Evaluation of toxic effects of a diet containing fish contaminated with methylmercury in rats mimicking the exposure in the Amazon riverside population

Environmental Research 111 (2011) 1074–1082

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Environmental Research

0013-93

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Evaluation of toxic effects of a diet containing fish contaminated withmethylmercury in rats mimicking the exposure in the Amazonriverside population

Denise Grotto a, Juliana Valentini a, Juliana Mara Serpeloni a, Patrıcia Alves Ponte Monteiro b,Elder Francisco Latorraca c, Ricardo Santos de Oliveira c, Lusania Maria Greggi Antunes a,Solange Cristina Garcia d, Fernando Barbosa Jra,n

a Departamento de Analises Clınicas, Toxicologicas e Bromatologicas, Faculdade de Ciencias Farmaceuticas de Ribeir~ao Preto, Universidade de S ~ao Paulo, 14040-903 Ribeir ~aoPreto,

S ~ao Paulo, Brazilb Departamento de Patologia, Faculdade de Medicina de Ribeir ~ao Preto, Universidade de S ~ao Paulo, Brazilc Departamento de Cirurgia E Anatomia, Faculdade de Medicina de Ribeir ~ao Preto, Universidade de S ~ao Paulo, Brazild Departamento de Analises Clınicas, Universidade Federal do Rio Grande do Sul, Brazil

a r t i c l e i n f o

Article history:

Received 13 May 2011

Received in revised form

14 September 2011

Accepted 21 September 2011Available online 13 October 2011

Keywords:

Fish consumption

Methylmercury

Oxidative stress

Genotoxicity

Inflammation

Blood pressure

51/$ - see front matter & 2011 Elsevier Inc. A

016/j.envres.2011.09.013

esponding author. Fax: þ55 16 36024725.

ail address: [email protected] (F. Barbosa

a b s t r a c t

This study was designed to evaluate the effects of a diet rich in fish contaminated with MeHg,

mimicking the typical diet of the Amazon riverside population, in rats. Animals were randomly

assigned to one of three groups with eight rats in each group: Group I—control, received commercial

ration; Group II—received a diet rich in uncontaminated fish; Group III—received a diet rich in fish

contaminated with MeHg. Treatment time was 12 weeks. Oxidative stress markers were evaluated, as

well as the effects of this diet on DNA stability, systolic blood pressure (SBP), nitric oxide (NO) levels

and histological damage in different tissues. There was a significant increase in SBP values in rats fed

with MeHg-contaminated fish diet after the 10th week of the treatment. As far as oxidative stress

biomarkers are concerned, no differences were observed in reduced glutathione and protein carbonyl

levels, glutathione peroxidase, catalase, superoxide dismutase or d-aminolevulinate dehydratase

activities between the groups of animals receiving contaminated and uncontaminated fish diets.

On the other hand, malondialdehyde levels increased significantly in rats fed with contaminated fish.

NO levels were similar in all groups. DNA migration showed augmented in rats exposed to

contaminated fish and histopathological analyses showed weak but significant leukocyte infiltration.

Thus, we conclude that the MeHg-contaminated fish diet induced a slight lipid peroxidation and

genotoxicity. However, these effects seem to be much less pronounced than when rats are exposed to

aqueous solution containing CH3HgCl.

Our findings support the contention that the chemical form of MeHg in fish or fish nutrients such as

polyunsaturated fatty acids, Se or vitamin E could minimize the toxic effects of MeHg exposure in fish-

eating communities.

& 2011 Elsevier Inc. All rights reserved.

1. Introduction

Mercury (Hg) is one of the most toxic pollutants, methylmer-cury (MeHg) being the most toxic forms of Hg. Its toxicity isparticularly linked to the nervous system, involving disturbancesof sensation in the extremities, ataxia, constriction of the visualfield and muscular weakness (WHO, 1996). Renal, immunologicaland cardiovascular effects after MeHg exposure have also been

ll rights reserved.

