Indian Institute of Toxicology Research (IITR), Lucknow 226 001, IndiaBanaras Hindu University (BHU), Varanasi, India
r t i c l e i n f o
rticle history:eceived 3 June 2010eceived in revised form3 September 2010ccepted 23 September 2010vailable online 1 October 2010
eywords:aneb
araquat
a b s t r a c t
Experimental studies have shown that toxicant responsive genes, cytochrome P450s (CYPs) and glu-tathione S-transferases (GSTs) play a critical role in pesticide-induced toxicity. CYPs play pro-oxidantrole and GSTs offer protection in maneb (MB) and paraquat (PQ)-induced brain and lung toxicities. Thepresent study aimed to investigate the effect of repeated exposures of MB and/or PQ on lipid peroxidation(LPO), glutathione content (GSH) and toxicant responsive genes, i.e., CYP1A1, 1A2, 2E1, GSTA4-4, GSTA1-1and GSTA3-3 in the liver and to correlate the same with polymorphonuclear leukocytes (PMNs). A signif-icant augmentation in LPO and reduction in GSH content was observed in a time of exposure dependentmanner in the liver and PMNs of MB and/or PQ treated animals. The expression and catalytic activity ofCYP2E1 and GSTA4-4 were significantly increased following MB and/or PQ exposure both in the liver and
ytochrome P450slutathione S-transferases
PMNs. Although the expression of GSTA3-3 was increased, the expression of GSTA1-1 was unaltered afterMB and/or PQ treatment in both the liver and PMNs. MB augmented the expression and catalytic activityof CYP1A1 in the liver, however, CYP1A2 was unaffected. PQ, on the other hand, significantly increasedhepatic CYP1A2 expression and catalytic activity. MB and/or PQ did not produce any significant changesin CYP1A1 and CYP1A2 in PMNs. The results of the study thus demonstrate that MB and PQ differentiallyregulate hepatic CYP1A1 and CYP1A2 while LPO, GSH, CYP2E1, GSTA4-4 and GSTA3-3 are modulated in
in th
the similar fashions both
. Introduction
1,1′-Dimethyl-4,4′-bipyridinium (paraquat; PQ), a commonlysed herbicide, is implicated in several pathological disorders
ncluding Parkinson’s disease (PD) [1]. Manganese N′, N′-diethylis-dithiocarbamate (maneb; MB), a fungicide, produces adverseffects in non-target organisms including, humans. MB enhanceshe PQ-induced neurotoxicity in PD phenotype in experimentalnimals [2,3]. Oxidative stress plays a critical role in MB and PQ-nduced toxicities [3–7]. PQ induces oxidative stress through direct
odulation of redox cycling, mitochondrial dysfunction, xenobi-
tics metabolizing enzymes and antioxidant defense system [8,9].owever, MB induces oxidative stress via the inhibition of complex
II of mitochondria, auto-oxidation of catecholamines and attenu-tion of antioxidant levels [5,10].
∗ Corresponding author at: Indian Institute of Toxicology Research (IITR), (Councilf Scientific and Industrial Research), Mahatma Gandhi Marg, Post Box- 80, Lucknow26 001, UP, India. Tel.: +91 522 2613618x342; fax: +91 522 2628227.
E-mail address: [email protected] (C. Singh).1 These authors contributed equally to this work.
Cytochrome P450 (CYP) isoforms especially CYP1A1, CYP1A2and CYP2E1 have been shown to facilitate formation of reac-tive oxygen species (ROS) during xenobiotic metabolism therebycontributing to oxidative stress-induced damage [11–14]. Directinvolvement of CYP-mediated free radical generation has beenreported in pesticides and ethanol-induced toxicities [3,15,16]. Therole of CYP2C isoforms in PQ-induced lung injury and involvementof CYP2E1-mediated lipid peroxidation in MB+PQ-induced PD inmouse model have been established [3,17]. Additionally, CYP2E1-mediated production of superoxide radicals and hydrogen peroxidein vitro and in transfected cultured cells has been documented inprevious studies [13].
GSTs are known to protect against oxidative stress andpesticides-induced toxicity [18–20]. Earlier studies have shownincreased expression of GSTA3-3, an isoform of GST-alpha in lungsand GSTA4-4 in brain following PQ and MB+PQ exposure, respec-tively in rodents [3,17,21]. Several studies have demonstrated that
GSTA4-4 provides protection against oxidative stress-mediateddamage [18–20,22]. Over-expression of GSTA4-4 by oxidativestress inducing chemicals including, MB and PQ further support theprotective role of GSTA4-4 against free radical-induced pathologies[3,21].
