Frequencies of X-ray induced chromosome aberrations in lymphocytes of xeroderma pigmentosum and Fanconi anemia patients estimated by Giemsa and fluorescence in situ hybridization staining techniques

Linfocitos sanguineos de pacientes com xeroderma pigmentosum (XP) e anemia de Fanconi (FA) foram avaliados quanto a sensibilidade a ionizacao radiante estimando-se a frequencia de aberracoes cromossomicas (CA) induzidas por raios-X (1 e 2 Gy). As frequencias de aberracoes no genoma inteiro foram estimadas em preparacoes de linfocitos irradiados nas fases G0 e G2 coradas com Giemsa. As frequencias de translocacoes e dicentricos envolvendo os cromossomos 1 e 3 e o cromossomo X foram determinadas em lâminas coradas por hibridizacao fluorescente in situ (FISH). Um aumento em todos os tipos de CA foi observado em linfocitos XP e FA irradiados na fase G0 quando comparados a controles. A frequencia de dicentricos e aneis foi 6-27% maior (com 1 e 2 Gy) em linfocitos XP e 37% maior (com 2 Gy) em linfocitos FA do que em controles, enquanto que as delecoes cromossomicas foram mais frequentes em linfocitos XP irradiados (30% com 1 Gy e 72% com 2 Gy) do que em controles e 28-102% mais frequentes em linfocitos FA. Em linfocitos irradiados na fase G2 a frequencia total de CA foi 24-55% mais elevada em linfocitos XP do que em controles. Na maior parte dos casos as frequencias de translocacoes foram maiores do que as frequencias de dicentricos (21/19).


INTRODUCTION
Ionizing radiation induces chromosome aberrations in G 0 human peripheral lymphocytes which can be classified as unstable (dicentrics, centric rings and acentric fragments) or stable (various kinds of translocations) (Natarajan et al., 1992(Natarajan et al., , 1994)).Unstable aberrations are lost during successive cell divisions whereas the stable ones can persist for a long time (Natarajan et al., 1991;Lucas et al., 1992;Boei et al., 1994).The frequencies of structurally aberrant chromosomes in peripheral lymphocytes of persons accidentally exposed to ionizing radiation have been used since the 1960s to estimate absorbed radiation dose (Bender and Gooch, 1966;Sasaki and Miyata, 1968;Natarajan, 1984).
According to classical models of formation of chromosome exchange aberrations, the ratio of radiation-induced symmetrical exchanges (reciprocal translocations, inversions) and asymmetrical exchanges (dicentrics, rings) should be 1:1 (Evans, 1962).A change in this ratio in favor of translocation frequencies has been observed (Natarajan et al., 1992;Schmid et al., 1992;Nakano et al., 1993), and it has been suggested that the higher estimate of translocations in these studies may be due to the misclassification of exchange aberrations.This problem could be overcome by combination of whole chromosome marking with centromere specific DNA probes (Weier et al., 1991).Straume and Lucas (1993) found a close correlation between dicentrics and reciprocal translocations even after the application of these techniques.Finnon et al. (1995) also observed that the yield of radiation-induced translocations was not significantly higher than that of dicentrics.Natarajan et al. (1996) explained this by suggesting that if one confines scoring only to reciprocal translocations, ignoring other types such as terminal and interstitial translocations, then the frequencies of dicentrics and translocations are almost equal.In general, the frequency of translocations has been found to be higher than the frequency of dicentrics.This shift may be dependent upon the structure of the chromosomes, position of the centromere (Natarajan et al., 1996), and the nature of the chromatin at the time of irradiation (Vyas et al., 1991).The formation of these two types of aberrations (translocations and dicentrics) may be the result of different mechanisms (Natarajan et al., 1996).
Fanconi anemia (FA) and xeroderma pigmentosum (XP) are rare repair-deficient, mutagen-sensitive, autosomal recessive human disorders with chromosome instability, which are cancer prone.Cells derived from FA patients have more spontaneous chromosomal damage than those of other instability syndromes (Schroeder et al., 1964(Schroeder et al., , 1989;;Natarajan et al., 1989).There exist inter-and intraindividual variations specifically with regard to susceptibility to cross-linking agents and X-rays (Sasaki and Tonomura, 1973).
XP is characterized by high sensitivity to sun exposure, susceptibility to skin cancer, cutaneous pigmentation, impaired DNA repair and, in some patients, neurological degeneration (Kraemer and Slor, 1985).These patients have a greater than 1000-fold-increased frequency of UV-induced skin cancer (Cleaver and Kraemer, 1989) when compared with the normal population.
Frequencies of X-ray induced chromosome aberrations in lymphocytes of xeroderma pigmentosum and Fanconi anemia patients estimated by Giemsa and fluorescence in situ hybridization staining techniques Radha Saraswathy and A.T. Natarajan