Jr).

demonstrated (Augusti et al., 2008; Clarkson, 2002; Fillion et al.,2006; Silbergeld et al., 2005; Virtanen et al., 2007).

Fish or seafood consumption is an important route for humanMeHg exposure (Clarkson and Magos, 2006; Malm et al., 1995).In the Amazon Basin, fish contaminated with Hg have beenrecognized as a problem affecting riparian people, whose mainsource of protein is fish. Many studies have shown a strongcorrelation between fish consumption and Hg exposure in thesepopulations (Cordier et al., 2002; Dolbec et al., 2000; Dorea et al.,2003; Pinheiro et al., 2008). Despite the pronounced high expo-sure to MeHg, little is known about possible toxic effects. In anepidemiologic study with adults living in a village along theTapajos River, Brazil, a decrease in near visual contrast sensitivity

D. Grotto et al. / Environmental Research 111 (2011) 1074–1082 1075

and manual dexterity was associated with hair Hg levels (Lebelet al., 1998). In a population of children exposed to MeHg inFrench Guiana, Cordier et al. (2002) observed a link betweenMeHg exposure and a number of perturbations in neurologicaland neuropsychological developments such as increased deeptendon reflexes, poorer leg coordination and decreased perfor-mance in a visuospatial test (Cordier et al., 2002). On the otherhand, an epidemiological study of female riparians from theNegro River, Brazil, did not detect symptoms of paraparesis,tremor or deadness of limbs, sensory disturbances associatedwith methylmercury exposure (Dorea et al., 2003). Additionally astudy assessing neurocognitive, language, memory, motor andbehavioral functions in children from Seychelles indicated nodetectable adverse effects in this population, which consumeslarge quantities of ocean fish (Myers et al., 2003).

Studies of MeHg effects in animal models are generally carriedout by exposing the animals to MeHg solutions. This does not byany means reflect or mimic the exposure conditions of riversideor other populations exposed to MeHg through contaminated fishor seafood. The controversial results of epidemiological studiesassociated with unrealistic exposure conditions in animal modelsprovide us with more questions than answers about the realeffects of MeHg exposure on fish-eating communities.

Thus, the present study aims to evaluate the effects of a dietrich in fish contaminated with MeHg in rats. Some biomarkers ofoxidative stress, DNA stability, systolic blood pressure (SBP) andnitric oxide (NO) levels were evaluated after sub-chronic expo-sure. Histological damage and Hg and Se levels were alsoevaluated in different tissues.

2. Materials and methods

2.1. Chemicals

Glycine, epinephrine, potassium phosphate monobasic (KH2PO4), potassium

phosphate dibasic (K2HPO4), hydrogen peroxide (H2O2), nicotinamide adenine

dinucleotide phosphate-oxidase (NADPH), reduced glutathione (GSH), 5-5-dithio-

bis-2-nitrobenzoic acid (DTNB), 2,4-dinitrophenylhydrazine (DNPH), sodium

dodecyl sulfate (SDS), trichloroacetic acid (TCA), reductase glutathione (GSHR),

thiobarbituric acid (TCA), malondialdehyde (MDA) bis(dimethylacetal), sodium

azide, d-aminolevulinic acid (ALA), ethidium bromide, agarose, Triton X-100 and

tetramethylammonium (TMAH) 25% (w/v) in water were purchased from Sigma-

Aldrich (St. Louis, MO, USA). All other reagents used were of analytical grade.

Aqueous solutions were prepared in Milli-Q water (Millipore, Bedford, MA, USA). A

clean laboratory and a laminar-flow hood capable of producing class 100 were

used for preparing solutions. All other operations were performed in a class-1000

clean room.

2.2. Preparation of the rats’ diet

To mimic the Amazon riverside population’s diet, we first measured the

amount of fish (in grams) contributing to the total mass ingested by an adult living

in this region during one whole day. According to Passos et al. (2008), an adult

male riparian consumes an average of 198.7 g of fish per meal, which means 397 g

per day (considering two meals with fish). Thus, fish contributes approximately

20% of the total mass of food ingested during one whole day. Based on this data,

the rat’s diet was a mix of 80% conventional food and 20% fish.

