e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 803–811
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etaine supplementation protects against renalnjury induced by cadmium intoxication in rats:ole of oxidative stress and caspase-3
anan Hagara,∗, Waleed Al Malkib
Department of physiology and Pharmacology, College of Medicine, King Khalid University Hospital,ing Saud University, Riyadh, Saudi ArabiaDepartment of Pharmacology, College of Pharmacy Umm Al-Qura University, Makkah, Saudi Arabia
r t i c l e i n f o
rticle history:
eceived 9 September 2013
eceived in revised form
January 2014
ccepted 14 February 2014
vailable online 23 February 2014
eywords:
admium
ephrotoxicity
etaine
xidative stress
ipid peroxidation
aspase-3
a b s t r a c t
Cadmium (Cd) is an environmental and industrial pollutant that can induce a broad spec-
trum of toxicological effects that affect various organs in humans and experimental animals.
This study aims to investigate the effect of betaine supplementation on cadmium-induced
oxidative impairment in rat kidney. The animals were divided into four groups (n = 10 per
group): control, cadmium, betaine and betaine + cadmium (1) saline control group; (2) cad-
mium group in which cadmium chloride (CdCl2) was given orally at a daily dose of 5 mg/kg
body weight for four weeks; (3) betaine group, in which betaine was given to rats at a dose of
250 mg/kg/day, orally via gavage for six weeks; (4) cadmium + betaine group in which betaine
was given at a dose of 250 mg/kg/day, orally via gavage for two weeks prior to cadmium
administration and concurrently during cadmium administration for four weeks. Cadmium
nephrotoxicity was indicated by elevated blood urea nitrogen (BUN) and serum creatinine
levels. Kidneys from cadmium-treated rats showed an increase in lipid peroxidation mea-
sured as thiobarbituric acid-reactive substances (TBARS) concentration and reductions in
total antioxidant status (TAS), reduced glutathione (GSH) content, glutathione peroxidase
(GSH-Px) activity, superoxide dismutase concentration (SOD) and catalase activity. Caspase-
3 activity, a marker of DNA damage was also elevated in renal tissues of cadmium-treated
rats. Pre-treatment of rats with betaine substantially attenuated the increase in BUN and
serum creatinine levels. Betaine also inhibited the increase in TBARS concentration and
reversed the cadmium-induced depletion in total antioxidant status, GSH, GSH-Px, SOD
and catalase concentrations in renal tissues. Renal caspase-3 activity was also reduced
with betaine supplementation. These data emphasize the importance of oxidative stress
and caspase signaling cascade in cadmium nephrotoxicity and suggest that betaine pre-
treatment reduces severity of cadmium nephrotoxicity probably via antioxidant action and
804 e n v i r o n m e n t a l t o x i c o l o g y a n
1. Introduction
Cadmium (Cd) is an abundant transition metal of worldwideconcern because it accumulates in the environment as a resultof its numerous industrial uses in electroplating, paints, dyestuffs, glass, metal alloys and batteries. Humans are exposedto cadmium, mainly through occupational and environmentalcontamination (Satarug and Moore, 2004). Non-occupationalexposure to cadmium predominantly results from smoking,air pollution and consumption of cadmium contaminatedseafood and water (Järup et al., 2000; Waisberg et al., 2003).Cadmium has an extremely long biological half-life of 15 yearsthat essentially makes it a cumulative toxin in the liver andkidney (Ercal et al., 2001). The kidney is the critical target organfor cadmium-induced toxicity and is well documented by anumber of studies in occupationally (Järup et al., 2000) andenvironmentally (Price et al., 1999) exposed human subjects,as well as in various experimental models (Ohta et al., 2000;Järup and Akesson, 2009). Cadmium can impair re-absorptionof proteins, glucose, and amino acids and produce a vari-ety of renal toxic effects involving the proximal tubules andglomerulus which is believed to be irreversible at an advancedstage (Ahn et al., 1999; Morales et al., 2006; Pari et al., 2007).Other effects of cadmium exposure are disturbances in cal-cium metabolism, hypercalciuria and formation of stones inthe kidney (Hu, 2000). Occupational exposure has been linkedto lung cancer and prostate cancer (Waalkes, 2000).
