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Experimental and Toxicologic Pathology 58 (2006) 203–208

www.elsevier.de/etp

Effect of monensin on the enzymes of oxidative stress, thiamine

pyrophosphatase and DNA integrity in rat testicular cells in vitro

Malti Singh, N.R. Kalla, S.N. Sanyal�

Department of Biophysics, Panjab University, Chandigarh 160014, India

Received 22 November 2005; accepted 27 June 2006

Abstract

Monensin, a sodium specific ionophore was evaluated for its in vitro effects on rat testis by studying changes atbiochemical parameters as well as at the DNA level. It was observed that monensin produced marked alterations in theactivities of various enzymes associated with the testicular functions. The significant inhibition of different enzymes ofoxidative defense system points toward the generation of reactive oxygen species (ROS) by monensin treatment. Thesignificant depletion of reduced glutathione and elevation in the level of lipid peroxidation further support the abovefindings. The significant inhibition of the activities of lactate dehydrogenase and adenosine triphosphatase shows theinterference of monensin with the normal energy supply in spermatogenesis. Moreover, the significant increase in theactivities of acid phosphatase and thiamine pyrophosphatase demonstrates the interference of monensin with theGolgi-lysosomal complex of the rat testis. Induced DNA fragmentation indicates towards the impact of monensin onthe DNA integrity and apoptosis. Further studies are needed to understand the important molecular mechanismsresponsible for these effects.r 2006 Elsevier GmbH. All rights reserved.

Keywords: Monensin; Oxidative defense system; DNA damage; Testis; Oxidative stress; Rat

Introduction

Monensin is a carboxyl polyether ionophore pro-duced by Streptomyces cinnamonensin (Mollenhauer etal., 1990). It has been known for many years in poultryindustry for its useful effect as food additive. It is alsowell known as a Na+/H+ exchanger across biologicaland model membranes. Being an ionophoric antibiotic,monensin is able to form lipophilic complexes withmonovalent cations, hence, can cause cation imbalances

e front matter r 2006 Elsevier GmbH. All rights reserved.

p.2006.06.006

ing author. Tel.: +91172 2534119;

7082.

esses: sanyal@pu.ac.in, sanyalpu@yahoo.co.in

which are known to produce different biochemical andhistological changes (Mollenhauer et al., 1990). Mon-ensin’s main action consists of exchanging protons forNa+ which leads to osmotic swelling of post Golgiendosomal structures and Golgi subcompartments byvirtue of its membrane-associated effects. This Na+/H+

antiport regulates different cellular phenomena includ-ing intracellular pH, cell volume, ion transport, trans-port of water and acid base equivalents (Grinstein et al.,1989; Zhu and Loh, 1995). Monensin is also known forits acute toxicity.

Atef et al. (1986) studied the effect of monensin on thefertility status of rats. It was noticed that the drugcaused oligospermia and azoospermia in male rats aswell as decreased activity of spermatogenic epithelium.

ARTICLE IN PRESSM. Singh et al. / Experimental and Toxicologic Pathology 58 (2006) 203–208204

Acrosome formation by Golgi bodies is one of thetransforming events during spermiogenesis, which is aprerequisite for the process of fertilization. Although itseems that the Golgi disturbing nature of monensin mayaccount for its antispermatogenic effect, some otherfactors like oxidative stress and DNA damage seem tobe important to understand the effects of the drug onmale fertility. Therefore, the present study was plannedto investigate the in vitro effects of monensin ontesticular function with emphasis on oxidative stressand DNA damage.

Materials and methods

Chemicals

All the chemicals used in the present study were ofA.R. grade. Monensin was purchased from SigmaChemicals Co., St. Louis. MO, USA.

Testicular cell culture

The testicular cell culture was done according to themethod of Steinberger (1975), in a medium thatcontained DMEM (with glutamine, without indicator)9.6 g/l, HEPES 10mM, gentamycin 10mg/l, fungizone1.25mg/l, sodium bicarbonate (NaHCO3) 2.0 g/l, fetalcalf serum (FCS) 5%. Rats were sacrificed afteranesthetizing with ether and testes were removed anddecapsulated. The tissues were gently teased (Hudsonand Hay, 1989) to get a uniform cell suspension of totaltesticular cell population, which was centrifuged at600 rpm for 10min. The cell pellet obtained was washedwith phosphate buffered saline (PBS) and recentrifuged.The resultant cell pellet was suspended in the mediumand centrifuged at 600 rpm for 10min and finallysuspended in the fresh medium. Cell viability waschecked by trypan blue exclusion test (Yip andAuersperg, 1972). The cells were plated at a concentra-tion of 0.5� 105 cells per well and monensin added atdifferent concentrations (10, 20, 40, 80 and 100 mM).This was incubated at 31 1C for 18 h in a humidifiedatmosphere of 5% CO2 and 95% air. After theincubation the cells were checked for viability anddifferent biochemical measurements were done. Also thegenomic DNA was isolated to perform the fragmenta-tion study.

