One-time ozone treatment improves the postharvest quality and antioxidant activity of Actinidia arguta fruit.

The major aim of this study was to check the effect of one-time ozonation on selected quality parameters and antioxidant status of Actinidia arguta fruit. For this purpose, A. arguta fruit was ozonated with gas at a concentration of 10 and 100 ppm, which was carried out successively for 5, 15 and 30 min. Next, the selected quality attributes, antioxidants level as well as NADPH and mitochondrial energy metabolism in mini-kiwi fruit after ozonation were analysed. Our research has shown that ozonation reduced the level of yeast and mould without affecting the content of soluble solids or acidity. In turn, ozonation clearly influenced the antioxidant activity and the redox status of the fruit. The ozonated fruit was characterised by a lower level of ROS due to the higher level of low molecular weight antioxidants, as well as the higher activity of superoxide dismutase and catalase. In addition, improved quality and antioxidant activity of the fruit were indirectly due to improved energy metabolism and NADPH level. The ozonated fruit showed a higher level of ATP, due to both higher activity of succinate dehydrogenase and higher availability of NADH. Moreover, the increased level of NAD+ and the activity of NAD+ kinase and glucose-6-phosphate dehydrogenase contributed to higher levels of NADPH in the fruit.

Self-coalescing flows in microfluidics for pulse-shaped delivery of reagents.

Microfluidic systems can deliver portable point-of-care diagnostics without the need for external equipment or specialist operators, by integrating all reagents and manipulations required for a particular assay in one device1. A key approach is to deposit picogram quantities of dried reagents in microchannels with micrometre precision using specialized inkjet plotters2-5. This means that reagents can be stored for long periods of time and reconstituted spontaneously when adding a liquid sample. But it is challenging to carry out complex operations using multiple reagents, because shear flow enhances their dispersion and they tend to accumulate at moving liquid fronts, resulting in poor spatiotemporal control over the concentration profile of the reconstituted reagents6. One solution is to limit the rate of release of reagents into the liquid7-10. However, this requires the fine-tuning of different reagents, conditions and targeted operations, and cannot readily produce the complex, time-dependent multireagent concentration pulses required for sophisticated on-chip assays. Here we report and characterize a capillary flow phenomenon that we term self-coalescence, which is seen when a confined liquid with a stretched air-liquid interface is forced to 'zip' back onto itself in a microfluidic channel, thereby allowing reagent reconstitution with minimal dispersion. We provide a comprehensive framework that captures the physical underpinning of this effect. We also fabricate scalable, compact and passive microfluidic structures-'self-coalescence modules', or SCMs-that exploit and control this phenomenon in order to dissolve dried reagent deposits in aqueous solutions with precise spatiotemporal control. We show that SCMs can reconstitute multiple reagents so that they either undergo local reactions or are sequentially delivered in a flow of liquid. SCMs are easily fabricated in different materials, readily configured to enable different reagent manipulations, and readily combined with other microfluidic technologies, so should prove useful for assays, diagnostics, high-throughput screening and other technologies requiring efficient preparation and manipulation of small volumes of complex solutions.

Comparison of glucose-6 phosphate dehydrogenase status by fluorescent spot test and rapid diagnostic test in Lao PDR and Cambodia.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy worldwide. Primaquine is the only licensed drug that effectively removes Plasmodium vivax hypnozoites from the human host and prevents relapse. While well tolerated by most recipients, primaquine can cause haemolysis in G6PD deficient individuals and is, therefore, underused. Rapid diagnostic tests (RDTs) could permit ascertainment of G6PD status outside of laboratory settings and hence safe treatment in remote areas. The performance of the fluorescent spot test (Trinity, Ireland; FST) and a G6PD RDT (Carestart, USA) against spectrophotometry were assessed. METHODS: Participants were enrolled during cross-sectional surveys in Laos and by purposive sampling in Cambodia. FST and RDT were performed during village surveys and 3 mL of venous blood was collected for subsequent G6PD measurement by spectrophotometry. RESULTS: A total of 757 participants were enrolled in Laos and 505 in Cambodia. FST and RDT performed best at 30% cut-off activity and performed significantly better in Laos than in Cambodia. When defining intermediate results as G6PD deficient, the FST had a sensitivity of 100% (95%CI 90-100) and specificity of 90% (95%CI 87.7-92.2) in Laos and sensitivity of 98% (94.1-99.6) and specificity of 71% (95%CI 66-76) in Cambodia (p < 0.001). The RDT had sensitivity and specificity of 100% (95%CI 90-100) and 99% (95%CI 97-99) in Laos and sensitivity and specificity of 91% (86-96) and 93% (90-95) in Cambodia (p < 0.001). The RDT performed significantly better (all p < 0.05) than the FST when intermediate FST results were defined as G6PD deficient. CONCLUSION: The interpretation of RDT results requires some training but is a good alternative to the FST. Trial registration clinicaltrials.gov; NCT01872702; 06/27/2013; https://clinicaltrials.gov/ct2/show/NCT01872702.