The composition of the conventional rat food is whole corn, soybean, wheat,

calcium carbonate, dicalcium phosphate, sodium chloride, vitamins, minerals and

amino acids. Values for vitamins, minerals and amino acids are given per kilo of

food: vitamin A (25,200 UI), vitamin D3 (2100 UI), vitamin E (60 mg), vitamin K3

(12.5 mg), vitamin B1 (14.4 mg), vitamin B2 (11 mg), vitamin B6 (12 mg), vitamin

B12 (60 mg), vitamin B3 (60 mg), vitamin B5 (112 mg), vitamin B9 (6 mg), vitamin

B7 (0.26 mg), choline (1100 mg), iron (50 mg), zinc (60 mg), copper (10 mg),

iodine (2 mg), manganese (60 mg), selenium (0.05 mg), cobalt (1.5 mg), lysine

(100 mg) and methionine (300 mg).

The fish used for the diet were carnivorous fish from the Tapajos River, Para, in

the north of Brazil (contaminated area) and from the Parana River in the south of

Brazil (uncontaminated area), which were used as an uncontaminated control.

Then, fish from both contaminated and uncontaminated areas were minced,

ground in a kitchen blender with a little water and lyophilized separately. Then

the resulting fish powder was sifted for homogeneity. Before incorporation into

food pellets, samples of lyophilized fish were randomly selected and analyzed to

determine total Hg and Se levels using an Inductively Coupled Plasma Mass

Spectrometer (ICP-MS; ELANDRC II, Perkin Elmer, SCIEX, Norwalk, CT, USA)

according to the method of Batista et al. (2009). Moreover, to determine the

amount of mercury in the form of MeHg in fish samples, chemical speciation of Hg

was done in samples according to the method of Rodrigues et al. (2010).

Finally, food pellets were prepared by mixing 80% of the commercial food

(Nuvital Nutrientes S/As) with 20% of the homogenate fish samples.

2.3. Animals

Male Wistar rats weighting 180–200 g from our Central Laboratory Animal

Facility (University of S~ao Paulo, Ribeir~ao Preto, Brazil) were used. The animals

were kept in 12 h light/dark cycles in an air-conditioned room at 22–25 1C, with

free access to water and food in accordance with the respective diets detailed

below. Animals were used in conformity with the guidelines of the Committee on

the Care and Use of Experimental Animal Resources, University of S~ao Paulo, Brazil

(approved protocol no. 07.1.1185.53.3).

Animals were randomly assigned to one of three groups with eight rats in each

group: Group I, the control group, received commercial food (Nuvital Nutrientes S/

As); Group II received a diet rich in uncontaminated fish and Group III received a

diet rich in fish contaminated with MeHg. The time of treatment was 12 weeks.

2.4. Systolic blood pressure

Systolic blood pressure (SBP) was measured weekly by tail cuff plethysmo-

graphy. The SBP value was determined as the mean of at least three measure-

ments in each rat, taken under resting conditions. The rat was placed in a rat

holder for 10 min before SBP measurement.

Variations in body weight were also analyzed weekly to compare differences

in body weight gain during the experiments.

2.5. Oxidative stress biomarkers

2.5.1. Reduced glutathione (GSH) assay

Reduced thiols such as GSH in total blood were determined by the method of

Ellman (1959). Blood (0.3 mL) was hemolyzed by 10% Triton X-100 (0.1 mL) and,

after 10 min, precipitated with 0.2 mL of 20% TCA. After centrifugation at

5000 rpm for 10 min, the supernatant aliquots were reacted to 50 mL of 10 mM

DTNB and the reaction product was read at 412 nm in a spectrophotometer. GSH

levels were expressed as mmol/mL blood.

2.5.2. Glutathione peroxidase (GSH-Px) activity

The activity of the antioxidant enzyme GSH-Px was determined in total blood

using GSH, GSHR, NADPH, sodium azide and H2O2. This method is based on the

oxidation of NADPH at 25 1C in the presence of the reagents above and diluted blood,

and GSH-Px activity was monitored in 340 nm absorbance, according to Paglia and

Valentine (1967). Data were expressed in nmol NADPH/min/mL blood.

