Epithelial chemerin-CMKLR1 signaling restricts microbiota-driven colonic neutrophilia and tumorigenesis by up-regulating lactoperoxidase.

Intestinal barrier immunity is essential for controlling gut microbiota without eliciting harmful immune responses, while its defect contributes to the breakdown of intestinal homeostasis and colitis development. Chemerin, which is abundantly expressed in barrier tissues, has been demonstrated to regulate tissue inflammation via CMKLR1, its functional receptor. Several studies have reported the association between increased expression of chemerin-CMKLR1 and disease severity and immunotherapy resistance in inflammatory bowel disease (IBD) patients. However, the pathophysiological role of endogenous chemerin-CMKLR1 signaling in intestinal homeostasis remains elusive. We herein demonstrated that deficiency of chemerin or intestinal epithelial cell (IEC)-specific CMKLR1 conferred high susceptibility to microbiota-driven neutrophilic colon inflammation and subsequent tumorigenesis in mice following epithelial injury. Unexpectedly, we found that lack of chemerin-CMKLR1 signaling specifically reduced expression of lactoperoxidase (LPO), a peroxidase that is predominantly expressed in colonic ECs and utilizes H2O2 to oxidize thiocyanates to the antibiotic compound, thereby leading to the outgrowth and mucosal invasion of gram-negative bacteria and dysregulated CXCL1/2-mediated neutrophilia. Importantly, decreased LPO expression was causally linked to aggravated microbiota-driven colitis and associated tumorigenesis, as LPO supplementation could completely rescue such phenotypes in mice deficient in epithelial chemerin-CMKLR1 signaling. Moreover, epithelial chemerin-CMKLR1 signaling is necessary for early host defense against bacterial infection in an LPO-dependent manner. Collectively, our study reveals that the chemerin-CMKLR1/LPO axis represents an unrecognized immune mechanism that potentiates epithelial antimicrobial defense and restricts harmful colonic neutrophilia and suggests that LPO supplementation may be beneficial for microbiota dysbiosis in IBD patients with a defective innate antimicrobial mechanism.

Dual oxidase 1 promotes antiviral innate immunity.

Dual oxidase 1 (DUOX1) is an NADPH oxidase that is highly expre-ssed in respiratory epithelial cells and produces H2O2 in the airway lumen. While a line of prior in vitro observations suggested that DUOX1 works in partnership with an airway peroxidase, lactoperoxidase (LPO), to produce antimicrobial hypothiocyanite (OSCN-) in the airways, the in vivo role of DUOX1 in mammalian organisms has remained unproven to date. Here, we show that Duox1 promotes antiviral innate immunity in vivo. Upon influenza airway challenge, Duox1-/- mice have enhanced mortality, morbidity, and impaired lung viral clearance. Duox1 increases the airway levels of several cytokines (IL-1ß, IL-2, CCL1, CCL3, CCL11, CCL19, CCL20, CCL27, CXCL5, and CXCL11), contributes to innate immune cell recruitment, and affects epithelial apoptosis in the airways. In primary human tracheobronchial epithelial cells, OSCN- is generated by LPO using DUOX1-derived H2O2 and inactivates several influenza strains in vitro. We also show that OSCN- diminishes influenza replication and viral RNA synthesis in infected host cells that is inhibited by the H2O2 scavenger catalase. Binding of the influenza virus to host cells and viral entry are both reduced by OSCN- in an H2O2-dependent manner in vitro. OSCN- does not affect the neuraminidase activity or morphology of the influenza virus. Overall, this antiviral function of Duox1 identifies an in vivo role of this gene, defines the steps in the infection cycle targeted by OSCN-, and proposes that boosting this mechanism in vivo can have therapeutic potential in treating viral infections.

Effects of oral administration of colostrum whey in peripartum goat on antimicrobial peptides in postpartum milk.

