X-ray induced mutation in Syrian hamster fetal cells

Mutation Research 500 (2002) 9–15

X-ray induced mutation in Syrian hamster fetal cells Paul J. Donovan a,∗ , George T. Smith a a Laboratory of Comparative Carcinogenesis, Department of Health and Human Services, National Cancer Institute at Frederick, Building 538, Room 205E, Frederick, MD 21702-1201, USA

Received 14 October 2001; accepted 12 November 2001

Abstract Transabdominal X-rays are a risk factor for childhood leukemia, and X-ray exposure of mouse fetuses has led to increases in both mutations and initiated tumors in offspring. However, fetal sensitivity and dose–response characteristics with regard to transplacental mutagenesis by X-rays have never been quantified. In the current experiment, pregnant Syrian hamsters at day 12 of gestation were irradiated with 300-kV X-rays. Twenty-four hours later, the fetuses were removed and their cells were allowed a 5 day expression time in culture. They were then seeded for colony formation and also for mutation selection by 6-thioguanine (6-TG). Mutation frequency was linear over the entire dose range, 10–600 R. The average induced 6-TG mutant frequency was 4.7 × 10−7 per R. These results suggest that fetal cells are highly sensitive to induction of mutations by X-rays, and that a no-effect threshold is not likely. The 10 R dose caused a 25-fold increase in mutation frequency over the historical control, 45 × 10−7 versus 1.8 × 10−7 , an increase per R of 2.5-fold. Increased risk of childhood cancer related to obstetrical transabdominal X-ray has also been estimated at 2.5-fold per R. Thus, our results are consistent with mutation contributing to this effect. Published by Elsevier Science B.V. Keywords: In vivo; 6-Thioguanine; Mutation; Mutagenicity test; Somatic cells

1. Introduction Transabdominal obstetric diagnostic X-rays have been clearly associated with increased risk of childhood cancer [1–3]. Stewart and Kneale [4] demonstrated a linear dose effect with increasing number of X-ray films. Dosage (R) delivered was calculated from the number of films, and the average frequency of cancer in children was estimated to be 5.73 × 10−4 per R [5]. Another calculation using the same data yielded a frequency of 4.6 × 10−4 per R [6]. The weighted mean exposure was 0.61 R for whole body dose in irradiated fetuses from obstetric radiography ∗ Corresponding author. Tel.: +1-301-846-5420; fax: +1-301-846-5946. E-mail address: [email protected] (P.J. Donovan).

0027-5107/02/$ – see front matter. Published by Elsevier Science B.V. PII: S 0 0 2 7 - 5 1 0 7 ( 0 1 ) 0 0 2 9 9 - 8

[6]. The odds ratio for excess childhood cancer death associated with obstetrical irradiation was 1.5 [7]. Since the doses employed in these studies were quite low, it is of interest to understand the mechanism of the effect and the reason for high fetal sensitivity. This information would be useful for extrapolation to other transplacental carcinogenesis risk situations. A mutagenic effect is one possibility. X-irradiation induced a high, dose-dependent incidence of somatic mutations in (PT X HT F1) mouse embryos at day 10.5 [8], with a control frequency of 2.8 and 6.5% at 30 R, and 14.1% at 103 R. There was a corresponding dose-dependent increase in skin tumors and hepatomas by post-natal administration of 12-O-tetradecanoylphorbol-13-acetate. Thus incidence of both mutations and tumors increased with dose of X-ray. In a second experiment [9] exposure

