Concomitant occurence of multiple autoantibodies against human cytochromes P450.

Cytochromes P450 (CYPs) are a large superfamily of heme-containing enzymes that are essential for the metabolism of a variety of endogenous and xenobiotic compounds. The role and the possible diagnostic or prognostic value of the occurrence of anti-CYP autoantibodies (aAbs) in cancer patients are essentially unclear. Recently we reported the monitoring of aAbs against CYP4Z1 and CYP19A1 in breast cancer patients and healthy controls. In the present study, we extended this investigation by screening the sera of 47 lung cancer patients (17 female and 30 male; age range 49-84) and 119 healthy controls (60 female and 59 male; age range 21-72) for the presence of aAbs directed against CYP2D6, CYP4Z1, or CYP17A1, respectively. Determination of anti-CYP aAb levels was done using our previously established ELISA method. Most sera gave low signals while a small fraction showed stronger responses; however, there were no statistically significant differences between the different test groups. Also, there was no significant difference in aAb signals between the various subtypes of lung cancer. Unexpectedly, sera from two female lung cancer patients (age 67 (adenocarcinoma) and 70 (small cell carcinoma)) and from four healthy controls (one female and three male; age range 34-48) showed significantly elevated signals for more than one of the three CYPs tested. These findings corroborate earlier reports that anti-CYP aAbs occur with low frequency in the general population and, moreover, suggest that the simultaneous presence of multiple aAbs targeting different CYPs should be taken into consideration when evaluating anti-CYP aAbs as biomarkers.

Dual effect of selenium loaded chitosan nanoparticles on growth, antioxidant, immune related genes expression, transcriptomics modulation of caspase 1, cytochrome P450 and heat shock protein and Aeromonas hydrophila resistance of Nile Tilapia (Oreochromis niloticus).

Nowadays there is a great attention for nanotechnology in aquaculture production. It has an efficient role in nutrients and drugs delivery, ponds sterilization, water treatment and aquatic diseases reduction. Till now, there is no available data on impact of selenite-loaded chitosan nanoparticles (SeChNPs) on Nile tilapia. Hence, the current study investigated the effects of selenite-loaded chitosan nanoparticles supplementation on the growth, immune, antioxidant and apoptotic related genes as well as resistance to Aeromonas hydrophila of Nile tilapia, Oreochromis niloticus. A total of 400 fish were randomly divided into four groups, and each group retained five replicates. The control group was fed a basal diet (with inorganic se), other groups fed diets supplemented with SeChNPs 0.5, 1 and 2 g/kg diet. The loading concentration of Se to ChNPs was 0.3, 0.6 and 1.2 mg/0.5, 1 and 2 gm respectively. Fish groups fed SeChNPs (0.5 and 1 g/kg) exhibited the highest final body gain, better feed utilization. Additionally, the expression of myostatin gene was down-regulated by 0.2 and 0.3 fold in group fed 0.5 and 1 g/kg SeChNPs when compared with control group. Dietary inclusion of SeChNPs increased serum lysozyme, alternative complement and myeloperoxidase activities and immunoglobulin type M level. Supplementation of SeChNPs at the level of 2 g/kg up-regulated glutathione peroxidase, superoxide dismutase and catalase expression by 1.12, 4.9 and 2.31 folds respectively, in comparison with control group. In contrast, the levels of C- reactive protein and malondialdehyde were reduced. The expression of IL-10, IL-8, TNF-α and IL-1ß genes was up-regulated after dietary inclusion of different levels of SeChNPs in a dose dependent manner. Post-challenge, the highest survival rate was detected in group fed 2 g/kg SeChNPs (93%) in contrast, the control group was displayed the lowest survival rate (45%). After challenge with A. hydrophila, the expression of caspase 1 was up-regulated in groups fed 1 and 2 g/kg of SeChNPs. Moreover, the maximum down-regulation of cytochromes P450 and heat shock protein were found in 2 g/kg SeChNPs supplemented group (reduced by 0.4 and 0.6-fold, respectively, when compared with control group). In conclusion, the ameliorative effects of SeChNPs on Nile tilapia growth resulted from immune stimulatory and free radicals scavenging effects of selenium loaded chitosan nano composite.

