protective effect of ethyl pyruvate on msp rat leukocytes damaged by alcohol intake

ALCOHOL DAMAGE AND ETHYL PYRUVATE 561

Copyright © 2007 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2007; 27: 561–570

DOI: 10.1002/jat

JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 2007; 27: 561–570Published online 9 March 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/jat.1236

Protective effect of ethyl pyruvate on msP ratleukocytes damaged by alcohol intake

Donatella Fedeli,1 Giancarlo Falcioni,1 Robert A. Olek,2 Maurizio Massi,3 Carlo Cifani,4 Carlo Polidori3

and Rosita Gabbianelli1,*

1 Department of M. C. A. Biology, University of Camerino, Italy2 Department of Bioenergetics, Jedrzej Sniadecki University School of Physical Education, Wiejska 1, 80-336 Gdansk, Poland3 Department of Experimental Medicine and Public Health, University of Camerino, Italy4 School of Advanced Studies, ‘Ageing and Nutrition’ Ph.D., University of Camerino, Italy

Received 6 December 2006; Revised 9 January 2007; Accepted 15 January 2007

ABSTRACT: Alcohol consumption for long periods negatively influences physiological functions of many cells, and leads

to organ damage. Reactive oxygen and nitrogen species produced by ethanol metabolism cause adverse effects that might

be alleviated by simultaneous treatment with various antioxidants. Here, the ability of ethyl pyruvate (EP) to reduce

ethanol-induced oxidative stress was evaluated.

Chemiluminescence studies show that EP has a higher capacity than pyruvate to scavenge hydrogen peroxide and

superoxide anions. In order to evaluate whether EP can exert a protective effect against ethanol, rats were offered 10%

ethanol in drinking burettes, containing or not different concentrations of EP (0.3%, 1% and 3%). The comet assay was

employed to quantify the alcohol-induced DNA damage in rat lymphocytes. This test is a promising tool for the estima-

tion of DNA damage at the single cell level. A significant protective effect of EP was observed in rat groups treated with

this antioxidant, compared with those drinking only ethanol.

Since EP has been shown to decrease the expression of numerous pro-inflammatory mediators, the monocyte respira-

tory burst was evaluated. The activation of monocyte NADPH oxidase by phorbol esters (PMA) showed that superoxide

anion production was higher in the ethanol group than in the control group. The presence of EP considerably reduced

superoxide anion production. In conclusion, hypotheses on possible mechanisms of action of EP on rat white blood cells

are proposed. Copyright © 2007 John Wiley & Sons, Ltd.

KEY WORDS: alcohol; rat; DNA damage; ethyl pyruvate; comet; respiratory burst

reactive hydroxyl radical (Halliwell and Gutteridge,

1989). The following enzymes afford the primary defence

system against ROS: superoxide dismutase, catalase,

glutathione peroxidase and glutathione reductase; they

receive additional support from metal binding proteins

such as transferrin, albumin, caeruloplasmin and hapto-

globin. The secondary defence system is provided by

vitamins E and C, β-carotenes as well as glutathione,

urates, bilirubin and other substances with scavenger

properties against overproduction of free radicals that

may occur under pathological conditions (Halliwell and

Gutteridge, 1989; Papas, 1999).

In recent years, a great deal of research has been

devoted to the study of natural and synthetic antioxidants

that may somehow mimic our natural endogenous ones or

at least minimize the deleterious effects produced by

ROS (Halliwell and Gutteridge, 1989; Gabbianelli et al.,

2004a, 2004b; Olek et al., 2005). There is a recent

renewal of interest in the antioxidant properties of

pyruvate and other alfa-ketoacids that have been used

in biological systems for their capacity to react non-

enzymatically with peroxides (Kim et al., 2006). In-

creased oxidation and degradation of cytosolic proteins

Introduction

Numerous experimental data indicate that free radicals

contribute to ethanol-induced cell injury (Montoliu et al.,

1995), and many pathways are suggested to play a key

role in ethanol-induced ‘oxidative stress’. Furthermore,

an ethanol-linked enhancement in free radical generation

can occur through nicotinamide adenine dinucleotide

phosphate (NADPH)-dependent electron transport chains,

membrane-bound oxidoreductase, cytosolic xanthine and

aldehyde oxidases (Cederbaum, 2001; Nordmann et al.,

1992; Little, 1999).

