Qingyi decoction attenuates intestinal epithelial cell injury via the calcineurin/nuclear factor of activated T-cells pathway.

BACKGROUND: Recent studies have demonstrated that dysfunction of the intestinal barrier is a significant contributing factor to the development of severe acute pancreatitis (SAP). A stable intestinal mucosa barrier functions as a major anatomic and functional barrier, owing to the balance between intestinal epithelial cell (IEC) proliferation and apoptosis. There is some evidence that calcium overload may trigger IEC apoptosis and that calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) signaling might play an important role in calcium-mediated apoptosis. AIM: To investigate the potential mechanisms underlying the therapeutic effect of Qingyi decoction (QYD) in SAP. METHODS: A rat model of SAP was created via retrograde infusion of sodium deoxycholate. Serum levels of amylase, tumor necrosis factor (TNF-α), interleukin (IL)-6, D-lactic acid, and diamine oxidase (DAO); histological changes; and apoptosis of IECs were examined in rats with or without QYD treatment. The expression of the two subunits of CaN and NFAT in intestinal tissue was measured via quantitative real-time polymerase chain reaction and western blotting. For in vitro studies, Caco-2 cells were treated with lipopolysaccharide (LPS) and QYD serum, and then cell viability and intracellular calcium levels were detected. RESULTS: Retrograde infusion of sodium deoxycholate increased the severity of pancreatic and intestinal pathology and the levels of serum amylase, TNF-α, and IL-6. Both the indicators of intestinal mucosa damage (D-lactic acid and DAO) and the levels of IEC apoptosis were elevated in the SAP group. QYD treatment reduced the serum levels of amylase, TNF-α, IL-6, D-lactic acid, and DAO and attenuated the histological findings. IEC apoptosis associated with SAP was ameliorated under QYD treatment. In addition, the protein expression levels of the two subunits of CaN were remarkably elevated in the SAP group, and the NFATc3 gene was significantly upregulated at both the transcript and protein levels in the SAP group compared with the control group. QYD significantly restrained CaN and NFATc3 gene expression in the intestine, which was upregulated in the SAP group. Furthermore, QYD serum significantly decreased the LPS-induced elevation in intracellular free Ca2+ levels and inhibited cell death. CONCLUSION: QYD can exert protective effects against intestinal mucosa damage caused by SAP and the protective effects are mediated, at least partially, by restraining IEC apoptosis via the CaN/NFATc3 pathway.

Formic acid induces hypertension-related hemorrhage in hSSAOTG in mice and human.

Hypertension is a confirmed risk factor for cerebral hemorrhage in humans. Which endogenous factor directly induces hypertension-related hemorrhage is unclear. In this study, 42 hemorrhagic patients with hypertension and hyperlipidemia and 42 age-matched healthy controls were enrolled. The contents of serum semicarbazide-sensitive amine oxidase (SSAO) and formic acid (FC, FC is a final product of SSAO through the oxidation of endogenous formaldehyde, which results from the enzymatic oxidative deamination of the SSAO substrate, methylamine) were examined in the patients after stroke. Hemorrhagic areas were quantified by computer tomography. In the animal study, hemorrhagic degree was assessed by hemotoxylin & eosin or tissue hemoglobin kits. The relationship between FC and blood pressure/hemorrhagic degree was examined in wild-type mice and hSSAOTG mice fed with high-fat diets or high-fat and -salt diets. The results showed that the levels of serum FC were positively correlated with blood pressure and hemorrhagic areas in hemorrhagic patients. Transfection of microRNA-134 could enhance SSAO expression in human vascular smooth muscle cells. Consistently, after treatment with high-fat and -salt diets, hSSAOTG mice exhibited higher levels of miR134 and FC, higher blood pressure, and more severe hemorrhage than wild-type mice. Interestingly, folic acid reduced hypertension and hemorrhage in hSSAOTG mice fed with high-fat diets. These findings suggest that FC is a crucial endogenous factor for hypertension and hemorrhage.

