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					UDK 576.3.32:615.014.425                          based on the right of manuscript




                          Altayeva Aliya Sagymbekovna




           Influence of heavy metals salts’ toxic on genetic apparatus


                                 03.00.16- ecology


                                   Autoabstract
   of the dissertation work submitted to the PhD degree the speciality by ecology
                                       science




                           The Republic of Kazakhstan
                                   Astana, 2010
The work was done at the Eurasian National University at the Department of
International faculty of environmental and engineering




Scientific advisor:                         PhD of biological science,
                                            Professor Khanturin М.R

Foreign scientific advisor:                 PhD, professor H.Habelhah
                                            USA

Official opponents:                         M.D, professor Ukbayeva Т.D.

                                            PhD of ecology Masalimov J.К




The defense of a thesis will take place on 23rd June, 2010 at 11.00 o’clock at the
State Qualification Commission meeting under the Eurasian National University n
L.N. Gumilyov, 5, Munaitpassov str., classroom 302, Astana, Republic of
Kazakhstan, 010008.



The thesis can be learnt at the “Otrar” library of the L.N. Gumilyov Eurasian
National University, 13 Pushkin str., Astana, Republic of Kazakhstan 010005.


The autoabstract was sent out on «_______» __________ 2010




Academic secretary of
State Qualification Commission                               I.A. Zaitseva




                                        2
    Relevance of an issue. Kazakhstan is among the biggest manufacturers and
exporters of copper, nickel, chrome, ores and ferroalloys in the world. During the
five past years the growth rates have increased. New industrial sites are being built
and put into operation; this contributes to air pollution and ecological degradation in
general. Major part of industrial run-offs is the result of activities of mining and
processing industry, iron and steel works, petrochemical and building materials
production.      The most dangerous sites are: lead and zinc plant in Ust-
Kamenogorsk, lead and phosphate industry of Shymkent, phosphorous industry of
Taraz, chrome plants of Aktubinsk. The most polluted air is registered in East
Kazakhstan and Karaganda regions.
     Heavy metals belong to the group of the strongest toxicants. They support
respiratory tract diseases, liver and blood diseases, failure of nervous and immune
system which quite often results in oncological diseases.
     Oxidizing damage of biological membranes takes main part in the process of
metal induced cell damage. This becomes possible due to reactive oxygen
compounds, generated under the influence of heavy metal ions. Many researches
demonstrated increased intensity of lipid peroxidation and decreased activity of
antioxidant ferments (Palmer H, Paulson, K.E, 1997) during the influence of heavy
metal ions where cells lose their protective functions.
       The purpose of this work is to determine the mechanism of developing
damage of laboratory animals’ cells induced by metals and tracing the
effectiveness of succinic acid protective action.

The objectives:
1. To study the change of resistance of erythrocyte membranes under the action of
salts of arsenic, chromium, and cadmium, nickel.
2. Set the number of micronuclei formed during intoxication with salts of heavy
metals under the combined protective effect of antioxidant - succinic acid
3. To study the effectiveness of succinic acid on the mechanisms of inhibition of
gene expression cIAP 10, controlled by the nuclear factor NF kB.
4. To determine the cytotoxicity of heavy metals in cell culture of mouse
fibroblasts.

Scientific originality of the paper.
       This work observes comparative influence of heavy metals on red-cells
resistance and intensity of micronuclei building within red-cells. Heavy metals being
tested are natrium hydroarsenates, cadmium chloride, nickel chloride. Cytotoxic
influence of heavy metal salts on the fibroblast cells of mice of pBA-TRAF-WT,
pBA- TRAF2- S11A line was demonstrated for the first time.
     Chromium oxide and natrium hydroarsenates influence on expression of gene
cIAP 10 was shown for the first time.
     It was discovered for the first time that succinic acid inhibits expression of gene
cIAP 10 of fibroblasts after intoxication with heavy metal salts.



                                          3
General statements for defense:
   1. Decrease of red cell membrane resistance under influence of arsenic, chrome,
      cadmium and nickel salts was discovered.
   2. Increase of amount of micronuclei under intoxication with heavy metal salts
      was determined. It was defined that number of micronuclei is dropping under
      joint effect of heavy metal salts and succinic acid.
   3. Demonstrated activation of expression of gene cIAP 10 under influence of
      heavy metal salts and possible inhibition of expression of gene cIAP 10 with
      the help of succinic acid.
   4. Cytotoxic effect of heavy metal salts on fibroblast cell culture of mice and
      protective activity of succinic acid was defined.

Approbation the study materials will be reported at the VII Moscow International
Scientific Conference «Biotechnology: Ecology of cities » and discussed at the
Department of Pathology, University of Iowa, at the Department of International
faculty of environmental and engineering , Eurasian National University.

       Theoretical and practical value of the work The study obtained new
scientific evidence on the impact of toxic agents (chromium salts, arsenic, nickel,
cadmium) on the processes of activation of transcription factor NF kB.
 The work results showed that the toxic effects of heavy metals succinic acid
inhibits activation of nuclear factor processes and significantly protects cells from
metal-induced damage.
       The data obtained contributes to biochemistry, microbiology, oncology
studying showing changes in genome under influence of heavy metals. Applied
character of this work is using of succinic acid as a protector of cell influenced by
heavy metal ions

   Publications By the theme of dissertation 8 manuscripts was published.

    Structure and the volume of thesis work.
     The thesis consist of introduction, basic part including literature review and the
results of own investigation, discussion of obtaining results, conclusion, list of using
literature. The work represented on the 102 pages, illustrated by 8 tables, 12 figures.
In the list of literature 103 sources was mentioned.


