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1

Effects of silver nanoparticles of different sizes on cytotoxicity and oxygen metabolism disorders in both reproductive and respiratory system cells

Zapór L.

Archives of Environmental Protection

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2016

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Vol. 42, no. 4

32--47

EN

Silver nanoparticles (AgNPs) are widely used in numerous industries and areas of daily life, mainly as antimicrobial agents. The particles size is very important, but still not suffi ciently recognized parameter infl uencing the toxicity of nanosilver. The aim of this study was to investigate the cytotoxic effects of AgNPs with different particle size (~ 10, 40 and 100 nm). The study was conducted on both reproductive and pulmonary cells (CHO-9, 15P-1 and RAW264.7). We tested the effects of AgNPs on cell viability, cell membrane integrity, mitochondrial metabolic activity, lipid peroxidation, total oxidative and antioxidative status of cells and oxidative DNA damage. All kinds of AgNPs showed strong cytotoxic activity at low concentrations (2÷13 μg/ml), and caused an overproduction of reactive oxygen species (ROS) at concentrations lower than cytotoxic ones. The ROS being formed in the cells induced oxidative damage of DNA in alkaline comet assay. The most toxic was AgNPs<10 nm. The results indicate that the silver nanoparticles, especially less than 10 nm, may be harmful to the organisms. Therefore, risk should be considered when using nanosilver preparations and provide appropriate protective measures when they are applied.

PL

Nanocząstki srebra (AgNPs), ze względu na silne właściwości bakteriobójcze, mają szerokie zastosowanie w wielu dziedzinach przemysłu, biomedycynie i produktach konsumenckich. Rozmiar cząstek jest istotnym, ale wciąż niewystarczająco zbadanym parametrem wpływającym na toksyczność nanosrebra. W pracy oceniono toksyczne działanie różnej wielkości cząstek srebra (~ 10, 40 i 100 nm) na komórki układu rozrodczego i oddechowego (CHO-9, 15P-1 i RAW264.7). Badano wpływ AgNPs na przeżywalność komórek, przepuszczalność błon komórkowych i aktywność metaboliczną komórek, zaburzenia metabolizmu tlenowego oraz odległe skutki działania w postaci uszkodzeń materiału genetycznego (DNA). Wszystkie badane AgNPs wykazywały silne działanie cytotoksyczne, w zakresie niskich stężeń (2÷13 μg/ml) oraz powodowały powstawanie stresu oksydacyjnego w komórkach w stężeniach niższych niż cytotoksyczne. Powstające w komórkach reaktywne formy tlenu powodowały oksydacyjne uszkodzenia DNA wykrywane w teście kometowym. Najsilniejsze działanie wykazywały cząstki o wielkości < 10 nm. Otrzymane wyniki wskazują, że nanocząstki srebra, zwłaszcza poniżej 10 nm, mogą stanowić zagrożenie dla organizmów. Dlatego też należy rozważyć ryzyko stosowania preparatów z nanosrebrem i zapewnić środki zapobiegające ich niekontrolowanemu uwalnianiu.

2

Nie tylko BRCA - geny umiarkowanego ryzyka w raku piersi

Pamuła-Piłat J., Tęcza K.

Laboratorium - Przegląd Ogólnopolski

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2015

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nr 7-8

26--33

PL

Ryzyko rozwoju procesu nowotworowego może być modulowane przez mutacje genetyczne bądź polimorfizmy wysokiej, umiarkowanej i niskiej penetracji. Choroba nowotworowa zatem niezmiernie rzadko jest efektem wystąpienia pojedynczej zmiany genetycznej, jest raczej rezultatem nagromadzenia określonych wariantów genów oraz ich wzajemną współzależnością.

EN

The risk of developing cancer is modulated by mutations or/and polymorphisms of low, intermediate or high penetrance. Therefore the cancer itself is very rarely caused by single genetic modification. Rather, it is a result of cumulation of several variants and is strongly depended on their mutual cooperation.

