Pioglitazone transiently stimulates paraoxonase-2 expression in male nonhuman primate brain: Implications for sex-specific therapeutics in neurodegenerative disorders.

Paraoxonase-2 (PON2) enhances mitochondria function and protects against oxidative stress. Stimulating its expression has therapeutic potential for diseases where oxidative stress plays a significant role in the pathology, such as Parkinson's disease. Clinical and preclinical evidence suggest that the anti-diabetic drug pioglitazone may provide neuroprotection in Parkinson's disease, Alzheimer's disease, and stroke, but the biochemical pathway(s) responsible has not been fully elucidated. To determine the effect of pioglitazone on PON2 expression we treated male African green monkeys with oral pioglitazone (5 mg/kg/day) for 1 and 3 weeks. We found that pioglitazone increased PON2 mRNA and protein expression in brain following 1 week of treatment, however, by 3 weeks of treatment PON2 expression had returned to baseline. This transient increase was detected in substantia nigra, striatum, hippocampus, and dorsolateral prefrontal cortex The short-term impact of pioglitazone on PON2 expression in striatum may contribute to the discrepancy in the potency of the drug between short-term animal models and clinical trials for Parkinson's disease. Both PON2 and pioglitazone's receptor, peroxisome proliferator-activated receptor gamma (PPARγ), possess sex- and brain region-dependent expression, which may play a role in the short-term effect of pioglitazone and provide clues to extending the beneficial effects of PON2 activation.

Investigation of pesticide exposure by genotoxicological, biochemical, genetic polymorphic and in silico analysis.

Soybean farmers are exposed to various types of pesticides that contain in their formulations a combination of chemicals with genotoxic and mutagenic potential. Therefore, the objective of this paper was to evaluate the genetic damages caused by this pesticide exposure to soybean producers in the state of Mato Grosso (Brazil), regarding biochemical, genetic polymorphic and in silico analyses. A total of 148 individuals were evaluated, 76 of which were occupationally exposed and 72 were not exposed at all. The buccal micronucleus cytome assay (BMCyt) detected in the exposed group an increase on DNA damage and cell death. No inhibition of butyrylcholinesterase (BchE) was observed within the exposed group. The detection of inorganic elements was made through the particle-induced X-ray emission technique (PIXE), which revealed higher concentrations of Bromine (Br), Rubidium (Rb) and Lead (Pb) in rural workers. A molecular model using in silico analysis suggests how metal ions can cause both DNA damage and apoptosis in the exposed cells. Analysis of the compared effect of X-ray Repair Cross-complement Protein 1 (XRCC1) and Paraoxonase 1 (PON1) genotypes in the groups demonstrated an increase of binucleated cells (exposed group) and nuclear bud (non-exposed group) in individuals with the XRCC1 Trip/- and PON1 Arg/- genes. There was no significant difference in the telomere (TL) mean value in the exposed group in contrast to the non-exposed group. Our results showed that soybean producers showed genotoxic effect and cell death, which may have been induced by exposure to complex mixtures of agrochemicals and fertilizers. In addition, XRCC1 Arg/Arg could, in some respects, provide protection to individuals.

A Possible Mechanism: Vildagliptin Prevents Aortic Dysfunction through Paraoxonase and Angiopoietin-Like 3.

The collected data have revealed the beneficial effects of dipeptidyl peptidase-4 (DPP-4) inhibitors on the vascular endothelium, including vildagliptin. However, the involved mechanisms are not yet clear. In this study, Sprague-Dawley rats were randomly divided into the following four groups: control, diabetic, diabetic + low-dose vildagliptin (10 mg/kg/d), and diabetic + high-dose vildagliptin (20 mg/kg/d). The diabetic model was created by feeding a high-fat diet for four weeks and injection of streptozotocin. Then, vildagliptin groups were given oral vildagliptin for twelve weeks, and the control and diabetic groups were given the same volume of saline. The metabolic parameters, endothelial function, and whole genome expression in the aorta were examined. After 12 weeks of treatment, vildagliptin groups showed significantly reduced blood glucose, blood total cholesterol, and attenuated endothelial dysfunction. Notably, vildagliptin may inhibit angiopoietin-like 3 (Angptl3) and betaine-homocysteine S-methyltransferase (Bhmt) expression and activated paraoxonase-1 (Pon1) in the aorta of diabetic rats. These findings may demonstrate the vasoprotective pathway of vildagliptin in vivo.

