Genotoxicity of malachite green and leucomalachite green in female Big Blue B6C3F1 mice

Malachite green, a triphenylmethane dye used in aquaculture as an antifungal agent, is rapidly reduced in vivo to leucomalachite green. Previous studies in which female B6C3F1 mice were fed malachite green produced relatively high levels of liver DNA adducts after 28 days, but no significant induction of liver tumors was detected in a 2-year feeding study. Comparable experiments conducted with leucomalachite green resulted in relatively low levels of liver DNA adducts but a dose-responsive induction of liver tumors. In the present study, we fed transgenic female Big Blue B6C3F1 mice with 450 ppm malachite green and 204 and 408 ppm leucomalachite green (the high doses used in the tumor bioassays) and evaluated genotoxicity after 4 and 16 weeks of treatment. Neither malachite green nor leucomalachite green increased the peripheral blood micronucleus frequency or Hprt lymphocyte mutant frequency at either time point; however, the 16-week treatment with 408 ppm leucomalachite green did increase the liver cII mutant frequency. Similar increases in liver cII mutant frequency were not seen in the mice treated for 16 weeks with malachite green or in female Big Blue rats treated with a comparable dose of leucomalachite green for 16 weeks in a previous study [Mutat. Res. 547 (2004) 5]. These results indicate that leucomalachite green is an in vivo mutagen in transgenic female mouse liver and that the mutagenicities of malachite green and leucomalachite green correlate with their tumorigenicities in mice and rats. The lack of increased micronucleus frequencies and lymphocyte Hprt mutants in female mice treated with leucomalachite green suggests that its genotoxicity is targeted to the tissue at risk for tumor induction.     

Biotransformation of malachite green by the fungus Cunninghamella elegans

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized the triphenylmethane dye malachite green with a first-order rate constant of 0.029 micromol x h(-1) (mg of cells)(-1). Malachite green was enzymatically reduced to leucomalachite green and also converted to N-demethylated and N-oxidized metabolites, including primary and secondary arylamines. Inhibition studies suggested that the cytochrome P450 system mediated both the reduction and the N-demethylation reactions.     

Biotransformation of Malachite Green by the Fungus Cunninghamella elegans

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized the triphenylmethane dye malachite green with a first-order rate constant of 0.029 μmol h⁻¹ (mg of cells)⁻¹. Malachite green was enzymatically reduced to leucomalachite green and also converted to N-demethylated and N-oxidized metabolites, including primary and secondary arylamines. Inhibition studies suggested that the cytochrome P450 system mediated both the reduction and the N-demethylation reactions.     

Analysis of mutations and bone marrow micronuclei in Big Blue rats fed leucomalachite green

Leucomalachite green (LMG) is the major metabolite of malachite green (MG), a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over MG and LMG is due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. In order to evaluate the risks associated with exposure to LMG, female Big Blue rats were fed up to 543 ppm LMG; groups of these rats were killed after 4, 16, or 32 weeks of exposure and evaluated for genotoxicity. We previously reported that this treatment resulted in a dose-dependent induction of liver DNA adducts, and that the liver lacI mutant frequency (MF) was increased, but only in rats fed 543 ppm LMG for 16 weeks. In the present study, we report the results from lymphocyte Hprt mutant assays and bone marrow micronucleus assays performed on these same rats. In addition, we have determined the types of lacI mutations induced in the rats fed 543 ppm LMG for 16 weeks and the rats fed control diet. No significant increases in the frequency of micronuclei or Hprt mutants were observed for any of the doses or time points assayed. Molecular analysis of 80 liver lacI mutants from rats fed 543 ppm LMG for 16 weeks revealed that 21% (17/80) were clonal in origin and that most (55/63) of the independent mutations were base pair substitutions. The predominant type of mutation was G:C --> A:T transition (31/63) and the majority (68%) of these involved CpG sites. When corrected for clonality, the 16-week lacI mutation frequency (36 +/- 10) x 10(-6) in treated rats was not significantly different from the clonally corrected control frequency (17 +/- 9 x 10(-6); P = 0.06). Furthermore, the lacI mutational spectrum in treated rats was not significantly different from that found for control rats (P = 0.09). Taken together, these data indicate that the DNA adducts produced by LMG in female rats do not result in detectable levels of genotoxicity, and that the increase in lacI MF observed previously in the liver of treated rats may be due to the disproportionate expansion of spontaneous lacI mutations.     

