Molecular and Cellular Biochemistry 178: 299–303, 1998.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

299

Melatonin reduces the increase in 8-hydroxy-

deoxyguanosine levels in the brain and liver of kainic
acid-treated rats
Lei Tang,1 Russel J. Reiter,1 Zhong-Ren Li,1 Genaro G. Ortiz,1
Byung Pal Yu2 and Joaquin J. Garcia1
Departments of 1Cellular and Structural Biology; 2Physiology, University of Texas Health Science Center, 7703 Floyd Curl
Drive, San Antonio, TX 78284-7762, USA

Received 9 April 1997; accepted 31 July 1997

Abstract
In the present study, the effect of melatonin on oxidative DNA damage induced by kainic acid (KA) treatment was investigated. 8-
hydroxy-deoxyguanosine (8-OH-dG) is a main product of oxidatively damaged DNA and was used as the endpoint in these studies.
The levels of 8-OH-dG were found to be elevated in the hippocampus and frontal cortex of rats treated with KA. These elevated
levels were significantly reduced in animals that were co-treated with melatonin. Thus, there was no difference in 8-OH-dG levels
in the brain of control rats compared to those treated with KA (10 mg/kg) plus melatonin (10 mg/kg). The levels of 8-OH-dG also
increased in the liver of rats treated with KA. This rise in oxidatively damaged DNA was also prevented by melatonin administration.
Melatonin’s ability to reduce KA-induced increases in neural and hepatic 8-OH-dG levels presumably relates to its direct free
radical scavenging ability and possibly to other antioxidative actions of melatonin. (Mol Cell Biochem 178: 299–303, 1998)