Prognostic value of prostaglandin I2 synthase and its correlation with tumor-infiltrating immune cells in lung cancer, ovarian cancer, and gastric cancer.

BACKGROUND: Prostaglandin I2 synthase (PTGIS) is a crucial gene for the synthesis of prostaglandin I2, which has multiple roles in inflammation and immune modulation. However, studies on the prognostic value of PTGIS and its correlation with tumor-infiltrating immune cells in multiple cancers are still rare. RESULTS: Multiple datasets of the Oncomine database showed that PTGIS was expressed at low levels in lung cancer and ovarian cancer compared to the levels in normal tissues. Kaplan-Meier plotter showed that high PTGIS was associated with poor overall survival and progression-free survival in lung, ovarian, and gastric cancers. Moreover, PTGIS expression was significantly positively correlated with infiltrating levels of macrophages and was strongly associated with a variety of immune markers, especially tumor-associated macrophages (TAMs) and T-regulatory cells (Tregs). CONCLUSIONS: High expression of PTGIS could promote the infiltration of TAMs and Tregs in the tumor microenvironment and deteriorate outcomes of patients with lung, ovarian, and gastric cancers. These findings suggest that PTGIS could be taken as a potential biomarker of prognosis and tumor-infiltrating immune cells. METHODS: PTGIS expression was investigated in different datasets of the Oncomine database, and its expression levels in various tumors and corresponding normal tissues were analyzed by the Tumor Immune Estimation Resource (TIMER). Then, the clinical prognostic value of PTGIS was assessed with online public databases. In addition, we initially explored the correlation between PTGIS and tumor-infiltrating immune cells by TIMER and Gene Expression Profiling Interactive Analysis (GEPIA).

Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals - Using Unconjugated Primary Antibodies from the Same Host Species.

Immunostaining is widely used in biomedical research to show the cellular expression pattern of a given protein. Multiplex immunostaining allows labeling using multiple primary antibodies. To minimize antibody cross-reactivity, multiplex immunostaining using indirect staining requires unlabeled primary antibodies from different host species. However, the appropriate combination of different species antibodies is not always available. Here, we describe a method of using unlabeled primary antibodies from the same host species (e.g., in this case both antibodies are from rabbit) for multiplex immunofluorescence on formalin-fixed paraffin-embedded (FFPE) mouse adrenal sections. This method uses the same procedure and reagents used in the antigen retrieval step to strip the activity of the previously stained primary antibody complex. Slides were stained with the first primary antibody using a general immunostaining protocol followed by a binding step with a biotinylated secondary antibody. Then, an avidin-biotin-peroxidase signal development method was used with fluorophore-tyramide as the substrate. The immunoactivity of the first primary antibody complex was stripped through immersion in a microwaved boiling sodium citrate solution for 8 min. The insoluble fluorophore-tyramide deposition remained on the sample, which allowed the slide to be stained with other primary antibodies. Although this method eliminates most false positive signals, some background from antibody cross-reactivity may remain. If the samples are enriched with endogenous biotin, a peroxidase-conjugated secondary antibody may be used to replace the biotinylated secondary antibody to avoid the false positive from recovered endogenous biotin.

DEHP induces immunosuppression through disturbing inflammatory factors and CYPs system homeostasis in common carp neutrophils.