Influence of oral contraceptives on lipid profile and paraoxonase and commonly hepatic enzymes activities.

BACKGROUND: The aim of the study was to verify the influence of oral contraceptives (OCs) on lipid profile and the arylesterase, lactonase and phosphotriesterase activities of paraoxonase 1 (PON1). Also commonly hepatic enzymes: aspartate aminotransferase (AST), alanine aminotransferase (ALT) and γ-glutamyltranspherase (GGT) were measured. METHODS: Lipid profile and hepatic enzymes were determined using commercial available reagents. Paraoxonase activities were estimated using earlier published procedures. Blood samples were collected from 120 women of similar age (22.6±1.0 years) with similar BMI (20.71±2.20 kg/m2 ). Participations were divided into two groups: 74 females do not take (group A) and 46 women taking OCs (group B). RESULTS: Higher triglycerides and lower low-density lipoproteins levels were observed in group B than in group A) (56.9±19.7 mg/dL. Castelli risk index I was significantly higher in group B when compared to group A (P<.0001), whereas we did not observe any statistically significant differences in Castelli risk index II value between studied groups. In group B, increase in ALT, AST and GGT activities were found, while the de Ritis ratio was lower in group B than in group A. In group B, lower phosphotriesterase activity and higher arylesterase and lactonase activities were found when compared to group A. CONCLUSION: Higher ALT, AST and GGT activities in serum as well as changes in lipid profile and PON activities can indicate that OCs usage can cause disorder in these parameters in the serum of women taking OCs.

Caffeine Increases Apolipoprotein A-1 and Paraoxonase-1 but not Paraoxonase-3 Protein Levels in Human-Derived Liver (HepG2) Cells.

BACKGROUND: Apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 are antioxidant and anti-atherosclerotic structural high-density lipoprotein proteins that are mainly synthesized by the liver. No study has ever been performed to specifically examine the effects of caffeine on paraoxonase enzymes and on liver apolipoprotein A-1 protein levels. AIMS: To investigate the dose-dependent effects of caffeine on liver apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 protein levels. STUDY DESIGN: In vitro experimental study. METHODS: HepG2 cells were incubated with 0 (control), 10, 50 and 200 µM of caffeine for 24 hours. Cell viability was evaluated by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 protein levels were measured by western blotting. RESULTS: We observed a significant increase on apolipoprotein A-1 and paraoxonase-1 protein levels in the cells incubated with 50 µM of caffeine and a significant increase on paraoxonase-1 protein level in the cells incubated with 200 µM of caffeine. CONCLUSION: Our study showed that caffeine does not change paraoxonase-3 protein level, but the higher doses used in our study do cause an increase in both apolipoprotein A-1 and paraoxonase-1 protein levels in liver cells.

Metals and Paraoxonases.

The paraoxonases (PONs) are a three-gene family which includes PON1, PON2, and PON3. PON1 and PON3 are synthesized primarily in the liver and a portion is secreted in the plasma, where they are associated with high-density lipoproteins (HDLs), while PON2 is an intracellular enzyme, expressed in most tissues and organs, including the brain. PON1 received its name from its ability to hydrolyze paraoxon, the active metabolite of the organophosphorus (OP) insecticide parathion, and also more efficiently hydrolyzes the active metabolites of several other OPs. PON2 and PON3 do not have OP-esterase activity, but all PONs are lactonases and are capable of hydrolyzing a variety of lactones, including certain drugs, endogenous compounds, and quorum-sensing signals of pathogenic bacteria. In addition, all PONs exert potent antioxidant effects. PONs play important roles in cardiovascular diseases and other oxidative stress-related diseases, modulate susceptibility to infection, and may provide neuroprotection (PON2). Hence, significant attention has been devoted to their modulation by a variety of dietary, pharmacological, lifestyle, or environmental factors. A number of metals have been shown in in vitro, animal, and human studies to mostly negatively modulate expression of PONs, particularly PON1, the most studied in this regard. In addition, different levels of expression of PONs may affect susceptibility to toxicity and neurotoxicity of metals due to their aforementioned antioxidant properties.

Esterase detoxication of acetylcholinesterase inhibitors using human liver samples in vitro.

