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Effects of copper on mammalian cell components
Chert-Biol. Interactions, 69 (1989) 1-16
1
Elsevier Scientific Publishers Ireland Ltd.
Review Article
EFFECTS OF C O P P E R ON MAMMALIAN CELL C O M P O N E N T S
K A L P A N A AGARWAL, A R C H A N A S H A R M A and GEETA T A L U K D E R
Human Genetics Unit, Centre for Advanced Study, (Cell and Chromosome Research), Department of Botany, University of Calcutta~ 35 Ballygunge Circular Road, Calcutta-700 019 (India)
(Received February 27th, 1988) (Revision received June 16th, 1988) (Accepted June 20th, 1988)
SUMMARY
Both deficiency and excess of copper induce toxic effects on mammalian cell s y s t e m s in vivo and in vitro. The effects can be related to the affinities of Cu(II) ions for specific cell components. The nucleus is a potential site for temporary Cu storage while primary targets for free Cu(II) ions are the thiol groups which reduce the ions to Cu(I). Cu(II) ions show a high affinity for nucleic acids, binding with DNA both at intrastrand and interstrand levels, possibly through intercalation between GC pairs. The ability to chelate Cu(II) ions is seen to be of the order: p u r i n e > p u r i n e ribonucleotides > purine ribonucleoside > pyrimidine ribonucleotides. Copper is an integral part of enzyme activation and enters into the molecular structure of several proteins, like ceruloplasmin. Cu(II) ion is a potential mutagenic agent as seen by its property of inducing infidelity in DNA synthesis in vitro. Teratogenic activities of copper have been reported but carcinogenicity is not yet confirmed. Copper is an essential component of chromatin and is known to accumulate preferentially in the heterochromatic regions. External application of higher doses, however, induces both clastogenic effects and spindle disturbances. In certain forms, inorganic copper enhances the clastogenic activity of other agents. The most widely studied h u m a n genetic maladies linked with copper metabolism are Menkes' and Wilson's diseases. Several mutations are k n o w n which influence Cu homeostasis in mammals. Such m u t a t i o n s in mice have been used extensively for biochemical studies. K e y words: Copper cytotoxicity - Metal cytotoxicity
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© 1989 Elsevier Scientific Publishers Ireland Ltd Printed and Published in Ireland
2 INTRODUCTION Copper is an essential micronutrient and is found in all mammalian tissues, bound to proteins or other organic molecules. Relatively higher a m o u n t s are present in liver, followed by brain and heart. Both enhancement and deficiency of the normal levels of copper have been related to adverse effects on the organism. The information on biological consequences of copper accumulation, at nuclear and cellular levels, following environmental exposure of h u m a n populations is however, meagre. The metal is of particular interest because of its preferential accumulation in the heterochromatic regions of the/chromosomes both in vivo and in vitro and its ability to destabilise DNA and to inhibit RNA polymerase activity in vitro. EFFECTS ON DNA The influence of metal ions upon the conformation of DNA was realised early, when it became apparent that these ions are involved in the stabilization of the Watson-Crick double helix [1,2]. Different metal ions, under a variety of conditions, were found to produce different effects upon the structure of polynucleotides [3-7]. These effects also included degradation of the ribose phosphate backbones by scission of the phosphate bonds [8-13]. Differential centrifugation analyses indicated that the nuclear fraction contains approximately 20% of the total hepatic copper in the adult mammals. Since the nucleus is made up of nucleic acids and several basic proteins, all of which bind copper, this organelle is a potential site for temporary copper storage. A portion of the nuclear Cu is involved in cellular metabolism and the polynucleo~ide strands are bridged by metal ions [14]. The biochemical basis for Cu toxicity is still not absolutely clear. Studies on isolated cells and organelles have shown that excessive a m o u n t s of Cu(II) ions labilized cellular membranes [15,16]. The primary targets for free cupric ions in the membranes were the thiol groups which are capable of reducing Cu(II) to Cu(I) form upon concomitant oxidation to disulfides in the membrane [17]. The Cu(I) ions formed could subsequently be re-oxidized to the Cu(II) form in the presence of molecular oxygen. Molecular oxygen, thereby converted to the toxic superoxide radical 02, could induce lipoperoxidation [16,18]. Ribonucleic acid, DNA and some of their constituents show a surprising strong affinity for cupric ion. The strength of the interaction of cupric ion with DNA and RNA m a y indicate an important role of these compounds in the biochemistry of copper and also m a y be significant in assessing the biological function of DNA and RNA in tissues. The formation of complexes by nucleotides is far superior to that of most amino acids and equivalent, if not superior, to many of the proteins [19]. In general, transition metals interact with DNA and polynucleotides both, by complexing with the phosphate group and under suitable conditions with
3 the electron donor sites on the bases as well [20]. The ultraviolet spectrum of native DNA was unaffected by Cu(II) ions, but the spectrum of denatured DNA shifted and enhanced, in line with the previous studies, indicating that Cu reacted with the bases in DNA. Increasing concentrations of Cu increased the spectral intensity upto a ratio of between one and two Cu ions per nucleotide base. The presence of the four nucleosides had no effect on the potentiometric titration of Cu, indicating that the binding of Cu to the bases proceeded without removal of protons. The presence of adenosine, did, however, eliminate the hysteris effect in the titration of Cu and the binding of the nucleoside to Cu was further indicated by the ability of the latter to shift the visible absorption of Cu to lower wavelengths. Cu was bound to the N7 positions of adenosine and guanine and to N1 of cytidine, but did not bind to thymidine. The phosphate resonance in deoxyadenosine monophosphate and deoxythymidine monophosphate was broadened by Cu ions. This indicated that Cu also binds phosphate, which was also demonstrated by potentiometric titration of Cu adenosine monophosphate and Cu DNA solution. The ability of Cu ions to cleave the phosphodiester bonds in polynucleotides, as well as to denature DNA, was thus explained by the demonstration that Cu could bind to both the phosphate and base moieties [21-26]. The binding of cupric ion with salmon sperm DNA was sensitive to changes in the metal concentration. It was time dependent at 25 ± 5°C and remained constant between pH 5.6 and 6.6 [24]. The stability of the Cu(ID nucleoside bond was not enough to overcome the attractive forces between the two DNA strands at room temperature. Rise in the temperature weakened