Detection of water toxicity using cytochrome P450 transgenic zebrafish as live biosensor: for polychlorinated biphenyls toxicity

Cytochrome P450 (CYPs) is significant in degradation of endogenous substrates and detoxification of carcinogens, therefore it is a biomarker for assessment of polycyclic aromatic hydrocarbons (PAHs) level in aquatic environment. In the present study, a transgenic line of zebrafish had been generated using a CYP-green fluorescence protein (CYP-GFP) construct, driven by CYP1A1 promoter. Polychlorinated biphenyls (PCBs) were used as toxicant, in concentrations of 0.02 μg/ml, 0.04 μg/ml, 0.08 μg/ml, 0.4 μg/ml, and 0.8 μg/ml. The transgenic control fish showed low intensity of fluorescence in the liver. After exposed to PCBs, zebrafish had morphological changes such as expansion of yolk, contortion of tails and inflation of pericardial area. Green fluorescence signals were found to express according to concentrations and time. The green fluorescence signal was most intense after treatment with 0.08 μg/ml PCBs. However, the maximum area of green fluorescent signal was found at 0.04 μg/ml PCBs. GFP started to express at 3h exposure to PCBs, increasing its intensity until 6 h exposure, and then level off. Since the GFP expression is fast responding and is sensitive to low PAHs concentrations, transgenic fish is a good tool for live imaging and monitoring of aquatic contamination.     

Features of cytochrome P450 evolution]

It is shown that the process of mutation in the CYP2 family of the superfamily of P450 cytochromes is species-specific (man, rat, and mouse). It is also shown that, within one species (rat), different families (CYP2 and CYP11) have different mutation spectra, indicating a high specificity of the mutation process for the families of cytochrome genes. A similar specificity was demonstrated for five families (CYP1, CYP2, CYP6, CYP7, CYP11) as compared with globins and prions. The analysis of the evolutionary mutation pattern, and the pattern of pseudogenes and damaged alleles of the CYP21 family (found in patients with congenital adrenal hyperplasia) does not confirm the widely accepted hypothesis that mutations arising in pseudogenes are transduced to normal alleles of the CYP21 gene through gene conversion.     

Use of bioconjugation with cytochrome P450 enzymes

Bioconjugation, defined as chemical modification of biomolecules, is widely employed in biological and biophysical studies. It can expand functional diversity and enable applications ranging from biocatalysis, biosensing and even therapy. This review summarizes how chemical modifications of cytochrome P450 enzymes (P450s or CYPs) have contributed to improving our understanding of these enzymes. Genetic modifications of P450s have also proven very useful but are not covered in this review. Bioconjugation has served to gain structural information and investigate the mechanism of P450s via photoaffinity labeling, mechanism-based inhibition (MBI) and fluorescence studies. P450 surface acetylation and protein cross-linking have contributed to the investigation of protein complexes formation involving P450 and its redox partner or other P450 enzymes. Finally, covalent immobilization on polymer surfaces or electrodes has benefited the areas of biocatalysis and biosensor design. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

Purification of cytochrome P450 BM-3 as a monooxygenase

After investigating two anion-exchange resins, the purification factor and activity yields of P450 BM-3 were higher with Resource Q than with DEAE-Sepharose FF. Screening of HIC media showed that Source 15ISO was the most suitable for purification of P450 BM-3. An effective isolation and purification procedure of P450 BM-3 was developed and included three steps: 35%-70% saturation (NH(4))(2)SO(4) precipitation, Source 15ISO hydrophobic interaction chromatograph and Sephacryl S-200 gel filtration chromatography. Using this protocol, the purification factor and P450 BM-3 activity recovery was 13.5 and 13.7%, respectively.     

Partial purification of human liver cytochrome P 450.

Human liver cytochrome P 450 was partially purified by hydrophobic chromatography on Octyl-Sepharose, followed by ion-exchange chromatography on DEAE-cellulose. Two fractions (A and B) were obtained; cytochrome P 450 of fraction A was purified sixfold, with an overall yield of about 6 %. Its spectral properties were similar to those previously described in animal cytochromes P 450. Moreover, p-nitroanisole-O-demethylase activity could be obtained in a reconstituted system involving cytochrome P 450 of fraction A, human NADPH-cytochrome $\text{c}$ reductase and phospholipids.     

