Rec Assay Research Articles

Toxicological assessment of Euglena gracilis strain Eu029 shows no adverse effects in vivo and in vitro

Euglena gracilis is a single-celled organism capable of photosynthesis and heterotrophy. Euglena sp. have long been studied in the laboratory for its metabolic pathways, cell motility, and ease of culture. The safety of E. gracilis strain eu029 (EG029) for use as a food ingredient was assessed in a bacterial reverse mutagenesis assay (Ames), rec assay, in vivo micronucleus assay, acute toxicity study in mice, 13-week toxicology in rats, and a teratology study in mice and rats. EG029 was not genotoxic. The No Observed Adverse Effect Level (NOAEL) in the 13-week study was greater than 1000 mg/kg/day, the highest dose tested. Teratogenicity studies did not find any defects in fetal development or effects to maternal health in rats at 1000 mg/kg/day, the highest dose tested.

Genotoxic potentials and eukaryotic DNA topoisomerase I inhibitory effects of some benzoxazine derivatives

Benzoxazines are heterocyclic compounds which have been used as intermediates in the synthesis of many heterocyclic structures of biological importance as it has been reported that some of the benzoxazines were effective in promoting apoptosis and inhibiting cell proliferation. Present study contains experimental data that showed genotoxic potentials and inhibitory effects on eukaryotic DNA topoisomerase I of 16 newly synthesized benzoxazine derivatives. By rec assay, the bacterial genotoxicity assay, only four tested compounds were found genotoxic at different concentrations and four compounds showed reverse effect. RC50 values evaluated by rec assay revealed that BS5 was the most genotoxic and BS4 was the most cytotoxic compound at micromolar concentration. Compounds were also tested for their inhibitory effects on eukaryotic DNA topoisomerase I enzyme and it was found that 14 of the compounds had inhibitory effects on eukaryotic DNA topoisomerase I enzyme. The most active compounds, BS18 and BS4, showed higher inhibitory activities than the positive control drug camptothecin which is a well-known commercial topoisomerase I inhibitor.

Tracking growth hormone abuse in sport: A comparison of distinct isoform-based assays

Detecting recombinant human growth hormone (rhGH) abuse in sport remains one of the major challenges in doping control. We have compared two different approaches to detect the hGH (human growth hormone) abuse. The first measures the concentrations of the 22kDa hGH isoform (rec assay) and pituitary derived isoforms (pit assay) and a ratio rec/pit is obtained. The second measures the concentrations of 22 and 20kDa hGH isoforms and also a ratio 22/20kDa is derived.Using a single set (nine healthy male subjects, 7 days, 0.026mg/kg/day of rhGH, 2 week wash out period) both approaches were compared. To quantify the agreement between the immunoassays, B.A. (Bland–Altman) analysis and P.r. (Pearson correlation) were used. To fully understand the assay readings, all relevant antibodies were characterised by surface plasmon resonance (SPR).In either approach the ratio numerator produces similar results and the denominator determines both signal-amplitude and time-frame of possible application. The rec vs pit approach displays a higher distinctive capacity to detect hGH abuse but the complex binding properties of the capture antibodies make it very difficult to evaluate the precise contributions of the individual hGH variants to the assay result. In the 22 vs 20 approach, the 20kDa hGH concentration measures determine its applicability.Both approaches are based on a different principle, should be preferably applied within 24h after rhGH administration, and are perfectly comparable given the results obtained. The reduced time frame of application indicates that their principle application should be preferably in an out-of-competition setting.

An investigation of antigenotoxic properties of plant extracts of Chelidonium majus L., Plantago major L. and Tussilago farfara L.

The antigenotoxic properties of the sap from three medicinal plants, the greater celandine (Chelidonium majus L.), the greater plantain (Plantago major L.), and coltsfoot (Tussilago farfara L.), were studied using two bacterial test systems (SOS chromotest and Rec assay). It was determined using the combined effect of the plant sap and model genotoxicants on the cells of test strains that the plant sap of the greater celandine reveals a dismutagenic effect decreasing the genotoxic effect of nalidixic acid in the SOS chromotest and furacilin in the Rec assay. The plants sap of the greater celandine and coltsfoot (in 1: 10 and 1: 100 dilutions) demonstrated a bioantimutagenic effect in the SOS chromotest because pre-incubation of the E. coli PQ37 cells test strain with the sap of these plants resulted in a significant decrease of the genotoxic effect of nalidixic acid. The antigenotoxic effect of the greater plantain sap was not statistically significant in both test systems. Possible mechanisms of determining the antigenotoxic properties of the plant sap from greater celandine and coltsfoot are discussed.