Examples of reactive oxygen species (ROS) of biologi-

cal importance include superoxide radical (O2−), hydrogen

peroxide (H2O2) and hydroxyl radical (•OH). The O2− can

be converted by the superoxide dismutase enzyme to

H2O2, which, via Fenton reaction, produces the highly

* Correspondence to: Dr Rosita Gabbianelli, Department of M. C. A.

Biology, University of Camerino, Via Camerini 2, I-62032 Camerino (MC)

Italy.

E-mail: [email protected]

562 D. FEDELI ET AL.


DOI: 10.1002/jat

were measured in alcohol-exposed mouse liver and

hepatoma cells (Kim et al., 2006). Recently, pyruvate

also has been shown to be capable of scavenging •OH

(Dobsak et al., 1999; Sappington et al., 2005). Pyruvate

can be transported into or secreted from cells by the

specific H+-monocarboxylate co-transporter (Harding

et al., 1994) thus, it could act both as an intracellular

and an extracellular H2O2 scavenger. Administration of

exogenous pyruvate can be helpful in numerous path-

ologic situations due at least in part to oxidative stress

conditions. Unfortunately, the use of pyruvate as a thera-

peutic agent is limited by its poor stability in H2O (Sims

et al., 2001). In fact, aqueous solutions of pyruvate spon-

taneously undergo an aldolic condensation reaction to

form 2-hydroxy-2-methyl-4-ketoglutarate (parapyruvate),

a compound that is a potent inhibitor of a critical step in

the mitochondrial tricarboxylic acid cycle (Montgomery

and Webb, 1956). To circumvent this issue, Sims et al.

formulated a derivative of pyruvic acid, namely ethyl

pyruvate, in a calcium- and potassium-containing bal-

anced salt solution (Sims et al., 2001).

Recently, lymphocyte DNA alteration was examined

in alcohol-preferring rats following sub-chronic ethanol

intake (Fedeli et al., 2003). The results showed lym-

phocyte DNA damage induced by ethanol intake (Fedeli

et al., 2003).

In this study, the antioxidant activity of ethyl pyruvate

versus hydrogen peroxide and superoxide anion was stud-

ied and compared with that of sodium pyruvate. These

determinations were performed by a chemiluminescence

(CL) technique with luminol and lucigenin as probes.

The possible antioxidant capacity of this compound was

monitored: experiments were made to determine whether

the treatment with EP can protect against ethanol-induced

lymphocyte DNA damage. The DNA damage was as-

sessed by a single-cell microgel electrophoretic method,

the ‘comet assay’ (Collins et al., 1997, 2001; Gabbianelli

et al., 2003). This technique offers many potential

applications in genotoxicity testing and biomonitoring

(Fairbairn et al., 1995; Hartmann and Speit, 1997). The

commonly used alkaline version of the test detects DNA

strand breaks and alkali labile lesions with sensitivity

(Singh et al., 1988). Relaxed and broken DNA fragments

stream further from the nucleus than intact DNA, so the

extent of DNA damage can be evaluated by the length of

the stream. With this technique it is possible to evaluate

reversible damage even at low levels in a single cell

using a very small sample. In this assay, the extent of

primary DNA damage can be measured by evaluating

three different comet parameters: tail length (TL—meas-

ured in μm from the head centre), tail intensity (TI—%

of fluorescence in the comet tail) and tail moment (TM)

(Collins et al., 1997, 2001) that considers both the tail

length and the fraction of the DNA in the comet tail.

Since ethanol can influence the immune system, the ac-

tivity of phagocytes, which are involved in the ingestion

and killing of microorganisms, was also investigated. In

particular, in monocytes the final step of phagocytosis,

known as the respiratory burst, was evaluated.