Organophosphorus (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxication can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON1) are considered factors underlying age-related sensitivity differences. We used an in vitro system to measure detoxication of AChE-inhibiting pesticides mediated via these esterases. Recombinant human AChE was used as a bioassay of inhibitor concentration following incubation with detoxifying tissue: liver plus Ca(+2) (to stimulate PON1s, measuring activity of both esterases) or EGTA (to inhibit PON1s, thereby measuring CaE activity). AChE inhibitory concentrations of aldicarb, chlorpyrifos oxon, malaoxon, methamidophos, oxamyl, paraoxon, and methylparaoxon were incubated with liver homogenates from adult male rat or one of 20 commercially provided human (11-83 years of age) liver samples. Detoxication was defined as the difference in inhibition produced by the pesticide alone and inhibition measured in combination with liver plus Ca(+2) or liver plus EGTA. Generally, rat liver produced more detoxication than did the human samples. There were large detoxication differences across human samples for some pesticides (especially malaoxon, chlorpyrifos oxon) but not for others (e.g., aldicarb, methamidophos); for the most part these differences did not correlate with age or sex. Chlorpyrifos oxon was fully detoxified only in the presence of Ca(+2) in both rat and human livers. Detoxication of paraoxon and methylparaoxon in rat liver was greater with Ca(+2), but humans showed less differentiation than rats between Ca(+2) and EGTA conditions. This suggests the importance of PON1 detoxication for these three OPs in the rat, but mostly only for chlorpyrifos oxon in human samples. Malaoxon was detoxified similarly with Ca(+2) or EGTA, and the differences across humans correlated with metabolism of p-nitrophenyl acetate, a substrate for CaEs. This suggests the importance of CaEs in malaoxon detoxication. Understanding these individual differences in detoxication can inform human variability in pesticide sensitivity.

Some Anticancer Agents Act on Human Serum Paraoxonase-1 to Reduce Its Activity.

Human serum paraoxonase (hPON1) is an important antioxidant enzyme. It protects low-density lipoproteins against oxidative stress and prevents atherosclerosis development. Anticancer agents have cardiotoxic effects, and this situation can lead to significant complications. Our aim was to evaluate the in vitro effects of some of the anticancer agents such as cetuximab, paclitaxel, etoposide, docetaxel, and ifosfamide on the activity of hPON1 in this study. For this reason, PON1 was purified from human serum with a specific activity of 3654.2 EU/mg and 16.84% yield using simple chromatographic methods. The five chemotherapeutic agents dose dependently decreased in vitro hPON1 activity. IC50 values for cetuximab, paclitaxel, etoposide, docetaxel, and ifosfamide were 0.0111, 0.042, 0.226, 0.665, and 23.3 mm, respectively. Ki constants were 0.0194, 0.0165, 0.131, 0.291, and 8.973 mm, respectively. The inhibition mechanisms of cetuximab, etoposide, docetaxel, and ifosfamide were non-competitive, and for paclitaxel was competitive. Consequently, inhibition of hPON1 by these anticancer agents may explain some of the cardiotoxic actions of these drugs.

The effect of PON1 enhancers on reducing acetylcholinesterase inhibition following organophosphate anticholinesterase exposure in rats.

Novel nucleophiles, a series of substituted phenoxyalkyl pyridinium oximes, have been previously shown by our laboratories to enhance in vitro paraoxonase 1 (PON1)-mediated degradation of a sarin surrogate (nitrophenyl isopropyl methylphosphonate, NIMP) and a VX surrogate (nitrophenyl ethyl methylphosphonate, NEMP). Five of the most efficacious of these nucleophiles were tested in rats for their ability to reduce the level of acetylcholinesterase (AChE) inhibition in brain and peripheral tissues following exposure to NIMP or NEMP. Following simultaneous administration of a nucleophile plus surrogate (at 3 dosages yielding about 10-50% AChE inhibition in the brain at 15 min), all five nucleophiles reduced the AChE inhibition in the brain at all 3 dosages, and reduced peripheral AChE inhibition at the lowest dosage. Protective effects were seen for only a short period of time, i.e., 15 min. Even though these nucleophiles are oximes, they are not effective AChE reactivators so it is unlikely that the resultant decreases in AChE inhibition are from appreciable AChE reactivation. It is likely that the protective effects seen are, at least in part, the result of enhancement of PON1-mediated surrogate degradation, an unprecedented mechanism of therapy that has the potential to be developed into a nerve agent countermeasure.

