Antimutagenic and antigenotoxic effects of vegetable matrices on the activity of pesticides

Two in vitro tests, one to detect bacterial mutagenicity (Ames test) on Salmonella typhimurium TA98, TA100, and TA1535 and the other the primary DNA damage (SOS Chromotest) on Escherichia coli PQ37, were applied to determine the overall genotoxic activity of 12 pesticides (azinphos methyl, chlorothalonil, chlorphyriphos ethyl, chlorphyriphos methyl, λ-cyhalothrin, cypermethrin, cyprodinil, fenazaquin, fludioxonil, indoxacarb, iprodione and penconazol). These were detected by gas chromatography (GC) analysis with electron capture (ECD) and nitrogen phosphorus detection (NPD) in 18 samples of vegetables. Some extracts of vegetables, found positive for pesticides with GC, were subjected to the Ames test and SOS Chromotest to evaluate the possible antimutagenic and/or antigenotoxic effects of vegetable matrices. The same bioassays were also performed on the mixtures of pesticides found in these samples to evaluate whether interactions could occur between pesticides and be responsible for the possible antimutagenic and/or antigenotoxic effects of the contaminated matrices. Experiments were also carried out to compare the results found for contaminated vegetables with their content of antioxidant components. Significant differences in mutagenicity and genotoxicity were found among the pesticides selected for this study. Of the 12 pesticides tested, only azinphos methyl, cyprodinil, fludioxonil and iprodione were found to be positive for both S. typhimurium and E. coli. No mutagenic/genotoxic activity was found in the extracts of vegetables contaminated by pesticides. S. typhimurium TA1535 showed a strong positive mutagenic effect for the mixtures of pesticides while they were not able to induce the SOS system. The data concerning the content of polyphenols and the total reducing activity of the contaminated vegetables indicated high amounts of antioxidants that could explain the inhibitory effect on the activity of pesticides shown by vegetables.     

Mutagenicity of Edible Palm Oil on the Ghanaian Market before and after Repeated Heating

Red palm oil produced in Ghana largely by village folks has never been tested for its mutagenic potential. The study aimed at determining the mutagenicity of high‐energy heated red palm oil (RRPO) and refined, bleached imported palm oil (PO) on the Ghanaian market. Samples of RRPO and PO were 1× and 5× heated for 10 min at 180 °C with a cooling period of 5 h in‐between. Unheated, together with heated samples, were tested for mutagenicity using Salmonella typhimurium TA 98 and TA 100 tester stains. Unheated PO was negative for the Ames mutagenicity test with TA 98 strain. However, 1× and 5× heated PO were mutagenic (P = 0.05, each). Testing PO, using TA 100 strain was negative. RRPO was mutagenic with TA 98 strain for heated oils (P = 0.05, each). Assays with TA 100 strain showed highly significant mutations (P = 0.001, each) that increased with increasing heating frequency. PO 1× and 5× heated samples caused significant frameshift mutation in the S. typhimurium TA 98 strain. RRPO caused highly significant point and frameshift mutations in heated samples. Furthermore, unheated RRPO mutagenic potential has serious health implications.     