Materials and Methods

Materials

All reagents were of pure analytical grade. Pyruvate,

ethyl pyruvate (EP), phorbol myristate acetate (PMA),

xanthine, xanthine oxidase, lucigenin and luminol were

obtained from Sigma. Hydrogen peroxide was obtained

from Baker, Deventer, Holland. Lymphoprep was from

Nycomed Pharma AS, Oslo, Norway.

Animals

Male Marchigian Sardinian alcohol-preferring rats (msP)

bred for 40 generations in the animal facility of the De-

partment of Experimental Medicine and Public Health

of the University of Camerino from Sardinian alcohol-

preferring rats of the 13th generation, provided by

the Department of Neurosciences of the University of

Cagliari (Italy) were employed. Alcohol-preferring rats

were employed as having a voluntary ethanol drinking in

pharmacologically relevant amounts, without overt signs

of intoxication. MsP rats, weighing 250–300 g, were

housed three animals per hanging stainless-steel cage

(grid floors) at constant room temperature (25 ± 1 °C)

and humidity (60 ± 5%) with an artificial 12:12 h light/

dark cycle (dark onset at 9:00 p.m.). They were offered

free access to chow pellets (Diet 4RF18, Mucedola,

Settimo Milanese, Italy) and the various solutions

described below.

All the animals were ethanol naïve at the beginning of

the experiments.

Experimental Procedure

For 10 weeks, eight groups of animals had, respectively,

access to only water (n = 3), water plus 0.3% EP solu-

tion (n = 3), water plus 1% EP solution (n = 3), water

plus 3% EP solution (n = 3), only 10% ethanol solution

(n = 6), 10% ethanol plus 0.3% EP solution (n = 6), 10%

ethanol plus 1% EP solution (n = 6) or 10% ethanol

plus 3% EP solution (n = 6). Animal body weight,

as well as food and fluid intake, were recorded weekly.

At the end of this period, the animals were lightly

anaesthetized with CO2 and then immediately a sample

of blood was withdrawn from the heart with a needle

attached to a 5 ml syringe previously filled with a drop

of heparin. The animals were immediately killed by

CO2 inhalation.



DOI: 10.1002/jat

Lymphocyte and Monocyte Separation

Lymphocyte and monocyte separation was performed by

using a Ficol density gradient. Whole blood diluted 1:1 with

phosphate buffer saline (PBS) was stratified on a solution

of lymphoprep and then centrifuged for 15 min at 2500 rpm.

Lymphocytes and monocytes, obtained by Ficol density

gradient, were washed three times with PBS and counted.

Chemiluminescence Measurement of Pyruvateand Ethyl Pyruvate Antioxidant Activity

Chemiluminescence measurements to evaluate the anti-

oxidant activity of pyruvate and EP were performed

using an Autolumat Berthold LB 953 (Berthold Co.,

Wilbard, Germany). In order to measure the scavenger

activity of these compounds against hydrogen peroxide,

a reaction mixture containing different concentrations of

pyruvate or EP, 100 μM luminol in 50 mM phosphate

buffer pH 7.0 were prepared. The reaction was initiated

by injecting 0.05 ml of H2O2 to a final concentration

50 mM. To assess the scavenger activity forward super-

oxide anion, a reaction mixture containing 0.9 U ml−1

xanthine oxidase, 150 μM lucigenin in 50 mM phosphate

buffer pH 7.0, and different concentrations of pyruvate or

EP were used. The reaction was started by injecting

xanthine at a final concentration of 50 μM. The data were

reported as the percentage (%) of inhibition of the CL

reaction and calculated as follows:

% I = 100 − (CLsample × 100/CLblank)

where CLsample is the chemiluminescence obtained for a

sample in the presence of pyruvate or ethyl pyruvate and

CLblank is the chemiluminescence obtained in the sample

without pyruvate or ethyl pyruvate.

Respiratory Burst

The respiratory burst (RB) was performed only on

monocytes obtained by Ficol density gradient.