                                     General Part
     Mutagenes or cancerogenes are inorganic: metal bivalent cations (Mo, Hg, Cu,
Mn, Cr, Ni, Co и др.), inorganic compounds of Co, Cd, Hg, As, Cr+3, Cr6+,
different compounds of Ni, bivalent salts of Pb; zinc acetate, oxide, sulphide and
chloride; some compounds of Se, Mo, Be, Al, Pl, Sb, Cu, Mn, Sn etc. Common
feature of the better part of cancerogenes is their ability of metabolic change into
strong electrophilic reagents which cooperate with nucleophilic centres of genetic

                                          4
apparatus of a cell. This plays a decisive role in a process of cell failure and
transformation (M.A. Mukasheva., 2006).
     Researches of the past years revealed that succinic acid possesses biological
activity with unique combination of such feature as: succinates acting like
adaptogenes and protectors; and in the presence of pathologies they show medical
effect unusually strong for adaptogenes. (A.Yu. Petrov, 2001).
     Studying of succinic based medicine development and widening the area of use
of known medicine is well promising. Thus being combination of high
pharmacological activity, succinic acid may be used for producing of medicine with
improved pharmacological features of original antioxidants. More over there are not
so many registered medical forms with succinic acid as a main compound
     Material and Methods.
     The investigation was done on 60 the white rat male, mass is 180-200 gr. At
the beginning of the experiment the animals were divided into 3 groups. The first
group consisted of the control rats (n=10). The second experimental group was the
animals, which were fed with per os sharp doses of the LD 50 metals chrome oxide-
25 mk/ kg (n=10), sodium arsenat - 15 mk/ kg (n=10). The third group was the
mice, fed with per os sharp doses LD 50 these heavy metals salts (n=10) when
acid is per os 20 mk/kg.
     The second aim of our research is to study the impact of succinic acid on the
fibroblast cells of mice in the context of chromium oxide intoxication. The work is
performed on cultivated cells of pBa -TRAF2- WT, pBa -TRAF2- S11A types.
Survival ability of cells was observed on the 24th, 48th and 72nd days. Solution of
chromium oxide of 7.5 µM and 10µM strength was used as xenobiotic.
     The destruction of the cells by free radicals is one of the toxic effects of
chrome oxide and sodium hydro arsenate. These metals were chosen, in the
concentration 10 µM (high toxicity for chrome oxide and sodium hydro arsenate).
Our work was directed to finding out molecular destructions in cells and
therapeutic effect of succinic acid.
     Cell Culture and Treatments The pBA –TRAF2-WT and S11A are mouse
fibroblast cell line were maintained in Eagle’s culture medium and incubated at 37º
C in a 5% CO2 incubator. Cells were growth to 80% confluency in 60 mm culture
dishes and were exposed to two carcinogenic chemicals. The exposure doses
mentioned above were chosen as high toxic concentrations the chemical
compounds used in this study were purchased from Sigma Aldrich. Thereafter, the
succinic acid loaded cells were washed with PBS and incubated in the culture
medium containing each test chemicals.
     Cells were harvested with trypsin (0.25%) solution. Real –Time Reverse
Transcriptase PCRTotal RNA extracted from cells was growth 80% confluency.
After 2, 6, 12, 20 h incubation total RNA extracted from treated cells according the
instruction of manufacturer and produced the synthesis of cDNA. PCR carried out
on amplification RTC-200 and run on 1.5% agarose gel in TBE buffer, pH 8.0 and
run at 100 V for 30 min at room temperature. For cDNA fragment with definition
sequences of nucleotide was carried out searching homological sequences in the
data base Gene NCBI bank using the program NCBI Sequence Viewer v2.0.
                                        5
                                                   Results and their discussion

      Red cell hemolyses under the strong intoxication with arsenic, chromium.
cadmium and nickel salts and the effect of succinic acid. Change of the condition
of free radical oxidation becomes a serious indicator of body’s reaction to the
influence of industrial and ecological environment. As a result of various toxic
influences, stress, hypoxia, inflammation, etc. control over the process of lipo-
peroxidation upsets resulting in weakening of barrier function and increasing of bio-
membrane penetrance. ( Rikans LE, Hornbrook KR., 1997).
Peroxidation of lipids is one of the main mechanisms for violation of membrane
integrity and necrocytosis. Ability of phagocytes to build free radicals of oxygen at
the contact areas of cytoplasmic membranes with corpuscular particle particles or
immune complexes is of great biological importance. Free radicals interfere with
mitosis and cell growth, inactivate thiolic ferments, take part in breathing,
glycolysis and other processes, disconnect breathing and oxidative
phosphorylation, and increase tenacity of membrane lipids.
         Thus under strong intoxication with heavy metals (chrome, arsenic,
cadmium, nickel) red cell membranes of laboratory mice were deteriorating. This
proves significant toxic effect on animal body. First of all functions of blood cells
fail, particularly of red cells. It is possible to suggest that toxic influence of heavy
metals resulted in decreased level of red cell hemolytic resistance and increased
content of phantom cells due to disorder of physiological condition of membrane
lipids. It was observed that under influence of antioxidant – succinic acid – toxic
effect of heavy metal salts on experimental animals’ bodies was growing down.

                                    14
       % of erythrocyte hemolyses




                                    12

                                    10

                                    8

                                    6

                                    4

                                    2

                                    0
                                         Blank   Arsenicum Arsenicum+    Chrome   Chrome+
                                                             Succinic             Succinic
                                                               acid                 acid


Figure 1- Number of erythrocyte hemolyses index under the severe intoxication of
chrome and arsenic, and against of succinic acid.


