Persistent oxidative stress in colorectal carcinoma patients

Int. J. Cancer: 101, 395–397 (2002) © 2002 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

SHORT REPORT PERSISTENT OXIDATIVE STRESS IN COLORECTAL CARCINOMA PATIENTS Daniel GACKOWSKI1, Zbigniew BANASZKIEWICZ2, Rafał ROZALSKI1, Arkadiusz JAWIEN2 and Ryszard OLINSKI1* 1 Department of Clinical Biochemistry, The Ludwik Rydygier Medical University in Bydgoszcz, Karlowicza 24, Bydgoszcz, Poland 2 Department and Clinic of Surgery, The Ludwik Rydygier Medical University in Bydgoszcz, Karlowicza 24, Bydgoszcz, Poland We examine whether the level of 8-oxo-2ⴕ-deoxyguanosine (8-oxodGuo) in lymphocytes DNA is higher in colon cancer when compared to the control group. Factors that may influence oxidative stress such as antioxidant vitamins and uric acid were also determined. Blood samples were obtained from a control group of 55 healthy persons and 43 colon cancers. 8-OxodGuo level and the vitamins concentration were measured by high-performance liquid chromatography. The levels of 8-oxodGuo were significantly higher whereas the concentrations of the vitamins and uric acid were significantly lower in colon cancer patients than in control group. Therefore, the decreased concentration of antioxidant vitamins together with lower amount of uric acid may be responsible for the formation of pro-oxidative environment in blood of colorectal carcinoma patients. © 2002 Wiley-Liss, Inc. Key words: colon cancer; antioxidants; 8-oxodGuo; oxidative stress

Many epidemiological studies have reported an inverse association between vegetable and fruit consumption and occurrence of cancer and other degenerative diseases.1–3 One of the possible mechanisms of this protective effect is by exerting the antioxidative activities of such plant food constituents as vitamins A, C and E. These antioxidant vitamins are effective free radical scavengers, therefore, they should protect biomolecules such as proteins, lipids and nucleic acids from oxidative damage. Another effective scavenger of ROS (reactive oxygen species) is uric acid. Uric acid in physiological concentration is regarded as a main antioxidant and not only does it efficiently scavenge free radicals but it has also been shown to stabilize ascorbic acid in human serum4 and reduce consumption of ␣-tocopherol and ␤-carotene.5 8-OxoGua, one of the oxidatively modified DNA bases is a typical biomarker of oxidative stress and may play some role in carcinogenesis.6 – 8 The presence of 8-oxoGua residues in DNA leads to GC3 TA transversion unless repaired before DNA replication.9 Therefore, the presence of 8oxoGua may lead to mutagenesis. Furthermore, many observations indicate a direct correlation between 8-oxoGua formation and carcinogenesis in vivo.6,7,10,11 We have demonstrated elevated levels of typical free radicalinduced DNA base modification, including 8-oxoGua, in human cancerous colon tissues when compared to cancer free surrounding tissues.6,7,12 These data were confirmed by Condo et al.13 who demonstrated higher amounts of 8-oxodGuo in colorectal carcinoma cells. The important question, however, has not yet been answered. Is oxidative stress in cancer patients restricted to cancer tissues only, or are some other tissues oxidative stressed as well? Lymphocytes are often used as surrogate cells, which should inform about oxidative stress, measured as a certain level of 8-oxodGuo, in other tissues.14,15 We examine whether this level in lymphocytes DNA is higher in colon cancer patients when compared to the control group. Factors that may influence oxidative stress such as antioxidant vitamins and uric acid were also determined.