the DNA structure, stabilising the Cu complex enough to compete effectively. At low ionic strength, the stability of the double helix did not exceed that of the Cu complex sufficiently to allow reformation of native DNA at an appreciable rate. A large increase in ionic strength, however, increased the stability of the double helix relative to the copper complex to such an extent that the native structure was again formed. Studies on absorbance and the optical rotation of the denatured product of the Cu(II) denatured DNA indicated that the two strands unwind. On the other hand, the sedimentation data showed that the denatured DNA was very highly aggregated. The apparently complex renaturation accompanying the addition of electrolyte can be best explained by assuming that the Cu ions, bound to the bases, keep the bases in register, even in the denatured state. The model which could fulfil these requirements indicated that in the denatured state Cu(II) ions were interposed between complementary strands by coordination to the bases in such a manner that all hydrogen bonds were broken and the secondary structure of each strand completely destroyed. Similar structures have been proposed for the Cu complexes of nucleotides [19]. A structure of this type represents a separation of the complementary strands of DNA [27]. At the present state of knowledge, the Cu(II) ions have been found to
show the highest affinity for nucleic acids amongst trace elements present in living organism. The specific interstrand binding of Cu(II) ions in the DNA-Cu(II) complexes has been suggested to be due to intercalation of Cu(II) between GC pairs, involving a change in deoxyguanosine conformation around the glycosyl bond from the anti to a syn position and formation of a G-Cu-C complex [28]. Another suggestion ascribed the specificity of Cu(II) binding to a G C c u C G structure [25]. Studies of the interaction of Cu(II) ions with native and denatured DNA as a function of ionic strength by the equilibrium dialysis method, confirmed the occurrence of interstrand and intrastrand binding of Cu(II) with DNA. Prasal [29] proposed a new molecular model of Cu(II)-DNA binding, assuming interstrand intercalation of one Cu(II) ion between two GC pairs, both in the successive even and odd groups, and interstrand binding of Cu(II) to the isolated GC pairs, with the exception of T-C-T and T-G-T sequences. This model indicates that the DNA-Cu(II) complex is most stable under the equilibrium with free Cu(II) ions at 4°C (pH 6.0). In these conditions, molar ratio of GC pairs to Cu(II) ions bound interstrandially is GC/Cu inter = 2 + 0.1 [29]. In this model, the chelates involving coordination of Cu(II) with N-7 and 0-6 of guanine bases, present in the model of F6rster et al. [25], are not included. On the other hand, the model contains mixed chelates involving N-3 of cytosine and phosphate residues, as well as the chelates proposed in the model of Zimmer et al. [28]. The reaction of mercury(II) with DNA resembled the Cu(II) DNA system, but much of the secondary structure remained in the denatured form. The mechanism of the reversal was quite different, since Hg(II) complex was formed in the presence of high electrolyte concentration and decomposed by competing ligands without change in the ionic strength [30-32]. Cu(II) ion increased the mutagenic potential of thymidine hydrogen peroxide and ascorbic acid. Soluble salts of Cu and other metals like silver, cadmium, cobalt, chromium, beryllium and others increased errors in cellfree DNA synthesis [33] and also the error frequency of avian myeloblastosis virus polymerase. It inhibited the human beta polymerase strongly at low concentrations. Cu reacted instantly and completely with sulfhydryl compounds such as mercaptoethanol to form Cu-S-C2HsOH. At 37°C, in presence of CuSO4, the rate of polymerisation decreased with time. The inhibition was constant at pH 8.6 to 7 and only 50% of reversal was possible. Inhibition was not initiated through the binding of the metal ion to the template or to the deoxynucleoside triphosphate but was probably due to a direct interaction of the metal with the enzyme [20]. In combination with Cu(II), isoniazid and other related hydrazine compounds induced unscheduled DNA synthesis (UDS) in cultured human fibroblasts [34]. The enhancement of DNA repair was lowered by Cu [34]. Copper acetate suppressed strongly the incorporation of tritiated thymidine
into rat liver DNA which was stimulated by injection of dimethylnitrosamine (DMN) [35]. Crystalline copper sulfide particles induced a considerable reduction in the molecular weight of DNA, as determined by alkaline sucrose gradient, following a 24-h treatment with 10/~g/ml of CuS [36]. Cu in biological systems is not thought to play an important part in the formation of free hydroxyl radical because of its tendency of binding strongly (tightly) to molecules containing amino groups. However, complex Cu in the presence of H202 c a n bring about site-specific damage [37]. DNA treated with ascorbic acid in the presence of Cu(II) ion or Cu tripeptide complex, under aerobic conditions, released the bases from the DNA molecule to a significantly high level. Addition of catalase or chemical scavengers of hydroxyl radical to the reaction mixture prevented the release of base. Total amount of base released was equivalent to the amount of phosphate in the reaction mixture and was caused by activated oxygen or hydroxyl radical from the H202, formed by auto-oxidation of ascorbic acid in presence of Cu [38-40]. Maximal fragmentation of DNA occurred at concentration ratio of DNA/Cu at 4: 1. The binding of Cu to the DNA molecule was essential for DNA breaking action of ascorbic acid. Cu bound to DNA was more effective than free Cu(II) ion. Proteins, nitrogen gas, hydroxyl radical scavengers and catalase inhibited Cu-dependent degradation of DNA [37,41-45]. Fragmentation of DNA in presence of ascorbatecopper was due to the induction of alkali-labile bonds in the DNA. Reduced Cu-salt autoxide in aerobic conditions formed hydroxyl radicals, which were capable of damaging most of the biological molecules. Cu ions readily ligated to amino groups of proteins and other molecules. Formation of the OH- radical was as follows:
Cu 2+ + H202 -->Cu + + H20 + H + 2H20 --, 202 + 2H--, H202 + 02 Cu2+ + O2-~ Cu÷+ O2, Cu + + H 2 0 2 --'> C u 2+ + O H - + OH.
Hydrozable tannins and 1,10-phenanthroline-copper complex under limited oxygen supply induced multiple single-stranded breaks of DNA in the spacer segment while core DNA segment in the chromatin remained intact. After the accumulation of single-stranded breaks in both strands, double-strand cleavage of DNA to fragments of nucleosomal size became apparent [46,47]. Cu(II) ions also bond with the nitrous base and phosphate group. Stability of the complex is increased due to hydrogen bonding of the partially hydrated ions of Cu(II) with the phosphate groups [48]. Deoxy derivatives interact with Cu(H) ions more strongly than the corresponding ribose compounds [19].
