oxidative stress biomarkers in cyprinus carpio exposed to commercial herbicide bispyribac-sodium

Oxidative stress biomarkers in Cyprinus carpioexposed to commercial herbicidebispyribac-sodium_JAT 590..595

Cândida Toni,a Charlene Cavalheiro de Menezes,a Vania Lucia Loro,a*Bárbara Estevão Clasen,a Roberta Cattaneo,a Adriana Santi,a

Alexandra Pretto,a Renato Zanellab and Jossiele Leitempergera

ABSTRACT: Cyprinus carpio were exposed under field conditions to 20.87 mg l-1 of commercial herbicide bispyribac-sodium(Nominee®, SC), during 7, 21 and 72 days. Enzymatic parameters such as catalase (CAT), glutathione S-transferase (GST) andacetylcholinesterase (AChE) activities, as well as thiobarbituric acid-reactive substances (TBARS) and protein carbonyl contentswere studied in different tissues. After 7 days of exposure, GST activity decreased. At the same period, brain AChE activityincreased, but a reduction of activity was observed in muscle tissue. Brain TBARS levels increased at 7 days. After 21 days ofexposure liver CAT levels and muscle AChE activities decreased. In the same period, liver protein carbonyl and muscle TBARSincreased. After 72 days of exposure in the field, AChE activity was reduced in both brain and muscle. Protein carbonyl contentsin liver and brain TBARS levels increased. Muscle AChE activity, TBARS and protein carbonyl can be used as biomarkers ofexposure to the herbicide bispyribac-sodium. This study demonstrates effects of exposure to bispyribac-sodium under ricefield conditions on oxidative stress parameters in tissues of Cyprinus carpio. Copyright © 2010 John Wiley & Sons, Ltd.

Keywords: fish; biomarkers; rice field; toxicology; herbicide

INTRODUCTION

Agricultural activity is the main factor leading to the increase ofcontaminants in aquatic ecosystems. Its toxicological effects canlead to the deterioration of water quality and adversely affectnon-target organisms, including fish. The sources of contami-nants are numerous and include pesticides, which represent alarge group of toxic chemicals. Bispyribac-sodium {sodium 2,6-bis[(4,6-dimethoxypyrimidin-2-yl) oxy] benzoate} is a pyrimidinylcarboxy herbicide, used to control grasses, sedges and broad-leaved weeds. This compound has solubility in water of 73.3 g l-1,is considered partially soluble and has a half-life of 60 days (Rod-rigues and Almeida, 1998). Its mechanism of action is to non-competitively inhibit the enzyme acetolactate synthase (ALS) onthe route of synthesis of the amino acids valine, leucine and iso-leucine, resulting in disruption of cell division and plant growth.Several studies have shown that pesticide exposure induces bio-chemical changes in fish (Crestani et al., 2006; Moraes et al., 2007;Fonseca et al., 2008).

Many pesticides are responsible for causing oxidative stress inaquatic organisms, because these contaminants may induce theformation of reactive oxygen species (ROS) and alterations in theantioxidant system (Üner et al., 2006). Of particular interest arethe reduction products of molecular oxygen, which may reactwith critical cellular macromolecules, possibly leading to enzymeinactivation, lipid peroxidation (LPO), DNA damage and, ulti-mately, cell death (Winston and Di Giulio, 1991).

Antioxidant enzymes activities, which defend organismsagainst ROS, are critically important in the detoxification of radi-

cals to non-reactive molecules (van der Oost et al., 2003). Defensesystems that prevent the formation of ROS include antioxidantenzymes, such as superoxide dismutase (SOD), glutathione per-oxidase (GPx) and catalase (CAT). Glutathione S-transferases(GSTs), a multigene superfamily of multifunctional, primarilysoluble, enzymes, catalyze the conjugation of electrophilic com-pounds (or phase I metabolites) with GSH, which is involved in thedetoxification of xenobiotics. GSTs play a critical role in protectingcells against oxidative damage and peroxidative products (vander Oost et al., 2003).

