Paraoxonase-1 and -3 Protein Expression in the Brain of the Tg2576 Mouse Model of Alzheimer's Disease.

BACKGROUND: Brain oxidative lipid damage and inflammation are common in neurodegenerative diseases such as Alzheimer's disease (AD). Paraoxonase-1 and -3 (PON1 and PON3) protein expression was demonstrated in tissue with no PON1 or PON3 gene expression. In the present study, we examine differences in PON1 and PON3 protein expression in the brain of a mouse model of AD. METHODS: we used peroxidase- and fluorescence-based immunohistochemistry in five brain regions (olfactory bulb, forebrain, posterior midbrain, hindbrain and cerebellum) of transgenic (Tg2576) mice with the Swedish mutation (KM670/671NL) responsible for a familial form of AD and corresponding wild-type mice. RESULTS: We found intense PON1 and PON3-positive staining in star-shaped cells surrounding Aß plaques in all the studied Tg2576 mouse-brain regions. Although we could not colocalize PON1 and PON3 with astrocytes (star-shaped cells in the brain), we found some PON3 colocalization with microglia. CONCLUSIONS: These results suggest that (1) PON1 and PON3 cross the blood-brain barrier in discoidal high-density lipoproteins (HDLs) and are transferred to specific brain-cell types; and (2) PON1 and PON3 play an important role in preventing oxidative stress and lipid peroxidation in particular brain-cell types (likely to be glial cells) in AD pathology and potentially in other neurodegenerative diseases as well.

Impaired paraoxonase-1 status in obese children. Relationships with insulin resistance and metabolic syndrome.

OBJECTIVES: To investigate the relationships between serum paraoxonase-1 (PON1), insulin resistance, and metabolic syndrome (MetS) in childhood obesity. DESIGN AND METHODS: We studied 110 obese children and 36 non-obese children with a similar gender and age distribution. We measured serum PON1 activity against 5-thiobutyl butyrolactone (TBBLase) and against paraoxon (paraoxonase). PON1 concentration was measured separately as were the levels of several standard metabolic variables. The homeostasis model assessment (HOMA) index was calculated as an estimate of insulin resistance. RESULTS: TBBLase was significantly decreased in obese children (P=0.008), while paraoxonase activity and PON1 concentrations showed non-significant trends towards decrease and increase, respectively (P=0.054 and P=0.060). TBBLase and paraoxonase specific activities were significantly decreased (P=0.004 and P=0.018, respectively). TBBLase specific activity was inversely associated with BMI, percentage body fat, insulin, HOMA, triglycerides, and C-reactive protein, and directly associated with HDL-cholesterol. Paraoxonase specific activity showed similar associations with BMI, percentage fat, HDL-cholesterol, and C-reactive protein. Obese children with MetS had lower TBBLase activities than obese children without MetS (P=0.018). Linear regression analyses showed that TBBLase was independently associated with HDL-cholesterol, BMI, percentage body fat and PON155 polymorphism, but paraoxonase activity was associated only with PON1192 polymorphism. CONCLUSIONS: Our results suggest that PON1 may play a role in the onset and development of metabolic alterations in childhood obesity leading to diabetes and cardiovascular disease later in life. However, being derived from statistical association study, this finding cannot be seen as showing cause-effect.

Paraoxonase-1 is associated with corneal endothelial cell alterations in patients with chronic obstructive pulmonary disease.

PURPOSE: To investigate the relationships between the levels of the antioxidant enzyme paraoxonase-1 (PON1) and corneal endothelial alterations in patients with chronic obstructive pulmonary disease (COPD) undergoing cataract surgery. METHODS: We studied 172 patients with cataract attending our ophthalmology clinic. Based on spirometric analysis, they were segregated into two groups, 110 (64%) with COPD and 62 (36%) without COPD. Corneal endothelial cell morphology was examined by widefield noncontact specular microscopy, which allows measurements of endothelial cell density (ECD), hexagonality, and endothelial cell size coefficient of variation (ECCV). Corneal thickness was measured by noncontact pachimetry. PON1 and plasma TNFα concentrations were analyzed by ELISA. Serum PON1 activity was analyzed by spectrophotometry. RESULTS: Patients with COPD had significant decreases in ECD, hexagonality, and corneal thickness, and a significant increase in ECCV. They also had significant decreases in serum PON1 activity but not in PON1 concentration. Serum PON1 activity showed a significant direct association with ECD, and an inverse association with corneal thickness. CONCLUSIONS: Results of the present study suggest that PON1 may be involved in the pathophysiology of corneal endothelial alterations in patients with COPD.

