Micronuclei In Human Lymphocytes Research Articles

LASU: An efficient and stable phthalocyanine dye with tolerable safety profile for self-disinfecting anti-COVID textiles activated by ambient light

BackgroundRecent COVID crisis has demonstrated that modern society urgently needs an accessible protection against mass infections, especially viruses, as the new strains are appearing at an ever-increasing pace and cause severe harm to the population and the world economy. MethodsWe have developed an efficient phthalocyanine photosensitizer LASU, that is suitable for dyeing textiles and allows to prepare reusable self-disinfecting fabrics with strong antiviral properties. The safety profile of LASU was evaluated in accredited laboratories by several in vitro assays according to the OECD-guidelines. ResultsThe textiles impregnated with LASU phthalocyanine showed a significant antiviral photodynamic effect even under moderate indoor and outdoor light. The dye did not show any genotoxic potential in human lymphocyte micronucleus assay. It showed a possible indication for eye irritation in human EpiOcular™ model and was phototoxic when tested in mouse BALB/c 3T3 cell test in the presence and absence of UVA-irradiation. ConclusionNovel phthalocyanine-dyed textiles are suitable for general use as self-disinfecting antiviral barriers and materials in hospitals, households, and public places. The safety profile of LASU is the phototoxic effect which is related to LASU´s mode of action.

Cladonia rangiformis Acetone Extract— New Insight into the Chemical Composition and Biological Activity

Introduction Cladonia rangiformis, also known as reindeer lichen, has been used for various remedies in folk medicine. The rich nutritional value of this lichen is reflected in the fact that it contains atranorin, which has been proven to have numerous biological activities. Objective The chemical composition, antioxidant, anticholinesterase, and antigenotoxic activities of the C. rangiformis acetone extract were investigated. The novelty of this work is that anticholinesterase activity, protective effect on human lymphocytes, and antioxidant activity by the cupric-reducing antioxidant capacity (CUPRAC) method of C. rangiformis extract were determined for the first time, as well as gas chromatography - mass spectrometry (GC-MS) profile. Methods Chemical composition was carried out by high-performance liquid chromatography (HPLC)-diode array detector and GC-MS. Antigenotoxic effect was evaluated in human lymphocytes by cytokinesis-block micronucleus assay. Total phenolic content, DPPH and ABTS radical scavenging capacities, CUPRAC, and total reducing power were determined spectrophotometrically. Determination of acetylcholinesterase activity was performed by Ellman's colorimetry assay. The statistical analysis of variance (One-way ANOVA) was performed using Origin software package version 7.0. Results HPLC method was used to identified 3 compounds with atranorin as predominant constituent (97.2%). Fumarprotocetraric and atraric acid were represented in a much smaller mutually approximate amount, 0.3 and 0.5%, respectively. Rangiformic acid was the most abundant volatile compound. Acetone extract of C. rangiformis (2 μg/mL) decreased the frequency of micronuclei by 15.5% while the radioprotectant amifostine reduced the frequency of occurrence of micronuclei by 11.4%. On the contrary, at a concentration of 1.0 mg/mL the extract exhibited an activating effect on cholinesterase (2.0%), while at a concentration of 10.0 mg/mL it showed a weak inhibitory effect (7.3%). The total phenol content was 132.71 μg gallic acid equivalents per mg of dry extract. The IC50 value for the DPPH experiment was 16.19 mg/mL and for ABTS was 11.80 mg/mL. Cupric reducing capacity and total reducing power were 17.81 μg Trolox equivalents per mg of dry extract and 0.45 μg ascorbic acid equivalents per mg of dry extract, respectively. Conclusion In addition to previously identified compounds in the acetone extract, atraric acid was identified for the first time by using HPLC method, and orcinol, β-orcinol, lauric alcohol, atranol, and rangiformic acid by using GC-MS method. The results of biological activity proved that acetone extract had antioxidant capacity and weak anticholinesterase activity. Reduction of the number of micronuclei in human lymphocytes, classified C. rangiformis as promising substances source with beneficial effects on DNA damage.