EFFECTS ON RNA The order of ability to chelate Cu(II) ion amongst the members of the ribose series was found to be purine>purine ribonucleotides >purine ribonucleoside > pyrimidine ribonucleotides [19]. Cu inhibited R N A polymerase under all conditions. At low concentrations, Cu(II) ion also inhibited ribonucleases. Cu has high affinity for these enzymes and acts directly on all stages of synthesis of R N A including initiation, condensation of nucleotides, detachment of newly formed RNA. These effects are not reversed by the presence of crysteine [49]. In rats, copper chloride injections induced the synthesis of a messenger R N A ( m R N A ) in the liver which coded for a protein with the properties of a metallothionein [50]. In lower organisms, for example, Escherichia coli, binding of Cu to the transfer R N A (tRNA) enhanced the intensity of characteristic absorption bands in the range 500-900 nm. Cu 2+ binding induced specific changes in t R N A electron spectrum in 230-450 range, indicating the direct binding of the metal ion to the nitrogen atom in the heterocyclic bases of t R N A [51].
EFFECTS ON PROTEINS Cu like m a n y other metals, zinc, cobalt, iron, nickel, magnesium and calcium, is a necessary component of enzyme activation and a disturbance in Cu content leads to poisoning of the cell [52]. Within the cells and vascular fluids of the organs, Cu forms stable complexes and chelates with organic molecules. Although ionic Cu is in dissociation equilibrium with most organic molecules, several proteins have been identified that require Cu as part of their molecular structure. Following absorption, Cu(II) is transported by serum albumin and transcuprein to the liver where it is incorporated into the plasma. Cu-protein, ceruloplasmin (Cp), is possibly stored as Cu-metallothionein or as superoxide dismutase. Ceruloplasmin is the long-term Cu transporter and carries Cu(II) to the tissues for the biosynthesis of key Cu(II) enzymes, especially cytochrome c oxidase, lysyl oxidase and others, acting as the centerpiece of Cu metabolism and function. It has a molecular weight of 132 000 D, has now been totally sequenced and the Cu-containing active sites located. There are seven possible functions for ceruloplasmin, which are expedited by ceruloplasmin receptors identified [53-57]. Metallothionein (MT) is often a major Cu-binding protein in the liver, particularly of Cu loaded animals. It plays an important role in the cellular detoxification of Cu and also is involved in the uptake, storage and transfer of Cu [58,59]. Metallothioneins are low molecular weight proteins (10 000 D) with a high cysteine content but without any aromatic amino acids or leucine [53,60]. These are difficultto isolate and characterize because they readily aggregate. There are conflicting reports concerning the effects of Cu
7 injections on metallothionein levels. Some authors have claimed clear evidence of increased M T levels in the liver of Cu-injected rats [61]. Others have suggested that the Cu-induced protein in rat liver is not metallothionein but another protein termed copper-chelatin [62].
MODE OF ACTIONOF COPPER
As mutagen Cu(II) ion is a potential mutagenic agent due to its property of inducing infidelity on DNA synthesis in vitro [63,64]. Copper compounds have been recorded to be mutagenic in animal cells in culture [65,66]. The results are not the same in bacteria, where copper sulphate was shown to be nongenotoxic using the bacterial colorimetric assay - the SOS chromotest [67]. In X174 single-stranded bacteriophage DNA, Cu(II) caused extensive damage and induced mutagenesis, possibly through the release of adenine residues [68]. As carcinogen There is as yet no proof for a positive correlation between Cu exposure and cancer. The increased incidence of lung cancer reported among workers in Cu refineries and Cu ore mines [80,101,102] has been attributed to the concomitant presence of arsenic compounds [103]. On the other hand, some Cu compounds seem to inhibit the development and growth of malignant tumour cells [104]. The Cu transport protein, Cp was suggested to be involved in carcinogenesis. Cp mRNA has been recorded in much higher amounts in tumor cells than normal ones in rats liver [105]. As teratogen Cu can penetrate the placental barrier into the fetus [106]. Low CuSO4containing diet fed to mice stimulated embryonic development. High levels however, increased fetal mortality, reduced the weight and produced malformation [107]. Four milligrams Cu/kg, given intramuscularly early in pregnancy, affected the development of the central nervous system (CNS) of the fetuses [108]. Deficiency of trace elements including Cu and proteins was associated with early abortion in goats [109] and a decrease in the fertilization rate and number of live offspring per dam with a concomitant increase in the gestation period, in rabbits [110]. Cu inhibited the 17fl-estradiol (E) binding to tissue cytoplasmic receptors in vitro and interfered with the hormone (steroid) delivery to the target cell nuclei in rat uterus [111]. Chronic administration (1 mg) to rabbits daily for 74 days, reduced the number of viable spermatozoa [112] and induced infertility in rats. The testicular Cu concentration increased significantly. The damage observed included vacuolation, karyorrhexis, pycnosis, cytolysis in the pachytene spermatocytes and early spermatids. Clumps of foreign particles, apparently metallic Cu, were found in the interstices of the caput together with the degenerative changes in the epithelial cells. The effect of
Cu appeared to be on the epididymal epithelium. Decrease in viability was marked. The major cause of infertility after Cu injection was due to the direct inhibition on the sperm. Damage to the seminiferous and epididymal epithelium was also recorded [113]. Incubation of human spermatozoa with metallic Cu, induced irreversible immobilization [113,115]. Microscopic analysis of sperm in copper-deficient mice revealed a higher sperm count as compared to the normal, accompanied by changes in the acrosomal and flagellum regions. Motility was lowered by 10 to 50%. Infertility in these mice was probably due to an impairment of testicular Cu transport which resulted in a decline in Cudependent processes [116]. Nicotine protected spermatozoa against the toxicity of Cu ions [117]. On the other hand, the teratogenicity of triethylenetetramine could be reduced by dietory Cu supplementation [118]. The widespread use of intrauterine devices containing metallic Cu is of relevance in this connection since these devices raise the endometrical Cu concentration 2-fold [119,120] and this excess might be transferred to the fetus [121].