Di-(2-ethylhexyl) phthalate (DEHP), a common pollutant in the water environment, has been reported to be associated with immune functions, especially aquatic organisms. However, whether DEHP exposure causes neutrophils toxicity in common carp is still unclear. To investigate the toxic effect of DEHP on immune functions, common carp neutrophils were exposed to DEHP (40 µmol/L and 200 µmol/L) for 2 h. The common carp neutrophils exposed to DEHP showed a decrease in neutrophil phagocytosis rate compared with control group. DEHP exposure induced a significant decrease in mRNA expression levels of inflammatory cytokines-related genes (Interleukin-6, Interleukin-8, transforming growth factor, tumor necrosis factor (TNF)-α, TNF-R1, TNF-T1, Interferon (IFN)-2a, IFN-g2b, IFN-g1) in common carp neutrophils, while the expression levels of IL-1ß and IL-10 were increased compared with control group (P < 0.05). Furthermore, the detection of cytochrome P450 enzyme related genes showed that the mRNA expression levels of CYP (cytochrome P450 proteins)-1A, CYP-1B1, CYP-C1, CYP-2K were significantly decreased, and the mRNA expression level of CYP-3A was significantly reduced (P < 0.05). The results indicated that DEHP could affect the phagocytic ability of neutrophils by regulating the expression of inflammatory cytokines and disrupting cytochrome P450 homeostasis, which caused the immunosuppression in common carp.

Hepatic cytochrome P450 metabolism suppressed by mast cells in type 1 allergic mice.

Mast cells and Kupffer cells secrete interleukin (IL)-1ß, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, which stimulate excess nitric oxide (NO) producing-inducible NO synthase (iNOS). Unlike Kupffer cells, immunoglobulin E-sensitized mast cells elicit sustained NO production. We investigated the participation of mast cell-released NO and cytokine-derived iNOS activation in type 1 allergy-suppressed hepatic cytochrome P450 (CYP) metabolism. Aminoguanidine, a selective iNOS inhibitor, completely suppressed serum nitrate plus nitrite (NOx) concentrations after primary and secondary sensitization of ICR mice and markedly attenuated allergy-suppressed hepatic CYP1A2, CYP2C, CYP2E1, and CYP3A activities. In the liver, primary and secondary sensitization enhanced iNOS-stimulating IFN-γ (5-15-fold) and TNF-α (3-5-fold) mRNA levels more than IL-1ß (2-fold) and F4/80-positive Kupffer cell (2-fold) mRNA levels. When mast cell-deficient (-/-) mice were sensitized, hepatic CYP activities were not suppressed. Serum NOx levels in the sensitized -/- mice were similar with those in saline-treated ICR and -/- mice. In the liver of -/- mice, secondary sensitization markedly enhanced mRNA expression of iNOS (20-fold), IFN-γ (15-fold), and TNF-α (3-fold). However, hepatic total NOS activities in -/- mice were not significantly different between saline treatment and sensitization. Similarly, primary and secondary ICR mice did not significantly enhance total NOS activities in the liver and hepatocytes. The total NOS activities observed did not relate to the high levels of iNOS, IFN-γ, and TNF-α mRNA in the liver. Hepatic c-kit-positive mast cells in sensitized ICR mice were maintained at control levels. Therefore, our data suggest that mast cell-released NO participates in type 1 allergy-suppressed CYP1A2, CYP2C, CYP2E1, and CYP3A metabolism.

Activity of Zearalenone in the Porcine Intestinal Tract.

This study demonstrates that low doses (somewhat above the No Observed Adverse Effect Level, NOAEL) of the mycoestrogen zearalenone (ZEN) and its metabolites display multispecificity towards various biological targets in gilts. The observed responses in gilts were surprising. The presence of ZEN and zearalenols (ZELs) did not evoke a response in the porcine gastrointestinal tract, which was attributed to dietary tolerance. Lymphocyte proliferation was intensified in jejunal mesenteric lymph nodes, and lymphocyte counts increased in the jejunal epithelium with time of exposure. In the distal digestive tract, fecal bacterial counts decreased, the activity of fecal bacterial enzymes and lactic acid bacteria increased, and cecal water was characterized by higher genotoxicity. The accompanying hyperestrogenism led to changes in mRNA activity of selected enzymes (cytochrome P450, hydroxysteroid dehydrogenases, nitric oxide synthases) and receptors (estrogen and progesterone receptors), and it stimulated post-translational modifications which play an important role in non-genomic mechanisms of signal transmission. Hyperestrogenism influences the regulation of the host's steroid hormones (estron, estradiol and progesteron), it affects the virulence of bacterial genes encoding bacterial hydroxysteroid dehydrogenases (HSDs), and it participates in detoxification processes by slowing down intestinal activity, provoking energy deficits and promoting antiporter activity at the level of enterocytes. In most cases, hyperestrogenism fulfils all of the above roles. The results of this study indicate that low doses of ZEN alleviate inflammatory processes in the digestive system, in particular in the proximal and distal intestinal tract, and increase body weight gains in gilts.

