Mutation Research, 319 (1993) 11-18
© 1993 Elsevier SciencePublishers B.V. All rights reserved 0165-1218/93/$06.00
Sister-chromatid exchanges induced by chloramphenicol on bovine lymphocytes J. Catalan a, C. Moreno b and M.V. Arruga
a Laboratory of Cytogenetics and b Quantitative Genetics Unit, Veterinary Faculty, Zaragoza, Spain
(Received 19 August 1992) (Revisionreceived31 march 1993) (Accepted 1 April 1993)
Keywords: Chloramphenicol;Sister-chromatidexchange;Proliferationrate index; Bovinelymphocytes
Summary The induction of sister-chromatid exchanges (SCE) was studied in bovine lymphocyte cultures treated with chloramphenicol (CAP), an antibiotic agent in wide use in human and animal therapy. A total of six individuals, matched for sex, race, age and environmental conditions, were used for the analysis. Chloramphenicol was tested at four different concentrations (5, 10, 20 and 40/xg/ml) and acted for the last 24 h of the culture. Each experiment included two animals, each of which was exposed to all chloramphenicol doses, for a total of three repetitions. The results of the corresponding analysis of variance showed that this chemical had a small but statistically significant effect on the SCE frequency. In addition, the lymphocyte cultures responded strangely to this chemical: the highest SCE induction was produced by the lowest dose. However, the study of high frequency cells did not show the presence of this kind of cell which could explain this chloramphenicol response. In addition, chloramphenicol induced a high delay in the cell cycle.
Chloramphenicol (CAP; CAS No. 56-75-7) is a broad-spectrum antibiotic employed in human and animal therapy. In spite of its widespread use, CAP shows various side effects, such as aplastic anemia in humans and animals (Nahata, 1987). An unusual association between aplastic anemia and leukemia made IARC (1989) condude that "chloramphenicol is probably carcino-
Correspondence: Dr. Julia Catal~In,Laboratoryof Cytogenetics, Facultyof Veterinary,MiguelServet 177,50013 Zaragoza, Spain. Tel. (976) 410466; Fax (976) 591994.
genic to humans". However, as pointed out by Rosenkranz (1988), CAP has not been systematically tested for genotoxicity, and the few available data are contradictory. From a cytogenetic point of view, CAP was found to induce chromosomal aberrations in plants (Yoshida and Yamaguchi, 1973), in cultured bovine and porcine lymphocytes (Queinnec et al., 1975; Babile et al., 1978), and in bone-marrow cells of mice (Manna and Bardhan, 1977; Sbrana et al., 1991). In human ceils, no induction (Byarugaba et al., 1975; Palmer, 1985) as well as an induction of chromosomal aberrations (Pant et al., 1976a, b; Sbrana et al., 1991) were reported. Chloramphenicol did
12 not increase the incidence of either rat micronucleated polychromatic erythrocytes or micronucleated hepatocytes in vivo (Martelli et al., 1991). Furthermore, while Pant et al. (1976a, b) reported no effect of CAP on sister-chromatid exchanges (SCEs) in human lymphocytes, and Sbrana et al. (1991) only found a slight increment of SCEs both in such cells and in V79 cells, both Romero (1987), in Chinese hamster ovary cells, and Arruga et al. (1992), in bovine fibroblasts, described a significant increment of SCEs. Bearing in mind these contradictory results, the induction by chloramphenicol of sister-chromatid exchanges in bovine lymphocytes was studied in the present work.
/~g/ml) for 1.5 h and hypotonic KCI solution (0.075 M) for 25 min and fixed three times, the first time overnight, with methanol-glacial acetic acid (3:1). The cells were dried on slides and stained with a modification of the fluorescence plus Giemsa technique (Perry and Wolff, 1974). Briefly, slides were stained for 30 min in Hoechst 33258 (33 /~g/ml) under the action of UV light, incubated in 2 x SSC buffer for 1 h at 55°C and then stained for 20 min with 4% Giemsa solution in phosphate buffer at pH 7.0. For each individual and dose, 20 mitoses were analyzed for SCE. In addition, the proliferation rate index (PRI) was calculated from 200 mitoses per individual and dose, following the calculations of Ivett and Tice (1982).
Material and methods Experimental design
Subjects Six male cattle of the same age (one year) and race (Pirenaica) and reared in the same farm were chosen for the analysis.
