Antibodies targeted against hypervariable and constant regions of cytochromes P450IIB1 and P450IIB2

Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli. Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein. With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins. Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes. Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2. This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1. A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2. Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively. These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily.     

Identification and location of alpha-helices in mammalian cytochromes P450

A model of the alpha-helical structure of mammalian cytochromes P450 is proposed. The location and sequence of alpha-helices in mammalian cytochromes P450 were predicted from their homology with those of cytochrome P450cam, and these sequences were generally confirmed as helical in nature by using a secondary structure prediction method. These analyses were applied to 26 sequences in 6 gene families of cytochrome P450. Mammalian cytochromes P450 consist of approximately 100 amino acid residues more than cytochrome P450cam. This difference was accounted for by three major areas of insertion: (1) at the N-terminus, (2) between helices C and D and between helices D and E, and (3) between helices J and K. Insertion 1 has been suggested by others as a membrane anchoring sequence, but the apparent insertions at 2 and 3 are novel observations; it is suggested that they may be involved in the binding of cytochrome P450 reductase. Only the mitochondrial cytochrome P450 family appeared to show a major variation from this pattern, as insertion 2 was absent, replaced by an insertion between helices G and H and between helices H and I. This may reflect the difference in electron donor proteins that bind to members of this cytochrome P450 family. Other than these differences the model of mammalian cytochromes P450 proposed maintains the general structure of cytochrome P450cam as determined by its alpha-helical composition.     

Membrane topology of the mammalian P450 cytochromes.

The membrane topology of the mammalian P450 cytochromes has been studied intensively by computational approaches, proteolysis, chemical modification, genetic engineering, and immunochemistry. Initial results for the cytochromes of the endoplasmic reticulum appeared to indicate a polytopic, four to eight transmembrane anchor model with an active site buried in the membrane. However, recent findings show that the microsomal P450s are bound to the endoplasmic reticulum by only one or two transmembrane peptides located at the NH2-terminal end, and that the active site is part of a large cytoplasmic domain that may have one or two additional peripheral membrane contacts. The membrane-bound state is viewed as rather rigid, and the plane of the heme lies between perpendicular and parallel to the plane of the endoplasmic reticulum. The mitochondrial P450 cytochromes lack a hydrophobic NH2 terminus in the mature form, and thus differ from the microsomal isozymes in this significant way. However, although the exact topology of cytochrome P450 in the inner mitochondrial membrane remains to be elucidated, certain features are clearly comparable to those of microsomal P450. Therefore, the membrane topology of the P450 gene superfamily may follow a similar pattern.     

Double Triton X-114 Phase Partitioning for the Purification of Plant Cytochromes P450 and Removal of Green Pigments

A double Triton X-114 phase partitioning procedure that separates plant cytochromes P450 from green pigments and provides an extract highly enriched in total cytochromes P450 has been developed. Upon phase partitioning in Triton X-114, plant cytochromes P450 have previously been found to partition to the pigmented detergent rich phase. These partitionings were carried out using phosphate buffer. We found that the partitioning of the cytochromes P450 could be shifted to a pigment-free Triton X-114 poor phase by changing the buffer component to borate. The protein extract containing the cytochromes P450 but devoid of green pigment was subjected to a second phase partitioning step before which the buffer was changed from borate to phosphate. This second phase partitioning step produced a Triton X-114-rich phase highly enriched in cytochromes P450 proteins compared to the microsomal starting material as monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, cytochrome P450 reconstitution assays, and Western blotting. The yield of the double phase partitioning purification procedure is about 26% which is high compared to the yields obtained at similar stages of purification using column chromatography. The double phase partitioning procedure takes 3–4 h to complete. This is very fast compared to traditional purification schemes for cytochromes P450 which involve multiple of column chromatographic steps. Plant cytochromes P450 are labile, low abundant proteins that are difficult to isolate. The double Triton X-114 phase partitioning here reported thus constitutes a versatile, efficient purification procedure circumventing many of the problems previously encountered.     

