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Oral microbiota has become an important factor in obesity, but its association with obesity-related diseases and serum 25-hydroxy vitamin D [25(OH)D] and B complex amounts is still uncertain. The main aim of the paper is to determine the variation in oral microbiota composition as a response to the vitamin status and obesity-related diseases in obese females from Croatia. We hypothesized that the prevalence of probiotic or pathogenic bacteria in the oral cavity of obese women in Croatia depends on the amounts of vitamin BTo test the defined research hypothesis, female individuals with body mass index (BMI)≥30 kg/mThe presented results do not support the hypothesis that the prevalence of probiotic or pathogenic bacteria in the oral cavity of obese women in Croatia depends on the amount of micronutrients. On the other hand, hypertension and diabetes/prediabetes favour the growth of oral pathogens, specifically increased levels ofTo the best of our knowledge, this is the first study showing the relationship between obesity, micronutrient amount, oral microbiota composition, and the incidence of obesity-related disease. We included only obese women from Croatia, so it is regionally specific. Also, we have shown that oral microbiota composition is not connected with micronutrient deficiencies but only with obesity-related diseases.

Waste landfills represent a global problem, which is more pronounced in developing countries because of the lack of resources to implement procedures that include separation and waste processing. The aim of this research was to analyze leachate and ground waters samples at the site, upstream and downstream from the landfill during different year seasons on a registered non-hazardous waste dump and to conduct physico-chemical and biological assays to determine potential risk for the ecosystem. Potential cytotoxic, prooxidative and mutagenic effects of leachates and water samples were evaluated on human laryngeal cell line (HEp2). Leachates collected at landfill site caused genotoxic effect and had a higher pH, chemical oxygen demand (COD), biochemical oxygen demand (BOD) and elevated concentrations of phosphorus, chloride, nitrogen compounds and sulphate. Genotoxicity of the leachate was increased in samples collected in dry and warm period of the year. These results are in accordance to the physico-chemical analysis which revealed that during summer period, because of intense degradation process at high temperatures increased concentrations of different chemicals can be found in leachate. Groundwater collected downstream and upstream from landfill did not show statistically significant (geno)toxic effect, irrespective of the sampling season. Chemical analysis revealed that all compounds in groundwater were below permitted values. Purification process at landfill is effective and compounds that reach groundwater do not represent a toxicological threat.

Obesity is composed of multifunctional interactions of eating habits, behaviors, microbiota, genetics, and other unknown factors. We hypothesize that correlations occur between the fat mass and obesity-associated single nucleotide polymorphisms (FTO SNPs), the composition of microorganisms in the saliva, and life habits in obese women, from Zagreb County. Our results of the analysis of three FTO SNPs showed a statistically significant positive correlation among the frequencies of the high- risk genotypes AA rs9939609 (P = .0367), CC rs1421085 (P = .0367), and GG rs17817449 (P = .0065) of the FTO gene in obese cases. Interestingly, 39.13% of obese women were triple homozygous for all three risk alleles. Furthermore, the composition of the oral microbiota in the obese group showed a higher occurrence of a major human pathogen, bacterium Staphylococcus aureus but a significantly low presence of bacteria Streptococcus oralis, Streptococcus mitis and Serratia ureilytica compared to the control group. The investigation also revealed that obese women prefer to consume candy and snacks and more meat and meat- derived products, they sleep less than six hours per day, and had higher hypertension, in comparison with the control group. These results support the hypothesis that female obesity is strongly related to all three variants of the FTO gene, and perhaps a specific composition of microbiota in saliva due to dietary habits. Considering the bimodal distribution of the single nucleotide polymorphisms and bacterial content of saliva in obese women taken together are factors to consider for risk of obesity.

