Biochimica et Biophysica Acta 1428 (1999) 201^208 www.elsevier.com/locate/bba
Bilirubin-Cu(II) complex degrades DNA S. Farhan Asad, Saurabh Singh, Aamir Ahmad, S.M. Hadi * Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202 002, India Received 19 January 1999; received in revised form 21 April 1999; accepted 28 April 1999
Abstract It has recently been reported that bilirubin forms a complex with Cu(II). In this paper we show that the formation of the complex results in the reduction of Cu(II) to Cu(I) and the redox cycling of the metal gives rise to the formation of reactive oxygen species, particularly hydroxyl radical. The bilirubin-Cu(II) complex causes strand breakage in calf thymus DNA and supercoiled plasmid DNA. Cu(I) was shown to be an essential intermediate in the DNA cleavage reaction by using the Cu(I) specific sequestering reagent neocuproine. Bilirubin-Cu(II) produced hydroxyl radical and the involvement of active oxygen species was established by the inhibition of DNA breakage by various oxygen radical quenchers. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Bilirubin ; Copper; Endogenous DNA damage; Prooxidant; Oxygen radical
1. Introduction Bilirubin is the end product of heme catabolism and has been the subject of interest because of its toxicity under conditions of hyperbilirubinemia. Under physiological conditions plasma bilirubin concentrations range from 5 to 17 WM [1], practically all of which is bound to albumin [2]. Under certain disease conditions such as kernicterus and jaundice plasma concentrations may reach considerably higher values. Concentrations greater than 300 WM are associated with the risk of development of neurological dysfunctions due to deposition of bilirubin in brain and its enhanced toxic e¡ects on cellular functions in this tissue [3,4]. Several other toxic e¡ects of
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bilirubin on cellular functions, particularly inhibition of several membrane bound enzymes, have also been reported [5]. The precise mechanism of bilirubin toxicity is uncertain. However, bilirubin may also have a bene¢cial role as albumin bound bilirubin is considered one of the naturally occurring antioxidants of human extracellular £uids [6]. Ames and coworkers have shown that albumin bound bilirubin is an e¤cient antioxidant against peroxyl radical induced oxidation in vitro [7]. Studies in our laboratory have shown that several of the proposed biological antioxidants, of both plant and animal origin, such as uric acid [8], £avonoids [9] and tannic acid [10,11] are themselves capable of acting as prooxidants either alone or in the presence of transition metal ions. The prooxidant reactions were shown to cause fragmentation of DNA and proteins. Copper is an essential constituent of many enzymes such as tyrosinase and superoxide dismutase. It has been reported to be a normal component of chromatin and such endogenous copper can be mo-
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 0 7 5 - 6
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bilized by chemical agents such as 1,10-phenanthroline to cause internucleosomal DNA fragmentation [12]. Copper has also been reported to be neurotoxic as evidenced by the brain pathology produced in patients with copper overload as a result of Wilson's disease [13]. In a recent report it was shown that bilirubin forms a complex with copper in the absence or presence of albumin in an aqueous solution [14]. In this paper we show that upon formation of the complex, Cu(II) is reduced to Cu(I) and this is accompanied by the generation of reactive oxygen species. Further, we also present evidence for the ¢rst time to show that the bilirubin-Cu(II) complex is capable of causing strand scission in DNA. 2. Materials and methods 2.1. Materials Calf thymus DNA (sodium salt; average molecular weight 1U106 ), S1 nuclease, bathocuproine and neocuproine were from Sigma (St. Louis, MO). Bilirubin was purchased from Aldrich (Milwaukee, WI). Supercoiled plasmid pBR322 DNA was prepared according to standard methods [15]. All reagents were prepared fresh. Bilirubin was dissolved in 5 mM NaOH as a 1^2 mM stock solution just prior to experimentation. Upon addition to reaction mixtures, in the presence of bu¡ers and at the concentrations used, bilirubin remained in solution. The volumes of stock solutions added did not lead to any appreciable change in the pH of the reaction mixtures. All the experiments were conducted in the dark to prevent photoisomerization of bilirubin. 2.2. Reaction of bilirubin with calf thymus DNA and digestion with S1 nuclease Reaction mixtures (0.5 ml) contained 10 mM TrisHCl (pH 7.5), 500 Wg of DNA, varying amounts of bilirubin and cupric chloride. Divalent metal ions or free radical scavengers were included in some experiments as indicated. Incubation was performed at 37³C. All solutions were sterilized before use. S1 nuclease digestion was performed as described previously [16]. Acid soluble deoxyribonucleotides were determined colorimetrically [17].
