plasma and erythrocyte lipid peroxidation levels in patients with testis tumor after orchiectomy

Plasma and Erythrocyte LipidPeroxidation Levels in Patients

with Testis Tumor After OrchiectomyBEKI

.R ÖZTÜRK,*,1 MEHMET GÜVEN,2 FI

.KRET ARPACI,1

SEREF KÖMÜRCÜ,1 AHMET ÖZET,1

AND MURAT BEYZADEOGLU3

1Department of Medical Oncology, Gülhane Military MedicalAcademy, Ankara, Turkey; 2Department of Medical Biology,

Cerrahpasa Medical School, Ankara, Turkey;and 3Department of Radiation Oncology, Gülhane Military

Medical Academy, Ankara, Turkey

Received April 8, 1999; Accepted May 11, 1999

ABSTRACT

Plasma and erythrocyte lipid peroxidation levels of 20 patientswith histopathologically confirmed testis cancer and 20 healthy con-trol individuals were studied between November 1995 and June 1997.The group with testis cancer had a mean age of 24.8 ± 8.2 yr and thecontrol group’s mean age was 28.3 ± 6.9 yr. Stage distribution of the tes-tis cancer cases were 4 of stage A, 10 of stage B, and 6 of stage C.Blood samples of the patients were drawn after orchiectomy and after12 h fasting before chemotherapy. Mean plasma and erythrocyte lipidperoxidation levels were detected to be 14.51 ± 5.30 nmol malondi-aldehide (MDA)/mL and 9.30 ± 2.06 nmol MDA/g hemoglobin (Hb),respectively, in the testis cancer group, whereas the healthy controlgroup had mean plasma and erythrocyte lipid peroxidation levels of10.7 ± 1.82 nmol MDA/mL and 6.18 ± 1.68 nmol MDA/g Hb, respec-tively. Plasma and erythrocyte lipid peroxidation values of the testiscancer patients were determined to be statistically significantly higherthan that of the health control group (p < 0.001, p < 0.001). No signif-icant correlation was determined between plasma, erythrocyte lipidperoxidation levels and tumor markers. In conclusion, it can be saidthat an increase in the lipid peroxidation may play a role in the patho-genesis of testis carcinomas in addition to the other causes.

© Copyright 2000 by Humana Press Inc.All rights of any nature, whatsoever, reserved.0163–4984/00/7302–0181 $11.75

Biological Trace Element Research 181 Vol. 73, 2000

*Author to whom all correspondence and reprint requests should be addressed.

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Index Entries: Testis tumor; lipid peroxidation; plasma; erythrocyte.

INTRODUCTION

Lipid peroxidation is the most prominent free-radical reaction char-acterized with biological injury; therefore, various methods were devel-oped to determine the lipid peroxidation level of a tissue, an organ, orwhole organism (1,2). Free oxygen radicals produced by tumor cells inan uncontrolled way pass over the defense mechanisms of the body,causing various lipid peroxidation reactions, and as the peroxidationreactions progress, new free-radical intermediate products occur. Theseradical intermediates are highly reactive in nature and damage theimmediate tissues. One of the last and most toxic products of lipid per-oxidation is malondialdehide (MDA) and is used as a measure of free-radical injury.

The role of oxidative mechanisms in carcinogenesis is recently a pop-ular topic of interest and great research related to oxidative mechanismis ongoing. Peroxide radicals caused by polyunsaturated fat acid peroxi-dation have been shown to be effective in carcinogenesis either directlyor via prostoglandin synthesis in various studies (3–8). Dormady et al.have acknowledged that the increased lipid peroxidation in the abnor-mally proliferated cells of the cancer patients led to increase in serumlipid peroxides (9). Hendrickse et al. determined the tumoral tissue ofcolorectal carcinoma patients to have a higher MDA content than that oftheir normal mucosa and the highest MDA level in Duke’s stage C (10).Zima et al. studied lipid peroxidation of nine multiple myeloma patientsand detected plasma and erythrocyte MDA levels to be significantlyhigher than that of the control group (11).

