Effect of Agrimonia eupatoria L. and Origanum vulgare L. Leaf, Flower, Stem, and Root Extracts on the Survival of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most antibiotic multi-resistant bacteria, causing chronic pulmonary disease and leading to respiratory failure and even mortality. Thus, there has been an ever-increasing search for novel and preferably natural antimicrobial compounds. Agrimonia eupatoria L. and Origanum vulgare L. shoots are commonly used as teas or alcoholic tinctures for their human health-promoting and antibacterial properties. Here, we explored the antimicrobial effects of all plant parts, i.e., leaf, flower, stem, and root extracts, prepared in water or in 60% ethanol, against P. aeruginosa. The impact of these extracts on bacterial survival was determined using a luminescent strain of P. aeruginosa, which emits light when alive. In addition, the antimicrobial effects were compared with the antioxidant properties and content of phenolic compounds of plant extracts. Ethanolic extracts of O. vulgare roots and flowers showed the highest antimicrobial activity, followed by A. eupatoria roots. In particular, chlorogenic acid, the ethanolic extract of O. vulgare roots contained high levels of protocatechuic acid, hesperidin, shikimic acid, rutin, quercetin, and morin. The synergistic effects of these phenolic compounds and flavonoids may play a key role in the antibacterial activity of teas and tinctures.

Co-Exposure to Aristolochic Acids I and II Increases DNA Adduct Formation Responsible for Aristolochic Acid I-Mediated Carcinogenicity in Rats.

The plant extract aristolochic acid (AA), containing aristolochic acids I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases associated with upper urothelial cancer. Recently (Chemical Research in Toxicology 33(11), 2804-2818, 2020), we showed that the in vivo metabolism of AAI and AAII in Wistar rats is influenced by their co-exposure (i.e., AAI/AAII mixture). Using the same rat model, we investigated how exposure to the AAI/AAII mixture can influence AAI and AAII DNA adduct formation (i.e., AA-mediated genotoxicity). Using 32P-postlabelling, we found that AA-DNA adduct formation was increased in the livers and kidneys of rats treated with AAI/AAII mixture compared to rats treated with AAI or AAII alone. Measuring the activity of enzymes involved in AA metabolism, we showed that enhanced AA-DNA adduct formation might be caused partially by both decreased AAI detoxification as a result of hepatic CYP2C11 inhibition during treatment with AAI/AAII mixture and by hepatic or renal NQO1 induction, the key enzyme predominantly activating AA to DNA adducts. Moreover, our results indicate that AAII might act as an inhibitor of AAI detoxification in vivo. Consequently, higher amounts of AAI might remain in liver and kidney tissues, which can be reductively activated, resulting in enhanced AAI DNA adduct formation. Collectively, these results indicate that AAII present in the plant extract AA enhances the genotoxic properties of AAI (i.e., AAI DNA adduct formation). As patients suffering from AAN and BEN are always exposed to the plant extract (i.e., AAI/AAII mixture), our findings are crucial to better understanding host factors critical for AAN- and BEN-associated urothelial malignancy.

Apoferritin/Vandetanib Association Is Long-Term Stable But Does Not Improve Pharmacological Properties of Vandetanib.

A tyrosine kinase inhibitor, vandetanib (Van), is an anticancer drug affecting the signaling of VEGFR, EGFR and RET protooncogenes. Van is primarily used for the treatment of advanced or metastatic medullary thyroid cancer; however, its usage is significantly limited by side effects, particularly cardiotoxicity. One approach to minimize them is the encapsulation or binding of Van in- or onto a suitable carrier, allowing targeted delivery to tumor tissue. Herein, we constructed a nanocarrier based on apoferritin associated with Van (ApoVan). Based on the characteristics obtained by analyzing the average size, the surface ζ-potential and the polydispersive index, ApoVan nanoparticles exhibit long-term stability and maintain their morphology. Experiments have shown that ApoVan complex is relatively stable during storage. It was found that Van is gradually released from its ApoVan form into the neutral environment (pH 7.4) as well as into the acidic environment (pH 6.5). The effect of free Van and ApoVan on neuroblastoma and medullary thyroid carcinoma cell lines revealed that both forms were toxic in both used cell lines, and minimal differences between ApoVan and Van were observed. Thus, we assume that Van might not be encapsulated into the cavity of apoferritin, but instead only binds to its surface.

