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1
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The role of labile iron pool in cardiovascular diseases.

100%
Kruszewski M.
|
|
vol. 51
|
issue 2
471-480
EN
Although multiple factors are associated with cardiovascular pathology, there is now an impressive body of evidence that free radicals and nonradical oxidants might cause a number of cardiovascular dysfunctions. Both direct damage to cellular components and/or oxidation of extracellular biomolecules, e.g. LDL, might be involved in the aetiology of cardiovascular diseases. The key molecules in this process seem to be iron and copper ions that catalyse formation of the highly reactive hydroxyl radical. Chelation of iron ions has a beneficial effect on the processes associated with the development of atherosclerosis and formation of post-ischemic lesions. These findings are indirectly supported by the increasing body of evidence that stored body iron plays a crucial role in pathogenesis of atherosclerosis and ischemia/reperfusion injury.
2
Content available remote

Induction of DNA breakage in X-irradiated nucleoids selectively stripped of nuclear proteins in two mouse lymphoma cell lines differing in radiosensitivity

64%
Kruszewski M., Iwaneńko T.
Acta Biochimica Polonica
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1998
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vol. 45
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issue 3
701-704
3
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Labile iron pool correlates with iron content in the nucleus and the formation of oxidative DNA damage in mouse lymphoma L5178Y cell lines.

64%
Kruszewski M., Iwaneńko T.
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2003
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vol. 50
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issue 1
211-215
EN
Labile iron pool (LIP) constitutes a crossroad of metabolic pathways of iron-containing compounds and is midway between the cellular need for iron, its uptake and storage. In this study we investigated oxidative DNA damage in relation to the labile iron pool in a pair of mouse lymphoma L5178Y (LY) sublines (LY-R and LY-S) differing in sensitivity to hydrogen peroxide. The LY-R cells, which are hydrogen peroxide-sensitive, contain 3 times more labile iron than the hydrogen peroxide-resistant LY-S cells. Using the comet assay, we compared total DNA breakage in the studied cell lines treated with hydrogen peroxide (25 μM for 30 min at 4°C). More DNA damage was found in LY-R cells than in LY-S cells. We also compared the levels of DNA lesions sensitive to specific DNA repair enzymes in both cell lines treated with H2O2. The levels of endonuclease III-sensitive sites and Fapy-DNA glycosylase-sensitive sites were found to be higher in LY-R cells than in LY-S cells. Our data suggest that the sensitivity of LY-R cells to H2O2 is partially caused by the higher yield of oxidative DNA damage, as compared to that in LY-S cells. The critical factor appears to be the availability of transition metal ions that take part in the OH radical-generating Fenton reaction (very likely in the form of LIP).
4
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Iron-sulfur cluster proteins: electron transfer and beyond

51%
Brzóska K., Męczyńska S., Kruszewski M.
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2006
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vol. 53
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issue 4
685-691
EN
Iron-sulfur clusters-containing proteins participate in many cellular processes, including crucial biological events like DNA synthesis and processing of dioxygen. In most iron-sulfur proteins, the clusters function as electron-transfer groups in mediating one-electron redox processes and as such they are integral components of respiratory and photosynthetic electron transfer chains and numerous redox enzymes involved in carbon, oxygen, hydrogen, sulfur and nitrogen metabolism. Recently, novel regulatory and enzymatic functions of these proteins have emerged. Iron-sulfur cluster proteins participate in the control of gene expression, oxygen/nitrogen sensing, control of labile iron pool and DNA damage recognition and repair. Their role in cellular response to oxidative stress and as a source of free iron ions is also discussed.
5
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Nonhomologous end-joining deficiency of L5178Y-S cells is not associated with mutation in the ABCDE autophosphorylation cluster

