Spontaneous sister chromatid exchanges in mitotic chromosomes of cattle (Bos taurus L)

Estimation des echanges interchromatidiques spontanes dans les chromosomes mitotiques de bovin (Bos taurus L). Des cultures de lymphocytes obtenus a partir du sang peripherique de 4 donneurs bovins ont ete realisees pendant 2 cycles cellulaires. Ces cultures sont effectuees en presence de 0,10, 0,25, 0,50, 1,0, 2,5 et 5,0 μg/ml de bromodeoxyuridine (BrdU). Nous avons etudie les relations reponse (echanges chromatidiques)- dose de BrdU afin, notamment, de connaitre le niveau spontane des echanges chromatidiques chez cette espece. Ces echanges ont ete comptes sur 50 plaques en metaphase II (par donneur et par dose) choisis par hasard. Le taux moyen d'echanges interchromatidiques (SCE: sister chromatid exchanges) par cellule (rapport SCE/cellule) pour une concentration de 0,1μg/ml de BrdU est de 2,48, valeur pouvant etre consideree comme le niveau spontane des echanges. La courbe reponse-dose de BrdU presente une rapide augmentation entre 0,10 et 0,50μg/ml de BrdU. Elle est plus rapide que pour des concentrations en BrdU plus elevees. Les distributions de frequence des SCE et des chromosomes portant un nombre variable d'echanges suivent une loi de Poisson seulement pour des concentrations de BrdU de 0,10 a 1,00μg/ml. L'utilite de determiner le niveau spontane des echanges chromosomiques chez les animaux domestiques, pour une application possible aux programmes de selection, est discutee.


INTRODUCTION
Spontaneous sister chromatid exchanges (SCEs) were first demonstrated by Kato (1974) and Wolff and Perry (1974) in Chinese hamster cell lines. Subsequently, other authors have extended these studies in in vitro (Tice et al, 1976) and in vitro systems (Mazrimas and Stetka, 1978;Tsuji and Kato, 1981;Morgan et al, 1986).
It is now well established that spontaneous SCEs occur as an integral part of DNA replication, even in the absence of agents known to induce SCEs (Tucker et al, 1986), and data are available for several species, including man, and various cell lines.
In the majority of these studies, bromodeoxyuridine (BUdR) was utilized at a final concentration of 10 !g/ml. Since BUdR is itself an inducer of SCE (Latt, 1973;Kligerman et al, 1982), the mean rates of SCE/cell detected so far reflect mostly induced exchanges. At the moment, nothing is known about the proportion of spontaneous versus induced SCEs.
For this reason, we decided to re-examine the SCE data obtained in domestic animals by focusing our attention on the spontaneous yield of SCEs which also provides an indirect estimation of the BUdR-induced SCEs.
The present paper refers to the spontaneous rate of SCEs in cattle (Bos taurus L).

