Paavo Honkakoski

Regulation of cytochrome P450 (CYP) genes by nuclear receptors.

Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks.

Inhibition and induction of human cytochrome P450 enzymes: current status

Variability of drug metabolism, especially that of the most important phase I enzymes or cytochrome P450 (CYP) enzymes, is an important complicating factor in many areas of pharmacology and toxicology, in drug development, preclinical toxicity studies, clinical trials, drug therapy, environmental exposures and risk assessment. These frequently enormous consequences in mind, predictive and pre-emptying measures have been a top priority in both pharmacology and toxicology. This means the development of predictive in vitro approaches. The sound prediction is always based on the firm background of basic research on the phenomena of inhibition and induction and their underlying mechanisms; consequently the description of these aspects is the purpose of this review. We cover both inhibition and induction of CYP enzymes, always keeping in mind the basic mechanisms on which to build predictive and preventive in vitro approaches. Just because validation is an essential part of any in vitro–in vivo extrapolation scenario, we cover also necessary in vivo research and findings in order to provide a proper view to justify in vitro approaches and observations.

Drug-activated nuclear receptors CAR and PXR

The metabolism and elimination of drugs is mainly mediated by cytochrome P450 (CYP) enzymes, aided by conjugative enzymes and transport proteins. An integral aspect of this elimination process is the induction of drug metabolism through activation of gene expression of metabolic and transport proteins. There is compelling evidence that induction is regulated by drug-activated nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR). This review outlines the basic properties of CAR and PXR, their ligands and target genes, and the mechanisms of the induction process. The implications of nuclear receptor-mediated induction for drug research are also discussed.

Immunochemical and catalytical studies on hepatic coumarin 7-hydroxylase in man, rat, and mouse

The cytochrome P-450-mediated coumarin 7-hydroxylase (COH) was studied in microsomal preparations from Wistar rat, DBA/2N mouse, and human liver. Human liver contained the highest constitutive COH activity of up to about 500 pmol/mg microsomal protein/min. The rat liver contained low levels of COH (about 3-5 pmol/mg protein/min) which could be demonstrated only with high substrate concentrations. Rabbit polyclonal antibody generated against P-450Coh (a P-450 isozyme purified from pyrazole-treated DBA/2N mouse liver showing high activity for coumarin 7-hydroxylation) inhibited COH activity by almost 100% in human liver microsomes and 86-99% in mouse liver microsomes. Also the deethylation of 7-ethoxycoumarin was inhibited somewhat by the antibody, whereas no inhibition was obtained in ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities. None of these enzyme activities was affected by the antibody in the rat liver microsomes. In Ouchterlony immunodiffusion analysis precipitin lines were obtained with human, mouse and rat liver microsomes. Complex coalescence patterns were obtained suggesting full identity between human and pyrazole-treated mouse antigens, partial identity between mouse and rat antigens, and no identity between human and rat antigens. Western blot analysis with the anti-P-450Coh antibody revealed a distinct 48-kDa protein in all four human samples tested. A 50-kDa protein comigrating exactly with P-450Coh was observed in microsomes from PB and pyrazole-treated mouse liver microsomes. No distinct protein bands appeared in rat liver samples. These data suggest that despite slightly differing molecular masses, the human and mouse P-450s supporting COH are structurally conserved at their active centers. The corresponding rat P-450 appears to differ from that of mouse and man.

Cell-surface glycosaminoglycans inhibit cation-mediated gene transfer

Cationic polymers and liposomes are used to wrap DNA into complexes that promote its cellular uptake. The mechanisms of the uptake and the intracellular fate of these complexes are obscure, as are reasons for an unpredictable and sometimes poor efficiency of the transgene expression. Polyanionic glycosaminoglycans (GAGs) on the cell surface interact with the cationic DNA complexes and influence transfection.