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of embryos and fetuses to 36 R X-ray at different days of gestation resulted, after birth, in a persistent increased tumor susceptibility to post-natal urethane, a carcinogen. This pertained to all stages irradiated from day 0 to 14 (except day 6), but was not observed when irradiation was given at late fetal stages. It is of interest that in these experiments no tumors appeared after irradiation only. In several other studies X-rays were found to have minimal effect on tumors in offspring, when these received no post-natal treatment [10–12]. In this context, maternal smoking may be synergistic with the effect of X-rays [13]. Mothers smoking one–nine cigarettes a day and X-rayed had children with a risk ratio of 4.0 for acute lymphoblastic leukemia. Children of mothers smoking more than nine cigarettes per day and X-rayed had a risk ratio of 5.4. The increased risk from abdominal X-rays alone was 1.3. It is possible that mutagenic damage due to X-rays requires additional cellular changes before neoplastic growth ensues. Further investigation of the role of fetal tissue mutagenesis in predisposing to cancer development could be facilitated by establishment of quantitative sensitivities and dose–response patterns. This report describes our investigation of the X-ray induction of 6-thioguanine (6-TG)-resistant mutants in Syrian hamster fetal cells. This was measured by an in vivo/in vitro mutation assay where the pregnant mothers were irradiated in vivo and 24 h later the fetuses were removed for in vitro measurement of 6-TG-resistant mutant frequency in somatic cells. The results indicate that cells of the developing Syrian hamster are very sensitive to the mutagenic effect of X-rays, with a linear dose–response curve.

2. Materials and methods 2.1. Animals Timed pregnant Syrian hamsters (Mesocricetus auratus) were obtained from the animal production area of NCI-Frederick, Frederick, MD. By convention, day 1 of gestation was taken as the day following overnight breeding. Animals usually weighed about 200 g and were multiparous. Animal use was in accordance with NIH guidelines and was approved by the NCI-Frederick Animal Care and Use Committee.

2.2. Irradiation Pregnant Syrian hamsters at day 12 of gestation were irradiated and allowed to proceed to day 13 of gestation when primary cultures were made. Animals were irradiated with a Phillips MG301 X-ray therapy unit (Phillips Electronic Instruments, Mount Vernon, NY) operated at 10 mA and 300 kV with a 2 mm aluminum filter and a dose rate of 105 R/min. Doses were checked with a Victoreen ionization meter (Victoreen Instrument Division, Cleveland, OH). 2.3. Preparation and cryo-preservation of primary cell suspensions On day 13 of gestation, cells were isolated for primary culture by the following method. Viable fetuses were aseptically removed; special care was taken to avoid selecting fetuses that were whitish or resorbing. Whole fetuses pooled from each litter were minced with curved iris scissors, the tissues were washed twice with Dulbecco’s phosphate buffered saline (PBS) (purchased from Biofluids, Rockville, MD) and the tissue fragments were dissociated in successive 100 ml volumes of 0.25% trypsin (Biofluids, Kankakee, IL). The trypsin was prewarmed to 37 ◦ C, but the 10 min period of gentle stirring in a fluted trypsinizing flask (Bellco Glass, Vineland, NJ) was at room temperature. The first trypsinate was discarded. The second and third trypsinates were then pooled with added fetal calf serum (FCS) to stop the trypsin action. After centrifugation at 100 × g for 10 min, the cells were resuspended at 0.25–0.5 × 108 ml−1 of Dulbecco’s modified Eagle’s medium (DMEM) containing 9% dimethylsulfoxide (DMSO) and 10% FCS in freezing tubes (A/S Nunk, Kanstrup, Denmark), frozen at 1 ◦ C/min in a programmed cell freezer (Minnesota Valley Engineering, Minneapolis, MN) and stored above liquid nitrogen. In general, the cells of one fetus were suspended in 1–1.5 ml. 2.4. General cell culture Tubes of frozen primary cells were quickly thawed at 37 ◦ C and the cells were added to 4 ml of FCS in a conical centrifuge tube (Costar, Cambridge, MA) and centrifuged at 100 × g for 10 min. All the plasticware was from Costar. The supernatant was aspirated and

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cells were plated in 25 ml of complete medium in a 150 cm2 tissue culture flask. Cells were routinely cultured in DMEM (low glucose formulation) made by BioFluids, Inc., Rockville, MD. The medium contained 10% v/v FCS (Sterile Systems, Logdon, UT), with penicillin and streptomycin (from Gibco Co., Grand Island, NY) at a final concentration of 100 U and 100 ␮g/ml, respectively. Cells were cultured at 37 ◦ C in a 10% CO2 atmosphere in a humidified water-jacketed incubator. For experiments or subculture, primaries were rinsed with Hank’s balanced salt solution (Gibco Co.), and then 10 ml of 0.05% trypsin (Biofluids) was added; the cells were removed by shaking when just starting to round up and centrifuged as above.