Paraoxonase-1 inhibits oxidized low-density lipoprotein-induced metabolic alterations and apoptosis in endothelial cells: a nondirected metabolomic study.

We studied the influence of PON1 on metabolic alterations induced by oxidized LDL when incubated with endothelial cells. HUVEC cells were incubated with native LDL, oxidized LDL, oxidized LDL plus HDL from wild type mice, and oxidized LDL plus HDL from PON1-deficient mice. Results showed alterations in carbohydrate and phospholipid metabolism and increased apoptosis in cells incubated with oxidized LDL. These changes were partially prevented by wild type mouse HDL, but the effects were less effective with HDL from PON1-deficient mice. Our results suggest that PON1 may play a significant role in endothelial cell survival by protecting cells from alterations in the respiratory chain induced by oxidized LDL. These results extend current knowledge on the protective role of HDL and PON1 against oxidation and apoptosis in endothelial cells.

Paraoxonase-1 status in patients with hereditary hemochromatosis.

Hereditary hemochromatosis (HH) is characterized by accumulation of iron, oxidative stress, inflammation, and fibrogenesis in liver tissue. In this setting, research on the protection afforded by intracellular antioxidants is of clinical relevance. Paraoxonase-1 (PON1) is an enzyme that degrades lipid peroxides. This study investigates the alterations in serum PON1 status, PON1 gene polymorphisms, and PON1 hepatic expression in patients with HH. We performed a case-control study in 77 patients with HH (80.5% men, 22-70 years of age) and 408 healthy individuals (43.1% men, 26-74 years of age). Serum PON1 activities against different substrates and PON1192 and PON155 polymorphisms were analyzed. PON1 protein expression was investigated in 20 liver biopsies. HH patients had significantly lower serum PON1 activity, which was inversely correlated with ferritin (marker of iron stores) and serum 8-isoprostane concentrations (index of oxidative stress). PON1 protein expression in liver tissue was higher in patients and showed stronger staining in hepatocytes surrounding the areas of inflammation. Our study provides preliminary evidence that PON1 may play a role in protecting against iron-induced oxidative stress in hereditary hemochromatosis.

Paraoxonase-1 deficiency is associated with severe liver steatosis in mice fed a high-fat high-cholesterol diet: a metabolomic approach.

Oxidative stress is a determinant of liver steatosis and the progression to more severe forms of disease. The present study investigated the effect of paraoxonase-1 (PON1) deficiency on histological alterations and hepatic metabolism in mice fed a high-fat high-cholesterol diet. We performed nontargeted metabolomics on liver tissues from 8 male PON1-deficient mice and 8 wild-type animals fed a high-fat, high-cholesterol diet for 22 weeks. We also measured 8-oxo-20-deoxyguanosine, reduced and oxidized glutathione, malondialdehyde, 8-isoprostanes and protein carbonyl concentrations. Results indicated lipid droplets in 14.5% of the hepatocytes of wild-type mice and in 83.3% of the PON1-deficient animals (P < 0.001). The metabolomic assay included 322 biochemical compounds, 169 of which were significantly decreased and 16 increased in PON1-deficient mice. There were significant increases in lipid peroxide concentrations and oxidative stress markers. We also found decreased glycolysis and the Krebs cycle. The urea cycle was decreased, and the pyrimidine cycle had a significant increase in orotate. The pathways of triglyceride and phospholipid synthesis were significantly increased. We conclude that PON1 deficiency is associated with oxidative stress and metabolic alterations leading to steatosis in the livers of mice receiving a high-fat high-cholesterol diet.

Monocyte chemoattractant protein-1 and paraoxonase-1 and 3 levels in patients with sepsis treated in an intensive care unit: a preliminary report.