Establishment of ex vivo calibration curve for X-ray induced “dicentric + ring” and micronuclei in human peripheral lymphocytes for biodosimetry during radiological emergencies, and validation with dose blinded samples

In the modern developing society, application of radiation has increased extensively. With significant improvement in the radiation protection practices, exposure to human could be minimized substantially, but cannot be avoided completely. Assessment of exposure is essential for regulatory decision and medical management as applicable. Until now, cytogenetic changes have served as surrogate marker of radiation exposure and have been extensively employed for biological dose estimation of various planned and unplanned exposures. Dicentric Chromosomal Aberration (DCA) is radiation specific and is considered as gold standard, micronucleus is not very specific to radiation and is considered as an alternative method for biodosimetry. In this study dose response curves were generated for X-ray induced “dicentric + ring” and micronuclei, in lymphocytes of three healthy volunteers [2 females (age 22, 23 years) and 1 male (24 year)]. The blood samples were irradiated with X-ray using LINAC (energy 6 MV, dose rate 6 Gy/min), in the dose range of 0–5Gy. Irradiated blood samples were cultured and processed to harvest metaphases, as per standard procedures recommended by International Atomic Energy Agency. Pooled data obtained from all the three volunteers, were in agreement with Poisson distribution for “dicentric + ring”, however over dispersion was observed for micronuclei. Data (“dicentric + ring” and micronuclei) were fitted by linear quadratic model of the expression YC + αD + βD2 using Dose Estimate software, version 5.2. The data fit has resulted in linear coefficient α = 0.0006 (±0.0068) “dicentric + ring” cell−1 Gy−1 and quadratic coefficient β = 0.0619 (±0.0043) “dicentric + ring” cell−1 Gy−2 for “dicentric + ring” and linear coefficient α = 0.0459 ± (0.0038) micronuclei cell−1 Gy−1 and quadratic coefficient β = 0.0185 ± (0.0010) micronuclei cell−1 Gy−2 for micronuclei, respectively. Background frequencies for “dicentric + ring” and micronuclei were 0.0006 ± 0.0004 and 0.0077 ± 0.0012 cell−1, respectively. Established curves were validated, by reconstructing the doses of 8 dose blinded samples (4 by DCA and 4 by CBMN) using coefficients generated here. Estimated doses were within the variation of 0.9–16% for “dicentric + ring” and 21.7–31.2% for micronuclei respectively. These established curves have potential to be employed for biodosimetry of occupational, clinical and accidental exposures, for initial triage and medical management.

Comprehensive Analysis of the Herbal Mixture Made of Juniperus oxycedrus L. Berries, Inner Bark of Betula pendula Roth., and Grains of Avena sativa L.

This is the first report of the high-performance liquid chromatography and gas chromatography–mass spectrometry profile of a herbal mixture (HM) made of Juniperus oxycedrus L. (redberry juniper) berries, inner bark of Betula pendula Roth. (silver birch), and grains of Avena sativa L. (oat), and its effect on the No. of micronuclei (MN) in human lymphocytes and toxicity toward Artemia salina. Constituents represented by over 1000 µg per g of methanol dry extract were gallic acid, protocatechuic acid, and amentoflavone. The methanol extract of the HM at a concentration of 2.0 μg/mL decreased MN frequency by 38.3%, which was more than 3 times greater than that of the radioprotectant amifostine. The essential oil isolated from the HM was composed mainly of β-myrcene (32%) and showed weaker toxicity toward Artemia salina than the positive control after both incubation periods (24 h and 48 h). These findings suggest that the examined HM, beside its ethnopharmacological relevance on the elimination of renal calculi, also significantly reduces the No. of MN in human lymphocytes.

