Mutation Research, 191 (1987)207 210 207 Elsevier

MTRL 016

Chromium chloride induces chromosomal aberrations in human lymphocytes via indirect action

Joseph Friedman, Fiorella Shabtai, Leonard S. Levy* and Meir Djaldetti Department of Medicine 'B', Hematologv Laboratory and B. Gattegno Research Institute, Hasharon Hospital, Petah-Tiqva, and

Tel A viv University Medical School (Israel)

(Accepted 27 Marcia 1987)

Kevwords: Chromium chloride; Chromosomal aberrations; Phytohemagglutinin; (Human lymphocytes).

Summary

The aim of this study was to examine the possible clastogenic effects of trivalent chromium chloride (CrC13) as the results in the literature are non-conclusive. Under the conditions used in this study Cr(IIl) induces chromosomal aberrations in phytohemagglutinin(PHA)-st imulated human lymphocytes. This ac- tivity, however, is suppressed by the antioxidants superoxide dismutase (SOD) (scavenger of 02), the SOD- like agents, catalase and mannitol (specific scavenger of OH' ) . The possibility that oxygen free radicals could evolve through stimulation of the arachidonic acid cascade is suggested using suitable inhibitors.

Environmental contamination by chromium (Cr) compounds may be a potential human hazard. Recent epidemiological studies confirmed earlier observations that workers exposed to chromate compounds have an increased risk of lung cancer (Alderson et al., 1981; Hayes et al., 1979; Satoh et al., 1981). While there is a general consensus that Cr(V1) compounds can exert carcinogenic, mutagenic and clastogenic effects, conflicting results have been obtained with Cr(II1) compounds

Correspondence: Dr. Joseph Friedman, Department of Microbiology, Faculty of Life Sciences, Tel Aviv University (Israel).

* Institute of Occupational Health, The University of Bir- mingham (Great Britain).

(IARC Monographs, 1982). According to some authors (Kaneko, 1976; Levis and Majone, 1979; Raffeto et al., 1977), they are clastogenic only when doses of Cr(III) greater by one or two orders of magnitude than Cr(V1) were used, whereas others claim they are not (Nakamuro et al., 1978; Tsuda and Kato, 1976, 1977; Umeda and Nishimura, 1979). The different effects of Cr(VI)

and Cr(llI) compounds may be due to the fact that Cr(VI) is readily taken up by cells, while Cr(III) is less capable of traversing cell membranes (Bianchi et al., 1980; Levis and Bianchi, 1982).

In the present study we showed that Cr(Il l) in- duces chromosomal aberrations in human lym- phocytes via oxygen free radical formation.

0165-7992/87/$ 03.50 (c" 1987 Elsevier Science Publishers B.V. (Biomedical Division)

208

Materials and methods

Chemicals" Phytohemagglutinin (HA 15 reagent-grade) was

obtained from Wellcome, England; CrCI3 .6H20

was purchased from BDH Chemicals, Ltd., Poole, England; superoxide dismutase (SOD, bovine erythrocytes 3200 units/rag protein), catalase

(40 000 units/mg protein, 1 720 000 units/ml),

mannitol, indomethacin, nordihydroguiaretic acid (NDGA) were from Sigma. 5,8,11,14-Eicosatetra-

ynoic acid (ETYA) was a gift from F. Hoffman-

La Roche & Co., Ltd. The SOD-like compounds

Cu(salicylate)2 and Cu(tyrosine) were a gift from Dr. M. Chevion (Department of Cellular Biochemistry, Hadassah Medical School, Jeru- salem, Israel).

Growth and cytogenetic procedures Lymphocyte cultures were grown up from the

buffy coats of freshly drawn heparinized venous

blood samples from healthy donors. The cells were

cultured in M-199 Hanks salts base supplemented

with 15% heat-inactivated fetal calf serum, an- tibiotics and glutamine (Biological Industries, Beth Haemek, Israel). The division of the lymphocytes

was stimulated by phytohemagglutinin (PHA) for 68-72 h. Other agents were added concomitantly with PHA.

