Indigo dyes: Toxicity, teratogenicity, and genotoxicity studies in zebrafish embryos.

Wastewater released by textile dyeing industries is a major source of pollution. Untreated wastewater released from indigo dyeing operations affects aquatic ecosystems and threatens their biodiversity. We have assessed the toxicity of natural and synthetic indigo dye in zebrafish embryos, using the endpoints of teratogenicity, genotoxicity, and histopathology. The zebrafish embryo toxicity test (ZFET) was conducted, exposing embryos to ten concentrations of natural and synthetic indigo dyes; the 96-hour LC50 values were approximately 350 and 300 mg/L, respectively. Both dyes were teratogenic, causing egg coagulation, tail detachment, yolk sac edema, pericardial edema, and tail bend, with no significant difference in effects between the natural and synthetic dyes. Both dyes were genotoxic (using comet assay for DNA damage). Real-time RT-PCR studies showed upregulation of the DNA-repair genes FEN1 and ERCC1. Severe histological changes were seen in zebrafish larvae following exposure to the dyes. Our results show that indigo dyes may be teratogenic and genotoxic to aquatic organisms, underscoring the need for development of sustainable practices and policies for mitigating the environmental impacts of textile dyeing.

Lethal and sub-lethal evaluation of Indigo Carmine dye and byproducts after TiO2 photocatalysis in the immune system of Eisenia andrei earthworms.

The Indigo carmine (IC) dye has been widely used in textile industries, even though it has been considered toxic for rats, pigs and humans. Owing to its toxicity, wastes containing this compound should be treated to minimize or eliminate their toxic effects on the biota. As an alternative to wastewater treatment, advanced oxidative processes (AOPs) have been highlighted due to their high capacity to destruct organic molecules. In this context, this study aimed to evaluate Indigo Carmine toxicity to soil organisms using the earthworm Eisenia andrei as a model-organism and also verify the efficiency of AOP in reducing its toxicity to these organisms. To this end, lethal (mortality) and sub-lethal (loss or gain of biomass, reproduction, behavior, morphological changes and immune system cells) effects caused by this substance and its degradation products in these annelids were evaluated. Morphological changes were observed even in organisms exposed to low concentrations, while mortality was the major effect observed in individuals exposed to high levels of indigo carmine dye. The organisms exposed to the IC during the contact test showed mortality after 72h of exposure (LC50 = 75.79mgcm-2), while those exposed to photoproducts showed mortality after 48h (LC50 = 243min). In the chronic study, the organisms displayed a mortality rate of 14%, while those exposed to the photoproduct reached up to 32.7%. A negative influence of the dye on the reproduction rate was observed, while by-products affected juvenile survival. A loss of viability and alterations in the cellular proportion was verified during the chronic test. However, the compounds did not alter the behavior of the annelids in the leak test (RL ranged from 20% to 30%). Although photocatalysis has been presented as an alternative technology for the treatment of waste containing the indigo carmine dye, this process produced byproducts even more toxic than the original compounds to E. andrei.

Ozonation of indigo enhanced by carboxylated carbon nanotubes: performance optimization, degradation products, reaction mechanism and toxicity evaluation.

As a promising disinfection technique to replace chlorination, ozonation has been demonstrated to be efficient in water treatment. This paper describes an effective way to enhance the ozonation of indigo by using carbon nanotubes functionalized with carboxyl groups (CNTs-COOH) as catalysts. The result of kinetic studies showed that the presence of CNTs-COOH dramatically increased the decolorization rate of indigo. Different types of catalysts were compared to further elucidate the internal mechanism of the catalytic reaction and the special nanostructure and the functional ­COOH groups are considered to play an important role in the catalytic ozonation process. Four aromatic intermediate products were identified using an electrospray time-of-flight mass spectrometer and further rationalized by the frontier electron density calculations. Ion chromatography analysis revealed that the nitrogen atom of indigo was released predominantly as ammonium and to a lesser extent as nitrate. The presence of the catalyst CNTs-COOH leads to a higher mineralization degree than single ozonation, as suggested by the total organic carbon (TOC) measurement. Three major carboxylic acids (i.e., oxalic, formic and acetic acids) were also identified as oxidation by-products, and they contributed significantly to the residual TOC after 2 h of ozonation. In addition, the toxicity evolution during the degradation was investigated through two aquatic model species to evaluate the potential ecological risks of the intermediate products.

Impact of excipients in the chronic toxicity of fluoxetine on the alga Chlorella vulgaris.