Antioxidant Action of an Aqueous Extract of Royal Sun Medicinal Mushroom, Agaricus brasiliensis (Agaricomycetes), in Rats with Adjuvant-Induced Arthritis.

The purpose of this study was to evaluate possible effects of the administration of an aqueous Agaricus brasiliensis extract on the oxidative state of the liver, brain, and plasma in adjuvant-induced arthritic rats, a model for human rheumatoid arthritis. Daily doses of 400 mg · kg-1 were administered by gavage for 23 days. This treatment produced significant diminutions in protein carbonylation and lipid damage in the liver, brain, and plasma. It also diminished the tissue reactive oxygen species and increased the antioxidant capacity of the plasma. Antioxidant defenses, which are diminished by arthritis, were improved by treatment with the A. brasiliensis extract, as revealed by preservation of the reduced glutathione and protein thiol levels and by the tendency of the activities of some antioxidant enzymes to normalize. The increased glucose-6-phosphate dehydrogenase activity was also almost normalized by the treatment. In addition, there were indications that A. brasiliensis can inhibit the initiation of structural events that can lead to hepatocyte necrosis. In conclusion, A. brasiliensis aqueous preparations can, in principle, be visualized as potential auxiliaries in the treatment of patients with rheumatoid arthritis as a result of their capacity to reduce oxidative stress. This effect was exerted in multiple organs, as can be judged from the results obtained in the liver, brain, and plasma. The continuous ingestion of A. brasiliensis as specific preparations or as a food supplement can possibly help to attenuate the systemic effects of rheumatoid arthritis and improve the quality of life of patients affected by this disease.

Ultra-performance liquid chromatography-tandem mass spectrometry-based multiplex enzyme assay for six enzymes associated with hereditary hemolytic anemia.

Deficiencies in erythrocyte metabolic enzymes are associated with hereditary hemolytic anemia. Here, we report the development of a novel multiplex enzyme assay for six major enzymes, namely glucose-6-phosphate dehydrogenase, pyruvate kinase, pyrimidine 5'-nucleotidase, hexokinase, triosephosphate isomerase, and adenosine deaminase, deficiencies in which are implicated in erythrocyte enzymopathies. To overcome the drawbacks of traditional spectrophotometric enzyme assays, the present assay was based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The products of the six enzymes were directly measured by using ion pairing UPLC-MS/MS, and the precision, linearity, ion suppression, optimal sample amounts, and incubation times were evaluated. Eighty-three normal individuals and 13 patients with suspected enzymopathy were analyzed. The UPLC running time was within 5min. No ion suppression was observed at the retention time for the products or internal standards. We selected an optimal dilution factor and incubation time for each enzyme system. The intra- and inter-assay imprecision values (CVs) were 2.5-12.1% and 2.9-14.3%, respectively. The linearity of each system was good, with R2 values >0.97. Patient samples showed consistently lower enzyme activities than those from normal individuals. The present ion paring UPLC-MS/MS assay enables facile and reproducible multiplex evaluation of the activity of enzymes implicated in enzymopathy-associated hemolytic anemia.

Waterborne cadmium and nickel impact oxidative stress responses and retinoid metabolism in yellow perch.