CYP4F18-Deficient Neutrophils Exhibit Increased Chemotaxis to Complement Component C5a.

CYP4Fs were first identified as enzymes that catalyze hydroxylation of leukotriene B4 (LTB4). CYP4F18 has an unusual expression in neutrophils and was predicted to play a role in regulating LTB4-dependent inflammation. We compared chemotaxis of wild-type and Cyp4f18 knockout neutrophils using an in vitro assay. There was no significant difference in the chemotactic response to LTB4, but the response to complement component C5a increased 1.9-2.25-fold in knockout cells compared to wild-type (P < 0.01). This increase was still observed when neutrophils were treated with inhibitors of eicosanoid synthesis. There were no changes in expression of other CYP4 enzymes in knockout neutrophils that might compensate for loss of CYP4F18 or lead to differences in activity. A mouse model of dextran sodium sulfate colitis was used to investigate the consequences of increased C5a-dependent chemotaxis in vivo, but there was no significant difference in weight loss, disease activity, or colonic tissue myeloperoxidase between wild-type and Cyp4f18 knockout mice. This study demonstrates the limitations of inferring CYP4F function based on an ability to use LTB4 as a substrate, points to expanding roles for CYP4F enzymes in immune regulation, and underscores the in vivo challenges of CYP knockout studies.

Cytochrome P450-Mediated Estrogen Metabolites and Autoimmunity: Relationship and Link to Free Radicals.

Cytochrome P450 enzymes are responsible for the hydroxylation of various endogenous estrogens of the Phase I metabolic pathway. Cytochrome P450s produce hormonally active estrogen metabolites that are typically reactive and mutagenic. Although these metabolites are known to have important roles in autoimmunity, the underlying mechanism of this remains unknown. Here we report that cytochrome P450-mediated estrogen metabolites produce high ROS concentrations that can result in DNA damage. Such DNA damage can alter its immunogenicity, resulting in the induction and elevation of autoantibody concentrations, thus generating various autoimmune conditions. Here we focus on the mechanisms through which cytochrome P450-catalyzed estrogen metabolites induce immune responses and subsequently produce the autoimmune phenomenon.

Xenobiotic and Endobiotic Mediated Interactions Between the Cytochrome P450 System and the Inflammatory Response in the Liver.

The liver is a unique organ in the body as it has significant roles in both metabolism and innate immune clearance. Hepatocytes in the liver carry a nearly complete complement of drug metabolizing enzymes, including numerous cytochrome P450s. While a majority of these enzymes effectively detoxify xenobiotics, or metabolize endobiotics, a subportion of these reactions result in accumulation of metabolites that can cause either direct liver injury or indirect liver injury through activation of inflammation. The liver also contains multiple populations of innate immune cells including the resident macrophages (Kupffer cells), a relatively large number of natural killer cells, and blood-derived neutrophils. While these cells are primarily responsible for clearance of pathogens, activation of these immune cells can result in significant tissue injury during periods of inflammation. When activated chronically, these inflammatory bouts can lead to fibrosis, cirrhosis, cancer, or death. This chapter will focus on interactions between how the liver processes xenobiotic and endobiotic compounds through the cytochrome P450 system, and how these processes can result in a response from the innate immune cells of the liver. A number of different clinically relevant diseases, as well as experimental models, are currently available to study mechanisms related to the interplay of innate immunity and cytochrome P450-mediated metabolism. A major focus of the chapter will be to evaluate currently understood mechanisms in the context of these diseases, as a way of outlining mechanisms that dictate the interactions between the P450 system and innate immunity.