Monocytes from the three groups (water, EtOH and

EtOH + 3% EP) were pooled together in order to obtain

three samples, each containing 1 × 106 cells. In every

sample, 150 μM lucigenin was added to 1 ml of Krebs-

Ringer phosphate solution plus glucose pH 7.4. Monocyte

suspensions were activated by 3 × 10−4M PMA and the

chemiluminescence was measured for 50 min. The meas-

urements were performed in duplicate twice.

Comet Assay

DNA damage in lymphocytes was evaluated using the

alkaline single-cell microgel electrophoresis (‘comet’

assay) (Gabbianelli et al., 2004b, Collins et al., 1997, 2001;

Gabbianelli et al., 2002, 2003). The comet assay was

carried out under yellow light. About 2 × 105 cells were

mixed with 65 μl of 0.7% low melting agarose (LMA) in

Ca2+- and Mg2+-free PBS to form a cell suspension. The

cell suspension was rapidly spread over a pre-cleaned

microscope slide previously conditioned by spreading a

1 ml aliquot of 1% NMA in Ca2+- and Mg2+-free PBS.

After solidification, the cells were protected with a top

layer of 75 μl of 0.7% LMA. To lyse the embedded cells

and to permit DNA unfolding, the slides were immersed

in freshly prepared ice-cold lysis solution (1% sodium N-

lauroyl-sarcosinate, 2.5 M NaCl, 100 mM Na2EDTA,

10 mM Tris–HCl, pH 10, with 1% Triton X-100 and 10%

DMSO added just before use) for 1 h at +4 °C in the

dark. After the lysis, the slides were placed on a horizon-

tal electrophoresis box. The unit was filled with freshly

made alkaline buffer (300 mM NaOH, 1 mM Na2EDTA,

pH > 13) and, to allow DNA unwinding and expression

of alkali labile damage, the embedded cells were left

in the solution for 20 min. Electrophoresis was performed

for 20 min by applying an electric field of 25 V and

adjusting the current to 200 mA. After the electro-

phoresis, the slides were washed gently with 0.4 M Tris-

HCl buffer pH 7.5 to neutralize the excess alkali and

remove detergents. Slides were stained by adding 20 μl

of ethidium bromide (2 μg ml−1) and the rate of DNA

damage was evaluated by analysing at a magnification of

×20 the images of 150 randomly selected cells (50 cells

from each of three replicate slides) by using an Axioskop

2 plus epi-fluorescence microscope (Carl Zeiss, Germany)

equipped with an excitation filter of 515–560 nm. Imag-

ing was performed using a specialized analysis system

(‘Metasystem’ Altlussheim, Germany) to determine tail

length, tail intensity and tail moment.

Statistical Analysis

Data are expressed as mean values ± SEM except for

chemiluminescence results, for which data are presented

as mean values ± SD. For the comet assay, at least 150

scores/sample were performed. Statistical analysis for

behavioural studies was carried out by two-way analysis

of variance (ANOVA), followed by the Newman Keuls

test. Statistical significance was set at P < 0.05.

Results

General Findings

The overall ANOVA showed a significant effect of treat-

ment (F(7,28) = 1.95; P < 0.01), time (F(9,252) = 522.3;

P < 0.0001) and treatment–time interaction (F(63,252) =2.79; P < 0.0001) on body weight gain between the eight



DOI: 10.1002/jat

Figure 2. Weekly fluid intake (ml kg−1) of rats exposed for 10 weeks to only water, water plus 0.3% ethylpyruvate solution, water plus 1% ethyl pyruvate solution, water plus 3% ethyl pyruvate solution, only 10%ethanol solution, 10% ethanol plus 0.3% ethyl pyruvate solution, 10% ethanol plus 1% ethyl pyruvate solution, or10% ethanol plus 3% ethyl pyruvate solution. Data are the mean ± SEM. * P < 0.05, ** P < 0.01

