**5. Conclusion**

432 Gel Electrophoresis – Advanced Techniques

*N*′- nitrosonornicotine (NNN) tumorigenicity in the rat esophagus by PEITC also appears to

The antimutagenic properties of ITCs have been reported towards NDMA and NPYRinduced oxidative stress before. In studies performed by **Knasmüller et al. (1996, 2003)**  using PEITC as a chemopreventive agent, the researchers observed a reduction in NDMAand NPYR-induced DNA damage in *Escherichia coli* K-12 and a considerable reduction in NDMA-induced micronuclei in HepG2 cells. The results of several studies demonstrated that ITCs exhibited strong antimutagenic effects against NDMA and NPYR in a dose dependent manner. In a study by **Smerák at al. (2009)**, the researchers investigated the effect of PEITC on the mutagenic activity of indirect-acting mutagens and carcinogens like aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) using the *Ames* bacterial mutagenicity test, the Comet assay, an *in vivo* micronucleus test, and direct-acting mutagen and carcinogen N-nitroso-N-methylurea (MNU). In the Ames test, the antimutagenic activity of PEITC was studied in the concentration range 0.3-300 g/plate. PEITC at concentrations of 0.3, 3 and 30 g/plate reduced dose-dependently mutagenicity of AFB1 and IQ in both *Salmonella typhimurium* TA98 and TA100 strains. In the case of the direct mutagen MNU, the antimutagenic effect of PEITC was detected only at concentration of 30 g/plate in the strain TA100. The PEITC concentration 300 g/plate was toxic in the Ames test. The 24 h pre-treatment of HepG2 cells with PEITC at concentration 0.15 g/ml resulted in a significant decrease of DNA breaks induced by MNU at concentrations 0.25 and 0.5 mM. Although a trend towards reduced strand break level were determined also at PEITC concentrations 0.035 and 0.07 g/ml**,** it did not reach the statistical significance. No effect, however, of PEITC on IQ-induced DNA breaks was observed. Chemopreventive effect of PEITC was revealed also *in vivo*. Pretreatment of mice with PEITC concentrations of 25 and 12.5 mg/kg bw administered to mice in three daily doses resulted in reduction of micronucleus formation in mice exposed to all three mutagens under study, with statistically significant effect at concentration of 25 mg/kg. Results of this study indicated that the strong PEITC antimutagenic properties may have an important role in the prevention of carcinogenesis and other chronic degenerative diseases that share some common pathogenetic mechanisms. In a recent study by **Tang et al. (2011),** PEITC was shown to induce a dose-dependent decrease in cell viability through induction of cell apoptosis and cell cycle arrest in the G2/M phase of DU 145 human prostate cells. Besides, PEITC induced morphological changes and DNA damage in DU 145 cells. The induction of G2/M phase arrest was mediated by the increase of p53 and Wee1 and it reduced the level of M-phase inducer phosphatase 3 (CDC25C) protein. The induction of apoptosis was mediated by the activation of caspase-8-, caspase-9- and caspase-3-depedent pathways. Results of this study also demonstrated that PEITC caused mitochondrial dysfunction, increasing the release of cytochrome c and Endo G from mitochondria, and led cell apoptosis through a mitochondria-dependent signaling pathway. The researchers concluded that PEITC might exhibit anticancer activity and become a potent agent for human prostate

There are a few studies on ITCs against nitrosamine-induced genotoxicity in literature. In a study by **Arranz et al. (2006)**, the protective effect of three ITCs was tested. ITC were highly protective against NPYR-induced oxidative DNA damage than against NDMA. The greatest protective effect towards NPYR-induced oxidative DNA damage was shown by I3C (1 M,

be due to inhibition of its metabolic activation **(Stoner et al., 1998)**.

cancer cells in the future.

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**0**

**22**

Veronika Kocurová

*250 68 Rež near Prague ˇ*

*Czech Republic*

**Gel Electrophoresis as Quality Control Method of**

Neurodegeneration is the leading term for the progressive loss of the neuron structure, including death of neurons. Many neurodegenerative diseases including the specific diseases such as Parkinson's, Alzheimer's, and Huntington's occur as a result of the neurodegenerative processes. As research progresses, many similarities appear which relate these diseases to one another on a sub-cellular level. Discovering these similarities offers hope for therapeutic advances that could ameliorate many diseases simultaneously. There are many parallels between different neurodegenerative disorders including atypical protein assemblies as well as induced cell death followed by an apoptosis. Apoptosis is a form of the programmed cell death in the multicellular organisms. It is one of the main types of the programmed cell death, and, last but not least, involves a series of the biochemical reactions leading to a characteristic cell morphology changes, and, finally, death. In according to the previously mentioned knowledge, there is a necessity to develop an imaging method which describes these cellular changes. The principal goal of the investigation monoclonal antibodies and their fragments is to examine the possibility of developing of an imaging radiotracer that would be specific for cytoskeleton of destructed dendrites and neuronal bodies. One of the suitable fitting marker, specific for neuronal tissue, performs anti III *β*-tubulin (bTcIII) antibody - TU-20 with molecular weight 150 kDa and its scFv fragment with molecular weight 27.7 kDa. The scFv fragment of TU-20 was synthesized for its higher mobility through tissue and vascular barriers. Biochemical characteristics (especially immunoaffinity) of the specific binding substance - anti III *β*-tubulin scFv fragment - is preserved, and, moreover, the biological availability is much better than in case of the whole antibody. See the structure in

To examine this hypothesis, it is necessary to radiolabel both substances with 125I and 123I. The next step is chemical analysis and, furthermore, biochemical properties are extensively investigated. The quality control, performed by gel filtration, electrophoresis, ELISA testing

Affinity coupling and RIA analytic methods occur under development with focusing on specifics of the antibody and its fragment behavior. *In vitro* experiment shows an extent of the preserved binding specifity of the species by incubation of the both radiolabeled substances

determines adequate properties of the radiolabeled substances for further studies.

with mice brain slices followed by an autoradiography.

**1. Introduction**

the Fig. 1.

**the Radiolabeled Monoclonal Antibodies**

*Nuclear Physics Institute, Academy of Sciences of the Czech Republic,*

