Prooxidant and antioxidant properties of iron-hydroquinone and iron-1,2,4-benzenetriol complex....

ELSEVIER SCIENCE IRELAND Toxicology 89 (1994) 25-33

IOXICOIW

Prooxidant and antioxidant properties of iron-hydroquinone and iron-l,2,4-benzenetriol

complex. Implications for benzene toxicity

Vinita Singh, Sarfaraz Ahmad, G.S. Rao*

Industrial Toxicology Research Centre, P.O. Box 80, Lucknow-226 001, India

(Received 1 June 1993; accepted 28 September 1993)

Abstract

Bleomycin-dependent degradation of DNA in bone marrow cells was studied in vitro in the presence of iron or iron polyphenol chelates which are formed during biotransformation of benzene. Iron polyphenol chelates markedly enhanced bleomycin-dependent DNA degradation in comparison to iron alone. About 1.5 and 2.5-fold increase was observed in the presence of iron hydroquinone (HQ) chelate and iron 1,2,4-benzenetriol (BT) chelate, respectively. Endogenous iron chelators such as glutamate, citrate, aspartate, glycine, cysteine, dithiothreitol, AMP, ADP and ATP did not enhance iron-catalysed bleomycin-dependent degradation of DNA. By bleomycin assay, the recovery of iron polyphenol chelate added externally to bone marrow lysate was complete. However, the presence of iron polyphenol chelate resulted in less thiobarbituric acid reactive products from glutamate or brain homogenate than with iron alone. The optical spectra of BT were modified in the presence of ferrous sulphate, reveal- ing a new absorption peak at 259 nm indicating complexation with iron. Thus, the iron polyphenol chelate, on one hand, is a more potent DNA cleaving agent in the presence of bleomycin, and on the other hand, it is a less effective free radical generator as compared to iron alone.

Key words." Bleomycin; Iron; Low molecular weight iron components; Hydroquinone; i,2,4- Benzenetriol

* Corresponding author. Abbreviations." BT, 1,2,4-Benzenetriol; DNA, deoxyribonucleic acid; HQ, hydroquinone, LMI, low molecular weight iron components; TBAR, thiobarbituric acid reactive products.

0300-483X/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0300-483X(93)02723-T

26 1I. Singh et al. / Toxicology 89 (1994) 25-33

1. Introduction

Accumulation of polyphenols in bone marrow against the concentration gradient has been reported in benzene exposed rabbits (Porteous and Williams, 1949; Greenlee et al., 1981 a). Male Fischer rats exposed to benzene (500 ppm) have been shown to accumulate about 1 mM polyphenolic metabolites in bone marrow (Rickert et al., 1979). Hydroquinone (HQ) and 1,2,4-benzenetriol (BT) have been shown to be toxic (Irons, 1985), and the suggested mechanism includes superoxide-derived free radical formation during their autooxidation and covalent binding of semiquinones to DNA, RNA and other cellular macromolecules (Greenlee et al., 1981b). BT has been shown to induce single strand breaks in DNA of cultured cells (Pellack- Walker and Blumer, 1986) and also in supercoiled DNA (Lewis et al., 1988).

The existence of an intracellular pool of low molecular weight iron components (LMI) maintaining an equilibrium between iron uptake, storage and incorporation into its final biochemical form has been described (Jacobs, 1977). It has been suggested that iron is released from ferritin under conditions in which oxidative stress is involved both in vivo and in isolated perfused organs (Mazur and Carleton, 1965; Healing et al., 1990). A fraction of cellular non-haem iron consisting of chelatable LMI represents iron species which is catalytically active in initiating free radical reactions (Gut- teridge, 1987). In various pathological conditions this decompartmentalized iron is 'mal-placed'. Bound to bleomycin and in the presence of oxygen, LMI can degrade DNA (Gutteridge et al., 1985). We have earlier demonstrated a preferential accumulation of bleomycin-detectable LMI in benzene-exposed rat bone marrow (Pandya et al., 1990). Contemplating that such LMI is possibly a chelate of polyphenolic metabolites of benzene with iron, we have presently investigated the efficiency of HQ or BT iron chelates in catalysing bleomycin-dependent DNA degradation, glutamate degradation and lipid peroxidation in vitro.

