3h2o ) and tq ( 2-isopropyl-5-methyl-1,4-benzoquinone ) were purchased from sigma - aldrich chemical co. ( st . louis , mo , usa ) .
healthy adult ( 4-month - old ) male wistar rats , weighing 200230 g , obtained from the tunisian society of pharmaceutical industries , were used in this study .
the animals were housed in plastic cages ( free from any source of chemical contamination ) with free access to tap water ( free from pb ) and standard diet .
the rats were kept at 223c , in a natural light / dark cycle , with 55% humidity and a ventilation system .
the experiments were started after the animals were allowed to adapt to the laboratory conditions for a week .
all experimental procedures in this study were in full compliance with the european council directive ( 86/609/eec ) and approved by the institutional bioethics committee . after the acclimation period , the rats were randomly divided into four groups of eight animals each and were treated for 5 weeks as follows : the control group received tap water ; the pb group received an aqueous solution containing 2,000 ppm of pb acetate ( 0.2% w / v ) ( 2022 ) ; the pb - tq group was cotreated with pb ( as in the pb group ) plus tq ( 5 mg / kg bw / day ) ; and the tq group received tap water and was given tq ( 5 mg / kg bw / day ) ( 2325 ) .
tq was administered by gastric tube daily between 8:00 and 9:00 am . at the end of the treatment period
the kidneys were removed quickly from the rats , cleared of adhesive tissue , washed in ice - cold 0.9% ( w / v ) nacl solution , and frozen at 80c until assayed .
the kidney tissue was homogenized in 10 volumes of ice - cold phosphate - buffered saline ( 136.75 mm nacl , 2.68 mm kcl , 10.14 mm na2hpo4 , 1.76 mm kh2po4 , ph 7.4 ) and the homogenates were centrifuged at 3,500g for 15 min at 4c .
sod ( 26 ) , gpx ( 27 ) , and gr ( 28 ) activities were determined in the kidney homogenates using commercial kits ( randox laboratories ltd . ,
the gsh level was determined spectrophotometrically using the method previously described by ellman ( 30 ) .
3h2o ) and tq ( 2-isopropyl-5-methyl-1,4-benzoquinone ) were purchased from sigma - aldrich chemical co. ( st . louis , mo , usa ) .
healthy adult ( 4-month - old ) male wistar rats , weighing 200230 g , obtained from the tunisian society of pharmaceutical industries , were used in this study .
the animals were housed in plastic cages ( free from any source of chemical contamination ) with free access to tap water ( free from pb ) and standard diet .
the rats were kept at 223c , in a natural light / dark cycle , with 55% humidity and a ventilation system .
the experiments were started after the animals were allowed to adapt to the laboratory conditions for a week .
all experimental procedures in this study were in full compliance with the european council directive ( 86/609/eec ) and approved by the institutional bioethics committee .
after the acclimation period , the rats were randomly divided into four groups of eight animals each and were treated for 5 weeks as follows : the control group received tap water ; the pb group received an aqueous solution containing 2,000 ppm of pb acetate ( 0.2% w / v ) ( 2022 ) ; the pb - tq group was cotreated with pb ( as in the pb group ) plus tq ( 5 mg / kg bw / day ) ; and the tq group received tap water and was given tq ( 5 mg / kg bw / day ) ( 2325 ) .
tq was administered by gastric tube daily between 8:00 and 9:00 am . at the end of the treatment period
the kidneys were removed quickly from the rats , cleared of adhesive tissue , washed in ice - cold 0.9% ( w / v ) nacl solution , and frozen at 80c until assayed .
the kidney tissue was homogenized in 10 volumes of ice - cold phosphate - buffered saline ( 136.75 mm nacl , 2.68 mm kcl , 10.14 mm na2hpo4 , 1.76 mm kh2po4 , ph 7.4 ) and the homogenates were centrifuged at 3,500g for 15 min at 4c .
sod ( 26 ) , gpx ( 27 ) , and gr ( 28 ) activities were determined in the kidney homogenates using commercial kits ( randox laboratories ltd . ,
the gsh level was determined spectrophotometrically using the method previously described by ellman ( 30 ) .
