Biochemical, Kinetic and Biological Properties of Group V Phospholipase A2 from Dromedary.

Secretory group V phospholipase A2 (PLA2-V) is known to be involved in inflammatory processes in cellular studies, nevertheless, the biochemical and the enzymatic characteristics of this important enzyme have been unclear yet. We reported, as a first step towards understanding the biochemical properties, catalytic characteristics, antimicrobial and cytotoxic effects of this PLA2, the production of PLA2-V from dromedary. The obtained DrPLA2-V has an absolute requirement for Ca2+ and NaTDC for enzymatic activity with an optimum pH of 9 and temperature of 45 °C with phosphatidylethanolamine as a substrate. Kinetic parameters showed that Kcat/Kmapp is 2.6 ± 0.02 mM-1 s-1. The enzyme was found to display potent Gram-positive bactericidal activity (with IC50 values of about 5 µg/mL) and antifungal activity (with IC50 values of about 25 µg/mL)in vitro. However, the purified enzyme did not display a cytotoxic effect against cancer cells.

A Proteinaceous Alpha-Amylase Inhibitor from Moringa Oleifera Leaf Extract: Purification, Characterization, and Insecticide Effects against C. maculates Insect Larvae.

The main objective of the current study was the extraction, purification, and enzymatic characterization of a potent proteinaceous amylase inhibitor from Moringa oleifera. The antimicrobial potential and insecticide effects against C. maculates insect larvae were also studied. The α-amylase inhibitor was extracted in methanol (with an inhibitory activity of 65.6% ± 4.93). Afterwards, the inhibitor αAI.Mol was purified after a heat treatment at 70 °C for 15 min followed by one chromatographic step of Sephadex G-50. An apparent molecular weight of 25 kDa was analyzed, and the N-terminal sequence showed the highest identity level (89%) with the monomeric α-amylase inhibitor from Triticum dicoccoides. αAI.Mol was found to tolerate pH values ranging from 5.0 to 11.0 and showed maximal activity at pH 9.0. Thermal stability was remarkably important, since the inhibitory activity was maintained at 55% after 1 h of incubation at 70 °C and at 53% after an incubation of 45 min at 80 °C. The potency of the current purified inhibitor against amylases from different origins indicates that αAI.Mol seems to possess the highest affinity toward human salivary α-amylase (90% inhibitory activity), followed by the α-amylase of insects Callosobruchus maculatus and Tribolium confusum (71% and 61%, respectively). The kinetic parameters were also calculated, and the Kmax and Vmax of the digestive amylase were estimated at 185 (mmol/min/mg) and 0.13 mM, respectively. The inhibitor possesses a strong bactericidal effect against Gram+ and Gram- strains, and the MIC values were >1 against B. cereus but >6 against E. coli. Interestingly, the rates of survival and pupation of C. maculates insect larvae were remarkably affected by the purified αAI.Mol from Moringa oleifera.

Molecular and Structural Characterizations of Lipases from Chlorella by Functional Genomics.

Microalgae have been poorly investigated for new-lipolytic enzymes of biotechnological interest. In silico study combining analysis of sequences homologies and bioinformatic tools allowed the identification and preliminary characterization of 14 putative lipases expressed by Chlorella vulagaris. These proteins have different molecular weights, subcellular localizations, low instability index range and at least 40% of sequence identity with other microalgal lipases. Sequence comparison indicated that the catalytic triad corresponded to residues Ser, Asp and His, with the nucleophilic residue Ser positioned within the consensus GXSXG pentapeptide. 3D models were generated using different approaches and templates and demonstrated that these putative enzymes share a similar core with common α/ß hydrolases fold belonging to family 3 lipases and class GX. Six lipases were predicted to have a transmembrane domain and a lysosomal acid lipase was identified. A similar mammalian enzyme plays an important role in breaking down cholesteryl esters and triglycerides and its deficiency causes serious digestive problems in human. More structural insight would provide important information on the enzyme characteristics.

A Novel Thermostable and Alkaline Protease Produced from Bacillus stearothermophilus Isolated from Olive Oil Mill Sols Suitable to Industrial Biotechnology.

