American Journal of Industrial Medicine 22243-247 (1992)
Biological Monitoring of Three 6oCoRadiation Incident Victims Sanja MilkoviC-Kraus, MD, PhD, Dragan Kubelka, MS, and Branko VekiC, PhD
Radiation-induced structural chromosomal aberrations were the only adverse effects found in three workers overexposed to 6oCo y-radiation. Chromosomal analyses were performed on the 5th and 75th days after exposure. Hematologic follow-up was carried out on the 5th, 12th, 16th, and 75th days after exposure. Hematologic findings did not differ over time in any of the three exposed workers. In two workers, we found dicentric chromosomes, which are conventional indicators for exposure to ionizing radiation. 0 1992 Wiley-Liss, Inc.
Key words: chromosomal aberrations, biological monitoring, y-radiation, ionizing radiation exposure
INTRODUCTION
On March 8, 1991, a radiation incident occurred at the y-ray facility located in Zagreb, Croatia. Three men ( A X ) were exposed to an unshielded 6oCo source of radiation. The object of this communication is to indicate the value of chromosomal aberrations analysis in comparison with hematologic and clinical parameters. The workers were at various distances from the source. Worker A was very close to the unshielded source, and the left side of his body, including the dosimeter, were closest to the 6oCo source. Consequently, the right side of his body, as well as his back, received considerably lower doses, but his feet were probably most irradiated. Workers B and C were at distances of 0.8 and 1.2 m, respectively, and they were irradiated more uniformly. Chromosomal aberration analysis (CAA) and hematologic follow-up were initiated on the fifth day after exposure, when the three exposed workers reported to an occupational health physician. No clinical signs of exposure were detected. Each of the exposed workers previously had regular annual checkups; we therefore had their CAA findings from 1986 and a hematologic follow-up for each subsequent year. Annual hematologic checkups are obligatory by law, in order to obtain work certification. Fortunately, in worker B, we had a CAA and hematologic report for 1 Institute for Medical Research and Occupational Health, University of Zagreb, Zagreb, Croatia (S.M.-K., D.K.). Ruder BoSkoviC Institute, Zagreb, Croatia (B.V.). Address reprint requests to Dr. Sanja M i k o v i f - b u s , Institute for Medical Research and Occupational Health, Ksaverska 2, Zagreb, Croatia. Accepted for publication November 3, 1991. 0 1992 Wiley-Liss, Inc.
244
MilkoviC-Kraus et al.
month prior to the incident. Workers A and C were due for their CAA 2 months later. We present CAA of all three workers for the 5 years prior to the incident and on the 5th and 75th day after the incident. DOSIMETRY
All three men were radiation workers and wore thermoluminescent dosimeters (TLDs) (CaF,:Mn produced at the Joief Stefan Institute, Ljubljana, Slovenia) on the left side of their chests. The readout of TLDs was made the day of the overexposure incident. A TOLEDO 654 reader (Vinten) was used. The radiation doses were 152.67 mSv for worker A, 33.70 mSv for worker B, and 29.70 mSv for worker C. In the conditions in which the incident occurred, scattered radiation of lower energies may have been present. The high sensitivity of the CaF2:Mn dosimeter shows considerable energy dependence. For this reason, the precision of the dose measurements was later checked by LiFMg, Ti (produced at the Institute for Nuclear Physics, Krakow, Poland), which has low energy dependence [Ranogajec Komor and Osvay, 1986; VekiC et al., 19901. The agreement between doses measured by CaF,: Mn and by LiF:Mg,Ti was satisfactory. DESCRIPTION OF THE INCIDENT
In the incident, three men were exposed to y-radiation emitted from a @ C o‘ source that was unintentionally moved from the container to the “up position.” The incident occurred during preparation for replenishment of 6oCo sources at the y-ray facility, which consists of a cylindrical irradiator with 24 source holders of the same number of the guiding tube of the irradiator. At the time of the incident, there was a complete system in every other guiding tube, including the source holder with 52 TBq (1,400 Ci) at the bottom of the container. One of the source holders with 52 TBg (1,400 Ci) of 6oCowas moved from the container to an unprotected part of the guiding tube. The exposure lasted approximately 2-5 sec. MATERIALS AND METHODS
We present hematologic and CAA data prior to the incident and immediately after, as well as a follow-up after the 5th and 75th days for CAA and the 5th, 12th, 16th, and 75th day for hematologic findings. Lymphocyte cultures were initiated immediately after blood samples of the three workers were collected and cultured for 48 hr. Fixation of the cultures and preparation of the slides were carried out according to conventional methods. Complete metaphases stained by Giemsa were used for analyses: 200 cells per person were scored for chromatid and dichromatic chromosomal aberrations [IAEA, 19861. Only well-distinguished metaphases with accompanying accentric fragments were scored for analysis of CAA. The material for the first total blood count was drawn from the same puncture. The following blood counts were analyzed in the same laboratory by the same technician approximately at the same time of day, in order to avoid daily variations in the lymphocyte (L) count. The last blood count and second cytogenetic analysis were similarly performed.
