Evaluation of occupational genotoxic risk in a Brazilian hospital

Many therapeutic, diagnostic and prophylactic procedures used in hospitals are of potential genetic risk. An evaluation was mad e of genotoxic occupational risk in 42 workers from the Hospital de Clinicas de Porto Alegre, RS, Brazil, who had been occupationall y exposed to lead (solder), ethylene oxide (sterilization area), antineoplastic drugs (nurses and pharmacists) or ionizing radiat ion. They were compared with 42 unexposed individuals. There was an increase in the frequency of binucleated cytochalasin-blocked lymphoc ytes with micronuclei, though it was not significant (P = 0.058). The groups exposed to antineoplastic drugs and radiation had a sig nificant increase in micronuclei frequency (P = 0.038 and P = 0.022, respectively). The high frequencies of dicentric bridges suggest th e action of clastogenics in these two groups. These results suggest that the safety procedures adopted were very important to protect worke rs from exposure to mutagenic agents and should be improved in the radiological and chemotherapeutical areas.


INTRODUCTION
There is increasing concern about the mutagenic and carcinogenic effects of genotoxic agents in humans occupationally or accidentally exposed (Carrano and Natarajan, 1988).Mutagenic agents are used in hospitals either for maintenance or for the diagnosis and treatment of patients.However, for professionals who are continually exposed to these agents, the risks need to be assessed in order to establish proper management.
The mutagenic effect of ionizing radiation has been studied extensively (Fenech et al., 1990;Balasem and Ali, 1991;Erexon et al., 1991) and an increase in chromosomal aberrations was observed in occupationally exposed workers when the permissible levels of radiation were exceeded (Jha and Sharma, 1991).
Chemicals, such as cytostatic agents, induce chromosomal damage, both experimentally (in vitro) and clinically in patients (Migliore et al., 1991;Gree et al., 1991).McDiarmid et al. (1992) reported a positive correlation between the time of occupational exposure to antineoplastic drugs and the frequency of sister chromatid exchanges.
Lead disturbs cell proliferation and DNA synthesis in vivo, and may be responsible for certain types of cancer, mainly in the kidneys (Gerber et al., 1980).Lerda (1992) demonstrated that lead causes chromosomal breaks.This phenomenon, which is very frequent, is the main chromosomal alteration caused by occupational intoxication with lead.
Workers from the Hospital de Clínicas de Porto Alegre (HCPA) were monitored using the cytokinesisblocked lymphocyte analysis (Fenech and Morley, 1985) to measure frequency of micronuclei, dicentric bridges and spindle anomalies.The main advantage of this method of evaluation is the reliable identification of cells that have completed only one nuclear division (Fenech, 1997).Our aim was to verify whether the safety procedures in use were enough to protect healthy workers from exposure to mutagenic agents.

MATERIAL AND METHODS
Peripheral blood samples were collected from 84 workers; 42 out of them were occupationally exposed to lead (N = 11), ionizing radiation (N = 11), ethylene oxide (N = 10) or cytostatic drugs (N = 10).The remaining 42 were controls from the same institution, matched for age, sex and smoking habits.The individuals exposed to lead were chosen based on their levels of delta-aminolevulinic acid and those exposed to ionizing radiation were chosen based on the level of radiation recorded by their dosimeters.The group exposed to cytostatic agents was formed by all individuals from this area and the individuals exposed to ethylene oxide were chosen based on their time of activity in the sterilization area.

Abstract
Many therapeutic, diagnostic and prophylactic procedures used in hospitals are of potential genetic risk.An evaluation was made of genotoxic occupational risk in 42 workers from the Hospital de Clínicas de Porto Alegre, RS, Brazil, who had been occupationally exposed to lead (solder), ethylene oxide (sterilization area), antineoplastic drugs (nurses and pharmacists) or ionizing radiation.They were compared with 42 unexposed individuals.There was an increase in the frequency of binucleated cytochalasin-blocked lymphocytes with micronuclei, though it was not significant (P = 0.058).The groups exposed to antineoplastic drugs and radiation had a significant increase in micronuclei frequency (P = 0.038 and P = 0.022, respectively).The high frequencies of dicentric bridges suggest the action of clastogenics in these two groups.These results suggest that the safety procedures adopted were very important to protect workers from exposure to mutagenic agents and should be improved in the radiological and chemotherapeutical areas.(Carrano and Natarajan, 1988).An aliquot of blood (0.3 ml) was added to 5 ml of standard supplemented RPMI 1640 medium containing 20% fetal calf serum and phytohemagglutinin (PHA, 0.2%).The flasks were cultured at 37°C.After 44 h, 4 µg of cytochalasin B/ml (Sigma Chemical Co., St. Louis, MO, USA) was added (Fenech, 1993).After 72 h, the cells were harvested and treated with sodium citrate (1%) and then fixed in methanol:glacial acetic acid (3:1, U/U).The slides for each individual were randomly numbered and stained with Giemsa.The presence of micronuclei and other anomalies was determined in 2000 binucleated cells per individual.The slides from exposed and non-exposed subjects were evaluated in parallel by "blind" analysis.Spindle anomalies were considered when two or more nuclei in the same cell clearly showed different sizes.The presence of dicentric bridges was scored when clearly separated nuclei were linked by nuclear material.Differences between groups were assessed using a two-sample t-test.The influence of age, smoking and drinking habits on the frequency of micronuclei was also examined.

