Spontaneous and mitomycin-C-induced micronuclei in human lymphocytes exposed to extremely low frequency pulsed magnetic fields

Fundamental and Molecular Mechanisms of Mutagenesis

ELSEVIER

Mutation Research 357 (1996) 183-190

Spontaneous and mitomycin-C-induced micronuclei in lymphocytes from subjects affected by Turner’s syndrome M.R. Scarf? a**, F. Prisco b, F. Bersani ‘, M.B. Lioi d, 0. Zeni d, R. Di Pietro a, C. Franceschi e, M. Motta ‘, D. Iafusco b, G. Stoppoloni by’ ’ CNR-IRECE. b Serrkio

’ Dipartimento ’ Dipartimmto ’ Dipartimento

Via Diockiano.

di Diabetologia

Pediatrica,

di Fisica,

Unilvrsitir

di Science delle Produzioni

di Science Biomediche.

Received 3 November

328-80124, II Unicersit~

Nap&. di Napoli.

di Bologna,

Animali.

Unicersitir

Sezione di Patologia

Generale.

Italy Napoli,

Bologna,

della Basilicata, Unioersith

1995; revised 19 April 1996: accepted

Ital)

Italy Potenza. Italy

di Modena,

Modena,

Italy

15 May 1996

Abstract Peripheral blood lymphocytes from 15 subjects affected by Turner’s syndrome (TS) and aged between 2 and 24 years (mean age 10.40 k 6.25) were tested to evaluate the spontaneous and Mitomycin-C-induced (MMC) micronucleus (MN) frequency. A group of 15 healthy subjects, in the same range of age (mean age 14.67 + 8.30), was also tested as control. As expected, statistically significant differences between spontaneous and MMC-induced MN were found either in TS and in healthy subjects. Unexpectedly, when the two groups of donors were compared, TS subjects showed a lower spontaneous and MMC-induced MN frequency, in comparison with healthy subjects. Cell proliferation kinetic and cytotoxicity were also measured applying the cytokinesis-block proliferation index (CBPI): the results show that MMC, at the employed concentration, does not induce cell cycle delay both in healthy and in TS donors. Whereas, when CBPI from TS and healthy donors were compared, a faster proliferation was found in TS patients in both untreated and MMC-treated cultures. Kewordst

Turner’s

syndrome;

Micronuclei;

Mitomycin-C;

Human lymphocyte

1. Introduction Since the introduction of the cytokinesis-block micronucleus (MN) method by Fenech and Morley in 1985, a variety of situations was tested applying this cytogenetic assay in order to evaluate the effects

* Corresponding author; 5705734. ’ Professor G. Stoppoloni

39-81-5707999:

died on 6th June, 1995.

Copyright SOO27-5107(96)00100-5

0027-5 107/96/$15.00 PIf

Tel.:

Fax:

39-81.

of many physical and chemical mutagens on one side and the spontaneous genomic instability on the other. In the last years cells from a number of different human subpopulations characterized by specific genetic anomalies, pathological conditions or by other situations, such as smoking (Larramendy and Knuutila, 199 1; Tomanin et al., 1991) or alcohol consumption (Da Cruz et al., 1994) and aging (Fenech and Morley, 1986; Fenech, 1993), were analysed. Indeed, in previous works we tested aged subjects (Franceschi et al., 1992) and subjects affected by a

