|
|
Determination of benzo(a)pyrene content in smoked meat products using modern isolation techniques
Suranová, Mária ; Šimon, Peter (referee) ; Šimko, Peter (advisor)
In this work a new simplified procedure for isolation and determination of PAH in smoked meat products was developed. This procedure is using ASE as selective extraction technique. Its principle is addition of silicagel activated during 18 hours at 140 °C directly to the extraction cell in ratio 10 g to 1 g of homogenized sample. In the next step the sample is extracted by n-hexane at 100 °C and 10 MPa for three 10 minutes static extraction cycles. The flush volume is 60 % and the purge time 120 sec. During the extraction silicagel adsorb koextracted lipids and thus the obtained extract is suitable for immediate HPLC-FLD analysis. On the contrary, the classical extraction procedures with nonpolar solvent require difficult purification of extract for removal of unwanted lipids. Mostly applied techniques here are GPC and SPE. The new procedure was validated for determination of B[a]P as well as for determination of 4 PAH (B[a]A, CHR, B[b]F, B[a]P) in smoked sausages. It meets all requirements to be set by Regulation (EC) No 333/2007, respective by new Regulation (EC) No 836/2011. After the method validation, the content of 4 PAH was investigated in twelve samples of various smoked sausages manufactured in Slovakia. Two analyzed products showed high PAH levels exceeding maximum legislation limits set for B[a]P as well as for sum of 4 PAH by Regulation (EC) No 835/2011.
|
|
|
Kinetics of benzo[a]pyrene destruction and identification of its products
Ryšavý, Jan ; Kolek, Emil (referee) ; Šimko, Peter (advisor)
This diploma thesis is focused on the study of conditions of benzo[a]pyrene, one of the major contaminant of foods, photodegradation under different conditions (solvents with different polarity, light sources, presence of antioxidants). In another part of the thesis, the degradation process of benzo[a]pyrene at various concentrations was studied, in order to characterise the kinetic aspects of photoinduced degradation. The attempt to identify the products of benzo[a]pyrene photodegradation was performed involving methods of gas chromatography and high performance liquid chromatography coupled with mass detectors, as well.
|
|
|
Metabolism of benzo [a] pyrene by the lung cells of a patient with cystic fibrosis
Skořepová, Adéla ; Indra, Radek (advisor) ; Wilhelm, Marek (referee)
Cancer is the most common disease, killing several million people worldwide each year. There are many reasons for its occurrence. The most common causes of cancer include alcohol and tobacco use. Nowadays, there is also a higher incidence of physical and chemical carcinogens that interfere with the environment. Poor eating habits and lack of exercise are also influential. Nowadays, thanks to good medical care, improved diagnostics and, above all, a number of existing vaccines, cancer is partly preventable. In the future, it is safe to say that cancer will increase. According to the IARC, over 19.3 million new cases will be diagnosed worldwide in 2020. By 2040, it is estimated that there will be another 10.9 million. Cystic fibrosis is an autosomal recessive inherited disease that affects 1 in 2500 newborns in the population. The main cause is impaired function of the CFTR protein as a transmembrane regulator of cystic fibrosis, leading to impaired fluid transport and impermeability of chloride ions, with a combination of excessive sodium absorption, leading to reduced water content in the periciliary fluid. People with cystic fibrosis are much more susceptible to colon cancer, especially those who have undergone lung transplantation. In recent years, the treatment of patients with cystic fibrosis...
|
|
|
Role of benzo[a]pyrene in cancer development
Vaňátková, Petra ; Moserová, Michaela (advisor) ; Kubíčková, Božena (referee)
4 ABSTRACT Cancer is nowadays one of the most serious diseases. Tumor development is a multistage process in which the effect of internal and external factors lead to failure of regulatory and defense mechanisms of the organism and to the accumulation of mutations which are generated by these organisms. Chemical carcinogens and also biological and physical factors can be regarded as the main external factors. Polycyclic aromatic hydrocarbons are large group of chemical carcinogens. One of them, benzo[a]pyren is the most studied polycyclic aromatic hydrocarbon. Carcinogenic, mutagenic and teratogenic effects of benzo[a]pyrene had been shown on laboratory animals. Benzo[a]pyrene is considered as the main carcinogen in tobacco smoke and is connected with lung cancer development among smokers. Benzo[a]pyrene is metabolized in activation or detoxication pathways by enzymes of mixed function monooxygenase systeme of cytochromes P450. The most important enzymes involved in the activation of these compounds are CYP1A1 and CYP1B1 with cooperation of epoxide hydrolase. The reactive species generated in its activation pathway are able to form covalent adducts with DNA. The most important carcinogenic product of benzo[a]pyrene is benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, which can caused irreversible ganges in...