−20 ◦ C. One hundred microliters of freshly thawed 6-TG stock solution were added to the 10 ml of medium already in the plates. Medium was replaced once a week with medium containing freshly dissolved 6-TG. Selection plates were fixed and stained with Giemsa (Fisher Scientific, Pittsburgh, PA) 3–4 weeks later. Colonies were counted without magnification. Colonies counted were at least 2 mm in diameter and contained at least 50 cells. Cloning efficiency of the cells was determined at the time of seeding for selection, by diluting the same to 20 cells/ml in complete medium and adding the suspension to a 100-mm plate.

2.5. Gestation stage and selection time

3.1. X-ray survival curve

We determined that cloning efficiency for fetal cells is optimal on gestation day 13 for the hamster. Primary cultures derived from fetuses and embryos at earlier times of gestation show lower cloning efficiencies [12,14]. Using N-ethyl-N-nitrosourea (ENU) as a transplacental mutagen in hamsters, we found that the optimum expression time in vitro for selection of mutants by 6-TG was 5 days, with a decrease in mutation frequency with longer selection times [15].

3. Results

Fig. 1 is a plot of the cloning efficiency of Syrian hamster fetal cells exposed to varying doses of X-ray,

2.6. Treatment of animals for positive controls In order to confirm that the procedures utilized were effective at detecting mutations, 10 pregnant hamsters were injected i.p. on gestation day 12 with the known mutagen ENU at a dose of 1 mmol/kg (117 mg/kg). Litters were removed and pooled on gestation day 13 (positive control). 2.7. Selection using 6-thioguanine The protocol for 6-TG selection was as follows. After an expression time of 5 days in culture, cells were seeded in 10 ml of complete medium at the rate of 2 × 105 per 100-mm plate. Twenty-four hours later, 6-TG was added, for a final concentration of 10 ␮g/ml. A stock solution of 6-TG was prepared as follows: 6-TG (Sigma Chemical Co., St. Louis, MO) was dissolved in 0.5% aqueous sodium carbonate, sterilized by filtration through a 0.22 ␮m filter and kept frozen at

Fig. 1. X-ray survival curve. Syrian hamster fetuses were irradiated at day 12 of gestation. At day 13 of gestation the fetuses were removed. Primary cultures were made and at day 5 in culture they were seeded at 300–200,000 cells per 100-mm plate. After 7 days the plates were fixed and stained for determination of cloning efficiency. Error bars represent standard errors of the mean.

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collected after 24 h in vivo and allowed a 5 day expression period in vitro. Values are relative to the control cells, which had a 16% cloning efficiency. Doses between 10 and 100 R had cloning efficiencies that were 30–50% of control, with decreases at the higher doses of 400 and 600 R. 3.2. 6-TG mutants per plate and per surviving cell The positive control ENU at 1 mmol/kg demonstrated the success of the selection procedure, yielding an average 3.56 ± 0.48 mutants per plate, a frequency per surviving cell of 1.3 × 10−4 . The concurrent control for this study, consisting of 39 plates of cells, presented no mutants. Historically, we have determined from a large data set (24 litters) that the baseline control frequency for mutation to 6-TG resistance in Syrian hamster cells is 0.01 per plate or 1.8 × 10−7

