JOURNAL OF

www.elsevier.com/locate/jinorgbio

Journal of Inorganic Biochemistry 98 (2004) 1696–1702

InorganicBiochemistry

Oxidative DNA damage induced by HEPES(2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)

buffer in the presence of Au(III)

Ahsan Habib, Masaaki Tabata *

Department of Chemistry, Faculty of Science and Engineering, Saga University, 1 Honjo-machi, Saga 840-8502, Japan

Received 16 April 2004; received in revised form 6 July 2004; accepted 7 July 2004

Available online 28 August 2004

Abstract

Oxidative DNA damage was investigated by free radicals generated from HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethane-

sulfonic acid) buffer, which is widely used in biochemical or biological studies, in the presence of Au(III). The effect of free radicals

on the DNA damage was ascertained by gel electrophoresis, electron spin resonance (ESR) spectroscopy and circular dichroism

(CD) spectroscopy. ESR results indicated the generation of nitrogen-centered cationic free radicals from the HEPES in the presence

of Au(III) which cause the DNA damage. No ESR spectra were observed for phosphate, tris(hydroxymethyl)aminomethane (Tris–

HCl) and acetate buffers in the presence of Au(III) or for HEPES buffer in the presence of other metal ions such as Mn(II), Fe(III),

Co(II), Ni(II), Cu(II), Zn(II) and Pd(II) or [Au(III)(TMPyP)]5+ and [Pd(II)(TMPyP)]4+, where [H2(TMPyP)]4+ denotes tetrakis(1-

methylpyridium-4-yl)porphyrin. Consequently, no DNA damage was observed for these buffer agents (e.g., phosphate, Tris–HCl or

acetate) in the presence of Au(III) or for HEPES in the presence of other metal ions or the metalloporphyrins mentioned above. No

detectable inhibitory effect on the DNA damage was observed by using the typical scavengers of reactive oxygen species (ROS) �OH,

O2�� and H2O2. This non-inhibitory effect indicated that no reactive oxygen species were generated during the incubation of DNA

with HEPES and Au(III). The drastic change in CD spectra from positive ellipticity to negative ellipticity � at 270 nm with increas-

ing concentration of Au(III) also indicated the significant damage of DNA. Only HEPES or Au(III) itself did not damage DNA. A

mechanism for the damaging of DNA is proposed.

� 2004 Elsevier Inc. All rights reserved.

Keywords: DNA damage; HEPES; Au(III); Free radicals; ESR

1. Introduction

During the last decades, much interest has been

paid on the studies of carcinogenic or mutagenic ef-

fects of chemicals or metal ions which are usually used

in laboratories of chemistry or biology and also some

aspects in industries [1–7]. Very recently, Kawanishiand co-workers [3,4] investigated that semicarbazide

causes oxidative DNA damage in the presence of

0162-0134/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.jinorgbio.2004.07.005

* Corresponding author. Tel./fax: +81 952 28 8560.

E-mail address: tabatam@cc.saga-u.ac.jp (M. Tabata).

Cu(II) or aromatic amine, 4-aminobiphenyl (4-ABP)

and its N-hydroxy (4-ABP(NHOH)) metabolites also

cause oxidative DNA damage in the presence of

Cu(II) and NADH. Moreover, it has been found that

chromium (VI), which has been confirmed to be a hu-

man carcinogen, induced oxidative DNA damage in

the presence of hydrogen peroxide (H2O2) throughthe generation of endogenous reactive oxygen species

(ROS) [1,2]. In addition, it has also been reported that

a number of metal compounds show their carcino-

genic effects due to damaging of DNA in the presence

of H2O2 [8–11].