2.3. Reaction of bilirubin with plasmid pBR322 DNA Reaction mixtures (30 Wl) contained 10 mM TrisHCl (pH 7.5), 0.50 Wg of plasmid DNA, varying amounts of bilirubin and cupric chloride as indicated in the legends. After incubation at 37³C, 10 Wl of a solution containing 40 mM EDTA, 0.05% bromophenol blue tracking dye and 50% (v/v) glycerol was added and the solutions were subjected to electrophoresis in 1% agarose gels. The gels were stained with ethidium bromide (0.5 Wg/ml), viewed and photographed on a transilluminator. 2.4. Assay of OH radicals generated by bilirubinCu(II) The method of Quinlan and Gutteridge [18] was followed without modi¢cation except that bilirubin replaced rifamycin S.V. The bu¡er was 10 mM Tris-HCl (pH 7.5) and contained increasing concentrations of bilirubin (10^200 WM). Final concentrations of CuCl2 and calf thymus DNA were 200 WM and 200 Wg respectively. After incubation for 2 h at 37³C in the dark, malondialdehyde formed from deoxyribose radicals was assayed by addition of thiobarbiturate and the resulting adduct was determined colorimetrically at 532 nm. 3. Results 3.1. Absorption spectra of bilirubin in the presence of Cu(II) Recently Adhikari et al. have shown that bilirubin forms a 1:1 complex with copper with a characteristic absorption maximum at 343 nm in aqueous solutions [14]. We have determined the spectral changes in bilirubin on addition of Cu(II) as a function of time (Fig. 1). Fig. 1 shows the absorption spectrum of bilirubin (15 WM) alone and after addition of Cu(II) (30 WM) at 0, 15 and 60 min. In agreement with the results of Adhikari et al. a hypsochromic shift in the absorption of bilirubin is seen on addition of Cu(II). However, it is also seen that the addition of Cu(II) to bilirubin has a quenching e¡ect on the absorption as seen in Fig. 1a where the spectrum has been recorded immediately after addition of Cu(II)
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(time 0). Secondly, the formation of the complex absorbing at 343 nm is a slow process and is possibly not complete even 1 h after the addition of Cu(II) to bilirubin. Also an intermediate species absorbing at 407 nm is formed before the eventual appearance of the maximum at 343 nm. 3.2. Reduction of Cu(II) to Cu(I) in the bilirubinCu(II) complex We employed bathocuproine as a selective Cu(I) sequestering agent. The Cu(I) chelates have absorption maxima at 480 nm [16,19]. Under our experimental conditions, neither Cu(II) nor bilirubin interferes with this maximum, whereas bilirubin and Cu(II) react to generate Cu(I) (Fig. 2). The implication of this ¢nding is that Cu(II) is reduced by bilirubin in the complex to generate Cu(I).
Fig. 2. Detection of bilirubin induced Cu(I) production by bathocuproine. The 2 ml reaction mixtures contained 10 mM Tris-HCl and indicated amounts of bilirubin, Cu(II), Cu(I) and bathocuproine. The spectra were recorded immediately on addition of components indicated. Bathocuproine was 300 WM in all cases. (^T^) bathocuproine+15 WM bilirubin+50 WM CuCl2 ; (...) bathocuproine+25 WM Cu(I); (^.^) bathocuproine+15 WM bilirubin; (9) bathocuproine+50 WM CuCl2 .
3.3. Production of hydroxyl radicals by the bilirubin-Cu(II) complex
Fig. 1. E¡ect of time on the absorption spectra of bilirubin in the presence of Cu(II). The concentrations of bilirubin and Cu(II) in a total of 2 ml reaction mixture containing 10 mM Tris-HCl (pH 7.5) were 15 WM and 30 WM respectively. (9) bilirubin alone; (^.^) bilirubin+Cu(II). Spectra recorded at time 0 (a), 15 min (b), 1 h (c).
We have previously shown that several metabolites including antioxidants such as uric acid [20], L-DOPA [21] and the £avonoid quercetin [22] are able to bind and reduce Cu(II) to Cu(I). It involves the reoxidation of Cu(I) by molecular oxygen to generate superoxide anion and other reactive oxygen species such as the hydroxyl radical [16]. Fig. 3a shows that bilirubin-Cu(II) complex generates hydroxyl radicals that react with calf thymus DNA. The assay is based on the fact that degradation of DNA by hydroxyl radicals results in the release of TBA reactive material which forms a colored adduct with TBA readable at 532 nm [18]. With increasing concentrations of bilirubin an increasing amount of TBA reactive material is produced. In the absence of Cu(II) the rate of formation of such material is considerably reduced. The experiment was also done with increasing copper concentrations at a ¢xed concentration of bilirubin. The results con¢rm the observation as a dose dependent response with copper is also seen (Fig. 3b).
