Adaptive response to ionizing radiation-induced chromosome aberrations in rabbit lymphocytes: Effect of pre-exposure to zinc, and copper salts

E L S E V I E R Mutation Research 369 (1996) 233-241 Genetic Toxicology

Adaptive response to ionizing radiation-induced chromosome aberrations in rabbit lymphocytes: Effect of pre-exposure to zinc,

and copper salts

Lu Cai a.b, M. George Cherian a,*

" Department of Pathology, The UnirersiO'of Western Ontario, London, Ontario, N6A 5C1, Canada hlstitute of Radiation Medicine, Norman Bethune Unil'ersiO" of Medical Sciences, Changchun, 130021, People's Republic of China

Received 19 December 1995: revised 1 April 1996; accepted 4 April 1996

Abstract

Various stress conditions including exposure to low-dose radiation and low concentrations of chemical mutagens can induce an adaptive response to subsequent radiation-induced chromosome damage. In this study, the effect of pretreatment of rabbit lymphocytes with zinc or copper salts on radiation-induced chromosome damage was investigated. Pretreatment of rabbit peripheral lymphocytes with Zn (50 ~M in vitro or 100 ~mol /g body weight in vivo) resulted in resistance to radiation (2.0 Gy)-induced chromosome aberrations such as dicentrics plus centric rings and cells with chromosome aberrations. On the other hand, pretreatment with Cu (50 p~M in vitro) did not show any protective effect on radiation-induced chromosome damage in rabbit lymphocytes. However, the concentration of metallothionein increased in activated lymphocytes 24 h after in vitro pretreatment with both Zn and Cu. In addition, ~/-radiation-induced calf thymus DNA damage could be prevented directly by the addition of Zn-metallothionein in the cell-free system. These results suggest that the induction of zinc-metallothionein synthesis may act as one of the defensive mechanisms to the induction of cytogenetic adaptive response to ionizing radiation while copper-metallothionein did not show any radioprotective effect.

Keywords: Copper; Zinc; Adaptive response: Chromosome aberration: Metallothionein; Ionizing radiation

1. Introduct ion

Olivieri et al. [1] first reported that a low-dose radiation exposure from [3H]TdR made human lymphocytes more resistant to subsequent large-dose radiation-induced chromatid aberrations, and they named this phenomenon cytogenetic adaptive response. Later, it was found that the cytogenetic

Cowespondence author: Tel.: (519) 661-2030: Fax: (519) 661-3370.

adaptive response could be induced not only by pretreatment with a low dose of radiation [2-9], but also by pretreatment with various other agents including anticancer drugs (bleomycin, mitomycin C and actinomycin D), free-radical generating chemi- cals such as hydrogen peroxide ( H 2 0 2) and mild hyperthermia in mammalian cells in vitro or in vivo [10-14].

Heavy metals, which constitute one of the important groups of environmental pollutants, can induce DNA or chromosome damage as mutagenic agents

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234 L. Cai, M.G. Cherian / Mutation Research 369 (1996) 233-241

[15,16]. As in the case of radiation, exposure to low concentrations of certain metals can also induce an adaptive response to subsequent exposure to large- dose mutagen-induced genotoxicity. In plant cells, for instance, pretreatment with metals such as Zn, Cu or Cd salts resulted in protecting cell from triethylen- emelamine-, maleic hydrazide- and methyl mercuric chloride-induced chromatid aberrations [17-19]. In mammalian cells, Zn- or Cd-pretreatment also protected cell from UV-induced cytotoxicity [20]. The mechanism of these metal-induced effects is not yet understood clearly.

Metallothionein (MT), an intracellular protein containing high amounts of thiol groups, is inducible in ceils or tissues by various metals such as Cd, Zn and Cu [21,22]. In general, the major biological function of MT is the detoxication of potentially toxic heavy metal ions, and regulation of the home- ostasis of essential trace metals. However, there is increasing evidence that MT can reduce the toxic effects of several types of free radicals including superoxide, hydroxyl and organic radicals [22,23]. The protective action of pre-induction of MT against Cd-induced DNA damage has been reported [24,25]. Although the role of MT in scavenging free radicals generated from ionizing radiation has been impli- cated, it has been demonstrated only in cytotoxicity and animal survival experiments [23,26,27]. In the present study, therefore, the effects of pretreatment of rabbit lymphocytes with inorganic salts (zinc sulphate and copper chloride) on radiation-induced chromosome aberrations were investigated to deter- mine whether metals can induce cytogenetic adaptive responses, by induction of MT protein synthesis.

2. Materials and methods

2.1. Blood samples

Venous blood from two healthy male white New Zealand rabbits (weighing 3 kg) was drawn into heparinized blood-collecting bottles. Whole blood (0.5 ml) was added to 5 ml of RPMI 1640 medium containing 100 units/ml of penicillin, 100 ~ g / m l of streptomycin and 0.02 units of phytohemagglutinin (PHA; Defico, M). The blood was cultured in 25 ml flat culture bottles at 37°C in an incubator in dupli-

care samples for each experimental group from each rabbit.