A. Habib, M. Tabata / Journal of Inorganic Biochemistry 98 (2004) 1696–1702 1697

HEPES (2-[4-(2-hydroxyethyl)piperazinyl]ethanesulf-

onic acid) is a popular pH buffer (Good�s buffer) that

is extensively used in laboratories of analytical, inor-

ganic, physical, biological, biochemical and also in tissue

culture [12,13]. This is because of its convenient pKa

(7.55), high solubility, and minimal complexation withmetal ions [14,15]. Recent studies, however, indicate that

HEPES is not inert as previously believed. Conse-

quently, it has been shown that HEPES can reduce

Cu(II) to Cu(I) in the presence of ligands that stabilize

Cu(I) [16]. Moreover, HEPES gives nitrogen-centered

cationic radicals in the presence of Fe(II)-, Fe(III)-poly-

mer and oxygen [17,18]. On the contrary, it is known

that Au(III) acts as inert metal due to its d8 electronicconfiguration. But very recently, it has been observed

that aqueous Au(III) shows higher toxicity to Trypano-

soma brucei brucei [19] in the HMI-9 medium (HMI-9

medium contains 0.05 M HEPES to maintain the phys-

iological pH 7.5), while [Au(III)(TMPyP)]5+ or Pd(II) or

its counterpart [Pd(II)(TMPyP)]4+ shows least toxic ef-

fects on T. b. brucei, where H2(TMPyP)4+ refers to tetra-

kis(1-methylpyridium-4-yl)porphyrin.In this study, we have investigated the ability of

HEPES to cause oxidative DNA damage in the presence

of Au(III). However, no DNA damage was observed

from only HEPES or only Au(III) but the mixture of

HEPES and Au(III) is able to damage DNA. To clarify

the DNA damaging mechanism, we have investigated

the formation of free radicals from the reaction of

HEPES with Au(III) using an electron spin resonance(ESR) spectrometer. Moreover, no detectable inhibitory

effect on the DNA damage (gel electrophoresis experi-

ments) by using scavengers for ROS also suggests the

DNA damaging mechanism.

2. Experimental

2.1. Materials

Sodium tetrachloroaurate(III) dihydrate; monoso-

dium dihydrogen phosphate; disodium monohydrogen

phosphate; sodium chloride; sodium hydroxide;

tris(hydroxymethyl) aminomethane (Tris); acetic acid;

sodium acetate; ethanol; dimethyl sulfoxide (DMSO);

superoxide dismutase (SOD); catalase; chloride salts ofiron(III), nickel(II), cobalt(II), zinc(II) and palla-

dium(II) were purchased from Wako Chemicals Co.

(Osaka, Japan); DD(�)mannitol was purchased from

TCI-GR (Tokyo Kasei Kogyo Co. Ltd., Japan); sodium

formate was purchased from Ishizu Pharmaceutical Co.

Ltd. (Osaka, Japan); manganese(II) chloride; copper(II)

chloride were purchased from Katayama Chemical Co.

(Japan); HEPES and EDTA (ethylenediaminetetraaceticacid, disodium salt) were purchased from Dojindo labo-

ratories Ltd., Japan. Moreover, H2(TMPyP)4+ was pur-

chased from Dojindo Laboratories as tosylate and its

Au(III)- and Pd(II)porphyrins were prepared by a

standard procedure [20]. The pBluescript II plasmid

DNA was prepared from a plasmid bearing Escherichia

coli strain using a standard procedure [21] and then dis-

solved in sterilized water. The base pairs concentrationof DNA was determined by absorbance measurements

using e260 = 1.32 · 104 mol�1 dm3 cm�1 at the absorp-

tion maximum of 260 nm [22,23]. Sterilized doubly

deionized water was used throughout the experiments.

2.2. Methods and apparatus

2.2.1. Studies on DNA damage by gel electrophoresis

The standard reaction mixture (in a microtube; 1.5

ml; Eppendorf) contained buffer agents (HEPES, phos-

phate, Tris–HCl and acetate), Au(III) and plasmid

DNA and then incubated at 37 �C for 60 min in a con-

stant temperature bath (Yamato, Japan). After incuba-

tion, the samples were stained with 1.0 · 10�3 ml of a

loading buffer (containing 30% glycerol, 0.1 M EDTA,

0.25% xylene cyanol and 0.25% bromophenol blue)and then run in 1% neutral agarose slab horizontal gel

