ELSEVIER Chemico-Biological Interactions 96 (1995) 103-I I 1

Release of iron from ferritin by 1,2,4-benzenetriol

Sarfaraz Ahmad, Vinita Singh, Gondi S. Rao*

Industrial Toxicology Research Cenrre, Post Box No. 80. M.G. Marg, Lucknow- 001, India

Received IO May 1994; revision received 29 August 1994; accepted 31 August 1994

Abstract

Release of iron from ferritin in the presence of polyhydroxy metabolites of benzene i.e., hydroquinone (HQ) or 1,2,4_benzenetriol (BT) was studied in acetate buffer, pH 5.6. The re- lease of iron from ferritin was quantitated by monitoring the formation of iron-ferrozine com- plex. The presence of hydroquinone (330 PM) did not result in the release of iron from ferritin, whereas the same concentration of BT resulted in the release of significant amounts of iron (3.2 pM/min) from ferritin. BT concentration-dependent increase in iron release from ferritin was observed although the increase was not linear with the concentration of BT. Under a N, atmosphere the presence of BT resulted in the release of iron (2.1 pM/min) from ferritin. The presence of oxyradical scavengers i.e., albumin, catalase or superoxide dismutase significantly inhibited iron release from ferritin by BT. The iron released from ferritin by BT enhanced lipid peroxidation in rat brain homogenate and released aldehydic products from bleomycin- dependent degradation of DNA. Addition of BT to bone marrow lysate resulted in an increase of iron release as a function of time. These studies indicate that BT is a potent reductant of ferric iron of ferritin and also mobilizes and releases iron from ferritin core. The release of iron from bone marrow lysate by BT may‘be of toxicological significance as this could lead to disruption of intracellular iron homeostasis in bone marrow cells.

Keywork 1,2,4_Benzenetriol; Ferritin; Autooxidation; Iron; Lipid peroxidation

1. Introduction

Iron is an essential element for living organisms because of its role in major bio- logical reactions such as the tricarboxylic acid cycle, electron transport, nitrogen

Abbreviations: BT, 1.2.4~benzenetriol; DNA, deoxyribonucleic acid; Ferrozine, 3-(2-pyridyl)-5,6- bi@phenylsulfonic acid)-l,2,4-triazine; HQ, hydroquinone; 6-OHDA, 6-hydroxydopamine; TBAR, thiobarbituric acid reactive products.

* Corresponding author.

0009-2797/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-2797(94)03575-S

104 S. Ahmad et al. / Chemico-Biological Inrrrtrcrions 96 f 1995 ) 103- I ii

fixation, DNA synthesis and toxication and detoxication reactions [l]. Iron also

generates oxygen species by promoting the conversion of superoxide anion and

hydrogen peroxide into the very reactive hydroxyl radical through the Haber-Weiss reaction [2]. Ferritin serves as an iron storage protein and can store 4500 atoms of ferric iron per molecule [3]. The mechanisms of iron uptake, mobilization, release

and utilization are well understood [4]. Low molecular weight iron chelates which

maintains a dynamic equilibrium between iron uptake, storage and utilization have

been described [5]. Mobilization of iron from ferritin requires reduction of ferric iron to ferrous iron [6]. The ferritin complex has six shallow ‘pockets’ through which

iron is deposited or mobilized [7] and this process requires reduction of ferric iron to ferrous iron and the potential physiological reductants have been identified [8]. Superoxide anion radical generated by xanthine-xanthine oxidase system [9] and many toxicological reductants which mobilize ferritin iron have been reported.

Redox cycling of paraquat [lo], the semiquinone radical of adriamycin [ 111, nitric

oxide [ 121, 6-hydroxydopamine [ 131, certain polyhydroxypyrimidines [14] and some therapeutic and physiological iron chelators [ 151 have been shown to promote mobi- lization and reductive release of iron from ferritin. Certain diphenols in the presence

of a reductant have also been shown to release iron from ferritin [ 161. Iron released from ferritin has been shown to catalyze lipid peroxidation [ 13,14,17].

Hydroquinone (HQ) and 1,2,4-benzenetriol (BT), the two principal polyphenolic metabolites of benzene, have been shown to be toxic. The suggested mechanism

includes free radical formation via superoxide and the covalent binding of the semi- quinones to DNA, RNA or other cellular macromolecules [18]. The hydroquinone moiety of the antibiotic rifamycin reacts with molecular oxygen to form reduced oxy- gen intermediates such as superoxide and hydrogen peroxide [19]. HQ or BT has

been shown to degrade glutamate, deoxyribose or DNA in the presence of transition metal ions via reactive oxygen species [20,21]. The aim of the present study is to investigate the potential of hydroquinone and 1,2,4-benzenetriol to release iron from

ferritin and whether the iron released can catalyze oxidation reactions.

