Reproductive Toxicology 28 (2009) 81–89

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Reproductive Toxicology

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Testicular toxicity in mercuric chloride treated rats: Association withoxidative stress

Mohamed Ali Boujbihaa, Khaled Hamdena, Fadhel Guermazib, Ali Bouslamac, Asma Omezzinec,Abdelaziz Kammound, Abdelfattah El Fekia,∗

a Lab. Ecophysiology of Animals, Faculty of Sciences, 3018 Sfax, BP 802, Tunisiab Lab. Biophysics, Faculty of Medicine & Nuclear Medicine, CHU Habib Bourguiba, Sfax, Tunisiac Lab. Biochemistry, CHU Sahloul Sousse, Tunisiad Lab. Physiologie of Animals, Faculty of Sciences, Tunis, Tunisia

a r t i c l e i n f o

Article history:Received 13 December 2007Received in revised form 3 March 2009Accepted 27 March 2009Available online 5 April 2009

Keywords:Mercuric chlorideTestisTestosteroneOxidative stressLipid peroxidationSuperoxide dismutaseCatalaseReproductive system toxicity

a b s t r a c t

Mercury has been recognized as an industrial hazard that adversely affects male reproductive systemsof humans and animals. However, less information is available concerning the underlying mechanism inthe pathogenesis of male reproductive dysfunction. The present study investigated the possible involve-ment of oxidative stress to induce oxidative deterioration of testicular functions in adult rats. Wistarmale rats (n = 132) were continuously exposed to HgCl2 at 0, 50 and 100 ppm during 90 days throughoral administration in the drinking water. Mercury exposure for 90 days resulted in an increase in theabsolute and relative wet weight of the testis and a decrease in the absolute and relative wet weightof the accessory sex glands, with respect to the matched control. Marked perturbation in testosteroneserum level was also detected during the treatment for the treated groups. Cauda epididymal spermcount/motility decreased significantly in the mercury-treated group and qualitative examination of tes-ticular sections revealed a fewer mature luminal spermatozoa in comparison to the control. When themercury-treated males were mated with normal cyclic females, mercury exposure resulted in a declineof the reproductive performance of male rats. These effects were associated with a significant increasein mercury content of testes and blood in time-dependent and dose-dependent fashion, respectively.The HgCl2 treatment was associated with oxidative stress. Evidence of induction of oxidative stress wasobtained in terms of perturbations in antioxidant defense and a significant dose-dependent increase inthe testicular lipid peroxidation as a consequence of pro-oxidant exposure. Taken together, the results

suggest that an increase in free radical formation relative to loss of antioxidant defense system aftermercury exposure may render testis more susceptible to oxidative damage leading to their functional

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. Introduction

Different models of experimental intoxication with mercury andts derivatives have shown that the extent of the resulting damager dysfunction depends on time, means of administration, and dose1]. On the other hand, the lack of a direct correlation between the

Abbreviations: HgCl2, mercuric chloride; ANOVA, analysis of variance; ROS,eactive oxygen species; SOD, superoxide dismutase; CAT, catalase; LPO, lipid peroxi-ation; MDA, malondialdehyde; TBARS, thiobarbituric acid reactive substances; TBA,hiobarbituric acid; PUFAs, polyunsaturated fatty acids; RIA, radioimmunoassay;PM, count per minute.∗ Corresponding author. Tel.: +216 74 276 400; fax 216 74 274 437.

E-mail address: Abdelfattah.Elfeki@fss.rnu.tn (A.E. Feki).

890-6238/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.reprotox.2009.03.011

© 2009 Elsevier Inc. All rights reserved.

mercury accumulation and the severity of lesions [2] suggests thatpotential protective mechanisms may act in such a way that nodysfunction can be detected during chronic treatment at low dose.Given this, experiments involving treatment with higher doses havebeen developed to characterize the mechanisms and possible fac-tors that may prevent or attenuate the dysfunctions induced bymercury [3]. Specifically, antioxidants play a crucial role in main-taining cell homeostasis and, when these defenses are impairedor surmounted, oxidative stress products, namely reactive oxygenspecies (ROS), may induce enzymatic inactivation and peroxidationof cell constituents. Moreover, mercury is a ubiquitous element in

the environment causing oxidative burst in the exposed individ-uals leading to tissue damage. Its contamination and toxicity hasposed a serious hazard to human health. Annually tons of mercuricchloride (HgCl2) are released into the atmosphere by industrial pro-cessing and municipal waste incineration. The principal sources of

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2 M.A. Boujbiha et al. / Reprod

xposure to inorganic mercury (Hg2+) are from water, food and air,hile exposure to other forms of mercury are from dental amal-

am in tooth filling (elemental Hg0) and from consumption of fishnd other seafood (organic mercury Hg+) [4]. Both the biologicalnd toxicological effects of mercury are dependent on the form ofercury. Inorganic ionic mercury, ingested in the diet is mainly

etained in the intestinal tract and is only slowly transferred tohe internal organs [5]. HgCl2 is one of the most toxic forms of

ercury because it easily forms organomercury complexes withroteins [4]. It is highly toxic and oxidative once absorbed intolood stream: inorganic mercury combines with proteins in thelasma or enters the red cells. The inorganic ionic mercury has greatffinity for SH groups of biomolecules, such as gluthatione (GSH)nd sulfhydryl proteins, which may contribute to its toxicity [6].nce bound to GSH, Hg can leave the cell to circulate in serum or