Another parameter frequently used to study pesticide toxicityis the measurement of acetylcholinesterase activity (AChE), whichis often decreased after pesticide exposure. The inhibition ofactivity can affect growth, survival, feeding, and reproductivebehavior of fish exposed to different pollutants (Dutta andArends, 2003). Lipid peroxidation (LPO)is a very important conse-quence of oxidative stress and used as a biomarker in a number ofstudies (Ando and Yanagida, 1999; Wilhelm Filho et al., 2001;Oakes and Van Der Kraak, 2003). Proteins also constitute a target

*Correspondence to: V. L. Loro, Departamento de Química, Universidade Federalde Santa Maria, 97105.900 Santa Maria, RS, Brazil.E-mail: [email protected]

aLaboratório de Bioquímica toxicológica e Adaptativa de Peixes, Programa de Pós-graduação em Ciências Biológicas, Bioquímica Toxicológica, Santa Maria, RioGrande do Sul, Brazil

bLaboratório de Análises de resíduos de Pesticidas, Departamento de Química,Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil

Research Article

Received: 30 September 2009, Revised: 16 February 2010, Accepted: 15 March 2010 Published online in Wiley InterScience: 30 April 2010

(www.interscience.wiley.com) DOI 10.1002/jat.1530

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of oxidative damage with subsequent alteration of their func-tions (Ferreira et al., 2005). Protein carbonyl has been used as abiomarker of exposure in fish because, when protein carbonyla-tion occurs, protein conformational changes, decreased catalyticactivity and breakdown of proteins by proteases also occur(Almroth et al., 2005).

Cyprinus carpio, a native fish of Eastern Europe and WesternAsia widely distributed in Brazil, is an omnivorous species thatfeeds on invertebrates, plants, algae, insect larvae, crustaceansand also on small fish (Querol et al., 2005). The effects of thecommercial formulation of bispyribac-sodium herbicide used inthe rice field have scarcely been studied in fish. Little is knownabout the changes in the antioxidant defense and cholinergicsystems in response to exposure to this herbicide in Cyprinuscarpio. Thus, the present study aimed to investigate the effects ofa commercial formulation of bispyribac-sodium herbicide underrice field conditions on parameters of oxidative stress in C. carpio,and also to determine possible biomarkers for fish exposure tothis herbicide.

MATERIALS AND METHODS

Fish

Cyprinus carpio of both sexes weighing of 20.0 � 1.0 g and mea-suring 11.0 � 1.0 cm in length were obtained from a fish farm (RS,Brazil). Fish were acclimated to laboratory conditions for 10 days,in tanks (250 l) prior to the experiments. They were kept in con-tinuously aerated water with a static system and with a naturalphotoperiod (12 h light/12 h dark). After the acclimation periodfish were transferred to points located in a rice field. During allexperimental period the average of water parameters were: tem-perature 24.5 � 2.0 oC, pH 6.5 � 0.2 units, dissolved oxygen4.21 � 2.0 mg l-1, non-ionized ammonia 0.8 � 0.01 mg l-1, nitrite0.06 � 0.01 mg l-1. Both in the period of acclimation and in theperiod of exposure, the fish were fed once a day with commercialfish pellets (42% crude protein, Supra, Brazil). After the exposureperiod, in the intoxicated fish there occurred an increase inweight of around 30%, while in the control group there was averified increase of 43%.

Chemicals

The commercial formulation of the herbicide bispyribac-sodiumsodium [2,6-bis (4,6-dimethoxypyrimidin-2-yloxy) benzoate],Nominee® (BASF) was used. Acethylthiocholine (ASCh),5,5’dithio-bis(2-nitrobenzoic acid) (DTNB), 1-chloro- 2,4 din-hitrobenzene (CDNB), bovine serum albumin, Triton X-100,hydrogen peroxide (H2O2), malondialdehyde (MDA), 2-thiobarbituric acid (TBA) and sodium dodecyl sulfate (SDS) wereobtained from Sigma Chemical Co. (St Louis, MO, USA).