2.2. Metal treatments

In the in vitro study, the lymphocytes in culture were exposed to 50 ~M zinc sulphate or 50 ~M copper chloride (both were prepared in sterilized saline) at 37°C immediately after addition of PHA. In another experiment, zinc chloride was injected subcutaneously to a rabbit at a dose of 30 m g / k g (about 100 ~ m o l / g weight) at 24 h prior to collec- tion of blood for cell culture experiments.

2.3. Irradiation

At 24 h after the start of lymphocyte culture, the cells were irradiated with 2 Gy of y-rays which was generated using a 6°Co source (Theratron Eldorado 6) at a dose rate of 0.5 Gy /min as determined by calibration using an air ionization chamber (Capinter PR-06C) connected to an electrometer (Capintex 192X). At the 46th hour after start of culture, 0.25 p~g/ml final concentration of colchicine was added to the cultures, and at the 52nd hour the cultured cells were harvested.

2.4. Chromosome preparation

The method used for preparation of chromosomes was described previously [3]. Briefly, cells were made hypotonic in 0.5% KCI for 15 min and then fixed in a solution of acetic acid/methanol (1:3) for 20 min. The fixed cells were then transferred to wet glass slides and stained with Giemsa for 20 min. After hypotonic treatment all red blood cells are lysed and only lymphocytes and mononuclear cells are retained. Since only T lymphocytes are able to respond to PHA and enter the cell cycle, all metaphase chromosome should be those of T lymphocytes in our experimental conditions. When cytogenetic analysis was carried out, 200 metaphases (e.g., 100 metaphases per sample in duplicative samples from each rabbit) were scored in each treated and control group.

2.5. MT protein assay

MT concentration in lymphocytes was measured in triplicate by a competitive enzyme-linked

L. Cai. M.G. Cherian / Mutation Research 369 (1996) 233-241 235

immunosorben t assay ( E L I S A ) [28]. The E L I S A em-

ploys the IgG fraction of a rabbit ant iserum to rat

l iver Cd MT-I I polymer . This antibody cross reacted

with both M T - I and MT-I I isoforms. A biot inylated

secondary ant ibody and a peroxidase conjugated

avidin [28] were used in the assay. Who le b lood (5

ml) with P H A was incubated with and without 50

txM Zn or Cu in vitro for 24 h at 37°C. Lymphocy te s

were isolated by centr i fugat ion through two layers o f

His topaque (1119 and 1077; S igma Chemica l Co.,

St, Louis, M O ) according to the manufac ture r ' s in-

struction. The const i tut ive and induced M T protein

was measured in 1.0 × 10 7 c e l l s / m l .

2.6. Measurement o]: DNA damage

D N A gel e lect rophores is and e th idium bromide

(EB) binding assay (based on the format ion of a

f luorescent complex be tween double-s t rand D N A and

EB) were used as descr ibed previous ly [29]. D N A

damage by exposure to ",/-radiation increases the

mobi l i ty of D N A in gel e lectrophoresis and also

interrupts intercalat ion by EB with a loss o f the

f luorescence. A l -ml 20 m M K-PBS (pH 7.0), 300

txM D N A with or wi thout MT. Immedia te ly after

irradiation, 100 Ixl o f the D N A solution was taken

out for D N A mobi l i ty analysis and 10 Ixl of a 1 m M

EB solution was added into the D N A solution for the

f luorescence measurement . For D N A mobi l i ty analy-

sis, 1% agarose gels were prepared in T A E buffer

(10 m M Tris base, 0.5 m M E D T A , 4.4 m M acetic

acid, pH 8.0). Twen ty - f ive Ixl D N A sample (includ-

ing 3 p~l loading buffer [0.25% bromopheno l blue,

0 .25% xylene cyanol FF and 30% glycerol in water])

were loaded, and electrophoresis was carried out in

T A E buffer plus 2% EB for 2 - 3 h at a vol tage of 70

V. Photographs were taken under a U V (312 nm)

t ransi l luminator to visual ize D N A mobil i ty. The flu-

orescence after EB interaction with D N A was mea-

sured using a Turner f luorescence spectrometer (ex-

ci tat ion at 510 nm and emiss ion at 590 nm). Since

the increase in the f luorescence o f EB was dose-de-

pendent fo l lowing interaction with D N A , it p rovided

a quanti tat ive measure o f the integrity of the DNA.

In controls, 100% f luorescence was assessed in a

D N A solution conta ining same concentrat ion of EB

without irradiation, represent ing 0% D N A damage.

Zero f luorescence was assessed in a solution contain-

ing EB without D N A , represent ing 100% D N A dam-

age.