2. Materials and methods

Femur bone marrow cells were isolated from female Wistar albino rats of the Industrial Toxicology Research Centre animal colony. Bone marrow cells were homogenized in distilled water and adjusted to pH 5.3 with 0.1 M acetate buffer. Bleomycin hydrochloride was obtained from Nippon Kayaku Co., Ltd., Japan, calf thymus DNA, L-glutamate monosodium were obtained from Sigma Chemical Company, St. Louis, USA. Hydroquinone and 1,2,4-benzenetriol were obtained from Aldrich, Milwaukee, USA. All other chemicals were either obtained from C.S.I.R. Centre for Biochemicals, New Delhi, India or were of analytical grade.

V. Singh et al. / Toxicology 89 (1994) 25-33 27

Iron-catalysed bleomycin-dependent DNA degradation was performed according to the method of Gutteridge (1987). Principally, bleomycin in the presence of LMI degrades DNA with the release of base propenals which react with 2-thiobarbituric acid (TBA) to give a chromogen similar to that given by malonaldehyde on reaction with TBA. Briefly, the assay system in a total volume of 3.0 ml consisted of 0.033 M acetate buffer pH 5.3, 500 #g calf thymus DNA, 50 #g bleomycin hydrochloride and the reaction was initiated by the addition of 20/~M iron or a complex of iron with polyphenol or of endogenous chelators (20 #M: 40 #M) which were made immediately before use. The contents were incubated at 37°C for 30 min and the reaction was terminated by the addition of 1.0 ml of 10% (w/v) TCA.

Release of thiobarbituric acid reactive products (TBAR) from glutamate was followed according to the method described (Gutteridge, 1981). Briefly, the reaction mixture (total volume 1.5 ml) contained 0.033 M sodium phosphate buffer, pH 7.4 in 0.15 M NaCI, 4 mM glutamate and the reaction was started by the addition of 20 #M iron or a complex of iron polyphenol (20 /~M: 40/~M). The contents were incubated for 15 min at 37°C and the reaction was terminated by the addition of 1.0 ml of 10% (w/v) TCA.

Lipid peroxidation in brain homogenate (2% in 0.15 M KC1) was assayed according to the method of Bernheim et al. (1948) by incubating either with 20/~M iron or iron polyphenol (20/~M: 40 ~M) or none in a metabolic shaker at 37°C. Aliquots (1.0 ml) were taken at intervals and the reaction was terminated by the addition of 1.0 ml of 10% (w/v) TCA.

The contents in the above experiments were centrifuged and to the clear supernatants, 0.67% (w/v) TBA was added and heated for 15 min in a boiling waterbath. The resultant TBA chromogen was read at 532 nm using a Spectronic-2001 spectrophotometer. The values are expressed as nmoles of malonaldehyde equivalents generated by using E532 - 1.56 x 105 cm -l M -1.

Recovery of iron polyphenol complex from bone marrow lysate (1 mg protein equivalent) was monitored by assaying iron polyphenol complex catalysed bleomycin-dependent degradation of DNA as compared to that without bone marrow lysate.

FeSOa.5H20 was used to prepare iron (20/~M) and iron chelates by the addition of iron and chelate in 1:2 ratio, i.e. 20/zM iron : 40 tzM chelate. Iron and iron chelate solutions were prepared just before use.

Optical spectra of polyphenols in freshly distilled water with or without the addition of FeSO4 • 5H20 were recorded on a Perkin-Elmer Lambda 15 UV/VIS spectrophotometer.