As clastogen Nuclei have been shown to respond to changes in the metal content of their environment [122]. The specificity of heavy metal binding in vivo and in vitro might be in part, a function of pre-existing states of condensation of the chromatin [123]. Thus, the loss of binding specificity observed at low ionic strength might be a reflection of decondensation phenomenon. Genetic implications of differential condensation are suggested by the fact that highly condensed chromatin has low template activity for RNA synthesis and DNA replication whereas less condensed chromatin, treated with various polyanions, shows release of template restrictions and marked increase in both RNA synthesis [124] and DNA replication [125]. Decay of radioactive isotope of e4Cu and 67Cu resulted in a high lethal efficiency in the mammalian cell line, possibly due to an injury inside the cellular DNA. Even though present in trace amounts, Cu atoms are essential chromatin components [126]. Cu accumulated in the liver in response to increased dietary intake. Since it is a normal constituent of liver nuclei and chromatin fractions, an elevation in total Cu was accompanied by significant increase in nuclear Cu. The metal preferentially accumulated in the heterochromatic portions of the nucleus in response to such an elevation both in vivo and in vitro, under isotonic conditions [127]. The biological implication of normal localization of Cu in heterochromatin and euchromatin and its accumulation in heterochromatin under experimental conditions remains to be clarified [127]. The presence of Cu, with other metals like iron and magnesium, in chromosomes has been reported by several workers. Cu acts on the chromatin substance, becoming tightly associated to form Cu-rich nucleoprotein complex [128]. Reports of the existence of metallic bonds linking DNA between carboxyl groups of proteins in the cell nuclei of mammalian
tissue have further strengthened the view of metal-chromatin interaction. Cu compounds induced chromosomal aberrations and abnormal cell divisions in animal cells [33,66]. It is reported that chronic administration of copper sulphate orally to Rattus norvegicus induced chromosomal breaks in proportions directly related to the concentrations used. The duration of treatment had very little effect. The effect of Cu was primarily at the G2 phase [129]. An impairing effect of Cu-salts on chromosomes in vitro in the presence of hydrogen peroxide and ascorbic acid has been reported [130132]. Cupric salts increased the frequency of non-complementary nucleotides in the synthesized DNA-double helix [133]. The practical relevance of these findings is not yet known [134]. Copper significantly (positive from 0.05 to 0.06 mM) enhanced the transformation of Syrian hamster ovary cells by Simian adenovirus 7 (SA7) [135]. Crystalline CuS particles were actively phagocytosed by cells and potently induced morphological transformation of the Syrian hamster embryo cells in a concentration-dependent manner. In contrast, the amorphous forms, at both cytotoxic and non-cytotoxic exposure levels, were relatively less toxic [136]. The CHO cells exposed to crystalline CuS (10 ftg/ml for I h) induced DNA breaks, indicative of the selective and specific effect of metal ion on DNA [137]. Cu als0 acts as an enhancer of other clastogens. Addition of Cu 2+ to the saliva sample of betel nut and Indian tobacco enhanced the clastogenic activity (chromosomal breaks and exchanges) in CHO cells [138] and the damage produced in plasmid DNA by microwave [139]. Phenylthiourea (PTU) enhanced the cytotoxicity of copper in chick embryo pigmented epithelial cells (PEC) as well as in other cell lines like human oral carcinoma RB cells and mouse neuroblastoma N-18 cells. Analogs of PTU had similar effects in presence of Cu. This enhancement was probably due to an increased uptake (approximately 6-fold) of the metal
[140]. Media depleted of Cu did not support normal T-lymphocyte proliferation but supported B-lymphocyte proliferation [141]. Cu had an inhibitory effect on volume regulating phase of Ehrlich ascites tumor cells. The primary effect of Cu was to increase the permeability of cell membrane to sodium [142]. In mouse glioma, in vitro, tubulin polymerization was completely inhibited by 2.5 × 10-4M Cu(II). Cu(II) at the growth inhibition concentrations showed no effect on microtubule network [143]. MUTATIONSAFFECTINGCOPPERMETABOLISM There are several known examples of mutations which influence Cu homeostasis in humans and animals. Pleiotropic effects are observed when the mutant gene disturbs Cu flux. In some cases, mutation alters the level of a specific Cu ligand (enzyme) and the clinical consequences are unique [69]. The two most widely studied genetic maladies linked with copper metabolism in humans are Menkes' and Wilson's diseases. Menkes' disease is an X-linked fatal disorder in which Cu accumulates in some organs
10 (intestine and kidney) and is low in others (liver and brain). It is characterized by the absence of metallothionein sequence from the X-chromosome [70-78] and has been suggested to be due to a defect in intracellular rather t h a n membrane t r a n s p o r t of copper [79]. Wilson's disease shows an autosomal recessive inheritance, in which Cu accumulates, if untreated in the liver and subsequently in the brain and kidney [80--86]. Pathophysiological consequences of Cu deficiency and toxicity characterize these two disorders. Specific mutations of h u m a n cupro-enzymes include over-production of Cu-Zn superoxide dismutase in the Down's syndrome; absence of tyrosinase in albinism, hereditary mitochondrial m y o p a t h y due to reduction in cytochrome c oxidase and altered lysyl oxidase in X-linked forms of cutis lax and Elders-Danlos syndrome [87]. Mutations altering Cu metabolism have also been identified in animals, including several murine ones. Such m u t a n t s have become valuable research tools for the study of specific h u m a n diseases of Cu metabolism and for elucidation of the biochemical role of copper [69]. The most extensively investigated ones are the mottled mice, in particular brindled ones, which have a m u t a t i o n analogous to t h a t of Menkes' disease [69,88-97]. A murine m u t a t i o n identified later is toxic milk (tx), an autosomal recessive disorder t h a t is characterized by Cu accumulation in liver [98]. Two other m u t a n t s , crinkled and quaking, earlier regarded to exhibit abnormal Cu metabolism, could not be confirmed from later observations. A m u t a t i o n has been found in Bedlingt0n terrier with very similar characteristics to Wilson's disease [99,100]. CONCLUSIONS
In mammalian cell components, both deficiency and excess of copper are shown to exhibit toxic effects, in vivo and in vitro. These effects can be related to the specific affinities of Cu(II) ions for individual components, including the nucleic acids and proteins and its preferential accumulation in the heterochromatic regions. ACKNOWLEDGEMENTS The authors are grateful to Professor A.K. Sharma, Programme Coordinator for facilities provided and to the Council of Scientific and Industrial Research and University Grants Commission for financial assistance. REFERENCES
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1
Elsevier Scientific Publishers Ireland Ltd.