smoking. The colorectal cancer patients’ group of 45 subjects comprised 26 males and 19 females. The median patient age was 63 years (range 44 –90). All the patients with newly recognised colon cancer (based on endoscopy; sigmo- or colonoscopy) were asked to fill out the dietary questionnaire. The majority of them reportedly consumed 3 servings of fruit and vegetables and about 250 g of meat and fat per day. Only these patients qualified. An additional criterion was non-smoking status. Average body weight of the patients was 71 kg. The patients were asked to abstain from vitamin supplementation for at least 1 month before the blood samples were collected. Then the blood was taken before surgery. Histopathological examination of the cancerous tissue confirmed the endoscopic diagnosis. All the patients had histologically proven adenocarcinomas with: A (n ⫽ 7), B1 (n ⫽ 28) and B2 (n ⫽ 10) of staging according to Duke’s scale with Astler-Coller modification. No differences in the investigated analytical parameters have been found between the staging groups. The patients were not treated with any drug during the time from the diagnoses up to the time of surgery (up to 4 weeks). The control group was chosen in such a way that the following criteria matched the patient group: eating habits, age, body weight, gender, and smoking status (all of the subjects were non-smokers). The study was approved by the medical ethics committee of The L. Rydygier Medical University, Bydgoszcz, Poland, (in accordance with Good Clinical Practice, Warsaw 1998) and all the patients gave informed consent. Determination of plasma vitamins A, E and C concentration by HPLC Quantification of vitamin E (␣-tocopherol), vitamin A (retinol) and vitamin C (ascorbic acid) by HPLC technique was described previously.16 Isolation of lymphocytes from venous blood Venous blood samples from the patients were collected. Each blood sample was divided into 3 aliquots by RPMI solution. The blood was carefully applied on top of Histopaque 1077 solution (Sigma, St. Louis, MO) and lymphocytes were isolated by centrifugation according to the procedure laid down by the manufacturer.

Grant sponsor: Committee for Science Research; Grant number: 4PO5D03017 and BW 72/2002. *Correspondence to: Department of Clinical Biochemistry, ul. Karlowicza 24, 85-092 Bydgoszcz, Poland. Fax: ⫹48-52-585-3771. E-mail: [email protected]

MATERIAL AND METHODS

Received 17 January 2002; Revised 27 May 2002; Accepted 25 June 2002

Patients Our study was conducted in 2 groups. The control group consisted of 55 healthy male (n ⫽ 25) and female (n ⫽ 30) with a median age 60 years (range 26 – 87 years). None had a history of

DOI 10.1002/ijc.10610 Published online 7 August 2002 in Wiley InterScience (www.interscience. wiley.com).

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DNA isolation and 8-oxo-2⬘-deoxyguanosine determination in DNA isolates DNA from lymphocytes was isolated using the method described by Miller et al.17 with some modifications. Briefly, lymphocytes were suspended in 3 ml of nuclei lysis buffer (10 mM Tris-HCl, 400 mM NaCl, 2 mM EDTA, pH 8.2, 1 mM desferrioxamine, 4 mM histidine, 3 mM GSH, pH 8.2) and after addition of 0.2 ml of 10% SDS and 0.5 ml solution containing 1 mg proteinase K in 1% SDS, 2 mM EDTA. The solution was digested for 1 hr in the dark. Then, proteins were precipitated by 1 ml of saturated NaCl followed by centrifugation for 15 min at 2,000g, 4°C. Supernatant containing nucleic acids was treated with 2 vol cold absolute ethanol to precipitate high molecular weight DNA. The precipitate was removed with a plastic spatula, washed with 70% ethanol and after centrifugation dissolved in 220 ␮l of water. Quantification of 8-oxodGuo by the mean of HPLC/ECD technique was described previously.18 RESULTS

General characteristic of the analytical parameters of the study groups is described in the Table I. The 8-oxodGuo level in lymphocytes of 36 patients ranged from 3.83–37.41 per 106 dG molecules (mean value ⫾ SD: 13.76 ⫾ 7.19). It was significantly higher (p ⫽ 0.0034) than in DNA of control group, where the level ranged from 2.86 –20.46 per 106 dG molecules (mean value 9.57 ⫾ 3.95). The mean levels of 8-oxodGuo are in the range of values reported recently by others.19,20 Interestingly, 8-oxodGuo level in lymphocytes, DNA may vary significantly according to country. In Ireland, the level is very close to that reported in our study.14 We did not, however, find any difference in 8-oxodGuo level between men and women in the studied groups. The mean endogenous levels of vitamin C in the plasma of the control group and in colon cancer patients reached the mean values of 49.76 ␮M (range 3.65–139.03) and 29.45 ␮M (range 1.96 – 144.01) respectively. This difference was statistically significant (p ⫽ 0.0006). As shown in Table I, the vitamin A and E level was reduced significantly in the plasma of cancer patients when compared to the control group. The mean values of vitamin A concentration were respectively 0.807 (range 0.074 –3.333) and 1.237 (range 0.193–2.692). Also the differences in uric acid level were statistically significant (p ⫽ 0.0277). The mean value of uric acid in control group was 4.28 mg/dL (range 1.10 –7.70) whereas in the patients group the level reach concentration of 3.73 mg/dL (range 1.86 –7.88). No correlation has been found between any of the plasma antioxidant and 8-oxodGuo level. DISCUSSION