Effect of the natural flavonoids myricetin and dihydromyricetin on the wound healing process in vitro.

Myricetin (MYR) and dihydromyricetin (DHM) are classified as natural flavonoids. Both substances are known for their anti-inflammatory and antioxidant properties. In this study, an in vitro model of inflammation was demonstrated on monolayers of scratched fibroblasts or keratinocytes exposed to LPS from Pseudomonas aeruginosa for six hours. MYR and DHM were subsequently applied to the cells for 24 hours at sub toxic concentrations (5-15 µM). Inflammatory parameters were analysed in collected cell medium and lysate after the incubation period using the Enzyme-Linked ImmuneSorbent Assay (ELISA) and Western blot. Both flavonoids inhibit the production of pro-inflammatory cytokines (IL-6, IL-8) in LPS-stimulated skin cells as well as the decreased level of MMP-1 in fibroblasts. However, the application of MYR and DHM dose dependently increased the level of MMP-1 in keratinocytes. In our experiments, we focused on the anti-glycation activity of MYR and DHM, where the higher concentration of MYR seems to be more effective.

In Vivo Metabolism of Aristolochic Acid I and II in Rats Is Influenced by Their Coexposure.

The plant extract aristolochic acid (AA), containing aristolochic acid I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy and Balkan endemic nephropathy, unique renal diseases associated with upper urothelial cancer. Differences in the metabolic activation and detoxification of AAI and AAII and their effects on the metabolism of AAI/AAII mixture in the plant extract might be of great importance for an individual's susceptibility in the development of AA-mediated nephropathies and malignancies. Here, we investigated in vivo metabolism of AAI and AAII after ip administration to Wistar rats as individual compounds and as AAI/AAII mixture using high performance liquid chromatography/electrospray ionization mass spectrometry. Experimental findings were supported by theoretical calculations using density functional theory. We found that exposure to AAI/AAII mixture affected the generation of their oxidative and reductive metabolites formed during Phase I biotransformation and excreted in rat urine. Several Phase II metabolites of AAI and AAII found in the urine of exposed rats were also analyzed. Our results indicate that AAI is more efficiently metabolized in rats in vivo than AAII. Whereas AAI is predominantly oxidized during in vivo metabolism, its reduction is the minor metabolic pathway. In contrast, AAII is mainly metabolized by reduction. The oxidative reaction only occurs if aristolactam II, the major reductive metabolite of AAII, is enzymatically hydroxylated, forming aristolactam Ia. In AAI/AAII mixture, the metabolism of AAI and AAII is influenced by the presence of both AAs. For instance, the reductive metabolism of AAI is increased in the presence of AAII while the presence of AAI decreased the reductive metabolism of AAII. These results suggest that increased bioactivation of AAI in the presence of AAII also leads to increased AAI genotoxicity, which may critically impact AAI-mediated carcinogenesis. Future studies are needed to explain the underlying mechanism(s) for this phenomenon.

Investigation of the Binding Affinity of a Broad Array of l-Fucosides with Six Fucose-Specific Lectins of Bacterial and Fungal Origin.

Series of multivalent α-l-fucoside containing glycoclusters and variously decorated l-fucosides were synthesized to find potential inhibitors of fucose-specific lectins and study the structure-binding affinity relationships. Tri- and tetravalent fucoclusters were built using copper-mediated azide-alkyne click chemistry. Series of fucoside monomers and dimers were synthesized using various methods, namely glycosylation, an azide-alkyne click reaction, photoinduced thiol-en addition, and sulfation. The interactions between compounds with six fucolectins of bacterial or fungal origin were tested using a hemagglutination inhibition assay. As a result, a tetravalent, α-l-fucose presenting glycocluster showed to be a ligand that was orders of magnitude better than a simple monosaccharide for tested lectins in most cases, which can nominate it as a universal ligand for studied lectins. This compound was also able to inhibit the adhesion of Pseudomonas aeruginosa cells to human epithelial bronchial cells. A trivalent fucocluster with a protected amine functional group also seems to be a promising candidate for designing glycoconjugates and chimeras.

Interaction of isoflavonoids with human liver microsomal cytochromes P450: inhibition of CYP enzyme activities.