Reduction of malachite green to leucomalachite green by intestinal bacteria.

Intestinal microfloras from human, rat, mouse, and monkey fecal samples and 14 pure cultures of anaerobic bacteria representative of those found in the human gastrointestinal tract metabolized the triphenylmethane dye malachite green to leucomalachite green. The reduction of malachite green to the leuco derivative suggests that intestinal microflora could play an important role in the metabolic activation of the triphenylmethane dye to a potential carcinogen.     

Particle beam liquid chromatography-mass spectrometry of triphenylmethane dyes: application to confirmation of malachite green in incurred catfish tissue

Eight triphenylmethane dyes (malachite green, leucomalachite green, gentian violet, leucogentian violet, brilliant green, pentamethyl gentian violet, N′,N′-tetramethyl gentian violet and N′,N″-tetramethyl gentian violet) have been characterized by particle beam liquid chromatography-mass spectrometry. The electron ionization spectra obtained of these dyes by this technique exhibit similar fragmentation, with the formation of phenyl and substituted phenyl radicals, and loss of alkyl groups from the amines. It was observed that the six cationic dyes are reduced in the mass spectrometer source to form the corresponding leuco compounds. This technique was evaluated for the confirmation of malachite green and leucomalachite green in incurred catfish (Ictalurus punctatus) muscle tissue.     

Determination of malachite green and leucomalachite green in edible goldfish muscle by liquid chromatography-ion trap mass spectrometry

A liquid chromatography-ion trap mass spectrometry method with three "time segments" has been developed to determine malachite green (MG) and its major metabolite, leucomalachite green (LMG) in edible goldfish muscle. By using the optimized "time segments", MG and LMG as well as the internal standard atrazine-d(5) were analyzed with good sensitivity with positive ESI-MS in a single run. The homogenized fish muscle tissues were extracted with a solution of perchloric acid and acetonitrile, followed by partitioning with dichloromethane. Strata-x polymeric solid-phase extraction column was used for the clean-up process. The determination of MG and LMG was achieved by using a reversed-phase HPLC gradient program coupled with MS/MS in multiple-reaction-monitoring mode. Matrix calibration curves were linear over the ranges of 5-500 ng/ml for MG and 1-100 ng/ml for LMG. Recoveries of the fish tissue extraction at three spiked levels (2, 10 and 30 ng/g for MG as well as 0.4, 2 and 6 ng/g for LMG) were better than 71% and 89%, respectively. Relative standard derivations from six determinations were less than 8%. The method detection limits were 0.13 ng/g for MG and 0.06 ng/g for LMG.     

Quirks of dye nomenclature. 6. Malachite green

Malachite green was discovered independently by two researchers in Germany in the 19^th century and found immediate employment as a dye and a pigment. Subsequently, other uses, such as staining biological specimens, emerged. A much later application was the control of fungal and protozoan infections in fish, for which the dye remains popular, although illegal in many countries owing to a variety of toxicity problems. In solution, malachite green can exist as five different species depending on the pH. The location of the positive charge of the colored cation on a carbon atom or a nitrogen atom is still debated. The original names of this dye, and their origins, are briefly surveyed.     