Abstract
Blood lymphocytes from xeroderma pigmentosum (XP) and Fanconi anemia (FA) patients were assessed for their sensitivity to ionizing radiation by estimating the frequency of X-ray (1 and 2 Gy)-induced chromosome aberrations (CA).The frequencies of aberrations in the whole genome were estimated in Giemsa-stained preparations of lymphocytes irradiated at G 0 or G 2 stages.The frequencies of translocations and dicentrics involving chromosomes 1 and 3 as well as the X-chromosome were determined in slides stained by fluorescence in situ hybridization (FISH) technique.An increase in all types of CA was observed in XP and FA lymphocytes irradiated at G 0 when compared to controls.The frequency of dicentrics and rings was 6 to 27% higher (at 1 and 2 Gy) in XP lymphocytes and 37% higher (at 2 Gy) in FA lymphocytes than in controls, while chromosome deletions were higher in irradiated (30% in 1 Gy and 72% in 2 Gy) than in control XP lymphocytes and 28 to 102% higher in FA lymphocytes.In G 2 -irradiated lymphocytes the frequency of CA was 24 to 55% higher in XP lymphocytes than in controls.In most cases the translocation frequencies were higher than the frequencies of dicentrics (21/19).
Several studies (Bigelow et al., 1979;Arlett and Harcourt, 1980;Parshad et al., 1983) have shown increased radiosensitivity of XP and FA cells in the G 2 phase of the cell cycle, but in other studies an increase in chromosomal aberrations was not observed in FA cells (Evans et al., 1978;Sasaki, 1978) and XP fibroblasts (Darroudi et al., 1995).
The present study was designed to estimate the frequencies of X-ray-induced chromosome aberrations in lymphocytes of XP and FA patients.

Culture conditions
Venous blood drawn in lithium heparin tubes was set up for whole blood cultures in Ham's F10 medium supplemented with 15% heat inactivated fetal calf serum (Gibco), phytohemagglutinin and antibiotics.The cultures were incubated at 37°C in a 5% CO 2 atmosphere.The FA blood sample was obtained from the Academic Hospital, Leiden, and XP from the Institute of Human Genetics, Budapest, Hungary; the complementation groups of these patients are not known.
X-Rays were generated by an ENRAF machine, operating at 200 kV, 6 mA at a dose rate of 2 Gy/min.

Treatment G 0 irradiation
Normal, XP and FA blood lymphocyte cultures were irradiated with a dose of 1 or 2 Gy and harvested 48 h after initiation of the cultures.Colcemid (Sigma; 0.3 µg/ml) was added to the cultures 2-3 h before harvest.5-Bromo-2deoxyuridine (BrdU, Sigma; 10 µM) was added to all the cultures after irradiation for identification of first and second division cells.Scoring of chromosomal aberrations was restricted to cells at first mitosis.

G 2 irradiation
Lymphocyte cultures were set up and grown for 69 h and then irradiated with doses of 0.5 or 1 Gy.Colcemid was added to these cultures 0.5 h after irradiation and the lymphocytes were harvested and fixed after 2.5 h.For harvesting the cultures, cells were subjected to hypotonic shock (0.075 mM KCl) for 25 min and fixed in acetic acid:methanol (1:3).Appropriate controls were prepared.
For G 0 irradiation studies, air-dried preparations were stained by the fluorescence plus Giemsa (FPG) technique (Perry and Wolff, 1974).Two hundred metaphases were analyzed for the presence of dicentrics, rings and chromosome fragments for each radiation dose.For G 2 irradiation studies, the slides were stained with 5% Giemsa solution.Chromatid gaps, chromatid breaks, isochromatid breaks and chromatid exchanges were scored in 200 cells at each dose.

