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Measurement of HPRT mutant frequencies in T-lymphocytes from healthy human populations
Mutation Research, 285 (1993) 267-279 © 1993 Elsevier Science Publishers B.V. All rights reserved 0027-5107/93/$06.00
267
MUT 05204
M e a s u r e m e n t of H P R T mutant frequencies in T-lymphocytes from healthy human populations R.F. Branda a, L.M. Sullivan a j.p. O'Neill a M.T. Falta a, J.A. Nicklas a B. Hirsch c, P.M. Vacek b and R.J. Albertini a a Genetics Laboratory, Vermont Cancer Center and b Medical Statistics Department, University of Vermont, Burlington, VT 05401, USA and c Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, M N 05455, USA
(Received 20 August 1992) (Accepted 21 August 1992)
Keywords: HPRT; 6-Thioguanine-resistant; T-lymphocytes; Human population monitoring
Summary Somatic cell mutant frequencies at the hprt locus of the X-chromosome were measured with the T-lymphocyte cloning technique in healthy human populations. A statistical analysis was performed of assays from 232 individuals (77 males and 155 females) ranging in age from 19 to 80 years. Data from 4 donor groups were compiled: (a) 132 participants in a study of identical and fraternal twins; (b) 17 health care workers studied as part of an assessment of the risks of handling chemotherapeutic drugs; (c) 62 women with benign breast masses; and (d) 21 normal laboratory and office personnel. The relationship between age and mutant frequency (MF) was expressed by the equation: In MF = 1.46 + 0.018 age (P < 0.001). Thus, MF increased by about 2% per year. Increases in cloning efficiency (CE) reduced the MF, as shown in the equation: In MF = 2.91-1.32 CE (P < 0.001). CE was significantly related to age (CE = 0.47-0.002 age, P = 0.038), and the interdependent relationship between MF, age and CE expressed by the equation: In MF = 1.99-1.13 CE + 0.016 age was significant at the P < 0.001 level. There was no statistically significant effect of donor gender or smoking history on MF in our population, but CE was significantly lower in males (P < 0.001). These findings confirm the importance of age and CE as factors which influence the thioguanine-resistant MF in circulating T-lymphocytes from normal adults.
Development of an assay to detect mutations at the hprt locus in lymphocytes has advanced our understanding of the mechanisms of genetic damage in human ceils. The initially described
Correspondence: Dr. Richard F. Branda, Genetics Laboratory, 32 N. Prospect St., Burlington, VT 05401, USA. Tel. (802)863-5716; Fax (802)656-8429.
autoradiographic assay (Strauss and Albertini, 1977, 1979; Morley et al., 1982) and its later refinement, the clonal assay (Albertini et al., 1982; Morley et al., 1983), have been employed by many laboratories to measure somatic mutation rates in normal individuals, in populations exposed to known or suspected mutagens, and in patients with inherited defective DNA repair (Ammenheuser et al., 1988; Caggana et al., 1991;
268
Cole et al., 1991; Davies et al., 1992; Dempsey et al., 1986; Hakoda et al., 1988; Messing et al., 1989; Seifert et al., 1987; Sala-Trepat et al., 1990; Tates et al., 1989, 1991; Vijayalaxmi and Evans, 1984). The clonal assay also has proven useful to characterize the specific molecular alterations and mutational spectrum in hprt- mutant cells (reviewed in Albertini et al., 1990) and a database of these mutations has been compiled (Cariello et al., 1992). As more experience with this assay system has accrued, it has become clear that the range of the mutant frequency at the hprt locus in peripheral blood lymphocytes from normal individuals is quite broad, spanning approximately one logv (Henderson et al., 1986; Albertini et al., 1988; Cole et al., 1991; Tares et al., 1991). Attempts have been made to define the factors which influence mutant frequency and thereby contribute to the differences observed between normal individuals. There is evidence that experimental variables such as cloning efficiency, as well as subject characteristics such as age and lifestyle (diet and exposure to cigarette smoke) may affect mutant frequency (Vijayalaxmi and Evans, 1984; Albert i n i e t al., 1988; Ammenheuser et al., 1988; Caggana et al., 1991; Cole et al., 1991, Tates et al., 1991; Davies et al., 1992; Branda et al., 1992). These studies are important not only to understand genetic damage in "normal" individuals, but also to identify elements which should be considered in studies of populations exposed to mutagens. One difficulty with identifying factors which modulate mutant frequency is sample size, since most published series are relatively small. Consequently, we have compiled our experience with the clonal assay in 232 normal individuals and compare our analysis with the published data from other laboratories. Materials and methods
The assay system used to measure hprt mutant frequencies has been previously described in detail (Albertini et al., 1988; O'Neill et al., 1987).
Study populations Clonal assay results were combined from 4 groups of normal individuals: (a) 132 participants
in the Minnesota twin study of adult development; (b) 17 women who participated in a study of health care personnel (Albertini et al., 1988); (c) 62 women with benign breast masses who served as controls in a study of breast cancer patients (Branda et al., 1992); and (d) 21 laboratory or office personnel (Nicklas et al., 1987; O'Neill et al., 1989). The 232 individuals consisted of 77 males and 155 females. The age range was 19-80 years.
T-lymphocyte cloning assay These studies utilized the mononuclear cell (MNC) fraction of peripheral blood samples. Peripheral blood was collected by venipuncture after informed consent was obtained, following procedures approved by the Committee on Human Research at the University of Minnesota (samples from Group a) or the University of Vermont (samples from Groups b, c and d). The MNC fraction from the heparinized blood was isolated by density sedimentation as described previously (O'Neill et al., 1987). The samples from Group a were shipped in 15 ml of medium RPMI 1640 containing 10% fetal bovine serum by overnight carrier to the University of Vermont. The samples from Groups b and c were cryopreserved as described previously (O'Neill et al., 1989). The samples from Group d were used immediately after isolation. We have previously shown that fresh and cryopreserved aliquots of a MNC sample yield similar unselected cloning efficiencies and hprt mutant frequencies (O'Neill et al., 1987, 1989). Either fresh or rapidly thawed samples were incubated at a density of 1 × 106 cells/ml in medium RPMI 1640 containing 20% medium HL-1 (Ventrex), 5% defined, supplemented bovine calf serum (BCS) (Sterile Systems), and 1 ~ g / m l phytohaemagglutinin (PHA, HA17, Wellcome Diagnostics) for 36-40 h to initiate lymphocyte activation. The cell number was then determined and cells plated at limiting dilution in 96-well (round bottom) microtiter dishes (Nunc). For the control plates, cells were plated at 1 and 2 cells/well in the absence of 6-thioguanine for all experiments (one 96-well plate at each density). In addition, most of the twin experiments and some normal healthy volunteer experiments
o
Z
Z
270 T A B L E 1 (continued) Donor
Sex
Age
- TG
+ TG
Mean CE
Number of positive wells
Total number of wells
MF ( x 10 6)
95% confidence limit Lower
Upper
Smoking status
Pack yrs.
1542 1581 1582 1591 1592
F M M M M
69 71 71 73 73
0.14 0.56 0.17 0.48 0.18
9 1 1 13 7
480 48 240 288 336
13.9 3.8 2.5 9.6 11.7
6.9 0.5 0.3 5.4 5.4
27.9 27.1 17.7 16.9 25.3
Non Ex Ex Ex Non
9.0 33.0 12.5 -
16ll 1612 1621 1622 1651
F F F F F
68 68 30 30 67
0.42 0.43 0.27 0.37 0.55
10 6 12 14 58
120 96 432 432 672
10.3 7.5 5.2 4.5 8.2
5.4 3.3 2.9 2.6 5.9
19.7 16.9 9.6 7.8 11.4
Non Non Non Non Ex
3.5
1652 1661 1662 1681 1682
F F F F F
67 69 69 71 71
0.43 0.22 0.36 0.52 0.50
31 13 10 45 9
384 360 312 432 336
9.8 8.4 4.5 10.6 5.4
6.7 4.7 2.4 7.4 2.7
14.5 15.1 8.5 15.2 10.8
Ex Non Cur Cur Ex
35.0 5.4 51.0 4.0
1731 1732 1761 1762 1791
F F M M F
73 73 64 64 67
0.33 0.33 0.54 0.18 0.05
12 8 15 2 4
120 120 96 240 96
31.9 20.9 31.3 2.4 39.0
17.6 10.2 18.4 0.6 12.9
58.0 42.8 53.3 9.7 117.8
Ex Non Ex Ex Ex
19.0 64.0 33.6 7.2
1792 1831 1832 1851 1852
F F F M M
67 67 67 67 67
0.24 0.52 0.32 0.25 0.19
28 51 28 13 3
360 432 216 324 96
17.2 12.1 22.0 16.2 17.0
11.3 8.6 14.5 9.1 5.4
26.3 17.1 33.3 28.9 54.0
Non Ex Ex Ex Ex
23.5 44.(/ 26.0 33.0
1891 1892 1901 1902 1911
M M F F F
66 66 32 32 33
0.30 0.44 1.02 0.70 0.42
12 17 24 18 9
288 360 384 336 120
14.4 10.9 6.3 3.9 9.3
7.9 6.6 4.1 2.4 4.7
26.1 18.1 9.8 6.4 18.3
Ex Non Non Non Non
1.0 -
1912 1921 1922 1941 1942
F F F M M
33 69 69 76 76
0.34 0.58 0.49 0.29 0.40
29 20 9 36 24
216 264 216 432 552
20.9 6.8 4.3 30.5 11.2
13.9 4.2 2.2 20.9 7.2
31.5 10.9 8.6 44.6 17.2
Non Ex Ex Non Non
36.8 50.0 -
1971 1972 2001 2002 2042
F F M M F
33 33 34 34 33
0.28 0.24 0.46 0.59 0.31
15 26 14 22 2
384 432 408 288 288
13.2 13.9 7.6 6.8 2.2
8.5 6.9 4.3 4.3 0.6
20.5 27.9 13.4 10.8 9.0
Cur Cur Non Non Cur
22.5 7.0 4.3
2051 2052 2061 2062 2072
M M M M F
68 68 33 33 62
0.19 0.17 0.49 0.34 0.40
30 26 19 15 91
672 672 432 480 612
12.2 11.8 4.6 9.2 20.3
8.0 7.6 2.8 5.3 15.5
l 8.6 18.5 7.5 16.2 26.5
Non Ex Non Non Non
0.5 -
271 TABLE 1 (continued) Donor
Sex
Age
- TG
+TG
Mean CE
Number of positive wells
Total number of wells
MF (XlO -6)
95% confidence limit Lower
Upper
Smoking status
Pack yrs.