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) widely used in the treatment of major depression. It has been detected in surface and wastewaters, being able to negatively affect aquatic organisms. Most of the ecotoxicity studies focused only in pharmaceuticals, though excipients can also pose a risk to non-target organisms. In this work the ecotoxicity of five medicines (three generic formulations and two brand labels) containing the same active substance (fluoxetine hydrochloride) was tested on the alga Chlorella vulgaris, in order to evaluate if excipients can influence their ecotoxicity. Effective concentrations that cause 50% of inhibition (EC50) ranging from 0.25 to 15 mg L⁻¹ were obtained in the growth inhibition test performed for the different medicines. The corresponding values for fluoxetine concentration are 10 times lower. Higher EC50 values had been published for the same alga considering only the toxicity of fluoxetine. Therefore, this increase in toxicity may be attributed to the presence of excipients. Thus more studies on ecotoxicological effects of excipients are required in order to assess the environmental risk they may pose to aquatic organisms.

Time- and dose-dependent cytotoxicities of ioxitalamate and indigocarmine in human nucleus pulposus cells.

BACKGROUND CONTEXT: Ioxitalamate (Telebrix 300) is an ionic iodinated contrast medium commonly used for discography or percutaneous endoscopic lumbar discectomy (PELD), though it has side effects such as anaphylactic shock and renal toxicity. Indigocarmine is an organic compound dye with a distinctive blue color that is commonly used during PELD to stain the acidic, degenerated nucleus pulposus (NP). Although ioxitalamate and indigocarmine are widely used in spinal surgery, there have been no reports on their effects on NP cells. We studied the toxicities of both ioxitalamate and indigocarmine to NP cells. PURPOSE: To determine the toxicities of both ioxitalamate and indigocarmine to NP cells in vitro. STUDY DESIGN: In vitro, controlled study of the toxicities of both ioxitalamate and indigocarmine to human NP cells. METHODS: Nucleus pulposus cells were obtained via discectomy from lumbar disc patients and isolated. Nucleus pulposus cells were cultured in three-dimensional (3D) alginate beads with 0.001, 0.1, 10, and 100 mg/mL ioxitalamate, 0.00001, 0.001, 0.1, and 10 mg/mL indigocarmine, or a mixture of both for 1, 2, or 3 days. The living cells were analyzed with trypan blue staining. Fluorescence Activated Cell Sorting analysis using Annexin V and propidium iodide and 3D alginate bead immunostaining was performed to identify live, apoptotic, and necrotic cells. RESULTS: Ioxitalamate, indigocarmine, and their combination induced statistically significant NP cell injury that was both time- and dose dependent (p<.05). Also, at the same concentration, ioxitalamate was more cytotoxic than was indigocarmine or the combination (p<.05). All three treatments also showed dose-dependent cytotoxicity according to flow cytometry and immunostaining. CONCLUSIONS: Ioxitalamate and indigocarmine are toxic to human NP cells in vitro in a time- and dose-dependent manner. We assume that ioxitalamate and indigocarmine may have similar effects in patients undergoing discography and PELD. Thus, we suggest that ioxitalamate and indigocarmine should be used carefully at low concentrations.

Toxicity of xanthene food dyes by inhibition of human drug-metabolizing enzymes in a noncompetitive manner.

The synthetic food dyes studied were rose bengal (RB), phroxine (PL), amaranth, erythrosine B (ET), allura red, new coccine, acid red (AR), tartrazine, sunset yellow FCF, brilliant blue FCF, and indigo carmine. First, data confirmed that these dyes were not substrates for CYP2A6, UGT1A6, and UGT2B7. ET inhibited UGT1A6 (glucuronidation of p-nitrophenol) and UGT2B7 (glucuronidation of androsterone). We showed the inhibitory effect of xanthene dye on human UGT1A6 activity. Basic ET, PL, and RB in those food dyes strongly inhibited UGT1A6 activity, with IC(50) values = 0.05, 0.04, and 0.015 mM, respectively. Meanwhile, AR of an acidic xanthene food dye showed no inhibition. Next, we studied the inhibition of CYP3A4 of a major phase I drug-metabolizing enzyme and P-glycoprotein of a major transporter by synthetic food dyes. Human CYP3A4 and P-glycoprotein were also inhibited by basic xanthene food dyes. The IC(50) values of these dyes to inhibit CYP3A4 and P-glycoprotein were the same as the inhibition level of UGT1A6 by three halogenated xanthene food dyes (ET, PL, and RB) described above, except AR, like the results with UGT1A6 and UGT2B7. We also confirmed the noninhibition of CYP3A4 and P-gp by other synthetic food dyes. Part of this inhibition depended upon the reaction of (1)O(2) originating on xanthene dyes by light irradiation, because inhibition was prevented by (1)O(2) quenchers. We studied the influence of superoxide dismutase and catalase on this inhibition by dyes and we found prevention of inhibition by superoxide dismutase but not catalase. This result suggests that superoxide anions, originating on dyes by light irradiation, must attack drug-metabolizing enzymes. It is possible that red cosmetics containing phloxine, erythrosine, or rose bengal react with proteins on skin under lighting and may lead to rough skin.