2.5.3. Catalase (CAT) activity

CAT activity was assayed by measuring the rate of decrease in 10 mM H2O2

absorbance in a spectrophotometer at 240 nm (Aebi, 1984). Total blood was

diluted in 50 mM of phosphate buffered saline (PBS); an aliquot of 20 mL was

added to 1910 mL of PBS and 70 mL of H2O2. CAT activity was monitored by the

consumption of H2O2. Data were expressed in k/g Hb. Routine laboratory methods

were used to analyze hemoglobin levels in blood (Hb) in order to correct CAT and

SOD enzyme values.

2.5.4. Superoxide activity (SOD) activity

SOD activity was assayed by quantifying the inhibition of superoxide-depen-

dent epinephrine self-oxidation by a spectrophotometer at 480 nm (McCord and

Fridovich, 1969). Data were expressed in USOD/mg Hb.

2.5.5. d-Aminolevulinate dehydratase (ALA-D) activity assay

ALA-D activity was assayed in heparinized total blood using Sassa’s method

(Sassa, 1982) with minor modifications by measuring the rate of phorphobilino-

gen formation within 1 h at 37 1C in the absence of the reductor agent dithio-

threitol. The enzyme reaction was initiated after 10 min of pre-incubation. The

reaction was started by adding ALA to a final concentration of 4 mmol/L in PBS pH

6.8, and incubation was carried out for 1 h at 37 1C. The reaction product was

measured at 555 nm.

2.5.6. Protein carbonyls (PC) assay

In the PC assay, serum was diluted, mixed with TCA, centrifuged at 5000 rpm

for 5 min and a DNPH solution was added to this precipitated protein. The

Table 1Analytical performance for the determination of trace elements in Standard

Reference Materials (SRM): SRM 1577a—Bovine Liver; SRM TORT-2—Lobster

Hepatopancreas; SeronormTM—Trace Elements Whole Blood. Values are denoted

as mean7SD, n¼3.

Referencematerial

Hg targetvalue (lg/g)

Hg foundvalue (lg/g)

Se targetvalue (lg/g)

Se foundvalue (lg/g)

SRM 1577a 0.00470.002 0.00570.002 0.7170.07 0.7870.05

SRM TORT-2 0.2770.06 0.2270.03 5.6370.67 5.2170.34

SeronormTM 14 (mg/L) 1372 (mg/L) 14 (mg/L) 1272 (mg/L)

Hg¼total mercury; Se¼total selenium.

D. Grotto et al. / Environmental Research 111 (2011) 1074–10821076

resulting mixture was vortex-mixed until homogeneous and incubated at room

temperature for 30 min. After that, the supernatant was discarded; the precipitate

was washed twice with 1 mL of ethanol/ethylacetate (1:1), to remove free DNPH.

The precipitate was dissolved in a solution containing SDS and EDTA and

incubated at 37 1C for 10 min. The color intensity of the supernatant was

measured in a spectrophotometer at 370 nm (Levine et al., 1990). Results were

expressed as nmol/mg protein.

2.5.7. Malondialdehyde (MDA) assay

MDA was quantified using the high performance liquid chromatography

(HLPC) technique in accordance with Grotto et al. (2007). The separation of the

MDA–(TBA)2 adduct was performed using a 150�9�4 mm3 reverse phase silica

based C18 column (Eurospher-100) with a particle size of 5 mm.

The mobile phase was a mixture of 2.5 mmol/L KH2PO4 pH 7.0 and methanol

(50:50 v/v). The sample run was 8 min, with a flow rate of 0.6 mL/min, maintained

isocratically throughout. The column was kept at 40 1C and the absorbance of the

eluent was monitored at 532 nm.