2081 2082 2092 2101 2102
M M M M M
74 74 68 65 65
0.12 0.55 0.33 0.36 0.41
18 35 27 10 40
480 312 288 240 432
32.4 10.7 29.6 11.7 23.8
18.9 7.3 19.4 6.2 16.7
55.6 15.8 45.1 22.4 34.0
Ex Ex Non Ex Ex
2121 2122 2141 2142 2162
M M F F M
67 67 74 74 65
0.14 0.58 0.47 0.39 0.37
3 18 18 34 17
168 288 288 576 360
13.0 11.2 6.9 7.9 13.2
4.1 6.8 4.2 5.4 7.8
41.6 18.5 11.3 11.5 22.4
Non Ex Non Non Cur
2221 2222 2231 2232 2271
F F M M M
31 31 64 64 65
0.41 0.58 0.15 0.24 0.28
15 22 1 7 25
384 528 240 288 287
9.6 3.7 2.8 10.1 16.5
5.5 2.3 0.4 4.7 10.6
16.7 5.9 20.4 21.7 25.5
Cur Cur Non Ex Ex
2272 2282 2291 2292 2311
M F M M F
65 68 63 63 33
0.37 0.36 0.41 0.35 0.73
25 2 12 17 10
480 360 384 336 288
14.6 0.8 7.7 14.7 2.4
9.2 0.2 4.3 8.8 1.3
23.0 3.1 13.9 24.3 4.7
Non Cur Cur Cur Non
2312 2341 2342 2381 2401
F F F F M
33 64 64 64 65
0.41 0.38 0.47 0.44 0.44
8 8 16 15 59
360 144 240 360 432
2.7 14.9 14.7 4.9 16.5
1.3 7.3 8.8 2.8 11.8
5.5 30.5 24.7 8.5 23.1
Non Non Non Non Ex
2402 2471 2472 2511 2512
M F F F F
65 62 62 64 64
0.27 0.53 0.39 0.76 0.65
32 63 21 19 24
360 384 216 360 384
17.4 17.0 13.2 7.1 9.9
11.0 12.4 8.3 4.4 6.3
27.4 23.4 20.9 11.6 15.4
Ex Non Non Non Ex
21.0
2531 2532 2551 2552 2561
M M F F M
64 64 64 64 63
0.23 0.20 0.23 0.22 0.33
28 31 15 16 14
372 480 192 288 240
17.4 17.1 35.2 26.5 18.1
11.4 11.2 20.4 15.5 10.4
26.5 26.0 60.7 45.2 31.5
Cur Ex Cur Cur Ex
12.5 6.5 32.3 NA
2562 2711 2712 2821 2951
M M M F F
63 62 62 61 65
0.40 0.16 0.12 0.20 0.42
18 7 4 18 12
216 192 120 240 456
21.8 23.9 27.6 38.7 6.4
13.3 10.9 9.7 23.2 3.5
35.7 52.1 78.8 64.7 11.5
Ex Non Ex Non Non
2952 9991 9992
F F F
65 69 69
0.11 0.19 0.29
12 22 12
312 336 456
17.8 35.0 9.4
8.6 21.7 5.1
36.7 56.5 17.0
Cur Non Non
43.0 75.0 58.8 60.0
1.8
75.0 0.1 1.7 9.0 45.0
52.5 82.5 147.0
33.6
15.0
102.0
20.0
272
were also plated at 5 cells/well (one plate) and all of the experiments on women with benign breast masses were plated at 5 and 10 cells/well (one plate at each density). For the selection plates, cells were plated at 1 or 2 × 104 cells/well in the presence of 10 /zM 6-thioguanine (TG). Cells were in 200 izl of RPMI 1640 medium containing 20% HL-1, 5% BCS, 20% growth factor supplement, 0.125 p.g/ml PHA and 1 x 1 0 4 irradiated human lymphoblastoid accessory cells. The growth factor supplement was the cell-free supernatant medium obtained after generation of LAK cell activity by incubating patient peripheral blood mononuclear cells in medium containing 1500 U / m l recombinant interleukin-2 (O'Neill et al., 1987; Branda et al., 1992). Following a 10-14 day incubation of the microtiter dishes, growth was determined by use of an inverted phase contrast microscope. Cloning efficiencies (CE) were calculated by use of the Poisson relationship, P0 = e x, where P0 is the fraction of wells without colony growth. The CE equals - l n Po/x, where x = the average number of cells/well. The variance is calculated as ( 1 - P o ) / [ ( P o ) ( t o t a l
number of wells)(ln /90)2]. The mean CE for the non-selection plates was calculated by averaging the non-selection CEs plated at different cell densities. Nearly all experiments had CE values for cells plated at 1 and 2 cells/well but many had values for cells plated at 5 and 10 cells/well as well. All obtained values were used except those where all wells were positive for cell growth (In 0 is undefined). The variance for this mean is the sum of the variances for each CE divided by the number of variances squared i.e. E u , / n 2. The mutant frequency is the ratio of the CE in the presence (selected) and mean CE in the absence (unselected) of TG. Confidence limits were calculated as in Furth et al. (1981) except that the mean variance was used for the nonselection variance part of the equation. In other words, the 95% confidence limits are: MF× exp[_ 1.96 × ( t ' + T G q- tJ TG)I/2].
Statistical method Because the distribution of mutant frequency is skewed, analyses were based upon the natural
TABLE 2 HEALTH CARE WORKERS Donor
Sex
Age
-TG
+TG
MF
95% c o n f i d e n c e limit
Smoking
M e a n CE
Number of positive wells
Total n u m b e r of wells
( × 10-6)
Lower
Upper
status
Pack yrs.
A01 A04 A05 A07 A12
F F F F F
41 41 61 36 37
0.57 0.12 0.44 0.73 0.40
16 2 59 40 34
288 192 384 480 576
5.0 4.2 19.1 6.0 7.7
3.0 1.0 13.7 4.2 5.1
8.5 17.6 26.7 8.6 11.5
Non Non Non Non Cur
15.0
AI3 A15 AI6 AI7 A21
F F F F F
43 35 55 35 58
0.21 0.47 0.66 0.33 0.52
27 51 12 33 20
288 278 191 768 276
23.5 21.7 4.9 6.7 7.2
14.6 15.3 2.7 4.4 4.4
37.8 30.6 9.0 10.2 11.6
Non Non Non Cur Cur
20 - 4 0 15.0
A24 A27 A32 A33 A34
F F F F F
52 63 54 32 39
0.31 0.14 0.27 0.25 0.21
24 9 53 13 4
384 192 480 288 96
10.5 17.6 21.9 9.2 10.4
6.6 8.4 15.0 5.0 3.7
16.8 37.1 31.9 16.8 28.8
Non Cur Off/On Non Non
18.0 -
A35 A40
F F
44 46
0.39 0.26
25 24
576 384
5.7 12.4
3.6 7.0
9.0 22.1
Non Non
-
-
273 TABLE 3 W O M E N WITH B E N I G N BREAST MASSES Donor
Sex
Age
- TG
+ TG
Mean CE
Number of positive wells
Total number of wells
MF (>(10 -6 )
95% confidence limit Lower
Upper
Smoking status
Pack yrs.