Fentanyl alleviates intestinal mucosal barrier damage in rats with severe acute pancreatitis by inhibiting the MMP-9/FasL/Fas pathway.

BACKGROUND: Fentanyl is an analgesic used against pancreatitis-related pain, while whether it ameliorates severe acute pancreatitis (SAP) has yet to be checked. This study aims to determine fentanyl-delivered effect on SAP and the mechanism underlying this effect. METHODS: Rat SAP models were established, following fentanyl treatment. The serum activity of amylase (AMY), lipase (LIP), and diamine oxidase (DAO) was detected by enzyme-linked immunosorbent assay (ELISA). Histological examination was performed in the pancreatic and intestinal tissues with hematoxylin-eosin staining. After transfection with matrix metalloproteinase (MMP) 9 overexpression plasmids, Caco-2 monolayers were treated with fentanyl and subsequently exposed to lipopolysaccharide (LPS). The transepithelial electrical resistance (TEER) value was determined in rat intestinal mucosa through an Ussing chamber assisted by Analyze & Acquire, and in Caco-2 cell monolayers through a voltohmmeter. Intestinal mucosa and paracellular permeabilities were determined by fluorescein isothiocyanate (FITC)-labeled dextran assay. The expressions of ZO-1, Occludin, MMP9, Fas and Fas ligand (FasL) in rat intestinal mucosa and/or Caco-2 monolayers were analyzed by qRT-PCR or/and western blot. RESULTS: Fentanyl alleviated SAP-related histological alterations in the pancreas and intestines, reduced the elevated levels of SAP-related AMY, LIP, and DAO, but promoted the levels of ZO-1 and Occludin. In SAP rats and Caco-2 monolayers, SAP-related or LPS-induced TEER value decreases, permeability increases, and increases in the expressions of MMP9, Fas, and FasL were reversed partly by fentanyl. Notably, MMP9 overexpression could reverse the above fentanyl-delivered in vitro effects. CONCLUSIONS: Fentanyl alleviates intestinal mucosal barrier damage in rats with SAP by inhibiting the MMP9/FasL/Fas pathway.

Enzymatic Spermine Metabolites Induce Apoptosis Associated with Increase of p53, caspase-3 and miR-34a in Both Neuroblastoma Cells, SJNKP and the N-Myc-Amplified Form IMR5.

Neuroblastoma (NB) is a common malignant solid tumor in children and accounts for 15% of childhood cancer mortality. Amplification of the N-Myc oncogene is a well-established poor prognostic marker in NB patients and strongly correlates with higher tumor aggression and resistance to treatment. New therapies for patients with N-Myc-amplified NB need to be developed. After treating NB cells with BSAO/SPM, the detection of apoptosis was determined after annexin V-FITC labeling and DNA staining with propidium iodide. The mitochondrial membrane potential activity was checked, labeling cells with the probe JC-1 dye. We analyzed, by real-time RT-PCR, the transcript of genes involved in the apoptotic process, to determine possible down- or upregulation of mRNAs after the treatment on SJNKP and the N-Myc-amplified IMR5 cell lines with BSAO/SPM. The experiments were carried out considering the proapoptotic genes Tp53 and caspase-3. After treatment with BSAO/SPM, both cell lines displayed increased mRNA levels for all these proapoptotic genes. Western blotting analysis with PARP and caspase-3 antibody support that BSAO/SPM treatment induces high levels of apoptosis in cells. The major conclusion is that BSAO/SPM treatment leads to antiproliferative and cytotoxic activity of both NB cell lines, associated with activation of apoptosis.

Lathyrus sativus diamine oxidase reduces Clostridium difficile toxin A-induced toxicity in Caco-2 cells by rescuing RhoA-GTPase and inhibiting pp38-MAPK/NF-κB/HIF-1α activation.