Fig. 2. X-ray induction of 6-TG mutants. Syrian hamster fetuses were exposed to X-ray on gestation day 12, and their cells were seeded at 2 × 105 cells per 100-mm plate, after an in vitro 5 day expression time. Twenty-four hours later 6-TG was added at 10 ␮g/ml. Plates were fixed and stained 3–4 weeks later. Inset: X-ray induction of 6-TG mutants after low doses. The historical control mutation frequency (- - -) was 1.8 × 10−7 , with a 95% upper population boundary of 7.3 × 10−7 (···) [15].

per surviving cell [15]. The upper 95% confidence bound was 6.3 × 10−7 or 3.5 times the overall mean [15]. Fig. 2 is a plot of the mutants per surviving Syrian hamster fetal cell exposed to varying doses of X-ray. All doses used in the current study led to mutations detected. The value for the lowest dose, 10 R, resulted in 0.05 mutants per plate, 45 × 10−7 per viable cell or 25 times the historical control. The increase in mutations with increasing X-ray dose was essentially linear up to 600 R. The average number of 6-TG mutants per R was 4.3 × 10−7 for 10 R; 4.3 × 10−7 for 25 R; 5.1 × 10−7 for 70 R; 3.9 × 10−7 for 200 R; 5.0 × 10−7 for 400 R; and 5.3 × 10−7 for 600 R. The average mutants per R for the whole curve was 4.7 × 10−7 . 4. Discussion Transplacental X-rays had a lethal effect on many cells of hamster fetuses late in gestation. This was already apparent at the lowest dose used, 10 R, but was surprisingly dose-independent up to 200 R. These results are consistent with findings for cultured cells, where the dose that allows 37% of the cells to survive was around 100 R for most cell types, including cell lines and primary cells [16]. In the surviving fetal cells assessed in vitro, the X-ray exposure had clearly been mutagenic, with the 10 R dose already causing a 25-fold increase in mutation rate over the historical control. Our assay was more sensitive than the coat color spot assay in mice, where 30 R yielded a 2.3-fold increase in mutations over controls [8]. In contrast to the effect on survival, the X-ray effect on mutation was quite linear. This suggests that a no effect threshold is not likely. If the dose–response was in fact linear below the 10 R dose, then an increased mutation rate of 1.5 would have been expected for 0.6 R, the mean exposure of human fetuses from transabdominal obstetrical X-ray [6] associated with a 1.5-fold increase in childhood cancer [7]. This remarkable concordance is quite consistent with mutagenesis contributing to the increased cancer risk observed in the children. In order to compare these effects in fetal hamster cells with the effects of X-rays in cells in culture, we have summarized in Table 1 published investigations which reported mutation in the HGPRT gene

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Table 1 Cell line

Curve

Doses (R)

Energy

(6-TG mutants × 10−7 )/Ra

Don [26] V79 [26] Human fetal lung [27] V79 [27] L5178Y [28] Human fetal lung [29] V79 [30] V79-4 [31] CHO [32] V79 [33] Human skin primary [34] CHO [35] CHO [36] L5178R [37] L5178S [37] Human lymphocytes [38] Human lymphocytes [39] TK6 human lymphoblasts [40] CHO [41] TK6 human lymphoblasts [42] TK6 human lymphoblasts [43] CHO AA8 [44] V79 [45] Human lymphocytes [46]

Curvilinear Curvilinear Linear Curvilinear Curvilinear Linear Linear Curvilinear Curvilinear Linear Linear Curvilinear Linear Linear Linear Linear Linear Linear Curvilinear Linear Linear Linear Curvilinear Linear

0–600 0–600 0–200 0–800 0–600 0–200 0–800 100–800 0–800 0–800 0–300 0–900 50–300 0–300 0–300 50–400 0–200 0–200 50–800 0–150 0–150 0–700 250–750 100–400

100 kV 100 kV 250 kV 250 kV 150 kV 250 kV 150 kV Not given X-ray 190 kV 150 kV 50 kVp 250 kV 250 kV 250 kV 100 kV 250 kV 100 kV 250 kV 220 kV 220 kV 250 kV 1.8 MeV 250 kV

0.5 3.6 4 3 3 3.1 1.4 1.1 2.5 2.3 2.6 6.1 0.34 0.95 0.35 2.4 2.0 0.62 1.7 0.81 0.92 2.7 2.5 1.3

a

For curvilinear responses, the value at the highest point on the curve is given.