The present study was conducted to examine whether colostrum supplementation in peripartum goats increases the antimicrobial peptides in their milk. Goats were orally administered 2 ml of colostrum whey products (colostrum group) or water (control group) daily, from 2 weeks before until 2 weeks after kidding. Body weights of mothers and kids were measured. Blood, milk, and fecal samples were collected from the mothers, and blood samples were collected from the kids. Concentrations of milk antimicrobial peptides (beta-defensin, cathelicidin, lactoferrin, S100A7, lactoperoxidase, and immunoglobulin A [IgA]) were determined. IgA and nutritional parameters (glucose, total cholesterol, triglyceride, ketone bodies, and non-esterified fatty acids) were also determined in the blood of mothers and kids. Milk IgA and lactoferrin concentrations were higher in the colostrum group than in the control group. Conversely, lower milk concentrations of S100A7 were observed in the colostrum group than that in the control group. Plasma IgA concentrations were higher for kids from the colostrum group than for those from the control group. These results suggest that oral administration of colostrum in pregnant goats increases IgA concentration in postpartum milk, which can subsequently improve the health of their kids.

Milk lactoperoxidase decreases ID1 and ID3 expression in human oral squamous cell carcinoma cell lines.

Milk consumption may modify the risk of squamous cell carcinoma. The role of milk to modulate the gene expression in oral squamous cell carcinoma cells has not been investigated so far. Here, HSC2 oral squamous carcinoma cells were exposed to an aqueous fraction of human milk and a whole-genome array was performed. Among the genes that were significantly reduced by human and cow milk were the DNA-binding protein inhibitor 1 (ID1), ID3 and Distal-Less Homeobox 2 (DLX2) in HSC2 cells. Also, in TR146 oral squamous carcinoma cells, there was a tendency towards a decreased gene expression. Upon size fractionation, lactoperoxidase but not lactoferrin and osteopontin was identified to reduce ID1 and ID3 in HSC2 cells. Dairy products and hypoallergenic infant formula failed to decrease the respective genes. These data suggest that milk can reduce the expression of transcription factors in oral squamous carcinoma cells.

In vitro effects of standard antioxidants on lactoperoxidase enzyme-A molecular docking approach.

Lactoperoxidase (LPO), an antioxidant enzyme, is a natural antimicrobial system that eliminates the harmful effects of microorganisms in milk. It has a wide range of applications and is also preferred in cosmetic and clinical applications, as well as used in foods. The use of antioxidants is well recognized in the food and feed industries to improve the shelf life of products. This study aimed to determine the in vitro inhibition effects of Trolox, α-tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, and propyl gallate, which are commonly used as antioxidants in food and pharmaceutical products. For this purpose, LPO was first purified in a single step using sepharose-4B-l-tyrosine-sulfanilamide affinity gel chromatography. Also, some inhibition parameters, including half-maximal inhibitory concentration (IC50 ), Ki values, and inhibition types, were calculated for each antioxidant molecule. The IC50 values of these molecules, which exhibited competitive inhibition, varied between 377.7 and 3397.8 nM. Molecular docking studies were also performed for all compounds. According to the binding scores, α-tocopherol was shown to exhibit the most effective inhibitor property (IC50 : 377.7 nM and Ki : 635.8 ± 16.8 nM) among the standard antioxidants used in this study. Inhibiting the LPO activity by standard antioxidants results in the weakening of the immune system during lactation, which is important for metabolism.

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines.

Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes.

Reactivation of peroxidase activity in human saliva samples by polyphenols.

OBJECTIVES: The enzyme lactoperoxidase (LPO), which is released into several body fluids like saliva, is an essential part to maintain the oral bacterial homeostasis by catalysing the oxidation of thiocyanate (SCN-) to hypo-thiocyanite (-OSCN). The formation of unreactive redox intermediates (like Compound II) leads to a decreased pseudo-halogenating enzyme activity, which is associated with a higher risk for oral infections. According to former studies with bovine LPO selected flavonoids were tested in respect to their potential to reactivate the enzymatic activity in a more physiological, human salivary system. DESIGN: Saliva samples from healthy donors were collected and characterized by using several gel staining methods and immunoblotting. Afterwards kinetic measurements were performed by applying the TNB-assay to evaluate the pseudo-halogenating salivary peroxidase (SAPX) activity. The measurements were performed in the presence of excess H2O2 to simulate pro-inflammatory conditions. Moreover selected flavonoids or an ethanolic extract of Tormentillae rhizoma were applied to test their regenerating effect on the LPO-derived -OSCN production. RESULTS: Despite the complex protein composition of the collected saliva samples, an SAPX-derived pseudo-halogenating activity could be identified. The -OSCN regenerating effects of the tested polyphenols were completely comparable to previous in vitro experiments with bovine LPO. Thus, we could show that phenolic substances are suitable to regenerate the peroxidase activity in human saliva samples after H2O2-induced inactivation. CONCLUSION: The studies provide new insights into the effect of pharmaceutical relevant polyphenols on salivary peroxidase activity and thus, suggest this enzyme as a new target for the prevention and therapy of oral inflammatory diseases.