3

Wykorzystanie spektrometrii mas do analizy modyfikacji nukleotydów i adduktów DNA

Hanus J., Jelonek K., Pietrowska M.

Wiadomości Chemiczne

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2011

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[Z] 65, 3-4

191-205

EN

Chemically modified nucleotides, which are not normally present in genetic material, are called DN A adducts. This type of DN A modifications (damage) is directly related to processes of mutagenesis and carcinogenesis. Elevated levels of DN A adducts present in genetic material reflect exposure of humans to carcinogenic factors and are markers of increased risk of cancer [1]. For this reason different methods useful for quantitative and qualitative analyses of DN A adducts are used in the field of cancer prevention and research (Tab. 1). Enzymatically-catalyzed methylation of cytosine, observed mostly in so called CpG islands, is a frequent endogenous modification of genetic material. Such a DN A methylation is a key factor involved in regulation of gene expression, and methylation status of oncogenes and tumor supressor genes is an important biomarker of carcinogenesis. As such, analytical methods for assessment of DN A methylation are of great importance for molecular diagnostics of cancer. During the last decade significant progress has been made in methods available for quantitative, qualitative and structural analyses of biological molecules. Among intensively developed tools for bioanalyses are methods of mass spectrometry. Spectrometers that are based on two methods of ionization, namely electrospray ionization (ESI ) [30] and matrix-assisted laser desorption-ionization (MALDI ) [48], are particularly suitable for analyses of biological macromolecules: proteins and nucleic acids. Currently available mass spectrometers, together with microscale methods for sample preparation and separation, significantly increased sensitivity and accessible mass range of analyses. New generation of “user-friendly” instruments is developed to bring the techniques directly into the workplaces of biological and clinical investigators. This review demonstrates representative examples of mass spectrometry techniques used for qualitative analyses of nucleotide modifications and adducts present in genetic material of humans. In this field several methods base on spectrometers with electrospray ionization. Generated ions are separated according to their mass-to-charge ratio in an analyzer by electric fields; among different ion analyzers frequently used in this methods are single or triple quadrupole and ion traps (Fig. 1). Among other methods available for assessment of DN A adducts is so called Accelerator Mass Spectrometry (Fig. 2) [41]. The most frequently applied method for the assessment of DN A methylation is based on methylation-specific PCR reaction. Products of such PCR reactions are analyzed using MALDI mass spectrometry [54] (Fig. 3). In summary, new powerful methods of mass spectrometry that made available qualitative analyses of damage and modifications of human genetic material found their important place in modern biological and medical laboratories.

4

5',8-cyklo-2'-deoksyadenozyna. Podwójne uszkodzenie w obrębie pojedynczego nukleozydu/nukleotydu

Karwowski B.T.

Wiadomości Chemiczne

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2010

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[Z] 64, 11-12

1013-1052

EN

Free radicals can react with different biomolecules present in the cells such as lipids, sugars or nucleic acid peptides. These free radicals initiate reactions with DNA or RNA molecules and then can lead to changes in the genome sequence. These mutations are most probably responsible for a number of different diseases (involving a change in the genome sequence) or, at least, can accompany them. Reactive oxygen species and more specifically - hydroxyl radical can react with DNA molecules and lead to changes in their structures. Formation of radicals at C5' and C8 atoms of 2'-deoxyadenosine leads, through intramolecular cyclisation, to 5',8-cyclo-2'-deoxyadenosine (cdA) derivatives. Frequency of DNA damage occurrence surges with an increase of an ionizing radiation dose. Different repair systems are however present in cellular machinery: BER, which exploits glycosylase and NER - a more complex process involving the removal of damaged oligonucleotides. The later is the basic mechanism for removal the 5',6-cyclo-2'-deoxynucleosides and 5',8-cyclo-2'-deoxynucleosides like cdA. Their defective activity may be responsible for many types of diseases, such as Parkinson, Alzheimer, chronic hepatitis, HCV, atopic dermatitis and different types of cancer. The mechanistic, structural and biochemical studies presented in this work produce quite clear answer as to the approximate range level of the (5'R)-cdA and (5'S)-cdA accumulation in the genome after the lesion period. Using quantum chemistry methods (DFT) paths of the cyclisation reaction have been determined. From the structural analysis point of view, it has been demonstrated that the covalent bond between C(5') and C(8) in nucleoside induces an unusual West conformation of the furanose ring. Based on the NMR data analysis it can be postulated that the rigid and fixed structure of cdA can strongly influence the global geometry of oligonucleosides. Moreover, using the quantum mechanic study of double strand DNA it has been demonstrated that the presence of (5'S)-cdA provokes a "domino effect" extending towards the 5'-end of the strand with this lesion. No domino effect is observed for the 3'-end. The obtained biological results indicate that the presence of (5'S)-cdA in the complementary strand to the strand under repair on the 5'-end side is the critical factor for the inhibition of the BER process of DNA.

