Batch and continuous biodegradation of Amaranth in plain distilled water by P. aeruginosa BCH and toxicological scrutiny using oxidative stress studies.

Bacterium Pseudomonas aeruginosa BCH was able to degrade naphthylaminesulfonic azo dye Amaranth in plain distilled water within 6 h at 50 mg l(-1) dye concentration. Studies were carried out to find the optimum physical conditions and which came out to be pH 7 and temperature 30 °C. Amaranth could also be decolorized at concentration 500 mg l(-1). Presence of Zn and Hg ions could strongly slow down the decolorization process, whereas decolorization progressed rapidly in presence of Mn. Decolorization rate was increased with increasing cell mass. Induction in intracellular and extracellular activities of tyrosinase and NADH-DCIP reductase along with intracellular laccase and veratryl alcohol oxidase indicated their co-ordinate action during dye biodegradation. Up-flow bioreactor studies with alginate immobilized cells proved the capability of strain to degrade Amaranth in continuous process at 20 ml h(-1) flow rate. Various analytical studies viz.--HPLC, HPTLC, and FTIR gave the confirmation that decolorization was due to biodegradation. From GC-MS analysis, various metabolites were detected, and possible degradation pathway was predicted. Toxicity studies carried out with Allium cepa L. through the assessment of various antioxidant enzymes viz. sulphur oxide dismutase, guaiacol peroxidase, and catalase along with estimation of lipid peroxidation and protein oxidation levels conclusively demonstrated that oxidative stress was generated by Amaranth.

Genotoxicity assessment of amaranth and allura red using Saccharomyces cerevisiae.

Amaranth (E123) and Allura red (E129), very important food azo dyes used in food, drug, paper, cosmetic and textile industries, were assessed for their genotoxic potential through comet assay in yeast cells. Comet assay was standardized by with different concentration of H(2)O(2). Concentrations of Amaranth and Allura red were maintained in sorbitol buffer starting from 9.76 to 5,000 µg/mL and 1 × 10(4) cells were incubated at two different incubation temperatures 28 and 37°C. Amaranth (E123) and Allura red (E129) were found to exhibit their genotoxic effect directly in Saccharomyces cerevisiae. No significant genotoxic activity was observed for Amaranth and Allura red at 28°C but at 37°C direct relation of Amaranth concentration with comet tail was significant and no positive relation was seen with time exposure factor. At 37°C the minimum concentration of Amaranth and Allura red at which significant DNA damage observed through comet assay was 1,250 µg/mL in 2nd h post exposure time. The results indicated that food colors should be carefully used in baking products as heavy concentration of food colors could affect the fermentation process of baking.

Evaluation of potential genotoxicity of five food dyes using the somatic mutation and recombination test.

In this study, different concentrations of five food dyes (amaranth, patent blue, carminic acid, indigotine and erythrosine) have been evaluated for genotoxicity in the Somatic Mutation and Recombination Test (SMART) of Drosophila melanogaster. Standard cross was used in the experiment. Larvae including two linked recessive wing hair mutations were chronically fed at different concentrations of the test compounds in standard Drosophila Instant Medium. Feeding ended with pupation of the surviving larvae. Wings of the emerging adult flies were scored for the presence of spots of mutant cells which can result from either somatic mutation or somatic recombination. For the evaluation of genotoxic effects, the frequencies of spots per wing in the treated series were compared to the control group, which was distilled water. The present study shows that carminic acid and indigotine demonstrated negative results while erythrosine demonstrated inconclusive results. In addition 25 mg mL(-1) concentration of patent blue and 12.5, 25 and 50 mg mL(-1) concentrations of amaranth demonstrated positive results in the SMART.

Differential colon DNA damage induced by azo food additives between rats and mice.

Azo dyes, amaranth, allura red and new coccine, which are currently used as food color additives in Japan, have been reported to cause colon specific DNA damage in mice. To examine species difference in the DNA damage between rats and mice, each of dyes was administered to male mice (1 and 10 mg/kg) and male rats (10, 100 and 1,000 mg/kg) by gavage. Brain, lung, liver, kidney, glandular stomach, colon, urinary bladder and bone marrow were sampled 3 hr (for mice) and 3, 6, 12 and 24 hr (for rats) after the treatment. The alkaline comet assay showed DNA damage in the mouse colon 3 hr after the administration of all of the dyes at 10 mg/kg. In rats, however, none of the dyes damaged DNA. Azo dyes should undergo metabolic reduction in the colon to be adducted to DNA. To determine transit time of the dyes to the colon after their administration, gastric emptying and intestinal transport in mice and rats were examined using brilliant blue FCF (BB) as an indicator. The half times of gastric emptying were 70 and 80 min for mice and rats, respectively; and about 60% of the BB was removed from the stomach 1 hr after the gastric intubation in both mice and rats. BB reached the mouse and rat colon 1 and 3 hr after the administration, respectively. Considering the wide dose range and sampling times well covering the transit time to the colon, rats may be insensitive to these azo dye-induced DNA damage.