The deleterious effects of cadmium reported to dateinclude increased generation of reactive oxygen species (ROS),altered antioxidant enzymes, modulation of apoptosis, andinhibition of DNA repair enzymes (Waalkes, 2003; Prozialeckand Edwards, 2012). Oxidative stress and reactive oxygenspecies (ROS) formed in the presence of cadmium could beresponsible for its toxic effects in many organs (Wang et al.,2004; Watjen and Beyermann, 2004). Although several che-lating agents and antagonists are established to reduce thecadmium toxicity, some of them are burned with undesirableside effects. Due to the intrinsic limitations and variability ofefficacy of heavy metal chelating agents, cadmium intoxica-tion therapy is looking for the development of new therapeuticagents with different mode of actions. Natural products havebeen the starting point for the discovery of many impor-tant modern drugs. A large number of natural products anddietary components have been evaluated as potential protec-tive agents to reduce the toxicity of contaminating cadmium(Fouad et al., 2009; Prabu et al., 2010; Saïd et al., 2010).
Betaine (glycine betaine or trimethylglycine) is one of nat-urally occurring antioxidants which can be obtained froma variety of foods including wheat, shellfish, spinach, andsugar beets (Sakamoto et al., 2002). In humans, betaine isobtained from the diet or from its metabolic precursor choline(Zeisel et al., 2003). The physiologic function of betaine iseither as an organic osmolyte to protect cells under stressor as a catabolic source of methyl groups via transmethyla-tion for use in many biochemical pathways. As an osmolyte,betaine protects cells, proteins, and enzymes from envi-
ronmental stress (e.g., low water, high salinity, or extremetemperature). As a methyl donor, betaine participates inthe methionine cycle—primarily in the human liver and
a r m a c o l o g y 3 7 ( 2 0 1 4 ) 803–811
kidney where it converts homocysteine into methionine viabetaine–homocysteine methyltransferase. In a renal context,betaine also plays a role in osmotic regulation in the kidneys,which are routinely exposed to high extracellular osmolar-ity during normal operation of the urinary concentratingmechanism (Craig, 2004; Kempson and Montrose, 2004). Theability of betaine to combat against oxidative stress has beendemonstrated in many situations (Ozturk et al., 2003). Further-more, dietary betaine has been shown to suppress nuclearfactor-�B (NF-�B) and pro-inflammatory molecules such ascyclooxygenase-2 (COX-2) and inducible nitric oxide (Go et al.,2007).
Although reactive oxygen species (ROS) has been impli-cated in the pathogenesis of cadmium-induced toxicity, theprotective effect of betaine against cadmium-induced nephro-toxicity was not yet investigated to the best of our knowledge.This study was carried out to examine the potential effects ofbetaine supplementation on cadmium-induced lipid peroxi-dation, oxidative stress, and nephrotoxicity in rats. In orderto find out the exact underlying mechanisms of the protectiveaction of betaine, the antioxidant activity and anti-apoptoticeffect were determined.
2. Materials and methods
2.1. Chemicals
Betaine, thiobarbituric acid, 5,5-dithiobis-(2-nitrobenzoic acid)were purchased from Sigma (St Louis, MO, USA). Hydrogenperoxide was obtained from Aldrich (USA). Cadmium was pur-chased by Pfizer, USA. Blood urea nitrogen and creatininelevels were measured using kits from Biomérieux Inc. (France).Total antioxidant status, glutathione peroxidase, and superox-ide dismutase were measured using diagnostic kits providedby Randox Chemical Co. (Antrim, United Kingdom). Caspase-3activity was measured using caspase-3 colorimetric assay (cat-alog number BF 3100) provided by R&D Company (MN, USA).
2.2. Animals
Adult male Wistar rats, weighing 220–250 g, were used in thisstudy. They were obtained from the Animal Care Centre, Col-lege of Pharmacy, King Saud University. All the animals werefed a standard rat chow and water ad libitum and kept in atemperature-controlled environment (20–22 ◦C) with an alter-nating cycle of 12-h light and dark. The animals used in thisstudy were handled and treated in accordance with the strictguiding principles of the National Institution of Health forexperimental care and use of animals.
2.3. Experimental design
The animals were divided into four groups (n = 10 per group)as follow:
(1) Saline control group.(2) Betaine group in which betaine (250 mg/kg/day) was given
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3) Cadmium group in which rats were given cadmium chlo-ride (5 mg/kg/day) orally via gavage for four weeks.