Fluorescence in situ hybridization
Slides to be processed for in situ hybridization were stored dry at -20°C.Chromosome specific libraries were obtained from blue-scribe plasmids.A mixture of three different chromosomes (1, 3 and X) was used.The in situ hybridization method routinely used in our laboratory (Natarajan et al., 1992) was adapted.In order to analyze the aberrations in the first mitosis, the slides were stained with Hoechst 33258 (Sigma), exposed to UV light and processed for in situ hybridization (Boei et al., 1994).
For immunological detection, after hybridization the slides were washed successively in 50% formamide/2X SSC (42°C), 0.1X SSC (60°C) and 4X SSC/0.05%Tween 20, pH 7, at room temperature.The first incubation was carried out with immunological buffer (NFDM) for 30 min at room temperature.After four washes with SSC, 0.05% Tween 20 and TNT (0.1 M Tris-HCl, 0.15 M NaCl and 0.05% Tween 20), the second incubation with antibodies avidin-FITC and mouse anti-digoxigenin diluted in TNB (0.1 M Tris-HCl, 0.15 M NaCl and 0.5% Boehringer blocking agent) was performed at 37°C for 30 min.The third incubation was carried out with goat-anti-avidin D and sheep anti-mouse dig diluted in TNB for 30 min at 37°C.The fourth incubation was carried out with avidin-FITC and sheep anti-dig TRITC for 30 min at 37°C.Each incubation was followed by washing with TNT.After the final wash with PBS the slides were counter-stained with DAPI (4,6-diamidino-2-phenylindole; Sigma) diluted in an anti-fading agent, vectashield (Vector Laboratories) (Figure 1).
In order to detect translocations, dicentrics and complex exchanges involving chromosome numbers 1 and 3 and the X-chromosome, 300 to 500 metaphases were analyzed for each radiation dose using a Zeiss fluorescence microscope equipped with triple filters.Due to variations in mitotic index the number of metaphases scored by FISH varied for each experimental point.The translocations and dicentrics were scored using the same slide.The background frequencies were subtracted to obtain the induced frequency of translocations and dicentrics.
The genomic translocation and dicentric frequencies were calculated using the formula for correction of the probed fraction of the genome (Lucas et al., 1992).This formula relates the translocation frequency measured by FISH, Fp, to the genomic translocation frequencies, F G , through the fraction of the genome covered by the probes, fp, as follows:

RESULTS AND DISCUSSION
Spontaneous chromosomal aberrations in normal, XP and FA lymphocytes The baseline aberration frequencies in normal and XP lymphocytes were in the range of 2 to 3%, whereas in FA the frequency was 23% (Table I).Darroudi et al. (1995) also observed a similar spontaneous aberration frequency in the range of 2 to 3% in the normal and XP-C cell lines, and 16 to 22% in FA cells, which is known to be characteristic of FA patients (Schroeder et al., 1964;Natarajan et al., 1989).However, the frequency of spontaneous aberrations in XP lymphocytes was similar to that of normal lymphocytes.To compare the aberration frequencies in XP, FA and normal lymphocytes the background aberration frequencies were subtracted from the induced aberration frequencies.The frequency of dicentrics and rings in XP and in FA lymphocytes was markedly high.There was a high frequency of chromosome deletions with a 30-72% increase in XP lymphocytes after 1 and 2 Gy of X-rays and a 28-102% increase in deletions in the FA lymphocytes as compared with normal lymphocytes.The ratio between the frequencies of dicentrics and chromosome deletions was 1:0.6 and 1:0.8 in normal lymphocytes, 1:1.9 and 1:1.6 in XP lymphocytes and 1:3.8 and 1:1.8 in FA lymphocytes (1 and 2 Gy, respectively).The frequencies of chromatid type aberrations in FA lymphocytes were more than 30 times higher when compared with XP lymphocytes and no chromatid aberration was observed in normal lymphocytes, while chro-matid exchanges were only observed in FA lymphocytes, a known characteristic of these cells.G 0 radiation produced chromatid breaks/gaps in FA lymphocytes (0.37 and 0.36 per cell in 1 Gy and 2 Gy, respectively) and were zero in normal lymphocytes and 0.01 in XP lymphocytes, which further confirms a positive defect in repair of radiation damage in FA cells.G 2 radiosensitivity in normal and XP lymphocytes The frequency of X-ray-induced total CA aberrations in XP lymphocytes was 0.79 and 2.17 per cell for 0.5 Gy and 1 Gy, respectively, while in the normal lymphocytes it was 0.55 and 1.62, indicating an increase (24-55%) in XP lymphocytes (Table II).The frequency of chromatid and isochromatid breaks in XP lymphocytes was 0.72 and 2.03 for 0.5 and 1 Gy, respectively, while in normal lympho-cytes the frequencies were 0.54 and 1.6, indicating an increase (18-43%) in the XP lymphocytes when compared to normal lymphocytes.The frequency of interchanges was seven times (6% at 0.5 Gy and 12% at 1 Gy) higher in the XP lymphocytes than in the controls.Isochromatid breaks induced by X-rays were observed only in XP lymphocytes and were dose related (0.15 and 0.22 per cell for 0.5 and 1 Gy, respectively).Even though Taylor (1978) observed an increase in chromatid aberrations in XP cells irradiated in G 2 , it was not as significant as it was in the case of ataxia telangiectasia cells.In XP-C lymphocytes an increase in ionizing radiation-induced chromosome aberrations has been observed in the G 2 phase of the cell cycle (Parshad et al., 1983).

