Cytogenetic analysis of chromosomal aberrations of peripheral lymphocytes in workers occupationally exposed to nickel

Ahtttrtiotl Rcsc~ctrch.279 ( 1902) I I I - I7Y ‘~1 lY92 Elsrvier

MUTGEN

171

Science Publishers B.V. All rights reserved OI~~-I~IX/O~/S~LS.IIII

01763

Cytogenetic analysis of chromosomal aberrations of peripheral lymphocytes in workers occupationally exposed to nickel V&Aav Senft ‘, FrantiSek LoSan ’ and Milan TuEek ’

(Received 7 November

1990)

(Revision received Y August 1991) (Accepted X November

Keywords: Chromosomal

aberrations of peripheral

1YYI

1

lymphocytes: Nickel exposure; Nickel in urine. serum and hair

Summary The authors carried out a cytogenetic examination of chromosomal aberrations of peripheral lymphocytes (100 cells evaluated in each sample) with simultaneous monitoring of the level of exposure by means of determination of nickel in the urine, serum and hair. The series included 21 workers occupationally exposed to nickel at two workshops producing NiO (6 persons) and NiSO, ( 15 persons) in a chemical plant. At the same time a comparable control group, i.e., 19 worker? of the same chemical plant but without any direct occupational nickel exposure (clerks, service men, etc.), were examined in the same way. In the exposed group chromosomal aberrations of peripheral lymphocytes were detected with an average value of 6.41 + 1.9% (range 2-14%); in the group producing NiO it was, on the average. 9.5 +_3.2% (range 7-14%) whereas in the NiSO, production workers it was only 5.2 + 1.9% (range 2-10%). There was a dependence of chromosomal aberrations of peripheral lymphocytes on the exposure time and on the nickel content of the I-iological material. Significantly increased values (in contrast to the normal value of chromosomal aberrations of peripheral lymphocytes, up to 2%) were detected in the control group as well (average value of 4.05 + 2.270/n,range l-10%,). The authors explain this fact by the nickel-polluted environment of the whole observed chemical plant.

The widespread pounds in various severe health risk cko, 1983; Bencko

Correspondence: Medicine,

Dr.

University

Plzeh (Czechoslovakia).

use of nickel and nickel comindustrial spheres represents a for the exposed workers (Benet al., 1986). It has been known

V.

Senft,

Hospital,

Alej

Clinic

of

Occupational

Svohody 80, CS-32318

for a long time that the metal nickel and its soluble compounds caused skin allergies (KluCik, 1969; Spruit and Bongarts, 1977). Moreover, the insoluble compounds are considered to be carcinogenic (IARC, 1976; Sunderman et al., 1981). Also the possible genotoxic effect of various nickel compounds demonstrated in many experiments (ChorvatoviEov& 1983; Newman et al., 1982; Nishimura and Umcda, 1979; Umeda and

~i~~imu~~.1979)is of great significance.

However, the relatively small number of studies of occupational exposure of people does not allow the definite conclusion that nickel is mutagenic at human exposure levels as well (Leonard et al., 1981). In our clinic we have been observing, for several years, a group of workers occupationally exposed to nickel in the production of various nickel compounds in a chemical plant and we have been investigating possible negative influences upon the health of the exposed persons (Senft et al., 1986, 199Oa,b, 1991). This report presents the results of cytogenetic analysis of chromosomal aberrations of peripheral lymphocytes in this group of people occupationally exposed to nickel. Material and methods Description of the workshops The production of the two most important products of the observed chemical plant has been chosen for observation: NiSO, (CAS No. lOlOl97-O) and NiO (CAS No. 1313-99-1). NiSO, production is a rather complicated technology including many production procedures (solution of the initial raw material, removal of undesired ingredients, thickening, filtration, c~stallization, centri~gation, product adjustment). Inhalation exposure of the workers, above all, is concerned, i.e., inhaling of fine aerosol drops during centrifugation. The technology of NiO production has several stages, the most significant being the grinding of NiO in a ball-shaped mill and the drying of the product in a hot air stream under a propanebutane burner. Exposure is mainly by inhalation, i.e., the workers inhale particles of NiO dust. The character of work is similar at both workshops, easy ph~i~l work. l-he group observed Exposed workers_ Altogether 21 occupationally exposed workers were examined (15 from NiSO, production and 6 from NiO production). Men and women were represented equally, the mean age was 41.7 years, range 20-57 years. The