2.7. Statistical analysis

Data were analysed according to the two-ta i led

Student ' s t-test. The ratio of M T proteins in treated-

groups to control, and the percentage of c h r o m o s o m e

Table 1 Percentage (%) of chromosome aberrations without and with 50 IxM Zn or Cu in vitro and 100 IXM Zn/g.weight in vivo pre-treatment for 24 hours after start of cell culture

Treatments Chromosomal aberrations t

Breaks Dics and Transl Rings

Min

Chromatid aberrations Total Cells with aberrations aberrations Breaks Exchanges

Control 0.5 _+ 0.3 2 0.0 0.0 0.0 1.3 + 0.5 0.5 _+ 0.5 1.8 _+ 0.3 1.8 _+ 0.5 Cu(II) 50 IXM 2.5 +_ 0.5 0.0 0.0 0.0 1.5 _+ 0.5 0.5 +_ 0.5 4.5 + 0.0 4.5 _+ 0.5 " Zn(II) 50 p~M 0.5 _+ 0.5 1.0 _+ 0.0 0.0 0.0 1.0 _+ 1.0 0.0 2.0 _+ 0.5 2.0 +_ 0.5 Zn(ll) 100 tx mol/g 3 0.5 _+ 0.5 0.0 0.0 0.0 1.5 _+ 0.5 0.0 2.0 _+ 0.0 2.0 _+ 0.0

2.0 Gy 34.0 _+ 1.5 39.3 _+ 2.4 0.0 0.5 _+ 0.3 1.8 _+ 0.5 3.0 _+ 0.9 78.5 _+ 1.2 57.8 _+ 3.5 Cu + 2.0 Gy 33.5 + 0.9 37.8 + 3.1 0.3 _+ 0.3 3.0 _+ 1.4 2.3 + 0.6 3.5 _+ 1.6 80.3 _+ 1.7 59.8 ___ 3.3 Zn + 2.0Gy 18.0_1.0 c 20.0_+2.3 c 0.0 1.0+__0.4 1.5_+0.6 0.5+_0.3 ~ 41.0_+3.0 " 32.5 ___ 1.9 ~

I Chromosomal aberrations including the chromosomal breaks (or fragments), dicentrics plus centric rings (i.e.. Translocation (Transl) and Minute (Min). 2 Mean + SEM, which is results of duplicative samples of one animal (n = 2). 3 Zn was pre-injected subcutaneously into whole body 24 h before blood was collected for cell culture. a P < 0.05 vs. control (two-tailed t-test). bx

p < 0.05 and p < 0.01. respectively, vs. 2.0 Gy group.

Dics and Rings),

236 L. Cai, M.G. Cherian /Mutation Research 369 (1996) 233-241

aberrations in treated and control groups were pre- sented as mean + SEM (n = 2).

3. Results

Cells treated with 50 IxM Zn did not show any significantly elevated chromosome aberrations, but

with 50 IxM Cu showed a slight increase in chromosome aberrations (Table 1). In most studies on cytogenetic adaptive response induced by low-dose radiation in human lymphocytes, the challenging radiation was given at 48 h (G 2 phase) after the start of cell culture [1-4,14]. However, in a few studies, the induction of cytogenetic adaptive response by low- dose radiation was performed with challenging dose

b

I'

t

0'

111 • G M

A

%o0

B

Fig. 1. Metaphases of rabbit lymphocyte with normal structure of chromosomes (A) and with dicentric chromosome aberrations after

exposure to radiation (B). Three dicentrics are indicated by arrows.

L. Cai, M.G. Cherian / Mutation Research 369 (1996) 233-241 237

of radiation given at G 1 phase of human lymphocytes [5-9]. In the present study, therefore, the challenging dose of radiation was given at 24 h after the start of cell culture, while most of lymphocytes are still at G n stage of cell cycle [2,3]. This will exclude the influence of cell cycle changes after Zn treatment [30]. When exposed to 2.0 Gy of ",/-rays the rabbit lymphocytes show significant increase in the number of chromosome aberrations and also in the number of cells affected (with chromosome aberrations). About 90% of the observed changes are chromosomal aberrations and only little chromatid aberrations (Table 1).

When 50 I, zM Zn was added immediately after the start of cell culture, it showed significant protective effects on the chromosomal aberrations (chromosomal breaks and dicentric plus centric tings) caused by 2 Gy ~-rays given at 24 h later (Table 1). But, Cu in the same conditions with Zn did not show any protective effects. Dicentric and centric rings are considered as the typical chromosome aberrations induced by ionizing radiation (Fig. 1). Therefore, the dicentric plus centric tings and the number of cells affected were compared in the in vivo experiment. The results shown in Fig. 2, indicate that the inci- dence of the dicentrics plus centric rings was

100,

/ [ ~ Cells with aberrations o~ Dicentrics & centric rings

. 75 O3 C- O

4 J 5O r~

@ _Q <

25

0 Zn (100 umol/g w)

2.O ~Y + 2.0 Gy

Fig. 2. Protection of ~-irradiation-induced chromosome aberra-

tions by Zn pretreatment in vivo. In radiation alone group, the

lymphocytes were collected before Zn injection and culture in vitro. They were stimulated with PHA and exposed to 2 Gy "/-radiation at 24 h. In Zn + radiation group, Zn (100 p~mol/g weight) was injected subcutaneously to rabbit and 24 h later, the lymphocytes were collected to culture in vitro and irradiated with 2 Gy 3,-radiation at 24 h. ~ * p < 0.01 vs. radiation alone group.