Expression and catalysis of sex-specific cytochrome P450 isozymes in rat liver

Research interest in the study of cytochromes P450 has recently been shifting to the characterization of "constitutively" expressed isozymes from that of the inducible forms. Several "constitutive" cytochrome P450 isozymes have been purified from rat liver including five immunochemically related proteins designated cytochromes P450f, P450g, P450h, P450i, and P450k. These hemoproteins have been identified as distinct isozymes on the basis of spectral, electrophoretic, and catalytic properties and NH2-terminal sequence analysis. Purification and immunoquantitation studies have indicated that these isozymes are expressed in a developmental as well as sex-related manner, and are relatively refractory to induction by xenobiotics. Cytochromes P450h and P450g are male-specific proteins, cytochrome P450i is a female-specific isozyme, while cytochromes P450f and P450k are present in both male and female adult rats. In addition, the expression of cytochrome P450g was shown to segregate into two phenotypes in outbred rats. Genetic studies utilizing inbred strains have indicated that the gene responsible for inheritance of high levels of cytochrome P450g is autosomal. Although considerable progress has been made in understanding the role of gonadal hormones and growth hormone in the hepatic regulation of cytochromes P450g, P450h, and P450i in particular, the physiological significance of the "constitutive" isozymes in the liver remains largely unresolved.     

Specificity of Isatin Interaction with Cytochromes P450

Cytochrome P450-dependent monooxygenase systems exist basically in all living organisms, where they perform various important functions. The coordinated functioning of these systems involves many proteins participating in different protein-protein interactions (PPI). Previously, we have found that the endogenous non-peptide bioregulator isatin (indoledione-2,3), synthesized from indole by means of certain cytochromes P450 (e.g. P450 2E1, P450 2C19, P450 2A6) regulates affinity of some PPI. In this study, an attempt has been undertaken to register a direct interaction of isatin with a set of different proteins related to the functioning of cytochrome P450-dependent monooxygenase systems: five isoforms of cytochromes P450, two isoforms of cytochrome b₅, cytochrome P450 reductase, adrenodoxin, adrenodoxin reductase and ferrochelatase. The study has shown high affinity specific binding of isatin only to cytochromes P450 (the equilibrium dissociation constant (K_d) is about 10^–8 M).     

Protein engineering of cytochromes P-450

The cytochromes P-450 are an immensely important superfamily of heme-containing enzymes. They catalyze the monooxygenation of an enormous range of substrates. In bacteria, cytochromes P-450 are known to catalyze the hydroxylation of environmentally significant substrates such as camphor, phenolic compounds and many herbicides. In eukaryotes, these enzymes perform key roles in the synthesis and interconversion of steroids, while in mammals hepatic cytochromes P-450 are vital for the detoxification of many drugs. As such, the cytochromes P-450 are of considerable interest in medicine and biotechnology and are obvious targets for protein engineering. The purpose of this article is to illustrate the ways in which protein engineering has been used to investigate and modify the properties of cytochromes P-450. Illustrative examples include: the manipulation of substrate selectivity and regiospecificity, the alteration of membrane binding properties, and probing the route of electron transfer.     

The cytochrome P450 complement (CYPome) of Streptomyces coelicolor A3(2)

In the present study we describe the complete cytochrome P450 complement, the "CYPome," of Streptomyces coelicolor A3(2). Eighteen cytochromes P450 (CYP) are described, in contrast to the absence of CYPs in Escherichia coli, and the twenty observed in Mycobacterium tuberculosis. Here we confirm protein identity as cytochromes P450 by heterologous expression in E. coli and measurement of reduced carbon monoxide difference spectra. We also report on their arrangement in the linear chromosome and relatedness to other CYPs in the superfamily. The future development of manipulation of antibiotic pathways and the use of streptomycetes in bioremediation and biotransformations will involve many of the new CYP forms identified here.     

