Single-strand breaks in deoxyribonucleic acid in fire fighters accidentally exposed to o-nitroanisole and

Single-strand breaks in deoxyribonucleic acid in fire fighters accidentally exposed to o-nitroanisole and other chemicals. Scand Health 1995; 21 :36-42. Objectives The aim of the study was to detect single-strand breaks in deoxyribonucleic acid (DNA) in ~noilonuclear blood cells of fire fighters exposed to o-nitroanisole and other substances released into the environment during an accident in a chemical plant. Methods The level of DNA single-strand brealts in mononuclear blood cells was detected by alkaline elution. The results were compared for 16 fire fighters who worked in a contaminated area for about 8 h and two reference groups (one of fire fighters who had not worlted in the conta~llinated area, group I, and one of persons without any apparent occupational exposure to genotoxic substances, group 11). Results The mean nonnalized elution rate (nER) 19 d after the accident was slightly but statistically significantly (P < 0.05) higher for the exposed fire fighters [mean 1.48 k 95% confidence interval (95% CI) 0.211 than for reference group I (mean 1.21 f 95% CI 0.21) or reference group 11 (mean 1.17 k 95% CI 0.18). No statistically significant difference was found between reference groups 1 and 11. Another analysis was performed three months after the first. The level of DNA single-strand breaks (mean nER 1.12 k 95% CI 0.1 I) was no longer increased in comparison with the levels of the reference groups. C O ~ ~ C ~ S DNA breaks in fire fighters exposed to o-nitroanisole and other substances. In comparison with the extent of DNA strand brealts found in other occupational groups the increase was only moderate. The observed decrease in DNA single-strand breaks to the reference level in exposed fire fighters three months later suggests a DNA repair mechanism for DNA single-strand brealts caused by o-nitroanisole. In this study we used a modified alkaline elution method to investigate DNA single-strand breaks in mononuclear blood cells of fire fighters who were working in the contaminated area. By alkaline elution the genotoxic

During an accident in a chemical factory in Germany (22 February 1993) about I 0 t of a vaporous mixture of substances was released from a methoxylation plant. The solid parts of the released vapor (3.5 t) precipitated in the area of the plant and in a suburb across the river Main (table 1). In the breathing air of this suburb, o-nitroanisole concentrations of up to 18 pg . m-"ere detected. In one street of the contaminated suburb 180-200 mg of o-nitroanisole precipitated per square meter of land. In a more distant street at the border of the contaminated area 5-1 0 mg of o-nitroanisole was detected per square meter of land. During the next few days this precipitate was removed mechanically with brushes, high-pressure cleaning machines, and so forth by workers from the chemical plant and by fire fighters. The fire fighters did not use protective masks or protective clothing at the beginning of their worlc in the contaminated area, because significant exposure levels were not expected. o-Nitroanisole, the most important substance emitted during the accident, is known to be mutagenic in the Ames test (1) and has caused cancer in experinlental animals (2). Exposure to o-nitroanisole can occur through inhalation, penetration of the skin (3), and ingestion. Several other chemicals released during the accident, such as o-anisidine and o-chloronitrobenzene, are also considered genotoxic (4)(5)(6)(7)(8).
In this study we used a modified alkaline elution method to investigate DNA single-strand breaks in mononuclear blood cells of fire fighters who were working in the contaminated area. By alkaline elution the genotoxic effect of all substances inducing DNA single-strand breaks or adducts which render the DNA alkali labile can bc detected. Thcrefore, it is not necessary to adapt the method for individual substances, and also the effect of unknown genotoxins can be determined. Therefore the alltaline elution method is suitable for bio~nonitoring the effect of rnixtures of various chemicals. DNA singlcstrand brealts or alkali labile sites are known to be induced by the majority of genotoxic animal carcinogens. Sina et a1 (8) used the alkaline elution assay for detecting DNA single-strand brealts in rat hepatocytes and reported that the assay had a high potential to identify genotoxic carcinogens. In humans, DNA single-strand breaks have previously been used as an indicator or genotoxic exposure in a few cases. Walles et a1 (9) and Maki-Paaltltanen et a1 (1 0) detected DNA single-strand breaks to biornonitor human exposure to styrene using the DNA unwinding method or the alkaline elution technique (1 1). Recently we described a modified alkaline elution procedure for bio~nonitoring DNA single-strand breaks in mononuclear blood cells 01 humans exposed to genotoxic substances (12)(13)(14). In the present study we have compared the nu~nbcr of DNA single-strand brealts in fire fighters accidently exposed to o-nitroanisole and several other chemicals with those of a matched group of fire lighters who were not in the contaminated area (relerence group I) and with other individuals without any apparent occupational exposure to genotoxic substances (reference group 11).