There are only very few experimental lipid peroxidation studiesdone on testis tumors. Vernie et al. studied on 15 teratoma patients anddetermined glutation peroxidase activity to be low after treatment witha cisplatin, including a chemotherapy regimen (12). Koizumi and Li haveshown an increase in hydrogen peroxide, iron and lipid peroxidation lev-els of rat testicle samples 12 h after a single carcinogenic subcutaneouscadium injection (13). Sugawara and Sugawara have shown that subcu-taneous selenium injection abolished cadmium-induced lipid peroxida-tion to a great extent in male mice testicles (14). The aim of this study isto analyze the serum and erythrocyte lipid peroxidation changes in testiscancer patients.

MATERIALS AND METHODS

From November 1995 to June 1997, 20 patients with testis carcinomaand 20 healthy controls were enrolled in to the study. Eligibility criteriaincluded histologically proven testis carcinoma, with orchiectomy for the

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patients and no prominent infection history within the last month beforeenrollment for the healthy controls.

The mean age of the patients was 24.8 ± 8.2 yr with an age range of20–57 yr. The stage distribution of patients was 4 cases of stage A, 10cases of stage B, and 6 cases of stage C (Table 1). Blood samples weretaken in the period after orchiectomy and before chemotherapy. Themean age of the 20 healthy male controls was 28.3 ± 3.9 yr with an agerange between 20 and 45 yr.

Blood samples drawn 12 h postfasting with EDTA from patients andhealthy control subjects were centrifuged for 10 min at 110g. Plasma hasbeen separated for plasma lipid peroxidation and erythrocytes for eryth-rocyte lipid peroxidation.

Plasma Lipid Peroxidation

To 0.5 mL of plasma, 2.5 mL of trichloroacetic acid (TCA) was addedand stirred. Then, 1 mL of thiobarbituric acid (TBA) was then added tothis mixture and the mixture was cooled after being kept in boiling waterbath for 30 min. Four milliliters of buthanol was added to the solutionand then the solution was centrifuged for 15 min at 200g. Supernatantwas read at 532 nm. Results were expressed in nmol MDA/mL plasmausing the molar extinction coefficient determined for MDA.

Erythrocyte Lipid Peroxidation

Erythrocytes were washed three times with 5 volumes of phosphatebuffer (PBS). Then, 0.2 mL erythrocyte was mixed with 0.8 mL PBS and25 µL butylyzed hydroxytoluen (BHT). One-half milliliter of 30% TCAwas added to this mixture and then the tubes were shaken and kept heldfor 2 h in ice. After incubation, the tubes were centrifuged for 15 minat 200g and then 1 mL of top liquid was transfered into other tubes.Seventy-five microliters EDTA (0.1M) and 0.5 mL 1% TBA solution wereadded to the tubes and the tubes were cooled after being kept at a boilingwater bath for 15 min. The absorbance values of samples at 532 and 600 nmwere subtracted from absorbance values at 532 nm and the results wereexpressed as nmol MDA/g hemoglobin (Hb) using the molar extinctioncoefficient (1.56 × 105M−1/cm) determined for MDA. Independent-samples(comparison of the mean values of two independent groups) t-test andcorrelation analysis were used in the statistical analyses of the results.

Lipid Peroxidation After Orchiectomy 183


Table 1Stage Distribution

Stage Number Percentage

A 4 %20B 10 %50C 6 %30

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RESULTS

Histologically, four patients had seminoma, six patients had terato-carcinoma, six patients had embryonal carcinoma, three patients hadmixed germ cell tumor, and one patient had a yolk sac tumor (Table 2).Plasma and erythrocyte lipid peroxidation values of 20 testicular cancerpatients and 20 healthy controls are shown in Tables 3 and 4, respectively.The mean plasma lipid peroxidation values of testicular cancer patients and

184 Öztürk et al.