Experimental Design

Fish were allocated in two groups as follows: control group, 45fish distributed in three tanks containing water free from herbi-cide (15 fish per tank); and exposure group with 45 fish exposedto initial measured concentration 20.87 mg l-1 of the herbicide (15fish per tank) for 7, 21 or 72 days. The control fish were in tankswith separate water supply from the exposure tanks, but condi-

tions and placing of ponds were similar for both groups. Theexperiment was carried out in the paddy field, with the fishtrapped in submerged cages, measuring 0.30 m (diameter) ¥1.05 m (length). Herbicide concentration in the water was moni-tored from the first day until it was not detected. Herbicide wasanalyzed by high-pressure liquid chromatography (HPLC) usingthe method described by Zanella et al. (2003). After each expo-sure period (7, 21 and 72 days), a sample of 15 individuals pertreatment (five fish per tank) was taken from the tanks andsubmitted to blood and tissue (brain, liver and white muscle)collection.

Catalase Activity Assay

Catalase (EC 1.11.1.6) activity was assayed by ultraviolet spectro-photometry (Nelson and Kiesow, 1972). Liver tissue were hom-ogenized in a Potter-Elvejhem glass/Teflon homogenizer with20 mM potassium phosphate buffer, pH 7.5 (1 : 20 w/v), centri-fuged at 10 000g for 10 min at 4 °C. The assay mixture consisted of2.0 ml potassium phosphate buffer (50 mM, pH 7.0), 0.05 ml H2O2

(0.3 M) and 0.05 ml homogenate. Change in H2O2 absorbance in60 s was measured by at 240 nm. Catalase activity was calculatedin picomoles and expressed in micromoles per minute per milli-gram protein.

Glutathione S-transferase

GST activity was measured according to Habig et al. (1974) usingCDNB as a substrate. The formation of S-2, 4-dinitrophenyl glu-tathione was monitored by the increase in absorbance at 340 nmagainst blank. The extinction coefficient used for CDNB was9.6 mM cm-1. The activity was expressed as mmol GS-DNBmin-1 mg-1 protein.

Acetylcholinesterase Activity Assay

The AChE (EC 3.1.1.7) activity was measured using the methoddescribed by Ellman et al. (1961) and modified by Miron et al.(2005). Brain and muscle tissues (30 mg) were weighted andhomogenized in a Potter-Elvejhem glass/Teflon homogenizerwith sodium phosphate buffer 50 mM pH = 7.2 and Triton X-1001%. The homogenate was than centrifuged for 10 min at 3000g at5 °C and the supernatant was used as enzyme source. Aliquots ofsupernatant (50 and 100 mL; brain and muscle, respectively) wereincubated at 30 °C for 2 min with a solution containing 0.1 M

sodium phosphate buffer pH 7.5 and 1 mM DTNB. After the incu-bation period, the reaction was initiated by the addition of ASCh(0.5 mM). The final volume was 2.0 ml. Absorbance was measuredby spectrophotometry (Femto Scan spectrophotometer) at412 nm during 2 min. Enzyme activity was expressed as micro-moles of ASCh hydrolyzed per minute per milligram protein.

Carbonyl Assay

The liver tissue was homogenized in 10 vols (w/v) of 10 mM Tris-HCl buffer pH 7.4 using a glass homogenizer. Protein carbonylcontent was assayed by the method described by Yan et al. (1995)with some modifications. Soluble protein (1.0 ml) was reactedwith 10 mM DNPH in 2 M hydrochloric acid. After incubation atroom temperature for 1 h in the dark, 0.5 ml of denaturing buffer

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(150 mM sodium phosphate buffer, pH 6.8, containing SDS 3.0%),2.0 ml of heptane (99.5%) and 2.0 ml of ethanol (99.8%) wereadded sequentially, vortexed for 40 s and centrifuged at 10 000gfor 15 min. Then, the protein isolated from the interface waswashed twice by resuspension in ethanol–ethyl acetate (1 : 1),and suspended in 1 ml of denaturing buffer, and the carbonylcontent was measured spectrophotometrically at 370 nm. Assaywas performed in duplicate and two tubes blank incubated with2 M HCl without DNPH were included for each sample. The totalcarbonylation was calculated using a molar extinction coefficientof 22 000 M cm-1.