containing tris(hydroxymethyl)aminomethane (TAE)

buffer of pH 8.3, 2.4 g; EDTA, 0.37 g; glacial acetic acid,

0.57 ml (99.7%) in 500 ml doubly deionized water for 33

min. The gel was stained in a solution of 0.5 lg l�1 of

ethidium bromide for 70 min. Gel electrophoresis was

performed by means of a Mupid-2 Cosmo Bio Company

apparatus (Japan) and DNA bands were photographedwith a Polaroid MP-4 land camera using a Polapan

black and white coatless film. Moreover, similar gel

electrophoresis experiments were carried out with

HEPES in the presence of: (i) different concentrations

of Au(III) (ii) other metal ions along with a few metal-

loporphyrins (e.g., Mn(II), Fe(III), Co(II), Ni(II),

Cu(II), Zn(II), Pd(II) and [Au(III)(TMPyP)]5+ and

[Pd(II)(TMPyP)]4+) and (iii) scavengers of ROS (e.g.,ethanol, mannitol, sodium formate, dimethyl sulfoxide

(DMSO), superoxide dismutase (SOD) and catalase).

Concentrations of the buffering agents and metal ions

including metalloporphyrins were maintained at

2.0 · 10�2 and 2.0 · 10�4 mol dm�3 (=M), respectively.

2.2.2. Electron spin resonance measurement

ESR spectra were recorded to detect radicals derivedfrom the respective buffering agents (e.g., HEPES, phos-

phate, Tris–HCl and acetate) in the presence of Au(III).

The spectra were measured at room temperature using a

JES-TE 300 (JEOL, Tokyo, Japan) spectrometer. The

spectra were recorded with a microwave power of 2

mW and a modulation amplitude of 0.63 mT. More-

over, a series of ESR experiments were carried out with

HEPES in the presence of [Au(III)(TMPyP)]5+ and[Pd(II)(TMPyP)]4+ and some other metal ions that have

already been mentioned in the above section (gel electro-

1698 A. Habib, M. Tabata / Journal of Inorganic Biochemistry 98 (2004) 1696–1702

phoresis). Concentrations of the buffering agents and

the metal ions including metalloporphyrins were main-

tained at 4.0 · 10�1 and 2.0 · 10�4 M, respectively.

2.2.3. Circular dichroism measurements

Circular dichroism (CD) measurements were con-ducted with a Jasco J-720 spectropolarimeter (Japan).

After each addition of Au(III), the spectra were scanned

five times at room temperature and then averaged. Con-

centration of the buffer agents (HEPES and phosphate)

was maintained at 2.0 · 10�2 M.

All experiments were conducted in the presence of

0.10 M sodium chloride. The main chemical species of

Au(III) are [AuCl(OH)3]� and [Au(OH)4]

� with a fewpercentage of [AuCl2(OH)2]

� at pH 7.4 [24,25]. In addi-

tion, the reagents were added in the order of NaCl,

HEPES, DNA and Au(III). Au(III) was the last since

radical formation occurred immediately after the addi-

tion of Au(III).

3. Results

3.1. Gel electrophoresis measurements

Fig. 1 shows the gel electrophoretogram of plasmid

DNA with different buffer agents (e.g., HEPES, phos-

phate, Tris–HCl and acetate) in the presence of Au(III)

(2.0 · 10�4 M). The results show that when plasmid

DNA is incubated with HEPES in the presence ofAu(III), Forms I (supercoiled) and II (circular) are con-

verted into Form III (linear) and other fragments as

shown in lane 2 (Fig. 1). But no DNA damage was ob-

served with phosphate, Tris–HCl or acetate in the pres-

ence of Au(III) (2.0 · 10�4 M) as shown, respectively, in

lanes 3–5 (Fig. 1) where lane 1 is DNA control. The ef-

fect of Au(III) concentrations on the DNA damage was

also carried out as shown in Fig. 2. Here, lane 1 is DNAcontrol and lanes 2–6 indicate increasing concentration

of Au(III). The degree of DNA damage depends on

the Au(III) concentration. Moreover, no DNA damage

Fig. 1. Gel electrophoresis of plasmid DNA (2.00 · 10�4 M base pairs) with

physiological pH 7.4. Lane 1, DNA alone; lane 2, HEPES; lane 3, phosphate;

and sodium chloride were maintained at 2.00 · 10�2 and 0.10 M, respectively.