Investigation of cyto-genotoxic effects of a food sweetener Acesulfame potassium

Acesulfame potassium (ACE-K) is an artificial sweetener widely used in many foods. This investigation assessed the cytotoxic effect of ACE-K using MTT assay in human hepatocellular carcinoma (HepG2) cell line and the genotoxic effect using chromosomal aberrations (CAs), micronucleus (MN), and comet assays in human lymphocytes. 7.5-240 μg/mL concentrations of ACE-K were applied to cells. ACE-K notably decreased the cell viability on HepG2 cells, especially at 120 and 240 μg/mL at 24 and 48 h. It also significantly reduced the mitotic index (MI) at 60, 120, and 240 μg/mL at both treatments (24 and 48 h) in human lymphocytes. The frequency of the CAs significantly increased at 60, 120, and 240 μg/mL for 48 h treatment compared to control. However, no difference was observed in the frequency of MN and nuclear division index (NDI) at all the treatments. ACE-K also induced comet tail length, tail intensity, and moment at 15 μg/mL in isolated human lymphocytes. Therefore, ACE-K showed a cytotoxic effect in HepG2 cells as well as human lymphocytes at higher concentrations. It also exhibits a mild genotoxic effect by increasing the frequency of CAs at long-term treatment and DNA damaging effect only at 15 μg/mL.

A genotoxicity assessment approach for botanical materials demonstrated with Poria cocos

Safety assessment of botanical materials often reveals genotoxicity data gaps. However, there are no harmonized regulatory genotoxicity testing approaches for botanical materials. Furthermore, literature genotoxicity testing reports often lack clear definition of the botanical materials (genus species, plant part, etc.) and/or analytical characterization. Here, upon a review of available regulatory testing batteries for botanicals, the authors conclude that an in vitro 2-test battery, consisting of the Ames test and the in vitro human lymphocyte micronucleus assay (HLM), is appropriate to assess the genotoxicity of botanical materials. This approach was then illustrated using a Poria cocos (PCS) botanical material as a case study. Before the genotoxicity testing, an analytical characterization coupled with in silico approach assured appropriate characterization of PCS and helped inform the genotoxic potential of the triterpenes that drive the genotoxicity assessment. The literature search and DEREK screening did not reveal a genotoxicity concern or a genotoxicity structural alert. PCS was then tested in OECD guideline compliant Ames and in vitro HLM and the negative results from this 2-test battery confirmed the absence of a genotoxic potential of the PCS. This fit-for-purpose approach is expected to be useful to fill genotoxicity data gaps for botanical materials.

Radioprotective effect of a novel and green bio-nanohybrid, chitosan/silver/cobalt complex, based on Ferulago angulate plant.

A novel bio-nanocomposite was developed by incorporating the extracted nanochitosan from shrimp wastes with Schiff base cobalt complex (Chit-Co complex). The phytosynthesis of Chit-Co complex/Ag bio-nanocomposite was designed utilizing Chit-Co complex at the presence of Ferulago angulate extraction and characterized by AFM, SEM, EDAX, TEM, FT-IR, and elemental analysis. The radioprotective application of this bio-nanocomposite on human lymphocyte cells was evaluated using micronucleus (MN) assay. Total antioxidant activities of it were evaluated using FRAP and DPPH assays. Chit-Co complex/Ag bio-nanocomposite significantly decreased the frequency of micronuclei in human lymphocytes exposed to ionization irradiation (IR). The highest protection was observed at 200μg/ml. Also, maximum antioxidant activities of bio-nanocomposite were provided at the same dose. These data exhibit the radioprotective effect of a bio-nanocomposite based on wastes of living organisms can be an excellent radioprotective agent, which can protect the normal cells of human against the genetic damage by IR.