ymph and be deposited in other organs or tissues [4]. The toxicityf HgCl2 via inhalation and oral exposure has been investigated inoth humans and animals. There have been numerous studies onhe effects of mercury on the immune system, renal system, cardio-ascular, reproductive system and the central nervous system [7].ccording to a number of review articles, mercury resulted in repro-uctive problems in humans and animals. Mercury compounds arenown to affect testicular spermatogenic and steroidogenic func-ions in experimental animals and men [8]. Mercury intoxicationas been reported to reduce secretory epididymal componentsecessary for sperm maturation [9]. Oral exposure of HgCl2 pro-uced adverse effects on the reproductive performance of mice10]. Mercury affects accessory sex gland function in rats and micehrough androgen deficiency [11]. Popescu [12] described repro-uctive toxicity among workers occupationally exposed to mercuryompounds. Keck et al. [13] reported elevated testicular mercury inn infertile man employed in the chloralkali industry. Decrementsn sperm count, motility, and viability have been reported in HgCl2xposed mice [14]. Keck et al. [13] also reported the possible effect ofnorganic mercury on the hypothalamic–pituitary–testicular axis.lthough, the effects of inorganic mercury on the male reproduc-

ive system have been investigated, less information is availableegarding the underlying mechanism in the pathogenesis of maleeproductive dysfunction. The transition metals act as catalysts inhe oxidative deterioration of biological macromolecules, there-ore, the toxicities associated with metals may be due at least inart to oxidative damage of tissues. A number of evidences advo-ate the role of oxidative stress in Hg (II) induced tissues toxicity15]. However, involvement of oxidative mechanisms in the toxic

anifestation induced by mercury compounds in male reproduc-ive system is quite insufficient. Mercury in the form of mercurichloride as used in this experiment is considered to be one ofhe pro-oxidants that induce oxidative stress [15]. Oxidative stressccurs when the production of ROS such as, superoxide anion (O−

2)ydrogen peroxide (H2O2), and the hydroxyl radical (•OH) exceedshe body’s defense mechanism, causing damage to macromoleculesuch as DNA, proteins and lipids [16] and trigger many pathologicalrocesses in the male reproductive system [17]. There is evidencehat ROS may have a detrimental effect on critical components ofhe steroidogenic pathway [18]. Moreover, various studies [15,19]ave suggested that a strong correlation exists between mercury-

nduced toxicity and the induction of lipid peroxidation which isonsidered as the most extensively studied manifestation of oxygenctivation in biology. Thus the present study was aimed to delineatehether oxidative mechanisms are involved in the defective testic-lar functions induced by mercury compounds. For this purpose

ose- or concentration- and time-dependent effects on testicular

ipid peroxidation, enzymatic antioxidants such as superoxide dis-utase and catalase, serum testosterone level, epididymal sperm

ount and percentage of sperm motility, histopathology of testiclend reproductive performance were examined in vivo .

Toxicology 28 (2009) 81–89

2. Materials and methods

One hundred thirty two sexually mature male albino rats of Wistar strain(Rattus rattus), aging three months old and weighing approximately 190 g, pur-chased from the breeding center of the Central Pharmacy of Tunis (Tunisia), wereused in the study. Animals were housed in stainless steel cages (six/cage) intemperature-controlled room (24–26 ◦C) with constant humidity (40–60%) and10 h/14 h light/dark cycle for at least one week prior to use in experimental proto-cols. Animals were maintained in accordance with guidelines for animals care of the“Faculté des Sciences de Sfax”, Tunisia. Rats were fed on commercial tablets (SociétéIndustrielle de Concentrés SICO; Sfax, Tunisia) and water ad libitum. The daily intakeof animal feed was monitored at least one week prior to start of mercury treatmentin order to determine the amount of water needed per experimental animal. Allforms of perceived stress were attenuated. Thereafter, animals were divided intothree groups keeping in account that each group had almost equal intake of animalfood. Drinking water and water used to prepare the solutions of mercury was ofdistilled quality and was supplied to the animals in Makrolon water bottles. The twodoses of mercuric chloride (Panreac, Quinica SA, Barcelona, Spain) were prepared asreported [20]. Groups of 44 male rats were allocated randomly and received mer-cury in the drinking water at concentrations of 0 (water control), 50 or 100 ppmthrough an exposure period of 90 days. We calculated the average dose of waterthat the treated rat drank along 90 days (11.66 ± 0.47 and 11.89 ± 0.47 ml/day forHG1 and HG2 groups; respectively) and this daily dose of water contained approx-imately 4 and 8 mg/kg-d in HG1 and HG2 animals, respectively. Body weight, dailyintake of food and water were determined three times/week throughout the study.At the end of each experimental stage (3, 7, 15, 30, 60 and 90 days), six rats fromeach group were decapitated under light ether anesthesia and fasting blood sam-ples were collected from the killed animals into dry and clean tubes. Serum sampleswere removed immediately after a centrifugation at 3000 g for 15 min and storedat −20 ◦C until assay. Also, testes, epididymis, prostate glands and seminal vesicleswere removed by micro-dissection from surrounding tissues and placed into pre-weighed tube. The organs were dried between two sheets of filter paper and theirwet weight was determined. The organ weight/body weight ratio × 100 was calcu-lated and expressed as relative organ weight. Histological study on testis was carriedout. After weighing, the organ was fixed in Bouin’s solution for 1–2 days, embeddedin paraffin by routine method, serially sectioned at 5 �m thickness and stained inhematoxylin/eosin for microscopic evaluation. The germinative cell layer thicknessof the seminiferous tubule from nine different areas of each testicle was measuredusing an ocular micrometer in a light microscope (×250), and the average thicknessof seminiferous tubule was calculated.