respectively.

was observed for other metal ions such as Mn(II),

Fe(III), Co(II), Ni(II), Cu(II), Zn(II) or Pd(II) even

for [Au(III)(TMPyP)]5+ or [Pd(II)(TMPyP)]4+ in the

presence of HEPES (data not shown). Fig. 3 shows

the gel electrophoretograms of DNA damage in the

presence of scavengers of ROS (a) for �OH and (b) forO2

�� and H2O2. The results show no detectable inhibi-

tory effects on the DNA damage.

3.2. Electron spin resonance measurements

Fig. 4 shows the ESR spectra of free radicals gener-

ated from the buffering agents (e.g., HEPES or phos-

phate) in the presence of Au(III). Figs. 4(a) and (b)show the ESR spectra of free radicals generated from

HEPES without and with DNA, respectively, while

Fig. 4(c) indicates no ESR spectra generated from the

phosphate buffer in the presence of Au(III). The same

ESR spectra were also observed at different pHs (pH

4.6, 5.4, 6.2 and 7.4), suggesting that the radical forma-

tion is independent of chemical species of Au(III). In

other words, hydrolysis of Au(III) species does not affecton the formation of free radicals from HEPES. No ESR

signal, however, was observed from Tris–HCl or acetate

buffer in the presence of Au(III) or for HEPES in the

presence of other metal ions including a few

metalloporphyrins.

3.3. Circular dichroism studies

To confirm the structural changes of plasmid DNA,

we measured the CD spectroscopy of DNA with various

buffer agents in the presence of Au(III). The results are

shown in Fig. 5. Figs. 5(a) and (b) indicate the CD spec-

tra of DNA with HEPES and phosphate in the presence

of Au(III), respectively. Drastic spectral change was ob-

served in case of HEPES whereas a systematic hypochr-

omicity of the CD spectra of plasmid DNA wasobserved with phosphate buffer. Similar spectral change

(like phosphate buffer) was also observed in the case of

Tris–HCl buffer.

different buffering agents in the presence of 2.00 · 10�4 M of Au(III) at

lane 4, Tris–HCl; lane 5, acetate. Concentrations of the buffering agents

Incubation temperature and time were maintained at 37 �C and 60 min,

Fig. 2. The effect of the concentrations of Au(III) in the presence of HEPES (2.00 · 10�2 M, pH 7.40) on the DNA damage. Lane 1, DNA alone; lane

2, HEPES + 2.00 · 10�6 M; lane 3, HEPES + 2.00 · 10�5 M; lane 4, HEPES + 2.00 · 10�4 M; lane 5, HEPES + 6.00 · 10�4 M; lane 6,

HEPES + 1.00 · 10�3 M Au(III). Concentrations of DNA and sodium chloride were maintained at 2.00 · 10�4 M base pairs and 0.10 M,

respectively. Incubation temperature and time were maintained at 37 �C and 60 min, respectively.

Fig. 3. Effects of scavengers of reactive oxygen species (ROS) on DNA damage induced by HEPES in the presence of Au(III). (a) DNA was

incubated with the �OH scavengers: lane 1, DNA alone; lane 2, HEPES + Au(III); lane 3, HEPES + Au(III) + 5% EtOH; lane 4,

HEPES + Au(III) + 0.1 M mannitol; lane 5, HEPES + Au(III) + 0.1 M HCOONa; lane 6, HEPES + Au(III) + 5% DMSO. (b) DNA was incubated

with different units of superoxide dismutase, scavenger of O2�� and catalase, scavenger of H2O2: lane 1, DNA alone; lane 2, HEPES + Au(III); lane 3,

HEPES + Au(III) + 60 unit superoxide dismutase (SOD); lane 4, HEPES + Au(III) + 60 unit catalase; lane 5, HEPES + Au(III) + 90 unit SOD; lane

6, HEPES + Au(III) + 90 unit catalase. Incubation temperature and time were maintained at 37 �C and 60 min, respectively. HEPES, 2.0 · 10�2 M

(pH 7.40); Au(III), 2.0 · 10�4 M; DNA, 2.0 · 10�4 M base pair; NaCl, 0.10 M.