Estimation of radiation-induced dicentrics
and translocations in normal, XP and FA lymphocytes using FISH

A. Genomic translocation frequency
The genomic translocation frequency (including reciprocal and terminal ones) calculated for individual chromosomes (1, 3, X) represents about 20% of the total genome.The translocation frequencies for chromosomes 1, 3 and the X-chromosome followed the order expected on the basis of their DNA contents in normal lymphocytes.The DNA contents of chromosomes 1, 3 and the X-chromosome are 8.15, 6.63 and 5.08% of the total genomic value, respectively (Mendelsohn et al., 1973).The translocation frequencies in individual chromosomes of XP lymphocytes when compared with those of the chromosomes of normal lymphocytes showed an increase of 73.2% in the X-chromosome (2 Gy) and 48.5% in chromosome 3 (at 2 Gy).At 1 Gy the translocation frequency was 3.2% and 10.3% higher than those of XP lymphocytes for chromosome 3 and the X-chromosome respectively (Table III).In FA lymphocytes, the translocation frequencies for chromosomes 1, 3 and the X-chromosome followed the order based on their DNA content.The translocation frequencies in individual chromosomes of FA lymphocytes were almost equal to the respective chromosomes in normal lymphocytes except for the X-chromosome (27.5% higher at 1 Gy).
Mean translocation frequency of the mixture of chromosomes 1 and 3 and the X-chromosome in normal, XP and FA lymphocytes At 1 Gy the mean translocation frequency in XP lymphocytes was similar to that of normal lymphocytes, while at a dose of 2 Gy the mean frequency was about twice that of normal lymphocytes.At 1 Gy the mean frequency of translocation in FA lymphocytes was lower than that of normal but at 2 Gy it was almost similar to normal (Table IV).

B. Genomic dicentric frequency
The genomic dicentric frequencies calculated using the same formula for calculating genomic translocation frequencies, for chromosomes 1, 3 and the X-chromosome in normal and XP lymphocytes at 2 Gy followed the order based on their DNA contents (Table III).The dicentric frequencies in individual chromosomes of XP lymphocytes when compared with the chromosomes from normal lymphocytes showed an increase of 15.1% for chromosome 1 (2 Gy) and almost equal for chromosomes 3 and X.In FA lymphocytes, the dicentric frequencies for individual chromosomes (1, 3 and X) followed the order based on their DNA contents.

Ratio between translocation and dicentric frequencies
The frequency of translocations in control, XP and FA lymphocytes increased 8.4, 10.8 and 10.6% at 1 Gy and 3.8, 53.9 and 25.9% at 2 Gy, respectively, over dicentrics (Table V).This confirms the observation of Natarajan et al. (1996) that the frequency of translocation is higher than the frequency of dicentrics, indicating that two different mechanisms may be involved in the production of translocations and dicentrics as observed in normal humans.

C. Complex exchanges and centric rings
In XP and FA lymphocytes a dose-dependent increase in the frequency of complex exchanges and centric rings was observed, ranging from 0.2-3.3%.These values were not used in our analysis.

CONCLUSIONS
The frequency of CA produced by G 0 and G 2 irradiation in XP and FA lymphocytes was higher than that of normal lymphocytes.The deletion type of chromosomal aberrations observed in XP and FA lymphocytes was several fold more abundant than in the normal lymphocytes, reflecting a possible defect in their capacity to repair dam-  Similarly, though FA cells are known to be sensitive to cross linking agents, implying a defect in the repair of cross links, they are also known to be sensitive to several classes of clastogenic agents (Sasaki and Tonomura, 1973).

ACKNOWLEDGMENTS
We would like to thank Dr. K.M. Marimuthu, Prof. Emeritus, Dept. of Genetics, Madras University, India, for his help in the preparation of this manuscript.The research was financially supported by an EU Nuclear Safety Programme grant to ATN.

Table I -
G 0 radiosensitivity of human peripheral lymphocytes in normal, xeroderma pigmentosum (XP) and Fanconi anemia (FA) individuals estimated by Giemsa staining*.
*Induced aberrations have been corrected for spontaneous aberrations.*Induced aberrations have been corrected for spontaneous aberrations.X-ray-induced chromosome aberrations in lymphocytes of XP and FA patients

Table III -
Genomic frequency of translocation and dicentrics in normal, xeroderma pigmentosum (XP) and Fanconi anemia (FA) lymphocytes following X-irradiation as estimated by FISH.

Table V -
Ratio between genomic translocation and dicentric frequencies in normal, xeroderma pigmentosum (XP) and Fanconi anemia (FA) lymphocytes following X-irradiation as estimated by FISH.