Measuring the genetic damage in cancer patients during radiotherapy with three genetic end-points

Abstract

In this paper the genetic damage for three genetic end-points was studied in cancer patients during radiotherapy using the micronucleus test, comet assay and hprt gene mutation test. The subjects were from: (i) a group of 24 patients suffering from various types of cancers; (ii) a group of 23 controls matched according to age, sex and smoking. Blood samples were collected from the controls and from the cancer patients prior to radiotherapy and after cumulative radiation doses of 10, 30 and 50 Gy. The results of the micronucleus test indicated that the mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in the cancer patients prior to radiotherapy were 12.46 and 11.29‰, respectively, which were significantly higher than those (6.65 and 5.96‰) in controls (P < 0.01). However, the results of the comet assay and hprt gene mutation test showed no significant difference between untreated cancer patients and controls (P > 0.05). The mean MNR/MCR at 0, 10, 30 and 50 Gy in cancer patients increased with the cumulative dose, being 12.46, 35.83, 64.63 and 85.00‰ for MNR and 11.29, 31.25, 53.63 and 67.28‰ for MCR, respectively. Similar results were obtained in the comet assay and hprt gene mutation test. Meanwhile, it was found that there was inter-individual variability in response to radiotherapy among patients.

Introduction

Ionizing radiation is widely and successfully used in oncology. The recent progress in cancer radiotherapy has improved the prognosis of cancer patients, but in turn it has brought complications (Sawada et al., 1998). Ionizing radiation primarily targets DNA molecules and produces an array of lesions that include single-strand breaks, base alterations (oxidative damage) and double-strand breaks (Li et al., 2001). Attention has focused on secondary cancer following radiotherapy, for which the risk is substantial (Byrne, 1999). There are studies of second tumors induced by radiotherapy for cervix, breast and childhood cancers, as well as for benign diseases of the skin, breast, stomach and female genital tract (Hendry, 2001).

In addition, patient-to-patient variability in response to radiotherapy can be observed even when the same treatment regimen is applied. Both patient- and therapy-related factors as well as intrinsic factors of individual radiosensitivity are considered to influence the variability of side-effects observed (Twardella et al., 2003). There is increasing evidence showing that the major factors determining these differences are related to intrinsic biological factors. Thus, the ability to predict the determinants of these differences would have important implications with regard to cancer treatment (Ruiz de Almodóvar et al., 2002), which would permit an individualization of radiotherapy dose to optimize results.

Therefore, simple and informative techniques to measure cytogenetic and molecular damage would be greatly valuable in studying genetic risk during radiotherapy. When physical dosimetry is unreliable, biological dosimetry based on the level of induced DNA strand breaks, chromosomal damage and gene mutations could be used (Cao et al., 2002). Determination of the most extensively used biomarkers involves cytogenetic methods (evaluation of chromosomal aberrations and/or sister chromatid exchange in mitogen-stimulated cells) or biochemical and immunological techniques, but these expensive and time-consuming methods are not suitable for large-scale study (Colleu-Durel et al., 2004).

To meet the challenge described above, in the present study three genetic end-points, i.e. chromosomal damage (micronucleus test), gene mutation (hprt gene mutation test) and DNA single-strand break (comet assay), were utilized to study: (i) whether there is a difference in background genetic damage between patients with various cancers and controls; (ii) whether there is a dose–response relationship between radiation dose and genetic damage in patients during radiotherapy; (iii) in spite of the small size of the sample, whether there is inter-individual variance in response to radiation.

Materials and methods

Subjects and blood sample collection

The peripheral blood was from 23 controls and 24 patients with various cancers in the same hospital. The cancer group without other complications consisted of 8 males and 16 females from 35 to 79 years old (mean age 55.5 years). The control group consisted of 7 males and 16 females from 27 to 85 years old (mean age 56.1 years). The general situation of cancer patients and controls is listed in Table I, which shows no significant differences between the cancer patient and control groups for sex, age and smoking habit. The blood samples of cancer patients were collected before and during radiotherapy at cumulative doses of 10, 30 and 50. Five patients (nos 1, 9, 14, 21 and 24) did not receive treatment at a cumulative dose of 50 Gy.

Table I.

