Cu(II) in PM2 DNA and in human fibroblasts

Toxicology Letters 94 (1998) 159 – 166

Synergistic DNA damaging effects of malondialdehyde/Cu(II) in PM2 DNA and in human fibroblasts M.-L. Vo¨hringer, T.W. Becker, G. Krieger, H. Jacobi, I. Witte * Carl-6on-Ossietzky Uni6ersita¨t Oldenburg, ICBM and FB7, Postfach 2503, D-26111 Oldenburg, Germany Received 8 September 1997; received in revised form 19 November 1997; accepted 24 November 1997

Abstract Malondialdehyde (MDA) is a product of lipid peroxidation (LPO). In combination with CuCl2 MDA induced single strand breaks in PM2 DNA whereas MDA or CuCl2 alone had no effect. Cu(II) oxidized MDA by a radical mechanism under formation of Cu(I). DNA strand break induction was inhibited by catalase (98%), neocuproine (76%) and DMSO (61%). The synergistic damaging effect of MDA and Cu(II) was also demonstrated in human fibroblasts measured by alkaline elution. The combination MDA/CuCl2 caused extensive DNA breakage while neither MDA nor CuCl2 alone induced DNA damage within the cell. Synergistic cytotoxic effects were observed 18 h after a simultaneous treatment of the cells with MDA and CuCl2 for 1 h. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Malondialdehyde; Copper; Combination effect; DNA strand breaks; Cytotoxicity; Human fibroblasts

1. Introduction Lipid peroxidation (LPO) is a complex radical chain reaction whereby unsaturated membrane lipids are oxidized. This process directly results in membrane damage (Richter, 1987) and indirectly in protein and DNA modifications provoked by reactive LPO products like lipid hydroperoxides (Inouye, 1984; Ueda et al., 1985; Yang and * Corresponding author. Fax: + 49 441 7983791; e-mail: [email protected]

Schaich, 1996) and aldehydes (Witz, 1989; Zollner et al., 1991; Yang and Schaich, 1996). Transition metals like iron and copper are able to enhance LPO. They act as initiators of LPO as well as catalysts of the propagation steps of the chain reaction (Halliwell and Gutteridge, 1989; Schaich, 1992). Lipid hydroperoxides which are formed as primary products of LPO react rapidly with transition metals like iron and copper ions to generate alkoxyl or peroxyl radicals. Thereby the reduced metal species are more effective in stimulating LPO than their oxidized form (Halliwell

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and Gutteridge, 1989). The termination of LPO results in non-radical fragmentation products, such as aldehydes. Malondialdehyde (MDA) or 4-hydroxynonenal are two of these toxicologically relevant final products of LPO (Brambills et al., 1986). Recently the oxidation of aliphatic aldehydes by Cu(II) under formation of Cu(I) was described (Becker et al., 1996). As a consequence of these radical reactions the formation of single and double strand breaks in PM2 DNA was observed, whereas neither the aldehydes alone nor Cu(II) possessed DNA breaking properties. If the aldehydes formed during LPO are also oxidized by Cu(II) via a radical process, they no longer may be looked at as final products of LPO. The resulting Cu(I) as well as the free radical species of the aldehydes may be capable of initiating new chains of LPO. Therefore beside the direct damage by radical species formed during the reaction of MDA/Cu(II) secondary cyto- and genotoxic effects by intermediates and products of LPO are possible. In this study we examined, if MDA may be oxidized by Cu(II) leading to radical reactions and DNA strand breaks. DNA damage was investigated in PM2 DNA and in human fibroblasts. Cytotoxic consequences of the reaction were examined by measuring cell viability directly after incubation with MDA/Cu(II) and after a recovery period of 18 h.

2. Materials and methods

2.1. Chemicals and reagents All salts and buffer substances were of analytical grade. Bromophenol blue, ethidium bromide and sodiumdodecylsulfate (SDS) were obtained from Serva, Heidelberg (FRG). Bathocuproinedisulfonic acid (BCS), dimethyl sulfoxid (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Ficoll were obtained from Sigma, Deisenhofen (FRG). Seakem agarose was purchased from FMC (Rockland, MI), and CuCl2 from Riedel de Hae¨n, Seelze (FRG). Catalase (specific activity: 65000 U/mg) was obtained from Boehringer Mannheim (FRG) and dimethyl-

formamide (DMF) from Busing and Fasch, Oldenburg (FRG). Malondialdehydetetrabutylammoniumchloride (MDA) and neocuproine hydrochloride were purchased from Fluka, Buchs (Switzerland). Malondialdehyde was stored under nitrogen. Supercoiled DNA from the phage PM2 was produced and isolated in our laboratory according to the method of Espejo and Canelo (1969).