This study was conducted to identify a new alkaline and thermophilic protease (Ba.St.Pr) produced from Bacillus stearothermophilus isolated from olive oil mill sols and to evaluate its culture conditions, including temperature, pH, carbon and nitrogen sources, and incubation time. The optimum culture conditions for cell growth (10 g/L) and protease production (5050 U/mL) were as follows: temperature 55 °C, pH 10, inoculation density 8 × 108 CFU/mL, and incubation time 24 h. The use of 3% yeast extract as the nitrogen sources and galactose (7.5 g/L) as the carbon sources enhanced both cell growth and protease production. Using reversed-phase analytical HPLC on C-8 column, the new protease was purified with a molecular mass of approximately 28 kDa. The N-terminal sequence of Ba.St.Pr exhibited a high level of identity of approximately 95% with those of Bacillus strains. Characterization under extreme conditions revealed a novel thermostable and alkaline protease with a half-life time of 187 min when incubated with combined Ca2+/mannitol. Ba.St.Pr demonstrated a higher stability in the presence of surfactant, solvent, and Ca2+ ions. Consequently, all the evaluated activity parameters highlighted the promising properties of this bacterium for industrial and biotechnological applications.

Purification and Biochemical Characterization of a New Protease Inhibitor from Conyza dioscoridis with Antimicrobial, Antifungal and Cytotoxic Effects.

The main objective of the current study was the extraction, purification, and biochemical characterization of a protein protease inhibitor from Conyzadioscoridis. Antimicrobial potential and cytotoxic effects were also examined. The protease inhibitor was extracted in 0.1 M phosphate buffer (pH 6-7). Then, the protease inhibitor, named PDInhibitor, was purified using ammonium sulfate precipitation followed by filtration through a Sephadex G-50 column and had an apparent molecular weight of 25 kDa. The N-terminal sequence of PDInhibitor showed a high level of identity with those of the Kunitz family. PDInhibitor was found to be active at pH values ranging from 5.0 to 11.0, with maximal activity at pH 9.0. It was also fully active at 50 °C and maintained 90% of its stability at over 55 °C. The thermostability of the PDInhibitor was clearly enhanced by CaCl2 and sorbitol, whereas the presence of Ca2+ and Zn2+ ions, Sodium taurodeoxycholate (NaTDC), Sodium dodecyl sulfate (SDS), Dithiothreitol (DTT), and ß-ME dramatically improved the inhibitory activity. A remarkable affinity of the protease inhibitor with available important therapeutic proteases (elastase and trypsin) was observed. PDInhibitor also acted as a potent inhibitor of commercial proteases from Aspergillus oryzae and of Proteinase K. The inhibitor displayed potent antimicrobial activity against gram+ and gram- bacteria and against fungal strains. Interestingly, PDInhibitor affected several human cancer cell lines, namely HCT-116, MDA-MB-231, and Lovo. Thus, it can be considered a potentially powerful therapeutic agent.

Efficient heterologous expression in Pichia pastoris, immobilization and functional characterization of a scorpion venom secreted phospholipase A2.

Industrial processes have expanded with the ability to clone and express recombinant immobilized enzymes in microorganisms such as Pichia pastoris that have commercially attractive amounts of the appropriate genes. This report describes the overexpression in Pichia pastoris, immobilization, and functional characterization of a secreted phospholipase A2 from scorpion venom Scorpio maurus: rPLA2(-5). After 48 h of culture, the recombinant rPLA2(-5) was secreted into the culture medium and expressed at about 9 mg/L. Comparative analyses of the kinetics and hydrolysis of rPLA2(-5) monolayers at various surface pressures were conducted with the same form produced in Escherichia coli. As a second part of the study, rPLA2(-5) overexpressed in Pichia pastoris was immobilized by adsorption on CaCO3, with about 78 percent of the activity. In comparison to the free enzyme, rPLA2(-5) was studied for stability. Immobilization improved the thermal stability of rPLA2(-5) and even the stability at acidic pH. Moreover, we found that the immobilization improved the stability of rPLA2(-5) towards bile salts, Tween 80, Triton X-100, and SDS, as well as its stability towards many organic solvents. Until now, this is the first study to describe the overexpression and immobilization of a scorpion venom phospholipase A2 that possesses an interesting stability characteristic that makes it useful for a wide range of biotechnological applications.

Purification and biochemical characterization of a secreted group IIA chicken intestinal phospholipase A2.