51%
Brzóska K., Kruszewski M., Szumiel I.
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2006
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vol. 53
|
issue 1
233-236
EN
Cells with mutated autophosphorylation sites in the ABCDE cluster of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are defective in the repair of ionising radiation-induced DSB, but show in an in vitro test the same DNA-PK activity as the cells possessing wild type enzyme. Nevertheless, the mutated DNA-PK is able to undergo ATP-dependent autophosphorylation and inactivation. This characteristics correspond well with the phenotypic features of the L5178Y-S (LY-S) cell line that is defective in DSB repair, shows a pronounced G1 phase radiosensitivity, but in which the level of DNA-PK activity present in total cell extracts is similar to that of its radioresistant counterpart L5178Y-R (LY-R) cell line. The purpose of this work was to examine the possible alterations in the sequence encoding the cluster of autophosphorylation sites in the DNA-dependent protein kinase in LY-S cells. Despite the presence of phenotypic features indicating the possibility of such alterations, no differences were found between the sequences coding for the autophosphorylation sites in L5178Y-R and L5178Y-S cells. In conclusion, the repair defect in LY-S cells is not related to the structure of the DNA-PK autophosphorylation sites (ABCDE casette).
6
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Content available

Tlenek węgla – trucizna czy potencjalny terapeutyk?

51%
Furman-Toczek D., Zagórska-Dziok M., Kruszewski M., Kapka-Skrzypczak L.
Medycyna Środowiskowa - Environmental Medicine
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2016
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vol. 19
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issue 4
59-69
EN
Carbon monoxide, a low molecular gaseous mediator with pleiotropic activity, arises as a result of heme degradation in reaction catalyzed by heme oxygenase. There are a variety of scientific reports confirming the participation of carbon monoxide and heme oxygenase in protection against cellular stress. It has been shown that inbiological systems, CO can display anti-inflammatory, anti-proliferative and anti-apoptotic effect. A better understanding of the function of this molecule has led to its use as a potential therapeutic agent in diseases associated with neurological, immune and vascular systems and transplantology. The aim of this study was to present the role of carbon monoxide in modulation of cellular processes, with particular consideration of potential application in pharmacotherapy.
PL
Tlenek węgla (CO), niskocząsteczkowy, gazowy mediator o wielokierunkowej aktywności, powstaje w komórce w reakcji rozpadu cząsteczki hemu na skutek aktywności oksygenazy hemowej. Istnieje wiele doniesień naukowych potwierdzających udział tlenku węgla oraz oksygenazy hemowej w zintegrowanym systemie ochrony przed stresem komórkowym. Wykazano, że w układach biologicznych CO wykazuje działanie przeciwzapalne, anty-proliferacyjne oraz anty-apoptotyczne. Dokładniejsze poznanie funkcji tej cząsteczki doprowadziło do stopniowego wykorzystywania jej, jako potencjalnego terapeutyku w chorobach układu nerwowego, immunologicznego, krwionośnego a także w transplantologii. Celem niniejszej pracy, było przedstawienie roli tlenku węgla w modulacji procesów komórkowych, ze szczególnym uwzględnieniem potencjalnego zastosowania tej cząsteczki w farmakoterapii.
7
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Repair of γ-ray-induced base damage in L5178Y sublines is damage type-dependent and unrelated to radiation sensitivity.

51%
Kruszewski M., Zastawny T. H., Szumiel I.
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2001
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vol. 48
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issue 2
525-533
EN
The L5178Y (LY) murine lymphoma sublines LY-R and LY-S are differentially sensitive to ionizing radiation. The high radiation sensitivity of LY-S cells is related to impaired rejoining of DNA double strand breaks. We found previously that the γ-ray-induced base damage is higher in the more radiosensitive LY-S subline. Here, we examine the role of the repair of ionizing radiation induced base damage in relation to the radiosensitivity difference of these sublines. We used the GS/MS technique to estimate the repair rates of six types of base damage in γ-irradiated LY cells. All modified DNA bases identified in the course of this study were typical for irradiated chromatin. The total amount of initial base damage was higher in the radiation sensitive LY-S subline than in the radiation resistant LY-R subline. The repair rates of 5-OHMeUra, 5-OHCyt, 8-OHAde were similar in both cell lines, the repair rates of FapyAde and 8-OHGua were higher in the radiosensitive LY-S cell line, whereas the repair of 5-OHUra was faster in its radioresistant counterpart, the LY-R Altogether, the repair rates of the γ-ray-induced DNA base damage in LY sublines are related neither to the initial amounts of the damaged bases nor to the differential lethal or mutagenic effects of ionizing radiation in these sublines.
8
Publication available in full text mode
Content available