|
|
|
Metabolism carcinogens and drugs by the system of monooxygenases
Moserová, Michaela ; Stiborová, Marie (advisor) ; Entlicher, Gustav (referee) ; Čeřovská, Noemi (referee)
Ellipticine, an alkaloid isolated from Apocynaceae plants, exhibits significant antitumor and HIV activities. Ellipticine is a pro-drug, whose pharmacological and genotoxic effects depend on activation by cytochromes P450 (CYP) and peroxidases (Px) to a reactive species generating DNA adducts. To elucidate contribution of CYPs (and which of them) and Px to ellipticine activation, we used rat and mouse models, mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in the liver (HRNTM ) and a control mouse line (WT), rats treated with ellipticine, and microsomal systems isolated from the liver of mouse lines and from the liver, kidney and lung of rats. The purified enzymes, CYP1A1 and 3A4, reconstituted with NADPH:CYP reductase were also used. The effect of cytochrome b5, a facultative component of the mixed function monooxygenase system, on ellipticine oxidation by CYP1A1 and 3A4 was also investigated. Carcinogenic benzo(a)pyrene (BaP), known to covalently bind to DNA after its activation with CYPs, was investigated for its potential to generate DNA adducts and to induce CYP and NADPH:CYP reductase enzymes in mouse livers. We investigated an influence of each of components of the mixed function oxidases (MFO) system on metabolism of BaP. CYP1A1 is widely accepted to be the...
|
|
|
Mechanism of enzymatic activation of carcinogens and drugs by the system of cytochrome P450
Indra, Radek ; Stiborová, Marie (advisor) ; Souček, Pavel (referee) ; Koblihová, Jitka (referee)
13 Abstract An environmental pollutant and a human carcinogen benzo[a]pyrene (BaP) is after its activation with cytochrome P450 (CYP) able to covalently bind to DNA. In the thesis, one of the target was to investigate an influence of individual components of mixed function monooxygenase (MFO) system on metabolism of benzo[a]pyrene and generation of adducts of activated BaP with DNA. The study was particularly focused to increase our knowledge on the effect of cyt b5 on metabolism of BaP by cytochrome P450 1A1 (CYP1A1) and its potential to serve as a donor of electrons during the reaction cycle of this cytochrome P450. The effect of cyt b5 on generation of BaP metabolites and adducts of BaP with DNA was investigated. In addition the effect of two different expression systems for cytochrome P450 1A1 (prokaryotic and eukaryotic) was also studied. The influence of cyt b5 on oxidation another xenobiotic compound, a plant alkaloid ellipticine that exhibit antitumor activities, was also investigated. Its pharmacological efficiency, as well as side effects depends on its metabolic activation by cytochrome P450. CYP3A4 is very important for ellipticine activation and therefore this enzyme was used in our experiments. Furthermore, a suitability of rat as a model organism mimicking the metabolic fate of BaP...
|
|
|
Effect of cytochrome b5 on activity of cytochromes P450
Ličko, Vojtech ; Indra, Radek (advisor) ; Feglarová, Tereza (referee)
ABSTRACT Cytochrome b5 (CYB5) is heme protein capable of reduction of cytochromes P450 (CYP) or some other enzymes. However, his regulative capability was also observed by his apo form, i.e. in absence of heme prosthetic group in the active center. CYB5 can accept electron from cytochrome b5 reductase (CYB5R) or from cytochrome P450 reductase (CYPOR). CYPOR by itself is reduced by NADPH and is also able to forward electron to CYP independently of CYB5. CYB5R on the other hand is reduced by NADH. Efficiency of CYB5 to accept and forward an electron was studied in vitro with five different substrates - testosterone, Sudan I, aristolochic acid I (AAI), ellipticine and vandetanib. These substrates were chosen considering their characteristic reactions, which are catalyzed by their respective isoforms of CYP. The experiments with these substrates were carried out in the medium with recombinant CYPs prepared in insect cells or E. coli or in the medium with hepatic microsomes isolated from different organisms. Rats, from which the majority of these microsomes was isolated, were premedicated by different CYP inducers. The experiments were carried out in medium with NADH or NADPH in order to assess the capability of CYB5 to reduce CYP independently of CYPOR. The capability of CYB5 and CYB5R to act as a...