Toxicological assessment of Opuntia dillenii (Ker Gawl.) Haw. cladode methanol extract, fractions and its alpha pyrones: Opuntiol and opuntioside

Ethnopharmacological relevanceThe edible plant Opuntia dillenii (Ker Gawl.) Haw. commonly known as Nagphana, belongs to the Cactaceae family. It is traditionally used to treat various ailments including inflammation, gastric ulcers, diabetes, hepatitis, asthma, whooping cough and intestinal spasm. Aim of the studyDespite its traditional use in various countries, detailed toxicological studies of O. dillenii cladode are few. Thus in the current study, toxicity of O. dillenii cladode derived methanol extract, fractions and its α-pyrones: opuntiol and opuntioside have been addressed. MethodsThe test agents were assessed using both in vitro and in vivo toxicity assays. MTT on human embryonic kidney cell line (HEK-293), tryphan blue exclusion in rat neutrophils, Cytokinesis-B block micronucleus (CBMN) in human lymphocytes and genomic DNA fragmentation using agarose gel electrophoresis were performed. In acute toxicity test, mice orally received extract (5 g/kg) for 7 days followed by measurements of relative organ weight, biochemical (blood profile, liver and kidney function test) and histological studies (liver and kidney) were carried out. Rat bone marrow micronucleus genotoxicity assay was also conducted. ResultsO. dillenii derived test agents were non-cytotoxic and had no effect on the integrity of DNA. Methanol extract (5 g/kg) orally administered in mice did not cause any significant change in relative organ weights, biochemical parameters and liver and kidney histology as compared to vehicle control. In parallel, extract did not stimulate micronuclei formation in rat bone marrow polychromatic erythrocytes. ConclusionThese results led to conclude that edible O. dillenii extract is non-toxic via the oral route and appears to be non-cyto-, hepato-, nephro- or genotoxic, thereby supporting its safe traditional use against various ailments. Therefore, opuntiol and opuntioside may serve as lead compounds in designing new drug(s) derived from edible plants.

The Genotoxic and Pro-Apoptotic Activities of Advanced Glycation End-Products (MAGE) Measured with Micronuclei Assay Are Inhibited by Their Low Molecular Mass Counterparts.

An association between the cancer invasive activities of cells and their exposure to advanced glycation end-products (AGEs) was described early in some reports. An incubation of cells with BSA–AGE (bovine serum albumin–AGE), BSA–carboxymethyllysine and BSA–methylglyoxal (BSA–MG) resulted in a significant increase in DNA damage. We examined the genotoxic activity of new products synthesized under nonaqueous conditions. These were high molecular mass MAGEs (HMW–MAGEs) formed from protein and melibiose and low molecular mass MAGEs (LMW–MAGEs) obtained from the melibiose and N-α-acetyllysine and N-α-acetylarginine. We have observed by measuring of micronuclei in human lymphocytes in vitro that the studied HMW–MAGEs expressed the genotoxicity. The number of micronuclei (MN) in lymphocytes reached 40.22 ± 5.34 promille (MN/1000CBL), compared to 28.80 ± 6.50 MN/1000 CBL for the reference BSA–MG, whereas a control value was 20.66 ± 1.39 MN/1000CBL. However, the LMW–MAGE fractions did not induce micronuclei formation in the culture of lymphocytes and partially protected DNA against damage in the cells irradiated with X-ray. Human melanoma and all other studied cells, such as bronchial epithelial cells, lung cancer cells and colorectal cancer cells, are susceptible to the genotoxic effects of HMW–MAGEs. The LMW–MAGEs are not genotoxic, while they inhibit HMW–MAGE genotoxic activity. With regard to apoptosis, it is induced with the HMW–MAGE compounds, in the p53 independent way, whereas the low molecular mass product inhibits the apoptosis induction. Further investigations will potentially indicate beneficial apoptotic effect on cancer cells.