A. Habib, M. Tabata / Journal of Inorganic Biochemistry 98 (2004) 1696–1702 1699

4. Discussion

The present study has demonstrated that HEPES,

which is widely used as a buffering agent, has the ability

to cause DNA damage in the presence of Au(III) as

shown in Fig. 1, lane 2. The damaging effect can be ex-

plained by considering the ESR spectroscopic study asshown in Fig. 4. The ESR results indicate that free rad-

icals were generated from the HEPES in the presence of

Au(III) which is responsible for the damaging of DNA

(Figs. 4(a) and (b)). By contrast, no DNA damage was

observed for phosphate, Tris–HCl or acetate buffer as

shown in Fig. 1 (lanes 3–5) or for the HEPES in the

presence of other metal ions such as Mn(II), Fe(III),

Co(II), Ni(II), Cu(II), Zn(II), Pd(II) and also for[Au(III)(TMPyP)]5+ or [Pd(II)(TMPyP)]4+. This non-

damaging behavior of DNA is due to the inabilities of

generation of any free radicals from the phosphate

(Fig. 4(c)), Tris–HCl or acetate buffers in the presence

of Au(III) or for the HEPES in the presence of other me-

tal ions or metalloporphyrins. The damaging of DNA

with HEPES in the presence of Au(III) is also explained

by the CD spectroscopic studies. The CD spectra of the

plasmid DNA for the HEPES with increasing the con-

centration of Au(III) show drastic change from positive

to negative ellipticity approximately at 270 nm (Fig.

5(a)). This drastic change in CD spectra is due to thedamage of DNA, resulting an irregular CD pattern.

The irregular CD spectra suggest the production of un-

wound DNA fragments. But in the case of other buffer

agents like phosphate, it was observed that the CD spec-

tra were decreased with increasing concentration of

Au(III) and stable positive and negative ellipticities were

attained although higher concentration of Au(III) was

maintained (Fig. 5(b) for phosphate buffer, data notshown for Tris–HCl or acetate buffer). These results sug-

gest that prevention of free radical formation is due to

the binding of Au(III) to these buffers.

The characterization of free radicals generated from

the mixture of HEPES and Au(III) has been confirmed

-1000

-700

-400

-100

200

500

800

(b)

x 2. 4

-400

-300

-200

-100

0

100

200

300

400

500

331 333 335 337 339 341

Inte

nsity

Inte

nsity

Inte

nsity

(c)

-1000

-700

-400

-100

200

500

800

Magnetic Field/mT

331 333 335 337 339 341

Magnetic Field/mT

331 333 335 337 339 341

Magnetic Field/mT(a)

x 1

Fig. 4. ESR spectra of free radicals generation in HEPES(a,b)/

phosphate(c) buffers at room temperature in the presence of Au(III).

Symbols a andbare for theHEPESwithout andwithDNA, respectively,

and symbol c for phosphate without DNA. DNA, 2.0 · 10�4 M base

pair: Au(III), 2.0 · 10�4 M (a), 3.0 · 10�4 M (b). Concentrations of the

buffering agents and sodium chloride were maintained at 4.0 · 10�1 and

0.10 M, respectively, and pH was maintained at 7.4. Instruments

settings: field set, 335 mT; sweep, 10 mT; scan rate, 4 min; modulation

amplitude, 0.63 mT; gain 790; and power, 2.0 mW (a, b); 4.0 mW (c).

