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The interest in the fate of pharmacologically active substances (PASs) in the aquatic environment continually increases. However, little is known about pharmacologically active dyes (PADs) as contaminants of water bodies. PADs are used in medicine, but due to their colouring properties are also applied in the textile, cosmetic and food industries. Their large-scale production and widespread applications have caused these dyes permeate to the aquatic environment. The pharmacological activity and toxicological properties of some of these dyes, caused their occurrence in water should be monitored. Up to now, PADs such as crystal violet, malachite green, methylene blue, rhodamine B, have been determined in the water of Greater China and Iran. However, there is no data on whether PADs pose an environmental problem for water bodies in Poland. Thus, different water samples were collected and analysed by the UPLC-MS/MS method allowing the determination of 20 PADs. The tests showed that dyes such as crystal violet, methyl violet 2 B and rhodamine B were found in 2 out of 36 water reservoirs (0.0122-0.0594 µgL-1). The environmental risk assessment indicated that determined dyes for most model organisms did not pose a risk. Only the presence of methyl violet 2 B (0.0571 µgL-1) was related to a low risk for rohu carp, and crystal violet (0.0122-0.0209 µgL-1) showed a moderate risk for medaka fish. The occurrence of PADs was tested on a larger scale in the water samples collected from different water reservoirs in Poland. Based on obtained results, 96.3% of water samples collected from different water bodies (94.5%) were free from dyes. Thus, it could be stated that generally environmental water of Poland is contaminated with PADs at a low level. On the other hand, the presence of dyes in two samples indicates that PADs permeate the water environment, and their occurrence should be monitored.

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In the literature, there is a lack of data on the effect of gentian violet (GV) and congo red (CR) dyes on the biosynthesis pathway of biogenic amines (BAs) in Lemna minor L. (common duckweed). This plant species is an important link in the food chain. Both dyes inhibited growth, biomass yield and the biosynthesis of chlorophyll a in common duckweed. The predicted toxic units demonstrated that GV had a more toxic effect on the growth rate and biomass yield of common duckweed than CR. Decarboxylase activity in the biosynthesis of BAs in common duckweed is also a useful indicator for evaluating the toxicity of both dyes. Gentian violet also exerted more phytotoxic effects on the analyzed biochemical features of common duckweed because it changed the putrescine (Put) biosynthesis pathway, increased tyramine content 1.6 fold, inhibited the activity of S-adenosylmethionine decarboxylase by 40% and the activity of ornithine decarboxylase (ODC) by 80%. Tyrosine decarboxylase (TDC) was most active in plants exposed to the highest concentration of GV. Similarly to control plants, in common duckweed exposed to CR, Put was synthesized from ornithine; however, spermidine content was 86% higher, Put content was 51% lower, and ODC activity was 86% lower.

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OBJECTIVE: Markers containing dyes such as crystal violet (CAS 548-62-9) are routinely used on the adventitia of vein bypass grafts to avoid twisting during placement. Because little is known about how these dyes affect vein graft healing and function, we determined the effect of crystal violet on cell migration and proliferation, which are responses to injury after grafting. METHODS: Fresh human saphenous veins were obtained as residual specimens from leg bypass surgeries. Portions of the vein that had been surgically marked with crystal violet were analyzed separately from those that had no dye marking. In the laboratory, they were split into easily dissected inner and outer layers after removal of endothelium. This cleavage plane was within the circular muscle layer of the media. Cell migration from explants was measured daily as either (1) percentage of migration-positive explants, which exclusively measures migration, or (2) number of cells on the plastic surrounding each explant, which measures migration plus proliferation. Cell proliferation and apoptosis (Ki67 and TUNEL staining, respectively) were determined in dye-marked and unmarked areas of cultured vein rings. The dose-dependent effects of crystal violet were measured for cell migration from explants as well as for proliferation, migration, and death of cultured outer layer cells. Dye was extracted from explants with ethanol and quantified by spectrophotometry. RESULTS: There was significantly less cell migration from visibly blue compared with unstained outer layer explants by both methods. There was no significant difference in migration from inner layer explants adjacent to blue-stained or unstained sections of vein because dye did not penetrate to the inner layer. Ki67 staining of vein in organ culture, which is a measure of proliferation, progressively increased up to 6 days in nonblue outer layer and was abolished in the blue outer layer. Evidence of apoptosis (TUNEL staining) was present throughout the wall and not different in blue-stained and unstained vein wall segments. Blue outer layer explants had 65.9 ± 8.0 ng dye/explant compared with 2.1 ± 1.3 for nonblue outer layer explants. Dye applied in vitro to either outer or inner layer explants dose dependently inhibited migration (IC50∼10 ng/explant). The IC50s of crystal violet for outer layer cell proliferation and migration were 0.1 and 1.2 µg/mL, whereas the EC50 for death was between 1 and 10 µg/mL. CONCLUSIONS: Crystal violet inhibits venous cell migration and proliferation, indicating that alternative methods should be considered for marking vein grafts.