Total mercury (Hgt) concentrations were determined in testes and whole bloodusing a modification of the method described by Shinyashiki et al. [21]. The sampleswere heated with 7 ml nitric acid and perchloric acid (2 v/1 v) for 1 h at 230–250 ◦Con a hot plate (Gerhardt, Germany). After digestion, mercury concentrations weremeasured by reducing vaporization-spectrometry atomic absorption method, usingNippon Instruments Mercury Analyzer, Model RA-2 (Tokyo, Japan). Data are pre-sented as total mercury (�g/g of tissue dry and �mol/L) corresponding to themercury content measured in the homogenized samples.

Epididymal sperm were counted by a modified method of Yokoi et al. [22]. Briefly,the cauda epididymis was minced with anatomical scissors in 2 ml of Earles buffer(0.8 g CaCl2, 0.4 g KCl, 0.2 g MgSO4 7H2O, 6.8 g NaCl, 2.2 g NaHCO3,/1 l of NaH2PO4

2H2O (50 mM, pH: 7.2–7.4)) placed in a rocker for 10 min at 35 ◦C. After dilution,the number of homogenization-resistant spermatozoon was counted in a hemocy-tometer and about 25 fields of view were examined under a light microscope at 40×magnification. Sperm motility was evaluated using the same method. Numbers ofmotile and non-motile sperms were counted. Motility was then expressed as thepercentage of motile spermatozoa.

Serum testosterone was assayed by radioimmunoassay (RIA). Testosterone levelswere analyzed using RIA kits purchased from IMMUNOTECH (Marseille, France), asper manufacture protocol. Fifty microliters of standard and rat serum samples weretransferred to RIA tubes, to which 500 �l testosterone 125I reagent was immediatelyadded. Tubes were gently shaken and placed in a water bath at 37 ± 1 ◦C. After 3 h, alltubes were decanted, aspirated and count/min (CPM) was analyzed in a multigammacounter connected to a computer. Experiments were repeated twice with biologicalreplicate samples and the mean result was reported in ng/ml serum testosterone.

Oxidative status in testicular tissues could be estimated from the concentrationsof malondialdehyde (MDA) and the activities of two representative anti-oxidantenzymes, superoxide dismutase (SOD) and catalase (CAT). To measure these indi-cators, testicular tissues frozen at −80 ◦C were thawed and homogenized in 2 ml oflysis buffer (50 mM Tris, 150 mM NaCl adjusted to pH 7.4); the homogenates werecentrifuged at 9000 rpm for 15 min; the supernatants were saved; and the proteinconcentrations was measured, according to the method of Lowry et al. [23], usingbovine serum albumin as standard.

As a marker of lipid peroxidation (LPO) production, the level of LPO in testes was

measured by the method of Essterbauer et al. [24] as thiobarbituric acid reactivesubstances (TBARS). Since malondialdehyde (MDA) is a degradation product of per-oxidized lipids, the development of pink color with the absorption characteristics(absorption maxima at 532 nm) of TBA–MDA chromophore is taken as an index ofLPO. The concentration of TBARS was expressed as n moles of malondialdehyde/mgof protein tissue.

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Testicular superoxide dismutase was assayed by method of Asada et al. [25],hich involves inhibition of photochemical reduction of nitroblue tetrazolium (NBT)

t pH 8.0. A single unit of enzyme is defined as the quantity of superoxide dismu-ase required to produce 50% inhibition of photochemical reduction of NBT. Thebsorbance was read at 580 nm against a blank using UV–vis spectrophotometer.he activity was expressed as units/mg protein.

Catalase activity was estimated in tetstes homogenate in a UV–vis spectropho-ometer as described by Aebi [26]. The reaction mixture (1 ml, vol.) contained.02 ml of suitably diluted cytosol in phosphate buffer (100 mM, pH 7.4) and.2 ml of 500 mM H2O2 in phosphate buffer. The specific activity of catalase haseen expressed as � moles of H2O2 consumed/min/mg protein. The difference inbsorbance at 240 nm/unit time is a measure of catalase activity.

For the reproductive performance, mercury-treated male rats (6/group) wereaged along with cyclic female rats (1:2) with proven fertility including vehicle con-rol. Vaginal smear samples were collected daily and examined for the presencef sperm. The day of sperm detection in vaginal smear was considered as day 0 ofregnancy. The dams were killed on day 10 post-conception, in the goal to counthe number of implanted pups and to determine the implantation efficiency. Theeproductive performance parameters from this study are expressed in terms of:

1. Mating index (%) = (the number of females showing evidence of mating/numberof females placed with males) × 100.

. Numbers of viable embryos/litter.

All the values are expressed as mean ± SD for six animals (n = 6) in each group.ignificant differences between the groups were determined with SPSS 13.0 softwareSPSS Inc., Chicago, IL, USA) using one-way analysis of variance (ANOVA), followedy post hoc Newman–Keuls test when necessary. Differences were considered sig-ificant when p < 0.05.

. Results

For the control rats, a slight increase in absolute testicular weightas noticed (Fig. 1a). For the HG1 group, a similar pattern wasbserved; except at 7 days when a significant decrease was detected

ig. 1. Alterations in the absolute/relative testis weight of rats subjected to dif-erent dose- and time-dependent mercury treatment. Dose group symbols: closedquares = 0 ppm, open squares = 50 ppm, closed triangles = 100 ppm. Letters a, b andindicate groups at means that are significant different at 5% significance level.