Structure-dependent relative toxic potencies of selected pyrrolizidine alkaloids

Pyrrolizidine alkaloids are naturally occurring secondary plant metabolites mainly found in plant families of Asteraceae, Boraginaceae, and Fabaceae. Chemically, Pas consist of a pyrrolizidine core bearing hydroxyl groups, the so-called necine base, and mono- or dicarboxylic necine acids bound to the pyrrolizidine core via ester linkages. 1,2-unsaturated PAs are hepatotoxic, genotoxic, and carcinogenic due to the highly reactive pyrrolic metabolites formed by cytochrome P450 monooxygenases (CYPs) primarily in the liver. The presence of PAs as frequent contaminants in the wide variety of food and feed products has to be considered a relevant safety issue. Based on the currently available data, the risk assessment of PAs was mainly approached using the two most toxic potent congeners, i.e., lasiocarpine and riddelliine. However, it is well recognized that toxicity is differing significantly between the congeners related to their structural features. The risk of PA-containing products is indeed overestimated, and a comprehensive risk assessment should take these differences into account. After analyzing the data of many PAs, Merz and Schrenk derived interim Relative Potency (iREP) factors to present the differences in their toxicity between the sub-groups of PA congeners concerning their structural features. The use of such iREP factors could probably provide a more scientific basis for PA risk assessment until sufficient experimental analysis of the toxicities of individual congeners is applied. To obtain a better understanding of the relationship between structure and toxicity of PA congeners and provide more evidence for further refinement of relative (toxic) potency factors, data of the in vitro cytotoxicity, genotoxicity, and mutagenicity of diverse individual PA congeners (lasiocarpine, monocrotalineφ, retrorsine, senecionine, seneciphyllineφ, echimidineφ, europineφ, heliotrineφ, indicine, and lycopsamine) has been generated in our project supported by Kooperation Phytopharmaka. Among them, lasiocarpine, retrorsine, senecionine, indicine and lycopsamine have been investigated on my part. Cytotoxicity was assessed using the Alamar blue assay in primary rat hepatocytes, HepG2 cells, and the HepG2 (CYP3A4) cell line. In HepG2 cells, none of the selected PAs exhibited cytotoxic effects, probably due to the lack of CYPs. In primary rat hepatocytes as well as in HepG2(CYP3A4) cells, a clear structure dependent cytotoxicity could be demonstrated. The role of CYP450 enzymes in metabolic activation was further confirmed using an inhibition assay. A kinetic assay analyzing 7-benzyloxyresorufin-O- dealkylation (BROD) was used for measuring the activity of CYP450 enzymes. Furthermore, the utilization of a glutathione-reductase-DTNB recycling assay indicated that glutathione might not play a critical role in PA-induced cytotoxicity. A micronucleus test was used for determining the PA-induced clastogenic genotoxicity. All selected PA congeners exhibited concentration-dependent toxicity in the HepG2 (CYP3A4) cells. The relative potencies of PA congeners estimated from Alamar blue assay and micronucleus assay are generally consistent with the following ranking: lasiocarpine > senecionine > seneciphylline > retrorsine > heliotrine (?) echimidine > europine = indicine = lycopsamine = monocrotaline. The relative toxic potencies evaluated based on our findings were not completely consistent with the iREP classification previously reported by Merz and Schrenk. Monocrotaline in both assays exhibited considerably lower toxic potency. Echimidine, however, was more toxic than expected. On the other hand, mutagenicity was measured in Ames fluctuation assay with Salmonella typhimurium strains TA98 and TA100. None of the selected PA congeners up to 300 μM showed mutagenic effects despite metabolic activation with S9-mix.     

Effect of Pectin and Lignin on Mutagenicity in the Ames Assay

The effect of incorporating varying concentrations of pectin and lignin into the Ames assay with the known mutagens, benzo(a) pyrene, 3-methyl cholanthrene, and N-methyl-N-nitro-N-nitrosoguanidine, on mutagenic activity was investigated. The addition of 50 μg, 150 μg, and 300 μg concentrations of pectin and lignin failed to block the mutagenicity of benzo-pyrene and 3-methyl cholanthrene in the Ames/mammalian microsome assay using Salmonella typhimurium TA 98 and TA 100 with and without preincubation. The incorporation of 50 μg, 150 μg, and 300 μg of pectin and lignin did not block the mutagenic activity of MNNG in the Ames Spot test with Salmonella typhimurium TA 100.     

Emission of polycyclic aromatic hydrocarbons, toxicity, and mutagenicity from domestic cooking using sawdust briquettes, wood, and kerosene