I--- 5--

i1) > r~

CK

[] Sor, trel ]

[ ] c~ (50 u,.)

3

2

'22 o

/ Fig. 3. Zn- or Cu-induced metallothionein protein expression in rabbit lymphocytes. Rabbit lymphocytes were cultured in vitro

with the presence of 50 I~M Zn or Cu immediately after PHA

stimulation. Metallothionein protein was determined using ELISA method as described in Section 2: Materials and Methods. Rela-

tive MT = the ratio of MT protein in treated to control. Control cells without any metal addition contained 0.79 ng M T / 1 × 107

cells. * *p < 0.01 vs. control.

markedly low in the cells collected from rabbit injected with 100 t-tmol Z n / g body weight than those in the cells collected from the same rabbit before Zn treatment. Thus Cu(II) pretreatment did not cause any protective or synergistic effects on radiation-induced dicentric and centric rings in rabbit lymphocytes in vitro; whereas pretreatment of cells with Zn(II) both in vitro and in vivo showed a significant protective effect against radiation-induced chromosome damage.

In order to understand the mechanism of zinc protection against radiation-induced chromosomal aberrations, the MT protein level was measured by ELISA in the cultured lymphocytes with or without treatments of Zn or Cu. After 24-h incubation, MT protein in control group was about 0.79 n g / l . 0 X 107

cell. While those treated with 50 p~M Zn or Cu had an increased MT protein level of about 2.5 or 2 times, respectively (Fig. 3).

The direct protective effect of Zn-MT against radiation-induced DNA damage was demonstrated using isolated calf thymus DNA. Exposure to 60 or 90 Gy ~/-radiation caused about 25-50% damage to DNA which was reduced significantly by addition of Zn-MT at 100 or 500 I~g/ml concentration (Fig. 4). The protective effect of Zn-MT was confirmed by DNA mobility changes in agarose gel electrophore-


Control calf thymus DNA

Zn-MT (500 ~tg/ml)

60 Oy

Zn-MT (500/zg/ml) + 60 Oy

Zn-MT (100 ,ug/ml) + 60 Gy

90Oy

Zn-MT (500 #g/ml) + 90 Gy

Zn-MT (100 #g/ml) + 90 Gy

I00 B

. [] Zn-MT (100 !~g/ml) + radiation ~) 75 O~ • Zn-MT (500 ~g/ml) + radiat,on

n~ E

50

-o

Z

60 90

Radiation doses (Gy)

Fig. 4. Protective effects of Z n - M T on radiat ion-induced DNA

damage . Isolated cal f thymus DNA was damaged by 60 or 90 Gy

of 3'-rays with and without the presence of 100 or 500 t x g / m l of

Zn-MT. The DNA mobili ty in gel electrophoresis (A) and the

f luorescence o f D N A interaction with EB (B) were compared , as

described in Section 2: Material and methods. ~ ~ p < 0.01 vs.

radiation alone group.

sis. The changes in mobility of DNA, induced by "y-radiation (60 or 90 Gy) were reduced by addition of Zn-MT.

4. Discussion

4.1. Induction of cytogenetic adaptice response to ionizing radiation and its relationship to induction of MT

This study demonstrated a significant chromosomal damage in rabbit lymphocytes exposed to 2 Gy of 3,-radiation and most of these effects can be protected by both in vitro Zn pretreatment (after PHA stimulation, i.e., Gj phase of cell cycle) and in vivo pretreatment (without PHA stimulation, i.e., G o phase) with zinc salts. These results are in agreement

with our previous and other studies that the cytogenetic adaptive response could be induced either in G 1 phase [2,3,14] or G O phase [3,13,14,31] by low-dose radiation, hyperthermia and low concentrations of H~O~. The results demonstrate that Zn can evoke the adaptive mechanisms to express the cytogenetic adaptive response against radiation-induced damage, similar to other stress conditions including exposure to low level of radiation [1-9,31], chemical mutagens [10-12], hydrogen peroxide [13] and mild hyperthermia [ 14].