1a through d , the kidney activities of sod , gpx , cat , and gr in the rats receiving tq alone were not significantly different ( p>0.05 ) from those of the control group ; following pb treatment , the activities of these enzymes were significantly decreased ( p<0.05 ) by 56.11 , 58.69 , 64.7 , and 54.78% , respectively . interestingly , tq coadministration significantly attenuated ( p<0.05 ) the deleterious effect of pb on the activities of these antioxidant enzymes . in fact , in the rats cotreated with pb and tq , the renal activities of sod , gpx , cat , and gr significantly increased ( p<0.05 ) by 70.57 , 78.85 , 106.5 , and 73.91% , respectively , in relation to the pb - intoxicated rats .
effects of lead ( pb ) , thymoquinone ( tq ) , and their coadministration on the kidney activities of superoxide dismutase ( sod , a ) , glutathione peroxidase ( gpx , b ) , catalase ( cat , c ) , and glutathione reductase ( gr , d ) in rats after five weeks .
student 's t - test : * p<0.05 versus control ; 
 p<0.05 versus tq - treated rats ; 
 p<0.05 versus pb - treated rats .
the results presented in fig . 2 indicate that the administration of tq alone had no significant effect ( p>0.05 ) on kidney gsh levels compared to those of the control rats .
by contrast , pb exposure caused a significant decrease ( p<0.05 ) of about 61.8% in the concentration of this non - enzymatic antioxidant in relation to the control rats .
this effect was significantly mitigated ( p<0.05 ) by 55.16% when the pb - treated animals simultaneously received tq .
effects of lead ( pb ) , thymoquinone ( tq ) , and their coadministration on the kidney level of reduced glutathione ( gsh ) in rats after five weeks .
student 's t - test : * p<0.05 versus control ; 
 p<0.05 versus tq - treated rats ; 
 p<0.05 versus pb - treated rats .
1a through d , the kidney activities of sod , gpx , cat , and gr in the rats receiving tq alone were not significantly different ( p>0.05 ) from those of the control group ; following pb treatment , the activities of these enzymes were significantly decreased ( p<0.05 ) by 56.11 , 58.69 , 64.7 , and 54.78% , respectively . interestingly , tq coadministration significantly attenuated ( p<0.05 ) the deleterious effect of pb on the activities of these antioxidant enzymes . in fact , in the rats cotreated with pb and tq , the renal activities of sod , gpx , cat , and gr significantly increased ( p<0.05 ) by 70.57 , 78.85 , 106.5 , and 73.91% , respectively , in relation to the pb - intoxicated rats .
effects of lead ( pb ) , thymoquinone ( tq ) , and their coadministration on the kidney activities of superoxide dismutase ( sod , a ) , glutathione peroxidase ( gpx , b ) , catalase ( cat , c ) , and glutathione reductase ( gr , d ) in rats after five weeks .
student 's t - test : * p<0.05 versus control ; 
 p<0.05 versus tq - treated rats ; 
 p<0.05 versus pb - treated rats .
the results presented in fig . 2 indicate that the administration of tq alone had no significant effect ( p>0.05 ) on kidney gsh levels compared to those of the control rats .
by contrast , pb exposure caused a significant decrease ( p<0.05 ) of about 61.8% in the concentration of this non - enzymatic antioxidant in relation to the control rats .
this effect was significantly mitigated ( p<0.05 ) by 55.16% when the pb - treated animals simultaneously received tq .
effects of lead ( pb ) , thymoquinone ( tq ) , and their coadministration on the kidney level of reduced glutathione ( gsh ) in rats after five weeks .
student 's t - test : * p<0.05 versus control ; 
 p<0.05 versus tq - treated rats ; 
 p<0.05 versus pb - treated rats .
pb is a pervasive environmental and industrial pollutant with no beneficial biological role , and its toxicity continues to be a major public health problem throughout the world .
recent studies point to the potential involvement of the cell 's antioxidant capacity failure in the pathogenesis of pb poisoning , suggesting that exogenous antioxidants may play an effective protective effect . in the present study
, we adopted an in vivo experimental animal model to investigate whether tq could maintain renal intracellular antioxidant reserves in pb subchronic treatment .
sod catalyzes the dismutation of o2 
  to h2o2 and o2 . because h2o2 is still harmful to cells , cat and gpx further catalyze the decomposition of h2o2 to water . in the reaction catalyzed by gpx ,
gsh is oxidized to gssg , which can then be reduced back to gsh by gr . in the present study
, we found that treatment with pb for 5 weeks significantly decreased the activities of sod , gpx , cat , and gr in the rat kidney .
these results are in concordance with previous findings ( 3 , 31 , 32 ) .
it has been shown that pb directly alters antioxidant activities by irreversible direct binding to functional sulfhydryl ( sh ) groups of several enzymes such as sod , gpx , cat , and gr ( 33 ) . because pb interferes with the metabolism of essential trace elements such as copper , zinc , selenium , and iron needed for proper molecular structure and enzymatic activity ( 2 )
the decrease in antioxidant enzyme activities may be explained by the downregulation of antioxidant enzyme mrna expression ( 34 ) .