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

210 260 310 360 410Wavelength/nm

Elli

ptic

ity/m

deg

(b)

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

210 260 310 360 410Wavelength/nm

Elli

ptic

ity/m

deg

(a)

6

11

12

1011

Fig. 5. CD spectral changes of DNA (1.4 · 10�4 M in base pairs) upon

addition of Au(III) in the presence of (a) HEPES and (b) phosphate

buffers of pH 7.40. Arrows indicate spectral decrease with addition of

Au(III) ion of (1) 0.0, (2) 0.8, (3) 1.5, (4) 3.0, (5) 4.5, (6) 7.0, (7) 8.5, (8)

10.0, (9) 11.5, (10) 13.0, (11) 15.0 · 10�5 M. Concentrations of the

buffer agents and sodium chloride were maintained at 2 · 10�2 and

0.10 M, respectively. Cell path length is 10 mm.

1700 A. Habib, M. Tabata / Journal of Inorganic Biochemistry 98 (2004) 1696–1702

by ESR spectroscopic study. The determined parameters

(g = 2.0074 ± 0.0003 and a = 2.40 ± 0.02 G) of the gener-

ated free radicals are in agreement with those reported for

HEPES in the presence of Fe(II)-, Fe(III)-polymer andoxygen [17] that suggest the formation of nitrogen-cen-

tered cationic free radicals [17,18]. Moreover, ESRmeas-

urement was further conducted to confirm the generation

of free radicals from the HEPES–Au(III) system in the

presence of DNA. The results show that identical nitro-

gen-centered free radicals were also generated in the pres-

ence of DNA but the spectral intensity was reduced to

approximately 50% although the Au(III) concentrationwas maintained at 1.5 times higher compared to without

DNA sample. The results are shown in Figs. 4(a) (with-

out DNA) and (b) (with DNA). This ESR intensity

A. Habib, M. Tabata / Journal of Inorganic Biochemistry 98 (2004) 1696–1702 1701

reduction is due to the consumption of free radicals by

DNA that causes DNA damage. Moreover, it is also

likely due to the interaction of Au(III) with DNA, result-

ing in the decrease of the concentration of free Au(III)

leading to the reduction of free radicals. To clarify the

generation of other free radicals, except nitrogen-cen-tered free radicals that could be responsible for the dam-

aging of DNA, a number of gel electrophoresis

experiments were conducted in the presence of typical

ROS scavengers, data shown in Fig. 3. The results show

that no detectable inhibitory effect was observed on the

DNA damage by using the �OH scavengers (Fig. 3(a))

as well as the O2�� and H2O2 scavengers (Fig. 3(b)). This

non-inhibitory effect on the DNA damage indicates thatnitrogen-centered cationic free radicals, therefore, are the

only responsible radicals for the damaging of DNA.

Gold nanoparticles are formed during the incubation

of DNA with HEPES and Au(III). The incubated DNA

sample shows an absorption peak at approximately 600

nm that supports the formation of gold nanoparticles

because it is a characteristic plasmon absorption by gold

nanoparticles [26,27]. Moreover, the strong Rayleighlight scattering and bluish color of the incubated sample

further confirm the formation of gold nanoparticles. The

gold nanoparticles formation is also related to the for-

mation of HEPES radicals.

On the basis of the results, a mechanism for the oxi-

dative damaging of DNA is proposed as shown in Fig.

6. Guanine is the most easily oxidized among the four

DNA bases because the oxidation potential of guanineis lower than that of the other DNA bases (e.g., guanine,

1.29 V; adenine, 1.42 V; cytosine, 1.6 V and thymine, 1.7

V versus normal hydrogen electrode, NHE) [3,28,29].