Preparation and application of anti-peptide antibodies for detection of orphan cytochromes P450.

OBJECTIVES: Cytochromes P450 (CYP) are monooxygenases, which metabolize mostly hydrophobic endogenous and exogenous compounds. CYPs without any clear connection to metabolism are called "orphans". Interestingly, these "orphan" CYPs are over-expressed in tumor tissues. Thus, the main aim of the paper is the development of antibodies for immunodetection of these CYPs as potential malignancy markers. METHODS: Unique sequences of CYP2S1 and 2W1 were selected and peptides synthesized. Chickens were immunized with peptides bound to hemocyanin (KLH). The antibodies were isolated from egg yolks and their reactivity was tested by ELISA. Antibodies were further affinity purified on immobilized peptides. Western blots containing CYP2S1 and 2W1 standards were developed with purified antibodies. RESULTS: Using unique peptide immunogens of CYP2S1 and 2W1 the antibodies were developed. As judged from ELISA all chickens produced specific antibodies against the respective peptides. Both affinity purified antibodies against CYP2S1 peptide recognized the CYP2S1 standard on Western blots, but only one of four anti-peptide antibodies against CYP2W1 reacted with CYP2W1 standard. The antibodies were used for the detection of CYPs in cancer cell lines and human tissues samples. Although both CYPs were frequently co-expressed in cancer cells, CYP2S1 was solely induced in the cell line BxPC3, while CYP2W1 was predominantly present in cell lines MCF7 and HeLa. Our data show that anti-peptide antibodies are an indispensable tool for detection of homologous CYPs. CONCLUSIONS: The anti-peptide antibodies successfully recognized CYP2S1 and 2W1 in the cancer cell lines and tissue samples.

Regulation of inflammation in cancer by eicosanoids.

Inflammation in the tumor microenvironment is now recognized as one of the hallmarks of cancer. Endogenously produced lipid autacoids, locally acting small molecule lipid mediators, play a central role in inflammation and tissue homeostasis, and have recently been implicated in cancer. A well-studied group of autacoid mediators that are the products of arachidonic acid metabolism include: the prostaglandins, leukotrienes, lipoxins and cytochrome P450 (CYP) derived bioactive products. These lipid mediators are collectively referred to as eicosanoids and are generated by distinct enzymatic systems initiated by cyclooxygenases (COX 1 and 2), lipoxygenases (5-LOX, 12-LOX, 15-LOXa, 15-LOXb), and cytochrome P450s, respectively. These pathways are the target of approved drugs for the treatment of inflammation, pain, asthma, allergies, and cardiovascular disorders. Beyond their potent anti-inflammatory and anti-cancer effects, non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 specific inhibitors have been evaluated in both preclinical tumor models and clinical trials. Eicosanoid biosynthesis and actions can also be directly influenced by nutrients in the diet, as evidenced by the emerging role of omega-3 fatty acids in cancer prevention and treatment. Most research dedicated to using eicosanoids to inhibit tumor-associated inflammation has focused on the COX and LOX pathways. Novel experimental approaches that demonstrate the anti-tumor effects of inhibiting cancer-associated inflammation currently include: eicosanoid receptor antagonism, overexpression of eicosanoid metabolizing enzymes, and the use of endogenous anti-inflammatory lipid mediators. Here we review the actions of eicosanoids on inflammation in the context of tumorigenesis. Eicosanoids may represent a missing link between inflammation and cancer and thus could serve as therapeutic target(s) for inhibiting tumor growth.