gsh is a tripeptide - containing cysteine that has a reactive sh group with reductive potency .
accordingly , gsh plays a vital role in the protection of cells against oxidative stress .
it can act as a non - enzymatic antioxidant by direct interaction of sh groups with reactive oxygen species , or it can be involved in the enzymatic detoxification reactions for reactive oxygen species , as a cofactor or a coenzyme . in agreement with recent investigations studying the effect of pb in the kidneys of rats and mice ( 35 , 36 ) ,
our data show that pb treatment significantly lowered the renal gsh level . as for antioxidant enzymes ,
the reduction in concentration of gsh may be due to the high affinity of pb to the sh groups of this tripeptide , thereby interfering with its antioxidant activity ( 33 ) .
pb can also decrease the level of gsh by inhibiting the activities of gsh metabolizing enzymes , such as gr , gst , and glucose-6-phosphate dehydrogenase , by blocking their sh groups ( 37 ) .
further , the reduction of gsh synthesis can be proposed as another explanation . despite extensive research now focusing on herbal products as alternative medicines ,
no evidence has been reported in the literature regarding the role of tq against pb - induced renal toxicity . in the present study , cotreatment of pb - exposed rats with tq significantly improved the altered antioxidant defense system in the kidneys .
our results are in consonance with recent literature data indicating that oral supplementation of tq ( 10 mg / kg / day , 15 days ) protects rat kidneys against sodium arsenite  induced depletion of antioxidant enzyme activities ( sod , gpx , and cat ) ( 38 ) .
furthermore , farag et al . ( 39 ) reported that tq ( 10 mg / kg / day , 28 days ) prevented reduction in kidney sod activity and gsh levels provoked by chronic treatment with cyclosporine a , an immunosuppressant drug , and by acute renal ischemia / reperfusion in rats .
( 40 ) reported that tq enhances the declined renal antioxidant status in gentamicin - treated rats .
tq ( 10 mg / kg / day , 10 days , po ) also reversed a renal decrease in cat activity and gsh concentration in rats receiving methotrexate , an anticancer drug ( 41 ) .
the restoration of tissue antioxidant function by tq clearly demonstrated in the current work could be attributed to its ability to upregulate antioxidant gene expression ( 42 , 43 ) .
the ability of tq to correct the disrupted antioxidant system , as demonstrated in the present research , does not precisely mean that kidney oxidative stress can be decreased .
numerous previous data showed that this component has powerful free radical scavenging activity ( 12 , 14 , 4446 ) , which may be related to the redox properties of the quinone structure of tq molecule and to its unrestricted crossing of morphophysiological barriers to access subcellular compartments ( 46 ) .
thus , in the current work we are limited to some antioxidant markers . in conclusion ,
our results clearly indicate that tq oral supplementation , at a safe dose , protects against pb - induced cellular antioxidant defense system depletion in rat kidneys .
am and hbc collected the data , and am also analyzed the data , designed the study , and wrote the paper .
this work was supported by funds allocated to the genetic , genotoxicity and childhood illness research unit ( ur12es10 ) by the tunisian ministry of higher education and scientific research .
 objectivealteration of the antioxidant status in the kidneys may be related to lead ( pb ) intoxication . 
 the present study aimed to investigate the possible beneficial effect of thymoquinone ( tq ) , the major active ingredient of the volatile oil of nigella sativa seeds , on pb - induced renal antioxidant defense system impairment.methodsa total of thirty two healthy adult male wistar rats were randomly divided into four equal groups as follows : a control group , which received no treatment ; a pb group , which was exposed to 2,000 ppm of pb acetate in drinking water ; a pb - tq group , which was cotreated with pb plus tq ( 5 mg / kg / day , per os ) ; and a tq group receiving only tq . 
 all treatments were applied for five weeks.resultstq alone did not induce any significant changes in the antioxidant defense system . 
 by contrast , pb exposure significantly decreased reduced glutathione level and superoxide dismutase , glutathione peroxidase , catalase , and glutathione reductase activities in the renal tissue . 
 interestingly , supplementation with tq significantly improved the affected antioxidant parameters.conclusionour data are the first to provide evidence on the protective effect of tq against pb - induced renal antioxidant capacity impairment and suggest that this component might be a clinically promising alternative in pb nephrotoxicity . 
Materials and methods
Chemicals
Animals
Experimental design
Tissue collection
Biochemical assays
Statistical analysis
Results
Antioxidant enzyme activities

GSH levels
Discussion
Authors' contributions
Conflict of interest and funding