                                                              6
     Micronuclei building induced by heavy metal intoxication and their
correction with succinic acid. In the course of experiment it was discovered that
under strong intoxication with natrium hydroarsenates number of red cell
micronuclei increased from 5,5±0,78% to 39,4±2,40% micronuclei per 2000 red
cells (p<0,001) in comparison with control animal group; under intoxication with
chromium oxide it increased from 5,5±0,78% to 38,2±5,9% (p<0,001).
     Changes of general and biochemical indications of animals’ blood, as in strong
experiment, demonstrate toxic effect of heavy metals ob different organs. To
determine mutagenic effect it is required to examine blood cells which can help to
discover genetic failures.
        It was determined that under strong heavy metal intoxication the number of
cells with micronuclei had increased. This shows rapid dissemination of metal over
tissues, organs and cells leading to development of toxic effect on the level of blast
hemopoietic cells. This toxic effect results in growth of cytogenetic failures.
Increase of micronuclei number defined within red cells of peripheral blood under
the influence of heavy metals is expressed more under continuous influence of toxic
agent. It also speaks about mutagenic activity of micronuclei caused by structure
failure of nucleic acid molecules. In comparative aspect of toxic rate metals in
peripheral blood were presented as follows: arsenic, chrome, cadmium, nickel.




                          45
                          40
                          35
       % of micronuclei




                          30
                          25
                          20
                          15
                          10
                          5
                          0

                               Blank   Arsenicum Arsenicum+   Chrome      Chrome+
                                                  Succinic                Succinic
                                                    acid                    acid


Figure 2 - Number of micronuclei index under the severe intoxication of chrome
and arsenic, and against of succinic acid.


                                               7
      Toxic metals increased cIAP10 gene expression. The destruction of the cells
by free radicals is one of the toxic effects of chrome oxide and sodium hydro
arsenate. These metals were chosen, in the concentration 10 µM (high toxicity for
chrome oxide and sodium hydro arsenate). Our work was directed to finding out
molecular destructions in cells and therapeutic effect of succinic acid. Chrome
oxide indicates the expression of the gens cIAP 10, besides, it make stronger the
synthesis TNF –α. As a positive control of the activation NF kB this activator of
this transcription factor was used. The increasing of the content TNF – α, cIAP 10
snows, that their concentration reflects the character and the deepness of the
destruction. The expression of cIAP 10 gen in case of the action of the investigated
inductors was analyzed by the RT-PCR.
      In many cells systems heavy metals salts cause the induction of apoptative
cascade; it acts on the activation of the transcription factor NF kB, which controls
the expression of many gens, regulating proliferation, inflammation and apoptosis.
The reactions, caused to the activation of NF kB, include phosphatating regulator
complex kinasis, which, in its turn, phosphate of transcriptive factor NF kB, which
caused its degradation. As a result, active subunits NF kB reveals and translocated
to the nucleus. It comes to the changing of some hundreds gens’ expression and,
respectively, to activity of the metabolite processes. As known, in some pathologic
cases, the main aim of NF kB is a transition of cells from one program to other to
save the function of the cell.
      It is t should be emphasized, that NF kB plays an important part in the
revealing of immune syndrome, so the inhibitions of this protein can be interesting
from this point of view. The extra activity of NF kB correlates with invasion
metastatic tumor cells and as a result inhibitor activity of this factor gives both
antiproliferative and anti metastative action. In norm NF kB protein is blocked by
specific cell protein inhibitor AP-1. But in case of activization this complex
dissociates and revealed NF kB transports to nucleus, where the control of the gen
expression is made. The inhibitors NF kB can hinder to dissociation of this
complex. It is interesting to notice, that, as NF kB is a main factor in the process of
inflammation, anti- inflammation medicines, which act as inhibitors Nf kB, can
show their effect and according to this mechanism, acting as oncoprotectors
(resveratrols, proantocyanydines, E vitamin, flavonoids) (H.J. Thompson, J.
Heimendinger, 1999). Besides, inhibitors of the NF kB action is flavonoides
(apigenin, luteolin) which appear as inhibitors and which act according to
antioxidant mechanism. It is necessary to pay a special attention to the their
influence on such important factors of cell functionality, including cancer cell, as
regulation of signal transduction processes, including the activity of the growth
factors, transcriptional factors, antioxidant system of cell and the processes which
are tightly connected with them as cell proliferation, angiogenesis and growth, and
in the case of cancer cells- invasion and metastasion.
      Among these nature connections the biologically active substances,
possessing antioxidant characteristics, occupy an important place. However, the
function of antioxidants in the preventive measures of the tumor growth, as, by the
way, in other processes, is often considered simplified and unilaterally (Costa M.
                                          8
1993). Their role is only considered as catching free radicals or connecting
prooxidant metals ions, reducing of the free radical processes, it is often considered
as the only mechanism of oncoprotective actions. In fact, from the point of view of
their influence on the tumor process these connections have other, non- antioxidant
functions, having a bigger effect on the cell systems. On the other side, antioxidant
action can be realized not only as a protective, but also as an important regulative
mechanism, controlling the tumor growth. The protection from oxidation action is
realized on many levels and structures and change according to differences of cell.
      Our investigation point out, that each of toxic agents has a specific effect on
transcription factor and expression of the known genetic construction, that can
provide the understanding between cell signals and cell regulation ways, connected
with their carcinogenetic effect.
      In the result of this research we have achieved the results, which are evidences
of revealing of NF kB from the complex and inhibitor in case of oxidant stress in
case of sharp inflammation. The function of NF kB can be transformed from
apoptative to proapoptative depending on nature of induced signal. However, the
realizing of transcription factor from the complex with inhibitor is the first phase
of their function. The second phase is a transportation of transcription function.
From all chrome forms, only chrome Cr (VI) does not act directly to DNA in vitro,
but causes the wide spectrum of DNA destructions, including Cr-DNA adducts
oxidant destructions and other DNA modifications. Chrome, being in low
concentration, is able to activate NF kB in T-cells, macrophages, epithelial cells.
The gens expression codes antiapoptative proteins, such as cIAP 10. The
expression of cIAP 10 in succinic acid- loaded cells comes to essentially inhibition
Cr- induced cell death. These results show, that NF kB plays a great role for
inhibiting ROS- depended cytotoxic being with antioxidant. Such a chrome
inductive cell death can be inhibits partly by some gens, antioxidants, and metals
itself.
        The result in the control sample showed the level of expression cIAP 10 is
less; the expression has increased in case of stimulation TNF- α. In two hours after
working up the cells with chrome oxide the level of expression cIAP10 had not
changed much (Fig 3), only by eight or twenty hours the expression had been
increasing. As we can see from the Fig 3 the treatment with chrome oxide makes
higher the level of the expression cIAP 10 in 8 and 20 hours. It is definitely that the
expression cIAP 10 goes through induction ROS. However, the connection
between metals and signal proteins changes the functionality of these proteins with
creation ROS can be not in all cases. In general, Cr (VI) is supposed to activate NF
kB in fibroblasts cells much more.
      In the samples with sodium hydro arsenate both metal- inducing of the gens’
expression cIAP 10 and in case of protective influence of succinic acid was
watched. From the Fig 4 in case of stimulation the cells with sodium hydro
arsenate (10 µM) in 2 or 6 the expression is not appear as much as in 8 or 20 hours.
The long – term influence of these metals changes oxidant- antioxidant
intracellular balance, oxidant surrounding cells components and as a result induced
either or keeps away from cells wreck.
                                          9
                                                                                  1 µМ succinic
                                          Cr2 O3 10 µM                                            Cr2 O3 10µM + 1 µM succinic acid
                        Blank