Lipid Peroxidation Estimation Assay

Lipid peroxidation was estimated by a TBARS assay, performed byan MDA reaction with TBA, which was optically measured. Liver,muscle and brain homogenates (250–400 mL) were added.Trichloroacetic Acid (TCA) 10% and 0.67% TBA were added toadjust to a final volume of 1.0 ml. The reaction mixture wasplaced in a microcentrifuge tube and incubated for 15 min at95 °C. After cooling, it was centrifuged at 5000g for 15 min andoptical density was measured using a spectrophotometer at535 nm. TBARS levels were expressed as nanomoles MDA permilligram protein according to Buege and Aust (1978).

Protein Determination

Protein was determined by the Coomassie blue method usingbovine serum albumin as standard. Absorbance of samples wasmeasured at 595 nm (Bradford, 1976).

Statistical Procedures

Comparison between two groups was made by the Studentt-test, paired test. The value of P � 0.05 was considered statisti-cally significant for all analyses (N = 15).

RESULTS

Table 1 shows the results of hepatic antioxidant enzymes fromfish exposed to bispyribac-sodium during 7, 21 and 72 days. CATactivity showed a significant decrease after 21 days of herbicideexposure, while no significant changes were found at 7 and 72days. In a similar manner, the herbicide showed a significantdecrease in GST activity after 7 days of exposure, while no statis-tically significant changes were observed after 21 and 72 dayswhen compared with the control group.

Results concerning TBARS are presented in Fig. 1. Brain TBARSlevels were increased in fish exposed to the herbicide at 7 and 72days (Fig. 1A). Similar results were observed in muscle (Fig. 1B)and liver (Fig. 1C) TBARS levels after 21 and 72 days of exposure,respectively. Figure 2 shows the protein carbonyl contents in theliver of fish exposed to bispyribac-sodium. The protein carbonylcontents were elevated after 21 and 72 days of exposure, whileno significant changes were observed at 7 days.

Acetylcholinesterase activity is presented in Fig. 3. AChE activ-ity in brain was significantly increased at 7 days of exposure, but,in contrast, a significant decrease was observed after 72 days ofexposure. At 21 days, no changes were recorded in brain AChEactivity (Fig. 3A). However, there was a significant decrease inmuscle AChE activity at all exposure periods (Fig. 3B).

Table 1. Catalase (CAT) and glutathione S-transferase (GST) activities in liver of Cyprinus carpio exposed to commercial herbicidecontaining bispyribac-sodium in rice field conditions after 7, 21 and 72 days. Data represent the mean � SD (n = 15)

CAT (mmol min-1 mg-1 protein) GST (mmol GS-DNB min-1 mg-1 protein)Time (days) Control 20.87 mg l-1 Control 20.87 mg l-1

7 0.0598 � 0.029 0.0568 � 0.015 0.3129 � 0.064 0.1762 � 0.045*21 0.0684 � 0.014 0.0250 � 0.026* 0.3382 � 0.134 0.4554 � 0.10072 0.0598 � 0.029 0.0568 � 0.015 0.5787 � 0.161 05671 � 0.141

*Significant difference between control and herbicide group (P � 0.05).

7 days 21 days 72 days0.0

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Figure 1. TBARS levels (nmol MDA mg-1 of protein) in brain (A), muscle(B) and liver (C) tissue of Cyprinus carpio exposed to commercial herbicidecontaining bispyribac-sodium in rice field conditions after 7, 21 and 72days. Data represent the mean � SD (n = 15). *Significant differencebetween control and herbicide group (P � 0.05).

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Bispyribac-sodium residues were monitored in the water of therice field system to verify the presence of the herbicide activeingredient in the water. These results are shown in Fig. 4.Bispyribac-sodium herbicide persisted in the water for 56 daysafter the first application in the rice field.