Cell cycle arrest (G~/S) permits the repair of DNA damage, preventing the fixation by DNA repli- cation. It also alters the sensitivity of cell population since G 2 phase of cell cycle is the most sensitive stage for cytotoxicity [32]. In the present study, the cycling status of the rabbit lymphocytes was not examined. Therefore, it is difficult to exclude the possibility that the adaptation induced by Zn, which reduces the cytogenetic damage caused by a chal- lenge ",/-irradiation dose, is mediated by a cell cycle delay. However, since the challenging dose of radiation was given at 24 h after PHA stimulation in vitro, most of the cells will be at G l phase of cell cycle [2,3]. It has been demonstrated that Zn treatment alone in vitro did not influence cell growth profile [33,34], and therefore the effects associated with the complexity of cell cycle perturbation can be ruled Out .

Although the exact mechanisms for induction of cytogenetic adaptive response by low-dose radiation are not understood clearly yet, several protective cellular activities may be involved. They include DNA repair, protein synthesis and scavenging the toxic-flee radicals [14]. As essential metals, both Zn and Cu are required for many important cellular functions. Pretreatment with Zn or Cu may stimulate the SOD(Cu-Zn) activity and reduce the damage induced by flee radicals generated from ionizing radiation exposure. Floersheim and Floersheim [35] observed the remarkable radioprotective effects of Zn(II) (o,L-aspartate), but did not lind a similar effect after i.p. injection with SOD(Cu-Zn) enzyme. Leccia et al. [20] also demonstrated the SOD-inde- pendent effect of Zn protection against radiation. Our results show that Zn pretreatment results in the cytogenetic radioadaptation but Cu pretreatment did not have any such effect.

L. Cai. M.G. Cherian / Mutation Research 369 (1996) 233-241 239

Incubation of freshly collected normal human lymphocytes with zinc salts resulted in synthesis of inducible protective low molecular weight proteins [36]. Similar effect was reported by Wolff et al. [37] with human lymphocytes after exposure to a low dose of radiation. These results suggest a role for MT in the induction of cytogenetic adaptive response to radiation. Injection of zinc salts has been shown to increase MT levels in several organs in rats [38]. In the present study, 24-h pretreatment with Zn both in vitro and in vivo increased MT concentration in activated lymphocytes (Fig. 3), and these cells showed high resistance to radiation-induced chromosome aberrations (Table 2, Fig. 2). In the in vitro cell-free system, the damage to isolated calf thymus DNA by ",/-radiation was markedly prevented by the addition of Zn-MT prior to irradiation n(Fig. 4). These results suggested that induced synthesis of MT may be one of the factors responsible for the high resistance to radiation in rabbit lymphocytes pretreatment with Zn. In the plant cells, Subhadra and Panda [19] have shown that phytochelatins (functionally similar to MT) were responsible for Zn-induced genotoxic adaptation in subsequent exposure to maleic hydrazide and methyl mercuric chloride. This is further supported by the finding that cytogenetic adaptive response induced by low-dose radiation required active protein synthesis [4,39]. In addition, MT protein synthesis and gene expression also can be induced by exposure to ionizing radiation [40,41].

4.2. The differential effects o f Zn- and Cu-MT for the induction of cytogenetic adaptit:e response to radiation

If induction of MT is one of the mechanisms for the cytogenetic adaptive response, the different results observed for Zn and Cu need to be explained. First of all, the anti-oxidant property of MT depends on its metal content. It has been accepted widely that metal-catalyzed production of highly reactive hydroxyl radical ( 'OH) by Fenton reaction play an important role in the toxicity of oxygen and hydrogen peroxide in vivo [42]. The formation of "OH by the Fenton reaction requires either Cu or Fe ions and it can cause DNA damage [29,42], and may also enhance the effects of radiation [43]. It has been reported that Cu-MT can increase lipid peroxidation

caused by organic hydroperoxide [44] and cause specific DNA base damage in vitro [45]. In addition, Cu /Zn-MT (mainly Cu) can stimulate lipid peroxidation, initiated by xanthine-xanthine oxidase reaction [46]. On the other hand, the DNA damage caused by free radical generated from Fe-EDTA [47], Cu [29] or radiation ([48], present data) in vitro was inhibited by Zn-MT or Zn/Cd-MT. These studies showed that the type of metals bound to MT can affect the ability of MT to scavenge free radicals and protect cells from oxidative damage.