General situation of 24 cancer patients and 23 controls

Patient no.	Sex	Age (years)	Smoking	Type of cancer	Dose (Gy)/fractions/weeks	Control no.	Sex	Age (years)	Smoking
1	F	48	NS	Brain	40/20/4	1	F	61	NS
2	F	73	NS	Leiomyosarcoma	66/33/7	2	M	57	S
3	F	61	NS	Brain	56/28/6	3	M	80	S
4	M	73	NS	Nasopharynx	70/35/7	4	M	78	S
5	M	73	NS	Esophagus	60/30/6	5	M	67	S
6	M	35	S	Testicle	50/25/5	6	M	41	NS
7	F	69	NS	Breast	50/25/5	7	M	66	S
8	M	38	S	Nasopharynx	70/35/7	8	M	85	S
9	F	53	NS	Breast	30/15/3	9	F	59	NS
10	M	65	NS	Rectum	60/30/6	10	F	39	NS
11	M	57	S	Lung	60/30/6	11	F	76	NS
12	M	46	NS	Testicle	50/25/5	12	F	49	NS
13	F	52	NS	Uterus	50/25/5	13	F	32	NS
14	M	79	NS	Esophagus	40/20/5	14	F	49	NS
15	F	44	NS	Breast	50/25/5	15	F	59	NS
16	F	43	NS	Breast	56/28/6	16	F	72	NS
17	F	55	NS	Breast	50/25/5	17	F	27	NS
18	F	39	NS	Breast	50/25/5	18	F	54	NS
19	F	55	NS	Breast	60/30/6	19	F	40	NS
20	F	59	NS	Breast	50/25/5	20	F	39	NS
21	M	73	NS	Rectum	41/23/4	21	F	54	NS
22	F	50	NS	Breast	50/25/5	22	F	53	NS
23	F	41	NS	Breast	50/25/5	23	F	54	NS
24	F	51	NS	Breast	40/20/4

F, female; M, male; S, smoker; NS, non-smoker.

Cytokinesis block micronucleus (CBMN) assay

The micronucleus test was conducted according to the method of Fenech and Morley (1985). Briefly, whole blood (0.5 ml) was added to RPMI 1640 (4.5 ml) (Gibco) containing 20% fetal calf serum and phytohaemagglutinin M (0.2 mg/ml) (PHA-M) (Gibco). Two independent cultures were incubated at 37°C for 72 h. Cytochalasin B (4.5 µg/ml) (Sigma) was added to each culture 28 h before harvesting cells. The cultures were harvested 72 h after initiation by centrifugation. Cells were then processed on the basis of the method described by Fenech and Morley (1985) as modified to enable the use of whole blood cultures. The lymphocytes were subjected to mild hypotonic treatment (0.075 M KCl) for 5 min, then fixed in fresh fixative solution (3:1 methanol:acetic acid) for 20 min. This fixation step was repeated twice after 20 min storage at 4°C. The cells were smeared on microscope slides, air dried and stained for 10 min with 10% Giemsa solution, pH 6.8. 1000 binucleated lymphocytes (500 cells per culture) were scored under light microscopy (400× magnification). The micronuclei and micronucleated cells were detected according to the criteria described by Fenech (2000). All the slides were examined by the same person. Micronucleated cell rate (MCR) and micronucleus rate (MNR), i.e. number of micronucleated cells and number of micronuclei per 1000 binucleated lymphocytes, respectively, served as indicators. Meanwhile, the nuclear division index (NDI) was calculated on the basis of the following formula (Zhang et al., 2002):  

\[\mathrm{NDI}\ =\ \left[1\mathrm{N}\ +\ \left(2\ {\times}\ 2\mathrm{N}\right)\ +\ \left(4\ {\times}\ {>}2\mathrm{N}\right)\right]/400\ \mathrm{cells}.\]

Statistical analyses used the Wilcoxon rank sum test and repeated measures ANOVA was applied to test the association between radiation dose and MNR or MCR. The statistical analysis was performed with the program SPSS 11.0 for Windows.

Comet assay

Human lymphocytes were isolated by the procedure described by Zhang et al. (2002) and were resuspended in phosphate-buffered saline (PBS). Following isolation, the cells were mixed with 0.4% Trypan blue solution. After 15 min the cells were counted and checked for viability. The remaining cells were immediately used for single cell gel electrophoresis. The assay was performed basically according to Singh et al. (1988). Roughened slides were cleaned with 100% ethanol and air dried. Two solutions, of 0.5% normal melting point agarose and 0.5% low melting point agarose, were prepared in Ca²⁺- and Mg²⁺-free PBS. An aliquot of 100 µl of normal melting point agarose was used for the first layer, while 75 µl of low melting point agarose+10 µl of PBS cell suspension (10 000 cells) was used for the second layer. Finally, the third layer of 75 µl of low melting point agarose was added. Slides were immersed in freshly prepared lysis solution (1% sodium sarcosinate, 2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Tris–HCl, pH 10, 1% Triton X-100 and 10% DMSO) at 4°C for 1 h. Then the slides were placed in a horizontal electrophoresis unit covered with fresh buffer (1 mM Na₂EDTA, 300 mM NaOH, pH 13) for 20 min to allow DNA unwinding and expression of alkali-labile sites. Electrophoresis was conducted for 20 min at 20 V and 300 mA. The above treatments were performed in an ice bath. Subsequently, slides were washed gently three times in neutralization buffer (0.4 M Tris–HCl, pH 7.5) to remove alkali and detergent. Each slide was stained with 50 µl of ethidium bromide (20 µg/ml). All the above steps were conducted under yellow light to prevent additional DNA damage.