Subjects
The exposed fire fighters were healthy men who had been exposed to a mixture of substances (table 1) released during an accident in a chemical plant. They had worlted in the containinated area for about 8 h. One fire fighter had been exposed for 40 h. Detailed information on the duties carried out in the containinated area is given in table 2. During the exposure, none of the fire fighters had worn protective clothing, nor had other safety provisions been applied.
Reference group I consisted of unexposed fire fighters who had not worlted in the contaminated area. They were matched with the exposed fire fighters for age, alcohol consumption, town of residence, and the number of cigarettes smoked in the group of sinolters. The exposed fire fighters were still in training and had not yet been involved in fire fighting, with the exception of subject F2. The firefighting activity of this subject and of the unexposed fire fighters (reference group I) was relatively low (less than two fires per month). Another group of referents (group 11) was constructed of male students and office e~nployees without any apparent occupational exposure to genotoxic substances.
An interviewer-administered questionnaire was used to collect data from each subject prior to the collection of the blood samnples. The questionnaire included information on cigarette sinolting (number of cigarettes currently sinolted per day, lifetime smolting history), age, health history, intake of alcohol and drugs, exposure to putative carcinogens or ionizing radiation, occupation, and length of occupation and occupational exposure. The fire fighters were asked to give the time they had worked in the contaminated area and the duties they had carried out, use or protective clothing, and coinplaints related to their exposure in the containinated area. The age, smoking habits, and occupation of the referents and fire fighters, as well as the coinplaints of the fire fighters in relation to exposure and their duty carried out in the contaminated region are summarized in tables 2-4. The exposed fire fighters and reference groups I and I1 were similar with respect to the distributions of age and the number of cigarettes smolted in the group of smolters. Alcohol consumption was similar for the exposed [4.2 (SD 2.2) g of alcohol a day] and unexposed [5.0 (SD 2.5) g a day] fire fighters but higher for reference group I1 [10.9 (SD 4.5) g a day]. None of the subjects used drugs or had apparently been exposed to genotoxic substances (besides the exposure studied) or ionizing radiation.     (16) was employed with some modifications. A suspension of two million inononuclear blood cells in 1 1n1 of cold phosphate buffered saline was poured onto a polycarbonate filter (Nucleopore, Tiibingen, Germany, 25 mm diameter, 2 ym pore size). The cells were lysed with 3 n11 of a solution of 10 mM ethyleliediaminetetraacetic acid (EDTA), 0.5% Triton X-100, and 2 M sodium chloride (NaCl) (adjusted to pH 10 with sodium hydroxide) for 60 min. The lysed cells were washed with 4.5 ml of 10 mM EDTA (adjusted to pH 10 with sodiilln hydroxide) using a flow rate of 3 in1 . h-I for 90 min. Before the sampling of the eluted DNA was started, one fraction equal to the volume of the tube (0.25 ml) was discarded. The elution was performed at 4°C in the dark using a 5 M NaC1, 2 mM EDTA solution adjusted to pH 12.6 with sodium hydroxide. With a pump speed of 1.5 ml . h-I, the eluate was collected over a period of 10 h. The filters were removed and sonicated in a 15-ml elution solution for 2 x 15 min (filter fraction). Quantification of the DNA was performed as described earlier (13). For standardization, DNA of untreated V79 cells, a Chinese hamster lung constant number of persons from each of the threc groups were analyzed per elution run. The U-test, according to Wilcoxon, Whitney, and Mann (two-sided) was applied to evaluate the statistical significailce of diffeerences in unpaired data, and Wilcoxon's signed rank test was used to evaluate paired data. Thc 95% confidence intervals (95% CI) were calculated as 1.96 . SD/no-5, where SD represents the standard deviation and n the number of persons. The mean values + 95% confidence intervals are given. The statistical significance of the correlation coefficients of the linear regression was evaluated with the use of the t-statistic.