Figure 1. Cumulative body weight gain of ratsexposed for 10 weeks to only water, water plus 0.3%ethyl pyruvate solution, water plus 1% ethyl pyruvatesolution, water plus 3% ethyl pyruvate solution, only10% ethanol solution, 10% ethanol plus 0.3% ethylpyruvate solution, 10% ethanol plus 1% ethyl pyruvatesolution, or 10% ethanol plus 3% ethyl pyruvate solu-tion. Data are the mean ± SEM

groups. Rats exposed to 10% ethanol plus 3% EP had a

lower body weight than animals that had only ethanol to

drink (see Fig. 1 for statistical significance). In particular,

the difference was statistically significant from week 3

to week 6, and from week 8 to week 10 of ethanol or

ethanol plus 3% EP access (Fig. 1). Weekly fluid intake

in ethanol drinking rats was lower than those not having

it. This might reflect the fact that ethanol intake also pro-

vides calories, so those animals might eat less food since

they get some calories from the ethanol solution.

The fluid intake of animals that had access to the 10%

ethanol plus EP was lower than that of rats who had access

to only 10% ethanol solution and was dosed by measurement

of the residual volume of the fluid content in the bottle.

Water intake in rats that had water + EP was not

significantly different from that of rats that had only

water to drink, without ethyl pyruvate.

The overall ANOVA has shown also that the fluid intake

in rats was significantly different between the eight groups

(F(7,28) = 68.1; P < 0.0001), for the time of treatment

(F(9,252) = 68.1; P < 0.0001). In particular, in the group

of rats that had 10% ethanol plus 3% or 1% EP, the dif-

ference from rats with just 10% ethanol to drink was stati-

stically significantly from the first week of treatment to

the last week, while the difference in intake of rats with

the lowest concentration of EP (0.3%) was statistically

significant only from week 6 until the last week (Fig. 2).



DOI: 10.1002/jat

Figure 5. Time course of lucigenin-amplified chemiluminescence of monocytes in rats treated with water (�),ethanol 10% (�) and ethanol 10% + EP 3% (�). Monocytes were stimulated with 3 × 10−4 mol l−1 phorbolmyristate acetate. Chemiluminescence was measured as counts per minute (cpm)

Figure 4. Inhibition of lucigenin-amplified chemilu-minescence (%) by pyruvate (�) or ethyl pyruvate (�)in the reaction system containing 50 μM xanthine,0.9 U ml−1 xanthine oxidase, and 150 μM lucigenin, in50 mM phosphate buffer pH 7.0. Data represent themean ± SD

minescence (Fig. 4). As can be observed, the scavenger

activity of EP was higher compared with pyruvate: the

50% inhibition was obtained with ~80 mM of EP, while

only ~10% inhibition was obtained with the same amount

of pyruvate (80 mM).

Respiratory Burst

Figure 5 shows the respiratory burst behaviour in mono-

cytes from rats treated with water, ethanol and ethanol +

Antioxidant Activity of Pyruvate and EthylPyruvate

Luminol amplified chemiluminescence was employed

to evaluate the scavenger activity of pyruvate and ethyl

pyruvate versus hydrogen peroxide (Fig. 3). Both com-

pounds can reduce the CL signal. In particular, a 50%

inhibition was obtained with ~12 mM EP and ~47 mM

pyruvate (Fig. 3). The antioxidant activity versus super-

oxide anion, generated by xanthine-xanthine oxidase

system, was measured by lucigenin-amplified chemilu-

Figure 3. Inhibition of luminol-amplified chemilu-minescence (%) by pyruvate (�) or ethyl pyruvate ( �) inthe reaction system containing 50 mM H2O2 and 100 μM

luminol in 50 mM phosphate buffer pH 7.0. Data repre-sent the mean ± SD



DOI: 10.1002/jat

Figure 6. Observed distributions of comet tail moment in white blood cells from rats treated for 10 weeks withonly water, water plus 0.3% ethyl pyruvate solution, water plus 1% ethyl pyruvate solution, water plus 3% ethylpyruvate solution, only 10% ethanol solution, 10% ethanol plus 0.3% ethyl pyruvate solution, 10% ethanol plus1% ethyl pyruvate solution, or 10% ethanol plus 3% ethyl pyruvate solution. Data (at least 150 scores/sample) aremean value ± SEM. § P < 0.05 compared with water. * P < 0.05 , ** P < 0.01, *** P < 0.001 compared with EtOH

3% EP. The cells were activated with PMA and the

superoxide anion was revealed by the presence of luci-

genin in the reaction system. The data obtained indicate

superoxide anion production in the three groups follow-

ing the activation of the NADPH oxidase system by

PMA. As can be observed in Fig. 5, superoxide anion

production was higher in the group treated with ethanol

compared with the control group. The ethanol + 3% EP

group produced fewer superoxide anions compared with

the control group.