1. The possibility of interaction of isoflavonoids with concomitantly taken drugs to determined isoflavonoids safety was studied. Inhibition of nine forms of cytochrome P450 (CYP3A4, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP2E1) by 12 isoflavonoids (daidzein, genistein, biochanin A, formononetin, glycitein, equol and six glucosides, daidzin, puerarin, genistin, sissotrin, ononin and glycitin) was studied systematically. 2. The most potent inhibitors were genistein and daidzein inhibiting noncompetitively the CYP2C9 with Ki of 35.95 ± 6.96 and 60.56 ± 3.53 µmol/l and CYP3A4 (inhibited by genistein with Ki of 23.25 ± 5.85 µmol/l also by a noncompetitive mechanism). Potent inhibition of CYP3A4 was observed also with biochanin A (Ki of 57.69 ± 2.36 µmol/l) and equol (Ki of 38.47 ± 2.32 µmol/l). 3. Genistein and daidzein inhibit noncompetitively CYP3A4 and CYP2C9. With plasma levels in micromolar range, a clinically important interaction with concomitantly taken drugs does not seem to be probable.

Photo-initiated crosslinking extends mapping of the protein-protein interface to membrane-embedded portions of cytochromes P450 2B4 and b5.

Protein-protein interactions play a central role in the regulation of many biochemical processes (e.g. the system participating in enzyme catalysis). Therefore, a deeper understanding of protein-protein interactions may contribute to the elucidation of many biologically important mechanisms. For this purpose, it is necessary to establish the composition and stoichiometry of supramolecular complexes and to identify the crucial portions of the interacting molecules. This study is devoted to structure-functional relationships in the microsomal Mixed Function Oxidase (MFO) complex, which is responsible for biotransformation of many hydrophobic endogenous compounds and xenobiotics. In particular, the cytochrome b5 interaction with MFO terminal oxygenase cytochrome P-450 (P450) was studied. To create photolabile probes suitable for this purpose, we prepared cytochrome b5 which had a photolabile diazirine analog of methionine (pMet) incorporated into the protein sequence, employing recombinant expression in Escherichia coli. In addition to wild-type cytochrome b5, where three methionines (Met) are located at positions 96, 126, and 131, six mutants containing only one Met in the sequence were designed and expressed (see Table 1). In these mutants, a single Met was engineered into the catalytic domain (at positions 23, 41, or 46), into the linker between the protein domains (at position 96), or into the membrane region (at positions 126 or 131). These mutants should confirm or exclude these portions of cytochrome b5 which are involved in the interaction with P450. After UV irradiation, the pMet group(s) in the photolabile cytochrome b5 probe was(were) activated, producing covalent crosslinks with the interacting parts of P450 2B4 in the close vicinity. The covalent complexes were analyzed by the "bottom up" approach with high-accuracy mass spectrometry. The analysis provided an identification of the contacts in the supramolecular complex with low structural resolution. We found that all the above-mentioned cytochrome b5 Met residues can form intermolecular crosslinks and thus participate in the interaction. In addition, our results indicate the existence of at least two P450:cytochrome b5 complexes which differ in the orientation of individual proteins. The results demonstrate the advantages of the photo-initiated crosslinking technique which is able to map the protein-protein interfaces not only in the solvent exposed regions, but also in the membrane-embedded segments (compared to a typical crosslinking approach which generally only identifies crosslinks in solvent exposed regions).

3D-printed biosensor with poly(dimethylsiloxane) reservoir for magnetic separation and quantum dots-based immunolabeling of metallothionein.

Currently, metallothioneins (MTs) are extensively investigated as the molecular biomarkers and the significant positive association of the MT amount was observed in tumorous versus healthy tissue of various types of malignant tumors, including head and neck cancer. Thus, we proposed a biosensor with fluorescence detection, comprising paramagnetic nanoparticles (nanomaghemite core with gold nanoparticles containing shell) for the magnetic separation of MT, based on affinity of its sulfhydryl groups toward gold. Biosensor was crafted from PDMS combined with technology of 3D printing and contained reservoir with volume of 50 µL linked to input (sample/detection components and washing/immunobuffer) and output (waste). For the immunolabeling of immobilized MT anti-MT antibodies conjugated to CdTe quantum dots through synthetic heptapeptide were employed. After optimization of fundamental conditions of the immunolabeling (120 min, 20°C, and 1250 rpm) we performed it on a surface of paramagnetic nanoparticles in the biosensor reservoir, with evaluation of fluorescence of quantum dots (λexc 400 nm, and λem 555 nm). The developed biosensor was applied for quantification of MT in cell lines derived from spinocellular carcinoma (cell line 122P-N) and fibroblasts (122P-F) and levels of the biomarker were found to be about 90 nM in tumor cells and 37 nM in fibroblasts. The proposed system is able to work with low volumes (< 100 µL), with low acquisition costs and high portability.