Toxicology 28 (2009) 81–89 83

as compared to that in the controls. For HG2 rats, the testicularweights at 3, 15 and 30 days appeared lower than those of theirmatched controls. However, slight and significant increases at 7, 60and 90 days of treatment, were noticed, respectively. Moreover, thegonad weight appeared higher, when compared to the HG1 animalswhatever the treatment days (except at 3, 15 and 30 days), espe-cially significant at 7 days (Fig. 1a). Testicular weight in relationto body weight decreased with age in control rats, whereas in theHG1 group an increase in relative weight (p < 0.001) was observedat 7 and 30 treatment days (Fig. 1b). In HG2 group, the testicular-relative-weights along treatment all periods studied were almostconstant except at 7 days, the difference was greater. It was alsoclear that relative testicular weight was significantly higher in 7-day-treatment HG2 rats than in HG1 animals.

Concerning accessory sex glands weights, mercury treatmentresulted in non-significant decrease in absolute weight in treated-rats whatever the treatment days, except at 60 and 90 days whena significant decrease was noticed in seminal vesicles and prostateweight among the two treated groups, With respect to the matchedcontrol. For epididymis, the decrease in absolute weight was onlysignificant among HG2 group (Figs. 2a, 3a and 4a). Relative acces-sory glands weights exhibited a non-significant variation in avarying degree during treatment periods (Figs. 2b, 3b and 4b).

The effects of mercury were further analyzed through histo-logical examination. Interstitial effusion in treatment groups HG1and HG2 was associated with changes in seminiferous tubules

(Fig. 5B1, C1). This effect was more pronounced in HG2-treatedgroup. In addition to increased space between the tubules, thelumen of the seminiferous tubules was enlarged in group HG2over the slight changes in group HG1 (Fig. 5B1, C1). Other observa-

Fig. 2. Alterations in the absolute/relative epidydimis weight of rats subjected todifferent dose- and time-dependent mercury treatment. Dose group symbols: closedsquares = 0 ppm, open squares = 50 ppm, closed triangles = 100 ppm. Letters a, b, cand d indicate groups at means that are significant different at 5% significance level.

84 M.A. Boujbiha et al. / Reproductive Toxicology 28 (2009) 81–89

Fig. 3. Alterations in the absolute/relative seminal vesicles weight of rats subjectedtcal

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were approximately 0.5 and 0.9 �M among HG1 and HG2 groups at

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o different dose- and time-dependent mercury treatment. Dose group symbols:losed squares = 0 ppm, open squares = 50 ppm, closed triangles = 100 ppm. Letters, b and c indicate groups at means that are significant different at 5% significanceevel.

ions included specific alterations in the histoarchitecture of testisn mercury-treated groups. Indeed, treatment with 50 ppm HgCl2aused initial histological alterations which were prominent withighest dose. The highest dose of HgCl2 (100 ppm) produced his-ological alterations that include moderate to severe degenerativehanges. Seminiferous tubules exhibited disintegration of germi-al epithelium, detachment and degenerative changes of liningells, reduction in the number of round spermatids, significantailure of their maturation to mature spermatids and thereforelmost absence of mature spermatozoa (∼ 70% of tubules) (Fig. 5C2).egenerated cellular material was sloughed into the lumen. The

owest dose of HgCl (50 ppm) resulted in less alterations of

2estes–histoarchitecture. Most tubules (∼ 48%) examined had nopermatozoa evident in contrast to controls. When compared withG2 group, the testes segment of HG1 treated rats shows lessegeneration of seminiferous tubules with partial loss of spermato-

able 1otal mercury (Hgt) concentration in testis and blood at different doses and time.

arameters 0 d 3 d

ercury concentration (�g/g tissue dry) 0 ppm 0 050 ppm 0 0100 ppm 0 0.13 ± 0.08(a) (a) (a)

otal blood mercury (�mol/L) 0 ppm 0 050 ppm 0 0.38 ± 0.03100 ppm 0 0.55 ± 0.03(a) (b) (c)

ata are presented as the mean ± SD, n = 6. One-way analysis of variance (ANOVA) test follt means that are significantly different at 5% significance level.

Fig. 4. Alterations in the absolute/relative prostate weight of rats subjected to dif-ferent dose- and time-dependent mercury treatment. Dose group symbols: closedsquares = 0 ppm, open squares = 50 ppm, closed triangles = 100 ppm. Letters a, b, cand d indicate groups at means that are significant different at 5% significance level.

genic cells and degenerated epithelial cells into the lumen (Fig. 5B1).However spermatogenesis proceeded normally with evidence ofspermatozoa contained in lumen of seminiferous tubules amongcontrol animals. Additionally, a dose decrease in both thicknesslayer (8.44 and 7.11 versus 9.77, p < 0.001) (Fig. 6) and density of ger-minative epithelium (Fig. 5B3, C3)/dose group was observed, withrespect to controls (Fig. 5A3).

The administration of Hg resulted in a time and dose-dependentincrease and in dose- dependent in its concentration in testes andblood, respectively (Table 1). The testis and blood Hg concentra-tion in rats no exposed to Hg was below the detection limits of themethod. Rats exposed to Hg reached testicular Hg concentrationsnear 1.8 and 3 �g/g dry tissue among HG1 and HG2 groups after 90days of administration, respectively. The Hg-blood concentrations

the end of exposure, respectively.Table 2 compares epididymal sperm number (ESN) in control

and after mercury exposure at two doses (50 and 100 ppm) inrats. The mean epididymal sperm number increased in a time-

7 d 15 d 30 d 60 d 90 d

0 0 0 0 00.10 ± 0.08 0.18 ± 0.08 0.47 ± 0.08 0.94 ± 0.08 1.29 ± 0.080.34 ± 0.08 0.79 ± 0.08 1.16 ± 0.08 1.81 ± 0.08 2.95 ± 0.08(b) (c) (d) (e)

