Full text of DOI:10.1385/BTER:77:1:1

©Copyright 2000 by Humana Press Inc.
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0163–4984/00/7701–0001 $13.00
Accelerated Article
Antioxidant Defense and Trace
Element Imbalance in Patients
with Postradiation Syndrome
First Report on Phase I Studies**
,1
1
AUSMA O. KUMEROVA,* ANNA G. LECE,
1
2
ANDREJS P. SKESTERS, GRIGORIJS A. ORLIKOV,
JURIS V. SELEZNEV, AND K. D. RAINSFORD3
2
1Department of Medical Biochemistry, Biochemical Laboratory
2
and Paul Stradins Clinical State Hospital, Medical Academy
of Latvia, 16 Dzirciema Str., LV-1007, Riga, Latvia;
3
and Division of Biomedical Sciences, Sheffield Hallam
University, Howard St., Sheffield, United Kingdom
Received March 10, 2000; Accepted March 15, 2000
ABSTRACT
More than 6000 residents of Latvia were involved in recovery
work in Chernobyl. They were healthy men exposed to substantial ion-
izing radiation (0.01–0.5 Gy). Now, these recovery workers suffer from
“postradiation syndrome”: dizziness and poor memory, headache, local
pains, and so forth. The biochemical mechanism of “postradiation
syndrome” has not been completely established. In this Phase I study,
we have investigated how exposure to radiation impacts antioxida-
tive defense and trace element concentrations in the blood of recov-
ery workers. Thirty-five patients with postradiation syndrome (men,
age range 33–50 yr) and 15 healthy men similar in age as control sub-
jects were studied for the effects on plasma chemiluminescence, the
activity of antioxidant enzymes, and the concentration of ceruloplas-
min and concentrations of selenium (Se), zinc (Zn), and copper (Cu)
in blood. The results revealed that plasma chemiluminescence was
significantly increased (3.5-fold to 5.5-fold), the activity of catalase in
erythrocytes was significantly elevated, and the activity of glutathione
*Author to whom all correspondence and reprint requests should be addressed.
**Presented in part on the SFRR (Europe) Summer Meeting in Dresden, July
2–5, 1999.
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Kumerova et al.
peroxidase in plasma was significantly reduced in examined patients.
Concentrations of Zn and Cu were significantly higher and the con-
centration of Se was lower in these patients. We conclude that the
patients exposed to ionizing radiation have diminished blood antioxi-
dant defense associated with pronounced Se deficiency and imbalance
of Zn and Cu.
Index Entries: Antioxidant defense; catalase; glutathione perox-
idase; trace elements; postradiation syndrome.
INTRODUCTION
Environmental factors (pollutants, radiation, ozone concentration)
have a strong influence on the human health. The concentration of reac-
tive oxygen species (ROS) in the human body increases significantly
with the progressive increase of pollution in the environment. These
environmental factors have been estimated to occur at approx 3 × 104
incidences per cell per day (1). Ionizing radiation significantly increases
the concentration of ROS (1–3). Because of this, we have been interested
in investigating those patients who were exposed to ionizing radiation
during the Chernobyl Accident. Apart from radiation-induced effects,
these people also have mental stress and shock, with additional adverse
effects on their physical and mental health. The WHO has paid particu-
lar attention to the accident recovery workers, who were exposed to
comparatively low doses of ionizing radiation (0.01–0.5 Gy). Of par-
ticular long-term significance is the impact following a longer term
(approx 10 yr) of ionizing radiation exposure. The clinical picture of
accident recovery workers most commonly observed includes com-
plaints of headache (70%), dizziness and poor memory, prostration, and
lowered work ability (76%), excessive nervousness and local pains in the
bones (70–72%), and disorders of the digestive system (2). It would
therefore appear that the Chernobyl Accident recovery workers suffer
from “postradiation syndrome.”
The exact role of ROS in the pathogenesis of postradiation syndrome
has not been established. Additional psycho-emotional stress may com-
plicate the influence of radiation-induced oxidant stress. The accumu-
lation of long half-life radioactive isotopes incorporated into recovery
workers’ bodies would be expected to increase the production of ROS.