The influence of ochratoxin A on DNA adduct formation by the carcinogen aristolochic acid in rats.

Exposure to the plant nephrotoxin and carcinogen aristolochic acid (AA) leads to the development of AA nephropathy, Balkan endemic nephropathy (BEN) and upper urothelial carcinoma (UUC) in humans. Beside AA, exposure to ochratoxin A (OTA) was linked to BEN. Although OTA was rejected as a factor for BEN/UUC, there is still no information whether the development of AA-induced BEN/UUC is influenced by OTA exposure. Therefore, we studied the influence of OTA on the genotoxicity of AA (AA-DNA adduct formation) in vivo. AA-DNA adducts were formed in liver and kidney of rats treated with AA or AA combined with OTA, but no OTA-related DNA adducts were detectable in rats treated with OTA alone or OTA combined with AA. Compared to rats treated with AA alone, AA-DNA adduct levels were 5.4- and 1.6-fold higher in liver and kidney, respectively, of rats treated with AA combined with OTA. Although AA and OTA induced NAD(P)H: quinone oxidoreductase (NQO1) activating AA to DNA adducts, their combined treatment did not lead to either higher NQO1 enzyme activity or higher AA-DNA adduct levels in ex vivo incubations. Oxidation of AA I (8-methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid) to its detoxification metabolite, 8-hydroxyaristolochic acid, was lower in microsomes from rats treated with AA and OTA, and this was paralleled by lower activities of cytochromes P450 1A1/2 and/or 2C11 in these microsomes. Our results indicate that a decrease in AA detoxification after combined exposure to AA and OTA leads to an increase in AA-DNA adduct formation in liver and kidney of rats.

Role of dihydromyricetin in cytochrome P450-mediated metabolism and carcinogen activation.

OBJECTIVES: Dihydromyricetin (DHM) is a flavonoid, which has been shown to antagonize effects of ethanol intoxication. As a potential pharmacological agent, its biological interactions with enzymes metabolizing foreign compounds should be tested. Thus, the aim of this study was to analyze the influence of DHM on the induction and metabolic activity of selected cytochromes P450 (CYPs). METHODS: After flavonoid administration by oral gavage to stomach the CYP expression at protein and mRNA levels was determined in rat liver and small intestine. The effects of flavonoids on CYP1A1/2, CYP1A2 or CYP2B1/2 enzyme activities in microsomes were measured using marker activities of these enzymes. Flavonoid-mediated inhibition of recombinant CYP1A2 was also assayed with luciferin-ME substrate. The flavonoid interaction with aryl hydrocarbon receptor (AhR) was assayed by reporter luciferase activity in Hep2G cells. RESULTS: The value of half maximal inhibitory concentration of DHM for CYP1A1/2, CYP1A2, and CYP2B1 were determined to be 4.1, 14.2, and 98.5 mmol.L(-1), respectively. With the exception of a weak induction of CYP2B1 and CYP1A2 in the middle part of small intestine and in the liver, respectively, DHM did not affect the CYP expression at protein levels. On the contrary, real-time PCR revealed elevated expression of CYP1A1 and CYP1A2 mRNA in proximal part of the small intestine while decreased in the middle part. In the study utilizing the HepG2 cells, DHM showed only an additive effect on the benzo[a]pyrene-mediated activation of Ah receptor. CONCLUSIONS: Dihydromyricetin doesn't significantly interfere with metabolic activity of CYP1A1/2 and CYP2B1 enzymes.

Cytotoxicity of and DNA adduct formation by ellipticine and its micellar form in human leukemia cells in vitro.