Biological Monitoring of 6oCoRadiation Incident Victims
245
RESULTS Table I lists all hematologic and cytogenetic findings prior to and after the incident. No marked hematologic changes were observed in any of the three workers, in contrast to cytogenetic findings. Worker A, who received three times the annual dose limit, had the most severe changes in chromosomes in comparison with his initial cytogenetic analysis taken in 1986. Worker B, who received a significantly lower dose of y-radiation than worker A, which amounted to less than the annual TLV, still had CAA findings that showed the ionizing radiation effect on circulating lymphocytes, with special emphasis on the findings one month prior to the incident, as well as the analysis taken in 1986 (Table I), which showed no chromosomal aberrations. Worker C, who received the smallest amount of y-radiation, showed no effect of the radiation on chromosomes. The total annual doses that these three workers received during the period 1986-1990 are presented in Table 11. In workers A and B, we found dicentric chromosomes, which are conventional indicators for exposure to ionizing radiation [Littlefield et al,. 19901. We found no dicentric chromosomes in worker C. No hematologic changes occurred in any of the three workers (see Table I). DISCUSSION
Whereas recommendations issued by WHO do not include cytogenetic analysis in pre-employment or periodic examinations, but only a medical history, physical examination, and blood count [WHO, 19861, we enforce cytogenetic analysis in both health examinations within a 5-year period. This is because ionizing radiation is one of the many occupational carcinogens that can activate ras-oncogenes frequently found in numerous types of human cancers, and different types of exposure induce different types of ras-oncogene activation [Balmain and Brown, 19881. Radiation damage increases with dose and is greater at high dose rates than at low dose rates. This can be recognized by the early effects, such as cell killing and chromosome breaks, and also in the late effects, such as induction of cancer and hereditary diseases [Erikson et al., 19831. Some studies show that the risk of radiation-induced cancers begins to decrease many years after exposure [Brandt-Rauf, 19901. Nevertheless, all populations occupationally exposed to ionizing radiation should be medically followed up throughout their lifetime, and current dose limits should be decreased from 50 mSv year-' to 20 mSv year-', as recommended by Hughes [1990]. Even worker B, who received less than the maximal permitted dose, had an increased number of chromosomal aberrations, including dicentric chromosomes. Chromosomal aberration analysis should be obligatory for such follow-up evaluations of occupationally exposed populations; when the gene becomes transposed from its original position, and when chromosome breaks occur by ionizing radiation, cell transformation may result, and proto-oncogenes could be activated [Ericson et al., 19831. It is well demonstrated in this work that no significant hematologic changes occurred, either during work in ionizing radiation exposure or after overexposure as described in this incident. However, cytogenetic analysis demonstrated excessive exposure to ionizing radiation in two workers. Prior to the incident, none of the three had dicentric or ring chromosomes, which were only found after the incident. On the 75th day after the excessive exposure to y-radiation, a smaller number of chromosomal aberrations were found, while whole blood count was still unchanged.