2. Materials and methods

Ferritin (horse spleen), ferrozine, dopamine, &hydroxydopamine, catalase (bovine liver), superoxide dismutase (bovine erythrocyte), bovine serum albumin and calf thymus DNA were obtained from Sigma (St. Louis, MO). HQ and BT were obtained from Aldrich, (Milwaukee, WI). Bleomycin was obtained from Nippon

Kayaku Co. Ltd., Japan. All other chemicals used were of analytical grade. Femur and brain were obtained after sacrificing albino rats maintained at ITRC animal col- ony. Bone marrow cells from four femurs were flushed out by 0.15 M NaCl. The cells of bone marrow pellet were lysed by resuspension in approximately 1.5 ml distilled water. This suspension was homogenized in Ultra Turrax T25 for 30 s and the volume was made up to 2.0 ml with acetate buffer to a final buffer concentration of 0.033 M. Brain homogenate (2%) was made in 0.15 M KCl.

Iron release from ferritin was investigated by using the assay procedure previously

described [16]. Briefly, the assay system (total volume 1.5 ml) contained 100 pg ferri-

S. Ahmad et al. / Chemico-Biological Interactions 96 (1995) 103-111 105

tin, 500 PM ferrozine and the desired concentration of HQ or BT (0.0-330 PM) in 0.033 M acetate buffer, pH 5.6. The reaction was started by the addition of HQ or BT and the formation of [Fe(ferrozine)J2+ was monitored by recording the increase in absorbance at 562 nm with time and the amount of iron released from ferritin is expressed as PM Fe*+/min.

The effect of oxyradical scavengers was studied by the addition of oxyradical scav- enger before the addition of BT (see Table 2). The time course of formation of [Fe(ferrozine)s] 2+ released from bone marrow cell lysate was assayed in a total volume of 1.5 ml containing 100 ~1 bone marrow cell lysate, 500 PM ferrozine, 330 PM BT in 0.033 M acetate buffer, pH 5.6. The amount of iron released is expressed as PM Fe*+/mg protein.

The effect of increasing concentrations of BT on lipid peroxidation of 2% rat brain homogenate in the presence of ferritin was followed. Briefly, the assay system (total volume 1.5 ml) contained 100 pg ferritin and BT (0.0-330 PM) in 0.033 M acetate buffer, pH 5.6. The contents were incubated for 30 min after which 2.0 ml of 2% brain homogenate was added and incubated for 1 h at 37°C. The thiobarbituric acid reactive products (TBAR) were measured as described [22]. The values are expressed as nmol malonaldehyde equivalents generated in 1 h by using E5s2 = 1.56 x lo5 cm-‘M-l.

The effect of increasing concentrations of BT on iron catalysed bleomycin- dependent degradation of DNA in the presence of ferritin was evaluated. Briefly, the assay system (total volume 1.5 ml) contained 100 pg ferritin and BT (0.0-165 PM) in 0.033 M acetate buffer, pH 5.6. The contents were incubated for 30 min after which 250 pg calf thymus DNA and 50 pg bleomycin HCl in 0.3 ml was added and incubated for 30 min at 37°C. The TBAR formed from DNA were measured and the values are expressed as described earlier.

Table I Effect of benzenetriol (BT), hydroquinone (HQ), 6-hydroxydopamine (6-OHDA) and dopamine (DA) on

the release of iron from ferritin

Addition Amount of iron released per min

(PM Fe’+imin)*

Benzenetriol

Hydroquinone

6-Hydroxydopamine

Dopamine

3.2 f 0.11

2.10 f 0.18 (anaerobic)

ND

4.6 + 0.15

0.3 f 0.01

Reaction mixture contained in a final volume of 1.5 ml, ferritin (100 agil .5 ml), BT (330 PM) /HQ (330

PM) /6-OHDA (125 PM) and ferrozine (500 PM) in 0.1 M sodium acetate buffer of pH 5.6. Measurements

of Fe2+-ferrozine complex followed at 562 nm due to iron mobilization from ferritin by different

polyphenols. Amount of iron released is in gM Fe’+/min.

*All values are averages f S.E. of two sets of experiments conducted in duplicate.

ND, None detectable.

