Long-term effects of combined brilliant blue G and xenon light induced retinal toxicity following macular hole repair surgery.

BACKGROUND: The purpose of this study was to look at the long-term effects of retinal phototoxicity after macular hole repair surgery using xenon endolight illumination and Brilliant blue G (BBG) dye. CASE PRESENTATION: An elderly man in his late seventies underwent para plana vitrectomy with BBG dye to repair an idiopathic full-thickness macular hole (MH) in his right eye. Prior to macular hole surgery, his visual acuity in the right eye was 6/60, N24 at the time of presentation. The MH closed with type 1 closure immediately after surgery, but there was extensive damage to the outer retinal layers and retinal pigment epithelium (RPE) at the macula, resulting in a reduction in visual acuity to 2/60. We presumed that the combination of BBG and xenon light, is the probable reason of retinotoxicity in the current patient. There was a progressive increase in the area of retinal and RPE layer damage and choroidal thinning over a 4-year period. CONCLUSION: Due to combined BBG-induced dye and endoilluminator toxicity, a rare case of continuously progressing RPE layer damage with choroidal thinning over a long follow-up interval was described. Such long-term effects of BBG and endolight induced retinotoxicity have not been reported in the literature, to the best of our knowledge.

Brilliant Blue G toxicity in macular hole surgeries: A report on combined phototoxicity and dye-induced macular damage.

PURPOSE: Vitrectomy with brilliant blue G (BBG) assisted internal limiting membrane (ILM) peeling is the standard operational technique in macular hole surgeries. However, BBG dye, though considered safe and nontoxic, can also occasionally lead to macular toxicity. This study aims to describe the clinical features and characteristics of four eyes who developed macular toxicity after following surgery for macular hole repair. METHODS: Retrospective review of four consecutive cases of macular toxicity after conventional BBG assisted ILM peeling. All the cases reviewed, their operative surgical notes were retrieved and analyzed. The ILM was stained twice during surgery with prolonged intraoperative surgical time. RESULTS: All four cases had a prolonged surgical time and the ILM was stained twice during surgery in all cases. The area of macular toxicity was corresponding to the area of ILM peeling which had been exposed to repeated staining by BBG dye. By the end of one month, all four cases had foveal thinning along with choriocapillary atrophy. The mean BCVA was 20/80 before surgery and the final mean visual acuity was <20/800. CONCLUSION: This report highlights the occurrence of macular and choriocapillary atrophy due to prolonged focal endoillumination and the increased risk of toxicity with repeated dye staining.

Oxidative Stress and Toxico-Pathic Branchial Lesions in Cyprinus carpio Exposed to Malachite Green.

Gill is the frontier tissue to come in direct contact with aquatic toxicants. Malachite green (MG) commercial textile dye was assessed for its impact on the gill cytoarchitecture. Cyprinus carpio were exposed to 0.087 and 0.146 mg/L of MG for 60 days. The tissue was processed, and HE stained slides revealed histo-pathic lesions such as lamellar curling, edema, necrosis, telangiectasia, aneurysm, and vacuolization. Scanning electron microscopy reported aberrations in lamellae and microridges of the epithelium. At the cellular level, transmission electron microscopy exhibited nuclear alterations in form of pyknosis and mitochondrial swelling followed by cristolysis. Pillar cells displayed cytoplasmic vacuolization and leukocyte infiltration, and goblet cell containing varied shaped and density mucous globules. The biochemical analysis supported the ultrastructural alterations and showed a negative impact of MG on the antioxidative enzymes (CAT, SOD, GSH), while levels of MDA were found to be significantly elevated. Thereby, concluding MG induced branchial toxicity in the fish.

Heterologous expression of bacterial CotA-laccase, characterization and its application for biodegradation of malachite green.

Malachite green (MG) is used as fungicide/parasiticide in aquaculture, its persistence is detrimental as it exhibits carcinogenic effects to aquatic organisms. Bacterial laccase evaluated as the best enzyme at extreme condition for aquatic MG removal. Study aims to increase laccase concentration, CotA-laccase from Bacillus subtilis was cloned and overexpressed in Escherichia coli. Optimal catalysis for purified CotA-laccase were at pH 5.0, 60 °C, and 1 mM of (2,2-azino-di-[3-ethylbenzothiazoline-sulphonate-(6)]) with Km and Kcat 0.087 mM and 37.64 S-1 respectively. MG biodegradation by CotA-laccase in clam and tilapia pond wastewaters and cytotoxic effect of biodegraded products in grouper fin-1 cells were determined. MG degradation by CotA-laccase was equally efficient, exhibiting upto 90-94% decolorization at freshwater and saline conditions and treated solution was non-toxic to GF-1 cells. Thus, recombinant-CotA-laccase could be an environmentally-friendly enzyme for aquaculture to remove MG, thereby effective to reduce its accumulation in aquatic organisms and ensuring safe aquaculture products.

