cytogenetic analysis in chinese hamster cells chronically exposed to low doses of x‐rays

Cytogenetic analysis in Chinese hamster cells chronically exposedto low doses of X-rays

A. GUERCI, F. DULOUT and A. SEOANE*

(Received 22 January 2003; accepted 19 June 2002)

Abstract.Purpose: It is important to develop simple experimental models toassess the induction of DNA damage and study the differentfactors involved under controlled conditions. This paper describesthe cytogenetic analysis carried out in Chinese hamster cells(CHO) sequentially exposed to very low doses of X-rays.Materials and methods: CHO cells were cultured for 14 passages.Irradiation treatment was performed once per passage, and threeirradiation doses were employed: 2.5, 5.0 and 10.0 mSv.Results: Sequential irradiation of CHO cells did not increase theyield of chomatid- or chromosome-type aberrations. However, asignificant increase of achromatic lesions (gaps) was found afterthe first or second X-ray dose, with all three irradiation dosesemployed.Conclusions: The variation in the frequency of gaps as well as thatin the mitotic index during the 14 cycles of radiation could be anindication of the induction of genomic instability. According tothis, continuous rises and falls in the frequency of gaps as well asin the mitotic index reflects the simultaneous induction ofendogenous DNA damage, cell death and cell survival.

1. Introduction

Most studies on the genetic effects of humanexposure to ionizing radiation are based upon theanalysis of chromosomal aberrations in peripheralblood lymphocytes after acute accidental exposures torelatively high doses (Sasaki and Miyata 1968, Schullet al. 1981, Croft 1989, Stephan and Oestreicher1989, Natarajan 1998). Cytogenetic investigation hastherefore become a routine tool for the assessment ofabsorbed radiation doses and their biological effectsafter occupational or accidental exposure to ionizingradiation. On the other hand, little information isavailable about chronic exposure to low doses ofradiation. Some authors have found chromosomaldamage in workers exposed to chronic low-levelionizing radiation. High frequencies of chromosomeaberrations, sister chromatid exchanges, micronucleusand even hyperploidy in peripheral blood lympho-cytes were reported in hospital workers in diagnostic

X-ray and nuclear medicine areas (Barquinero et al.1993, Hagelstrom et al. 1995, Paz y Mino et al. 1995,Bonassi et al. 1997, Vera et al. 1997, Pincheira et al.1999). In addition, increases in the relative risk ofchromosomal aberrations and cancer were found incivil aviation pilots, aircrew and astronauts (Heimerset al. 1995, Bagshaw et al. 1996, Obe et al. 1997,Ballard et al. 2000, Picco et al. 2000, Rafnsson et al.2000, Cavallo et al. 2002) as well as in people living inthe proximity of nuclear reactors (Schmitz-Feuerhakeet al. 1993, 1997). On the other hand, negative resultsin relation to the increase of chromosomal aberra-tions were found in female cabin attendants inGermany (Wolf et al. 1999) as well as in odontologistsin Londrina, Brazil (Miyaji and Syllos Colus 2002). Inaddition, negative results were found using the cometassay to detect base damage in hospital workers(Kruszewski et al. 1998).

Although information obtained from studies car-ried out in exposed populations is important for theestimation of genetic damage and evaluation ofcancer risk, it is not easy to elucidate the mechanismsinvolved in the induction of DNA lesions. In thissense, it should be important to develop simpleexperimental models to assess the induction of DNAdamage that identify the different factors involvedunder controlled conditions. This paper describes thecytogenetic analysis carried out in Chinese hamstercells sequentially exposed to very low doses of X-rays.

2. Materials and methods

Chinese hamster ovary (CHO) cells originallyobtained from American Type Culture Collection(ATCC) were employed. Cells were cultured in HamF10 medium (Gibco) supplemented with 10%inactivated foetal calf serum, 50 IU ml21 penicillinand 50mg ml21 streptomycin sulfate at 37‡C in ahumidified atmosphere with 5% CO2. Cells werecultured in Falcon T-25 flasks with 10 ml culturemedium. Cell cycle duration of CHO cells underthese conditions varies between 12 and 15 h, asdetermined when using BrdU.

Cells were cultured for 14 passages. Irradiation

International Journal of Radiation Biology ISSN 0955-3002 print/ISSN 1362-3095 online # 2003 Taylor & Francis Ltd

http://www.tandf.co.uk/journals

DOI: 10.1080/09553000310001600916

*Author for correspondence; e-mail: [email protected]

CIGEBA (Centro de Investigaciones en Genetica Basica yAplicada), Facultad de Cs. Veterinarias, Universidad Nacionalde La Plata, Calle 60 y 118 s/n, CC 296, B-1900-AVW LaPlata, Argentina.

