Ecotoxicology and Environmental Safety 97 (2013) 1–9

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Ecotoxicology and Environmental Safety

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larissa.bbebel_alucenabviniciusdebora.ealmeidclaudiab

journal homepage: www.elsevier.com/locate/ecoenv

Pollution-induced metabolic responses in hypoxia-tolerantfreshwater turtles

Larissa Paola Rodrigues Venancio a,n, Maria Isabel Afonso Silva a, Tiago Lucena da Silva a,Vinicius Augusto Gobbe Moschetta a, Débora Aparecida Pires de Campos Zuccari b,Eduardo Alves Almeida c, Claudia Regina Bonini-Domingos a

a Department of Biology, Centro de Estudo de Quelônios (CEQ) and Laboratório de Hemoglobinas e Genética das Doenças Hematológicas (LHGDH),IBILCE, UNESP – Sao Paulo State University, Sao Jose do Rio Preto, SP 15054-000, Brazilb Sao Jose do Rio Preto School of Medicine, FAMERP, Sao Jose do Rio Preto, SP 15090-000, Brazilc Department of Chemistry and Environmental Science, Laboratório de Biomarcadores de Contaminação Ambiental (LABCA), IBILCE, UNESP – Sao Paulo StateUniversity, Sao Jose do Rio Preto, SP 15054-000, Brazil

a r t i c l e i n f o

Article history:Received 27 February 2013Received in revised form19 June 2013Accepted 21 June 2013Available online 29 August 2013

Keywords:Freswater turtlesPhrynops geoffroanusContamination biomarkersAntioxidant capacityContaminated effluents

13/$ - see front matter & 2013 Elsevier Inc. Alx.doi.org/10.1016/j.ecoenv.2013.06.035

esponding author.ail addresses: larissa_biorp@yahoo.com.br,luerose@gmail.com (L.P.R. Venancio),fonso@yahoo.com.br (M.I.A. Silva),io@hotmail.com (T.L. da Silva),.moschetta@gmail.com (V.A.G. Moschetta),zuccari@famerp.br (D.A.P. de Campos Zuccari)a@ibilce.unesp.br (E.A. Almeida),onini@sjrp.unesp.br (C.R. Bonini-Domingos).

a b s t r a c t

The physiological control to support the absence of O2 for long periods of diving, and oxidative damageimpact caused by the whole process of hypoxia/reperfusion in freshwater turtles is well known. However,effects of contaminants may act as co-varying stressors and cause biological damage, disrupting thehypoxia/reperfusion oxidative damage control. In order to investigate the action of environmental stressorspresent in domestic or industrial wastewater effluent, we performed a biochemical analysis of biotrans-formation enzymes, oxidative stress, as well as neuromuscular, physiological and morphological para-meters in Phrynops geoffroanus, an hypoxic-tolerant freshwater turtle endemic of South America, usinganimals sampled in urban area, contaminated by sewage and industrial effluents and animals sampled incontrol area. Here we demonstrate the physiological and biochemical impact caused by pollution, and theeffect that these changes cause in antioxidant activity. Animals from the urban area exhibited higher EROD(ethoxyresorufin-O-deethylase, CYP1A1), GST (glutathione S-transferase), G6PDH (glucose-6-phosphatedeshydrogenase), AChE (acetilcholinesterase) activities and also TEAC (trolox-equivalent antioxidantcapacity) and TBARS (thiobarbituric acid reactive substances) values. We examined whether twomorphometric indices (K – condition factor and HIS – hepatosomatic index) which help in assessing thegeneral condition and possible liver disease, respectively, were modified. The K of the urban animals wassignificantly decreased compared to the control animals, but the HIS value was increased in animals fromthe urban area, supporting the idea of an impact in physiology and life quality in the urban freshwaterturtles. We propose that this freshwater turtle specie have the ability to enhance its antioxidants defensesin order to protect from tissue damage caused by hypoxia and reperfusion, but also that caused byenvironmental contamination and that the oxidative damage control in hypoxic conditions has resulted inan adaptive condition in hypoxic-tolerant freshwater turtle species, in order to better tolerate the release ofcontaminated effluents resulting from human activity.

& 2013 Elsevier Inc. All rights reserved.

1. Introduction

Brazil has the richest fauna and flora of the entire world, withabout 1.8 million species, and is second in regards to wealth of

l rights reserved.

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reptilian species, though most information on reptiles is stillpreliminary (Rodrigues, 2005; Comunicado Ipea, 2011; Bérnilis(org), 2010). The Order Testudines are very important in an ecoto-xicological context; however, the use of reptiles as bioindicators ofenvironmental contamination is a recent development. There aremany reasons for this: the difficulty in sampling specimens insufficiently large numbers, a lack of perceived economic value andthe difficulty of acclimatizing them to the laboratory. However, theirpersistence in a variety of habitats, wide geographic distributionand longevity, actually make them very suitable bioindicators ofcontamination (Meyers-Schöne and Walton, 1994).