Table 2Histologic Distribution of Patients

Cancer type Cases %

Seminoma 4 %20Teratocarcinoma 6 %30Embryonal carcinoma 6 %30Mixed germ cell tumor 3 %15Yolk sac tumor 1 %5

Table 3Plasma and Erythrocyte Lipid Peroxidation Levels of Patients

with Testicular Carcinoma

Age Erythrocyte lipid Plasma lipidCase (year) Stage peroxidation(nmol MDA/gHb) peroxidation(nmol MDA/ml plasma)

1 21 B 8.10 12.322 20 B 8.3 12.303 33 A 6.82 13.214 26 B 9.14 14.575 20 B 9.68 18.256 22 A 10.61 21.547 22 A 11.60 22.558 57 B 12.66 18.879 21 B 10.89 27.35

10 22 C 9.67 19.8011 24 A 7.94 11.7512 23 B 7.53 9.9213 24 C 7.73 7.8014 24 C 6.80 11.7815 22 C 12.79 6.4716 22 C 6.27 14.3217 23 B 7.79 12.2718 28 C 8.91 8.1019 21 B 9.87 14.1820 20 B 12.95 11.90

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healthy controls were determined to be 14.51 ± 5.36 and 10.07 ± 1.82nmol MDA/mL plasma, respectively, whereas the mean erythrocyte lipidperoxidation values determined for patient and healthy control groupswere 9.30 ± 2.06 and 6.18 ± 1.68 nmol MDA/L gHb, respectively.

Erythrocyte lipid peroxidation and plasma lipid peroxidation valuesof the patient group were determined to be statistically significantlyhigher than that of the healthy control group (p < 0.001, Table 5). No sig-nificant correlation was determined between erythrocyte and plasma



Table 4Plasma and Erythrocyte Lipid Peroxidation Levels of Controls

Erythrocyte lipid Plasma lipidCase Age peroxidation (nmol MDA/gHb) peroxidation (nmol MDA/ml plasma)

1 31 4.89 9.682 20 6.52 12.683 45 6.41 10.424 42 3.20 8.895 32 5.20 10.926 31 3.29 13.287 38 4.57 11.998 28 6.21 9.619 22 4.64 11.29

10 29 6.55 9.2311 29 9.68 10.9212 28 5.05 6.3813 21 7.23 9.6814 30 8.19 9.1315 23 7.93 7.0516 24 7.26 7.4617 21 7.02 10.0518 23 5.83 12.1419 24 5.56 11.1720 25 8.29 9.31

Table 5Mean Lipid Peroxidation Levels of Patients and Controls

Patient (n = 20) Control (n = 20)Mean ± SD Mean ± SD p value

Age (year) 24.8 ± 8.2 28.3 ± 6.9 >0.05Erythrocyte lipid peroxidation 9.30 ± 2.06 6.8 ± 1.68 <0.001

(nmol MDA/mL plasma)Plasma lipid peroxidation 14.51 ± 5.36 10.07 ± 1.82 <0.001

(nmol MDA/gHb)

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lipid peroxidation levels and age and tumor markers. On the basis ofstages, erythrocyte and plasma lipid peroxidation levels revealed no sta-tistically significant difference; for stage A versus stage C, stage A versusstage B, stage B versus stage C, they were p = 0.26 and p = 0.07, p = 0.33and p = 0.28, and p = 0.11 and p = 0.07, respectively.

DISCUSSION

Testicular cancer is the commonest form of malignancy in menbetween the ages of 20 and 40 yr (15). It ranks third in order of death forthis age group. The tumor etiology is not known. Some studies pointedout the role of malignant changes independent of “external growth fac-tors” and regulators.

Lipid peroxidation is a process that causes the oxidation of unsatu-rated fatty acids in the membrane phospholipids leading to membranelipid structure change, resulting in structural and functional cell injury(3). Cell membranes subject to oxygen radicals stimulate lipid peroxida-tion reactions. Unsaturated fatty acids are particularly sensitive to per-oxidation. Lipid peroxidation starts with the abstraction of a hydrogenatom from the target fatty acid to form a lipid radical.

The extremely rapid addition of oxygen to the fatty acid radicalforms a lipid peroxyl radical. This is capable of reacting with otherpolyunsaturated fatty acids, beginning a new chain of oxidation and thusforming a lipid hydroperoxide on the original polyunsaturated fatty acidand generating a new lipid radical.

Aldehydes are the most toxic products. MDA and other end prod-ucts of lipid peroxidation are either stable; they can reach the other cellsor distant cell sites and can cause toxic injury to the tissues that are notsubject to direct peroxidative damage.