BACKGROUND: There is considerable interest in the research of molecules modulating the acute inflammatory response in patients with sepsis. Paraoxonases (PON) are antioxidant and anti-inflammatory enzymes that inhibit the production of the monocyte chemoattractant protein-1 (MCP-1). This preliminary study investigated changes in PON status and MCP-1 concentrations in critically ill patients with severe sepsis treated in an ICU and their relationship with the evolution of disease. METHODS: This was a longitudinal, prospective and observational study on 15 patients with sepsis, studied at baseline and on days 1, 2, 5, 7 and 10 of their stay in the ICU. In all the patients we measured serum PON1 and PON3 concentrations, PON1 paraoxonase and lactonase activities, serum MCP-1 concentrations, and several standard biochemical and haematological parameters. RESULTS: MCP-1 concentration significantly decreased with the resolution of sepsis, and this decrease was especially important during the first 5 days of hospitalisation. PON1 and PON3 tended to decrease during the first 5 days in ICU and significantly increased in days 7 and 10. Linear regression analysis showed significant and direct correlations among serum MCP-1 concentration and lactate levels at baseline. At the end of stay, PON1 paraoxonase and lactonase activities were significantly correlated with organ system function measurements. CONCLUSIONS: We observed an inverse pattern between changes in MCP-1, and PON1 and PON3 levels in patients with sepsis, this was related to the resolution of their infection after receiving treatment in an ICU.

Serum paraoxonase-3 concentration is associated with insulin sensitivity in peripheral artery disease and with inflammation in coronary artery disease.

OBJECTIVE: There are no data on the relationship between serum paraoxonase-3 (PON3) concentration and atherosclerosis in humans. Our aim was to investigate possible associations, using recently developed methods, in patients with peripheral artery disease (PAD) or coronary artery disease (CAD). METHODS: We studied 118 PAD and 72 CAD patients and 175 healthy volunteers. Serum PON3 was determined by in-house ELISA using polyclonal antibodies generated against a synthetic peptide with a sequence specific to PON3. Polymorphisms of the PON3 promoter were analysed by the Iplex Gold MassArray™ method. RESULTS: There was a significant increase in serum PON3 concentration in both groups of patients with respect to the control group. In PAD patients, we observed significant positive correlations between PON3, insulin levels and HOMA index. These associations were not observed in CAD. There were significant positive associations between serum PON3 and ß-2-microglobulin, CCL2 and high-sensitivity C-reactive protein in CAD patients, but not in PAD. We did not find any significant differences in PON3 gene promoter polymorphisms and their haplotypes between patients and controls, indicating that associations were not genetically determined. CONCLUSION: In both atherosclerotic phenotypes, serum PON3 concentration was increased, but this was associated with decreased insulin sensitivity in PAD and with inflammation in CAD.

Serum paraoxonase-3 concentration in HIV-infected patients. Evidence for a protective role against oxidation.

We investigated the influence of the HIV infection on serum paraoxonase-3 (PON3) concentration and assessed the relationships with lipoprotein-associated abnormalities, immunological response, and accelerated atherosclerosis. We studied 207 HIV-infected patients and 385 healthy volunteers. Serum PON3 was determined by in-house ELISA, and PON3 distribution in lipoproteins was investigated by fast-performance liquid chromatography (FPLC). Polymorphisms of the PON3 promoter were analyzed by the Iplex Gold MassArray(TM) method. PON3 concentrations were increased (about three times) in HIV-infected patients with respect to controls (P < 0.001) and were inversely correlated with oxidized LDL levels (P = 0.038). Long-term use of nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy was associated with a decrease of PON3 concentrations. In a multivariate linear regression analysis, these relationships were still strong when the main confounding covariates were considered. PON3 was mainly found in HDL in HIV-infected patients, but a substantial amount of the protein was detected in LDL particles. This study reports for the first time an important increase in serum PON3 concentrations in HIV-infected patients that is associated with their oxidative status and their treatment with NNRTI. Long-term, prospective studies are needed to confirm the possible influence of this enzyme on the course of this disease and its possible utility as an analytical biomarker.

Serum paraoxonase-3 concentration is associated with the severity of hepatic impairment in patients with chronic liver disease.

OBJECTIVES: Research on paraoxonase-3 (PON3) has been hampered by the lack of methods for measurement. This is a pilot study aimed at exploring whether chronic liver impairment is associated with changes in serum PON3 concentrations, and to know whether this measurement may provide useful information to investigate this derangement. DESIGN AND METHODS: We studied 110 patients with chronic liver disease (21 minimal changes, 79 chronic hepatitis, 10 cirrhosis) and 356 healthy volunteers. Serum PON3 concentration was determined by ELISA using polyclonal antibodies generated against a synthetic peptide with a sequence specific to PON3. RESULTS: Serum PON3 concentrations were increased in patients with chronic hepatitis or cirrhosis and showed significant direct correlations with the degree of periportal abnormalities including fibrosis, and with serum FAS (a marker of antiapoptosis) concentrations. CONCLUSION: These results suggest that PON3 may play a hepatoprotective role against histological alterations and hepatic cell apoptosis leading to liver disease.