Results
The mean normalized elution rate of the fire fighters working in the contaminated area was 1.48 (+ 95% CI 0.21) in the first measurement, which was performed 19 d after the exposure (table 2). For the unexposed fire fighters (reference group I) living in the same town a mean normalized elution rate of 1.21 (f 95% CI 0.21) was obtained (table 3). The difference between the exposed fire fighters and reference group I was statistically sigilificant (P < 0.05, U-test). The mean normalized elution rate of reference group I1 was 1.17 (+ 95% CI 0.18) (table 4), and it did not differ statistically significantly from that of reference group I, but was statistically significantly smaller than the mean normalized elution rate of fire fighters exposed to o-nitroanisole (P < 0.05, U-test).
Another analysis of DNA single-strand breaks was performed for the same exposed fire fighters 88 d after the first analysis (table 2). The lnean normalized elution rate in this investigatio~l was 1.1 2 (+95% CI 0.1 I), which was similar to that obtained for the reference groups and was statistically sigilificantly smaller than the mean elution rate in the first analysis (P < 0.01, Wilcoxon's signed rank test).
In several other groups occupationally exposed to genotoxic substances, we detected a higher increase in DNA single-strand breaks in nonsmokers than in smokers (12,14). Therefore all persons tested in this study were analyzed according to their smolung habits (tables 2-4). No statistically significant differences could be detected between the smolters and nonsmolters in reference groups I and 11. In the first analysis, the mean normalized elution rate of the nonsmolung fire fighters who had worked in the contaminated area was significantly higher [I .60 (+ 95% CI 0.241 compared with that of nonsmoking persons from reference group I [1.29 (+ 95% CI 0.25)] and reference group TI [1.16 (f 95% CI 0.23)] (P<0.05, U-test). No statistically significant difference in the normalized elution rates could be found between the smoking exposed fire fighters [1.17 (f 95% CI 0.25)] and the smol<ers frorn reference groups I 10.97 (+ 95% CI 0.30)J and I1 (1.1 8 + 95% CI 0.28). However, it should be mentioned that the number of individuals was very small in these subgroups. The mean normalized elution rate was 27% higher in the total group of smolting and nonsmoking fire fighters when compared with that of reference group I1 and 38% higher if only nonsn~okers were considered.
No statistically significant correlations (1-statistics of correlation coefficients) were found between the extent of DNA damage and age or alcohol consun~ption for the individuals in reference groups I and I1 and for the exposed fire fighters.
An analysis of the elutioil rates of the exposed fire fighters with respect to the length of exposure could not be performed because almost all of the fire fighters were exposed for 8 h with the exception of one person who was exposed for 40 h. Nevertheless, it is striking that this particular fire fighter (F2) also showed the highest mean normalized elution rate (2.26) within the group of fire fighters exposed to o-nitroanisole.
Twelve of the 16 fire fighters complained of symptoms occurring after their work in the contaminated region (table 2). Slun irritation of the neck or around the mouth was reported by three persons. Three fire fighters complained of burning of the eyes, and four noted an irritation of the upper respiratory tract. Three fire fighters reported nose bleeding and four complained about headache.

Discussion
An increase in DNA single-strand brealts of 22% was detected in fire fighters exposed to o-nitroanisole compased with unexposed fire fighters matched for age, cigarette smoking, and alcohol consumption. Liou et a1 (17) detected an increased number of benzo[a]pyrene diol epoxide-ENA adducts in fire fighters. Therefore an increased number of DNA single-strand breaks in mononuclear blood cells of fire fighters could also be expected, possibly due to skin contact or f~lmes inhaled during fire fighting. In this study, however, similar numbers of DNA single-strand breaks were detected in fire fighters not exposed to o-nitroanisole (reference group I) and persons without any apparent occupational exposure to genotoxic substances (rcfercnce group 11).
