During the life cycle aerobic organisms are exposed to a number of endogenous
and exogenous sources of ROS. Present in low to moderate concentrations these
reactive molecules play an important role ...in many physiological processes
such as regulation of signaling cascades and gene expression. In high
concentrations, ROS can oxidize cellular proteins, lipids and DNA, causing
changes in structure and function, damage and even cell death. When the
concentration of free radicals exceeds the physiological level, a cell is
said to be in a state of oxidative stress. To prevent the onset and reduce
the consequences of oxidative stress, living organisms have developed
powerful antioxidant system (AOS). This system includes a set of mechanisms
to maintain the level of free radicals in the narrow range between
physiological and toxic concentrations. The most important enzyme components
in AOS are: superoxide dismutase (SOD), catalase (CAT), glutathione
peroxidase (GPx) and glutathione reductase (GR). These enzymes are involved
in the removal of ROS and participate in maintaining level of reduced
glutathione, thus ensuring the preservation of physiological functions and
inhibit the development of cell damage and disease. Because of such an
important role of AOS, the regulation of the functional expression of its
components is of particular importance for physiological and pathological
processes in aerobic organisms. One of the most important regulatory
molecules in this system is Nrf2 (NF-E2 related factor 2). Nrf2 is a
transcription factor that induces expression of many cytoprotective proteins
including antioxidant enzymes, and therefore play an important role in the
regulation of oxidative stress. Previous studies have clearly demonstrated a
link between oxidative stress and carcinogenesis. Cancer is a multistage
process that develops over three stages: initiation, promotion and
progression and oxidative stress is associated with each of them. Moreover,
antioxidant profile is altered in cancer cells compared with healthy, normal
tissue, so these molecules may be important biomarkers in the assessment of
risk and the degree of carcinogenesis. Endometrial cancer is one of the three
most common diseases of female reproductive organs. It has been shown that
benign and premalignant changes precede the malignant transformation of the
uterus, which is why these conditions may be considered as stages of
carcinogenesis. Despite numerous studies, the molecular processes involved in
multi-stage development of endometrial cancer are not yet fully known.
Therefore, the aim of this dissertation was to examine the changes in
expression of the four most important antioxidant enzymes in the blood and
tissues of patients with benign, premalignant and malignant endometrial
transformations, as well as the mechanism of their regulation by
transcription factor Nrf2. In this research we used endometrial tissue and
venous blood of patients diagnosed with: polypus endometrii, uterus
myomatosus, hyperplasia simplex endometrii, hyperplasia complex endometrii
and adenocarcinoma endometrii. Polypus endometrii and uterus myomatosus were
considered as benign, while hyperplasia simplex and hyperplasia complex
endometrii were considered as premalignant transformation of the uterus.
After adequate sample preparation, we initially determined the total protein
concentration in blood and endometrial tissue of all five groups of patients.
Then we performed SDS PAGE electrophoresis, transfer to nitrocellulose
membrane and chemiluminescent detection of CuZnSOD, CAT, GPx, GR and Nfr2
protein molecules. The protein levels were normalized with respect to
β-actin. Furthermore, in endometrial samples of all five groups of patients,
RNA was extracted and underwent reverse transcription into cDNA, followed by
the TaqMan Real-Time PCR method by which the amounts of CuZnSOD, CAT, GPx, GR
and Nfr2 mRNA were determined in respect to POLR2 as endogenous control. The
results of these experiments showed the following: 1. In comparison to
patients with polyps and myomas, in endometrium of women with hyperplasia
simplex and hyperplasia complex decreased levels of Nrf2 were recorded, while
in adenocarcinoma tissue Nrf2 protein level was increased. The observed
changes were regulated at the transcriptional level, except in adenocarcinoma
tissue in which increase in Nrf2 level was regulated by some
post-transcriptional mechanism. 2. CuZnSOD mRNA level and the amount of
CuZnSOD protein in the endometrium of patients with hyperplasia simplex and
hyperplasia complex were decreased compared to patients with polyps and
myomas. In adenocarcinoma tissue, however, the values of these parameters
were significantly increased compared to both benign and both premalignant
transformations. Observed transcriptional and translational CuZnSOD
variability in different stages of endometrial transformation were the
consequences of changes in the level of transcription factor Nrf2. 3.