Role of paraoxonase-1 in the protection of hydrogen sulfide-donating sildenafil (ACS6) against homocysteine-induced neurotoxicity.

ACS6, a novel hydrogen sulfide (H2S)-releasing sildenafil, has been demonstrated to inhibit superoxide formation through donating H2S. We have previously found that ACS6 antagonizes homocysteine-induced apoptosis and cytotoxicity. The aim of the present study is to explore the molecular mechanisms underlying ACS6-exerted protective action against the neurotoxicity of homocysteine. In the present work, we used PC12 cells to explore whether paraoxonase-1 (PON-1) is implicated in ACS6-induced neuroprotection against homocysteine neurotoxicity. We show that ACS6 treatment results in prevention of homocysteine-caused neurotoxicity and overproduction of reactive oxygen species (ROS). Homocysteine downregulates the expression and activity of PON-1; however, this effect is significantly blocked by co-treatment with ACS6. The specific inhibitor of PON-1 2-hydroxyquinoline reverses the inhibitory effect of ACS6 on homocysteine-induced neurotoxicity and intracellular ROS accumulation. These results indicate that ACS6 protects PC12 cells against homocysteine-induced neurotoxicity by upregulating PON-1 and suggest a promising role of PON-1 as a novel therapeutic strategy for homocysteine-induced toxicity.

Paraoxonase enzyme activity is enhanced by zinc supplementation in hemodialysis patients.

BACKGROUND AND AIMS: Patients on maintenance hemodialysis (HD) face an increased risk of atherosclerosis, a crucial problem and the leading cause of cardiovascular morbidity and mortality. This study was designed to evaluate the effects of zinc supplementation on paraoxonase (PON) enzyme activity in patients on HD. METHODS: This double-blind randomized controlled trial was conducted from June 2005 to June 2007. Sixty HD patients were enrolled and divided into two groups: treatment (case) and control. The treatment and control groups were treated with 100 mg/day zinc or placebo, respectively, for 2 months. Serum zinc concentration was measured by atomic absorption spectrophotometry. PON activity was evaluated by spectrophotometric method. Lipid profile was determined using commercial kits, and apolipoprotein AI (Apo-AI) and B (Apo-B) levels were measured by commercial immunoturbidimetric kits. RESULTS: In the case group, there was no significant change in the serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and Apo-B levels, while the serum levels of high-density lipoprotein (HDL), Apo-AI, and PON activity were significantly increased (p = 0.02). In the control group, although significant increases were observed in the serum levels of TC, TG, and Apo-B (p = 0.009, 0.019, and 0.001, respectively), the serum PON activity was significantly decreased (p = 0.025) and the serum levels of HDL, LDL, and Apo-AI were not changed. At the end of intervention period, the serum level of Apo-AI and PON activity were significantly higher in the case group. CONCLUSIONS: Zinc supplementation increased both the activity of PON and the serum level of Apo-AI in the HD patients.

Knockdown of paraoxonase 1 expression influences the ageing of human dermal microvascular endothelial cells.

Skin is one of the most commonly studied tissues for microcirculation research owing to its close correlation of cutaneous vascular function, ageing and age-related cardiovascular events. To elucidate proteins that determine this correlation between endothelial cell function and ageing in the vascular environment of the skin, we performed a proteomic analysis of plasma samples from six donors in their 20s (young) and six donors in their 60s (old). Among identified proteins, paraoxonase 1 (PON1) was selected in this study. To elucidate the role of PON1 on skin ageing and determine how it controls cellular senescence, the characteristics of PON1 in human dermal microvascular endothelial cells (HDMECs) were determined. When the expression of endogenous PON1 was knocked-down by small interfering RNA (siRNA) targeting PON1, HDMECs showed characteristic features of cellular senescence such as increases in senescence-associated ß-galactosidase stained cells and enlarged and flattened cell morphology. At 48 h post-transfection, the protein expression of p16 in PON1 siRNA-treated HDMECs was higher than that in scrambled siRNA-treated HDMECs. In addition, the expressions of moesin and rho GTP dissociation inhibitor, additional age-related candidate biomarkers, were decreased by PON1 knock-down in HDMECs. In conclusion, these results suggest that PON1 functions as an ageing-related protein and plays an important role in the cellular senescence of HDMECs.

The growing importance of PON1 in cardiovascular health: a review.