0003-1 0008-1 0010-1 0011-1 0019-1
F F F F F
34 63 34 41 40
0.14 0.61 0.30 0.44 0.55
21 31 12 4 14
240 480 144 96 192
33.2 5.5 14.3 4.8 6.9
19.2 3.7 7.9 1.8 4.0
57.5 8.0 25.7 13.0 12.0
Ex Cur Non Non Non
3.5 51.0
0020-1 0026-1 0031-1 0033-1 0036-1
F F F F F
54 41 56 41 45
0.25 0.31 0.10 0.18 0.46
29 30 19 38 21
456 348 192 480 156
13.0 14.6 53.5 22.9 15.7
8.7 9.8 32.3 15.9 10.0
19.4 21.8 88.7 33.0 24.9
Cur Cur Ex Ex Non
13.5 NA 0.5 0.1
0038-1 0045-1 0051-1 0053-1 0054-1
F F F F F
38 40 50 41 35
0.36 0.49 0.44 0.17 0.15
76 14 39 10 19
612 132 480 240 384
18.5 11.5 9.6 12.7 16.8
13.9 6.6 6.7 6.6 10.2
24.7 19.8 13.6 24.3 27.8
Cur Cur Cur Non Non
1.5 12.0 17.0
0055-1 0056-1 0058-1 0059-1 0060-1
F F F F F
44 38 42 42 56
0.90 0.58 0.34 0.90 0.56
55 76 26 68 88
672 768 576 384 1056
4.7 8.9 6.8 10.8 7.8
3.4 6.7 4.5 8.0 6.0
6.5 11.8 10.3 14.6 10.2
Non Non Ex Cur Ex
13.0 15.0 30.0
0061-1 0063-1 0064-1 0065-1 0066-1
F F F F F
40 60 42 39 42
0.15 0.40 0.14 0.17 0.61
29 29 12 11 104
480 280 480 192 480
21.1 13.8 8.8 17.4 20.0
13.9 9.2 4.8 9.4 15.6
31.9 20.6 15.9 32.4 25.6
Non Ex Cur Ex Cur
3.5 20.0 0.1 42.0
0067-1 0068-1 0069-1 0072-1 0074-1
F F F F F
36 39 43 62 64
0.25 0.40 0.23 0.28 0.71
7 17 11 28 87
192 432 288 276 768
7.4 5.0 8.3 19.4 8.5
3.5 3.0 4.5 13.0 6.5
15.8 8.2 15.4 29.1 11.2
Ex Non Non Ex Ex
0079-1 0084-1 0085-1 0087-1 0091-1
F F F F F
39 22 52 65 39
0.45 0.30 0.19 0.27 0.40
28 102 13 116 38
384 576 264 864 336
8.4 32.1 13.4 26.3 7.6
5.6 25.3 7.5 20.5 5.3
12.7 40.9 24.0 33.7 10.9
Non Non Non Cur Ex
0092-1 0093-1 0094-1 0096-1 0098-1
F F F F F
55 45 44 39 53
0.32 0.44 0.15 0.22 0.27
63 150 24 22 15
480 864 672 480 120
22.0 21.6 12.4 10.5 25.1
16.1 17.1 7.9 6.7 14.8
29.9 27.3 19.5 16.6 42.5
Cur Non Non Ex Ex
0099-1 0100-1 0101-1 0104-1 0105-1 0106-1
F F F F F F
52 48 40 56 37 53
0.32 0.39 0.63 0.62 0.23 0.32
46 17 35 53 38 19
636 348 384 336 960 288
11.8 6.4 7.6 13.9 8.7 10.6
8.5 3.9 5.3 10.0 6.1 6.6
16.6 10.5 11.0 19.2 12.4 17.0
Non Ex Cur Cur Non Non
4.0
0.1 5.2
53.8 1.5 57.0
14.0 12.8
21.0 60.0
274 TABLE 3 (continued) Donor
Sex
Age
- TG
+ TG
Mean CE
Number of positive wells
Total number of wells
MF (×10
6)
95% confidence limit
Smoking
Lower
status
U ppe r
Pack yrs.
0110-1 11111- I 0113-1 0116-1
F F F F
44 38 37 41
0.43 0.54 0.51 0.75
30 53 24 29
960 960 624 576
3.7 5.3 3.8 3.4
2.5 3.8 2.5 2.3
5.5 7.2 5.9 5.2
Non Ex Ex Ex
10.0 12.11 -
0118-1 1/120-1 0125-1 11126-1 0128-1
F F F F F
54 35 42 40 43
0.68 0.42 0.34 0.63 0.45
65 4 11 28 40
384 252 288 1344 288
27.1 1.9 5.7 2.3 16.4
19.4 0.7 3.1 1.5 11.6
38.0 5.1 10.5 3.6 23.4
Non Cur Non Non Non
13.0 -
0129-1 0130-1 0132-1 11134-1 013%1
F F F F F
46 36 36 44 38
0.81 0.56 0.78 0.84 0.64
104 44 85 48 55
672 288 480 672 336
10.4 14.9 12.4 4.4 13.9
7.9 10.7 9.4 3.2 10.1
13.7 20.8 16.5 6.2 19.1
Non Ex Cur Ex Ex
1.0 31.5 < 4.11 20-40
0138-1 0139-1
F F
50 60
0.48 0.41
26 43
288 288
9.8 19.6
6.5 13.9
14.9 27.6
Non Ex
9.(1
logarithms of mutant frequencies (Henderson et al., 19861. Groups were compared using analysis of variance to assess differences in In MF, CE and age, and a Chi-square test was used to assess smoking status differences. Gender differences were analyzed by t-tests. Regression analyses were used to examine the relationships between In MF, CE and age and to assess the effect of smoking status on In MF, adjusting for age and CE. Results and discussion
The data obtained on the 4 groups of donors are shown in Tables 1-4. Each table presents information regarding the age, sex, mean unselected CE, number of wells containing TG-resistant colonies, total number of wells analyzed, MF, 95% confidence limits of the MF, and smoking history, expressed as pack-years. The data are presented in a format similar to that used in other recent reports (Henderson et al., 1986; Cole et al., 1988; Sala-Trepat et al., 1990; Tates et al., 1991; Davies et al., 1992) to facilitate the development of a large historical database on normal individuals.
The first group (Table 1) includes 132 participants in a study of identical and fraternal twins. The second group (Table 2) consists of 17 health care workers studied as part of an assessment of the risks of handling cancer chemotherapeutic agents. Some data derived from this second group of individuals have been published previously (A1bertini et al., 1988). The third group (Table 3) is composed of 62 women with benign breast masses who were enrolled in a study of environmental factors which modulate genetic damage caused by treatment for breast cancer (Branda et al., 1992). The diagnoses which brought them to medical attention were: fibrocystic changes (17), fibroadenoma (10), microcalcification (4), pigmented nevus (2), lipoma (2), mammographic density without pathologic diagnosis (2), acute and chronic mastitis (1), fatty benign breast tissue (1), abscess (1) and cellulitis (1). The last group (Table 4) contains 21 normal volunteers recruited from laboratory and office personnel. Some data obtained from these individuals have been published (Nicklas et al., 1987; O'Neill et al., 1987, 1989; Branda et al., 1992). A comparison between groups is shown in Table 5. Group 1 (healthy identical and fraternal
275
TABLE 4 NORMAL HEALTHY VOLUNTEERS
Donor
Sex
Age
+ TG
MF
95% confidence limit Smoking
Mean CE
Number of positive wells
Total number of wells
( × 10- 6)
Lower
- TG
Upper
status
Pack yrs.
VT1 VT2 VT3 VT4 VT5
M M F F F
19 21 22 25 34
0.28 0.37 0.30 0.39 0.15
19 22 19 35 5
460 480 672 1056 192
7.6 6.3 4.8 4.3 8.5
4.6 3.9 2.9 2.9 3.4
12.8 10.1 8.0 6.5 21.2
NA NA NA NA NA
-
VT6 VT7 VT8 VT9 VT10
M M M M F
37 29 22 27 25
0.10 0.26 0.11 0.49 0.70
40 8 12 28 45
864 180 672 288 927
24.8 8.8 8.3 10.5 3.5
14.8 4.3 4.2 6.9 2.5
41.8 18.0 16.4 16.1 5.0
NA NA NA NA NA
-
VTll VT12 VT13 VT14 VT15
M F M M M
23 43 27 38 26
0.36 0.41 0.39 0.19 0.35
13 52 33 9 7
817 384 480 467 480
2.2 17.7 9.3 5.2 2.1
1.2 12.9 6.1 2.5 1.0
4.1 24.3 14.0 10.7 4.6
NA NA NA NA NA
-
VT16 VT17 VT18 VT19 VT20
M M M M M
34 23 30 25 37
0.41 0.51 0.54 0.29 0.56
33 16 13 47 33
768 871 565 960 288
5.4 1.8 2.2 8.7 10.9
3.6 1.1 1.2 5.9 7.5
8.1 3.1 3.8 12.7 16.0
NA NA NA NA NA
-
VT21
M
22
0.50
14
464
3.1
1.7
5.4
NA
-
-
twins) was significantly older, and Group 4 (normal healthy volunteers) was significantly younger, than the other 3 groups. The In MF for Group 4 was significantly lower than the other 3 groups.
in Fig. 1 for the combined group of 232 donors and is expressed by this equation:
Age
with R 2 = 0.151, P < 0.001. Thus, MF increases by about 2% per year in our series.
The relationship between age and MF is shown
In MF = 1.46 + 0.018 age
TABLE 5
COMPARISON BETWEEN GROUPS 1-4 Group
N
Mean age
Mean In MF ( ± std. dev.)
( _+std. dev.) 0.37 + 0.18 0.385:0.18 0.43 + 0.21 0.365:0.16
1 2 3 4
132 17 62 21
61.7 ± 13.3 a 45.45:9.9 44.6 5 : 8 . 8 28.05:6.7 a
2:53 + 0.73 2.275:0.58 2.37 ± 0.66 1.77_+0.71
Total
232
52.9 5:16.0
2.40 ± 0.73
a Significantly different from other 3 groups at p < 0.05.
a
Mean CE
0.39 5:0.18
276
These results are similar to previously reported rates of 2.18% per year in normal adults by Cole and colleagues (1991) and 3% per year by Tates et al. (1991). The subjects included in the present report spanned the age range 19-80 years. Although we have studied and described elsewhere (McGinniss et al., 1989) assays in normal newborns, these data were not included in the present analysis. The cord-blood group differs from the others with respect to CE and MF, and all the ages in this group are zero. Cole et al. similarly reported a low MF in cord blood, which increased about 10-fold during early childhood (1991).