Compared to the control groups with polyps and myomas, levels of CAT mRNA and
CAT protein were significantly decreased in the tissues of patients with
premalignant and malignant lesions of the uterus. It is also shown that Nrf2
had no direct effect on the CAT gene transcription, but acting on other
translational and posttranslational processes can probably influence the
level of this enzyme in the endometrium. 4. During carcinogenesis, increase
of GPx protein level was detected in premalignant endometrium, while decline
in the the amount of this enzyme was measured in adenocarcinoma tissue. GPx
level was positively correlated with the amount of transcription factor Nrf2,
but mechanism that regulates the expression of this enzyme varied with the
degree of tissue transformation. 5. In the course of malignant endometrial
transformation, increased expression of glutathione reductase was observed.
Consequent increased level of GR protein was the result not only of the
intensified transcription under the influence of Nrf2, but also of an
additional, posttranscriptional mechanism activated in transformed cells of
the uterus. 6. As regards AOE expression in the blood of examined patients,
it was observed that in comparison to the control group, in groups of women
with benign endometrial changes a decline in Nrf2 and GR levels were
measured, GPx level was increased, while the relative amounts of CuZnSOD and
CAT protein did not change significantly. Somewhat more pronounced changes
were observed in the blood of patients diagnosed with hyperplasia simplex,
hyperplasia complex and adenocarcinoma, characterized by declined protein
levels of Nrf2, CuZnSOD and GR, increased level of GPx and unaltered
expression of CAT. It was also shown that the blood expression levels of all
investigated AOE correlated positively with the amount of Nrf2. The results
of this dissertation indicate the existence and importance of the specific
expression pattern of antioxidant enzymes and transcription factor Nrf2 in
gynecological patients diagnosed with: polypus endometrii, uterus myomatosus,
hyperplasia simplex endometrii, hyperplasia complex endometrii and
adenocarcinoma endometrii. The observed changes confirm the important role of
the examined enzymes in complex molecular interactions that underlie the
transformation of endometrial cells. It is obvious that these AO molecules
not only influence the development of lesions in the uterine tissue, but they
are also an important factor for the progression of benign changes in
premalignant and malignant phenotype. It can be concluded that the results of
this dissertation contribute significantly to a better understanding of the
molecular processes involved in carcinogenesis. Implementation of this
knowledge in clinical practice could contribute to the quality of prevention,
diagnosis and treatment of gynecological patients, may lead to to improvement
in the disease course and prognosis and, consequently, may result in
increased survival rate of cancer patients.
Tokom čitavog životnog ciklusa aerobni organizmi su izloženi brojnim
endogenim i egzogenim faktorima koji indukuju povećanje produkcije ROS-a.