0 1996 Elsevier Science B.V. All rights reserved

syndrome of accelerated aging. such as Down patients. and we found a difference in spontaneous MN frequency, as well as an increase in mitomycin-C (MMC) induced MN. which was age-dependent (Scarf? et al.. 1990). However, in other pathological conditions we recently investigated. such as diabetes. negative results concerning spontaneous and MMCinduced MN frequency were found (Scarfi et al.. manuscript in preparation). In this study we have investigated the MN frequency and cell proliferation kinetics in untreated and MMC-treated lymphocyte cultures from subjects affected by Turner’s syndrome (TS) compared to healthy donors. Cytokinesis-block micronucleus assay was employed to evaluate MN frequency (Fenech and Morley. 1985). while cytokinesis-block proliferation index was used to evaluate cell kinetics (Surralles et al.. 199Sa.b). TS consists of bilateral gonadal dysgenesis with streak gonads, female phenotype. sexual infantilism and a pattern of distinctive somatic stigmata. In most of patients the disorder is specifically associated with 45X0 chromosomes. The incidence of gonadal dysgenesis varies in new-borns from I per 2500 to 1 per 10000 phenotypic females (Hook and Hamertton, 1977; Robinson. 1990). The most consistent features for people bearing this syndrome are short stature and gonadal dysgenesis. A smaller number of individuals has abnormalities of the heart, especially coarctation of the aorta, and renal abnormalities such as horseshoe kidney. The interest of this study lies in the fact that TS is apparently a very complex genetic disorder that nevertheless allows the subjects to live an apparently normal life. However. TS. together with the other two more frequent chromosome aneuploidies. i.e.. Down’s syndrome and Klinefelter’s syndrome, is considered one of those genetic syndromes in humans that are high-ranking candidates for categorisation as ‘segmental progeroids syndromes’. i.e.. syndromes which show the highest number of concordances with the major phenotype criteria of aging (Martin, 1978). Moreover. an increased frequency of tumours and a number of immunological abnormalities are present in subjects affected by TS (Cacciari et al.. 1981). These data would suggest the presence in TS subjects of genetic instability and/or alterations of DNA repair capability. However. no data

I Age. therapy and karyotype of the subjects affected by Turner‘s

Table

hyndrome Subject

A&years

)

Therapy

II

GH GH GH GH

II I3 I3 18 I8 21

GH GH + EST EST EST EST

h IO I0

GH, growth hormone;

Karyotype 35x0 15X0/16xX 45x0 ISXO/-rSXY 46XiCXq) 45x0 46XiCXq)isoX 45X/46Xx1, 45x0 45x0 45X0/46Xx 15x0 36Xi(Xq) 45x0 45x0

EST. estrogena.

are available on this topic, except those of Herds and Coca (1986) suggesting a normal sensitivity to X-rays of lymphocytes from TS patients and our preliminary data on an increased sensitivity to low frequency electromagnetic fields. which are unable to increase MN frequency in lymphocytes from normal subjects (manuscript in preparation). Thus, we thought it worthwhile to measure spontaneous and MMC-induced MN in TS lymphocytes even if, as far as we know, no reports on genomic instability in this syndrome exist in the literature.

2. Material and methods

Peripheral blood lymphocyte cultures from I.5 TS subjects aged between 2 and 24 years (mean age: 10.40 & 6.25) were tested. All patients were in good health: some of them were treated with growth hormone and/or estrogens. In Table 1 age, therapy and karyotype for all of them are reported. A group of IS healthy donors, in the same range of age (mean age: 14.67 + 8.30). was also tested as control. For each subject an untreated and a MMC-treated culture was set up.

M.R. Scarfi et al./Mutation

2.2. Cell cultures and micronucleus

test

Venous blood samples were collected by venipuncture into heparinized tubes. Whole blood lymphocyte cultures were established in Falcon plastic flasks by mixing 0.8 ml whole blood with 9.2 ml RPM1 1640 medium (Gibco, New York, USA) containing 15% heat-inactivated fetal calf serum (FCS, Gibco), 2 mM L-glutamine (Gibco) and 100 pl Table 2 Spontaneous Donor (healthy control)

and MMC-induced Age (years)

1

I

2

4

3

5

4

9

5

11

6

11

I

12

8

12

9

13

10

22

II

23

12

23

13

24

14

24

15

26

Mean k SD

MN frequency

and citotoxicity

Research 357 (1996) 183-190

185

phytohemagglutinin (PHA-M form, Gibco). Cultures were maintained at 37°C for 72 h in a 5% CO, incubator. 44 h after PHA-stimulation, cytochalasinB (Sigma, St. Louis, MO, USA, 2 mg/ml dissolved in dimethyl sulfoxide) was added to give a final concentration of 3 kg/ml, according to Fenech and Morley (1985). 72 h after culture initiation cells were harvested and slides were made up, fixed and stained as previously described (Scat-t? et al., 1991).