Cocaine N-demethylation and the metabolism-related hepatotoxicity can be prevented by cytochrome P450 3A inhibitors

Cocaine is eliminated and detoxified principally through the metabolism of nonspecific plasma and tissue esterases. Microsomal oxidative metabolism is of importance in cocaine N-demethylation, this being a principal pathway of cocaine bioactivation and hepatotoxicity. The contribution of different cytochrome P450 (CYP) enzymes to cocaine N-demethylase activity was studied in vitro with DBA/2 mouse and human liver microsomes, and cocaine hepatotoxicity was examined in vivo in DBA/2 male mice. Species dependent enzyme kinetics was observed. Cocaine N-demethylase displayed two Km values in murine liver (40-60 microM and 2-3 mM), whereas only one Km value was observed in human liver microsomes (2.3-2.7 mM). We suggest that CYP3A plays a prominent role in the N-demethylation of cocaine for the following reasons: (i) pregnenolone-16 alpha-carbonitrile, an inducer of CYP3As increases cocaine N-demethylase in parallel with testosterone 6 beta-hydroxylase activity and immunoreactive 3A protein in mouse liver; (ii) human and mouse cocaine N-demethylase and testosterone 6 beta-hydroxylase activities can be inhibited by triacetyloleandomycin, cannabidiol, or gestodene, all selective inhibitors of CYP3A P450s; (iii) antibodies directed against P450s within subfamilies 1A, 2A, 2B, 2C, or 2E inhibited cocaine N-demethylase activity only marginally, and finally, (iv) treatment of mice with triacetyloleandomycin or cannabidiol in vivo significantly attenuated the cocaine-elicited hepatotoxicity as assessed by the serum alanine aminotransferase activity and liver histology in parallel with decreased cocaine N-demethylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse liver phenobarbital-inducible P450 system: Purification, characterization, and differential inducibility of four cytochrome P450 isozymes from the D2 mouse

Three novel cytochrome P450 isozymes were purified from phenobarbital (PB)-treated D2 mouse liver microsomes and compared to the previously characterized coumarin 7-hydroxylase, P450Coh. The molecular masses were 56.5, 55, 51, and 49.5 kDa, and the peaks of the reduced CO complexes were at 450, 447.5, 451.5, and 449 nm for P450PBI, P450PBII, P450PBIII, and P450Coh, respectively. The NH2-terminal sequences suggest that these isozymes belong to the P450 gene subfamilies 2B, 1A, 2C, and 2A, respectively. On the basis of reconstituted activities and microsomal immunoinhibition studies, P450Coh was the sole catalyst of coumarin 7-hydroxylation. P450PBI was the major isozyme catalyzing the high Km 7-pentoxyresorufin O-dealkylation. This reaction was also mediated at a slower rate by the low Km isozyme, P450PBII. P450PBIII contributed significantly to the microsomal O-deethylation of 7-ethoxyresorufin and N-demethylation of benzphetamine. Western blotting and dot immunobinding analyse of microsomes showed that the induction patterns of the isozymes were different. PB and TCPO-BOP induced all isozymes variably: P450PBI (19- and 31-fold), P450PBII (2- and 3-fold), P450PBIII (9- and 4-fold), and P450Coh (about 2-fold). Pyrazole induced only P450Coh, while all other isozymes were decreased by 30 to 60%. The changes in the microsomal amounts of these isozymes correlated generally well with the variation in the immunoinhibitable enzyme activities. On the basis of the structural and catalytic properties, immunochemical characteristics, and induction profiles, all three isozymes were different from each other and from the previously characterized P450Coh. This mouse PB-inducible P450 model may be valuable in further studies on the induction mechanisms of PB and TCPOBOP.

A lipid carrier with a membrane active component and a small complex size are required for efficient cellular delivery of anti-sense phosphorothioate oligonucleotides.

Anti-sense oligonucleotides are potential therapeutic agents that are used to block protein expression from mRNA. To assess the essential properties for an efficient cellular delivery system of phosphorothioate oligonucleotides (PS-ODNs), different cationic carriers were compared. The carriers were complexed with oligonucleotides at various +/- charge ratios in MES-Hepes buffer. Cationic polymers, polylysines (PLL, mean MWs 4000, 20000, 200000 kDa), polyethyleneimines (PEI, mean MWs 25 and 800 kDa) and fractured sixth-generation polyamidoamine dendrimer (PAMAM) were tested for ODN delivery into a D 407 cell line (human retinal pigment epithelial cells) with stably transfected luciferase gene. Anti-sense ODN was directed against the luciferase gene, and the anti-sense effect was determined using a luminometric method. Lipid-based vehicles included DOTAP, DOTAP/DOPE (1/1 by mol), DOTAP/Chol (1/1 by mol), DOTAP/DOPE/Chol (2/1/1 by mol), DOGS and Cytofectin GS/DOPE (2/1 by mol). Additionally a membrane-active peptide JTS-1 (NH(2) -GLFEALLELLESLWELLLEA-COOH) was added to the complexes containing DOTAP, PEI or PLL. In D 407 and CV-1 cells, the anti-sense effect was seen only with lipid-based carriers with a membrane-active component (DOPE or JTS-1). The polymeric systems were ineffective. The effect of the complexation medium was further studied on CV-1 cells. Complexes were prepared in either water, MES-Hepes buffer or cell growth medium (DMEM). Complexes prepared in water were generally most effective and the greater activity is probably due to the smaller complex size. Complex sizes differed greatly in buffer and DMEM, especially in the case of DOPE containing complexes. In conclusion, lipid carrier with a membrane active component and small complex size are required for an efficient cellular delivery of phosphorothioate oligonucleotides.