Systemic exposure to MB and PQ at low doses causes neurode-eneration and alters the expression levels of toxicant responsiveenes [2,3,21]. MB and PQ at neurotoxic doses increase theusceptibility of lung injury in aged animals [23]. Oxidative stress-ediated PQ-induced hepatotoxicity after acute/sub-acute high
ose exposures is reported in earlier studies. However, its tox-city at low/neurotoxic doses and moderate exposure is not yetssessed. Similarly, the effect of systemic exposure at these dosesf PQ and MB in combination is not yet investigated. Therefore, thetudy was performed using the same doses of PQ and MB as opti-ized to cause neurodegeneration and induce PD like features in
nimals. The present study was undertaken to assess the effects ofhe same doses on oxidative stress and toxicant responsive genesn the liver of exposed rats. MB and PQ increase lipid peroxidationt neurotoxic doses in PMNs, however, their effects on GSH andoxicant responsive genes have not yet been deciphered [24]. Theffect of systemic exposure of MB and/or PQ on CYP1A1, CYP1A2,YP2E1, GSTA4-4, GSTA1-1 and GSTA3-3, lipid peroxidation andSH content in the rat liver and their correlation with the poly-orphonuclear leukocytes (PMNs) were assessed in this study.
aneb (MB), nicotinamide adenine dinucleotide phosphate-educed form (NADPH), N-nitrosodimethylamine (NDMA), p-itrophenol, 4-nitrocatechol, paraquat (PQ), potassium chloride,esorufin ethyl ether (ERF), resorufin methyl ether (MRF), resorufinetra sodium salt, sodium dihydrogen orthophosphate, sodiumodecyl sulfate (SDS), thiobarbituric acid (TBA) and Tween-20 wererocured from Sigma–Aldrich, USA. 5,5′-dithibis 2-nitrobenzoiccid (DTNB), formaldehyde, glutathione (GSH) reduced form, mag-esium chloride (MgCl2) and zinc sulphate (ZnSO4) were purchased
rom SRL Ltd., Mumbai, India. 4-Hydroxynonenal (4-HNE) was pur-hased from Cayman chemicals, USA. RT-PCR kits, Taq polymerase,NTPs and 100 bp DNA ladder were procured from MBI Fermen-as, USA. The forward and reverse primers for CYP1A1, CYP1A2,YP2E1, GSTA1-1, GSTA3-3, GSTA4-4 and �-actin were purchased
rom Metabion GmbH, Germany. Mouse anti-rat CYP1A1, 1A2nd �-actin monoclonal antibodies were procured from Santaruz Biotechnology Inc., USA. Rabbit anti-rat CYP2E1 and mousenti-human GSTA4-4 were procured from Chemicon Interna-ional, USA and Abnova Corporation, USA respectively. Immobilon-membrane was procured from Millipore Corporation, USA.
.2. Animal treatment
Male Wistar rats (180–200 g) were obtained from the ani-al colony of the Indian Institute of Toxicology Research (IITR),
ucknow. The study was approved by the Institutional Ethics Com-ittee for use of laboratory animals. Animals were maintained
nder standard conditions of temperature (22 ± 1 ◦C), humidity45–55%) and light (12/12-h light/dark cycle). The animals wereed with standard pellet diet comprising of 22.5% wheat flour, 60%oasted Bengal gram flour, 4% casein, 5% skimmed milk powder, 4%
efined groundnut oil, 4% salt mixture and 0.5% vitamin mixtures per the source formula of National Institute of Nutrition, Hyder-bad, India and water ad libitum. Rats were divided into four groups:ontrol/vehicle, MB, PQ and MB+PQ treated groups. MB (15 mg/kg)nd/or PQ (5 mg/kg) were administered intraperitoneally twice a
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week for 1, 3 and 6 weeks. Control animals were treated with anequal volume of normal saline.
2.3. Isolation of PMNs
Blood was collected in sodium citrate (0.129 mol/L, pH 6.5, 9:1v/v) from control and treated groups through cardiac punctureunder ether anesthesia. PMNs were isolated as described elsewhere[24,25]. In brief, PMNs were isolated from the buffy coat by dextransedimentation, further purified with histopaque density gradientcentrifugation and finally washed with Hankes’ Balanced Salt Solu-tion (HBSS; pH 7.4). The cell viability was tested by trypan blueexclusion test, which was never less than 95%. Cell counting wasdone using hemocytometer.
2.4. Preparation of liver microsomes
Hepatic microsomes were prepared by standard protocolas described elsewhere [26]. In brief, the liver was perfusedusing ice cold saline, excised and homogenized using ice cold50 mmol/L phosphate buffer (pH 7.4) containing 0.15 mol/L KCl.The homogenate was centrifuged for 20 min at 10,000 × g at 4 ◦Cand the supernatant obtained was further centrifuged for 60 minat 105,000 × g at 4 ◦C. The microsomal pellet was washed onceand resuspended in 0.1 mol/L phosphate buffer (pH 7.4) contain-ing EDTA (1 mM) and glycerol (20%). Microsomes were stored inaliquots at −80 ◦C till further use.