(CBPI) in lymphocytes

from healthy subjects

Treatment

MN frequency

Distribution of cells according to No. nuclei/500 cells 1

2

3

4

control MMC control MMC

1.62 3.35 1.02 2.28 1.01 I .87 1.28 2.37 1.16 2.11 0.46

437 450 193 182 205 233 393 297 433 419 369 375 319 311 323 346 323 326 178 258 144 193 288 289 163 174 352 217 206 208

59 48 215 196 204 179 82 158 60 71 115 116 145 144 151 135 166 161 217 187 225 212 156 159 210 210 126 182 207 219

1 0 38 56 41 45 10 21 2 1 4 5 20 22 11 10 5 7 38 21 55 23 23 22 44 43 11 20 38 35

3 2 54 66 50 43 15 24 5 9 12 4 16 23 15 9 6 6 67 24 76 72 33 30 83 73 11 21 49 38

MMC control MMC control MMC control MMC control MMC control MMC

(928) (1044) (2065) (965) (1972) (2833) (1405) (1183) (2235) (1234) (1538) 1.27 (1256) 0.89 (1889) 2.93 (1125) 1.00 (1004) 2.02 (1088) 0.87 (1490) 2.28 (1096) 1.00 (4616) 3.37 (2460) 0.74 (2716) 2.65 (2675) 0.86 (2561) 2.35 (894) 1.08(2975) 2.57 (2376) 0.32 (2720) 2.52 (3649) 1.01 (1786) 2.3 I (3243)

14.61

control

I a2 f 0.27

* 8.30

MMC

2.42 + 0.54 * ’

MMC MMC MMC control MMC control MMC MMC control MMC

* Numbers in brakets refer to the total number of CB cells scored. ’ * Significantly different in comparison with spontaneous MN frequency, and Wilcoxon test (P < 0.02).

CBPI

1.134 1.104 1.798 1.880 1.772 1.710 1.264 1.496 1.148 1.182 1.294 1.268 1.434 1.468 1.406 1.346 1.376 1.374 1.854 1.534 1.974 1.804 1.536 1.526 I.928 1.884 I .340 1.528 I .762 1.730 1.53 + 0.29 to.29 1.52 f 0.24

as assessed by two tailed paired Student’s

t-test (f’ < 0.0005)

2.3. Cell scoring and index calculatiotts FOJ each culture MN in a minimum of 800 CB cells were scored but, in many instances, more than 1000 CB cells were observed with a light microscope at 1000 X magnification. Cells were classified following standard criteria summarized by Country-

man and Heddle (1976) and MN frequency was evaluated as the ratio between cytokinesis-blocked (CB) cells presenting MN and the total number of CB cells scored, expressed as percentage. Cytotoxicity and cell proliferation kinetic were measured on the same slides by scoring the number of nuclei in 500 cells and determining the cytokine-

Table 3

Spontaneousand MMC-induced Donor (TS subjects)

kc (years)

I 7 3 _I 5

MN frequency and cilotoxicity (CBPI) Treatment

MN Frequency

in lymphocyte< from TS Distribution

subjects

of cells according

I&) No. nuclei/500

CBPI

cells

control

0.75

I09

5.1

53

I.992

MMC

I.55 (lOi?)

I67

26

I .76J

control

0.36 (212)

203

79

23 16

MMC

0.w

207

38

61

I.784

control

0.63 (3992)

2x0

6

28

I .50X

MMC

I .‘3

231

20

36

I.630

control

0.87

277

3’

50

I.710

MMC

l.ZY

(1516)

22X

3x

52

I.701

conlrol

0.7x

(X09)

Z-II

10

32

I .6-l?