144
151 144 144
4.30 4.36 3.98 4.02 4.06 4.32 120 131 202
160
220 224
171 158 225 21 1 165 160 193
172 165 I62 161 155 166
5.95 4.65 7.10 7.95 9.30 7.50
6.30 5.30 6.15 6.60 6.30 6.45 5.00
3.55 5.65 4.25 3.85 4.45 5.30
Platelets Leukocytes (nu (nl)
0.27 0.38 0.36 0.33 0.29 0.37
0.30 0.39 0.35 0.43 0.41 0.42 0.37
0.38 0.38 0.38 0.39 0.32 0.45
(a)
Lymphocytes
-
1.5 3 2.5
6.5
2 1.5 5.5 -
1 7 5
(%)
Chromosomal aberrations
~~
Accentric
Ring
Dicentric
"Baseline, 1990 and 1991 (mandatory yearly), and postincident. bBaseline and postincident, except for worker B, whose 5-year mandatory 1991 data had been obtained 1 month prior to incident. 'One month prior to the incident.
1991 5th 12th 16th 75th
1990
144 139
149 143 153 152 153 156 142
Worker B 1986 1990 1991' 1991 5th 12th 16th 75th
Worker C 1986
4.41 4.71 4.5 1 4.40 4.28 4.56
142 148 156 147 152 145
Yearlday Worker A 1986 1990 1991 5th 12th 16th 75th 4.50 4.55 4.70 4.79 4.66 4.61 4.24
Erythrocytes (PIL)
Hemoglobulin (g/L)
TABLE I. Hematologic' and CytogeneticbData for Three Workers Overexposed to Ionizing Radiation: 1991 Chromatid breaks
Chromosomal breaks
Biological Monitoring of “Co Radiation Incident Victims
247
TABLE 11. Total Annual Doses of mSv During the Period 19861990 Year
Worker A (mSv)
Worker B (mSv)
Worker C (mSv)
1986 1987 1988 1989 1990
0.00 0.33 0.07 0.25 0.22
0.00 0.11 0.30 0.01 0.00
0.00 0.03 0.60 0.01 0.12
CONCLUSION
Following an incident of overexposure to y-radiation, the cytogenetic and hematologic parameters of three workers were assessed and compared with prior data. The worker closest to the source showed the greater changes in CAA; none showed hematologic changes. We recognize that the number of cells analyzed was too small to estimate the dose on the basis of frequency of bicentric chromosomes, and that individual and interlaboratory differences exist. However, the aim of this study was not to estimate dose, but rather an attempt to demonstrate that analysis of chromosomal aberration indicates the changes occurring in the organism prior to changes in the blood count, the parameter recommended by WHO. As clearly shown in Table I, exposure to ionizing radiation at doses even lower than the TLV affected chromosomes, but not the hematologic values. We believe that cytogenetic analysis should be enforced in pre-employment, as well as in periodic examinations, in order to evaluate the biological effect of ionizing radiation in occupationally exposed populations. REFERENCES Balmain A, Brown K (1988): Oncogene activation in chemical carcinogenesis. Adv Cancer Res 51: 147-182. Brandt-Rauf PW ( 1990): Oncogene proteins as biomarkers in molecular epidemiology of occupational carcinogenesis. Int Arch Occup Environ Health 63:l-8. Ericson J, Rushdu A, Drawings HL, Nowel PC, Croec CH (1983): Transcriptional activation of the translocated C-miyc oncogene in Burkitt lymphoma. Proc Natl Acad Sci USA 80320-824. Hughes D (1990): The revision of dose limits for exposure to ionizing radiation. Ann Occup Hyg 34535-539. Littlefield LG, Jonier EE, Colyer SP, Ricks RC, Lushbaugh CC, Hurdato-Monroy R (1991): The 1989 San Salvador 6oCo radiation accident: Cytogenetic dosimetry and follow-up evaluation in three accident victims. Radiat Protect Dosim 35:115-123. Ranogajec-Komor M, Osvay M (1986): Dosimetric characteristics of different TL phosphorus. Radiat Prot Dosim 17:379-384. Vekie B, Ranogajec-Komor M, &danan M (1990): Comparison of the responses of several TLDs to various radionuclides. Radiat Prot Dosim 33:335-338. World Health Organisation (1986): “Early Detection of Occupational Diseases.” Geneva: WHO.