4) Cadmium + betaine group in which betaine(250 mg/kg/day) was given orally via gavage for two weeksprior to and concurrently during cadmium administrationfor four weeks (5 mg/kg/day).
The doses of cadmium and betaine were chosen depend-ng upon our preliminary experiments and on the literatureGanesan et al., 2010; Renugadevi and Prabu, 2009). At thend of experiments, the animals were sacrificed; blood andidneys were collected. Blood samples were centrifuged at000 × g for 10 min at 4 ◦C to separate the serum and sera wereollected and assessed for renal functions. All samples weretored at −80 ◦C until analyzed.
.4. Estimation of blood urea nitrogen and creatinineevels
he levels of urea and creatinine in serum were estimatedpectrophotometrically using the commercial diagnostic kits.
.5. Assessment of thiobarbituric acid-reactiveubstances concentration
he amount of renal thiobarbituric acid-reactive substancesTBARS) was measured by the thiobarbituric acid assay (TBA)s previously described by Buege and Aust (1978). Briefly, 0.5 mlf homogenates was added to 2 ml of TBA reagent containing.375% TBA, 15% trichloroacetic acid and 0.25 N HCl. Samplesere boiled for 15 min., cooled and centrifuged. Absorbancesf the supernatants were spectrophotometrically measuredt 532 nm. TBARS concentrations were calculated by the usef 1,1,3,3-tetraethoxypropane as a standard. The results werexpressed as nmol/g wet tissue weight. All assays were donesing samples in duplicate from each animal.
.6. Determination of SOD activity
otal (Cu–Zn and Mn) SOD activity was determined using RAN-OD kit from Randox Company and according to the methodf Sun et al. (1988). The principle of the method is based onhe inhibition of NBT reduction by the xanthine–xanthine oxi-ase system as a superoxide generator. One unit of SOD wasefined as the enzyme amount causing 50% inhibition in theBT reduction rate. SOD activity was expressed as units perilligram protein.
.7. Measurement of catalase activity
atalase activity was measured using the method of Aebi1984). Briefly, the assay mixture of 1.5 ml contained 980 �lf 50 mM sodium phosphate buffer pH 7.0 and 20 �l ofomogenate (10–15 �g protein). Reaction was started by addi-
ion of 500 �l of 30 mM hydrogen peroxide. The decompositionf hydrogen peroxide (H2O2) was followed spectrophotomet-ically at 240 nm. Catalase activity is expressed as �mol H2O2
ecomposed/min/mg protein.
m a c o l o g y 3 7 ( 2 0 1 4 ) 803–811 805
2.8. Determination of reduced glutathioneconcentration
Reduced glutathione (GSH) was determined as non-proteinand total sulphydryl contents in rat kidneys using the methodby Ellman (1959) and modified by Nagi et al. (1992). Briefly, tis-sues were homogenized in 5.0 ml of cold KCl (1.15%), and thesamples were precipitated with trichloroacetic acid. The reac-tion mixture containing 0.5 ml supernatant, 2.0 ml Tris-EDTAbuffer (pH 8.9), and 0.1 ml 5,5′-dithio-bis-2-nitrobenzoic acid(DTNB). The solution was read at 412 nm on a spectropho-tometer (Genesys, Spectronic Instruments). The results wereexpressed as �mol/g wet tissue weight.
2.9. Determination of glutathione peroxidase activity
Renal glutathione peroxidase (GSH-Px) was estimated accord-ing to the method described by Paglia and Valentine (1967)using RANSEL kit. GPH-Px activity was defined as the numberof �mol NADPH oxidized/min/g wet tissue weight.
2.10. Determination of total antioxidant status
Total antioxidant status (TAS) was determined using kit fromRandox Company. The principle of the method is based onthe generation of the ABTS radical cation (ABTS• + ) fromthe interaction between metmyoglobin, 2,2′-azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid) (ABTS) and a stabilizedform of hydrogen peroxide. The TAS assay was performedusing a 20 �l sample and assay read time of 3 min. Absorbancewas measured at 600 nm. The results were expressed asmmol/min/mg protein.