Clostridium difficile toxin A (TcdA) impairs the intestinal epithelial barrier, increasing the mucosa permeability and triggering a robust inflammatory response. Lathyrus sativus diamino oxidase (LSAO) is a nutraceutical compound successfully used in various gastrointestinal dysfunctions. Here, we evaluated the LSAO (0.004-0.4 µM) ability to counter TcdA-induced (30 ng/mL) toxicity and damage in Caco-2 cells, investigating its possible mechanism of action. LSAO has improved the transepithelial electrical resistance (TEER) score and increased cell viability in TcdA-treated cells, significantly rescuing the protein expression of Ras homolog family members, A-GTPase (RhoA-GTPase), occludin, and zonula occludens-1 (ZO-1). LSAO has also exhibited an anti-apoptotic effect by inhibiting the TcdA-induced expression of Bcl-2-associated X protein (Bax), p50 nuclear factor-kappa-B (p50), p65nuclear factor-kappa-B (p65), and hypoxia-inducible transcription factor-1 alpha (HIF-1α), and the release of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and vascular endothelial growth factor (VEGF) in the cell milieu. Our data showed that LSAO exerts a protective effect on TcdA-induced toxicity in Caco-2 cells, placing itself as an interesting nutraceutical to supplement the current treatment of the Clostridium difficile infections.

Vegetal diamine oxidase alleviates histamine-induced contraction of colonic muscles.

Excess of histamine in gut lumen generates a pronounced gastrointestinal discomfort, which may include diarrhea and peristalsis dysfunctions. Deleterious effects of histamine can be alleviated with antihistamine drugs targeting histamine receptors. However, many antihistamine agents come with various undesirable side effects. Vegetal diamine oxidase (vDAO) might be a relevant alternative owing to its histaminase activity. Mammalian intestinal mucosa contains an endogenous DAO, yet possessing lower activity compared to that of vDAO preparation. Moreover, in several pathological conditions such as inflammatory bowel disease and irritable bowel syndrome, this endogenous DAO enzyme can be lost or inactivated. Here, we tested the therapeutic potential of vDAO by focusing on the well-known effect of histamine on gut motility. Using ex vivo and in vitro assays, we found that vDAO is more potent than commercial anti-histamine drugs at inhibiting histamine-induced contraction of murine distal colon muscles. We also identified pyridoxal 5'-phosphate (the biologically active form of vitamin B6) as an effective enhancer of vDAO antispasmodic activity. Furthermore, we discovered that rectally administered vDAO can be retained on gut mucosa and remain active. These observations make administration of vDAO in the gut lumen a valid alternative treatment for histamine-induced intestinal dysfunctions.

Copper­containing amine oxidase purified from Lathyrus sativus as a modulator of human neutrophil functions.

Over the last few decades, copper­containing amine oxidase (Cu­AO) from vegetal sources, and belonging to the class of diamine oxidase, has been documented to exhibit beneficial effects in both in vivo and ex vivo animal models of inflammatory or allergic conditions, including asthma­like reaction and myocardial or intestinal ischemia­reperfusion injuries. The aim of the present study was to assess the potential of vegetal Cu­AO as an anti­inflammatory and an antiallergic agent and to clarify its antioxidant properties. In cell­free systems, the reactive oxygen species and reactive nitrogen species scavenging properties of Cu­AO that is purified from Lathyrus sativus were investigated. Its effect on the formyl­methionyl­leucyl­phenylalanine peptide (fMLP)­activated cellular functions of human neutrophils were subsequently analyzed. The obtained results demonstrated that Cu­AO is not a scavenger of superoxide or nitric oxide, and does not decompose hydrogen peroxide. However, it inhibits the fMLP­dependent superoxide generation, elastase release and cell migration, and interferes with the process of calcium flux, supporting the idea that plant Cu­AO can interact with human neutrophils to modulate their inflammatory function. Therefore, the importance of these properties on the possible use of vegetal Cu­AO to control inflammatory conditions, particularly intestinal inflammation, is discussed in the current study.

Lathyrus sativus diamine oxidase counteracts histamine-induced cell proliferation, migration and pro-angiogenic mediators release in human colon adenocarcinoma cell line Caco-2.

