179
Cancer Letters, 50 (1990) 179- 181 Elsevier Scientific Publishers Ireland Ltd.
Xanthine
oxidase levels in human
brain tumors
E. KijkogW, A. BeIce”, E. &yurtb and Z. Tepeler” “Department
ofBiochemistry and
Weurosurgery,
Cerrahpasa Medical Faculty, Istanbul (Turkey)
25 May 1989) (Revision received 23 November 1989) (Accepted 18 December 1989)
(Received
Summary Xanthine oxidase is most recognized for its role as the rate-limiting enzyme in nucleic acid degradation through which all purines are channelled for terminal oxidation. The enzyme serves as a source of oxygen-derived free radicals which induce both cellular injury and edema as well as changes in vascular permeability. In the study we compared xanthine oxidase levels of human brain tumors with normal brain tissues. Statistical eualuation of our results shows significantly higher xanthine oxi-
dase levels in tumoral
brain tissues. However,
xanthine oxidase has not any significance for the differentiation of tumor types among each
others. The oncotypes gioma and astrocytoma.
Keywords: cancer.
xanthine
studied
were
menin-
oxidase;
brain
tissue;
Introduction Xanthine oxidase is a highly versatile enzyme that is widely distributed among species (from bacteria to man) and within the various tissues of mammals. The enzyme participates in the oxidation of a wide variety of endogenous (purines) and exogenous (ethanol) substrates. However, xanthine oxidase is most recognized for its role as the rate0304-3835/90/$03.50 Published and Printed
0 1990 Elsevier Scientific Publishers in Ireland
limiting enzyme in nucleic acid degradation through which all purines are channelled for terminal oxidation. Although the main physiological function of xanthine oxidase remains unclear, there is growing interest in the ability of this enzyme to serve as a source of oxidizing agents such as hydrogen peroxide and superoxide radical. Indeed, xanthine oxidase has the distinction of being the best documented biologic source of oxygen-derived free radicals [lo]. Interest in the enzyme as a source of oxidizing agents has increased markedly since it has been implicated in the pathogenesis of ischemia-reperfusion injury to tissues such as the intestine, kidney and heart [5,6,8]. There is growing evidence that oxygen readicals generated by xanthine oxidase, are primarily responsible for the cellular deterioration associated with reoxygenation of hypoxic tissues [lo]. Superoxide anion (0;)) hydrogen peroxide (H202) and hydroxyl radicals (.OH) are produced in biological systems by the singleelectron reduction of oxygen. These highly reactive oxygen-derived free radicals may play a role in cerebral ischemia and trauma [4,111. Enzymatically generated oxygen-derived free radicals, when infused intracerebrally into rats, induce both cellular injury and edema as well as changes in vascular permeability [ 11. Although the elucidation of the physiological role of xanthine oxidase has gained much Ireland Ltd
180
interest, there is a lack of knowledge on the behaviour of this enzyme at pathological conditions. Since xanthine oxidase is responsible for production of oxyen-derived free radicals which cause tissue damage, we wanted to study the levels of this enzyme in various types of tumoral brain tissues. The oncotypes studied were meningioma and astrocytoma. Materials and methods
Tumoral tissue samples were obtained from the Department of Neurosurgery immediately after surgical intervention. Macroscopically homogenous pieces of tumor were separated and samples were sent to the Department of Pathology for identification. Normal brain tissues were obtained at autopsy within five hours following death. Tumoral and normal tissue samples were homogenized in phosphate buffer (pH 7.5) at 4OC forming a 10% (w/v) homogenate. Homogenization was performed with a tissue grinder which had been fitted with a teflon pestle at speed of 3000 rev. /min for 10 min. Hashimoto’s xanthine oxidase method was used with a slight modification. Xanthine oxidase levels were measured spectrophotometrically by the formation of uric acid from xanthine through the increase in absorbancy at 292 nm [7]. Tissue homogenate was incubated (0.2 ml) for 30 min at 30°C in 3 ml of medium containing phosphate buffer (pH 7.5)) 150 pmol; xanthine, 0.2 pmol; and as uricase inhibitor, xanthine, 0.3 pmol. The reaction was stopped by the addition of 0.1 ml 100% (w/v) TCA and the mixture was centrifuged at 10,000 x g for 15 min. With resultant clear supernatant, absorbtion at 292 nm was measured against blank. A calibration curve was constructed by using lo-50 munits/ml concentrations of standard xanthine oxidase solutions (Sigma). Protein levels were determined by the Lowry procedure [9].
