Journal of Environmental Radioactivity 64 (2003) 19–25www.elsevier.com/locate/jenvrad

The effects of exposure of60Co on theoxidant/antioxidant status among radiation

victimsMustafa Demira,∗, Dildar Konukoglu b, Levent Kabasakala,

Hakan Kadir Yelkeb, Kadir Ergenc, Sabbir Ahmedaa Department of Nuclear Medicine, Istanbul University, Istanbul, Turkey

b Department of Biochemistry, Istanbul University, Istanbul, Turkeyc Haseki Public Hospital, Section of Internal Medicine of the Cerrahpasa Medical Faculty, Istanbul

University, Istanbul, Turkey

Received 3 December 2001; received in revised form 17 March 2002; accepted 24 March 2002

Abstract

This retrospective study has been performed with radiation victims who were accidentallyexposed to a60Co source and its release into the environment. The aim of the study was toassess the effects of elevated radiation exposures on plasma level, on erythrocyte thio barbituricacid reactive substance (TBARS) level and on erythrocyte glutathione (GSH) levels. Patientswere treated in different hospitals with different symptoms such as nausea, vomiting, dizziness,along with severe anemia in some patients. Blood samples were collected 3–5 days followingthe radiation accident. Increases in plasma (6.25±0.90 nmol ml�1) and erythrocyte TBARSlevels (330.5±30.5 µmol gHb�1) were found in comparison to a healthy group (3.72±0.68nmol ml�1 and 150.7±20.5µmol gHb�1, respectively) at a significant level (p�0.001). Erythro-cyte GSH levels (5.2±0.30 µmol gHb�1) were found to be decreased among the victims(healthy group: 10.2±0.7 µmol gHb�1) at the same significance level (p�0.001). These obser-vations confirm a significant change induced by radiation in the oxidant/antioxidant statusamong the victims. It is suggested here that antioxidant supplementation therapy might beeffective in preventing the harmful effects of60Co radiation among radiation victims. 2002 Elsevier Science Ltd. All rights reserved.

Keywords: 60Co; Radiation exposure; Oxidative stress

∗ Corresponding author. Cerrahpas¸a Tıp Faku¨ltesi, Nukleer Tıp ABD, 34303 Cerrahpas¸a, Istanbul,Turkey. Tel.:+90-212-586-02-82; fax:+90-212-587-82-30.

E-mail address: mmdemir@e-kolay.net (M. Demir).

0265-931X/02/$ - see front matter. 2002 Elsevier Science Ltd. All rights reserved.PII: S0265 -931X(02)00037-1

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1. Introduction

Irradiation of organisms with electromagnetic radiation (X–rays and γ-rays) andparticulate radiation (electrons, protons, neutrons, deuterons, α and β particles) gen-erates primary radicals by transferring their energy to cellular components such aswater. Radiation causes breakage of one of the oxygen–hydrogen covalent bonds inwater, leaving a single electron on the hydrogen atom and one on the oxygen atomand creates radicals, especially hydroxyl radicals. These radicals then undergo sec-ondary reactions with dissolved O2 or with cellular solutes (Freeman & Crapo, 1982).Perhaps the most characteristic biological damage caused by radicals is their abilityto initiate the free radical chain reaction known as lipid peroxidation. This occurswhen the hydroxyl radicals are generated close to or within membranes and thenattack the fatty acid-side chains of the membrane phospholipids (Halliwell, 1987).On the other hand, radicals are detoxified by antioxidant systems including gluta-thione (GSH) and related enzymes (GSH-peroxidase, GSH-reductase), vitamin E orvitamin C, superoxide dismutase or catalase, and numerous polphenolic compounds(Liebler, 1993). When the rate of generation of free radicals exceeds the ability ofthe defense system to detoxify free radicals, this causes tissue damage (Halliwell &Chirico, 1993).

60Co is one of the most efficient radionuclides used as a source in therapeutictreatment. Normally, the activity of the sources used in radiotherapy ranges between3000 and 12 000 Rh�1 m (84 730–33 890 MBq) and the specific activities of thesesources range between 5550 and 11 100 GBq g�1. The activity of 60Co sourcesdecreases by 13.2% per year and at the end of 5 years the sources become mostlyinefficient for therapeutic purposes. Then the source from the unit is replaced byanother one and the original source is disposed of following the codes of practice(Johns & Cunningham, 1969).

Improper management of an old 60Co radiotherapy unit in Istanbul, Turkey causedsevere environmental contamination followed by a group of people receiving con-siderable radiation exposure. The lead shield of the radiotherapy unit was disposedof at an iron dealer without removing the source; this caused contamination of theenvironment and the workers and others were exposed externally and internally. Thevictims showing critical symptoms of acute radiation exposure were taken to differenthospitals within 3–5 days.

