New insight into the antigenotoxic activity of Gentiana lutea extracts - Protective effect against food borne mutagens.

Food mutagens formed from amino acids during heating of meat have the potential to induce serious consequences on human health. As a result, the identification of naturally occurring, genoprotective agents, is of great importance. The aim of this study was to chemically characterize a root and leaf extracts of Gentiana lutea and to investigate the antigenotoxic effects of extracts and pure constituents (gentiopicroside and mangiferin). Antigenotoxic effects were shown for combinations with the food borne mutagens IQ and PhIP using hepatoma HepG2 cells. Furthermore, their antioxidant activity and their capacity to modulate Nrf2 expression and affect the glutathione redox status were tested. Chemical analyses showed that the most abundant constituents found in root extract are gentiopicroside and sweroside. On the other hand, homoorientin and isovitexin were the dominant ones in leaf extract. Strong genoprotective activities of all tested compounds against both mutagens were observed in alkaline comet assays (up to 77% of tail intensity inhibition, p < 0.001). The protection against glutathione depletion was partially due to the radical scavenging activity and up-regulation of Nrf2 expression by the substances. The results of this study strongly encourage further investigations of the antimutagenic properties of G. lutea.

The presence of periopathogenic bacteria in subgingival and atherosclerotic plaques - An age related comparative analysis.

INTRODUCTION: There is a known connection between periodontitis and atherosclerosis and the presence of periopathogens in blood vessels. However, changes of the oral microflora related to the aging process and its possible effects on atherosclerosis, have yet to be analyzed. The aim of this study was to assess temporal changes in the frequency of periodontal bacteria in the subgingival plaque and in atherosclerotic blood vessels of patients with atherosclerosis. METHODOLOGY: The study included 100 patients with atherosclerosis and periodontitis, divided into two groups, below and over 60 years of age. Clinical examinations were performedand subgingival plaque specimens were collected as well as biopsy specimens from the following arteries: coronary (34), carotid (29), abdominal (10), femoral (10), mammary (13) and iliac (4). Subgingival and artery specimens were subjected to PCR detection of 5 major periodontal pathogens: Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Aggregatibacter actinomycetemcomitans (Aa), Tannerella forsythensis (Tf) and Treponema denticola (Td). RESULTS: Tf was the most and Td the least frequent bacteria in both age groups and in both types of samples. The frequencies of bacteria in subgingival versus atherosclerotic samples were: Tf (76%:53%), Pi (71%:31%), Pg (60%:38%), Aa (39%:14%) and Td (21%:6%). Only Aa and Pi showed a significant difference of prevalence between younger and older patients. The most colonized artery was a. coronaria, followed by a. carotis, a. abdominalis, a. mammaria, and a. femoralis. CONCLUSIONS: Patient's age and the distance of a given blood vessel from the oral cavity influenced microbiological findings in the atherotic plaque.

Chemical characterization, antioxidant, genotoxic and in vitro cytotoxic activity assessment of Juniperus communis var. saxatilis.

Chemical composition and antioxidative, genotoxic and cytotoxic potential of essential oil (EO) and post-distillation waste (PDW) of Serbian Juniperus communis L. var. saxatilis Pall. was studied in human lung carcinoma (A549) and normal lung fibroblast (MRC-5) cells. GC-MS analysis identified 93.95% of total EO content and determined α-pinen as a dominant component (23.61%). LC-MS/MS analysis of PDW pointed at rutin (12.2 mg g-1) and quinic acid (11.1 mg g-1) as the most abundant. Antioxidativity of PDW was strong in DPPH (IC50 was 5.27 µg mL-1), and moderate in TBA and FRAP assays. Both substances were more cytotoxic to A549 than to MRC-5 cells. Obtained IC50 values were 69.4 µg mL-1 and 120 µg mL-1 for EO, and 1.27 mg mL-1 and 2.86 mg mL-1 for PDW, respectively. PDW was genotoxic (0.3 mg mL-1 and 1 mg mL-1 in A549 and MRC-5 cells, respectively) and induced apoptosis and arrested cell cycle in G2/M phase in A549 cells (0.3 mg mL-1). In mixtures with doxorubicin cytotoxicity of EO and PDW increased, and combination index values (0.12-0.18) revealed clear synergistic effect, stronger in cancer cells. This indicates that J. communis var. saxatilis could decrease the chemotherapeutic doses of doxorubicin, potentially reducing its side effects.