0 0 0 0 00.38 ± 0.03 0.38 ± 0.03 0.38 ± 0.03 0.45 ± 0.03 0.47 ± 0.030.84 ± 0.03 0.84 ± 0.03 0.82 ± 0.03 0.84 ± 0.03 0.88 ± 0.03(c) (c) (c) (c)

owed by Newman–Keuls test was performed. Letters a, b, c, d and e indicate groups

M.A. Boujbiha et al. / Reproductive Toxicology 28 (2009) 81–89 85

F . (A) Ra pm fom n (×4

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ig. 5. Photomicrographs of paraffin-embedded H&E-stained rat testicular sectionsnimals (50 ppm for 90 days). (C) Testis section from mercury-treated animals (100 picroscopic magnification (×250) and Panel C represents microscopic magnificatio

ependent manner; except in HG1 and HG2 groups when a decreaseas observed at the end of exposure. A dose-related reduction in

he ESN/dose group was recorded with respect to matched con-rols (p < 0.001). The maximum effect was showed after 7 days of

able 2ffect of mercury at different doses and time on sperm count and sperm motility/cauda e

arameters 0 d 3 d 7 d

perm count (×106/ml) 0 ppm 17.23 ± 0.78 17.87 ± 0.78 20.7250 ppm 17.23 ± 0.78 16.00 ± 0.78 15.80100 ppm 17.23 ± 0.78 11.57 ± 0.64 11.05(b, c) (a) (b) (c)

perm motility (%) 0 ppm 46.57 ± 4.04 52.91 ± 4.04 65.7850 ppm 46.57 ± 4.04 42.50 ± 4.04 36.11100 ppm 46.57 ± 4.04 22.64 ± 3.33 27.65(b) (a) (b) (c)

ata are presented as the mean ± SD, n = 6. One-way analysis of variance (ANOVA) test follt means that are significantly different at 5% significance level.

at testicular section from control animals. (B) Testis section from mercury-treatedr 90 days). Panel A represents microscopic magnification (×100), Panel B represents00).

exposure; the ESN was reduced by 24% and 47% in HG1 and HG2groups as compared to respective controls, respectively (Table 2).Concerning the epididymal sperm motility (ESM), a similar patternwas observed (p < 0.001). The most ESM decrease was recorded after

pididymis.

15 d 30 d 60 d 90 d

± 0.73 21.92 ± 0.67 22.54 ± 0.73 25.45 ± 0.68 26.00 ± 0.68± 0.74 16.00 ± 0.78 19.04 ± 0.74 23.07 ± 0.67 17.92 ± 0.65± 0.83 14.78 ± 0.71 16.00 ± 0.71 20.00 ± 0.71 16.13 ± 0.67

(d) (e) (d)

± 3.76 78.57 ± 3.46 72.58 ± 3.76 78.14 ± 3.53 73.84 ± 3.53± 3.85 47.36 ± 4.04 72.00 ± 3.85 61.33 ± 3.46 57.75 ± 3.39± 4.28 44.70 ± 3.68 70.65 ± 3.68 44.00 ± 3.68 50.48 ± 3.46

(d) (c) (c)

owed by Newman–Keuls test was performed. Letters a, b, c, d and e indicate groups

86 M.A. Boujbiha et al. / Reproductive Toxicology 28 (2009) 81–89

Table 3Effect of mercury at different doses and times on serum testosterone level in rats.

Parameters 0 d 3 d 7 d 15 d 30 d 60 d 90 d

Testosterone (ng/ml serum) 0 ppm 2.92 ± 0.43 2.89 ± 0.43 2.67 ± 0.43 2.36 ± 0.43 2.40 ± 0.43 2.25 ± 0.43 2.02 ± 0.4350 ppm 2.92 ± 0.43 2.53 ± 0.43 1.59 ± 0.43 1.13 ± 0.43 4.86 ± 0.43 4.34 ± 0.43 1.72 ± 0.43100 ppm 2.92 ± 0.43 2.27 ± 0.43 5.53 ± 0.43 2.89 ± 0.43 2.08 ± 0.43 1.76 ± 0.43 1.62 ± 0.43

(b, c) (a, b, c) (c

Data are presented as the mean ± SD, n = 6. One-way analysis of variance (ANOVA) test fomeans that are significantly different at 5% significance level.

Fig. 6. Germinative cell layer thickness of seminiferous tubules (�m) in testes tis-sue. Letters a and b indicate groups at means that are significant different at 5%significance level.

Table 4Effect of mercury at different doses and times on fertility parameters in rats.

Parameters Dose (ppm)

0 50 100

Viable embryos/litter 12 ± 1.08 8.5 ± 1.08 4.5 ± 1.08Mating index (%) 100 100 50

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ata are presented as the mean ± SD, n = 6. One-way analysis of variance (ANOVA)est followed by Newman–Keuls test was performed. Letters a, b and c indicateroups at means that are significantly different at 5% significance level.

days by 55% and 58% in HG1 and HG2 groups with respective con-rols; respectively. Statistically equal ESM was recorded at 30 daysith control (Table 2).

Serum testosterone level was measured to determine the pos-ible adverse effect of HgCl2 on steroidogenesis. During 90 daysf exposure, Table 2 showed significant variations of the testos-erone serum level for treated rats in comparison with the controlsp < 0.001). This rate appeared decreased on day 3 of the exposureor the HG1 and HG2 groups, and then this decrease seemed to beompensated by significant increases on day 7 of the treatment for

G2 and on day 30 for HG1. At the end of the exposure period, it hadstatistically equal serum testosterone levels with control (Table 3).