RESULTS AND DISCUSSION
There was a slight non-significant increase (P = 0.058) in the frequency of micronucleated lymphocytes (16.9 ± 6.9) (in 2000 analyzed cells per individual) in exposed workers compared to the controls (14.2 ± 5.8).This slight increase in the frequency of micronuclei was attributable to those exposed to cytostatic drugs and ionizing radiation.The frequency of dicentric bridges was increased in the same groups.There was no increase in spindle disorders.
The frequency of micronucleated lymphocytes in individuals exposed to antineoplastic drugs was significantly greater (P = 0.038) than in the paired controls.The frequency of dicentric bridges was also increased, although not significantly (P = 0.0545), while the frequency of spindle anomalies was similar in both groups (Table II).An increased frequency of SCE has also been found, following low level exposures in nurses working with cancer patients compared to office workers (Norppa et al., 1980;Waksvik et al., 1981;Sorsa et al., 1982).Structural chromosomal aberrations were also significantly greater in nurses exposed to antineoplastic drugs compared to nonexposed laboratory workers and hospital clerks (Nikula et al., 1984).Similarly, nurses who handled cytostatic drugs without using safety covers had more dicentric chromosomes than those who used safety covers, but there was no difference in the frequency of chromosomal breaks and SCE (Oestreicer et al., 1990).Our sample consisted of nurses and pharmacists from a hospital considered to have well-established safety rules.
The group exposed to ionizing radiation had an increased frequency of micronucleated cells relative to controls (P = 0.0217), and also a non-significantly higher frequency of dicentric bridges (P = 0.1172).The spindle anomalies were similar in both groups (Table III).Among hospital workers, such as physicians, nurses and technicians, who are exposed to very low levels of X-or γrays, there is an increased frequency of chromosomal aberrations, including dicentrics (Bigatti et al., 1988).In our assay, dicentric chromosomes were indicated by the frequency of bridges.Medical workers exposed to diagnostic levels of X-rays show a higher frequency of dicentric and acentric chromosomes compared to normal controls (Jha and Sharma, 1991).The frequency of micronuclei did not show a correlation with the radiation doses recorded by the dosimeters.This result is in agreement with the results reported by Barquinero et al. (1993) in a study with 26 workers with an accumulated dose range from 2.3 to 131.7 mSv.As pointed out by Barquinero et al. (1993), it is difficult to establish doseeffect relationships for low radiation levels.
The lead-exposed group showed a similar micronuclei frequency and dicentric bridges to that of the con-  IV).Lead-exposed individuals had a significant increase in the frequency of spindle disorders, with no parallel increase in micronuclei frequency.Previous studies have also demonstrated a genotoxic effect of lead (Gerber et al., 1980;Lerda, 1992).Based on the low levels of aminolevulinic acid in our subjects (Table IV), there apparently was no dangerous exposure to lead in these individuals.Levels of aminolevulinic acid higher than 15 mg/l can be considered dangerous.
Although other studies have demonstrated a significant relationship between the frequency of genetic alterations and the degree of exposure to ethylene oxide (Lerda and Rizzi, 1992;Ribeiro et al., 1994), we observed no such relationship (Table V), thus confirming the effectiveness of the safety procedures adopted.
There was no significant correlation between the genetic abnormalities detected and non-occupational factors such as age, sex and smoking or drinking habits, although the lack of influence of the latter on the micronuclei frequency could be related to the fact that these were not frequent habits in our subjects.
The concomitant analysis of dicentric bridges and spindle disorders when determining the micronucleus frequency does not involve much extra work, and may serve as a reference to the type of mutagen (clastogenic or aneugenic).Based on our results, we suggest clastogenic activity in the individuals with an increased frequency of micronuclei.
Workers handling antineoplastic drugs can be exposed through inhalation of aerosolis, transdermal absorption and accidental ingestion.An increase in genetic damage in workers occupationally exposed to antineoplastic drugs has been associated with careless handling (Grummt et al., 1993;Machado-Santelli et al., 1994;Sorsa and Anderson, 1996;Undeger et al., 1999).In our study, pharmacists and nurses used safety covers and followed the guidelines for working with antineoplastic drugs.Based on the results of the analysis of genetic damage by the micronuclei assay, the group of workers handling antineoplastic drugs was advised to modify their work schedule to reduce exposure time.
The safety procedures adopted were very important to protect workers from exposure to mutagenic agents and should be improved in the radiological and chemotherapeutical areas.The cytokinesis-blocked micronucleus assay appears to be useful for monitoring populations chronically exposed to genotoxic agents.δ-ala, Delta-aminolevulinic acid levels in urine (mg/l).For other abbreviations see Table II.

Table II -
Frequency of binucleated lymphocytes with micronuclei (MN) and other nuclear anomalies in 2000 cells analyzed per hospital worker potentially exposed to cytostatic drugs, and their respective controls.

Table I -
Age and sex of the hospital workers studied.

Table IV -
Frequency of binucleated cells with micronuclei (MN) and other nuclear anomalies in 2000 cells analyzed per hospital worker potentially exposed to lead, and their respective controls.

Table V -
Frequency of binucleated cells with micronuclei (MN) and other nuclear anomalies in 2000 cells analyzed per hospital worker potentially exposed to ethylene oxide, and their respective controls.

Table III -
Frequency of binucleated cells with micronuclei (MN) and other nuclear anomalies in 2000 cells analyzed per hospital worker potentially exposed to ionizing radiation, and their respective controls.Radiation dose in millisieverts.For other abbreviations see TableII.