Molecular Interactions of Carcinogenic Aromatic Amines, 4-Aminobiphenyl and 4,4'-Diaminobiphenyl, with Lactoperoxidase - Insight to Breast Cancer.

BACKGROUND/AIM: Lactoperoxidase (LPO) is an antimicrobial protein present in milk, saliva, gastric secretions, tears and upper respiratory tract secretions. LPO constitutes an important enzyme of the human immune defense system. However, LPO has also been suggested to be involved in breast cancer etiology through production of reactive free radicals and activation of carcinogenic aromatic compounds. Aromatic compounds are generally highly lipophilic and thus accumulate in highly fatty breast tissues. The aromatic compounds 4-aminobiphenyl (ABP) and 4,4'-diaminobiphenyl (BZ) are known to have carcinogenic properties. LPO catalyzes their oxidation and converts them into reactive products which bind to DNA and form adducts. These DNA adducts subsequently lead to breast cancer. MATERIALS AND METHODS: The crystal structure of LPO was obtained from Protein Data Bank. Structures of ABP and BZ were retrieved from PubChem database. Induced Fit Docking was performed using glide module from Schrodinger. RESULTS: The present study reports the structural binding of ABP and BZ with LPO using in silico approaches. The amino acid residues of LPO involved in the binding with the two aromatic ligands were characterized and binding energy values were calculated. CONCLUSION: Both ABP and BZ were placed in the substrate binding site present in the distal heme cavity of LPO with good affinity. The binding mode mimicked that of the natural substrate since these compounds did not disturb the water molecule that plays an important role in the oxidation reaction. Thus, the water molecule is potentially available for facilitating the subsequent activation of the aromatic amines to reactive species which may form DNA adducts leading to breast cancer.

Lactoperoxidase, an Antimicrobial Milk Protein, as a Potential Activator of Carcinogenic Heterocyclic Amines in Breast Cancer.

BACKGROUND: Lactoperoxidase (LPO) is an antimicrobial protein secreted from mammary, salivary and other mucosal glands. It is an important member of heme peroxidase enzymes and the primary peroxidase enzyme present in breast tissues. In addition to the antimicrobial properties, LPO has been shown to be associated with breast cancer etiology. Heterocyclic amines, an important class of environmental and dietary carcinogens, have been increasingly associated with breast cancer etiology. Heterocyclic amines undergo activation in breast tissue as a result of oxidation by LPO. The current study includes three important heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methy-6-phenylimidazo[4,5-b]-pyridine (PhIP), that have carcinogenic activity. MATERIALS AND METHODS: The structural binding characterization of IQ, MeIQx and PhIP with LPO was done using in silico approaches. Their binding pattern and interactions with LPO amino acid residues were analyzed. RESULTS: The three compounds bound in the distal heme cavity of LPO without replacing the important water molecule required for oxidation of substrate compounds. PhIP displayed lesser binding affinity for LPO in comparison to IQ and MeIQx. The binding mode of heterocyclic amines in distal heme cavity of LPO resembled to that of substrate binding pattern. CONCLUSION: The three heterocyclic amines are suggested to act as LPO substrate. The undisturbed water molecule present in distal heme cavity of the LPO is expected to facilitate the oxidation and activation of the three heterocyclic amines. These activated compounds may potentially bind with DNA in breast tissues forming DNA adducts and may subsequently lead to breast cancer initiation.

High-affinity von Willebrand factor binding does not affect the anatomical or hepatocellular distribution of factor VIII in rats.