Observations were made at 400× magnification using a fluorescence microscope (Olympus, BX51) equipped with a 530 nm excitation filter, a 590 nm emission filter, a camera (Olympus DP50) and a computer-based image analysis system (Media Cybernetics, USA). The data were based on 50 randomly selected cells per sample, i.e. 25 cells from each of the two replicate slides. The mean tail length and mean tail moment served as indicators. Statistical analyses used the Wilcoxon rank sum test in the program SPSS 11.0 for Windows. Repeated measures ANOVA was applied to test the association between radiation dose and mean tail length (MTL) or mean tail moment (MTM).

The hprt gene mutation test

The hprt gene mutation test was performed according to Cao et al. (2002). Two sets of cultures were prepared, each set of culture containing 0.5 ml of heparinized blood and 4.5 ml of RPMI 1640 with 20% fetal calf serum and 0.2 mg/ml PHA-M. One set of cultures was added with 0.2 mM 6-thioguanine (Sigma). After 33 h incubation cytochalasin B (final concentration 4.5 µg/ml) was added to the two sets of cultures. After 72 h incubation lymphocytes were harvested by centrifugation and fixed with methanol:acetic acid (3:1). Slides were prepared and stained with 10% Giemsa solution, pH 6.8. Binucleated and multinucleated cells per 1000 lymphocytes in the two sets of cultures were scored under light microscopy (400× magnification). Mutant frequency of the hprt gene (Mf-hprt) was calculated using the following formula:  

Statistical analyses used the Wilcoxon rank sum test and the repeated measures ANOVA was applied to test the association between radiation dose and Mfs-hprt. The statistical analysis was performed with the program SPSS 11.0 for Windows.

Results

The results of micronucleus test, comet assay and hprt gene mutation test in untreated cancer patients and controls

Table II indicates the results of the CBMN test, comet assay and hprt gene mutation test in 24 untreated cancer patients and 23 controls. In the CBMN test, the ranges of MNRs in 23 controls and 24 untreated cancer patients were 0–16 and 3–29‰, respectively, while the mean MNRs were 6.65 ± 0.82 and 12.46 ± 1.36‰, respectively. The mean MNR of untreated cancer patients was significantly higher than that of the controls (P < 0.01). The ranges of MCRs of controls and untreated cancer patients were 0–14 and 3–25‰, respectively. The average MCR of patients was 11.29 ± 1.22‰, which was significantly higher than that (5.96 ± 0.70‰) of controls (P < 0.01). The distribution of micronuclei per cell and the NDIs of controls and patients are listed in Table III. The NDIs of controls and untreated cancer patients were 1.90 and 1.81, respectively. In the comet assay, DNA damage of lymphocytes was shown by tail length and tail moment. The MTL and the MTM were 1.77 ± 0.10 µm and 0.37 ± 0.04 in untreated cancer patients, respectively, while the MTL and MTM in controls were 1.81 ± 0.07 µm and 0.41 ± 0.03, respectively. No significant differences between the untreated cancer patient group and the control group were found for these two indicators (P > 0.05). In the hprt gene mutation test the mutant frequency of the hprt gene (Mf-hprt) was used as an indicator. The average Mf-hprt in untreated cancer patients was 0.82 ± 0.02‰, which was not significantly higher than that (0.79 ± 0.03‰) in controls (P > 0.05).

Table II.

The results of micronucleus test, comet assay and hrpt gene mutation test in untreated cancer patients and controls