High voltage electrical discharge (gas and liquid) plasmas are new developing techniques used for food and water decontamination. This study investigates the influence of high voltage gas phase plasma treatment on several parameters: inactivation of Escherichia coli K12, recovery, proteomic analyses, cellular leakage and influence of pH and generated H2O2 species on inactivation of the E. coli cells. Samples were treated in a glass reactor with a point-to-plate electrode configuration. Electrical conductivity (100 and 800 μS/cm), polarity (+/−), time (5 and 10 min) and frequency (60, 90 and 120 Hz) were chosen in order to determine their influence on listed parameters. Statistical analyses were obtained to optimize conducted treatments. Treatment regime of 90 Hz, positive polarity, 100 μS/cm and 10 min resulted in the highest reduction (7.8 log10 CFU/mL) of E. coli. Recovery of cells after the treatment suggested oxidative stress overcome. Industrial relevance Plasma processing is an energy effective process, comparing to traditional thermal processes like pasteurization and sterilization. Plasma treated liquid remains at low temperature, which lowers electrical energy consumption during industrial processes. The proposed study investigates the possible application of high voltage gas phase electrical discharge plasmas for inactivation of Escherichia coli K12 MG1655 in water. The main aim was to confirm effectiveness of plasma treatment conducted in argon (which is uninfected by plasma and can be recycled) on inactivation and cell recovery of E. coli K12. This research is a first step towards the development of the gas phase plasma technology for disinfection of liquids. However, future development, optimization and application of this technology for food sterilization will lead to acceptable ecological regulations like lowering greenhouse gas emissions.

Today, polyphenols represent one of the most well-known secondary plant metabolites characterized by a large and diverse array of unique bioactive properties, which makes them highly appreciated for their beneficial effects on both plants and humans. Polyphenols are compounds that are structurally extremely different, but essential for a variety of functions in plants, responsible for organoleptic and nutritional properties of plant-derived foods, and useful for numerous practical applications. In this introductory chapter, the basic information on polyphenols is presented, covering their historical background, evolution of the chemical definition of polyphenols, and different ways of their classification. The structural diversity of polyphenols is elaborated and related to their common food sources. The basic physicochemical properties of polyphenols, especially the reducing capacity and binding properties (metal chelating activity and protein–polyphenol complexation), are thoroughly elaborated. Different mechanisms of the main bioactive feature of polyphenols and their antioxidant properties are outlined, as well as mechanisms governing their oxidation, reactivity with dioxygen (autoxidation), and modulation of oxidative damage. Additionally, recent progress in the models for probing the antioxidant activity of polyphenols in the gastrointestinal tract and the effects of controlled polyphenol-rich foods on selected parameters of oxidative stress after consumption are presented.

RATIONALE We describe a novel negative chemically activated fragmentation/positive chemically activated fragmentation (CAF-/CAF+) technique for protein identification. The technique was used to investigate Lactobacillus brevis adaptation to nutrient deprivation. METHODS The CAF-/CAF+ method enables de novo sequencing of derivate peptides with negative and positive ion mode matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS). Peptide sequences obtained from MS/MS spectra were matched against the National Center for Biotechnology Information (NCBI) non-redundant (nr) database and confirmed by the mass spectrometry data of elucidated peptide mass sequences derived from the annotated genome. This improved protein identification method highlighted 36 differentially expressed proteins in the proteome of L. brevis after 75 days of starvation. RESULTS The results revealed the key differences in the metabolic pathways that are responsible for the survival of L. brevis in a hostile environment. Proteomics analysis demonstrated that numerous proteins engaged in glucose and amino-acid catabolizing pathways, glycerolipid metabolizing pathways, and stress-response mechanisms are differentially expressed after long-term starvation. Amino acid and proteomics analysis indicated that starved L. brevis metabolized arginine, glycine, and histidine from dead cells as alternative nutrient sources. The production of lactic acid also varied between the parent cells and the starved cells. CONCLUSIONS Differentially expressed proteins identified exclusively by peptide sequence reading provided promising results for CAF-/CAF+ implementation in a standard proteomics workflow (e.g., biomarker and mutation discovery and biotyping). The practical performance of a reliable de novo sequencing technique in routine proteomics analysis is emphasized in this article. Copyright © 2013 John Wiley & Sons, Ltd. Copyright

Obesity has become one of the major threats for public health in industrialised world among adults, but also among adolescents and children. It is infl uenced by the interaction of genes, nutrition, environment, and lifestyle. Environmental and lifestyle risk factors include foetal and lifelong environment, nutrient quality, chemical and microbial exposure, and psychical stress, all of which are important contributing infl uences. Removing or limiting chemical and pharmaceutical obesogens from human environment could make a difference in the growing epidemic of obesity. Additionally, nutrigenomics describes how modifi cations in individual diets can improve health and prevent chronic diseases, as well as obesity, by understanding the effects of a genetic profi le in the interaction between food and increase in body weight. Furthermore, individual genetic variations in genome represent an individual′s predisposition for obesity. Therefore, the use of individual genetic information, avoiding obesogens, and a healthy lifestyle could help to improve the management of obesity and maintain a healthy weight.