Review Article
EFFECTS OF C O P P E R ON MAMMALIAN CELL C O M P O N E N T S
K A L P A N A AGARWAL, A R C H A N A S H A R M A and GEETA T A L U K D E R
Human Genetics Unit, Centre for Advanced Study, (Cell and Chromosome Research), Department of Botany, University of Calcutta~ 35 Ballygunge Circular Road, Calcutta-700 019 (India)
(Received February 27th, 1988) (Revision received June 16th, 1988) (Accepted June 20th, 1988)
SUMMARY
Both deficiency and excess of copper induce toxic effects on mammalian cell s y s t e m s in vivo and in vitro. The effects can be related to the affinities of Cu(II) ions for specific cell components. The nucleus is a potential site for temporary Cu storage while primary targets for free Cu(II) ions are the thiol groups which reduce the ions to Cu(I). Cu(II) ions show a high affinity for nucleic acids, binding with DNA both at intrastrand and interstrand levels, possibly through intercalation between GC pairs. The ability to chelate Cu(II) ions is seen to be of the order: p u r i n e > p u r i n e ribonucleotides > purine ribonucleoside > pyrimidine ribonucleotides. Copper is an integral part of enzyme activation and enters into the molecular structure of several proteins, like ceruloplasmin. Cu(II) ion is a potential mutagenic agent as seen by its property of inducing infidelity in DNA synthesis in vitro. Teratogenic activities of copper have been reported but carcinogenicity is not yet confirmed. Copper is an essential component of chromatin and is known to accumulate preferentially in the heterochromatic regions. External application of higher doses, however, induces both clastogenic effects and spindle disturbances. In certain forms, inorganic copper enhances the clastogenic activity of other agents. The most widely studied h u m a n genetic maladies linked with copper metabolism are Menkes' and Wilson's diseases. Several mutations are k n o w n which influence Cu homeostasis in mammals. Such m u t a t i o n s in mice have been used extensively for biochemical studies. K e y words: Copper cytotoxicity - Metal cytotoxicity
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2 INTRODUCTION Copper is an essential micronutrient and is found in all mammalian tissues, bound to proteins or other organic molecules. Relatively higher a m o u n t s are present in liver, followed by brain and heart. Both enhancement and deficiency of the normal levels of copper have been related to adverse effects on the organism. The information on biological consequences of copper accumulation, at nuclear and cellular levels, following environmental exposure of h u m a n populations is however, meagre. The metal is of particular interest because of its preferential accumulation in the heterochromatic regions of the/chromosomes both in vivo and in vitro and its ability to destabilise DNA and to inhibit RNA polymerase activity in vitro. EFFECTS ON DNA The influence of metal ions upon the conformation of DNA was realised early, when it became apparent that these ions are involved in the stabilization of the Watson-Crick double helix [1,2]. Different metal ions, under a variety of conditions, were found to produce different effects upon the structure of polynucleotides [3-7]. These effects also included degradation of the ribose phosphate backbones by scission of the phosphate bonds [8-13]. Differential centrifugation analyses indicated that the nuclear fraction contains approximately 20% of the total hepatic copper in the adult mammals. Since the nucleus is made up of nucleic acids and several basic proteins, all of which bind copper, this organelle is a potential site for temporary copper storage. A portion of the nuclear Cu is involved in cellular metabolism and the polynucleo~ide strands are bridged by metal ions [14]. The biochemical basis for Cu toxicity is still not absolutely clear. Studies on isolated cells and organelles have shown that excessive a m o u n t s of Cu(II) ions labilized cellular membranes [15,16]. The primary targets for free cupric ions in the membranes were the thiol groups which are capable of reducing Cu(II) to Cu(I) form upon concomitant oxidation to disulfides in the membrane [17]. The Cu(I) ions formed could subsequently be re-oxidized to the Cu(II) form in the presence of molecular oxygen. Molecular oxygen, thereby converted to the toxic superoxide radical 02, could induce lipoperoxidation [16,18]. Ribonucleic acid, DNA and some of their constituents show a surprising strong affinity for cupric ion. The strength of the interaction of cupric ion with DNA and RNA m a y indicate an important role of these compounds in the biochemistry of copper and also m a y be significant in assessing the biological function of DNA and RNA in tissues. The formation of complexes by nucleotides is far superior to that of most amino acids and equivalent, if not superior, to many of the proteins [19]. In general, transition metals interact with DNA and polynucleotides both, by complexing with the phosphate group and under suitable conditions with
3 the electron donor sites on the bases as well [20]. The ultraviolet spectrum of native DNA was unaffected by Cu(II) ions, but the spectrum of denatured DNA shifted and enhanced, in line with the previous studies, indicating that Cu reacted with the bases in DNA. Increasing concentrations of Cu increased the spectral intensity upto a ratio of between one and two Cu ions per nucleotide base. The presence of the four nucleosides had no effect on the potentiometric titration of Cu, indicating that the binding of Cu to the bases proceeded without removal of protons. The presence of adenosine, did, however, eliminate the hysteris effect in the titration of Cu and the binding of the nucleoside to Cu was further indicated by the ability of the latter to shift the visible absorption of Cu to lower wavelengths. Cu was bound to the N7 positions of adenosine and guanine and to N1 of cytidine, but did not bind to thymidine. The phosphate resonance in deoxyadenosine monophosphate and deoxythymidine monophosphate was broadened by Cu ions. This indicated that Cu also binds phosphate, which was also demonstrated by potentiometric titration of Cu adenosine monophosphate and Cu DNA solution. The ability of Cu ions to cleave the phosphodiester bonds in polynucleotides, as well as to denature DNA, was thus explained by the demonstration that Cu could bind to both the phosphate and base moieties [21-26]. The binding of cupric ion with salmon sperm DNA was sensitive to changes in the metal concentration. It was time dependent at 25 ± 5°C and remained constant between pH 5.6 and 6.6 [24]. The stability of the Cu(ID nucleoside bond was not enough to overcome the attractive forces between the two DNA strands at room temperature. Rise in the temperature weakened the DNA structure, stabilising the Cu complex enough to compete effectively. At low ionic strength, the stability of the double helix did