Patient no.	MCR (‰)	MNR (‰)	MTL (µm)	MTM	Mf-hprt (‰)	Control no.	MCR (‰)	MNR (‰)	MTL (µm)	MTM	Mf-hprt (‰)
1	14	15	2.25	0.71	0.82	1	10	12	2.07	0.50	0.94
2	5	5	1.49	0.39	0.58	2	10	10	2.08	0.45	0.89
3	17	18	1.46	0.25	0.67	3	6	6	1.07	0.13	0.83
4	25	29	1.55	0.31	0.75	4	7	7	2.05	0.36	0.84
5	7	7	2.30	0.73	0.74	5	4	4	1.95	0.36	0.97
6	21	24	1.98	0.41	1.03	6	6	8	2.04	0.48	0.92
7	21	22	2.17	0.48	0.74	7	5	5	1.39	0.35	0.64
8	7	8	2.09	0.43	0.79	8	7	8	2.1	0.47	0.84
9	11	11	1.90	0.48	0.89	9	4	4	1.58	0.39	0.94
10	9	10	1.83	0.29	0.82	10	2	2	1.79	0.57	0.95
11	5	6	1.76	0.30	0.91	11	3	3	1.78	0.37	0.96
12	3	3	2.33	0.61	0.76	12	4	8	1.87	0.52	0.88
13	11	13	0.91	0.13	0.93	13	0	0	1.88	0.27	0.59
14	9	9	1.14	0.12	0.77	14	2	2	1.05	0.13	0.86
15	9	10	1.28	0.18	0.79	15	5	5	2.09	0.43	0.56
16	12	14	1.14	0.21	0.96	16	4	4	1.52	0.32	0.56
17	5	6	1.26	0.14	0.87	17	3	3	–	–	0.64
18	8	9	1.29	0.11	0.93	18	5	5	1.6	0.39	0.54
19	7	7	1.47	0.16	0.91	19	14	16	2.04	0.56	0.73
20	15	17	1.66	0.22	0.58	20	10	12	1.71	0.44	0.95
21	4	6	2.43	0.47	0.83	21	10	11	1.67	0.34	0.65
22	13	15	2.46	0.65	0.82	22	7	7	1.6	0.44	0.81
23	14	15	2.29	0.57	0.77	23	9	11	1.56	0.37	0.75
24	19	20	2.04	0.49	0.95
Mean ± SE	11.29 ± 1.22a	12.46 ± 1.36a	1.77 ± 0.10	0.37 ± 0.04	0.82 ± 0.02		5.96 ± 0.70	6.65 ± 0.82	1.75 ± 0.07	0.39 ± 0.03	0.79 ± 0.03

MCR, micronucleated cell rate; MNR, micronucleus rate; MTL, mean tail length; MTM, mean tail moment; Mf-hprt, mutant frequency of hprt gene; –, data lost.

a

P < 0.01, patients compared with controls.

Table III.

The results of micronucleus test in 23 controls and 24 cancer patients during radiotherapy