Although it is generally accepted that genetic factors are important determinants for the genesis of colon cancer, it appears that differences in colon cancer incidence are mainly attributable to environmental factors.21 Because oxidative stress may be involved in cancer development, one group of such factors may be antioxidant vitamins that have been suggested as limiting oxidative

damage in humans.22 There are, however, conflicting data concerning the relationship between colon cancer incidence and antioxidant vitamin level or fruit and vegetable consumption.4 One of the possible mechanisms of protective effect of antioxidant vitamins against cancer development may be by decreasing the amount of potentially mutagenic oxidatively modified DNA bases. Duthie et al.23 using single cell gel electrophoreses (comet assay) found that supplementation of healthy volunteers with vitamin C (100 mg/day) vitamin E (280 mg/day) and ␤-carotene (25 mg/day) significantly reduced base damage in lymphocyte DNA. Collins et al.24 demonstrated significant negative correlation between basal concentration of serum carotenoids and oxidatively modified pyrimidines. Supplementation of patients with carotenoids did not influence oxidative DNA damage. The authors did not find any correlation between the damage and concentration of vitamins E and C. We have found that the endogenous levels of the analyzed antioxidant vitamins in plasma of carcinoma patients were significantly lower than that in the control group. The simple explanation of this finding may be differences in feeding habits between the studied groups. It is noteworthy, however, that the members of the studied groups were chosen randomly and, according to the interview, the groups were consisted of in such a way that the members of the both could match feeding habits and living conditions (Material and Methods). Therefore, it is rather unlikely that the different concentration of vitamins in their blood was a result of lifestyle. Presumably, severe oxidative stress, characteristic for colon cancer, resulting in the production of ROS is responsible for consumption of the antioxidant vitamins. The decreased amount of uric acid also supports this assumption. The precise mechanism(s) of the oxidative stress remains unknown. Some mechanisms may be suggested. It has been documented recently that cancer patients showed signs of extensive granulocyte activation with a release of reactive oxygen species.25 Still another reason of the observed phenomenon may be that some tumors may stimulate the defense systems of the body so that they react against the tumor to produce cytokines.26 Some of the cytokines can produce large amounts of ROS.27,28 It has been shown that elevated plasma level of TNF is responsible for increase oxidative DNA damage of CD 34⫹ cells.29 It has been shown that malignant cells can produce hydrogen peroxide at levels as large as those characteristic for stimulated polymorphonuclear leukocytes.30,31 Therefore, one of the reasons for the observed oxidative stress in advanced stages of cancer may be a release of the large number of cancer cells into the blood stream32 and their penetration into other tissues. In this context it is noteworthy, that we have demonstrated that exposure to the activated leukocytes causes oxidative DNA base modifications (among them 8-oxoguanine) in target cells.30 Our finding that 8-oxodGuo level is elevated in DNA isolated from carcinoma patients’ lymphocytes is in good agreement with aforementioned results and strengthen the suggestion that oxidative stress may be characteristic not only for the diseased tissue but for some other tissues of the patients with carcinoma as well. It has been shown recently that oxidative stress represented by dramatic

TABLE I – ANALYTICAL PARAMETERS OF THE STUDY GROUP

Plasma ascorbic acid concentration [␮M] Plasma uric acid concentration [mg/dL] Plasma ␣-tocopherol concentration [␮M] Plasma retinol concentration [␮M] 8-oxodGuo/106dG in lymphocytes