|
|
|
The effects of endocrine disruptors on the expression and the activity of cytochromes P450 2B in laboratory rat as a model organism
Měkotová, Barbora ; Dračínská, Helena (advisor) ; Levová, Kateřina (referee)
Endocrine disrupting chemicals are compounds that interfere with natural hormones and they are responsible for functional changes which may lead to damage of the endocrine system. Their presence in the environment is associated with a number of diseases whose extent is hard to predict. As endocrine disrupting chemicals, a wide range of exogenous and endogenous compounds is present in the environment. Important exogenous endocrine disrupting chemicals include benzo[a]pyrene (BaP) and 17α-ethinylestradiol (EE2); the female sex hormone 17β-estradiol (E2) can act like endogenous endocrine disruptor. In this thesis, the effect of these three compounds and their combinations on the expression and the activity of rat biotransformation enzymes cytochromes P450 2B is studied. The gene expression was determined by quantitative PCR, the expression of the protein itself was studied using Western blot method and consecutive immunodetection. The results show that CYP2B expression is almost unchanged after BaP premedication, whereas estrogenic compounds, E2, EE2, their combination and their combinations with BaP, significantly decrease the expression. The enzyme activity of CYP2B was also studied in rat liver microsomes using the marker substrate 7-pentoxyresorufin. EE2, E2 and their combination decrease the...
|
|
|
Expression and activity of rat cytochromes P450 3A after exposure to benzo[a]pyrene and Sudan I
Ličko, Vojtech ; Dračínská, Helena (advisor) ; Ptáčková, Renata (referee)
The aim of this Bachelor thesis is the study of the effect of two carcinogenic compounds, benzo[a]pyrene and Sudan I, co-administered to rats individually or in combination, on the expression and the activity of important biotransformation enzymes cytochromes P450 of subfamily 3A in liver - a main organ of xenobiotic metabolism, in which the amount of CYP3A is especially high. Using the quantitative PCR method, the decrease of the gene expression of CYP3A1/2 in the livers of rats exposed to benzo[a]pyrene and Sudan I individually or in combination, was observed. Using the Western Blot method with a consecutive immunodetection, we found the decrease of the protein expression of CYP3A in the livers of rats treated with benzo[a]pyrene and Sudan I alone. Specific activity of CYP3A, determined by marker reaction of CYP3A, which is 6β-hydroxylation of testosterone, did endorse the previous results only in some of the premedicated groups of rats. It can be concluded that the exposure of rats to both studied compounds with carcinogenic potential resulted in a decrease in the expression of hepatic CYP3A in vivo. (In Czech) Keywords: cytochromes P450, benzo[a]pyrene, Sudan I, expression, enzyme activity
|
|
|
The influence of endocrine disruptors on the expression of cytochrome P450 1A2
Orlovská, Ľubica ; Dračínská, Helena (advisor) ; Ječmen, Tomáš (referee)
The term endocrine disruptor is used for chemical compounds which imitate or antagonize the effects of endogenic hormones, alter hormone synthesis and metabolism or modify levels of hormonal receptors. Synthetic estrogen 17α-ethinylestradiol (EE2) and carcinogenic substance benzo[a]pyrene (BaP) belong to the group of chemicals described as exogenic compounds with endocrine destruction, while estrogenic hormone 17β- estradiol (EST) figures as natural endogenic endocrine disruptor. The bachelor thesis focuses on study of the influence of these endocrine disruptors and their combinations on expression and specific activity of CYP1A2. RNA was isolated from the lungs of rats treated with the endocrine disruptors and from untreated rats. RNA was converted to cDNA by reverse transcription. Relative amount of CYP1A2 in livers, kidneys and lungs was quantified by real-time PCR. The protein expression of CYP1A2 was studied using the Western blot with consecutive immunodetection. Finally, the specific activity of hepatic CYP1A2 was determined by measuring 7-methoxyresorufin O- demethylation. It was confirmed that BaP induces gene expression of CYP1A2 in livers, kidneys and lungs, even in combination with EE2 and EST. However, both estrogens decrease the induction potential of BaP. When given individually,...
|
guest ::