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0.8
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Cloning efficiency The relationship between MF and CE for these 232 adults is shown in Fig. 2 and is described by the equation:
Fig. 2. Regression line showing the relationship between lymphocyte cloning efficiency and mutant frequency.
In MF = 2.91-1.32 CE
with R 2 = 0.019 and P = 0.038, indicating that CE was significantly inversely proportional to age in our subjects. This relationship supports the observations of Tates and colleagues (1991), who found also a significant decrease from one year to the next.
with R 2 = 0 . 1 0 9 and P < 0 . 0 0 1 . This result is comparable to the observation of Tates et al. (1991), who reported that doubling the CE reduced the MF by 37% in his series, and of Cole and coworkers (1991), who found a 40% reduction of MF when the CE doubled. The relationship between cloning efficiency and age is described by the equation:
CE = 0.47-0.002 age 4.6 4.0 • .3.6
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0
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Donor gender Males and females did not differ with respect to MF, but CE was significantly lower in males ( P < 0.001), even after adjustment for age differences. Previous studies which have included a consideration of donor gender have similarly found no significant effect on MF (Caggana et al., 1991; Cole et al., 1988; Davies et al., 1992; Vijayalaxmi and Evans, 1984; Tates et al., 1989).
: I
1,0
Interrelationships between mutant frequency, age and cloning efficiency The inter-relationship between mutant frequency, age and cloning efficiency in these 232 donors is expressed by the equation:
0,5
0.0
In MF = 1.99 - 1.13 CE + 0.016 age
-0.5
0.229 and P < 0.001. Thus, MF values increase with increasing age and with decreasing CE. This relationship is portrayed graphically in Fig. 3.
with R 2 = Fig. 1. Regression line indicating the effect of donor age on the mutant frequency at the hprt locus in 232 healthy individ uals.
277 5.0
j
4,5 4.0
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2.5
3.0
3.5
4.0
4.5
In M F = 1.99 - 1.13 C E + 0.016 A G E
Fig. 3. Regression line indicating the interrelationship between mutant frequency, cloning efficiency and age in 232 donors. The dotted lines represent 95% confidence limits.
Effect of smoking cigarettes The effect of smoking on MF was assessed by including current smoking status (smoker, nonsmoker) in the regression analysis in addition to CE and age. 22 subjects for whom no smoking information was available were excluded from this analysis. The following equation was obtained: In MF = 2.29 - 1.16 CE + 0.012 age - 0.09 s where s = 0 for non-smokers and s = 1 for smokers. By this analysis, the effect of smoking was not statistically significant ( P = 0.413). Other analyses found no significant relationship between In MF and number of years smoked for current smokers or pack-years. Our results differed from other recent analyses of mutant frequency at the hprt locus. For example, Tates and colleagues (1991) reported that smoking had a significant effect on the In MF, with P = 0.025, and that smoking increased the MF by 36% for any combination of CE and age. Similarly, Cole et al. (1991) found that smoking caused a highly significant increase in MF whether or not CE was considered. However, these workers found that the smoking effect was age-dependent, and that the increase in MF per year for smokers was 2.9% compared to 0.8% for non-smokers (Cole et al., 1990). Smoking significantly affected the MF
in the series of Ammenheuser et al. (1988) and in the control group reported by Caggana and colleagues (1991). In contrast smoking habits had no effect on MF in the healthy donors studied by Davies et al. (1992) and was associated with lower MFs than in non-smokers in the report of SalaTrepat (1990). The basis for this diverse effect of smoking history on MF in various laboratories is obscure at present. In conclusion, our investigations with the Tlymphocyte clonal assay confirm that somatic cell mutant frequency varies widely in normal individuals. Most laboratories experienced with this technique agree that donor age and lymphocyte CE are important factors which influence MF and contribute to this variability. The effect of cigarette smoking history has been less consistent. Many authors report that MF tends to be higher in smokers, but this elevation is not always statistically significant, as was the case in the present study. Recently, we found that serum levels of a micronutrient, folic acid, may modify genetic damage at the hprt locus, and that folic acid deficiency was associated with higher mutant frequencies following exposure to chemotherapy (Branda et al., 1991, 1992). These latter studies suggest that dietary components also may be important contributing factors to the variability observed in the MF. Finally, it is likely that differences in DNA-repair efficiencies will also affect mutant frequency values (Tates et al., 1989; Cole et al., 1991).
Acknowledgements Supported by grants from the National Cancer Institute (CA 41843, CA 30688 and P30 CA22435), the National Institute of Health (AG06886) and by EPA Cooperative Agreement No. CR-812837. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. This article has not been subjected to U.S. Environmental Protection Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
278
References Albertini, R.J., K.L. Castle and W.R. Borcherding (1982) T-cell cloning to detect the mutant 6-thioguanine-resistant lymphocytes present in human peripheral blood, Proc. Natl. Acad. Sci. (U.S.A.), 79, 6617-6621. Albertini, R.J., L.M. Sullivan, J.K. Berman, C.J. Greene, J.A. Stewart, J.M. Silveira and J.P. O'Neill (1988) Mutagenicity monitoring in humans by autoradiographic assay for mutant T lymphocytes, Mutation Res., 204, 481-492. Albertini, R.J., J.A. Nicklas, J.P. O'Neill and S.H. Robison (1990) In vivo somatic mutations in humans: Measurement and analysis, Annu. Rev. Genet., 24, 305-326. Ammenheuser, M.M., J.B. Ward, E.B. Whorton, J.M. Killian and M.S. Legator (1988) Elevated frequencies of 6-thioguanine-resistant lymphocytes in multiple sclerosis patients treated with cyclophosphamide: a prospective study, Mutation Res., 204, 509-520. Branda, R.F., J.P. O'Neill, L.M. Sullivan and R.J. Albertini (1991) Factors influencing mutation at the hprt locus in T-lymphocytes: women treated for breast cancer, Cancer Res., 51, 6603-6607. Branda, R.F., J.P. O'Neill, D. Jacobson-Kram and R.J. Albertini (1992) Factors influencing mutation at the hprt locus in T-lymphocytes: studies in normal women and women with benign and malignant breast masses, Environ. Mol. Mutagen., 19, 274-281. Caggana, M., H.L. Liber, P.M. Mauch, C.N. Coleman and K.T. Kelsey (1991) In vivo somatic mutation in lymphocytes of Hodgkin's disease patients, Environ. Mol. Mutagen., 18, 6-13. Cariello, N.F., T.R. Craft, H. Vrieling, A.A. van Zeeland, T. Adams and T.R. Skopek (1992) Environ. Mol. Mutagen., 20, 81-83. Cole, J., M.H.L. Green, S.E. James, L. Henderson and H. Cole (1988) A further assessment of factors influencing measurements of thioguanine-resistant mutant frequency in circulating T-lymphocytes, Mutation Res., 204, 49835007. Cole, J., M.H.L. Green, G. Stephens, A.P.W. Waugh, D. Beare, H. Steingrimsdottir and B.A. Bridges (1990) HPRT somatic mutation data, in: M.L. Mendelsohn and R.J. Albertini (Eds.), Mutation and the Environment, WileyLiss, New York, pp. 25-35. Cole, J., A.P.W. Waugh, D.M. Beare, M. Sala-Trepat, G. Stephens and M.H.L. Green (1991) HPRT mutant frequencies in circulating lymphocytes: population studies using normal donors, exposed groups and cancer-prone syndromes, in: B.L. Gledhill and F. Mauro (Eds.), New Horizons in Biological Dosimetry, Wiley-Liss, New York, pp. 319-328. Davies, M.J., D.P. Lovell and D. Anderson (1992) Thioguanine-resistant mutant frequency in T-lymphocytes from a healthy human population, Mutation Res., 265, 165-171. Dempsey, J.L., and A.A. Morley (1986) Measurement of in vivo mutant frequency in lymphocytes in the mouse, Environ. Mutagen., 8, 385-391.