S-adenosyl-l-methionine (SAM) is an important molecule in the cellular metabolism of mammals. In this study, we examined several of the physiological characteristics of a SAM-accumulating strain of the yeast Scheffersomyces stipitis (M12), including SAM production, ergosterol content, and ethanol tolerance. S. stipitis M12 accumulated up to 52.48 mg SAM/g dry cell weight. Proteome analyses showed that the disruption of C-24 methylation in ergosterol biosynthesis, a step mediated by C-24 sterol methyltransferase (Erg6p), results in greater SAM accumulation by S. stipitis M12 compared to the wild-type strain. A comparative proteome-wide analysis identified 25 proteins that were differentially expressed by S. stipitis M12. These proteins are involved in ribosome biogenesis, translation, the stress response, ubiquitin-dependent catabolic processes, the cell cycle, ethanol tolerance, posttranslational modification, peroxisomal membrane stability, epigenetic regulation, the actin cytoskeleton and cell morphology, iron and copper homeostasis, cell signaling, and energy metabolism.

Cereulide (CER) intoxication occurs at relatively high doses of 8 µg/kg body weight. Recent research demonstrated a wide prevalence of low concentrations of cereulide in rice and pasta dishes. However, the impact of an exposure to low doses of cereulide was never studied before. In this research, we investigate the effect of low concentrations of cereulide on the behavior of intestinal cells using Caco-2 cell line. The MTT (mitochondrial 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide) and the SRB (Sulforhodamine B) reactions were used to measure the mitochondrial activity and the measurement of cellular protein content, respectively. Both assays showed that differentiated Caco-2 cells were sensitive to low concentrations of CER (in MTT reaction 1 ng/mL after 3 days of treatment ; in SRB reaction 0.125 ng/mL after 3 days of treatment). Cell counts revealed that cells were released from the differentiated monolayer at 0.5 ng/mL of cereulide. Additionally, 0.5 and 2 ng/mL of CER increased lactate presence in the cell culture medium. Proteomic data showed that cereulide at concentration of 1 ng/mL led towards a significant decrease in energy managing and H2O2 detoxification proteins and to an increase in cell death markers. This is amongst first reports to describe the influence of sub-emetic concentrations of cereulide on a differentiated intestinal monolayer model showing that low doses may induce an altered enterocyte metabolism and membrane integrity.

U Europskoj uniji propisane kontrole za sigurnost hrane prema zakonskim aktima određuju provjeru hrane prije stavljanja na tržište. Tijekom proteklog desetljeća provjera kakvoće, autohtonosti i sljedivosti hrane poboljšana je ubrzanim razvojem osjetljivih, robusnih, učinkovitih i cjenovno prihvatljivih analitičkih metoda. Ovaj rad opisuje najučinkovitije analitičke metode za forenziku hrane i prikazuje njihovu primjenu u određivanju: (1) kvalitativnih i kvantitativnih sastojaka hrane, (2) sigurnosti hrane obzirom na patogene mikroorganizme, nutritivne alergene, ostatke pesticida i toksina, (3) sljedivosti sukladno zemljopisnom i botaničkom podrijetlu te utjecaju tehnološke obrade i skladištenja. Obzirom na način ispitivanja, opisane analitičke metode podijeljene su u četiri skupine: biokemijske, molekularno-genetske, spektroskopske i spektrometrijske te separacijske metode., The European Union food safety legislation prescribes controls in order to implement the testing of food before putting it on the market. Over the past decade checking quality, originality and the traceability of food has improved the rapid development of sensitive, robust, efficient and affordable analytical methods. This paper describes the most effective analytical methods for forensic food and shows their use in determining: (i) qualitative and quantitative food ingredients, (ii) food safety due to pathogens, allergens, nutritional, residues of pesticides and toxins, (iii) traceability according to geographical and botanical origin and the impact of technological processing and storage. Given the way tests are described analytical methods are divided into four groups as following: biochemical, molecular-genetic, spectroscopic and spectrometric, and separation methods