Group	Subject no.	No. of cells analyzed	No. of cells carrying one or more micronuclei							MCR (‰)	MNR (‰)	NDI
			1	2	3	≥4
Controls	1	1000	8	2	0	0	10	12	1.74
	2	1000	10	0	0	0	10	10	1.81
	3	1000	6	0	0	0	6	6	1.87
	4	1000	7	0	0	0	7	7	2.19
	5	1000	4	0	0	0	4	4	1.76
	6	1000	4	2	0	0	6	8	1.80
	7	1000	5	0	0	0	5	5	1.92
	8	1000	6	1	0	0	7	8	1.88
	9	1000	4	0	0	0	4	4	1.84
	10	1000	2	0	0	0	2	2	1.87
	11	1000	3	0	0	0	3	3	1.86
	12	1000	1	2	1	0	4	8	2.05
	13	1000	0	0	0	0	0	0	1.98
	14	1000	2	0	0	0	2	2	1.95
	15	1000	5	0	0	0	5	5	1.90
	16	1000	4	0	0	0	4	4	2.03
	17	1000	3	0	0	0	3	3	2.01
	18	1000	5	0	0	0	5	5	1.96
	19	1000	12	2	0	0	14	16	1.95
	20	1000	8	2	0	0	10	12	1.84
	21	1000	9	1	0	0	10	11	1.84
	22	1000	7	0	0	0	7	7	1.90
	23	1000	7	2	0	0	9	11	1.81
Mean ± SE		1000	5.31 ± 0.61	0.61 ± 0.19	0.04 ± 0.04	0	5.96 ± 0.70	6.65 ± 0.82	1.90 ± 0.02
Patients (0 Gy)	1	1000	13	1	0	0	14	15	1.71
	2	1000	5	0	0	0	5	5	1.52
	3	1000	16	1	0	0	17	18	1.81
	4	1000	21	4	0	0	25	29	1.68
	5	1000	7	0	0	0	7	7	1.71
	6	1000	18	3	0	0	21	24	1.77
	7	1000	20	1	0	0	21	22	1.73
	8	1000	6	1	0	0	7	8	1.89
	9	1000	11	0	0	0	11	11	1.94
	10	1000	8	1	0	0	9	10	1.93
	11	1000	4	1	0	0	5	6	1.85
	12	1000	3	0	0	0	3	3	1.60
	13	1000	9	2	0	0	11	13	1.77
	14	1000	9	0	0	0	9	9	1.56
	15	1000	8	1	0	0	9	10	1.95
	16	1000	10	2	0	0	12	14	2.26
	17	1000	4	1	0	0	5	6	1.80
	18	1000	7	1	0	0	8	9	1.80
	19	1000	7	0	0	0	7	7	1.84
	20	1000	13	2	0	0	15	17	1.80
	21	1000	2	2	0	0	4	6	1.93
	22	1000	11	2	0	0	13	15	1.88
	23	1000	13	1	0	0	14	15	1.86
	24	1000	18	1	0	0	19	20	1.80
Mean ± SE		1000	10.12 ± 1.11	1.17 ± 0.21	0	0	11.29 ± 1.20a	12.46 ± 1.36a	1.81 ± 0.03
Patients (10 Gy)	1	1000	26	3	1	0	30	35	1.58
	2	1000	16	0	0	0	16	16	1.45
	3	1000	27	1	0	0	28	29	1.65
	4	1000	32	5	0	0	37	42	1.57
	5	1000	21	2	0	0	23	25	1.69
	6	1000	38	10	0	0	48	58	1.72
	7	1000	38	0	0	0	38	38	1.66
	8	1000	10	3	0	0	13	16	1.74
	9	1000	21	0	1	0	22	24	1.92
	10	1000	25	2	0	0	27	29	1.89
	11	1000	15	2	0	0	17	19	1.88
	12	1000	15	4	0	0	19	23	1.54
	13	1000	32	5	2	0	39	48	1.74
	14	1000	28	0	0	0	28	28	1.44
	15	1000	28	4	3	0	35	45	1.86
	16	1000	48	10	1	0	59	71	2.08
	17	1000	32	5	3	0	40	51	1.76
	18	1000	42	3	0	0	45	48	1.61
	19	1000	25	3	0	0	28	31	1.72
	20	1000	36	6	0	0	42	48	1.63
	21	1000	10	4	0	0	14	18	1.75
	22	1000	26	2	2	0	30	36	1.84
	23	1000	27	3	1	0	31	36	1.86
	24	1000	36	5	0	0	41	46	1.7
Mean ± SE		1000	27.25 ± 2.00	3.42 ± 0.55	0.58 ± 0.20	0	31.25 ± 2.37b	35.83 ± 2.89b	1.72 ± 0.03
Patients (30 Gy)	1	1000	35	3	2	0	40	47	1.53
	2	1000	33	2	2	0	37	43	1.43
	3	1000	35	2	1	0	38	42	1.60
	4	1000	38	5	2	0	45	54	1.53
	5	1000	32	2	0	0	34	36	1.60
	6	1000	45	11	2	0	58	73	1.41
	7	1000	50	7	2	0	59	70	1.65
	8	1000	56	19	4	0	79	106	1.68
	9	1000	48	3	2	0	53	60	1.79
	10	1000	45	5	1	0	51	58	1.65
	11	1000	47	5	5	0	57	72	1.51
	12	1000	42	8	1	0	51	61	1.54
	13	1000	48	4	2	0	54	62	1.34
	14	1000	59	14	6	0	79	105	1.61
	15	1000	57	6	2	0	65	75	1.99
	16	1000	54	13	2	0	69	86	1.74
	17	1000	57	17	0	0	74	91	1.59
	18	1000	43	9	0	0	52	61	1.64
	19	1000	67	15	3	0	85	106	1.59
	20	1000	45	10	0	0	55	65	1.56
	21	1000	21	5	1	0	27	34	1.48
	22	1000	35	4	2	0	41	49	1.78
	23	1000	32	5	0	0	37	42	1.53
	24	1000	42	4	1	0	47	53	1.65
Mean ± SE		1000	44.42 ± 2.19	7.42 ± 1.02	1.79 ± 0.31	0	53.63 ± 3.15c	64.63 ± 4.39c	1.60 ± 0.03
Patients (50 Gy)	1	Untreated	Untreated	Untreated	Untreated	0	Untreated	Untreated	Untreated
	2	1000	40	4	0	0	44	48	1.46
	3	1000	42	5	2	0	49	58	1.35
	4	1000	43	5	3	0	51	62	1.53
	5	1000	40	2	1	0	43	47	1.54
	6	–	–	–	–	0	–	–	–
	7	1000	60	11	3	0	74	91	1.56
	8	1000	74	25	10	0	109	154	1.60
	9	Untreated	Untreated	Untreated	Untreated	0	Untreated	Untreated	Untreated
	10	1000	53	18	0	0	71	89	1.41
	11	1000	58	4	0	0	62	66	1.6
	12	1000	48	15	6	0	69	96	1.48
	13	1000	59	25	9	0	93	136	1.52
	14	Untreated	Untreated	Untreated	Untreated	0	Untreated	Untreated	Untreated
	15	1000	69	18	2	0	89	111	1.94
	16	1000	52	15	2	0	69	88	1.64
	17	1000	60	20	2	0	82	106	1.51
	18	1000	45	13	2	0	60	77	1.63
	19	1000	68	17	4	0	89	114	1.51
	20	1000	48	14	2	0	64	82	1.46
	21	Untreated	Untreated	Untreated	Untreated	0	Untreated	Untreated	Untreated
	22	1000	44	3	3	0	50	59	1.41
	23	1000	40	3	0	0	43	46	1.60
	24	Untreated	Untreated	Untreated	Untreated	0	Untreated	Untreated	Untreated
Mean ± SE		1000	52.39 ± 2.56	12.06 ± 1.82	2.83 ± 0.68	0	67.28 ± 4.55d	85.00 ± 7.28d	1.54 ± 0.03

MCR, micronucleated cell rate; MNR, micronucleus rate; –, data lost.

a

P < 0.01 compared with control.

b

P < 0.01 compared with 0 Gy.

c

P < 0.01 compared with 10 Gy.

d

P < 0.01 compared with 30 Gy.