Biochemical measurements

Superoxide dismutase (SOD). The activity of SODwas measured by the method of Kono (1978), byobserving the reduction of nitroblue tetrazolium (NBT)dye by superoxide anions.

Catalase. The enzyme activity was measured accord-ing to the method of Luck (1963), where the decom-position of H2O2 was measured by decrease inabsorbance at 240 nm.

Glutathione peroxidase (GSH-PX). The assay wasperformed by the method of Paglia and Valentine(1967), the enzyme activity was calculated by thesimultaneous oxidation of NADPH at 340 nm.

Glutathione reductase (GR). The activity of GR wasestimated by the method of Massey and Williams(1965), the utilization of NADPH at 340 nm was directlyrelated to the enzyme activity.

Glutathione-S-transferase (GST). The method ofHabig et al. (1974) was applied using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate.

Reduced glutathione (GSH). GSH content wascalculated by the method of Moron et al. (1979) 5,50-dithiobis-(2-nitrobenzoic acid) was reduced to nitromer-captobenzoic acid, which was used to measure the –SHgroups at 412 nm.

Lipid peroxidation (LPO). LPO levels were checkedaccording to the method of Buege and Aust (1978),where malonyldialdehyde, produced by peroxidativereactions, was measured at 532 nm.

Lactate dehydrogenase (LDH). The enzyme wasmeasured by the method of Schatz and Segel (1969) byfollowing the change in absorbance at 340 nm due to thesimultaneous oxidation of NADH to NAD+.

ATPase. The enzyme activity was assessed by themethod of Quigley and Gotterer (1969), and theliberated phosphorous was calculated according to themethod of Fiske and Subbarow (1925).

Acid phosphatase. The enzyme was measured by themethod of Linhart and Walter (1965), using p-nitrophe-nyl phosphate as the substrate.

Thiamine pyrophosphatase (TPPase). The enzymewas measured by the method of Allen and Slater(1961), and for the determination of inorganic phos-phate, the method of Berenblum and Chain as modifiedby Martin and Doty (1956) was applied.

Proteins. These were measured by the method ofLowry et al. (1951).

DNA fragmentation

DNA from testicular cells was isolated using cetyltrimethyl ammonium bromide (CTAB)/NaCl by themethod of Sambrook and Russell (2001). Briefly the cellpellet was suspended in Tris–EDTA (pH ¼ 8) bufferand treated with SDS and proteinase K. After incubat-ing at 65 1C for 1 h, the samples were treated withCTAB/NaCl and again incubated for 1 h. After this, anequal volume of chloroform: isoamyl alcohol (24:1) wasadded and vortexed. DNAwas precipitated by adding iso-propanol at �20 1C for 30min. For gel electrophoretic

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Table 1. The effect of different concentrations of monensin added in vitro in the testicular cell culture on the activities of different

oxidative defense enzymes

Monensin

treatment

(mM)

SOD (unit)a Catalase (mmole

H2O2 decomposed/

min/mg protein)

GPX (mmole

NADPH oxidized/

min/mg protein)

GR (n moles

NADPHoxidized/

min/mg protein)

GST (n moles

conjugate formed/

min/mg protein)

0 5.5870.36 16.1172.5 0.24170.04 12.8371.3 9.1871.5

10 3.3670.7*** 9.972.0*** 0.22270.04 15.8873.1* 5.2171.0***

20 5.7770.8 9.2171.0*** 0.22370.02 11.572.0 9.5171.7

40 2.1870.5*** 4.6771.1*** 0.18270.02** 6.4670.2*** 5.0670.2***

80 1.4370.3*** 2.7870.24*** 0.15970.007** 4.2371.2*** 4.7171.25***

100 1.570.7*** 3.2870.76*** 0.18870.03* 5.870.9*** 5.5871.5***

Values are mean7SD of six observations; *po0.05, **po0.01, ***po0.001 represent the comparison between the control and treated sample.aOne unit of enzyme is expressed as the amount of protein (mg) required to inhibit the reduction rate of NBT by 50%.