Characterization of a new cytochrome P450 enzyme, CYP2S1, in rats: its regulation by aryl hydrocarbon receptor agonists.

In the present study, we examined the expression of CYP2S1 mRNA and protein in tissues from male and female rats and investigated aryl hydrocarbon receptor (AhR)-mediated regulation. CYP2S1 mRNA was detected by RT-PCR in all rat tissues examined, except for the adrenal gland, and no sex-dependent differences were observed. To study the regulation of CYP2S1 mRNA expression by AhR agonists, rats were treated with 3-methylcholanthrene (3-MC; 25mg/kg/dayx3 days) or with a single intraperitoneal injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at various dosages (0, 1, 5, 10, 50, 100mug/kg). CYP2S1 mRNA levels were increased in lung, stomach, jejunum and ileum following treatment with 3-MC and in lung, liver and kidney tissues following treatment with TCDD. Induction of CYP2S1 mRNA was greater with TCDD than 3-MC treatment and was more pronounced in lung than other tissues. Antiserum raised against a peptide corresponding to the C-terminus of CYP2S1 was used to measure relative CYP2S1 protein expression by immunoblot analysis. An immunoreactive CYP2S1 protein band with an approximate molecular weight of 50kDa was detected in microsomes of rat lung, stomach and kidney, but not other tissues. Unlike CYP2S1 mRNA, CYP2S1 protein levels were not increased after treatment with 3-MC or the highest dosage of TCDD, indicating that CYP2S1 protein expression was less sensitive than mRNA expression to AhR-mediated regulation. Our study is the first to characterize CYP2S1 mRNA and protein expression in rats, and from the results obtained, we conclude that AhR is involved in the transcriptional regulation of CYP2S1 in rats.

In vitro metabolism and inhibitory effects of pranlukast in human liver microsomes.

We investigated the metabolism of pranlukast, a selective leukotriene agonist, and the potential for drug-drug interactions. Although cytochrome P450 (CYP) 3A4 appeared to be the major cytochrome P450 isoform involved in the metabolism of pranlukast, the results suggested that pranlukast metabolism was inhibited less than 50% by ketoconazole, a reversible CYP3A4 inhibitor, or by anti-CYP3A4 antibodies. Irreversible macrolide CYP3A4 inhibitors, clarithromycin, erythromycin and roxithromycin, exhibited little effect on pranlukast metabolism. On the other hand, pranlukast reversibly inhibited CYP2C8 and/or 2C9, and CYP3A4, with K(i) values of 3.9 and 4.1 micromol/l, respectively. The [I](in,max,u)/K(i) ratios were 0.004 and 0.003, respectively. The K(i) values were about 300-fold greater than the [I](in,max,u), therefore it is suggested that, at clinical doses, pranlukast will not affect the pharmacokinetics of concomitantly administered drugs that are primarily metabolized by CYP2C8 and/or 2C9 or CYP3A4.

Antigenic stimulation with cytochrome P450 2J expressed in mouse hepatocellular carcinoma cells regulates host anti-tumour immunity.

Cytochrome P450 2J subfamily (CYP2J) enzymes expressed in mouse hepatocellular carcinoma (HCC) cells were identified as an antigen recognized by specific CD4(+) T cells and the structure of its T cell epitope was determined by proteomics-based exploration. The major histocompatibility complex (MHC) class II binding peptides were isolated from I-A(k)/peptide complex of dendritic cells (DCs) loaded or unloaded with MIH-2 mouse HCC cells. MHC class II-binding peptides found in MIH-2-loaded DCs but not in unloaded DCs were determined by tandem mass spectrometric analysis. The peptide, consisting of amino acid 276-290 (DFIDAFLKEMTKYPE) of mouse CYP2J enzymes, was identified as an antigenic peptide presented in the context of MHC class II. Preventive treatment of mice with CYP2J peptide stimulated interferon (IFN)-gamma production of splenocytes and suppressed the growth of implanted CYP2J-positive MIH-2 cells but not CYP2J-negative murine bladder tumour cells. However, continuous treatment of MIH-2-bearing mice with CYP2J peptide significantly suppressed IFN-gamma production of splenocytes and accelerated the growth of implanted MIH-2 tumours in vivo. Increased frequencies of CD4(+)forkhead box P3 regulatory T cells and CD11b(+)Gr-1(+) myeloid suppressor cells were observed in splenocytes from the continuously immunized mice. These results indicate that antigenecity of CYP2J isoforms expressed in HCC cells activate host anti-tumour immunity at an initial stage of HCC, but suppress host anti-tumour immunity with excessive antigenic stimulation at an advanced stage.