minimum period of employment with exposure was 6 months, the maximum was 20 years. There were 7 smokers in the group (on average 10 cig.jday), the others were non-smokers. Cuntrol group. This group consisted of 19 workers of this chemical plant without occupational nickel exposure (clerks, service men, etc.) matched for sex, age and smoking status. The normal, spontaneous chromosomal levels of aberrations in Czechoslovakia in persons not occupationally exposed to nickel were considered and evaluated, in accordance with the literature (Svandovj. and Riissner, 1989) as well as our own long-term experience, to range up to 2%. None of the exposed or control persons had been X-rayed during at least 3 months before the blood collection. Only one exposed worker and two control persons had had influenza during the month before the collection. They were not excluded from the observation as the chromosomal aberration values found in these persons did not exceed normal values. No-one in either the exposed or the control group consumed significant quantities of alcohol. Sample co~~e&tion 5 ml of blood was collected from the exposed and control persons into sterile heparinized test tubes for ~togenetic analysis of chromosomal aberrations of peripheral lymphocytes. Urine, serum, and hair samples for the determination of nickel concentrations were also collected. The samples of urine and serum were collected into bottles washed out with diluted HNO, and rinsed out properly with deionized water. Whole hairs were collected (0.2-0.3 g) and put into plastic bags. Sample analysis Cytogenetic examination of chromosomal aberrations of peripheral lymphocytes was carried out using standard methods, 100 cells were evaluated in each sample (Riissner and Cern& 1988). Nickel in the biological materia1 was determined using the method of atomic absorption spectrophotometry (AAS) (Senft et al., 199Ob). In case of hair a standard washing procedure was used before the analysis (Rjabuchin, 1978). The

173

nickel concentration in the air (air samples were collected in the respiratory zone of the workers) was determined according to Seflovi and Sponar (1983) using the AAS method as well. Evaluation of the results

For interpolating the dependences (curves presented in Figs. l-4), the authors used a computer working on the basis of the method of multiplex regression (model Y = aXb). Besides, in Figs. 2-4 straight lines were interpolated with the model Y = a + bX. The statistical significance of the differences among mean values of chromosomal aberrations of peripheral lymphocytes in the exposed group, the control group and the normal values was evaluated by means of the bilateral t-test. Results

In the N&O, workshop the mean nickel concentration in the air was 1.3 mg/m” (range 0.312.86). In the NiO workshop it was 0.77 mg/m” (range 0.28-1.52). In the work places of the matched controls, the nickel concentration in the air was not measured for technical reasons. Other results are presented in the form of tables and graphs. Table 1 presents the raw data, i.e., age, sex, smoking status, chromosomal aberrations of peripheral lymphocytes, nickel in the urine, serum and hair, observed in the workers from the exposed group. Table 2 gives the raw data of persons from the control group. Table 3 presents the nickel concentrations in the urine, serum, and hair in the exposed and control groups, as well as normal values presented in the literature. Table 4 presents chromosomal aberrations in the exposed workers and in the control group. Table 5 presents the occurrence of aberrant cells in peripheral lymphocytes in the exposed and the control groups. Fig. 1 presents the dependence of the number of chromosomal aberrations on the period of employment in the plant. Persons from the control group, from NiO production, and from NiSO, production are distinguished, and the curves corresponding to the character of the dependence in individual groups are interpolated. Figs. 2, 3 and 4 present in a similar way the dependences of chromosomal aberrations on the

nickel concentration in the serum, urine and hair in the exposed persons. There was a statistically significant increase (P < 0.001) in the mean value of chromosomal aberrations in the exposed group compared not only with the normal value but also with the control group. Similarly significant (P < 0.01) was the increase in the control group compared with the normal value. Discussion