Because histamine is a modulator of cancer cell proliferation and invasiveness, this study aimed at investigating the effect of Lathyrus sativus-derived diamine oxidase (LSAO) and its mechanism of action on Caco-2 cell line, considering that LSAO catalizes the oxidative deamination of histamine to the corresponding aldehyde, NH3 and H2 O2 . Histamine (0.01-1 µM) caused a proliferative effect on Caco-2 cells promoting cell migration, invasion and nitric oxide and vascular endothelial growth factor release. Histamine (1 µM) stimulus also down regulated occludin expression, favouring up regulation of pro-proliferative nuclear protein Ki67. Incubation with LSAO (0.004-0.4 µM) resulted in a significant inhibition of histamine-induced effects. LSAO rescued occludin expression and down regulated Ki67, and it inhibited histamine-induced increase of both MMP-2 and 9 expression. Histamine effects were mediated by RhoA-GTP down regulation and inversely related to phospho-p38MAPK/p50/65 up regulation. These effects were counteracted by LSAO incubation. Histamine catabolism by LSAO accounts for a significant down regulation of proliferation and invasiveness of Caco-2 cells. This study highlights the importance to control histamine levels in contrasting pro-angiogenic and metastatization capability of colon cancer cells and expands the knowledge about the diamine oxidase from L. sativus seeding as a phytotherapeutic approach for colon cancer.

Diamine Oxidase from White Pea (Lathyrus sativus) Combined with Catalase Protects the Human Intestinal Caco-2 Cell Line from Histamine Damage.

Diamine oxidase (DAO) administration has been proposed to treat certain gastrointestinal dysfunctions induced by histamine, an immunomodulator, signaling, and pro-inflammatory factor. However, H2O2 resulting from the oxidative deamination of histamine by DAO may be toxic. The purpose of this study was to investigate to which extent DAO from white pea (Lathyrus sativus), alone or in combination with catalase, may modulate histamine toxicity in the human intestinal Caco-2 cell line. The results show that histamine at concentrations higher than 1 mM is toxic to the Caco-2 cells, independently of the cell differentiation status, with a LC50 of ≅ 10 mM following a 24-h exposure. Depending on its concentration, DAO increased histamine toxicity to a greater extent in differentiated cells compared to undifferentiated cultures. In the presence of catalase, the DAO-induced increase in histamine toxicity was completely abolished in the undifferentiated cells and only partially decreased in differentiated cells, showing differences in the sensitivity of Caco-2 cells to the products resulting from histamine degradation by DAO (H2O2, NH3, or imidazole aldehyde). It appears that treatment of food histaminosis using a combination of vegetal DAO and catalase would protect against histamine toxicity and prevent H2O2-induced damage that may occur during histamine oxidative deamination.

The plant histaminase: a promising enzyme with antioxidant properties versus histamine release in isoprenaline-induced myocardial infarction in rats.

The present study was designed to evaluate possible protective effects of purified histaminase from Lathyrus sativus L. seedling on the myocardial injuries upon isoprenaline-induced myocardial infarction in rats. In this regard, blood histamine concentration, creatine kinase-MB (CK-MB) activity, antioxidant status, and histopathological changes of the hearts were measured. A total of 40 adult male Sprague-Dawley rats were divided into five equal groups and treated in the following order: control (normal saline), isoprenaline (isoproterenol 110 mg/kg BW), Isopren.-H1 (isoprenaline plus histaminase 80 U/kg BW), Isopren.-H2 (isoprenaline plus histaminase 120 U/kg BW), and Isopren.-H3 (isoprenaline plus histaminase 160 U/kg BW). Myocardial infarction was manifested by a significant elevation in the level of CK-MB and histopathological findings in isoprenaline group when compared to controls. In contrast, histaminase pretreatment at dose of 160 U/kg prevented isoprenaline-induced histamine release and significantly decreased CK-MB activity as well as histopathological changes in Isopren.-H3 group. A significant increase in the catalase (CAT) and superoxide dismutase (SOD) activities was also observed by histaminase treatment in Isopren.-H2 and Isopren.-H3 groups. Although the activity of glutathione peroxidase (GPx) increased significantly to suppress oxidative stress in isoprenaline group, it was not able to prevent lipid peroxidation (as shown by TBARS concentration) in the heart of rats. In conclusion, the plant-originated histaminase presented as a promising enzyme with antioxidant properties against histamine release and myocardial infarction in rats, and it seems be a suitable therapeutic agent for future clinical trials in humans.