Biological Monitoring of Three 6oCoRadiation Incident Victims Sanja MilkoviC-Kraus, MD, PhD, Dragan Kubelka, MS, and Branko VekiC, PhD
Radiation-induced structural chromosomal aberrations were the only adverse effects found in three workers overexposed to 6oCo y-radiation. Chromosomal analyses were performed on the 5th and 75th days after exposure. Hematologic follow-up was carried out on the 5th, 12th, 16th, and 75th days after exposure. Hematologic findings did not differ over time in any of the three exposed workers. In two workers, we found dicentric chromosomes, which are conventional indicators for exposure to ionizing radiation. 0 1992 Wiley-Liss, Inc.
Key words: chromosomal aberrations, biological monitoring, y-radiation, ionizing radiation exposure
INTRODUCTION
On March 8, 1991, a radiation incident occurred at the y-ray facility located in Zagreb, Croatia. Three men ( A X ) were exposed to an unshielded 6oCo source of radiation. The object of this communication is to indicate the value of chromosomal aberrations analysis in comparison with hematologic and clinical parameters. The workers were at various distances from the source. Worker A was very close to the unshielded source, and the left side of his body, including the dosimeter, were closest to the 6oCo source. Consequently, the right side of his body, as well as his back, received considerably lower doses, but his feet were probably most irradiated. Workers B and C were at distances of 0.8 and 1.2 m, respectively, and they were irradiated more uniformly. Chromosomal aberration analysis (CAA) and hematologic follow-up were initiated on the fifth day after exposure, when the three exposed workers reported to an occupational health physician. No clinical signs of exposure were detected. Each of the exposed workers previously had regular annual checkups; we therefore had their CAA findings from 1986 and a hematologic follow-up for each subsequent year. Annual hematologic checkups are obligatory by law, in order to obtain work certification. Fortunately, in worker B, we had a CAA and hematologic report for 1 Institute for Medical Research and Occupational Health, University of Zagreb, Zagreb, Croatia (S.M.-K., D.K.). Ruder BoSkoviC Institute, Zagreb, Croatia (B.V.). Address reprint requests to Dr. Sanja M i k o v i f - b u s , Institute for Medical Research and Occupational Health, Ksaverska 2, Zagreb, Croatia. Accepted for publication November 3, 1991. 0 1992 Wiley-Liss, Inc.
244
MilkoviC-Kraus et al.
month prior to the incident. Workers A and C were due for their CAA 2 months later. We present CAA of all three workers for the 5 years prior to the incident and on the 5th and 75th day after the incident. DOSIMETRY
All three men were radiation workers and wore thermoluminescent dosimeters (TLDs) (CaF,:Mn produced at the Joief Stefan Institute, Ljubljana, Slovenia) on the left side of their chests. The readout of TLDs was made the day of the overexposure incident. A TOLEDO 654 reader (Vinten) was used. The radiation doses were 152.67 mSv for worker A, 33.70 mSv for worker B, and 29.70 mSv for worker C. In the conditions in which the incident occurred, scattered radiation of lower energies may have been present. The high sensitivity of the CaF2:Mn dosimeter shows considerable energy dependence. For this reason, the precision of the dose measurements was later checked by LiFMg, Ti (produced at the Institute for Nuclear Physics, Krakow, Poland), which has low energy dependence [Ranogajec Komor and Osvay, 1986; VekiC et al., 19901. The agreement between doses measured by CaF,: Mn and by LiF:Mg,Ti was satisfactory. DESCRIPTION OF THE INCIDENT
In the incident, three men were exposed to y-radiation emitted from a @ C o‘ source that was unintentionally moved from the container to the “up position.” The incident occurred during preparation for replenishment of 6oCo sources at the y-ray facility, which consists of a cylindrical irradiator with 24 source holders of the same number of the guiding tube of the irradiator. At the time of the incident, there was a complete system in every other guiding tube, including the source holder with 52 TBq (1,400 Ci) at the bottom of the container. One of the source holders with 52 TBg (1,400 Ci) of 6oCowas moved from the container to an unprotected part of the guiding tube. The exposure lasted approximately 2-5 sec. MATERIALS AND METHODS
We present hematologic and CAA data prior to the incident and immediately after, as well as a follow-up after the 5th and 75th days for CAA and the 5th, 12th, 16th, and 75th day for hematologic findings. Lymphocyte cultures were initiated immediately after blood samples of the three workers were collected and cultured for 48 hr. Fixation of the cultures and preparation of the slides were carried out according to conventional methods. Complete metaphases stained by Giemsa were used for analyses: 200 cells per person were scored for chromatid and dichromatic chromosomal aberrations [IAEA, 19861. Only well-distinguished metaphases with accompanying accentric fragments were scored for analysis of CAA. The material for the first total blood count was drawn from the same puncture. The following blood counts were analyzed in the same laboratory by the same technician approximately at the same time of day, in order to avoid daily variations in the lymphocyte (L) count. The last blood count and second cytogenetic analysis were similarly performed.