                                ТNF




                                                                                  acid
                                          ________________                                        ________________
                                           +      +   +  +                                         +      +  +    +
                                                                                                  ______
                                                                                                  _____________________________
 cIAP10                                                                                           _______


Hours                                      2       6   12                20           30              2        6   12   20
                                                                                      min

         Figure 3 - Analyze of Cr- induced expression the gen cIAP10 was studied by
         method RT-PCR. The cells was treated by Chrome oxide (10 µМ ) for different
         intervals. To determine the effect on the expression of succinic acid, the cells were
         treated with 1 μМ succinic acid for 30 min.
                                                            1 µМ succinic acid




                                                                                 Na2HAsO3 10 µМ+ 1µM
                Blank

                          ТNF




                                      Na2HAsO3 10 µМ                             succinic acid
                                      +    +   +       +                         +                +       +    +


 cIAP10


                                  2        6   12 20 30min 2                                 6        12      20
 Hours

         Figure 4- PCR analysis of Cr-induced cIAP10 gen expression in mouse
         fibroblast pBA-TRAF2-WT cells and its prevention by succinic acid.

              NF kB activation has been linked to the carcinogenesis process because of its
         role in differentiation, inflammation, and cell growth. Carcinogens and tumour
         promoters involving toxic metals, UV radiation, phorbol esters are among the
         external stimuli that activate NF kB. In tumor cells, NF-κB is active either due to
         mutations in genes encoding the NF-κB transcription factors themselves or in
         genes that control NF-κB activity (such as IκB genes); in addition, some tumor
         cells secrete factors that cause NF-κB to become active.


                                                            10
     It has been reported that the high dose chrome and arsenic exposure
stimulated the signaling pathway associated with apoptosis; whereas the low dose
of these metals exposure activated the cell proliferation pathway ad might be
carcinogenic. The expression of cIAP 10 observed in this study may correspond to
the carcinogenesis of low doses of Cr and As.
     In this study we performed the RT-PCR analyses for the As and Cr exposed
cells that were loaded with succinic acid beforehand to examine the effects of
antioxidant molecule on metal exposure. The metal exposures inhibited the cIAP10
gen in succinic acid – loaded cells.
     Blocking NF-κB can cause tumor cells to stop proliferating, to die, or to
become more sensitive to the action of anti-tumor agents. Thus, NF-κB is the
subject of much active research among pharmaceutical drugs as a target for anti-
cancer therapy. Because NF-κB controls many genes involved in inflammation, it
is not surprising that NF-κB is found to be chronically active in many
inflammatory diseases, such as inflammatory bowel disease, arthritis, sepsis,
asthma, among others (Grilli M, Pizzi M, Memo M, Spano P. Science, 1996).
     The importance of metals and other reactive oxygen species on NF-kB
activation is further supported by studies demonstrating that activation of NF –kB
by nearly all stimuli can be blocked by antioxidants, including N-acetylcysteine
(NAC), thiols, green tea, polyphenols and vitamine E [14]. Among the wide
spectrum of antioxidant facilities such as tocopherol, carotin, vitamin C, selenium
and other- succinic acid – natural metabolite of cycle Krebs acids proved well.
Studying of protective action of succinic acid can be used for preventive treatment
against different disease, even cancer.
     In our work we suppose that protective action of succinic acid shows its
capability to regulate the factors which can influence on the gens expression and
other cell functions. The possible mechanism of protective action of succinic acid
is connected with its antioxidant feature, defining the catch of free radicals. The
expression levels of cIAP10 at each metal were shown in Fig 1,2. the preliminary
treatment of the cells with succinic acid in case of toxic metals had to reducing
gens’ expression cIAP 10 (which are activated by nucleus factor NF kB),
inhibiting NF kB signal ways.
     Defining of cytotoxicity of heavy metal salts on fibroblast cell culture.
Cytotoxicity level of chromium oxide and natrium hydroarsenate, cadmium
chloride and nickel chloride was defined using method of cell coloring with Gyms
solution.
     After natrium hydroarsenate and chromium oxide treatment with concentration
of 7.5 µM increase of dead cells was observed; concentration of 10 µM was toxic
enough for cells. Number of cells survived of WT line under influence of chromium
oxide concentration of 7,5 µM for 24th day decreased from 100,3±0,14% to
74,0±0,21%, and with use of succinic acid number of cells survived increased from
74,0±0,21% to 85,1±0,94 %. With concentration of 10 µM chromium oxide for cells
of WT line number of cells survived for the 24th day decreased from 100,3±0,14%
to 52,3±0,77 %, and with use of succinic acid number of cells survived increased
from 52,3±0,77 % to 70,0±0,70 % (p<0,001). Number of cells survived of WT line
                                       11
under the influence of small concentration of natrium hydroarsenate for the 24th day
decreased from       100,3 ±0,12 to 64,5±0,21%, and with use of succinic acid
increased from 64,5±0,21% to 72,1±0,04 % (p<0,001).
     Under the treatment WT cells with natrium hydroarsenate (10 µM) the number
of cells survived for the 24 th day decreased from 100,3 ±0,12 to 55,1±0,07 %, and
with use of succinic acid increased from 55,1±0,07 % to 67,0±0,06 % (p<0,001).