Fertility parameters and reproductive performance of male ratreated with HgCl2 are summarized in Table 4. While, the mat-ng index was only affected in the HG2 group (50%) in contrast of

able 5ffect of mercury at different doses and times on testicular lipid peroxidation (LPO) level

arameters 0 d 3 d

BARS (nmol MDA/mg protein) 0 ppm 0.05 ± 0.01 0.05 ± 0.0150 ppm 0.05 ± 0.01 0.07 ± 0.01100 ppm 0.05 ± 0.01 0.18 ± 0.01

(a) (b)

ata are presented as the mean ± SD, n = 6. One-way analysis of variance (ANOVA) test foeans that are significantly different at 5% significance level.

) (a, b) (c) (b, c) (a)

llowed by Newman–Keuls test was performed. Letters a, b and c indicate groups at

control; there was statistically significant decrease (p < 0.01) in thenumber of viable embryos/litter in both mercury-treated groups.36%, 76% decreases were recorded in HG1 and HG2 group respec-tively.

Infertility is a problem with large magnitude. Oxidative stresshas been investigated as a causative factor. The dose-dependentincrease in lipid peroxidation in testicular tissue suggests acausative role of the pro-oxidant activity of HgCl2 in testicular tox-icity. Testicular LPO levels were significantly (p < 0.001) higher inboth treated groups in comparison to control (Table 5). Significant(p < 0.001) difference in LPO levels was also observed between HG1and HG2 groups. A time-course study showed that maximum LPOresponse was evident at 7 and 90 days of treatment (Table 5). Theincrease of TBARS level was by 200% and 400% in HG1 and HG2groups compared to control at day 7, respectively. A dramatic andsignificant increase with a range of 300–650% in HG1 and HG2groups with respect to the 90-day-controls was recorded, respec-tively. Further evidence of oxidative stress such as alterations in theactivity of antioxidant enzymes (SOD and CAT) was recorded dur-ing 90 days of exposure (p < 0.001) (Table 6). Indeed, in HG1 group,the activity of SOD and CAT was reduced by 18% and 27% comparedto control at day 7, respectively. The decrease in enzymes activitywas compensated by a significant increase at 30 days (32% and 105%with respect to the 30-day-controls, respectively) of the treatment.For the HG2 group, a similar pattern was observed (the SOD and CATactivity was low by 29% and 57% with respect to the 7-day-controls,respectively), but the significant compensatory response was takenat 15 days. It is noticeable that the adaptative-CAT response amongHG2 rats was lower than the HG1 rat’s one. Beyond 60 days, HgCl2-treatment resulted in a significant dose-decreased-manner of theradical scavengers’ activity (−11%, −46% in SOD activity and −42%,−55% in CAT activity among HG1 and HG2, with respect to 90-day-controls, respectively).

4. Discussion

Laboratory studies on analysis of stress responses in tissues oforganisms exposed to metal can help to understand the mechanismthrough which metals exert their toxicity in organisms and hencethe results can be used to explain the impact of heavy metal toxicity

on organisms in field. To date, mercuric chloride has been studiedalmost extensively as model of nephrotoxicity in a number of ani-mal species. However, little information is currently available on thetoxic effect of HgCl2 on testis. Indeed, the mammalian testis pro-vides a suitable and necessary environment for spermatogenesis

in testicular rats.

7 d 15 d 30 d 60 d 90 d

0.05 ± 0.01 0.04 ± 0.01 0.04 ± 0.01 0.04 ± 0.01 0.04 ± 0.010.14 ± 0.01 0.09 ± 0.01 0.09 ± 0.01 0.06 ± 0.01 0.09 ± 0.010.26 ± 0.01 0.17 ± 0.01 0.12 ± 0.01 0.14 ± 0.01 0.27 ± 0.01(c) (b) (b) (b) (c)

llowed by Newman–Keuls test was performed. Letters a, b and c indicate groups at

M.A. Boujbiha et al. / Reproductive Toxicology 28 (2009) 81–89 87

Table 6Effect of mercury at different doses and times on superoxide dismutase (SOD) and catalase (CAT) activities in testicular rats.

Name of the antioxidant enzymes Activities of the antioxidant enzymes

0 d 3 d 7 d 15 d 30 d 60 d 90 d

SOD (Unit/mg protein) 0 ppm 0.89 ± 0.03 0.84 ± 0.03 0.73 ± 0.03 0.78 ± 0.03 0.76 ± 0.03 0.77 ± 0.03 0.84 ± 0.0450 ppm 0.89 ± 0.03 0.74 ± 0.03 0.60 ± 0.03 0.53 ± 0.03 1.01 ± 0.03 0.87 ± 0.03 0.74 ± 0.04100 ppm 0.89 ± 0.03 0.58 ± 0.03 0.52 ± 0.03 1.05 ± 0.03 0.93 ± 0.03 0.74 ± 0.04 0.52 ± 0.04(c) (b) (a) (b) (c) (b) (b)

CAT (�mol/min/mg protein) 0 ppm 4.39 ± 0.30 4.79 ± 0.30 4.59 ± 0.30 4.89 ± 0.30 5.05 ± 0.30 4.76 ± 0.30 5.29 ± 0.3050 ppm 4.39 ± 0.30 3.15 ± 0.30 2.93 ± 0.30 7.45 ± 0.30 8.11 ± 0.30 4.52 ± 0.30 2.69 ± 0.30100 ppm 4.39 ± 0.30 2.98 ± 0.30 1.74 ± 0.30 5.59 ± 0.30 3.53 ± 0.30 3.41 ± 0.30 2.07 ± 0.30

(b) (a) (a) (c) (c) (b) (a)