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Crystal Violet (CV), a triphenylmethane dye, has been extensively used in human and veterinary medicine as a biological stain, as a textile dye in textile processing industries and also used to provide a deep violet color to paints and printing ink. CV is also used as a mutagenic and bacteriostatic agent in medical solutions and antimicrobial agent to prevent the fungal growth in poultry feed. Inspite of its many uses, CV has been reported as a recalcitrant dye molecule that persists in environment for a long period and pose toxic effects in environment. It acts as a mitotic poison, potent carcinogen and a potent clastogene promoting tumor growth in some species of fish. Thus, CV is regarded as a biohazard substance. Although, there are several physico-chemical methods such as adsorption, coagulation and ion-pair extraction reported for the removal of CV, but these methods are insufficient for the complete removal of CV from industrial wastewaters and also produce large quantity of sludge containing secondary pollutants. However, biological methods are regarded as cost-effective and eco-friendly for the treatment of industrial wastewaters, but these methods also have certain limitations. Therefore, there is an urgent need to develop such eco-friendly and cost-effective biological treatment methods, which can effectively remove the dye from industrial wastewaters for the safety of environment, as well as human and animal health.

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The enzyme triphenylmethane reductase (TMR) reduces toxic triphenylmethane dyes into colorless, nontoxic derivatives, and TMR-producing microorganisms have been proposed as bioremediation tools. Analysis of the genome of Listeria monocytogenes H7858 (1998-1999 hot dog outbreak) revealed that the plasmid (pLM80) of this strain harboring a gene cassette (bcrABC) conferring resistance to benzalkonium chloride (BC) and other quaternary ammonium disinfectants also harbored a gene (tmr) highly homologous to TMR-encoding genes from diverse Gram-negative bacteria. The pLM80-associated tmr was located two genes downstream of bcrABC as part of a putative IS1216 composite transposon. To confirm the role of tmr in triphenylmethane dye detoxification, we introduced various tmr-harboring fragments of pLM80 in a pLM80-cured derivative of strain H7550, from the same outbreak as H7858, and assessed the resistance of the constructs to the triphenylmethane dyes crystal violet (CV) and malachite green. Transcriptional and subcloning data suggest that the regulation of TMR is complex. Constructs harboring fragments spanning bcrABC and tmr were CV resistant, and in such constructs tmr transcription was induced by sublethal levels of either BC or CV. However, constructs harboring only tmr and its upstream intergenic region could also confer resistance to CV, albeit at lower levels. Screening a panel of BC-resistant L. monocytogenes strains revealed that all those harboring bcrABC and adjacent pLM80 sequences, including tmr, were resistant to CV and decolorized this dye. The findings suggest a potential role of TMR as a previously unknown adaptive attribute for environmental persistence of L. monocytogenes.

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PURPOSE: To evaluate whether the gentian violet staining of the anterior lens capsule during the cataract surgery is cytotoxic for the human lens epithelial cells, as an indirect indication of possible toxicity towards the corneal endothelium and the safety of gentian violet application. MATERIALS AND METHODS: Two groups of anterior lens capsules obtained during the cataract surgery, gentian violet stained and non-stained, were incubated with fluorescent dye Fura-2. Their fluorescence, upon excitation at 360 and 380 nm, was imaged to monitor changes in free intracellular calcium concentration ([Ca(2+)]i) in response to pharmacological stimulation by acetylcholine. The [Ca(2+)]i homeostasis is the indicator of cellular function. The changes in [Ca(2+)]i were compared between the two groups. RESULTS: Epithelial cells responded to acetylcholine in both groups of capsules - gentian violet stained (n = 17) and non-stained ones (n = 33). No significant differences of the elicited responses were found in rise time (p = 0.89), decay time (p = 0.61) or amplitude of [Ca(2+)]i (p = 0.96 for 63× and p = 0.26 for 40× objectives) between the two groups of capsules (Student t test). CONCLUSIONS: The staining of the anterior lens capsule with gentian violet during phacoemulsification in concentration of 0.01%, does not have detectable cytotoxic effects, which would affect the [Ca(2+)]i homeostasis in lens epithelial cells. The data, if extrapolated to corneal endothelium, exposed to the same concentration, suggest that gentian violet in concentration of 0.01% is safe as an adjunct for capsule visualization in cataract surgery.