5

Specific for DNA damages GFP microbial biosensor as a tool for genotoxic action assessment of environmental pollution

Matejczyk M.

Budownictwo i Inżynieria Środowiska

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2010

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Vol. 1, no. 4

319-326

EN

In the presented paper, autofluorescent reporter of Escherichia coli K-12 recA::gfpmut2 strain, which contained a plasmid-borne transcriptional fusion between DNA-damage inducible recA promoter involved in the SOS regulon response and fast folding GFP variant reporter gene-gfpmut2, have been used. GFP-based bacterial biosensors allowed the detection of bacterial cells response to selected tested genotoxic compounds such as mitomycin C (MMC), actinomycin D, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and formaldehyde (CH2O). Experiment indicated that E. coli K-12 recA::gfpmut2 biosensor strain is more specific and sensitive for especially two genotoxins: actinomycin D and MNNG and with very low response to other agents. So it was concluded that for formaldehyde and MMC E. coli K-12 recA::gfpmut2 genetic system is disqualified for genotoxicity screening.

6

DNA damage in subpopulations of human lymphocytes irradiated with doses in the range of 0-1 Gy of X-radiation

Wojewódzka M., Machaj E. K., Goździk A., Iwaneńko T., Ołdak T., Kruszewski M., Pojda Z.

Nukleonika

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2008

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Vol. 53, No. 4

145-149

EN

We compared three methods usually applied in biological dosimetry for estimation of radiation-induced DNA damage in human T and B lymphocytes: alkaline comet assay, micronucleus (MN) test and formation of histone gamma-H2AX foci. Human peripheral blood lymphocytes were fractionated using T cells and B cells isolation kits. Cells were irradiated with doses in the range of 0-1 Gy of X-rays. Induction of DNA damage was assessed by the standard alkaline comet assay, MN test and histone gammaH2AX foci immunofluorescence assay. Notwithstanding different end-points measured by the applied methods, all tests revealed a similar induction of DNA damage in B lymphocytes as compared with T lymphocytes. The results indicated that all three tests detect DNA damage with similar sensitivity, the lowest dose being approximately 0.3 Gy. The difference between irradiated and control cells was expressed as the ratio of the value obtained for irradiated cells (1 Gy) to that for control cells. The highest ratio was obtained for formation of gammaH2AX foci and was 6.2 for T and 13.8 for B lymphocytes, whereas those for comet assay and micronucleus test were 3.5; 3.6 and 5.6; 4.8, respectively.

7

Comparison of the effects of bleomycin and ionizing radiation in two sublines of murine lymphoma L5178Y

Kruszewski M., Zaim J., Grądzka I., Szumiel I.