DISCUSSION

The results of the present study show that CAT and GST activities,TBARS levels and protein carbonyl contents were affected after allexperimental periods as compared with the control group. Takentogether, these results indicate the development of oxidativestress that represents an increase in TBARS and carbonyl levelsand at same time a decrease in or lack of response of the antioxi-dant system. Under rice field conditions, after 7 days of exposure,GST activity in the liver decreased. GST plays an important role inprotecting tissues from oxidative stress and its activity was sup-pressed in this study, probably due to the oxidative stress situa-tion generated by herbicide exposure. This reduction on activity

may have occurred because the liver is one of the first organsexposed to pesticides or other pollutants. These results clearlyindicate a disruption in compensatory mechanisms to help fishtissues to detoxify the herbicide. Thus, results concerning GSTactivity suggest possible liver oxidative damage that disruptedthe detoxification process. In agreement with our result, Moraeset al. (2009b) observed decreased liver GST activity in carp afterexposure to imazethapyr and imazapic in a similar rice field con-dition. Similarly, GST activity was decreased in Oreochromis niloti-cus exposed to oxyfluorfen (Peixoto et al., 2006). In the sameperiod the brain TBARS levels were increased and no alterationsin this parameter were observed in liver and muscle tissue. TheTBARS measurement is considered a biomarker for intoxication,and consideration of environmental context biomarkers offerspromise as a sensitive indicator demonstrating that toxicantshave entered organisms, have been distributed between tissuesand are eliciting a toxic effect at critical targets. In this study liverand brain TBARS increase after 72 days of exposure, thus TBARSmeasurement could be considered a biomarker for bispyribac-sodium poisoning. The increased brain TBARS levels might haveresulted from an increase in free radicals due to stress conditionscaused by herbicide intoxication. Miron et al. (2008) observedincreased TBARS levels in brain of Leporinus obtusidens afterexposure to clomazone herbicide considering a similar period tothat used in this study. Liver CAT activity as well as protein carbo-nyl did not show significant results after 7 days of herbicide expo-sure. The lack of response of catalase is probably due to tissueoxidative damage, and also could represent a response againstherbicide toxicity, helping liver tissue to prevent protein oxida-tion and lipid damage.

After 21 days’ exposure under rice field conditions, TBARSlevels were increased in white muscle, but were not altered incerebral and hepatic tissues. Probably the increase in muscleTBARS levels was due to the failure of antioxidant defenses in thistissue. In contrast, CAT enzyme activity was decreased in liver.CAT is a primary antioxidant defense component that protectsfish from oxidative stress by converting hydrogen peroxide tooxygen and water (Atli and Canli, 2007). Decreased CAT activitywas also observed in the liver of Leporinus obtusidens exposed toclomazone and propanil in a field study similar to ours (Moraeset al., 2009a). Protein carbonyl formation can occur as a result ofoxidative stress. In the present study, after 21 days of herbicideexposure, protein carbonyl contents were found to be signifi-cantly increased and this coincided with a depletion of antioxi-dant enzymes in the liver. Similar to our results, a significantincrease in protein carbonyls was observed in freshwater fish

7 days 21 days 72 days0

2

4

6

8

10

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14 0.0 µg/L20.87 µg/L

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*

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Figure 2. Protein carbonyl levels in liver of Cyprinus carpio exposed tocommercial herbicide containing bispyribac-sodium in rice field condi-tions after 7, 21 and 72 days. Data represent the mean � SD (n = 15).*Significant difference between control and herbicide group (P � 0.05).


0.1

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Figure 3. Acetylcholinesterase activity in brain (A) and muscle (B) tissueof Cyprinus carpio exposed to commercial herbicide containingbispyribac-sodium at rice field condition after 7, 21 and 72 days. Datarepresent the mean � SD (n = 15). *Significant difference between controland herbicide group (P � 0.05).

0 8 16 24 32 40 48 560

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Figure 4. Bispyribac-sodium concentration (mg l-1) in water fromrice field.

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(Channa punctata) exposed to different toxic compounds (Parvezand Raisuddin, 2005). After 21 days of herbicide exposure, thepicture of liver tissue changed and in this context the antioxidantsystem was able to prevent TBARS formation, but not proteincarbonyl increase. In addition, in this period a lack of response ofGST activity, an important enzyme for the detoxification process,contributed to fish poisoning. The herbicide was detected inwater in this period and probably the fish are able to absorb it.Considering all the effects of herbicide, including undesirableeffects, more studies are need to better understand bispyribac-sodium toxicity from carps. The measurement of herbicide in fishtissues will be considered in the future studies.