Comet Assay

The comet assay was employed to evaluate DNA damage

in lymphocytes following ethanol treatment and to

explore the potential protective effect of EP. Figures 6

and 7 show the data obtained after 10 weeks of water

or ethanol ± EP intake. When rats were treated with

ethanol, a significant increase of tail moment was meas-

ured (Fig. 6). No change was measured among the water

group and the groups treated with water + (0.3%, 1%,

3%) EP (Fig. 6). When rats drank ethanol + EP, a signifi-

cant reduction of TM, TL and TI was detected (Figs 6

and 7). In particular, as can be observed in Fig. 6, the

TM decreased with the increase of percentage of EP

added to ethanol.

In order to quantify the ethanol DNA damage, the 95th

percentile was used as a cut-off point for the considered

tail parameters (TL, TI, TM) in the control cells (water

and ethanol), Cells with tail parameter values below the

cut-off were classified as ‘undamaged’ and those with

higher values as ‘damaged’. The cut-off (95th percentile)

values observed in this study are reported in Table 1.

The protective effect of EP can also be observed in

Fig. 7 (B–E): the distributions of tail intensity and tail

length show a lower percentage of cells with higher

cut-off values of TL and TI compared with samples

treated with ethanol alone.

Discussion

Ethanol consumption is linked to several pathologies,

such as liver injury, neurotoxicity, cardiomyopathy, fetal

alcoholic syndrome and cancer. Ethanol toxicity can be

correlated with its metabolism to acetaldehyde and with

Table 1. Values corresponding to the 95th percentilefor the considered tail parameters (tail length, tailintensity and tail moment) evaluated in white bloodcells from rats treated with only water and only 10%ethanol solution

Tail parameter Water EtOH

Tail length (μm) 14.00 26.25

Tail intensity (%) 8 371 11 789

Tail moment 1.824 3.69



DOI: 10.1002/jat

oxidative stress, which occurs in different tissues, follow-

ing ROS formation (Russo et al., 2001; Zima and

Kalousova, 2005). Being lipophilic, ethanol can cross the

plasma membrane and exert its effect on cellular com-

ponents; in particular, previous studies show that increas-

ing alcohol concentrations can induce impairment of the

respiratory chain, producing reactive oxygen species

(ROS) (Russo et al., 2001). ROS cause lipid peroxida-

tion, protein oxidation and DNA damage (Halliwell and

Gutteridge, 1989). Several studies on various cells have

demonstrated that ethanol can induce DNA alterations,

which are dose dependent (Russo et al., 2001; Zima and

Kalousova, 2005; Berrettini et al., 2004). Previous work

showed that ethanol is able to interact directly and/or

indirectly with DNA, leading to disruption of genomic

function by DNA strand break formation, DNA adduct

formation, and changes in the expression of transcription

factors, immediate-early genes that promote apoptosis,

and mRNA (Lamarche et al., 2004). Ethanol metabolism

is linked to the activity of two enzymes: alcohol dehydro-

genase (ADH), a cytosolic enzyme, and the cytochrome

P4502E1 (CYP2E1), a membrane-bound component of

the ethanol oxidizing microsomal system (Lieber, 2004).

Both enzymes lead to the generation of acetaldehyde.