Quantitative whole-cell cytochrome P450 measurement suitable for high-throughput application

The recombinant expression of cytochrome P450 enzymes involved in drug metabolism is of interest to the pharmaceutical and biotechnological industries due to the versatile catalytic properties of these enzymes. Accurate quantification of cytochrome P450 enzymes expressed in bacterial culture generally depends on disruption and fractionation of cells to prepare membranes for spectral analysis. Although whole-cell methods for spectral determination have been reported, problems with poor reproducibility and low signal-to-noise ratio confound the use of such techniques where P450 hemoprotein expression levels are relatively low, such as in cultures of certain mammalian forms. In particular, interference from bacterial hemoproteins often obscures the P450 peak. In the current study, the combination of culture concentration, incubation under microaerobic conditions, and a modified method of baseline correction enabled reproducible quantification of cytochrome P450s in whole cells. This whole-cell method is well suited to high-throughput application, as large sets or libraries of enzymes can be expressed in parallel and relative expression levels measured without downstream cell processing.     

Identification of cytochrome P450IA2 as a human autoantigen

Autoantibodies occurring in a patient with idiopathic autoimmune type chronic active hepatitis (CAH) were found to react with purified rabbit cytochrome P450IA2 and to a much lesser extent with P450IA1. Both cytochrome P450s are known to be inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rabbit, and the expression of the microsomal protein recognized by the patient serum was induced in adult rabbit livers after treatment with TCDD. This protein is only weakly detected in liver microsomes from neonatal rabbits exposed to TCDD in utero, which is consistent with the age-dependent induction of P450IA2 by TCDD. The serum specifically reacted with a protein of similar size in microsomes prepared from COS-1 cells transfected with an expression vector containing the full length human P450IA2 cDNA. This reactivity was not detected in the cells transfected with the vector alone, indicating that the antibody recognizes human P450IA2. In addition, the serum extensively inhibited 7-ethoxyresorufin O-deethylation catalyzed by isolated human liver microsomes. This catalytic activity is associated with class IA P450s in other species. A screen of sera from patients with various hepatic and nonhepatic diseases indicates that the autoantibody to P450IA2 occurs rarely in CAH. Cytochrome P450IA2 becomes the third P450 identified as an autoantigen in inflammatory liver diseases.     

The direct measurement of cytochrome P450 in unfixed tissue sections

Summary A method for the measurement of cytochrome P450 in unfixed cryostat sections is described. The sections are incubated for 10 minutes at room temperature in a buffered solution containing polyvinyl alcohol and sodium dithionite. Two incubations are performed on serial sections, one in nitrogen and the other in carbon monoxide. Readings are taken on a Vickers M85 microdensitometer fitted with a high sensitivity photomultiplier amplifier system, the measurements being made on corresponding fields in the serial sections. Subtraction of the nitrogen values from the carbon monoxide values, after allowing for an absorption shift, gives the absolute spectrum of cytochrome P450. The subtraction corrects for the tissue content of other haem-containing proteins. The cytochrome P450 spectrum shows a sharp maximum at 450 nm, and two other minor components absorbing at 444 nm and 458 nm. The content of cytochrome P450 in animals fed with phenobarbitone was 2.4 times greater than in control animals.     

Ontogenesis of rabbit liver cytochrome P450. Evidence for a cytochrome P450-IIE (3a)-related form prevailing during the post-natal period

The liver hydroxylating system, mainly composed of cytochromes P450, is not highly active during foetal life. If develops after birth and reaches the adult level several weeks post-partum. We have studied the ontogenesis of rabbit cytochrome P450 during the post-natal period. Total P450 as well as isozymes 2, 3b, 3c, 4 and 6 were measured. The evolution of these proteins with ageing, together with qualitative modification of an electrophoretic profile, produced evidence of an early developing P450 prevailing from one week to three weeks after birth. We isolated and characterized a cytochrome, called P450 2y, from two-week liver microsomes. It is closely related to P450 3a, an adult form of rabbit P450 induced by ethanol. They have similar molecular masses, the same lambda max of CO-reduced spectrum and exhibit immunological cross-reactivity. However, we cannot conclude that the two proteins are identical from N-terminal amino acid analysis or the two-dimensional gel electrophoresis pattern. These results, as well as the recent evidence of two different genes coding for the P450 3a family, strengthen the idea that P450 2y and 3a are distinct proteins. P450 2y seems to be an early developing form abundant soon after birth, while P450 3a is a delayed form appearing like most P450 isozymes during the fourth post-natal week. Besides the quantitative development during perinatal life, there is an important qualitative modification of liver cytochrome P450 content.     