Biological Monitoring of 6oCoRadiation Incident Victims
245
RESULTS Table I lists all hematologic and cytogenetic findings prior to and after the incident. No marked hematologic changes were observed in any of the three workers, in contrast to cytogenetic findings. Worker A, who received three times the annual dose limit, had the most severe changes in chromosomes in comparison with his initial cytogenetic analysis taken in 1986. Worker B, who received a significantly lower dose of y-radiation than worker A, which amounted to less than the annual TLV, still had CAA findings that showed the ionizing radiation effect on circulating lymphocytes, with special emphasis on the findings one month prior to the incident, as well as the analysis taken in 1986 (Table I), which showed no chromosomal aberrations. Worker C, who received the smallest amount of y-radiation, showed no effect of the radiation on chromosomes. The total annual doses that these three workers received during the period 1986-1990 are presented in Table 11. In workers A and B, we found dicentric chromosomes, which are conventional indicators for exposure to ionizing radiation [Littlefield et al,. 19901. We found no dicentric chromosomes in worker C. No hematologic changes occurred in any of the three workers (see Table I). DISCUSSION
Whereas recommendations issued by WHO do not include cytogenetic analysis in pre-employment or periodic examinations, but only a medical history, physical examination, and blood count [WHO, 19861, we enforce cytogenetic analysis in both health examinations within a 5-year period. This is because ionizing radiation is one of the many occupational carcinogens that can activate ras-oncogenes frequently found in numerous types of human cancers, and different types of exposure induce different types of ras-oncogene activation [Balmain and Brown, 19881. Radiation damage increases with dose and is greater at high dose rates than at low dose rates. This can be recognized by the early effects, such as cell killing and chromosome breaks, and also in the late effects, such as induction of cancer and hereditary diseases [Erikson et al., 19831. Some studies show that the risk of radiation-induced cancers begins to decrease many years after exposure [Brandt-Rauf, 19901. Nevertheless, all populations occupationally exposed to ionizing radiation should be medically followed up throughout their lifetime, and current dose limits should be decreased from 50 mSv year-' to 20 mSv year-', as recommended by Hughes [1990]. Even worker B, who received less than the maximal permitted dose, had an increased number of chromosomal aberrations, including dicentric chromosomes. Chromosomal aberration analysis should be obligatory for such follow-up evaluations of occupationally exposed populations; when the gene becomes transposed from its original position, and when chromosome breaks occur by ionizing radiation, cell transformation may result, and proto-oncogenes could be activated [Ericson et al., 19831. It is well demonstrated in this work that no significant hematologic changes occurred, either during work in ionizing radiation exposure or after overexposure as described in this incident. However, cytogenetic analysis demonstrated excessive exposure to ionizing radiation in two workers. Prior to the incident, none of the three had dicentric or ring chromosomes, which were only found after the incident. On the 75th day after the excessive exposure to y-radiation, a smaller number of chromosomal aberrations were found, while whole blood count was still unchanged.