Comparative effects of commonly used intraocular dyes on the viability of human retina Müller cells.

This study investigated the in vitro effect of various vital dyes in common clinical use on human Müller cell viability, and it compared the toxicity of these dyes using a cell culture model. Müller cells were exposed to a series of concentrations (1 %, 0.5 %, 0.25 %, and 0.125 % or 12.9 mM, 6.45 mM, 3.22 mM and 1.61 mM) of Indocyanine green (ICG) for 2, 24, 48, and 72 h. Similarly, groups of Müller cells were stained with "Heavy" brilliant blue G (HBBG), Trypan blue (TB) (0.15 %, or 1.56 mM), Membrane-blue-dual (MBD), and ICG (0.25 %, or 3.22 mM) or BBG (0.025 %, or 0.3 mM) with glucose (GS) (50 %, 66 % and 75 % or 2.78 M, 3.67 M and 4.17 M) for 30, 60, and 120 s. Cell viability was measured with the Cell Counting Kit-8 (CCK-8) and Lactate Dehydrogenase (LDH) release assays. We found that high stain concentration and long exposure time resulted in increased toxicity to Müller cells. Nevertheless, ICG seemed to be safe at the clinically relevant concentration of 0.25 % (3.22 mM) in the short time of exposure. TB was safer than both HBBG and MBD, especially HBBG. Hypertonic GS as a dilution was not safe for Müller cells, and the negative effect was more obvious in 0.025 % (0.3 mM) BBG than that in 0.25 % (3.22 mM) ICG. This is the first report to observe the cytotoxicity of commonly used stains in clinical on human Müller cells in vitro, and to provide some basis for further studies, including in vivo investigation.

pH-triggered solubility and cytotoxicity changes of malachite green derivatives incorporated in liposomes for killing cancer cells.

Three different malachite green leuco derivatives (MG-Xs) are incorporated in liposomes. In all three cases, a substituent (X) is covalently linked to the central carbon atom, abbreviated as MG-OH, MG-OCH3, and MG-CN. The three MG-X compounds are solubilized separately in liposome membranes and become cationic (MG+) and water soluble under acidic conditions. MG+ is consequently released from the liposome to the aqueous exterior. Their release behavior corresponds to their ionization ability: MG-OH > MG-OCH3 > MG-CN. The cellular uptake of the liposomes, the cytotoxic effect, and the location of MG+ in cancer cells are investigated using murine cells derived from colon cancer (Colon 26 cells) and human embryonic kidney cells (HEK 293 cells). The toxic effect on cancer cells is correlated to the ionization ability of MG-Xs. The liposomes effectively deliver MG+via the endocytic pathway, resulting in the cytotoxicity of liposomes containing MG-OH which is higher than that of free MG-OH and MG+. The difference in the phospholipids constituting the liposome membranes barely had an effect on the ionization ratio and the cytotoxicity of MG-OH. Confocal fluorescence microscopic observations revealed that MG+ is ultimately transported into the nuclei after being released in acidic cellular compartments.

Bioprocessing strategies for cost-effective simultaneous removal of chromium and malachite green by marine alga Enteromorpha intestinalis.

A large number of industries use heavy metal cations to fix dyes in fabrication processes. Malachite green (MG) is used in many factories and in aquaculture production to treat parasites, and it has genotoxic and carcinogenic effects. Chromium is used to fix the dyes and it is a global toxic heavy metal. Face centered central composite design (FCCCD) has been used to determine the most significant factors for enhanced simultaneous removal of MG and chromium ions from aqueous solutions using marine green alga Enteromorpha intestinalis biomass collected from Jeddah beach. The dry biomass of E. intestinalis samples were also examined using SEM and FTIR before and after MG and chromium biosoptions. The predicted results indicated that 4.3 g/L E. intestinalis biomass was simultaneously removed 99.63% of MG and 93.38% of chromium from aqueous solution using a MG concentration of 7.97 mg/L, the chromium concentration of 192.45 mg/L, pH 9.92, the contact time was 38.5 min with an agitation of 200 rpm. FTIR and SEM proved the change in characteristics of algal biomass after treatments. The dry biomass of E. intestinalis has the capacity to remove MG and chromium from aquatic effluents in a feasible and efficient manner.