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PURPOSE: Gentian violet (GV) is an antimicrobial and antifungal agent that has been used widely to treat intractable discharge in the ear. The purpose of this report is to warn clinicians about the ototoxic effect of GV in the middle ear. MATERIALS AND METHODS: GV ototoxicity was evaluated by measuring compound action potentials (CAPs) in the VIIIth nerve in adult Hartley guinea pigs. The middle ear cavities of the animals were filled with GV solution (0.5% or 0.13%), and CAPs were measured after intervals of 5 and 30 minutes and 1, 2, 6, and 24 hours. After all measurements were completed, the temporal bones were harvested for histopathologic evaluation. Celloidin-embedded specimens were cut into 20-µm slices and examined using light microscopy. The bacteriostatic activity of GV was evaluated using a disk-diffusion assay. RESULTS: A 0.5% GV solution produced a mild elevation in the CAP threshold at 30 minutes, a greater reduction at 1 hour, and complete abolishment of CAP at 24 hours. A 0.13% GV solution caused mild elevation in the CAP threshold at 2 hours and severe elevation at 6 hours. Massive new bone formation was found in the middle ear cavity at 6 weeks. GV concentrations of 0.13% and 0.06% were effective against all bacteria tested, with the exception of Pseudomonas aeruginosa. CONCLUSIONS: Although GV has marked antibacterial and antifungal activities, its use should be limited to the external ear canal. GV exerts an ototoxic effect in a concentration- and time-dependent manner, and so the use of this drug in the middle ear cavity is not recommended.

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Activated-carbon-supported iron oxides were prepared and used as a catalyst in an integrated ultrasound/heterogeneous Fenton process for the decolorization of Crystal Violet. A synergistic effect was observed when ultrasound was combined with the heterogeneous Fenton process. The decolorization efficiency increased with the increasing power density and catalyst dosage, but decreased with the increase of initial pH value. There exists an optimal hydrogen peroxide concentration for decolorization. Catalyst stability was evaluated by measuring iron leaching in solution. The decolorization efficiency was 88% under the optimal conditions. Toxicity test with Daphnia magna showed that the acute toxicity of dye solution decreased significantly after the treatment by the heterogeneous sono-Fenton process.

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El Violeta de Genciana (GV) se usa como aditivo en la sangre para eliminar el Trypanosoma cruzi en la quimioprofilaxis de la infección por enfermedad de Chagas vía transfusión sanguínea, cuando no es posible un control previo de laboratorio o bajo situaciones de emergencia. En estos estudios se encontraron efectos genotóxicos del GV con el ensayo Cometa, cuando se lo agregó a la sangre bajo las condiciones empleadas para esterilizarla para transfusión. El efecto genotóxico fue aún más intenso si la sangre se mantenía con GV por 48 horas. Los resultados obtenidos con el ensayo Cometa sugieren la formación de bases hidroxiladas de ADN como resultado de un ataque de especies reactivas de oxígeno y apoyan la genotoxicidad del GV y su potencial carcinogénico ya informado previamente. Los efectos genotóxicos observados en el ensayo Cometa fueron parcialmente prevenidos por administración de antioxidantes que ya tienen uso clínico seguro, como á-tocoferol, ácido lipoico o N-acetilcisteína. El ácido lipoico fue capaz también de reaccionar in vitro con GV. Los resultados sugieren un uso potencial de estos antioxidantes para prevenir los efectos secundarios no deseados del GV para el individuo receptor de la sangre.