Smoke samples, in both gas and particulate matter (PM) phases, of the three domestic stoves were collected using U.S. EPA modified method 5 and were analyzed for 17 PAH (HPLC-UV), acute toxicity (Microtox test), and mutagenicity (Amestest). The gas phase of smoke contributed > or = 95% of 17 PAH, > or = 96% of toxicity, and > or = 60% of mutagenicity. The highest emission factor of 17 PAH was from sawdust briquettes (260 mg/kg), but the highest emission of 11 genotoxic PAH was from kerosene (28 mg/kg). PM samples of kerosene smoke were not toxic. The total toxicity emission factor was the highest from sawdust, followed by kerosene and wood fuel. Smoke samples from the kerosene stove were not mutagenic. TA98 indicated the presence of both direct and indirect mutagenic activities in PM samples of sawdust and wood fuel but only direct mutagenic activities in the gas phase. TA100 detected only direct mutagenic activities in both PM and gas-phase samples. The higher mutagenicity emission factor was from wood fuel, 12 x 10(6) revertants/kg (TA100-S9) and 3.5 x 10(6) (TA98-S9), and lower from sawdust, 2.9 x 10(6) (TA100-S9) and 2.8 x 10(6) (TA98-S9). The low burning rate and high efficiency of a kerosene stove have resulted in the lowest PAH, toxicity, and mutagenicity emissions from daily cooking activities. The bioassays produced toxicity and mutagenicity results in correspondence with the PAH content of samples. The tests could be used for a quick assessment of potential health risks.     

Untersuchungen zur Mutagenit?t von Zuckercouleuren

Zusammenfassung In der vorliegenden Arbeit wurden vier kaustische und drei Ammoniakcouleure auf ihre mutagene Wirkung mit Hilfe des Ames-Tests untersucht. Dazu wurden die StandardteststämmeSalmonella typhimurium TA 1535, TA 1537, TA 98 und TA 100 eingesetzt. Sowohl mit als auch ohne metaboler Aktivierung konnte kein signifikanter Anstieg in der Mutationsrate festgestellt werden. Auch eine Vorinkubation erbrachte für die hier getesteten Zuckercouleure keine Hinweise auf eine mögliche Mutagenität im Ames-Test.

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On the in-vitro mutagenicity of caramel colours

Summary In the present study commercially available caustic and ammoniated caramel colours were tested for their mutagenic potential using the Ames assay. The test was performed using the standard test strainsSalmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without a metabolic activation system (S9-mix). Furthermore, a special preincubation procedure without metabolic activation system was applied. None of the tested caramel colours showed any mutagenic effect in the Ames test.

     

Mutagenicity and Identification of the Reaction Products of Aqueous Chlorine or Chlorine Dioxide with L-Tryptophan

Nonvolatile products generated from reactions of graded molar ratios of aqueous chlorine or chlorine dioxide with L-tryptophan (1:1, 3:1 and 7:1) were shown to be direct-acting mutagens to Salmonella typhimurium TA100 and TA98. Increasing the ratio of disinfectant relative to amino acid led to increased mutagenic activity, with mutagenicity highest at the 7:1 molar ratio. Several fluorescent bands obtained after thin layer chromatographic fractionation of the reaction mixtures were shown to be more mutagenic than the reaction mixtures. GC/MS analysis of the compounds in a highly mutagenic fraction of the aqueous chlorine reaction products identified 1,1,3-trichloropro-panonc, 1,1,3,3-tetrachloropropanone and dichloroquinoline.     

Formation of protein adducts of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine in cooked foods

Heterocyclic amines (HCAs) are mutagenic and carcinogenic compounds found in cooked meat and fish. Although HCAs are known to form adducts with protein after metabolic activation, adduct formation during cooking has not been elucidated. In this study, we showed that 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is released from high molecular weight compounds by acid or enzymatic hydrolysis of cooked foods. Formation of free and protein adduct forms of PhIP was dependent on cooking temperature and time, and PhIP–protein adducts were estimated to form after formation of free PhIP. We also demonstrated that PhIP–protein adduct is formed by heating of PhIP and albumin as a model protein. A new adduct peak including [M+H]⁺ (m/z=225) of PhIP as a fragment ion was detected in the high molecular weight fraction of heat‐treated protein by LC–MS analysis. From model experiments by heating of PhIP and amino acids, the adduct was estimated to be produced by condensation of the amino group of PhIP and the carboxyl group of protein. PhIP–protein adducts were detected in several cooked meat and fish at ng/g food level as PhIP content. These results suggest that food‐borne protein adducts of HCAs may influence human HCA exposure and carcinogenic risk.     