Semicarbazide-sensitive amine oxidase kills African trypanosomes in vitro.

The African trypanosome Trypanosoma brucei is the cause of sleeping sickness in humans and Nagana in animals. Here we report that semicarbazide-sensitive amine oxidases (SSAOs), enzymes that are abound in T. brucei mammal hosts, eliminate trypanosomes by oxidation of its substrate in vitro. SSAO and its endogenous substrate methylamine are not toxic to T. brucei, but parasites were killed in the presence of both of them. SSAO inhibitors antagonized the SSAO-methylamine induced toxicity on T. brucei. The trypanocidal activity was mainly associated with formaldehyde generated in the SSAO mediated oxidation of methylamine. This finding suggests that SSAO may play some roles in non-specific defense of trypanosome infection in mammals.

Bovine serum amine oxidase and spm potentiate docetaxel and interferon-alpha effects in inducing apoptosis on human cancer cells through the generation of oxidative stress.

It was previously demonstrated that bovine serum amine-oxidase (BSAO) and SPM (SPM) addition to cancer cells induces cell growth inhibition and over-run the multi-drug resistance (MDR) phenotype through the oxidative stress caused by polyamine metabolites. In this study, it is reported that BSAO/SPM enzymatic system antagonizes the survival pathway induced by either docetaxel (DTX) or interferon alpha (IFNalpha) in human epidermoid cancer KB cells. The combination of BSAO/SPM with either DTX or IFNalpha had a synergistic effect on cell growth inhibition through apoptosis in both human epidermoid KB and breast cancer MCF-7 cell lines. The effects of the BSAO/SPM-DTX combination on apoptosis were caspase 3 and 9-dependent and were paralleled by the enhancement of intracellular O(2-), nitric oxide levels and of lipo-oxidation. The scavenger moiety N-acetyl-cysteine antagonized the effects on apoptosis and cell growth inhibition induced by the combination suggesting a role of the oxidative products of SPM. These effects occurred together with a decrease of the physiological scavenger MnSOD and an increase of both p38 kinase activity and DNA damage. The results suggest that DTX and IFNalpha could sensitize tumour cells to the oxidative stress and apoptosis induced by BSAO/SPM through the induction of a survival ras-dependent pathway and the consequent elevation of the intracellular polyamine pool. These data allow the design of new therapeutic strategy based on the use of this combination in human neoplasms.

Histamine excites rat lateral vestibular nuclear neurons through activation of post-synaptic H2 receptors.

Through whole-cell patch recordings in brainstem slices, the effects of histamine on neuronal activity of the lateral vestibular nucleus (LVN) were investigated. Bath application of histamine elicited a concentration-dependent excitation of both spontaneous firing (n=19) and silent (n=7) LVN neurons. Moreover, histamine induced a stable inward current in the LVN neurons (n=5) and the histamine-induced depolarization of membrane potential persisted in the presence of tetrodotoxin (n=4), indicating a direct post-synaptic effect of the histamine on the LVN neurons. Selective histamine H2 receptor antagonist ranitidine effectively blocked the histamine-evoked excitatory responses on the LVN neurons (n=4), but selective histamine H1 receptor antagonist triprolidine did not (n=4). In addition, selective histamine H2 receptor agonist dimaprit (n=3) rather than 2-pyridylethylamine (n=4), a selective histamine H1 receptor agonist, mimicked the excitatory action of histamine on LVN neurons. The results demonstrate that histamine excites the LVN neurons via post-synaptic histamine H2 receptors and suggest that the central histaminergic projection arising from the hypothalamus may modulate LVN neurons activity and actively influence the vestibular reflexes and functions.

Plant histaminase as an investigational drug in splanchnic artery occlusion and reperfusion.