DNA damaging effects of the dyes used in sentinel node biopsy: possible implications for clinical practice.

OBJECTIVE: This study investigates whether methylene blue (MB), patent blue V (PBV), and indigo carmine (IDC) commonly used in sentinel node biopsy cause DNA damage to breast epithelial cells in vitro. METHODS: MCF-7 and HB-2 cells were exposed for 5 minutes to the above dyes at the same concentrations used in clinical practice. Following exposure, the comet assay was performed to detect DNA damage. The enzyme, Fapy-DNA glycosylase (FpG) was incorporated to enable the detection of additional oxidative damage. RESULTS: Both PBV and MB stimulated DNA strand breaks in both MCF-7 and HB2 cell lines (P < 0.05). Levels were elevated over 3-fold (P < 0.05) in MCF-7 and HB2 cells treated with 2.5% PBV and 1% MB, compared with untreated control cells. In contrast, IDC did not stimulate DNA strand break damage at clinically relevant concentrations in either cell line. Addition of Fapy-DNA glycosylase enzyme also revealed significantly (P < 0.05) increased levels of oxidative DNA lesions (ODL) in MCF-7 cells treated with PBV (17.6% ODL) compared with control cells (5.9% ODL). CONCLUSIONS: This study shows, for the first time, that certain dyes (MB and PBV) commonly used in SLNB have genotoxic effects on breast cells at clinically relevant concentrations in vitro. In vivo studies are now warranted to assess and minimize DNA damage caused by these dyes during SLNB.

[Stereum hirsutum (Wild) Pers. action in dye degradation]. / Acción de Stereum hirsutum (Wild) Pers. en la degradación de colorantes.

Stereum hirsutum, a white rot fungus, has a good growth in solid state fermentation. This was carried on with wheat bran, soy bran and a mixture of both. Mycelia grown on soy bran showed the highest decolorization activity on Ponceau 2R (xylidine), indigo carmine and malachite green. Optimal relationship between decolorization and detoxification of malachite green was 30 g of fresh weight (mycelia plus substrate) in 500 ml malachite green solution, 42 U/l of laccase was measured in this solution. Decolorization was carried on without the addition either of nutrients or mediators. Conditions for maximal decolorization did not agree with those for maximal ligninolytic enzyme production, but effectiveness of laccase activity on decolorization was evidenced by electrophoretic analysis, that allowed laccase identification and its decolorization activity in gels stained with indigo carmine and malachite green, with ABTS as mediator. Detoxification was assayed using the sensible fungus Phanerochaete chrysosporium.

Adhesion-promoting properties of dyes routinely used during fertility surgeries.

PURPOSE: With the link between peritoneal adhesions and infertility well established, it is critical that materials used in pelvic surgery be tested for their adhesion-forming properties. The current study examined the adhesion-inducing properties of two dyes routinely used for visualization during pelvic surgery. DESIGN: In vivo and in vitro examination of the effects of the dyes methylene blue and indigo carmine on adhesion formation in a mouse model. METHOD: A series of three experiments was conducted. In the first, dyes were injected directly into the peritoneal cavity. The mice were then sacrificed at one of two time points and the peritoneal cavity examined for adhesion formation. In addition, because of their purposed role in adhesion formation, macrophages from the cavity were examined for signs of dye-induced activation. Further studies of macrophage activation were then conducted in vitro to determine the effects of dye concentration and exposure time on the activation process. RESULTS: Both methylene blue and indigo carmine appeared to induce adhesion formation as well as macrophage activation in vivo. Further, long-term exposure to visual concentrations of both dyes appeared to induce macrophage activation. However, only those macrophages exposed to methylene blue exhibited signs of activation when the exposure time was limited to times equivalent to those which might be expected during surgery. CONCLUSION: Of the two dyes tested, indigo carmine might be the dye of choice in surgeries where fertility is to be maintained.

Methylene blue but not indigo carmine is toxic to human luteal cells in vitro.