2.5. Lipid peroxidation (LPO) and glutathione (GSH) content
LPO in the liver and PMNs was estimated according to themethod described by Okhawa and coworkers [27] with slightmodifications. In brief, assay mixture containing 10% homogenate(0.1 mL) or cell lysate (107 cells/0.1 mL) was mixed with 10% SDSsolution and incubated for 5 min at room temperature followedby the addition of 20% acetic acid (0.6 mL) and further incubatedfor 2–5 min. Finally 0.8% TBA (0.6 mL) was added and the reactionmixture was incubated in a boiling water bath for 1 h. The assaymixture was cooled, centrifuged and absorbance of the supernatantwas read at 532 nm against control without lysate. LPO levels areexpressed as nmoles malondialdehyde (MDA)/mg tissue (liver) andnmoles MDA/107 cells (PMNs).
GSH content of the liver and PMNs was measured according toMoron et al. [28] using 5,5′-dithiobis 2-nitrobenzoic acid (DTNB).Briefly, the sample (0.1 mL) was mixed with DTNB (2 mL in phos-phate buffer pH 8.0) and the volume was made up to 3.0 mL withphosphate buffer. The absorbance was read immediately at 412 nmand the GSH content was calculated in �M/mg tissue (liver) and�M/107 cells (PMNs) using standard curve of GSH.
Catalytic activities of CYP1A1/EROD and CYP1A2/MROD weremeasured following literature reported protocols [29] with slightmodifications. In brief, liver microsomal or cell lysate protein(50–250 �g) was mixed with 0.1 M phosphate buffer (pH 7.4)containing magnesium sulphate, BSA and 7-ethoxy resorufin (sub-strate for CYP1A1, 1.5 �M) or 7-methoxy resorufin (substrate forCYP1A2, 1.5 �M). The reaction was initiated by the addition of
NADPH (250 �M) followed by incubation at 37 ◦C for 20 min andstopped by the addition of methanol (2.5 mL). The reaction mix-ture was centrifuged, and fluorescence of the supernatant wasmeasured at 550 nm excitation and 585 nm emission wavelengths.Enzyme activities are expressed as pmoles/min/mg protein.
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Fig. 1. Effect of MB and/or PQ on lipid peroxidation (LPO). Upper panel depicts LPOlevels in the liver of control and exposed rats and lower panel shows LPO levels inthe PMNs after MB and/or PQ exposure for 1, 3 and 6 weeks. First, second, thirdand fourth bar in each panel represents control, MB, PQ and MB+PQ-treated groupsrespectively (***p < 0.001 as compared with controls; ##p < 0.01 and ###p < 0.001 ascompared with MB-treated group; $p < 0.05, $$p < 0.01 and $$$p < 0.001 as comparedwith PQ-treated group).
Fig. 2. Effect of MB and/or PQ on glutathione (GSH) content. Upper panel depictsGSH content in liver of control and exposed groups and lower panel shows GSH con-tent in the PMNs following 1, 3 and 6 weeks of MB and/or PQ exposures. First, second,
68 I. Ahmad et al. / Chemico-Biolog
.6.2. CYP2E1 activityCYP2E1 activity was determined by measuring the hydroxy-
ation of 4-nitrophenol to 4-nitrocatechol by using the methodescribed elsewhere [30] with slight modifications. In brief, 4-itrophenol (0.2 mM) was mixed with Tris–HCl (50 mM; pH.4) containing MgCl2 (25 mM) and hepatic microsomal protein250 �g) and the assay mixture was incubated for 5 min at 37 ◦C.he reaction was initiated by the addition of NADPH (1 mM)ollowed by incubation at 37 ◦C for 30 min. The reaction was ter-
inated by the addition of 0.6 N perchloric acid (500 �L) andamples were centrifuged at 825 × g for 20 min. The supernatantas mixed with 10 N sodium hydroxide (9:1 ratio) and absorbanceas read at 510 nm. CYP2E1 activity is expressed in nmol/min/mgrotein.