Evaluation of antigenotoxic effects of juices of plants Chelidonium majus L., Plantago major L. и Tussilago farfara L.

The antigenotoxic effects of juices of three medicinal plants, Chelidonium majus L., Plantago major L. and Tussilago farfara L. has been studied in two bacterial tests — SOS chromotest and Rec assay. Antigenotoxic effect was determined against known genotoxic substances — nalidixic acid in SOS chromotest and furacilin in Rec assay. Preparations obtained from the leaves of Ch. majus L. exhibited significant antigenotoxic effect in both the SOS chromotest and the Rec assay. It was shown that dilution of the herb juice of T. farfara L resulted in high bioantimutagenic activity in SOS chromotest. P. major L. preparations did not display statistically significant antigenotoxic activity in the both tests used. Possible mechanisms of antigenotoxic effects of Ch. majus L. and T. farfara L. plants obtained are discussed.

Mutagenic and Antimutagenic Effects of the Traditional Herb Used for Treating Erectile Dysfunction,Butea superbaRoxb.

Butea superba is a traditional tuberous Thai plant enriched with flavonoids that is used for treating erectile dysfunction. We investigated the mutagenic and antimutagenic potentials of a B. superba extract by using the pre-incubation method of the Ames test. Salmonella typhimurium strains TA 98 and TA 100 were applied as the tester strains in the presence and absence of an S9 mixture. Prior to the mutagenic and antimutagenic tests, the survival of the tester strains was measured by treating with the B. superba extract. The results show that the B. superba extract exhibited dose-dependent cytotoxic effects. Data from the Ames test revealed that the B. superba extract to be non-mutagenic in the presence and absence of the S9 mixture. In contrast, the B. superba extract showed antimutagenic potential towards either or both of the tested mutagens: 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2) and benzo(a)pyrene (B(a)P) in the respective presence and absence of the S9 mixture, respectively. The plant antimutagenic activity was confirmed by a rec assay. A further study by micronucleus test demonstrated that the B. superba extract at the maximum loading volume could induce acute micronucleus formation in the tested animals. The in vitro mutagenic and antimutagenic assays confirmed the safe consumption of B. superba products at low dose (not more than 781.25 microg/ml of the plant extract), but the in vivo genotoxic assay demonstrated the unsafe consumption at a high dose (300 mg/kg of the BW plant extract or 16 g/kg of the BW plant powder).

Free radical scavenging activities and inhibition of inflammatory enzymes of phenolics isolated from Tripodanthus acutifolius

Ethnopharmacological relevance Leaf extracts from Tripodanthus acutifolius (Ruiz and Pavón) Van Tieghem have long been used in argentinean traditional medicine as anti-inflammatory, however, there is no scientific evidence which supports this use in the literature. Aim of the study The present study was conducted to evaluate the ability of five phenolic compounds purified from infusion prepared from Tripodanthus acutifolius leaves to inhibit key enzymes in inflammatory processes. As anti-inflammatory compounds frequently possess free radical scavenging activities, purified substances were comparatively evaluated to asses their free radical scavenging properties. Genotoxic effects were also evaluated. Materials and Methods Compounds were evaluated on their ability to inhibit hyaluronidase and cyclooxygenase-2 (COX-2) activities to assess their anti-inflammatory capacities. Free radical scavenging activity was assessed by: 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH), superoxide anion assay and the inhibition on lipid peroxidation. Genotoxicity was evaluated by Bacillus subtilis rec assay. Results Fractionation of Tripodanthus acutifolius infusion yielded a novel phenylbutanoid derivative (tripodantoside) and four known flavonoid glycosides (rutin, nicotiflorin, hyperoside and isoquercitrin). Flavonoids produced higher inhibition on hyluronidase activity (IC 50 ≈ 1.7 mM) than tripodantoside (IC 50 = 27.90 mM). A similar COX-2 inhibition activity was exerted by tripodantoside and monoglycosilated flavonoids (IC 50 ∼ 50 μM). Compounds were strong radical scavengers, with effective concentration 50 (EC 50) values for DPPH in the range of 2.7–6.3 μg/mL, and for superoxide anion in the range of 3.9–8.7 μg/mL. All compounds scavenged peroxyl radicals in the lipid peroxidation assay. The substances showed no genotoxic effects. Conclusions The anti-inflammatory effects, free radical scavenging activities and lack of genotoxicity of purified compounds may support the folk use of infusion from Tripodanthus acutifolius leaves as anti-inflammatory.