In this experiment, we studied the transcriptional and functional (enzymatic) responses of yellow perch (Perca flavescens) to metal stress, with a focus on oxidative stress and vitamin A metabolism. Juvenile yellow perch were exposed to two environmentally relevant concentrations of waterborne cadmium (Cd) and nickel (Ni) for a period of 6 weeks. Kidney Cd and Ni bioaccumulation significantly increased with increasing metal exposure. The major retinoid metabolites analyzed in liver and muscle decreased with metal exposure except at high Cd exposure where no variation was reported in liver. A decrease in free plasma dehydroretinol was also observed with metal exposure. In the liver of Cd-exposed fish, both epidermal retinol dehydrogenase 2 transcription level and corresponding enzyme activities retinyl ester hydrolase and lecithin dehydroretinyl acyl transferase increased. In contrast, muscle epidermal retinol dehydrogenase 2 transcription level decreased with Cd exposure. Among antioxidant defences, liver transcription levels of catalase, microsomal glutathione-S-transferase-3 and glucose-6-phosphate dehydrogenase were generally enhanced in Cd-exposed fish and this up-regulation was accompanied by an increase in the activities of corresponding enzymes, except for microsomal glutathione-S-transferase. No consistent pattern in antioxidant defence responses was observed between molecular and biochemical response when fish were exposed to Ni, suggesting a non-synchronous response of antioxidant defence in fish exposed to waterborne Ni. There was a general lack of consistency between muscle transcription level and enzyme activities analyzed. The overall findings from this investigation highlight the usefulness of transcriptional and biochemical endpoints in the identification of oxidative stress and vitamin A metabolism impairment biomarkers and the potential use of multi-level biological approaches when assessing environmental risk in fish.

Platelet enzyme abnormalities in leukemias.

AIM OF THE STUDY: The aim of this study was to evaluate platelet enzyme activity in cases of leukemia. MATERIALS AND METHODS: Platelet enzymes glucose-6-phosphate dehydrogenase (G6PD), pyruvate kinase (PK) and hexokinase (HK) were studied in 47 patients of acute and chronic leukemia patients, 16 patients with acute myeloid leukemia (AML)(13 relapse, three in remission), 12 patients with acute lymphocytic leukemia (ALL) (five in relapse, seven in remission), 19 patients with chronic myeloid leukemia (CML). RESULTS: The platelet G6PD activity was significantly low in cases of AML, ALL and also in CML. G6PD activity was normalized during AML remission. G6PD activity, although persistently low during ALL remission, increased significantly to near-normal during remission (P < 0.05) as compared with relapse (P < 0.01). Platelet PK activity was high during AML relapse (P < 0.05), which was normalized during remission. Platelet HK however was found to be decreased during all remission (P < 0.05). There was a significant positive correlation between G6PD and PK in cases of AML (P < 0.001) but not in ALL and CML. G6PD activity did not correlate with HK activity in any of the leukemic groups. A significant positive correlation was however seen between PK and HK activity in cases of ALL remission (P < 0.01) and CML (P < 0.05). CONCLUSIONS: Both red cell and platelet enzymes were studied in 36 leukemic patients and there was no statistically significant correlation between red cell and platelet enzymes. Platelet enzyme defect in leukemias suggests the inherent abnormality in megakaryopoiesis and would explain the functional platelet defects in leukemias.

Pentose phosphate pathway flux analysis for glycopeptide antibiotic vancomycin production during glucose-limited cultivation of Amycolatopsis orientalis.

In vivo pentose phosphate pathway (PPP) enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and transaldolase (TAL) activities as well as ATP- and ADP-level variations of Amycolatopsis orientalis were investigated with respect to glucose concentration and incubation period. G6PDH, 6PGDH, and TAL activities of A. orientalis reached maximum levels at 48 hr for all glucose concentrations used, after which the levels began to decline. G6PDH, 6PGDH, and TAL activities showed positive correlation with the glucose concentration up to 15 g/L, while further increases had an opposite effect. Intracellular ATP level showed a positive correlation with glucose concentrations, while ADP level increased up to 15 g/L. ATP concentration of A. orientalis increased rapidly at 48 hr of incubation, as was the case also for G6PDH, 6PGDH, and TAL activities, although the incubation period corresponding to maximum values of ADP shifted to 60 hr. Production of the glycopeptide antibiotic vancomycin increased with the increases in glucose concentrations up to 15 g/L, by showing coherence in the rates of oxidative and nonoxidative parts of the PPP.

The efficiacy of bismuth subnitrate against genotoxicity and oxidative stress induced by aluminum sulphate.