D est fom

aclRtrivdmgHdatcvasttvsaoommstdotsamod[otdbtsulcgt

ata are presented as the mean ± SD, n = 6. One-way analysis of variance (ANOVA) teans that are significantly different at 5% significance level.

nd maturation of spermatozoa; the damage induced by mercuryompounds appears of great importance in understanding the bio-ogical effects of mercury in regard to male reproductive function.esults of the present study suggest that the exposure of rats tohe two doses of HgCl2 through oral administration promoted maleeproductive toxicity in rats. Mercury treatment showed a decreasen accessory sex organ weight. The weight reduction of seminalesicle, epididyms and prostate may be due to mercury-inducedecrease in the formation or storage of their fluids. Mercury treat-ent also resulted in a decrease in testicular as well as accessory sex

lands weight whereas an increase, especially significant amongG2 group, in both relative and absolute testis weight at the 90thays of treatment was noticed. This increase may have been due tocompensatory change or to fluid accumulation in the testis. On

he other hand, histological observation following 90 days of toxi-ity by mercury displayed a severe interstitial effusion with bloodessel dilation in the treated testes. The pathophysiological mech-nism involved in gonad damage caused by mercury intoxicationeems to be linked to an edema. As can be seen in Fig. 5, edema-ous testes were very flaccid and might render the seminiferousubules hypoxic because of the increased separation between theasculature and the tubules. As, testosterone plays a major if notole role in the maintenance of structural integrity and functionalctivities of the accessory sex organs, reduction in accessory sexrgan weight, from 30 days, was a reflection of the interruptionf testicular flow to the reproductive tract in mercury-treated ani-als. Moreover, it was reported that edematous interstitial fluiday contain proteins or other factors or generate reactive oxygen

pecies (ROS) [27] that directly suppress spermatogonial differen-iation or induce apoptosis of differentiating spermatogonia. Theata on testicular antioxidant status in mercury-treated animalsbtained in the present study are in corroboration with the lat-er finding indicating an imbalance in pro-oxidant and antioxidantystem, leading to oxidative stress in mercury exposed rats. ROSre highly reactive molecules that are produced during normaletabolism in the body or after exposure to environmental pro-

xidants. Excess ROS cause a dangerous chain reaction that canestroy nucleic acid, proteins, lipids and other cellular compounds28]. In the present study, mercury treatment to normal rats at dosesf 50 and 100 ppm during 90 days caused a marked increase in theesticular LPO. Lipid peroxidation is the process of oxidative degra-ation of polyunsaturated fatty acids (PUFAs) and its occurrence iniological membranes causes impaired membrane function, struc-ural integrity, decrease in membrane fluidity and inactivation of aeveral membrane bound enzymes [16]. Thus, it is plausible to spec-

late that mercury treatment may result in peroxidation of PUFAs,

eading to the degradation of phospholipids and ultimately result inellular deterioration in the testis. Various studies [15,19] also sug-ested that a strong correlation exists between mercury-inducedoxicity and the induction of LPO.

llowed by Newman–Keuls test was performed. Letters a, b and c indicate groups at

Antioxidant enzymes are essential part of the cellular defenseagainst ROS. SOD and catalase are primary antioxidant enzymespresent in mammalian cells. SOD, the first enzyme in the detox-ifying process, catalyses the dismutation of superoxide radicalsto hydrogen peroxide and molecular oxygen and CAT mediatesthe cleavage of hydrogen peroxide [29]. The antioxidant enzymescatalase protects superoxide dismutase against inactivation byhydrogen peroxide. Reciprocally, the superoxide dismutase pro-tects the catalase against inhibition by superoxide anion. Thus,these enzymes work together to eliminate active oxygen speciesand small deviations in physiological concentrations may have adramatic effect on the resistance of cellular lipids, proteins andDNA to oxidative damage [29]. During the first seven days of expo-sure, the present study indicated a dose-dependent decrease in theactivity of both testicular antioxidant enzymes in mercury-treatedanimals. Further, the induction of these antioxidant enzymes activ-ities at days 15 and 30 indicates an adaptative onset of the redoxdefense system. The enhanced SOD activity observed in this studymight support the last hypothesis that hydrogen peroxide resultedfrom oxygen free radicals including superoxide anion. Thereafterthe H2O2 accumulation associated with the increased activity ofSOD for O2

− conversion might be produced in a dose manner andresulted in an inadequate availability of enzyme. Thus, as comparedto HG1 group the antioxidant potential of CAT in HG2 tissues mightnot be enough to block the lipid peroxidation process and indicatedthe critical role of the enzyme to removal of H2O2 induced by Hg.In parallel, the degree of the peroxidative damage appears to varywith severity of the dose since LPO levels were significantly higherin HG2 in comparison to HG1. Although Hg depressed CAT activity,the enzyme can take part in an efficient defense mechanism againstmercury-induced oxidative stress in testes. This result suggests therole of superoxide dismutase as major enzyme in protecting mam-malian testis [30]. The depressed activity of CAT following 90 days oftoxicity by HgCl2 could possibly be owed to the dramatic consump-tion of this enzyme in converting the H2O2 to H2O. The observeddecrease in testicular SOD activity in mercury exposed rat, suggest-ing increased superoxide radicals production. Thus, the changes inoxidant defense systems associated with mercury exposure may beattributed to ineffective scavenging of ROS and thus increasing thesteady-state level of oxidants in the testis leading to increased lipidperoxidation.