We describe a novel negative chemically activated fragmentation/positive chemically activated fragmentation (CAF-/CAF+) technique for protein identification. The technique was used to investigate Lactobacillus brevis adaptation to nutrient deprivation.The CAF-/CAF+ method enables de novo sequencing of derivate peptides with negative and positive ion mode matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS). Peptide sequences obtained from MS/MS spectra were matched against the National Center for Biotechnology Information (NCBI) non-redundant (nr) database and confirmed by the mass spectrometry data of elucidated peptide mass sequences derived from the annotated genome. This improved protein identification method highlighted 36 differentially expressed proteins in the proteome of L. brevis after 75 days of starvation.The results revealed the key differences in the metabolic pathways that are responsible for the survival of L. brevis in a hostile environment. Proteomics analysis demonstrated that numerous proteins engaged in glucose and amino-acid catabolizing pathways, glycerolipid metabolizing pathways, and stress-response mechanisms are differentially expressed after long-term starvation. Amino acid and proteomics analysis indicated that starved L. brevis metabolized arginine, glycine, and histidine from dead cells as alternative nutrient sources. The production of lactic acid also varied between the parent cells and the starved cells.Differentially expressed proteins identified exclusively by peptide sequence reading provided promising results for CAF-/CAF+ implementation in a standard proteomics workflow (e.g., biomarker and mutation discovery and biotyping). The practical performance of a reliable de novo sequencing technique in routine proteomics analysis is emphasized in this article.

Lactic acid bacteria (LAB) as starter culture in food industry must be suitable for large-scale industrial production and possess the ability to survive in unfavorable processes and storage conditions. Approaches taken to address these problems include the selection of stressresistant strains. In food industry, LAB are often exposed to metal ions induced stress. The interactions between LAB and metal ions are very poorly investigated. Because of that, the influence of non- toxic, toxic and antioxidant metal ions (Zn, Cu, and Mn) on growth, acid production, metal ions binding capacity of wild and adapted species of Leuconostoc mesenteroides L3, Lactobacillus brevis L62 and Lactobacillus plantarum L73 were investigated. The proteomic approach was applied to clarify how the LAB cells, especially the adapted ones, protect themselves and tolerate high concentrations of toxic metal ions. Results have shown that Zn and Mn addition into MRS medium in the investigated concentrations did not have effect on the bacterial growth and acid production, while copper ions were highly toxic, especially in static conditions. Leuc. mesenteroides L3 was the most efficient in Zn binding processes among the chosen LAB species, while L. plantarum L73 accumulated the highest concentration of Mn. L. brevis L62 was the most copper resistant species. Adaptation had a positive effect on growth and acid production of all species in the presence of copper. However, the adapted species incorporated less metal ions than the wild species. The exception was adapted L. brevis L62 that accumulated high concentration of copper ions in static conditions. The obtained results showed that L. brevis L62 is highly tolerant to copper ions, which allows its use as starter culture in fermentative processes in media with high concentration of copper ions.