Nepeta rtanjensis (Lamiaceae), a plant endemic to the Balkans: Phenolic composition, antioxidant activity, and in vitro antigenotoxic effects in triiodothyronine-induced DNA damage in human lymphocytes.

The success of antioxidant therapy in hyperthyroidism implies that disease is mediated by oxidative stress, which is known as one of the causing agents of ageing, degenerative diseases, and cancer. The main objective of our study was to determine possible protective effects of methanolic extract of N. rtanjensis in triiodothyronine (T3)-induced DNA breaks of human lymphocytes under in vitro conditions, based upon plant antioxidant capacity related to its phytochemical profile, mainly its polyphenolic complex. The total phenolic and flavonoid content and the antioxidant activity using in vitro 1,1-dyphenyl-2- picrylhydrazyl reagent (DPPH) was determined in methanolic extracts of plant leaves and flowers. The phenolic compound content of 62.73±1.80mg of GaA/g, exhibited solid antioxidant activity (IC50= 112.59±0.95µg/ml). The antigenotoxic activity of 0.2, 0.5 and 1.0mg/ml N. rtanjensis methanol extracts mixture with 100µM of T3 was studied in human lymphocytes in vitro using the Comet assay. It is supposed that the antigenotoxicity of N. rtanjensis methanol extracts was caused by high presence of chlorogenic acid, rosmarinic acid and rutin, all known as efficient antioxidant bioactive compounds, which were determined by ultrahigh-pressure liquid chromatograph with MS/MS Mass Spectroscopy (UHPLC/-HESI-MS / MS).

The Protective Effects of Probiotic Bacteria on Cadmium Toxicity in Rats.

One of the useful properties of probiotic bacteria is their capacity to bind different targets, thus eliminating them through feces. It is supposed that one of these targets could be cadmium, a widespread environmental toxicant that causes various disturbances in biological systems. This study examined the protective effects of probiotic supplementation against cadmium-induced toxicity in the rat. The experiment was conducted in the course of 5 weeks. Animals were divided into four groups: (1) controls, (2) probiotics treated, (3) cadmium treated, and (4) probiotics + cadmium treated. The cadmium concentration was measured in the blood, liver, kidney, and feces, as well as the blood alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as biomarkers of the liver function. Histomorphological changes in the liver and kidney were also determined. Our results revealed that probiotics combined with cadmium increase this metal concentration in feces. As a result, blood, liver, and kidney Cd levels, as well as blood ALT and AST activities were lessened compared to the rat group treated with cadmium only. Besides, probiotics consumed simultaneously with cadmium attenuated histomorphological changes in the liver and kidney caused by cadmium. The rise in lactobacilli number in feces of rats treated simultaneously with cadmium and probiotics results in strong correlation with the increase of Cd concentration in their feces and the decrease of Cd concentration in their blood. We speculate that probiotics actively contribute to cadmium excretion through feces, probably, by its binding to their bacterial cell wall.

Flooding modifies the genotoxic effects of pollution on a worm, a mussel and two fish species from the Sava River.

Extreme hydrological events, such as water scarcity and flooding, can modify the effect of other stressors present in aquatic environment, which could result in the significant changes in the ecosystem functioning. Presence and interaction of various stressors (genotoxic pollutants) in the environment can influence the integrity of DNA molecules in aquatic organisms which can be negatively reflected on the individual, population and community levels. Therefore, in this study we have investigated the impact of flooding, in terms of genotoxicity, on organisms belonging to different trophic levels. The study was carried out on the site situated in the lower stretch of the Sava River which faced devastating effects of severe flooding in May 2014. The flooding occurred during our field experiment and this event provided a unique opportunity to assess its influence to the environment. The in situ effects of this specific situation were monitored by measuring physical, chemical and microbiological parameters of water, and by comparing the level of DNA damage in coelomocytes and haemocytes of freshwater worms Branchiura sowerbyi, haemocytes of freshwater mussels Unio tumidus and blood cells of freshwater fish Abramis bjoerkna/Abramis sapa, by means of the comet assay. Our study indicated that the flooding had a significant impact on water quality by decreasing the amount and discharge rate of urban wastewaters but simultaneously introducing contaminants from the nearby fly ash disposal field into river by runoff, which had diverse effects on the level of DNA damage in the studied organisms. This indicates that the assessment of genotoxic pollution in situ is strongly affected by the choice of the bioindicator organism.