Gentian violet (GV) is being used as blood additive to eliminate Trypanosoma cruzi in the chemoprophylaxis of Chagas disease infection via blood transfusion when prior laboratory control is not possible or under emergency circumstances in endemic areas. In these studies genotoxic effects of GV were found employing the Comet assay when GV was added to rat blood under the cisconditions employed to sterilize it for transfusion. The genotoxic effect was even more intense if blood was kept with GV for 48 hours. The positive results obtained in the Comet assay suggest the formation of DNA hydroxylated bases as result of a reactive oxygen species (ROS) attack and further confirm GV genotoxicity and its potential carcinogenic effects previously reported. Genotoxicity effects observed in the Comet assay were partially but significantly prevented by prior administration of antioxidants having safe clinical use such as á-tocopherol; lipoic acid or N-acetylcysteine. Lipoic acid was also able to chemically react in vitro with GV (eg. the one remaining in the transfusion mixture after it had enough time to eliminate the parasite from blood). Results would suggest the potential use of these antioxidants to prevent unwanted side effects of GV for the blood recipient.

O Violeta de Genciana (GV) é utilizado como aditivo no sangue para remover o Trypanosoma cruzi da sangue em quimioprofilaxia da infecção por doença de Chagas através de transfusão de sangue, quando não é possível controle prévio de laboratório ou em situações de emergência. Nestes estudos encontraram-se efeitos genotóxicos do GV utilizando o ensaio Cometa, quando o GV foi adicionado ao sangue sob as condições utilizadas para a esterilização para transfusão. O efeito genotóxico foi ainda mais intenso se o sangue era mantido durante 48 horas com GV. Os resultados obtidos com o ensaio Cometa sugerem a formação de bases de DNA hidroxiladas, como resultado de um ataque de espécies reativas de oxigênio e apoiam a genotoxicidade do GV e seu potencial carcinogênico já informado anteriormente. Efeitos genotóxicos observados no ensaio do Cometa foram parcialmente prevenidos por administração de antioxidantes que já têm uso clínico seguro, como á-tocoferol, o ácido lipoico ou N-acetilcisteína. O ácido lipoico também foi capaz de reagir in vitro com GV. Os resultados sugerem um uso potencial destes antioxidantes para prevenir os efeitos colaterais não desejados de GV para o indivíduo receptor do sangue.

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PURPOSE: To assess tight junction (TJ) integrity in cultured human fetal retinal pigment epithelium (HFRPE) after exposure to clinically relevant novel vital dyes. METHODS: HFRPE floater cells were harvested from RPE primary cultures of 4 donor eyes and seeded on polyester Transwell® for 4-6 weeks. The apical compartments of well-differentiated cultures were exposed to 0.005 mg/ml Coomassie violet R200 (CVR), methyl 2B (M2B) or Orange II. Periods of 30-300 s were chosen to mimic surgical exposure times, while 3 h was used for toxicity assays, with subsequent washout. Cell-cell junctions were studied by immunofluorescence (zonula occludens-1, ZO-1). Transepithelial electrical resistance (TER) was measured regarding blood-retina barrier (BRB) function. RESULTS: At 4-6 weeks after confluence, HFRPE had grown into pigmented hexagonal monolayers with stable TER values (451-1,520 Ω·cm(2)). After 300-second dye treatments, a continuous ZO-1 signal was detected in all vital dye-treated groups 1.5 h after exposure, whereas trypsin controls showed patchy loss of the TJ stain. TER of CVR-, M2B- and Orange-II-treated groups had dropped 1.5 h after exposure to 148 ± 58.4, 162 ± 23.7 and 164 ± 18.5 Ω·cm(2), respectively, compared to 73 ± 44.9 Ω·cm(2) in positive controls. After 3 h of exposure to 0.005 mg/ml vital dyes in thick drops, TER maintained similar levels to those prior to exposure (90.8 ± 4.7% of the original values, 93.8 ± 6.5 and 91.9 ± 3.6%, respectively), together with no difference from the vehicle controls (94.8 ± 6.6%). TER values recovered in all groups to prior levels within 3 days. CONCLUSION: Novel vital dyes (CVR, M2B and Orange II) caused no outer BRB function alteration.

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Gentian violet (GV) is a well-known triarylmethane dye that is used in aquacultural, industrial and medicinal fields. But concerns in growing number have been paid to its potential health problems to human beings and its hazardous effects to environment. Herein, the toxic interaction of GV with bovine hemoglobin (BHb) was investigated by a series of spectroscopic methods and molecular modeling method. The fluorescence emission profile exhibited a remarkable quenching upon addition of GV to the buffered aqueous solution of BHb and the analysis of results revealed the dominant role of static quenching mechanism in GV-BHb interaction. The negative ΔH and positive ΔS values demonstrated that the electrostatic interactions mainly stabilized this toxicantprotein complex. Synchronous fluorescence, UV-Vis absorption and CD spectroscopic studies proved that the conformational change of BHb was induced by GV's combination. Molecular modeling studies exhibited the binding mode of GV-BHb complex and the detailed information of related driving forces. During the (1)H nuclear magnetic resonance spectra ((1)H NMR) study, the chemical shift perturbation and spin-lattice relaxation times of different protons were further used to investigate the interaction of GV with BHb and the results indicated that GV bound orientationally to BHb.