In order to study the character number of SCEs, which is expressed as No. SCEs/cell, a randomized block design was chosen, according to the following model:
Analyzed product A solution of chloramphenicol (Sigma) in RPMI medium was used. Four different doses (5, 10, 20 and 40/~g/ml) were tested with two series without the drug: zero dose and a positive control. The former contained only 5 tzg/ml of bromodeoxyuridine (BrdU), enough to obtain the harlequin staining, while the latter contained 30 tzg/ml of BrdU, which was previously shown to differ significantly from the zero dose. The positive control was included in each experiment in order to verify the analysis.
Yiikl=/X -t- B i q-/ij q- Dk + eijkl
where /x = general mean; B i = block effect; Iii = individual effect; D k = dose effect; eiiva= residual. One SCE/cell was chosen as being the minimal observed significant difference between doses. Therefore, considering the variability of the character, the minimal sample size which it was necessary to analyze to show this difference with errors a = 5% (error type I) and /3 = 20% (error type II) was about 100 cells per dose. This sample size was calculated by the methodology developed by Steel and Torrie (1985). Three blocks, which included two individuals, were analyzed. In each individual, four chloramphenicol
Peripheral blood was cultured for about 72 h at 38°C. A final concentration of 7 × 105 lymphocytes per ml was added to RPMI 1640 medium (Flow) with 15% fetal bovine serum (Sero-lab), 1% antibiotic-antimicotic (Gibco) and 2% phytohemagglutinin (Wellcome). BrdU, in a final concentration of 5 /~g/ml, was added 26 h before harvest. Furthermore, chloramphenicol acted for the last 24 h of the culture, in accordance with the observation of Sbrana et al. (1991) that a prolonged treatment seems necessary for CAP to act. The cells were treated with colcemid (0.05
E IijYijkleijkl= /Z + B ik+0D0
= si +
S2 0 0 S2
doses, in addition to the zero dose and the positive control cultures, were tested. The model employed here was solved by an analysis of variance, using the HARVEY program (version 1987). Furthermore, the relationship between chloramphenicol doses and PRI values was studied by a simple regression analysis. Results
Effect of chloramphenicol on the SCE frequency The mean values of SCE/cell as well as number of analyzed cells, range and proliferation rate indexes are given in Table 1.
After checking the correct working of the SCE assay by means of the comparison between cultures containing only 5 and 30 /zg/ml of BrdU (data not shown), an analysis of variance was applied to the previously established model. As Table 2 shows, individual and chloramphenicol dose factors seemed to have a significant effect, whereas block factor did not. Later, the validation of the model was made. Thus, residues of the variable under study were calculated and evaluated by the following tests: normality test, independence test and homogeneity of variance test. The frequency distribution of the SCEs residues is shown in Fig. 1. When the Kolmogorov-Smirnov normality test was applied, the statistical
TABLE 1 MEAN FREQUENCIES OF SCE PER CELL AND PRI VALUES Dose
Mean + SE
Control Control Control Control Control Control
1 1 2 2 3 3
1 2 3 4 5 6
20 20 20 8 20 20
6.40 + 0.438 7.75 + 0.516 6.30 + 0.572 7.37 + 0.625 5.45 + 0.591 4.95 + 0.596
3-10 4-12 2-11 6-11 0-11 1-12
36.0 9.5 25.0 33.3 34.5 32.0
64.0 70.0 50.0 64.4 60.5 62.5
0.0 20.5 25.0 2.3 5.0 5.5
1.640 2.110 2.000 1.690 1.705 1.735
5/zg/ml 5/xg/ml 5 p~g/ml 5/zg/ml 5/~g/ml 5/.Lg / m l
1 1 2 2 3 3
1 2 3 4 5 6
20 20 20 9 20 20
5.85 + 0.466 7.95 + 0.705 8.20 + 0.655 7.56 + 1.056 6.20+0.479 6.45+0.500
3-9 3-16 5-14 3-15 3-11 2-10
37.0 18.0 30.0 41.1 23.6 34.0
63.0 68.5 62.0 56.4 66.7 61.5
0.0 13.5 8.0 2.5 9.7 4.5
1.630 1.955 1.780 1.613 1.861 1.705
10/zg/ml 10/zg/ml 10 ~ g / m l 10/~g/ml 10/.~g/ml 10/zg/ml
1 1 2 2 3 3
1 2 3 4 5 6
20 20 20 6 20 20