OBJECTIVES: The improvements of cancer treatment are the major challenge in oncology research. Nanocarriers are one of the promising approaches to selectively target tumor cells, frequently leading to improve drug therapeutic index. Ellipticine is an anticancer agent that functions through multiple mechanisms. Here, the toxic effects of an anticancer drug ellipticine encapsulated in a micellar nanotransporter and free ellipticine on human HL-60 leukemia cells and formation of ellipticine-derived DNA adducts by both forms of the drug in these cells were investigated. METHODS: The toxicity of modified ellipticine on cells was compared to that of free ellipticine using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide cytotoxicity assay. 32P-postlabeling was utilized to determine ellipticine-DNA adducts in treated cells. RESULTS: The comparison of efficiencies of free ellipticine and ellipticine-micelles [the poly(ethylene oxide)-block-poly(allyl glycidyl ether) block copolymer] to form ellipticine-derived DNA adducts in leukemia HL-60 cells and to act as cytotoxic agent on these cells was performed. Exposure of HL-60 cells to ellipticine in micelles resulted in formation of ellipticine-DNA adducts and caused the cytotoxic effect on these cells. The influence of ellipticine in micelles on HL-60 cells was very similar to that of free ellipticine. The ellipticine half maximal inhibition concentration was determined as 1.3±0.3 µmol.L(-1) and 1.4±0.3 µmol.L(-1) for ellipticine and ellipticine in micelles, respectively. Likewise, the levels of ellipticine-DNA adducts generated in HL-60 cells by both forms of ellipticine were analogous. CONCLUSION: The results found in this work demonstrate similar cytotoxicity and DNA-damaging effects of ellipticine and its micellar form on leukemia HL-60 cells in vitro.

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.

The effects of heavy metal ions, phthalates and ochratoxin A on oxidation of carcinogenic aristolochic acid I causing Balkan endemic nephropathy.

OBJECTIVES: Balkan endemic nephropathy (BEN) is a chronic progressive fibrosis associated with upper urothelial carcinoma (UUC). Aetiology of BEN is still not fully explained. Although carcinogenic aristolochic acid I (AAI) was proven as the major cause of BEN/UUC, this nephropathy is considered to be multifactorial. Hence, we investigated whether other factors considered as potential causes of BEN [a mycotoxin ochratoxin A (OTA), Cd, Pb, Se and As ions and organic compounds (i.e. phthalates) released from lignite deposits in BEN areas] can influence detoxication of AAI, whose concentrations are crucial for BEN development. METHODS: Oxidation of AAI to 8-hydroxyaristolochic acid I (AAIa) in the presence of Cd, Pb, Se, As ions, dibutylphthalate (DBP), butylbenzylphthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP) and OTA by rat liver microsomes was determined by HPLC. RESULTS: Only OTA, cadmium and selenium ions, and BBP inhibited AAI oxidation by rat liver microsomes. These compounds also inhibited activities of CYP1A1 and/or CYP2C6/11 catalysing AAI demethylation in rat livers. Therefore, these CYP inhibitions can be responsible for a decrease in AAIa formation. When the combined effects of these compounds were investigated, the most efficient inhibition was caused by OTA combined with BBP and selenium ions. CONCLUSION: The results show low effects of BBP, cadmium and selenium ions, and/or their combinations on AAI detoxication. No effects were produced by the other metal ions (Pb, As) and phthalates DBP and DEHP. This finding suggests that they do not influence AAI-mediated BEN development. In contrast, OTA might influence this process, by inhibition of AAI detoxication.

Fluorescent cellular assay for screening agents inhibiting Pseudomonas aeruginosa adherence.

Antibodies against Pseudomonas aeruginosa (PA) lectin, PAIIL, which is a virulence factor mediating the bacteria binding to epithelium cells, were prepared in chickens and purified from egg yolks. To examine these antibodies as a prophylactic agent preventing the adhesion of PA we developed a well plate assay based on fluorescently labeled bacteria and immortalized epithelium cell lines derived from normal and cystic fibrosis (CF) human lungs. The antibodies significantly inhibited bacteria adhesion (up to 50%) in both cell lines. In agreement with in vivo data, our plate assay showed higher susceptibility of CF cells towards the PA adhesion as compared to normal epithelium. This finding proved the reliability of the developed experimental system.

A Mechanism of O-Demethylation of Aristolochic Acid I by Cytochromes P450 and Their Contributions to This Reaction in Human and Rat Livers: Experimental and Theoretical Approaches.