3. Results

Iron-catalysed and bleomycin-dependent degradation of DNA was

28 V. Singh et al. / Toxicology 89 (1994) 25-33

Table 1 Comparison of TBAR formation from DNA, glutamate or brain homogenate by iron (20 #M) and iron:polyphenol (20:40 #M) complex

Iron Iron:HQ Iron:BT

Bleomycin-dependent degradation of DNA 1.52 2.23 3.58 Iron-dependent degradation of glutamate 8.25 1.56 2.83 Iron-dependent lipid peroxidation in brain 67.14 41.13 38.05

homogenate

Experimental conditions as described in the Methods section. Values are represented as nmoles of TBAR. All values are averages of two sets of experiments conducted in duplicate with variations of 10% or less.

markedly greater in the presence of iron polyphenol complex than in the presence of iron alone (Table 1). Further, DNA degradation was faster with iron BT complex (2.4-fold) than with iron HQ complex (1.5-fold). The degradation of DNA showed an increase with increasing concentration of iron or iron polyphenol complex (Fig. 1).

3 . 0 -

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2.0- o

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ge2+(pM) Fig. l. Linearity of TBAR formation from DNA with increase in concentration of Fe 2+. The reaction mixture (total volume 3.0 ml) contained 0.033 M phosphate buffer, pH 7.4, 500 #g calf thymus DNA, 50 ~g bleomycin hydrochloride and the reaction was initiated by the addition of iron (O - - O), iron HQ complex (O - - O) or iron BT complex (A - - A). For details see Materials and methods. The means of at least two experiments are shown with the variation

of 10% or less.

v. Singh et al./ Toxicology 89 (1994) 25-33 29

Table 2 Recovery of iron: polyphenol (24/zM: 48/~M) complex added to bone marrow lysate for the bleomycin dependent degradation of DNA

Iron:HQ Iron:BT

(-) Bone marrow lysate 2.71 5.73 (+) Bone marrow lysate 3.33 7.67

Iron polyphenol complex was added to bone marrow lysate (1 mg protein equivalent) and the values are expressed as nmoles of TBAR formed from DNA. All values are averages of two sets of experiments conducted in duplicate with variations of 10% or less.

The i ron-dependen t release o f T B A R f rom glu tamate exhibited a decrease in the presence o f e i ther H Q or BT. The inhibi t ion o f i ron-dependen t release o f T B A R f rom glu tamate was 81% in the presence o f H Q and 66% in the presence o f BT (Table 1). Similarly, the i ron-dependen t lipid perox ida t ion in

brain homogena tes was substant ial ly inhibi ted in the presence o f H Q where it was 39% or BT where it was 43% (Table 1).

The iron po lypheno l complex added to bone m a r r o w lysate was 100% recoverable when assayed by the b leomyc in -dependen t degrada t ion o f D N A (Table 2). F o r m a t i o n o f T B A R f rom D N A by b leomycin in the presence o f iron, i ron po lypheno l or i ron with potent ia l endogenous i ron chelators is recorded in Table 3. The endogenous chelators viz. glucose, fructose,

Table 3 Formation of TBAR from DNA by bleomycin in the presence of iron (20 #M) or iron with the potential iron chelator (1:2 ratio)

nmoles of TBAR from DNA/30 min

Iron 1.52 Iron + HQ 2.67

+ BT 3.52 + Citrate 0.19 + Glucose 1.40 + Fructose 1.29 + Glycine 1.03 + Glutamic acid 1.53 + Cysteine 1.59 + Aspartic acid 1.09 + AMP 0.82 + ADP 0.92 + ATP 1.49 + Dithiothreitol 0.49

Experimental conditions as described in the Methods section. All values are averages of two sets of experiments conducted in duplicate with variations of 10% or less.


glutamic acid, cysteine and ATP did not affect the iron catalysed bleomycin- dependent TBAR formation from DNA. Whereas glycine, aspartic acid, AMP and ADP caused a significant inhibition, the presence of citrate and dithiothreitol resulted in a drastic inhibition of bleomycin-dependent iron catalysed TBAR formation from DNA (Table 3).

The addition of FeSO4.5H20 to HQ or BT in freshly distilled water revealed no change in the optical spectra of HQ. However, that of BT revealed a new absorption peak at 259 nm (Fig. 2A, 2B).