The increase in apoptosis or programmed cell death has been considered as one of the adaptive mechanisms for the induction of cytogenetic adaptive response, because cell death occurs more in the low-dose radiation range [49,50]. Under these conditions, the major cause of cell death was found to be through apoptosis mechanism [50,51]. Furthermore, pretreatment with low-dose radiation or mild hyperthermia sensitized cells to become apoptotic after subsequent large dose of radiation exposure which may remove the genetically damaged cells [52]. Zn appears to have a dual effect on apoptosis in lymphocytes, acting both as an inhibitor at high concentrations (500 IxM or greater) and as an activator at low concentration [53,54]. Other biologically rele- vant metals, such as copper, iron and nickel, did not induce apoptosis in mouse thymocytes at 80-200 ~M range, a dose level of zinc which was effective in inducing apoptosis in the same cells [54]. In our study, the doses of Zn used is within the effective dose range (50 ~M in vitro and 100 Ixmol/g body weight in vivo) in inducing apoptosis. Tumour suppressor protein, p53, is an important factor in the regulation of radiation-induced apoptosis. In cells or animals with deficient p53 gene, the ability to initiate apoptosis after exposure to ionizing radiation was decreased, leading to high susceptibility to radiation-induced DNA or chromosome damage [55,56]. Zn plays an important structural role to stabilize the DNA-binding domain of p53. In con- trast, Cu binds directly to p53 resulting in a confor- mational change, analogous to that of oncogenic mutants of p53 and thus inhibiting its DNA-binding activity [57,58]. Therefore, there are certain differential roles for Zn and Cu in regulating the structural function of p53 and the apoptotic process.


References

[1] Olivieri, G., J. Bodycote and S. Wolff (1984) Adaptive response of human lymphocytes to low concentrations of radioactive thymidine. Science, 233, 594-597.

[2] Shadley, J.D., V. Afzal and S. Wolff (1987) Characterization of the adaptive response to ionizing radiation induced by low dose of X-rays to human lymphocytes. Radiation Res., 111, 511-517.

[3] Cai, L. and S.Z. Liu (1990)Induction of cytogenetic adaptive response of somatic and germ cells in vivo and in vitro by low-dose X-radiation. Int. J. Radiat. Biol., 58, 187-194.

[4] Cal, L. and S.Z. Liu (1992) Effect of cycloheximide on the adaptive response induced by low dose radiation. Biomed. Environ. Sci., 5, 46-52.

[5] Wang, Z.Q., S. Saigusa and M.S. Sasaki (1991) Adaptive response to chromosome damage in cultured human lymphocytes primed with low doses of X-rays. Mutation Res., 246, 179-186.

[6] Shadley, J.D. and G. Dai (1992) Cytogenetic and survival adaptive responses in G I phase human lymphocytes. Muta- tion Res., 265, 273-281.

[7] Shadley, J.D. and G. Dai (1993) Evidence that the adaptive response of human lymphocytes to ionizing radiation acts on lethal damage in nonaberrant cells. Mutation Res., 301, 171-176.

[8] Barquinero, J.F., L. Larrios, M.R. Caballin, R. Miro, M. Ribas, A. Sublas and J. Egozcue (1995) Occupational exposure to radiation induces an adaptive response in human lymphocytes. Int. J. Radiat. Biol., 67, 187-191.

[9] Khandogina, E.K., G.R. Mutovin, S.V. Zvereva, A.V. An- tipov, D.O. Zverev and A.P. Akifyev (1991) Adaptive response in irradiated human lymphocytes: radiobiological and genetical aspects. Mutation Res., 251, 181-186.

[10] Wolff, S., V. Afzal, J.K. Wiencke, G. Olivieri and A. Michaeli (1988) Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA. In. J. Radiat. Biol., 53, 39-48.

[11] Vijayalaxmi and W. Burkart (1989) Resistance and cross-resistance to chromosome damage in human lymphocytes adapted to bleomycin. Mutation Res., 211. 1-5.

[12] Mozdarani, H. and Saberi, A.H. (1994) Induction of cytogenetic adaptive response of mouse bone marrow cells to radiation by therapeutic doses of bleomycin sulfate and actinomycin D as assayed by the micronucleus test. Cancer Res., 78, 141-150.

[13] Cortes, F., 1. Domingquez, M.J. Flore, J. Pinero and J.C. Mateos (1994) Differences in the adaptive response to radiation damage in G 0 human lymphocytes conditioned with hydrogen peroxide or low-dose X-rays. Mutation Res., 311, 157-163.

[14] Cai, L. and Jiang J. (1995) Mild hyperthermia can induce adaptation to cytogenetic damage caused by subsequent X irradiation. Radiation Res., 143, 26-33.

[15] Agarwal, K., A. Sharma and G. Talukder (1990) Clastogenic

effects of copper sulphate on the bone marrow chromosomes of mice in vivo. Mutation Res., 243, 1-6.

[16] Kappus, H. and Ch. Reinhold (1994) Heavy metal-induced cytotoxicity to cultured human epidermal keratinocytes and effects of antioxidants. Toxicol. Lett., 71, 105-109.

[17] Michaelis, A., S. Takehisa, R. Rieger and O. Aurich (1986) Ammonium chloride and zinc sulfate pretreatments reduce the yield of chromatid aberrations induced by TEM and maleic hydrazide in Viciafaba. Mutation Res., 173, 187-191.

[18] Michaelis, A. and R. Rieger (1993) Inhibition of protein synthesis by cycloheximide does not prevent adaptive response triggered by heavy metal salts in Viciafaba. Mutation Res., 302, 157-160.