INT. J. RADIAT. BIOL, OCTOBER, 2003, VOL. 79, NO. 10, 793–799

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treatment was performed once per passage when thecells were 90–95% confluent. Fifteen hours afterirradiation, cytogenetic analysis was carried out onthe first post-irradiation metaphases. Colchicine(1 mg ml21 final concentration) was added to all thecultures 2 h before fixation. Air-dried slides wereprepared by following routine protocols.

Three irradiation doses were employed: 2.5, 5.0 and10.0 mSv, taking into account the dosimetry reportedin epidemiological expositions (Barquinero et al. 1993,Paz y Mino et al. 1995, Balakrishnan and Rao 1999,Heimers 2000, Cardoso et al. 2001, Cavallo et al. 2002).Irradiation was performed with a X-ray apparatusDSJ 65 kV at 5 mA. Doses were determined by using adosimeter Keithley Digital 35617 EBS micro chamberPTW N 2336/414. After treatment, cells were dividedinto two fractions. One was contained in culture as the‘chronically’ exposed population, the other was culturedfor cytogenetic analysis. A control group remaineduntreated during its 14 passages.

Each experiment was repeated twice. At least 200metaphases per experimental point were scored. Theidentification of chromosomal aberrations was carriedout following the criteria recommended by Archeret al. (1981) and WHO (1985). Non-staining or verylightly stained chromosome regions in one or bothchromatids were considered as achromatic lesions(gaps) when there was no displacement of the chro-matid fragment(s) distal to the lesion. If there wasdisplacement of the distal chromatid fragment or thenon-staining region was wider than the width of achromatid, the aberration was scored as a deletion(break).

Statistical analysis was performed by using thex2-test.

3. Results

Tables 1–3 summarize the results obtained for cellsirradiated with 2.5, 5.0 and 10 mSv, respectively, for

Table 1. Frequencies of structural chromosome aberrations in CHO cells irradiated with 2.5 mSv X-rays.

TreatmentAbnormal

metaphases1 (%)

Chromosomal aberrations per 100 cellsMitotic

index (%)AL2 B3 DIC4 RIN5

– 0.00 2.00 0.00 0.00 0.00 32.67(0.00) (0.27) (0.00) (0.00) (0.00)

1st Irradiation 0.75 5.00 0.25 0.50 0.00 26.00(0.17) (0.4) (0.09) (0.14) (0.00)

2nd Irradiation 0.25 4.75 0.25 0.00 0.00 35.00(0.09) (0.39) (0.09) (0.00) (0.00)

3rd Irradiation 1.00 3.75 0.00 0.75 0.25 24.00(0.19) (0.35) (0.00) (0.17) (0.09)

4th Irradiation 0.50 4.75 0.25 0.25 0.00 42.33(0.14) (0.39) (0.09) (0.09) (0.00)

5th Irradiation 0.50 4.50 0.25 0.25 0.00 32.67(0.14) (0.38) (0.09) (0.09) (0.00)

6th Irradiation 0.50 2.25 0.50 0.00 0.00 39.33(0.14) (0.28) (0.14) (0.00) (0.00)

7th Irradiation 0.50 5.00 0.00 0.50 0.00 43.00(0.14) (0.4) (0.00) (0.14) (0.00)

8th Irradiation 0.25 7.00 0.00 0.25 0.00 16.00(0.09) (0.44) (0.00) (0.09) (0.00)

9th Irradiation 0.75 5.00 0.00 0.50 0.25 16.33(0.17) (0.4) (0.00) (0.14) (0.09)

10th Irradiation 0.50 4.25 0.25 0.25 0.00 10.66(0.14) (0.37) (0.09) (0.09) (0.00)

11th Irradiation 1.00 2.75 0.00 1.00 0.00 29.67(0.19) (0.31) (0.00) (0.19) (0.00)

12th Irradiation 1.75 4.50 0.75 1.00 0.00 26.33(0.25) (0.38) (0.17) (0.19) (0.00)

13th Irradiation 1.25 4.50 0.00 1.25 0.00 24.00(0.21) (0.38) (0.00) (0.21) (0.00)

14th Irradiation 0.25 3.75 0.00 0.25 0.00 17.66(0.09) (0.35) (0.00) (0.09) (0.00)

1Metaphases with al least one chromosomal aberration (figures with achromatic lesions are not scored as abnormal); 2achromaticlesions; 3chromatid or chromosome breaks; 4dicentrics; 5rings.

Standard error of the mean is in parentheses.

794 A. Guerci et al.

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each of the 14 passages. Sequential irradiation ofCHO cells did not increase the yield of chomatid- orchromosome-type aberrations. However, a significantincrease of achromatic lesions (AL gaps) was foundafter the first or second irradiation with the threedoses employed. This increment persisted along the14 cycles, although a decrease was observed in thesixth cycle with the lower dose and in the fourth cyclewith a dose of 5 mSv. With the highest dose (10 mSv),the frequency of gaps increased after the firstirradiation and remained at the same level until theseventh cycle. In the eighth cycle, an abrupt increasewas observed followed by a decrease to the previouslevels in the two next cycles, and another increase inthe 11th and 12th cycles. Only a few metaphaseswere found after the 13th irradiation hit.