Cancer1 (n ⫽ 45)

Control1 (n ⫽ 55)

p-value (Student’s t-test) cancer vs. control

29.457 ⫾ 27.414 3.734 ⫾ 1.327 18.87 ⫾ 14.50 0.807 ⫾ 0.752 13.76 ⫾ 7.19 (n ⫽ 36)2

49.76 ⫾ 29.24 4.285 ⫾ 1.136 24.69 ⫾ 14.55 1.237 ⫾ 0.610 9.57 ⫾ 3.95 (n ⫽ 35)2

0.00063 0.0283 0.0493,4 0.00213 0.00343

1 Values are means ⫾ SD.–2Because of technical difficulties (insufficient amount of DNA) only part of the samples were analysed for the presence of 8-oxodGuo in lymphocytes.–3Statistically significant differences (p ⬍ 0.05 Student’s t-test).–4These differences were independent on cholesterol concentration since serum cholesterol level was similar in both the groups (5.18 mM and 5.05 mM respectively).

PERSISTENT OXIDATIVE STRESS IN COLON CANCER

increase of 8-isoprostane plasma level is associated with advanced stages of adenocarcinomas.25 Therefore it is possible that persistent oxidative stress may be characteristic for many types of cancers. It has been estimated that at least 11,000 individual DNA mutations exist in a single carcinoma cell of colorectal tumors.33,34 It is possible that part of these mutations may arise during development of the disease. Our results suggest that one potential source of this unusually large number of mutations may be damage to

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DNA by reactive oxygen species. Oxidative stress may be also responsible for impaired T cell function in cancer patients.25 Therefore it is possible that people with advance stages of cancer development may be helped by treatment with antioxidants. Supplementation of antioxidants may slow dawn the progression of the disease. It is also possible, however, that pro-oxidants environment found in our study is characteristic for advance stages of colon cancer and that oxidative stress is rather a result of the disease development.

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14.

15. 16.

17. 18.

Block G, Patterson B, Subar A. Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr Cancer 1992;18: 1–29. Willet WC. Diet and health: what should we eat? Science 1994;264: 532–7. Ames BN, Gold LS, Willett WC. The causes and prevention of cancer. Proc Natl Acad Sci USA 1995;92:525865. Sies H. Oxidative stress: oxidants and antioxidants. New York: Academic Press, 1991. Schlotte V, Sevanian A, Hochstein P, Weithmann KU. Effect of uric acid and chemical analogues on oxidation of human low density lipoprotein in vitro. Free Radic Biol Med 1998;25:839 – 47. Jaruga P, Zastawny TH, Skokowski J, Dizdaroglu M, Olinski R. Oxidative DNA base damage and antioxidant enzyme activities in human lung cancer. FEBS Lett 1994;341:59 – 64. Olinski R, Zastawny TH, Budzbon J, Skokowski J, Zegarski W, Dizdaroglu M. DNA base modifications in chromatin of human cancerous tissues. FEBS Lett 1992;193:193– 8. Loft S, Poulsen HE. Markers of oxidative damage to DNA: antioxidants and molecular damage. Methods Enzymol 1995;300:166 – 84. Cheng KC, Cahill DS, Kasai H, Nishimura S, Loeb LA. 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G3 T and A3 C substitutions. J Biol Chem 1992;267:166 –72. Feig DI, Reid TM, Loeb LA. Reactive oxygen species in tumorigenesis. Cancer Res 1994;54:1890 – 4. Floyd RA. The role of 8-hydroxyguanine in carcinogenesis. Carcinogenesis 1990;11:1447–50. Foksinski M, Kotzbach R, Szymanski W, Olinski R. The level of typical biomarker of oxidative stress: 8-hydroxy-2⬘-deoxyguanosine is higher in uterine myomas than in control tissues and correlates with the size of the tumour. Free Radic Biol Med 2000;29:597– 601. Kondo S, Toyokuni S, Iwasa Y, Tanaka T, Onodera H, Hiai H, Imamura M. Persistent oxidative stress in human colorectal carcinoma, but not in adenoma. Free Radic Biol Med 1999;27:401–10. Collins AR, Gedik CM, Olmedilla B, Southon S, Bellizzi M. Oxidative DNA damage measured in human lymphocytes: large differences between sexes and between countries, and correlations with heart disease mortality rates. FASEB J 1998;12:1397– 400. Lenton KJ, Therriault H, Fulop T, Payette H, Wagner JR. Glutathione and ascorbate are negatively correlated with oxidative DNA damage in human lymphocytes. Carcinogenesis 1999;20:607–13. Gackowski D, Kruszewski M, Jawien A, Ciecierski M, Olinski R. Further evidence that oxidative stress may be a risk factor responsible for the development of atherosclerosis. Free Radic Biol Med 2001; 31:542–7. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215. Foksinski M, Bialkowski K, Skiba M, Ponikowska I, Szmurlo W, Olinski R. Evaluation of 8-oxodeoxyguanosine, typical oxidative DNA damage, in lymphocytes of ozone treated arteriosclerotic patients. Mutat Res 1999;438:23–7.

