Human cell death in relation to DNA damage after exposure to the untreated and biologically treated pharmaceutical wastewater.

Among all the pharmaceutical drugs that contaminate the environment, antibiotics occupy an important place due to their high consumption rates in both veterinary and human medicine. The present study examined the ability of Pseudomonas putida to grow on the antibiotic wastewater, currently expanding in Tunisia, containing amoxicillin and cefadroxil. P. putida was very efficient to grow quickly in pharmaceutical wastewater (PW) and in reducing the total dissolved solids to 80.1 %. Cytotoxicity of PW, before and after biodegradation with P. putida mt-2, was evaluated in vitro, using the MTT assay, against four human tumor cell lines such as A549 (lung cell carcinoma), HCT15 (colon cell carcinoma), MCF7 (breast adenocarcinoma), and U373 (glioma cell carcinoma). The PW reduced all human cell lines viability in a dose-dependent manner. This activity was very remarkable against U373 cell line. For this reason, we have tested the genotoxicity of PW using comet assay for quantification of DNA fragmentation. In fact, PW has statistically significant (p<0.001) influence on DNA. Indeed, the percentage of genotoxicity was 66.87 and 87.5 %, after 24 and 48 h of treatment, respectively. However, cytotoxicity and genotoxicity decreased strongly when tested the PW obtained after incubation with P. putida mt-2. Our results indicate that P. putida is a promising and improved alternative to treating industrial-scale effluent compared to current chemical treatment procedures used by the industrials.

Effect of zinc sacrificial anode degradation on the defence system of the Pacific oyster, Crassostrea gigas: chronic and acute exposures.

Two types of exposures were performed to assess the effects of zinc released from sacrificial anode degradation: a chronic exposure, in which oysters were exposed to 0.53±0.04 mg Zn L(-1) for 10 weeks, and an acute exposure, where oysters were exposed to 10.2±1.2 mg Zn L(-1) for 1 week. At the end of the acute exposure experiment, 81.8% mortality was recorded. In contrast, no mortality was detected after 10 weeks exposure. Moreover, all of the immune system biomarkers studied, except the number of circulating haemocytes, were stimulated by a moderate level of zinc and inhibited by a high level. Our exposure conditions did not induce SOD or MXR mRNA expression in gills and digestive gland. However, an increase of MT mRNA is observed in these tissues. The results indicate that oysters are sensitive to acute zinc toxicity but are only moderately affected by a mild zinc concentration.

Bioremediation of industrial pharmaceutical drugs.

Recently, attention has been drawn toward the occurrence of pharmaceuticals in the environment. In recent years, many reports have been made on the occurrence of the large, differentiated group of pharmaceuticals in wastewater (PW), surface water, ground water, and in soil. The pharmaceutical sector is currently expanding in Tunisia, with more than 34 industries. The aim of this work was to evaluate the ability of Pseudomonas putida mt-2 to treat PW. P. putida was very efficient in reducing chemical oxygen demand (COD), total dissolved solids (TDS), and turbidity of solution (85.5, 89.1, and 81.5%, respectively). Genotoxicity of effluent, before and after biodegradation, was evaluated in vivo in mouse bone marrow by assessing the percentage of cells bearing different chromosome aberrations. Results indicated that PW showed a significant ability to induce DNA damage. In addition, PW induced a remarkable lipid peroxidation (LPO) effect, however, activities of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were unchanged when treated with PW, compared to nontreated PW. This toxicity was imputed to the presence of pharmaceutical compounds in wastewater. However, chromosome aberration, as well as LPO of PW, were significantly reduced after bioremediation. Thus, the use of this strain for testing on the industrial scale seems possible and advantageous.

Impact of a sacrificial anode as assessed by zinc accumulation in different organs of the oyster Crassostrea gigas: results from long- and short-term laboratory tests.