超声波辅助抽提法在孔雀石绿分子印迹固相萃取中的应用

采用沉淀聚合法制备孔雀石绿分子印迹聚合物(MG-MIPs),以洗脱效率及吸附量为指标,考察超声波辅助抽提法对MIPs中MG洗脱效果及吸附性能的影响,通过扫描电镜观察MIPs的表面形态,并对其吸附性能进行研究。结果表明:模板分子MG在超声30 min、超声10次、料液比m(MG-MIPs)∶V(甲醇-乙酸溶液)为1∶10(g/m L)、温度为25℃、超声功率为270 W的条件下,洗脱效果最好,MIPs在固相萃取柱中的吸附效率较高,达到198μg/g。     

A confirmatory analysis of malachite green residues in rainbow trout with liquid chromatography-electrospray tandem mass spectrometry

A quantitative liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed for the determination of malachite green (MG) and its metabolite leucomalachite green (LMG) in fish. Residues were extracted with an acetonitrile-acetate buffer and purified using the automated solid-phase extraction (ASPEC). Residues were analyzed with a reversed-phase LC-MS/MS using a positive-ion electrospray ionisation (ESI). Isotope-labelled leucomalachite green (LMG-D5) was used as an internal standard for the quantification of LMG residues. The related dye, brilliant green (BG) was used as an instrumental standard. Identification and quantification of analytes were based on the ion transitions monitored by multiple reaction monitoring (MRM). The decision limit (CCalpha) for MG and LMG was 0.13 and 0.16 microgkg(-1). The respective detection capabilities (CCbeta) were 0.22 and 0.27 microgkg(-1). The absolute recovery (repeatability SD(r)) was in the range of 58-65% (7.8-11.2%) for MG and 59-68% (9.7-16.9%) for LMG. LMG was quantified also based on the internal standard, giving a recovery (repeatability SD(r)) of 103-110% (4.8-9.3%). The method was further evaluated by analyzing a total of 34 fish residue monitoring samples, of which eight samples were found to be non-compliant containing low residues of LMG.     

Dyes as fungal inhibitors: effect on colony enumeration

The effects of three dyes on the colony enumeration of nine fungal strains (including members of the Deuteromycetes and Zygomycetes) in pure and mixed cultures were investigated. Using malt extract agar as basal and control medium, the following dyes and concentrations were assayed: auramine (25 ppm), gentian violet (5 ppm) and malachite green (1 ppm). The chemicals commonly used in commercial media dichloran (2 ppm) and rose bengal (50 ppm) were included in the study as reference mould-spreading inhibitors. Higher counts were usually obtained in the media containing dichloran, rose bengal or auramine, including the control medium in the absence of chemical when the mixed-conidium inocula did not include a spreading mould. Nevertheless in most cases no significant differences were observed between them. Malachite green (1 ppm) performed mainly as a strong inhibitor of spreading moulds, only allowing adequate colony development and recoveries of both Fusarium and Aspergillus strains tested.     

Lack of effect of butylparaben and methylparaben on the reproductive system in male rats

BACKGROUND: Parabens are widely used preservatives in cosmetics and pharmaceutical products, and approved as food additives. Parabens have been considered safe for these uses for many years. Recently, adverse effects on male reproductive parameters in rats have been reported when parabens were given orally for 8 weeks starting at three weeks of age. Our studies used two representative parabens, methyl‐ and butylparaben, to try to replicate these studies and thereby evaluate potential reproductive effects in male Wistar rats. METHODS: Diets containing 0, 100, 1000 or 10,000 ppm of either butyl‐ or methylparaben were fed to male rats for eight weeks. Rats were 22 days of age at the start of exposure. Parameters evaluated included organ weights, histopathology of reproductive tissues, sperm production, motility, morphology and reproductive hormone levels (butylparaben only). RESULTS: None of the parameters evaluated for either paraben showed compound‐ or dosage‐dependent adverse effects. Metabolism experiments of butylparaben indicate that it is rapidly metabolized by non‐specific esterases to p‐hydroxybenzoic acid and butanol, neither of which is estrogenic. CONCLUSIONS: Exposure to methyl‐ or butylparaben in the diet for eight weeks did not affect any male reproductive organs or parameters at exposures as high as 10,000 ppm, corresponding to a mean daily dose of 1,141.1±58.9 or 1,087.6±67.8 mg/kg/day for methyl‐ and butylparaben, respectively. The rapid metabolism of parabens by esterases probably explains why these weakly estrogenic substances elicit no in vivo effects when administered by relevant exposure routes (i.e., topical and oral). Birth Defects Research (Part B) 2008. 2008 Wiley‐Liss, Inc.     