Micronucleus test in cancer patients during radiotherapy

The results of the CBMN test in 24 cancer patients during radiotherapy are presented in Table III. It was found that MNR and MCR increased with cumulative radiation dose (0, 10, 30 and 50 Gy) for each patient and that the average MNRs and MCRs were significantly elevated with incresed cumulative radiation dose. The average MNRs at 0, 10, 30 and 50 Gy were 12.46, 35.83, 64.63 and 85.00‰, respectively. The average MCRs at 0, 10, 30 and 50 Gy were 11.29, 31.25, 53.63 and 67.28 ‰, respectively. The average MCR and MNR at 10, 30 and 50 Gy were significantly elevated as compared with those at 0 Gy (P < 0.01). The dose–response relationships for MCR and MNR are shown in Figure 1. Table III shows that the NDIs of cancer patients decreased with radiation dose, being 1.81, 1.72, 1.60 and 1.54, respectively.

Fig. 1.

The dose–effect relationships between radiation dose and average MNR/MCR in 24 cancer patients during radiotherapy. Vertical bars, standard deviation.

Comet assay in cancer patients during radiotherapy

Table IV displays the results of the comet assay as MTL and MTM in 24 cancer patients during radiotherapy. The MTLs and MTMs at 10, 30 and 50 Gy were significantly higher than the MTL and MTM at 0 Gy for each patient. The average MTLs of 24 cancer patients at 10, 30, 50 Gy were 1.96, 2.26 and 2.63 µm, respectively, which were significantly higher than that (1.77) at 0 Gy (P < 0.05). A similar phenomenon was observed when MTM acted as the indicator, the average MTMs of 24 cancer patients at 10, 30, 50 Gy being 0.43, 0.56 and 0.70, respectively, which were significantly higher than that (0.37) at 0 Gy (P < 0.05). The dose–response relationships for MTL and MTM are shown in Figure 2.

Fig. 2.

The dose–effect relationships between radiation dose and average MTL/MTM in 24 cancer patients during radiotherapy. Vertical bars, standard deviation.

Table IV.

The results of the comet assay in 24 cancer patients during radiotherapy

Patient no.	MTL (µm)							MTM
	0 (Gy)	10 (Gy)	30 (Gy)	50 (Gy)	0 (Gy)	10 (Gy)	30 (Gy)	50 (Gy)
1	2.25	–	–	Untreated	0.71	–	–	Untreated
2	1.49	1.60	2.27	3.07	0.39	0.73	0.77	0.80
3	1.46	1.58	2.18	3.66	0.25	0.28	0.46	1.28
4	1.55	1.67	2.59	2.05	0.31	0.43	1.06	1.20
5	2.30	–	–	–	0.73	–	–	–
6	1.98	2.27	2.41	3.25	0.41	0.81	0.75	0.85
7	2.17	2.27	2.26	2.94	0.48	0.53	0.57	0.89
8	2.09	2.49	2.55	2.34	0.43	0.44	0.51	0.69
9	1.90	2.53	–	Untreated	0.48	0.53	–	Untreated
10	1.83	2.00	2.06	2.29	0.29	0.30	0.45	0.56
11	1.76	1.77	1.88	2.35	0.30	0.33	0.39	0.41
12	2.33	2.35	2.54	–	0.61	0.76	0.68	–
13	0.91	1.16	1.13	1.41	0.13	0.13	0.16	0.18
14	1.14	1.68	1.83	Untreated	0.12	0.19	0.36	Untreated
15	1.28	1.53	1.68	2.65	0.18	0.17	0.22	0.64
16	1.14	1.18	1.35	2.49	0.21	0.14	0.14	0.49
17	1.26	1.95	2.24	2.44	0.14	0.32	0.50	0.57
18	1.29	1.40	2.38	2.58	0.11	0.16	0.62	0.67
19	1.47	1.98	2.82	–	0.16	0.29	0.84	–
20	1.66	1.74	2.13	2.32	0.22	0.40	0.59	0.54
21	2.43	2.44	2.53	Untreated	0.47	0.56	0.57	Untreated
22	2.46	2.88	3.62	3.76	0.65	0.65	0.74	0.80
23	2.29	2.33	2.35	2.39	0.57	0.69	0.62	0.60
24	2.04	2.33	2.66	Untreated	0.49	0.52	0.78	Untreated
Mean ± SE	1.77 ± 0.10	1.96 ± 0.10a	2.26 ± 0.11b	2.62 ± 0.15c	0.37 ± 0.04	0.43 ± 0.05d	0.56 ± 0.05b	0.70 ± 0.07c