Effect of Allium flavum L. and Allium melanantherum Panc. Extracts on Oxidative DNA Damage and Antioxidative Enzymes Superoxide Dismutase and Catalase.

Allium flavum L. and Allium melanantherum Panc. are wild growing plants used in traditional diet in Balkan region. While chemical composition and some biological activities of A. flavum have been reported, A. melanantherum, as an endemic in the Balkan Peninsula, has never been comprehensively examined. After chemical characterization of A. melanantherum, we examined the protective effect of methanol extracts of both species against t-butyl hydro-peroxide (t-BOOH)-induced DNA damage and mutagenesis. The bacterial reverse mutation assay was performed on Escherichia coli WP2 oxyR strain. DNA damage was monitored in human fetal lung fibroblasts (MRC-5) with alkaline comet assay. Obtained results indicated that extracts reduced t-BOOH-induced DNA damage up to 70 and 72% for A. flavum and A. melanantherum extract, respectively, and showed no effect on t-BOOH-induced mutagenesis. Since the results indicated modulatory effect on cell-mediated antioxidative defense, the effect of extracts on total protein content, and superoxide dismutase (SOD) and catalase (CAT) amounts and activities were monitored. Both extracts increased total protein content, while the increase of enzyme amount and activity was obtained only with A. melanantherum extract and restricted to CAT. The activity of CuZnSOD family was not affected, while SOD1 and SOD2 amounts were significantly decreased, indicating potential involvement of extracellular CuZnSOD. Obtained results strongly support the traditional use of A. flavum and A. melanantherum in nutrition and recommend them for further study.

Assessment of the genotoxic potential along the Danube River by application of the comet assay on haemocytes of freshwater mussels: The Joint Danube Survey 3.

In this study we assessed the level of genotoxic pollution along the Danube River by measuring the level of DNA damage in the haemocytes of freshwater mussels of Unio sp. (Unio pictorum/Unio tumidus) and Sinanodonta woodiana. The comet assay was used for the assessment of DNA damage. The research was performed on 34 out of 68 sites analysed within the Joint Danube Survey 3 - the world's biggest river research expedition of its kind in 2013. During research, 2285 river kilometres were covered with an average distance of 68 km between the sites. The complex data set on concentrations of various substances present in water, suspended particulate matter and sediment on investigated sites gave the opportunity to identify the groups of xenobiotics which mostly affect the studied biomarker - DNA damage. The highest levels of DNA damage were recorded in the section VI (Panonnian Plain), which is under the impact of untreated wastewater discharges. Both positive and negative influences of the large tributaries on the level of genotoxicity in the Danube River were evident. Significant correlation in response was detected between the studied species of freshwater mussels. The level of DNA damage in mussels correlated with concentrations of compounds from the group of hazardous priority substances (polycyclic aromatic hydrocarbons), persistent organic pollutants (dioxins) and emerging pollutants (Oxazepam, Chloridazon-desphenyl).

Comparative study of genotoxic, antigenotoxic and cytotoxic activities of monoterpenes camphor, eucalyptol and thujone in bacteria and mammalian cells.

Genotoxic/antigenotoxic, mutagenic/antimutagenic and cytotoxic effects of monoterpenes camphor, eucalyptol and thujone were determined in bacteria and mammalian cells using alkaline comet assay, Escherichia coli K12 reversion test and MTT assay, respectively. When applied in low doses (up to 200 µM in bacterial assay and 50 µM in comet assay) monoterpenes protected repair proficient E. coli and Vero cells against UV-induced mutagenesis and 4NQO-induced DNA strand breaks, respectively. Antimutagenic response was not detected in nucleotide excision repair (NER) deficient bacteria. When monoterpenes were applied in higher doses, a weak mutagenic effect was found in mismatch repair (MMR) and NER deficient E. coli strains, while induction of DNA strand breaks was evident in human fetal lung fibroblasts MRC-5, colorectal carcinoma HT-29 and HCT 116 cells, as well as in Vero cells. Moreover, the involvement of NER, MMR and RecBCD pathways in repair of DNA lesions induced by monoterpenes was demonstrated in E. coli. Camphor, eucalyptol and thujone were cytotoxic to MRC-5, HT-29 and HCT 116 cells. The most susceptible cell line was HCT 116, with IC50 values of 4.5 mM for camphor, 4 mM for eucalyptol and 1 mM for thujone. Observed effects of monoterpenes are consistent with hormesis response, characterized by a low dose beneficial effect and a high dose adverse effect of a stressor agent, and provide a basis for further study of both chemopreventive and chemotherapeutic potential of camphor, eucalyptol and thujone.