In the second analysis, which was performed 88 d after the first, the number of DNA single-strand brealts was no longer higher in the exposed fire fighters than in the unexposed fire fighters or the persons from reference group 11. Possible mechanisms for the observed decrease in DNA single-strand breaks could be DNA repair or the turnover of rnononuclear blood cells. Mononuclear blood cells of healthy donors consist of T lymphocytes (about 81%), B lynlphocytes (about 3%), and rnonocytes (about 16%). T lymphocytes consist of a larger long-living subpopulation (at least 90%) and of a smaller subpopulation (about 10%) with half-times of 300-1000 d and less than two weeks, respectively. The majority of B lympl~ocytes is short-lived (half-time shorter than two weelts), and monocytes leave the vascular system with a mean half-time of 8.4 h (18). With the use of these numbers, it can be calculated that about 66% of the mononuclear blood cells present in the first analysis were also present in the second analysis 88 d later. According to these data the disappearance of DNA single-strand breaks during a period of 88 d was not likely to be due exclusively to the turnover of mononuclear cells. Therefore the decrease in DNA single-strand breaks seeins to have been caused by a slow DNA repair mechanism, which could not remove all of the single-strand breaks induced by o-nitroanisole within the 19 d, but was able to reduce DNA single-strand breaks to reference levels 88 d later.
Little is known about huinan genotoxic effects of the substances released during the accident, o-Nitroanisole, which represented the main fraction of substances released (25.3%), was mutagenic in the Alnes test (1) and caused cancer in animals (2). o-Chloronitrobenzene, which represented 1.1 % of the substances released, was mutagenic in the presence of rat liver lnicrosomes as the metabolic activating system (6), and caused tumors in rats (7). o-Anisidine, representing 0.6% of the substances released, caused DNA single-strand breaks in mouse lymphoma cells in the presence of rat liver microsolnes (4), and also caused bladder cancer in rats and inice (5).
In earlier studies, we analyzed DNA single-strand breaks in mononuclear blood cells of persons occupationally exposed to putative carcinogens. For nonsmolting workers, cornpared with referents, the increase in DNA single-strand breaks was found to be 60% for painters, 70% for workers exposed to ethylene oxide, and 69% for metal workers exposed to cutting fluids (1 2, 14). In smokers consuming more than 10 cigarettes per day, 13% more single-strand breaks could be detected than in nonsmokers (12). In comparison with the results of earlier studies, the mean normalized elution rate of the fire fighters exposed to o-nitroanisole was increased to a relatively low extent (38% in the nonsmoking fire fighters) in comparison with that of the nons~noking referents. Furthermore, individuals from the aforementioned occupational groups are usually exposed to the substances continuously during their occupational life, whereas the fire fighters were exposed to the accidentally emitted o-nitroanisole and other compounds of the mixture for a short period only.
The determination of an equivalent concentration of a well-known genotoxic substance such as ethylene oxide could help one assess the risk for the exposed fire fighters. However, this kind of risk assessment is indirect because it is not known whether DNA damage caused by ethylene oxide or o-nitroanisole has similar biological relevance. The detection of DNA single-strand brealts in inononuclear blood cells of persons exposed to ethylene oxide revealed an increase in iiormalized elution rates of 0.19 if the concentration of ethylene oxide was elevated by 1 ing . m-? (4-h time-weighted average) (14). Therefore an increase in norlnalized elution rates by 0.27, calculated for the fire fighters compared with persons without any apparent occupational exposure to genotoxic substances, would be equivalent to exposure to an ethylene oxide concentration of 1.4 mg . m-?. Huinan exposure to ethylene oxide concentrations of almost 2 mg . m-3 (occupational exposure limit) may be tolerated during their complete occupational life, whereas the fire fighters were exposed to the accidentally emitted substances for a short period of time. Therefore we estiinate only a minor genotoxic hazard for the fire fighters due to their service in the accidentally contaminated area, compared with the genotoxic hazard of other occupational groups. However, these considerations have to be treated with caution since, at present, it is not known whether DNA single-strand breaks or alkali labile sites caused by ethylcne oxide or o-nitroanisole have similar biological consequences.