1 h prior to harvesting, 0.3 #g/ml colchicine was added. The cells were hypotonically treated with 20% phosphate buffer solution, pH 7.0, for 12 rain and fixed by acetic acid and methanol (1:3 by

volume). At least 50 mitoses were examined for

each experimental point for each donor on coded

slides, by a blind study. Aberrations such as gaps (achromatic lesions without dislocation in the chromatid), breaks (lesions with some distortion in

the continuity of the chromatid) were considered

and in addition fragments, rings, dicentrics and polyploids, which, however, were very rare.

Results and discussion

Table 1 shows that lymphocytes of 6 out of the 7 donors showed increased chromosomal aberra-

tions in the presence of 2.5 #g/ml of CrCI3. This concentration was found to be optimal in our system. (Higher concentrations were toxic, data

not shown). There are significant individual dif-

ferences in the response to CrC13. Since there has

always been a debate as to whether to score gaps as true aberrations, we list the overall aberration frequencies also after exclusion of gaps. The con- clusions remain the same for both sets of data.

The nature of the chromosomal aberrations observed in pooled results of control and

CrC13-treated cultures is shown in Table 2. In the

presence of CrCI3 there was an increase in gaps as well as in breaks. Dicentrics and fragments were

seen only in CrCl3-treated cultures. The literature suggests that results with Cr(lll)

are controversial but it may just be the conse- quence of different experimental conditions. In

our investigations, CrCI3 was present during the 3 days of incubation, while other investigators added the CrCI3 for the last 24 h (Nakamuro et al., 1978). Hexavalent Cr (KzCr207) which is known to be clastogenic was examined in our system. As 2.5

/zg/ml were toxic, lower concentrations were used: 0.01 ~tg/ml was found to allow growth and caused

a 2-3-fold increase in aberrations compared to control cultures.

Table 3 shows the effect of different antiox- idants on chromosomal aberrations induced by

CrC13. All antioxidants inhibited or reduced the in-

TABLE 1

INDUCTION OF CHROMOSOMAL ABERRATIONS IN HUMAN LYMPHOCYTES IN THE PRESENCE OF 2.5 ;tg/ml CrCls. PERCENTAGE OF MITOSES WITH ABERRATIONS

Donor No. 1 2 3 4 5 6 7 Control 5.8(3.6) 9 .6(4 .8) 12.6(3.6) 6.3(2.9) 8.5(2.3) 7 .0 (2 .8 ) 5.5(1.2) CrCI3 2.5 /zg/ml 24.7(11.8) 20.0(15.0) 13.0(6.1) 15.8(3.6) 13.6(3.7) 13.3(3.9) 13.0(2.8)

The values oul of parentheses include gaps, the values in parentheses exclude gaps.

TABLE 2

TYPES OF CHROMOSOMAL ABERRATIONS OBSERVED

IN POOLED RESULTS OF CONTROL (561 MITOSES) AND

CrCI3-TREATED CULTURES (615 MITOSES), NUMBER

OF ABERRATIONS PER 100 MITOSES

Chromosome aberration Control CrCI~

Gaps 10.3 22.9

Breaks 3.3 5.36

Diccntrics 0.32

Fragments 0.16

Centromeric disrupture 0.17 0.32

Polyploids 0.17 0.16

cidence of chromosomal aberrations. Catalase in all experiments reduced the aberrations close to control levels. H202 which is formed from O2 (superoxide ion) by SOD is uncharged and diffuses readily into the extracellular medium. Therefore, a large portion of H 2 0 2 c a n be inactivated b y

catalase without the need for the enzyme to penetrate the cell. Preincubation of the cells with mannitol for 20 min before addition of CrCI3 in- hibited completely the Cr(l l l ) effect. When it was added concomitantly with Cr(Ill), the Cr(Ill) ef- fect was only partially inhibited. Mannitol is

TABLE 3

INHIBITIONS BY ANTIOXIDANTS OF CHROMOSOMAL

ABERRATIONS INDUCED BY CrCIs AT 2.5 #g/ral

Percentage of mitoses with aberrations relative

to control (100°7,0) a

Control 100

CrCIs 245.0 + 85.7

CrCls + SOD 100pg/ml 181.5 _+ 63.8

CrCI3 + Cu(salicylate)2 10 / ,g/ml 136.0 _+ 29.7

CrCI3 + Cu(tyrosine)2 10,ug/ml 141.5 _+ 27.6

CrCI3 + catalase 25 #g/ml 129.5 + 20.1

CrCI3 + catalase 25 #g/ml

+ SOD 100,ug/ml 101.6 + 33.7

CrC13 + mannitol 0.1 M:

no preincubation 169.4 + 26.7

CrC13 + mannitol t' 0.1 M:

preincubation 104.4 + 35.6

~'Mean values and standard deviations are based on pooled results for 3- 6 Expts. for each treatment. The aberration fre- quencies include gaps. bMannitol was preincubated with lymphocyte cell cultures for

20 min before adding CrCls.