106 S. Ahmad et al. / Chemico-Biological Inreracrions 96 { 199s) 103-f I I

Fig. 1. BT concentration dependent iron release from ferritin. The total assay mixture of 1.5 ml contained

0.033 M acetate buffer, pH 5.6, ferritin 100 gg, ferrozine 500 pM and the reaction was started by the addi-

tion of 0.0-330 pM BT. Measurements of iron released from ferritin was calculated in terms of pM

Fe’+/min after monitoring the absorbance of [Fe(ferrozine)J]2c at 562 nm with time.

3. Results

Release of iron from ferritin in the presence of polyphenols i.e., hydroquinone or 1,2,4-benzenetriol was recorded in Table 1. Addition of HQ (330 PM) did not result in the release of iron from ferritin. However, the same concentration of BT resulted in a significant release of iron (3.2 pM/min) from ferritin. The release of iron was found to be linear up to 15 min and about 20% of total ferritin iron was released. Similarly, addition of dopamine (DA) failed to release iron from ferritin, whereas

Table 2

Release of iron from ferritin by BT and the inhibition by oxygen radical scavengers

Reaction mixture pM Fe2+ released/min”

Complete reaction mixWeb 3.2 f 0.1 I

+ Urea (5 mM)’ 2.88 f 0.24 (lO)d

+ Thiourea (5 mM) 2.64 f 0.08 (18)

+ Mannitol (50 mM) 2.72 f 0.10 (15)

+ Albumin (150 &I.5 ml) 2.32 f 0.02 (28)

+ Catalase (150 &I.5 ml) 2.00 * 0.05 (38)

+ SOD (150 pgIl.5 ml) 2.16 zrz 0.12 (33)

aAll values are averages f SE. of two sets of experiments conducted in duplicate.

bComplete reaction mixture contained in a final volume of I .5 ml, ferritin (100 ag), BT (330 pM), fer-

rozine (500 PM), 0.1 M sodium acetate buffer of pH 5.6 (500 )rl) and oxygen radical scavengers.

CNumbers in parentheses are concentration of oxyradical scavenger.

dNumbers in parentheses are percent inhibition.

S. Ahmud et al. / Chemico-Biological Interacrions 96 (1995) 103-111 107

- 6.0-

.

62.5 165 2475 330

BT (/Al)

Fig. 2. Effect of increasing concentration of BT on lipid peroxidation of 2% rat brain homogenate in the presence of ferritin. Experimental details are given in Materials and methods.

6-hydroxydopamine (6-OHDA) released large amounts of iron (4.6 rM/min) from ferritin as was reported earlier [ 131. The release of iron from ferritin was concentra- tion dependent on BT as is shown in Fig. 1, although the increase was not linear with the concentration of BT. Significant amounts of iron release from ferritin was observed in a N2 atmosphere, although to a lesser extent than in aerobic conditions (Table 1).

Table 2 represents the effect of oxyradical scavengers on the release of iron from ferritin in the presence of BT. The release of iron was substantially inhibited as was evident from the decreased formation of [Fe(ferrozine)s]*+ in the presence of albu- min, catalase and superoxide dismutase. Thiourea and mannitol which are hydroxyl radical scavengers were able to inhibit iron release to an extent of about 15%. This

5.0 -

33 66 99 132 165

BTWI)

Fig. 3. Effect of increasing concentration of BT on iron catalysed bleomycin-dependent degradation of DNA. Experimental details are given in Materials and methods.

108 S. Ahmad et al. / Chemico- Biological Inreracrions 96 ( 1995) 103-I I I

indicates that BT mediated release of iron was not completely due to the mediation of active oxygen species generated during autooxidation of BT.

The effect of addition of increasing concentrations of BT to ferritin and the effect on lipid peroxidation of rat brain homogenate is shown in Fig. 2. The increase in lipid peroxidation as measured by TBAR formation was almost linear with the increase in BT concentration which shows that there is BT concentration dependent increase in iron release. Similarly, the iron released from ferritin in the presence of increasing concentrations of BT was tested for the iron catalysed bleomycin- dependent degradation of DNA. There was a linear increase in TBAR formation from the deoxyribose moiety of DNA with the increase in concentration of BT (33-165 PM) (Fig. 3).

Release of iron from bone marrow cell lysate in the presence of HQ and BT show- ed that HQ did not release any iron from bone marrow cell lysate (results not shown) whereas BT (330 PM) resulted in the release of iron from bone marrow cell lysate. The increase in the release of iron from bone marrow lysate in the presence of BT as a function of time was also observed (Fig. 4).