A study on the genotoxic activities of some new benzoxazoles

The Bacillus subtilis rec assay has been specially developed to detect DNA-damaging potential in chemicals, with the rationale based on the relative difference of survival of a DNA repair combination proficient strains and its defi- cient strain, which is interpreted as genotoxicity. The genotoxic activities of newly (1-6) and previously (7-18) synthesized various benzoxazoles and benzimidazoles were analyzed via the B. subtilis rec assay. Newly obtained benzoxazole and benzimidazole derivatives (1-6) were synthesized in the presence of polyphosphoric acid (PPA) and 6 N HCl, respectively to detect their DNA-damaging activities. Among the tested compounds, 6-methyl-2-(o-chlorophenyl)benzoxazole (9), 5- amino-2-(p-methylbenzyl)benzoxazole (4), 5-(p-fluorobenzamido)-2-phenylbenz- oxazole (13), and 2-(p-methylaminophenyl)benzoxazole (18) showed genotoxic activities having Rec50 values of 1.85, 1.74, 1.60, and 1.50 or S-probit values of 0.534, 0.482, 0.460, and 0.357, respectively. On the other hand, 2-(p-bromobenzyl)- 5-methylbenzimidazole (6) and 2-benzyl-5-(p-fluorophenylacetamido)-benzoxazole (15) were exhibited a reverse effect that displayed a bacterial growth in the rec - strains while there was no any bacterial growth in rec + strains at the same con- centration.

SOS-red fluorescent protein (RFP) bioassay system for monitoring of antigenotoxic activity in plant extracts

An optical antigenotoxicity assay using genetically engineered red fluorescent bacteria is presented. Exposure of Escherichia coli RS4U to genotoxicants [mitomycin C (MMC), nalidixic acid (NA) and hydrogen peroxide (HP)] resulted in phenotypic red fluorescence proportional to the concentration of the inducer. Except for tannic acid (TA), co-treatment of the genotoxicant-activated bacteria with ascorbic acid (AA) and aqueous plant extracts (Mangifera indica, Psidium guajava and Syzygium cumini) afforded protection against all three genotoxicants. TA was effective in suppressing the genotoxic effect of MMC and HP. The antigenotoxic effect is seen as inhibition of the genotoxicant-triggered red fluorescence. The IC50 of the plant extracts and AA varied with the genotoxicant used. Rec assay verified the antigenotoxic activity of the plant extracts. Folin-Ciocalteu test, FeCl3 test and DPPH assay confirmed the presence of polyphenolic compounds and hydrolyzable tannins in the plant extracts and the antioxidant capacity of the plant samples.

Normalization of DNA Damage Indices in Comet Assay for Comparing with Various Mutagenicity Tests

The comet assay has been widely used in genetic toxicology, environmental biomonitoring and clinical investigations. However, many indices are typically used to evaluate degree of DNA damage in comet assay and various bioassays have also been employed for the mutagenicity test of environmental waters. In this study, the DNA damage evaluation characteristics of each index were examined and the numerical data obtained were normalized on the basis of untreated controls to compare with data obtained under different experiment conditions. For confirmation of detection characteristics and sensitivity, comet assay were compared with Ames test, umu test and Rec assay. Among the comet assay indices, tail moment most effectively represented the sensitivities of individual cells and dose-dependent DNA damage was detected. By normalization of tail moment, comparison of experimental results in different conditions was simplified. The comet assay did not have the capacity to detect DNA cross-linking agent. The comparison of data revealed that mutagenicity detection sensitivity for comet assay is greater than Ames test and Rec assay and as high as that of umu test.

Water-soluble cationic polymers and their polymer-metal complexes with biocidal activity: A genotoxicity study

Water-soluble polyelectrolytes containing ammonium and sulfonic groups, their polymer–Ag(I) complexes, and silver nitrate were investigated as bactericidal compounds for Staphylococcus aureus (Collection No. ATCC 28922) and Escherichia coli (Collection No. 6538P) according to the National Committee for Clinical Laboratory Standards (NCCL) method. All the compounds, except Ag(I), showed bactericidal activity only for S. aureus. Ag(I) showed high bactericidal activity for both bacteria. No important effect of the molecular weight or macromolecular size on the maximum bactericidal concentration value was observed. The genotoxicity was studied using the rec assay. None of the macromolecular compounds showed genotoxicity, except silver ions, whose value was borderline. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 87: 452–457, 2003