Proteins and enzymes subjected to lipid peroxidation in solutionmay undergo polymerization, polypeptid chain breakages, and aminoacid structure changes. The DNA damage resulting from free radicals inthe living organisms may be related to main or side-chain breakings andhydrogen-bond separations. All nucleic acid components may be subjectto free-radical injury. These injuries may be permanent or may berepaired by special mechanisms. Irrepairable damage in the bases maycause mutation resulting in malignant transformation and changed geneexpression (8). Research done thus far implicates lipid peroxidation andrelease of free radicals in both initiation and promotion stages of cancer.The malignant character of cells is gained either by structural changes inthe cell’s DNA or by a series of changes occurring in the cell membraneand cytoplasm.

A high proliferative rate of cancer cells causes excessive superoxideformation in cancer tissue, which leads to oxidative inactivation of serumantiproteases, resulting in cancer cell growth with increased metastatic

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potential. The lipid peroxidase increase in erythrocytes shows an anti-oxidase enzyme system defect in those cells subject to oxidative stress.

In this study with 20 testicle carcinoma cases of various stages, wehave determined MDA levels in plasma and erythrocytes to be signifi-cantly higher than those of controls.

This outcome corresponds to the results of Sugawara and Sugawara’sexperimental studies revealing testicular peroxidation level rise resultingfrom oxidative stress in mice given cadmium at a carcinogenic dose(13,14). No significant correlation was noted for plasma and erythro-cyte lipid peroxidation levels of testicular carcinoma cases on a stage-comparison basis. In a study done for Duke’s C colon carcinomas, MDAwas determined to be high (10). It is hard to comment since there werefew patients with stage A and C testis tumor in our study.

As a conclusion, increased plasma and erythrocyte lipid peroxida-tion levels that reveal oxidative stress may play a role in the etiopatho-genesis of testis tumors in addition to other factors.

REFERENCES

1. M. Miyake and S. Fuchimato, Production of hydroxyl radicals by tumor cells mea-sured by electron spin resonance spectrometry. Res. Commun. Chem. Pathol. Pharmacol.71(3), 293–307 (1990).

2. K. Yagi, Lipid pereoxides and related radicals in clinical medicine. Bioassays 1,58–60 (1984).

3. P. A. Cerutti, Prooxidant state and tumor promotion. Science 227, 375–382 (1985).4. K. H. Cheesman, Mechanism and effects of lipid peroxidation. Mol. Aspects Med. 14,

191–197 (1993).5. K. H. Cheesman and T. F. Slatter, An introduction to free radical biochemistry. Br.

Med. Bull. 49, 481–493 (1993).6. M. U. Dianzani, Lipid peroxidation and cancer. Crit. Rev. Oncol. Hemat. 15, 125–147 (1993).7. E. N. Frankel, Lipid oxidation: mechanism, products and biological significance.

JAOCS 61, 1909–1916 (1984).8. R. E. Pacifici and K. J. Davies, Protein, lipid and DNA repair systems in oxidative

stress: the free-radical theory of aging revisited. Gerontology 37, 166–180 (1991).9. T. L. Dormady, An approach to free radicals. Lancet 2, 1010–1014 (1983).

10. C. W. Hendrickse, R. W. Kelly, and S. Radley, Lipid peroxidation and prostoglandinsin colorectal cancer. Br. J. Surgery 81, 1219–1223 (1994).

11. T. Zima, I. Spickal, and S. Stipek, Antioxidant enzymes and lipid peroxidation inpatients with multiple myeloma. Neoplasma 43(2), 69–73 (1996).

12. L. N. Vernie, C. Zegers, and C. S. Baldew, Cisplatin induced changes of selenium lev-els and glutathione peroxidase activities in blood of testis tumor patients. Cancer Lett.40, 83–91 (1988).

13. T. Koizumi and Z. G. Li, Role of oxidative stress in single-dose, cadmium inducedtesticular cancer. J. Toxicol. Environ. Health 37(1), 25–36 (1992).

14. N. Sugawara and C. Sugawara, Selenium protection against testicular lipid peroxi-dation from cadmium. J. Appl. Biochem. 6, 199–204 (1984).

15. J. P. Richie, Advances in the diagnosis and treatment of testicular cancer. CancerInvest. 11(6), 670–675 (1993).



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plasma and erythrocyte lipid peroxidation levels in patients with testis tumor after orchiectomy

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