Nanocząstki złota jako detektory pestycydów w żywności i wodzie pitnej

51%
Matysiak M., Kruszewski M., Kapka-Skrzypczak L.
|
EN
Due to the high toxicity and harmful effects of pesticides on human health, it is necessary to constantly monitor their presence in food and drinking water. The current standard in the determination of pesticides is instrumental analysis, such as chromatography and mass spectrometry. These techniques are expensive, laborious and require use of specialized laboratory equipment and trained personnel. Furthermore, appropriate sample preparation is needed, which significantly increases the overall measurement time. Thus, pesticide detectors based on other principle have become popular over the past few years, especially metallic gold nanoparticles (AuNPs). AuNPs based detectors are faster and less expensive than traditional methods. In this review we describe a potential use of AuNPs in the detection of pesticides. Additionally, methods for quantitation of the detector response are illustrated, starting with a direct colorimetric and fluorescence detection, through enzymatic and immunological methods, to Raman spectroscopy and electrochemical techniques.
PL
Ze względu na wysoką toksyczność i szkodliwe działanie pestycydów na zdrowie człowieka, konieczny jest stały monitoring obecności ich pozostałości w żywności i wodzie pitnej. Obecnie standardem w oznaczaniu pestycydów jest analiza instrumentalna, a przede wszystkim techniki takie jak chromatografia i spektrometria mas. Są to jednak metody drogie, wymagające użycia specjalistycznego sprzętu laboratoryjnego i przeszkolonego personelu. Ponadto niezbędne jest odpowiednie przygotowanie próbki, co znacząco wydłuża czas pomiarów. Od kilku lat na popularności zyskują detektory oparte na nanocząstkach złota metalicznego (AuNPs), które umożliwiają znacznie szybsze i mniej kosztowne oznaczenia. W pracy przedstawiono możliwości wykorzystania AuNPs w detekcji pestycydów. Opisano także metody oznaczania odpowiedzi w detektorach AuNPs, poczynając od bezpośredniej detekcji kolorymetrycznej i fluorescencyjnej, przez metody enzymatyczne i immunologiczne, kończąc na spektroskopii ramanowskiej i technikach elektrochemicznych.
9
Content available remote

DNA damage induced by hydrogen peroxide treatment at 4°C and 37°C in murine lymphoma L5178Y sublines

51%
Kruszewski M., Szumiel I.
Acta Biochimica Polonica
|
1994
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vol. 41
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issue 2
120-121
10
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Lymphocyte labile iron pool, plasma iron, transferrin saturation and ferritin levels in colon cancer patients.

45%
Gackowski D., Kruszewski M., Banaszkiewicz Z., Jawien A., Olinski R.
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2002
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vol. 49
|
issue 1
269-273
EN
Patients with colorectal carcinoma showed statistically significant lower values of transferrin saturation, total iron binding capacity and serum iron level as compared with control group, while the level of ferritin and the size of labile iron pool in carcinoma patients were higher, although this difference was not statistically significant. Our observations are in favour of the hypothesis which suggests that changes in iron metabolism restrict iron availability for tumour cells and as consequence, slow their growth.
11
Content available remote

Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood

45%
Ołdak T., Kruszewski M., Machaj E. K., Gajkowska A., Pojda Z.
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2002
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vol. 49
|
issue 3
625-632
EN
Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34+ hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34+ hematopoietic cells were separated on immunomagnetic MiniMACS columns. Pure population of CD34+ cells was incubated in a serum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of various pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 μF, 550 V/cm, and 10 μg of DNA per 500 μl. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capacitance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34+ hematopoietic cells derived from human umbilical cord blood.
12
Content available remote

The response of L5178Y lymphoma sublines to oxidative stress: Antioxidant defence, iron content and nuclear translocation of the p65 subunit of NF-κB.