MTL, mean tail length; MTM, mean tail moment; –, data lost.

a

P < 0.01 compared with 0 Gy.

b

P < 0.01 compared with 10 Gy.

c

P < 0.01 compared with 30 Gy.

d

P < 0.05 compared with 0 Gy.

hprt gene mutation test in cancer patients during radiotherapy

Mf-hprt for 24 cancer patients during radiotherapy are shown in Table V. Mf-hprt of each patient increased with radiotherapy dose. The average Mf-hprt of 24 cancer patients were 0.91 ± 0.03, 1.07 ± 0.05 and 1.15 ± 0.05‰ at radiation doses of 10, 30 and 50 Gy, respectively, which were significantly higher than that (0.82 ± 0.02‰) at 0 Gy (P < 0.01). The dose–response relationship is shown in Figure 3.

Fig. 3.

The dose–effect relationship between radiation dose and average Mf-hprt in 24 cancer patients during radiotherapy. Vertical bars, standard deviation.

Table V.

The results of hrpt gene mutation test in 24 cancer patients during radiotherapy

Patient no.	Mf-hprt (‰)
	0 (Gy)	10 (Gy)	30 (Gy)	50 (Gy)
1	0.82	0.91	1.87	Untreated
2	0.58	0.61	0.73	0.95
3	0.67	0.71	0.89	0.92
4	0.75	0.79	0.84	0.95
5	0.74	0.81	0.90	1.03
6	1.03	1.14	1.20	–
7	0.74	0.8	0.91	1.13
8	0.79	0.86	1.22	1.20
9	0.89	1.01	1.23	Untreated
10	0.82	0.93	0.97	1.04
11	0.91	0.95	1.00	1.29
12	0.76	0.84	0.90	1.14
13	0.93	0.94	1.06	1.15
14	0.77	0.79	0.91	Untreated
15	0.79	0.82	0.83	1.02
16	0.96	0.96	0.98	1.08
17	0.87	0.88	1.03	1.12
18	0.93	1.27	1.27	1.75
19	0.91	1.07	1.11	1.21
20	0.58	0.82	0.93	1.06
21	0.83	0.89	0.99	Untreated
22	0.82	1.09	1.73	1.55
23	0.77	0.86	0.95	1.02
24	0.95	1.11	1.26	Untreated
Mean ± SE	0.82 ± 0.02	0.91 ± 0.03a	1.07 ± 0.05b	1.15 ± 0.05c

Mf-hprt, mutant frequency of hprt gene; –, data lost.

a

Compared with 0 Gy P < 0.01.

b

Compared with 10 Gy P < 0.01.

c

Compared with 30 Gy P < 0.01.

Discussion

The baselines for three end-points and chromosomal instability of 24 cancer patients

Cytogenetic methods have been used extensively to monitor baseline genetic damage in healthy donors (Kubota et al., 2000) and cancer patients prior to therapy (Sawada et al., 1998; Gil et al., 2000; Colleu-Durel et al., 2004). Human tumours are characterized by numerical and structural chromosomal alterations and chromosomal instability. Chromosomal instability is the occurrence of gains and losses of whole chromosomes or chromosomal segments at an increased rate in a population of cells (Gollin, 2004). In the present study genetic damage in 24 patients with various cancers was detected for three end-points. The results of the CBMN test showed that the MCR and MNR of cancer patients were significantly higher than those of controls. However, the results of the comet assay and hprt gene mutation test were negative. The biological significance of the three tests is different. DNA single-strand and double-strand breaks and alkali-labile sites can be detected by the alkaline comet assay (Kopjar et al., 2002); the hprt gene mutation test is used to detect somatic cell mutation (Kubota et al., 2000); the CBMN test is utilized to measure micronuclei, which originate mainly from chromosome break or whole chromosomes that fail to engage with the mitotic spindle when the cell divides (Fenech, 2002). So, an increased MCR or MNR reflects chromosomal instability of human peripheral lymphocytes in cancer patients. It was found that the results of the CBMN and hprt gene mutation tests in our study were similar to those of previous studies. Baciuchka-Palmaro et al. (2002) reported that the MCR and MNR in 10 untreated patients with different kinds of cancers were 19.0 and 21.75‰, respectively, which were significantly higher than those (9.2 and 11.1‰) in 10 healthy controls. Venkatachalam et al. (1999) studied the frequency of micronulei in 25 patients with various cancers and Palyvoda et al. (2003) detected the level of micronuclei in a head and neck cancer patient group, and obtained similar results. Sawada et al. (1998) discovered that the mutant frequency at the hprt gene in 32 children with various malignancies before radiotherapy were well within the range for non-exposed healthy controls. Zwingmann et al. (1999) also reported similar results in 23 patients with solid malignancies at various sites. However, our results for 24 cancer patients using the comet assay were inconsistent with the results reported by other studies. Kopjar et al. (2002), Palyvoda et al. (2003) and Colleu-Durel et al. (2004) found that the level of background DNA damage measured by the comet assay in untreated cancer patients was significantly higher than that in controls. The contradictory comet assay results between our experiment and these other studies may be due to the sample (size, cancer type, etc.). Hence, in future investigations the number of patients will be increased and only one or two types of cancers will be studied.