The level of CYP2E1 is known to be higher in other tissues asompared with peripheral tissues, such as blood and blood cells. Inhe peripheral tissues, p-nitrophenol method could not work dueo its less sensitivity. Therefore, in the beginning, p-nitrophenol
ethod was used for liver and CYP2E1 activity in PMNs was mea-ured using more sensitive N-nitrosodimethylamine demethylaseNDMA) assay as reported earlier [31]. The colorimetric assays based on the measurement of formaldehyde formed by Nasheaction. Briefly, the assay mixture (0.5 mL) contained Tris–HCl50 mM; pH 7.4), MgCl2 (10 mM), KCl (150 mM), NADPH (1.0 mM),ell lysate protein (250 �g) and NDMA (4.0 mM). The reactionas initiated by the addition of NDMA or NADPH. The reac-
ion was terminated by the addition of 25% ZnSO4 and saturateda(OH)2 (0.05 mL each), mixed thoroughly and the samples wereentrifuged. The supernatant was then mixed with Nash reagentnd incubated at 50 ◦C for 30 min. The absorbance was read at12 nm and enzyme activity was calculated using standard curvef formaldehyde. The results are expressed as nmoles/min/mg pro-ein.
.6.3. GSTA4-4 activityGSTA4-4 activity towards 4-hydroxynonenal (HNE) was mea-
ured as described earlier [22]. The enzyme assay was performed inhe supernatant obtained by centrifugation of the liver homogenatet 28,000 × g for 30 min at 4 ◦C. In brief, assay system contained 4-NE (0.1 mM) and glutathione (0.5 mM). The conjugation of the-HNE with GSH was monitored spectrophotometrically at 224 nmor 3 min. The enzyme activity was calculated using molar extinc-ion coefficient (D = 13750 M−1 cm−1) and the results are expresseds �moles/min/mg protein.
GSTA4-4 activity in rat PMNs was quantified using HPLC methods described elsewhere [32]. In brief, the reaction mixture con-aining cell lysate (5–20 �g), GSH (500 �M) and 4-HNE (100 �M)n 0.1 M phosphate buffer was incubated at 30 ◦C for 15 min andnalysed by HPLC for 4-HNE content. The mobile phase flow rateas 1 mL/min and consisted of 40% acetonitrile and 60% of water;
bsorbance of the effluent was monitored at 224 nm. Concentra-ion of 4-HNE in the samples was calculated by standard curvebtained at different concentrations of 4-HNE.The enzyme activitys expressed as �moles/min/mg protein.
.7. Protein estimation
Protein content was measured in the liver homogenate/icrosomes and cell lysate of control and treated animals using
owry’s method [33]. Protein concentration was calculated usinghe standard curve of bovine serum albumin (BSA).
.8. Gene expression using RT-PCR
Total RNA was extracted from the liver and PMNs of controlnd treated groups using Trizol reagent by standard procedure and
third and fourth bar in each panel represents control, MB, PQ and MB+PQ-treatedgroups respectively (*p < 0.05, and ***p < 0.001 as compared with controls; ##p < 0.01and ###p < 0.001 as compared with MB-treated animals; $p < 0.05 and $$p < 0.01 ascompared with PQ-treated group).
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DNA was synthesized using reverse transcriptase (RT) cDNA syn-hesis kit as per manufacturer’s protocol. Briefly, total RNA (5 �g)ample was mixed with oligo dT18, incubated at 70 ◦C for 5 min andhilled on ice followed by the addition of 5× reaction buffer (4 �L)or RT-PCR. The dNTP mixture (10 mM) was then added and incu-ated at 37 ◦C for 5 min. Reverse transcriptase was added to theeaction mixture, incubated for 60 min at 42 ◦C followed by 10 mint 70 ◦C.
PCR amplification of CYP1A1, CYP1A2, CYP2E1, GSTA1-1, GSTA3-, GSTA4-4 and �-actin genes was performed as reported earlier
sing cDNA from the liver and PMNs of control and treated animals21,34–37]. The amount of template cDNA for each target gene wastandardized before selecting its amount for PCR amplification andas within the linear range of amplification. PCR products were
isualized by agarose gel electrophoresis using ethidium bromide
ig. 3. (A) Upper, middle and lower panels demonstrate the effect of MB and/or PQ exposun hepatic microsomes (n = 5). (B) Effect of MB and/or PQ exposure on CYP1A1 catalytic aanels respectively in rat PMNs (n = 5). First, second, third and fourth bar in each panel*p < 0.01 as compared with control group).
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under UV light. The band density was quantified using Alpha Imagerand normalized using �-actin as a reference.