Antibodies as a probe in cytochrome P450 research.

Cytochrome P450 (P450) is the superfamily of enzymes responsible for biotransformation of endobiotics and xenobiotics. However, their large isoform multiplicity, inducibility, diverse structure, widespread distribution, polymorphic expression, and broad overlapping substrate specificity make it difficult to measure the precise role of each individual P450 to the metabolism of drugs (or carcinogens) and hamper the understanding of the relationship between the genetic/environmental factors that regulate P450 phenotype and the responses of the individual P450s to drugs. The antibodies against P450s have been useful tools for the quantitative determination of expression level and contribution of the epitope-specific P450 to the metabolism of a drug or carcinogen substrate in tissues containing multiple P450 isoforms and for implications in pharmacogenetics and human risk assessment. In particular, the inhibitory antibodies are uniquely suited for reaction phenotyping that helps to predict human pharmacokinetics for clinical drug-drug interaction potential in drug discovery and development.

Characterization of a single peptide derived from cytochrome P450 1B1 that elicits spontaneous human leukocyte antigen (HLA)-A1 as well as HLA-B35 restricted CD8 T-cell responses in cancer patients.

Cytochrome P450 1B1 (CYP1B1) is widely expressed in human malignancies, but silent in most normal tissues. Importantly, the protein is believed to play an important role in the survival and growth of cancer cells in a stressed environment, e.g., as a result of hypoxia or chemotherapy. Thus, targeting of CYP1B1 represents a potentially successful strategy in the treatment of metastatic cancer, e.g., by therapeutic vaccination. Herein, we describe the characterization of a novel peptide from the CYP1B1 protein (CYP240), which is spontaneously recognized by CD8 T cells in cancer patients. Interestingly, the peptide binds to both human leukocyte antigen (HLA)-A1 and HLA-B35. Hence, peripheral blood lymphocytes from a total of 49 cancer patients (25 melanoma, 13 RCC, and 11 breast cancer; 41 HLA-A1 positive, 8 HLA-B35 positive) were analyzed for reactivity taking advantage of the EliSpot assay. Rare but strong responses were detected in HLA-A1-positive patients, and more frequent responses were detected in HLA-B35-positive patients. No reactivity against the peptide could be detected in healthy donors. Furthermore, we demonstrated that peptide-specific T cells were able to lyze target cells presenting the peptide on the surface. The characterized CYP240 peptide presented herein opens the avenue for more broader recruitment of patients in vaccination trials targeting CYB1B1.

Utility of polyclonal antibodies targeted toward unique tryptic peptides in the proteomic analysis of cytochrome P450 isozymes.