The biological effects of pollution and the anthropogenicimpact on Brazilian aquatic systems are poorly evaluated, despite

L.P.R. Venancio et al. / Ecotoxicology and Environmental Safety 97 (2013) 1–92

the importance of Brazil's natural wealth. Only 3.7 percent of theTurvo/Grande Watershed, located in south east Brazil, is coveredwith natural vegetation, and has significant levels of organicdomestic pollutants, as well as a high risk of chemical contamina-tion to both the soil and water, and a very poor water quality indexneeded to protect aquatic life (Comitê da Bacia Hidrográfica Turvo/Grande, 2010). Despite the Turvo/Grande Watershed's poor waterquality, Geoffroy's side-necked turtles, Phrynops geoffroanus(Schweigger, 1812) specimens, an hypoxic-tolerant freshwaterturtle endemic to South America are commonly found, thoughthe specie's natural history is poorly understood. This specie arefrequently found in rivers, lakes and pounds with slow currentsand also can be found in unusual habitat, such a polluted urbanrivers (Pritchard and Trebbau, 1994; Souza and Abe, 2001; Zagoet al., 2010a, 2010b), attracted mainly by the wide food availabilityderived from organic effluents, as occurs in Turvo/Grande Water-shed. The freshwater turtles species are a group with a minimumtolerance of hypoxia/anoxia of more than 4.5 h (Belkin, 1963). Thephysiological control to support the absence of O2 for long periodsof diving, and oxidative damage impact caused by the wholeprocess of hypoxia/reperfusion in freshwater turtles is well known(Storey, 1996; Willmore and Storey, 1997; Hermes-Lima andZenteno-Savín, 2002; Gorr et al., 2010). However, effects ofcontaminants may act as co-varying stressors and cause biologicaldamage, disrupting the hypoxia/reperfusion oxidative damagecontrol. This raises an intriguing question of how contaminantsmay influence the control of oxidative damage caused by theevents of hypoxia and reperfusion in hypoxic-tolerant freshwaterturtles.

To investigate the action of environmental stressors derivedfrom sewage wastewater effluents in oxidative damage control,which contain constituents considered complex mixtures ofunknown composition, we have conducted an evaluation of EROD(CYP1A), glutathione S-transferase (GST), glucose 6-phophatedehydrogenase (G6PDH), acetylcholinesterase (AChE) activities aswell as an evaluation of thiobarbituric acid reactive substances(TBARS) and trolox-equivalent antioxidant capacity (TEAC) inP. geoffroanus collected in both urban and controlled areas, thusanalyzing the main elements associated whith phase I and II ofdetoxification, antioxidant capacity and lipid peroxidation. We alsodetermined the condition factor (K) and hepatosomatic index (HIS)as indicators of physiological status that summarizes their growth,reproduction under a given environmental condition. We reportthe physiological and biochemical impact caused by pollution andthe effects of these changes in antioxidant activity and in oxidativedamage control produced by hypoxia and reperfusion events,beyond the specie adaptation to adverse condition generated byenvironmental contamination.

2. Materials and methods

2.1. Samples

We collected ten animals from the urban area (Felicidade stream, Preto river,Turvo/Grande Watershed, Sao Paulo State, 20146′25.99"S; 49121′17.63" W). Thisarea receives domestic effluents and also receives pollutants of diffuse source fromflooding and agriculture runoffs. We collected ten animals in the controlled area(“Reginaldo Uvo Leone” breeding farm, Sao Paulo State, 20159′47.5"S, 49107′16.6"W). This breeding farm works with wild and exotic reptiles, amphibians andbirds for commercial purposes. The animals are fed with fish food (45 percent ofprotein), and the water of artificial lakes is from an artesian well, devoid ofcontamination. The number of samples was chosen in an effort to obtain anacceptable balance between statistical power and any adverse effects on the naturalturtle population which is already under anthropogenic pressure, and about whichthe population size was unknown and only adults (males and females) are sampled.All specimens were collected in the same period (February and March, matingperiod) to avoid deviations in the morphometric parameters caused by influencesfrom the breeding period and season. The collection activities, of animals and

biological material, were authorized and approved by IBAMA/ICMBio (register no.28387/16488-1 and 16488-2) and the Ethics Committee for Animal Experimenta-tion of Sao Jose do Rio Preto School of Medicine (FAMERP no. 5517/2008). Captureof animals in the urban area was done with traps with netting used in thecontrolled area. From all specimens were collected two morphological parameters(1) maximum over-the-curve carapace length, in centimeters, from the anterior-most part of the carapace to the posterior-most tip of the carapace on the same side(Wyneken, 2001) and (2) body mass in grams. All animals were euthanizedimmediately after the capture.

2.2. Chemicals

All reagents were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA).

2.3. Ecotoxicology evaluation

For ecotoxicological assays, we used liver, brain and peripheral blood fromeuthanized animals in accordance with procedures recommended by CONCEA.For the AChE analyses, brain tissue was homogenized (1:4, w/v) in a 0.1 M Trisbuffer, at pH 8.0, and then centrifuged for 30 min at 30,000g. The AChE activity wasmeasured and then characterized following methodology previously described(Ellman et al., 1961). The enzymatic activity is performed based on the followingequation:

Acetylcholine+H2O-Acetic Acid+Thiocholine

The spectrophotometric assay is performed at 25 1C, λ¼412 nm, εmM¼13.6 mM �1 cm�1, following the DTNB reaction with thiocholine in 2 min. Thereaction was performed using a Tris–HCl buffer (100 mM, pH 8.0), acetylthiocholineiodide 100 mM, DTNB 1 mM and the extract in a final volume of 1 mL.