2.5.8. Total nitric oxide (NO) assay

Plasma samples were analyzed for their nitrate/nitrite content using Nitric

Oxide Colorimetric Assay Kit, Assay Designs, Stressgen. First of all, nitrate is

converted to nitrite utilizing nitrate reductase. After that, Griess reagent (sulfani-

lamide in acid medium) converts nitrite to a deep purple azo die compound,

measured at 540 nm. Results were expressed in mmol/L.

2.5.9. Comet assay

The alkaline version of the comet assay was performed according to guidelines

proposed by Singh et al. (1988), and in vivo assay recommendations by Hartmann

et al. (2003). Heparinized periphery blood (20 mL) was mixed with 120 mL of 0.5%

low-melting-temperature agarose in PBS and applied to microscope slides pre-

coated with 1.5% normal-melting-temperature agarose in PBS. The slides were

covered with microscope coverslips and refrigerated for 5 min to gel. This was

followed by immersion in ice-cold alkaline lysing solution (final pH 10.0) for at

least 1 h. The slides were then incubated for 20 min in an ice-cold electrophoresis

solution (pH413), followed by electrophoresis for 20 min. After that, the slides

were neutralized and stained with ethidium bromide (20 mg/mL). One hundred

cells per animal (two slides of 50 cells each) were analyzed at 400� using a

fluorescence microscope (Zeiss, Axiostarpluss) equipped with a 515–560 nm

excitation filter and a 590 nm barrier filter connected to an Axiocam camera

(Zeiss). Comet ScoreTM software was obtained from the public domain (http://

www.tritekcorp.com/products_cometscore.php), and DNA damage was quantified

by measuring the percentage of DNA in the tail (% DNA).

2.5.10. Determination of Hg and Se levels in tissues and blood

Total Hg and Se in kidney, liver, heart and brain were determined using ICP-

MS. For this analysis we adopted the method proposed by Batista et al. (2009).

Briefly, 50–75 mg of each tissue was weighed and transferred to a conical tube

(15 mL). Then, 1 mL of 50% (v/v) TMAH solution was added to the samples,

incubated at room temperature for 12 h and the volume was made up to 10 mL

with a solution containing 0.5% (v/v) HNO3 and 0.01% (v/v) Tritons X-100.

Analytical calibration standards were prepared daily over the range of 0–20 mg/L

in a diluent containing 5% (v/v) TMAH, 0.5% (v/v) HNO3 and 0.01%

(v/v) Tritons X-100. The correlation coefficient for calibration curves was better

than 0.9999.

Total Hg and total Se in whole blood were determined in accordance with Palmer

et al. (2006). For the calibration curves, ovine whole blood was homogenized and

diluted 50 times with a solution containing 0.01% (v/v) Tritons X-100 and 0.5% (v/v)

HNO3 (Matrix-matching calibration). Blood samples collected from animals were

prepared and diluted 1:50 with a solution containing 0.01% (v/v) Tritons X-100 and

0.5% (v/v) HNO3. The correlation coefficient for calibration curves was better than

0.9999.

In order to verify data accuracy, standard reference materials (SRM) and reference

materials were analyzed. All found values were in good agreement with the certified

or reference values (Table 1).

For the speciation analysis in fish, 50 mg of sample was placed in 15-ml

polypropylene test tubes with 4.90 ml of a solution containing 0.10% v/v HClþ0.05%

m/v L-cysteineþ0.10% v/v 2-mercaptoethanol and then sonicated for 15 min in an

ultrasonic bath. The resulting solution was centrifuged and then filtered through

0.20 mm Nylons filters (Millipore, USA). For data validation, SRM TORT-2 from the

National Research Council Canada was analyzed and the results were in good

agreement with the certified value.

2.5.11. Histopathological analysis

A histopathological analysis of liver, kidney, heart and brain was carried out.