MMC

I.‘)5

(71X)

21x

32

I6

I ,600

( 1736)

( I 12-1) (275-I) (C)76)

I .74-l

control

I.15 (2266)

273

70

IS

I.511

MMC

7.37 ( 1266)

192

11

IO

I .16-l

control

0.73

(X17)

I 67

44

53

I .X60

MMC

I.64

(‘)I11

223

7X

ix

I .6X6

control

0.7x (10721

I95

17

13

1.790

MMC

I .54 ( 14’9)

212

21

37

I.618

control

0.w

( 1277)

317

6

I2

I.102

MMC

_._. “5

(137X)

266

IX

I6

I.536

control

0.55 ( 1080)

I96

28

35

I.734

MMC

2.02 (1086)

215

34

39

I.716

II

control

0.89 ( I 127)

16X

43

60

I .x70

MMC

2.13

(93X)

168

35

71

I .8Y6

I2

control

0.56 ( 1239)

309

27

45

1.7’6

MMC

I .X6 ( Iil‘L)

2-H

‘8

I9

I .59X

control

0.58 (1212)

206

15

51

I.730

MMC

I.(>3 (1177)

239

31

53

I.710

control

0.X0 (13%)

1’):

3X

39

I.770

MMC

I.38

(796)

194

30

37

I .766

control

(1.X’) ( 1800)

207

31

?Y

1.706

MM’Z

1.x3 (I?OJ)

256

I7

I7

I.556

control

0.7 I i 0.23

MMC

I .71 & 0.3’~

6 7 x 9 I0

13 I4 I5

mean f SD

I.71 iO.15 I .67 i-o.1 I

I Numbers in hraket> refer to the total number of CB cells scored. I * Significantly

different in comparison with spontaneous MN frequency. as awessed by two railed paired Student’s t-test (P < 0.0005) and

Wilcoxon test (P < 0.02).

M.R. %a$

et d./

Mutation

sis block proliferation index (CBPI). It indicates the average number of cell cycles per cell and, according to Surralles et al. (1995a,b), is defined as follows: CBPI=

[(M,

+2M?+3(Mj+Mq)]/N

where M, to M, represent the number of cells with 1 to 4 nuclei, respectively, and N the total number of cells scored.

2.4. Mitomyciil-C

treatment

A dose-response curve of MMC was established in our laboratory, taking into account previous data from other authors, in order to obtain an adequate number of MN without affecting cell growth (data not shown). 0.033 pg/ml of MMC (Sigma), sampled from a solution freshly prepared in sterile distilled water, was added at the start of the culture and left in the medium throughout the whole culture period.

2.5. Statistical analysis To compare spontaneous and MMC-induced MN frequency in each subject, the Z-test for proportions was applied (Glantz, 1987; Scarf? et al., 1991, 1993, 19941, where the proportion here considered was just MN frequency. In order to compare the global difference between spontaneous and MMC-induced MN for each group of subjects (healthy people or TS subjects) the twotailed paired Student’s t-test was used. As a further control, a non-parametric test, i.e., the Wilcoxon test for rank comparison was used. To assess whether spontaneous and MMC-induced MN formation was affected by TS in comparison to healthy people, the Student’s t-test and the Mann-Whitney non-parametric U-test were applied. Similar results were obtained by using other statistical tests, including a two-way analysis of variance with interaction and multiple range analysis taking as variation sources the MMC treatment and the health status (data not reported). CBPI was compared both for each health status and for each treatment (untreated/MMC-treated) by means of the two-tailed paired Student’s t-test.