Table 1. Xanthine oxidase levels tumoral brain tissues (P < 0.001).
in normal
and
Tissue
No. of samples
Xanthine oxidase activity (munits/mg protein)
Normal brain tissue Tumoral brain tissue
10
1.0645
20
2.339
+ 0.42 + 0.44
Results and discussion In this study, xanthine oxidase levels were found to be significantly increased in the tumoral brain tissues as compared with normal brain tissues (P < 0.001) (Table 1). There was no significant difference between the enzyme levels of meningioma and astrocytoma (P > 0.05) (Table 2). Chan et al., demonstrated that cellular injury and edema were induced by xanthine oxidase/hypoxanthine/ADP-Fe3+ (a widelyused in vitro free radical generating system), using brain slices as an in vitro bioassay system [2]. The degree of brain edema and injury was depended on the dose of xanthine oxidase [ 11. The present studies demonstrate that both neurons and glia are affected by free radicals ]2,31. According to our findings we suggest that the increased xanthine oxidase levels in Table 2. Xanthine oxidase astrocytoma (P> 0.05).
levels in meningioma
and
Tissue
No. of samples
Xanthine oxidase activity (munits/mg protein)
Astrocytoma Meningioma
10 10
2.345 2.335
+ 0.448 + 0.454
181
tumoral brain tissues cause to increase in the formation of oxygen-derived free radicals, which in turn may cause brain tissue damage. In conclusion, we suggest that the levels of xanthine oxidase in brain tissues can be used as a biochemical marker for the differentiation of tumoral brain tissues from normal tissues but cannot be used to differentiate the tumor types. References Chan, P.H. Schimidley, J.W., Fishman, R.A. and Longar, S.M. (1984) Brain injury, edema and vascular permeability changes induced by oxygen-derived free radicals. Neurology, 34,315-320. Chan. P.H., Yurko, M. and Fishman, R-A. (1982) Phospholipid degradation and cellular edema induced by free radicals in brain cortical slices. J. Neurochem., 38, 525531. Chan, P.H. and Fishman, R.A. (1982) Alterations of membrane integrity and cellular constituents by arachidonic acid in neuroblastoma and gjioma cells. Brain Res., 284.151-157.
4
5
6
7
8
9
10
11
Flamm, ES., Demopoulos, H.B., Seligman, M.L., Poser, R.G. and Ransohoff, J. (1978) Free radicals in cerebral &hernia. Stroke, 9,445-447. Granger, D.N., Rutili, G. and MC Cord, J.M. (1981) Role of Superoxide radicals in intestinal ishcemia. Gastroenterology, 81, 22-29. Hansson. R.O., Jonsson, O., Lundstam, S., Pettersson, S. and Schersten, T. (1983) Effects of free radical scavengers on renal circulation after ischemia in the rabbit. Clin. Sci., 65.605-610. Hashimoto, S. (1974) A new spectrophotometric assay method of xanthine oxidase in crude tissue homogenate. Analytical Biochemistry, 62,426-435. Hearse, D.J., Manning, AS., Downey, J.M. and Yellon, D.M. (1986) Xanthine oxidase: A critical mediator of myocardial injury during ischemia and reperfusion. Acta Physiol Stand. (Suppl), 548,65-78. Henry, R.J., Cannon, D.C. and Winkelman, J.W. (1974) Clinical Chemistry: Principles and Techniques, pp. 424428, New York. Parks, D.A. and Granger, D.N. (1986) Xanthine oxidase: biochemistry, distribution and physiology. Acta Physiol. Stand. (Suppl), S48,87-99. Yoshida, S., Abe, K., Busto, R., Watson, B.D., Kogurek, K. and Ginsberg, M.D. (1982) Influence of transient ischemia on lipid-soluble antioxidants, free fatty acids and energy metabolites in rat brain. Brain Res.. 245, 307316.