Evaluating the nickel values measured in the atmosphere, the maximal permissible concentration (in Czechoslovakia 0.05 mg/m”) is significantly exceeded. High values of nickel in the biological material of the exposed group exceeding the normal value correspond to this significant exposure (Senft et al., 1991). In the work places of persons from the control group the nickel concentration was not measured, for technical reasons, but the concentration in the urine, serum and especially in the hair (Table 3) is significantly increased, over normal values, though not so much as in the exposed group. Some studies (Chorvatovieov& 1983; Nishimura and Umeda, 1979; Waksvik and Boysen, 1982; Waksvik et al., 1984) have shown an increase in chromosomal aberrations during niche1 exposure. Hence the increased chromosomal aberrations in the occupationally exposed group (on average 6.41%) did not surprise us. On the other hand, we were taken aback by the increased level (on average 4.05%) in the control group compared to the normal value, i.e., up to 2% (Svandovh and Riissner, 1989). That is why we directed our attention to the values in more detail and compared them with other known facts, i.e., with the work anamnesis above all (production of soluble or insoluble nickel compounds, duration of exposure or employment in the chemical plant), and nickel contents in the biological material, which can be considered an indicator of exposure level. In Fig. 1 there are interesting relations between chromosomal aberrations and the exposure period (in the exposed group) or the employment period (in the control group). The exposure to NiO was rather significant. Chromosomal aberra-

Sex

M F F F F

M M F F M

M M F F F

M M M M F

M

Initials

B.R. T.I. N.H. L.H. R.M.

R.R. C.A. B.M. V.D. B.L.

B.T. G.J. H.I. K.M. S.B.

B.J. D.J. U.J. W.M. L.R.

U.F.

8

51

20

9

8 2 8 1 10

42 43 57 20 42

3 2 20 20 5

2 14 2

0.5

1s 0.5 1 10 1

5

-

Exposure period (years)

40 29 36 46 36

53 57 54 54 29

32 27 31 46 50

Smoking

(cig./ day)

Age

(years)

NiO

NiO NiO NiO NiO NiO

NiS04 NiSO, NiSO, NiSO, NiS04

NiSO, NiSO, NiSO, NiSO, NiSO,,

NjS04 NiSO, NiSO, NiS04 NISO,

of Ni

Form

10

8 12 14 6 6

7 2 4 6 5

7 6 3 6 6

6 3 3 10 5

somal aberrations (%)

Chromo-

2

4 7 3 1 11

a

2 2 s 4

7 4 10 2 2

8 10 8 6

5

Gaps

RAW DATA OBSERVED IN THE WORKERS FROM THE EXPOSED GROUP

TABLE 1

Chromo-

4 1

6

3 11 7

5 i

1 1

2 4 1 5 2

2 1 3 7 3

some breaks

3 4 3 1 3

5 1 3 3 4

7 3 1 1 4

1

I 5

3

matid breaks

Chromatid exchanges

Chro-

Chromo-

2

1

some exchanges

Ni

0.9

4.6 2.9 4.8 6.0 10.1

33.4 16.6 2.2 25.5 0.8

20.7 21.4 19.8 27.3

5.7

8.8 8.2 8.5 8.9 28.3

serum (yg/l)

Ni

3.5

7.3 1.9 2.9 10.5 4.5

8.5 13.4 7.9 19.7 19.0

3.6 17.3 14.9 17.9 16.2

ICI.2 8.5 10.9 14.1 8.5

urine @g/II

43.1

67.3 48.0 70.1 49.4 53.9

45.1 70.1 61.8 X1.6 86.2

64.2 114.5 63.8 72.6 94.7

63.5 110.3 78.0 59.3 65.0

Ni hair (p&‘&t)

F F M

M M

M M F M M

F F F F

T.V. V.K. J.J.

S.J. P.L.

S.F. V.V. H.H. J.V. B.M.

S.E. S.L. K.R. S.V.

35 28 4X 46

51 27 46 55 27

48 51

31 53 43

42 40 42 46 54

5

1

Chtomatid exchanges

2

I

1

1

Chromosome exchanges

1.0 2.3 2.1 5.9 2.0

4.6 3.5

0.8 2.9 1.9

1.4 4.4 2.1 3.0 1.6

3.x 7.x

1.7 7.5 2.7

3.5 5.0 6.9 1.3 3.2

(/.G/B

(cr&!/B 2.4 7.3 4.0 t&9 0.X

Ni urine

Ni serum

I.2

I 1 1 3 1

Chromosome breaks

4,x 1,s 5.0

7 3 1 8 5

2 1

3 1 2

3 1 2 7 4

1 3 2 1 7

3 4 3 4 I

Chromatid breaks

2 5 14

3 2 3 5 1

6 I

2 6 6

3 4 6 5 10

Gap5

GROUP

9

21 2 2Q 32 4

20

10 20

32 27

11 34 8

22 23 20 26 38

10

10

15

M F M F F

P.M. V.O. D.Z. P.J. P.O.