Aristolochic acid I (AAI) is a plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is detoxified by cytochrome P450 (CYP)-mediated O-demethylation to 8-hydroxyaristolochic acid I (aristolochic acid Ia, AAIa). We previously investigated the efficiencies of human and rat CYPs in the presence of two other components of the mixed-functions-oxidase system, NADPH:CYP oxidoreductase and cytochrome b5, to oxidize AAI. Human and rat CYP1A are the major enzymes oxidizing AAI. Other CYPs such as CYP2C, 3A4, 2D6, 2E1, and 1B1, also form AAIa, but with much lower efficiency than CYP1A. Based on velocities of AAIa formation by examined CYPs and their expression levels in human and rat livers, here we determined the contributions of individual CYPs to AAI oxidation in these organs. Human CYP1A2 followed by CYP2C9, 3A4 and 1A1 were the major enzymes contributing to AAI oxidation in human liver, while CYP2C and 1A were most important in rat liver. We employed flexible in silico docking methods to explain the differences in AAI oxidation in the liver by human CYP1A1, 1A2, 2C9, and 3A4, the enzymes that all O-demethylate AAI, but with different effectiveness. We found that the binding orientations of the methoxy group of AAI in binding centers of the CYP enzymes and the energies of AAI binding to the CYP active sites dictate the efficiency of AAI oxidation. Our results indicate that utilization of experimental and theoretical methods is an appropriate study design to examine the CYP-catalyzed reaction mechanisms of AAI oxidation and contributions of human hepatic CYPs to this metabolism.

Chicken and rabbit antibodies against porcine pepsinogen A.

Isolated porcine pepsinogen A was used for the preparation of polyclonal rabbit and polyclonal chicken anti-pepsinogen A antibodies. Immunochemical properties of both immunoglobulin fractions were compared. The rabbit anti-serum was further purified using immobilized porcine pepsinogen A on magnetic cellulose beads and the resulting anti-pepsinogen A fraction proved to be applicable for the separation and the determination of porcine pepsinogen A. In contrary, antibodies prepared from chicken eggs by the same way have been found not suitable for the evaluation of the pepsinogen A level. Unexpectedly, the pre-immune fraction of chicken antibodies showed reactivity against porcine pepsinogen A and the affinity separation of specific polyclonal chicken anti-pepsinogen A antibodies on immobilized porcine pepsinogen A did not result in an enrichment of anti-pepsinogen A antibodies.

Effect of chicken antibodies on inflammation in human lung epithelial cell lines.

OBJECTIVES: As an alternative therapeutic approach for the prevention and treatment of bacterial infections with P. aeruginosa of cystic fibrosis (CF) patients, chicken yolk antibodies (IgY) could be used. The most significant advantage of IgY, in contrast to mammalian IgG, consists in the fact, that when bound to the antigen, they usually do not induce inflammatory reaction. In addition, the simplicity of egg production and the ease of IgY preparation makes this kind of antibody an excellent tool for passive immunization. Thus, the aim of our project was to study the effect of IgY and its Fab fragment on the potential induction of pro-inflammatory reactions in lung epithelial cells. METHODS: Chicken IgY were prepared from pooled egg yolks. Fab fragmens of IgY were purified from the papain digest of IgY using DEAE-Sephacel ion exchange chromatography. Their purity was verified by SDS electrophoresis in polyacrylamide gel. Immortalized human cell lines, CuFi (CF patient) and NuLi (healthy subject), and A549 (human adenocarcinoma cells) were exposed to IgY, Fab, OVA, LPS (positive control), PBS (negative control), and human and goat IgG for 24 hours. The concentration of pro-inflammatory cytokines TNF-α, IL-1ß, IL-6 and GM-CSF were determined in cell media using the BioPlex method, which enables the quantification of multiple analytes simultaneously in one sample. RESULTS: Our results show that i) the Fab fragment induced levels of some proinflammatory cytokines, when compared to the PBS control, whereas ii) chicken IgYs did not induce any notable production of pro-inflammatory cytokines in contrast to intense effect of LPS on TNF-α and GM-CSF. In summary, our data show that levels of all cytokines are comparable with physiological values in human serum except of IL-1ß, which concentration in cell medium was markedly elevated by Fab fragment. CONCLUSIONS: The present data indicate that IgY are not inflammatory for lung cells and thus they are possibly applicable for prevention of airway bacterial infections.

Quantification of interactions between cytochrome P450 2B4 and cytochrome b5 in a functional membrane complex.