Essential oil of Eucalyptus gunnii hook. As a novel source of antioxidant, antimutagenic and antibacterial agents.

The present study describes radical scavenging capacity (RSC), antimutagenic and antibacterial properties of the essential oil (EO) of the leaves of Eucalyptus gunnii Hook. (Southern Montenegro). Chemical composition was evaluated by gas chromatography-mass spectrometry (GC-MS). In oil, 1,8-cineole (67.8%) and α-pinene (14.12%) were the major compounds comprising almost 82% of total EO. EO exhibited moderate DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity, with IC50 value of 7.19 µL/mL. The antimutagenic properties were assayed against the spontaneous and t-BOOH-induced mutagenesis in Escherichia coli IC202 oxyR mutant strain, deficient in removing radical oxygen species (ROS). Reduction of the spontaneous mutagenesis in the presence of E. gunnii EO was only slight, up to 12% at the highest concentration tested. However, when the oxidative mutagen was used, EO displayed more significant reduction of mutagenesis (maximum 23%) in a concentration dependent manner. Antibacterial activity was tested against the selected strains from ATTC and NCIB collections: Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Micrococcus flavus, Klebsiella pneumoniae, and the two Escherichia coli strains from our laboratory collection (SY252 and IB112) using both the disk-diffusion and MIC assays. The greatest sensitivity was shown by M. flavus, K. pneumoniae and E. coli lpcA (MIC = 0.83 mg/mL), while the highest resistance was shown by E. coli (ATTC 25922) and S. epidermidis. This study represents the first report on chemical composition and biological activity of the Eucalyptus gunnii in the South Balkan region and beyond.

The impact of in vivo and in vitro exposure to base analogue 5-FU on the level of DNA damage in haemocytes of freshwater mussels Unio pictorum and Unio tumidus.

The impact of in vivo and in vitro exposure to anticancer drug 5-Fluorouracil (5-FU) on the level of DNA damage was evaluated using comet assay on haemocytes of freshwater mussels Unio pictorum and Unio tumidus. For the in vivo experiment, the groups of 5 mussels per concentration were exposed for 72 h to 5-FU (0.04, 0.4, 4, 40 and 100 µM) with 0.4 µM being the lowest concentration to induce significant DNA damage. For the in vitro experiment, the primary cultures of haemocytes were treated with 0.04, 0.4, 4 and 40 µM 5-FU for 22 h and the treatment with CdCl2 was used as a positive control. In contrast to in vivo exposure, 5-FU did not induce significant increase of DNA damage in vitro, possibly because of the absence of haemocytes proliferation in primary cultures.

Monitoring of DNA damage in haemocytes of freshwater mussel Sinanodonta woodiana sampled from the Velika Morava River in Serbia with the comet assay.

This study was undertaken to investigate the potential of the freshwater mussel Sinanodonta woodiana for detection of genotoxic pollution of the environment. Study was performed at two sites in the Velika Morava River, from May 2010 to February 2011. The alkaline comet assay on haemocytes was used, and the olive tail moment (OTM) was chosen as a measure of DNA damage. The specimens held on acclimation under controlled laboratory conditions for 10d were used as a control. Chemical analysis revealed the presence of phosphates and increased concentrations of zinc, copper and nickel at both sites during the entire sampling period. The values of OTM in mussels collected from the environment, significantly correlated with the concentration of zinc (r=0.6248), temperature (r=0.7006) and dissolved oxygen (r=0.7738). Seasonal variations in genotoxic response were observed, with the highest OTM values obtained during summer months. Preliminary results of the in vitro study indicated the effect of water temperature on genotoxic response to zinc and cadmium in S. woodiana suggesting that the presence of genotoxic pollutants during months with lower temperature could be under-estimated. Obtained results indicate that S. woodiana could be a valuable tool for active biomonitoring of aquatic environments and emphasizes the importance of seasonal genotoxic monitoring with this organism.