P450s are key enzymes responsible for biotransformation of numerous endogenous and exogenous compounds and are located in almost every tissue. This superfamily is the largest group of enzymes (>6000) that share a high degree of similarity in protein sequence. The human genome contains 57 CYP genes and 58 pseudogenes. A major gap exists in our knowledge about differences in CYP expression on a protein level. DNA and mRNA information are not sufficient because transcription and particularly translation events are not necessarily correlated with levels of expressed proteins. The data reported in this study complete the framework of an integrated proteomic method for the simultaneous qualitative and quantitative analysis of CYP isozyme composition using MALDI-TOF-MS and immunochemistry that has been developed in our laboratory over the last several years (Alterman et al., 2005a,b) and is based on the fact that each P450 isozyme possesses unique tryptic peptide(s) (UTP) that could be used for differential analysis of human CYP expression. Here we demonstrate that three different immunochemical techniques (ELISA, Western blot, and peptide affinity enrichment on magnetic beads with attached antibodies) have potential to be incorporated in an integrated proteomic method combining mass spectrometry and immunochemistry. Fundamentally, this approach is based on the measurement of the same chemical entity (isozyme-specific UTP) in the tryptic digest by two orthogonal analytical techniques, mass spectrometry and immunochemistry. The application of this approach is illustrated with two human CYP isozymes--CYP1A2 and CYP2E1.

P450 phenotyping of the metabolism of selegiline to desmethylselegiline and methamphetamine.

Although there is evidence in the literature of the participation of CYP2B6 in the metabolism of selegiline, it is not clear which other CYP isoforms contribute to its metabolism. The aim of this study was to investigate the P450 isozymes (CYPs) involved in the metabolism of selegiline to desmethylselegiline (DMS) and methamphetamine (MA) using four assays: incubation of selegiline with cDNA expressed CYPs, inhibition of DMS and MA formations in human liver microsomes by CYP-selective chemical inhibitors or CYP-specific antibodies, and correlation analysis. Correlation analysis, performed in a bank of 15 individual human liver microsomes, yielded correlation coefficients for DMS and MA formation of 0.769 and 0.792, respectively, for CYP2B6 (p<0.0001) and 0.333 and 0.349, respectively, for CYP3A4 (p<0.05). These results were supported by chemical/specific antibody inhibition assays. The results of correlation analysis and chemical inhibition also indicated that CYP2A6 seems to play a small role in the metabolism of selegiline. These findings confirm that CYP2B6 plays a major role in the metabolism of selegiline and also suggest the involvement of CYP3A4 and CYP2A6.

Characterization of human cytochrome P450 isoenzymes involved in the metabolism of vinorelbine.

Vinorelbine (VRL) (IV Navelbine) is a semi-synthetic vinca alkaloid, used in therapeutics for the treatment of non-small-cell lung cancer and advanced breast cancer. The aim of this study was to characterize the cytochrome P450 (CYP) isoenzymes involved in VRL metabolism. VRL was incubated at 1.28 x 10(-5) m for 90 min with human hepatic microsomes prepared from 14 donors (one woman and 13 men aged from 27 to 76 years old) and characterized for CYP1A2, CYP2D6, CYP2E1 and CYP3A4 activities. A four-combined-approach study was performed, including correlation between CYP activities and VRL metabolism among the 14 batches of microsomes, inhibition of VRL biotransformation by isoform-selective substrates and by specific inhibitory antibodies, and incubation with supersomes. Analysis of unchanged VRL and its metabolites was performed using an HPLC method coupled with both radioactive and UV detections. No correlation between CYP1A2 or CYP2E1 and VRL metabolism was observed in the 14 batches of microsomes used. A correlation was shown between VRL metabolism and CYP3A4 activity as determined with the dextromethorphan N-demethylase and nifedipine oxidase activities (r(2)=0.80 and 0.59, respectively). These results were strengthened by a correlation between the metabolism extent of VRL and CYP3A4 protein content determined by immunoblotting (r(2)=0.75). Furthermore, VRL biotransformation was inhibited by troleandomycine, the CYP3A4-specific inhibitor substrate (80% of inhibition) and by anti-CYP3A antibodies (36% of inhibition). On the contrary, a low correlation with CYP2D6 activity as determined by dextrometorphan O-demethylation (r(2)=0.31) was established. CYP2D6 supersomes did not metabolize the drug whereas 63.4% of VRL were metabolized by microsomes overexpressing CYP3A4 isoform. These data indicated that CYP3A4 is the main enzyme involved in the hepatic metabolism of VRL in human, whereas CYP2D6 is not involved.