UNLABELLED: Essentials Von Willebrand factor (VWF) stabilizes factor VIII (FVIII) and prevents its premature clearance. Rat anatomical and hepatocellular distribution studies assessed the VWF effect on FVIII clearance. Hepatocytes and liver sinusoidal endothelial cells play a key role in FVIII clearance. Anatomical and hepatocellular distribution of FVIII is independent of high-affinity VWF binding. ABSTRACT: Background Von Willebrand factor (VWF) stabilizes factor VIII in the circulation and prevents its premature clearance. Objective To study the effects of VWF on FVIII clearance in rats with endogenous VWF. Methods Anatomical and hepatocellular distribution studies were performed in rats following intravenous administration of glycoiodinated recombinant FVIII (rFVIII) and a FVIII variant, FVIII-Y1680F, lacking high-affinity VWF binding. Radioactivity was quantified in organs, and in distinct liver cell populations. The role of VWF binding was also studied by immunohistochemical staining of rat livers perfused ex vivo with rFVIII alone or with a FVIII-binding VWF fragment. Results The liver was the predominant organ of rFVIII distribution, and a radioactivity peak was also observed in the intestines, suggesting FVIII secretion to the bile by hepatocytes. In the liver, ~60% of recovered radioactivity was associated with hepatocytes, 32% with liver sinusoidal endothelial cells (LSECs), and 9% with Kupffer cells (KCs). When calculated per cell, 1.5-fold to 3-fold more radioactivity was associated with LSECs than with hepatocytes. The importance of hepatocytes and LSECs was confirmed by immunohistochemical staining; strong staining was seen in LSECs, and less intense, punctate staining in hepatocytes. Minor staining in KCs was observed. Comparable anatomical and hepatocellular distributions were observed with rFVIII and FVIII-Y1680F, and the presence of the VWF fragment, D'D3A1, did not change the FVIII staining pattern in intact livers. Conclusions The present data support FVIII clearance via the liver, with hepatocytes and LSECs playing a key role. High-affinity VWF binding did not alter the anatomical or hepatocellular distribution of FVIII.

Gut Microbiota Conversion of Dietary Ellagic Acid into Bioactive Phytoceutical Urolithin A Inhibits Heme Peroxidases.

Numerous studies signify that diets rich in phytochemicals offer many beneficial functions specifically during pathologic conditions, yet their effects are often not uniform due to inter-individual variation. The host indigenous gut microbiota and their modifications of dietary phytochemicals have emerged as factors that greatly influence the efficacy of phytoceutical-based intervention. Here, we investigated the biological activities of one such active microbial metabolite, Urolithin A (UA or 3,8-dihydroxybenzo[c]chromen-6-one), which is derived from the ellagic acid (EA). Our study demonstrates that UA potently inhibits heme peroxidases i.e. myeloperoxidase (MPO) and lactoperoxidase (LPO) when compared to the parent compound EA. In addition, chrome azurol S (CAS) assay suggests that EA, but not UA, is capable of binding to Fe3+, due to its catechol-like structure, although its modest heme peroxidase inhibitory activity is abrogated upon Fe3+-binding. Interestingly, UA-mediated MPO and LPO inhibition can be prevented by innate immune protein human NGAL or its murine ortholog lipocalin 2 (Lcn2), implying the complex nature of host innate immunity-microbiota interactions. Spectral analysis indicates that UA inhibits heme peroxidase-catalyzed reaction by reverting the peroxidase back to its inactive native state. In support of these in vitro results, UA significantly reduced phorbol myristate acetate (PMA)-induced superoxide generation in neutrophils, however, EA failed to block the superoxide generation. Treatment with UA significantly reduced PMA-induced mouse ear edema and MPO activity compared to EA treated mice. Collectively, our results demonstrate that microbiota-mediated conversion of EA to UA is advantageous to both host and microbiota i.e. UA-mediated inhibition of pro-oxidant enzymes reduce tissue inflammation, mitigate non-specific killing of gut bacteria, and abrogate iron-binding property of EA, thus providing a competitive edge to the microbiota in acquiring limiting nutrient iron and thrive in the gut.

The sodium iodide symporter is unlikely to be a thyroid/breast shared antigen.