Dose-effect relationships of 12C6+ ions-induced dicentric plus ring chromosomes, micronucleus and nucleoplasmic bridges in human lymphocytes in vitro

Purpose The objective of this research was to explore the dose-effect relationships of dicentric plus ring (dic + r), micronucleus (MN) and nucleoplasmic bridges (NPB) induced by carbon ions in human lymphocytes. Materials and methods Venous blood samples were collected from three healthy donors. 12C6+ ions beam was used to irradiate the blood samples at the energy of 330 MeV and linear energy transfer (LET) of 50 keV/μm with a dose rate of 1 Gy/min in the spread-out Bragg peak. The irradiated doses were 0 (sham irradiation), 1, 2, 3, 4, 5 and 6 Gy. Dic + r chromosomes aberrations were scored in metaphases. The cytokinesis-block micronucleus cytome (CBMN) was conducted to analyze MN and NPB. The maximum low-dose relative biological effectiveness (RBEM) values of the induction of dic + r, MN and NPB in human lymphocytes for 12C6+ ions irradiation was calculated relative to 60Co γ-rays. Results The frequencies of dic + r, MN and NPB showed significantly increases in a dose-depended manner after exposure to 12C6+ ions. The distributions of dic + r and MN exhibited overdispersion, while the distribution of NPB agreed with Poisson distribution at all doses. Linear-quadratic equations were established based on the frequencies of dic + r and MN. The dose-response curves of NPB frequencies followed a linear model. The derived RBEM values for dic + r, MN and NPB in human lymphocytes irradiated with 12C6+ ions were 8.07 ± 2.73, 2.69 ± 0.20 and 4.00 ± 2.69 in comparison with 60Co γ-rays. Conclusion The dose-response curves of carbon ions-induced dic + r, MN and NPB were constructed. These results could be helpful to improve radiation risk assessment and dose estimation after exposed to carbon ions irradiation.

Potassium Sorbate Induces Oxidative Stress and Genotoxicity in Human Lymphocytes

Potassium sorbate, a potassium salt of sorbic acid, has been used as food preservative and antimicrobial agent.Some observations in rat and hamster cells suggest its toxicity. However, observations in human cells arelimiting. Therefore, it has been intended to investigate impacts of potassium sorbate on human lymphocytes.The lymphocytes were cultured and treated with 0.5, 1.0, 1.5 and 2.0 mg/ml of potassium sorbate for 24 and48 h. Evaluations of its impacts were done through the MTT assay, analysis of chromosomal aberrations,formation of micronucleus and activities of superoxide dismutase. The results revealed that potassiumsorbate induced oxidative stress, genotoxicity, damages to chromosome, formation of micronucleus inhuman lymphocytes.

Genotoxic properties of Betonica officinalis, Gratiola officinalis, Vincetoxicum luteum and Vincetoxicum hirundinaria extracts

Genotoxicity of B. officinalis, G. officinalis, V. luteum and V. hirundinaria extracts, which demonstrated strong antioxidant capacity, was tested using chromosome aberration, sister chromatid exchange (SCE), cytokinesis-block micronucleus and alkaline single-cell gel electrophoresis (comet) assays in human lymphocytes in vitro and Ames Salmonella/microsome test. All tested extracts were not mutagenic in S. typhimurium strains TA98 and TA100 with and without metabolic activation and did not induce chromosome aberrations in human lymphocytes in vitro. Extract from G. officinalis was the only one, which induced significant increase in micronuclei, indicating possible aneugenic effect. All investigated plant extracts induced DNA damage evaluated by the comet assay, while B. officinalis and V. luteum extracts induced slight increase in SCE values. The determined variation in response might be due to the plant extract tested and donor susceptibility.

Inclusion of an extended treatment with recovery improves the results for the human peripheral blood lymphocyte micronucleus assay.