2.2. Chemical treatment of PM2 DNA and determination of DNA strand breaks All solutions were freshly prepared before use. PM2 DNA (0.18 mg) was incubated with CuCl2 (0.1 mM) and MDA in various concentrations up to 20 mM in sodium phosphate buffer (100 mM, pH 7.25) for 60 min at 37°C. For inhibition of single strand breaks catalase (10 or 70 mg/ml), DMSO (10%) or neocuproine (0.4 mM) were added to the incubation mix. Strand break formation was stopped by adding a solution containing 10% SDS, 10% Ficoll, 25% DMSO and 0.04% bromophenol blue in bidistilled water and by chilling on ice. Gels were formed using 0.5% Seakem agarose dissolved in electrophoresis buffer (890 mM Tris– HCl, 890 mM boric acid, 25 mM EDTA, pH 8.4). Electrophoresis was run at a constant voltage of 2.7 V/cm for 3 h. The gel was stained with ethidium bromide (1 mg/l in water) for 1 h in the dark and then treated with 1 mM MgSO4 for 15 min to remove excess ethidium bromide. The gel was illuminated from below with UV light (Biometra fluo-link TFL-20M, emitting predominantly at 312 nm) and photographed on Polaroid type 667 films. The intensities of different PM2 DNA forms were measured directly from the photographs with a Chromoscan scanning densitometer (Joyce and Loebl, UK). Peaks were then integrated and the number of single strand breaks per PM2 DNA molecule were calculated as described by Buschfort and Witte (1994).

2.3. Determination of Cu(I) Cu(I) generation was determined by using the Cu(I) binding reagent BCS according to the

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method of Rahman et al. (1990). To determine the amount of all Cu(I) ions generated during the reaction, BCS was added to the MDA/CuCl2 mixture for the entire incubation time. MDA (final concentration 10 mM) was mixed with CuCl2 (final concentration 20 mM) and BCS solution (final concentration 80 mM) in sodium phosphate buffer (100 mM, pH 7.25) and incubated at 37°C. Directly after the incubation the stable BCS-Cu(I) complex was determined by measuring its absorbance at 480 nm. To determine the actual Cu(I) concentration in the MDA/CuCl2 mixture at specific times, BCS was added after distinct time intervals of the reaction.

2.4. Cell culture Human fibroblasts, cell line GM 05757, from the Human Genetic Mutant Cell Repository (Camden, NJ) were used. Monolayer cultures (passage 9–15) were grown in Eagle’s minimum essential medium (MEM) supplemented with 12% fetal calf serum, vitamins, essential and non-essential amino acids and with 100 U/ml of both penicillin and streptomycin. The cells were grown at 37°C in an atmosphere of 5% CO2/95% air with more than 95% humidity.

2.5. Determination of cell 6iability Cell viability was measured by the MTT assay which was performed according to Hansen et al. (1989). Malondialdehyde was freshly dissolved in serum-free medium (SFM, without antibiotics) at pH 7.2. CuCl2 (200 mM, dissolved in bidistilled water) was diluted with SFM. The pH was adjusted to 7.2 by addition of NaOH. Eight thousand cells in 200 ml medium were seeded in each well of a 96-well tissue culture microtiter plate. Two days later after the cells had achieved confluency the medium was removed and the cells were washed twice with SFM. Cells were incubated for 1 h with various concentrations of MDA alone and in combination with 2.5 mM CuCl2. Controls were incubated with SFM or with 2.5 mM CuCl2. During 1 h of incubation with 2.5 mM CuCl2 the copper content within the fibroblasts was increased from 39 to 84 ng/mg protein. This was

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measured by atomic absorption spectroscopy as described earlier (Hartmann et al., 1995). At least four wells were used for every concentration tested. After incubation the test solutions were removed and the cells were washed once. Subsequently directly after, or after a recovery period of 18 h in serum containing medium, 100 ml of a MTT containing medium were added to each well. The solution was freshly prepared by a 1:5 dilution of an MTT stock solution (5 mg/ml PBS buffer). The plate was incubated for an additional 3 h. Thereafter 100 ml lysis solution (20 g/l SDS, 50% v/v DMF, pH adjusted to 4.7 by adding 2.5% (v/v) of a 80% acetic acid and 2.5% 1 N HCl) was added to each well and the plate was shaken overnight in the dark to extract and solubilize the formazan. Formation of formazan was measured with a Biorad microplate reader using a 570 nm test wavelength and a 655 nm reference wavelength. Cell viability was calculated as the percent ratio of the absorbance of the samples to the referring control.