Modern methods of cytochrome P450 analysis

The review describes recent approaches used for evaluation of cytochrome P450 concentration and activity. It considers employment of modern methods of proteomic analysis including electrophoresis and chromatography/mass-spectrometry (MS) for investigation of the particular group of proteins. Special attention is paid to targeted quantitative MS analysis of cytochromes P450 in biological samples. Finally, we analyze methods used for assay of cytochromes P450 activities and problems of correlation between content of certain P450 isoforms and their specific enzymatic activities.     

Orientation of cytochromes P450 in the endoplasmic reticulum

The orientation of eukaryotic cytochromes P450, with respect to the membrane of the endoplasmic reticulum, has been investigated. There is now good evidence that the tertiary structure of these proteins is essentially the same as that of the soluble bacterial isoenzyme cytochrome P450CI, with the exception of an extension at the N-terminus which is thought to form a membrane-anchoring sequence. The remainder of the molecule protrudes from the cytosolic face of the membrane so that it can interact with substrates and electron-donating proteins. Two models based on this structure have been considered, in which the plane of the heme of cytochrome P450 is oriented either parallel with or perpendicular to the plane of the membrane of the endoplasmic reticulum. The validity of these models has been assessed from the results of studies involving the binding of antipeptide antibodies directed toward known regions of cytochromes P450, modeling of the interaction of cytochrome P450 with cytochrome b5, proposed intramolecular movements of cytochrome P450 during its catalytic cycle, and the partitioning of substrates for cytochrome P450 between the cytosol and membrane. It is concluded that cytochrome P450 is most likely oriented such that the heme is not fixed horizontal to the plane of the membrane of the endoplasmic reticulum and may well lie with the heme perpendicular to the membrane.     

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.     

Occurrence and biological function of cytochrome P450 monooxygenases in the actinomycetes

Many species within the order Actinomycetales contain one or more soluble cytochrome P450 monooxygenases, often substrate-inducible and responsible for a variety of xenobiotic transformations. The individual cytochromes exhibit a relatively broad substrate specificity, and some strains have the capacity to synthesize large amounts of the protein(s) to compensate for low catalytic turnover with some substrates. All three of the Streptomyces cytochromes sequenced to date are exclusive members of one P450 family, CYP105. In several instances, monooxygenase activity arises from induction of a P450 and associated ferredoxin, or of a P450 only, suggesting that some essential electron donor proteins (reductase and ferredoxin) are not co-ordinately regulated with the cytochrome. The overall properties of these systems suggest an adaptive strategy whose twofold purpose is to maintain a competitive advantage via the production of secondary metabolites, and, whenever possible, to utilize unusual growth substrates by introducing metabolites from these reactions into the more substrate-specific primary metabolic pathways.     

Functional expression of mammalian cytochromes P450IIB in the yeast Saccharomyces cerevisiae

Three mammalian cytochromes P450 from the IIB subfamily, P450IIB11 from canine and P450IIB4 and P450IIB5 from rabbit, have been expressed in the yeast Saccharomyces cerevisiae by use of an autonomously replicating vector containing the galactose-inducible gal10 promoter. Cytochromes P450IIB4 and P450IIB5 are closely related proteins, with only 11 amino acid substitutions between them. P450IIB11 is a homologous protein, likely orthologous with IIB4 or IIB5, with 102 amino acid substitutions compared with the P450IIB4 protein and 106 compared with the P450IIB5 protein. The expressed proteins are functional in yeast microsomes, exhibiting activity toward androstenedione, 7-ethoxycoumarin, and, in some cases, progesterone. Expressed cytochromes P450IIB4 and P450IIB11 hydroxylate androstenedione with regio- and stereoselectivity characteristic of the purified, reconstituted proteins. A striking difference in the androstenedione metabolite profiles of IIB4 and IIB5 was observed, with IIB4 producing almost exclusively the 16 beta-hydroxy metabolite and IIB5 producing the 16 alpha-hydroxy and 15 alpha-hydroxy products. This is the first time that 15 alpha-hydroxylase activity has been associated with IIB4/IIB5. This activity has also been detected in liver microsomes from some, but not all, individual phenobarbital-induced rabbits tested and is largely inhibited by anti-rabbit P450IIB immunoglobulin G. These studies illustrate the utility of the yeast expression system for defining catalytic activities of individual mammalian cytochromes P450 and identifying new marker activities that can be utilized in liver microsomes.     