2.9. Western blot analysis
Effects of MB and/or PQ on the protein levels of CYP1A1,CYP1A2, CYP2E1 and GSTA4-4 in the liver microsomes and PMNswere analysed by western blot technique. PMNs were suspendedin HBSS buffer containing EDTA (1 mM), EGTA (1 mM), PMSF(1 mM), sodium fluoride (50 mM), sodium orthovanadate (2 mM)
and protease inhibitors cocktail in ice-cold conditions and soni-cated using three pulses of 10 s. The samples were then centrifugedat 10,000 × g for 20 min at 4 ◦C and the supernatant obtained wasused for western blotting. Proteins from hepatic microsomes/PMNswere resolved by SDS-polyacrylamide gel electrophoresis and
re on CYP1A1/EROD activity, mRNA expression and protein expression respectivelyctivity, mRNA expression and protein level is depicted by upper, middle and lowerrepresents control, MB, PQ and MB+PQ-treated groups respectively (*p < 0.05 and
570 I. Ahmad et al. / Chemico-Biological Interactions 188 (2010) 566–579
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lectro-blotted onto PVDF membranes using standard method. Theembranes were blocked using Tris buffered saline containing
ween-20 buffer (TBST; 50 mM Tris–HCl, 135 mM NaCl and 0.1%ween-20; pH 7.5) containing 5% non-fat dry milk at 4 ◦C overnight.he blots were incubated with specific anti-rat monoclonal anti-odies for CYP1A1 (1:5000), CYP1A2 (1:5000), CYP2E1 (1:5000),STA4-4 (1:8000) and �-actin (1:5000) for 2 h at room temper-ture and washed 3–4 times with TBST buffer. The blots werehen incubated with secondary antibodies conjugated to alkalinehosphatase and developed using BCIP/NBT substrate. The bandensities were captured and densitometric analysis was performedy taking �-actin as a reference.
.10. Statistical analysis
Results are expressed as means ± standard error of meansS.E.M.). Two-way analysis of variance (ANOVA) was used fortatistical analyses. Bonferroni post-test was used for multipleomparisons. The differences were considered statistically signifi-ant when ‘p’ value was less than 0.05.
nued).
3. Results
3.1. Effect of MB and/or PQ on LPO and GSH in PMNs and liver
Repeated exposures of MB and/or PQ significantly augmentedLPO in the liver and PMNs of exposed animals after 1, 3 and 6 weeksof exposure. Combined treatment resulted in more pronouncedeffect than alone both in the liver and PMNs of treated animals.In case of the liver, co-exposure resulted in an additive effect afterone week and increase in exposure time showed more pronouncedeffect but it was less than the additive. In PMNs, MB+PQ treatmentshowed synergistic increase in LPO levels after one week of expo-sure and 3 weeks onwards the effect was more pronounced thanthe individual but it was neither synergistic nor additive. However,in both the liver and PMNs maximum effect was obtained after 6
weeks of combined treatment (Fig. 1A and B).
MB and/or PQ exposure reduced GSH content in the liver as wellas in PMNs of treated animals. Combined treatment yielded morepronounced decrease in GSH content as compared with MB or PQalone, however, it was neither synergistic nor additive. Six weeks
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f combined treatment resulted in maximum attenuation of GSHontent (Fig. 2A and B).
.2. MB and PQ-induced alterations in CYP1A1
MB increased catalytic activity and protein/mRNA expression of
epatic CYP1A1 after 1 week of exposure, which was maintainedp to 6 weeks without further increase. PQ did not produce anyignificant change in any of these parameters. Combined treatmentid not produce any further increase as compared with MB aloneFig. 3A). MB and/or PQ exposure did not alter CYP1A1 activity in
ig. 4. (A) Effect of MB and/or PQ exposure on CYP1A2 activity, protein and mRNA exprhe CYP1A2 activity, mRNA expression and protein level in the liver microsomes respectiv
iddle and lower panels demonstrate the CYP1A2 catalytic activity, mRNA expression andanel represents control, MB, PQ and MB+PQ-treated groups respectively (*p < 0.05 and *roup).
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the rat PMNs. CYP1A1 protein and mRNA expression analyses inPMNs also showed the similar patterns (Fig. 3B).
3.3. Effect of MB and PQ on CYP1A2
PQ treatment increased hepatic CYP1A2 activity after 3 weeks,
which was augmented significantly following 6 weeks of treatment.PQ significantly augmented CYP1A2 protein and gene expressionsonly after 6 weeks of exposure. MB did not produce any alterationsin CYP1A2 activity and protein/mRNA expressions. Combined treat-ment did not enhance the PQ-mediated increase in CYP1A2 as
ession in the liver microsomes. The upper, middle and lower panels demonstrateely (n = 5). (B) Effect of MB and/or PQ exposure on CYP1A2 in rat PMNs. The upper,protein level in PMNs, respectively (n = 5). First, second, third and fourth bar in each
**p < 0.001 as compared to controls and ###p < 0.001 as compared with MB-treated
572 I. Ahmad et al. / Chemico-Biological Interactions 188 (2010) 566–579
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ompared with PQ. CYP1A2 protein and gene expression in MB+PQreated group showed the similar patterns as observed with PQ-reated animals (Fig. 4A).