Increased levels of ROS have direct impact on antioxidant defense and
are risk factors for the development of several diseases. Enhanced con-
centrations of ROS have been implicated in over 100 human and animal
diseases (3). The antioxidant defense system protects tissues against at-
tack by ROS (4). Some authors have investigated the role of antioxidant de-
fense on the formation of postradiation syndrome (5,6), but the numbers of
studies are relatively few and the results obtained have been contradictory.
In view of this, we have decided to undertake long-term investigations
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Trace Elements in Postradiation Syndrome
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on the role of changes in the antioxidant defense system in manifesting
the development of postradiation syndrome in individuals from Latvia,
who were involved in recovery work following the Chernobyl Accident.
We report here the first, or Phase I, studies, these being part of long-term
longitudinal investigations that we are undertaking on these subjects.
MATERIAL AND METHODS
Patients
The study group comprised 35 residents of Latvia (men, age range
35–50 yr), who participated in the recovery work after the Chernobyl
Accident (1986–1991), admitted to the P. Stradin’s Clinical State Hospital
at Medical Academy of Latvia. All patients had clinical symptoms of
postradiation syndrome. We divided these into two subgroups compris-
ing those patients with polyneuropathy (PN, n = 25) and patients with
gastric ulcer (GU, n = 10). Fifteen healthy men (age range 25–50 yr) were
recruited as age-matched control subjects. The research design was
approved by the Ethics Committee of the Medical Academy of Latvia; all
subjects gave informed consent before participating in this study.
Reagents
All reagents used were analytical grade. Na2HPO4 · 2H2O, NaH2PO4
· H2O, KH2PO4, CCl3COOH, (NH4)6Mo7O24 · 4H2O, Tris-HCl, NaCl, H2O2
30%, and dimethyl sulfoxide were obtained from Merck (Darmstadt, Ger-
many). EDTA, luminol, and chloroform were purchased from Serva Fein-
biochemica GmbH&Co (Heidelberg, Germany) and reduced L-glutathione
from Fluka Chemie AG (Buchs, Switzerland). The water used was double
distilled (Sanyo Gallenkamp PLC, Leicester, UK).
Blood Samples
After overnight fasting, venous blood samples were collected from
all study subjects into Venoject II tubes with lithium heparin between 8
and 10 AM. Within 2 h after sampling, the blood was centrifuged at 1000g
for 10 min to separate the plasma. The erythrocytes were washed three
times in cold saline (8.6 g/L NaCl). For the assay of catalase activity,
the erythrocytes were lysed in cold (4°C) 5 mM Tris-HCl, pH 7.4 (1/9 by
volume). After 15 min, 0.4 mL ice-cold chloroform–ethanol (15/25 by vol-
ume) was added. The solutions were thoroughly mixed and placed in an
acetone-ethanol (50/50 by volume) liquid-nitrogen bath (−30°C to −25°C)
for 30 min, then centrifuged at 10,000g for 10 min. The upper aqueous
phase was retained for assay. For the assay of glutathione peroxidase
the washed erythrocytes were lysed in ice-cold ultrapure water (1/9 by
volume).
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Luminol-Enhanced Chemiluminescence
Chemiluminescence intensity was determined as described (7). The
reaction mixture comprised 10 µL blood plasma, 2 mL 0.2M phosphate
buffer, pH 7.8, and 100 µL of 10−5M luminol. After incubation at 37°C for
3 min, the reaction was initiated by the addition of 500 µL of 0.03% H2O2.
The time-dependent chemiluminescence responses were recorded on a
portable tube luminometer EMILITE EL 1105 (BioChimMak, Russia/Latvia)
using a chart recorder Servogor 310 (BBC, Goers, Austria). Total light emis-
sion was estimated as the area under the curve, and the maximal intensity
of chemiluminescence flash was calculated. The total light emission gives a
measure of the total oxidizability of plasma (S ox), the chemiluminescence
flash reflects hydroperoxides concentration (H max) (8).
Assay of Catalase Activity
Catalase (CAT) activity was determined using the method described
by Korolyuk et al. (9). The 50 µL of sample was incubated in the pres-
ence of 2 mL of 0.03% H2O2. The decomposition of H2O2 was stopped
after 10 min with 1000 µL of 4% NH4MoO7. The absorbance of colored
products was detected at 410 nm on the spectrophotometer (SPECORD
M-40, Jena, Germany).