Obesity has become one of the major threats for public health in industrialised world among adults, but also among adolescents and children. It is influenced by the interaction of genes, nutrition, environment, and lifestyle. Environmental and lifestyle risk factors include foetal and lifelong environment, nutrient quality, chemical and microbial exposure, and psychical stress, all of which are important contributing influences. Removing or limiting chemical and pharmaceutical obesogens from human environment could make a difference in the growing epidemic of obesity. Additionally, nutrigenomics describes how modifications in individual diets can improve health and prevent chronic diseases, as well as obesity, by understanding the effects of a genetic profile in the interaction between food and increase in body weight. Furthermore, individual genetic variations in genome represent an individual′s predisposition for obesity. Therefore, the use of individual genetic information, avoiding obesogens, and a healthy lifestyle could help to improve the management of obesity and maintain a healthy weight., U industrijaliziranom svijetu među odraslim osobama, adolescentima i djecom pretilost je postala jedna od glavnih prijetnja za javno zdravlje ljudi. Njezina je pojavnost pod utjecajem međudjelovanja gena, prehrane, okoliša i načina života. Važni čimbenici rizika vezani su uz okolinu i način života, uključujući čimbenike prisutne već u okruženju fetusa te one prisutne tijekom cijeloga života kao što su kvaliteta prehrane, izloženost kemikalijama, mikroorganizmima i psihičkom stresu. Uklanjanje ili ograničavanje kemijskih tvari i lijekova koji uzrokuju pretilost iz ljudske okoline moglo bi utjecati na opadanje epidemije pretilosti. Dodatno, nutrigenomika opisuje kako se promjenama u prehrani pojedinca može poboljšati zdravstveno stanje i spriječiti razvoj kroničnih bolesti, uključujući i pretilost, a pritom je potrebno poznavati utjecaj genskog profila na međudjelovanje hrane i porasta tjelesne mase. Nadalje, genske varijacije u genomu pojedinih osoba stvaraju i njihovu predispoziciju za razvoj pretilosti. Stoga se zahvaljujući informacijama o genskom profilu pojedinca, izbjegavanjem tvari koje uzrokuju pretilost i zdravim načinom života može poboljšati kontrola pretilosti i održavati optimalna tjelesna masa.

Low-fat diets increase serum triacilglycerol and decrease HDL-cholesterol concentrations, thereby potentially adversely affecting cardiovascular disease (CVD) risk. The present study compared the CVD risk profi le of a high- MUFA diets (olive oil) and high-PUFA diets (pumpkin seed oil). The most signifi cant difference between the two groups was that the atherogenic index in groups given olive oil was signifi cantly lower (for approximately 60 %) than atherogenic index recorded in control group, while in the group receiving pumpkin seed oil this reduction was approximately 40 %. Collectively, these findings point to the fact that both a high MUFA and PUFA diet may be preferable to a low-fat diet because of more favorable effects on the CVD risk profile.

Metagenomics and advances in molecular biology methods have enhanced knowledge of microbial evolution, metabolism, functions, their interactions with other organisms and their environment. The ability to persist and adapt to changes in their environment is a common lifestyle of 1 % of the known culturable bacteria. Studies in the variety of species have identified an incredible diversity of bacterial lifespan. The holy grail of molecular biology is to understand the integrated genetic and metabolic patterns of prokaryotic organisms like the enteric bacterium Escherichia coli. The usual description of E. coli life cycle comprises four phases: lag, logarithmic, stationary, and death phase, omitting their persistence and evolution during prolonged stationary phase. During prolonged stationary/starvation period, in batch bacterial culture, selected mutants with increased fitness express growth advantage in stationary phase (GASP), which enables them to grow and displace the parent cells as the majority population. The analyses of growth competition of Gram-negative and/or Gram-positive mixed bacterial cultures showed that GASP phenomenon can result in four GASP phenotypes: strong, moderate, weak or abortive. Bacterial stress responses to starvation include functions that can increase genetic variability and produce transient mutator state, which is important for adaptive evolution., Metagenomika i suvremene metode molekularne biologije omogućili su razumijevanje evolucije, metabolizma i funkcije mikroorganizama te njihovih interakcija s drugim organizmima u okolišu. Otpornost i prilagodba na promjene u okolišu uobičajeni su za 1 % poznatih bakterija što se mogu uzgajati u laboratoriju. Istraživanjem različitih bakterijskih vrsta uočena je njihova velika raznolikost. Escherichia coli je „sveti gral“ molekularne biologije u razumijevanju genetike i metaboličkih modela. Životni se ciklus E. coli sastoji od četiri faze: lag, logaritamske, stacionarne i faze odumiranja, zanemarujući bakterijsku postojanost i evoluciju tijekom produljene stacionarne faze. U šaržnoj bakterijskoj kulturi, tijekom produljene stacionarne faze ili vremena izgladnjivanja, preživjele stanice mutanata brže rastu (engl. growth advantage in stationary phase - GASP), pa prerastaju i zamjenjuju većinu roditeljskih stanica. Analiza kompetitivnoga rasta Gram-pozitivnih i/ili Gram-negativnih bakterija, tijekom produljene stacionarne faze u mješovitim kulturama, pokazala je postojanje četiriju GASP fenotipova: jaki, umjereni, slabi i nerazvijeni. Bakterijski odgovor na izgladnjivanje obuhvaća stanične funkcije koje mogu povećati genetičku raznolikost i stvarati mutator stanice bitne za adaptivnu evoluciju bakterija.