The in vitro micronucleus (IVMN) test was endorsed for regulatory genotoxicity testing with adoption of the Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 487 in 2010. This included two equally acceptable options for extended treatment in the absence of metabolic activation: a treatment for 1.5-2.0 cell cycles with harvest at the end of treatment (Option A) or treatment for 1.5-2.0 cell cycles followed by recovery for 1.5-2.0 cell cycles prior to harvest (Option B). Although no preferences were discussed, TG 487 cautions that Option B may not be appropriate for stimulated lymphocytes where exponential growth may be declining at 96 h after phytohaemagglutinin (PHA) stimulation. Following revision of TG 487 in 2014 and 2016, emphasis has been placed on using Option A. Given the purpose of the IVMN assay is to determine both clastogenic and aneugenic potential, the authors believe the assay is compromised if an extended treatment with recovery is not included for sensitive detection of certain classes of chemical. In this study, average generation time (via bromodeoxyuridine incorporation) of human peripheral blood lymphocytes (HPBL) was measured up to 144 h after PHA stimulation. In addition, the HPBL micronucleus (MN) assay was performed using Option A and B treatment schedules. Cytotoxicity (replication index) and MN induction were determined following treatment with 14 chemicals. The data demonstrate that lymphocytes actively divide beyond 96 h after PHA stimulation. Furthermore, MN induction was only observed with some aneugenic chemicals and nucleoside analogues in HPBLs following extended treatment with a recovery period. For the majority of chemicals tested the magnitude of MN induction was generally greater and MN induction was observed across a wider concentration range following the Option B treatment schedule. In addition, steep concentration-related toxicity following treatment without recovery is more common, making selection of suitable concentrations (within regulatory toxicity limits) for MN analysis challenging.

Evaluation of in vitro and in vivo genotoxic and antigenotoxic effects of Rhus coriaria

Rhus coriaria has been important in the treatment of many diseases in traditional use. In this content, the genotoxic, antigenotoxic, and oxidative stress effects of methanol extract of R. coriaria (RCE) were investigated in this study. Two hundred fifty, 500, or 750 µg/mL concentrations of RCE were not found to have DNA damaging effect on pET22-b(+) plasmid and were unable to induce micronuclei in human lymphocytes (24 or 48 h treatment period). However, it did not inhibit the genotoxic effect of mitomycin-c (0.25 µg/mL). Cytotoxic effects of RCE were investigated using mitotic index (MI) and nuclear division index (NDI). Five hundred, 1000, and 2000 mg/kg concentrations of RCE did not induce chromosome aberrations in rat bone marrow cells for 12 or 24 h treatment period. In addition, 2000 mg/kg concentration of RCE showed an antigenotoxic effect by decreasing to genotoxic effect of 400 mg/kg urethane at 12 and 24 h treatment periods. RCE showed cytotoxic effects by significantly decreasing NDI. Moreover, RCE increased cytotoxic effect of Mitomycin C (MMC). However, RCE did not induce cytotoxicity in rat bone marrow cells. The highest concentration of RCE reduced total oxidant level in 12 h treatment. Interestingly, the lowest total oxidant level was found in rats blood treated with the lowest concentration RCE and urethane together. Thousand and 2000 mg/kg concentrations of RCE decreased total antioxidant levels of rat blood at 24 h treatment period. Our results showed that RCE possess cytotoxic effect in short-term treatments in vitro. However, it does not demonstrate genotoxic or cytotoxic effects in vivo.