Sacrificial anodes made of zinc are currently used in marine environments to mitigate marine corrosion as part of CP systems of immerged metallic structures. The aim of this work was to study zinc bioconcentration in the oyster Crassostrea gigas by performing two in vivo tests during different time periods and at different zinc concentrations. The first test was conducted during a period of 10 weeks at a concentration of 0.53 ± 0.04 mg Zn L(-1) to simulate long-term exposure, and a second test was conducted during a 168-hour period at a concentration of 10.2 ± 1.2 mg Zn L(-1) to reproduce short-term exposure. In these experiments, the zinc source was an electrochemical device that included a sacrificial anode to mimic the in situ conditions. During the first 14 days of the long-term experiment, digestive glands of C oysters exhibited bioaccumulation of zinc that varied according to the oysters' reproductive cycle. Both a bioconcentration factor (BCF) of ≤ 13,397 and a zinc accumulation percentage of +297% of zinc occurred in this organ after 10 weeks. The results obtained from the short-term test showed a lower BCF of 405 but a faster bioaccumulation of zinc (starting from the first day) in the same organ. No mortality was observed in long-term assay, but 81.8% of the oysters died at the end of the short-term assay. These results demonstrate the great capacity of C. gigas to accumulate zinc released from the anode, especially when low concentrations are released, as in the case of anode dissolution used as CP. This study confirmed the necessity to monitor this zinc-contamination source in marine environments in relation to the usual oyster consumption by humans (especially in France). No implication for human health of this zinc-contamination source was demonstrated until now, and this was not the purpose of this study; however, zinc remains one of the most abundant nutritionally essential elements in the human body that may affect the human immune system at high-level uptake.

Implications of Lactobacillus collinoides and Brettanomyces/Dekkera anomala in phenolic off-flavour defects of ciders.

Different Lactobacillus collinoides and Brettanomyces/Dekkera anomala cider strains were studied for their ability to produce volatile phenols in synthetic medium. All strains were able to produce 4-ethylcatechol (4-EC), 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) from caffeic, p-coumaric and ferulic acids, respectively. Interestingly, D. anomala and L. collinoides were also able to produce 4-EC, 4-EP and 4-EG in cider conditions. The quantities of ethylphenols produced by these two species were similar in both tested ciders. The impact of precursor quantities was studied and it showed that the addition of caffeic and p-coumaric acids in ciders allowed for higher 4-EC and 4-EP production by D. anomala and L. collinoides. In parallel, D. anomala and L. collinoides strains were isolated from a phenolic off-flavour defective bottled cider after ethylphenol production hence confirming the implication of these two species in this cider spoilage. Finally, detection thresholds of the main ethylphenols were determined in ciders by orthonasal and retronasal sampling. The 4-EC and 4-EP detection thresholds (close to 20-25mg/l and 1.5-2.0mg/l, respectively) were matrix dependant.

Assessment of sacrificial anode impact by aluminum accumulation in mussel Mytilus edulis: a large-scale laboratory test.

Since the early 1960s, the application of aluminum alloy sacrificial anodes to mitigate marine corrosion has been well known. The aim of this work was to study aluminum bioconcentration in Mytilus edulis by an in vitro test performed in two tanks: the first containing non-contaminated water (NCW) and the second containing aluminum-contaminated water (CW) (530 µg L(-1)) released by sacrificial anode. The mussels were collected and examined over a period of 8 weeks. A comparison between the aluminum concentrations in the digestive glands of mussels from the CW and NCW tanks shows that the highest value (1700 mg/kg d.w.) was found in the CW mussels collected after 13 days. In NCW, the mean aluminum concentration in digestive glands during the test was 281 mg/kg d.w. The rapid concentration decrease in digestive glands is probably due to the inhibition of filtering activity due to valve closure at the high concentration as well as the induction of the detoxification response.

Screening of representative cider yeasts and bacteria for volatile phenol-production ability.

Representative cider microorganisms (47 yeast strains and 16 bacterial strains) were studied for their ability to produce volatile phenols in a synthetic medium simulating cider conditions and supplemented with the necessary precursors. The various strains were tested for cinnamoyl esterase activity and only Lactobacillus collinoides were able to hydrolyse chlorogenic acid. Phenolic acid decarboxylase (PAD) activities were observed for 6 yeasts and 4 bacterial species allowing them to produce vinylphenols from hydroxycinnamic acids. On the other hand, 4 bacterial species exhibited phenolic acid reductase (PAR) activities leading to the formation of hydroxyphenylpropionic acids. Brettanomyces/Dekkera anomala and L. collinoides were able to produce 4-ethylcatechol (4-EC) and 4-ethylphenol (4-EP) from caffeic and p-coumaric acid, respectively, indicating that both species exhibit PAD and vinylphenol reductase (VPR) activities. In the experimental conditions used, the production of ethylphenols by L. collinoides was faster than the one observed for D. anomala.

