Hemin-mediated DNA Strand Scission*

Hemin (ferric protoporphyrin IX chloride) has been shown to cause strand scission in DNA in a reaction which requires the presence of oxygen and the reducing agent, 2-mercaptoethanol. In model studies, circu- lar supercoiled plasmid DNA is converted within 30 min to the open circle and linear forms. With longer incubation times the DNA is degraded to small pieces. The reaction is markedly influenced by the addition of divalent cations; Mg2+ and Ca2+ inhibit the reaction while the transition metals Go2+, Zn2+, Ni2+, and Cu2+ promote the degradation. These observations are dis-cussed in relation to the role of hemin in the modulation of gene expression during cell differentiation.

*This work was supported by Grants CA-07175 and CA-23076 from the National Cancer Institute, United States Public Health Service, and National Institutes of Health Training Grants CA-09020 and CA-09230. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisemnt" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Incubations were carried out at 20 "C in uncapped Microfuge tubes.
The reactions were initiated by adding 2-mercaptoethanol and terminated with 5 pl of sample buffer consisting of 50% glycerol, 3 mM EDTA, and 0.1% bromphenol blue. The samples were immediately loaded on either a 1% neutral agarose gel containing 20 mM Tris-HCl, 1.5 mM sodium acetate, 50 M M EDTA (pH 8.8), or a 1% alkaline agarose gel containing 20 mM glycine, 20 mM NaCl, 75 mM NaOH, and 2 mM EDTA and electrophoresed in a horizontal slab gel apparatus. The gel was stained with a solution of 1 pg/ml of ethidium bromide for 20 min. The alkaline agarose gel was first soaked in a solution of 0.5 M Tris-HC1 (pH 7.0), 0.1 M NaCI, 1 mM EDTA for 45 min prior to staining with the ethidium bromide. The DNA bands were detected and photographed using a long wavelength ultraviolet light source.
The linear form of pBR322 DNA which was used as a marker was prepared by digesting pBR322 with the restriction enzyme HindIII.

RESULTS
The initial observation in this study was that the incubation of supercoiled pBR322 DNA in the presence of hemin and 2mercaptoethanol produced strand breaks in the DNA. introduce breaks in the supercoiled plasmid DNA at concentrations as low as 10 PM (Fig. 2, lane 3). As the hemin concentration is increased, DNA is converted more rapidly to the open circle and linear forms.
Reduced oxygen species have been shown to be involved in DNA degradation (11,12). To determine whether oxygen plays a role in the hemin-mediated nicking of DNA, the reaction was carried out under either oxygen, air, or nitrogen atmospheres. As shown in Fig. 3, an atmosphere of nitrogen essentially precluded scission of DNA (lanes 5-7). In an atmosphere of 100% oxygen, the decrease of the supercoiled DNA (lanes 8-10), and the appearance of the linear form is more rapid than in air in which case some supercoiled DNA still remains after a 15-min incubation ( l a n e 4). Hemin alone or 2-mercaptoethanol alone, even in an atmosphere of 100% oxygen, failed to cause measurable strand scission (lanes 12 and 11, respectively). These data clearly demonstrate the dependence of the hemin-mediated scission of DNA on the presence of all three reagents: oxygen, hemin, and 2-mercaptoethanol.
The hemin-mediated degradation of DNA is time-dependent and progressive (Fig. 4). In the first 10 min the DNA damage is largely reflected in a conversion of supercoiled DNA to the open circle form (compare lanes 3 and 4). By 20 min ( l a n e 5 ) this conversion appears complete, and further nicking leads to the appearance of linear forms and smaller DNA fragments that migrate as a smear in the gel. By 70 min of incubation ( l u n e 9), only small DNA fragments remain.
In search of additional insights into the mechanism of the hemin interaction with DNA, various cations were tested for their effects on the hemin-mediated DNA degradation reaction. Fig. 5 and Table I show the results of adding monovalent and divalent cations to the DNA, hemin, and mercaptuethanol reaction mixture. NaCl and KC1 at 50 and 100 PM levels had little or no effect on the amount of DNA degraded (lanes 5-7). Magnesium and calcium added at 50 PM (lanes 10  degradation of these products to smaller fragments was largely inhibited. At higher concentrations of magnesium and calcium, all of the DNA could be recovered as the nicked open circle and linear forms (data not shown). The reason for the reaction stopping at this point is not clear from available data; however, the possibility exists that calcium or magnesium precipitate or otherwise condense the DNA structure.