[19] Subhadra, A.V. and B.B. Panda (1994) Metal-induced genotoxic adaptation in barley (Hordeum ~'ulgare L.) to maleic hydrazide and methyl mercuric chloride. Mutation Res., 321, 93-102,

[20] Leccia, M.T., M.J. Richard, J.C. Beani, H. Faure, A.M. Monjo, J. Cadet, P. Amblard and A. Favier (1993) Protective effect of selenium and zinc on UV-A damage in human skin fibroblasts. Photochem. Photobiol., 59, 548-553.

[21] Cherian, M.G. and R.A. Goyer (1978) Metallothioneins and their role in the metabolism and toxicity of metals. Life Sci., 23, 1-10.

[22] Cherian. M.G. and H.M. Chan (1993) Biological functions of metallothionein, in: K.T. Suzuki, N. Imura and M. Kimura (Eds.), Metallothionein III, Biological Roles and Medical Implications, Birkhauser Verlag, Basel. Boston, Berlin, pp. 87-110.

[23] Sato, M. and I. Bremner (1993) Oxygen free radicals and metallothionein. Free Radical Biol. Med., 14, 325-337.

[24] Coogan, T. P., R.M. Bare, E.J. Biornson and M.P. Waalkes (1994) Enhanced metallothionein gene expression is associated with protection from cadmium-induced genotoxicity in cultured rat liver cells. J. Toxicol. Environ. Health. 41. 233-245.

[25] Shiraishi, N., J.F. Hochadel, T.P. Coogan, J. Koropatnick and M.P. Waalkes (1995) Sensitivity to cadmium-induced genotoxicity in rat testicular cells is associated with minimal expression of the metallothionein gene. Toxicol. Appl. Phar- macol., 130, 229-236.

[26] Matsubara. J.. Y. Tajima and M. Karasawa (1987) Metalloth- ionein induction as a potent means of radiation protection in mice. Radiation Res., 111,267-275.

[27] Renan, M.J. and P.I. Dowman (1989) Increased radioresis- tance of tumor cells exposed to metallothionein-inducing agents. Radiation Res., 120, 442-455.

[28] Chan, H.M., M.G. Cherian and I. Bremner (1992) Quantifi- cation of metallothionein isoforms using an enzyme-linked immunosorbent assay (ELISA) with two specific antisera. Toxicol. Appl. Pharmocol.. 116, 267-270.

[29] Cai, L., J. Koropatnick and M.G. Cherian (1995) Metalloth- ionein protects DNA from copper-induced, but not iron-induced cleavage in vitro. Chem.-Biol. Interact., 96, 143-155.

[30] Sasaki, M.S. (1995) On the reaction kinetics of the radioad- aptive response in cultured mouse cells. Int. J. Radiat. Biol.. 68, 281-291.

L. Cai, M.G. Cherian / Mutation Research 369 (1996) 233-241 241

[31] Liu, S.Z., L. Cai and S.Q. Sun (1992) Induction of cytogenetic adaptive response by chronic exposure of rabbit to very low dose-rate gamma-irradiation. Int. J. Radiat. Biol., 62, 187-190.

[32] Hall, E.J. (1994) Radiobiology for the Radiologist, Fourth edition, Lippincott company, Philadelphia, pp. 91-105.

[33] Perkins, D.M. and J.R. Duncan (1991) The effect of gamma- linolenic acid and zinc supplementation on the grwth of normal and tumor cells in vitro. Prostagland. Leukotr. Essen- tial Fatty Acids, 43, 43-48.

[34] Hatayama, T., Y. Tsukimi, T. Wakatsuki, T. Kitamura and H. Imahara (1992) Characteristic induction of 70,000-Da heat-shock protein and metallothionein by zinc in HeLa cells. Mol. Cell. Biochem., 112, 143-153.

[35] Floersheim, G.L. and P. Floersheim (1986) Protection against ionizing radiation and synergism with thiols by Zn(II) (aspartate). Br. J. Radiol., 59, 597-602.

[36] Phillipes, J.L. (1979) Zinc-induced synthesis of low molecular weight zinc-binding protein by human lymphocytes. Biol. Trace Element. Res., 1, 359-371.

[37] Wolff, S., J.K. Wiencke, V. Afzal, J. Youngbloom and F. Cortes (1989) The adaptive response of human lymphocytes to very low doses of ionizing radiation: a case of induced chromosomal repair with the induction of specific proteins, in: K.F. Baverstock and J.W. Strachter (Eds.), Low Dose Radiation: Biological Bases of Risk Assessment, Taylor and Francis, London, pp. 446-454.

[38] Onosaka, S. and M.G. Cherian (1982) The induced synthesis of metallothionein in various tissues of rats in response to metals. 11 Influence of zinc status and specific effect on pancreatic metallothioneins. Toxicology 23, 11-20.