Degradation and detoxification of acid orange 52 by Pseudomonas putida mt-2: a laboratory study.

INTRODUCTION: Acid orange 52 (AO52), extensively used in textile industries, was decolorized by Pseudomonas putida mt-2. AO52 azoreduction products such as N,N'-dimethyl-p-phenylenediamine (DMPD) and 4-aminobenzenesulfonic acid (4-ABS), were identified in the static degradation mixture. These amines were identified only in media of static incubation, which is consistent with their biotransformation under shaken incubation (aerobic conditions). MATERIALS AND METHODS: Tests with azo products were carried out, and whole cells were found able to easily degrade DMPD contrary to 4-ABS. However, this last could be attacked by cell extract, and an oxygen uptake was observed during the reaction. RESULTS: Degradation of DMPD by entire cells led to the formation of catechol. These results show that P. putida was able to decolorize AO52 and metabolize its derivative amines. In addition, the ability of tested compounds was evaluated in vitro to reduce human plasma butyrylcholinesterase (BuChE) activity. CONCLUSION: Azoreduction products seem to be responsible for BuChE inhibition activity observed in static biodegradation extract. However, toxicity of AO52 completely disappears after shaken incubation with P. putida, suggesting that bacterium has a catabolism which enables it to completely degrade AO52 and especially, to detoxify the dye mixture.

Alteration of in vitro and acute in vivo toxicity of textile dyeing wastewater after chemical and biological remediation.

INTRODUCTION: Textile industry is one of the most common and essential sectors in Tunisia. However, the treatment of textile effluents becomes a university because of their toxic impacts on waters, soils, flora, and fauna. MATERIALS AND METHODS: The aim of this work was to evaluate the ability of Pseudomonas putida mt-2 to decolorize a textile wastewater and to compare the biologic decolorization process to the chemical one currently used by the textile industry. RESULTS: P. putida exhibited a high decolorizing capacity of the studied effluent, compared to the coagulation-flocculation method with decolorization percentage of 86% and 34.5%, respectively. Genotoxicity of the studied effluent, before and after decolorization by P. putida mt-2, was evaluated in vitro, using the SOS chromotest, and in vivo, in mouse bone marrow, by assessing the percentage of cells bearing different chromosome aberrations compared to not treated mice. In addition, textile effluent statistically significant influenced acetylcholinesterase and butyrylcholinesterase activities and lipid peroxidation (p < 0.01) when compared to not-treated mice. Coagulation-flocculation treatment process used by industry was revealed to be ineffective. Indeed toxicities persisted after treatment and the effluent did not show any statistically significant decrease in toxicities compared to non-treated effluent. Our results indicate that P. putida is a promising and improved alternative to treating industrial scale effluent compared to current chemical decolorization procedures used by the Tunisian textile industry.

Comparative toxicities of aluminum and zinc from sacrificial anodes or from sulfate salt in sea urchin embryos and sperm.

The toxicity of aluminum or zinc from either sacrificial anodes (SA) or their sulfate salts (SS) was evaluated in sea urchin (Paracentrotus lividus) embryos or sperm exposed to Al(III) or Zn(II) (SA or SS, 0.1-10 microM), scoring developmental defects (DDs), fertilization rate (FR), and mitotic abnormalities. A significant DD increase was observed in SS, but not SA Al(III)- and Zn(II)-exposed embryos vs. controls. Both Al(III) and Zn(II), up to 10 microM, from SA and SS, inhibited mitotic activity and induced mitotic aberrations in exposed embryos. SA-Al(III)-exposed sperm displayed a significant FR increase, unlike Al(III) sulfate overlapping with controls. Both SA-Zn(II) and Zn(II) sulfate sperm exposure resulted in a significant FR increase. The offspring of SA-Al(III)-exposed sperm displayed a significant DD decrease, unlike Al(III) sulfate exposure. Zinc sulfate sperm exposure resulted in a significant increase in offspring DDs, whereas SA-Zn(II) sperm exposure decreased DDs. Together, exposures to SA-dissolved Al(III) or Zn(II) resulted in lesser, if any toxicity, up to hormesis, compared to SS. Studies of metal speciation should elucidate the present results.