A proteolytically sensitive region common to several rat liver cytochromes P450: effect of cleavage on substrate binding.

Limited proteolysis of rat liver microsomes was used to probe the topography and structure of cytochrome P450 bound to the endoplasmic reticulum. Three cytochromes P450 from two families were examined. Monoclonal antibodies to cytochrome P450 forms 1A1, 2B1, and 2E1 were used to immunopurify these proteolyzed cytochromes P450 from microsomes from rats treated with 3-methylcholanthrene, phenobarbital, and acetone, respectively. Electrophoretic and immunoblot analysis of tryptic fragments revealed a highly sensitive cleavage site in all three cytochromes P450. N-Terminal sequencing was performed on the fragments after transfer onto poly(vinylidene difluoride) membranes and showed that this preferential cleavage site is at amino acid position 298 of P450 1A1, position 277 of P450 2B1, and position 278 of P450 2E1. Multiple sequence alignment revealed that these positions are at the amino terminal of a highly conserved region of these cytochromes P450. The important functional role implied by primary sequence conservation along with the proteolytic sensitivity at its amino terminal suggests that this region is a protein domain. Comparison with the known structure of the bacterial cytochrome P450cam predicts that this proteolytically sensitive site is within an interhelical turn region connected to the distal helix that partially encompasses the heme-containing active site. Substrate binding to the cleaved cytochromes P450 was examined in order to determine whether the newly added conformational freedom near the cleavage site functionally altered these cytochromes P450. Cleavage of P450 2B1 abolished benzphetamine binding, which indicates that the cleavage site contains an important structural determinant for binding this substrate. However, cleavage did not affect benzo[a]pyrene binding to P450 1A1.     

Isolation of four forms of acetone-induced cytochrome P-450 in chicken liver by h.p.l.c. and their enzymic characterization.

The purpose of this study was to purify and characterize the forms of cytochrome P-450 induced in chicken liver by acetone or ethanol. Using high performance liquid ion-exchange chromatography, we were able to isolate at least four different forms of cytochrome P-450 which were induced by acetone in chicken liver. All four forms of cytochrome P-450 proved to be distinct proteins, as indicated by their N-terminal amino acid sequences and their reconstituted catalytic activities. Two of these forms, also induced by glutethimide in chicken embryo liver, appeared to be cytochromes P450IIH1 and P450IIH2. Both of these cytochromes P-450 have identical catalytic activities towards benzphetamine demethylation. However, they differ in their abilities to hydroxylate p-nitrophenol and to convert acetaminophen into a metabolite that forms a covalent adduct with glutathione at the 3-position. Another form of cytochrome P-450 induced by acetone is highly active in the hydroxylation of p-nitrophenol and in the conversion of acetaminophen to a reactive metabolite, similar to reactions catalysed by mammalian cytochrome P450IIE. Yet the N-terminal amino acid sequence of this form has only 30-33% similarity with cytochrome P450IIE purified from rat, rabbit and human livers. A fourth form of cytochrome P-450 was identified whose N-terminal amino acid sequence and enzymic activities do not correspond to any mammalian cytochromes P-450 reported to be induced by acetone or ethanol.     