BACKGROUND: Secretory phospholipase A2 group IIA (IIA PLA2) is a protein shown to be highly expressed in the intestine of mammals. However, no study was reported in birds. RESULTS: Chicken intestinal group IIA phospholipase A2 (ChPLA2-IIA) was obtained after an acidic treatment (pH.3.0), precipitation by ammonium sulphate, followed by sequential column chromatographies on Sephadex G-50 and mono-S ion exchanger. The enzyme was found to be a monomeric protein with a molecular mass of around 14 kDa. The purified enzyme showed a substrate preference for phosphatidylethanolamine and phosphatidylglycerol, and didn't hydrolyse phosphatidylcholine. Under optimal assay conditions, in the presence of 10 mM NaTDC and 10 mM CaCl2, a specific activity of 160 U.mg⁻¹ for purified ChPLA2-IIA was measured using egg yolk as substrate. The fifteen NH2-terminal amino acid residues of ChPLA2-IIA were sequenced and showed a close homology with known intestinal secreted phospholipases A2. The gene encoding the mature ChPLA2-IIA was cloned and sequenced. To further investigate structure-activity relationship, a 3D model of ChPLA2-IIA was built using the human intestinal phospholipase A2 structure as template. CONCLUSION: ChPLA2-IIA was purified to homogeneity using only two chromatographic colomns. Sequence analysis of the cloned cDNA indicates that the enzyme is highly basic with a pI of 9.0 and has a high degree of homology with mammalian intestinal PLA2-IIA.

Antibacterial properties of chicken intestinal phospholipase A2.

BACKGROUND: The presence of chicken group-IIA PLA2 (ChPLA2-IIA) in the intestinal secretion suggests that this enzyme plays an important role in systemic bactericidal defence. We have analyzed the bactericidal activity of purified ChPLA2-IIA, on several gram-positive and gram-negative bacteria by using the diffusion well and dilution methods. RESULTS: ChPLA2-IIA displays potent bactericidal activity against gram-positive bacteria but lacks bactericidal activity against gram negative ones. We have also demonstrated a synergic action of ChPLA2-IIA with lysozyme when added to the bacteria culture prior to ChPLA2-IIA. The bactericidal efficiency of ChPLA2-IIA was shown to be dependent upon the presence of calcium ions and then a correlation could be made to its hydrolytic activity of membrane phospholipids. Interestingly ChPLA2-IIA displays a higher dependence to Ca²+ ions than to Mg²+ ions. CONCLUSION: We conclude that the main physiological role of ChPLA2-IIA could be the defence of the intestine against bacterial invasions.

Purification and characterization of the first recombinant bird pancreatic lipase expressed in Pichia pastoris: the turkey.

BACKGROUND: The turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Some biochemical properties and kinetic studies were determined using emulsified system and monomolecular film techniques. Those studies have shown that despite the accumulation of free fatty acids at the olive oil/water interface, TPL continues to hydrolyse efficiently the olive oil and the TC4 in the absence of colipase and bile salts, contrary to most classical digestive lipases which denaturate rapidly under the same conditions. The aim of the present study was to express TPL in the methylotrophic yeast Pichia pastoris in order to get a large amount of this enzyme exhibiting interesting biochemical properties, to purify and characterize the recombinant enzyme. RESULTS: The recombinant TPL was secreted into the culture medium and the expression level reached about 15 mg/l after 4 days of culture. Using Q-PCR, the number of expression cassette integrated on Pichia genomic DNA was estimated to 5. The purified rTPL, with molecular mass of 50 kDa, has a specific activity of 5300 U/mg on emulsified olive oil and 9500 U/mg on tributyrin. The optimal temperature and pH of rTPL were 37°C and pH 8.5. The stability, reaction kinetics and effects of calcium ions and bile salts were also determined. CONCLUSIONS: Our results show that the expressed TPL have the same properties as the native TPL previously purified. This result allows us the use of the recombinant enzyme to investigate the TPL structure-function relationships.

Purification and biochemical characterization of pancreatic phospholipase A2 from the common stingray Dasyatis pastinaca.

BACKGROUND: Mammalian sPLA2-IB are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes. RESULTS: A marine stingray phospholipase A2 (SPLA2) was purified from delipidated pancreas. Purified SPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 14 kDa. A specific activity of 750 U/mg for purified SPLA2 was measured at optimal conditions (pH 8.5 and 40 °C) in the presence of 4 mM NaTDC and 8 mM CaCl2 using PC as substrate. The sequence of the first twenty first amino-acid residues at the N-terminal extremity of SPLA2 was determined and shows a close similarity with known mammal and bird pancreatic secreted phospholipases A2. SPLA2 stability in the presence of organic solvents, as well as in acidic and alkaline pH and at high temperature makes it a good candidate for its application in food industry. CONCLUSIONS: SPLA2 has several advantageous features for industrial applications. Stability of SPLA2 in the presence of organic solvents, and its tolerance to high temperatures, basic and acidic pH, makes it a good candidate for application in food industry to treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds.