5.40+0.499 8.15+0.751 5.75 + 0.403 6.00+0.577 6.45+0.583 6.20:t:0.574
2-10 2-17 3-10 4-8 3-12 2-11
47.5 19.5 29.5 57.1 18.5 25.0
52.5 53.5 60.5 41.3 59.0 71.0
0.0 3.0 10.0 1.6 22.5 4.0
1.525 1.783 1.805 1.444 2.040 1.790
20/~g/ml 20/.~g/ml 20/xg/ml 20/zg/ml 20 p~g/ml 20/zg/ml
1 1 2 2 3 3
1 2 3 4 5 6
20 20 20 14 20 20
6.05 + 0.387 6.40+0.467 7.05 + 0.643 5.00 + 0.378 6.70+0.624 4.50 + 0.450
3-9 3-10 2-16 2-7 3-13 1-8
38.0 42.0 25.5 54.6 37.0 46.5
62.0 55.5 62.5 45.4 62.5 53.0
0.0 2.5 12.0 0.0 0.5 0.5
1.620 1.605 1.865 1.454 1.635 1.540
40/.t g / m l 40 ~ g / m l 40/zg/ml 40/~g/ml 40 ~ g / m l 40/.~g/ml
1 1 2 2 3 3
1 2 3 4 5 6
20 20 20 8 . 20
6.45+0.473 5.65+0.534 7.20+0.655 4.37 + 0.680 . . 5.95 + 0.630
3-12 1-11 3-15 2-8 . 1-13
55.0 50.0 41.5 57.3
45.0 48.8 66.5 42.7
0.0 1.2 2.0 0.0
1.450 1.512 1.605 1.427
14 TABLE 2 RESULTS OF THE ANALYSIS OF VARIANCE OF THE SCE FREQUENCIES (ORIGINAL DATA)
Source of variation
Block Individual Dose
1.086 5.363 2.593
0.4418 0.0013 0.0358
Source of variation Block Individual Dose
value obtained (0.07, p < 0.01) indicated that the SCE frequency did not follow a normal distribution. The independence requirement, evaluated by the U p - a n d - D o w n test, was satisfied. Finally, the homogeneity of the residual variance was not satisfied, as was demonstrated by the relationship between the m e a n and the standard deviation from each analyzed combination of individual and dose. Therefore, the corresponding regression line ( Y = 0.562 + 0.285X, where Y = standard deviation and X = mean) was highly significant ( p < 0.01). The results of validating the model indicated that normality and homogeneity of variance requirements were not met when working with the observable scale of data and, consequently, data transformation had to be applied. The square root transformation has been specifically recomm e n d e d for these cases (Erexson et al., 1983; Steel and Torrie, 1985); therefore, the character to be analyzed was Y = ( S C E / c e l I ) 1/2. The application of the analysis of variance to the explained model using transformed data produced the results given in Table 3. As can be seen, individual and dose factors showed an im-
F 1.550 4.515 2.652
P 0.3449 0.0041 0.0325
portant influence on the SCE frequencies, while the block factor did not. The evolution of SCE frequencies with increasing chloramphenicol doses is shown in Fig. 2. T h e r e was an initial increment in SCE frequencies from zero to the lowest dose, but this frequency decreased at higher doses. In order to elucidate whether this response was based on the existence of different lymphocyte populations, which differ in their sensitivity to chloramphenicol, a study of high frequency cells was made. A tolerance level of 90% was considered, and we analyzed separately cells whose SCE frequency was in the 10% u p p e r SCE frequencies (high frequency cells) and cells whose SCE frequency was in the remaining 90%. T h e application of one-way variance analysis to these two groups, considering the dose as the only variation factor, showed the results given in Table 4. Thus, the chloramphenicol effect was significant in both lymphocyte populations. Furthermore, Fig. 3 shows the response of SCEs with increasing chloramphenicol doses in the total, 10% and 90% populations. As can be seen, the response curve was very similar in the three cases. Therefore, the
ANALYSIS OF VARIANCE CORRESPONDING TO TRANSFORMED DATA (Y = SCE/cell) 1/2)
i m I
70. 60 50. 40. 30. 20.
10. 0 -8
Fig. 1. Histogram of the distribution of residuals.
Fig. 2. Evolution of the chloramphenicol-induced SCE frequencies.
15 TABLE 4 ONE-WAY ANALYSIS OF VARIANCE OF THE SCE FREQUENCIES OF DIFFERENT LYMPHOCYTE SUBPOPULATIONS ANOVA
Frequency 90% Frequency 10%
SCE total SCE 10% SCE 90%
< 0.01), as was their correlation value (r = -0.98, p < 0.01). Therefore, the more the chloramphenicol dose is increased, the longer the cell cycle-is delayed.