Age- and sex-related expression of cytochromes p450f and P450g in rat liver

We have previously shown that rat hepatic cytochromes P450f, P450g, P450h, and P450i possess a high degree of immunochemical and, presumably, structural relatedness. Polyclonal antibodies directed against cytochromes P450f and P450g were made monospecific by immunoabsorption against the cross-reactive proteins. The specificity of the immunoabsorbed antibodies was established by using Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays (ELISA), and immunoblots. Since factors regulating the expression of cytochromes P450f and P450g are unknown, a competitive ELISA employing the monospecific antibodies was developed to quantitate each of these isozymes in hepatic microsomes from control and treated rats. The results obtained showed that expression of cytochrome P450f is developmentally regulated in both male and female rat liver. Cytochrome P450f levels rise from less than 1% in young animals to approximately 7 and 14% of total cytochrome P450 in adult male and female rats, respectively. Cytochrome P450g is sex-specific since it is expressed only in male rat liver where it also is developmentally regulated. Levels of cytochrome P450g rise from less than 1% in 3-week-old male rats to an average value of 17% of total cytochrome P450 in 6-week-old adult animals. However, there appear to be at least two subpopulations of adult male Long Evans rats, one of which expresses low levels (less than 1%) of cytochrome P450g and the other high levels (greater than or equal to 10%). This expression appears to be independent of serum testosterone levels. Treatment of immature and adult male rats with 20 xenobiotics that are known inducers of certain cytochrome P450 isozymes revealed that cytochromes P450f and P450g are relatively refractory to induction, although Kepone appears to be a weak inducer of cytochrome P450f.     

Purification of two isozymes of rat liver microsomal cytochrome P450 with testosterone 7 alpha-hydroxylase activity

Cytochrome P450a was purified to electrophoretic homogeneity from liver microsomes from immature male Long-Evans rats treated with Aroclor 1254. Rabbit polyclonal antibody raised against cytochrome P450a cross-reacted with cytochromes P450b, P450e, and P450f (which are structurally related to cytochrome P450a). The cross-reacting antibodies were removed by passing anti-P450a over an N-octylamino-Sepharose column containing these heterologous antigens. The immunoabsorbed antibody recognized only a single protein (i.e., cytochrome P450a) in liver microsomes from immature male rats treated with Aroclor 1254 (i.e., the microsomes from which cytochrome P450a was purified). However, the immunoabsorbed antibody recognized three proteins in liver microsomes from mature male rats, as determined by Western immunoblot. As expected, one of these proteins (Mr 48,000) corresponded to cytochrome P450a. The other two proteins did not correspond to cytochromes P450b, P450e, or P450f (as might be expected if the antibody were incompletely immunoabsorbed), nor did they correspond to cytochromes P450c, P450d, P450g, P450h, P450i, P450j, P450k, or P450p. One of these proteins was designated cytochrome P450m (Mr approximately 49,000), the other cytochrome P450n (Mr approximately 50,000). Like cytochrome P450a, cytochrome P450n was present in liver microsomes from both male and female rats. However, whereas cytochrome P450a was detectable in liver microsomes from 1-week-old rats, cytochrome P450n was barely detectable until the rats were at least 3 weeks old. Furthermore, in contrast to cytochrome P450a, the levels of cytochrome P450n did not decline appreciably with age in postpubertal male rats. Cytochrome P450m was detectable only in liver microsomes from postpubertal (greater than 4 week-old) male rats. Cytochromes P450m and P450n were isolated from liver microsomes from mature male rats and purified to remove cytochrome P450a. When reconstituted with NADPH-cytochrome P450 reductase and lipid, cytochrome P450n exhibited little testosterone hydroxylase activity, whereas cytochrome P450m catalyzed the 15 alpha-, 18-, 6 beta-, and 7 alpha-hydroxylations of testosterone at 10.8, 4.6, 2.0, and 1.9 nmol/nmol P450/min, respectively. The ability of cytochrome P450m to catalyze the 7 alpha-hydroxylation of testosterone was not due to contamination with cytochrome P450a, which catalyzed this reaction at approximately 25 nmol/nmol P450a/min. Cytochrome P450m also converted testosterone to several minor metabolites, including androstenedione and 15 beta-, 14 alpha-, and 16 alpha-hydroxytestosterone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of cytochrome P450 isozymes from beta-naphthoflavone-induced adult hen liver