Evaluation of the DNA damaging effects of amitraz on human lymphocytes in the Comet assay.

Amitraz is formamidine pesticide widely used as insecticide and acaricide. In veterinary medicine, amitraz has important uses against ticks, mites and lice on animals. Also, amitraz is used in apiculture to control Varroa destructor. It this study, the alkaline Comet assay was used to evaluate DNA damaging effects of amitraz in human lymphocytes. Isolated human lymphocytes were incubated with varying concentrations of amitraz (0.035, 0.35, 3.5, 35 and 350 mu g/mL). The Comet assay demonstrated that all concentrations of amitraz caused statistically significant increase in the level of DNA damage, thus indicating that amitraz possesses genotoxic potential. The concentration of amitraz that produced the highest DNA damage (3.5 mu g/mL) was chosen for further analysis with the antioxidant catalase. The obtained results showed that co-treatment with antioxidant catalase (100 IU/mL or 500 IU/mL) significantly reduced the level of DNA damage, indicating the possible involvement of reactive oxygen species in DNA damaging effects of amitraz. Flow cytometric analysis revealed increase of the apoptotic index following treatment with amitraz. However, co-treatment with catalase reduced the apoptotic index, while treatment with catalase alone reduced the percentage of apoptotoc cells even in comparison with the negative control. Therefore, catalase had protective effects against ROS-mediated DNA damage and apoptosis.

Heavy metal accumulation and the genotoxicity in barbel (Barbus barbus) as indicators of the Danube river pollution.

The aim of this study was to analyze 16 trace elements (Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Li, Mn, Mo, Ni, Pb, Sr, and Zn) in different barbel (Barbus barbus) tissues and to detect the presence of genotoxic effects in erythrocytes with the alkaline comet assay. Barbel specimens were collected in the Danube river near Belgrade, Serbia, where the discharge of untreated communal and industrial wastewaters is likely to produce negative effects on fish residing in this area. The highest concentrations of Sr, Mn, Fe, Ba, B, and Al were found in gills, Mo and Cu in liver, and As and Zn in gonads. Concentrations of Zn and Fe were above maximum acceptable concentrations (MACs) in a number of gonad, gill, and liver samples. Three-year-old barbel specimens had higher tail moment and Zn concentrations in gills (1.71 and 51.20 µg/g dw, resp.) than 5-year-old specimens (0.85 and 42.51 µg/g dw, resp.). Results indicate that the younger barbel specimens might be more suitable for the monitoring of environmental pollution.

Modulation of genotoxicity and DNA repair by plant monoterpenes camphor, eucalyptol and thujone in Escherichia coli and mammalian cells.

The aim of this work was to examine the antigenotoxic potential of plant monoterpenes: camphor, eucalyptol and thujone in prokaryotic and eukaryotic cells and to elucidate their effect on DNA repair. We compared the effect of monoterpenes on spontaneous, UV- and 4NQO-induced mutagenesis in Escherichia coli K12 repair proficient, and MMR and NER deficient strains. Positive controls tannic acid and vanillin were included in bacterial tests. We also examined protective effect of monoterpenes against 4NQO-induced genotoxicity in Vero cell line by alkaline comet assay. The results obtained in repair proficient strain indicated antimutagenic potential of monoterpenes against UV- and 4NQO-induced mutagenesis, which was diminished with NER deficiency. Camphor and eucalyptol maintained UV-induced SOS response longer than in controls, while thujone decreased SOS response and reduced general protein synthesis and the growth rate. The three monoterpenes increased spontaneous and UV-induced recombination in recA730 and camphor additionally in recA(+) cells. Incubation of 4NQO-pretreated Vero cells with monoterpenes resulted in significant reduction of tail moment. However, higher concentrations of monoterpenes induced DNA strand breaks. Obtained results indicate that by making a small amount of DNA lesions camphor, eucalyptol and thujone can stimulate error-free DNA repair processes and act as bioantimutagens.