PURPOSE: Anti-thyroid peroxidase (TPO) autoantibodies (TPOAb) seem to be protective for patients with breast cancer (BC). Thyroid and breast tissues both express the sodium iodide symporter (NIS), similarly both have a peroxidase activity, TPO and lactoperoxidase (LPO) respectively. We hypothesize a common immune response to a thyroid/breast shared antigen suggesting three putative mechanisms: (1) TPOAb react to both TPO and LPO, (2) TPO could be expressed in BC and (3) patients with TPOAb could have autoantibodies to NIS (NISAb). Previous studies excluded NISAb that block NIS activity in sera of patients with thyroid autoimmunity (TA) and/or BC. This study investigates neutral NISAb (binding without affecting function). METHODS: Clones of CHO cells stably expressing human NIS (hNIS; CHO-NIS) were isolated following transfection of hNIS in pcDNA3 vector. Expression of hNIS mRNA and surface protein was confirmed by PCR and flow cytometry respectively using a hNIS-mouse-monoclonal-antibody. CHO-NIS and controls transfected with the empty pcDNA3 vector (CHO-Empty) were incubated with 42 heat-inactivated human sera followed by an anti-human-IgG-AlexaFluor488-conjugate: 12 with BC, 11 with TA, 10 with both BC and TA and 9 with non-autoimmune thyroid diseases. The Kolmogorov-Smirnov Test was used to compare the fluorescence intensity obtained with CHO-NIS and CHO-Empty, using sera from six young males as a negative control population. RESULTS: None of the 42 sera were positive for NISAb. CONCLUSIONS: NISAb are rare and NIS is unlikely to be a common thyroid/BC shared antigen. We have recently demonstrated TPO expression in BC tissue and are currently investigating TPOAb cross-reactivity with TPO/LPO.

The genes Scgb1a1, Lpo and Gbp2 characteristically expressed in peri-implant epithelium of rats.

OBJECTIVES: The peri-implant epithelium (PIE) plays an important role in the prevention against initial stage of inflammation. To minimize the risk of peri-implantitis, it is necessary to understand the biological characteristics of the PIE. The aim of this study was to investigate the characteristic gene expression profile of PIE as compared to junctional epithelium (JE) using laser microdissection and microarray analysis. METHODS: Left upper first molars of 4-week-old rat were extracted, and titanium alloy implants were placed. Four weeks after surgery, samples were harvested by laser microdissection, and total RNA samples were isolated. Comprehensive analyses of genes expressed in the JE and PIE were performed using microarray analysis. Confirmation of the differential expression of selected genes was performed by quantitative real-time polymerase chain reaction and immunohistochemistry. RESULTS: The microarray analysis showed that 712 genes were more than twofold change upregulated in the PIE compared with the JE. Genes Scgb1a1 were significantly upregulated more than 19.1-fold, Lpo more than 19.0-fold, and Gbp2 more than 8.9-fold, in the PIE (P < 0.01). Immunohistochemical localization of SCGB1A1, LPO, and GBP2 was observed in PIE. CONCLUSION: The present results suggested that genes Scgb1a1, Lpo, and Gbp2 are characteristically expressed in the PIE.

Hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular H1N2 influenza A virus.

OBJECTIVE AND DESIGN: Our aim was to study whether an extracellular, oxidative antimicrobial mechanism inherent to tracheal epithelial cells is capable of inactivating influenza H1N2 virus. MATERIAL OR SUBJECTS: Epithelial cells were isolated from tracheas of male Sprague-Dawley rats. Both primary human and rat tracheobronchial epithelial cells were differentiated in air-liquid interface cultures. TREATMENT: A/swine/Illinois/02860/09 (swH1N2) influenza A virions were added to the apical side of airway cells for 1 h in the presence or absence of lactoperoxidase or thiocyanate. METHODS: Characterization of rat epithelial cells (morphology, Duox expression) occurred via western blotting, PCR, hydrogen peroxide production measurement and histology. The number of viable virions was determined by plaque assays. Statistical difference of the results was analyzed by ANOVA and Tukey's test. RESULTS: Our data show that rat tracheobronchial epithelial cells develop a differentiated, polarized monolayer with high transepithelial electrical resistance, mucin production and expression of dual oxidases. Influenza A virions are inactivated by human and rat epithelial cells via a dual oxidase-, lactoperoxidase- and thiocyanate-dependent mechanism. CONCLUSIONS: Differentiated air-liquid interface cultures of rat tracheal epithelial cells provide a novel model to study airway epithelium-influenza interactions. The dual oxidase/lactoperoxidase/thiocyanate extracellular oxidative system producing hypothiocyanite is a fast and potent anti-influenza mechanism inactivating H1N2 viruses prior to infection of the epithelium.