Furth, E.M., W.G. Thilly, B.A. Penham, H.L. Liber and W.M. Rand (1981) Quantitative assay for mutation in diploid human lymphoblasts using microtiter plates, Anal. Biochem., 110, 1-8. Hakoda, M., M. Akiyama, S. Kyoizumi, A.A. Awa, M. Yamakido and M. Otake (1988) Increased somatic cell frequency in atomic bomb survivors, Mutation Res., 201, 39-48. Henderson L., H. Cole, J. Cole, S. Elizabeth James and M. Green (1986) Detection of somatic mutations in man: evaluation of the microtitre cloning assay for T-lymphocytes, Mutagenesis, 1, 195-200. Messing, K., J. Ferraris, W.E.C. Bradley, J. Swartz and A.M. Seifert (1989) Mutant frequency of radiotherapy technicians appears to be associated with recent dose of ionizing radiation, Health Phys., 57, 537-544. Morley, A.A., S. Cox, D. Wigmore, R. Seshadri and J.L. Dempsey (1982) Enumeration of thioguanine-resistant lymphocytes using autoradiography, Mutation Res., 95, 363-375. Morley, A.A., K.J. Trainor, R. Seshadri and R.G. Ryall (1983) Measurement of in vivo mutations in human lymphocytes, Nature (London), 302, 155-156. McGinniss, M.J., M.T. Falta, L.M. Sullivan and R.J. Albertini (1989) In vivo hprt mutant frequencies in T-cells of normal human newborns, Mutation Res., 240, 117-126. O'Neill, J.P., M.J. McGinniss, J.K. Berman, L.M. Sullivan, J.A. Nicklas and R.J. Albertini (1987) Refinement of a T-lymphocyte cloning assay to quantify the in vivo thioguanine-resistant mutant frequency in humans, Mutagenesis, 2, 87-94. O'Neill, J.P., L.M. Sullivan, J.K. Booker, B.S. Pornelos, M.T. Falta, C.J. Greene and R.J. Albertini (1989) Longitudinal study of the in vivo hprt mutant frequency in human T-lymphocytes as determined by a cell cloning assay, Environ. Mol. Mutagen., 13, 289-293. Sala-Trepat, M., J. Cole, M.H.L. Green, O. Rigaud, J.R. Vilcoq and E. Moustacchi (1990) Genotoxic effects of radiotherapy and chemotherapy on the circulating lymphocytes of breast cancer patients, III. measurement of mutant frequency to 6-thioguanine resistance, Mutagenesis, 5, 593-598. Seifert, A.M., W.C. Bradley and K. Messing (1987) Exposure of nuclear medicine patients to ionizing radiation is associated with rises in H P R T - mutant frequency in peripheral T-lymphocytes, Mutation Res., 191, 57-63. Strauss, G.H., and R.J. Albertini (1977) 6-Thioguanine resistant lymphocytes in human peripheral blood, in: D. Scott, B.A. Bridges and F.H. Sobels (Eds.), Progress in Genetic Toxicology: Developments in Toxicology and Environmental Sciences, Vol. 2, Elsevier, Amsterdam, p. 327. Strauss, G.H. and R.J. Albertini (1979) Enumeration of 6thioguanine resistant peripheral blood lymphocytes in man as a potential test for somatic cell mutations arising in vivo, Mutation Res., 61,353-379. Tares, A.D., L.F. Bernini, A.T. Natarajan, J.S. Ploem, N.P. Verwoerd, J. Cole, M.H.L. Green, C.F. Arlett and P.N.
279 Norris (1989) Detection of somatic mutants in man: HPRT mutations in lymphocytes and hemoglobin mutations in erythrocytes, Mutation Res., 213, 73-82. Tates, A.D., F.J. van Dam, H. van Mossel, H. Schoemaker, J.C.P. Thijssen, V.M. Woldring, A.H. Zwinderman and A.T. Natarajan (1991) Use of the clonal assay for the measurement of frequencies of HPRT mutants in T-
lymphocytes from five control populations, Mutation Res., 253, 199-213. Vijayalaxmi and H.J. Evans (1984) Measurement of spontaneous and X-irradiation-induced 6-thioguanine-resistant human blood lymphocytes using a T-cell cloning technique, Mutation Res., 125, 87-94.
267
MUT 05204
M e a s u r e m e n t of H P R T mutant frequencies in T-lymphocytes from healthy human populations R.F. Branda a, L.M. Sullivan a j.p. O'Neill a M.T. Falta a, J.A. Nicklas a B. Hirsch c, P.M. Vacek b and R.J. Albertini a a Genetics Laboratory, Vermont Cancer Center and b Medical Statistics Department, University of Vermont, Burlington, VT 05401, USA and c Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, M N 05455, USA
(Received 20 August 1992) (Accepted 21 August 1992)
Keywords: HPRT; 6-Thioguanine-resistant; T-lymphocytes; Human population monitoring
Summary Somatic cell mutant frequencies at the hprt locus of the X-chromosome were measured with the T-lymphocyte cloning technique in healthy human populations. A statistical analysis was performed of assays from 232 individuals (77 males and 155 females) ranging in age from 19 to 80 years. Data from 4 donor groups were compiled: (a) 132 participants in a study of identical and fraternal twins; (b) 17 health care workers studied as part of an assessment of the risks of handling chemotherapeutic drugs; (c) 62 women with benign breast masses; and (d) 21 normal laboratory and office personnel. The relationship between age and mutant frequency (MF) was expressed by the equation: In MF = 1.46 + 0.018 age (P < 0.001). Thus, MF increased by about 2% per year. Increases in cloning efficiency (CE) reduced the MF, as shown in the equation: In MF = 2.91-1.32 CE (P < 0.001). CE was significantly related to age (CE = 0.47-0.002 age, P = 0.038), and the interdependent relationship between MF, age and CE expressed by the equation: In MF = 1.99-1.13 CE + 0.016 age was significant at the P < 0.001 level. There was no statistically significant effect of donor gender or smoking history on MF in our population, but CE was significantly lower in males (P < 0.001). These findings confirm the importance of age and CE as factors which influence the thioguanine-resistant MF in circulating T-lymphocytes from normal adults.
Development of an assay to detect mutations at the hprt locus in lymphocytes has advanced our understanding of the mechanisms of genetic damage in human ceils. The initially described
Correspondence: Dr. Richard F. Branda, Genetics Laboratory, 32 N. Prospect St., Burlington, VT 05401, USA. Tel. (802)863-5716; Fax (802)656-8429.
autoradiographic assay (Strauss and Albertini, 1977, 1979; Morley et al., 1982) and its later refinement, the clonal assay (Albertini et al., 1982; Morley et al., 1983), have been employed by many laboratories to measure somatic mutation rates in normal individuals, in populations exposed to known or suspected mutagens, and in patients with inherited defective DNA repair (Ammenheuser et al., 1988; Caggana et al., 1991;
268
Cole et al., 1991; Davies et al., 1992; Dempsey et al., 1986; Hakoda et al., 1988; Messing et al., 1989; Seifert et al., 1987; Sala-Trepat et al., 1990; Tates et al., 1989, 1991; Vijayalaxmi and Evans, 1984). The clonal assay also has proven useful to characterize the specific molecular alterations and mutational spectrum in hprt- mutant cells (reviewed in Albertini et al., 1990) and a database of these mutations has been compiled (Cariello et al., 1992). As more experience with this assay system has accrued, it has become clear that the range of the mutant frequency at the hprt locus in peripheral blood lymphocytes from normal individuals is quite broad, spanning approximately one logv (Henderson et al., 1986; Albertini et al., 1988; Cole et al., 1991; Tares et al., 1991). Attempts have been made to define the factors which influence mutant frequency and thereby contribute to the differences observed between normal individuals. There is evidence that experimental variables such as cloning efficiency, as well as subject characteristics such as age and lifestyle (diet and exposure to cigarette smoke) may affect mutant frequency (Vijayalaxmi and Evans, 1984; Albert i n i e t al., 1988; Ammenheuser et al., 1988; Caggana et al., 1991; Cole et al., 1991, Tates et al., 1991; Davies et al., 1992; Branda et al., 1992). These studies are important not only to understand genetic damage in "normal" individuals, but also to identify elements which should be considered in studies of populations exposed to mutagens. One difficulty with identifying factors which modulate mutant frequency is sample size, since most published series are relatively small. Consequently, we have compiled our experience with the clonal assay in 232 normal individuals and compare our analysis with the published data from other laboratories. Materials and methods
The assay system used to measure hprt mutant frequencies has been previously described in detail (Albertini et al., 1988; O'Neill et al., 1987).
Study populations Clonal assay results were combined from 4 groups of normal individuals: (a) 132 participants
in the Minnesota twin study of adult development; (b) 17 women who participated in a study of health care personnel (Albertini et al., 1988); (c) 62 women with benign breast masses who served as controls in a study of breast cancer patients (Branda et al., 1992); and (d) 21 laboratory or office personnel (Nicklas et al., 1987; O'Neill et al., 1989). The 232 individuals consisted of 77 males and 155 females. The age range was 19-80 years.
T-lymphocyte cloning assay These studies utilized the mononuclear cell (MNC) fraction of peripheral blood samples. Peripheral blood was collected by venipuncture after informed consent was obtained, following procedures approved by the Committee on Human Research at the University of Minnesota (samples from Group a) or the University of Vermont (samples from Groups b, c and d). The MNC fraction from the heparinized blood was isolated by density sedimentation as described previously (O'Neill et al., 1987). The samples from Group a were shipped in 15 ml of medium RPMI 1640 containing 10% fetal bovine serum by overnight carrier to the University of Vermont. The samples from Groups b and c were cryopreserved as described previously (O'Neill et al., 1989). The samples from Group d were used immediately after isolation. We have previously shown that fresh and cryopreserved aliquots of a MNC sample yield similar unselected cloning efficiencies and hprt mutant frequencies (O'Neill et al., 1987, 1989). Either fresh or rapidly thawed samples were incubated at a density of 1 × 106 cells/ml in medium RPMI 1640 containing 20% medium HL-1 (Ventrex), 5% defined, supplemented bovine calf serum (BCS) (Sterile Systems), and 1 ~ g / m l phytohaemagglutinin (PHA, HA17, Wellcome Diagnostics) for 36-40 h to initiate lymphocyte activation. The cell number was then determined and cells plated at limiting dilution in 96-well (round bottom) microtiter dishes (Nunc). For the control plates, cells were plated at 1 and 2 cells/well in the absence of 6-thioguanine for all experiments (one 96-well plate at each density). In addition, most of the twin experiments and some normal healthy volunteer experiments
o
Z
Z
270 T A B L E 1 (continued) Donor
Sex
Age
- TG
+ TG
Mean CE
Number of positive wells
Total number of wells
MF ( x 10 6)
95% confidence limit Lower
Upper
Smoking status
Pack yrs.