Measurement of serum PON-3 concentration: method evaluation, reference values, and influence of genotypes in a population-based study.

Experimental studies showed that paraoxonase-3 (PON3) retards lipoprotein oxidation. Our objective was to describe a new assay to measure serum PON3 concentrations and report their reference values in a population-based study. The influence of PON3 promoter polymorphisms and their relationships with PON1 and lipid profile were also studied. We generated an anti-PON3 antibody by inoculating rabbits with a synthetic peptide specific to mature PON3. This antibody was used to develop an ELISA. The average regression line of standard plots (n = 8) was y = 0.9587 (0.3392) log(10)x + 1.9466 (0.0861) [r(2) = 0.924 (0.0131); P < 0.001]. There was no cross reaction with PON1. Detection limit was 0.24 mg/l. Imprecision was ≤ 13.2%. Reference interval (n = 356) was 1.00-2.47 mg/l. PON3 was observed in HDL particles containing apolipoprotein (apo)A-I and PON1, but not apoA-II or apoE. Serum PON3 concentrations showed a moderate influence (about 10% variation) by PON3 promoter polymorphisms. Our study describes for the first time a method to measure serum PON3 concentrations. This method offers new opportunities in the investigation of the properties and role of PON3 in cardiovascular disease, with possible implications in clinical practice.

'Dippers' flu' and its relationship to PON1 polymorphisms.

OBJECTIVES: Sheep-dippers report an acute flu-like condition (dippers' flu: DF) but the cause and relation to chronic disability are unknown. METHODS: In a case-referent study previously reported, 175 sheep dippers with chronic disability and 234 referents, sheep dippers in good health, completed an interview with information on dipping, type of pesticide used and health for each year 1970-2000 and gave blood for typing of PON1 polymorphisms. RESULTS: Reports of DF were much higher (66.3% 116/175) in the chronically unwell than in those without chronic ill-health (18.0% 42/234: OR=8.99 95% CI 5.69-14.21). No significant relation was seen between reported exposures and DF in those with chronic illness, but risk was higher with concentrate handling in those without. An R allele at position 192 on PON1 related to reports of DF both in those with chronic illness (OR=2.04 95% CI 1.08-3.87) and in those who started dipping after 1969 and were not chronically unwell (OR=2.52 95%CI 1.00-6.37). Interaction between handling diazinon concentrate and PON1 (192R) increased the risk of DF. No precipitating factor was identified in a case-crossover analysis. In the group without chronic illness those with 192R developed DF earlier (risk ratio 2.49 95%CI 1.03-6.02). CONCLUSION: 'Dippers' flu' and chronic ill-health attributed to dipping share a common polymorphism (192R). The interaction between handling diazinon concentrate and PON1 genotype supports the conclusion that organophosphates may cause DF. Sheep dippers who are still healthy but experience 'dippers' flu' may be wise to further limit exposures to organophosphates.

Tissue distribution and expression of paraoxonases and chemokines in mouse: the ubiquitous and joint localisation suggest a systemic and coordinated role.

A vicious cycle between oxidation and inflammation leads to complications in a growing number of disease states. Knowledge on tissue distribution of chemokines, mediators of inflammatory response, and paraoxonases, with antioxidant and anti-inflammatory actions, may be relevant. Using immunohistochemistry and quantitative real-time PCR we have investigated the distribution of PON1, 2 and 3, CCL2, 7, 8 and 12 and the chemokine receptor CCR2 protein and mRNA in 23 tissues from C57BL/6J mice. As expected, PON1, 2 and 3, CCL2, 7, 8 and 12 and CCR2 proteins were present in the vast majority of tissues investigated. Surprisingly, mRNA for these proteins was also expressed in most of these tissues suggesting local production and the ability to respond in situ to inflammatory stimuli. The wide distribution and expression of mRNA for paraoxonases and CC-chemokines suggest a systemic, probably coordinated, role in the overall inflammatory response.

Decreased paraoxonase-1 activity is associated with alterations of high-density lipoprotein particles in chronic liver impairment.