Fig. 3. Evolution of SCE frequencies in different lymphocyte populations.
strange behavior of chloramphenicol-induced SCEs on cattle lymphocytes is not due to the existence of high frequency cells.
Effect of chloramphenicol on the cellular kinetics The relationship between chloramphenicol concentrations and PRI values, evaluated by a simple regression analysis, is shown in Fig. 4, where the corresponding regression line (Y= 1.797-0.007X, where Y= PRI value and X = chloramphenicol dose) was highly significant (p
Fig. 4. Relationship between PRI values and chloramphenicol doses.
The results of the variance analysis indicated that while individual and dose factors had a significant effect on SCE frequencies, block factor did not. Since factors such as race, sex, age or environmental conditions can have a significant effect on SCE frequencies, it is important, as pointed out by Margolin and Shelby (1985), to mitigate their influence as much as possible when an agent of unknown action is tested. Because of this, all the individuals were matched for these factors. Thus, the block factor represented the uncontrolled variability existing among experiments. Its non-significant effect indicates that this variability is not important, so there is very good repeatability. Furthermore, we wanted to consider the individual variance in our model separately, because of its well reported influence (Di Berardino and Shoffner, 1979; Leibenguth and Thiel, 1986; Tucker et al., 1987, 1988; Miller, 1991), which has also been proved in the present work. In this way, the effect of individual variance could not interfere with the chloramphenicol dose effect, the testing of which was the main objective of our work. Not many studies are available in the literature about the analysis of chloramphenicol genotoxicity by the SCE assay. In addition, their results are contradictory, as was noted in the introduction. In the present work, as was shown in Table 3, chloramphenicol dose has a significant effect on the SCE frequencies. However, the maximal difference between doses was about 1 SCE/cell in the original scale, which cannot be considered genotoxically important since, according to Scott (1983), this kind of minor increase in SCEs (less than twice the control level) should be treated with caution, especially if the concentration giving a positive effect is subtoxic. Our results agree with those obtained recently by Sbrana et al. (1991), who observed a very slight increment of SCEs over the baseline value both in human lymphocytes, with a dose range of 2.4-3.2 mg/ml
of chloramphenicol, and in V79 cells, with a dose range of 3-12 m g / m l of chloramphenicol. However, these authors found a very different dose response depending on the cell type, since V79 cell SCE frequency increased progressively with increasing chloramphenicol doses, whereas lymphocyte SCE frequency increased greatly with the 2.8 m g / m l dose, but decreased with the higher dose. Although for a much lower dose range, these responses were similar to those we obtained with bovine cells. In our previous work (Arruga et al., 1992), we found that SCE frequency increased continuously from 0 /zg/ml (6.67 + 0.47 SCE/cell) to 60 /xg/ml (15.50 + 0.84 SCE/celI) of chloramphenicol in bovine fibroblasts; this response is statistically and genotoxically significant in this case. On the other hand, in the present work, we also found an increment of SCE frequency in bovine lymphocytes, followed by a progressive decrement. But in this case, the maximum SCE frequency was produced by the lowest dose. Bearing in mind the above discussion, there seems to be a peculiar behavior of lymphocyte cultures in relation to their SCE response to chloramphenicol treatment. A possible explanation could be the heterogeneity of lymphocyte populations. This heterogeneity can be due to the existence of high frequency cells (HFCs), which show a higher SCE frequency but also a higher sensitivity than the other lymphocyte populations. The analysis of HFCs was used on the study of persons exposed first to ethylene oxide and later to another agent, i.e., mitomycin C. The first dose of mitomycin C killed the ethylene oxide-induced HFCs, showing a lower SCE frequency than the control, whereas with greater doses the increment produced on the other lymphocyte populations was bigger than the total of HFCs and normal cell frequencies in the control dose (Nichols et al., 1988; Kelsey et al., 1988). Thus, similar behavior would be possible in the present work, although as there was no previous exposure to another agent, the control population would not have HFCs. These would appear as a consequence of the first chloramphenicol dose, but increasing doses would kill them. According to this theory we should obtain a scatter in which the SCE frequency of the higher population de-