2.11. Estimation of caspase-3 activity
Renal tissues were placed in 0.15 M KCl and homogenizedin a polytron homogenizer for 10 strokes. The resultinghomogenates were centrifuged at 3000 rpm for 15 min, and thesupernatant fractions were used for caspase-3 activity mea-surement. Caspase-3 activity was estimated in 96-well platesaccording to the manufacturer’s protocol by measuring enzy-matic cleavage of the substrate Ac-DEVD-AMC (R&D System).50 �l of homogenate (200 �g of total protein) was added to50 �l of the assay buffer followed by 5 �l of caspase-3 col-orimetric substrate (DEVD-pNA) and incubated at 37 ◦C for2 h. Absorbance was measured at 405 nm using a microplatereader (Bmg Lab Technologies, Chicago, USA). Experimentswere performed in duplicate. The caspase-3 activity wasexpressed as nmol/mg protein.
2.12. Measurement of protein content
Total protein concentration was estimated according to themethod of Lowry et al. (1951) with bovine albumin as astandard.
2.13. Statistical analysis
The results were expressed as the mean ± S.E. in each group.Statistical analysis was performed using analysis of variance
Renal GSH-Px activity was reduced in cadmium-treatedrats (2.19 ± 0.18 �mol/min/g wet tissue, P < 0.001) as
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Fig. 1 – (A) Effect of betaine supplementation (250 mg/kg/d,orally via gavage) for two weeks on serum creatinine levelin cadmium-induced nephrotoxicity (Cd, 5 mg/kg/d, orally)for four weeks in adult male rats. Values are expressed asmean ± S.E.M., n = 10. ***P < 0.001 compared with controlgroup. ##P < 0.01 compared with cadmium-treated group. (B)Effect of betaine supplementation (250 mg/kg/d, orally viagavage) for two weeks on blood urea nitrogen level in
806 e n v i r o n m e n t a l t o x i c o l o g y a n
(ANOVA) followed by Tukey–Kramer multiple comparisonstest. The data were analyzed with Graph prism statistical soft-ware and a statistical probability of P < 0.05 is considered to besignificant.
3. Results
3.1. Effect of betaine on renal functions in cadmiumintoxication
Cadmium administration induced nephrotoxicity that wasreflected by the significant (P < 0.001) increase in serum creat-inine and blood urea nitrogen levels (P < 0.001). Betaine alonehas no effect on kidney function of normal rats. Betainesupplementation improved renal functions in cadmium-treated rats as manifested by the significant (P < 0.001)reduction in levels of serum creatinine and BUN levels com-pared with cadmium group alone (P < 0.001) (Fig. 1A and B).
3.2. Effect of betaine on renal lipid peroxidesconcentration in cadmium intoxication
Renal TBARS concentration, as an index of lipid peroxida-tion, was increased in cadmium-treated rats (178 ± 5 nmol/gwet tissue, P < 0.001) as compared to normal control rats(60 ± 3 nmol/g wet tissue) (Fig. 2). Treatment with betaineinhibited cadmium-induced lipid peroxidation and resulted ina significant decrease in TBARS level (92 ± 4 nmol/g wet tissue,P < 0.001) as compared to cadmium group alone.
3.3. Effect of betaine on renal total antioxidant statusin cadmium intoxication
As depicted in Fig. 3, renal total antioxidant activity in cad-mium group was significantly reduced (0.048 ± 0.001 mmol/mgprotein, P < 0.001) as compared to normal control group(0.092 ± 0.002 mmol/mg protein, P < 0.001). Betaine supple-mentation was able to attenuate the reduction in total antioxi-dant activity in cadmium-treated rats (0.088 ± 0.012 mmol/mgprotein, P < 0.001) as compared to cadmium group alone. Nochange was observed in the renal total antioxidant activity inrats treated with betaine alone (0.099 ± 0.023 mmol/mg pro-tein) as compared to normal control group.
3.4. Effect of betaine on renal GSH concentration incadmium intoxication
Cadmium administration produced a reduction in renalGSH content (0.80 ± 0.088 �mol/g wet tissue, P < 0.001) ascompared to normal control group (2.2 ± 0.067 �mol/g wet
tissue) (Fig. 4A). Treatment with betaine abrogated cadmium-induced GSH depletion and increased GSH concentration upto (1.8 ± 0.02 �mol/g wet tissue, P < 0.001) as compared to cad-mium group alone.
a r m a c o l o g y 3 7 ( 2 0 1 4 ) 803–811
3.5. Effect of betaine on renal GSH-Px activity incadmium intoxication
cadmium-induced nephrotoxicity (Cd, 5 mg/kg/d, orally) forfour weeks in adult male rats. Values are expressed asmean ± S.E.M., n = 10. ***P < 0.001 compared with controlgroup. ##P < 0.01 compared with cadmium-treated group.