Furthermore, reduction in sperm number and motility/caudaepididymis was observed in dose-dependent manner, which indi-cates an interference with spermatogenesis. It is worth mentioningthat only specific chemical forms of a metal can pass through the

blood–testis barrier and are more likely to target organs of thereproductive track and concentrate in the semen. For instance,methyl mercury, contrarily to mercury inorganic species, is ableto pass through this natural barrier affecting testicular tissues[31]. However, our results showed that a dose and time-dependent

8 uctive

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8 M.A. Boujbiha et al. / Reprod

ncrease of mercury occurred in evaluated testes (Table 1). There-fter, the decrease in cauda epididymal number/motility spermuggests that inorganic mercury has crossed the blood–testis bar-ier and gained access to germinal cells. The adverse effects of HgCl2n mammalian testicular tissue have been reported with markedesticular spermatogenic degeneration at the spermatocyte leveln rats [11]. Rao [9] also mentioned suppression of testicular func-ion in mice treated within organic and methyl mercury, in supportf our present data. Similar observations have been made in theperm of men who were occupationally exposed to mercury [12].he reduction in viability of sperm in our study is consistent withuch effects. With regard to the exceedingly susceptibility of testeso oxidative stress, ample evidence has implied that the fragile char-cteristics of the sperm cells in response to oxidative stress shoulde largely attributable [32]. The sperm membranes are uniquelyich in PUFAs so are liable to lipid peroxidation [32]. As a conse-uence of lipid peroxidation, PUFAs in plasma membranes undergoegradation by a chain reaction and ultimately lead to the produc-ion of cytotoxic aldehydes including malondialdehyde [33]. Arabi34] reported a strong negative correlation between LPO rate andercentage of viable sperm. Loss of integrity can also lead to an

ncrease in membrane permeability and a loss in the capacity to reg-late the intracellular concentrations of ions involved in the controlf sperm movement [35]. Mercury compounds have been reportedo cause DNA breaks by means of free-radical mediated reactions36] that may attack the integrity of DNA and accelerate the processf germ cell apoptosis [17]. Thus the testicular histoarchitecture ofhe mercury-treated animals showed marked damages character-zed by the presence of degenerating cells and germinal epitheliumisruption in the seminiferous tubules. As can be seen in Fig. 6, Hg-dministration induced a dose-dependent loss of spermatogenicell layers. Thus the overall changes in the histoarchitecture of testisn response to mercury could be due to its toxic effects primarilyy the generation of ROS causing damage to the various membra-ous components of the cell. The degenerative conditions observed

n testis of mercury-treated animals are in corroboration with thebserved biochemical changes, wherein an increased level of lipideroxidation was noticed.

Further, hypospermatogenesis may be induced as a consequencef mercury exposure because mercury doses significantly disrupterum testosterone levels in treated animals. Since Leydig cellsre the only source of testosterone production in testis; mercuryreatment had modulated Leydig cell function to disrupt steroidroduction in treated animals. The disruption of serum testosterone

evel in mercury-treated rats may be due to impaired synthesis ornhanced metabolism, both of which may be influenced by mercuryxposure. Such disruption in serum testosterone level by methylercury has been reported [37].Administration of HgCl2 at 100 ppm resulted in more pro-

ounced toxic effect as compared to lower dose in the impairmentf reproductive performance as indicated by reproductive tractesions and infertile mating. A significant decrease in number ofiable embryos/litter in pregnancies sired by males kept on HgCl2-reatment (HG1 and HG2 groups) was observed. Decreased matingndex and viable-embryos number might result from increased ROSecause sperm that have been exposed to a very high level of oxida-ive stress exhibited a significant decline in the quality of sperm

ovement. Another plausible explanation for the alteration of theeproductive performance is the role of sperm acrosome reaction.perm are presumed to carry on their acrosome-reacted head aydrolytic enzyme that causes digestion of the zona pellucida. It has

een suggested that the acrosome reaction plays an important role

n the fusion of sperm and oocyte [38]. These changes could haveccurred during spermiogenesis in the gonad or sperm maturationn the epididymis. Peroxidative damage to the sperm plasma haseen shown to result in loss of membrane fluidity and integrity and,

[

Toxicology 28 (2009) 81–89

as a result, sperm lose their competence to participate in membranefusion events associated with the fertilization [39]. Severe oxidativestress can lead to infertility because of the negative impact on fusionevents such as acrosome reaction and sperm–oocyte fusion [40].Thus emphasizing redox-regulated pathways by HgCl2 can havephysiologic and pathologic effects in regulation of sperm functionand thus reproductive ability.

In conclusion, the results presented in this study clearly demon-strate the susceptibility of testicular tissue to oxidative stressinduced by mercuric chloride, and suggest the effectiveness ofantioxidant enzymes as key regulators of basic testicular-cells pro-cesses, during the intoxication by mercuric chloride. The detailedmechanism of mercury toxicity on the male reproductive sys-tem has not been elucidated and will be the subject of futureexperiments. In light of evidence that some complications of Hg-reproductive toxicity, as demonstrated along this experiment, maybe caused and exacerbated by metal-induced oxidative stress, it willbe useful to screen the antioxidant activities of natural products,which may be specific to the germinal tissue (such preparationsare advocated to humans with fertility problems in the ayurvedicsystem of medicine). The present study raises concerns about thereproductive health of those who are occupationally exposed tomercury.

Conflict of interest

None.

Acknowledgements

We thank Dr. Ahmed RebaÏ for his assistance in statistical analy-ses. We also express thanks to Pr. Tarek RebaÏ for his assistance withmicroscopy. This study is part of a doctoral thesis by Mohamed AliBoujbiha. This research was financially supported by Minister ofScientific-Recherche, Tunisia, through a grant to lab. Ecophysiologyanimals, faculty of Sciences of Sfax (FSS).

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