Change at three end-points of cancer patients during radiotherapy

Genetic damage induced by radiotherapy in cancer patients has been detected using different tests in several previous investigations (Barber et al., 2000; Jagetia et al., 2001; Cao et al., 2002; Ruiz de Almodóvar et al., 2002; Borgmann et al., 2002; Twardella et al., 2003). Jagetia et al. (2001) found a 3-fold enhancement of the micronucleus frequency in patients with various cancers after radiotherapy (at a dose of 66 Gy), without a dose–response relationship. Cao et al. (2002) reported similar results, with the mean micronucleus frequency in patients with nasopharyngeal cancer increasing 2.5-fold after radiotherapy (at a dose of 68 Gy), with a linear dose–effect relationship. The results of the CBMN test in our experiment showed that the mean MCRs and MNRs of patients with various cancers (at doses of 10, 30 and 50 Gy) increased 2.8-, 4.8- and 6.0-fold and 2.9-, 5.2- and 6.8-fold, respectively, after radiotherapy, with a clear dose–response relationship. Moreover, the mean NDIs decreased with radiotherapy dose, which suggested that division of human lymphocytes was reduced. The comet assay is usually utilized to measure genetic damage induced by radiotherapy. The investigation of Cao et al. (2002) showed that the average MTL increased only 1.75-fold in 9 patients with nasopharyngeal cancer after radiotherapy (at a dose of 68 Gy), without an obvious dose–response relationship. In our study the mean MTL and MTM of cancer patients after radiotherapy (at doses of 10, 30 and 50 Gy) increased 1.1-, 1.3- and 1.5-fold and 1.2-, 1.5- and 1.9-fold, respectively, and showed a dose–response relationship. In addition, Zwingmann et al. (1999) reported a 7-fold increase in mean Mf-hprt in cancer patients after radiotherapy (at a dose of 61 Gy); Cao et al. (2002) found that the average Mf-hpr of 9 cancer patients increased 3-fold after radiotherapy (at a dose of 68 Gy). However, in the present study the mean Mf-hpr of 24 cancer patients increased 1.1-, 1.3- and 1.4-fold, respectively, after radiotherapy (at doses of 10, 30 and 50 Gy). As shown above, the indices for three end-points increased with radiotherapy dose in our investigation, so it is felt that indices at three end-points can serve as biomarkers for radiation exposure. Moreover, the three end-points have different biological significances. Although clinical irradiation doses were used as exposure doses in this study, the irradiated volume for different patients should also be considered, because the patients did not all have the same type of cancer.

Inter-individual variation in response to radiotherapy among 24 cancer patients

Following radiotherapy, patients have shown a wide variation of response of both tumour and normal tissues (Ruiz de Almodóvar et al., 2002). Similarly, the results of our study indicated that wide individual variability in response to radiotherapy occurred in 24 cancer patients. For example, the MCR and MNR at the dose of 50 Gy each increased 2-fold for patient 4, but 23- and 32-fold for patient 12 in the CBMN test. The MTL and MTM at the dose of 50 Gy increased 1.05- and 1.04-fold for patient 23, but 2.5- and 5-fold for patient 3 in the comet assay. However, the individual variability in the hprt gene mutation test was not as obvious as that in the CBMN test and comet assay. For example, Mfs-hprt at the dose of 50 Gy increased 1.13-fold for patient 17 and 1.89-fold for patient 23. Some of the variation may be accounted for by known factors, such as dose distribution and patient size, but it has been estimated that up to 70% of the observed inter-individual differences in radiosensitivity may be due to genetic predisposition (Barber et al., 2000).

This research work was supported by the International Cooperative Foundation of the Science-Technique Bureau of Zhejiang Province (grant no. 012104).

Author notes

1Zhejiang University Medical College, Institute of Occupational and Environmental Health, Hangzhou 310006, Zhejiang and 2Jiaxing University Medical College, Jiaxing, 314001, Zhejiang, People's Republic of China