Rec effect of certain textile dyes in Bacillus subtilis

A large number of compounds are toxic, genotoxic, mutagenic, teratogenic and/or carcinogenic. The genotoxicity of four textile dyes commonly used in India namely Sulphur Red Brown 360 (SRB), Jade Green 2G (JG), Reactofix Turquoise Blue 5GFL (RTB) and Direct Scarlet 4BS (DS) was determined by Bacillus subtilis spore Rec assay, both in the presence and absence of metabolizing activation mixture (S9 mix). Each dye was toxic at higher dose levels. A dose-dependent increase in the depth of growth inhibition zones was observed for all dyes. Zones of inhibition were usually clearer at higher doses of the dyes and with Rec− bacteria, but were translucent with Rec+ bacteria. SRB and DS were toxic to Rec+ and Rec− bacteria. JG was less genotoxic in the absence of S9 mix, however, its genotoxic potential increased in the presence of S9 mix. Reactofix T blue was more genotoxic in the absence of S9 mixture.

STUDIES ON THE REC EFFECTS OF CHROMIUM(VI) COMPOUNDS IN BACILLUS SUBTILIS

Bacterial systems are subjected to cellular repair of different types. A recombination (rec) repair test applied to chemicals/ carcinogens produces positive results in the rec assay. Two chromium com pounds, chromium trioxide (CrO3, MW 100.01) and potassium dichrom ate (K2Cr2O7, MW 294.21) have been subjected to rec assay. The rec effect was greater with K2Cr2O7 than with CrO3. The S9 metabolic activation mixture had a detoxifying effect for both the compounds.

Evaluation of the genotoxicity of stevioside and steviol using six in vitro and one in vivo mutagenicity assays.

Stevioside, a constituent of Stevia rebaudiana, is commonly used as a non-caloric sugar substitute in Japan. The genetic toxicities of stevioside and its aglycone, steviol, were examined with seven mutagenicity tests using bacteria (reverse mutation assay, forward mutation assay, umu test and rec assay), cultured mammalian cells (chromosomal aberration test and gene mutation assay) and mice (micronucleus test). Stevioside was not mutagenic in any of the assays examined. The aglycone, steviol, however, produced dose-related positive responses in some mutagenicity tests, i.e. the forward mutation assay using Salmonella typhimurium TM677, the chromosomal aberration test using Chinese hamster lung fibroblast cell line (CHL) and the gene mutation assay using CHL. Metabolic activation systems containing 9000 g supernatant fraction (S9) of liver homogenates prepared from polychlorinated biphenyl or phenobarbital plus 5,6-benzoflavone-pretreated rats were required for mutagenesis and clastogenesis. Steviol was weakly positive in the umu test using S.typhimurium TA1535/pSK1002 either with or without the metabolic activation system. Steviol, even in the presence of the S9 activation system, was negative in other assays, i.e. the reverse mutation assays using S.typhimurium TA97, TA98, TA100, TA102, TA104, TA1535, TA1537 and Escherichia coli WP2 uvrA/pKM101 and the rec-assay using Bacillus subtilis. Steviol was negative in the mouse micronucleus test. The genotoxic risk of steviol to humans is discussed.

Mutagenic and membranal effect of a phytotoxic molecule isolated from olive leaves parasitized by the fungusCycloconium oleaginum Cast

A phytotoxic substance (C23H44O3) which is named 'Substance A', was purified from olive leaves infected with Cycloconium oleaginum Cast. The mutagenic effect of this substance was detected using TA 100 and TA 102 strains of Salmonella in the Ames test using Bacillus subtilis strains M45 rec-, H17 rec+ in the rec assay. Another substance manifesting the mutagenic effect was found in the extract from the Cycloconium oleaginum culture. This substance was not detected in the extract from contaminated olive leaves. Substance A increased electrolytes leakage from tissue of olive leaves, thus manifesting its phytotoxicity.

Mitotic toxicity, sister chromatid exchange, and rec assay of pesticides.

Genotoxicity of 10 pesticides (chlornitrofen, chlomethoxyfen, molinate, thiobencarb, simazine, simetryn, diazinon, iprofenfos, piperofos and oxadiazone) was studied by mitotic toxicity, sister chromatid exchange, and rec assay. The pesticides are detected frequently at high levels in the Yodo River water in Osaka, Japan, which is used for drinking water by thirteen million people. Mitotic toxicity was evaluated by mitotic index (MI) and second mitosis index (SI), using a Chinese hamster cell line V79. SI is the rate of twice divided metaphases in chromosome preparation for sister chromatid exchange. All the pesticides decreased the two indices dose-dependently. MI50 and SI50, the concentrations of pesticides which lowered the indices to 50% of the solvent control, was determined. The MI50 and SI50 of each pesticide were very similar, and the pesticides did not hinder cell division specifically. None of the pesticides induced more sister chromatid exchanges than 1.5 times the solvent control. Chlomethoxyfen and simazine induced sister chromatid exchanges significantly in V79 cells, but the dose dependencies were poor. Simetryn had rec effect and was concluded to have DNA damaging activity.