Inactivating effects of the lactoperoxidase system on bacterial lyases involved in oral malodour production.

The main components of oral malodour have been identified as volatile sulfur compounds (VSCs), including hydrogen sulfide (H(2)S) and methyl mercaptan (CH(3)SH). The lactoperoxidase (LPO) system (consisting of LPO, glucose oxidase, glucose and thiocyanate) was previously shown to exhibit antimicrobial activities against some oral bacteria in vitro and suppressive effects on VSCs in mouth air in a clinical trial. Here, we examined the in vitro effects of the LPO system on the activities of the bacterial lyases involved in the production of VSCs by oral anaerobes. The exposure of crude bacterial extracts of Fusobacterium nucleatum and Porphyromonas gingivalis or purified methionine γ-lyase to the LPO system resulted in the inactivation of their lyase activities through l-cysteine and l-methionine, which was linked to the production of H(2)S and CH(3)SH, respectively. The exposure of living F. nucleatum and P. gingivalis cells to the LPO system resulted in the suppression of cell numbers and lyase activities. The inactivation of the crude bacterial extracts of F. nucleatum and purified methionine γ-lyase by the LPO system was partly recovered by the addition of DTT. Therefore, the LPO system may inactivate bacterial lyases including methionine γ-lyase by reacting with the free cysteine residues of lyases. These results suggested that the LPO system suppresses the production of VSCs not only through its antimicrobial effects, but also by its inactivating effects on the bacterial lyases of F. nucleatum and P. gingivalis.

(99m)Tc-amitrole as a novel selective imaging probe for solid tumor: In silico and preclinical pharmacological study.

Lactoperoxidase (LPO) inhibitors are very selective for solid tumor due to their high binding affinity to the LPO enzyme. A computational study was used to select top-ranked LPO inhibitor (alone and in complex with (99m)Tc) with high in silico affinity. The novel prepared (99m)Tc-amitrole complex demonstrated both in silico and in vivo high affinity toward solid tumors.(99m)Tc-amitrole was radio-synthesized with a high radiochemical yield (89.7±3.25). It showed in vitro stability for up to 6h. Its preclinical evaluation in solid tumor-bearing mice showed high retention and biological accumulation in solid tumor cells with a high Target/Non-Target (T/NT) ratio equal to 4.9 at 60min post-injection. The data described previously could recommend (99m)Tc-amitrole as potential targeting scintigraphic probe for solid tumor imaging.

Metabolomic profiling unravels DNA adducts in human breast that are formed from peroxidase mediated activation of estrogens to quinone methides.

Currently there are three major hypotheses that have been proposed for estrogen induced carcinogenicity, however exact etiology remains unknown. Based on the chemical logic, studies were undertaken to investigate if estrogens could generate quinone methides in an oxidative environment which then could cause DNA damage in humans. In presence of MnO2 estrogens were oxidized to quinone methides. Surprisingly quinone methides were found to be stable with t1/2 of 20.8 and 4.5 min respectively. Incubation of estrogens with lactoperoxidase (LPO) and H2O2 resulted in formation of respective quinone methides (E1(E2)-QM). Subsequent addition of adenine to the assay mixture lead to trapping of E1(E2)-QM, resulting in formation of adenine adducts of estrogens, E1(E2)-9-N-Ade. Targeted ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based metabolomic analysis of the breast tissue extracts showed the presence of adenine adducts of estrogens, E1(E2)-9-N-Ade, along with other estrogen related metabolites. Identity of E1(E2)-N-Ade in LPO assay extracts and breast tissue extracts were confirmed by comparing them to pure synthesized E1(E2)-9-N-Ade standards. From these results, it is evident that peroxidase enzymes or peroxidase-like activity in human breast tissue could oxidize estrogens to electrophilic and stable quinone methides in a single step that covalently bind to DNA to form adducts. The error prone repair of the damaged DNA can result in mutation of critical genes and subsequently cancer. This article reports evidence for hitherto unknown estrogen metabolic pathway in human breast, catalyzed by peroxidase, which could initiate cancer.