After 56 days, the herbicide was not detected in paddy water,but its effects or metabolites derived from it probably continuedto cause disorders in fish tissues. Thus, after 72 days of exposure,brain and liver TBARS were increased. Similarly, Oropesa et al.(2009) observed an increase of TBARS levels in Cyprinus carpio,collected in simazine-polluted water under field conditions (90days). In addition protein carbonyl contents were significantlyincreased after 72 days of herbicide exposure. A significantincrease in protein carbonyl contents was observed by Almrothet al. (2005) in eelpount (Zoarces viviparous). In our study, theincrease in protein carbonyl contents in fish liver exposed for 21and 72 days may indicate that normal protein metabolism wasaltered, resulting in the accumulation of damaged molecules. Theresults concerning TBARS and carbonyl after 72 days of exposureshowed the long-term effects of herbicide on fish tissues.

With respect to neural and neuromuscular function, studieshave indicated that the long-standing biomarker, AChE, mayrespond to low levels of contaminants in the environment (Payneet al., 1996). Some effects on the non-target organism areexpected, including alterations in AChE activity, especially aftermedium- or long-term exposure. In this study, AChE activityincreased significantly in brain after 7 days of exposure to com-mercial herbicide bispyribac-sodium as compared with thecontrol. Our results are in agreement with those obtained byMoraes et al. (2009b), who found increased brain AChE activityupon fish exposure to imazethapyr and imazapic herbicidesunder rice field conditions. However, the enzyme activation islittle understood in fish and the present result needs more inves-tigation considering a laboratory model to compare effects andunderstand this increase. AChE activity in muscle was inhibitedduring all experimental periods (7, 21 and 72 days). Similarly, after72 days of exposure, the brain AChE activity showed a significantdecrease. The most commonly found effect of pesticide toxicity isthe inhibition of enzyme activity. Our results are agreement withsimilar experiments using rice field conditions, where Leporinusobtusidens were exposed to commercial herbicides clomazoneand quinclorac (Moraes et al., 2007) and clomazone and propanil(Moraes et al. 2009a). For both studies, brain and muscle AChEwere inhibited after a long period of exposure to pesticides. Asignificant depression of AChE activity in fish was also observedin brain and muscle tissues of common carp (Cyprinus carpio)exposed to simazine during 90 days (Oropesa et al., 2009). Theinhibition of AChE may influence the process of cholinergic neu-rotransmission and promote undesirable effects. The reductionsin AChE activity in both brain and in muscle found in our inves-tigation may have been caused by the herbicide in the short-termperiod and persisted over a long time. The effects may also becumulative due to herbicide retention in tissues or caused bysurfactants used in the commercial formulations. As can be seenin Fig. 4, bispyribac-sodium residues were found in the water for

56 days. Taken together, it can be suggested that the resultsobserved after prolonged exposure may have been due to thecommercial formulation and not bispyribac-sodium per se.

CONCLUSION

The present study shows that the commercial formulation con-taining bispyribac-sodium at rice field concentrations may causechanges in toxicology and oxidative stress parameters in tissuesof Cyprinus carpio. Decreased GST, CAT and AChE activities maybe the result of severe oxidative stress generated by herbicideexposure. Oxidative damage in lipids and proteins could be thecause of the suppressed enzyme activity. The inhibition of GSTactivity reflects a failure of herbicide detoxification mechanisms.Increased TBARS levels indicate oxidative damage in lipids. Thesechanges coincided with the persistent increase in protein carbo-nyl levels, reflecting a severe disorder in the metabolism due tofish poisoning. In conclusion, our findings indicate that TBARS,protein carbonyl and muscle AChE activity can be used as biom-arkers for bispyribac-sodium herbicide exposure. Brain AChE acti-vation needs more investigation in order to explain the possibletoxicity mechanism.

Acknowledgments

We would like to thank the Universidade Federal de Santa Mariafor the support and the facilities, CNPq/CT-HIDRO 552546/2007-09 for the financial support, CNPq for the student assistant-ship and the undergraduate student salary. Vania Lucia Lororeceived a research fellowship from CNPq.

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