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Fig. 2 – Effect of betaine supplementation (250 mg/kg/d,orally via gavage) for two weeks on renal thiobarbituric acidreactive substances (TBARS) concentration incadmium-induced nephrotoxicity (Cd, 5 mg/kg/d, orally) forfour weeks in adult male rats. Values are expressed asmean ± S.E.M., n = 10. ***P < 0.001 compared with controlgroup. ##P < 0.001 compared with cadmium-treated group.
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Fig. 3 – Effect of betaine supplementation (250 mg/kg/d,orally via gavage) for two weeks on renal total antioxidantstatus in cadmium-induced nephrotoxicity (Cd, 5 mg/kg/d,orally) for four weeks in adult male rats. Values areexpressed as mean ± S.E.M., n = 10. ***P < 0.001 comparedwith control group. ##P < 0.001 compared withcadmium-treated group.
m a c o l o g y 3 7 ( 2 0 1 4 ) 803–811 807
compared to normal control rats (6.33 ± 0.18 �mol/min/gwet tissue) (Fig. 4B). Betaine alone caused no change inGSH-Px activity (6.19 ± 0.11 �mol/min/g wet tissue) comparedto control group. Pre-treatment of cadmium-intoxicated ratswith betaine corrected the decline in renal GSH-Px activity(5.62 ± 0.37 �mol/min/g wet tissue, P < 0.001) as compared tocadmium group alone.
3.6. Effect of betaine on renal SOD activity incadmium intoxication
Administration of cadmium alone caused a significant inhibi-tion in renal SOD activity (4.2 ± 0.14 U/mg protein, P < 0.001)as compared to normal control rats (7.82 ± 0.22 U/mg pro-tein, P < 0.001) (Fig. 4C). Betaine alone caused no change inSOD activity (7.94 ± 0.18 U/mg wet tissue) compared to con-trol group. Treatment with betaine blunted cadmium-induceddecreased SOD activity (7.34 ± 0.21 U/mg wet tissue, P < 0.001)as compared to cadmium group alone.
3.7. Effect of betaine on renal catalase activity incadmium intoxication
As depicted in Fig. 4D, renal catalase activity in cadmiumgroup was significantly reduced (42.34 ± 2.79 �mol/min/mgprotein, P < 0.001) as compared to normal control group(89.25 ± 3.42 �mol/min/mg protein). Betaine was able to atten-uate the reduction in catalase activity in cadmium-treated rats(78.55 ± 2.31 �mol/min/mg protein, P < 0.001) as compared tocadmium group alone.
3.8. Effect of betaine on renal caspase-3 activity incadmium intoxication
A significant increase in renal caspase-3 activity was observedin cadmium-treated group (198.9 ± 7.45 nmol/mg protein,P < 0.001) as compared to control group (49.8 ± 2.82 nmol/mgprotein, P < 0.001). Betaine administration resulted in a sig-nificant reduction in renal caspase-3 level (67 ± 4.32 nmol/mgprotein, P < 0.001) in cadmium-treated rats in comparisonwith cadmium group alone (Fig. 5). Betaine administra-tion to normal rats produced no change in renal caspase-3activity.
4. Discussion
Cadmium induces a broad spectrum of toxicological effectsand biochemical dysfunctions constituting serious hazards tohealth (Järup and Akesson, 2009). In the current investiga-tion, cadmium administration elicited renal dysfunction thatwas evident by elevation in BUN and serum creatinine levels.Oxidative stress was indicated by increased lipid peroxidationmeasured as TBARS and by declines in concentrations of totalantioxidant activity, reduced glutathione, SOD, catalase andGSH-Px in renal tissues. Caspase-3, a marker of DNA dam-
age was also elevated in renal tissues of cadmium-intoxicatedrats. Prior-administration of betaine was able to abrogate thedecline in kidney function, protect against lipid peroxidationand ameliorate the decrement in the antioxidant enzymes.