not exceed that of the Cu complex sufficiently to allow reformation of native DNA at an appreciable rate. A large increase in ionic strength, however, increased the stability of the double helix relative to the copper complex to such an extent that the native structure was again formed. Studies on absorbance and the optical rotation of the denatured product of the Cu(II) denatured DNA indicated that the two strands unwind. On the other hand, the sedimentation data showed that the denatured DNA was very highly aggregated. The apparently complex renaturation accompanying the addition of electrolyte can be best explained by assuming that the Cu ions, bound to the bases, keep the bases in register, even in the denatured state. The model which could fulfil these requirements indicated that in the denatured state Cu(II) ions were interposed between complementary strands by coordination to the bases in such a manner that all hydrogen bonds were broken and the secondary structure of each strand completely destroyed. Similar structures have been proposed for the Cu complexes of nucleotides [19]. A structure of this type represents a separation of the complementary strands of DNA [27]. At the present state of knowledge, the Cu(II) ions have been found to
show the highest affinity for nucleic acids amongst trace elements present in living organism. The specific interstrand binding of Cu(II) ions in the DNA-Cu(II) complexes has been suggested to be due to intercalation of Cu(II) between GC pairs, involving a change in deoxyguanosine conformation around the glycosyl bond from the anti to a syn position and formation of a G-Cu-C complex [28]. Another suggestion ascribed the specificity of Cu(II) binding to a G C c u C G structure [25]. Studies of the interaction of Cu(II) ions with native and denatured DNA as a function of ionic strength by the equilibrium dialysis method, confirmed the occurrence of interstrand and intrastrand binding of Cu(II) with DNA. Prasal [29] proposed a new molecular model of Cu(II)-DNA binding, assuming interstrand intercalation of one Cu(II) ion between two GC pairs, both in the successive even and odd groups, and interstrand binding of Cu(II) to the isolated GC pairs, with the exception of T-C-T and T-G-T sequences. This model indicates that the DNA-Cu(II) complex is most stable under the equilibrium with free Cu(II) ions at 4°C (pH 6.0). In these conditions, molar ratio of GC pairs to Cu(II) ions bound interstrandially is GC/Cu inter = 2 + 0.1 [29]. In this model, the chelates involving coordination of Cu(II) with N-7 and 0-6 of guanine bases, present in the model of F6rster et al. [25], are not included. On the other hand, the model contains mixed chelates involving N-3 of cytosine and phosphate residues, as well as the chelates proposed in the model of Zimmer et al. [28]. The reaction of mercury(II) with DNA resembled the Cu(II) DNA system, but much of the secondary structure remained in the denatured form. The mechanism of the reversal was quite different, since Hg(II) complex was formed in the presence of high electrolyte concentration and decomposed by competing ligands without change in the ionic strength [30-32]. Cu(II) ion increased the mutagenic potential of thymidine hydrogen peroxide and ascorbic acid. Soluble salts of Cu and other metals like silver, cadmium, cobalt, chromium, beryllium and others increased errors in cellfree DNA synthesis [33] and also the error frequency of avian myeloblastosis virus polymerase. It inhibited the human beta polymerase strongly at low concentrations. Cu reacted instantly and completely with sulfhydryl compounds such as mercaptoethanol to form Cu-S-C2HsOH. At 37°C, in presence of CuSO4, the rate of polymerisation decreased with time. The inhibition was constant at pH 8.6 to 7 and only 50% of reversal was possible. Inhibition was not initiated through the binding of the metal ion to the template or to the deoxynucleoside triphosphate but was probably due to a direct interaction of the metal with the enzyme [20]. In combination with Cu(II), isoniazid and other related hydrazine compounds induced unscheduled DNA synthesis (UDS) in cultured human fibroblasts [34]. The enhancement of DNA repair was lowered by Cu [34]. Copper acetate suppressed strongly the incorporation of tritiated thymidine
into rat liver DNA which was stimulated by injection of dimethylnitrosamine (DMN) [35]. Crystalline copper sulfide particles induced a considerable reduction in the molecular weight of DNA, as determined by alkaline sucrose gradient, following a 24-h treatment with 10/~g/ml of CuS [36]. Cu in biological systems is not thought to play an important part in the formation of free hydroxyl radical because of its tendency of binding strongly (tightly) to molecules containing amino groups. However, complex Cu in the presence of H202 c a n bring about site-specific damage [37]. DNA treated with ascorbic acid in the presence of Cu(II) ion or Cu tripeptide complex, under aerobic conditions, released the bases from the DNA molecule to a significantly high level. Addition of catalase or chemical scavengers of hydroxyl radical to the reaction mixture prevented the release of base. Total amount of base released was equivalent to the amount of phosphate in the reaction mixture and was caused by activated oxygen or hydroxyl radical from the H202, formed by auto-oxidation of ascorbic acid in presence of Cu [38-40]. Maximal fragmentation of DNA occurred at concentration ratio of DNA/Cu at 4: 1. The binding of Cu to the DNA molecule was essential for DNA breaking action of ascorbic acid. Cu bound to DNA was more effective than free Cu(II) ion. Proteins, nitrogen gas, hydroxyl radical scavengers and catalase inhibited Cu-dependent degradation of DNA [37,41-45]. Fragmentation of DNA in presence of ascorbatecopper was due to the induction of alkali-labile bonds in the DNA. Reduced Cu-salt autoxide in aerobic conditions formed hydroxyl radicals, which were capable of damaging most of the biological molecules. Cu ions readily ligated to amino groups of proteins and other molecules. Formation of the OH- radical was as follows:
Cu 2+ + H202 -->Cu + + H20 + H + 2H20 --, 202 + 2H--, H202 + 02 Cu2+ + O2-~ Cu÷+ O2, Cu + + H 2 0 2 --'> C u 2+ + O H - + OH.
Hydrozable tannins and 1,10-phenanthroline-copper complex under limited oxygen supply induced multiple single-stranded breaks of DNA in the spacer segment while core DNA segment in the chromatin remained intact. After the accumulation of single-stranded breaks in both strands, double-strand cleavage of DNA to fragments of nucleosomal size became apparent [46,47]. Cu(II) ions also bond with the nitrous base and phosphate group. Stability of the complex is increased due to hydrogen bonding of the partially hydrated ions of Cu(II) with the phosphate groups [48]. Deoxy derivatives interact with Cu(H) ions more strongly than the corresponding ribose compounds [19].