Cancer Letters, 50 (1990) 179- 181 Elsevier Scientific Publishers Ireland Ltd.
Xanthine
oxidase levels in human
brain tumors
E. KijkogW, A. BeIce”, E. &yurtb and Z. Tepeler” “Department
ofBiochemistry and
Weurosurgery,
Cerrahpasa Medical Faculty, Istanbul (Turkey)
25 May 1989) (Revision received 23 November 1989) (Accepted 18 December 1989)
(Received
Summary Xanthine oxidase is most recognized for its role as the rate-limiting enzyme in nucleic acid degradation through which all purines are channelled for terminal oxidation. The enzyme serves as a source of oxygen-derived free radicals which induce both cellular injury and edema as well as changes in vascular permeability. In the study we compared xanthine oxidase levels of human brain tumors with normal brain tissues. Statistical eualuation of our results shows significantly higher xanthine oxi-
dase levels in tumoral
brain tissues. However,
xanthine oxidase has not any significance for the differentiation of tumor types among each
others. The oncotypes gioma and astrocytoma.
Keywords: cancer.
xanthine
studied
were
menin-
oxidase;
brain
tissue;
Introduction Xanthine oxidase is a highly versatile enzyme that is widely distributed among species (from bacteria to man) and within the various tissues of mammals. The enzyme participates in the oxidation of a wide variety of endogenous (purines) and exogenous (ethanol) substrates. However, xanthine oxidase is most recognized for its role as the rate0304-3835/90/$03.50 Published and Printed
0 1990 Elsevier Scientific Publishers in Ireland
limiting enzyme in nucleic acid degradation through which all purines are channelled for terminal oxidation. Although the main physiological function of xanthine oxidase remains unclear, there is growing interest in the ability of this enzyme to serve as a source of oxidizing agents such as hydrogen peroxide and superoxide radical. Indeed, xanthine oxidase has the distinction of being the best documented biologic source of oxygen-derived free radicals [lo]. Interest in the enzyme as a source of oxidizing agents has increased markedly since it has been implicated in the pathogenesis of ischemia-reperfusion injury to tissues such as the intestine, kidney and heart [5,6,8]. There is growing evidence that oxygen readicals generated by xanthine oxidase, are primarily responsible for the cellular deterioration associated with reoxygenation of hypoxic tissues [lo]. Superoxide anion (0;)) hydrogen peroxide (H202) and hydroxyl radicals (.OH) are produced in biological systems by the singleelectron reduction of oxygen. These highly reactive oxygen-derived free radicals may play a role in cerebral ischemia and trauma [4,111. Enzymatically generated oxygen-derived free radicals, when infused intracerebrally into rats, induce both cellular injury and edema as well as changes in vascular permeability [ 11. Although the elucidation of the physiological role of xanthine oxidase has gained much Ireland Ltd
180
interest, there is a lack of knowledge on the behaviour of this enzyme at pathological conditions. Since xanthine oxidase is responsible for production of oxyen-derived free radicals which cause tissue damage, we wanted to study the levels of this enzyme in various types of tumoral brain tissues. The oncotypes studied were meningioma and astrocytoma. Materials and methods
Tumoral tissue samples were obtained from the Department of Neurosurgery immediately after surgical intervention. Macroscopically homogenous pieces of tumor were separated and samples were sent to the Department of Pathology for identification. Normal brain tissues were obtained at autopsy within five hours following death. Tumoral and normal tissue samples were homogenized in phosphate buffer (pH 7.5) at 4OC forming a 10% (w/v) homogenate. Homogenization was performed with a tissue grinder which had been fitted with a teflon pestle at speed of 3000 rev. /min for 10 min. Hashimoto’s xanthine oxidase method was used with a slight modification. Xanthine oxidase levels were measured spectrophotometrically by the formation of uric acid from xanthine through the increase in absorbancy at 292 nm [7]. Tissue homogenate was incubated (0.2 ml) for 30 min at 30°C in 3 ml of medium containing phosphate buffer (pH 7.5)) 150 pmol; xanthine, 0.2 pmol; and as uricase inhibitor, xanthine, 0.3 pmol. The reaction was stopped by the addition of 0.1 ml 100% (w/v) TCA and the mixture was centrifuged at 10,000 x g for 15 min. With resultant clear supernatant, absorbtion at 292 nm was measured against blank. A calibration curve was constructed by using lo-50 munits/ml concentrations of standard xanthine oxidase solutions (Sigma). Protein levels were determined by the Lowry procedure [9].