In vitro genotoxic and antigenotoxic effects of cynarin

Ethnopharmacological relevanceCynarin is an artichoke phytochemical that possesses a variety of pharmacological features including free-radical scavenging and antioxidant activity. The origin of artichoke species appears to be Mediterranean region. Two of these species, globe artichoke (Cynara cardunculus var. scolymus L.) and cardoon (Cynara cardunculus var. altilis DC), are widely cultivated and consumed. This vegetable, as the basis of the mediterranean diet, has been used as herbal medicine for its therapeutic effects since ancient times. Therefore, this study was performed to determine genotoxic and antigenotoxic effects of cynarin against MMC (mitomycin C) and H2O2 (hydrogen peroxide) induced genomic instability using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronucleus (MN), and comet assays in human lymphocytes. Materials and methodsLymphocytes obtained from two healthy volunteers (1 male and 1 female) were exposed to different concentrations of cynarin (12–194 μM) alone and the combination of cynarin and MMC (0.60 μM) or cynarin and H2O2 (100 μM, only for comet assay). ResultsCynarin alone did not induce significant genotoxic effect in the CA, SCE (except 194 μM), MN, and comet assays. The combination of some concentrations of cynarin and MMC decreased the frequency of CAs, SCEs and MN induced by MMC. Furthermore, the combination of cynarin and H2O2 reduced all comet parameters at all the concentrations compared to H2O2 alone. While the highest concentrations of cynarin significantly decreased mitotic index (MI), the combination of cynarin and MMC increased the reduction of MI induced by MMC alone. ConclusionAll the results obtained in this study demonstrated that cynarin exhibited antigenotoxic effects rather than genotoxic effects. It is believed that cynarin can act as a potential chemo-preventive against genotoxic agents.

X-ray Structures and Feasibility Assessment of CLK2 Inhibitors for Phelan-McDermid Syndrome.

CLK2 inhibition has been proposed as a potential mechanism to improve autism and neuronal functions in Phelan-McDermid syndrome (PMDS). Herein, the discovery of a very potent indazole CLK inhibitor series and the CLK2 X-ray structure of the most potent analogue are reported. This new indazole series was identified through a biochemical CLK2 Caliper assay screen with 30k compounds selected by an in silico approach. Novel high-resolution X-ray structures of all CLKs, including the first CLK4 X-ray structure, bound to known CLK2 inhibitor tool compounds (e.g., TG003, CX-4945), are also shown and yield insight into inhibitor selectivity in the CLK family. The efficacy of the new CLK2 inhibitors from the indazole series was demonstrated in the mouse brain slice assay, and potential safety concerns were investigated. Genotoxicity findings in the human lymphocyte micronucleus test (MNT) assay are shown by using two structurally different CLK inhibitors to reveal a major concern for pan-CLK inhibition in PMDS.

Assessment of cytotoxic and genotoxic effects of enniatin-A in vitro

ABSTRACTEnniatin A (EN-A) is a Fusarium mycotoxin which is a common contaminant in grains and especially in maize and it causes serious loss of product. The aim of this study was to investigate the cytotoxic effects using 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT) assay in human cervix carcinoma (HeLa) cell line, and genotoxic effects of EN-A using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (MN) and comet assays in human lymphocytes. The cells were treated with 0.07, 0.14, 0.29, 0.57, 1.15, 2.29, 4.59 and 9.17 μM concentrations of EN-A. It exhibited cytotoxic effects in HeLa cell lines especially when the concentrations were increased. The half-inhibitory value (IC50) was determined as 1.15 μM concentration for 24 h and 0.57 μM concentration for 48 h. However, EN-A failed to affect the frequency of CAs, SCEs and MN in human lymphocytes. Only a slight increase was observed in the frequency of SCEs at 0.57 μM concentration over 48 h. The replication (RI) and nuclear division (NDI) indices were not affected. On the contrary, EN-A decreased the mitotic index (MI) significantly at all concentrations compared to the negative control and solvent control (except at 0.29 μM for 24 h, and except at 0.14, 0.29 and 0.57 μM for 48 h). Treatments over 2.29 μM showed toxic effects in human lymphocytes. EN-A significantly increased comet tail intensity (except at 0.07 and 0.57 μM) in isolated human lymphocytes. The results of this study demonstrate that EN-A has an obvious cytotoxic effect especially when the EN-A concentration was increased. In addition, EN-A could exhibit a mild genotoxic effect.

Safety evaluation of the food enzyme α-amylase from a genetically modified Aspergillusniger (strain NZYM-SB).