creases drastically after the first dose while the lower population shows the same frequency for all doses because it is less sensitive to CAP. However, the scatter in Fig. 3 shows a similar response in both cases as well as for the total population. In addition, the analysis of variance in Table 4 indicates that the significant effect of the lowest dose appears also in both lymphocyte populations. Thus, the strange SCE response by chloramphenicol treatment in bovine cultured lymphocytes cannot be explained by the existence of HFCs. Another possible source of heterogeneity could be the different cellular kinetics of lymphocyte subpopulations. In fact, there are several works in the literature which show a high negative correlation between SCE frequency and kinetics of cell proliferation in lymphocyte cultures (Lindblad and Lambert, 1981; Erexson et al., 1983; Crossen et al., 1986; Santesson, 1986; Miller, 1991). As the CAP treatment and sample time were the same for all the doses, only cells which had been through two complete S phases in the presence of the drug were included in the analysis. As Fig. 4 shows, increasing doses of chloramphenicol induces a clear delay in cell cycle. If, as the authors mentioned above propose, fast cells carry fewer SCEs than slow ceils, then the earliest second-division cells seen will carry a lower frequency. At the lower doses, these cells might get through the mitosis and pass to the next phase, while they will just reach mitosis at the higher doses as a consequence of the CAP-induced delay. However, other authors who also find differences among proliferation rates in lymphocyte subpopulations find no relationship between them and SCE frequencies, at least in relation to basal SCE values (Giulotto et al., 1980; Deknudt, 1986). However, considering induced SCE values, Deknudt (1986) shows that if the mutagenic agent is added to the cultures 48 h after starting (as in the present work) there are differences in SCE frequencies according to the stimulated lymphocyte subpopulation, but these differences do not appear if the mutagenic treatment is administered before mitogenic stimulation. Concerning PRI values, as has just been mentioned, increasing doses of chloramphenicol produced a longer delay in the cell cycle, as the high
correlation between dose and PRI values (r = 0.98, p < 0.01) indicates. These results agree with those found by Verma and Lin (1978) in maize meristematic ceils, Mittwoch et al. (1974) in human fibroblasts, and Sbrana et al. (1991) in human lymphocytes, Chinese hamster cells and bone marrow cells of treated mice. However, it is still not clear whether this inhibitory effect of CAP is produced by an indirect action on D N A replication, as Yunis et al. (1980) and Murray et al. (1983) propose, or by interference only with the processes developing during the G 2 phase, as is supported by Sbrana et al. (1991). However, the latter recognize that a short treatment with CAP in G 2 phase is unable to break chromosomes; a prolonged treatment for a whole cell cycle is necessary to observe this effect. In conclusion, chloramphenicol has a clear inhibitory effect on cellular kinetics, which, in the case of lymphocyte populations, could perhaps explain the strange response of chloramphenicol-induced SCE frequencies observed in this kind of cells. References Arruga, M.V., J. Catal~,n and C. Moreno (1992) The effect of chloramphenicol on sister chromatid exchange (SCE) in bovine fibroblasts, Res. Vet. Sci., 52, 256-259. Babile, R., G. Queinnec, H.M. Berland and R. Darre (1978) Structure chromosomique des lymphocytes du porc et additifs alimentaires, C. R. Soc. Biol., 172, 546-553. Byarugaba, W., H.W. Riidiger, T. Koske-Westphal, W. W6hler and E. Passarge (1975) Toxicity of antibiotics on cultured human skin fibroblasts, Humangenetik, 28, 263-267. Crossen, P.E., J.M. Godwin and M.P. Bodger (1986) Sister chromatid exchange in immature haemopoietic cells, Tand B-lymphocytes, Hum. Genet., 72, 101-103. Deknudt, G. (1986) Chemical induction of sister-chromatid exchanges in human lymphocyte: treated in GO prior to stimulation by different mitogens and revealed 72 h later in second division cells, Mutation Res., 174, 67-70. Di Berardino, D., and R.N. Shoffner (1979) Sister chromatid exchange in chromosomes of cattle (Bos taurus), J. Dairy Sci., 62, 627-632. Erexson, G.L., J.L. Wilmer and A.D. Kligerman (1983) Analyses of sister-crhomatid exchange and cell-cycle kinetics in mouse T- and B-lymphocytes from peripheral blood cultures, Mutation Res., 109, 271-281. Giulotto, E., A. Mottura, R. Giorgi, L. de Carli and F. Nuzzo, (1980) Frequencies of sister-chromatid exchanges in relation to cell kinetics in lymphocyte cultures, Mutation Res., 70, 343-350.
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