        120
        100
         80
         60
         40
         20
          0
               24 hours    48 hours     72 hours        24 hours         48 hours        72 hours
                Cr2O3       Cr2O3        Cr2O3        Cr2O3+ 1µM       Cr2O3+ 1µM      Cr2O3+ 1µM
                                                      succinic acid    succinic acid   succinic acid
                                                   hours


                 Blank                  Cr2O3 7.5 µM WT               Cr2O3 10 µM WT
                 Cr2O3 7.5 µM S11\55A   Cr2O3 10 µM S11\55A


     Figure 5 - Influence of chromium oxide in the 7.5 µM and 10µM
concentration on the рBA-TRAF2- WT/ S11A cells, and against of succinic acid 1
µM.

      Number of cells survived of S11A line under influence of with natrium
hydroarsenate concentration of 7,5 µM for 24th day decreased from 100,3 ±0,12 % to
23,2±0,70 %, and with use of succinic acid increased from 23,2±0,70 % to
41,8±0,20 % (p<0,001). Under the treatment S11A cells with chromium oxide (10
µM) the number of cells survived for the 24 th day decreased from 100,3 ±0,12 to
55,1±0,07 %, and with use of succinic acid increased from 55,1±0,07 % to
67,0±0,06 % (p<0,001).
      Number of cells survived of S11A line under influence of chromium oxide
concentration of 7,5 µM for 24th day decreased from 100,3 ±0,12 to 40,0±1,05 %,
and with use of succinic acid increased from 40,0±1,05 %, to 59,8±0,06%
(p<0,001). Under the treatment S11A cells with chromium oxide (10 µM) the
number of cells survived for the 24 th day decreased from 100,3 ±0,12 to
55,1±0,07 %, and with use of succinic acid increased from 55,1±0,07 % to
67,0±0,06 % (p<0,001). The treatment of chromium oxide with concentration 10
µM for S11\A cells the number of cells survived for the 24 th day decreased from

                                        12
100,3±0,14% to 24,8±0,24 %, and with use of succinic acid increased from
24,8±0,24 % to 37,7±0,39 % (p<0,001).




                                     120


                                     100
       % of viability of the cells




                                     80


                                     60


                                     40


                                     20


                                      0
                                            24 hours    48 hours      72 hours     24 hours     48 hours     72 hours
                                           Na2HAsO4    Na2HAsO4      Na2HAsO4    Na2HAsO4+ Na2HAsO4+ Na2HAsO4+
                                                                                 1µM succinic 1µM succinic 1µM succinic
                                                                                     acid         acid         acid

                                             Blank                    Na2HAsO4 7.5 µM WT       Na2HAsO4 10 µM WT
                                             Na2HAsO4 7.5 µM S11\A    Na2HAsO4 10 µM S11\A



       Figure 6- Influence of natrium hydro arsenates in the 7.5 µM and 10µM
concentration on the рBA-TRAF2- WT/ S11A cells, and against of succinic acid 1
µM.
     Number of cells survived of WT line under influence of cadmium chloride
concentration of 7,5 µM for 24th day decreased from 99,7 ±0,08% to 63,1±0,27 %,
and with use of succinic acid number of cells survived increased from 63,1±0,27 %
to 70,3±0,08%. Under the treatment WT cells with cadmium chloride (10 µM) the
number of cells survived for the 24 th day decreased from 99,7 ±0,08% to
50,1±0,09 % and with use of succinic acid increased from 50,1±0,09 % to
64,0±0,06 % (p<0,001).
     For the S11/A cells line under influence of cadmium chloride concentration of
7,5 µM for 24th day decreased from 99,7 ±0,08% to 63,2±0,16 %, and with use of
succinic acid increased from 63,2±0,16 % to 72,8±0,27% (p<0,001). Number of
S11A cells survived under the treatment with cadmium chloride (10 µM) number of
cells survived for the 24 th day decreased from 99,7 ±0,08 % to 45,8±0,20%, and
with use of succinic acid increased from 45,8±0,20% to 54,8±0,19 % (p<0,001).
     Number of cells survived of WT line under influence of nickel chloride (250
µM) for 24th day decreased from 99,7 ±0,08% to 89,5±0,28%, and with succinic
acid acquired control meaning 99,3±0,08 (p<0,001). Number of WT survived
                                                                     13
cells treated with nickel chloride (500 µM) for 24th day decreased from 99,7
±0,08% to 84,1±0,37 %, and with use of succinic acid increased from 84,1±0,37 %
to 92,0±0,06 % (p<0,001).
     For the S11/A cells treated with nickel chloride concentration (250 µM) the
number of survived cells for 24th day decreased from 99,7 ±0,08 % to 73,2±0,76%,
and with use succinic acid increased from 73,2±0,76% to 87,8±0,27 % (p<0,001).
Under treated with 500 µM concentration nickel chloride the number of S11/A
survived cells for 24 th day decreased from 99,7 ±0,08% to 65,8±0,26%, and with
use succinic acid increased from 65,8±0,26% to 74,8±0,19 % (p<0,001) .