(6-TG selects for hypoxanthine-guanine phosphoribosyl transferase (HGPRT)-deficient cells and is a recessive X-linked trait [17–20]). It is apparent from the comparison that the Syrian hamster fetal cells are more sensitive to the mutagenic effect of X-ray than are most cultured cells. This may be due to rapid rate of cell divisions in the fetus, which is much higher than cells in vitro, low antioxidant or DNA repair capacity in the fetus, or other factors. It is also of interest that the somatic mutation rate for mice in the mouse spot test is 7 × 10−7 per cell per locus per R [21]. Also for comparison the germinal rate for the specific locus test for spermatogonia is 2.4 × 10−7 per cell per locus (the same four loci as the mouse spot test) [22]. Thus, even though sensitivity to mutations [23] or tumors [24,25] caused by chemicals is greatest on a per cell basis early in gestation, the late fetus is still a highly sensitive target for a direct-acting mutagenic agent such as X-ray. The lower cell division rate in the late fetus, compared with the early embryo, is

counterbalanced by a reduction in lethal effects and an increase in numbers of cells at risk. Our results underscore the importance of shielding fetuses and embryos from mutagenic insults at all stages of gestation.

Acknowledgements The authors thank Drs. Lucy M. Anderson and Larry K. Keefer for critical reading of the manuscript, Larry Claggett for technical assistance, and Kathy Breeze for assistance with manuscript preparation.

References [1] A. Stewart, J. Webb, D. Hewitt, A survey of childhood malignancies, Br. Med. J. 1 (1958) 1495–1508. [2] B. MacMahon, Prenatal X-ray exposure and childhood cancer, J. Natl. Cancer Inst. 28 (1962) 1173–1191.

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P.J. Donovan, G.T. Smith / Mutation Research 500 (2002) 9–15

[3] D.D. Ford, J.C.S. Paterson, W.L. Treuting, Fetal exposure to diagnostic X rays, and leukemia and other malignant diseases in childhood, J. Natl. Cancer Inst. 22 (1959) 1093–1104. [4] A. Stewart, G.W. Kneale, Radiation dose effects in relation to obstetric X-rays and childhood cancers, Lancet 1 (1970) 1185–1188. [5] A. Stewart, Low dose radiation cancers in man, Adv. Cancer Res. 14 (1971) 359–390. [6] R.H. Mole, Childhood cancer after prenatal exposure to diagnostic X-ray examinations in Britain, Br. J. Cancer 62 (1990) 152–168. [7] J.F. Bithell, A.M. Stewart, Pre-natal irradiation and childhood malignancy: a review of British data from the Oxford survey, Br. J. Cancer 31 (1975) 271–287. [8] T. Nomura, H. Nakajima, T. Hatanaka, M. Kinuta, T. Hongyo, Embryonic mutation as a possible cause of in utero carcinogenesis in mice revealed by post-natal treatment with 12-O-tetradecanoylphorbol-13-acetate, Cancer Res. 50 (1990) 2135–2138. [9] T. Nomura, Induction of persistent hypersensitivity to lung tumorigenesis by in utero X-radiation in mice, Environ. Mutagen. 6 (1984) 33–40. [10] A.C. Upton, T.T. Odell Jr., E.P. Sniffen, Influence of age at time of irradiation on induction of leukemia and ovarian tumors in RF mice, Proc. Soc. Exp. Biol. Med. 104 (1960) 769–772. [11] S. Sasaki, T. Kasuga, F. Sato, N. Kawashima, Late effects of fetal mice X-irradiated at middle or late intrauterine stage, Gann 69 (1978) 167–177. [12] R. Rugh, L. Duhamel, L. Skaredoff, Relation of embryonic and fetal X-irradiation to life time average weights and tumor incidence in mice, Proc. Soc. Exp. Biol. Med. 121 (1966) 714–718. [13] M. Stjernfeldt, K. Berglund, J. Lindsten, J. Ludvigsson, Maternal smoking and irradiation during pregnancy as risk factors for child leukemia, Cancer Detect. Prev. 16 (1992) 129–135. [14] H.Z. Hill, C.L. Miller, Plating efficiency of mouse embryo cells as a function of gestational age, Experientia 32 (1976) 1054–1055. [15] P.J. Donovan, G.T. Smith, C.W. Riggs, Hamster and rat fetal cells have low spontaneous mutation frequencies and rates, Mutat. Res. 478 (2001) 51–63. [16] T.T. Puck, The mammalian cell as a microorganism, Genetic and Biochemical Studies in Vitro, Holden-Day, San Francisco, CA, 1972, pp. 102–130. [17] J.T. Stout, C.T. Caskey, HGPRT: gene structure, expression, and mutation, Annu. Rev. Genet. 19 (1985) 127–148. [18] C.F. Arlett, Mutagenicity testing with V79 Chinese hamster cells, in: B.J. Kilbey, M. Legator, W. Nichols, C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, 1977, pp. 175–192. [19] M.O. Bradley, B. Bhuyan, M.C. Francis, R. Langenbach, A. Peterson, E. Huberman, Mutagenesis by chemical agents in V79 Chinese hamster cells: a review and analysis of the literature. A report of the gene-tox program, Mutat. Res. 87 (1981) 81–142.