Methylene blue (MB) is reported to be teratogenic when injected intra-amniotically. Indigo carmine (IC) appears to be a safe alternative. To determine if MB has potential detrimental effects on ovarian tissue, we compared the effect of MB and IC on human granulosa luteal cell (GC) function in vitro. Human oocyte-cumulus complexes were obtained during in vitro fertilization cycles and one to three were placed in an organ culture dish. After insemination with sperm, oocytes were removed the day after retrieval and the attached GC were washed daily for 3 more days by changing 2 mL of culture medium. All the dishes were treated with human chorionic gonadotropin (hCG) for the next 24 h and progesterone (P) production during this interval was taken as baseline. Test chemicals were added with hCG for the next 48 h with daily media changes. The P production during the last 24 h of chemical treatment was expressed as a percentage of the baseline. MB significantly reduced P production whereas IC did not appear to have any effect. Moreover, under inverted microscopy more than 90% of the GC cells contained several small bluish intracellular granules when exposed to 0.01% MB but not 0.01% IC. These results indicate that MB may be taken up and processed by GC cells and inhibits P production. This finding adds to previous reports on the use of in vitro GC assay to identify potential reproductive toxicants. The clinical significance of this preliminary study needs further investigation.

Evaluation of the potential teratogenicity of FD & C Blue No. 2 in rats and rabbits.

Charles River CD rats (20 pregnant rats/group) received by gavage on days 6-15 of gestation 0.5% Methocel (controls, A, B and C), retinoic acid at 7.5 mg/kg/day or FD & C Blue No. 2 in doses of 25, 75 or 250 mg/kg/day. Pregnant Dutch belted rabbits (ten pregnant does/group) received by gavage on days 6-18 of gestation 0.5% Methocel (controls A, B and C), thalidomide at 150 mg/kg/day or FD & C Blue No. 2 in doses of 25, 75 or 250 mg/kg/day. All animals were observed twice daily during gestation for signs of toxicity. The animals were killed 1 day before term and appropriate maternal and foetal parameters were evaluated. There were no consistent, significant compound-related adverse effects on any of these parameters. Foetal malformations occurred in both positive control groups. Under the conditions of this study, FD & C Blue No. 2 did not exert any teratogenicity or other developmental toxicity in either rats or rabbits.

Chronic toxicity/carcinogenicity study of FD & C Blue No. 2 in mice.

Charles River CD-1 mice were fed FD & C Blue No. 2 in the diet levels of 0.5, 1.5 and 5.0% in a long-term toxicity/carcinogenicity study. Each group consisted of 60 males and 60 females. Two concurrent control groups each of 60 males and 60 females received the basal diet. Maximum exposure was 23 months. No consistent compound-related or statistically significant biologically adverse effects were noted.

Chronic toxicity/carcinogenicity study of FD & C Blue No. 2 in rats.

FD & C Blue No. 2 was fed to rats in the diet in a long-term toxicity/carcinogenicity study. The study included an in utero phase in which the compound was administered to groups of 60 male and 60 female Charles River CD albino rats at levels of 0.5, 1.0 and 2.0%. Two concurrent control groups, each containing 60 rats of each sex, received the basal diet. After random selection of the F1 animals, the long-term phase was initiated at the same dietary levels, with 70 rats of each sex in each dose group and in each of two control groups. Maximum exposure was 30 months. No consistent compound-related biologically adverse effects were noted. There were random statistically significant differences from the controls with respect to body weight, food consumption and clinical chemistry tests. Food consumption by the test groups showed a dose-related increase. This was probably due to the non-nutritive character of the colouring. A statistically significant increase in gliomas in the high-dose male rats was not found to be biologically significant, since none of the criteria for determining the neurocarcinogenic potential of chemical substances was met. The overall brain-tumour incidence in this study was within the range typical for 2-yr-old CD rats. Under the conditions of this study, FD & C Blue No. 2 did not produce evidence of any toxicity, including carcinogenicity.

[Mutagenic effect of the food-coloring agents tartrazine and indigo carmine]. / Izuchenie mutagennogo deistviia pishchevykh krasitelei tartrazina i indigokarmina.

The authors studied the mutagenic action of the food dyes, tartrazine (both Soviet and imported) and indigocarmine in a microbial model and in warm-blooded animals (linear mice). Determined the toxicity and mutagenic action of the dyes on E. coli, strain K-12, carried out chromosomal analysis of the bone marrow, examined the dominant lethals in CBA X C57BL/6 mice. The recommended daily dose amounts to 400 mg/kg for tartrazine and to 50 mg/kg for indigocarmine with regard to the safety factor equal to 100. The data derived as a result of studying the mutagenic activity of tartrazine manufactured in the USSR and CSSR and indigocarmine paste in 3 experimental models allow the conclusion to be made that the doses of these dyes applied in food industry are fairly safe.