Immunohistochemical localization of hepatopancreatic phospholipase A2 in Hexaplex trunculus digestive cells.

BACKGROUND: Mammalian sPLA2-IB localization cell are well characterized. In contrast, much less is known about aquatic primitive ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes and the mode of digestion of lipid food. RESULTS: The marine snail digestive phospholipase A2 (mSDPLA2) has been previously purified from snail hepatopancreas. The specific polyclonal antibodies were prepared and used for immunohistochimical and immunofluorescence analysis in order to determine the cellular location of mSDPLA2. Our results showed essentially that mSDPLA2 was detected inside in specific vesicles tentatively named (mSDPLA2+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion in the snail might occur in specific granules inside the digestive cells. CONCLUSION: The cellular location of mSDPLA2 suggests that intracellular phospholipids digestion, like other food components digestion of snail diet, occurs in these digestive cells. The hepatopancreas of H. trunculus has been pointed out as the main region for digestion, absorption and storage of lipids.

Biochemical properties of pancreatic colipase from the common stingray Dasyatis pastinaca.

BACKGROUND: Pancreatic colipase is a required co-factor for pancreatic lipase, being necessary for its activity during hydrolysis of dietary triglycerides in the presence of bile salts. In the intestine, colipase is cleaved from a precursor molecule, procolipase, through the action of trypsin. This cleavage yields a peptide called enterostatin knoswn, being produced in equimolar proportions to colipase. RESULTS: In this study, colipase from the common stingray Dasyatis pastinaca (CoSPL) was purified to homogeneity. The purified colipase is not glycosylated and has an apparent molecular mass of around 10 kDa. The NH2-terminal sequencing of purified CoSPL exhibits more than 55% identity with those of mammalian, bird or marine colipases. CoSPL was found to be less effective activator of bird and mammal pancreatic lipases than for the lipase from the same specie. The apparent dissociation constant (Kd) of the colipase/lipase complex and the apparent Vmax of the colipase-activated lipase values were deduced from the linear curves of the Scatchard plots. We concluded that Stingray Pancreatic Lipase (SPL) has higher ability to interact with colipase from the same species than with the mammal or bird ones. CONCLUSION: The fact that colipase is a universal lipase cofactor might thus be explained by a conservation of the colipase-lipase interaction site. The results obtained in the study may improve our knowledge of marine lipase/colipase.

Spirulina platensis, Punica granatum peel, and moringa leaves extracts in cosmetic formulations: an integrated approach of in vitro biological activities and acceptability studies.

The selection of suitable natural raw materials in the cosmetic research and development is a key point, in order not only to obtain the expected results but also to avoid undesirable side effects. In this study, spirulina platensis, pomegranate (Punica granatum) peel, and moringa leaves alone were evaluated for anti-oxidant and antimicrobial properties. The chemical composition (moisture, dry matter, protein, lipid, and ash) and total polyphenols, flavonoids, and carotenoids content were evaluated in the three extracts. Total antioxidant capacity and ferric reducing activity power of extracts were also studied. Using agar diffusion method, the anti-Micrococcus luteus, Staphylococcus aureus, E. coli, Listeria monocytogenes, Salmonella typhimurium, and Enterococus faecalis activities were measured. Interestingly, after combinations, pomegranate peel/spirulina (A), and moringa/spirulina (B): 25%/75% and 50%/50%, we have found that pomegranate peel can be incorporated into cosmetic formulations as an excellent preservative due to its exceptionally amount of phenolic compounds, powerful antioxidant activity, and its antibacterial activity against pathogenic strains.

Production and structure prediction of amylases from Chlorella vulgaris.

Amylases are enzymes required for starch degradation and are naturally produced by many microorganisms. These enzymes are used in several fields such as food processing, beverage, and medicine as well as in the formulation of enzymatic detergents proving their significance in modern biotechnology. In this study, a three-stage growth mode was applied to enhance starch production and amylase detection from Chlorella vulgaris. Stress conditions applied in the second stage of cultivation led to an accumulation of proteins (75% DW) and starch (21% DW) and a decrease in biomass. Amylase activities were detected and they showed high production levels especially on day 3 (35 U/ml) and day 5 (22.5 U/ml) of the second and third stages, respectively. The bioinformatic tools used to seek amylase protein sequences from TSA database of C. vulgaris revealed 7 putative genes encoding for 4 α-amylases, 2 ß-amylases, and 1 isoamylase. An in silico investigation showed that these proteins are different in their lengths as well as in their cellular localizations and oligomeric states though they share common features like CSRs of GH13 family or active site of GH14 family. In brief, this study allowed for the production and in silico characterization of amylases from C. vulgaris.