Reactions of Aqueous Chlorine and Chlorine Dioxide with Tryptophan, N-Methyltryptophan, and 3-Indolelactic Acid: Kinetic and Mutagenicity Studies

Reactions of tryptophan, N-methyltryptophan and 3-indolelactic acid with aqueous chlorine or chlorine dioxide (ClO₂) in 0.1M potassium phosphate buffer, pH 6.0, were investigated to determine any structural relationships with regards to kinetics and mutagenicity. The reaction with ClO₂ followed pseudo-first order kinetics, with the half-life of the respective compounds being 36, 22, and 8 milliseconds. The formation of a dark precipitate in the reaction of tryptophan with HOCl precluded any kinetic comparison. The reaction products of tryptophan with hypochlorous acid (HOCl) or ClO₂ were mutagenic to Salmonella typhimurium TA98 and TA100; while those of N-methyltryptophan with HOCl and ClO₂ were more mutagenic toward TA98. Higher recoveries of the reaction products were achieved by passing the acidified (pH 2.5) mixture through an XAD-8/XAD-2 resin column.     

Presence of insecticides in shrimp farms adjacent to the sea of cortes: detection, quantification, and toxicity testing

The presence of insecticides in a shrimp farming park in Sonora, Mexico, was investigated. Sediment and water were from an estuary adjacent to the farming park and sediment, water, and shrimp from ponds were sampled and analyzed for insecticides using gas chromatography. Artemia salina acute toxicity assay and the Salmonella mutagenicity test were performed on insecticides found in shrimp and on insecticide-containing shrimp, respectively. All insecticides searched were found in shrimp samples, as well as in sediment and water from the estuary. Chlorpyrifos was found to cause >50% mortality at levels below those found in ponds. All insecticide-containing shrimp were not mutagenic towards Salmonella TA98 and TA100 tester strain. Results suggest that insecticides levels found in the farming environment might contribute to reduced production. Although insecticide-containing shrimp were no mutagenic, further toxicity testing is necessary to evaluate the risk for the consumer.     

Antimutagenic activities of some foods

There has been a recent increase in research into the possible ways of protecting individuals from cancer by making changes in dietary habits. In this study raw and boiled juice and leaf and dried seeds of Urtica sp, Stachys annula, sage, rosehip, grape molasses and tarhana were not found mutagenic in S typhimurium TA 100. All of the foods decreased sodium azide mutagenicity in S typhimurium TA 100. Among the foods tested, raw Urtica sp (46.32%) and rosehip (44.03%) showed the highest antimutagenic effect followed by leaf of Urtica sp (41.25%), boiled juice of Urtica sp (37.22%), Stachys annula (36.42%), grape molasses (33.03%) and tarhana (28.60%), respectively. The antimutagenic activities of Urtica sp and rosehip were found to be significantly higher than the antimutagenic activity of tarhana (p<0.01). © 1999 Society of Chemical Industry     

Acrolein induced DNA damage, mutagenicity and effect on DNA repair

Acrolein (Acr) is a ubiquitous environmental contaminant; it also can be generated endogenously by lipid peroxidation. Acr contains a carbonyl group and an olefinic double bond; it can react with many cellular molecules including amino acids, proteins and nucleic acids. In this review article we focus on updating information regarding: (i) Acr-induced DNA damage and methods of detection, (ii) repair of Acr-DNA damage, (iii) mutagenicity of Acr-DNA adducts, (iv) sequence specificity and methylation effect on Acr-DNA adduct formation and (v) the role of Acr in human cancer. We have found that Acr can inhibit DNA repair and induces mutagenic Acr-dG adducts and that the binding spectrum of Acr in the p53 gene in normal human bronchial epithelial cells is similar to the p53 mutational spectrum in lung cancer. Since Acr-DNA adduct has been identified in human lung tissue and Acr causes bladder cancer in human and rat models, we conclude that Acr is a major lung and bladder carcinogen, and its carcinogenicity arises via induction of DNA damage and inhibition of DNA repair.     