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Fig. 3. Formation of hydroxyl radicals as a function of bilirubin and Cu(II) concentrations. (a) E¡ect of increasing concentrations of bilirubin : (R) in the presence of 200 WM CuCl2 ; (b) in the absence of Cu(II). (b) E¡ect of increasing concentrations of Cu(II): (R) in the presence of 200 WM bilirubin; (b) in the absence of bilirubin. The reaction mixtures were incubated at 37³C for 2 h in darkness. All points represent triplicate samples and mean values are plotted. See text for details.
3.4. Breakage of calf thymus and plasmid pBR322 DNA In Figs. 4a,b and 5a,b we directly show that degradation of DNA occurs when incubated in the presence of bilirubin and Cu(II). Bilirubin and Cu(II) generated S1 -sensitive sites in calf thymus DNA [16]. The reaction was assessed by recording the proportion of double stranded DNA converted to acid soluble nucleotides by S1 nuclease. Control experiments established that heat denatured DNA resulted in 100% conversion whereas native DNA resulted in V10% conversion in the presence of Cu(II) (0.5 mM). Bilirubin generated a dose dependent increase in calf thymus DNA-S1 sensitive sites (Fig. 4a). A similar increase in DNA hydrolysis was observed when calf thymus DNA was incubated with 0.5
mM bilirubin and with increasing concentrations of Cu(II) (Fig. 4b). Supercoiled plasmid pBR322 DNA was examined as a substrate as the relaxation of such a molecule is a sensitive test for just one nick per molecule which results in its conversion to the open circular form. Bilirubin converted supercoiled DNA to relaxed open circles and linear forms in a copper dependent reaction and at higher concentrations the molecules were converted to progressively smaller heterogeneously sized fragments (Fig. 5a). Similar results were obtained with increasing Cu(II) concentrations (Fig. 5b). The full conversion to relaxed forms is seen at a 20 WM concentration of Cu(II) as compared to a 10 WM concentration of bilirubin. Further, it was of interest to establish whether Cu(I) played a role in the DNA breakage reaction. Fig. 6 shows the e¡ect of increasing concentrations of neocuproine (2,9-dimethyl 1,10-phenanthroline), another Cu(I) sequestering agent, on bilirubinCu(II) mediated DNA cleavage. The concentration of Cu(II) in the reactions was 50 WM. Complete inhibition of the conversion of supercoiled molecules to the relaxed form is seen only at neocuproine concentrations of 200 WM and above, whereas the stoichiometry of the copper-neocuproine complex is copper1 (neocuproine)2 [Cu1 (Ne)2 ] [12]. At relatively lower concentrations of 25 WM and 50 WM an enhancement in the extent of DNA degradation is observed (lanes 3 and 4) as suggested by the formation of heterogeneously sized small molecules. We are unable to explain this result at present. However, it has been suggested by Burkitt et al. that a complex of Cu(I) with 1,10-phenanthroline binds to the DNA minor groove and is available for the generation of Table 1 S1 nuclease hydrolysis following damage to DNA by bilirubin and transition metal ions using 0.2 mM bilirubin and 0.2 mM metal ion solutions Metal ion Cu(II) Fe(II) Ca(II) Mn(II) Mg(II)
DNA hydrolyzed (%) Metal ion+bilirubin
Metal ion
23.8 2 ^ ^ ^
^ ^ ^ ^ ^
Experiments were carried out in triplicate and mean values are reported.
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Fig. 4. Degradation of calf thymus DNA by the bilirubin-Cu(II) complex. (a) E¡ect of bilirubin concentration in the presence of 500 WM CuCl2 . (b) E¡ect of CuCl2 concentration in the presence of 500 WM bilirubin. The reaction mixtures were incubated at 37³C for 4 h in darkness.
hydroxyl radicals resulting in DNA strand cleavage [12]. Possibly, this is the case when the concentrations of the [Cu1 (Ne)2 ] complex are relatively lower. Irrespective of the precise interpretation of results it is clear that sequestration of Cu(I) by neocuproine inhibits the DNA cleavage reaction.
dismutase and catalase remove superoxide anion (O3c 2 ) and hydrogen peroxide (H2 O2 ) respectively, whereas sodium benzoate, mannitol, potassium io-
3.5. E¡ect of alternative metal ions Of the several metal ions tested only Cu(II) complemented bilirubin in the DNA breakage reaction (Table 1). 3.6. Involvement of free radicals in the reaction The bilirubin-Cu(II) DNA breakage reaction was inhibited by various radical scavengers (Table 2). Sodium azide is a singlet oxygen scavenger, superoxide
C
Fig. 5. Agarose gel electrophoretic pattern of ethidium bromide stained pBR322 DNA after treatment with bilirubin and Cu(II). Reaction mixtures were incubated at 37³C for 1 h in darkness. (a) E¡ect of increasing concentrations of bilirubin in the presence of Cu(II). Lane 1, DNA alone; lanes 2^6, 10, 20, 50, 100 and 200 WM bilirubin in the presence of 100 WM Cu(II). (b) Effect of increasing concentrations of Cu(II) in the presence of bilirubin. Lane 1, DNA alone; lanes 2^6, 10, 20, 50, 100 and 200 WM Cu(II) in the presence of 100 WM bilirubin.