1542 1581 1582 1591 1592
F M M M M
69 71 71 73 73
0.14 0.56 0.17 0.48 0.18
9 1 1 13 7
480 48 240 288 336
13.9 3.8 2.5 9.6 11.7
6.9 0.5 0.3 5.4 5.4
27.9 27.1 17.7 16.9 25.3
Non Ex Ex Ex Non
9.0 33.0 12.5 -
16ll 1612 1621 1622 1651
F F F F F
68 68 30 30 67
0.42 0.43 0.27 0.37 0.55
10 6 12 14 58
120 96 432 432 672
10.3 7.5 5.2 4.5 8.2
5.4 3.3 2.9 2.6 5.9
19.7 16.9 9.6 7.8 11.4
Non Non Non Non Ex
3.5
1652 1661 1662 1681 1682
F F F F F
67 69 69 71 71
0.43 0.22 0.36 0.52 0.50
31 13 10 45 9
384 360 312 432 336
9.8 8.4 4.5 10.6 5.4
6.7 4.7 2.4 7.4 2.7
14.5 15.1 8.5 15.2 10.8
Ex Non Cur Cur Ex
35.0 5.4 51.0 4.0
1731 1732 1761 1762 1791
F F M M F
73 73 64 64 67
0.33 0.33 0.54 0.18 0.05
12 8 15 2 4
120 120 96 240 96
31.9 20.9 31.3 2.4 39.0
17.6 10.2 18.4 0.6 12.9
58.0 42.8 53.3 9.7 117.8
Ex Non Ex Ex Ex
19.0 64.0 33.6 7.2
1792 1831 1832 1851 1852
F F F M M
67 67 67 67 67
0.24 0.52 0.32 0.25 0.19
28 51 28 13 3
360 432 216 324 96
17.2 12.1 22.0 16.2 17.0
11.3 8.6 14.5 9.1 5.4
26.3 17.1 33.3 28.9 54.0
Non Ex Ex Ex Ex
23.5 44.(/ 26.0 33.0
1891 1892 1901 1902 1911
M M F F F
66 66 32 32 33
0.30 0.44 1.02 0.70 0.42
12 17 24 18 9
288 360 384 336 120
14.4 10.9 6.3 3.9 9.3
7.9 6.6 4.1 2.4 4.7
26.1 18.1 9.8 6.4 18.3
Ex Non Non Non Non
1.0 -
1912 1921 1922 1941 1942
F F F M M
33 69 69 76 76
0.34 0.58 0.49 0.29 0.40
29 20 9 36 24
216 264 216 432 552
20.9 6.8 4.3 30.5 11.2
13.9 4.2 2.2 20.9 7.2
31.5 10.9 8.6 44.6 17.2
Non Ex Ex Non Non
36.8 50.0 -
1971 1972 2001 2002 2042
F F M M F
33 33 34 34 33
0.28 0.24 0.46 0.59 0.31
15 26 14 22 2
384 432 408 288 288
13.2 13.9 7.6 6.8 2.2
8.5 6.9 4.3 4.3 0.6
20.5 27.9 13.4 10.8 9.0
Cur Cur Non Non Cur
22.5 7.0 4.3
2051 2052 2061 2062 2072
M M M M F
68 68 33 33 62
0.19 0.17 0.49 0.34 0.40
30 26 19 15 91
672 672 432 480 612
12.2 11.8 4.6 9.2 20.3
8.0 7.6 2.8 5.3 15.5
l 8.6 18.5 7.5 16.2 26.5
Non Ex Non Non Non
0.5 -
271 TABLE 1 (continued) Donor
Sex
Age
- TG
+TG
Mean CE
Number of positive wells
Total number of wells
MF (XlO -6)
95% confidence limit Lower
Upper
Smoking status
Pack yrs.
2081 2082 2092 2101 2102
M M M M M
74 74 68 65 65
0.12 0.55 0.33 0.36 0.41
18 35 27 10 40
480 312 288 240 432
32.4 10.7 29.6 11.7 23.8
18.9 7.3 19.4 6.2 16.7
55.6 15.8 45.1 22.4 34.0
Ex Ex Non Ex Ex
2121 2122 2141 2142 2162
M M F F M
67 67 74 74 65
0.14 0.58 0.47 0.39 0.37
3 18 18 34 17
168 288 288 576 360
13.0 11.2 6.9 7.9 13.2
4.1 6.8 4.2 5.4 7.8
41.6 18.5 11.3 11.5 22.4
Non Ex Non Non Cur
2221 2222 2231 2232 2271
F F M M M
31 31 64 64 65
0.41 0.58 0.15 0.24 0.28
15 22 1 7 25
384 528 240 288 287
9.6 3.7 2.8 10.1 16.5
5.5 2.3 0.4 4.7 10.6
16.7 5.9 20.4 21.7 25.5
Cur Cur Non Ex Ex
2272 2282 2291 2292 2311
M F M M F
65 68 63 63 33
0.37 0.36 0.41 0.35 0.73
25 2 12 17 10
480 360 384 336 288
14.6 0.8 7.7 14.7 2.4
9.2 0.2 4.3 8.8 1.3
23.0 3.1 13.9 24.3 4.7
Non Cur Cur Cur Non
2312 2341 2342 2381 2401
F F F F M
33 64 64 64 65
0.41 0.38 0.47 0.44 0.44
8 8 16 15 59
360 144 240 360 432
2.7 14.9 14.7 4.9 16.5
1.3 7.3 8.8 2.8 11.8
5.5 30.5 24.7 8.5 23.1
Non Non Non Non Ex
2402 2471 2472 2511 2512
M F F F F
65 62 62 64 64
0.27 0.53 0.39 0.76 0.65
32 63 21 19 24
360 384 216 360 384
17.4 17.0 13.2 7.1 9.9
11.0 12.4 8.3 4.4 6.3
27.4 23.4 20.9 11.6 15.4
Ex Non Non Non Ex
21.0
2531 2532 2551 2552 2561
M M F F M
64 64 64 64 63
0.23 0.20 0.23 0.22 0.33
28 31 15 16 14
372 480 192 288 240
17.4 17.1 35.2 26.5 18.1
11.4 11.2 20.4 15.5 10.4
26.5 26.0 60.7 45.2 31.5
Cur Ex Cur Cur Ex
12.5 6.5 32.3 NA
2562 2711 2712 2821 2951
M M M F F
63 62 62 61 65
0.40 0.16 0.12 0.20 0.42
18 7 4 18 12
216 192 120 240 456
21.8 23.9 27.6 38.7 6.4
13.3 10.9 9.7 23.2 3.5
35.7 52.1 78.8 64.7 11.5
Ex Non Ex Non Non
2952 9991 9992
F F F
65 69 69
0.11 0.19 0.29
12 22 12
312 336 456
17.8 35.0 9.4
8.6 21.7 5.1
36.7 56.5 17.0
Cur Non Non
43.0 75.0 58.8 60.0
1.8
75.0 0.1 1.7 9.0 45.0
52.5 82.5 147.0
33.6
15.0
102.0
20.0
272
were also plated at 5 cells/well (one plate) and all of the experiments on women with benign breast masses were plated at 5 and 10 cells/well (one plate at each density). For the selection plates, cells were plated at 1 or 2 × 104 cells/well in the presence of 10 /zM 6-thioguanine (TG). Cells were in 200 izl of RPMI 1640 medium containing 20% HL-1, 5% BCS, 20% growth factor supplement, 0.125 p.g/ml PHA and 1 x 1 0 4 irradiated human lymphoblastoid accessory cells. The growth factor supplement was the cell-free supernatant medium obtained after generation of LAK cell activity by incubating patient peripheral blood mononuclear cells in medium containing 1500 U / m l recombinant interleukin-2 (O'Neill et al., 1987; Branda et al., 1992). Following a 10-14 day incubation of the microtiter dishes, growth was determined by use of an inverted phase contrast microscope. Cloning efficiencies (CE) were calculated by use of the Poisson relationship, P0 = e x, where P0 is the fraction of wells without colony growth. The CE equals - l n Po/x, where x = the average number of cells/well. The variance is calculated as ( 1 - P o ) / [ ( P o ) ( t o t a l
number of wells)(ln /90)2]. The mean CE for the non-selection plates was calculated by averaging the non-selection CEs plated at different cell densities. Nearly all experiments had CE values for cells plated at 1 and 2 cells/well but many had values for cells plated at 5 and 10 cells/well as well. All obtained values were used except those where all wells were positive for cell growth (In 0 is undefined). The variance for this mean is the sum of the variances for each CE divided by the number of variances squared i.e. E u , / n 2. The mutant frequency is the ratio of the CE in the presence (selected) and mean CE in the absence (unselected) of TG. Confidence limits were calculated as in Furth et al. (1981) except that the mean variance was used for the nonselection variance part of the equation. In other words, the 95% confidence limits are: MF× exp[_ 1.96 × ( t ' + T G q- tJ TG)I/2].