Cytogenetic evaluation and DNA interaction studies of the food colorants amaranth, erythrosine and tartrazine.

Food coloring agents, amaranth, erythrosine and tartrazine have been tested at 0.02-8mM in human peripheral blood cells in vitro, in order to investigate their genotoxic, cytotoxic and cytostatic potential. Amaranth at the highest concentration (8mM) demonstrates high genotoxicity, cytostaticity and cytotoxicity. The frequency of SCEs/cell was increased 1.7 times over the control level. Additionally, erythrosine at 8, 4 and 2mM shows a high cytotoxicity and cytostaticity. Finally, tartrazine seems to be toxic at 8 and 4mM. No signs of genotoxicity were observed. Reversely, tartrazine showed cytotoxicity at 1 and 2mM. Furthermore, spectroscopic titration studies for the interaction of these food additives with DNA showed that these dyes bind to calf thymus DNA and distinct isosbestic points are observed clearly suggesting binding of the dyes to DNA. Additionally DNA electrophoretic mobility experiments showed that these colorants are obviously capable for strong binding to linear dsDNA causing its degradation. PCR amplification of all DNA fragments (which previously were pre-treated with three different concentrations of the colorants, extracted from agarose gel after separation and then purified), seems to be attenuated with a manner dye concentration-dependent reflecting in a delayed electrophoretic mobility due to the possible binding of some molecules of the dyes. Evaluation of the data and curves were obtained after quantitative and qualitative analysis of the lanes of the gel by an analyzer computer program. Our results indicate that these food colorants had a toxic potential to human lymphocytes in vitro and it seems that they bind directly to DNA.

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.

Lack of genotoxic effect of food dyes amaranth, sunset yellow and tartrazine and their metabolites in the gut micronucleus assay in mice.

The food dyes amaranth, sunset yellow and tartrazine were administered twice, at 24h intervals, by oral gavage to mice and assessed in the in vivo gut micronucleus test for genotoxic effects (frequency of micronucleated cells) and toxicity (apoptotic and mitotic cells). The concentrations of each compound and their main metabolites (sulfanilic acid and naphthionic acid) were measured in faeces during a 24-h period after single oral administrations of the food dyes to mice. Parent dye compounds and their main aromatic amine metabolites were detected in significant amounts in the environment of colonic cells. Acute oral exposure to food dye additives amaranth, sunset yellow and tartrazine did not induce genotoxic effect in the micronucleus gut assay in mice at doses up to 2000 mg/kg b.w. Food dyes administration increased the mitotic cells at all dose levels when compared to controls. These results suggest that the transient DNA damages previously observed in the colon of mice treated by amaranth and tartrazine by the in vivo comet assay [Sasaki, Y.F., Kawaguchi, S., Kamaya, A., Ohshita, M., Kabasawa, K., Iwama, K., Taniguchi, K., Tsuda, S., 2002. The comet assay with 8 mouse organs: results with 39 currently used food additives. Mutat. Res. 519, 103-119] are unable to be fixed in stable genotoxic lesions and might be partly explained by local cytotoxicity of the dyes.

Investigation of the toxicity of the products of decoloration of Amaranth by Trametes versicolor.

Trametes versicolor decolorized 2000 mg L(-1) of the mono-azo substituted naphthalenic dye Amaranth with no dye sorption observed visually. The changes in the toxicity were assessed over a period of 30 d for the dye-treated viable culture, control (no dye added), and a boiled culture treated with dye, using the Microtox Acute Toxicity assay. Before dye addition, the culture filtrate had some toxicity, which increased after the dye addition. The toxicity of the dye-treated culture decreased during the treatment. The loss of toxicity occurred at the same time, with the loss of color suggesting that detoxification is associated with decoloration. The change in pH was due to natural metabolic processes and had a small effect on detoxification. Because the toxicity of the treatment was similar to that of the control at the end of the treatment, the effluent seems to be safe for release into the environment, potentially rendering this treatment suitable for industrial application.

Immunological aspects of the common food colorants, amaranth and tartrazine.