2.6. Determination of DNA strand breaks by alkaline elution The alkaline elution technique is used to determine single strand breaks and alkali-labile sites in cellular DNA which may be the result of a chemical attack or of an enzymatic reaction during the repair process. The procedure was performed according to Doerjer et al. (1988). Subconfluently growing cells (about 0.5 × 106 cells per dish, 57 cm2) were incubated with the test solutions as described for the MTT assay under 2.5. After 1 h of incubation the test solutions were removed and the cells were washed twice with ice-cold Saline A (8 g/l NaCl; 0.4 g/l KCl; 1 g/l glucose-1-hydrate; 0.35 g/l NaHCO3) and trypsinized. Two identical samples were combined in a total volume of 6 ml ice-cold saline A. The following procedure was performed in the dark. For determination of the total DNA content an aliquot of 0.5 ml was taken. The remaining cell suspension was loaded onto the surface of a polycarbonate filter (25 mm-diameter, 2 mm-pore size, Nucleopore) held in a Millipore Swinnex filter holder. The outflows from the filters were connected with vinyl tubing

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(0.5 mm diameter) over a peristaltic pump to a fraction collector (LKB Bromma 2211, Superrac, Sweden). After the solvent had disappeared from the filter 3.5 ml lysis solution (10 mM EDTA, 0.5% Triton X-100, 2 M NaCl, pH 10) was added to the cells and pumped with a velocity of 3 ml/h through the membrane. For determination of DNA-protein crosslinks proteinase K was added to the lysis solution at a concentration of 0.5 mg/ml. After lysis the remaining DNA on the filter was washed with 4.5 ml buffer containing 20 mM EDTA at pH 10. Afterwards the pump speed was reduced to 1.5 ml/h and DNA elution was started with 27 ml elution buffer (20 mM EDTA, adjusted with 20% tetraethylammoniumhydroxide to pH 12.6). The eluate of the first 20 min was discarded. The elution was continued for further 10 h and ten fractions were collected. For determining the mass of DNA 1 ml of each fraction was diluted with 2 ml of elution solution and neutralized by the addition of 170 ml 1 M KH2PO4. The samples were treated with 100 ml Hoechst dye 33258 (40.4 ml/ml in KH2PO4-buffer from stock solution of 0.224 mg/ml in distilled water). After 30 min in the dark fluorescence was measured at an excitation of 360 nm and an emission of 450 nm. DNA remaining on the filter was calculated from the difference of the total DNA content and the DNA content in the collected fractions.

formed immediately after starting the incubation of 10 mM MDA and 20 mM CuCl2. After 1 min about a half of the total amount of Cu(II) in the incubation mixture was reduced in the presence of BCS. After 20 min the Cu(I) formation was completed whereby nearly all the Cu(II) ions in the mixture were reduced. In the absence of BCS a concentration of about 6 mM Cu(I) was detected at each time interval measured.

3.2. Induction of DNA strand breaks by MDA/CuCl2 in PM2 DNA The combination of 0.1 mM CuCl2 with various concentrations of MDA induced single strand breaks (ssbs) in superhelical PM2 DNA in a concentration dependent manner (Fig. 2). At CuCl2 concentrations as low as 1 mM in combination with MDA strand scissions occured to a lower extent (data not shown). CuCl2 alone induced up to 0.3 ssbs/PM2 DNA molecule. MDA alone, its counterion tetrabutylammonium, or tetrabutylammoniumchloride/CuCl2 did not show any additional strand breaking properties even at the highest concentration used.

3. Results

3.1. Formation of Cu(I) during the MDA/CuCl2 incubation The formation of Cu(I) was photometrically determined during the MDA/CuCl2 incubation either in the presence of bathocuproine (BCS) by continuous scavenging of the Cu(I) ions or discontinuously by addition of BCS after distinct time intervals of the reaction. In the latter case the actual Cu(I) concentration in the mixture was determined whereas in the former case the summation of all Cu(I) ions formed during the incubation was obtained. As seen in Fig. 1 Cu(I) was

Fig. 1. Cu(I) production measured as Cu(I)-bathocuproinedisulfonic acid (BCS) complex in MDA/CuCl2 mixture. (“) Cu(I) formation during continuous incubation with 80 mM BCS, () Cu(I) concentration after addition of BCS at distinct time intervals. The MDA concentration was 10 mM, the CuCl2 concentration was 20 mM. Incubation was carried out in 100 mM sodium phosphat buffer, pH 7.25.