Effect of in vitro exposure to zinc on immunological parameters of haemocytes from the marine gastropod Haliotis tuberculata.

Environmental pollutants such as heavy metals exert immunotoxic effects on aquatic organisms. The immune defence of molluscs is comprised of cell-mediated and humoral mechanisms, in which haemocytes play a key role. In this study, a model based on primary cultured haemocytes from the gastropod mollusc Haliotis tuberculata was established to investigate the effects of zinc in vitro. Cells were exposed for 24 h to ZnCl(2) concentrations of 0, 10, 100 or 1000 microM. The effects of zinc on haemocyte parameters were investigated using morphological, spectrophotometric and flow cytometry analysis. Immunotoxicity was reflected by a significant decrease in the number of viable haemocytes (LC(50)(24 h) = 314 microM). Moreover, the cell area was dramatically reduced, and the percentage of rounded cells increased with increasing zinc concentrations. Exposure to 1000 muM zinc induced a significant reduction in acid phosphatase activity, phagocytic activity and reactive oxygen species production in haemocytes. However, several haemocyte parameters increased significantly after 24 h of zinc exposure. In response to a 1000 microM exposure, the phenoloxidase level was 26-fold higher than that of the control, and non-specific esterase activity was increased by 69% above that of the control. These results suggest a relationship between zinc exposure and alterations in the functional responses of haemocytes from H. tuberculata.

Differences in the volatile compositions of French labeled brandies (Armagnac, Calvados, Cognac, and Mirabelle) using GC-MS and PLS-DA.

A total of 207 volatile compounds were identified in extracts of four French labeled brandies: Armagnac, Cognac, Calvados, and Mirabelle. Relative levels of all components were determined using GC-MS after integration of a selected peak of the mass spectrum of each. Each type of brandy could be clearly discriminated using PLS-DA statistical analyses based on these levels. French Mirabelle spirit, which was studied for the first time, was characterized by higher levels of many aldehydes and acetals and by the presence of compounds having an odd number of carbons together with benzaldehyde and some of its derivatives. Many possible derivatives of acrolein and high amounts of butan-2-ol were rather specific for the volatile composition of Calvados. The most important difference between the two wine-based samples seemed to be directly linked to the distillation system used. Many furanic compounds are specific to Cognac, whereas two or three compounds such as 1-(ethoxyethoxy)-2-methylbutane and gamma-eudesmol were specific to Armagnac. These two brandies presented rather high distributions of isobutanol and isopentanols, whereas Mirabelle and Calvados compositions offer more concentrated aliphatic linear alcohols.

Acid violet 7 and its biodegradation products induce chromosome aberrations, lipid peroxidation, and cholinesterase inhibition in mouse bone marrow.

INTRODUCTION: Acid violet 7 (AV7), mostly used in food, paper, cosmetic, and especially in textile industries, was degraded by Pseudomonas putida mt-2 at concentrations up to 200 mg/l. MATERIALS AND METHODS: In this study, toxicity of AV7, before and after biodegradation, was evaluated in vivo, in mouse bone marrow, by assessing the percentage of cells bearing different chromosome aberrations, membrane lipid peroxidation, and acetylcholinesterasic activity inhibition. The studies included same conditions for animal treatment, corresponding to increasing doses by intraperitoneal (ip) injection. RESULTS: Results indicated that AV7 showed a significant ability to induce chromosome aberrations, lipid peroxidation, and acetylcholinesterase inhibitory effect. The toxicity of AV7 increased significantly after static biodegradation with P. putida mt-2 and totally disappeared after shaken incubation. In addition, the toxicity generated by the pure azo dye and the corresponding azoreduction metabolites (4'-aminoacetanilide (4'-AA) and 5-acetamido-2-amino-1-hydroxy-3,6-naphtalene disulfonic acid (5-ANDS)) were compared. 4'-AA and 5-ANDS would be responsible of static biodegradation medium toxicity. The present study demonstrates that P. putida mt-2, incubated under aerobic condition, has a catabolism which enables it to degrade AV7, and especially to completely detoxify the dye mixture.