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Chitin grafted poly (acrylic acid) (chi-g-PAA) is synthesized and characterized as an adsorbent of toxic organic compounds. Chi-g-PAA copolymers are prepared using of ammonium cerium (IV) nitrate (Ce(4+)) as the initiator. The highest grafting percentage of AA in chitin obtained using the traditional technique is 163.1%. A maximum grafting percentage of 230.6% is obtained using central composite design (CCD). Experimental results are consistent with theoretical calculations. The grafted copolymer is characterized by Fourier transform Infrared spectroscopy and solid state (13)C NMR. A representative chi-g-AA copolymer is hydrolyzed to a type of sodium salt (chi-g-PANa) and used in the adsorption of malachite green (MG), methyl violet (MV), and paraquat (PQ) in aqueous. The monolayer adsorption capacities of these substances are 285.7, 357.1, and 322.6 mg/g-adsorbent, respectively. Thermodynamic calculations show that the adsorption of MG, MV, and PQ are more favored at diluted solutions. The high adsorption capacity of chi-g-PANa for toxic matter indicates its potential in the treatment of wastewater and emergency treatment of PQ-poisoned patients.

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Green tea polyphenols (GTP) are widely used as food preservatives and are considered to be extremely safe. However, the bacterial response to GTP has not been well studied. Here we investigated whether short exposure of Pseudomonas aeruginosa to sub-lethal dose of GTP could lead to cross-resistance to some environmental stresses. One-hour exposure of P. aeruginosa to 1 mg/ml GTP significantly increased the tolerance to oxidants (2 mM H(2)O(2), 4 mM tert-butylhydroperoxide), low pH solution (pH 4.0) containing various organic acids (60 mM citric, acetic, propionic or lactic acid) and other stress conditions (47 °C, 25 % NaCl, 12 % ethanol and 150 µg/ml crystal violet). The development of H(2)O(2) tolerance in GTP-exposed cells was prevented by chloramphenicol, a well-known inhibitor of protein synthesis in prokaryotic cells. Furthermore, we observed significantly increased catalase activity after GTP exposure, suggesting that P. aeruginosa develops GTP-induced cross-resistance by increasing synthesis of protective protein. These observations raise concerns over the underlying risks associated with using GTP as food preservatives.

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PURPOSE: To determine the endothelium damage resulting from the application of "dry" gentian violet (GV) stromal markings on corneas precut for Descemet stripping automated endothelial keratoplasty (DSAEK) using a Moria "S" Stamp. METHODS: Five precut corneas had the stromal graft bed marked with GV using the Moria "S" stamp and 6 precut corneas were left unmarked as controls. After applying the ink to the stamp, care was taken to allow the alcohol carrier to dry for 10 seconds before applying the dry dye to the stromal surface. Tissue was then trephinated and stained with calcein AM to assess endothelial viability. Grafts were photographed and digital pixel planometry, using an established analysis technique, was used to compare the damage between the control and experimental groups. RESULTS: The mean percent cell damage of corneas treated with GV "S" stamp (n = 5) was 8.6% (range 4.4-12.9), and it was 8.1% (range 3.9-15.1) in the DSAEK control set (n = 6). Median percent cell damage was 6.7% among GV-treated corneas and 7.4% among control corneas. The distributions were not significantly different between groups (Mann-Whitney U test = 15.0, two-tailed P = 1.0). Moreover, no "S" pattern of damage was seen in any study eye. CONCLUSIONS: There were no significant differences in endothelial damage between the 2 groups. GV stromal markings may be applied without undue damage to the endothelium using the dry-ink technique described.

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This work demonstrated that Shewanella decolorationis NTOU1 decolorized 200 mg l(-1) of crystal violet, malachite green, or methyl violet B within 2-11h under anaerobic conditions at 35 degrees C. The initial color removal rate of malachite green was highest, while that of methyl violet was lowest. GC/MS analyses of the intermediate compounds produced during and after decolorization of malachite green and methyl violet B suggested that biodegradation of these dyes involved reduction to leuco form, N-demethylation, and reductive splitting of the triphenyl rings. The number of N-methylated groups of these dyes might have influenced decolorization rates and the reductive splitting of the triphenyl rings of these dyes. Cytotoxicity and antimicrobial test data showed that malachite green and methyl violet B solution (100 mg l(-1)) were toxic. Toxicity of the dyes decreased after their decolorization, but further incubation resulted in increased toxicity.