Table 1. Xanthine oxidase levels tumoral brain tissues (P < 0.001).
in normal
and
Tissue
No. of samples
Xanthine oxidase activity (munits/mg protein)
Normal brain tissue Tumoral brain tissue
10
1.0645
20
2.339
+ 0.42 + 0.44
Results and discussion In this study, xanthine oxidase levels were found to be significantly increased in the tumoral brain tissues as compared with normal brain tissues (P < 0.001) (Table 1). There was no significant difference between the enzyme levels of meningioma and astrocytoma (P > 0.05) (Table 2). Chan et al., demonstrated that cellular injury and edema were induced by xanthine oxidase/hypoxanthine/ADP-Fe3+ (a widelyused in vitro free radical generating system), using brain slices as an in vitro bioassay system [2]. The degree of brain edema and injury was depended on the dose of xanthine oxidase [ 11. The present studies demonstrate that both neurons and glia are affected by free radicals ]2,31. According to our findings we suggest that the increased xanthine oxidase levels in Table 2. Xanthine oxidase astrocytoma (P> 0.05).
levels in meningioma
and
Tissue
No. of samples
Xanthine oxidase activity (munits/mg protein)
Astrocytoma Meningioma
10 10
2.345 2.335
+ 0.448 + 0.454
181
tumoral brain tissues cause to increase in the formation of oxygen-derived free radicals, which in turn may cause brain tissue damage. In conclusion, we suggest that the levels of xanthine oxidase in brain tissues can be used as a biochemical marker for the differentiation of tumoral brain tissues from normal tissues but cannot be used to differentiate the tumor types. References Chan, P.H. Schimidley, J.W., Fishman, R.A. and Longar, S.M. (1984) Brain injury, edema and vascular permeability changes induced by oxygen-derived free radicals. Neurology, 34,315-320. Chan. P.H., Yurko, M. and Fishman, R-A. (1982) Phospholipid degradation and cellular edema induced by free radicals in brain cortical slices. J. Neurochem., 38, 525531. Chan, P.H. and Fishman, R.A. (1982) Alterations of membrane integrity and cellular constituents by arachidonic acid in neuroblastoma and gjioma cells. Brain Res., 284.151-157.
4
5
6
7
8
9
10
11
Flamm, ES., Demopoulos, H.B., Seligman, M.L., Poser, R.G. and Ransohoff, J. (1978) Free radicals in cerebral &hernia. Stroke, 9,445-447. Granger, D.N., Rutili, G. and MC Cord, J.M. (1981) Role of Superoxide radicals in intestinal ishcemia. Gastroenterology, 81, 22-29. Hansson. R.O., Jonsson, O., Lundstam, S., Pettersson, S. and Schersten, T. (1983) Effects of free radical scavengers on renal circulation after ischemia in the rabbit. Clin. Sci., 65.605-610. Hashimoto, S. (1974) A new spectrophotometric assay method of xanthine oxidase in crude tissue homogenate. Analytical Biochemistry, 62,426-435. Hearse, D.J., Manning, AS., Downey, J.M. and Yellon, D.M. (1986) Xanthine oxidase: A critical mediator of myocardial injury during ischemia and reperfusion. Acta Physiol Stand. (Suppl), 548,65-78. Henry, R.J., Cannon, D.C. and Winkelman, J.W. (1974) Clinical Chemistry: Principles and Techniques, pp. 424428, New York. Parks, D.A. and Granger, D.N. (1986) Xanthine oxidase: biochemistry, distribution and physiology. Acta Physiol. Stand. (Suppl), S48,87-99. Yoshida, S., Abe, K., Busto, R., Watson, B.D., Kogurek, K. and Ginsberg, M.D. (1982) Influence of transient ischemia on lipid-soluble antioxidants, free fatty acids and energy metabolites in rat brain. Brain Res.. 245, 307316.