Table 2. The effect of different concentrations of monensin

added in vitro in the testicular culture on reduced glutathione

and lipid peroxidation

Monensin

treatment (mM)

GSH (mmoles/mg

protein)

LPO (n moles

MDA formed/

min/mg protein)

0 0.55570.06 20.674.34

10 0.40370.05*** 2372.94

20 0.52970.08 24.1674.29

40 0.40470.02*** 26.575.6*

80 0.38370.03*** 3074.3***

100 0.41870.01*** 25.374.2*

Values are mean7SD of six observations; *po0.05, ***po0.001

represent the comparison between the control and treated sample.

M. Singh et al. / Experimental and Toxicologic Pathology 58 (2006) 203–208 205

separation of DNA 1.2% Agarose gel containingethidium bromide (1 mg/ml) was prepared in Tris–Bor-ate–EDTA (TBE) buffer (pH ¼ 8). After completion ofelectrophoresis, the bands obtained were visualised onan UV transilluminator.

Statistical analysis

All experiments have been repeated six times (n ¼ 6)as reported in the present paper. Results were expressedas mean7the standard deviation of the mean (SD) andthe data was analyzed using Student’s t-test. p-valuesless than 0.05*, 0.01** and 0.001*** were consideredsignificant.

Results

Cell viability was found to be more than 95% in allthe samples at the time of culture. At the end of theculture 90% cells were viable in control cells, however,more than 75% cells were noticed to be viable in variousmonensin treated samples.

Monensin at various concentrations significantlyinhibited the activities of SOD and Catalase (Table 1),however, the enzyme activities of GSH-PX and GR weredecreased significantly with the higher concentrations ofmonensin (Table 1). It was noticed a significant increasein GR activity with the lowest concentration ofmonensin. GST activity was found to be significantlyinhibited with 10, 40, 80 and 100 mM of monensintreatment (Table 1). Monensin except 20 mM, signifi-cantly depleted the GSH contents in comparison to thecontrol, however, LPO levels were enhanced at thehigher concentrations of the drug (Table 2).

Monensin except 20 mM, significantly decreased theLDH activity, ATPase was found to be inhibitedsignificantly with 80 and 100 mM of monensin treatment(Table 3). The activity of acid phosphatase was elevatedwith 20–80 mM monensin, however, the activity of

TPPase was elevated with 20–100 mM of monensintreatment (Table 3). Monensin treatment at variousconcentrations also induced fragmentation of DNA incomparison to the control cells (Fig. 1).

Discussion

The in vitro treatment of testicular cells withmonensin at various concentrations resulted in markedalterations in the activities of various oxidative defenseenzymes. The significant inhibition in their activitiesmay be due to the induction of oxidative stress by thedrug. The cellular systems are equipped with effectivedefense system and SOD provides the first line ofdefense against ROS (Yoo et al., 1999). It has also beensuggested that SOD provides the major component ofthe antioxidant defense of human spermatozoa from thedamaging effects of ROS (Alvarez et al., 1987; Peltola etal., 1992). In the present study, it has been observed asignificant decrease in the activity of SOD at variousconcentrations of monensin, leading to the impairmentin tackling of the ROS. In the testicular tissue thedetoxification of H2O2 to H2O is done by GSH-PX and

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Table 3. The effect of different concentrations of monensin added in vitro on the activities of several enzymes of oxidative

metabolism and thiamine pyrophosphatase

Monensin treatment

(mM)

LDH (mmoles of

NADH oxidized/min/

mg protein)

ATPase (mmoles/min/

mg protein)

Acid phosphatase

(mmoles/min/mg

protein)

TPPase (mmoles/min/

mg protein)

0 0.42670.009 0.23070.03 0.43270.04 0.41470.01

10 0.31570.03*** 0.20970.02 0.47370.07 0.42870.09

20 0.43170.06 0.25570.04 0.57370.13* 0.46970.06*

40 0.30870.04*** 0.22270.02 0.54670.09* 0.47270.06*

80 0.18070.03*** 0.18970.01** 0.57370.04*** 0.50270.09*

100 0.21570.04*** 0.19770.01* 0.43370.06 0.52870.06**

Values are mean7SD of six observations; *po0.05, **po0.01, ***po0.001 represent the comparison between the control and treated sample.

Fig. 1. Agarose gel electrophoresis of genomic DNA isolated

from testicular cells treated with different concentrations of

Monensin. (From left to right) Lane 1: 100mM monensin, lane

2: 80mM monensin, lane 3: 40 mM monensin, lane 4: 20 mMmonensin, lane 5: 10 mM monensin, lane 6: control and lanes 7

and 8: positive control.