We describe a sensitive and reproducible microassay model using human peripheral blood lymphocytes (PBL) for discrimination between the cytotoxic and immunosuppressive effects of food colorants such as amaranth and tartrazine. The cytotoxic effects of a wide range of concentrations of these substances were studied on human PBL by the colorimetric in vitro cytotoxicity assays, neutral red uptake (NR) and thiazolyl blue tetrazolium bromide (MTT). The immunotoxic properties of these 2 substances were determined by a [3H]-thymidine DNA incorporation assay on phytohemagglutinin stimulated or non-stimulated lymphocytes, as well as by a Cr51 release Natural Killer assays. The results showed clear immunosuppressive effects from the 2 substances tested, although the concentrations chosen for this study proved to be non-cytotoxic by NR and MTT cytotoxic endpoints.

Buckwheat protein extract suppression of the growth depression in rats induced by feeding amaranth (Food Red No. 2).

Dietary fiber has an ameliorative effect on the toxicity of amaranth (Food Red No. 2). To test the possibility that a buckwheat protein extract (BWPE) has dietary fiber-like activity by virtue of its low digestibility, we examined the influence of BWPE on amaranth toxicity in rats. The results show that BWPE-containing diet suppressed the growth depression induced by the dietary addition of 5% amaranth.

Genotoxicity testing of two red dyes in the somatic and germ line cells of Drosophila.

Two red dyes, rhodamine B and amaranth, were tested for their genotoxic effects in the somatic (wing primordia) and germ line cells of Drosophila melanogaster following the wing spot and the sex-linked recessive lethal tests. Second- and third-instar larvae, carrying suitable genetic markers, were subjected to chronic exposure to different concentrations of the test dyes. The results indicate that rhodamine is genotoxic in both somatic and germ line cells and amaranth is non-genotoxic.

Interlaboratory evaluation of embryotoxicity in the postimplantation rat embryo culture.

The embryotoxicity of eight xenobiotic compounds in rat postimplantation whole embryo culture was blindly tested in four laboratories according to a standard protocol. The results show that the four nonteratogens amaranth, penicillin, isoniazid, and saccharin did not affect embryogenesis apart from general toxicity at very high concentrations in culture for amaranth and isoniazid. There was good concordance of results across the laboratories. The four teratogens (retinoic acid, 6-aminonicotinamide, acetylsalicylic acid, and vincristine) induced a variety of specific embryotoxic effects, which were in most cases similar in all laboratories. These results indicate that the definition for specific embryotoxicity used, as well as the culture duration and embryonic age are crucial for concordant scoring. Other methodologic differences did not significantly influence scoring of embryotoxicity. Therefore, within the limits of the end points and embryonic stage represented in the method, embryo culture appears as a useful method for embryotoxicity screening, which can be reproducibly applied in different laboratories.

Reproductive and neurobehavioral effects of amaranth administered to mice in drinking water.

The color additive amaranth was given in the drinking water at levels of 0 (control), 0.025, 0.075, and 0.225% from 5 weeks of age in F0 generation until F1 generation mice were weaned, with selected reproductive, developmental and behavioral parameters being measured. Amaranth had little adverse effect upon litter size, litter weight and sex ratio. Average body weight in both sexes of the F1 mice was significantly increased in the 0.025% group in both sexes. Survival index at postnatal day (PND) 21 was reduced in the 0.025% amaranth group. For the neurobehavioral parameters, surface righting at PND 4 in female offspring and olfactory orientation in both sexes were significantly affected by treatment. Several parameters of movement activity of male offspring at 3 weeks of age were affected in amaranth 0.075% group, but those of female offspring were similar in all groups. The dose levels of amaranth in this study produced a little adverse effect on behavioral development in mice.

Effects of amaranth on F1 generation mice.

The color additive, amaranth, was given in the diet to provide dietary levels of 0 (control), 0.03, 0.09 and 0.27%, from 5 weeks of age in F0 generation mice to 9 weeks of age in F1 generation mice, and some reproductive, developmental and behavioral parameters were measured. There was no effect on the parameters of litters, litter size, pup weight and litter weight. The body weight of pups during the lactation period in the treatment groups increased less significantly, and the survival index at postnatal day (PND) 21 of the amaranth 0.27% group was reduced. Developmental parameters, direction of swimming on PND 4 in male pups and olfactory orientation in each sex were significantly reduced in the treatment groups. The dose levels of amaranth in this study influenced some reproductive, developmental and behavioral parameters in mice.

[Mutagenicity testing of some azo dyes used as food additives]. / Gidalara katilan bazi azo boyalarinin mutajenik etkilerinin test edilmesi.