In the present study, changes in blood parameters are described, plasma anderythrocyte thiobarbituric acid substance (TBARS) levels and erythrocyte GSH levelsare monitored after being externally irradiated by 60Co.

2. Materials and methods

Ten male patients were selected for the study, aged 35±10 years, depending onthe severity of their symptoms and under their written consent. The duration and therate of radiation exposure could not be precisely reconstructed; however, all patientswere working for 3–5 days close to the source, at a distance of 1–2 m. According

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to the International Atomic Energy Agency (IAEA), the radiation dose to the victimswas within the range of 3.1±0.3–0.6±0.2 Gy (IAEA, 2000), with an approximateaverage individual dose of 1.7±0.3 Gy. As a control group, 10 male persons werechosen, aged 31±9 years, with good health status and free from any complicationsin liver and renal function.

Following 3–5 days of 60Co irradiation, blood samples (10 ml) were collected fromthe victims in tubes containing EDTA. After a whole blood sample was separated forthe determination of hemoglobin content and erythrocytes count, the remaining bloodwas centrifuged at 2000 rev min�1 for 10 min. The plasma was separated and buff-coat was discarded. Erythrocytes were washed three times with a cold sterile 9 gl�1 w/v sodium chloride solution in one to ten dilutions.

Plasma and erythrocyte lipid peroxidation levels were determined by measuringthe formation of malondialdehyde as thiobarbituric acid reactive materials (Buege &Aust, 1978). One volume of sample was mixed thoroughly with two volumes of astock solution of 15% w/v trichloroacetic acid, 0.375% w/v thiobarbituric acid and0.25 mol hydrochloric acid containing 0.001% butylated hydroxytoluene. The combi-nation of sample and stock solution was heated for 30 min in a boiling water bath.After cooling, the precipitate was removed by centrifugation for 10 min. The absorb-ency of the clear sample was determined at 535 nm and the TBARS concentrationwas calculated using 1.56×105 M�1 cm�1 as the molar absorption coefficient. Theintra- and inter-assay coefficients of variation for TBARS were 4.7 and 4.9%,respectively. Results were expressed as nmol of TBARS per ml of plasma (nmolml�1) or nmol of TBARS per g hemoglobin (nmol gHb�1). Hemoglobin levels weredetermined by the cyanmethaemoglobin reagent (Sigma Chemical Co., St Louis,MO).

Erythrocyte GSH levels were determined according to Beutler, Duran, and Kelly(1963), using metaphosphoric acid for protein precipitation and 5�5�-dithiobis-2nitrobenzoic acid for color development at 412 nm. The intra- and inter-assay coef-ficients of variation for GSH were 4.7 and 4.8%, respectively. The values wereexpressed as micromoles of GSH per gram of hemoglobin (µ mol gHb�1).

The levels of plasma urea, creatinine, glucose, total protein, fibrinogen, transamin-ases (SGOT and SGPT) and total bilirubin were measured using commercial enzy-matic kits (Sigma Chemical Co.).

All statistical comparisons were carried out by unpaired student’s t-test. Theunpaired student’s t-test was also validated by the non-parametric Wilcoxon test.Analysis of variance (ANOVA) was used to compare multiple-group means. p�0.05was considered statistically significant. All data are expressed as mean±SD.

3. Results and discussions

All the patients had common clinical symptoms such as nausea, vomiting tendencyand gingival bleeding. Two of them were suffering from lopecia, three patients weresuffering from pharyngeal erythema and one patient had labial erythema. In fourpatients, subfebril fever was observed.

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A comparison of the general blood levels of the victims with those of the healthycontrols is shown in Table 1. Blood glucose, urea, creatinine, SGOT, SGPT, totalprotein, and fibrinogen levels are within normal ranges. However, white blood celland platelet counts are found to be lower and a subsequent increase in the totalbilirubin levels is noted at a very significant level (p�0.001). Hematocrit and hemog-lobin levels are found to be lower at a significant level (p�0.01).

Table 2 shows a comparison of the plasma and erythrocyte TBARS levels, erythro-cyte GSH levels and protracted dose by dicentrics (IAEA, 2000) of victims withthose of healthy controls. Among the victims, a significant increase (p�0.001) inplasma and erythrocyte TBARS levels could be observed. At the same time, erythro-cyte GSH levels are found to decrease with corresponding values at the same levelof significance (p�0.001).

Oxidative stress may be defined as the measure of steady-state level of reactiveoxygen or oxygen radicals in a biological system. Increased oxidative stress mayresult from an overproduction of reactive precursors and/or a decreased efficiencyof the inhibitory and scavenger system. The increased number of oxygen radicalscan activate an autocatalytic cycle of metabolic stress, tissue damage, and cell deaththat further stimulates free radical production and compromises the inhibitory andscavenger mechanism (Menendez, Hacker, Sonnenfeld, Mc Connel, & Rivlin, 1984).Erythrocyte studies represent an early concept for studying oxidative stress. Erythro-cytes are subject to oxidative reactions because of relatively high oxygen tensions,the presence of hemoglobin and a rich plasma membrane in polyunsaturated lipid(Stocks, Offerman, & Modell Ormandy, 1972). Therefore, we have used erythrocytesas an indicator for the determination of oxidative stress of the radiation victims.