Key words: kainic acid, melatonin, free radical, 8-hydroxy-deoxyguanosine

Introduction
Kainic acid (KA) is a nondegradable analog of glutamate
which was first isolated from the seaweed Digenea simplex.
KA possesses potent neuroexcitatory and neurotoxic pro-
perties [ 1]. KA binds and activates the KA/AMPA (a-amino-
3-hydroxy-5-methyl-4-isoxazole propionate) receptor
subtype of the glutamate receptor family. In addition to
inducing lesions directly, KA also triggers epileptiform
activity and secondary brain injury which are inhibited by
anticonvulsant drugs [2]. Recently, it was proposed that the
activation of excitatory amino acid receptors triggers the
formation of reactive oxygen species (ROS) which can lead
to neuronal damage [3]. ROS are known to damage a variety
of critical biological molecules, including DNA, cellular
proteins and membrane lipids [4, 5].
Address for offprints: R.J. Reiter, Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio,
TX 78284-7762, USA
ROS also increase the release of glutamate [6], which in turn
augments ROS formation, and thus a positive feed back loop
develops, thereby producing progressively more oxidative
damage. In a variety of experiments designed to identify the
mechanisms that underlay the neurotoxic action of KA, it has
been shown that a significant attenuation in the ability of KA
to induce neuronal damage is obtained with various free radical
scavengers and antioxidants [3, 7].
Melatonin is well known for its functional interactions with
both the neuroendocrine axis and with the circadian system [8].
In recent years, a considerable amount of evidence also has
accumulated showing that melatonin is a direct scavenger of
the highly toxic hydroxyl radical (·OH) and that it possesses
substantial antioxidant activity [4, 5, 9]. Being highly lipophilic
[10] as well as hydrophilic [11], melatonin has the potential
to provide oxidative protection in many organs and all
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subcellular compartments [4, 5, 8]. Moreover, once oxidized,
melatonin is not readily reduced and it is not involved in
regenerating processes that may cause toxic recycling and
formation of free radicals [5].
In the present study, we investigated the ability of mela-
tonin to resist KA-induced DNA oxidative damage in rats.
Since 8-hydroxy-deoxyguanosine (8-OH-dG) is a major
product of oxidatively damaged DNA [12], the level of 8-
OH-dG was used as an index of KA toxicity and the asso-
ciated DNA damage.
Materials and methods
Chemicals
KA, melatonin and deoxyguanosine (dG) were purchased
from Sigma (St. Louis, MO). RNase A, RNase T1, proteinase
K, nuclease P1 and alkaline phosphatase were from Boeh-
ringer Mannheim. High purity phenol was purchased from
Life Technologies (Gaithersburg, MD). 8-OH-dG was from
WAKO Chemicals USA (Richmond, VA). All the other
reagents were of the highest quality available.
Animals
Adult male Sprague-Dawley rats (body weight 250 ± 10 g)
were obtained from Harlan (Houston, Texas).
Treatments
After one week of acclimatization to the animal facilities,
rats were divided into 4 groups. KA was dissolved in double
distilled water and intraperitoneally administered at a dose of
10 mg/kg. Melatonin was dissolved in ethanol and then diluted
with double distilled water (the diluent had a final concentration
of ethanol of 4%) and injected i.p. at a dose of either 4 mg/kg
or 10 mg/kg. Group 1 (controls ) animals received an injection
of alcoholic saline (diluent) followed 15 min later by a distilled
water injection. Group 2 received a injection of diluent
followed 15 min later by an injection of KA. Group 3 was
injected with melatonin (4 mg/kg) and KA 15 min later. Group
4 received an injection of melatonin (10 mg/kg) followed by
an injection of KA 15 min later. After 8 h (09:00–17:00 h), rats
were anesthetized and subjected to intracardiac perfusion with
ice-cold saline in order to remove excess of iron that could be
released from intracellular storage sites thereby artificially
increasing free radical formation. The brain and liver were
removed and two brain regions were dissected, the hippocampus
and frontal cortex.All samples were immediately frozen on solid
CO2 and then stored at –80°C.
8-OH-dG assay
Tissue samples were homogenized in 1 ml ice-cold homo–
genization buffer [0.1 M NaCl, 30 mM Tris, 10 mM EDTA,
10 mM 2-mercaptoethanol, 0.5% (v/v) Triton X-100, pH 8.0].
The homogenates were centrifuged at 4°C for 10 min at 1,000 g
to pellet the nuclei. The supernatant was discarded and the crude
nuclear pellet was resuspended in 0.5 ml lysis solution (120
mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, 0.5% SDS, pH 8.0).
20 microliter butylated hydroxytoluene (BHT, 5% in methanol)
was added as an antioxidant.
RNA was digested by incubating the samples with 20 µl
RNase (50 U/ml RNase A, 100 U/ml RNase T1) for 1 h at 50°C.
Protein was digested on incubation for 1 h at 50°C with 100 µl
proteinase K (5 mg/ml in 10 mM Tris/1 mM EDTA, pH 7.4).
Five hundred µl buffer saturated phenol was added to the
samples and they were shaken gently by hand. The samples were
centrifuged at room temperature to separate the phases. The
upper aqueous phase was transferred to a Phase Lock Gel tube
(5 Prime→3 Prime Inc, Boulder, CO). Four hundred µl PCI
(buffer saturated phenol:chloroform:isoamyl alcohol, 25:24:1,
v/v, stood overnight) were added and the mixture was shaken
gently by hand. Thereafter it was centrifuged at 13,000 g for
5 min.After transferring the upper aqueous phase to a new Phase
Lock Gel tube, 400 µl Sevag (chloroform:isoamyl alcohol,
24:1, v/v) were added. The mixture was shaken gently followed
by centrifugation at 13,000 g for 5 min. After transferring the
aqueous phase (600 µl) from the last extraction to a new 2 ml
Eppendorf tube, the DNA was precipitated by adding 60 µl 3 M
sodium acetate and 1.2 ml ethanol (–20°C). The DNA pellet
was washed with 70% ethanol (–20°C) and the DNA samples
were stored in 100% ethanol with 0.01% BHT.
DNA samples were dried by vacuum. DNA was dissolved in
180 µl H2O. Twenty µl 200 mM sodium acetate (pH 5.0) was
added. DNA was hydrolyzed to nucleotides by incubation for
10 min at 65°C with 4 µl of 3.3 mg/ml nuclease Pi (in 20 mM
sodium acetate, pH 5.0). The pH was adjusted by adding 20 µl
1 M Tris-HCl buffer (pH 8.5) and DNA was hydrolyzed to the
corresponding nucleosides during incubation with 4 µl alkaline
phosphatase (AP, 1 U/µl) for 1 h at 37°C. After AP digestion,
20 µl 3 M sodium acetate (pH 5.0) was added followed by the
addition of 20 µl of 50 mM EDTA. The hydrolysate was filtered
through Microcon microconcentrators [membrane molecular
weight cut-off at 3,000 MW (Beverly, MA)] prior to HPLC
analysis to remove enzymes and other macromolecules. HPLC
analysis was performed with a Waters 510 pump equipped with
high sensitivity filter units (Milford, MA). The column used
was supelcosil LC-18-DB [4.6 × 250 mm (Bellefonte, PA)]. An
isocratic eluent [50 mM KH2PO4 buffer (pH 5.5) with 5%
methanol] was used at a flow rate of 1 ml/min. Detection was
performed with a Waters 486 UV/Visible detector at 290 nm
for dG. 8-OH-dG was determined using a Waters 464 Pulsed
Electrochemical Detector (DC mode, +700 mV). Peaks from