For GST (Keen et al., 1976), G6PDH (Glock and McLean, 1953) and EROD (Burkeand Mayer, 1974) (with modifications previously described in Nogueira et al., 2010to GST and EROD, and Sáenz et al., 2010 to G6PDH) analyses, the liver washomogenized (1:4, w/v) in a Tris 20 mM buffer (pH 7.4), sucrose 0.5 mM, KCl0.15 mM and 1 mM protease inhibitor (PMSF). The samples were then centrifugedat 9000g for 20 min at 4 1C, and the upper phase was collected and centrifugedonce more at 50,000g for 60 min. The cytosolic fraction was then collected for GSTand G6PDH analysis, while EROD activity (indicative of CYP1A) was measured inthe pellet, which was resuspended in 100 mL Tris 100 mM buffer, at pH 7.5,containing EDTA 1 mM, DTT 1 mM, KCl 100 mM and 20 percent glycerol.

The GST assay is performed based on the reaction below:

GSH+CDNB2CDNB-SG+HCl

The spectrophotometric evaluation was performed at 30 1C, λ¼340 nm. Thereaction was conducted with a potassium phosphate buffer 0.2 M, pH 6.5, GSH200 mM, CDNB 200 mM and sample.

The spectrophotometric evaluation of G6PDH activity was performed at 1 min.,λ¼340 nm. The reaction was conducted with 50 mL of Tris–HCl buffer 0.1 M, pH7.4, MgCl2 0.2 mol/L, 10 mg of NADP and water (final volume: 100 mL). 21.25 mg ofG6P at 50 mL of the reaction medium was added. The assay was performed using asolution without G6P and a sample and two aliquots of reaction medium contain-ing G6P and sample.

The EROD evaluation was performed in fluorometer. The λEx (excitation)¼537 nm and the λEm (emission)¼583 nm performed in 180 s. The assay isperformed with potassium phosphate buffer 80 mM, pH 7,4, 7-etoxiresorufin332 mM, NADPH 20 mM and homogenized sample, totaling 2 mL of reaction. Thereaction was monitored for 3 min at 30 1C and EROD activity was calculated basedon a resorufin standard curve that had been prepared. The protein evaluation wasperformed using the Bradford (1976) method.

The plasma level of thiabarbituric acid reactive species (TBARS) (Yagi, 1976)(with modifications) was used to evaluate the samples' lipid peroxidation. Themethod is based on the reaction of malondyaledehyde and other aldehydes withtiobarbituric acid (TBA) at a low pH and high temperature to form a complex with amaximum absorption at 535 nm. The final TBARS amount in each sample wasobtained in ng/mL. The antioxidant potential of the samples was determinedaccording to their equivalence to a potent known antioxidant, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble synthetic analog ofvitamin E, in plasma samples. This is a colorimetric method based on the reactionof ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic-acid)) with potassiumpersulfate (K2S2O8), producing the radical cation ABTS+, a chromophore of green-ish/blue, with maximum absorbance at wavelengths 645, 734 and 815 nm. Thesamples addition containing this preformed cation radical was reduced once againin ABTS and the decolorization assessed at 734 nm. Final results were expressed inmM/L, corresponding to the concentration of the studied sample's trolox equivalentantioxidant capacity, called TEAC (Re et al., 1999).

Table 1Summary of variable statistical data (morphometric data, biomarkers values andmorphometric indices). (t-independent test for all comparisons).

Variables Controlled area(n¼10)

Urban area(n¼10)

p (o0.05) – two-tailed

Mean7S.E.M. Mean7S.E.M.

Total weight (g) 1420.07370.66 2334.07134.20 o0.05Liver weight (g) 35.3374.71 115.9717.63 o0.0001Length (cm) 27.572.9 30.771.41 40.05nn

EROD activitya 0.470.049 0.8570.21 o0.05GST activityb 2.070.17 2.7370.15 o0.05G6PDH activityc 19.8774.94 34.6274.74 o0.05AChE activityc 42.1173.45 55.0572.74 o0.05Kd 0.1570.006 0.01270.0003 o0.0001HIS (%) 2.3970.18 5.1870.37 o0.0001TBARSe 310.34711.02 564.68798.16 o0.0001TEACf 1.6170.08 1.9870.09 o0.05

g: grams; cm: centimeters; %: percentage symbol; S.E.M.: standard error of mean.a Activity expressed in pmol/min/mg protein.b Activity expressed in U/mg protein.c Activity expressed in mU/mg protein.d No unit expressed (index).e Expressed in ng/mL.f Expressed in nM/L.nn No statistic difference.