Aluminum (Al) is commonly used in industrial processes and drugs and is thought to induce erythrocytes damage via activation of oxidative stress. Recently, bismuth (Bi)-containing drugs are used in the treatment of various diseases. However, uncertain effects of Bi in blood tissue may participate in the therapeutic efficacy of Bi compounds as related to metals. Hence, this study aimed to determine the roles on human blood cells of the various concentrations of aluminum sulphate (Al(2)(SO(4))(3)) and bismuth subnitrate (BSN), separate and together. With this aim, oxidative status was assessed on erythrocytes by measuring following oxidative stress markers: reduced glutathione (GSH), superoxide dismutase (SOD), glucose-6-phosphate dehydrogenase (G-6-PDH) and catalase (CAT). Two chemicals were tested for their ability to induce cytogenetic change in human lymphocytes using assays for chromosome aberrations (CAs) and sister chromatid exchanges (SCEs). Our results showed that high dose of Al(2)(SO(4))(3) (20 µg/mL) caused oxidative stress and increased CA and SCE frequencies. Whereas, BSN doses did not change CA and SCE rates. Moreover, it led to changes of antioxidant capacity at different concentrations. After concomitant treatment with Al(2)(SO(4))(3) and BSN, the effects of BSN doses were different on enzyme activities and decreased the genotoxic damage. However, the high dose of BSN and Al(2)(SO(4))(3) was shown to enhance the frequencies of CAs and SCEs in a synergistic manner. In conclusion, BSN could be effective in the protection against the blood toxicity of Al(2)(SO(4))(3).

[Glutathione-dependent enzymes and glucose-6-phosphate dehydrogenase in breast tumors].

The authors made a comparative analysis of the activity of antioxidative enzymes and a search for a possible relationship between the activity of glutathione reductase and that of glucose-6-phosphate dehydrogenase in the blood and saliva of patients with breast tumors with varying activity of the pathological process.

[A new reagent for methemoglobin reduction test].

OBJECTIVE: To develop a new stable and efficient reagent for methemoglobin reduction test. METHODS: The results of methemoglobin reduction test using the new reagent were compared with those by G6P/6PG ratio method and classic methemoglobin reduction test. RESULTS: The new reagent was stable for at least 6 months at room temperature and 12 months at 2-8 degrees celsius;. The results of the test using this new reagent were stable and reliable. CONCLUSION: The new reagent for methemoglobin reduction test allows easy operation with well reproducible results and can be used in clinical screening of glucose-6-phosphate dehydrogenase deficiency.

Assessment of Doñana National Park contamination in Procambarus clarkii: integration of conventional biomarkers and proteomic approaches.

Aquatic ecosystems of Doñana National Park (DNP) were monitored using Procambarus clarkii as bioindicator in four campaigns carried out between 2003 and 2004 to assess environmental quality possibly threatened by agrochemicals used in nearby areas. An integrated approach was carried out, by combining the responses of well-established biomarkers and the massive analysis of biological effects at the proteomic level. In sites potentially polluted, lower catalase, glucose-6-P dehydrogenase, and esterase activities, and higher malondialdehyde, metallothionein and glutathione levels were found. Two-dimensional gel electrophoresis resolved >2500 gill spots, and image analysis detected that 35 showed significant intensity differences between the reference site and the other seven sites studied. The superiority of proteomic approaches was clearly recognized in our study since four different protein expression patterns were established based in the fold-number of up-/down-regulation of the 35 differentially expressed proteins. Sites located within Doñana Biological Reserve were essentially free of contaminants and those near the DNP limits were only slightly polluted. The higher proteomic responses found at the upper "Rocina" and "Partido" courses indicate that non-persistent agrochemicals are mainly used in Doñana surroundings. The highest responses corresponded to rice growing areas placed between the Guadiamar stream and the Guadalquivir River, according to the extended and intensive use of agrochemicals in such areas.

Changes in incidence and sex ratio of glucose-6-phosphate dehydrogenase deficiency by population drift in Taiwan.

We analyzed data from a single screening center in Taiwan from January 1, 1996 to December 31, 2005 to evaluate the change in incidence and female to male ratio of G6PD deficiency. During the study period, 1,211,632 of 2,667,922 (45.41%) neonates delivered in Taiwan were screened at the National Taiwan University Hospital. Of these, 21,997 neonates (1.82%) were confirmed to have G6PD deficiency. The annual incidence has decreased since 2002, from 1.94% to 1.61%. During this period, the male to female ratio in the screened population was 1.091 (range 1.073-1.098), the incidences in male and female neonates were 2.81% (2.57-3.07%), and 0.7% (0.45-0.95%), respectively. The change in sex ratio of the disease was unrelated to the change in incidence. During 2000-2005, 15-25% of newborns were born from newly immigrated females. G6PD deficiency screening has confirmed a subtle genetic flow in Taiwan. Besides the psychosocial effects, medical issues caused by population movements should be carefully watched in the future in Taiwan.

Protein partitioning in poly(ethylene glycol)/sodium polyacrylate aqueous two-phase systems.