EFFECTS ON RNA The order of ability to chelate Cu(II) ion amongst the members of the ribose series was found to be purine>purine ribonucleotides >purine ribonucleoside > pyrimidine ribonucleotides [19]. Cu inhibited R N A polymerase under all conditions. At low concentrations, Cu(II) ion also inhibited ribonucleases. Cu has high affinity for these enzymes and acts directly on all stages of synthesis of R N A including initiation, condensation of nucleotides, detachment of newly formed RNA. These effects are not reversed by the presence of crysteine [49]. In rats, copper chloride injections induced the synthesis of a messenger R N A ( m R N A ) in the liver which coded for a protein with the properties of a metallothionein [50]. In lower organisms, for example, Escherichia coli, binding of Cu to the transfer R N A (tRNA) enhanced the intensity of characteristic absorption bands in the range 500-900 nm. Cu 2+ binding induced specific changes in t R N A electron spectrum in 230-450 range, indicating the direct binding of the metal ion to the nitrogen atom in the heterocyclic bases of t R N A [51].
EFFECTS ON PROTEINS Cu like m a n y other metals, zinc, cobalt, iron, nickel, magnesium and calcium, is a necessary component of enzyme activation and a disturbance in Cu content leads to poisoning of the cell [52]. Within the cells and vascular fluids of the organs, Cu forms stable complexes and chelates with organic molecules. Although ionic Cu is in dissociation equilibrium with most organic molecules, several proteins have been identified that require Cu as part of their molecular structure. Following absorption, Cu(II) is transported by serum albumin and transcuprein to the liver where it is incorporated into the plasma. Cu-protein, ceruloplasmin (Cp), is possibly stored as Cu-metallothionein or as superoxide dismutase. Ceruloplasmin is the long-term Cu transporter and carries Cu(II) to the tissues for the biosynthesis of key Cu(II) enzymes, especially cytochrome c oxidase, lysyl oxidase and others, acting as the centerpiece of Cu metabolism and function. It has a molecular weight of 132 000 D, has now been totally sequenced and the Cu-containing active sites located. There are seven possible functions for ceruloplasmin, which are expedited by ceruloplasmin receptors identified [53-57]. Metallothionein (MT) is often a major Cu-binding protein in the liver, particularly of Cu loaded animals. It plays an important role in the cellular detoxification of Cu and also is involved in the uptake, storage and transfer of Cu [58,59]. Metallothioneins are low molecular weight proteins (10 000 D) with a high cysteine content but without any aromatic amino acids or leucine [53,60]. These are difficultto isolate and characterize because they readily aggregate. There are conflicting reports concerning the effects of Cu
7 injections on metallothionein levels. Some authors have claimed clear evidence of increased M T levels in the liver of Cu-injected rats [61]. Others have suggested that the Cu-induced protein in rat liver is not metallothionein but another protein termed copper-chelatin [62].
MODE OF ACTIONOF COPPER
As mutagen Cu(II) ion is a potential mutagenic agent due to its property of inducing infidelity on DNA synthesis in vitro [63,64]. Copper compounds have been recorded to be mutagenic in animal cells in culture [65,66]. The results are not the same in bacteria, where copper sulphate was shown to be nongenotoxic using the bacterial colorimetric assay - the SOS chromotest [67]. In X174 single-stranded bacteriophage DNA, Cu(II) caused extensive damage and induced mutagenesis, possibly through the release of adenine residues [68]. As carcinogen There is as yet no proof for a positive correlation between Cu exposure and cancer. The increased incidence of lung cancer reported among workers in Cu refineries and Cu ore mines [80,101,102] has been attributed to the concomitant presence of arsenic compounds [103]. On the other hand, some Cu compounds seem to inhibit the development and growth of malignant tumour cells [104]. The Cu transport protein, Cp was suggested to be involved in carcinogenesis. Cp mRNA has been recorded in much higher amounts in tumor cells than normal ones in rats liver [105]. As teratogen Cu can penetrate the placental barrier into the fetus [106]. Low CuSO4containing diet fed to mice stimulated embryonic development. High levels however, increased fetal mortality, reduced the weight and produced malformation [107]. Four milligrams Cu/kg, given intramuscularly early in pregnancy, affected the development of the central nervous system (CNS) of the fetuses [108]. Deficiency of trace elements including Cu and proteins was associated with early abortion in goats [109] and a decrease in the fertilization rate and number of live offspring per dam with a concomitant increase in the gestation period, in rabbits [110]. Cu inhibited the 17fl-estradiol (E) binding to tissue cytoplasmic receptors in vitro and interfered with the hormone (steroid) delivery to the target cell nuclei in rat uterus [111]. Chronic administration (1 mg) to rabbits daily for 74 days, reduced the number of viable spermatozoa [112] and induced infertility in rats. The testicular Cu concentration increased significantly. The damage observed included vacuolation, karyorrhexis, pycnosis, cytolysis in the pachytene spermatocytes and early spermatids. Clumps of foreign particles, apparently metallic Cu, were found in the interstices of the caput together with the degenerative changes in the epithelial cells. The effect of
Cu appeared to be on the epididymal epithelium. Decrease in viability was marked. The major cause of infertility after Cu injection was due to the direct inhibition on the sperm. Damage to the seminiferous and epididymal epithelium was also recorded [113]. Incubation of human spermatozoa with metallic Cu, induced irreversible immobilization [113,115]. Microscopic analysis of sperm in copper-deficient mice revealed a higher sperm count as compared to the normal, accompanied by changes in the acrosomal and flagellum regions. Motility was lowered by 10 to 50%. Infertility in these mice was probably due to an impairment of testicular Cu transport which resulted in a decline in Cudependent processes [116]. Nicotine protected spermatozoa against the toxicity of Cu ions [117]. On the other hand, the teratogenicity of triethylenetetramine could be reduced by dietory Cu supplementation [118]. The widespread use of intrauterine devices containing metallic Cu is of relevance in this connection since these devices raise the endometrical Cu concentration 2-fold [119,120] and this excess might be transferred to the fetus [121].