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2.4. K and HIS evaluation

The condition factor (K, Le Cren, 1951) and length–weight relationship weredetermined by measuring total weight and total length, and the curves parametersa and b were determined by log transformation of raw data (Froese, 2006). Growthcurves obtained from straight line for regression between total weight and totallength are logW¼ log a+b log L. The theoretical equation of the length–weightrelationship followed the equation W¼aLb. Condition factor was assessed by thefollowing expression: K¼W/Lb, where K is the condition factor; W is the totalweight; L is the total length; b is the coefficient of regression. The HIS wascalculated by liver weight/total body weight�100 (Oguri, 1978).

2.5. Statistical analysis

For the statistical analysis of the evaluation of ecotoxicological assays, the datawere assessed for normality and homogeneity of variances through Shapiro–WilkW and Levene's tests, respectively. Log transformation of data was employed wherenecessary, in order to allow the use of parametric statistical methods. Theprobability of significance was set at αo0.05, and data reported as mean7S.E.M.Data were subjected to parametric testing (t-test), two-tailed, and for independentsamples. Associations between biomarker variables and morphometric indiceswere examined by Pearson's correlation procedure using individual turtle data.After confirmation of the homogeneity of slopes, analysis of covariance (ANCOVA)was used to compare the intercepts of these relationships between the areas ofstudy. Biochemical and morphometric parameters variation was further summar-ized in the principal component analysis (PCA). The statistical analyses wereperformed in STATISTICAs version 8.0.

3. Results

3.1. Assessment of ecotoxicological parameters

Under normal physiological conditions, animals maintain abalance between generation and neutralization of reactive oxygenspecies (ROS). However, when organisms are subjected to xeno-biotic compounds, the production rate of ROS, such as superoxideanion radicals (O2

d�), hydrogen peroxide (H2O2), hydroxyl radicals(dOH) and peroxyl radical (ROO�) exceeds their scavengingcapacity (Halliwell and Gutteridge, 1999). All organisms have theirown cellular antioxidative defense system, composed of bothenzymatic and non-enzymatic components.

To identify the impact of xenobiotic compounds resulting fromsewage wastewater effluent exposure, we performed several bioch-emical analyses of contamination biomarkers.

Liver, plasma and brain samples collected from both the urbanand controlled area animals were evaluated in order to assessvariations in hepatic EROD activity (1A isoform of CytochromeP450, a phase I biotransformation enzyme), GST (Phase II), G6PDHand TEAC (antioxidant capacity), encephalic AChE (neurotoxicity)and TBARS (lipid peroxidation) (Table 1).

Animals from the urban area exhibited significantly higherEROD activity when compared to those from the controlled area,indicating a twofold increase in liver CYP1A, which may be relatedto the presence of anthropogenic organic contaminants in theurban area, such as polycyclic aromatic hydrocarbons (PAHs) andpolychlorinated biphenyls (PCBs) (Whyte et al., 2000; Van der Oostet al., 2003; Pathiratne and Hemachandra, 2010), derived fromburning of fossil fuel, drag and disposal of the abrasion productsbetween asphalt and tires, air emissions that contribute signifi-cantly to the HPA input to the aquatic environment, urban runoffand domestic effluents (Martins et al., 2007). All these events arecommon in the Felicidade stream region, indicating possiblecontamination by these elements.

The GST activity evaluation showed values significantly higher(1.36 times) in animals from the urban area. The primary reactioncatalyzed by GSTs is the nucleophilic attack by reduced GSH on adiverse group of hydrophobic compounds which contain an elec-trophilic carbon, nitrogen, or sulfur atom (Hayes and Pulford, 1995).

The evaluation of G6PDH activity presented a significant differencewith higher activity in animals from the urban area (1.76 times),

suggesting an increased action in the antioxidant defenses of animalsfrom the contaminated area. It is known that G6PDH activity canincrease during stressful situations in order to guarantee the NADPHsupply which is key to sustaining antioxidant defenses (Ramnananand Storey, 1986). This action is confirmed by the TEAC analysis, whichshowed elevated values in the urban area (1.23 times higher).We therefore expected low rates of lipid peroxidation (LPO). However,the LPO analysis showed higher values in the urban area (1.76 times)in comparison to the controlled area. It is known that TBARS levelsmay be elevated following exposure to metals, such as aluminum (Al)and cadmium (Cd), as well as diesel fuel exposure and heat stress(Downs et al., 2001; Kaiser et al., 2005).

In this study, the animals of the urban area exhibited signifi-cantly higher values (1.3 times) of AChE activity compared to thecontrol animals. This result indicates that there are elements in theenvironment that stimulate AChE activity. The water present in theurban area showed annual lower O2 dissolved levels (1.42–3.71 mg L�1, unpublished data), high concentrations of cadmium(Cd, 2.0–3.0 mg/L, Maschio et al., 2009) and aluminum (Al, 174.8–707.85 mg/L, unpublished data)., when compared with referencesite and parameters of Brazilian legislation to superficial waterquality. It has been proposed that the high Cd levels can beassociated with high TBARS levels (Downs et al., 2001), and highAl levels with increased AChE activity (Kaiser et al., 2005).

These results indicate a consistent picture of a strong environ-mental impact in the urban area. However, the TEAC evaluationdemonstrates that urban freshwater turtles have a high antiox-idant capacity. We propose that this freshwater turtle specie havethe ability to enhance its antioxidants defenses in order to protectfrom tissue damage caused by hypoxia and reperfusion, but alsothat caused by environmental contamination.