BACKGROUND: Paraoxonase-1 (PON1), a lactonase synthesized by the liver, circulates in blood bound to high-density lipoproteins (HDL). This enzyme is thought to degrade oxidized phospholipids and play an important role in the organism's antioxidant and anti-inflammatory system. Chronic liver diseases are characterized by decreased serum PON1 activity. The aim of the present study was to investigate the compositional changes in HDL that could influence PON1 activity in liver impairment. METHODS: The study was performed in samples from five patients with advanced liver cirrhosis and with preserved renal function, chosen on the basis of having low serum PON1 activity and high serum PON1 concentration. As a control group, we accessed five healthy volunteers from among our hospital staff. Lipid and protein compositional analysis of lipoprotein particles were done by high-performance liquid chromatography, gel electrophoresis, and Western-Blot. RESULTS: HDL particles from cirrhotic patients had an increased phospholipid content that was inversely correlated to PON1 activity. The HDL particles contained high levels of PON1 that corresponded, in part, to an immunoreactive protein of high molecular weight (55 kDa) not present in control subjects. This protein was identified as glycosylated PON1 and was also present in biopsies from patients with steatosis and from rats with CCl(4)-induced hepatic impairment. These changes were associated with an increased plasma concentration of markers of oxidative stress, inflammation and fibrogenesis. CONCLUSION: Abnormalities in the composition of lipids and proteins of HDL particles, including PON1 glycosylation, are associated with the decrease in serum PON1 activity in patients with liver disease. These alterations may adversely affect the protective role of HDL against oxidative stress and inflammation in these patients.

Paraoxonase-1 gene haplotypes are associated with metabolic disturbances, atherosclerosis, and immunologic outcome in HIV-infected patients.

BACKGROUND: Oxidative stress is associated with human immunodeficiency virus (HIV) infection. Paraoxonase-1 (PON1) is an antioxidant enzyme that is bound to high-density lipoproteins (HDLs). We evaluated whether PON1 gene haplotypes influence the metabolic disturbances, presence of subclinical atherosclerosis, and virologic outcome associated with the infection. METHODS: DNA from blood samples collected from 234 HIV-infected patients and 633 healthy control subjects had single-nucleotide polymorphisms of PON1(192), PON1(55), PON1(-162), PON1(-832), PON1(-909), PON1(-1076), and PON1(-1741) analyzed using the Iplex Gold MassArray method. Subsequently, the influence of these single-nucleotide polymorphisms on measured biochemical and clinical variables was assessed. RESULTS: We observed significant differences in the haplotype distribution between the control subjects and the HIV-infected patients. Haplotype H10 (GTCCGTC) was more prevalent in the HIV-infected patients (6.41% vs 0.64%; P < .001), and haplotype H5 (GACCGTC) was less prevalent in HIV-infected patients (27.7% vs 42.9%; P = .001). In HIV-infected patients, haplotype H7 (AATTCCT) was associated with better CD4(+) cell count recovery, higher levels of HDL cholesterol (P = .048) and apolipoprotein A-I (P = .019), lower levels of triglycerides (P = .004), and lower rates of subclinical arteriosclerosis (P < .001). CONCLUSIONS: PON1 haplotypes segregate with HIV infection, HDL metabolism, the presence of subclinical atherosclerosis, and CD4(+) cell recovery after treatment.

Paraoxonase-1 is related to inflammation, fibrosis and PPAR delta in experimental liver disease.

BACKGROUND: Paraoxonase-1 (PON1) is an antioxidant enzyme synthesized by the liver. It protects against liver impairment and attenuates the production of the pro-inflammatory monocyte chemoattractant protein-1 (MCP-1). We investigated the relationships between hepatic PON1 and MCP-1 expression in rats with liver disease and explored the possible molecular mechanisms involved. METHODS: CCl4 was administered for up to 12 weeks to induce liver damage. Serum and hepatic levels of PON1 and MCP-1, their gene and protein expression, nuclear transcription factors, and histological and biochemical markers of liver impairment were measured. RESULTS: High levels of PON1 and MCP-1 expression were observed at 12th week in the hepatocytes surrounding the fibrous septa and inflammatory areas. CCl4-administered rats had an increased hepatic PON1 concentration that was related to decreased gene transcription and inhibited protein degradation. Decreased PON1 gene transcription was associated with PPARdelta expression. These changes were accompanied by increased hepatic MCP-1 concentration and gene expression. There were significant direct relationships between hepatic PON1 and MCP-1 concentrations (P = 0.005) and between PON1 and the amount of activated stellate cells (P = 0.001). CONCLUSION: Our results from this experimental model suggest a hepato-protective role for PON1 against inflammation, fibrosis and liver disease mediated by MCP-1.