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Fig. 2. Single strand break formation by MDA alone () and MDA/0.1 mM CuCl2 (“) in PM2 DNA. Incubation was carried out for 1 h at 37°C in sodium phosphat buffer (100 mM, pH 7.25).

To investigate if Cu(I) and/or reactive oxygen species (ROS) are involved in DNA breakage, catalase, the Cu(I) scavenger neocuproine, and DMSO were added to the incubation mixtures. The effects of these additives are shown in Table 1. While catalase nearly completely abolished strand break formation, the inhibitory effect of DMSO was only 61%. Neocuproine inhibited strand break formation of MDA/CuCl2 by 76%. Table 1 Inhibition of DNA single strand break formation by catalase, neocuproine and DMSO Inhibitor

ssbs/PM2 DNA molecule

Inhibition (%)

None

1.02

0

Catalase 10 mg/ml 70 mg/ml 70 mg/ml inactivated

0.07 0.02 0.87

93 98 15

Neocuproine 0.4 mM

0.24

76

DMSO 10%

0.4

61

The data represent the values of two determinations. Incubation was carried out with 6 mM MDA in combination with 0.1 mM CuCl2 in 100 mM sodium phosphat buffer.

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Fig. 3. Alkaline elution profile of DNA from human fibroblasts treated with MDA alone and in combination with CuCl2. Four samples (filled symbols) were treated with proteinase K (0.5 mg/ml) during lysis. (x) serum-free medium; (2) 2.5 mM CuCl2; (") 2.5 mM CuCl2 and proteinase K; () 10 mM MDA; (“) 10 mM MDA and proteinase K; ( ) 10 mM MDA with 2.5 mM CuCl2; ( ) 10 mM MDA with 2.5 mM CuCl2 and proteinase K; () 7.5 mM MDA with CuCl2; () 7.5 mM MDA with 2.5 mM CuCl2 and proteinase K.

3.3. Genotoxic effects of MDA/CuCl2 in human fibroblasts Genotoxic effects of MDA/CuCl2 were determined by the alkaline elution technique measuring DNA strand break formation and alkali labile sites within the cellular DNA. As presented in Fig. 3 neither 2.5 mM CuCl2 alone nor a concentration of 10 mM MDA induced any DNA strand breaks or alkali labile sites within the cell. The same results were obtained by adding proteinase K to the MDA or CuCl2 treated cells showing that no DNA-protein crosslinks were formed by the single substances. The combination of 7.5 and 10 mM MDA with 2.5 mM CuCl2 caused extensive DNA strand breakage. At 7.5 mM MDA in combination with 2.5 mM CuCl2 DNA-protein crosslinks were additionally detected which is demonstrated by an increase of DNA strand breakage in the presence of proteinase K.

3.4. Cytotoxic effects of MDA/CuCl2 in human fibroblasts The effect of Cu(II) on cytotoxicity of MDA was investigated by measuring cell viability of

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human fibroblasts after 1 h of incubation with MDA in the presence and absence of CuCl2. CuCl2 was used in all combinations at its EC10 value (EC10 = concentration which reduces cell viability by 10%), whereas MDA was added in various concentrations. Cell viability was measured either subsequently after incubation with MDA/CuCl2, or 18 h later. When cell viability was measured right after treatment, the MDA toxicity was only slightly enhanced by CuCl2 at the two highest concentrations used (Fig. 4). At 18 h after treatment with the toxicants, a drastic enhancement of toxicity by MDA/CuCl2 compared to MDA alone was observed (Fig. 5). Whereas 15 mM MDA alone reduced cell viability by 20% and 2.5 mM CuCl2 by 10% the combination of both compounds inhibited cell viability by 95%. Tetrabutylammoniumchloride did not contribute to the toxicity of MDA, neither alone nor in combination with CuCl2 (data not shown).

4. Discussion Our results demonstrate synergistic DNA damaging effects of MDA in combination with Cu(II). The observed DNA strand break formation is

Fig. 4. Cell viability of human fibroblasts after incubation (1 h) with various concentrations of MDA alone () and in combination with 2.5 mM CuCl2 (“). The data represent the mean9S.D. of four determinations. Control samples were incubated with SFM and 2.5 mM CuCl2 and set at 100% viability.