Highly reactive species such as �OH cause DNA damage

at every nucleotide, whereas less reactive species e.g.,

HOH2CH2CN NCH2CH2SO3-

HOH2CH2CN NCH2CH2SO3

DNA damage at G in 5’-AG

Au(III)(HEPES)

Au(I) Gold Nanoparticles

HEPES free radicalsHEPES

(HEPES free radical)

DNA

Fig. 6. A proposed mechanism of oxidative DNA damage induced by

HEPES in the presence of Au(III).

nitrogen-centered radicals cause DNA damage specifi-

cally at guanines [3,30]. Possibly in the present case,

HEPES-derived radical induces the guanine-specific

DNA damage. Particularly, the 5 0-G in GG sequence

is considered to have the lowest oxidation potential, be-

cause this guanine has the lowest ionization potentialamong the guanine-containing dinucleotides [31]. The

nitrogen-centered cationic radicals may lead to the for-

mation of the oxidative products of guanine such as 8-

oxodGuo [3,28,32] and piperidine-labile products (e.g.,

imidazolone and oxazolone) [3,28,33–35]. HEPES-de-

rived radicals may also play important roles in DNA

damage in vivo under certain conditions because these

organic radicals are not scavenged by catalase.

5. Conclusion

HEPES, a non-mutagenic carcinogen, induces DNA

damage in the presence of Au(III) through the generation

of nitrogen-centered cationic free radicals, but not other

radicals such as �OH,O2�� orH2O2. TheDNAdamage by

the radicals was ascertained by gel electrophoresis and

the measurements of ESR and CD spectra. The radical

species were also confirmed by the use of radical scaven-

gers. HEPES or Au(III) separately did not damageDNA.

The nitrogen-centered cationic radicals would participate

to guanine-specific DNA damage and lead to mutations,

such as G.C ! T.A or G.C ! C.G transversions. The

DNA damage, therefore, may be relevant to the toxicityor carcinogenecity of HEPES.

6. Abbreviations

HEPES 2-[4-(2-hydroxyethyl)-1-piperazinyl]

ethanesulfonic acid

ESR electron spin resonanceCD circular dichroism

Tris tris(hydroxymethyl)aminomethane

[H2(TMPyP)]4+ tetrakis(1-methylpyridium-4-yl)por-

phyrin

SOD superoxide dismutase�OH hydroxyl radical

O2�� superoxide radical anion

H2O2 hydrogen peroxideDMSO dimethyl sulfoxide

EDTA ethylenediaminetetraacetic acid

TAE Tris–Acetate–EDTA

Acknowledgments

The authors thank Professor K. Watanabe of the

Agriculture Department, Saga University, for his help

in the preparation of plasmid DNA. This work was sup-

1702 A. Habib, M. Tabata / Journal of Inorganic Biochemistry 98 (2004) 1696–1702

ported in part by a PSJP (Postgraduate Special Joint

Program) scholarship (M.A. Habib) and Grants-in-

Aid, B (No. 15350046) (M. Tabata), from the Ministry

of Education, Science, Technology, Sports and Culture

of Japan.

References

[1] S. Kawanishi, Y. Hiraku, M. Murata, S. Oikawa, Free Radic.

Biol. Med. 32 (2002) 822–832.

[2] S. Kawanishi, D. Inoue, S. Sano, J. Biol. Chem. 261 (1986) 5952–

5958.

[3] K. Hirakawa, K. Midorikawa, S. Oikawa, S. Kawanishi, Mutat.

Res. 536 (2003) 91–101.

[4] M. Murata, A. Tamura, M. Tada, S. Kawanishi, Free Radic.

Biol. Med. 30 (7) (2001) 765–773.

[5] IARC Working Group, 4-Aminobiphenyl, in: IARC (Ed.), IARC

Monographs on the Evaluation of the Carcinogenic Risk of

Chemicals to Man, vol. 1, Lyon, IARC, 1972, pp. 74–79.

[6] IARC Working Group, Tobacco habits other smoking; betelquid

and areca nut chewing; and some related nitrosamines, in: IARC

(Ed.) IARC Monographs on the Evaluation of the Carcinogenic

Risk of Chemicals to Man, vol. 38, Lyon, IARC, 1985, p. 392.

[7] M. Murata, S. Kawanishi, J. Biol. Chem. 275 (2000) 2003–2008.

[8] S. Inoue, S. Kawanishi, Cancer Res. 47 (1987) 6522–6527.

[9] K. Yamamoto, S. Inoue, A. Yamazaki, T. Yoshinaga, S.