Statistical method Because the distribution of mutant frequency is skewed, analyses were based upon the natural
TABLE 2 HEALTH CARE WORKERS Donor
Sex
Age
-TG
+TG
MF
95% c o n f i d e n c e limit
Smoking
M e a n CE
Number of positive wells
Total n u m b e r of wells
( × 10-6)
Lower
Upper
status
Pack yrs.
A01 A04 A05 A07 A12
F F F F F
41 41 61 36 37
0.57 0.12 0.44 0.73 0.40
16 2 59 40 34
288 192 384 480 576
5.0 4.2 19.1 6.0 7.7
3.0 1.0 13.7 4.2 5.1
8.5 17.6 26.7 8.6 11.5
Non Non Non Non Cur
15.0
AI3 A15 AI6 AI7 A21
F F F F F
43 35 55 35 58
0.21 0.47 0.66 0.33 0.52
27 51 12 33 20
288 278 191 768 276
23.5 21.7 4.9 6.7 7.2
14.6 15.3 2.7 4.4 4.4
37.8 30.6 9.0 10.2 11.6
Non Non Non Cur Cur
20 - 4 0 15.0
A24 A27 A32 A33 A34
F F F F F
52 63 54 32 39
0.31 0.14 0.27 0.25 0.21
24 9 53 13 4
384 192 480 288 96
10.5 17.6 21.9 9.2 10.4
6.6 8.4 15.0 5.0 3.7
16.8 37.1 31.9 16.8 28.8
Non Cur Off/On Non Non
18.0 -
A35 A40
F F
44 46
0.39 0.26
25 24
576 384
5.7 12.4
3.6 7.0
9.0 22.1
Non Non
-
-
273 TABLE 3 W O M E N WITH B E N I G N BREAST MASSES Donor
Sex
Age
- TG
+ TG
Mean CE
Number of positive wells
Total number of wells
MF (>(10 -6 )
95% confidence limit Lower
Upper
Smoking status
Pack yrs.
0003-1 0008-1 0010-1 0011-1 0019-1
F F F F F
34 63 34 41 40
0.14 0.61 0.30 0.44 0.55
21 31 12 4 14
240 480 144 96 192
33.2 5.5 14.3 4.8 6.9
19.2 3.7 7.9 1.8 4.0
57.5 8.0 25.7 13.0 12.0
Ex Cur Non Non Non
3.5 51.0
0020-1 0026-1 0031-1 0033-1 0036-1
F F F F F
54 41 56 41 45
0.25 0.31 0.10 0.18 0.46
29 30 19 38 21
456 348 192 480 156
13.0 14.6 53.5 22.9 15.7
8.7 9.8 32.3 15.9 10.0
19.4 21.8 88.7 33.0 24.9
Cur Cur Ex Ex Non
13.5 NA 0.5 0.1
0038-1 0045-1 0051-1 0053-1 0054-1
F F F F F
38 40 50 41 35
0.36 0.49 0.44 0.17 0.15
76 14 39 10 19
612 132 480 240 384
18.5 11.5 9.6 12.7 16.8
13.9 6.6 6.7 6.6 10.2
24.7 19.8 13.6 24.3 27.8
Cur Cur Cur Non Non
1.5 12.0 17.0
0055-1 0056-1 0058-1 0059-1 0060-1
F F F F F
44 38 42 42 56
0.90 0.58 0.34 0.90 0.56
55 76 26 68 88
672 768 576 384 1056
4.7 8.9 6.8 10.8 7.8
3.4 6.7 4.5 8.0 6.0
6.5 11.8 10.3 14.6 10.2
Non Non Ex Cur Ex
13.0 15.0 30.0
0061-1 0063-1 0064-1 0065-1 0066-1
F F F F F
40 60 42 39 42
0.15 0.40 0.14 0.17 0.61
29 29 12 11 104
480 280 480 192 480
21.1 13.8 8.8 17.4 20.0
13.9 9.2 4.8 9.4 15.6
31.9 20.6 15.9 32.4 25.6
Non Ex Cur Ex Cur
3.5 20.0 0.1 42.0
0067-1 0068-1 0069-1 0072-1 0074-1
F F F F F
36 39 43 62 64
0.25 0.40 0.23 0.28 0.71
7 17 11 28 87
192 432 288 276 768
7.4 5.0 8.3 19.4 8.5
3.5 3.0 4.5 13.0 6.5
15.8 8.2 15.4 29.1 11.2
Ex Non Non Ex Ex
0079-1 0084-1 0085-1 0087-1 0091-1
F F F F F
39 22 52 65 39
0.45 0.30 0.19 0.27 0.40
28 102 13 116 38
384 576 264 864 336
8.4 32.1 13.4 26.3 7.6
5.6 25.3 7.5 20.5 5.3
12.7 40.9 24.0 33.7 10.9
Non Non Non Cur Ex
0092-1 0093-1 0094-1 0096-1 0098-1
F F F F F
55 45 44 39 53
0.32 0.44 0.15 0.22 0.27
63 150 24 22 15
480 864 672 480 120
22.0 21.6 12.4 10.5 25.1
16.1 17.1 7.9 6.7 14.8
29.9 27.3 19.5 16.6 42.5
Cur Non Non Ex Ex
0099-1 0100-1 0101-1 0104-1 0105-1 0106-1
F F F F F F
52 48 40 56 37 53
0.32 0.39 0.63 0.62 0.23 0.32
46 17 35 53 38 19
636 348 384 336 960 288
11.8 6.4 7.6 13.9 8.7 10.6
8.5 3.9 5.3 10.0 6.1 6.6
16.6 10.5 11.0 19.2 12.4 17.0
Non Ex Cur Cur Non Non
4.0
0.1 5.2
53.8 1.5 57.0
14.0 12.8
21.0 60.0
274 TABLE 3 (continued) Donor
Sex
Age
- TG
+ TG
Mean CE
Number of positive wells
Total number of wells
MF (×10
6)
95% confidence limit
Smoking
Lower
status
U ppe r
Pack yrs.
0110-1 11111- I 0113-1 0116-1
F F F F
44 38 37 41
0.43 0.54 0.51 0.75
30 53 24 29
960 960 624 576
3.7 5.3 3.8 3.4
2.5 3.8 2.5 2.3
5.5 7.2 5.9 5.2
Non Ex Ex Ex
10.0 12.11 -
0118-1 1/120-1 0125-1 11126-1 0128-1
F F F F F
54 35 42 40 43
0.68 0.42 0.34 0.63 0.45
65 4 11 28 40
384 252 288 1344 288
27.1 1.9 5.7 2.3 16.4
19.4 0.7 3.1 1.5 11.6
38.0 5.1 10.5 3.6 23.4
Non Cur Non Non Non
13.0 -
0129-1 0130-1 0132-1 11134-1 013%1
F F F F F
46 36 36 44 38
0.81 0.56 0.78 0.84 0.64
104 44 85 48 55
672 288 480 672 336
10.4 14.9 12.4 4.4 13.9
7.9 10.7 9.4 3.2 10.1
13.7 20.8 16.5 6.2 19.1
Non Ex Cur Ex Ex
1.0 31.5 < 4.11 20-40
0138-1 0139-1
F F
50 60
0.48 0.41
26 43
288 288
9.8 19.6
6.5 13.9
14.9 27.6
Non Ex
9.(1
logarithms of mutant frequencies (Henderson et al., 19861. Groups were compared using analysis of variance to assess differences in In MF, CE and age, and a Chi-square test was used to assess smoking status differences. Gender differences were analyzed by t-tests. Regression analyses were used to examine the relationships between In MF, CE and age and to assess the effect of smoking status on In MF, adjusting for age and CE. Results and discussion
The data obtained on the 4 groups of donors are shown in Tables 1-4. Each table presents information regarding the age, sex, mean unselected CE, number of wells containing TG-resistant colonies, total number of wells analyzed, MF, 95% confidence limits of the MF, and smoking history, expressed as pack-years. The data are presented in a format similar to that used in other recent reports (Henderson et al., 1986; Cole et al., 1988; Sala-Trepat et al., 1990; Tates et al., 1991; Davies et al., 1992) to facilitate the development of a large historical database on normal individuals.
The first group (Table 1) includes 132 participants in a study of identical and fraternal twins. The second group (Table 2) consists of 17 health care workers studied as part of an assessment of the risks of handling cancer chemotherapeutic agents. Some data derived from this second group of individuals have been published previously (A1bertini et al., 1988). The third group (Table 3) is composed of 62 women with benign breast masses who were enrolled in a study of environmental factors which modulate genetic damage caused by treatment for breast cancer (Branda et al., 1992). The diagnoses which brought them to medical attention were: fibrocystic changes (17), fibroadenoma (10), microcalcification (4), pigmented nevus (2), lipoma (2), mammographic density without pathologic diagnosis (2), acute and chronic mastitis (1), fatty benign breast tissue (1), abscess (1) and cellulitis (1). The last group (Table 4) contains 21 normal volunteers recruited from laboratory and office personnel. Some data obtained from these individuals have been published (Nicklas et al., 1987; O'Neill et al., 1987, 1989; Branda et al., 1992). A comparison between groups is shown in Table 5. Group 1 (healthy identical and fraternal
275
TABLE 4 NORMAL HEALTHY VOLUNTEERS
Donor
Sex
Age
+ TG
MF
95% confidence limit Smoking
Mean CE
Number of positive wells
Total number of wells
( × 10- 6)
Lower
- TG
Upper
status
Pack yrs.