Research 357 (1996) 183-190

187

3. Results In Tables 2 and 3, the MN frequency and CBPI in untreated and MMC-treated cultures from healthy and TS subjects, respectively, are reported. In healthy subjects a MMC-induced MN frequency increase was found in comparison with untreated cultures (Table 2), as expected (mean frequencies 2.42 + 0.54 and 1.02 k 0.24, respectively). The statistical analysis was performed applying the two tailed paired Student’s t-test (p < 0.0005) and the Wilcoxon test ( p < 0.02). These results agree with our previously reported findings (Scarfi et al., 1990, 1991, 1993) and with those of other authors (Fenech and Morley, 1986; Surralles et al., 1995a). As can be seen in Table 3, a statistically significant difference between untreated and MMC-treated cultures was also found in lymphocytes from TS donors, the mean frequency being 0.71 + 0.23 and 1.72 + 0.39, respectively (t-test: p < 0.0005; Wilcoxon test: p < 0.021. From the comparison between the two groups of donors. it comes out that MN frequency in TS subjects is lower than in healthy ones for both treatments: the mean frequency was 0.71 & 0.23 and 1.02 & 0.27 for untreated cultures and 1.72 & 0.39 and 2.42 + 0.54 for MMC-treated ones. These data were statistically analyzed applying the Student’s t-test and the Mann-Whitney non-parametric U-test ( p < 0.02). The results mentioned above are also depicted in Fig. 1. Moreover, in Table 4 we report Z test for proportions in order to compare MN frequency in control and MMC-treated cultures in each subject: a significant difference ( p < 0.05) was found in 13 out of 15 healthy donors and in 10 out of 15 TS subjects. Cell kinetics. in untreated and MMC-treated samples, was evaluated by means of CBPI. The results reported in Tables 2 and 3 show that MMC-treatment, at the chosen concentration, does not affect cell proliferation neither in healthy subjects (1.53 f 0.29 and 1.52 * 0.24) nor in TS patients (1.7 1 rt_0.15 and 1.67 + 0.1 1). Statistical analysis was performed applying two tailed paired Student’s t-test Cp > 0.05). On the other hand, by comparing CBPI between the two groups of subjects, it comes out that cell

188 2.6

Finally. different karyotypes and growth hormone and/or estrogens administration gave no apparent effect on spontaneous and MMC-induced MN frequency and CBPI. even if more data for each subgroup are required to perform a statistical analysis.

2

\

I I

1.8

1.6 I

T1

/

’

(

4. Discussion

I

1.4 I 1.2

1

i

1

0 1‘-. -..__

0.8

L ~._

0.6

0

Health status: O=normal, I=Turner Fig.

I.

Spontaneous and mitomycin-C-induced

micronucleus t’re-

quency in healthy subjects and in TS patients. Ah~cksa: status. 0.

normal:

MMC-induced

I.

Turner.

Ordinate:

health

spontaneous (0)

and

(1) MN frequency (average f SE).

proliferation results higher in TS patients in both untreated and MMC-treated cultures (t-test: 17< 0.05). Table 3 Z test for proportionh Healthy donors Donor

Turner’s sydrome donors

2

I’

Z

I’

I

2.296

0.022

Donor

I.805

0.07

2

2.562

0.010 0.023 0.052

2.090

0.037

I

3

2.379

4

I.941

5 6

2.059

0.039

I .843

0.065

2.136

0.031

1.63X

0.00X

7

4.103

0.000

I.508

0.131

8

1.720

0.085

1.93 I

0.003

I

2.116

0.0 I6

I.102

0.270

9

2.78

0.005

2.518

O.Ol?