The mutagenicity of 4 azo dyes (Ponceau 4R, Amaranth, Sunset Yellow FCF and Tartrazine) that are widely used to color food has been evaluated. They were tested for mutagenicity in the Salmonella typhimurium plate-incorporation and preincubation assays. The standard plate-incorporation and preincubation assays performed directly on the dyes in the absence and presence of rat-liver S9. No mutagenic activity was seen for any of the azo dyes tested by using the standard tester strains, TA 98 and TA 100.

Further validation of FETAX: evaluation of the developmental toxicity of five known mammalian teratogens and non-teratogens.

The developmental toxicity of five compounds was evaluated with the Frog Embryo Teratogenesis Assay: Xenopus (FETAX). Late Xenopus laevis blastulae were exposed to 5-azacytidine, methotrexate, pseudoephedrine, aspartame, and amaranth for 96 h. Three separate static-renewal assays were conducted for each compound. Based on Teratogenic Index [LC50/EC50 (malformation)] values, types and severity of induced malformations, and embryo growth, 5-azacytidine and methotrexate tested as having strong teratogenic potential. Pseudoephedrine scored as having moderate teratogenic potential, but amaranth and aspartame had little or no teratogenic potential. Results support the use of FETAX for the screening of developmental toxicants.

Long-term toxicity study of amaranth in rats using animals exposed in utero.

Groups of 90 (control) and 54 (treated) rats of each sex were given amaranth in their diet to provide daily intakes of 0 (control), 50, 250 or 1250 mg/kg for 111 wk (male) and 112 wk (female) after weaning. The rats had also been exposed to the same dose levels in utero, and their parents were exposed for 60 days before mating. The colouring had no adverse effects on fertility, haematological parameters, serum chemistry or incidence of tumours. All treated animals showed contamination of the fur and red colouring of the faeces and at the high dose only the faecal pellets were poorly formed. Rats in the high-dose group produced more pups, and the average pup weight was lower than that of the controls. Rats of the F1 generation given the highest dose level were slightly lighter than the controls despite a small increase in food and water intake. Both sexes given the highest dose level and males given 250 mg/kg/day had increased caecal weight. High-dose females excreted more protein in the urine after 18 months and on histopathological examination females in all treated groups showed an increased incidence of renal calcification and pelvic epithelial hyperplasia with degenerative changes. It is concluded that amaranth fed to rats at dose levels of up to 1250 mg/kg/day in the diet did not have any carcinogenic effect. However, because of the effect on the kidneys of the females it was not possible to establish a no-untoward-effect level in this study.

The weight of evidence: regulatory toxicology in Canada.

The legislative application of regulatory toxicology in Canada is reviewed, together with the sources of experimental evidence used for action. Examples are given of the critical toxicological information that led to a regulatory decision. Risk numbers have only been used to a limited extent in Canada. Some possibilities for future research are offered.

Configurational changes in rat liver nuclear chromatin caused by azo dyes.

The effects of azo dyes on the chromatin configuration of isolated rat-liver nuclei were examined by electron microscopy for elucidation of the mechanism of dye-stimulated in vitro RNA synthesis. Sunset Yellow FCF had no effect on the incorporation of [3H]UTP into RNA but amaranth and Ponceau 3R stimulated incorporation approximately two- and 3.5-fold, respectively. Sunset Yellow FCF did not produce any major alteration in liver nuclei. Isolated liver nuclei treated with amaranth or Ponceau 3R showed dissociation of heterochromatin and the nucleoli lost their fine-granular structure acquiring a coarse-granular configuration. The dense structure of heterochromatin in liver nuclei treated with Ponceau 3R was completely dissociated and appeared as a lace-like configuration. These results suggest that both Ponceau 3R and amaranth stimulate in vitro RNA synthesis by causing the dissociation of heterochromatin in isolated rat-liver nuclei.

Mechanisms of adverse effect of amaranth feeding in the rat.

In order to clarify the mechanism of the adverse effects of dietary amaranth, trisodium 1-(4-sulfo-1-naphthylazo)-2-naphthyl-3,6 disulfonic acid, the effects of amaranth in vitro and in a jejunum perfusion in vivo on intestinal sucrase were investigated in rats. The inhibitory effect of amaranth in vitro on the sucrase activity was not detected even at the concentration of 1%, whereas the remarkable release of intestinal sucrase from intestine was observed with the jejunum perfusion of Ringer bicarbonate solution (RBS) containing amaranth at the 1% level. On the other hand, the perfusion of RBS containing tris(hydroxymethyl)-aminomethane, a strong inhibitor of intestinal disaccharidase activities, did not produce the release of intestinal alkaline phosphatase. These findings suggest that the toxicity of dietary amaranth is due to the exfoliating or solubilizing effects of amaranth on the brush border membrane of the small intestine.