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Fig. 6. E¡ect of increasing concentrations of neocuproine on the bilirubin-Cu(II) induced breakage of pBR322 DNA. Reaction mixtures were incubated at 37³C for 1 h in darkness. Lane 1, DNA alone; lane 2, DNA+bilirubin (50 WM)+Cu(II) (50 WM); lanes 3^8, lane 2+25, 50, 100, 200, 400 and 600 WM neocuproine.
dide and thiourea eliminate hydroxyl radicals (OHc ). Thiourea completely inhibited the DNA breakage; sodium benzoate, potassium iodide and mannitol also showed varying degrees of appreciable inhibitions, indicating the involvement of hydroxyl radicals in the reaction. Sodium azide and catalase also substantially inhibited the reaction indicating that H2 O2 and singlet oxygen (1 O2 ) may also be involved. 4. Discussion The principal ¢ndings of the above experiments may be stated as follows: (i) the binding of bilirubin to Cu(II) results in its reduction to Cu(I) and the redox recycling gives rise to the formation of reactive
oxygen species, particularly hydroxyl radical; (ii) the reaction is capable of causing strand breakage in DNA. Bilirubin is considered an antioxidant of the human extracellular £uid capable of scavenging free radicals such as peroxide radical [7]. However, there are numerous reports on the cytotoxicity and DNA damaging ability of bilirubin on photoillumination [23,24]. Rosenstein et al. have shown that bilirubin in the presence of light generates hydrogen peroxide and possibly other peroxides that can cause DNA damage [25]. As already mentioned, antioxidants are known to act as prooxidants under certain conditions. Thus, bilirubin appears to have similar properties. It cannot be said whether the bilirubin-Cu(II) mediated DNA damaging reaction is possible in vivo. In order for such a reaction to occur several conditions have to be met. Bilirubin should be available in a free form, it should be able to traverse the cell and nuclear membranes and free or loosely bound copper is present. Most bilirubin in plasma is found either tightly bound to albumin or as a conjugate with glucuronic acid. The latter reaction is catalyzed by the enzyme glucuronyl transferase. In the neonate the activity of the glucuronydating enzyme in liver is low and bilirubin accumulates. Because of its lipophilicity and membrane permeability it is able to traverse membrane barriers naked and alone and unassociated with proteins [26]. Further, it has been observed that when the molar concentration of bilirubin is considerably greater than its normal physiological concentration, it is not very tightly bound to albumin [27]. This would suggest that in pathological conditions such as hyperbilirubinemia of the newborn or
Table 2 Inhibition of S1 nuclease sensitivity of calf thymus DNA after treatment with bilirubin and Cu(II) (0.2 mM each) in the presence of radical scavengers Quencher
DNA hydrolyzed (%)
Inhibition (%)
Control Control+catalase (0.1 mg/ml) Control+SOD (0.1 mg/ml) Control+sodium azide (50 mM) Control+potassium iodide (50 mM) Control+mannitol (50 mM) Control+thiourea (50 mM) Control+sodium benzoate (50 mM)
24.1 14.4 21.8 10.9 12.5 17.7 0 13.5
^ 40.2 9.5 54.7 48.1 26.5 100 43.9
SOD: superoxide dismutase. Control: bilirubin (200 WM)+Cu(II) (200 WM). Experiments were carried out in triplicate and mean values are reported.
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certain liver diseases some of the excess bilirubin may be available for secondary reactions. It has been reported that normal serum contains up to 8 WM loosely bound copper. Other biological £uids may also contain similar amounts in the low micromolar ranger [28,29]. Loosely bound copper is de¢ned by Gutteridge as that copper which is available for binding to the chelating agent 1,10-phenanthroline. It is possible that such loosely bound copper can be mobilized by bilirubin. As already mentioned, copper is a normal component of chromatin and can be mobilized by metal chelating agents.
[10]
Acknowledgements
[13]
Two of the authors (S.F.A. and S.S.) gratefully acknowledge the ¢nancial assistance rendered by the University Grants Commission (UGC), New Delhi. A.A. would like to acknowledge the Council of Scienti¢c and Industrial Research (CSIR), New Delhi for the same. CSIR is also acknowledged for ¢nancial help vide Grant 37(0982)/98/EM-II.
[9]
[11]
[12]
[14]
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