Assay of Glutathione Peroxidase Activity
The activity of glutathione peroxidase (GSH-Px) in both plasma and
hemolysate was determined by the method described in ref. 10. This assay
requires hydrogen peroxide as a substrate and is based on the detection
of oxidized glutathione. The reaction mixture contains 1000 µL of 0.3M
potassium–sodium phosphate buffer, pH 7.4, with 12 mM NaN3 and
6 mM EDTA, 200 µL of the sample (diluted fourfold), and 500 µL 2.5 mM
reduced glutathione were incubated at 37°C for 5 min, then added to
500 µL of 1.8 mM H2O2. Reaction was stopped after 2 min by adding
1000 µL of cold (4°C) 10% CCl3COOH. Samples were centrifuged at 2500g
for 15 min, and the absorbance was detected at 260 nm on the spectro-
photometer (SPECORD M-40, Jena, Germany). The specific enzyme activ-
ities were expressed as international units per liter (IU/L) or per gram of
hemoglobin (IU/g Hb). All measurements were performed in duplicate.
Hb Detection
Hemoglobin concentrations were determined spectrophotometrically
by the standard colorimetric cyanomethaemoglobin method (11).
Determination of Ceruloplasmin
Ceruloplasmin concentration in serum was detected with a Dade
Behring standard kit based on immunochemical reaction, using a neph-
elometer BN 100 (Dade Behring GmbH, Germany).
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Trace Elements in Postradiation Syndrome
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Determination of Trace Elements
A highly sensitive analytical method of using inductively coupled
plasma emission–mass spectrometry was used for the detection sele-
nium, zinc, and copper concentrations in whole-blood samples (12).
Statistical Analysis
Using the SPSS 6.0 software standard version, statistical analyses
were performed. Differences in means between groups were analyzed by
the independent-sample t-test. Spearman’s correlation coefficient was
used to investigate association between parameters. A p-value of 0.05 or
less was considered statistically significant.
RESULTS
Luminol-Enhanced Chemiluminescence
We used chemiluminescence for the detection of plasma oxidizabil-
ity that illustrates total antioxidant status or total antioxidant defense of
plasma. Figure 1 shows the maximal intensity of chemilumenescence
flash and oxidizability of plasma in irradiated patients. It is clear that
hydroperoxides concentration in plasma of irradiated patients is signifi-
cantly increased (approx 5.5 times), and elevating was more expressed
for patients with a gastric ulcer (GU) than for patients with PN. Figure 1
shows that oxidizability of plasma of irradiated patients is elevated
(approx 3.5 times). Similar to the concentration of hydroperoxides, the total
oxidizability of plasma on patients with GU is elevated more convincing
that on patients with polyneuropathy (PN).
Activities of Antioxidant Enzymes
The increase of plasma oxidizability and corresponding decrease of
total antioxidant defense could be associated with changes of antioxidant
enzyme activities. Therefore, we assayed the activity of GSH-Px in
plasma. Figure 2 illustrates the obtained data for GSH-Px activity in plasma
of irradiated patients. GSH-Px activity was significantly decreased (by
30–40%) on the entire patient group as well as on patients with GU and
PN as compared with healthy controls (p < 0.05). No significant alter-
ations were observed between groups of patients. It was interesting to note
how diminishing plasma total antioxidative defense affects the anti-
oxidant system of the blood cells (erythrocytes). For this purpose, we
assessed the activities of the following antioxidant enzymes: GSH-Px,
superoxide dismutase (SOD), and CAT. The activity of GSH-Px in ery-
throcytes was not different from the controls either on the whole patient
group or on patients with GU and PN (Fig. 3), and corresponded to the
normal values obtained on healthy subjects. Erythrocyte SOD activity
also was similar to that from normal apparently healthy subjects (data
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Fig. 1. Chemiluminescence of plasma: hydroperoxide concentration (H max)
and total oxidizability (S ox) in examined patients and healthy controls. Data are
presented as mean value SEM of each separate group. The asterisk represents
a statistically significant difference versus controls. Samples were collected and
analyzed as described in the section Materials and Methods. Abbreviations:
HC—healthy control subjects; Patients—the entire study group comprised irra-
diated patients; PN—irradiated patients with polyneuropathy; GU—irradiated
patients with gastric ulcer.
not shown); the activity of CAT in erythrocytes of irradiated patients was
significantly higher than in the control: for the entire group the increase
was 3.2-fold, but for patients with GU, it was 4.2-fold (p < 0.05) (Fig. 3).