Chromosomal aberrations (%‘o)

FROM THE CONTROL

Employment period (years)

Smoking tcig./ day)

Sex

Initials

Age (years)

IN THE PERSONS

RAW DATA OBSERVED

TABLE 2

12.7 10.3 14.7

Y.4

12.4 30.4

8.5 27.X 11.5,

17.6 28.9 2x.3 6.Y 11.3

(/G/g)

Ni hair

T.QLE 3 ~~NCENTR~~TION OF Ni IN BIOLOGICAL MATERIAL tN THE GROUPS EXPOSED TO NiO AND TO NiSO, AND IN THE CONTROL CROUP AND NORMAL VALUE FROM THE LITERATURE Material

Normal value from the literature

Control group

Group exposed to NiSO,

Group exposed to NiO

Urine tpg/ll

s.1,

3.1

12.7% 4.6

3.6 +_2.5

Serum &$I)

4.9+ 3.1

15.7 &-18.3

2.9, I.9

Hair ( pg/g)

55.0+ 11.0

75.5 * 19.1

l5.3f 7.7

tions in the NiO production workers increase in dependence on the exposure period, much more significantly than in the NiSO, production workers. However, the ch~mo~mal aberrations increase to a certain extent in the control group as well. This can be explained by the fact that the persons from the control group were ‘exposed’ to nicket (even though not immediately in their work) in the rather polluted en~ro~ment in the observed chemical plant, as the situation with respect to environmental protection is very bad in this plant. The reduction of the exposure in the individual work places is being solved by simply blowing off the emissions out of the workshop. Moreover, the nickel shops are situated in the western part of the chemical plant area, while western winds prevail in that region. That is why other workshops of the chemical plant are significantly exposed, too. This hypothesis, that the high levels of chromo~mal aberrations in the control group can be caused by increased nickel exposure from the polluted environment of the chemical plant, is supported by the fact that the single low finding (1% chromosomal aberrations) in a worker

2.7f 1.6 Nomoto and Sunderman, 1970 2.6 f 0.8 Nomoto and Sunderman, 1970 0.6-3.6 Iyengar et al., 1978

with a long employment period (37 years) in the chemical plant was for a car-driver who was practically continuously on business trips outside the chemical plant. The increased concentration of nickel in the biological material (see Table 3) of persons from the control group also supports the hypothesis of environment exposure. The significantly increased levels of chromosoma1 aberrations in the NiO production workers are in harmony with the literature (Costa and Mollenhauer, 1980; Senft et al., 1990a; Sunderman, 1976; Waksvik et al., 1984). The carcinogenic and mutagenic risks of nickel probably correlate with the solubility or insolubili~ of nickel compounds. It may be explained since during prevalently inhalation exposure the lungs are mostly affected. The soluble compounds are being absorbed rather quickly (biological half-life 11 h; Christensen and Lagesson, 1981; Sunderman et al., 1989) from the lungs into the organism and quickly secreted in the urine (Senft et al., 1991) whereas the insoluble compounds are being absorbed only slowly (biological half-life 4-7 months; Tanaka et al., 1988) and that is why they

TABLE 4 CHROMOSOMAL ABERRATIONS

OF PERIPHERAL

LYMPHOCYTES IN THE EXPOSED AND CONTROL GROUPS

Group

Number of persons

Chromosomal aberrations (%I

Gaps

~hromatid breaks

Chromosome breaks

Chromatid exchanges

Chromosome exchanges

Exposed workers Controls

21 I9

h.41 4.05

11.68 7.15

2.95 1.84

3.04 2.0

0.04 0.1

0.52 0.52

Exposed workers Controls

Group

OCCURRENCE

TABLE 5

21 19 3

1 1 2

2 3 3

3 1 3

4 2 3

5 7 4

6

Number of aberrant cells (from 100 evaluated cells) 0

persons 2 -

7 1 -

8

-

9

2 1

10

-

11

LYMPHOCYTES IN THE EXPOSED AND CONTROL GROUPS

Number of

OF ABERRANT CELLS IN PERIPHERAL

1 _

12

_

13

1 -

14

AB’B

k---z---

2’0

-----3i

40

YEARS

Fig. 1. Curves expressing the relation between chromosomsi aberrations of peripheral ~~rnpho~tes (AB.B% f and empfvy-