MB and/or PQ did not alter CYP1A2 activity and protein/genexpressions in PMNs of exposed animals as compared with controlsFig. 4B).
.4. Effect of MB and PQ on CYP2E1
CYP2E1 activity was estimated using p-nitrophenol and NDMAethod in the liver and PMNs respectively. However, NDMA
ethod was also used for the liver to confirm the values obtained by
-nitrophenol method and no significant changes were observed inhe results. MB and/or PQ exposure resulted in significant augmen-ation of CYP2E1 activity along with increased protein and mRNAxpression in the liver of exposed animals. The effect was time of
nued).
exposure dependent and the combined treatment resulted in morepronounced increase than MB or PQ alone, however, it was less thanthe additive effect. Maximum elevation was obtained after 6 weeksof co-exposure (Fig. 5A).
PMNs exhibited similar results, i.e., CYP2E1 activity was aug-mented following MB and/or PQ exposure. The combined treatmentshowed more pronounced increase than individual treatment,however, it was less than the additive. Protein and gene expres-sion analyses showed time dependent increase after MB and/or PQexposures as observed in the case of activity (Fig. 5B).
3.5. Modulation of GSTA4-4 in the rat liver and PMNs
GSTA4-4 activity was augmented significantly in the liver of MBand/or PQ exposed rats in a time of exposure dependent man-ner. Combined treatment resulted in an additive effect after 1
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eek of exposure. More pronounced augmentation than individ-al treatment was observed after 3 and 6 weeks of treatment but
t was less than the additive. Western blot analysis and mRNAxpression showed similar patterns as observed with the activityFig. 6A).
MB and/or PQ exposure elevated GSTA4-4 activity and itsxpressions in PMNs in a time of exposure dependent man-er. Combined exposure yielded results similar to the liver,
.e., initially the additive effect was observed and althoughncrease in exposure time showed more pronounced increase thanhe individual pesticide, the value was less than the additiveFig. 6B).
ig. 5. (A) Effect of MB and/or PQ exposure on CYP2E1 activity, protein and mRNA exprYP2E1 activity, mRNA expression and protein level in the liver microsomes, respectivelRNA expression and protein expression by upper, middle and lower panels, respective
ontrol, MB, PQ and MB+PQ-treated groups (*p < 0.05, **p < 0.01 and ***p < 0.001 as comparroup; $p < 0.05 as compared with PQ-treated group).
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3.6. Effect of MB and/or PQ on GSTA1-1 and GSTA3-3 geneexpression
No significant alteration was observed in the gene expressionpattern of hepatic GSTA1-1 after MB and/or PQ exposures whereastime dependent increase was obtained in the expression of hepaticGSTA3-3 gene after MB and/or PQ exposure (Fig. 7A). Combinedtreatment resulted in more pronounced increase than the individ-
ual alone but it was neither additive nor synergistic. Maximumaugmentation was obtained after 6 weeks of MB+PQ exposure(Fig. 7A). Similar results were obtained with GSTA1-1 and GSTA3-3gene expression in the PMNs of exposed rats. GSTA1-1 gene expres-
ession in liver microsomes. The upper, middle and lower panels demonstrate they (n = 5). (B) Effect of MB and/or PQ exposure is demonstrated on CYP2E1 activity,ly in rat PMNs (n = 5). First, second, third and fourth bar in each panel represents
ed with controls; #p < 0.05, ##p < 0.01 and ###p < 0.001 as compared with MB-treated
574 I. Ahmad et al. / Chemico-Biological Interactions 188 (2010) 566–579
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ion was unaltered, however, GSTA3-3 showed time of exposureependent increase in the mRNA expression (Fig. 7B).
. Discussion
MB and PQ are commonly and concurrently used by the farm-rs leading to their co-exposure in real life situations [38]. Highose acute exposures to PQ have been shown to produce toxic-
ty in humans. Systemic exposure with low doses of MB and PQn combination results in synergistic effect on the nigrostriatalopaminergic neurodegeneration in experimental animals and iseported to be critical in humans [3,21,38,39]. The doses selected
n this study, are shown to cause neurodegeneration in rodents withncreased susceptibility to lung injury in aged animals [2,3,21,33].lthough the liver is the main site of metabolism of these pesticides,o such studies have yet been conducted in the liver. Secondly,cute exposure to these pesticides at high doses induces the liver
nued).
injury via oxidative stress; however, low doses are reported to benon-toxic. The present study investigated the effect of the neu-rotoxic doses of MB and/or PQ on oxidative stress and toxicantresponsive genes in the liver of exposed animals and correlatedthe changes with the changes observed in the peripheral system(PMNs) in order to develop biomarker to predict the toxicity. Theincreased LPO levels observed in the liver and PMNs of MB and/orPQ exposed animals are in concurrence with the previous studiesdocumenting the increased LPO level in the lungs and liver afteracute PQ exposure and brain following systemic exposure of PQalone and in combination with MB [3,6,21,40,41]. The present studyshowed that repeated doses of MB and PQ induce oxidative stress
in the liver and PMNs in a time of exposure dependent mannershowing the similarity between them.