3.2. Morphometric indices evaluation

Next, we examined whether two morphometric indices(K and HIS) which help in assessing the general condition andpossible liver disease, respectively, were modified. These indicescan provide important information on potential pollution impacton aquatic animals (Van der Oost et al., 2003).

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To test this question, these indices were calculated through therelationship between total body length and weight (K), and thetotal liver weight and total body weight (HIS). The K of the urbananimals was significantly decreased (12.9 times) compared to thecontrol animals, but the HIS value was significantly increased (2.17times) in animals from the urban area, supporting the idea of animpact in physiology and life quality in the urban freshwaterturtles. In this study, animals in the urban area proved to beheavier than the animals from controlled area (Table 1), andANCOVA (analysis of covariance) showed that mass varied sig-nificantly between areas, considering the liver weight as a covari-ate (F¼33.74, po0.0001). We suggested that the increased weightseen in the urban area is due to fat accumulation from theconsumption of large amounts of decomposing organic materialavailable to these animals. In relation to HIS, the values indicate anassociation with effluent rich in organic materials, and enhanceddetoxification activities in response to the presence of contami-nants, with an increase in cell length and number, which wasreflected in overall liver weight (Yeom et al., 2007; Sol et al., 2008).

3.3. Biomarkers, K and HIS indices relationship

Next, we performed multivariate exploratory techniques withall variables analyzed. The cluster analysis indicated two distinctgroups corresponding to samples collected in the controlled areaand another of samples collected in the urban area (Fig. 1).Biochemical and morphometric parameter variation was furthersummarized in the principal component analysis (PCA). The PCAanalysis and component loading plots analysis exhibited a separa-tion of components in morphometric variables (PC 1, 38.29percent), biomarkers and elements associated with antioxidantcapacity (PC 2, 25.27 percent) in the controlled area (Fig. 2A), and aseparation of components in biomarkers (PC 1, 32.80 percent) andweight/length (PC 2, 30.77 percent) in the urban area (Fig. 2B). Inthe controlled area a strong positive correlation was observedbetween AChE activity (brain) and TBARS levels (plasma) (Fig. 2C).In the urban area, positive correlation was observed between HISand G6PDH activity (liver) (Fig. 2D), and a negative correlation wasobserved between K and EROD activity (liver) (Fig. 2E). Thisanalysis reflects the existence of an important effect of phase I ofxenobiotic detoxification in the ability of animals to tolerate toxic

0 1 2

10u9u8u6u4u3u5u2u7u1u6c7c5c4c9c8c

10c2c3c1c

Con

trol a

rea

Urb

an a

rea

Fig. 1. Urban and controlled area cluster analysis. The analysis indicates the presence of(biochemical and morphometric) were used and evaluated in the study, and all these p

challenge and environmental stress (Whyte et al., 2000). Thehigher values in HIS were associated with higher values ofG6PDH activity reflecting the possible exposure of turtles to asub-lethal concentration of pollutants, resulting in an increase inthe number of hepatic cells, as a consequence of induction oractivation of biotransformation enzymes (Yeom et al., 2007; Solet al., 2008; Kopecka-Pilarczyk and Correia, 2009). The ANCOVAanalyses showed that HIS varied significantly between areas afterallowing for G6PDH activity as a covariate (F¼40.4, po0.0001).

3.4. Discussion

In this study we have shown that environmental contaminationaffects antioxidant capacity and the network of elements involvedin the protection of oxidative stress caused by hypoxia/reperfusionin hypoxic-tolerant freshwater turtles (Figs. 3 and 4).

The results found in this study can be associated with twohypotheses: (1) development of tolerance and cellular adaptationand (2) development of hormesis due to environmental stressors.The hormesis concept comes from numerous findings haveemerged in recent years indicating that ROS may evoke cellularsignaling that promotes metabolic health and longevity (Barja,1993; Gems and Partridge, 2008; Ristow and Schmusser, 2011;Woo and Shadel, 2011; Dolci et al., 2013). The biotransformationsystems (phase I and phase II) are very susceptible to hormeticinduction; this induction provides protection against moleculardamage and can be achieved by administration of xenobiotics atnontoxic levels (Gems and Partridge, 2008).

The hormesis has been used to explain the antioxidant defensesin mammals and fish, but nothing was previously reported tofreshwater turtles. In fishes, Bengtsson (1979) proposed the devel-opment of hormesis during exposure of fishes to low concentrationsof environmental pollutants. Laughtin et al. (1981) showed anenhanced growth rate of crustacean larvae exposed to petroleumpollutants for short periods as compared to unexposed ones, andthen, considered as hormesis.

However, most studies of oxidative stress adaption have beenlimited to adaptation induced by acute stress. In other hand,environmental and physiological stresses are repeated or chronic.The more recent study evaluating the capacity of adapting tochronic or repeated stress in both cultured mammalian cells and

3 4 5

two groups, which relate to the two areas of study. For this analysis all parametersarameters were responsible for the groupings (c¼controlled area; u¼urban area).