Dimemorfan N-demethylation by mouse liver microsomal cytochrome P450 enzymes

Dimemorfan (d-3-methyl-N-methylmorphinan), an analogue of dextromethorphan, is commonly used as a non-opioid antitussive. To clarify the contribution of cytochrome P450 (P450) in dimemorfan N-demethylation, effects of selective inducers and inhibitors were studied in ICR mice. Phenobarbital (PB)- and dexamethasone (Dex)-treatments caused 5-fold increases of liver microsomal dimemorfan N-demethylation activity. In untreated mouse liver microsomes, demethylation activity was strongly inhibited by a CYP3A inhibitor, ketoconazole. In PB-and Dex-treated mouse liver microsomes, ketoconazole caused strong inhibition, whereas orphenadrine caused a decrease of less than 20%. Pretreatment of control mouse liver microsomes with anti-CYP3A inhibited demethylation activity, whereas pre-treatment with anti-CYP2B had no effect. In PB-and Dex-treated mouse liver microsomes, the demethylation activity was inhibited by both anti-CYP3A and anti-CYP2B. In control mice, the intrinsic clearance of dimemorfan from N-demethylation was 5.8 microl min(-1)mg protein(-1). In PB- and Dex-treated mice, the correlation coefficient of fitting using one-enzyme and two-enzyme models were similar. The intrinsic clearances of induced mouse liver microsomes were similar. These results revealed that CYP3A played a major role in hepatic demethylation in untreated mice. Both CYP3A and CYP2B were involved in this demethylation in PB- and Dex-treated mice.     

细胞色素P450scc在人早期胎盘中的表达（简报）

人妊娠期间,胎盘合成大量的类固醇激素,与妊娠的启动、维持、分娩以及胎儿的发育均存在密切的关系。阐明胎盘类固醇激素特别是孕酮合成与分泌的调节机制对于寻找理想的生育调控技术和生殖保健方法具有重要的意义。因此,胎盘类固醇激素合成与分泌的调节向来是生殖生物学与妇产科学领域所关注的焦点问题之一,     

In vitro oxidation of oxicam NSAIDS by a human liver cytochrome P450.

The nature of the enzyme(s) catalyzing the major metabolic pathway (5'-hydroxylation) of oxicam NSAIDs was investigated in subcellular preparations of human liver tissue. Microsomal, but not cytosolic, fractions catalyzed the 5'-hydroxylation of tenoxicam. This reaction required NADPH and was inhibited by various nonselective P450 inhibitors (CO, SKF-525A, ketoconazole), but not by the peroxidase inhibitor NaN3. Tenoxicam 5'-hydroxylation exhibited simple Michaelis-menten kinetics compatible with catalysis by a single enzyme, but it strongly inhibited its own oxidation at concentrations higher than 100-150 microM. Piroxicam competitively inhibited tenoxicam 5'-hydroxylation and, conversely, tenoxicam competitively inhibited piroxicam 5'-hydroxylation. Tenoxicam 5'-hydroxylation kinetics were similar in microsomes from one poor and five extensive metabolizers of debrisoquin (CYP2D6). Dextromethorphan (CYP2D6 prototype substrate) and midazolam (CYP3A prototype substrate) had no influence on tenoxicam 5'-hydroxylation, whereas mephenytoin, tolbutamide and sulfaphenazole (Ki = 0.1 microM) inhibited it. This indicates that the 5'-hydroxylation of both piroxicam and tenoxicam is predominantly catalyzed by at least one cytochrome P450 isozyme of the CYP2C subfamily.     

Thermophilic cytochrome P450 enzymes

Thermophilic cytochrome P450 enzymes are of potential interest from structural, mechanistic, and biotechnological points of view. The structures and properties of two such enzymes, CYP119 and CYP175A1, have been investigated and provide the foundation for future work on thermophilic P450 enzymes.     

Moonlighting cytochrome P450 monooxygenases

Recently, cytochrome P450 170A1 (CYP170A1) has been found to be a bifunctional protein, which catalyzes both monooxygenase activity and terpene synthase activity by two distinct active sites in the well-established P450 protein structure. Therefore, CYP170A1 is identified clearly as a moonlighting protein. The known activities of a small number of the 13,000 members of the P450 superfamily fall into two general classes: promiscuous enzymes that are not considered as moonlighting and forms that participate in biosynthesis of endogenous compounds, such as steroids, vitamins and play different roles in different tissues, sometimes being moonlighting enzymes. Here, we review examples of moonlighting P450, which add to our understanding of the large CYP superfamily.     

Reductase and oxidase activity of rat liver cytochrome P450 with 2,3,5,6-tetramethylbenzoquinone as substrate.