OBJECTIVES: The mammalian mixed function oxidase (MFO) system participates in hydroxylation of many hydrophobic endogenous compounds as well as xenobiotics such as drugs and carcinogens. This biotransformation system, located in a membrane of endoplasmic reticulum, consists of cytochrome P-450 (P450), NADPH:P450 oxidoreductase and a facultative component, cytochrome b5. The knowledge of the interactions among the individual components of the MFO system is essential to understand the relationships between the structure and function of this system that finally dictate a qualitative and quantitative pattern of produced metabolites (e.g. detoxified xenobiotics and/or activated carcinogens). To elucidate the quantitative aspects of the interactions within the MFO system we acquired the photo-initiated cross-linking approach. METHODS: The photo-initiated cross-linking employing cytochrome b5 as a protein nanoprobe [an amino acid analogue of methionine (pMet) was incorporated into cytochrome b5 sequence during recombinant expression] was used to quantify its interaction with P450 2B4 in a functional membrane complex. The cross-linking was initiated by UV-irradiation that formed from a pMet photolabile diazirine group highly reactive carbene biradical. This biradical is able to covalently bind amino acids in the close proximity and to form cross-link. The Met 96 of cytochrome b5 is situated in a linker region between its catalytic and membrane domains, while Met 126 and 131 are located in its membrane domain. The combination of several methods (electrophoresis in polyacrylamide gel, isoelectric focusing, Edman N-terminal degradation and amino acid analysis) was employed to characterize the molar ratio of P450 2B4 to cytochrome b5 in formed covalent cross-links to quantify their transient interactions. RESULTS: The successfully produced cytochrome b5 nanoprobe (with confirmed pMet incorporation by mass spectrometry) stimulates the catalytical activity of P450 2B4 when reconstituted with NADPH:P450 oxidoreductase in vitro in dilauroylphosphatidylcholine (DLPC) vesicles. The cross-linking was carried out in similar reconstituted system without NADPH:P450 oxidoreductase, and at least three products were separated on 1D SDS-PAGE. The molar ratio of P450 to cytochrome b5 in each complex was estimated using the above-mentioned combination of methods as 1:1, 1:2 and 2:1. CONCLUSIONS: The results demonstrate the utility of cytochrome b5 nanoprobe to study the interactions in MFO system. Using this nanoprobe, heterodimer with P450 2B4 and in addition also heterooligomers were identified, suggesting rather complex interactions of both proteins in this system that suppose the formation of such multimeric structures in the membrane of endoplasmic reticulum.

Dicoumarol inhibits rat NAD(P)H:quinone oxidoreductase in vitro and induces its expression in vivo.

OBJECTIVES: Dicoumarol is known to act as an inhibitor of NAD(P)H: quinone oxidoreductase (NQO1). This cytosolic reductase significantly contributes to the genotoxicity of the nephrotoxic and carcinogenic alkaloid aristolochic acid I (AAI). Aristolochic acid causes aristolochic acid nephropathy (AAN), and Balkan endemic nephropathy (BEN), as well as associated urothelial malignancies. NQO1 is the most efficient enzyme responsible for the reductive bioactivation of AAI to species forming covalent AAI-DNA adducts. However, it is still not known how dicoumarol influences the NQO1-mediated reductive bioactivation of AAI. METHODS: AAI-DNA adduct formation was determined by 32P-postlabeling. Expression of NQO1 mRNA and NQO1 protein was determined by real-time polymerase chain reaction and Western blotting, respectively. RESULTS: In this study, dicoumarol inhibited AAI bioactivation to form AAI-DNA adducts mediated by rat and human NQO1 in vitro as expected. We however, demonstrated that dicoumarol acts as an inducer of NQO1 in kidney and lung of rats treated with this NQO1 inhibitor in vivo, both at protein and activity levels. This NQO1 induction increased the potency of kidney cytosol to bioactivate AAI and elevated AAI-DNA adduct levels were found in ex-vivo incubations of AAI with renal cytosols and DNA. NQO1 mRNA levels were induced in liver only by dicoumarol. CONCLUSION: Our results indicate a dual role of dicoumarol in NQO1-mediated genotoxicty of AAI. It acts both as an NQO1 inhibitor mainly in vitro and as an NQO1 inducer if administered to rats.