CYP2E1 catalyses the generation of free radicals and

other reactive species that can lead to the formation

of lipid peroxides, including malondialdehyde and 4-

hydroxynonenal, which is responsible for liver oxidative

damage (Donohue et al., 2006). Studies conducted with

recombinant Hep G2 cells expressing ADH and CYP2E1

and responding to ethanol in a similar ways to liver

parenchymal cells, show that clearance of ethanol and

generation of acetaldehyde were highly dependent on the

expression of ADH (Donohue et al., 2006). Moreover,

Badger et al. showed that rats exposed to chronic ethanol

treatment present increased gene expression of ADH I,

which is the main liver enzyme responsible for ethanol

oxidation to acetaldehyde (Badger et al., 2000). Blood–

brain barrier alterations following alcohol consumption

have been attributed to acetaldehyde or ROS produc-

tion (Haorah et al., 2005). Some reports indicate that

acetaldehyde induces DNA-protein crosslink in liver,

brain and other organs in the rat; this interaction is

Figure 7. Correlation between % of migrated DNAtail intensity and tail length in white blood cellsfrom rats treated for 10 weeks with only water (A),only 10% ethanol solution (B), 10% ethanol plus 0.3%ethyl pyruvate solution (C), 10% ethanol plus 1% ethylpyruvate solution (D), or 10% ethanol plus 3% ethylpyruvate (E) solution. The lines indicate the 95thpercentile values for tail length (vertical) and tailintensity (horizontal), respectively. Percentile valueswere calculated in the control cells (water and EtOH)and were used as cut-off points to classify the cells as‘undamaged’ or ‘damaged’



DOI: 10.1002/jat

thought to play an important role in ethanol-induced

DNA damage (Niemela, 2001; Xi et al., 2004; IARC,

2002).

Our studies in alcohol-preferring rat lymphocytes

are in accordance with previous studies in which DNA

damage occurs after ethanol intake. In fact, a significant

increase of tail moment (1.64 times) was measured,

indicating lymphocyte DNA damage following ethanol

intake. In order to take advantage of pyruvate ability to

scavenge reactive oxygen species, avoiding the problems

associated with its instability in solution, it was deter-

mined whether the derivative EP would be protective in

an animal model of ethanol mediated cellular damage

(O’Donnell-Tormey et al., 1987; Varma et al., 1998).

Three EP doses (0.3%, 1% and 3%) were employed in

eight rat groups treated with water or 10% ethanol. No

significant DNA alterations were observed when EP was

added to water at all concentrations used, indicating that

the amount of EP used does not modify the DNA. When

EP was added to ethanol in 10% ethanol solution, a sig-

nificant dose dependent reduction of TM was measured.

This behaviour indicates that EP can oppose the damage

induced by alcohol. It can be hypothesized that the

mechanism by which EP exerts its effect involves differ-

ent pathways linked to its scavenger activity. In particu-

lar, the ROS scavenger action might be correlated either

directly with the EP or with the free pyruvate obtained

from its de-esterification (Woo et al., 2004). Pyruvate,

like other α-keto acids, can react rapidly and non-

enzymatically with hydrogen peroxide (Kim et al., 2006).

Like pyruvate, EP can also rapidly scavenge hydrogen

peroxide. As reported by Wang et al., EP directly decom-

poses hydrogen peroxide in a concentration-dependent

manner (Li-Zhen et al., 2005). These data can also be

confirmed in our chemiluminescence results, where EP

reduces the luminol amplified CL signal four times more

than pyruvate. Moreover, our lucigenin amplified CL

results show that EP can scavenge superoxide anion

more efficiently than pyruvate. Chemiluminescence was

employed both to quantify the hydrogen peroxide and

superoxide anion scavenging activities of pyruvate and

EP, and to measure the monocyte respiratory burst in

different rat groups. This technique allows the determina-

tion of the light produced following a chemical reaction

(Murphy and Sies, 1990). When the light signal obtained

from the excited state of the chemical reaction product is

low, it is necessary to amplify it using a probe that does

not interfere with the reaction (Murphy and Sies, 1990).