4. Discussion

During benzene exposure of experimental animals many oxidation-reduction reactions have been shown to occur (Irons, 1985; Lewis et al., 1988), which may release ferrous iron from ferritin. The ferrous iron may be chelated to the polyhydroxy metabolites of benzene, viz. HQ and BT. The

1.0-

0 .8 "

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< 0.4-

0.2-

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< o.4

0.2.

o.o. 200.00

o:oo .o' o s oo )~ (rim)

2.00 m.® . no .oo

( ~

Fig. 2. A. Optical spectra of HQ (100 #M) in freshly distilled water. Incubation mixtures contained 100 #M HQ alone or with 200 #M Fe 2+ and scans were recorded every minute up to 3 min. B. Optical spectra of BT (100 #M) in freshly distilled water. Incubation mixtures contained 100/~M BT alone or with 200/zM Fe 1+ and scans were recorded every minute up

to 4 min.

V. Singh et al. /Toxicology 89 (1994) 25-33 31

+ Fe(m) ~ ( I ) + 2H

i T BT iron complex

Fig. 3. The tentative chemical structure of BT iron complex.

tentative chemical structure of polyphenol iron complex for BT:iron(III) is diagrammatically presented in Fig. 3.

We propose that the complex of polyphenol with iron (Fig. 3) may be the LMI in bone marrow which has been reported to accumulate selectively during the exposure to benzene (Pandya et al., 1990). In the present study we observed that the presence of polyphenolic metabolites of benzene enhanced iron-catalyzed bleomycin-dependent degradation of DNA. The iron polyphenol complex appears to be stable and intact as the recovery from bone marrow lysate was complete. This is consistent with the earlier reports that certain phenolic compounds such as the flavonoids (rutin and quercetin) reveal an affinity for iron ions and act as iron chelators (Afanas'ev et al., 1989). These flavonoids were found to enhance iron ion-dependent damage to DNA by bleomycin and this was assumed to be due to reduction of ferric bleomycin to the ferrous form (Afanas'ev et al, 1989; Laughton et al., 1991). It is also possible that the HQ or BT possess combined iron-chelating and iron ion-reducing properties to stimulate DNA degradation in the presence of bleomycin. Further, the iron polyphenol complexes have shown an antioxidant activity by inhibiting iron-dependent lipid peroxidation in brain homogenate and glutamate degradation. This is similar to the antioxidative properties exhibited by flavonoids due to their ability to chelate iron (Afanas'ev et al., 1989; Laughton et al., 1991). Thus the flavonoids and polyphenolic chelates limit the availability of iron for lipid peroxidation and at the same time facilitate bleomycin-dependent degradation of DNA.

It has been shown recently that upon mixing HQ with Cu(II) the absor- bance of HQ at 289 nm decreased with a corresponding increase in absor- bance at 247 nm due to the formation of BQ (Li and Trush, 1993). However, the optical spectra of HQ did not show any change in the presence of ferrous sulphate. This indicates that HQ is not converted to 1,4-benzoquinone (BQ), and iron is kept in the ferrous state to catalyse oxidation reactions. The addition of ferrous sulphate to BT, on the other hand, revealed a new absorption peak at 259 nm with a slight decrease at 286 nm. These observations suggest a complex formation of polyphenol with iron rather than reduction of iron and oxidation of polyphenol.

It is concluded from the present study that the phenolic metabolites of


benzene enhance the availability of Fe(II) to bleomycin. Such Fe(II) is possibly made available due to reduction of Fe(III) by polyphenols, thus protecting the Fe(II) from hydrolysis to hydroxides and consequent autooxidation. The formation of such a complex in vivo is not known. Further studies are in progress to characterize such complexes in vivo and evaluate their toxicological significance in benzene-induced hematotoxicity.

5. Acknowledgements

The authors wish to thank the Indian Council of Medical Research for a Grant-in-aid, Mr. Lakshmi Kant for typographical assistance and Mr. Musleh Ahmad for microphotography.