The yield of gaps in the different cycles with thethree doses employed can be observed in figure 1A–C, where the frequency of abnormal metaphases includ-ing or not including achromatic lesions is represented.

Discussion

In the scoring of induced chromosomal aberra-tions, gaps are not considered as true chromosomebreaks and, in general, metaphases carrying only gapsare not considered as abnormal ones. However, thedifferences between gaps and breaks are not so clear.Several years ago, the structure of X-ray-inducedgaps and breaks was studied by means of acombination of light microscopy, transmission elec-tron microscopy of whole-mount preparations andsectioned material, and scanning electron microscopy.Results obtained showed that gaps and breaks werenot separate phenomena and could be considered asdifferent manifestations of the same events and thatgaps may be incomplete breaks (Brecher 1977). Inother studies of the clastogenic effects of Junin virus, asignificant increase of gaps was found in bonemarrow chromosomes of guinea pigs inoculatedwith an attenuated virus strain. When animals weretreated with a very high doses of caffeine 24 h before

Table 2. Frequencies of structural chromosome aberrations in CHO cells irradiated with 5 mSv X-rays.

TreatmentAbnormal

metaphases1 (%)

Chromosomal aberrations per 100 cellsMitotic

index (%)AL2 B3 DIC4 RIN5

– 0.00 1.50 0.00 0.00 0.00 36.00(0.00) (0.23) (0.00) (0.00) (0.00)

1st Irradiation 1.00 1.50 1.00 0.00 0.00 25.66(0.19) (0.23) (0.19) (0.00) (0.00)

2nd Irradiation 1.25 4.50 0.50 0.75 0.00 34.00(0.21) (0.38) (0.14) (0.17) (0.00)

3rd Irradiation 1.50 4.00 0.75 0.75 0.00 36.66(0.23) (0.36) (0.17) (0.17) (0.00)

4th Irradiation 0.25 2.25 0.00 0.25 0.00 48.33(0.09) (0.28) (0.00) (0.09) (0.00)

5th Irradiation 1.00 3.75 0.75 0.25 0.00 30.67(0.19) (0.35) (0.17) (0.09) (0.00)

6th Irradiation 1.00 4.00 0.50 0.50 0.00 44.66(0.19) (0.36) (0.14) (0.14) (0.00)

7th Irradiation 0.75 9.00 0.50 0.25 0.00 41.33(0.17) 0.48) (0.14) (0.09) (0.00)

8th Irradiation 0.75 2.50 0.75 0.00 0.00 30.67(0.17) (0.30) (0.17) (0.00) (0.00)

9th Irradiation 0.25 5.00 0.00 0.25 0.00 38.66(0.09) (0.40) (0.00) (0.09) (0.00)

10th Irradiation 1.75 2.25 1.00 0.75 0.00 33.00(0.25) (0.28) (0.19) (0.17) (0.00)

11th Irradiation 0.50 7.75 0.00 0.50 0.00 33.00(0.14) (0.46) (0.00) (0.14) (0.00)

12th Irradiation 1.00 4.50 0.75 0.25 0.00 29.33(0.19) (0.38) (0.17) (0.09) (0.00)

13th Irradiation 2.00 5.75 1.50 0.50 0.00 20.00(0.27) (0.42) (0.23) (0.14) (0.00)

14th Irradiation 0.50 5.00 0.25 0.25 0.00 2.00(0.09) (0.40) (0.09) (0.09) (0.00)



Cytogenetic analysis in Chinese hamster cells chronically exposed to low doses of X-rays 795

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sacrifice, a decrease of the frequency of gaps and acorrelative increase of the frequency of breaks wasfound. These findings were also interpreted asdifferent manifestations of the same phenomenon inthe sense that gaps are transformed into breaks by anunspecific inhibition of repair mechanisms by caffeine(Dulout et al. 1983, 1985). Recent studies ofperipheral blood lymphocytes of long-haul aircrewshowed a significant increase in gaps and breaks aswell as of translocations (Cavallo et al. 2002). Inaddition, similar results were found in hospitalworkers with high frequencies of stable aberrationsas well as chromatid gaps and breaks. The increase ofsuch types of aberrations was attributed to chronicexposure to low level ionizing radiation (Hagelstromet al. 1995).