The main objective of the present study was to investigate the proposed role of cytochrome P450 in the reductive metabolism of quinones as well as in the formation of reduced oxygen species in liver microsomes from phenobarbital (PB-microsomes) and beta-naphthoflavone (beta NF-microsomes) pretreated rats. In the present study, 2,3,5,6-tetramethylbenzoquinone (TMQ) was chosen as a model quinone. Anaerobic one-electron reduction of TMQ by PB-microsomes showed relatively strong electron spin resonance (ESR) signals of the oxygen-centered semiquinone free radical (TMSQ), whereas these signals were hardly detectable with beta NF-microsomes. Under aerobic conditions TMSQ formation was diminished and concomitant reduction of molecular oxygen occurred in PB-microsomes. Interestingly, TMQ-induced superoxide anion radicals, measured by ESR (using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide), and hydrogen peroxide generation was found to occur with beta NF-microsomes as well. Furthermore, SK&F 525-A (a type I ligand inhibitor of cytochrome P450) inhibited TMQ-induced hydrogen peroxide formation in both PB- and beta NF-microsomes. However, metyrapone and imidazole (type II ligand inhibitors of cytochrome P450) inhibited molecular oxygen reduction in beta NF-microsomes and not in PB-microsomes. The present study indicates that cytochrome P450-mediated one-electron reduction of TMQ to TMSQ and subsequent redox cycling of TMSQ with molecular oxygen constitutes the major source for superoxide anion radical and hydrogen peroxide generation in PB-microsomes (i.e. from the reductase activity of cytochrome P450). However, most of the superoxide anion radical formed upon aerobic incubation of TMQ with beta NF-microsomes originates directly from the dioxyanion-ferri-cytochrome P450 complex (i.e. from the oxidase activity of cytochrome P450). In conclusion, both the one-electron reduction of TMQ and molecular oxygen were found to be cytochrome P450 dependent. Apparently, both the reductase and oxidase activities of cytochrome P450 may be involved in the reductive cytotoxicity of chemotherapeutic agents containing the quinoid moiety.     

Peroxisome proliferator activated receptor alpha regulates a male-specific cytochrome P450 in mouse liver

We set out to find if the strain-specific, male-specific hepatic expression of Cyp4a protein in mouse was due to expression of Cyp4a12 and to understand the genetic basis for reported differences in expression. 12-Lauric acid hydroxylase (LAH) activity was found to show higher levels in male ddY, but not C57Bl/6, mouse liver microsomes. The expression of Cyp4a12 mRNA was studied using RNAase protection assays in male and female liver and kidney of nine mouse strains. Cyp4a12 was found to be highly expressed in male liver and kidney, but at much lower levels in female liver and kidney, in all strains studied. Western blotting with an antibody specific for Cyp4a12 confirmed that Cyp4a12 was expressed in a male specific fashion in C57Bl/6 mouse liver. RNAase protection analysis for Cyp4a10 and 14 in ddY mice revealed that neither of these genes showed male-specific expression. To further investigate genetic factors that control male-specific Cyp4a12 expression, PPARalpha+/+ and -/- mice were studied, showing that total P450 and 12-LAH activity was male-specific in +/+, but not -/- mice. RNAase protection assays were used to confirm that Cyp4a12 was lower in -/- mice. However, the male-specific Slp and MUP-1 genes retained hepatic male-specific levels of expression in +/+ and -/- mice, showing that the decrease in Cyp4a12 was not a general effect on male-specific expression. Thus, PPARalpha has a specific effect on constitutive expression of Cyp4a12.     

The differentiation of rat liver endoplasmic reticulum membranes: Apo–cytochrome P450 as a membrane protein

The proteins of washed microsomal membranes from adult rat liver were solubilized by 2% SDS and electrophoresed on polyacrylamide gels. Confirming earlier reports, a large Coomassie-Blue staining band in the ～50,000 MW region was identified as cytochrome P₄₅₀ by four criteria: similar electrophoretic mobility to a purified cytochrome P₄₅₀ preparation, an increase in this band after in vivo phenobarbital administration, a decrease in this band after in vivo allylisopropylacetamide administration, and direct specific binding of added purified heme to this region of a washed, unfixed gel. Although cyt P₄₅₀ is not spectrally evident until just at the time of birth of the rats, a large band in this region was detectable in gels of microsomal membrane protein at all times, from three days before birth onward; this band also bound added heme after membrane proteins from fetal rat liver microsomes were electrophoresed on the gels. The conclusion was that apo-cyt P₄₅₀ is present in microsomal membranes at these times during differentiation, and that, regarding this protein, during differentiation heme is bound to the apo-protein already there, concomitant with a synthesis of more cyt P₄₅₀ molecules. The process of differentiation of this membrane type is also discussed.