Kawanishi, Chem. Res. Toxicol. 2 (1989) 234–239.

[10] S. Kawanishi, S. Inoue, K. Yamamoto, Carcinogenesis 10 (1989)

2231–2235.

[11] S. Inoue, S. Kawanishi, Biochem. Biophys. Res. Commum. 159

(1989) 445–451.

[12] A. Fisk, S. Pathak, Nature (London) 224 (1969) 1030–1031.

[13] T.F. Dyer, D.A. Cocks, Ann. Clin. Biochem. 17 (1980) 214–

215.

[14] N.E. Good, K. Izawa, in: Methods in Enzymology, vol. 24,

Academic Press, New York, 1972, pp. 53–68.

[15] N.E. Good, G.D. Winget, W. Winter, T.N. Connolly, K. Izana,

R.M.M. Singh, Biochemistry 5 (1966) 467–468.

[16] K. Hegetschweiler, P. Saltman, Inorg. Chem. 25 (1986) 107–109.

[17] J.K. Grady, N.D. Chasteen, D.C. Harris, Anal. Biochem. 173

(1988) 111–115.

[18] W.C. Danen, R.C. Rickard, J. Am. Chem. Soc. 94 (1972) 3254–

3256.

[19] E. Nyarko, T. Hara, D.J. Grab, A. Habib, Y. Kim, O. Nikolskaia,

T. Fukuma, M. Tabata, Chemico-Biological Interact. 148 (2004)

19–25.

[20] M. Tabata, A.K. Sarker, K. Watanabe, Chem. Lett. (1998) 325–

326.

[21] J.S. Heilig, in: F.M. Ausubel, R. Brent, R.E. Kingston, D.D.

Moore, J.G. Seidman, J.A. Smith, K. Struhl (Eds.), Current

Protocols in Molecular Biology, Large-scale Preparation of

Plasmid DNA, vol. 1, Greene Publishing Associates and Wiley–

Interscience, New York, 1987, p. 1.7.1.

[22] E.J. Gibbs, M.C. Maurer, J.H. Zhang, W.M. Reiff, D.T. Hill, M.

Malicka-Blaszkiewick, R.E. Mckinnie, H.Q. Liu, R.F. Paster-

nack, J. Inorg. Biochem. 32 (1988) 39.

[23] J.A. Patcher, C.H. Huang, V.H. DuVernay Jr., A.W. Prestayko,

S.T. Crooke, Biochemistry 21 (1982) 1541.

[24] A. Habib, M. Tabata, Y. Wu, J. Porphyrins Phthalocyanines

(accepted).

[25] C.F. Baes Jr., R.E. Mesmer, The Hydrolysis of Cations, Krieger

Publishing Company, Florida, 1986.

[26] M. Kerker, The Scattering of Light and Other Electromagnetic

Radiation, Academic Press, New York, 1969.

[27] F. Bohren, D.R. Huffman, Absorption and Scattering of Light by

Small Particles, Wiley, New Work, 1983.

[28] C.J. Burrows, J.G. Muller, Chem. Rev. 98 (1998) 1109–1151.

[29] S. Steenken, S. Jovanovic, J. Am. Chem. Soc. 119 (1997) 617–

618.

[30] S. Kawanishi, Y. Hiraku, S. Oikawa, Mutat. Res. 488 (2001) 65–

76.

[31] H. Sugiyama, I. Saito, J. Am. Chem. Soc. 118 (1996) 7063–

7068.

[32] H. Kasai, Z. Yamaizumi, M. Berger, J. Cadet, J. Am. Chem. Soc.

114 (1992) 9692–9694.

[33] S. Raoul, M. Berger, G.W. Buchko, P.C. Joshi, B. Morin, M.

Weinfeld, J. Cadet, J. Chem. Soc., Perkin Trans. 2 (1996) 371–

381.

[34] J. Cadet, G. Remaud, J.L. Ravanat, Biochem. Biophys. 374

(2000) 118–127.

[35] K. Kino, H. Sugiyama, Chem. Biol. 8 (2001) 369–378.