39%
Boużyk E., Grądzka I., Iwaneńko T., Kruszewski M., Sochanowicz B., Szumiel I.
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2000
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vol. 47
|
issue 4
881-888
EN
We examined the response to hydrogen peroxide of two L5178Y (LY) sublines which are inversely cross-sensitive to hydrogen peroxide and X-rays: LY-R cells are radioresistant and hydrogen peroxide-sensitive, whereas LY-S cells are radiosensitive and hydrogen peroxide-resistant. Higher initial DNA breaks and higher iron content (potentially active in the Fenton reaction) were found in the hydrogen peroxide sensitive LY-R cells than in the hydrogen peroxide resistant LY-S cells, whereas the antioxidant defence of LY-R cells was weaker. In particular, catalase activity is twofold higher in LY-S than in LY-R cells. The content of monobromobimane-reactive thiols is 54% higher in LY-S than in LY-R cells. In contrast, the activity of glutathione peroxidase (GPx) is about two times higher in LY-R than in LY-S cells; however, upon induction with selenium the activity increases 15.6-fold in LY-R cells and 50.3-fold in LY-S cells. Altogether, the sensitivity difference is related to the iron content, the amount of the initial DNA damage, as well as to the efficiency of the antioxidant defence system. Differential nuclear translocation of p65-NF-κB in LY sublines is due to the more efficient antioxidant defence in LY-S than in LY-R cells.
13
Content available remote

The rapid interphase chromosome assay (RICA) implementation: comparison with other PCC methods

39%
Sommer S., Buraczewska I., Sikorska K., Bartłomiejczyk T., Szumiel I., Kruszewski M.
|
|
issue 4
933-941
EN
A report is presented on the advantages of the rapid interphase chromosome assay (RICA) and the difficulties that may be met while implementing this method for application in biological dosimetry. The RICA test can be applied on unstimulated human lymphocytes; this is an advantage in comparison with the dicentric chromosomes or micronucleus tests. In the former two tests, stimulated lymphocytes are examined and hence, 48 h more are needed to obtain cells traversing the cell cycle. Due to the use of unstimulated nondividing cells, higher numbers of cells are available for RICA analysis than for dicentric chromosomes or micronuclei tests. Moreover, the method can be applied after exposure to ionizing radiation doses in excess of 5 Gy. Such doses cause a significant cell cycle delay or result in the loss of G2 phase and mitotic cells because of apoptosis. Therefore, the traditional biodosimetry based on the evaluation of the incidence of damage to chromosomes is very difficult to carry out. This is due to the lack of an adequate number of mitotic cells for analysis. RICA is free of this disadvantage. An automatic microscope can be used to retrieve cell images; automatic image analysis can also be used.
14
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Sirtuin inhibition increases the rate of non-homologous end-joining of DNA double strand breaks

39%
Wojewódzka M., Kruszewski M., Buraczewska I., Xu W., Massuda E., Zhang J., Szumiel I.
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2007
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vol. 54
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issue 1
63-69
EN
Sirtuins (type III histone deacetylases) are an important member of a group of enzymes that modify chromatin conformation. We investigated the role of sirtuin inhibitor, GPI 19015, in double strand break (DSB) repair in CHO-K1 wt and xrs-6 mutant cells. The latter is defective in DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end-joining (D-NHEJ). DSB were estimated by the neutral comet assay and histone γH2AX foci formation. We observed a weaker effect of GPI 19015 treatment on the repair kinetics in CHO wt cells than in xrs6. In the latter cells the increase in DNA repair rate was most pronounced in G1 phase and practically absent in S and G2 cell cycle phases. The decrease in the number of histone γH2AX foci was faster in xrs6 than in CHO-K1 cells. The altered repair rate did not affect survival of X-irradiated cells. Since in G1 xrs6 cells DNA-PK-dependent non-homologous end-joining, D-NHEJ, does not operate, these results indicate that inhibition of sirtuins modulates DNA-PK-independent (backup) non-homologous end-joining, B-NHEJ, to a greater extent than the other DSB repair system, D-NHEJ.
15
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Cytotoxic and mutagenic effects of hydrogen peroxide in murine L5178Y sublines