Mutagenicity of Smoke Flavorings

Six homemade and five commercial smoke flavorings were tested for mutagenic activities by means of the Ames assay using Salmonella typhimurium strains TA98 and TA100. All the smoke flavorings were mutagenic. Homemade smoke flavorings were mutagenic in TA100, and the activities were weakened by metabolic activation. On the other hand, in TA98, homemade smoke flavorings were mutagenic only in the presence of S-9 mix. Commercial smoke flavorings were mutagenic in TA100 in the absence of S-9 mix. Further examination of a commercial smoke flavoring revealed a weak dose-dependent mutagenicity which was partly masked by cytotoxicity. However, when the toxicity was reduced by removal of volatile compounds in vacuo, the mutagenicity became obvious. A similar phenomenon was observed in a homemade smoke flavoring. In TA100 and TA98 in the presence of S-9 mix, mutagenicity of a commercial flavoring was dose-dependent but very weak. All the smoke flavorings showed DNA-damaging activities in Rec assay using Bacillus subtilis strains H17 and M45. In order to characterize the mutagens in smoke flavoring, a commercial smoke flavoring was fractionated by use of diethyl ether. In TA100, the aqueous fraction was as mutagenic in the absence of S-9 mix as in the presence of S-9 mix. On the other hand, the ether fraction was also mutagenic but the activity was weakened by addition of S-9 mix. These findings suggested that there were at least two mutagens in the smoke flavoring.

Genotoxicity of beryllium, gallium and antimony in short-term assays

The genotoxicity of beryllium, gallium and antimony compounds was studied with the rec, Salmonella mutagenicity and SCE assays. In the rec assay, all the salts of the metals, BECl 2, Be(NO 3) 2, GaCI 3, Ga(NO 3) 3, SbCl 3, SbCl 5, and an oxide, Sb 2O 3, had DNA-damaging activity. None of the compounds was mutagenic to Salmonella. In the SCE assays using V79 cells, 2 antimony(III) compounds, SbCl 3 and Sb 20 3, and 2 beryllium compounds, BeCl 2 and Be(NO 3) 2, induced SCEs significantly. Sb 2O 3, slightly soluble in water, was positive in both the rec assay and the SCE assay at very low doses.

Lymphocyte proliferation kinetics and genotoxic findings in a pilot study on individuals chronically exposed to arsenic in Mexico

In the search for early biological markers to detect genetic damage, a pilot study on a hydroarsenicism-exposed group was designed. Blood and urine samples were taken from 11 individuals chronically exposed and from 13 individuals with lower exposure to the metal. Lymphocyte cultures for cytogenetic studies and HGPRT assay were done with coded peripheral blood samples, while arsenic levels and the “rec assay” in B. subtilis were determined in urine samples. The highly exposed group excreted greater amounts of As, nevertheless the rec assay showed negative results. An interesting finding is that the cell-cycle kinetics exhibited a significant difference between the groups studied, the average generation time (AGT) was longer in the highly exposed group. The percentages of chromosomal aberrations and the frequencies of sister-chromatid exchanges were similar in both populations, although complex aberrations were more frequent in the highly exposed group, which also showed a higher average variation frequency in the HGPRT assay, but the 2 latter observations were not statistically significant. The lag in lymphocyte proliferation could mean an impairment of the immune response due to arsenic exposure.

Mutagenic activity of various kinds of cheese on the Ames, rec and umu assays

Some kinds of cheese were found to be mutagenic when some inhibiting substances were removed through extraction with MeOHH 2O and then treated with ion exchange resin. After this procedure, 16 types of commercial cheese out of 45 types tested showed significant mutagenicity on Salmonella typhimurium TA104, TA102 and TA97 without S9 mix. Almost all of these cheese also showed genotoxicity on the rec assay and the umu assay. The appearance of mutagenicity in these cheeses seemed to be related to the type of aging during the production process; most of the cheeses with high potency belonged to the blue mold and Propionibacteria-type cheeses and only a few belonged to the white mold, Lactobacillus-type or fresh-type cheeses. The substances inhibiting mutagenicity were isolated and identified as long-chain fatty acids.