Expression of lactoperoxidase in differentiated mouse colon epithelial cells.

Lactoperoxidase (LPO) is known to be present in secreted fluids, such as milk and saliva. Functionally, LPO teams up with dual oxidases (DUOXs) to generate bactericidal hypothiocyanite in the presence of thiocyanate. DUOX2 is expressed in intestinal epithelium, but there is little information on LPO expression in this tissue. To fill the gap of knowledge, we have analyzed Lpo gene expression and its regulation in mouse intestine. In wild-type (WT) C57BL/6 (B6) mouse intestine, an appreciable level of mouse Lpo gene expression was detected in the colon, but not the ileum. However, in B6 mice deficient in glutathione peroxidase (GPx)-1 and -2, GPx1/2-double-knockout (DKO), which had intestinal pathology, the colon Lpo mRNA levels increased 5- to 12-fold depending on mouse age. The Lpo mRNA levels in WT and DKO 129S1/SvlmJ (129) colon were even higher, 9- and 5-fold, than in B6 DKO colon. Higher levels of Lpo protein and enzymatic activity were also detected in the 129 mouse colon compared to B6 colon. Lpo protein was expressed in the differentiated colon epithelial cells, away from the crypt base, as shown by immunohistochemistry. Similar to human LPO mRNA, mouse Lpo mRNA had multiple spliced forms, although only the full-length variant 1 was translated. Higher methylation was found in the 129 than in the B6 strain, in DKO than in control colon, and in older than in juvenile mice. However, methylation of the Lpo intragenic CpG island was not directly induced by inflammation, because dextran sulfate sodium-induced colitis did not increase DNA methylation in B6 DKO colon. Also, Lpo DNA methylation is not correlated with gene expression.

Inactivation of the potent Pseudomonas aeruginosa cytotoxin pyocyanin by airway peroxidases and nitrite.

Pyocyanin (1-hydroxy-N-methylphenazine, PCN) is a cytotoxic pigment and virulence factor secreted by the human bacterial pathogen, Pseudomonas aeruginosa. Here, we report that exposure of PCN to airway peroxidases, hydrogen peroxide (H(2)O(2)), and NaNO(2) generates unique mononitrated PCN metabolites (N-PCN) as revealed by HPLC/mass spectrometry analyses. N-PCN, in contrast to PCN, was devoid of antibiotic activity and failed to kill Escherichia coli and Staphylococcus aureus. Furthermore, in contrast to PCN, intratracheal instillation of N-PCN into murine lungs failed to induce a significant inflammatory response. Surprisingly, at a pH of ∼7, N-PCN was more reactive than PCN with respect to NADH oxidation but resulted in a similar magnitude of superoxide production as detected by electron paramagnetic resonance and spin trapping experiments. When incubated with Escherichia coli or lung A549 cells, PCN and N-PCN both led to superoxide formation, but lesser amounts were detected with N-PCN. Our results demonstrate that PCN that has been nitrated by peroxidase/H(2)O(2)/NO(2)(-) systems possesses less cytotoxic/proinflammatory activity than native PCN. Yield of N-PCN was decreased by the presence of the competing physiological peroxidase substrates (thiocyonate) SCN(-) (myeloperoxidase, MPO, and lactoperoxidase, LPO) and Cl(-) (MPO), which with Cl(-) yielded chlorinated PCNs. These reaction products also showed decreased proinflammatory ability when instilled into the lungs of mice. These observations add important insights into the complexity of the pathogenesis of lung injury associated with Pseudomonas aeruginosa infections and provide additional rationale for exploring the efficacy of NO(2)(-) in the therapy of chronic Pseudomonas aeruginosa airway infection in cystic fibrosis.