Health risk assessment of toxicologically relevant residues in emerging countries: A pilot study on Malachite Green residues in farmed freshwater fish of Armenia.

Malachite Green (MG) has a worldwide application in aquaculture as a therapeutic agent; however, its use in food producing animals is illegal, due to potential carcinogenicity and persistence of residues. This pilot study, the first conducted in Armenia, aimed to determine the concentration of MG residues in flesh of fish grown in artificial ponds of Armenia and conduct dietary exposure assessment to characterize possible health risks to consumers. Detection of MG residues, including the major metabolite leucomalachite, was carried out in 29 fish composite samples by ELISA. The results were confirmed by LC-MS/MS. To determine fish consumption values, a food frequency questionnaire was used. Possible health risks were evaluated by calculating the Margin of Exposure (MOE) based on BMDL of 13 (neoplastic effects) and 6 (non-neoplastic effects) mg/kg bw. In 34.5% of the investigated fish samples MG residues exceeded the minimum required performance limit. For BMDL10 and BMDL0.5, the MOEs ranged 3.36E+06-3.37E+07 and 1.55E+06-1.55E+07, respectively. The MOE for neoplastic effects was more than 10,000 and for non-neoplastic effects was more than 100. The results do not indicate public health concerns. However, the results highlight issues concerning the illegal use of MG in Armenian aquaculture, which deserves further attention.

Microbial degradation, spectral analysis and toxicological assessment of malachite green by Streptomyces chrestomyceticus S20.

Malachite green (MG), a triphenylmethane dye is extensively used for coloring silk, aquaculture and textile industries, it has also has been reported toxic to life forms. This study aimed to investigate the biodegradation potential of MG by actinobacteria. The potent actinobacterial strain S20 used in this study was isolated from forest soil (Sabarimala, Kerala, India) and identified as Streptomyces chrestomyceticus based on phenotype and molecular features. Strain S20 degraded MG up to 59.65 ± 0.68% was studied in MSM medium and MG (300 mg l-1) and degradation was increased (90-99%) by additions of 1% glucose and yeast extract into the medium at pH 7. The treated metabolites from MG by S20 characterized by FT-IR and GC-MS. The results showed MG has been degraded into nontoxic compounds evaluated by (1) phytotoxic assay on Vigna radiata, (2) microbial toxicity on Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Streptococcus sp. and Escherichia coli, (3) cytotoxicity assay in a human cell line (MCF 7). The toxicity studies demonstrated that the byproducts from MG degradation by S. chrestomyceticus S20 were no toxic to plants and microbes and less toxic to human cells as compared to the parent MG. Perhaps this is the first work reported on biodegradation of MG by S. chrestomyceticus which could be a potential candidate for the removal of MG from various environments.

Extracellular expression of mutant CotA-laccase SF in Escherichia coli and its degradation of malachite green.

In this study, mutant CotA-laccase SF was successfully expressed in Escherichia coli by co-expression with phospholipase C. The optimized extracellular expression of CotA-laccase SF was 1257.22 U/L. Extracellularly expressed CotA-laccase SF exhibits enzymatic properties similar to intracellular CotA-laccase SF. CotA-laccase SF could decolorize malachite green (MG) under neutral and alkaline conditions. The Km and kcat values of CotA-laccase SF to MG were 39.6 mM and 18.36 s-1. LC-MS analysis of degradation products showed that MG was finally transformed into 4-aminobenzophenone and 4-aminophenol by CotA-laccase. The toxicity experiment of garlic root tip cell showed that the toxicity of MG metabolites decreased. In summary, CotA-laccase SF had a good application prospect for degrading malachite green.

Sandalwood-derived carbon quantum dots as bioimaging tools to investigate the toxicological effects of malachite green in model organisms.