The wide acceptance of the oxidation theory of atherogenesis has prompted attention to antioxidant mechanisms, particularly the prevention of lipid oxidation by high-density lipoprotein-associated proteins like paraoxonase 1 (PON1) enzyme. PON1 is a calcium-dependent enzyme that has interested toxicologists since its discovery as an organophosphate hydrolase. There is a growing interest in the enzyme's importance in cardiovascular health prompted by evidence that it may have a role in lipid metabolism and the development of atherosclerosis via its antioxidant effects. PON1 is capable of hydrolyzing homocysteine thiolactone, a metabolite of homocysteine that can impair protein function leading to endothelial dysfunction and vascular damage. Although this lactonase activity has been suggested to be PON1's native activity, the enzyme's specific physiological role and substrate remain unclear. In this review, we summarize developments in the field of PON1 research relating to cardiovascular health, and highlight those that perhaps warrant further research.

[Paraoxonase: its multiple functions and pharmacological regulation]. / Paraoxonasa: sus múltiples funciones y regulación farmacológica.

Homocysteine, a non-protein amino acid, important risk factor for atherosclerosis and thrombosis, causes dysfunction of vascular endothelial cells traduced in inadequate vasodilatation mechanism, is pro-inflammatory and induces endoplasmic reticulum stress. The more reactive conformation is the homocysteine thiolactone (HcyT), product to the nonspecific action of methionyl-tRNA synthetase, which is incorporated into proteins by disulfide bonds (S-homocysteinilation) or amide bonds (N-homocysteinilation) affecting protein structure and function leading to cell toxicity, autoimmune responses and atherogenesis. The enzyme paraoxonase-1 (PON1), part of high density lipoprotein (HDL), had been studied only for its ability to hydrolyze organophosphate derivatives. But, more recently it has been attributed other important role. The enzyme activities are involving in protecting against the development of atherosclerosis, by preventing oxidation of lipoproteins and hydrolyze HcyT. There is growing evidence about the protective role of PON1 in vascular disease. Genetic factors (polymorphisms of the PON1), environmental and lifestyle influence their concentration and biological activity, but drugs used as cardioprotectives and lipid-lowering or others, such as antibiotics and steroids, are also important modulators. This review is an updated of the most prominent information on clinical and experimental studies for understanding the role of the PON-1 in the protection against development of atherosclerosis.

Paraoxonasa: sus múltiples funciones y regulación farmacológica / Paraoxonase: its multiple functions and pharmacological regulation

La homocisteína, aminoácido no-proteico, es un importante factor de riesgo de aterosclerosis y trombosis, afecta la vasodilatación y la función normal del endotelio vascular, es pro-inflamatoria e induce estrés de retículo endoplásmico. Su conformación más reactiva, la homocisteína tiolactona, producto de la acción no específica de la metionil-t RNA sintetasa, se incorpora a proteínas mediante puentes disulfuro (S-homocisteinilación) o uniones amida (N-homocisteinilación) produciendo graves efectos sobre la estructura y función proteica conduciendo a toxicidad celular, respuestas autoinmunes y aterogénesis. La enzima paraoxonasa-1, integrante de la lipoproteína de alta densidad, fue inicialmente considerada por su capacidad de hidrolizar derivados organofosfato, pero luego se le atribuyó un importante papel protector contra la aterosclerosis por prevenir la oxidación de lipoproteínas e hidrolizar homocisteína tiolactona. Existen evidencias acerca del papel de paraoxonasa-1 en la enfermedad vascular. Los factores genéticos (polimorfismos de la paraoxonasa-1), ambientales y el estilo de vida influyen sobre su concentración y actividad biológica, pero distintos fármacos como hipolipemiantes o cardioprotectores y otros, como antibióticos y esteroides, son también importantes moduladores. En la presente revisión se actualiza la más destacada información sobre los estudios clínicos y experimentales que permiten entender el papel que cumple esta enzima en la protección ante el desarrollo de la aterosclerosis.