M. Singh et al. / Experimental and Toxicologic Pathology 58 (2006) 203–208206

catalase (Surai et al., 1998). The inhibition of catalaseactivity in the present study may thus be responsible forthe production of ROS by monensin. The enzymecatalase does not seem to be responsible for the removalof all the cellular hydrogen peroxide, but GSH-PXremoves most of it (Orrenius et al., 1982) and isconsidered as more efficient in protecting the cell from

the damage of lipid peroxidation. It is largely distributedin male germ cells (Zini and Schlegel, 1997). It seemsthat monensin treatment is causing peroxidative damageby inhibiting the activities of SOD, catalase and GSH-PX .The functioning of GSH-PX is dependent on thelevels of GSH which are maintained by the enzyme GR(Chow, 1987), thus GR protects the cell indirectly fromthe ROS damage. Male germ cells exhibit appreciableGR activity (Kaneko et al., 2002). The inhibition of itsactivity may be responsible for the GSH depletion andgeneration of lipid peroxidation reaction products whichwill affect the proper recycling of glutathione in thetestis. In the present investigations a significant deple-tion in GSH content was noticed along with a markedelevation in LPO levels. GSTs provide the protectionagainst the products of oxidative damage (Hayes andPulford, 1995). The in vitro treatment of monensinsignificantly reduced the GST activity, which may bedue to the less availability of GSH, which is also foundto be depleted in the present study. A significantdepression in GST activity in monensin treated ratswas also reported by Dalvi (1992). The depletion inGSH levels may occur due to the induction of oxidativestress by monensin. GSH related enzymes viz. GSH-PX,GR and GST and are known to play a major role in freeradical scavenging. GSH-PX and GR are involved in theredox-cycling of GSSG and GSH whereas, GSTcatalyses detoxification through the formation of GSHconjugates (Sies and Ketterer, 1988). In the presentstudy the activities of these enzymes were found to bereduced, hence, making the system more prone to thedamaging effects of the free radicals.

Testicular LDH is an essential component of themetabolic machinery of spermatozoa and involved inthe energy generation processes. The decreased enzymeactivity in monensin-treated cells points toward theinterference of monensin with the energy metabolism ofthe cell. The decrease in ATPase activity may be due tothe damage/depletion of membranes of germ cellsresulting into low enzyme activity in testis. Monensinresults into cation imbalances and cause mitochondrial

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swelling thereby decreasing the ATPase activity(Mollenhauer et al., 1990). A close association has beennoticed between ATPase activity and Golgi acidification(Yeh and Rossum, 1991). Therefore, the decrease in theenzyme activity may be due to the disturbance of Golgi bymonensin. Thiamine pyrophosphatase is the trans-Golgimarker enzyme (McGuinness and Orth, 1992; Morre etal., 1994). In germ cells the enzyme activity is stronglyassociated with the spermatogonial population (Passia etal., 1985). The present results show an overall increase inthe enzyme activity in different monensin treatmentswhich may be an indication of the interference of the drugwith the Golgi apparatus. Therefore the increasedactivities of acid phosphatase and thiamine pyropho-sphatase further support the idea that monensin may beinterfering with the Golgi-lysosomal complex of rat testis.

The effect of monensin was generally found dosedependent except for the 20 mM concentration for GR,GST, GSH and LDH. However, the change was notstatistically significant. LPO and acid phosphataseactivity showed an increase at all the concentration ofmonensin, although the change is less at 100 mM than80 mM, the reason for which is not clear, but probablyindicate to a saturating dose for the drug effect.

The results from the DNA fragmentation studyindicate that DNA damage is caused by monensin.Oxidative stress in testicular milieu is associated withoxidative damage and can cause significant effect onmale fertility (Ames et al., 1995; Kumar et al., 2002).Enhanced lipid peroxidation may also result into DNAdamage (Higuchi, 2003). The results from the presentstudy are indicative of induction of oxidative stress bymonensin treatment, which might be responsible forDNA damage. DNA damage could also be due toapoptosis (Gorczya et al., 1993) and monensin isreported to regulate the cellular phenomenon likeapoptosis (Zhu and Loh, 1995; Park et al., 2003).

Results obtained from the present study stronglysuggest that monensin interferes with the spermatogen-esis by inducing oxidative stress, DNA damage and alsoby affecting the Golgi-lysosomal system of rat testis.However, further studies are needed to explore themolecular mechanisms responsible for such effects.

Acknowledgements

Thanks are due to Indian Council of MedicalResearch for providing the financial support.

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