Malachite green is an N-methylated diaminophenylmethane dye that has generated much concern over its suggestive carcinogenic nature. After its excessive use in aquaculture industry as an effective ectoparasitide, much debate was raised over its toxicological effects leading to scientific studies conducted on animal models. Even after several bans, malachite green is still easily available in many parts of the world and unscrupulously even used to give green vegetables a fresher look. This study aims to address this concern by systematically studying the toxicological effects of malachite green through bioimaging in plant and animal cell and tissue. Sandalwood-derived carbon quantum dots have been used as a bioimaging tool since they are non-cytotoxic and show excellent fluorescence properties. Onion tissues demonstrate the translocation of the dye inside cells having high affinity for the nuclei and cell walls. Toxicological effects on the growth of Vigna radiata (mung beans) have been studied methodically. Bioimaging of the transverse cross-section of the dye-treated plant root shows a significant difference from the control. In animal cells, dose-dependent decrease in cell viability of MG-63 cells was observed with MG. CQD showed good fluorescence in both cytoplasm and nucleus of MG63 cells. In addition, CQDs were employed as a great tool for bioimaging of the histopathologically adverse effects of MG in Golden hamster animal model. This study showed CQDs could be used as an alternative non-site specific fluorescent probe for cell and tissue imaging for better visualization of cell and tissue architectural changes.

The gut microbiota: a new perspective on the toxicity of malachite green (MG).

Gut microbiome critically contributes to host health status. Thus, investigating the relationship between the gut microbiome and toxic chemicals is a hot topic in toxicology research. Exposure to malachite green (MG) has been linked to various health disorders. Thus, exploring the gut microbiota changes in response to MG would provide a new perspective on the toxicity effects of this chemical substance. MG exposure resulted in the significantly lower alpha diversity (Mann-Whitney U test, z = - 6.83, p = 0.00) but higher beta diversity (Mann-Whitney U test, z = - 1.98, p = 0.04) of gut microbiota, and significantly decreased ecosystem stability (alpha and beta variability; Mann-Whitney U test, all p < 0.05) of gut microbial communities. Gut bacterial networks showed that the interactions became more complex and stronger after MG exposure, which could decrease the stability of the network. Changes in gut microbiota composition were mainly reflected in the enrichment of opportunistic bacteria (i.e., Aeromonas and Vibrio) and the depression of fermentative bacteria (i.e., Bacteroides and Paludibacter). MG exposure leads to a significantly increased gut permeability (lipopolysaccharide-binding protein; Mann-Whitney U test, z = - 6.92, p = 0.00), which could reduce the host selective pressures on particular bacterial species (such as members in Aeromonas and Vibrio). This result was further supported by the weakened importance of a deterministic microbial assembly after MG exposure. All these findings indicated that MG exposed fishes might have more possibilities to be infected, as demonstrated by the enrichment of opportunistic pathogenic bacteria, high-level immune responses, and increased gut permeability. These findings greatly improve our understanding of the toxicity effects of MG.

Evaluation of Patent Blue as the Vital Dye in an Animal Model of Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To evaluate the safety and feasibility of patent blue (PB) as the vital dye in Descemet membrane endothelial keratoplasty (DMEK). METHODS: Bovine corneal endothelial cells were incubated with different concentrations (0.02%-2.5%) of PB. The cell viability, which was assessed by Cell Counting Kit-8 assay, was compared with that of untreated control and 0.06% to 0.4% trypan blue. The dyes were also used for graft preparation and implantation in the porcine eye model to evaluate stain quality, dye retention, and the feasibility of using PB in DMEK surgery. RESULTS: No obvious increase in cytotoxicity was detected for 0.06% to 0.4% trypan blue and PB at concentrations up to 1.0%, but the cell viability after incubating with 1.5% to 2.5% PB was significantly reduced. PB at 0.5% to 1.0% generated good staining quality that can be used to facilitate graft implantation. Although the staining quality of 0.5% to 1.0% PB faded to an intermediate level after a 30-minute wash in phosphate-buffered saline, dye retention persisted for up to 24 hours. CONCLUSIONS: PB at 0.5% to 1.0% is biocompatible and can stain the graft sufficiently, making it an alternative for DMEK surgery.

Malachite Green Induced Ultrastructural Corneal Lesions in Cyprinus carpio and Its Amelioration Using Emblica officinalis.

Malachite green, a multi-purpose dye induces cyto-toxicity upon its entry and bioaccumulation in tissues. A semi-static chronic (60 days) bioassay was conducted by exposing Cyprinus carpio to sublethal concentration of the dye and Emblica officinalis in four experimental groups viz control, malachite green, E. officinalis, and malachite green + E. officinalis. Effect of dye on the cornea was investigated considering ultra-structural alterations owing to its direct contact to the pollutant in the aquatic medium. SEM studies on corneal epithelium revealed broken continuity of pavement cells, shrunk microplicae, increased intra-microplicae distance, globularization and epithelial uplifting, thereby affecting the integrity of corneal surface and tear film adherence. Whereas dietary supplementation with the plant extract served to restore cytoarchitecture with appearance of large number of regenerating cells. Both lesions and restoration were found to be duration dependent. Thus, E. officinalis can be considered as an effective ameliorant against malachite green induced toxicity.