Mouse liver P450Coh: Genetic regulation of the pyrazole-inducible enzyme and comparison with other P450 isoenzymes

Genetic experiments with two inbred strains of mice, AKR/J and DBA/2N, show a single major gene inheritance of additive mode for pyrazole-inducible coumarin 7-hydroxylase. Intragroup variation in the enzyme activity further suggests the contribution of minor modifying genes to the final enzyme activity. Western blot analysis with a polyclonal antibody raised against the purified isozyme P450Coh (highly active in the 7-hydroxylation of coumarin) showed that a difference in the amounts of P450Coh protein between the D2 and AKR mice is the reason for the differences in the enzyme activity between the two mouse strains. Accordingly, changes at the regulatory level rather than at the structural gene would explain the genetic difference in the activity of coumarin 7-hydroxylase. This hypothesis is further supported by the identical Km values of the basal and induced enzyme. The inducibility of coumarin 7-hydroxylase by phenobarbital (PB) and its genetic regulation have been previously studied by A. W. Wood and colleagues ((1974) Science 185, 612-614; (1979); J. Biol. Chem. 254, 5641-5646 and 5647-5651). Our present experiments show that the regulation is the same for the pyrazole-inducible enzyme. Furthermore the experiments with anti-P450Coh antibody show that the PB- and pyrazole-inducible proteins have the same molecular weight and are immunologically indistinguishable. This suggests that PB and pyrazole may induce the same enzyme. Immunoinhibition of microsomal coumarin 7-hydroxylase is practically 100% for control animals and after pretreatment with pyrazole or PB. This suggests that in each case the same or immunologically closely related proteins are metabolizing coumarin and that the P450Coh may be the only P450 isoenzyme in mouse liver microsomes catalyzing the 7-hydroxylation of coumarin. The N-terminal amino acid sequence of P450Coh was found to be identical with those from Type I and Type II genes of the mouse P45015 alpha family for the first 21 amino acids. With rat PB-inducible P450b the homology is only 33%. Also the immunological properties of P450Coh are different from those of P450b. This may suggest that P450Coh has a closer association to the steroid 15 alpha-hydroxylase gene family than to the P450IIB subfamily of phenobarbital-inducible isoenzymes.

Characterization of human cytochrome P450 induction by pesticides.

Pesticides are a large group of structurally diverse toxic chemicals. The toxicity may be modified by cytochrome P450 (CYP) enzyme activity. In the current study, we have investigated effects and mechanisms of 24 structurally varying pesticides on human CYP expression. Many pesticides were found to efficiently activate human pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR). Out of the 24 compounds tested, 14 increased PXR- and 15 CAR-mediated luciferase activities at least 2-fold. While PXR was predominantly activated by pyrethroids, CAR was, in addition to pyrethroids, well activated by organophosphates and several carbamates. Induction of CYP mRNAs and catalytic activities was studied in the metabolically competent, human derived HepaRG cell line. CYP3A4 mRNA was induced most powerfully by pyrethroids; 50 μM cypermethrin increased CYP3A4 mRNA 35-fold. CYP2B6 was induced fairly equally by organophosphate, carbamate and pyrethroid compounds. Induction of CYP3A4 and CYP2B6 by these compound classes paralleled their effects on PXR and CAR. The urea herbicide diuron and the triazine herbicide atrazine induced CYP2B6 mRNA more than 10-fold, but did not activate CAR indicating that some pesticides may induce CYP2B6 via CAR-independent mechanisms. CYP catalyzed activities were induced much less than the corresponding mRNAs. At least in some cases, this is probably due to significant inhibition of CYP enzymes by the studied pesticides. Compared with human CAR activation and CYP2B6 expression, pesticides had much less effect on mouse CAR and CYP2B10 mRNA. Altogether, pesticides were found to be powerful human CYP inducers acting through both PXR and CAR.