19. Lodovici M, Casalini C, Cariaggi R, Michelucci L, Dolara P. Levels of 8-hydroxydeoxyguanosine as a marker of DNA damage in human leukocytes. Free Radic Biol Med 2000;28:13–7. 20. Chen L, Bowen PE, Berzy D, Aryee F, Stacewicz-Sapuntzakis M, Riley RE. Diet modification affects DNA oxidative damage in healthy humans. Free Radic Biol Med 1999;26:695–701. 21. Maclennan R. Diet and colorectal cancer. Int J Cancer 1997; 10(Suppl):10 –2. 22. Halliwell BM, Gutterige JMC. Free radicals in biology and medicine, 3rd edition. Oxford: Oxford Science Publication; 1999. 23. Duthie SJ, Ma A, Ross MA, Collins AR. Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res 1996;56:1291–5. 24. Collins AR, Olmedilla B, Southon S, Granado F, Duthie SJ. Serum carotenoids and oxidative DNA damage in human lymphocytes. Carcinogenesis 1998;19:2159 – 62. 25. Shmielau J, Finn OJ. Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanisms of suppression of T cell function in advanced cancer patients. Cancer Res 2001;61: 4756 – 60. 26. Franks LM, Teich NM, eds. Introduction to cellular and molecular biology of cancer. New York: Oxford University Press; 1997. 27. Ohba M, Shibanuma M, Kuroki T, Nose K. Production of hydrogen peroxide by transforming growth factor-␤ 1 and its involvement in induction of egr-1 in mouse osteoblastic cells. J Cell Biol 1994;126: 1079 – 88. 28. Kayanoki Y, Fujii J, Suzuki K, Kawata S, Matsuzawa Y, Taniguchi N. Suppression of antioxidative enzyme expression by transforming growth factor-␤ 1 in rat hepatocytes. J Biol Chem 1994;269:15488 – 92. 29. Peddie C-M, Wolf C-R, McLellan L-I, Collins A-R, Bowen D-T. Oxidative DNA damage in CD34⫹ myelodysplastic cells is associated with intracellular redox changes and elevated plasma tumour necrosis factor-␣ concentration. Br J Haematol 1997;99:625–31. 30. Dizdaroglu M, Olinski R, Doroshow JH, Akman SA. Modification of DNA bases in chromatin of intact target human cells by activated human polymorphonuclear leukocytes. Cancer Res 1993;53:1269 –72. 31. Szatrowski TP, Nathan CF. Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res 1991;51:794 – 8. 32. de Vita VT Jr., Hellman S, Rosenberg SA. Cancer, principles and practice of oncology. 6th Ed. New York: Lippincott Williams and Wilkins; 2001. 33. Ionov Y, Peinado MA, Malkhosyan S, Shibata D, Perucho M. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature (Lond) 1993;363:558 – 61. 34. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y, White R, Smits AM, Bos JL. Genetic alterations during colorectal-tumor development. N Engl J Med 1988; 319:525–32.