       Conclusion
       Thus level of red-cells hemolytic resistance was decreasing due to failure of
physiological condition of membrane lipids under strong heavy metal intoxication
(chrome, arsenic, cadmium, nickel).
       It was discovered that animals affected by strong heavy metal intoxication had
more cells micronuclei. This points to development of toxic effect on the level of
blast blood cells resulting in cytogenetic failures. It defines their mutagenic
influence caused by damage of structure of nucleinic acid molecules. In peripheral
blood during toxic level comparative aspect metals were observed in the following
sequence: arsenic, chrome, cadmium, nickel. It was noticed that under the influence
of succinic acid, which is an antioxidant agent, toxic effect of heavy metal salts on
red cells was decreasing.
     Protective activity of succinic acid demonstrates its ability to regulate factors
influencing genes expression and other functions of cells. Possible mechanism of
protective activity of succinic acid is connected with its antioxidant features which
define capturing of free radical. Preliminary treatment of cells with succinic acid
under the influence of toxic metals resulted in inhibition of NF-kB signal ways and
decreased expression of genes cIAP10 (activated by NF-kB nuclear factor).
       Succinic acid has antioxidant and cytoprotective features. Transformation of
succinate in a body concerns producing of energy required for life support.
     Succinic acid inhibits erythrocyte hemolyses, micronuclei building,
cytotoxicity of fibroblast, and activation of gene with cIAP 10 that testifies the
protective activity of succinic acid.
     At the present time we study succinic acid as a possible inductor of the
protection mechanism in the presence of oxidation stress and for regulation of a
number of vital processes in the cells. Thus further investigation in this field will
enable us to explore mechanisms of succinic acid activity in DNA repairing and its
role in antioxidation systems.

     Conclusions:
   1. Under sever intoxication by heavy metals the red- cell membranes of the
laboratory rats were destroyed, where succinic acid weakened metal influence on
red-cell membrane. Intensity of erythrocyte hemolyses resulted from acute
intoxication with natrium hydroarsenates was 271%, chromium oxide 237%,
cadmium chloride 258 %, nickel chloride 216%. Under combined influence of
                                         14
metal and succinic acid erythrocyte hemolyses was registred as follows: with
natrium hydroarsenates was 179 %, chromium oxide - 195%, cadmium chloride-
151%, nickel chloride -184%.
    2. Number of red- cells with micronuclei after acute intoxication with natrium
hydroarsenates was 716 % from the check value, chromium oxide - 694%,
cadmium chloride- 558%, nickel chloride -541%. Under combined influence
succinic acid and metal salts number of red-cells with micronuclei was registered
as follows: with natrium hydroarsenates- 436 %, chromium oxide - 412%,
cadmium chloride- 389%, nickel chloride - 345%.
    3. Number of WT line cells survived, affected by metals was decreasing
depending on concentration and period of influence. Succinic acid was increasing
the number of cells survived in the course of every observation. Thus on the 24 th
day of affecting cells with metals under succinic acid influence the number of cells
was registered correspondingly: with natrium hydroarsenates- 63 % and 72%,
chromium oxide – 74% and 85%, cadmium chloride- 63% and 70%, nickel
chloride - 89% and 99%.
    4. Number of S11A line cells affected with metals decreased more then WT
line cells. Thus on the 24 th day of affecting cells with metals, their number was
registered correspondingly: natrium hydroarsenates- 22 % and 41%, chromium
oxide – 40% and 59%, cadmium chloride- 63% and 72%, nickel chloride - 74%
and 87%.Under the influence of succinic acid number of cells was growing up.
    5. Under the influence of chromium oxide and natrium hydroarsenites the
activation of gene with cIAP 10 with more than 50% was registered, it indicates
carcinogenic activity of these heavy metals salts. Natrium hydroarsenites was
noticed to have more of this activity. Expression of gene with cIAP 10 has been
significantly inhibited and showed approaching the check value under the
preliminary stimulation of the cells with the succinic acid.

    A list of publications on the dissertation

        1. Altayeva A.S. Natural antioxidants as one of the main mechanisms of
           correction of destructive processes in organs and cells of animals
           exposed to toxic agents. Journal of L.N Gumilyov Eurasian National
           University, № 6, 67, 2008 pp. 90-95
        2. Altayeva A.S Cytogenetic effects of toxic agents and the protective
           role of antioxidants. Journal of Hygiene, Epidemiology and
           Immunology № 2, Almaty, 2009, pp.129-134
        3. Altayeva A.S The survival rate of fibroblast cells of mice under the
           influence of chromium oxide on the background of succinic acid.
           Moscow International Scientific Conference Biotechnology: Ecology of
           large cities on March 15-17, 2010 pp. 456.
        4. Altayeva A.S, Khanturin M.R. Differential cytotoxic effects of heavy
           metals on the NF kB signal pathway and effect of antioxidant.
           Messenger of KazNU. Al - Farabi № 3 (42), 2009, pp 97-101.