Bread has been an essential part of human nutrition and culture for thousands of years. Every step of it’s production, from careful raw material selection to dough preparation, processing and baking, could be considered as art that ensure unlimited abundance of color, taste and aroma. Diversity of bakery products is result of the diversities in tradition, culture and geographic origin. All around the world alluring and aromatic bakery products always contain sourdough. Sourdough is a complex microbial ecosystem that is composed of lactic acid bacteria and yeasts. The use of lactic acid bacteria in the bread production has long tradition, from spontaneous fermentations (leavening the dough only), to the development and use of defi ned and functional starter cultures. This review covers the sourdough technology, with major emphasis on production of wheat bread with enhanced nutritional value, prolonged shelf life, better taste and aroma., Kruh je od pamtivijeka neizostavni dio ljudske prehrane i kulture. Svaki korak u njegovoj proizvodnji, od pažljivog izbora sirovina do pripreme i obrade tijesta te procesa pečenja, predstavlja dio umjetnosti koja u konačnici osigurava neograničeno bogatstvo boje, okusa i arome kruha. Raznolikost pekarskih proizvoda rezultat je različitosti tradicije, kulture te geografskog podrijetla. Međutim, san svakog pekara, primamljivi i mirisni pekarski proizvodi diljem svijeta, uvijek sadržavaju kiselo tijesto. Kiselo tijesto je kompleksni mikrobni ekosustav kojeg čine bakterije mliječne kiseline i kvasci. Upotreba bakterija mliječne kiseline u pripremi kruha ima dugu tradiciju, od spontanih fermentacija gdje je njihova uloga bila samo dizanje tijesta, preko defi niranih starter kultura, do razvoja i primjene funkcionalnih starter kultura. U ovom radu dat je prikaz tehnologije proizvodnje kiselog tijesta s naglaskom na proizvodnju pšeničnog kruha povećane nutritivne vrijednosti i trajnosti, bogatijeg okusa i arome.

Uspjehom projekta sekvencioniranja ljudskog genoma, povećalo se razumijevanje uzroka, a time i prevencije različitih bolesti u ljudi. Spoznalo se da mijenjajući prehrambene navike možemo spriječiti pojavu nekih bolesti. Tako se na području istraživanja prehrane razvija nova disciplina, nutrigenomika. Taj novi smjer molekularne prehrane omogućuje upoznavanje pozadine interakcije hrane koju konzumiramo i našeg genetičkog profi la, a time nam daje i mogućnost razvoja novih načina liječenja i prevencije bolesti. U ovom radu opisani su osnovni ciljevi i metode nutrigenomike te njena praktična primjena u razvoju koncepta individualne prehrane, The success of sequencing the human genome has led to the increasing understanding of causes and thus the prevention of various human illnesses. It is understood that by adjusting ones dieting habits one can prevent disease appearance. Such knowledge has made way for a new discipline in the fi eld of nutrition research, nutrigenomics. This novel direction of molecular nutrition provides insight into the interaction of the food we consume and our genetic profi le, and therefore grants the possibility to develop new methods of treatment and disease prevention. This article encloses the description of the primary objective and the methods of nutrigenomics as well as its practical implementation in the development of an individual diet concept.

The role of nucleotide excision repair in the mutagenicity of the monofunctional alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), N-ethyl-N'- nitro-N-nitrosoguanidine (ENNG), and N-ethyl-N- nitrosourea (ENU) in Salmonella typhimurium was examined. The mutagenic potential of the mutagenic agents used increased in the following order: MMS less than ENU less than ENNG less than MNNG. The results obtained confirm the involvement of nucleotide excision repair in the removal of mutagenic lesions from the DNA of S. typhimurium cells exposed to high doses of methylating as well as ethylating agents. At the low doses of all the alkylating agents used, the nucleotide excision repair-proficient strain was mutagenized more efficiently than the uvrB mutant. This phenomenon, a consequence of competition between nucleotide excision-repair enzymes and constitutive O6- methylguanine-DNA methyltransferase, is discussed.