Chebulinic acid and Boeravinone B act as anti-aging and anti-apoptosis phyto-molecules during oxidative stress.

INTRODUCTION: Aquatic pollutant Malachite green (MG) induces oxidative stress by producing intracellular H2O2 and associated hydroxyl, hydroxymethyl or hydroperoxide radicals in Saccharomyces cerevisiae. These radicals disturb cellular functions leading to early aging. Exogenous supply of natural antioxidants may play a crucial role as anti-aging by ensuring the cellular survival. METHODS: Protective effect of Chebulinic acid (CA) and Boeravinone B (BB) was biochemically evaluated by measuring the expression levels of antioxidant enzymes. Intracellular oxidants generation, nuclear damage, necrosis, apoptosis, reduction in caspase 3/7 activity studied microscopically, spectrofluorometrically and biochemically along with growth dynamics and relative quantitation of Yap1, Sir2 and Bir1 expression using RT-PCR. RESULTS: Malachite green (MG) showed adverse effect on S. cerevisiae showing 400.83% enhancement in accumulation of intracellular H2O2 and associated hydroxyl, hydroxymethyl or hydroperoxide radicals. Independent supplementation of CA (5 µg/ml) and BB (3 µg/ml) significantly reduced the accumulation by 385.78 and 372.68%, respectively. Presence of MG extended the lag phase of growth curve and also reduced colony forming units (CFUs)/ml to 3 × 108 from 15 × 108. Whereas, CA and BB maintained the normal growth curve, CFUs and proved as anti-aging. Elevation in the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) by 241.35, 539.02 and 432.60% was observed after 2 h MG exposure. However, CA and BB significantly reduced the CAT, SOD and GPx activities. Microscopic observation of CA and BB augmented cells revealed protection from H2O2 and associated hydroxyl, hydroxymethyl or hydroperoxide radicals accumulation, nuclear disorganization, morphological distortion, apoptosis and necrosis contrary to MG exposed cells. An enhancement of 112.78% in caspase 3/7 activity was noted in MG exposed cells over control. Both CA and BB supplementation reduced the caspase 3/7 activity by 106.06 and 105.82%, respectively which was almost near normal. MG was found to induce the expression of yeast transcription factor Yap1; while presence of CA and BB restored expression of Yap1. Expression of longevity responsible gene Silent Information Regulator (Sir2) was also found to be reduced during MG exposure. However, CA and BB triggered the expression of Sir2. Similarly, MG lowered the expression of Baculoviral IAP repeat (Bir1) which is the inhibitor of apoptosis while CA and BB aided the over expression of Bir1. CONCLUSIONS: CA and BB supplementation could significantly decrease oxidative stress, enhance cell viability and ultimately protected S. cerevisiae cells form aging.

Foetal bovine serum can reduce toxicity of indocyanine green, brilliant blue G and trypan blue in ARPE-19 cellular model that suggests new surgical staining protocols for internal limiting membrane peeling procedure.

BACKGROUND: To investigate and compare the cytotoxicity of indocyanine green (ICG), brilliant blue G (BBG) and trypan blue (TB) using ARPE-19 cells that have been pre-treated/post-treated with balanced salt solution (BSS) or foetal bovine serum (FBS). METHODS: The cultured human retina pigment epithelium ARPE-19 cells were pre-treated/post-treated with BSS or FBS (represent the autologous serum in clinic) in parallel with cells being soaked with various concentrations of ICG, BBG and TB. The cells were then assessed for viability, growth rate, reactive oxygen species (ROS) level, mitochondrial membrane potential (Δψ) and mitochondrial mass as cytotoxic indices. For the FBS pre-treated cells, only ROS was examined. RESULTS: Using the MTT assay, cytotoxicity seemed to appear when the dye concentration was above 2.5 mg/mL for ICG but no cytotoxicity for BBG and TB at the concentrations used. Cell growth was arrested at a concentration 1 mg/mL when ICG or BBG were present but no arrest at any of the tested concentrations was found for TB with the cell-growth curve was slowest for ICG. Cellular ROS levels increased at all concentrations of all dyes, but the increasing slopes were decreased after FBS post-treatment washout. CONCLUSIONS: As a rinse buffer FBS performs much better than BSS in terms of cell rescue, which agrees with a clinical report when autologous whole blood was applied to macular hole surgery. However, FBS pre-treatment seems to be much better than FBS use as washout buffer in post-treatment.