Protective effect of basil (Ocimum basilicum L.) against oxidative DNA damage and mutagenesis.

Mutagenic and antimutagenic properties of essential oil (EO) of basil and its major constituent Linalool, reported to possess antioxidative properties, were examined in microbial tests. In Salmonella/microsome and Escherichia. coli WP2 reversion assays both derivatives (0.25-2.0 microl/plate) showed no mutagenic effect. Salmonella. typhimurium TA98, TA100 and TA102 strains displayed similar sensitivity to both basil derivatives as non-permeable E. coli WP2 strains IC185 and IC202 oxyR. Moreover, the toxicity of basil derivatives to WP2 strains did not depend on OxyR function. The reduction of t-BOOH-induced mutagenesis by EO and Linalool (30-60%) was obtained in repair proficient strains of the E. coli K12 assay (Nikolic, B., Stanojevic, J., Mitic, D., Vukovic-Gacic, B., Knezevic-Vukcevic, J., Simic, D., 2004. Comparative study of the antimutagenic potential of vitamin E in different E. coli strains. Mutat. Res. 564, 31-38), as well as in E. coli WP2 IC202 strain. EO and Linalool reduced spontaneous mutagenesis in mismatch repair deficient E. coli K12 strains (27-44%). In all tests, antimutagenic effect of basil derivatives was comparable with that obtained with model antioxidant vitamin E. Linalool and vitamin E induced DNA strand breaks in Comet assay on S. cerevisiae 3A cells, but at non-genotoxic concentrations (0.075 and 0.025 microg/ml, respectively) they reduced the number of H(2)O(2)-induced comets (45-70% Linalool and 80-93% vitamin E). Obtained results indicate that antigenotoxic potential of basil derivatives could be attributed to their antioxidative properties.

Subspecies-specific distribution of intervening sequences in the Bacillus subtilis prophage ribonucleotide reductase genes.

A collection of 212 gram-positive bacilli isolated from natural habitats was screened for the presence of intervening sequences (introns and intein-coding sequences) in the SPbeta prophage-related ribonucleotide reductase genes bnrdE and bnrdF. Three novel configurations were identified on the basis of the presence of (i) intervening sequences in bnrdE and bnrdF, and (ii) an ORF in the bnrdE-bnrdF spacer. Analysis of the cell wall genetic determinants as well as of the incorporation of radio-labelled glycerol into cell wall allowed newly and previously identified B. subtilis strains with different configurations of bnrdE/bnrdF intervening sequences to be assigned to one of two subspecies. Strains apparently belonging to the subsp. subtilis contain three intervening sequences many of which are associated with the putative homing endonuclease activity. Strains of the subsp. spizizenii contain only one or two ORF-less group I introns. Introns occupying bnrdF are confined to the subspecies subtilis.

Comparative study of the antimutagenic potential of Vitamin E in different E. coli strains.

The antimutagenic potential of Vitamin E due to its antioxidative properties was studied. The new Escherichia coli K12 assay-system designed in our laboratory was employed in order to detect the antimutagenic potential of Vitamin E and to determine its molecular mechanisms of action. The assay is composed of three tests. In Test A, we examine the influence of the antioxidant on induced oxidative mutagenesis in a repair-proficient strain. Spontaneous mutagenesis is monitored in Test B, which is performed with two mutator strains, one mismatch repair-deficient (mutS) and another deficient in 8-oxo-dGTP-ase activity (mutT). In Test M, a repair-proficient strain and its mismatch repair-deficient counterpart (mutH), both carrying a plasmid with microsatellite sequences, are used to measure the level of microsatellite instability. To examine the antimutagenic potential of Vitamin E we also used the WP2 antimutagenicity test. Protective properties of Vitamin E against oxidative mutagenesis were detected in all tests with the E. coli K12 assay-system as well as in the WP2 antimutagenicity test. This study confirms that mismatch repair is essential for repair of oxidative DNA damage. The results obtained indicate that Vitamin E prevents the formation of DNA adducts by lipid peroxidation products rather than those formed by direct oxidation of DNA bases. Moreover, it can reduce microsatellite instability. After further validation, the new E. coli K12 assay-system can be used to test the antimutagenic potential of antioxidants.