Malachite green: applications in electron microscopy.

Incorporation of malachite green into a glutaraldehyde fixative results in enhanced staining of a number of cellular elements. Ribosomes and myofilaments exhibit increased electron density, but cell membranes generally are not stained. In certain tissues, lipid inclusions are uniformly and heavily stained. Other populations of lipid droplets exhibit differential affinity for malachite green, facilitating their division into subclasses. In addition to its function as a dye, malachite green has previously been reported to stabilize lipid elements soluble in aqueous glutaraldehyde. Such a component was observed in the stroma of uterine endometrium. The variety of cell components which exhibit increased contrast after preparation with malachite green suggests that this technique may find widespread application in fine structure studies.     

Effects of malachite green on leucocyte abundance in rainbow trout, Salmo gairdneri (Richardson)

Blood counts from more than 1000 young-of-the-year rainbow trout, Salmo gairdneri (Richardson), were examined to determine if static exposure to malachite green at 1·35,13·5, or 21·0 mg/1 for 25–30 min or 42·0 or 72·0 mg/1 for 5 min caused chronic leucopaenia. The major changes in fish exposed for 25–30 min came during the first 24 h. After an initial lag of 3·4 h, total leucocyte-thrombocyte counts in both treated and control fish rapidly declined. Recovery was essentially complete 1·4 days after exposure, and no leucopaenia was noted after 14 or 28 days. Thrombocytosis developed during the first 24 h in fish exposed to the higher concentrations. Lymphopaenia and neutrophilia also developed, but abated after the 4th post-treatment day. Leucocyte numbers in control and exposed groups were virtually the same by the 14th day. The total leucocyte-thrombocyte counts in fish exposed for 5 min declined after 24 h, but counts were not as depressed as those in fish exposed for 30 min.
Because leucocyte changes similar to those in exposed fish were evident in the controls in both experiments, we believe that the leucocyte changes in rainbow trout exposed to malachite green were a result of a nonspecific vertebrate stress syndrome, rather than of specific leucocytotoxic effect of this chemical.     

High accuracy determination of malachite green and leucomalachite green in salmon tissue by exact matching isotope dilution mass spectrometry

A high accuracy method for the quantification of malachite green (MG) and leucomalachite green (LMG) in salmon is described. Analytical challenges including the effects of analyte instability and matrix suppression were minimised by the use of exact matching isotope dilution mass spectrometry. The developed method included overnight extraction in acidified acetonitrile/ammonium acetate buffer and analysis by LC-MS/MS utilising isotopic internal standards. This method was used to determine the level of MG and LMG in a sample of salmon used in an international inter-comparison organised by the Comité Consultatif pour la Quantité de Matière (CCQM). The sum of MG and LMG was found to be 9.32+/-0.98ngg(-1) at the 95% confidence interval (relative expanded uncertainty 10.5% (k=2)). This encompassed the mean and median of the CCQM inter-comparison.     

Effect of estrogen on the affinity of malachite green for staining cardiac lipid inclusions in mice

The effect of estradiol-17-beta on lipids of the ventricular myocardium of mice has been studied with a cytochemical technique in which malachite green was added to glutaraldehyde. This malachite green-glutaraldehyde fixative enhances the visualization of certain phospholipid-related elements. Estrogen induces an affinity of ventricular cardiac lipid inclusions for the cationic dye malachite green. The staining affinity is evidenced only in the estrous female, not in diestrus. In oophorectomized animals, malachite green staining is seen only following estradiol injection, but this effect is blocked by progesterone. In the male, ventricular lipids do not stain, nor do they develop malachite green affinity with estrogen stimulation. These results imply a blockade of the estradiol-mediated dye affinity by progesterone and testosterone. This reinforces the concept of the heart as a target organ for sex steroids and expands the previously described estrogen effects on myocardium.     