[39] Youngblom, J.H., J.K. Wiencke and S. Wolff (1989) Inhibi- tion of the adaptive response to ionizing radiation induced by low doses of X-rays to human lymphocytes. Radiation Res., 111,511-517.

[40] Shiraishi, N., H. Hayashi, Y. Hiraki, K. Aono. Y. hano, F. Kosaka, S. Noji and S. Taniguchi (1989) Elevation in metal- Iothionein messenger RNA in rat tissues after exposure to X-irradiation. Toxicol. Appl. Pharmacol.. 98. 501-506.

[41] Koropatnick, J., M. Leibbrand and M.G. Cherian (1989) Organ-specific metallothionein induction in mice by X-irradiation. Radiation Res., 119, 356-365.

[42] Goldstein, S., D. Meyerstein and G. Czapski (1993) The Fenton reagents. Free Radical Biol. Med.. 15. 435-445.

[43] Lloyd, R.E., R.A. Larson, T.L. Adair and R.W. Tuveson (1993) Cu(II) sensitizes pBR322 plasmid DNA to inactiva- tion by UV-B (280-315). Photochem. Photobiol., 57, 1011- 1017.

[44] Stephenson. G.F., H.M. Chan and M.G. Cherian (1994) Copper-metallothionein from the toxic milk mutant mouse enhances lipid peroxidation initiated by an organic hydroperoxide. Toxcol. Appl. Pharmacol.. 125, 90-96.

[45] Oikawa, S., M. Kurasaki, Y. Kojima and S. Kawanishi

(1995) Oxidative and nonoxidative mechanisms of site- specific DNA cleavage induced by copper-containing metallothioneins. Biochemistry 34, 8763-8770.

[46] Arthur, J.R., I. Bremner, P.C. Morrice and C.F. Mills (1987) Stimulation of peroxidation in rat liver microsomes by (copper, zinc)-metallothioneins. Free Radical Res. Commun., 4, 15-20.

[47] Abel, J. and N. Ruiter (1989) Inhibition of hydroxyl-radical- generated DNA degradation by metallothionein. Toxicol. Lett., 47, 191-196.

[48] Greenstock. C.L., C.P. Jinot, R.P. Whitehouse and M.D. Sargent (1987) DNA radiation damage and its modification by metallothionein. Free Radical Res. Commun., 2, 233-239.

[49] Singh, B., J.E. Arrand and M.C. Joiner (1994) Hypersensi- tive response of normal human lung epithelial cells at low radiation doses. Int. J. Radiat. Biol., 65, 457-464.

[50] Mothersill, C., J. Harne, F. Lyng, D. Cottell, K. Parsons, D.M. Murphy and C.B. Seymour (1995) Primary explants of human uroepthelium show an unusual response to low-dose irradiation with cobalt-60 gamma rays. Radiation Res., 142, 181-187.

[51] Normura. T., M. Kinuta, T. Hongyo, H. Nakajima and T. Hatanaka (1992) Programmed cell death in whole body and organ system by low dose radiation. J. Radiat. Res., 33 (Suppl.), 109-123.

[52] Cregan, S.P.. D.R. Boreham, P.R. Walker, D.L. Brown and R.E.J. Mitchel (1994) Modification of radiation-induced apoptosis in radiation- or hyperthermia-adapted human lymphocytes. Biochem. Cell Biol., 72, 475-482.

[53] Zalewski, P.D. and L.J. Forbes (1993) lntracellular zinc and the regulation of apoptosis, in: M. Lavin and D. Watters (Eds.), Programmed Cell death: The Cellular and Molecular Biology of Apoptosis. Harwood Academic Publishers, Chur. Switzerland, pp. 73-85.

[54] Telford W.G. and P.J. Fraker (1995) Preferential induction of apoptosis in mouse CD4 + CD8 + a[3TCR I° CD3 t~' thymocytes by zinc. J. Cell. Physiol., 164, 259-270.

[55] Lee, J.M., J.L. Abrahamson and A. Bernstein (1994) DNA damage, oncogenesis and the p53 tumour-suppressor gene. Mutation Res., 573-581.

[56] Lee, J.M., J.L. Abrahamson, R. Kandel, L.A. Donehower and A. Bernstein (1994) Susceptibility to radiation-carcino- genesis and accumulation of chromosomal breakage in p53 deficient mice. Oncogene 9, 3731-4736.

[57] Cho, Y., S. Gorina, P.D. Omichinski, K. Sakaguchi, N. Zambrano, H. Sakamoto, E. Appella and A.M. Gronenborn (1994) Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 265, 386-390.

[58] Hainaut. P., N. Rolley, M. Davies and Jo Milner (1995) Modulation by copper of p53 conformation and sequence- specific DNA binding: role for Cu(II)/Cu(1) redox mechanism. Oncogene 10, 27-32.

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Adaptive response to ionizing radiation-induced chromosome aberrations in rabbit lymphocytes: Effect of pre-exposure to zinc, and copper salts