Response surface methodology optimization for sorption of malachite green dye on sugarcane bagasse biochar and evaluating the residual dye for phyto and cytogenotoxicity.

In the present study, sorption and detoxification of malachite green (MG) dye was executed using biochar resulting after pyrolysis of agro-industrial waste at 400, 600 and 800 °C. Maximum sorption of MG dye (3000 mg/L) was observed on the sugarcane bagasse biochar (SCB) prepared at 800 °C. The interactive effects of different factors like dye concentration, time, pH and temperature on sorption of MG dye were investigated using response surface methodology (RSM). Optimum MG dye concentration, contact time, temperature and pH predicted through Box-Behnken based RSM model were 3000 mg/L MG dye, 51.89 min, 60 °C and 7.5, respectively. ANOVA analysis displayed the non-significant lack of fit value (0.4566), whereas, the predicted correlation coefficient values (R2 0.8494) were reasonably in agreement with the adjusted value (R2 0.9363) demonstrating highly significant model for MG dye sorption. The applicability of this model was also checked through F- test (30.39) with lower probability (0.0001) value. Furthermore, the characterization of SCB was performed using fourier transform infra-red spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM), Brunauer-Emmett-Teller surfaces (BET), total organic carbon (TOC) and atomic absorption spectroscopy (AAS). Phyto-toxicity and cytogenotoxicity studies showed successful removal of MG dye using SCB. In addition, the batch sorption studies for reutilization of SCB revealed that the SCB was effective in removal of MG for five repeated cycles. This technology would be effective for treating the toxic textile effluent released from the textile industries.

Adsorption of malachite green from aqueous solution by using novel chitosan ionic liquid beads.

Chitosan ionic liquid beads were prepared from chitosan and 1-butyl-3-methylimidazolium based ionic liquids to remove Malachite Green (MG) from aqueous solutions. Batch adsorption experiments were carried out as a function of initial pH, adsorbent dosage, agitation time and initial MG concentration. The optimum conditions were obtained at pH 4.0, 0.008g of adsorbent dosage and 20min of agitation time were utilized in the kinetic and isotherm studies. Three kinetic models were applied to analyze the kinetic data and pseudo-second order was found to be the best fitted model with R2>0.999. In order to determine the adsorption capacity, the sorption data were analyzed using the linear form of Langmuir, Freundlich and Temkin equations. The isotherm was best fitted by Langmuir isotherm model. The maximum adsorption capacity (qmax) obtained from Langmuir isotherm for two chitosan beads 1-butyl-3-methylimidazolium acetate A and 1-butyl-3-methylimidazolium B are 8.07mgg-1 and 0.24mgg-1 respectively.

In vitro interactions of malachite green and leucomalachite green with hepatic drug-metabolizing enzyme systems in the rainbow trout (Onchorhyncus mykiss).

Malachite green (MG) has been widely used in aquaculture to treat a number of microbial and parasitic diseases. It is currently banned in the EU because of the high cytotoxicity and carcinogenic activity, which is also shared by leucomalachite green (LMG), a reduced MG metabolite that can persist in fish tissues for months. There is scant information about the ability of either compound to interact with drug metabolizing enzymes in fish. Therefore we evaluated the in vitro effects of MG and LMG (25, 50 and 100µM) on some DMEs and glutathione (GSH) content in rainbow trout liver subfractions. LMG did not affect any of the examined parameters. In contrast, MG proved to deplete GSH and to depress to a various extent the activities of NAD(P)H cytochrome c reductase, 7-ethoxycoumarin O-deethylase, 1-naphthol uridindiphosphoglucuronyl-transferase and maximally those of 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) accepting 1-chloro2,4-dinitrobenzene (CDNB) as substrate. The inhibition mechanisms of EROD and GST were investigated by means of non-linear Michaelis-Menten kinetics and Lineweaver-Burk plots using 0.175-8µM MG. The calculated IC50 for EROD was 7.1µM, and the inhibition appeared to be competitive (Ki 2.78±0.24µM). In the case of GST, the calculated IC50 was 0.53µM. The inhibition was best described as competitive toward GSH (Ki 0.39±0.02µM) and of mixed-type toward CDNB (Ki 0.64±0.06µM). Our findings indicate that, contrary to LMG, MG behaves as a relatively strong inhibitor of certain liver DMEs and can reversibly bind GSH.