808 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 803–811
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Fig. 4 – Effect of betaine supplementation (250 mg/kg/d, orally via gavage) for two weeks on renal concentrations of (A)reduced glutathione, (B) glutathione peroxidase, (C) superoxide dismutase, and (D) catalase in cadmium-inducednephrotoxicity (Cd, 5 mg/kg/d, orally) for four weeks in adult male rats. Values are expressed as mean ± S.E.M., n = 10.
ed w
***P < 0.001 compared with control group. ##P < 0.001 compar
Several studies have demonstrated that cadmium-inducednephrotoxicity is associated with the proximal tubular dam-age in the kidney of experimental animals (Morales et al., 2006;Renugadevi and Prabu, 2009; Pari et al., 2007).
In our study we have observed that renal TBARS, an index oflipid peroxidation, was markedly increased in rats exposed tocadmium, thus suggesting increased reactive oxygen speciesproduction. Although cadmium itself does not generate freeradicals directly, it indirectly generates various radicals suchas superoxide radical, hydroxyl radical, and nitrogen speciessuch as peroxynitrite, nitric oxide thus causing damage con-
sistent with oxidative stress (Stohs et al., 2000). These reactiveoxygen species attack the cell membrane and leads to desta-bilization and disintegration of cell membrane as a result ofperoxidation of membrane lipids (Stajn et al., 1997).
ith cadmium-treated group.
The degree of cell damage under heavy metal stressdepends on the rate of reactive oxygen species formation andon the efficiency of detoxification and repair mechanisms. Thecellular defense system against toxicity from ROS includessuperoxide dismutase, catalase and glutathione peroxidase.In the present study, renal total antioxidant capacity wasmarkedly decreased in cadmium-treated animals and this wascorrected back to normal by betaine supplementation. Dis-turbance of renal total antioxidant capacity could be explainedby its consumption to neutralize the increased reactive oxy-gen species production induced by cadmium. In agreement
with this, we have observed that renal content of intracel-lular scavengers as SOD, GSH-Px, catalase and reduced GSHare decreased in rats exposed to cadmium. These data indi-cate that cadmium can induce its nephrotoxic actions via the
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Fig. 5 – Effect of betaine supplementation (250 mg/kg/d,orally via gavage) for two weeks on renal caspase-3 activityin cadmium-induced nephrotoxicity (Cd, 5 mg/kg/d, orally)for four weeks in adult male rats. Values are expressed asmean ± S.E.M., n = 10. ***P < 0.001 compared with controlgroup. ##P < 0.001 compared with cadmium-treated group.
paa
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roduction of reactive oxygen species and interference withntioxidant defense mechanisms (Waisberg et al., 2003; Fouadnd Jresat, 2011).
Reduced GSH, catalase, SOD and GSH-Px constitute a majorupportive team of defense against ROS-induced oxidativeamage. Superoxide dismutase is considered as the first linef defense against the deleterious effects of oxygen radicals
n the cells where it scavenges ROS by catalyzing the dismu-ation of superoxide to H2O2 and O2. The inhibitory action ofadmium on SOD may be due to competition between Cd andn or Cu that are required for activity of SOD (Huang et al.,006). Catalase is a hemeprotein which catalyses the reductionf H2O2 to water and oxygen and thus protects the cell fromxidative damage by H2O2 and OH (Chance et al., 1952). Theecrease in catalase activity by cadmium may be attributed tohe decreased absorption of iron, an essential trace elementequired for the activity of catalase.
Reduced glutathione is a sulphur-containing nucleophilicubstance found in high concentration in kidney (Meister andnderson, 1983). It plays a pivotal role in the protection ofells against oxidative stress and metals detoxification. By par-icipating in the glutathione redox cycle, GSH together withSH-Px convert lipid peroxides to non-toxic products thusaintain the integrity of mitochondria and cell membranes. In
he current study, reduced GSH level was declined in the cad-ium group compared to the normal group. Depletion of renal
SH stores by cadmium in the present study can account forhe inhibition of renal GSH-Px activity. Cadmium binds exclu-ively to sulfhydryl groups of GSH leading to its inactivation
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(Sunitha et al., 2001). Cadmium may also exhibit an antagonis-tic effect with selenium and lowers its availability to GSH-Pxwhich is selenium dependent (Gambhir and Nath, 1992). Ourfindings are in consonance with the other published reports(Fouad and Jresat, 2011).