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Natural organic coagulants (NOCs) such as chitosan and Moringa oleifera seeds have been extensively characterized for potential application in water treatment as an alternative to metal-based coagulants. However, the action of both chitosan and M. oleifera seeds is mainly restricted to anionic organic pollutants because of their cationic functional groups affording poor cationic pollutant coagulation by electrostatic repulsion. In this study, we employed ethanolic grape seed extract (GSE) and grape seed-derived polyphenols such as tannic acid and catechin in an effort to find novel NOCs showing stable anionic forms for removal of cationic organic pollutants. The target substances tested were malachite green (MG) and crystal violet (CV), both mutagenic cationic dyes. Polyphenol treatment induced fast decolorization followed by gradual floc formation concomitant with red or blue shifts in maximum absorbance wavelengths of the cationic dyes. Liquid chromatography analysis of flocs formed by polyphenols directly showed that initial supramolecular complexes attributed mainly to electrostatic attraction between polyphenol hydroxyphenyl groups and cationic dyes further progressed into stronger aggregates, leading to precipitation of dye-polyphenol complexes. Consistent with the results obtained using catechin and tannic acid, use of GSE also resulted in effective decolorization and coagulation of soluble MG and CV in aqueous solutions. Screening of several organic GSE components for NOC activity strongly suggested that natural polyphenols are the main organic ingredients causing MG and CV removal via gradual floc formation. The treatment by natural polyphenols and GSE decreased toxicity of MG- or CV-contaminated water.

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Rhizobium radiobacter MTCC 8161 completely decolorized methyl violet (10 mg l(-1)) within 8 h both at static and shaking conditions. The decolorization time increased with increasing dye concentration. The effect of different carbon and nitrogen sources on the decolorization of methyl violet was studied. The maximum decolorization was observed in the presence of sucrose (1%) and urea (1%). UV-Visible, HPLC and FTIR analysis of extracted products confirmed biodegradation of methyl violet. The significant increase in the activities of lignin peroxidase and aminopyrine N-demethylase in the cells obtained after decolorization indicated involvement of these enzymes in the decolorization process. In addition to methyl violet, this strain also shows an ability to decolorize various industrial dyes, (red HE7B, yellow 4G, blue 2B, navy blue HE22, red M5B and red HE3B).

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Basic Violet 3, Basic Violet 1, and Basic Violet 4 are triphenylmethane dyes that function as direct (nonoxidative) hair colorants. No current uses or use concentrations in cosmetics are reported. The term Gentian Violet is used synonymously with Basic Violet 1 and Basic Violet 3, although the chemical structures of these 2 dyes are not the same. The Cosmetic Ingredient Review Expert Panel noted that Basic Violet 1, 3, and 4 contain quaternary ammonium ions, and therefore the rate of penetration across the epidermis is expected to be slow. The panel concluded that because of the carcinogenic potential of these dyes, insufficient data exist to support the safety of Basic Violet 1, 3, and 4 in cosmetic formulation. Dermal absorption data and a risk assessment are needed to complete this safety assessment.

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PURPOSE: To qualitatively assess the endothelial damage on Descemet-stripping automated endothelial keratoplasty (DSAEK) donor tissue resulting from a gentian violet marking pen. METHODS: An in vitro model was used, by using corneoscleral rims, DSAEK quality corneal donor tissue, and a gentian violet marking pen. After making a mark on the stromal side of a microkeratome-prepared DSAEK corneal button to confirm appropriate orientation of the donor tissue after insertion into the anterior chamber, the corneal tissue was returned to Optisol GS solution for 1 hour. A vital dye assay was used to identify devitalized and necrotic endothelial cells with alizarin red S and trypan blue. RESULTS: Corneal donor tissue evaluated with the gentian violet marking pen showed positive trypan blue staining, limited to the area marked with the gentian violet ink. CONCLUSIONS: Marking the DSAEK donor stromal surface with a gentian violet marker damages the endothelium. Surgeons should limit the size of the mark or use an insertion technique that avoids confusion about orientation of the donor cornea.

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