Cytochromes P450 beta NF-A, beta NF-B, and beta NF-C were purified from beta-naphthoflavone-treated adult hens. Cytochrome P450 beta NF-A, however, appeared at two places in the purification scheme. They were designated as cytochromes P450 beta NF-A1 and beta NF-A2 for property comparison. The cytochromes beta NF-A1 and beta NF-A2 were induced by both phenobarbital and beta-naphthoflavone treatment and were similar to P450 PB-A (previously purified from phenobarbital-induced hen livers) in molecular weights, isoelectric pH, spectral properties, behavior on chromatography columns, catalysis of substrates, immunological cross-reactivity on Ouchterlony plates and by immunoblotting, and NH2-terminal amino acid sequence. However, P450 PB-A differed from beta NF-A1/beta NF-A2 in peptide pattern after partial proteolysis by alpha-chymotrypsin and Staphylococcus aureus V8 protease, and complete digestion of 125I-labeled cytochromes by trypsin. The cytochrome P450 PB-A also differed from beta NF-A1/beta NF-A2, in that its antibodies cross-reacted with P-450 of normal, PB-, and beta-NF-induced rabbit liver microsomes. The cytochromes beta NF-B and beta NF-C, although immunochemically cross-reactive with each other, were distinct enzymes on the basis of molecular weights, spectral characteristics, isoelectric pH, peptide pattern on partial proteolysis, tryptic peptide pattern, cross-reactivity of their antibodies with other species, and NH2-terminal amino acid sequence. The most notable difference between beta NF-B and beta NF-C was that the anti-beta NF-C IgG completely inhibited O-dealkylation of 7-methoxyresorufin and 7-ethoxyresorufin by beta-NF-induced microsomes. These activities increased 40- to 50-fold in beta-NF-induced microsomes as compared to only 2- to 4-fold in PB-treated hens. The amino-terminal sequences of beta NF-B and beta NF-C were different from those of mammalian and other nonmammalian species.     

New findings in studies of cytochromes P450

Cytochromes P450 represent a numerous family of heme-containing enzymes belonging to the group of monooxygenases. In prokaryotes, cytochromes P450 usually perform a plastic function, whereas in eukaryotes their functions are very diverse. Mammalian cytochromes P450 are components of membranes and are involved in biosynthesis and metabolism of many physiologically active substances; moreover, these cytochromes are unique in their ability to catalyze biotransformation of xenobiotics, i.e. metabolize substances of foreign origin (drugs, toxins, environmental pollutants). The latter promotes elimination of xenobiotics, but sometimes intermediates of their metabolism are even more toxic and dangerous than the original xenobiotics per se. Some catalytic features of cytochromes P450 still need unambiguous explanation, i.e. broad substrate specificity, diversity of catalytic reactions, and unusual kinetics. Under some conditions, cytochromes P450 can produce reactive oxygen species, and this is another problem attracting increasing attention. In this respect, a recent finding in mitochondria of analogs of microsomal cytochromes P450 seems especially intriguing; it was postulated that P450 can be responsible for mitochondrial dysfunction, cell apoptosis, and pathogenesis of some diseases. In this paper the present state of the art concerning these problems is considered.     

Ventilator associated pneumonia and infection control

Rifampicin, an important drug in the treatment of tuberculosis, is used extensively despite its broad effects on drug-drug interactions, creating serious problems. The clinical importance of such interactions includes autoinduction leading to suboptimal or failed treatment. The concomitantly administered effects of rifampicin on other drugs can result in their altered metabolism or transportation that are metabolised by cytochromes P450 or transported by p-glycoprotein in the gastrointestinal tract and liver. This review paper summarises recent findings with emphases on the molecular mechanisms used to explain these broad drug-drug interactions. In general, rifampicin can act on a pattern: rifampicin activates the nuclear pregnane X receptor that in turn affects cytochromes P450, glucuronosyltransferases and p-glycoprotein activities. This pattern of action may explain many of the rifampicin inducing drug-drug interactions. However, effects through other mechanisms have also been reported and these make any explanation of such drug-drug interactions more complex.