Fig. 5. Cell viability of human fibroblasts 18 h after incubation (1 h) with various concentrations of MDA alone () and in combination with 2.5 mM CuCl2 (“). The data represent the mean9 S.D. of four determinations. Control samples were incubated with SFM or 2.5 mM CuCl2 and set at 100% viability.

supposedly the consequence of a redox reaction of MDA with Cu(II). Formation of Cu(I) was shown by detection of the Cu(I)-BCS complex during the MDA/Cu(II) incubation. Ninety-five percent of all copper added to the reaction mix was scavenged during the first 20 min, showing a nearly complete reduction of Cu(II) to Cu(I) in the mixture. Nevertheless, only one third of the added copper was found as Cu(I) in the mixture during the entire incubation time. This was shown by adding BCS to the mixture at distinct time intervals immediately before photometrical measurement. In conclusion of these two results we suppose, that at least a part of the generated Cu(I) is reoxidized during the reaction. Redox cycling of Cu(I)/Cu(II) can be assumed. It is known that Cu(I) is able to reduce molecular oxygen under formation of Cu(II) and reactive oxygen species (O2− , H2O2 and · OH). Strand break formation was completely prevented by catalase and effectively suppressed by neocuproine. Catalase as well as neocuproine are potent inhibitors of the copper driven Fenton reaction so that DNA damaging · OH radicals cannot be formed (Mello-Filho and Meneghini,

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1984, 1991). The inhibitory effects of catalase and neocuproine point to a function of Cu(I) as a Fenton reagent. DMSO prevented single strand breaks induced by MDA/Cu(II) by 61%. Because of its property as a specific · OH-scavenger (Klein et al., 1981; Rao et al., 1988) a complete inhibitory effect of DMSO is expected when · OH radicals are the cause of DNA strand breakage. If other than · OH radicals with DNA damaging properties are formed a lower scavenging effect of DMSO should be expected. The incomplete inhibitory effect of DMSO indicates that · OH radicals are not the only DNA breaking species produced in the MDA/Cu(II) reaction. However, the complete inhibitory effect of catalase cannot be understood on the basis of the inhibition of the Fenton reaction. Another substrate than H2O2 is suspected. Combination effects of MDA/Cu(II) were additionally investigated in human cell culture. In this case we used copper concentrations of 2.5 mM CuCl2, which approximately doubles the copper content within the cells from 39 to 84 ng/mg protein after 1 h of incubation and did not reduce cell viability by more than 10%. This is a relevant copper concentration found in fibroblasts of patients with a genetic disturbance of the copper metabolism (Wilson disease; Hartmann et al., 1995). Organs like liver, brain and kidney also retain higher copper concentrations than other cells (Venugopal and Luckey, 1978). MDA strongly reduced cell viability in combination with Cu(II) which was pronounced at 18 h after removal of the chemicals. Synergistic DNA damage of MDA/Cu(II) was demonstrated in human fibroblasts by induction of single strand breaks and to a smaller extend by DNA-protein crosslinks. This is possibly due to reactive intermediates or products formed during the redox reaction of MDA/Cu(II) as it was assumed for strand break induction in isolated DNA. But DNA damage in the cellular DNA may also be a secondary event as a consequence of lipid peroxidation. The radical species formed during the redox reaction of MDA/Cu(II) are known to induce lipid peroxidation under formation of lipid hydroperoxides. Resulting Cu(I) propagates LPO by cleavage of the lipid hydroperoxides whereby

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reactive alkoxyl radicals are formed. Alkoxyl radicals like other intermediates and products formed during LPO are known to damage the DNA (Vaca et al., 1988; Yang and Schaich, 1996). Both possibilities would explain the observed delayed cytotoxicity which is observed 18 h after the incubation with MDA/Cu(II). Cytotoxic consequences of a lipid peroxidation are observed only several hours after initiation while LPO can be measured within minutes of intoxication (Recknagel et al., 1989). Direct DNA damage by reaction products of MDA/Cu(II) should not immediately result in cell death in non-dividing confluently growing cells, which are used in the experiments. Only when protein pools of the cell are exhausted and proteins have to be replaced by new synthesis, damaged DNA may inhibit DNA transcription. The identification of the radical species formed during the MDA/Cu(II) reaction is at present under investigation in our laboratory.

Acknowledgements We thank Dr Ursula Juhl-Strauss for helpful critical discussions. We wish to thank the ‘Deutsche Forschungsgesellschaft’ for the financial support.

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