Inhibition of mutagenic N-nitroso compound formation in sausage samples by using -ascorbic acid and α-tocopherol

In this study 24 samples of sausage with different amounts of nitrite, -ascorbic acid and α-tocopherol were prepared in order to determine the inhibitory effects of -ascorbic acid and α-tocopherol as reductants against formation of mutagenic N-nitroso compounds that form in cured meat products. These mutagenic N-nitroso compounds were extracted by phosphate buffer and ethylacetate. The mutagenicity of extracts were investigated by salmonella/microsome assay. The number of revertants indicated the N-nitroso compounds content. Among the Salmonella typhimurium strains tested, the revertants of S. typhimurium TA100 were significantly reduced (P<0.5) by 60% when reductants were added to the samples.     

Screening for mutagens in norwegian foods by the ames test

Twelve Norwegian food products prepared in an ordinary domestic manner, were screened for mutagenic activity towards Salmonella typhimurium strains. Mutagenic activity towards the frameshift mutations strains (TA1538 and TA98) was observed in the basic organic extracts from fried fish cakes and fish pudding. Metabolic activation with rat liver homogenate (S9) was required. The ten other food items studied were found to be devoid of mutagenic activity.     

Evaluation of antioxidative and mutagenic properties of 50% ethanolic extract from red beans fermented by Aspergillus oryzae

Various bean products fermented by microorganisms are commonly consumed in Asian diets; however, the safety or functional properties of fermented beans can vary with different microbial species and with different processes being applied to different beans. The objectives of this study were to evaluate the antioxidative and mutagenic properties of 50% ethanolic extracts from red beans fermented by Aspergillus oryzae. The extracts' antioxidative activities, including alpha,alpha;-diphenyl-beta-picryl-hydrazyl (DPPH) radical-scavenging effects, Fe(2+)-chelating ability, and reducing power, were studied in vitro. The antioxidative effects provided by the extracts depended strongly on their concentrations. In general, antioxidative activity increased with extract concentration to a certain point and then leveled off as the concentration further increased. The fermented red bean extracts showed less of a scavenging effect on the DPPH radical and less reducing power than the commercial antioxidants alpha-tocopherol and butylated hydroxytoluene, but better Fe(2+)-chelating ability. No mutagenicity or toxicity effect on any of the tested strains (Salmonella Typhimurium TA97, TA98, TA100, TA102, and TA1535) was found for the 50% ethanolic extracts of fermented red beans with the Ames mutagenicity assay. These results suggest that the 50% ethanolic extracts were not mutagenic.     

In Vivo Genotoxicity Evaluation of an Artichoke (Cynara scolymus L.) Aqueous Extract

The Cynara scolymus (artichoke) is widely consumed as tea or food and shows important therapeutic properties. However, few studies have assessed the possible toxic effects of artichoke extracts. This study evaluates genotoxic and mutagenic activities of artichoke leaf aqueous extract in mice using the comet assay and the micronucleus test. Leaf extracts were given by gavage (500 mg/kg, 1000 mg/kg, and 2000 mg/kg) for 3 consecutive days. Extract composition was investigated using phytochemical screening and high‐performance liquid chromatography (HPLC). In addition, antioxidant capacity was analyzed through the diphenyl‐picrylhydrazyl (DPPH) and xanthine oxidase assay. Phytochemical screening detected the presence of phenolic compounds, flavonoids, and saponins. HPLC analyses indicated the presence of chlorogenic acid, caffeic acid, isoquercetrin, and rutin. Extracts showed a dose‐dependent free radical scavenging effect of DPPH and an inhibitory effect of xanthine oxidase. The genotoxic results showed that leaf extracts did not increase micronuclei in peripheral blood cells. Compared to the control group, a significant increase in comet assay values was observed only in bone marrow of group treated with 2000 mg/kg, the highest dose tested, indicating that artichoke tea should be consumed with moderation. Practical Application: This is the first report of in vivo mutagenic and genotoxic evaluation with C. scolymus. The present study revealed leaf aqueous extract from artichoke shows lack of mutagenicity in vivo, and low genotoxicity and antioxidant activity; indicating that artichoke tea should be consumed with moderation.     