VT1 VT2 VT3 VT4 VT5
M M F F F
19 21 22 25 34
0.28 0.37 0.30 0.39 0.15
19 22 19 35 5
460 480 672 1056 192
7.6 6.3 4.8 4.3 8.5
4.6 3.9 2.9 2.9 3.4
12.8 10.1 8.0 6.5 21.2
NA NA NA NA NA
-
VT6 VT7 VT8 VT9 VT10
M M M M F
37 29 22 27 25
0.10 0.26 0.11 0.49 0.70
40 8 12 28 45
864 180 672 288 927
24.8 8.8 8.3 10.5 3.5
14.8 4.3 4.2 6.9 2.5
41.8 18.0 16.4 16.1 5.0
NA NA NA NA NA
-
VTll VT12 VT13 VT14 VT15
M F M M M
23 43 27 38 26
0.36 0.41 0.39 0.19 0.35
13 52 33 9 7
817 384 480 467 480
2.2 17.7 9.3 5.2 2.1
1.2 12.9 6.1 2.5 1.0
4.1 24.3 14.0 10.7 4.6
NA NA NA NA NA
-
VT16 VT17 VT18 VT19 VT20
M M M M M
34 23 30 25 37
0.41 0.51 0.54 0.29 0.56
33 16 13 47 33
768 871 565 960 288
5.4 1.8 2.2 8.7 10.9
3.6 1.1 1.2 5.9 7.5
8.1 3.1 3.8 12.7 16.0
NA NA NA NA NA
-
VT21
M
22
0.50
14
464
3.1
1.7
5.4
NA
-
-
twins) was significantly older, and Group 4 (normal healthy volunteers) was significantly younger, than the other 3 groups. The In MF for Group 4 was significantly lower than the other 3 groups.
in Fig. 1 for the combined group of 232 donors and is expressed by this equation:
Age
with R 2 = 0.151, P < 0.001. Thus, MF increases by about 2% per year in our series.
The relationship between age and MF is shown
In MF = 1.46 + 0.018 age
TABLE 5
COMPARISON BETWEEN GROUPS 1-4 Group
N
Mean age
Mean In MF ( ± std. dev.)
( _+std. dev.) 0.37 + 0.18 0.385:0.18 0.43 + 0.21 0.365:0.16
1 2 3 4
132 17 62 21
61.7 ± 13.3 a 45.45:9.9 44.6 5 : 8 . 8 28.05:6.7 a
2:53 + 0.73 2.275:0.58 2.37 ± 0.66 1.77_+0.71
Total
232
52.9 5:16.0
2.40 ± 0.73
a Significantly different from other 3 groups at p < 0.05.
a
Mean CE
0.39 5:0.18
276
These results are similar to previously reported rates of 2.18% per year in normal adults by Cole and colleagues (1991) and 3% per year by Tates et al. (1991). The subjects included in the present report spanned the age range 19-80 years. Although we have studied and described elsewhere (McGinniss et al., 1989) assays in normal newborns, these data were not included in the present analysis. The cord-blood group differs from the others with respect to CE and MF, and all the ages in this group are zero. Cole et al. similarly reported a low MF in cord blood, which increased about 10-fold during early childhood (1991).
5.o1
4.5~" 4.o] 3.5,
3.oi
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o** . " •
mQ
o- o* . , ei
o
• ".o,Oo.
I
•
:
~
.
I.
"° L : "
,"
.,-,: - . - . . : . • ; ...
:,...,
2.0
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1.5
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•
- :,,: •..
•.•.,...-
.
*~e, ".;~.'
,** ,:
. •
".'.
•
, :
•
lO o51 oo
o51
i
. . . . . . . . . . .
0.0
0.1
0.2
.... 0.3
• .... 0.4
, . . . . . . . . 0.5
0.6
0,7
0.8
0.9
1.0
1.1
CE
Cloning efficiency The relationship between MF and CE for these 232 adults is shown in Fig. 2 and is described by the equation:
Fig. 2. Regression line showing the relationship between lymphocyte cloning efficiency and mutant frequency.
In MF = 2.91-1.32 CE
with R 2 = 0.019 and P = 0.038, indicating that CE was significantly inversely proportional to age in our subjects. This relationship supports the observations of Tates and colleagues (1991), who found also a significant decrease from one year to the next.
with R 2 = 0 . 1 0 9 and P < 0 . 0 0 1 . This result is comparable to the observation of Tates et al. (1991), who reported that doubling the CE reduced the MF by 37% in his series, and of Cole and coworkers (1991), who found a 40% reduction of MF when the CE doubled. The relationship between cloning efficiency and age is described by the equation:
CE = 0.47-0.002 age 4.6 4.0 • .3.6
," !
0
3.o~
. •
,.o 1,5
""
.
.
,o
•
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",
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jill
,,=''"
l
: "
: ~_- ~ ,~:,: : :~:.,.: ~ " " ."~ . -~' ~' ~' ~' ~' ~' ~' ~"' ~'. . = :' ". ' . : .
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Donor gender Males and females did not differ with respect to MF, but CE was significantly lower in males ( P < 0.001), even after adjustment for age differences. Previous studies which have included a consideration of donor gender have similarly found no significant effect on MF (Caggana et al., 1991; Cole et al., 1988; Davies et al., 1992; Vijayalaxmi and Evans, 1984; Tates et al., 1989).
: I
1,0
Interrelationships between mutant frequency, age and cloning efficiency The inter-relationship between mutant frequency, age and cloning efficiency in these 232 donors is expressed by the equation:
0,5
0.0
In MF = 1.99 - 1.13 CE + 0.016 age
-0.5
0.229 and P < 0.001. Thus, MF values increase with increasing age and with decreasing CE. This relationship is portrayed graphically in Fig. 3.
with R 2 = Fig. 1. Regression line indicating the effect of donor age on the mutant frequency at the hprt locus in 232 healthy individ uals.
277 5.0
j
4,5 4.0
3,5 ]
•
I
O'o• ..-... _c
-.
,
#.:,*
•
g. • ¢,~ y'.~:
a.oJ
2.01
~ . ~ . . ,~
"o t
. .:.~'~.:. . . ,~.,~ " .
1.61
o°oO
1,0i
•
0.6
•
°.%°
.
•
-.o" ..
." .•
•
,* •
•
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
In M F = 1.99 - 1.13 C E + 0.016 A G E
Fig. 3. Regression line indicating the interrelationship between mutant frequency, cloning efficiency and age in 232 donors. The dotted lines represent 95% confidence limits.
Effect of smoking cigarettes The effect of smoking on MF was assessed by including current smoking status (smoker, nonsmoker) in the regression analysis in addition to CE and age. 22 subjects for whom no smoking information was available were excluded from this analysis. The following equation was obtained: In MF = 2.29 - 1.16 CE + 0.012 age - 0.09 s where s = 0 for non-smokers and s = 1 for smokers. By this analysis, the effect of smoking was not statistically significant ( P = 0.413). Other analyses found no significant relationship between In MF and number of years smoked for current smokers or pack-years. Our results differed from other recent analyses of mutant frequency at the hprt locus. For example, Tates and colleagues (1991) reported that smoking had a significant effect on the In MF, with P = 0.025, and that smoking increased the MF by 36% for any combination of CE and age. Similarly, Cole et al. (1991) found that smoking caused a highly significant increase in MF whether or not CE was considered. However, these workers found that the smoking effect was age-dependent, and that the increase in MF per year for smokers was 2.9% compared to 0.8% for non-smokers (Cole et al., 1990). Smoking significantly affected the MF
in the series of Ammenheuser et al. (1988) and in the control group reported by Caggana and colleagues (1991). In contrast smoking habits had no effect on MF in the healthy donors studied by Davies et al. (1992) and was associated with lower MFs than in non-smokers in the report of SalaTrepat (1990). The basis for this diverse effect of smoking history on MF in various laboratories is obscure at present. In conclusion, our investigations with the Tlymphocyte clonal assay confirm that somatic cell mutant frequency varies widely in normal individuals. Most laboratories experienced with this technique agree that donor age and lymphocyte CE are important factors which influence MF and contribute to this variability. The effect of cigarette smoking history has been less consistent. Many authors report that MF tends to be higher in smokers, but this elevation is not always statistically significant, as was the case in the present study. Recently, we found that serum levels of a micronutrient, folic acid, may modify genetic damage at the hprt locus, and that folic acid deficiency was associated with higher mutant frequencies following exposure to chemotherapy (Branda et al., 1991, 1992). These latter studies suggest that dietary components also may be important contributing factors to the variability observed in the MF. Finally, it is likely that differences in DNA-repair efficiencies will also affect mutant frequency values (Tates et al., 1989; Cole et al., 1991).
Acknowledgements Supported by grants from the National Cancer Institute (CA 41843, CA 30688 and P30 CA22435), the National Institute of Health (AG06886) and by EPA Cooperative Agreement No. CR-812837. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. This article has not been subjected to U.S. Environmental Protection Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
278
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