Detoxification of malachite green and textile industrial effluent by Penicillium ochrochloron

Malachite green was detoxified into p-benzyl-N,N-dimethylaniline and N,N-dimethyl-aniline hydrochloride by Penicillium ochrochloron. Degradation metabolites were analyzed by TLC, HPLC, and FTIR and identified by GCMS analysis. Phytotoxicity testing revealed the nontoxic nature of these metabolites. The percentage decolorization of malachite green (50 mg/L) was 93% in czapek dox broth after 14 h with an optimum pH of 7 at 30°C. The induction in the activity of lignin peroxidase after degradation suggested that the degradation of malachite green was peroxidase-mediated. Fungal culture was also found to have detoxified the textile effluent. The values of TDS, TSS, COD, and BOD were reduced in the treated samples compared to the control effluent. The treated effluent was non-toxic to the plants of Triticum aestivum and Ervum lens Linn, and the amount of total chlorophyll was higher in plants with treated effluent when compared to control effluent.     

Isolation of a malachite green-degrading Pseudomonas sp. MDB-1 strain and cloning of the tmr2 gene

The release of malachite green, a commonly used triphenylmethane dye, into the environment is causing increasing concern due to its toxicity, mutagenicity, and carcinogenicity. A bacterial strain that could degrade malachite green was isolated from the water of an aquatic hatchery. It was identified as a Pseudomonas sp. based on the morphological, physiological, and biochemical characteristics, as well as the analysis of 16S rRNA gene sequence and designated as MDB-1. This strain was capable of degrading both malachite green and leucomalachite green, as well as other triphenylmethane dyes including Crystal Violet and Basic Fuchsin. The gene tmr2, encoding the triphenylmethane reductase from MDB-1, was cloned, sequenced and effectively expressed in E. coli. These results highlight the potential of this bacterium for the bioremediation of aquatic environments contaminated by malachite green.     

Electron microscopy of malachite green--glutaraldehyde fixed bacteria.

Malachite green combined with glutaraldehyde has been used recently as a fixative for preserving and revaling lipid complexes in thin sections of eukaryotic cells examined by electron microscopy. When bacteria were prefixed with the above mixture granular electron dense inclusions were revealed in all cultures tested. These inclusions were replaced by electron transparent areas in cells fixed with glutaraldehyde alone. The structures were frequently located near to or within the nucleoid and adjacent to the cell membrane in Gram-negative bacteria and were associated with the nucleoid and mesosomes in Gram-positive species. Polyhydroxybutyrate granules, generally poorly preserved in thin sections of Aquaspirillum serpens, were well preserved by the malachite green-glutaraldehyde fixative. Malachite green complexes were observed outside of the cells in all preparations. Capsules were neither preserved nor stained.     

Electron Microscopy of Malachite Green— Glutaraldehyde Fixed Bacteria

Malachite green combined with glutaraldehyde has been used recently as a fixative for preserving and revealing lipid complexes in thin sections of eukaryotic cells examined by electron microscopy. When bacteria were prefixed with the above mixture granular electron dense inclusions were revealed in all cultures tested. These inclusions were replaced by electron transparent areas in cells fixed with glutaraldehyde alone. The structures were frequently located near to or within the nucleoid and adjacent to the cell membrane in Gram-negative bacteria and were associated with the nucleoid and mesosomes in Gram-positive species. Polyhydroxybutyrate granules, generally poorly preserved in thin sections of Aquaspirillum serpens, were well preserved by the malachite green-glutaraldehyde fixative. Malachite green complexes were observed outside of the cells in all preparations. Capsules were neither preserved nor stained.