Prisutni u fiziološkim koncentracijama, reaktivni molekuli ROS-a imaju
značajnu ulogu u važnim ćelijskim procesima kao što su regulacija signalnih
kaskada i genske ekspresije. U visokim koncentracijama ROS mogu oksidovati
ćelijske proteine, lipide i DNK i time dovesti do promena strukture i
funkcije, oštećenja, pa i smrti ćelije. Kada koncentracija slobodnih radikala
premaši fiziološki nivo, smatra se da se ćelija nalazi u stanju oksidativnog
stresa. Kako bi sprečili nastanak i umanjili posledice ove vrste stresa, živi
organizmi su razvili moćan antioksidativni sistem zaštite (AOS). Ovaj sistem
uključuje seriju mehanizama kojima se nivo slobodnih radikala održava u uskom
opsegu između fiziološke i toksične koncentracije. Najvažnije enzimske
komponente AOS su: superoksid dismutaze (SOD), katalaza (CAT), glutation
peroksidaza (GPx) i glutation reduktaza (GR). Ovi enzimi uklanjaju višak
ROS-a i učestvuju u održavanju nivoa redukovanog glutationa, čime obezbeđuju
očuvanje osnovnih životnih funkcija i sprečavaju nastanak oštećenja i bolesti
ćelije. Zbog tako važne uloge AOS, regulacija funkcionalne ekspresije
njegovih komponenti je naročito značajna za fiziološke i patološke procese u
aerobnim organizmima. Jedan od najvažnijih regulatornih molekula u tom
sistemu je Nrf2 (NF-E2 related factor 2). Nrf2 je transkripcioni faktor koji
indukuje ekspresiju mnogih citoprotektivnih proteina uključujući i
antioksidativne enzime, zbog čega ima značajnu ulogu u regulaciji
oksidativnog stresa. Savremena istraživanja na različitim model-sistemima
ukazuju na povezanost oksidativnog stresa i kancerogeneze. Razvoj kancera je
višestepeni proces koji se razvija kroz tri faze: inicijaciju, promociju i
progresiju, a oksidativni stres je povezan sa svakim od ova tri stadijuma
transformacije ćelije. Osim toga i sam antioksidativni p
Butil p-hidroksibenzoična kiselina, poznata i pod nazivom butilparaben (BP), najčešći je oblik parabena, koji se apsorbira putem kože i probavnoga sustava te razgrađuje u jetri i bubrezima. Najnovija ...istraživanja in vivo i in vitro upozoravaju na to da BP djeluje toksično na reproduktivne organe, plod (teratogeno djelovanje) i razvoj organizma. No dosad nije detaljno istražen oksidacijski stres koji on izaziva niti njegova povezanost s oštećenjem tkiva. Stoga je cilj ovoga istraživanja bio utvrditi djelovanje BP-a na aktivnost enzima koji sudjeluju u pentoza fosfatnom putu i onih ovisnih o glutationu poput glukoza-6-fosfat dehidrogenaze (G6PD), 6-fosfoglukonat dehidrogenaze (6-PGD), glutation reduktaze (GR), glutation peroksidaze (GPx) i glutation-S-transferaze (GST) u tkivu bubrega, jetre, mozga i testisa. U tu su svrhu muški štakori bili nasumce raspodijeljeni u četiri skupine: tri su skupine četrnaest dana primale oralne doze BP-a od 200, 400 ili 800 mg/kg na dan, a kontrolna skupina kukuruzno ulje. Nakon tretmana životinje su žrtvovane i u njihovim su tkivima izmjerene aktivnosti G6PD, GR, GST, 6-PGD i GPx te su napravljene histopatološke pretrage tkiva. BP je poremetio ravnotežu enzimskih aktivnosti te doveo do oštećenja svih ispitanih vrsta tkiva. Ovo su prvi nalazi koji potvrđuju degenerativnu ulogu BP-a na staničnoj razini. Zabilježeni poremećaji homeostaze i obrambenoga antioksidacijskoga sustava upućuju na oksidacijski stres kao mehanizam u podlozi oštećenja tkiva izazvanoga BP-om.
Sorta jagoda Elsanta uzgajana je u tresetnom supstratu u plasteniku. Puna doza i 50% smanjena doza dušika i kalija primjenjene su tijekom proljetnoga plodonošenja, uz primjenu biostimulatora Viva®, ...Megafol® i njihove kombinacije. Specifične aktivnosti gvajakol peroksidaze (GPXs; EC 1.11.1.7), katalaze (CATs; EC 1.11.1.6), askorbat peroksidaze (APXs; EC 1.11.1.11) i glutation reduktaze (GRs; EC 1.6.4.2) u listu jagode bile su značajno više poslije tretiranja biostimulatorima i reduciranom gnojidbom. Najjača povezanost uočena u ovom istraživanju, bila je između enzima askorbat-glutation ciklusa (APXs i GRs), koji su bili u pozitivnoj
korelaciji s masom svježega lista (TLFM). Najveća masa svježega lista (TLFM) uočena je na biljkama tretiranim Megafolom®.
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