High CAT activity provides adequate antioxidative defense in erythro-
cytes to fulfill these cells’ functions.
Trace Element Concentrations
Because the data showed that there was a significant decrease of
GSH-Px activity in plasma, it seems to be important to assess Se concen-
tration (four Se atoms are necessary to ensure GSH-Px activity). Data
obtained show that Se levels were low in blood from the investigated
patients (Fig. 4). In the entire patient group, the Se concentration in blood
was, on average, twofold lower compared with reference normal values
for the European population (13). We emphasize that patients with GU
had very low Se concentrations in the blood. The lowest Se concentration
we detected in an irradiated patient was 17 µg/L. We found statistically
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Fig. 2. Activity of GSH-Px in plasma. Data are presented as mean value
SEM of each separate group. The asterisk represents a statistically significant
difference versus controls. Samples were collected and analyzed as described in
the section Materials and Methods. Abbreviations are as for Fig. 1.
Fig. 3. Activity of CAT and GSH-Px in erythrocytes of examined patients
and controls. There were no significant differences in activities of intracellular
GSH-Px between the patients and controls. Data are presented as mean value
SEM of each separate group. The asterisk represents a statistically significant dif-
ference versus controls. Samples were collected and analyzed as described in the
section Materials and Methods. Abbreviations are as for Fig. 1.
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Fig. 4. Selenium concentration in whole blood of irradiated patients. Data
are presented as mean value
SEM of each separate group. Samples were
collected and analyzed as described in the section Materials and Methods.
Abbreviations: Patients—the entire study group comprised irradiated patients;
PN—irradiated patients with polyneuropathy; GU—irradiated patients with
gastric ulcer; Norm—normal values (13).
significant positive correlation between GSH-Px activity in plasma and
Se concentration in blood for the whole patient group (r = 0.474, p < 0.05)
and for patients with PN (r = 0.589, p < 0.05).
Our previous study showed that apparently healthy residents of
Latvia have marginal Se deficiency because of the poor availability of soil
Se for plants (acid, poorly aerated soil with high humidity and iron con-
tent). It is known that low Se content influences other trace element
concentrations in blood. From this point of view, it was significant to
determine the concentration of Zn and Cu in the blood of irradiated
patients. Data obtained showed the Zn concentration increased by 50%
in the entire patient group (Fig. 5). At the same time, the Zn concen-
tration in the blood of patients with GU tended to decrease as compared
to patients with PN. Cu is an essential element involved in a variety of
enzyme systems, including SOD. Our results demonstrated that the Cu
concentration in blood of examined patients was very high (Fig. 6).
The increase was more than threefold for the whole patient group as
compared to normal referent values (13). These undoubtedly impact on
the Cu/Zn ratio in serum. The observation of hypercupremia leads to the
question: Are the Cu ions in blood bound to ceruloplasmin (the main
transport protein for Cu in blood)? Usually, in different pathological con-
ditions, the increase of Cu concentration in blood is accompanied with a
corresponding increase of ceruloplasmin concentration in plasma. The
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Trace Elements in Postradiation Syndrome
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Fig. 5. Concentration of zinc in whole blood of irradiated patients. Data
are presented as mean value SEM of each separate group. Samples were col-
lected and analyzed as described in the section Materials and Methods. Abbre-
viations are as in Fig. 4.
Fig. 6. Concentration of copper in whole blood of irradiated patients. Data
are presented as mean value SEM of each separate group. Samples were col-
lected and analyzed as described in the section Materials and Methods. Abbre-
viations are as in Fig. 4.
ceruloplasmin concentration in the serum of irradiated patients was not
different from accepted normal values or in the entire patient group, nor
for patients with PN or GU (mean SD, g/L: 0.37 0.095, 0.37 0.10,
0.38 + 0.10, respectively).
