Borovic , Andreas Meinitzer , Iva Loncaric , Senka
Sabolovic , Renate Wildburger , Manfred Tillian , Pero
Martinac , Igor Stipancic  and Neven Zarkovic
- Department of Molecular Medicine, "Ruder Bo�kovic" Institute,
- Department of Laboratory Medicine, Blocklabor 1, University of
Graz, Graz, Austria
- Department of Neuropathology, Clinical Hospital Center "Rebro",
- Department of Surgery, Clinical Hospital "Dubrava", Zagreb,
- University Clinic of Traumatology, University of Graz, Graz,
- Institute of Biochemistry, Karl-Franzens University of Graz,
Keywords: oxidative stress, hydroxyl radical, salicylic acid,
surgery, tumor, rat
Mr. Sc. Suzana Borovic
Department of Molecular Medicine, "Ruder Bo�kovic" Institute
10000 Zagreb, Croatia.
Phone: ++385/14561130; Fax: ++385/14680094
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Hydroxyl radical (HO�) is one of the most harmful reactive
oxygen species (ROS), which are formed in excess during oxidative
stress (shock, sepsis, trauma, surgery, hypoxia,
ischemia-reperfusion, etc.) (1,2,3). For measurement of HO�
indirect methods are used determining aromatic acids hydroxylation
products. The most used is salicylic acid whose hydroxylation
products 2,3-dihydroxybenzoic acid (2,3-DHBA) and
2,5-dihydroxybenzoic acid (2,5-DHBA) can be separated and
quantified by HPLC method with electrochemical detection (4,5,6).
As 2,5-DHBA can be produced also by enzymatic pathway, only
2,3-DHBA serves as a measure for HO� radical production (7).
The purpose of this work was to investigate HO� formation in
surgically treated tumor bearing rats and to see whether difference
in HO� production can be seen between healthy and tumor bearing
Walker carcinoma tumor cells (107 live cells) were injected i.m.
in the hind limb of male Wistar rats. Experiment was performed 6
days later when tumor was visible. Rats were divided in three
groups: 1) anaesthetised only; 2) anaesthetised and operated
(laparatomy); 3) anaesthetised and operated by laparatomy followed
by ischemia-reperfusion (I/R) of tumor tissue caused by clamping
ipsilateral iliac artery. The same treatment was done on healthy
rats without tumor. Rats were treated per os with acetylsalicylic
acid (ASA), 20 mg/kg body weight given through probe, anaesthetised
with chloralhydrate (300 mg/kg) and operated (laparatomy). Plasma
samples were collected after 60 minutes of ischemia and 30 minutes
For DHBAs determination samples were extracted with hydrochloric
acid and diethylether with 3,4-DHBA as internal standard.
Separation was performed on 150 x 4,6 Waters Spherisorb ODS2 3 �m
column using 7,48 mM sodium citrate/acetic acid mobile phase pH 4,6
with 3 % of methanol. Detection was done with electrochemical
detector (ESA Detector Coulochem 2, with 5040 analytical cell
model) set on 400 mV and flow on 0,6 ml/min. Chromatogram showing
separation of DHBAs was presented in Figure 1. Samples for SA determination were
extracted with ethanol with 2,6-DHBA as internal standard.
Separation was done on the same column with UV detector set on 296
nm and flow of 1 ml/min. Mobile phase consisted of 7,48 mM sodium
citrate/acetic acid pH 5,4 with 15 % of methanol. All analyses were
done with Merck-Hitachi L-7100 HPLC system. Chromatogram showing
separation of salicylic acid was presented in Figure 2.
Results were calculated as a percentage of salicylic acid from
plasma and compared according to the Mann-Whitney test. Values with
p<0,05 were considered as significant.
The results obtained are summarised on Figure 3. Only minimal amount, i.e.
approximately 0,01 - 0,05 % of SA was hydroxylated to 2,3-DHBA
while 0,7 - 1,4 % was metabolised to 2,5-DHBA. In normal, healthy
rats increase in 2,3-DHBA production was measured when they were
surgically treated by laparatomy only (p<0,05) or by laparatomy
followed by I/R (p<0,05). Such response was not noticed in tumor
bearing rats where increase in 2,3-DHBA production was not observed
neither in animals exposed to laparatomy only nor if laparatomy was
followed by the tumor I/R. The difference observed between operated
controls and tumor bearing rats was significant (p<0,05). For
2,5-DHBA production, there was no difference observed between
operated and non-operated rats for both control and tumor bearing
It is supposed that tumor cells are under persistent oxidative
stress which seems to be beneficial to them, increasing metastatic
potential and genetic instability, thus helping tumor cells to
survive and progress (8,9,10). It is often assumed that mild
oxidative stress caused by surgery can intensify metastases
formation (11,12), although severe oxidative stress is not
beneficial to the tumor cells and may even cause their destruction.
Thus, additional ROS production in the I/R injury might be
cytotoxic and cause cellular destruction (13,14). With our work we
have not been able to see the increase in HO� production in tumor
bearing animals caused by laparatomy, which was seen in healthy
surgically treated rats. Thus, it appears that systemic stress
response caused by laparatomy was different in healthy and tumor
bearing animals. Difference in scavenger's levels and composition,
and ROS production between normal and tumor bearing rats cannot be
excluded, while final appearances of these interactions can
resemble perhaps even steady state as in unstressed animals. Since
initial values of HO� measured by 2,3-DHBA production were the
same, we suppose that compensatory mechanisms (15), such as
increased scavenger activity in tumor bearing organisms can be the
cause of such response.
Finally, due to the difficulties with per os application of ASA and
consequently relatively ununiform 2,3-DHBA values present in the
same group of animals, further evaluation of this model and a new
approach with intravenous application seems reasonable. These
experiments are already in progress.
The authors would like to thank IFCC for providing Professional
Scientific Exchange Scholarship to Ms. Suzana Borovic that made
this work possible. Many sincere thanks to Univ. Prof. Dr. Gerhard
Lanzer for generous hospitality in Department of Laboratory
Medicine, Landeskrankenhaus, Graz, in which was done analytical
part of this work. The support of the Croatian Ministry of Science
and Technology is kindly acknowledged.
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