Homocysteine, a non-protein amino acid, important risk factor for atherosclerosis and thrombosis, causes dysfunction of vascular endothelial cells traduced in inadequate vasodilatation mechanism, is pro-inflammatory and induces endoplasmic reticulum stress. The more reactive conformation is the homocysteine thiolactone (HcyT), product to the nonspecific action of methionyl-tRNA synthetase, which is incorporated into proteins by disulfide bonds (S-homocysteinilation) or amide bonds (N-homocysteinilation) affecting protein structure and function leading to cell toxicity, autoimmune responses and atherogenesis. The enzyme paraoxonase-1 (PON1), part of high density lipoprotein (HDL), had been studied only for its ability to hydrolyze organophosphate derivatives. But, more recently it has been attributed other important role. The enzyme activities are involving in protecting against the development of atherosclerosis, by preventing oxidation of lipoproteins and hydrolyze HcyT. There is growing evidence about the protective role of PON1 in vascular disease. Genetic factors (polymorphisms of the PON1), environmental and lifestyle influence their concentration and biological activity, but drugs used as cardioprotectives and lipid-lowering or others, such as antibiotics and steroids, are also important modulators. This review is an updated of the most prominent information on clinical and experimental studies for understanding the role of the PON-1 in the protection against development of atherosclerosis.

Paraoxonase-2 modulates stress response of endothelial cells to oxidized phospholipids and a bacterial quorum-sensing molecule.

OBJECTIVE: Chronic infection has long been postulated as a stimulus for atherogenesis. Pseudomonas aeruginosa infection has been associated with increased atherosclerosis in rats, and these bacteria produce a quorum-sensing molecule 3-oxo-dodecynoyl-homoserine lactone (3OC12-HSL) that is critical for colonization and virulence. Paraoxonase 2 (PON2) hydrolyzes 3OC12-HSL and also protects against the effects of oxidized phospholipids thought to contribute to atherosclerosis. We now report the response of human aortic endothelial cells (HAECs) to 3OC12-HSL and oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) in relation to PON2 expression. METHODS AND RESULTS: Using expression profiling and network modeling, we identified the unfolded protein response (UPR), cell cycle genes, and the mitogen-activated protein kinase signaling pathway to be heavily involved in the HAEC response to 3OC12-HSL. The network also showed striking similarities to a network created based on HAEC response to Ox-PAPC, a major component of minimally modified low-density lipoprotein. HAECs in which PON2 was silenced by small interfering RNA showed increased proinflammatory response and UPR when treated with 3OC12-HSL or Ox-PAPC. CONCLUSION: 3OC12-HSL and Ox-PAPC influence similar inflammatory and UPR pathways. Quorum sensing molecules, such as 3OC12-HSL, contribute to the proatherogenic effects of chronic infection. The antiatherogenic effects of PON2 include destruction of quorum sensing molecules.

Paraoxonase-1 activity, malondialdehyde and glutathione peroxidase in non-alcoholic fatty liver disease and the effect of atorvastatin.

BACKGROUND AND STUDY AIMS: The prevalence of non-alcoholic fatty liver disease (NAFLD) appears to be increasing. The aim of the present study was to investigate the relationship between hepatic antioxidant paraoxonase 1 (PON1) activity, lipid peroxidation and antioxidant enzymes in patients with NAFLD and the effect of atorvastatin. PATIENTS AND METHODS: This study was conducted on 50 patients with NAFLD and 20 normal subjects matched for age and sex. All of them were subjected to the following investigations: abdominal ultrasonography, serum PON1 activity level, liver function tests, serum lipid profile, fasting and postprandial blood glucose and serum levels of malondialdehyde (MDA) and glutathione peroxidase (GP). NAFLD patients were further randomly classified into two groups (25 patients each), groups Ia and Ib. Only group Ia received atorvastatin 40mg tablet for 8months. RESULTS: Obesity, dyslipidaemia and impaired glucose tolerance were prevalent in group I. There was a significant decrease in serum PON1 activity with a significant increase in MDA and GP activity (i.e., there is a significant increase in lipid peroxidation rate) in group I compared with group II. After atorvastatin therapy, there was a significant increase in serum PNO1 activity and significant decrease in serum MDA levels. CONCLUSION: Patients with NAFLD show enhanced oxidative stress which may lead to non-alcoholic steatohepatitis (NASH). Reduced PON1 activity and increased MDA could be considered a biochemical marker for lipid peroxidation, which require follow-up in patients with NAFLD. Atorvastatin may have a role in prevention of, or delay, the transformation of liver steatosis into NASH.

Esterase metabolism of cholinesterase inhibitors using rat liver in vitro.