MATERIALS AND METHODS
Peripheral blood samples were obtained from 4 (2 males and 2 females) clinically healthy, unrelated, cattle of the Italian Friesian breed nearly 6 months of age.
From each animal, 0.5 ml aliquots of whole blood (3 x 10 6 lymphocytes) were added to each of 6 culture flasks containing 9.5 ml of RPMI 1640 medium (Gibco, Dutch modification New York, USA) together with 1 ml of fetal calf serum (Gibco), 0.1 ml of L -glutamine, 30 !1 of antibiotic/antimycotic mixture (Gibco) and 0.1 ml of Pokeweed mitogen (Gibco). All cultures were grown at 37.5 °C. After 36 h from initiation, BUdR (Sigma, Saint Louis, MO, USA) was added to each culture flask at final concentrations of 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 !g/ml, respectively. The cultures were protected from light and allowed to grow for an additional 36 h. Colcemid was added for the final 2 h. Harvested cells were treated with hypotonic solution (KCI, 0.075 M) for 20 min at 37.5 °C and fixed 3 times with methanol/acetic acid solution (3:1). Air-dried slides were stained with a 0.2% acridine orange solution in phosphate buffer (pH 7.0) for 10 min, washed thoroughly in tap water, mounted in phosphate buffer and sealed with paraffin. SCEs were counted on 50 s cycle metaphase spreads, randomly scored for each animal, for each BUdR level. To avoid possible individual bias, all scoring was performed by the same person. In our experimental conditions it was not possible to utilize BUdR doses lower than 0.1 wg/ml because of the poorly defined sister chromatid differential. Table I reports mean number and standard deviation of the SCEs per cell scored at each BUdR level in the 4 animals tested. At the lowest dose of 0.1 !g/ml of BUdR the individual SCE/cell rates varied from 1.64 ! 1.16 to 3.62 t 2.1, with an average of 2.48 f 1.75. At the dose of 5.0 !g/ml of BUdR, the individual SCE/cell rates varied from 3.64 f 1.9 to 6.58 ! 3.05, with an average of 5.16 ! 2.75. The analysis of variance performed on these data indicated significant differences at each BUdR level (P < 0.001) among the 4 animals investigated. Figure 1 shows the mean rate of SCE/cell in the 4 donors tested. The overall mean rate of SCE/cell increased more rapidly between 0.1 and 0.5 !g/ml of BUdR and less rapidly at further concentrations, thus indicating a saturation level. This trend was found to be logistic (y = a/I + e bx , where a = 5.05 and b = -1.31).

RESULTS
When the data were examined by using quartile statistics (fig 2), 50% of the SCE/cell values observed between 0.1 (D1) and 0.25 (D2) !g/ml of BUdR remained fairly stable, starting to rise at higher concentrations. This would suggest that (in our laboratory conditions) up to 0.25 !g/ml of BUdR the exchanges could be considered as spontaneous, being little affected by the analogue.
In order to characterize the distribution of the exchanges in the cell population,  Figure 3 shows the frequency distributions of the SCEs!cell observed at 0.1 (D1) and 5.0 (D2) !g/ml of BUdR and the Poisson expected frequencies. It is quite evident that in D6 the Poisson expectations do not fit the observed frequencies ( X 2 = 30.2 > X5 . 05 ) ' However, when 2 subpopulations of lymphocytes are considered instead of 1 (Exp. D6a line), the Poisson distribution fits well the observed frequencies, with a chisquare of 13.33 (x) 05 = 18.3; df = 10). Table III reports the overall number of chromosomes with 0, 1 and 2 or more exchanges at each BUdR level and the number expected on the basis of the Poisson distribution. The chi-square analysis revealed that from 0.1 to 1.0 wg/ml of BUdR the expected frequencies were close (P < 0.05) to the observed, whereas at higher concentrations they were not, thus confirming to a large extent that low BUdR levels provide a better fit to the Poisson expectations, as previously suggested by Tucker et al (1986).