The partition of hemoglobin, lysozyme and glucose-6-phosphate dehydrogenase (G6PDH) in a novel inexpensive aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The effect of NaCl and Na(2)SO(4), pH and PEG molecular size on the partitioning has been studied. At high pH (above 9), hemoglobin partitions strongly to the PEG-phase. Although some precipitation of hemoglobin occurs, high recovery values are obtained particularly for lysozyme and G6PDH. The partitioning forces are dominated by the hydrophobic and electrochemical (salt) effects, since the positively charged lysozyme and negatively charged G6PDH partitions to the non-charged PEG and the strongly negatively charged polyacrylate enriched phase, respectively.

Level of oxidative stress in the red blood cells of patients with liver cirrhosis.

BACKGROUND & OBJECTIVE: Liver cirrhosis is associated with gastrointestinal haemorrhage and oesophageal variceal bleeding. Altered platelet functions has been reported to be a cause of bleeding complication. We carried out this study to find out the level of oxidative stress in the red blood cells of patients with liver cirrhosis. METHODS: Fifty patients admitted with the complication of liver cirrhosis (with bleeding complications, n=30 and without bleeding complications, n=20) were included in the study. Age and sex matched normal healthy volunteers (n=45) served as controls. The levels of oxyhaemoglobin and methaemoglobin were assayed in the red blood cells. Oxidative stress markers such as lipid peroxides, lipid hydroperoxides and nitric oxide were determined along with enzymatic antioxidants. Membrane bound adenosine triphosphatases, cytosolic glucose-6-phosphate dehydrogenase and NADHmethaemoglobin reductase were also measured. The levels of cholesterol and total phospholipids were assessed in red blood cell membrane. The osmotic fragility of red blood cells was monitored using different concentrations of sodium chloride. RESULTS: The level of methaemoglobin was significantly higher (P < 0.001) in the red blood cells of liver cirrhotic patients with bleeding complication compared to that of non bleeding patients. The activity level of NADH-methaemoglobin reductase was significantly lower (P<0.001) compared to that of normal subjects. Levels of oxidative stress markers including nitric oxide were found to be higher in patients. The levels of enzymatic antioxidants were low except of glutathione peroxidase. The activity levels of adenosine triphosphatases were also found to be significantly lower (P<0.001) in patients compared to normal subjects. A significant alteration (P<0.05) was found in membrane cholesterol/phospholipid ratio of cirrhotic bleeders. Osmotic fragility of red blood cells was also altered in patients. INTERPRETATION & CONCLUSION: In cirrhotic condition red blood cells are subjected to severe oxidative stress with significant alterations in the membrane properties.

Catalase deficiency may complicate urate oxidase (rasburicase) therapy.

Patients with low (inherited and acquired) catalase activities who are treated with infusion of uric acid oxidase because they are at risk of tumour lysis syndrome may experience very high concentrations of hydrogen peroxide. They may suffer from methemoglobinaemia and haemolytic anaemia which may be attributed either to deficiency of glucose-6-phosphate dehydrogenase or to other unknown circumstances. Data have not been reported from catalase deficient patients who were treated with uric acid oxidase. It may be hypothesized that their decreased blood catalase could lead to the increased concentration of hydrogen peroxide which may cause haemolysis and formation of methemoglobin. Blood catalase activity should be measured for patients at risk of tumour lysis syndrome prior to uric acid oxidase treatment.

Effects of paraquat on anti-oxidant system in rats.

The toxic effects of paraquat on the anti-oxidant defense system of male albino rats were evaluated, after administering either a single dose (1.5 and 7.5 mg/kg of body weight) or continuous daily doses (same as above, i.e., 1.5 mg/kg and 7.5 mg/kg of body weight) for 3 and 7 days. Glutathione levels in blood cells, liver, lung and kidney tissues decreased in a dose and time dependent manner. Glutathione reductase and glucose-6-phosphate dehydrogenase activity decreased, whereas the activity of glutathione-S-transferase, glutathione peroxidase, catalase and superoxide dismutase increased in paraquat exposure. Malondialdehyde formation also increased in a dose and time dependent manner. The alterations of anti-oxidant system particularly glutathione can be utilized as biomarkers during management of paraquat poisoning.

[Impact of antioxidative therapy on the activity of salivary glutathione-dependent enzymes in patients with fluorosis].

