Cytogenetic studies in human populations exposed to gas leak at Bhopal, India.

Frequencies of chromosomal abnormalities, sister chromatid exchanges, and replicative index were assessed following peripheral lymphocyte culture in 129 individuals from Bhopal, India. Of these, 83 persons (40 male and 43 female) had been exposed directly to the methyl isocyanate (MIC) gas after the accident at the Union Carbide plant on December 2 and 3, 1984. The remaining 46 samples were taken from age-matched unexposed persons in the same city. Chromosome aberrations were recorded at first cycle metaphase (M1) and sister chromatid exchanges, at second cycle metaphase (M2), following standard schedule. The frequency of chromosomal aberrations was, in general, higher in individuals from the exposed populations, with the females showing a higher incidence. Nondisjunction of chromosomes or laggard was rare. The frequencies of sister chromatid exchanges and depression in mitotic and replicative indices could not be related to exposure or sex. The persistence of chromosomal abnormalities in the form of replicating minutes and exchange configurations, even 1114 days after exposure to the gas, may indicate a residual effect on T-cell precursors.


Introduction
The disaster in the Union Carbide plant at Bhopal, India, between December 2 midnight and the early morning of December 3, 1984, affected more than 14,000 individuals (1). The inhabitants in the township of Bhopal were exposed in different degrees, depending on their proximity to the plant and atmospheric factors. A major lacuna in treating the exposed persons was the lack of adequate information on the toxicological effects of MIC (2). The unprecedented mortality and morbidity rates initiated detailed studies in experimental animals (3) and in some exposed individuals (4). The resultant publications show the wide spectrum covered by the toxic effects of MIC (1)(2)(3)(4). Individual publications are now available on the chemistry of the reaction (5) as well as its genotoxic and clastogenic effects on laboratory test systems, ranging from Salmonella, Drosophila, and mice in vivo to Chinese hamster ovary cells in vitro (6)(7)(8)11,12). Sister chromatid exchanges (SCEs) have been reported in some exposed persons after the accident (4,9,10,13).
In the present case, chromosomal analysis was carried out in cases randomly selected in the exposed area up to 1114 days after exposure to the gas in order to study the residual effects, if any, on the chromosomes.

Material and Methods
The 129 individuals studied were categorized into those persons directly exposed with history of exposure and those who were not exposed, that is, residents in neighboring areas where the gas did not spread. The group of exposed persons was chosen through a random computer-based selection of listed survivors. The control group was taken from the unexposed population and was matched by age and sex.
Peripheral venous blood was collected in heparinized vials and plasma was separated out by gravity sedimentation. After an hour, leukocytes were inoculated to RPMI-1640 medium (GIBCO, Grand Island, NY), supplemented with 20% heat-inactivated fetal calf serum (Sera Lab., England) and phytohemagglutinin (M Form, GIBCO) at 0.2 mL/5 mL of culture.
In the set for the study of sister chromatid exchanges, 6.0 ,ug of 5-bromo-2-deoxyuridine (BrdU, Sigma Chemical Co., St. Louis, MO) was added per 1.0 mL of the culture medium in the dark to prevent photoinactivation.
Four cultures were maintained for each subject in two replicate sets in the dark at 37°C. The period of incubation was 48 hr for the study of chromosome aberrations and 72 hr for SCEs; 2 hr before termination ofthe culture, colchicine (40 ,ug/mL) was added to each cul-After further incubation for 2 hr, cells were centrifuged into pellets and treated successively in hypotonic solution (0.09% NaCl in deionized water) and fixative (methanol acetic acid 3:1) for 25 to 30 min at 37°C. The cells were centrifuged and resuspended in fixative three times. Air-dried slides were prepared and stained in Giemsa according to the standard procedure (14)(15)(16) with slight modifications as required. The slides were coded and scored blind by two observers. The parameters recorded in each subject were (17)(18)(19)(20)(21)(22): a) chromosomal abnormalities (including damaged cells; total aberrations with and without gaps, breaks per damaged cell) recorded in 100 first-cycle scattered metaphases (M1) per subject; b) sister chromatid exchanges in 50 scattered complete second-cycle metaphases (M2) per subject; and c) cell cycle kinetics in 200 metaphases per subject. Parameters included replicative index (RI) and cell cycle metaphases in first (M1), second (M2), and third (M3) cycles (23). The aberrations for the first three sets of parameters were compared by Student's t test between exposed and unexposed populations of both sexes.

Results and Discussion
Chromosomal aberrations were found to occur in statistically higher frequencies in the exposed group as compared to the control, especially in female subjects ( Table 1). The types of abnormalities recorded were chromosome breaks, gaps, dicentrics, rings, and triradial and quadriradial configurations. Persistent, replicating minute chromosomes and quadriradial configurations were seen even after 1114 days (Figs. 1-4). In general, the number of breaks per cell was higher in exposed females as compared to unexposed ones. In males the difference was not significant. These observations are contradictory to the record of the higher incidence of micronuclei in male mice subjected to MIC in vivo (7,8).   The frequencies of SCEs did not differ markedly between exposed and unexposed populations. In general, the range was above the baseline ranges of 4 to 14 per cell reported earlier (24). The range of SCEs, number of breaks per cell, and percentage of cells with SCE could not be related to exposure or the sex ofthe person ( Table  2). These observations do not support an earlier isolated report of increased frequency of SCEs in individuals exposed to MIC during the Bhopal accident (10). In another communication, no relation has been observed to factors such as smoking, alcohol intake, and pregnancy (13). The replicative index was not altered significantly between the exposed and unexposed populations.
The study indicates an apparent increase in the frequency of chromosomal aberrations in exposed females 1114 days after exposure to the gas. Since the selection was made as double blind in order to facilitate independent checks by two observers, the history and clinical features (if any) of these cases have not yet been fully related to the observations. The incidence of chromosomal alterations like dicentrics, rings, and quadriradial configurations even after 1114 days may indicate persistent clastogenic effects. Because the lesions induced by chemicals are mostly S dependent for expression in the subsequent divisional cycle, the damaged Tlymphocytes may remain circulating for long periods, and these aberrations can be observed only if the cells are stimulated to divide in vitro (25). The results are comparable to those obtained following exposure to pdioxane (26).
Some of the females with persistent chromosomal aberrations have a history of fetal loss, which may have been the consequence of exposure to MIC. None of them has been exposed to any other known clastogenic agent except a single chest X-ray. The majority ofthe exposed females studied were housewives and the males were day laborers.
At present the observations are not complete and more needs to be done. In assessing the effects of exposure to toxic chemicals such as MIC, the sex of the subject and the physiological and nutritional status as well as other compounding factors such as genetic composition have to be taken into account (27,28). The persistence ofaberrations even after 1114 days indicates the possibility of a higher susceptibility to chromosome damage of persons exposed to MIC.