As clastogen Nuclei have been shown to respond to changes in the metal content of their environment [122]. The specificity of heavy metal binding in vivo and in vitro might be in part, a function of pre-existing states of condensation of the chromatin [123]. Thus, the loss of binding specificity observed at low ionic strength might be a reflection of decondensation phenomenon. Genetic implications of differential condensation are suggested by the fact that highly condensed chromatin has low template activity for RNA synthesis and DNA replication whereas less condensed chromatin, treated with various polyanions, shows release of template restrictions and marked increase in both RNA synthesis [124] and DNA replication [125]. Decay of radioactive isotope of e4Cu and 67Cu resulted in a high lethal efficiency in the mammalian cell line, possibly due to an injury inside the cellular DNA. Even though present in trace amounts, Cu atoms are essential chromatin components [126]. Cu accumulated in the liver in response to increased dietary intake. Since it is a normal constituent of liver nuclei and chromatin fractions, an elevation in total Cu was accompanied by significant increase in nuclear Cu. The metal preferentially accumulated in the heterochromatic portions of the nucleus in response to such an elevation both in vivo and in vitro, under isotonic conditions [127]. The biological implication of normal localization of Cu in heterochromatin and euchromatin and its accumulation in heterochromatin under experimental conditions remains to be clarified [127]. The presence of Cu, with other metals like iron and magnesium, in chromosomes has been reported by several workers. Cu acts on the chromatin substance, becoming tightly associated to form Cu-rich nucleoprotein complex [128]. Reports of the existence of metallic bonds linking DNA between carboxyl groups of proteins in the cell nuclei of mammalian
tissue have further strengthened the view of metal-chromatin interaction. Cu compounds induced chromosomal aberrations and abnormal cell divisions in animal cells [33,66]. It is reported that chronic administration of copper sulphate orally to Rattus norvegicus induced chromosomal breaks in proportions directly related to the concentrations used. The duration of treatment had very little effect. The effect of Cu was primarily at the G2 phase [129]. An impairing effect of Cu-salts on chromosomes in vitro in the presence of hydrogen peroxide and ascorbic acid has been reported [130132]. Cupric salts increased the frequency of non-complementary nucleotides in the synthesized DNA-double helix [133]. The practical relevance of these findings is not yet known [134]. Copper significantly (positive from 0.05 to 0.06 mM) enhanced the transformation of Syrian hamster ovary cells by Simian adenovirus 7 (SA7) [135]. Crystalline CuS particles were actively phagocytosed by cells and potently induced morphological transformation of the Syrian hamster embryo cells in a concentration-dependent manner. In contrast, the amorphous forms, at both cytotoxic and non-cytotoxic exposure levels, were relatively less toxic [136]. The CHO cells exposed to crystalline CuS (10 ftg/ml for I h) induced DNA breaks, indicative of the selective and specific effect of metal ion on DNA [137]. Cu als0 acts as an enhancer of other clastogens. Addition of Cu 2+ to the saliva sample of betel nut and Indian tobacco enhanced the clastogenic activity (chromosomal breaks and exchanges) in CHO cells [138] and the damage produced in plasmid DNA by microwave [139]. Phenylthiourea (PTU) enhanced the cytotoxicity of copper in chick embryo pigmented epithelial cells (PEC) as well as in other cell lines like human oral carcinoma RB cells and mouse neuroblastoma N-18 cells. Analogs of PTU had similar effects in presence of Cu. This enhancement was probably due to an increased uptake (approximately 6-fold) of the metal
[140]. Media depleted of Cu did not support normal T-lymphocyte proliferation but supported B-lymphocyte proliferation [141]. Cu had an inhibitory effect on volume regulating phase of Ehrlich ascites tumor cells. The primary effect of Cu was to increase the permeability of cell membrane to sodium [142]. In mouse glioma, in vitro, tubulin polymerization was completely inhibited by 2.5 × 10-4M Cu(II). Cu(II) at the growth inhibition concentrations showed no effect on microtubule network [143]. MUTATIONSAFFECTINGCOPPERMETABOLISM There are several known examples of mutations which influence Cu homeostasis in humans and animals. Pleiotropic effects are observed when the mutant gene disturbs Cu flux. In some cases, mutation alters the level of a specific Cu ligand (enzyme) and the clinical consequences are unique [69]. The two most widely studied genetic maladies linked with copper metabolism in humans are Menkes' and Wilson's diseases. Menkes' disease is an X-linked fatal disorder in which Cu accumulates in some organs
10 (intestine and kidney) and is low in others (liver and brain). It is characterized by the absence of metallothionein sequence from the X-chromosome [70-78] and has been suggested to be due to a defect in intracellular rather t h a n membrane t r a n s p o r t of copper [79]. Wilson's disease shows an autosomal recessive inheritance, in which Cu accumulates, if untreated in the liver and subsequently in the brain and kidney [80--86]. Pathophysiological consequences of Cu deficiency and toxicity characterize these two disorders. Specific mutations of h u m a n cupro-enzymes include over-production of Cu-Zn superoxide dismutase in the Down's syndrome; absence of tyrosinase in albinism, hereditary mitochondrial m y o p a t h y due to reduction in cytochrome c oxidase and altered lysyl oxidase in X-linked forms of cutis lax and Elders-Danlos syndrome [87]. Mutations altering Cu metabolism have also been identified in animals, including several murine ones. Such m u t a n t s have become valuable research tools for the study of specific h u m a n diseases of Cu metabolism and for elucidation of the biochemical role of copper [69]. The most extensively investigated ones are the mottled mice, in particular brindled ones, which have a m u t a t i o n analogous to t h a t of Menkes' disease [69,88-97]. A murine m u t a t i o n identified later is toxic milk (tx), an autosomal recessive disorder t h a t is characterized by Cu accumulation in liver [98]. Two other m u t a n t s , crinkled and quaking, earlier regarded to exhibit abnormal Cu metabolism, could not be confirmed from later observations. A m u t a t i o n has been found in Bedlingt0n terrier with very similar characteristics to Wilson's disease [99,100]. CONCLUSIONS
In mammalian cell components, both deficiency and excess of copper are shown to exhibit toxic effects, in vivo and in vitro. These effects can be related to the specific affinities of Cu(II) ions for individual components, including the nucleic acids and proteins and its preferential accumulation in the heterochromatic regions. ACKNOWLEDGEMENTS The authors are grateful to Professor A.K. Sharma, Programme Coordinator for facilities provided and to the Council of Scientific and Industrial Research and University Grants Commission for financial assistance. REFERENCES
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