6. References

Afanas'ev, I.B., Dorozhoko, A.I., Brodskii, A.V., Kostyuk, V.A., and Potapovitch, A.I. (1989) Chelating and free radical scavenging mechanisms of inhibitory action of rutin and quercetin and lipid peroxidation. Biochem. Pharmacol. 38, 1763.

Bernheim, F., Bernheim, M.L.C. and Wilbur, K.M. 0948) The reaction between thiobarbituric acid and the oxidation products of certain lipids. J. Biol. Chem. 174, 257.

Greenlee, W.F., Gross, E.A. and Irons, R.D. (1981a) Relationship between benzene toxicity and the disposition of 14C-labelled benzene metabolites in the rat. Chem.-Biol. Interact. 33, 285.

Greenlee, W.F., Sun, J.D. and Bus, J.S. (1981b) A proposed mechanism of benzene toxicity: Formation of reactive intermediates from polyphenol metabolites. Toxicol. Appl. Phar- macol. 59, 187.

Gutteridge, J.M.C. (1981) Thiobarbituric acid-reactivity following iron-dependent free- radical damage to amino acids and carbohydrates. FEBS Lett. 128, 343.

Gutteridge, J.M.C. (1987) Bleomycin-detectable iron in knee joint synovial fluid from arthritic patients and its relationship to the extracellular antioxidant activities of caeruloplasmin, transferrin and lactoferrin. Biochem. J. 245, 415.

Gutteridge, J.M.C., Rowley, D.A., Griffiths, E. and Halliwell, B. (1985) Low-molecular weight iron complexes and oxygen radical reactions in idiopathic hemochromatosis. Clin. Sci. 68, 463.

Healing, G., Gower, J., Fuller, B. and Green, C. (1990) Intracellular iron redistribution. An important determinant of reperfusion damage to rabbit kidneys. Biochem. Pharmacol. 39, 1239.

Irons, R.D. 0985) Quinones as toxic metabolites of benzene. J. Toxicol. Environ. Health 16, 673.

Jacobs, A. (1977) Low molecular weight intracellular iron transport compounds. Blood 50, 433.

Laughton, M.J., Evans, P.J., Moroney, M.A., Hoult, J.R.S. and Halliwell, B. (1991) Inhibi- tion of mammalian 5-1ipoxygenase and cyclo-oxygenase by flavonoids and phenolic dietary additives. Relationship to antioxidant activity and to iron ion-reducing ability. Biochem. Pharmacol. 42, 1673.

Lewis, J.G., Steward, W. and Adams, D.O. (1988) Role of oxygen radicals in induction of DNA damage by metabolites of benzene. Cancer Res. 48, 4762.

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Li, Y. and Trush, M.A. (1993) Oxidation of hydroquinone by copper: Chemical mechanism and biological effects. Arch. Biochem. Biophys. 300, 346.

Mazur, A. and Carleton, A. (1965) Hepatic xanthine oxidase and ferritin iron in the develop- ing rat. Blood 26, 317.

Pandya, K.P., Rao, G.S., Khan, S. and Krishnamurthy, R. (1990) Accumulation of low molecular weight (bleomycin detectable) iron in bone marrow cells of rats after benzene exposure. Arch. Toxicol. 64, 339.

Pellack-Walker, P. and Blumer, J.L. (1986) DNA damage in LSI78YS cells following exposure to benzene metabolites. Mol. Pharmacol. 30, 42.

Porteous, J.W. and Williams, R.T. (1949) Studies in detoxication 20. The metabolism of benzene II. The isolation of phenol, catechol, quinol and hydroxyquinol from the etheral sulphate fraction of the urine of rabbits receiving benzene orally. Biochem. J. 44, 56.

Rickert., D.E., Baker, T.S., Bus, J.S., Barrow, C.S. and Irons, R.D. (1979) Benzene disposition in the rat after exposure by inhalation. Toxicol. Appl. Pharmacol. 49, 417.

Prooxidant and antioxidant properties of iron-hydroquinone and iron-1,2,4-benzenetriol complex....

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