Influence of growth phase and geographic origin on the essential oil composition of Pituranthos chloranthus from Tunisia.

The chemical compositions of the essential oils of Pituranthos chloranthus harvested at the vegetative, flower budding, flowering and fruiting stages from three distinct geographical areas of Tunisia were investigated using GC-FID and GC-MS. One hundred and fifty compounds were identified in which alpha-pinene, beta-pinene, alpha-phellandrene, beta-myrcene, beta-phellandrene, p-cymene, 8-methyldecanal, exo-2-hydroxycineole acetate and carvacrol could reach more than 10% of the total amount. However, this composition varied with respect to both the geographical area and the season. A clear discrimination of samples could be achieved by submitting the results to PLS discriminant analysis. p-Cymenene was only detected at the floral budding stage (February), whereas high amounts of exo-2-hydroxycineole and exo-2-hydroxycineole acetate were specific for the flowering period (April). Carvacrol was showed to be characteristic mainly of the fruiting period (August), whereas the vegetative state (November) could be distinguished from the others by the presence of alpha- and beta-pinene. Limonene, camphene, geraniol and beta-damascenone were likely to be specific for the essential oils of this species collected from the different regions of Tunisia.

A comparative evaluation of mutagenic, antimutagenic, radical scavenging and antibacterial activities of essential oils of Pituranthos chloranthus (Coss. et Dur.).

The Salmonella typhimurium/microsome assay is a widely used bacterial genotoxicity assay to test potential carcinogens. The aim of this work was to evaluate the mutagenic and antimutagenic activities with and without the addition of an extrinsic metabolic activation system of essential oils obtained from an aerial part of Pituranthos chloranthus harvested from different stations in Tunisia. The oils showed no mutagenicity when tested with S. typhimurium strains TA98, TA100, and TA1535. On the other hand, we showed that these essential oils reduced significantly Benzo [a] pyrene (B[a] P) and sodium-azide-induced mutagenicity. The scavenging capacity of these essential oils was also estimated by evaluating the inhibition of DPPH radical. Essential oils harvested at Medenine and Gabes in November were more effective in scavenging activity. The essential oils were tested for their antimicrobial properties against five different bacteria, and were found to be weakly active, with MIC and MBC values in the range 0.6-4 and 2.2-5 mg/mL, respectively.

Elaboration, characterization and application of polysulfone and polyacrylic acid blends as ultrafiltration membranes for removal of some heavy metals from water.

Polysulfone (PSf)/polyacrylic acid ultrafiltration (PSf/PAA) membranes were prepared from a polymer blend in dimethylformamide by coagulation in water according to the wet phase inversion method. Immobilization of water-soluble PAA within the non-soluble PSf matrix was proven by the increase of ion exchange capacity and the intensity of the carboxyl groups' peak with the increase of PAA content as shown by Fourier transform infrared spectra. These results lead to consider that PSf and PAA form a semi-interpenetrating polymer networks. The obtained membranes showed a decrease of mean surface-pore sizes, the overall porosity and the hydraulic permeability with the increase in PAA content. Such results were imputed to the morphologic modifications of PSf film with the immobilization of increasing PAA amount. PSf/PAA membranes showed high lead, cadmium and chromium rejection which reaches 100% at pH superior to 5.7 and a low rejection at low pH. Moreover, the heavy metal rejection decreases with feed solution concentration and applied pressure increases. These behaviors were attributed to the role of carboxylic groups in ion exchange or complexation. As a matter of fact, the strong lead ion-PAA interactions were revealed by the scanning electron microscopy with energy dispersive X-rays (SEM-EDX).

Mutagenicity and genotoxicity of acid yellow 17 and its biodegradation products.