209

known to diffuse into the cell, albeit slowly (Novogrodsky et al., 1982). The partial effect of SOD may be due to the inability of the enzyme to penetrate the cell membrane and to reach the loca- tions of 62 production. Therefore, we have used biomimetic SOD-like compounds Cu(salicylate)2 (De Alvare et al., 1976) and Cu(tyrosine)2 (Joester et al., 1972) which penetrate the cell easily. Indeed these compounds inhibited markedly the Cr(l l l ) effect. The results indicate that oxygen free radicals 62, H202 and OH" are responsible, or mediate the chromosomal aberrations induced by Cr(lll).

The possibility that Cr(IlI) could prodt~ce free radicals through activation of the arachidonic acid cascade was considered. As shown in Table 4 the Cr(IIl) effect was inhibited partially by in- domethacin (an inhibitor of cyclo-oxygenase pathway), and more dramatically by ETYA (an in- hibitor of cyclo-oxygenase and lipoxygenase) and N D G A (an inhibitor of lipoxygenase). N D G A may affect the radical scavenging potential of the cell and act as a metal chelator (Kaplan et al., 1978).

Generally, the results with Cr(Ill) in the present study resemble those o f Emerit et al. (1981, 1983) with 12-O-tetradecanoyl phorbol- 13-acetate (TPA). They showed that TPA, which does not enter the cells and is not mutagenic, hence similar to Cr(Ill), caused chromosomal aberrations in

TABLE 4

THE EFFECTS OF ARACHIDONIC ACID METABLISM

1NHIBITORS ON CHROMOSOMAL ABERRATIONS

INDUCED BY CrCI3 AT 2.5 ,~g/ml

Percentage of mitoses with aberrations relative

to control (100%)"

Control 100

CrCls 197.8 + 39.1

CrCI3 + indomethacin 30 #M 207.2 +_ 43.6

CrCls + indomethacin 60 p.M 141.8 _+ 35.7

CrCh + ETYA 50,uM 128.3 _+ 48.9

CrCI3 + NDGA 10 #M 84.1 + 18.3

Indomethacin, ETYA and NDGA were dissolved in ethanol. Maximal concentration was 0.2%. Controls with the same con- cemration of ethanol were used. Means with standard deviation

of 3 Expts. are listed. The aberration frequencies include gaps.

210

h u m a n l y m p h o c y t e s . T h e y f u r t h e r s h o w e d t h a t an-

t i o x i d a n t s a n d a r a c h i d o n i c ac id c a s c a d e i n h i b i t o r s

were ab le to r e d u c e c h r o m o s o m a l a b e r r a t i o n s in-

d u c e d by T P A . H o w e v e r , in c o n t r a s t to o u r sy s t em

wi th C r ( l l l ) , E m e r i t et al. (1983) f o u n d no e f f ec t o f

c a t a l a se on T P A - i n d u c e d c h r o m o s o m a l a b e r -

r a t i ons .

T h e gene ra l r e s e m b l a n c e b e t w e e n ou r s y s t e m

wi th C r ( l l l ) a n d the T P A s y s t e m c i ted fi ts the

m o d e l o f ' m e m b r a n e m e d i a t e d c h r o m o s o m a l

d a m a g e ' p r o p o s e d by E m e r i t et al. (1983) a n d

Ce ru t t i et al. (1983). T h u s , we sugges t t h a t C r ( [ I I ) ,

l ike T P A , ° m a y pos se s s t u m o r p r o m o t i n g - l i k e p r o p -

er t ies . It fnay well be t h a t C r ( I I l ) a n d T P A pa r -

t i c ipa te in a c t i v a t i o n o f p r o t o - o n c o g e n e s to

o n c o g e n e s t h r o u g h c h r o m o s o m a l a b e r r a t i o n s

(R.owley, !984).

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Communicated by R.J. Preston