144
151 144 144
4.30 4.36 3.98 4.02 4.06 4.32 120 131 202
160
220 224
171 158 225 21 1 165 160 193
172 165 I62 161 155 166
5.95 4.65 7.10 7.95 9.30 7.50
6.30 5.30 6.15 6.60 6.30 6.45 5.00
3.55 5.65 4.25 3.85 4.45 5.30
Platelets Leukocytes (nu (nl)
0.27 0.38 0.36 0.33 0.29 0.37
0.30 0.39 0.35 0.43 0.41 0.42 0.37
0.38 0.38 0.38 0.39 0.32 0.45
(a)
Lymphocytes
-
1.5 3 2.5
6.5
2 1.5 5.5 -
1 7 5
(%)
Chromosomal aberrations
~~
Accentric
Ring
Dicentric
"Baseline, 1990 and 1991 (mandatory yearly), and postincident. bBaseline and postincident, except for worker B, whose 5-year mandatory 1991 data had been obtained 1 month prior to incident. 'One month prior to the incident.
1991 5th 12th 16th 75th
1990
144 139
149 143 153 152 153 156 142
Worker B 1986 1990 1991' 1991 5th 12th 16th 75th
Worker C 1986
4.41 4.71 4.5 1 4.40 4.28 4.56
142 148 156 147 152 145
Yearlday Worker A 1986 1990 1991 5th 12th 16th 75th 4.50 4.55 4.70 4.79 4.66 4.61 4.24
Erythrocytes (PIL)
Hemoglobulin (g/L)
TABLE I. Hematologic' and CytogeneticbData for Three Workers Overexposed to Ionizing Radiation: 1991 Chromatid breaks
Chromosomal breaks
Biological Monitoring of “Co Radiation Incident Victims
247
TABLE 11. Total Annual Doses of mSv During the Period 19861990 Year
Worker A (mSv)
Worker B (mSv)
Worker C (mSv)
1986 1987 1988 1989 1990
0.00 0.33 0.07 0.25 0.22
0.00 0.11 0.30 0.01 0.00
0.00 0.03 0.60 0.01 0.12
CONCLUSION
Following an incident of overexposure to y-radiation, the cytogenetic and hematologic parameters of three workers were assessed and compared with prior data. The worker closest to the source showed the greater changes in CAA; none showed hematologic changes. We recognize that the number of cells analyzed was too small to estimate the dose on the basis of frequency of bicentric chromosomes, and that individual and interlaboratory differences exist. However, the aim of this study was not to estimate dose, but rather an attempt to demonstrate that analysis of chromosomal aberration indicates the changes occurring in the organism prior to changes in the blood count, the parameter recommended by WHO. As clearly shown in Table I, exposure to ionizing radiation at doses even lower than the TLV affected chromosomes, but not the hematologic values. We believe that cytogenetic analysis should be enforced in pre-employment, as well as in periodic examinations, in order to evaluate the biological effect of ionizing radiation in occupationally exposed populations. REFERENCES Balmain A, Brown K (1988): Oncogene activation in chemical carcinogenesis. Adv Cancer Res 51: 147-182. Brandt-Rauf PW ( 1990): Oncogene proteins as biomarkers in molecular epidemiology of occupational carcinogenesis. Int Arch Occup Environ Health 63:l-8. Ericson J, Rushdu A, Drawings HL, Nowel PC, Croec CH (1983): Transcriptional activation of the translocated C-miyc oncogene in Burkitt lymphoma. Proc Natl Acad Sci USA 80320-824. Hughes D (1990): The revision of dose limits for exposure to ionizing radiation. Ann Occup Hyg 34535-539. Littlefield LG, Jonier EE, Colyer SP, Ricks RC, Lushbaugh CC, Hurdato-Monroy R (1991): The 1989 San Salvador 6oCo radiation accident: Cytogenetic dosimetry and follow-up evaluation in three accident victims. Radiat Protect Dosim 35:115-123. Ranogajec-Komor M, Osvay M (1986): Dosimetric characteristics of different TL phosphorus. Radiat Prot Dosim 17:379-384. Vekie B, Ranogajec-Komor M, &danan M (1990): Comparison of the responses of several TLDs to various radionuclides. Radiat Prot Dosim 33:335-338. World Health Organisation (1986): “Early Detection of Occupational Diseases.” Geneva: WHO.