I 4

8

12

ment period. 0. control grvup: *. NiO prvduction workers: x . NiSQ production workers.

Nt

URINE

Psi’

Fig. 3. Dependence

of chromosomal

aberrations of peripheral

lymphocytes on the nickel concentration

remain in the organism for a long period and can affect the cell nuclei in the sensitive phases of development. fn accordance with this opinion findings are presented in Figs. 2, 3 and 4 which can be summarized in the following way. Nickel exposure in forms allowing easy penetration into the serum and from it into the urine and hair is probably less significant from the point of view of chromosomal aberrations than the exposure to hardly absorbable nickel forms. Fig. 3 suggests that the occurrence of chromosomal aberrations is related indirectly to the amount of nickel absorbed into the organism and secreted from the organism immediately after the

16

in the urine. Symbols

as in Fig. 1.

exposure. This means that the more nickel is secreted after exposure in the urine, the higher the nickel concentration determined in the urine and the fewer chromosomal aberrations are found. Nevertheless, this dependence can be understood as an indication of direct dependence of the occurrence of chromosomal aberrations upon the amount of nickel remaining in the organism for a long time. That is, the more nickel remains for a long time after exposure, e.g., in the lungs (the lower the concentration of nickel in the

:*&T.?. .__.. .:: .. : ,,,..., x

I.......

X

3.

X

IL

X

X

X

i

10

20

30

Ni

SERUM

I 43

63

83

103

w/1

Fig. 2. Dependence

of chromosomal aberrations

lymphacytes on nickel concentration in Fig. I.

of peripheral

in the serum. Symbols as

NI

HAIR

Kvg

Fig. 4. Dependence

of chromosomal

aberrations of peripheral

lymphocytes on the nickel concentration as in Fig. 1.

in the hair. Symbols

179

urine), the higher is the finding of chromosomal aberrations. We may conclude that an increased occurrence of chromosomal aberrations was observed especially after exposure to insoluble NiO, in direct dependence on the exposure period or in indirect dependence on the nickel concentration in the biological material. A significant increase in chromosomal aberrations in the control group was evidently caused by increased exposure to nickel in the much polluted environment of the plant. On the basis of the detected significant findings the inconvenient production of NiO has been stopped and an extensive modification of technology has been started aimed at a significant reduction of the exposure.

Nishimura.

M.. and M. Umeda (lY7Yt Induction of chromoso-

mai aberrations in cultured mammalian compounds. Mutation Res.. 6X. 337-343. Nomoto.

S.. and F.W. Sunderman

Rjabuchin.

J.S. (lY7X) Activation

tor of contamination Riissner.

P.. and M. Cerni

monitoring. 14. 120-127

Nickel:

Immunol..

a review of its occupational

toxicology.

J. Hyg. Epidemiol.

and

Microbial.

27. 237-242.

Bencko. V.. V. Wagner.

Vienna.

(19Xx) Genotoxic environmental

Acta Hyg. Epidemiol. (in Czech).

Microbial.

Immunol..

Seflovri. A.. and J. Sponar (lYX3) The analysis of trace elements in solid aerosol. Cs. Hyg.. 2X. 423-42X. Senft.

V..

F. HBzl.

nephrotoxicity

and J. Sykora (19X6) Comparison

of

of cadmium and nickel by professional ex-

posure. Pracov. L&k.. 3X. 453-457

(in Czech).

Senft. V.. F. Hiizl. J. Sykora and V. Eybl (199t)a) Exposure limits of soluble and insoluble

nickel compounds.

Biol.

in press. and F. HBzl (lY9Oh) The determination

ing and immunobiochemical 3YY-4Oh. Costa. M.. and H.H. of particulate

and V. Zavlizal(lYX6)

response, Environ.