The food enzyme is an α‐amylase (4‐α‐d‐glucan glucanohydrolase; EC 3.2.1.1), produced with the genetically modified Aspergillus niger strain NZYM‐SB by Novozymes A/S. The food enzyme does not contain the production organism or its DNA; therefore, there is no safety concern for the environment. The α‐amylase is intended for use in starch processing, beverage alcohol (distilling) processes and baking processes. Residual amounts of total organic solids (TOS) are removed by distillation and by the purification steps applied during the production of glucose syrups (by > 99%). Consequently, dietary exposure was not calculated for these two uses. Based on the maximum use levels recommended for the baking processes and individual consumption data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme–TOS was estimated to be up to 3.075 mg TOS/kg body weight per day in European populations. The food enzyme did not induce gene mutations in bacteria or micronuclei in human lymphocytes. Subchronic toxicity was assessed by means of a repeated‐dose 90‐day oral toxicity study in rodents. A no observed adverse effect level (NOAEL) was derived that, compared with the dietary exposure, resulted in a sufficiently high margin of exposure (MOE). Similarity of the amino acid sequence to those of known allergens was searched and two matches were found. The Panel considered that the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered low. Based on the genetic modifications, the manufacturing process, the compositional and biochemical data, the findings in the toxicological and genotoxicity studies, as well as the estimated dietary exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Mutagenicity and genotoxicity of ClearTaste.

The present study investigates whether ClearTaste is mutagenic/genotoxic by employing it as a test article in bacterial reverse mutation (Ames test) and in vitro human peripheral blood lymphocyte micronucleus assays conducted by a Good Laboratory Practice certified third party as parameterized by the United States Food and Drug Administration. ClearTaste is a taste modulator derived from the filtrate of submerged Cordyceps sinensis and is typically processed into a powder. It functions as a bitter, sour, astringency, metallic and lingering aftertaste mitigator/blocker. The Ames test includes revertant colony counts almost exclusively less than 100/plate and significantly fewer ClearTaste counts as opposed to known mutagen counts. The micronucleus assay reported cytotoxicity exclusively < 25% for doses up to 2,000 μg/L with Cytokinesis Block Proliferation Indices less than water and statistically significant differences between micronucelated cells post dosing compared to cyclophosphamide and vinblastine controls. The conclusion of these data is that ClearTaste is neither muta- nor carcinogenic.

Ramalina capitata (Ach.) Nyl. acetone extract: HPLC analysis, genotoxicity, cholinesterase, antioxidant and antibacterial activity.

In the present investigation, effects of Ramalina capitata acetone extract on micronucleus distribution on human lymphocytes, on cholinesterase activity and antioxidant activity (by the CUPRAC method) were examined, for the first time as well as its HPLC profile. Additionally, total phenolic compounds (TPC), antioxidant properties (estimated via DPPH, ABTS and TRP assays) and antibacterial activity were determined. The predominant phenolic compounds in this extract were evernic, everninic and obtusatic acids. Acetone extract of R. capitata at concentration of 2 μg mL-1 decreased a frequency of micronuclei (MN) for 14.8 %. The extract reduces the concentration of DPPH and ABTS radicals for 21.2 and 36.1 % (respectively). Values for total reducing power (TRP) and cupric reducing capacity (CUPRAC) were 0.4624 ± 0.1064 μg ascorbic acid equivalents (AAE) per mg of dry extract, and 6.1176 ± 0.2964 μg Trolox equivalents (TE) per mg of dry extract, respectively. The total phenol content was 670.6376 ± 66.554 μg galic acid equivalents (GAE) per mg of dry extract. Tested extract at concentration of 2 mg mL-1 exhibited inhibition effect (5.2 %) on pooled human serum cholinesterase. The antimicrobial assay showed that acetone extract had inhibition effect towards Gram-positive strains. The results of manifested antioxidant activity, reducing the number of micronuclei in human lymphocytes, and antibacterial activity recommends R. capitata extract for further in vivo studies.