Fluorosis caused by long-term intake of high fluoride levels is characterized by clinical bone and tooth manifestations. The adverse impact of high fluoride intake was also observed in soft tissues. Although fluorosis is irreversible it could be prevented by appropriate and timely interventions through the understanding of the process at biochemical and molecular levels. Increased production of reactive oxygen forms and lipid peroxidation are considered to play an important role in the pathogenesis of chronic fluoride toxicity. Saliva is a biological fluid of the human organism may be a reflection of the metabolic state. Salivary indices are clinical diagnostic indicators. The purpose of this investigation was to comparatively study the salivary antioxidative defense system, including glutathione, glutathione reductase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase in adult patients with fluorosis before and after complex antioxidative therapy. Analysis indicated that there was a negative correlation between the level of glutathione and the clinical characteristics of the disease in patients with fluorosis. There was a direct relationship between the activity of glutathione-S-transferase and the clinical manifestations in the patients. These results reflected dose-dependent fluoride intoxication and metabolic imbalance. The imbalanced salivary antioxidative defense system was in part corrected by complex antioxidative therapy.

RCE1 corneal epithelial cell line: its variability on phenotype expression and differential response to growth factors.

BACKGROUND: By serial transfer of rabbit corneal epithelial cells, the spontaneous RCE1 cell line was previously established. These cells mimic the stage-dependent differentiation of the corresponding cell type. METHODS: RCE1 cells were cultured either on plastic culture dishes or on collagen rafts to compare the epithelial ultrastructure after growth on these substrata. Phenotypic variability was studied after subcloning of cells. The differentiation ability of each subclone was determined by Western blot with antibodies against the differentiation-linked keratin pair K3/K12 and by measuring LDH activity and LDH isozymes in cytosolic extracts. The proliferative response of RCE1 cells to EGF, TGFalpha, amphiregulin, bFGF or IL-6 was determined under serum-free culture conditions. RESULTS: Cells grown on collagen rafts formed 5- to 7-layered epithelia with characteristics closer to those found in normal corneal epithelium than cells cultivated on plastic substrata, which formed 3- to 5-layered epithelia. Subcloning experiments demonstrated that every proliferative cell is able to grow and constitute stratified epithelia expressing K3/K12 keratins. LDH levels in RCE1 epithelia were similar to those of cultured or freshly harvested corneal epithelia; however, they showed a slightly altered LDH isozyme set, with prevalence of LDH-3 isoform. Whereas EGF and TGF-alpha were equipotent, amphiregulin elicited a 4-fold lower proliferative response. Also, bFGF was 10-fold less mitogenic than EGF, and IL-6 had the lowest effect with an ED(50) 20-fold lower than EGF. CONCLUSIONS: The results demonstrate that every RCE1 proliferative cell has the ability to generate epithelial sheets. We conclude that EGF and TGF-alpha are the major effectors of RCE1 cell proliferation.

Toxicological effects of the lipid regulator gemfibrozil in four aquatic systems.

Gemfibrozil is a lipid-regulating agent widely used in patients at risk of coronary disease. Pharmaceutical products, such as gemfibrozil, are found in municipal effluents and represent a major source of contamination. To date, there is little available information about the adverse effects of gemfibrozil in aquatic organisms. For this reason, the toxic effects were investigated using model systems from four trophic levels. The most sensitive system was the immobilization of Daphnia magna, with a non-observed adverse effect level of 30 microM and a mean effective concentration of 120 microM after 72 h, followed by the inhibition of bioluminescence of Vibrio fischeri, the hepatoma fish cell line PLHC-1 line and the inhibition of the growth of Chlorella vulgaris. Although protein content, neutral red uptake, methylthiazol metabolization and lysosomal function were reduced in PLHC-1 cells, stimulations were observed for lysosomal function, metallothionein levels and succinate dehydrogenase, glucose-6-phosphate dehydrogenase and acetylcholinesterase activities. No changes were observed in ethoxyresorufin-O-deethylase activity. The main morphological alterations were hydropic degeneration and loss of cells. Modulation studies on gemfibrozil toxicity were also carried out. General antioxidants and calcium chelators did not modify the toxicity of gemfibrozil, whereas a Fe(III) chelator, a membrane permeable sulphydryl-protecting compound and glutathione level modifying agents did change the toxicity. One of the possible mechanisms of gemfibrozil toxicity seems to be the binding to sulphydryl groups, including those of glutathione. According to the result, gemfibrozil should be classified as harmful to aquatic organisms. However, comparing the concentrations in water and the toxicity quantified in the assayed systems, gemfibrozil is not expected to represent acute risk to the aquatic biota.