The genotoxic effects of tryptophol (indole-3-ethanol), an aromatic alcohol and known secondary metabolite of the opportunistic yeast Candida albicans and other Candida spp., were studied using a battery of short-term assays on human white blood cells in vitro . The concentration range of tryptophol tested was 0.25 mM to 2.00 mM. Lymphocyte viability and induction of apoptosis/necrosis were studied by simultaneous use of a fluorescent assay with ethidium bromide and acridine orange. Levels of primary DNA damage and dynamics of DNA repair were evaluated using the alkaline comet assay while the levels and nature of residual DNA damage were assessed by the analysis of structural chromosome aberrations, the sister chromatid exchange test and the cytokinesis-block micronucleus assay. The results obtained suggest cytotoxic, cytostatic and genotoxic effects of the tryptophol treatment in vitro that were mainly dose-dependent. The type and the extent of DNA lesions detected in tryptophol-treated samples indicate the possibility that observed damage is mediated by highly reactive aldehyde metabolite and/or free radicals produced by treatment. The results show that mortality of lymphocytes in tryptophol-treated samples was primarily caused by apoptosis. The generation of additional DNA strand breaks and cytogenetic consequences (chromosome aberrations, sister chromatid exchanges and micronuclei), as observed in this study, sustain the possibility that tryptophol toxicity is mediated by the formation of DNA cross-links and aldehyde-protein adducts. In conclusion, this preliminary study elucidates only a part of tryptophol toxicity to human cells. Because current evidence is not sufficient to obtain information relevant for human risk assessment, further in vitro and in vivo studies are essential in order to clarify remaining issues, especially to elucidate the exact mechanisms and nature of the damage produced following treatment as well to estimate possible interindividual variability in genotoxic responses to the chemical.

Gliotoxin is a potent immunosuppressive and cytotoxic myctoxin from the epidithiodioxopiperazine group of secondary metabolites. Its production in situ by medically important molds Asperegillus fumigatus Fres. and A. terreus Thom contribute to invasive apergillosis of mammals. Our previous research found potent cytotoxic effects on epithelial, fibroblast-like, and monocyte cell lines (with IC50 from 2 to 11 microM). In this study we analysed in vitro genotoxicity of gliotoxin (CAS 67-99-2) on epithelial (A549, Hep G-2, CHO), fibroblast-like (COS-7), and monocyte (THP-1) cell lines as well as on human leukocytes ex situ using alkaline comet assay (single cell gel electrophoresis). After 18h-exposure of cells to the gliotoxin, comet tail length (TL, in micrometers), tail intensity (DNA%) and tail moment (TM) were recorded using fluorescence microscope and Comet Assay II software. At 2 microM gliotoxin expresses genotoxic effects with the most differences noted in THP-1 cell line (p

The role of nucleotide excision repair and 3- methyladenine DNA glycosylases in removing cytotoxic lesions induced by N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) in Salmonella typhimurium and Escherichia coli cells was examined. Compared to the E. coli wild-type strain, the S. typhimurium wild-type strain was more sensitive to the same dose of MNNG. Nucleotide excision repair in both bacterial species does not contribute significantly to the survival after MNNG treatment, indicating that the observed differences in survival between S. typhimurium and E. coli should be attributed to DNA-repair systems other than nucleotide excision repair. The survival of the E. coli alkA mutant strain is seriously affected by the lack of 3-methyladenine DNA glycosylase II, accentuating the importance of this DNA-repair enzyme in protecting E. coli cells against the lethal effects of methylating agents. Following indications from our experiments, the existence of an alkA gene analogue in S. typhimurium has been questioned. Dot-blot hybridisation, using the E. coli alkA gene as a probe, was performed, and such a nucleotide sequence was not detected on S. typhimurium genomic DNA. The existence of constitutive 3- methyladenine DNA glycosylase, analogous to the E. coli Tag gene product in S. typhimurium cells, suggested by the results is discussed.