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DISCUSSION
The chemiluminescence method is highly sensitive and suitable for
investigators. Reported data (14) demonstrate that ionizing radiation
increases plasma chemiluminescence. Our results confirmed this change.
Using the chemiluminescence method, we found that hydroperoxide con-
centration in plasma was markedly increased in the patient group. Oxi-
dizability of plasma also was enhanced, and this indicates a diminished
antioxidative defense. These results are in accordance with that reported
by Ukrainian and Russian investigators (15,16), who observed a reduction
in antioxidant defense after ionizing radiation exposure.
Antioxidant enzymes are an important part of the defense mecha-
nism against free-radical damage. The decrease of enzyme GSH-Px ac-
tivity in plasma we observed may have two causes. At first, increased
concentrations of ROS in blood of examined patients affect the activity of
GSH-Px. The second cause may be connected with decline of Se concen-
tration, because GSH-Px contains Se as a functional component (17). The
latter is confirmed by the positive correlation between GSH-Px activity in
plasma and Se concentration in blood. The correlation between these
variables has been reported by other authors (18). Se is incorporated into
the plasma protein P structure, which seems to have an antioxidative
function (19). Thus, the decline in Se concentration in blood significantly
affects the blood total antioxidative defense. The decrease of Se concen-
tration in blood following exposure to ionizing radiation was previously
reported by Golubkina and co-workers (20).
Cu,Zn-SOD, which catalyzes dismutation of the superoxide anion
radical, is a very important enzyme for ensuring antioxidative defense.
In some pathological conditions, Cu,Zn-SOD activity is enhanced at the
beginning of the disease, followed by a decrease of enzyme activity (21).
Conversely, we have not found changes in the activity of Cu,Zn-SOD in
erythrocytes of recovery workers as compared with healthy subjects. We
propose that this is the result of increased concentration of Zn and Cu in
the blood of examined patients.
Among intracellular antioxidant enzymes we have studied, only ery-
throcyte CAT activity was increased. Recovery workers had not only
been exposed to external radiation but also exposed to internal radiation
sources from cesium and strontium isotopes, which may have caused the
increase formation of ROS, including H2O2. CAT detoxifies H2O2, which
seems to be very important for the prevention of extremely reactive
hydroxyl radical formation from H2O2. We propose that increased CAT
activity in erythrocytes on irradiated patients serves as defense mecha-
nism for the decrease of H2O2 concentration to protect cells from damage
caused by an aggressive hydroxyl radical.
Iron and Cu ions are known to catalyze hydroxyl radical formation.
The enhanced Cu concentration in the blood of irradiated patients may
be very important from this point of view. Increased Cu concentration in
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Trace Elements in Postradiation Syndrome
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blood was found in infectious diseases, blood pathologies, neurologic
syndromes, and cancer. In these cases, hypercupremia correlates with
increased concentration of the copper-binding protein ceruloplasmin in
blood plasma (22). Conversely, we have not found an increase in cerulo-
plasmin content in recovery workers; values of this protein were not dif-
ferent from reference normal values. Savitskij and Gotsuljak previously
reported a correlation between ceruloplasmin and Cu concentrations in
blood as an effect of radiation exposure (23). Ceruloplasmin carries about
80% of Cu present in serum, and other plasma proteins (especially albu-
min) also can bind Cu ions. As far as we know, there are no reports in
the literature concerning the significant increase of the plasma proteins
in blood in persons participating in recovery works following the Cher-
nobyl Accident. The results obtained indicate that it is possible to
hypothesize that patients may have free Cu ions, not bound to proteins,
in blood. Perhaps, these Cu ions provoke hydroxyl radical formation via
the Fenton reaction and promote the increase of ROS concentration in
blood of recovery workers.
In conclusion, our data indicate that relatively low doses of ionizing
radiation (0.01–0.5 Gy), received by Chernobyl Accident recovery work-
ers, after a 10-yr period caused Se deficiency and trace elements (Zn, Cu)
overload in blood that provoke the decrease of the blood antioxidant
defense.
ACKNOWLEDGMENTS
The study was partly supported by grants 93.927 and 96.0763 from
the Science Council of Latvia. We wish to thank Dr. I. Jaunalksne for
ceruloplasmin detection.
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