BACKGROUND: Amine oxidases are ubiquitous enzymes involved in the metabolism of biogenic amines. Copper amine oxidases catalyze the oxidative deamination of primary amine groups of several biogenic amines, such as putrescine, cadaverine and histamine. OBJECTIVE: In the present review the effects of a plant amine oxidase (histaminase, EC1.4.3.6), purified from pea seedlings, in the prevention of splanchnic postischemic reperfusion damage are reported. CONCLUSION: Various studies have clearly indicated that the use of histaminase will offer a good perspective for a novel therapeutic approach in the medical treatment of intestinal ischemia.

Mechanism of cell death induced by spermine and amine oxidase in mouse melanoma cells.

Polyamines such as spermine, spermidine and putrescine are necessary for cell proliferation and are detected at higher concentrations in most tumor tissues, compared to normal tissues. The amine oxidase enzymes can generate cytotoxic products such as hydrogen peroxide and aldehydes from these polyamines. This study investigates the mechanisms of cell death in B16-F0 mouse melanoma tumor cells exposed to bovine serum amine oxidase and exogenous spermine. The bovine serum amine oxidase/spermine enzymatic system induced inhibition of cell proliferation in B16-F0 melanoma cells and cell death by both apoptotic and necrotic processes. Bovine serum amine oxidase or spermine, alone, did not induce cytotoxicity or cell death by apoptosis, indicating that the enzymatic reaction products were responsible. Catalase and NAD-dependent aldehyde dehydrogenase, inhibitors of hydrogen peroxide and aldehydes, respectively, decreased cell death by apoptosis and necrosis. This further confirms that the cytotoxic products are responsible for causing cell death. Use of inhibitors of different caspases showed that melanoma cells were sensitive to processes involving caspase-3 and -9, but were insensitive to caspase-6. Bovine serum amine oxidase in the presence of spermine could be useful as a promising new tool for anticancer treatment by the selective generation of toxic compounds from polyamines in tumors.

Soluble semicarbazide sensitive amine oxidase (SSAO) catalysis induces apoptosis in vascular smooth muscle cells.

Semicarbazide sensitive amine oxidase (SSAO) metabolizes oxidative deamination of primary aromatic and aliphatic amines. It is selectively expressed in vascular cells of blood vessels, but it is also circulating in blood plasma. SSAO activity in plasma is increased in some diseases associated with vascular complications and its catalytic products may cause tissue damage. We examined the effect of the oxidation of the SSAO substrate, methylamine, on cultured smooth muscle cells. Cell incubation with methylamine plus soluble SSAO, contained in bovine serum, resulted toxic to rat aorta A7r5 and human aortic smooth muscle cells, as measured by MTT reduction. This effect was completely reverted by specific SSAO inhibitors, indicating that the toxicity was mediated by the end products generated. Moreover, SSAO-mediated deamination of methylamine induced apoptosis in A7r5 cells, detected by chromatin condensation, Caspase-3 activation, PARP cleavage and cytochrome c release to cytosol. Formaldehyde, rather than H2O2, resulted to be a strong apoptotic inducer to A7r5 cells. Taken together, the results suggest that increased plasma SSAO activity in pathological conditions, could contribute to apoptosis in smooth muscle cells, leading to vascular tissue damage.

Reactions of plant copper/topaquinone amine oxidases with N6-aminoalkyl derivatives of adenine.