Enhancement of Aeribacillus pallidus strain VP3 lipase catalytic activity through optimization of medium composition using Box-Behnken design and its application in detergent formulations.

Lipases are hydrolytic enzymes owing much importance in industrial applications. These enzyme-based detergents are ecofriendly and produce a wastewater with low level of COD (chemical oxygen demand). In the present work, a novel halophilous, thermoalkaline, and detergent-tolerant lipase produced by a newly isolated Aeribacillus pallidus strain VP3 was studied. Considerable interest has been given to this lipase by the improvement of its catalytic activity through the optimization of the pH, the (C/N) ratio, and the inoculum size, using the response surface methodology based on the Box-Behnken design of experiments. A total of 16 experiments were conducted, and the optimized pH, (C/N) ratio, and inoculum size were 10, 1, and 0.3, respectively. The results of the analysis of variance (ANOVA) test indicated that the established model was significant (p value < 0.05). The optimization of the production conditions leads to 2.83-fold of increase in the catalytic activity calculated as the ratio of the activity obtained after optimization (68 U) and the initial activity before optimization (24 U). All in all, the lipase of Aeribacillus pallidus could be considered as a potential candidate to be incorporated in detergent formulations since it shows a good stability towards detergents and wash performance.

Hydrolysis of three different head groups phospholipids by chicken group V phospholipase A2 using the monomolecular film technique.

The kinetic aspects of lipolysis by pulmonary phospholipase A2 (ChPLA2-V), chicken intestinal phospholipase A2 (ChPLA2-IIA) and chicken pancreatic phospholipase A2 (ChPLA2-IB), from chicken have been compared using the monomolecular films technique, on short-chain phospholipids (with three different head groups) and on long-chain phospholipids. The main conclusions from our experimental data indicate that the maximum catalytic activities of ChPLA2-V on 1,2 phosphatidylcholine and 1,2 phosphatidylethanolamine reached 15.26 and 36.12 moles/cm2.min.mM, respectively, at a pressure of 15 and 35 dynes/cm, respectively. Whereas, those of ChPLA2-IB were 3.58 (at the pressure of 20 dynes/cm) and 4.9 moles/cm2.min.mM. However, hydrolysis of phosphatidylglycerol monolayers (C12PG), were very much higher compared with all the substrates tested with 122 moles/cm2.min. Surprisingly, the hydrolysis rate of ChPLA2-V on long-chain phosphatidylglycerol (C18PG) was very low (1.45 moles/cm2.min) compared with all tested substrates, even with the use of p-cyclodextrin. And thus, the fatty acid preference of ChPLA2-V was 2-decanoyl > 2-oleoyl with a PG head group. In order to gain significant correlations between enzyme's structures and their relative functions, we tried to examine the surface electrostatic potentials of the various secreted phospholipase 2 (sPLA2) from chicken. In the present study, we detailed that the substrate affinity, specificity and the hydrolysis rates of sPLA2 at each interface is governed by the surface electrostatic potentials and hydrophobic interactions operative at this surface.

Purification, biochemical and molecular study of lipase producing from a newly thermoalkaliphilic Aeribacillus pallidus for oily wastewater treatment.

Treatment of oily wastewater is constantly a challenge; biological wastewater treatment is an effective, cheap and eco-friendly technology. A newly thermostable, haloalkaline, solvent tolerant and non-induced lipase from Aeribacillus pallidus designated as GPL was purified and characterized of biochemical and molecular study for apply in wastewater treatment. The GPL showed a maximum activity at 65°C and pH 10 after 22 h of incubation, with preference to TC4 substrates. Pure enzyme was picked up after one chromatographic step. It displayed an important resistance at high temperature, pH, NaCl, at the presence of detergents and organic solvents. In fact, GPL exhibited a prominent stability in wide range of organic solvents at 50% (v/v) concentration for 2 h of incubation. The efficiency of the GPL in oil wastewater hydrolysis was established at 50°C for 1 h, the oil removal efficiency was established at 96, 11% and the oil biodegradation was confirmed through fourier transform infrared (FT-IR) spectroscopy. The gene that codes for this lipase was cloned and sequenced and its open reading frame encoded 236 amino acid residues. The deduced amino acids sequence of the GPL shows an important level of identity with Geobacillus lipases.

Antibacterial, antifungal and anticoagulant activities of chicken PLA2 group V expressed in Pichia pastoris.

