Sensitivity of lymphocytes from vulcanizers to the in vitro induction of sister chromatid exchanges.

Spontaneous frequencies of sister chromatid exchanges (SCEs) and SCEs induced in vitro by chemicals with different mechanisms of action such as mitomycin C, 4-nitroquinoline oxide, and 3-aminobenzamide were examined in phytohemagglutinin-stimulated peripheral blood lymphocytes from a group of workers in a rubber plant and a control group, both of which had been analyzed for levels of spontaneous SCEs 2 years earlier. An interindividual variability in the induction of SCEs was found after in vitro treatments with the different mutagens, which did not correlate with occupational exposure. This variability in the sensitivity to the induction of SCEs might be correlated to genetic differences among individuals, which have to be taken into account in environmental monitoring programs.


Introduction
The level of chromosomal aberrations in long-lived circulating lymphocytes is considered a reliable biological dosimeter of the absorbed radiation dose (1). With chemical mutagens the situation is more complex because the frequencies of chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) may be affected by interindividual differences in metabolism and susceptibility to chemicals. However, cytogenetic tests on chemically exposed populations have been able to detect different genotoxic exposures (2)(3)(4).
In previous work (5), the frequencies of chromosomal aberrations and SCEs in peripheral blood lymphocytes of vulcanizers working in the rubber industry were studied. This is a typical case where human biomonitoring may be justified, considering the organization of the working process and the complex interactions among the several hundred chemicals used, which may result in the formation of new carcinogenic materials, making it difficult to search 'Centro Genetica Evoluzionistica, c/o Dipartimento Genetica e Biologia Molecolare Universita "La Sapienza" 00185 Roma, Italy.
for specific carcinogenic agents. Known genotoxic agents such as benzene, benzo[a]pyrene, and nitrosamines have been detected in the effluents from rubber vulcanization (6), and epidemiological studies have shown that cancer risks still exist in the rubber industry (6).
No differences were found in our investigation between 34 vulcanizers and 16 control individuals for CAs or SCEs. Cigarette smoking was clearly associated with increased frequencies of SCEs in both exposed workers and controls. In a parallel study, Benigni et al. (7) estimated DNA repair ability in the same population by measuring in vitro UVinduced unscheduled DNA synthesis (UDS) in peripheral blood lymphocytes. In spite of the considerable interindividual variability, decreased UDS values were observed among vulcanizers compared to controls.
Therefore, in spite of the lack of detectable cytogenetic alterations and the uncertainties concerning the correlation between DNA excision repair and the genetic end points studied, it could be argued that vulcanizers could be at higher risk for mutagenesis and carcinogenesis.
Two years later, we further investigated a smaller group of the same vulcanizers and controls for both UDS and SCEs. In this second survey, the UDS was not different between the two populations, which may be explained by the high interindividual variability in the UV-induced UDS response.
Blood samples ofvulcanizers and controls were exposed in vitro to known mutagens with different modes of action to detect whether the in vivo exposure to chemical agents even at levels that did not produce cytogenetic effects could modify the sensitivity of lymphocytes to other mutagenic agents and to verify whether vulcanizers had lower DNA repair ability. Mitomycin C (MMC) was chosen as a directacting crosslinking agent; 4-nitroquinoline oxide (4-NQO) was used because it mimics effects of ultraviolet radiation and may be useful to detect UV sensitive individuals. 3-Aminobenzamide (3-AB) is known to increase the spontaneous frequency of SCEs when present during the S-phase of the cell cycle. It is a very potent inhibitor of poly(ADP-ribose)polymerase (8).

Exposed Individuals and Controls
Blood samples were obtained from eight vulcanizers and seven employees ofa rubber plant in central Italy. Working conditions and selection ofthe subjects were presented in a previous paper (5). Table 1 shows the demographic data used for statistical correlations.
Duplicate cultures were exposed 24 hr after stimulation to 0.009 ,uM MMC (Kyowa, Tokyo, Japan), 1 ,uM 4-NQO (Sigma), or 5 mM 3-AB (Sigma) until harvesting (51 hr later). Colchicine at the final concentration of 5 x 10 -7 M was added 71 hr after PHA addition, and cultures were fixed 3 hr later. Chromosome preparations were produced using the air-drying technique. Slides were differentially stained as previously described (9). For each experimental point, 100 cells were scored for M1, M2, M3 ratio to calculate the proliferation rate index (PRI) as follows: PRI = (M1 + 2 x M2 + 3 x M3)/total methaphases scored. Forty second-division metaphases were scored for SCEs. Figure 1 and Table 2 show that no differences in the yields of SCEs were found in the two repeated measurements between vulcanizers and controls. Therefore, the frequencies of spontaneous SCEs in 1982 and 1984 in the total number of individuals are also reported in Table 2. The lower frequency of SCEs observed in the whole population 2 years later could be ascribed to technical differences in culture conditions in the second examination as confirmed by the higher proliferation rate index shown in Table 3. Table 2 shows the induction of SCEs by different mutagenic agents. The data show that there are no differences in the induction of SCE by the three chemicals between vulcanizers and controls. PRI values following MMC,   4-NQO, and 3-AB are shown in Table 3 sensitivity in the induction of SCEs in the same individual depends on the chemical. Individuals exceeding two standard deviations are shown in Figure 2. As can be seen from Figure 2, interindividual variability was present both in vulcanizers and in controls. This result indicates that the variability in response to in vitro mutagenic treatment is not correlated to occupational exposure but is more characteristic of the individual and the chemical used for treatment.

Results and Discussion
To verify whether individuals with low spontaneous yields of SCEs are less sensitive to mutagen-induced SCEs, we have graphically correlated the two variables as shown in Figure 3. No correlations were found, suggesting that the response in SCEs after mutagen treatment is not directly related to the spontaneous levels of SCEs. This finding of differences in the frequencies of SCEs among individuals following in vitro exposure to mutagens has also been found by other authors (10)(11)(12) and might be related more to genetic differences among individuals than to other factors (lifestyle, workplace, smoking, etc.). These observations have to be taken into account in environmental biomonitoring programs.