Betaine administration significantly corrected kidney func-tions, increased renal total antioxidant activity and markedlyattenuated the increase in renal TBARS, as well as the decreasein renal content of reduced GSH, catalase, GSH-Px and SOD.Thus these data demonstrate that the protective effect ofbetaine against cadmium nephrotoxicity may be attributed atleast in part to its antioxidants properties. In agreement withthis suggestion, the ability of betaine to protect against oxida-tive stress induced by many insults has been demonstrated inother studies (Ozturk et al., 2003; Ganesan et al., 2010). Betainewas able to protect against isoprenaline-induced myocardialdysfunction and this was attributed to its antioxidant, itspreservative effects on mitochondrial function (Ganesan et al.,2007) and lysosomal integrity (Ganesan et al., 2010). In astudy by Váli et al. (2007), betaine as a constituent of naturalantioxidant-rich diet such as table beet (Beta vulgaris var. rubra)has been shown to have a positive effect on redox homeostasisduring ischemia–reperfusion injury to liver in rats.
The ability of betaine to protect against oxidative stressis attributed to the fact that betaine is highly lipotropic and,when administered exogenously, it can readily pass across themembrane lipid bilayer and diffuses into intracellular com-partments (Kanabak et al., 2001). One hypothesis regardingthe lipotropic properties of betaine is that it contains anelectrophilic methyl group that ameliorates pathologic statesinduced by reductive and oxidative stress (Ghyczy and Boros,2001). Betaine is also involved in the synthesis of methionine,which serves as a major supplier of cellular cysteine via thetrans-sulfuration pathway for the synthesis of reduced glu-tathione that protects the cell from reactive metabolites (Kimand Kim, 2005). This is supported by our results that showedthat betaine was effective in correcting the decline in reducedglutathione concentration in renal tissues induced by cad-mium. The ability of betaine to replenish the thiol pool hasbeen demonstrated by other studies (Go et al., 2007).
The present study revealed that caspase-3 level, an exe-cutioner of cell apoptosis, was increased by cadmium inrenal tissues, an action that was corrected by betaine admin-istration. These results support the hypothesis that themechanism of nephrotoxicity of cadmium is related not onlyto oxidative stress but also to apoptosis of kidney tissues. Cad-mium exposure can induce DNA damage and cell apoptosisvia activation of reactive oxygen species, pro-inflammatorycytokines that ultimately culminate in activation of caspasefamily of proteases (Yang et al., 2007; Lee and Thévenod, 2008).Caspase-induced cadmium toxicity has been described in cul-tured cells as kidney proximal tubules (Lee and Thévenod,2008), and murine macrophages (Kim and Sharma, 2006) andin vivo studies in liver (Fouad et al., 2009) and kidney (Gobeand Craneb, 2010).
Betaine supplementation significantly inhibited thecadmium-induced increase in caspase-3. Inhibition of
caspase-3 by other antioxidants protected against cadmium-induced oxidative stress and cytotoxicity (Kim and Sharma,2006; Lee et al., 2006), suggesting that caspase-3 activation
810 e n v i r o n m e n t a l t o x i c o l o g y a n
also contributes to generation of reactive oxygen species(ROS). Betaine was able to inhibit the induction of caspase-3,8, and 9 activities after osmotic stress in Madin Darley caninekidney cells (Horio et al., 2001). Betaine supplementationsignificantly ameliorated hepatocytes apoptosis in vivo andin vitro following bile duct ligation and its antiapoptotic actionwas largely attributed to an inhibition of the pro-apoptoticmitochondrial pathway (Graf et al., 2002). Moreover, theprotein stabilizing effect of betaine on macromolecules maycontribute to the protection from cadmium-induced DNAdamage and apoptosis (Horio et al., 2001).
In conclusion, betaine exerts its protective effect bydecreasing lipid peroxidation, improving antioxidants statusand inhibiting caspase-3 activity to prevent renal tubular dam-age induced by cadmium administration. Further study onhuman subjects would be beneficial as it may be worthy toconsider the supplementation of betaine as a part of renopro-tective strategies against cadmium intoxication.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Transparency document
The Transparency document associated with this article canbe found in the online version.
Acknowledgments
This work was funded by a grant from Deanship of ScientificResearch, College of Medicine Research Center (CMRC), andKing Saud University.
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