                                        15
5. Altayeva A.S Activating with natrium hidroarsenate and chromium
   oxide transcription factor NF kB and the possibility of inhibition of
   succinic acid in a cell culture of fibroblasts. Bulletin of the Kazakh
   Agrarian University № 1, (56) 2010, Astana, pp. 374-380
6. Altayeva A.S, Khanturin M.R .The protective effect of succinic acid in
   case of poisoning the organism with salts of heavy metals. Russian
   Academy of Natural Sciences № 6, Internet publication, www.rae.ru
7. Altayeva A.S . Protective role of succinic acid in free radical processes.
   Biotechnology and Biomedical Engineering. Proceedings of the III All-
   Russian scientific-practical conference with international participation,
   dedicated to the 75 th anniversary of the Kursk State Medical
   University. - Kursk, 2010 pp. 166-167.
8. Altayeva A.S, Khanturin M.R. The protective role of succinic acid on
   the NF kB signaling pathway of mouse fibroblast cells under the
   influence of chromium oxide and sodium arsenite. David Publishing.
   Journal of Environmental Science and Engineering №6, Vol 4, June
   2010, USA




                                 16
                       Алтаева Әлия Сағымбековна

     Ауыр метал тұздарының генетикалық аппаратқа токсикалық әсері

                         03.00.16- экология
 Доктор Ph.D академикалық дәрежесіне ізденуге арналған диссертацияның
                           авторефераты

                                Түйіндеме

      Зерттеу нысаны. Мышьяк, хром, кадмий, никель тұздарының әрекеті
кезінде эритроциттердің мембранасы резистенттілігінің төмендеуі байқалды.
Ауыр металдардың тұздарымен улану кезінде пайда болған микроядролар
санының артқаны белгіленді. Металл тұздары мен кәріптас қышқылының
ортақ әрекеттесуі кезінде микроядролар санының азайғандығы анықталды.
Ауыр металдар тұзының әрекеті кезінде cIAP 10 генінің белсенділігі және
cIAP 10 генін кәріптас қышқылының әсерімен тежеу мүмкіндігі көрсетілді.
Ауыр металдар тұзының тышқан талшықтарының жасушыларын улауы және
кәріптас қышқылының қорғаушы әрекеті анықталды.
      Жұмыстың мақсаты. Жұмыстың мақсаты болып зертхана жануарлары
жасушаларының металды индукциялық зақымдауының даму механизмін
белгілеу және кәріптас қышқылының өнімнің қорғаушы әрекетінің
тиімділігін анықтау болып табылады.
       Зерттеу әдістері. Зерттеулер 60 ақ тұқымсыз еркек егеуқұйрықтартарға
жасалды. Жануарлар металдардың (хром оксиді, натрий гидроарсенаты,
кадмий хлориді және никель хлориді) ЛД 50 тұздарының per os үлкен
мөлшерімен уланған және ауыр метал тұздарымен бірге қосымша кәріптас
қышқылының per os 20 мг/кг реңінде уланған топтардан құралды..
Тәжірибеде рBA –TRAF2 WT және S11A жасуша дақылдары қолданылды.
Жасушалар 10% эмбриондық бұқа ерітіндісі, 5% CO2 атмосферасында 37° С
температурасы кезінде 1% пеницилин мен стрептомицин бар Dulbeco
Modified Eagle’s Medium ортасында өсірілді. Канцерогенді металл ретінде Cr2
O3 хром оксидін, Na2НAsO4 натрия гидороарсенаті, никел хлоридін NiCl2,
кадмий      хлоридін     CdCl2    пайданылды.     Тышқандардың      жасуша
талшықтарының рBA –TRAF 2 WT және S11A гендер дәлдігін кері
транскрипциясымен полимеразды тізбекті реакция тәсілі арқылы анықталды.
2,4,8,20 сағат инкубациядан кейін өңделген жасушалардан жиынтық
жасушалы РНҚ өндірушінің нұсқауына сәйкес бөлініп алынды және ДНҚ-ға
синтез жүргізілді.
       Зерттеу нәтижелері.
1. Ауыр металдармен қатты улану кезінде зертхана егеуқұйрықтарының
эритроциттері мембранысының бұзылуы байқалды, кәріптас қышқылы
металдардың эритроциттер мембранасына әсерін әлсіретті. Гемолиздің
қарқындылығы натрий гидроарсентаымен улуну кезінде 271%- ды, хром
окисидімен 237%- ды, кадмий хлоридімен 258 %- ды никель хлоридімен
                                    17
216%- ды құрады. Металл мен кәріптас қышқылының ортақ әрекеті кезінде
гемолиз натрий гидроарсенатымен 179 %- ды, хром окисидімен 195%- ды,
кадмий хлоридімен 151 %- ды, никель хлоридімен 184%- ды құрады.
2. Металдармен қатты улану кезінде эритроциттердің микроядролармен
саны натрий гидроарсенатымен улуну кезінде қорытынды шаманың 716 %-
ын, хром окисидімен 694%- ды, кадмий хлоридімен 558 %- ды, никель
хлоридімен 541%- ды құрады. Кәріптас қышқылының ортақ әрекеті кезінде
микроядролар санықорытынды шамадан натрий гидроарсенатымен 436 %-
ды, хром окисидімен 412%- ды, кадмий хлоридімен 389 %- ды, никель
хлоридімен 345 %- ды құрады.
3. Тірі қалған WT жасушаларының саны металдардың әрекеті кезінде
әсердің ұзақтылығы мен қоюлығына байланысты азая берді, кәріптас
қышқылы барлық бақылауларда тірі қалған жасушалардың санын артып
отырды, осылайша 24- тәулікке қарай металдармен улау кезінде және
кәріптас қышқылының әсерімен жасушалар саны сәйкесінше: натрий
гидроарсенатымен 63 % мен 72%, хром окисидімен 74% мен 85%-ды, кадмий
хлоридімен 63 % мен 70% - ды, никель хлоридімен 89 % мен 99 %-ды
құрады. Тірі қалған S11A жасушаларының саны WT жасушаларына
қарағанда металдардың әрекеті кезінде үлкен мөлшермен азая бастады,
осылайша 24- тәулікке қарай металдармен улау кезінде жасушалар саны
сәйкесінше: натрий гидроарсенатымен 22 % мен 41%, хром окисидімен 40%
мен 62%-ды, кадмий хлоридімен 63 % мен 72% - ды, никель хлоридімен 74 %
мен 87 %-ды құрады,         кәріптас қышқылының әсерімен тірі қалған
жасушалардың саны арта түсті.
4. Хром оксиді мен натрий гидроарсенатының әсері кезінде cIAP 10
генінің белсенділігі 50%- дан артты, бұл ауыр металдардың көрсетілген
тұздарының канцерогенді әрекетін көрсетеді. Ең күшті канцерогенді әрекет
натрий гидроарсенатының әрекетінде байқалды. Жасушаларды кәріптас
қышқылымен алдын ала ынталандыру кезінде cIAP 10 гені тежелді және
қорытынды мәнге жақындай түсті.
5. Кәріптас қышқылы эритроциттер гемолизін, микроядролардың пайда
болуын, талшықтардың цитоулағыштығын, cIAP 10 генінің белсенділігін
тежейді, бұл кәріптас қышқылының қорғаушы әрекетін көрсетеді. Осы
жұмысымызда кәріптас қышқылын тотықтырғыш стресі кезіндегі қорғаныш
механизмдерінің мүмкін индикаторы ретінде, сондай-ақ жасушалардағы
көптеген тіршілік процестерін реттеу үшін қарастырдық.
    Сондықтан бұл бағытта әрі қарай зерртеудің ДНҚ үдестерінде және
антиоксиданттық жүйелер рөліндегі кәріптас қышқылы әрекетінің
механизмін ашуға мүмкіндік береді.
    Практикалық маңызы. Алынған деректер биохимия, молекулярлық
биология және онкология ғылымдарына үлес қосады, себебі ауыр
металдардың әсерімен NF kB дабыл жолдарын тежеуін және оны
антиоксидант дәрмектермен әлсіретуін көрсетеді. Жұмыстың қолданбалы
сипаты жасушаларға ауыр металдар ионының әсері кезінде кәріптас
қышқылын жасушалардың қорғаушысы ретінде қолдану болып отыр.
                                   18
                      Алтаева Алия Сагымбековна
  Токсическое действие солей тяжелых металлов на генетический аппарат
                           03.00.16 – экология
                              Автореферат
диссертации на соискание академической степени доктора философии (PhD)