[20] A.W. Hsie, D.A. Casciano, D.B. Couch, D.F. Krahn, J.P. O’Neill, B.L. Whitfield, The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals, Mutat. Res. 86 (1981) 193–214. [21] L.B. Russell, M.H. Major, Radiation-induced presumed somatic mutations in the house mouse, Genetics 42 (1957) 161–175. [22] W.L. Russell, X-ray-induced mutations in mice, Cold Spring Harbor Symp. Quant. Biol. 16 (1951) 327–336. [23] P.J. Donovan, G.T. Smith, Cell sensitivity to transplacental mutagenesis by N-ethyl-N-nitrosourea is greatest during early gestation in the Syrian hamster, Mutat. Res. 427 (1999) 47– 58. [24] P.J. Donovan, Cell sensitivity to transplacental carcinogenesis by N-ethyl-N-nitrosourea is greatest in early post-implantation development, Mutat. Res. 427 (1999) 59–63. [25] T. Nomura, Sensitivity of a lung cell in the developing mouse embryo to tumor induction by urethan, Cancer Res. 34 (1974) 3363–3372. [26] A.A. van Zeeland, J.W.I.M. Simons, Ploidy level and mutation to hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) deficiency in Chinese hamster cells, Mutat. Res. 28 (1975) 239–250. [27] J. Thacker, R. Cox, Mutation induction and inactivation in mammalian cells exposed to ionising radiation, Nature 258 (1975) 429–431. [28] A.G.A.C. Knaap, J.W.I.M. Simons, A mutational assay system for L5178Y mouse lymphoma cells, using hypoxanthineguanine-phosphoribosyl-transferase (HGPRT)-deficiency as marker. The occurrence of a long expression time for mutations induced by X-rays and EMS, Mutat. Res. 30 (1975) 97–110. [29] R. Cox, W.K. Masson, X-ray-induced mutation to 6-thioguanine resistance in cultured human diploid fibroblasts, Mutat. Res. 37 (1976) 125–136. [30] A.A. van Zeeland, J.W.I.M. Simons, Linear dose–response relationships after prolonged expression times in V-79 Chinese hamster cells, Mutat. Res. 35 (1976) 129–137. [31] J. Thacker, A. Stretch, M.A. Stephens, The induction of thioguanine-resistant mutants of Chinese hamster cells by ␥-rays, Mutat. Res. 42 (1977) 313–326. [32] A.W. Hsie, J.P. O’Neill, D.B. Couch, J.R. SanSebastian, P.A. Brimer, R. Machanoff, J.C. Fuscoe, J.C. Riddle, A.P. Li, N.L. Forbes, M.H. Hsie, Quantitative analyses of radiationand chemical-induced lethality and mutagenesis in Chinese hamster ovary cells, Radiat. Res. 76 (1978) 471–492. [33] D. Jenssen, C. Ramel, Relationship between chemical damage of DNA and mutations in mammalian cells. Part I. Dose–response curves for the induction of 6-thioguanineresistant mutants by low doses of monofunctional alkylating agents, X-rays and UV radiation in V79 Chinese hamster cells, Mutat. Res. 73 (1980) 339–347. [34] Y.C.E.M. de Ruijter, J.W.I.M. Simons, Determination of the expression time and the dose–response relationship for mutations at the hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) locus induced by X-irradiation in human diploid skin fibroblasts, Mutat. Res. 69 (1980) 325–332.

