB and 2,5-DHB, consistent with an .OH-dependent attack on salicylate. The data indicate that the microsomal P-450 system catalyses hydroxylation of salicylate to 2,5-DHB, but not formation of 2,3-DHB. Hence measurement of 2,3-DHB might provide a means of monitoring .OH formation. Care must be taken in studies of substrate hydroxylation by microsomes or reconstituted P-450 systems to avoid artefacts resulting from .OH generation.

Cytochrome P-450-dependent formation of reactive oxygen radicals: isozyme-specific inhibition of P-450-mediated reduction of oxygen and carbon tetrachloride.

1. Ethanol-inducible P450 IIE1 exhibits a high rate of oxygen consumption and oxidase activity. The enzyme is selectively distributed in the liver centrilobular area, the acinar region specifically destroyed after treatment with P450 IIE1 substrates/inducers such as ethanol, carbon tetrachloride, chloroform, N-nitrosodimethylamine and paracetamol. 2. Twenty substrates and ligands for cytochrome P450 IIB4 and P450 IIE1 were evaluated for their ability to inhibit microsomal and reconstituted NADPH-dependent oxidase activity, and the P450 IIE1-catalysed reduction of carbon tetrachloride to chloroform. Type I ligands and substrates did not inhibit the processes whereas nitrogen-containing compounds such as octylamine, cimetidine, imidazole and tryptamine inhibited NADPH oxidation and H2O2 formation in microsomes from starved and acetone-treated rats by around 50%. 3. Tryptamine, octylamine, isoniazid and p-chloroamphetamine inhibited reconstituted P450 IIE1-dependent oxidase activity with half maximal effects at 14-170 microM. 4. Isoniazid, cimetidine and tryptamine inhibited the P450 IIE1-dependent reduction of carbon tetrachloride, whereas acetone was without effect. 5. The oxygen dependency of microsomal oxidase activity exhibited high-affinity and low-affinity phases, with partial saturation at 20 microM of O2. 6. It is concluded that microsomal oxidase activity takes place at physiological concentrations of O2 and that isozyme-specific type II ligands compete with oxygen or carbon tetrachloride for reduction by P-450 haem.

Cytochrome P-450-dependent oxidase activity and hydroxyl radical production in micellar and membranous types of reconstituted systems.

In view of conflicting results in the literature regarding the contribution of cytochrome P-450 to hydrogen peroxide production and formation of hydroxyl radicals in the microsomal electron transport chain, experiments were undertaken to evaluate this problem using reconstituted micellar and membranous systems containing NADPH-cytochrome P-450 reductase and cytochrome P-450 LM2 purified from rabbit liver. It was found that P-450 LM2 increased the rate of NADPH consumption in the vesicular system, reconstituted with microsomal phospholipids, much more than in the micellar system, based on dilauroylphosphatidylcholine (DLPC) under otherwise similar conditions. At small amounts of Fe(III)-EDTA (1-5 microM), the enhanced oxidase activity was manifested in a much higher dependency on P-450 LM2 for the production of hydroxyl radicals, as determined by the oxidation of dimethylsulphoxide (Me2SO) or 2-keto-4-thiomethylbutyric acid (KMBA), in the vesicular than in the micellar system. In the presence of high amounts of Fe(III)-EDTA (10-50 microM), the relative increase due to P-450 LM2 was less pronounced in both types of reconstituted systems, although the increase in absolute terms was about the same as at small Fe(III)-EDTA concentrations. The data indicate that in the presence of no or small amounts of chelated iron in negatively-charged membranous systems, most of the hydrogen peroxide and superoxide anions necessary for generation of hydroxyl radicals, are produced by cytochrome P-450 LM2. This appears to be due to a higher affinity between the reductase and P-450 LM2 in this system. In reconstituted micellar systems or in the presence of high amounts of chelated iron, "uncoupling" at the level of the reductase appears to take place, with a resulting production of hydroxyl radicals and other forms of reactive oxygen species.

Metabolism of n-pentane by ethanol-inducible cytochrome P-450 in liver microsomes and reconstituted membranes.

The cytochrome P-450-dependent metabolism of n-pentane was studied by head-space gas chromatographic analysis of incubations with liver microsomes and reconstituted systems from rat and rabbit liver. The alkane was metabolized in liver microsomes from imidazole-treated rabbits at an apparent Vmax of 3 nmol mg-1 min-1 and a Km of 35 microM and in liver microsomes from acetone-treated rats at an apparent Vmax of 10 nmol mg-1 min-1 and a Km of 9 microM. The rate of microsomal n-pentane metabolism was enhanced fivefold by acetone treatment of rats, compared to the rate observed in liver microsomes from control rats. The reaction was inhibited in microsomes by compounds that are known to interact with the acetone and ethanol-inducible form of liver microsomal cytochrome P-450, like acetone, imidazole, ethanol and benzene. Effective inhibition was also accomplished when microsomal incubations were performed in the presence of IgG against this form of cytochrome P-450 from either rat or rabbit liver. In reconstituted membrane vesicles containing NADPH--cytochrome P-450 reductase, ethanol-inducible P-450 from rat liver (P-450j) was a fivefold more efficient catalyst of pentane metabolism than was the corresponding P-450 form from rabbit liver (P-450 LMeb). The rabbit enzyme metabolized the hydrocarbon with an apparent Vmax of 4 nmol nmol-1 min-1 and a Km of 8 microM, By contrast, phenobarbital-inducible P-450 LM2 or 3-methylcholanthrene-inducible P-450 LM4 from rabbit liver were quite ineffective catalysts of n-pentane metabolism. It is concluded that n-pentane constitutes a good substrate for cytochrome P-450 and, in particular, for the ethanol-inducible form of this hemoprotein. It is suggested that n-pentane is an important substrate for P-450 under in vivo conditions and that pentane measurements in expired air as an indicator of lipid peroxidation must be interpreted with caution.