Despite individual clinical symptoms, it is clear from the results that the plasmaand erythrocyte lipid peroxidation levels of all the radiation victims are significantly

Table 1Blood levels of the victims and healthy persons

Healthy control (n=10) Victims (n=10)

White blood cells ( 103/mm3) 6.3±2.5 0.43±0.16∗∗Platelets (103/mm3) 150.0±350.0 19.6±2.3∗∗Hemoglobin 47±5 29.8±2.3∗Hematocrit (%) 41±3 25.8±1.3∗Total bilrubin (mg/dl) 0.52±0.41 2.22±0.51∗∗Glucose (% mg) 85±20 90±15Urea (% mg) 35±13 30±12Creatinine (% mg) 0.48±0.32 0.69±0.31SGOT (U/l) 32±11 27±9SGPT( U/l) 28±12 36±5Total protein (% mg) 7.9±2.6 7.9±0.6Fibrinogen (% mg) 295±73 355±105

Values are means±SD. ∗∗Significantly different from the controls at p�0.001 level. ∗Significantly differ-ent at p�0.01 level.

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Table 2Plasma and erythrocyte TBARS levels, erythrocyte GSH levels and protracted dose by dicentrics (IAEA,2000) among radiation victims and healthy persons

Patient No. Protracted dose by Plasma TBARS Erythrocyte TBARS Erythrocyte GSHdicentrics±S.D. (Gy) (nmol ml�1) (nmol gHb�1) (µmol Hb�1)

1 2.2±0.3 7.5 380 4.752 2.3±0.4 7.25 370.62 4.953 3.1±0.3 7.18 345.42 54 2.5±0.2 6.85 341.95 5.15 2.5±0.5 6.6 332.97 5.126 1.8±0.2 5.7 328.83 5.257 0.9±0.1 5.5 313.98 5.258 0.6±0.1 5.2 312.42 5.339 0.8±0.2 5.3 290.5 5.4910 0.6±0.2 5.4 288.45 5.82Average for 1.73±0.3patients 6.25±0.91∗∗ 330.51±30.53∗∗ 5.21±0.30∗∗Average for 3.72±0.68 150.6±20.5 10.2±0.7control(n=10)

Values are means±SD. ∗∗Significantly different from the control at p�0.001 level.

higher than in the corresponding healthy control group. Until now, studies on bio-chemical oxidative stress in radiation victims, especially those externally exposed to60Co, are rare. Here, the results can be explained in terms of oxidative stress asgenerated by radiation exposure. Radiation may lead to the intensification of lipidperoxidation, which is expressed by a significant increase in TBARS values. Anincrease in erythrocyte lipid peroxidation among thyroid cancer patients afterradioiodine treatment has been reported (Konukoglu, Hatemi, Arıkan, Demir, &Akcay, 1998; Bartoc, Dumeitrescu, Belgun, & Olinescu, 1993).

Simultaneously in this study, erythrocyte GSH levels are found to be decreasedafter 60Co irradiation. It is well known that in physiological processes, glutathioneacts as a protective agent against the generation of reactive oxygen species. Theincrease in oxidative stress may increase the production of GSH for the removal ofperoxides. The export of GSH from the erythrocyte membranes to the plasma dueto membrane damage causes a decrease in GSH levels (Bump & Brown, 1990). Itis suggested that antioxidant protection is determined by the cellular glutathionestatus and redox cycles delivery, reducing equivalents to cellular oxidants and hencecontrol glutathione status (Reed, 1990). Additionally, it is also reported that patientswith anemia have elevated lipid peroxidation (Kumerova, Lece, Skesters, Silova, &Petuhous, 1998). All the radiation victims in this study suffered from severe anemiadue to the depression of bone marrow.

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4. Conclusions

In the present study, a significant increase in plasma and erythrocyte TBARSlevels and a significant decrease in GSH levels have been demonstrated among radi-ation victims in comparison with normal healthy controls. These observations suggestsignificant changes in oxidant/antioxidant status in erythrocytes due to radiationamong the victims causing anemia. The results may constitute a biochemical basisfor therapeutic treatment among these victims with antioxidant nutrients, includingvitamin C, vitamin E and carotenoids (Maxwell, 1995). These compounds directlyscavenge reactive oxidants and enable a vital endogenous defense system to be con-structed against oxidative cell and tissue injury caused by toxic and carcinogenicirradiation among subjects exposed to radiation.

Acknowledgements

This work was supported by the research fund of the University of Istanbul, Tur-key. Project No. O-993/27042001.

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