Mollenhauer

nickel

Res.. 40.

(19X0) Carcinogenic

compounds

in proportion

activity to their

cellular uptake. Science. 20Y. 515-520. Chorvatovi?ov&

D. (19X3) The influence of NiCI,

on chromo-

1112 (in Czech). D.B..

and V. Lagesson (19X1) Nickel concentra-

tion of blood and urine after oral administration.

Ann.

Clin. Chem. Sci., I I. IIY-124. JARC (lY7h)

Monographs

on the Evaluation

of the Carcino-

genic Risk of Chemicals to Man. Vol. II: Cadmium. some epoxides.

miscellaneous

considerations

industrial

on volatile

nickel,

chemicals

anesthetics,

and

Interna-

tional Agency for Research on Cancer. Lyon, pp. 39-44. lyengar. G.V., Elemental

W.E.

Kollmer

Composition

ids. Verlag Chemie.

and H.J.M.

Bowen (197X) The

of Human Tissues and Body Flu-

Weinheim,

16X pp.

fUuc?k, 1. (1969) Biological and hygienic significance of nickel. Pracov. Lek.. 13, 243-250 A.,

G.B.

Gerber

genicity. mutagenicity

(in Czech). and P. Jacquet

and teratogenicity

(19X1) Carcinoof nickel, Muta-

tion Res.. X7. l-7. Newman, SM..

of nickel

D..

and P.J.H.

Bongarts

(1977)

Nickel

content

plasma. urine and hair in contact dermatitis. Chemistry

and Chemical

of

in: Clinical

Toxicology of Metals.

Elsevier.

Amsterdam. pp. Zhl-267. Sunderman. F.W. (19X1) Recent research on nickel carcinogenesis. Environ. Health Sunderman,

Perspect.. 40. 131-135.

F.W.. S.M. Hopfer.

K.R. Sweeney, A.H.

kinetics in human volunteers,

spontaneous level of chromosomal of Czechoslovakia,

and

Proc. Sot. Exp. Biol. Med..

191. S-IO. Svandovii. E.. and P. Rossner (19X9) The tion

Marcus.

Most and J. Creason (19X9) Nickel absorption

Pracov.

Czech). Tanaka. 1.. S. Ishimatsu. J. Haratake.

contribution

aberration Lek..

41. 216-218

A. Horie

to

in popula(in

and Y. Ko-

dama (198X) Biological half time in rats exposed to nickel monosulfide (amorphous) aerosol by inhalation. Biol. Trace Elem. Res.. 17. 237-241. Umeda, M.. and M. Nishimura somal aberrations

(1979) lnducihility of chromo-

by metal compounds in cultured mam-

malian cells. Mutation Res.. 67. 121-2X. Waksvik. H.. and M. Boysen (19x2) Cytogenetic lymphorytes

from workers in a nickel refinery.

analysis of Mutation

Res.. 103. 185-191. Waksvik. H.. M. Boysen and A.Ch. Hsgetveit (19x4) Jncreased incidence of chromosomal aherrations in peripheral lymphocytes of retired nickel workers. Carcinogenesis.

R.L. Summit and L.J. Nunez (19X2) Incidence

of nickel induced chromatid exchange. Mutation

Monitoring

exposure. Pracov. Lek.. 43. S-9 tin Czech).

B.B.

somal aberrations by Crirecrtlus g&errs, Biologia. 3X. I lO7Christensen.

Senft. V.. F. Hfizl and J. Sykora (19Yl) Spruit.

M. Wagnerovi

Human exposure to nickel and cobalt: biological monitor-

67-74.

trace ele-

IAEA.

of Ni in biological material by AAS in monitoring of nickel exposure. Pracov. Lek.. 42. 433-435 (in Czech).

environmental

Leonard.

analysis of hair as an indica-

of man by environmental

ment pollutants. Report IAEA/RL/SO.

Senft, V.. V. Jirotovi

Bencko. V. (IYX3)

general

abx>rption

(lY7tt) Atomic

spectrometry of nickel in serum. urine and other biological material. Clin. Chem., lo. 477-485.

Monitor..

References

cells by nickel

Res.. 101.

5. 1525-1531.