E 3 6 9 12 15 k

Time{ min)

Fig. 4. Time course of formation of [Fe(ferrozine)j] ?+ followed at 562 nm due to iron mobilization from

bone marrow cell lysate by BT. The total assay volume of 1.5 ml contained 0.033 M acetate buffer, pH

5.6, ferrozine 500 pM, femur bone marrow cell lysate 100 pl and the reaction was initiated by adding BT

(330 pM) and the iron released was calculated in terms of PM Fe2+/mg protein.

S. Ahmad et al. / Chemico-Biological Interactions 96 (1995) 103-111 109

4. Discussion

Benzene exposure interferes with iron metabolism which includes, decreased in- corporation of 59Fe into circulating erythrocytes [23] and accumulation of iron in bone marrow which has been shown to catalyze bleomycin-dependent degradation of DNA [24]. However, the mechanism of how benzene interferes with iron metabo- lism is not known. Polyphenolic metabolites of benzene have been shown to accumu- late in bone marrow against the concentration gradient [25,26]. Autooxidation of BT has been shown to generate superoxide radicals [27]. Superoxide radical is a potent reductant of ferritin iron and causes release of iron from ferritin [9]. In the present investigation it was observed that the presence of BT induced iron release from ferri- tin in a concentration dependent manner. The release of iron from ferritin during autooxidation of BT in vivo may result in increased intracellular concentrations of free iron. The inability of HQ or DA to release iron in comparison to BT and 6- OHDA suggests that the hydroxyhydroquinone moiety is essential. Similarly, dialuric acid, a tetrahydroxy pyrimidine and isouramil, a trihydroxy-pyrimidine have been shown to release iron from ferritin [14]. Although, the physiological mechanism of iron mobilization from ferritin is poorly understood, the observed iron release from ferritin by BT could occur through the direct reduction of iron by the hydroxyhydroquinone form or via its autooxidation intermediates i.e., super- oxide radical and semiquinone.

The presence of oxyradical scavengers i.e., albumin, catalase or SOD caused only 28,38 or 33% inhibition, respectively of BT dependent iron release from ferritin with the enzymatic activities of catalase and SOD being of little importance relative to the protein effect of albumin. The semiquinone radicals of several antitumorigenic compounds have been shown to reduce and release ferritin iron [ 11,281. The ox- yradical scavenger insensitive rate of iron release from ferritin by BT is probably due to direct reduction of ferritin iron by BT, as substantial amounts of iron were releas- ed in a Nz atmosphere also. Based on the present investigation, it is proposed that probably BT releases iron from ferritin after direct reduction and the iron released may facilitate the reduction of 0, by BT resulting in the generation of superoxide radical. The autooxidation rate of BT has been shown to be 92-fold higher than that of the parent hydroquinone lacking an -OH substituent [29]. The presence of HQ or DA did not result in iron release from ferritin, probably due to the lack of an -OH substituent and the resulting slow autooxidation.

The results of the present study demonstrate that iron released from ferritin in the presence of BT is capable of inducing lipid peroxidation and catalyzing bleomycin- dependent DNA degradation. The mechanism by which iron is released from bone marrow cells and whether it acts as a prooxidant during benzene toxicity is not known. However, earlier studies had demonstrated that enhanced lipid peroxidation was observed in experimental animals exposed to benzene [30,31]. Finally, polyphe- nolic metabolites of benzene have been shown to accumulate in bone marrow in millimolar concentrations during benzene exposure [25]. The present study indicated that a much lower concentration of BT (66 PM) is able to release iron from ferritin. Involvement of a similar mechanism in vivo as BT dependent reduction and release

110 S. Ahmad et al. / Chemico-Biological Interactions 96 ( 1995) IO3- I I I

of iron from storage sites could result in increased generation of active oxygen species which in turn damage cellular macromolecules and also can interfere in maturation and differentiation of erythroblasts. In a recent study [32] we have shown that iron in the presence of BT was a potent agent to catalyze bleomycin- dependent degradation of DNA. Similarly, the iron released from ferritin by BT could catalyze bleomycin-dependent degradation of DNA.

In summary, the present study offers a new mechanism that the iron released from ferritin by BT could contribute to the better understanding of the toxicity of ben- zene. In the light of the present results, it is believed that the BT-induced iron release in bone marrow cells in vivo leads to generation of superoxide radicals, peroxidation of lipids and probably affects maturation and differentiation of bone marrow cells.

Acknowledgements

The authors wish to thank Dr. R.C. Srimal, Director, Industrial Toxicology Research Centre for his encouragement and interest in the work. Thanks are due to ICMR, New Delhi for grant-in-aid, Mr. Lakshmi Kant for typographical assistance and Mr. Ram La1 for microphotography.

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