Genetic effects of accidental or occupationalexposure to radiation are usually analysed incirculating lymphocytes in the G0 period and theeffects are observed in the next mitosis mainly aschromosome-type aberrations (Natarajan et al. 1996).In order to simulate these conditions, in the present

study CHO cells were treated when the cultures were90–95% confluent, when it was supposed that90–95% were in a quiescent state, in the G0/G1

period of the cell cycle (Volkmer and Virsik-Peuckert1990). But instead of chromosome-type aberrations,only the frequency of gaps was increased in the nextmetaphase. This fact is in agreement with the above-mentioned results obtained in aircrew and hospitalworkers, but some other factors must be taken intoaccount. First, the fact that some cells could be in anyphase of the cell cycle must be considered. Actually,BrdU experiments carried out simultaneously (datanot shown) showed that the confluent state was notindicative of quiescence in CHO cells. However, thelapse of 15 h between irradiation and fixation wastoo long to suppose that cells exhibiting gaps inmetaphase were in prophase at the moment ofexposure. On the other hand, the variation in thefrequency of gaps as well as the variation in themitotic index along the 14 cycles could be anindication of the induction of genomic instability inthe population of CHO cells. Genomic instability has

Table 3. Frequencies of structural chromosome aberrations in CHO cells irradiated with 10 mSv X-rays.

Treatment Abnormal metaphases1 (%)

Chromosomal aberrations per 100 cells

Mitotic index (%)AL2 B3 DIC4 RIN5

– 0.00 1.50 0.00 0.00 0.00 36.00(0.00) (0.23) (0.00) (0.00) (0.00)

1st Irradiation 1.75 4.75 0.75 1.00 0.00 21.66(0.25) (0.39) (0.17) (0.19) (0.00)

2nd Irradiation 0.75 3.75 0.25 0.50 0.00 33.33(0.17) (0.35) (0.09) (0.14) (0.00)

3rd Irradiation 0.50 4.50 0.50 0.00 0.00 39.66(0.14) (0.38) (0.14) (0.00) (0.00)

4th Irradiation 1.00 5.00 0.50 0.50 0.00 38.00(0.19) (0.40) (0.14) (0.14) (0.00)

5th Irradiation 1.50 5.00 1.00 0.50 0.00 43.67(0.23) (0.40) (0.19) (0.14) (0.00)

6th Irradiation 0.75 4.25 0.50 0.25 0.00 33.33(0.17) (0.37) (0.14) (0.09) (0.00)

7th Irradiation 1.00 4.00 1.00 0.00 0.00 33.33(0.19) (0.36) (0.19) (0.00) (0.00)

8th Irradiation 1.25 10.0 0.75 0.50 0.00 31.97(0.21) (0.48) (0.17) (0.14) (0.00)

9th Irradiation 0.75 4.50 0.50 0.25 0.00 53.33(0.17) (0.38) (0.14) (0.09) (0.00)

10th Irradiation 0.00 5.00 0.00 0.00 0.00 18.33(0.00) (0.40) (0.00) (0.00) (0.00)

11th Irradiation 2.50 9.50 1.50 1.00 0.00 43.33(0.30) (0.48) (0.23) (0.19) (0.00)

12th Irradiation 0.50 15.5 0.50 0.00 0.00 49.00(0.14) (0.48) (0.14) (0.00) (0.00)

13th Irradiation – – – – – 1.6614th Irradiation – – – – – 2.33




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been defined as the increased rate of acquisition ofalterations in the genome due to a process initiated byradiation within a cell that is perpetuated in theprogeny of that irradiated cell (Morgan et al. 1996,2002, Kaplan et al. 1997, Nagar et al. 2003). Theresults described could reflect a complex response torepeated irradiations. If genomic instability is pro-duced by factors secreted by unstable cells thatstimulate the production of reactive oxygen species(Morgan et al. 2002), the induction of gaps could bethe consequence of the direct effect of very low dosesof X-rays plus the effect of the factors secreted byunstable cells. According to this, continuous rises andfalls in the frequency of gaps as well as in the mitoticindex could reflect the simultaneous induction ofendogenous DNA damage, cell death and cell

survival. In fact, with the doses of 5.0 and10.0 mSv, the mitotic index abruptly fell after the13th and 14th irradiation, respectively, indicatingperhaps an accumulative effect proportional to theradiation dose. However, further studies are necessaryto confirm these assumptions.

Acknowledgements

This work was part of the Proyecto integrado demutagenesis y carcinogenesis ambiental of thePrograma de Incentivos para Docentes-Investigadoresde Universidades Nacionales. A. Guerci had afellowship from the National University of La Plata.The authors are grateful to Professor Juan Andrieufor calibration of the irradiation equipment and to

Figure 1. Abnormal metaphases in CHO cells irradiated with 2.5 (A), 5.0 (B) and 10.0 mSv (C). AM, abnormal metaphases; AMzG,abnormal metaphases including gaps.


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Professor Susana J. Barani for revision of themanuscript.

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cytogenetic analysis in chinese hamster cells chronically exposed to low doses of x‐rays

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