Advancing excellence in laboratory medicine for better healthcare worldwide

Monitoring influence of surgical stress on formation of hydroxyl radicals in tumor bearing rats



Suzana Borovic [1], Andreas Meinitzer [2], Iva Loncaric [3], Senka Sabolovic [4], Renate Wildburger [5], Manfred Tillian [6], Pero Martinac [4], Igor Stipancic [4] and Neven Zarkovic [1]

  1. Department of Molecular Medicine, "Ruder Bo�kovic" Institute, Zagreb, Croatia
  2. Department of Laboratory Medicine, Blocklabor 1, University of Graz, Graz, Austria
  3. Department of Neuropathology, Clinical Hospital Center "Rebro", Zagreb, Croatia
  4. Department of Surgery, Clinical Hospital "Dubrava", Zagreb, Croatia
  5. University Clinic of Traumatology, University of Graz, Graz, Austria
  6. Institute of Biochemistry, Karl-Franzens University of Graz, Graz, Austria

Keywords: oxidative stress, hydroxyl radical, salicylic acid, surgery, tumor, rat

Correspondence to:
Mr. Sc. Suzana Borovic
Department of Molecular Medicine, "Ruder Bo�kovic" Institute
Bijenicka 54
10000 Zagreb, Croatia.
Phone: ++385/14561130; Fax: ++385/14680094

Download as a PDF here


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 animals.

Materials and methods

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 of reperfusion.

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 animals.


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.


1. Braughler JM, Hall ED. Central nervous system trauma and stroke: I. Biochemical considerations for oxygen radical formation and lipid peroxidation. Free Rad Biol Med 1989; 6:289-301.
2. Hall ED, Braughler JM. Central nervous system trauma and stroke: II. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid peroxidation. Free Rad Biol Med 1989; 6:303-313.
3. Halliwell B. Antioxidants in human health and disease. Ann Rev Nutrition 1996; 16:33-50.
4. McCabe DR, Maher TJ, Acworth IN. Improved method for the estimation of hydroxyl free radical levels in vivo based on liquid chromatography with electrochemical detection. J Chrom Biochem Applic 1997; 691:23-32.
5. Choudray C, Talla M, Martin S, Fat�me M, Favier A. High-performance liquid chromatography - electrochemical determination of salicylate hydroxylation products as an in vivo marker of oxidative stress. Anal Biochem 1995; 227:101-111.
6. Liu L, Leech JA, Urch RB, Silverman FS. In vivo salicylate hydroxylation - a potential biomarker for assessing acute ozone exposure and effects in humans. American J Resp Crit Care Med 1997; 156:1405-1412.
7. Ingelman-Sundberg M, Kaur H, Terelius Y, Persson JO, Halliwell B. Hydroxylation of salicylate by microsomal fractions and cytochrome P-450: lack of production of 2,3-dihydroxybenzoate unless hydroxyl radical formation is permitted. Biochem J 1991; 276:753-757.
8. Toyokuni S, Okamoto K, Yodoi J, Hiai H. Persistent oxidative stress in cancer. FEBS Lett 1995; 358:1-3.
9. Kundu N, Zhang SZ, Fulton AM. Sublethal oxidative stress inhibits tumor cell adhesion and enhances experimental metastasis of murine mammary carcinoma. Clin Exper Metastasis 1995; 13:16-22.
10. Dreher D, Junod AF. Role of oxygen free radicals in cancer development. Eur J Cancer 1996; 32A:30-38.
11. Page GG, Beneliyahu S. Increased surgery-induced metastasis and suppressed natural killer cell activity during proestrus/estrus in rats. Breast Canc Res Treatment 1997; 45:159-167.
12. Freiregarabal M, Nuneziglesias MJ, Balboa JL, et al. Inhibitory effects of alprazolam on the enhancement of lung metastases induced by operative stress in rats. Int J Oncol 1993; 3:513-517.
13. Yoshikawa T, Kokura S, Oyamada H, et al. Antitumour effect of ischemia-reperfusion injury induced by tranzient embolization. Cancer Res 1994; 54:5033-5035.
14. Jessup JM, Battle P, Waller H, et al. Reactive nitrogen and oxygen radicals formed during hepatic ischemia-reperfusion kill weakly metastatic colorectal cancer cells. Cancer Res 1999; 59:1825-1829.
15. Lin SL, Shi DY, Pan JH, Yao SK. Observations on the compensatory effects of superoxide dismutase under hypoxic or ischemic stress in rats and rabbits. Med Sci Res 1998; 26:741-743.

Copyright © 2001 International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). All rights reserved.

Website developed by Insoft Digital