Fig. 2. PCA loading plots and correlation analysis between significant variables. (A) Controlled area PCA plot. (B) Urban area PCA plot. The analysis indicated a correlationbetween TBARS assessment (9) and AChE activity (7) (black arrow) in controlled area. (C) The correlation analyses between AChE and TBARS parameters showed a strongpositive relationship. The urban area PCA indicated a correlation between G6PDH activity (8) and HIS (2) (gray arrows), and EROD activity (5) and K (1) (black arrows).(D) Correlation between HIS and G6PDH. (E) Correlation analysis between EROD activity and K.

L.P.R. Venancio et al. / Ecotoxicology and Environmental Safety 97 (2013) 1–9 5

Fig. 3. Physiological mechanism of protection to environmental contamination associated with hypoxia. (A) With the increase in GST activity and in turn the consumption ofGSH, an imbalance occurs in the GSH/GSSG ratio in favor of GSSG. To maintain the reduced cellular status, G6PDH activity increases in order to maintain high levels of NADPHfor GR activity that restores the GSH levels in an attempt to maintain physiological balance. The increase in antioxidant capacity directly reflects the increased hepatocytesrequest, which can be seen in the HIS increase. (B) In the brain, with the increased AChE activity, there is a possible complementary increase in LPO. The increasedconcentration of LPO products such as MDA can lead to imbalance in the [GABA]/[Glu] ratio, breaking homeostasis between GABAergic and glutamatergic activities (Gouldand Gross, 2002), which reduces brain activity in an attempt to withstand oxidative stress. Also, there is the possibility of cell replacement in specific brain areas (Gould andGross, 2002; Radmilovich et al., 2003). (C) The increase in enzyme activity may lead to increased ATP consumption in situations where consumption is to be basal. The highermetabolic activity and cell replacement in brain and liver tissue, combined with possible behavioral changes (e.g. sleep-like and fatigue-like behaviors), can change theanimal's condition. However, the physiological adjustments to protect against oxidative damage have proved effective for maintenance and protection in highlycontaminated environments.

Fig. 4. Adaptation of freshwater turtles to polluted environments associated with the reduction or prevention of oxidative stress caused by reoxygenation/hypoxia. Anoxia/reperfusion in mammals results in an increase in ROS that causes damage in proteins, lipids and nucleotides, resulting in cellular dysfunction and death (purple box). Turtlessurvive the successive situations of hypoxia and re-oxygenation and this physiological condition also confers survival in polluted areas by increasing antioxidant capacity (A).Furthermore, physiological support for hypoxia promotes an increase in elements associated with cellular damage protection (B) such as increasing the stability ofnucleotides, proteins and lipid structures associated with the upregulation of repair functions. An important adaptation seen in these animals is neurogenesis (C), whichallows cell division and neuronal replacement (adapted from Milton and Prentice, 2007).

L.P.R. Venancio et al. / Ecotoxicology and Environmental Safety 97 (2013) 1–96

fruit fly Drosophila melanogaster concluded that, in mammaliancells, the chronic oxidant exposure at low oxidant levels canactually potentiate and extend adaptive responses, but chronicexposure to higher oxidant levels prevents adaptation. For fruit fly,repeated oxidative stress adaptation at intervals of 1 or 3 days istoxic, and the life span and short-term survival seems to benegatively influenced by all repeated stress adaptation regimens(Pickering, 2013).Our findings point towards the development ofhormesis, but in a minor proportion. The hormesis-inducedadaptation is associated with acute stress exposure and lowconcentration of environmental contaminants. The animals of urbanarea are in a condition of chronic exposure to high concentration

contaminants, therefore living in a highly eutrophic area, whosephysico-chemical parameters show impact by diffuse source ofpollutants from flooding and agriculture runoffs and organic effluentsnot allowing the maintenance of aquatic life (Campanha et al., 2010).Based on physiology and molecular mechanisms of hypoxia/anoxiacontrol and relationship of this mechanisms with detoxification ofROS, the inducibility of key protective mechanisms associated withturtle's exposure to oxygen variability tends to be more relevant inadapting to environmental contamination.

Biotransformation plays a critical role in the elimination ofxenobiotics and that different CYPs can catalyze the directinsertion of oxygen atoms into several organic compounds

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(Letcher et al., 2000). The intermediary elements generated inphase I of biotransformation and other biochemical reactions canbe conjugated with glutathione (GSH) in a reaction catalyzed byGST. Thus, GST expression is of importance when consideredsusceptible to environmental chemical toxicity (Richardson et al.,2009). The increased GST activity in animals from naturalpopulations is directly related to the increase in oxidative stress.Like other hypoxia-tolerant reptiles, freshwater turtles must dealwith a significant amount of ROS generated as a consequence ofthe ischemia-reperfusion associated with diving (Storey, 1996).Turtles have mechanisms that prevent the formation of ROS orthat minimize oxygen free radical damage during oxygen reper-fusion in the recovery period (Belkin, 1963; Willmore and Storey,1997; Willmore and Storey, 2005; Willmore and Storey, 2007;Reischl, 1986). Such mechanisms could involve the use of bothenzymatic and nonenzymatic antioxidant defense systems. Themost important of the nonenzymatic antioxidants is glutathione.Because GSH is present in high intracellular concentrations, thereis a high probability that any ROS will be immediately quenchedon formation. Glutathione dissulfide (GSSG) is formed but issubsequently reduced by glutathione reductase (GR) to restoreGSH. When ROS are present in large amounts, GSSG formationcan exceed its clearance and the ratio of reduced to oxidizedglutathione (GSH/GSSG) decreases (Whyte et al., 2000). Themaintenance of GSH levels, and thereby the reducing environ-ment of the cell, is crucial to organisms that periodically undergooxidative stress (Willmore and Storey, 1997). This maintenance isguaranteed by glutathione reductases and G6PDH, that have beenshown to increase as the GSH/GSSG decreases (Xu et al., 2003).