PQ and MB-mediated GSH reduction in the lungs, liver and brainand increase in PQ-induced toxicity in GSH depleted animals haveimplicated the protective role of GSH against pesticides induced
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oxicities [40,42–44]. Reduced GSH content after MB and PQ expo-ure in both the liver and PMNs of exposed animals and combinedxposure exhibited more pronounced effect than the individuallone. This further indicates the generation of oxidative stress ands in concurrence with earlier reports [3,40,42–44]. The decreasedSH level could be due to increased consumption to combat MBnd/or PQ-induced ROS generation or due to direct effect of pesti-ides on glutathione metabolism.
Roles of CYPs and GSTs in the biotransformation and pesticides-nduced toxicity are well established [45–49]. Although the rolef xenobiotics metabolizing enzymes in PQ-induced lung toxicitynd MB+PQ-induced neurotoxicity is known, a few reports havehown the effect of MB and PQ on hepatic xenobiotic metabolizing
nzymes after acute or sub acute exposures. However, the effectf repeated exposures of MB and/or PQ is not yet fully deciphered50,51]. The involvement of CYP1A1 and CYP1A2 in the metabolismf xenobiotics/pesticides and also in the ROS generation has been
ig. 6. (A) Effect of MB and/or PQ exposure on GSTA4-4 activity, mRNA and protein exespectively (n = 5). (B) Effect of MB and/or PQ exposure on GSTA4-4 activity, mRNA andespectively (n = 5). First, second, third and fourth bar in each panel represents control, Ms compared with controls; #p < 0.05, ##p < 0.01 and ###p < 0.001 as compared with MB-tr
teractions 188 (2010) 566–579 575
reported [11,12]. CYP1A2 is also involved in the metabolism ofMPTP, a neurotoxin, which is structurally similar to PQ [52]. In thepresent study, MB mediated increase in CYP1A1 activity, gene andprotein levels was observed after 1 week treatment, which wassustained up to 6 weeks without any further changes. Although MBshowed statistically significant increases in CYP1A1 expression andEROD activity, the same may or may not be of biological relevancein real life situation, as these values were only slightly higher ascompared with respective controls. PQ did not alter the activity andgene and protein expressions of CYP1A1 in the exposed group andcombined exposure showed the results similar to MB treated group.PQ increased the activity of hepatic CYP1A2 after 3 weeks, whichwas significantly elevated after 6 weeks. However, the increased
protein and gene expressions were observed only after 6 weeks ofPQ exposure. MB alone or in combination with PQ did not alter hep-atic CYP1A2, which indicates that the effect was solely due to PQ.No alterations were observed in the activity, protein and gene level
pression in the liver homogenate is depicted by upper, middle and lower panels,protein expression in PMNs is demonstrated by upper, middle and lower panels,B, PQ and MB+PQ-treated groups respectively (*p < 0.05, **p < 0.01 and ***p < 0.001eated group).
576 I. Ahmad et al. / Chemico-Biological Interactions 188 (2010) 566–579
(Conti
iPtb
iarScCiotstr
Fig. 6.
n CYP1A1 and CYP1A2 in PMNs of exposed groups after MB and/orQ treatment. The increased hepatic CYP1A2 could be involved inhe metabolism of PQ or contribute in the oxidative stress inducedy the pesticide.
A time of exposure dependent augmentation of CYP2E1 activ-ty and its protein and gene expressions in hepatic microsomesfter MB and PQ exposure was observed. Combined treatmentesulted in more pronounced effect than the individual exposure.ince CYP2E1 is known to be one of the most active CYP isoformsontributing in induction of oxidative stress therefore, increasedYP2E1 could be associated with oxidative stress. However, the
nvolvement of elevated CYP2E1 in oxidative stress or metabolism
f MB and PQ needs to be investigated. The results obtained inhis study are supported by the previous observations, which havehown increased CYP2E1 expression in brain [3,21]. Augmenta-ion of CYP2E1 after MB exposure is contradictory to a previouseport where acute exposure of MB resulted in the inhibition of
nued).
basal and induced CYP2E1 activity and protein expression [50].This disparity could be due to the differences in dose, treatmentregime and exposure time or MB might act as both inducer andinhibitor of CYP2E1 depending upon the dose as reported for zineb,another pesticide belonging to same class as MB [53]. PMNs alsoshowed the similar results, i.e., MB and PQ elevated CYP2E1 activity,increased CYP2E1 protein and gene expressions and more pro-nounced increase was observed after combined exposure. CYP2E1contributes to the metabolism of xenobiotics and endogenous sub-stances, including lipid hydroperoxides and ketone bodies resultingin ROS generation [13,16,54]. A parallel increase observed in theLPO levels and CYP2E1 suggest that CYP2E1 could possibly be
involved in MB and PQ-induced oxidative stress in the liver andPMNs of exposed rats. However, further investigation is needed toelucidate the exact mechanism.