Plant copper/topaquinone-containing amine oxidases (CAOs, EC 1.4.3.6) are enzymes oxidising various amines. Here we report a study on the reactions of CAOs from grass pea (Lathyrus sativus), lentil (Lens esculenta) and Euphorbia characias, a Mediterranean shrub, with N6-aminoalkyl adenines representing combined analogues of cytokinins and polyamines. The following compounds were synthesised: N6-(3-aminopropyl)adenine, N6-(4-aminobutyl)adenine, N6-(4-amino-trans-but-2-enyl) adenine, N6-(4-amino-cis-but-2-enyl) adenine and N6-(4-aminobut-2-ynyl) adenine. From these, N6-(4-aminobutyl) adenine and N6-(4-amino-trans-but-2-enyl)adenine were found to be substrates for all three enzymes (Km approximately 10(-4)M). Absorption spectroscopy demonstrated such an interaction with the cofactor topaquinone, which is typical for common diamine substrates. However, only the former compound provided a regular reaction stoichiometry. Anaerobic absorption spectra of N6-(3-aminopropyl)adenine, N6-(4-amino-cis-but-2-enyl)adenine and N6-(4-aminobut-2-ynyl)adenine reactions revealed a similar kind of initial interaction, although the compounds finally inhibited the enzymes. Kinetic measurements allowed the determination of both inhibition type and strength; N6-(3-aminopropyl)adenine and N6-(4-amino-cis-but-2-enyl)adenine produced reversible inhibition (Ki approximately 10(-5) - 10(-4) M) whereas, N6-(4-aminobut-2-ynyl)adenine could be considered a powerful inactivator.

Effect of a plant histaminase on asthmalike reaction induced by inhaled antigen in sensitized guinea pig.

This study evaluates the effects of a copper amine oxidase (histaminase) purified from the pea seedling as a free or immobilized enzyme on asthmalike reactions to inhaled antigen in actively sensitized guinea pig in vivo. Male albino guinea pigs, sensitized with ovalbumin, were challenged with the antigen given by aerosol; free histaminase or CNBr-Sepharose immobilized histaminase was given intraperitoneally (20 microg, 3 or 24 h before antigen challenge) or by aerosol (4 microg, 30 min before or during ovalbumin aerosol). The following parameters were examined: latency time for the onset of respiratory abnormalities, cough severity score, and occurrence and duration of dyspnea. We also evaluated lung histopathology, mast cell degranulation, and lung myeloperoxidase and malonydialdehyde levels. Histaminase significantly reduced the severity of cough and the occurrence of dyspnea and delayed the onset of respiratory abnormalities. Both enzymes prevented bronchial constriction, pulmonary air space inflation, leukocyte infiltration (evaluated as myeloperoxidase activity), and lipoperoxidation of cell membranes (evaluated as malonyldialdehyde production). No relevant differences in pharmacological potency were noted between free or immobilized enzyme. This study provides evidence that histaminase counteracts acute allergic asthmalike reaction in actively sensitized guinea pigs, raising the possibility of new therapeutic strategies for allergic asthma in humans.

Mitochondrial alterations induced by serum amine oxidase and spermine on human multidrug resistant tumor cells.

Multidrug resistance (MDR) has been studied extensively because it is one of major problems in cancer chemotherapy. The MDR phenotype is often due to overexpression of P-glycoprotein (P-gp), that acting as an energy-dependent drug efflux pump exports various anticancer drugs out of cells. The major goal of our investigation is to establish whether bovine serum amine oxidase (BSAO), which generates the products H(2)O(2) and aldehyde(s), from the polyamine spermine, is able to overcome MDR of human cancer cells. The cytotoxicity of the products was evaluated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. A clonogenic cell survival assay demonstrated that LoVo DX cells were more sensitive than LoVo WT cells. Exogenous catalase protected cells against cytotoxicity mainly due to the formation of H(2)O(2). However, spermine-derived aldehyde(s) still induced some cytotoxicity. The cytotoxic effect was totally inhibited in the presence of both enzymes, catalase and NAD-dependent aldehyde dehydrogenase (ALDH). Transmission electron microscopy investigations showed that BSAO and spermine induced evident mitochondria alterations, more pronounced in MDR than in LoVo WT cells. The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1, that displayed a basal hyperpolarized status of the mitochondria in multidrug-resistant cells. After treatment with amine oxidase in the presence of polyamine-spermine, the cells showed a marked increase in mitochondrial membrane depolarization higher in LoVo DX than in LoVo WT cells. Our findings suggest that toxic oxidation products formed from spermine and BSAO could be a powerful tool in the development of new anticancer treatments, mainly against MDR tumor cells.