10

7.002

0.000

2.844

0.004

II

5.333

0.000

2.160

0.03 I

IZ

3.313

0.000

2.809

0.005

I3

4.035

0.000

2.35 I

0.019

I4

3.287

0.00 I

I.046

0.296

IS

3.175

0.002

7.155

0.03 I

In the present study we have collected data on MN frequency, either spontaneous and induced by a potent genotoxic agent, such as MMC, on peripheral blood lymphocyte cultures from a group of 15 subjects aged between 2 and 24 years and affected by Turner’s syndrome. Cell proliferation was also measured by means of CBPI. The data were compared with those obtained from a control group of 15 healthy subjects in the same range of age. The results indicate that the two groups of subjects show a similar response to MMC in terms of MN frequency: in addition, the employed MMC concentration (0.033 kg/ml) does not affect cell proliferation, as assessed by CBPI. This last result agrees with those reported by other authors on healthy subjects (Fenech and Morley. 1985) who evaluated cell kinetics using a different technique. that is the autoradiographic method. Unexpectedly, when a comparison between the groups of donors were made. statistically different results were obtained both for spontaneous and MMC-induced responses with the two employed tests. Indeed, TS subjects showed a lower MN frequency. suggesting a greater genetic stability. This phenomenon can not be simply ascribed to an excess of damaged cells dying in excess as the proliferation index (CBPI) is higher in TS subjects than in healthy donors. Our data are in apparent contrast with those reported by Heras and Coca (1986) who showed that the sensitiveness of lymphocytes from TS patients to X-rays. assessed by the chromosomal aberration test. was the same as in the control healthy subjects. These authors reported that the spontaneous frequency in TS subjects was not significantly different from that of normal subjects. The apparent discrepancy between our data and those of Heras and Coca

M.R. Scarf et al/Mutation

can be due to the different test applied. Indeed, the two tests have different sensitivities and measure different cytogenetic endpoints. The MN assay was proved to be a simple and very sensitive technique, more sensitive than the chromosome aberration test with respect to the cytogenetic effects of ionizing radiation (Fenech and Morley, 1986). The unexpected genomic stability and higher proliferation in TS patients could be due to cytochalasin-B (Cyt-B) dose used: this concentration, 3 Kg/ml, was first recommended by Fenech and Morley (1985) and then widely utilized by many authors to block cytokinesis in human blood lymphocytes. Recently, other authors suggested to use a Cyt-B concentration of 6 kg/ml since 3 pg/ml can be insufficient in blocking cytokinesis, yielding an underestimation of cell cycle (CBPJ) and, therefore, an overestimation of MN frequency, as well explained by Surralles et al. (1994). It comes out that the use of Cyt-B 6 p,g/ml is probably more suitable, especially when groups of donors with supposed different cell proliferation and MN frequency are compared. Another possible important variable is sex. In a recent work, Fenech et al. (1994) showed that in the range of age 2 l-90 years the spontaneous MN frequency is higher in the females than in males. Unfortunately, the range of ages analysed by Fenech et al. is different from ours (l-26 years), so that a direct comparison cannot be made, even if the mean of our spontaneous MN frequency is of the order of that of the female group of Fenech et al. in the range of 21-30 years. On the other hand our control group was composed by 6 males and 9 females in which the difference in MN frequency between the two sexes was not significant. This lower frequency of spontaneous MN is not unprecedented, as we reported a similar phenomenon in subjects affected by another major chromosomal aneuploidy, i.e., Down’s syndrome (Scat-B et al., 1990). The biological significance of this phenomenon is unclear, and it could be related to unspecific and unknown effects of aneuploidy. However, at variance with Down syndrome, lymphocytes from TS show a lower frequency also of MMC-induced MN. This lower sensitivity to a classical genotoxic agent, such as MMC, implies a consistent genomic stability and is even more unexpected taking into account that lymphocytes from TS seems to be more

Research 357 (1996) 183-190

189

sensitive to extremely low frequencies electromagnetic fields as far MN induction is concerned (manuscript in preparation). Although the results of the present work are not directly explainable, they are interesting, also in consideration that TS subjects are more prone than normal subjects to tumor risk, mainly gonadal tumours (Lobaccaro et al., 1994; Pierga et al., 1994), but also extragonadal (Pawliger et al., 1970; Mulvihill, 1975).

Acknowledgements This work was partially supported by CNRIRECE, grants to M.R.S. and by Regione Emilia e Romagna, Progetto Sanitario Finalizzato to F.B. and 40% MURST to F.B. and C.F.

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