Nukleonika

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2001

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Vol. 46, nr 3

81-86

EN

We compared the effects of bleomycin (BLM) and ionizing radiation on two sublines of murine lymphoma L5178Y (LY): LY-R, radiation resistant and LY-S, radiation sensitive. This radiosensitivity difference is related to the ability to rejoin DNA double strand breaks. LY-S cells were about two times more sensitive to BLM than LY-R, similarly as in the case of sensitivity to X rays. Since there was no difference in the P-glycoprotein-related drug transport system between the sublines, it could be expected that the enhanced sensitivity of LY-S cells to BLM was caused by the DNA repair defect. Growth disturbances in BLM treated cell populations were proportional to the lethal effect and their duration was observed until elimination of dead cells (3-6 days after 50 ěM BLM, 1 h at 37oC). There was no slow growth phase accompanied by normal viability, as previously described for X-irradiated LY-S cells. Initial DNA damage, estimated with the single cell gel electrophoresis method was linearly related to BLM dose in LY-S cells; in LY-R cells - in the low dose range (up to 10 ěM) - there was more damage than in LY-S cells, however, at higher doses the dose - effect curves became identical. The doseeffect relationship for ă rays was linear and identical in both cell sublines. DNA damage distribution in BLM treated cells was much less uniform as compared to that in irradiated cells and indicated the presence of cells with severely damaged DNA, a feature typical for BLM action in vitro.

8

Signal transfer in the cellular response to ionizing radiation

Szumiel I., Sochanowicz B.

Nukleonika

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1998

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Vol. 43, nr 2

133-146

EN

Numerous factors have recently been identified that are involved in the control of cell cycle checkpoints. It was found that the DNA damage-induced G1 and G2 delays are associated with a regulatory system consisting of cyclin-dependent protein kinases (CDKs) associated with cell cycle phase specific cyclins. This article reviews the recently discovered associations between cell cycle regulation and cell death or recovery in the in vitro cellular systems exposed to ionizing radiation. The concept of alarm signal is explained. A brief comment on the role of apoptosis in the postirradiation cell fate is included.

PL

W ostatnich latach zidentyfikowano liczne czynniki mające udział w regulacji przechodzenia komórek przez punkty kontrolne cyklu komórkowego. Okazało się, że indukowane przez uszkodzenie DNA bloki w fazach G1 i G2 regulowane są przez cyklinozależne kinazy białkowe, CDK. Praca jest przeglądem nowo odkry1ych powiązań między śmiercią lub odnową popromienną komórek a układem regulującym cykl komórkowy w układach in vitro. PQnadto wyjaśnione jest pojęcie sygnału alarmowego oraz skomentowana rola apoptozy w losie napromienionej komórki.

9

DNA and chromosomal damage estimate in blood of people suspected of exposure to radiation

Cebulska-Wasilewska A., Niedźwiedź W., Nowak D., Kasper E., Wierzewska A., Wójcik A., Boużyk E.

Nukleonika

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1998

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Vol. 43, nr 1

65-72

EN

In 1996 four persons were suspected of accidental exposure to ionizing radiation. In order to estimate the absorbed doses, peripheral lymphocytes were analyzed for the presence of unstable chromosomal aberrations. Additionally, the comet assay was applied for the analysis of DNA damage. Chromosomal aberrations were scored in the first postirradiation metaphase and the absorbed doses reconstructed on the basis of a calibration curve established for lymphocytes irradiated in vitro. All absorbed doses were found to be below 1 Gy. In the comet assay, the absorbed doses were assessed on the basis of dose response relationship and calibration coefficient obtained from the comparison of two DNA damage detection systems. In the case of two persons who received the highest doses, a good agreement was found between chromosomal aberrations and the comet assay. In the case of the other persons, a high level of DNA damage observed in the comet assay did not correlate with a low level of chromosomal aberrations. In the case of one person additional analysis of the sister chromatid exchange (SCE) suggested exposure to chemicals (e.g. long medical treatment or smoking). On the basis of the obtained results we assume that, although the comet assay is sensitive to DNA damage, it is not appropriate for a low radiation dose estimate because, unlike in cytogenetic analysis, one cannot distinguish between DNA damage caused by radiation and other agents.