38%
Kruszewski M., Szumiel I.
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vol. 40
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issue 1
42-45
16
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Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy

33%
Markowicz S., Niedzielska J., Kruszewski M., Ołdak T., Gajkowska A., Machaj E. K., Skurzak H., Pojda Z.
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2006
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vol. 53
|
issue 1
203-212
EN
Dendritic cells (DC) generated from human umbilical cord blood might replace patients' DC in attempts to elicit tumor-specific immune response in cancer patients. We studied the efficiency of transfection of human cord blood DC with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene, to test if nonviral gene transfer would be a method to load DC with protein antigens for immunotherapy purposes. Cord blood mononuclear cells were cultured in serum-free medium in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) and Flt-3 ligand (FL), to generate DC from their precursors, and thereafter transfected by electroporation. Maturation of DC was induced by stimulation with GM-CSF, SCF, FL and phorbol myristate acetate (PMA). Transfected DC strongly expressed EGFP, but transfection efficiency of DC, defined as HLA-DR+ cells lacking lineage-specific markers, did not exceed 2.5%. Expression of the reporter gene was also demonstrated in the DC generated from transfected, purified CD34+ cord blood cells, by stimulation with GM-CSF, SCF, FL, and tumor necrosis factor α (TNF-α). Transfection of CD34+ cells was very efficient, but proliferation of the transfected cells was much reduced as compared to the untransfected cells. Therefore, the yield of transgene-expressing DC was relatively low. In conclusion, nonviral transfection of cord blood DC proved feasible, but considering the requirements for immunotherapy in cancer patients, transfection of differentiated DC or generation of DC from transfected hematopoietic stem cells provide only a limited number of DC expressing the transgene.
17
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Cu,Zn-superoxide dismutase deficiency in mice leads to organ-specific increase in oxidatively damaged DNA and NF-κB1 protein activity

27%
Siomek A., Brzoska K., Sochanowicz B., Gackowski D., Rozalski R., Foksinski M., Zarakowska E., Szpila A., Guz J., Bartlomiejczyk T., Kalinowski B., Kruszewski M., Olinski R.
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2010
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vol. 57
|
issue 4
577-583
EN
Earlier experimental studies have demonstrated that: i) Cu,Zn-superoxide dismutase deficiency leads to oxidative stress and carcinogenesis; ii) dysregulation of NF-κB pathway can mediate a wide variety of diseases, including cancer. Therefore, we decided, for the first time, to examine the level of oxidative DNA damage and the DNA binding activity of NF-κB proteins in SOD1 knockout, heterozygous and wild-type mice. Two kinds of biomarkers of oxidatively damaged DNA: urinary excretion of 8-oxodG and 8-oxoGua, and the level of oxidatively damaged DNA were analysed using HPLC-GC-MS and HPLC-EC. The DNA binding activity of p50 and p65 proteins in a nuclear extracts was assessed using NF-κB p50/p65 EZ-TFA transcription factor assay. These parameters were determined in the brain, liver, kidney and urine of SOD1 knockout, heterozygous and wild-type mice. The level of 8-oxodG in DNA was higher in the liver and kidney of knockout mice than in wild type. No differences were found in urinary excretion of 8-oxoGua and 8-oxodG between wild type and the SOD1-deficient animals. The activity of the p50 protein was higher in the kidneys, but surprisingly not in the livers of SOD1-deficient mice, whereas p65 activity did not show any variability. Our results indicate that in Cu,Zn-SOD-deficient animals the level of oxidative DNA damage and NF-κB1 activity are elevated in certain organs only, which may provide some explanation for organ-specific ROS-induced carcinogenesis.
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