Acid yellow 17 (AY17), a very important commercial azo dye used in the textile industry, was degraded by Pseudomonas putida mt-2 at a concentration of up to 200 mg/L. High-performance liquid chromatography analysis of the biodegradation media revealed the presence of 4-aminobenzensulfonic acid (4-ABS) derived from AY17 azoreduction, which attests the expression of an azoreductase by this bacterium. This amine was identified only in the medium of static incubation, which is consistent with its biotransformation under shaken incubation (i.e., aerobic conditions). The mutagenicity of AY17 and its biodegradation products was evaluated by using Salmonella typhimurium TA102 and TA104. No mutagenicity was observed in the presence or absence of a metabolic activation system (S9). In addition, the ability of tested compounds to induce DNA damage in vitro with the DNA strand scission assay was evaluated. Results showed that only static decolorization culture of AY17 showed a significant ability to induce the pKS plasmid DNA opening. The present study showed that P. putida mt-2, cultivated under aerobic conditions, was able to decolorize, and especially to detoxify, AY17.

Genotoxic and antibutyrylcholinesterasic activities of acid violet 7 and its biodegradation products.

Acid violet 7, a sulfonated azo dye was degraded by Pseudomonas putida mt-2 in mineral medium at concentrations up to 200 mg/L. The genotoxicity of AV7 and its biodegradation extracts was evaluated by using the DNA-strand scission assay. No genotoxicity was observed, even with or without exposition to UV irradiation, for biodegradation under shaking conditions, but increased significantly after biodegradation under static conditions. In addition, the ability of tested compounds to reduce human plasma butyrylcholinesterase (BuChE) activity was evaluated in vitro. Genotoxicity and anti-BuChE activity generated by the azoreduction products [4'-aminoacetanilid (4'-AA) and 5-acetamido-2-amino-1-hydroxy-3,6-naphtalene disulfonic acid (5-ANDS)] were assessed and compared with that of the parent unsubstituted amines. 4'-AA exhibited a strong genotoxicity, which was imputed to the presence of the acetoxy (COCH3) substituent on the aromatic amine; however, the presence of sulphonic groups in 5-ANDS seems to be responsible for its BuChE inhibition activity. The present study demonstrates that P. putida mt-2, incubated under aerobic conditions, has a catabolism that enables it to degrade AV7 and, especially, to detoxify the dye mixtures.

In vitro study of DNA damage induced by acid orange 52 and its biodegradation derivatives.

Mutagenicity of acid orange 52 (AO52) and its degradation products by Pseudomonas putida mt-2 was evaluated with the use of Salmonella Typhimurium TA102 and TA104 with and without the metabolic activation system (S9). No mutagenicity was observed in the absence of S9 and in the presence of S9 for biodegradation under shaking conditions, but it increased significantly in the presence of S9 after biodegradation under static conditions. In addition, the ability of tested compounds to induce DNA damage in vitro was evaluated with the DNA strand scission assay. The toxicity generated by the pure azo dye and the corresponding azoreduction products (4-aminobenzenesulfonic acid and N,N'-dimethyl-p-phenylenediamine) were compared. We suggest that the mutagenicity mechanism of these molecules occurs through free radical generation processes. In this study, we demonstrate that P. putida mt-2 incubated under aerobic conditions undergoes catabolism that enables it to degrade AO52 completely and, especially, to detoxify the dye mixtures.

In vitro mutagenicity of Acid Violet 7 and its degradation products by Pseudomonas putida mt-2: Correlation with chemical structures.

Acid Violet 7 (AV7), a very important commercial azo dye used in the textile, food, paper and cosmetic industries, was degraded by Pseudomonas putida mt-2 at a concentration up to 200mg/l. HPLC analysis of the biodegradation media revealed the presence of either 4'-aminoacetanilide (4'-AA) or 5-acetamido-2-amino-1-hydroxy-3,6-naphthalene disulfonic acid (5-ANDS) deriving from AV7 azoreduction which attests the expression of an azoreductase by this bacterium. These amines were identified only in media of static incubation, which is consistent with their biotransformation under shaken incubation (aerobic conditions). Pure azo dye, pure azoreduction products and total lyophilized biodegradation extracts were assayed for their mutagenic properties using Ames test. Mutagenicity of AV7 even with or without the S9 metabolizing system increased significantly after static biodegradation and totally disappeared after shaken incubation. In addition, mutagenicity of pure azo reduction products of AV7 was assessed and compared with that of the parent unsubstituted amines. 4'-AA exhibited a strong mutagenicity which was imputed to the presence of the acetoxy (COCH(3)) substituent on the aromatic amine; however, the presence of sulphonic groups in 5-ANDS limited its mutagenicity.