GSTs are involved in pesticides metabolism and increased lev-els of GSTs are reported in response to pesticides-induced oxidative
ical In
ssMibtmtieieathiw
l
Feo(
I. Ahmad et al. / Chemico-Biolog
tress [3,48,50]. Increased GST activity along with increased expres-ion of GSTA4-4 and GTS-yc/GSTA3-3 have been reported in PQ andB+PQ-induced toxicities [17,3,55]. GSTA4-4 shows highest affin-
ty against 4-hydroxynonenal (4-HNE) and other alkenals formed asy-products during lipid peroxidation and protects against oxida-ive stress [22,31]. Increased GSTA4-4 levels might be an adaptive
echanism to protect against MB and PQ-induced lipid peroxida-ion and could account for the decreased GSH content due to anncreased consumption. GSTA4-4 was augmented in the time ofxposure dependent manner in PMNs as observed in the liver show-ng the similarity between the two. Similarly an increased GSTA3-3xpression observed in the liver and PMNs could be an indicative ofn adaptive response to combat MB and/or PQ-induced the oxida-ive stress. This is in concurrence with the previous study, which
as shown an increased GSTA3-3 expression as an early response
n PQ-induced lung toxicity [17]. However, no significant changeas observed in GSTA1-1 after MB and/or PQ exposure.
Although the doses recommended for agricultural use are veryow (the total daily doses of PQ for ground and arial application are
ig. 7. (A) Upper and lower panels demonstrate the effect of MB and/or PQ exposure onxposed animals (n = 5). (B) Upper and lower panels demonstrate the effect of MB and/orf control and MB and/or PQ exposed animals (n = 5). First, second, third and fourth bar*p < 0.05, **p < 0.01 and ***p < 0.001 as compared to controls; #p < 0.05, ##p < 0.01 and ###
teractions 188 (2010) 566–579 577
reported to be 3.2 × 10−4 mg/kg/day and 1.4–4.8 × 10−3 mg/kg/dayrespectively), in case of the developing countries where recom-mended precautionary measures are not properly followed orrepeated pesticide spraying is performed manually, the risk of get-ting exposed to higher doses in recurrent fashion is high. Acutehigh dose PQ poisoning cases in humans have been documentedand severity assessed by PQ-plasma concentrations [56]. Since longterm low dose exposures to these pesticides are already reported toincrease the risk for PD, therefore, the study might prove to be rele-vant to humans exposed to recurrent low doses of these pesticidesnot capable of showing immediate toxic effects.
Thus, it can be concluded from this study that MB and PQ differ-entially modulate hepatic CYP1A1 and CYP1A2 respectively, whiletheir status in PMNs was unaltered. MB and/or PQ induce the simi-
lar changes in CYP2E1, GSTA4-4 and GSTA3-3 in the liver and PMNs.The increase in LPO, CYPs and GSTs and decrease in GSH in the liverand PMNs observed suggest the involvement of CYPs and GSTs inMB and/or PQ-induced injury. Since MB and/or PQ exhibited similarpatterns in CYP2E1, GSTA4-4 and GSTA3-3 in the liver and PMNs,
mRNA expression of GSTA1-1 and GSTA3-3 respectively in the liver of control andPQ exposure on mRNA expression of GSTA1-1 and GSTA3-3 respectively in PMNs
in each panel represents control, MB, PQ and MB+PQ-treated groups respectivelyp < 0.001 as compared with MB-treated group).
578 I. Ahmad et al. / Chemico-Biological Interactions 188 (2010) 566–579
(Conti
tb
C
A
(apmBc
R
Fig. 7.
hese genes could act as potential candidates for developing asiomarkers to assess the MB and PQ-induced hepatotoxicity.
onflict of interest
The authors declare that there are no conflicts of interest.
cknowledgements
We sincerely thank the Indian Council of Medical ResearchICMR) for providing financial support for the study. Authorscknowledge Council of Scientific and Industrial Research (CSIR) forroviding fellowship to Israr Ahmad and University Grants Com-ission (UGC) for providing fellowships to Ashutosh Kumar and
rajesh Kumar Singh. The IITR communication number of this arti-le is 2848.
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