A variety of chemicals, such as organophosphate (OP) and carbamate pesticides, nerve agents, and industrial chemicals, inhibit acetylcholinesterase (AChE) leading to overstimulation of the cholinergic nervous system. The resultant neurotoxicity is similar across mammalian species; however, the relative potencies of the chemicals across and within species depend in part on chemical-specific metabolic and detoxification processes. Carboxylesterases and A-esterases (paraoxonases, PON) are two enzymatic detoxification pathways that have been widely studied. We used an in vitro system to measure esterase-dependent detoxification of 15 AChE inhibitors. The target enzyme AChE served as a bioassay of inhibitor concentration following incubation with detoxifying tissue. Concentration-inhibition curves were determined for the inhibitor in the presence of buffer (no liver), rat liver plus calcium (to stimulate PONs and thereby measure both PON and carboxylesterase), and rat liver plus EGTA (to inhibit calcium-dependent PONs, measuring carboxylesterase activity). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (chlorpyrifos oxon, paraoxon, methyl paraoxon, malaoxon) were in agreement with the literature, serving to support the use of this assay. Only a few other inhibitors showed slight or a trend towards detoxification via carboxylesterases or PONs (mevinphos, aldicarb, oxamyl). There was no apparent PON- or carboxylesterase-mediated detoxification of the remaining inhibitors (carbofuran, chlorfenvinphos, dicrotophos, fenamiphos, methamidophos, methomyl, monocrotophos, phosphamidon), suggesting that the influence of esterases on these chemicals is minimal. Thus, generalizations regarding these metabolic pathways may not be appropriate. As with other aspects of AChE inhibitors, their metabolic patterns appear to be chemical-specific.

Glabridin, a phytoestrogen from licorice root, up-regulates manganese superoxide dismutase, catalase and paraoxonase 2 under glucose stress.

The risk of death from cardiovascular diseases (CVDs), which are exacerbated by oxidative stress, is higher in diabetic women. This phenomenon has been attributed to the loss of estradiol-vascular protection. Such knowledge led us to examine the potential of glabridin, a phytoestrogen, to substitute estradiol up-regulation of antioxidant enzymes under high glucose conditions. Chronic glucose stress was found to down-regulate catalase (CAT) and paraoxonase 2 (PON2) mRNA expression by 20% and 17%, respectively, and to decrease PON2 activity by 83% in macrophages. Inflammatory conditions had an additive effect on PON2 expression in a time-dependent manner. Treatment with glabridin, under high glucose stress, increased PON2 activity by 60% and up-regulated its mRNA expression by 3.5 fold. Furthermore, glabridin up-regulated the expression of manganese superoxide dismutase (Mn-SOD) and CAT in monocytes. In conclusion, glabridin has the potential of strengthening the antioxidant defense mechanism and may serve as an antiatherogenic agent in diabetes.

Macrophage endoplasmic reticulum (ER) proteins and reducing elements stabilize paraoxonase 2 (PON2).

OBJECTIVE: To analyze the ability of macrophage sub-cellular fractions to stabilize paraoxonase 2 (PON2). METHODS: Nuclei, mitochondria, lysosomes, endoplasmic reticulum (ER) and cytosol were isolated from J774A.1 macrophage cell line and incubated with recombinant PON2. RESULTS: Among the fractions analyzed the ER contains the highest PON2 lactonase activity, and was the most potent one in stabilizing recombinant PON2 (rePON2). Whereas control rePON2 activity was decreased by 40% after 20 h of incubation at 37°C, in the presence of ER it decreased by only 15%. This effect could be attributed to the ER aqueous phase, and not to the ER lipids. The ER proteins fraction was responsible for PON2 stabilization, since heated ER or proteinase K-treated ER was not able to protect rePON2 from inactivation, while the protein fraction (after ammonium sulfate precipitation) completely prevented rePON2 inactivation. Since in the macrophage ER, there are increased levels of NADPH, secondary to glutathione reductase deficiency, we next studied the effect of the redox environment on PON2 inactivation. Incubation of rePON2 with DTT protected PON2 from inactivation. Similarly, NADPH, but not NADP, significantly increased rePON2 lactonase activity by up to 19%, after 20h of incubation as compared to control rePON2. Unlike ER from non-treated macrophages, ER harvested from oxidized-, or from cholesterol loaded-macrophages showed a significant lower basal PON2 lactonase activity, and did not protect PON2 from inactivation but rather increased it. CONCLUSION: Under normal conditions macrophage ER stabilizes PON2 activity, and this effect could be attributed to ER proteins and redox status.