Previous studies have shown that the total activity of turtleliver GSTs decreases by 25 percent during hypoxia/anoxia(Willmore and Storey, 2005). Purification and characterization ofturtle liver GSTs showed a decrease in activity of all isoformsduring anoxia from 7.3 U/mg protein in aerobic conditions to2.8 U/mg protein under anoxic conditions (Willmore and Storey,2005; Willmore and Storey, 2007). The implication of this is thatthe turtle liver relies on a fixed pool of glutathione duringprolonged hypoxia/anoxia (with lower turnover), which it canuse for buffering redox changes, associated with changes in GSTactivity in support of anaerobiosis (Willmore and Storey, 2007).Therefore, the glutathione function analysis in hypoxia-tolerantturtles shows that a reduction in enzyme activity is associatedwith use/degradation (i.e. GST) and an increase in the activities ofenzymes involved in the production of GSH in key tissues (kidney,liver, heart and brain) during hypoxia, prevents the formation of,or minimizes damage by, ROS during transitions between hypoxicand aerobic states (Willmore and Storey, 1997). High redox buffercapacity associated with high basal levels of total thiols in redblood cells attributed to increased GSH levels, and has beenpostulated as a mechanism preventing reperfusion injury in thehypoxia-resistant turtle Phrynops hilarii (Reischl, 1986).

Influxes of oxyradical-generating compounds due to environ-mental contaminants may alter GSH status and/or metabolism inseveral ways. Increased fluxes of oxyradicals can impose anintracellular drain on equivalent reductors with potentially pro-found consequences on a variety of metabolic processes. Theconsumption of GSH due to the direct scavenging of oxyradicals,or as a cofactor for GSH-dependent enzymes (i.e. GSTs andglutathione peroxidases), may represent such a drain, and NADPHmust be continuously oxidized to maintain GSH levels via glu-tathione reductase (GR) (Di Giulio et al., 1995). The results ofG6PDH activity and NADPH production derived from enzymeactivity are directly associated with the generation of elementsrelated to the increase of antioxidant activity (Figs. 3A and 4A). Inanimals from the urban area, the G6PDH requirement increasedcompared to the controlled area, indicating the presence of

exogenous elements that are changing the activity of the enzy-matic and nonenzymatic factors associated with oxidative pro-cesses in P. geoffroanus. In addition, there was strong relationshipbetween G6PDH activity and HIS. The increase in G6PDH activity isclosely related to lipid biosynthesis (Zomeño et al., 2010), whichalso influences the HIS increase, as the index also identifiesincreased liver fat. The disruption of this balance by the presenceof xenobiotics may be more accentuated during re-oxygenationrecovery. However, it is known that in tissues from organs such asthe heart, kidney and brain, due to glutathione status, little or nooxidative stress occurs during anoxia or recovery from anoxia(Willmore and Storey, 1997). Thus, the changes impact on enzymeactivity involved in detoxification and antioxidant activity infreshwater turtles, due to the presence of environment pollutants,and can alter the entire mechanism of tolerance to hypoxia andnormoxia.

The increase in antioxidant activity might reflect a generalmetabolic expression response to anoxia/hypoxia that may affectprotein synthesis. Indeed, translation is known to be a highly ATPconsuming process (Hochachka and Lutz, 2001), requiring anincrease in metabolic activity, and higher physiological efforts,reflected in the fitness of the impacted population, that may beevidenced by K and HIS evaluation. However, the increased G6PDHactivity ensures increased NADPH production and a direct responsein GR production and recycling of GSH levels, to support the redoxbuffering capacity of tissues protecting against oxidative stress.Moreover, NADPH also provides the reducing power for thedetoxification and xenobiotic transformation reactions catalyzedby the cytochrome P540 family of enzymes (Di Giulio et al., 1989)(Fig. 3A).