DISCUSSION
Spontaneous SCEs provide an indication of the extent of somatic recombination occurring in untreated cells and allow estimation of the proportion of induced SCEs (Tucker et al, 1986). Detection of the spontaneous level of SCEs in cattle has never been attempted. The only report available is that of McFee and Sherrill (1979) who detected a mean value of 5.95 SCE/cell in cattle lymphocytes exposed to 0.5 !g/ml of BUdR. The present paper reports a mean value of 2.48 SCEs/cell at a concentration of 0.1 wg/ml of BUdR. Below this level, sister chromatid differential was not satisfactory for SCE detection. Even though BUdR concentrations less than 0.1 !g/ml could be used in other laboratory conditions (Tucker et al, 1986), the mean value of 2.48 SCEs/cell can be considered very close to the spontaneous yield of SCEs in cattle. Since the exchange frequency observed is the sum of exchanges formed during 2 subsequent cell cycles, the average frequency of SCEs per cell generation is 1.24. By considering that cattle somatic cells have a diploid number of 60 and a diploid DNA content of 6.4 pg (Green and Bahr, 1975), the corresponding values are 0.02 SCEs per chromosome per cell generation and 0.19 SCEs per picogram of DNA.
The spontaneous yield of 2.48 SCEs/cell in cattle chromosomes is very close to that reported by Kato (1974) in a pseudodiploid Chinese hamster cell line exposed to the same BUdR concentration (2.3 SCEs/cell) but considerably lower than that reported by Tucker et al (1986) in human and mouse blood lymphocytes exposed to 30 nM of BUdR (7.2 and 4.9 SCEs/cell, respectively). McFee and Sherrill (1979) also reported higher values of SCEs/cell in cattle, pig, sheep and human lymphocytes but they used 0.5 wg/ml of BUdR. The dose-response curve of cattle chromosomes exposed to increasing doses of BUdR was found to be logistic. The yield of SCEs rises quite steeply between 0.1 and 1.0 wg/ml of BUdR. Above this concentration the curve rises slowly, indicating a saturation level. This pattern is very similar to that found by Wolff and Perry (1974) in Chinese hamster ovary cells grown under BUdR concentrations varying from 0.25 to 20 !g/ml, and also to that reported by McFee and Sherrill (1979) on cattle lymphocytes grown at concentrations varying from 0.5 to 20 !g/ml of BUdR.
However, a different pattern of the dose-response curve has been reported by other authors who found a plateau of the SCE frequencies at low BUdR doses in a Chinese hamster pseudodiploid cell line (Kato, 1974), in an in vivo rat system (Tice et al, 1976), in HeLa cells (Tsuji and Kato, 1981), and in human and mouse lymphocytes (Tucker et al, 1986). These contradictory results can be accounted for by a variety of factors such as species, individuals, cell source and laboratory conditions (Das and Sharma, 1983). However, when quartile statistics were applied to our data, a fairly steady line was found between 0.1 and 0.25 !g/ml of BUdR, thus indicating that 50% of the cells scored were little affected in their SCE response by the increase of BUdR. Furthermore, when the data reported in table I are examined within the range from 0.1 to 1.0 wg/ml of BUdR, donors n3 and n4 show a plateau of SCE values, while donors nl and n2 display a rapid increase of SCE/cell frequencies. This finding indicates that individual variations play an important role in determining the overall dose-response pattern. Our data also indicate that when Pokeweed is used as mitogen for SCE studies, BUdR concentrations higher than 1.0 !g/ml may provide SCE responses which do not follow Poisson expectations, due to the presence of 2 subpopulations of B and T lymphocytes which are known to differ in their SCE response, proliferation rate and sensitivity to chemically induced damage (Lindblad and Lambert, 1981;Erexson et al, 1983;Bloom et al, 1993). Recently, Catalan et al (1994) studied a group of 24 cattle of different breeds, ages and farms, and reported a spontaneous incidence of 5.77 ! 0.082 (se) SCEs by using 5 R g/ml of BUdR. While this value is in close agreement with ours (5.16!2.75 (sd) SCEs/cell), we can hardly believe that SCEs obtained at 5 !g/ml of BUdR can be considered as spontaneous, as claimed by those authors.
The results of the present investigation demonstrated that in cattle the mean rates of SCE/cell reported by various authors so far include a spontaneous level of 2.5 SCEs/cell, the remaining SCEs being induced by BUdR.
The SCE test is commonly used to assess chromosome instability (Chaganti et al, 1974) and genetic damage under mutagenic exposure (Carrano et al, 1978). Spontaneous SCEs have been related to the 'unscheduled' DNA synthesis occurring in mammalian cells to repair apurinic DNA sites (Verly et al, 1973;Kato, 1974).
Even though the molecular mechanism of SCE formation still remains to be elucidated, both types of SCEs seem to be related to the DNA repair activity of the cells. It seems, therefore, important to ascertain the proportion of spontaneous versus induced SCEs, rather than the overall SCE response alone. Variations among species, breeds, and individuals in the proportion of spontaneous/induced SCEs would reflect different DNA repairing efficiencies which should be taken into consideration when breeding animals, especially those destined for artificial insemination, are evaluated and selected for animal production improvement. This aspect, however, is worthy of further investigation.