PL

W 1996 r. cztery osoby były podejrzane o przypadkową ekspozycję na promieniowanie jonizujące. Stosując elektroforezę pojedynczych komórek w żelu agarozowym oraz cytogenetykę klasyczną oceniono potencjalną dawkę pochłoniętego promieniowania na podstawie anlizy uszkodzeń DNA oraz aberracji chromosomowych. Dawki pochłonięte przez poszczególne osoby zostały oszacowane na podstawie krzywych kalibracyjnych wykreślonych dla limfocytów napromieniowanych in vitro. Wszystkie pochłonięte dawki nie przekraczały 1 Gy. W przypadku metody kometowej dawki oszacowano na podstawie współczynnika kalibracji uzyskanego z porównania dwu metod oceny uszkodzeń DNA. Dla dwu osób, które otrzymały najwyższe dawki, zaobserwowano bardzo dużą zgodność w ocenie pochłoniętej dawki na podstawie aberracji chromosomowych i uszkodzeń DNA. W przypadku pozostałych osób poziom uszkodzeń DNA nie był skorelowany z niską frekwencją aberracji chromosomowych. Dla jednej z tych osób dodatkowa analiza wymian chromatyd siostrzanych wskazywała na ekspozycję na czynniki chemiczne (leki, papierosy). Słaba korelacja pomiędzy niskimi dawkami oszacowanymi z wykorzystaniem tych dwu metod sugeruje ograniczenia w zastosowaniu metody kometowej w środowiskowej dozymetrii biologicznej.

10

Application of the DNA comet assay for detection of irradiated meat

Kruszewski M., Malec-Czechowska K., Dancewicz A. M., Iwaneńko T., Szot Z., Wojewódzka M.

Nukleonika

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1998

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Vol. 43, nr 2

147-160

EN

Radiation induces damage to the DNA. This damage (fragmentation) can be assessed in the irradiated food using Single Cell Gel Electrophoresis (SCGE), known as DNA comet assay. Fragmentation of DNA may also be caused by improper storage of meat and repeated freezing and thawing. This makes identification of irradiated meat by this assay not reliable enough. In order to know the scale of the processes imitating irradiation effects in DNA of the comets, their shape and lenghts were examined in both unirradiated and irradiated fresh meat (D = 1.5 or 3.0 kGy) stored at 4°C or frozen (-21°) up to 5 months. Comets formed upon SCGE were stained with DAPI or silver and examined in fluorescent or light microscope. They were divided arbitrarily into 4 classes. Comets of class IV were found quite often in fresh meat stored at 4°C. In meat samples that were irradiated and stored frozen, comets of class, I, II and III were observed. The negative comet test is univocal. Positive comet test, however, needs confirmation. The meat should be subjected to further analysis with other validated methods.

PL

Promieniowanie powoduje uszkodzenia DNA. Te uszkodzenia (fragmentację) można ocenić w napromieniowanej żywności stosując elektroforezę w żelu pojedynczej komórki, zwaną także testem kometowym. Fragmentację DNA w mięsie mogą także wywoływać: nieprawidłowe przechowywanie mięsa oraz powtarzane zamrażanie i rozmnażanie. Czyni to identyfikację napromieniowania mięsa mniej wiarygodną. W celu poznania procesów imitujących napromieniowanie, tj. powodujących powstawanie kometek DNA, oceniano ich kształt i długość w mięsie nie napromieniowanym i napromieniowanym dawką 1.5 lub 3.0 kGy przechowywanym w 40C lub w stanie zamrożenia do 5 miesięcy. Otrzymane kometki DNA barwiono barwnikiem fluorescencyjnym DAPI lub srebrem i badano w mikroskopie fluorescencyjnym lub zwykłym. Kometki podzielono na 4 klasy. Kometki IV klasy znajdowano często w mięsie przechowywanym w 40C. W próbkach mięsa napromieniowanego i przechowywanego w stanie zamrożenia obserwowano kometki klasy l, II i III. Negatywny test kometowy jest jednoznaczny. Test dodatni wymaga potwierdzenia przez zastosowanie innych metod dających miarodajne wyniki.