P.J. Donovan, G.T. Smith / Mutation Research 500 (2002) 9–15 [35] H.J. Burki, Ionizing radiation-induced 6-thioguanine-resistant clones in synchronous CHO cells, Radiat. Res. 81 (1980) 76–84. [36] B. Singh, R.S. Gupta, Mutagenic response to ultraviolet light and X-rays at five independent genetic loci in Chinese hamster ovary cells, Environ. Mutagen. 4 (1982) 543–551. [37] J.Z. Beer, E.D. Jacobson, H.H. Evans, I. Szumiel, X-ray and UV mutagenesis in two L5178Y cell strains differing in tumorigenicity, radiosensitivity, and DNA repair, Br. J. Cancer 49 (Suppl. VI) (1984) 107–111. [38] B.J.S. Sanderson, J.L. Dempsey, A.A. Morley, Mutations in human lymphocytes: effect of X- and UV-irradiation, Mutat. Res. 140 (1984) 223–227. [39] Vijayalaxmi, H.J. Evans, Measurement of spontaneous and X-irradiation-induced 6-thioguanine-resistant human blood lymphocytes using a T-cell cloning technique, Mutat. Res. 125 (1984) 87–94. [40] A.J. Grosovsky, J.B. Little, Evidence for linear response for the induction of mutations in human cells by X-ray exposures below 10 rad, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 2092–2095.

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[41] J.P. O’Neill, K.B. Flint, X-ray induction of 6-thioguanineresistant mutants in division arrested, G0 /G1 phase Chinese hamster ovary cells, Mutat. Res. 150 (1985) 443–450. [42] H.L. Liber, P.-M. Leong, V.H. Terry, J.B. Little, X-rays mutate human lymphoblast cells at genetic loci that should respond only to point mutagens, Mutat. Res. 163 (1986) 91–97. [43] B.W. Corn, H.L. Liber, J.B. Little, Differential effects of radical scavengers on X-ray-induced mutation and cytotoxicity in human cells, Radiat. Res. 109 (1987) 100–108. [44] L.F. Fuller, R.B. Painter, A Chinese hamster ovary cell line hypersensitive to ionizing radiation and deficient in repair replication, Mutat. Res. 193 (1988) 109–121. [45] C.B. Klein, T.G. Rossman, Transgenic Chinese hamster V79 cell lines which exhibit variable levels of gpt mutagenesis, Environ. Mol. Mutagen. 16 (1990) 1–12. [46] N. Mei, N. Kunugita, S. Nomoto, T. Norimura, Comparison of the frequency of T-cell receptor mutants and thioguanine resistance induced by X-rays and ethylnitrosourea in cultured human blood T-lymphocytes, Mutat. Res. 357 (1996) 191–197.