Secretory class V phospholipase A2 (PLA2-V) has been shown to be involved in inflammatory processes in cellular studies, but the biochemical and physical properties of this important enzyme have been unclear. As a first step towards understanding the structure, function and regulation of this PLA2, we report the expression and characterization of PLA2-V from chicken (ChPLA2-V). The ChPLA2-V cDNA was synthesized from chicken heart polyA mRNA by RT-PCR, and an expression construct containing the PLA2 was established. After expression in Pichia pastoris cells, the active enzyme was purified. The purified ChPLA2-V protein was biochemically and physiologically characterized. The recombinant ChPLA2-V has an absolute requirement for Ca2+ for enzymatic activity. The optimum pH for this enzyme is pH 8.5 in Tris-HCl buffer with phosphatidylcholine as substrate. ChPLA2-V was found to display potent Gram-positive and Gram-negative bactericidal activity and antifungal activity in vitro. The purified enzyme ChPLA2-V with much stronger anticoagulant activity compared with the intestinal and pancreatic chicken PLA2-V was approximately 10 times more active. Chicken group V PLA2, like mammal one, may be considered as a future therapeutic agents against fungal and bacterial infections and as an anticoagulant agent.

A novel bactericidal homodimeric PLA2 group-I from Walterinnesia aegyptia venom.

A novel non-toxic phospholipase A2 was purified to homogeneity in a single chromatography step from the venom of Walterinnesia aegyptia, a monotypic elapid snake caught in Saudi Arabia, and its antimicrobial and hemolytic properties were evaluated as well. This enzyme, namely WaPLA2, is a homodimer with an estimated molecular mass of 30 kDa, and its NH2-terminal sequence exhibits a significant degree of similarity with PLA2 group-I. At optimal pH (8.5) and temperature (45 °C), the purified PLA2 exhibited a specific activity of 2100 U/mg, and it requires bile salts and Ca2+ for its activity. However, other cations such as Cd2+ and Hg2+ diminished the enzyme activity remarkably, thereby suggesting that the catalytic site arrangement has an exclusive structure for Ca2+ binding. Furthermore, WaPLA2 maintained almost 100% and 60% of its full activity in a pH range of 6.0-10 after 24 h incubation or after 60 min treatment at 70 °C, respectively. In the biological activity assays, WaPLA2 displayed potent indirectly hemolytic and antimicrobial activities that were strongly correlated. These promising findings encourage further in-depth research to understand the molecular mechanism of WaPLA2's antimicrobial properties for its possible use as a potential therapeutic lead molecule for treating infections.

Maneb disturbs expression of superoxide dismutase and glutathione peroxidase, increases reactive oxygen species production, and induces genotoxicity in liver of adult mice.

Maneb (MB), a fungicide largely used in agriculture throughout the world including Tunisia, protects many vegetables, fruits and field crops against a wide spectrum of fungal diseases. However there is a lack of informations regarding the risks arising from MB exposure on non target organisms, especially mammals. The aim of this study was to investigate the morphological, biochemical and molecular aspects of liver injury after exposure of mice to MB. Four doses of MB corresponding to 1/8 (group D1), 1/6 (group D2), 1/4 (group D3), and 1/2 (group D4) of lethal dose (DL50 = 1500 mg/kg body weight) were administered to adult mice. Oxidative stress parameters were also objectified by molecular and histological endpoints in the liver. Maneb caused hepatotoxicity as characterized by the significant increase in the levels of malondialdehyde and protein oxidation marker, advanced oxidation protein products (AOPP). The activities of catalase, glutathione peroxidase, superoxide dismutase and the levels of glutathione decreased significantly in all treated mice, while vitamin C levels decreased only in group D4. We also noted a significant decrease in gene expression of superoxide dismutase and glutathione peroxidase enzymes. Maneb caused nucleic acids degradation testifying its genotoxicity. Yet, biochemical markers in plasma showed a decrease in total protein and an increase in aspartate, alanine amino transferases and bilirubin levels in all treatment groups. Moreover, plasma levels of cholesterol, triglycerides and low density lipoprotein-cholesterol significantly increased, while those of high density lipoprotein-cholesterol decreased. These biochemical alterations were correlated with significantly histological changes. Our data showed, for the first time, that intraperitoneal injection of very high non environmentally relevant MB concentrations to adult mice resulted in oxidative stress leading to hepatotoxicity and the impairment of defense systems, confirming the pro-oxidant and genotoxic effects of this fungicide.