    В работе изучено сравнительное действие тяжелых металлов: оксида
хрома, гидроарсената натрия, хлорида кадмия и хлорида никеля на
резистентность эритроцитов и на интенсивность образования микроядер в
эритроцитах. Изучено, цитотоксическое действие солей тяжелых металлов на
клетки фибробластов мышей линии pBA-TRAF-WT, pBA- TRAF2- S11A.
Показано действие оксида хрома и гидроарсената натрия на экспрессию гена
cIAP 10. Обнаружено, что янтарная кислота ингибирует экспрессию гена cIAP
10 фибробластов при отравлении солями тяжелых металлов.
    Цель работы Установить механизмы развития метал- индуцированного
повреждения клеток лабораторных животных и выявление эффективности
защитного действия янтарной кислоты.
     При острой интоксикации тяжелыми металлами (хром, мышьяк, кадмий,
никель) снижался уровень гемолитической резистентности эритроцитов за счет
нарушения физиологического состояния липидов мембраны.
     Было выявлено, что при острой интоксикации животных тяжелыми
металлами увеличилось количество клеток с микроядрами. Что указывает на
развитие токсического эффекта на уровне бластных кроветворных клеток,
следствием чего является развитие цитогенетических нарушений. Это
характеризует их мутагенное действие, вызванное нарушением структуры
молекул нуклеиновых кислот. В периферической крови в сравнительном
аспекте по степени токсичности металлы были в такой последовательности:
мышьяк, хром, кадмий, никель. При действии антиоксидантного препарата –
янтарной кислоты сокращался токсический эффект солей тяжелых металлов на
эритроциты.
     Протекторное действие янтарной кислоты показывает ее способность
регулировать факторы, которые могут воздействовать на экспрессию генов и
другие клеточные функции. Возможный механизм протекторного действия
янтарной кислоты        связан с его противооксидантным свойством,
определяющим улавливание свободных радикалов. Предварительная
обработка клеток янтарной кислотой под действием токсических металлов
приводила к снижению экспрессии генов cIAP10 (которые активизируются
ядерным фактором NF-kB), ингибируя NF-kB сигнальные пути.
    Янтарная кислота обладает антиоксидантными и цитопротекторными
свойствами. Превращение сукцината в организме связано с продукцией
энергии, необходимой для обеспечения жизнедеятельности.
    Таким образом, предположительно, что защитное свойство янтарной
кислоты для клеток осуществляется за счет окисления янтарной кислоты,

                                   19
которая активизирует мощную систему энергопродукции сукцината в клетке и
за счет ингибирования янтарной кислотой процессов перекисного окисления.
     По результатам исследования получены новые научные данные о влиянии
токсических агентов (солей хрома, мышьяка, никеля, кадмия) на процессы
активации транскрипционного фактора NF kB.
      Результаты работы показали, что при токсическом действии солей
тяжелых металлов      янтарная кислота ингибирует процессы активации
ядерного фактора и существенно защищает клетки от метал- индуцированного
повреждения.
      Полученные данные вносят вклад в биохимию, молекулярную биологию,
онкологию, так как показывают изменения в геноме под влиянием тяжелых
металлов. Прикладной характер работы заключается в применении янтарной
кислоты в качестве протектора клеток при действии на них ионов тяжелых
металлов.




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