Among biomarkers, the measurement of AChE activity isconsidered of great interest in evaluating the effects of exposureto neurotoxic compounds in aquatic animals (Cajaraville et al.,2000). AChE plays an important role in the functioning of theneuromuscular system, preventing continuous muscular contrac-tion (Fulton and Key, 2001). Analysis of metal concentration inwater samples of the urban area (unpublished data) showed highaluminum (Al) levels. Evaluation of in vivo and in vitro effects ofaluminum (Al) on the activity of mouse brain AChE revealed ahigher effect of Al in vivo in relation to in vitro tests, with acrescent activity of AChE along the exposure period (Zatta et al.,2002). Indeed, chronic Al exposure reveals effects on brainphysiology, including alteration of the lipid composition and theactivities of various membrane-bound enzymes. The effect of Al onAChE activity may be due to a direct neurotoxic effect of the metalor perhaps a disarrangement of the plasmatic membrane causedby increased LPO (Kaiser et al., 2005). This result indicates theimportant impact of metal contaminants, like Al, in brain physiologyof freshwater turtles. With the increase of AChE activity in the brain,acetylcholine hydrolysis (ACh) in synaptic clefts is probablyincreased. Consequently, the reduction of this neurotransmitter andneuromodulator in the central nervous system could lead to cerebraldysfunctions that affect the behavior, locomotor system, balance andorientation (Silva et al., 2011). In hypoxia-tolerant turtles, the increaseof LPO and malondialdehyde (MDA) in the brain can induce sleep-like and fatigue-like behaviors, as well as a glutamate and GABAimbalance, resulting in turtle brain suppression and an increase inthe protective response of the brain (Fig. 3B). These demonstrate thatthe turtle's brain has a stronger capacity to fight stress thanmammals (Li et al., 2010). Also, the neurons and glial cells in certainparts of the central nervous system continue to be producedthroughout a turtle's life span (Gould and Gross, 2002). In the brainthese cells proliferate more rapidly under warm conditions than atcolder temperatures (Radmilovich et al., 2003), providing a potentialmeans to replace anoxia-damaged cells during the warmer months(Milton and Prentice, 2007) (Fig. 3C). We suggest that the disruption

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in normal AChE activity could be associated with aluminum expo-sure; however the condition of cell replacement in brains of hypoxia-tolerant turtles suggests evidence of the maintenance of normalbrain conditions (Figs. 3B, C and 4B, C).

In addition to supporting environmental contamination, themechanisms that turtles employ to survive hipoxia/anoxia mightbe linked to longevity. Resistance to stress is generally correlatedwith longevity and organisms that are tolerant of one kind ofstress are typically more likely to be tolerant to others, since manycytoprotective mechanisms operate over a wide variety of stresses(Söti and Csermly, 2007; Krivoruchko and Storey, 2010). In relationto turtle brain, many mechanisms involved in brain hypoxia-tolerance are also linked to age-related neurodegeneration, andas such may contribute to turtle longevity (Lutz et al., 2003).

4. Conclusions

Our results reveal the important influence that industrial anddomestic effluent contamination plays in hypoxic-tolerant fresh-water turtles. Despite the increase in EROD activity and the effecton GST activity, the mean GST values in the urban area are withinthose expected under hypoxia conditions. This observation sug-gests that the GST increase in response to ROS production due tothe presence of pollutants increases the antioxidant defense net-work, controlling the oxidative damage caused by hypoxia andreperfusion and oxidative damage caused by pollutants. Despitethe increase in the antioxidant capacity in urban area, themorphometric indices indicate an impact on health and physiolo-gical condition, probably due to the increased physiological strainof maintaining high levels of antioxidant capacity to protectagainst hypoxia damage caused by contamination. The increasein antioxidant capacity in the urban animals reflects an adaptationto the adverse environmental conditions caused by pollution,reinforcing the observation that reproduction in P. geoffroanusdoes not seem to be affected in polluted areas, suggesting that thisspecies is very tolerant to pollution impact. However, a significantinfluence on fitness can be observed. There is no data about thetime scale of metal contamination in the areas studied, suggestingthat the effects could currently still be subclinical, and yet lead todisruption in the population's reproductive success over time (Piñaet al., 2009). Furthermore, it is also suggested that the robuststress-tolerance mechanisms that permit long term anaerobiosisby turtles may also support the longevity of these animals (Krivo-ruchko and Storey, 2010). Thus, the oxidative damage control inhypoxic conditions associated with spectacular levels of antiox-idant capacity would have introduced a strong condition oftolerance in hypoxic-tolerant freshwater turtles species, allowingthem to withstand the adverse environmental situation, such ascontaminated effluent released as a result of human activity.

Author's contributions

LPRV designed the study, conducted the experiments and thedata analysis, and wrote the manuscript, with contributions fromall authors. MIAS conducted the TBARS and TEAC experiments.LPRV, MIAS, TLS and VAGM conducted the collecting of animalsand tissues from both the urban and controlled areas. DAPCZconducted the veterinary evaluation of the animals and alsoconducted the euthanasia process. EAA with LPRV conducted theecotoxicology assay and provided the reagents for the experiments.CRBD coordinated the study. All authors discussed the experi-ments, edited and approved the final manuscript.

Acknowledgments

We are grateful to Luis Dino Vizotto, Ph.D., for his importantsuggestions, and Lilian Nogueira, Camila Trídico and Aline Rodri-gues for their technical assistance. This study was supported byCNPq – Brazil fellowship to LPRV (MCT/CNPq no27/2007 – 143419/2008-0). This research was also supported in part by Laboratóriode Hemoglobinas e Genética das Doenças Hematológicas (LHGDH)and Laboratório de Biomarcadores de Contaminação Ambiental(LABCA), Unesp, Brazil.

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