Usually, luminol and lucigenin are employed to detect

hydrogen peroxide and superoxide anion, respectively

(Olek et al., 2005; Gabbianelli et al., 1997). The latter

ROS is an important phagocyte respiratory burst pro-

duct (Babior, 1984, 1987). Alterations in phagocyte

activity have been detected in heavy drinkers (Morland

et al., 1985). Defects in the function and number of

phagocyte cells were observed (Morland et al., 1985).

Ethanol-related immunosuppression is manifested by

increased susceptibility to bacteria or viral pathogen

infections, as well as impaired clearance of these organ-

isms during the infections (Napolitano et al., 1995). An

important component of the altered immunity after alco-

hol use is aberrant monocyte function (Mandrekar et al.,

1996; Szabo et al., 1998). Changes in the production of

tumor necrosis factors (TNF-α, interleukins (IL-1β and

IL6, IL-10 and IL-12) cytokines resulted in macrophages/

monocyte after acute ethanol exposure (Mandrekar et al.,

1996; Szabo et al., 1998). Human monocyte IL-10

production is increased by acute ethanol treatment

(Mandrekar et al., 1996). Interleukin 10 has been shown

to prolong cell survival, inhibit polymorphonuclear

leukocyte cytokine production and, most importantly,

regulate macrophage/monocyte functions (Mandrekar

et al., 1996). Our data on monocyte superoxide anion

production after PMA activation show that the rat group

treated with ethanol produced more superoxide anion

compared with the control group. This effect could be

linked to the priming effect induced by IL-10. This event

consists of increased phagocyte response when interact-

ing with different activators (platelet-activating factor,

substance P, tumor necrosis factors, interleukins, hydro-

xylated derivatives of arachidonic acid, phorbol esters)

(Gabbianelli et al., 1995; Kitagawa et al., 1988). In par-

ticular, the increased responsiveness, known as priming,

can be differentiated from stimulation obtained with only

one agonist. The priming effect is induced by sublimit-

ing concentrations of activating compounds. When the

primed phagocytes interact with a different agonist, their

response is amplified (Gabbianelli et al., 1995; Kitagawa

et al., 1988; Kono et al., 2000). Different pathways

can be related with the priming effect, such as an

increase of free cytosolic Ca++, protein kinase C activa-

tion, NADPH oxidase activation and translocation of cell

surface receptors (Kitagawa et al., 1988; Kono et al.,

2000; Ferrante, 1992). Moreover, when rats were treated

with 10% ethanol plus 3% EP, a great reduction in the

CL signal was observed.

Some possible EP mechanisms of action may be pro-

posed: a scavenger effect of EP on superoxide anion, in

accordance with our CL results, produced by NADPH

oxidase system, an alteration in signal transduction or an

interference on NADPH oxidase complex formation. As

known, the NADPH oxidase complex is composed of

various membrane and cytoplasmatic subunits that inter-

act together when the cells are activated. The lack of one

subunit or an alteration in the formation of the complex

produced a low level of superoxide anion. Moreover, the

reduced superoxide production by EP could be correlated

with a decrease of the expression of numerous pro-

inflammatory mediators as reported by Sappington in

many in vitro and in vivo model systems (Sappington

et al., 2005). Our results on the monocyte respiratory

burst indicate that EP is able to reduce the excessive



DOI: 10.1002/jat

response of phagocytes, but this effect should be limited,

since a reduced respiratory burst leads to a decrease of

immune defense. For this reason, the amount of EP

should be optimized in order to contain its effect.

In summary, the above data indicate that the treat-

ment with EP can reduce the ethanol lymphocyte DNA

damage and the abnormal-increased answer of monocyte

during the respiratory burst. These results could be inter-

esting in order to investigate whether EP, following its

scavenger effect, can increase the lifetime of rats drink-

ing ethanol. Moreover, longer time exposure to EP could

permit the evaluation of the long-term effect of EP on the

health of animals drinking ethanol. Further studies will be

performed in order to measure the influence of EP on the

other respiratory burst steps and to optimize the best dose

for maintaining normal phagocyte activity. Moreover, the

ability of EP to contain the oxidative stress induced by

chemical and physical sources should be investigated in

various cell types.

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