Nonclinical Safety Assessment of Morus alba L. Fruits: Study of 90‐D Toxicity in Sprague Dawley Rats and Genotoxicity in Salmonella

Morus alba L. is a traditional herb with a long history of consumption, both as an edible fruit and as medicine. However, its safety evaluation has not yet been established. The objective of this study was to evaluate subchronic oral toxicity and genotoxicity of M. alba L. fruits (MFE). The subchronic toxicity after daily oral administration of MFE at 0, 40, 200, and 1000 mg/kg for 90 d was examined in Sprague Dawley (SD) rats. MFE administration did not lead to death, adverse effects, change in food and water consumption, and body weight gain. Significant toxic effects were not found within the parameters of organ weight, biochemical values, and hematological and urine analysis between the control and the MFE group. The genotoxicity of MFE was assayed by Ames test in Salmonella typhimurium strains TA98, TA102, and TA1535. No genotoxicity was found in all the tested strains. Thus in this study, a no‐observed‐adverse‐effect level for MFE in 90 d repeated oral toxicity study in rats was determined to be greater than 1000 mg/kg regardless of gender. The results also suggested that MFE does not have a genotoxicity potential.     

Efficacy and Safety Evaluation of Ozonation to Degrade Aflatoxin in Corn

ABSTRACT: This study determined the efficacy and safety of ozonation in degrading aflatoxin in corn. Ozonation (10 to 12 wt%) reduced aflatoxin levels by 92% and no reversion to the parent compound was observed. Ozonation had minimal effect on fatty acids of uncontaminated corn, but had significant effect on fatty acids of contaminated corn. Crude extracts showed no mutagenic potential in the Ames assay using TA98 and TA100. Clean-up using hexane increased their mutagenic potentials. Clean-up using Mycosep columns increased the mutagenic potentials 18 to 617%. Hexane extracts from ozone-treated contaminated corn had lower inhibitory effect. This suggested that a fat-soluble mutagen is being formed or natural inhibitors of mutagenicity are being destroyed.     

Ames Test to Detect Mutagenicity of 2‐Alkylcyclobutanones: A Review

Food irradiation is an effective and safe method for preservation and long‐term storage, and it is approved for use in over 60 countries for various applications in a wide variety of food products. This process is performed by use of accelerated electron beams, X‐rays, or gamma radiation (⁶⁰Co or ¹³⁷Cs). 2‐Alkylcyclobutanones (2‐ACBs) are the only known radiolytic products generated from foods that have fatty acids (triglycerides) and are subjected to irradiation. Since the 1990s toxicological safety studies of 2‐ACBs have been conducted extensively through synthetic compounds, then and tests to determine if the compounds have any mutagenic activity are strictly necessary. The Ames test was chosen by many researchers to assess the mutagenicity of 2‐ACBs. The test uses distinct bacterial cell lines Salmonella typhimurium to detect point mutations at sites guanine–cytosine (G–C) and Escherichia coli to detect point mutations at sites adenine–thymine (A–T). This bibliographic research aims to bring together all the results obtained and a comparison and cell lines used, type of plates, and solvents. This research showed that no mutagenic activity was observed in any of the cell lines and concentrations evaluated by the works of authors, so the 2‐ACBs compounds showed no mutagenic substance in concentrations detectable by the Ames test.     

Caffeic acid derivatives,total phenols,antioxidant and antimutagenic activities of Echinacea purpurea flower extracts

This study evaluated the effects of ethanol concentration and extraction temperature on the quality of extracts obtained from freeze-dried Echinacea purpurea flowers. The antioxidant and antimutagenic effects of flower extract were also examined. The optimal extraction conditions for freeze-dried flowers were 50% aqueous ethanol and 65 °C extraction temperature, with extraction yield of 37.4%. The contents of total phenols, caffeic acid derivatives, and cichoric acid in freeze-dried extracts were 473.34 mg chlorogenic acid equivalents/g, 302.20 mg/g and 217.61 mg/g, respectively. The 50% ethanolic flower extract did not show toxicity and mutagenicity toward Salmonella typhimurium TA98 and TA100 with or without S9 mix. The ethanolic extract at 0.25–5 mg/plate exhibits a dose-dependent inhibitory effect against the mutagenicity of 2-aminoanthracene. Thus, freeze-dried E. purpurea flower ethanolic extract exhibits good antioxidant and antimutaginic activities.