Effect of divalent transition cations on the level of hemin required for nicking of DNA
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Samples, in an atmosphere of air, were incubated for 15 min as described in the legend to Fig. 5 with a 50 p~ level of the indicated cation and hemin concentrations ranging from 0.5 to 50 pM. The DNA products were analyzed by electrophoresis in 1% neutral agarose gels. The level of hemin required to produce a destruction of DNA comparable to that obtained with 50 p~ hemin and 10 mM 2mercaptoethanol in the absence of cation additions was ascertained In contrast, the transition metal cations, zinc, cobalt, and copper, strikingly promoted both the initial nicking reaction and the subsequent degradation process leading to a total conversion of the plasmid DNA to small fragments in 15 min (luanes 9, 11, and 13). The addition of these metals alone or with 2-mercaptoethanol had no effect on the integrity of the DNA. A provocative aspect of these experiments was the finding that in the presence of these metals the efficacy of hemin in the reaction was increased, allowing for a significant reduction of the hemin concentration to achieve an equivalent amount of nicking and degradation of DNA as seen in a standard reaction mixture without the metaIs. Table I shows that Co2+ was the most effective in that a 50 PM level of this cation permitted a reduction of the hemin concentration to %OO the usual level. Zn2+ and Ni2+ were similarly effective in reducing the amount of hemin required for a comparable DNA degradation; Cu2' was the least effective of the transition cations tested.

DISCUSSION
This study demonstrates that hemin in the presence of 2mercaptoethanol and oxygen can introduce nicks into DNA. The reaction is rapid and leads to an extensive degradation of DNA to oligonucleotides. Although the chemical mechanism of the reaction was not elucidated, the requirement for a reducing compound and molecular oxygen suggests that a reactive reduced oxygen species, such as a hydroxyl radical, or an iron oxygen complex may be generated. Earlier findings have already demonstrated that oxygen radicals cause DNA strand scission (13,14).
In such a process it is projected that the ferric iron in hemin is first reduced to the ferrous form which can then react with molecular oxygen to produce the reactive species that attack the DNA. While not proven in these experiments, this oxidation-reduction process may well be cyclic. In this view, the mechanism of the hemin-mediated DNA strand scission is similar to the ferrous oxidase cycle proposed for iron chelated with bleomycin. In the latter reaction, ferrous-bleomycin can bind oxygen leading to the production of reduced oxygen species (15,16).
The contrasting effects of the different divalent cations on the degradative process is especially interesting when viewed from the aspect of their influence on DNA structure. While all of the tested divalent cations can interact with the phosphate residues in the DNA backbone, they have different affinities for the bases themselves (17). For example, Mg2' does not readily interact with the bases in double-stranded DNA whereas the transition metals do. Thus, in contrast to Mg+, whose interaction is suggested to contribute to a more compact structure of the DNA, Co2+ and related transition metal ions might open up the DNA structure for a more effective interaction or intercalation of hemin with this macromolecule. It is significant to this discussion that cations can alter DNA conformation to a more condensed form (18,19) or cause the helix to change from the B form to a lefthanded Z DNA structure (20,21). While we are not aware of any data showing that hemin can intercalate into DNA, this phenomenon has been demonstrated for certain synthetic porphyrins (22,23). In this connection, it is important to note that while this paper was in preparation, Fiel et al. (24) published data showing that synthetic metalloporphyrins with the capacity to intercalate into DNA can cause DNA strand scission; however, they did not report the dependency of the process on the presence of molecular oxygen.
Chelated iron complexes, such as the antitumor antibiotics bleomycin and doxorubicin have been shown previously to nick DNA in vitro (11,12) and in uiuo (25). This study with hemin, however, appears to be the first example of a physiological iron chelator that can nick DNA. Hemin possesses two charged carboxylic groups, and these could also be involved in the selective actions of the different divalent cations. However, it seems unlikely that a simple neutralization of the negatively charged carboxylic acid groups of the hemin and phosphates of the DNA underlies the cation effects since Na' and K' had no effect on the degradation process.
With respect to the possible relevance of the DNA nicking action by hemin in the differentiation of Friend erythroleukemia cells and immature red blood cells, it should be noted that heme accumulates in the nucleus as these cells differentiate (5, 26, 27), and becomes covalently linked to both proteins and DNA of the nucleus (28). Heme is also essential for the erythroid differentiation process (5,6,28). In Friend cells, nicks occur in the DNA during differentiation (8, 9) and the number of topological turns in the DNA appears to decrease (29). As the result of these correlations we are led to propose that hemin may implement the changes in gene expression associated with erythroid differentiation by the generation of localized reactive oxygen species with localized damaging effects on the chromatin structure.