Organs were fixed in 10% formalin and dehydrated in an ascending graded ethanol

series, cleared in xylene and embedded in paraffin; 5-mm sections were obtained

with a standard microtome and were stained with hematoxylin and eosin. The

sections (three sections, ten fields by section) were examined by a pathologist

with no knowledge of the experimental groups and scored according to a

predetermined severity scoring system, adapted from Rumbeiha et al. (2000). A

normal organ was given a score of 0. Organs with weak leukocyte infiltration were

given a score of þ1. Organs with moderate leukocyte infiltration were given a

score of þ2. Those with severe leukocyte infiltration were given a score of þ3.

2.5.12. Statistical analyses

Data from SPB, oxidative stress biomarkers, NO levels, DNA damage, and Hg

and Se levels were reported as mean7standard deviation (SD). Results from

histopathological analysis were expressed as a leukocyte infiltration score.

Differences among the treatments were evaluated by Kruskal–Wallis or one-way

ANOVA, followed by Duncan’s post-hoc. Moreover, SBP values were analyzed as

longitudinal data. Thus, SBP (normally distributed data) were considered at each

time point and also in the same treated group over 3 months. For that, repeated

measures ANOVA were carried out, followed by Duncan’s post-hoc; p values

o0.05 were considered significant. Data were analyzed in Statisticas 8.0 (Statsoft

software—USA) and Graph-Pad Prism 5 (Graph-Pad Software—USA).

3. Results

Fig. 1 provides the result of the speciation analysis of mercuryin the fish samples used to prepare the diet and feed the animals.The first chromatogram represents the separation and retentiontime of three different analytical standards of Hg: inorganic Hg,MeHg and ethylmercury—EtHg. The second represents a chroma-togram of lyophilized and homogenate fish from the TapajosRiver, Brazil, showing the exclusive presence of MeHg inthese fish.

MeHg levels found in fish from the Tapajos River and the southof Brazil were, respectively, 1.9570.09 mg/g (dry weight) and0.02370.002 mg/g (dry weight). Total Se levels of 2.5770.23 mg/g and 2.3270.19 mg/g were found in fish from the Tapajos Riverand in fish from the south, respectively, showing a similarity in Selevels even among fish from different locations.

Gain and loss of body weight were monitored over the entirecourse of the study and are shown in Fig. 2. All rats gained weightgradually and no significant differences were found when allgroups are compared (p40.05).

SBP values are presented in Fig. 3. Comparing the threetreatments over time – control, MeHg-uncontaminated fish andMeHg-contaminated fish diets – SBP values remained constantuntil the 10th week. In the following weeks (11th and 12th), therewas a significant increase in SBP values in rats fed the dietcontaining MeHg-contaminated fish when compared to the groupwithout fish in the diet and the uncontaminated-fish group.Moreover, the data of SBP being longitudinal, the measures wereanalyzed in the same groups over the 3 months. The mainoutcome was related with SBP of the rats that received MeHg-contaminated fish diet. Comparing the beginning (week ‘‘0’’) andthe end of the experiment (11th and 12th weeks), SBP signifi-cantly increased in the last 2 weeks. Besides, SBP values in MeHg-contaminated fish group were different in 3rd and 4th weekscompared to the 12th week.

Oxidative stress biomarker results are shown in Table 2. Ingeneral, no differences in oxidative stress induction were found

3200016000MeHgMeHg

2400012000

160008000 Hg-i

80004000 EtHgIn

tens

ity (c

ount

s/s)

Inte

nsity

(cou

nts/

s)

002 0 2 4 6 8 100 4 6 8 10

Time (min)Time (min)

Fig. 1. Mercury (Hg) speciation chromatograms. In A), separation of Hg analytical standards: inorganic Hg (Hg-i), methylmercury (MeHg) and ethylmercury (EtHg). In B)

fish used to prepare the diet (from the Tapajos River, Para, Brazil).

Fig. 2. Body weight gain of rats treated for twelve weeks with diets rich in fish

either contaminated or uncontaminated with methylmercury (MeHg). Values are

expressed as mean7SD. No significant differences were found (p40.05).

D. Grotto et al. / Environmental Research 111 (2011) 1074–1082 1077

among the three groups. GSH levels, GSH-Px, CAT, SOD and ALA-Dactivities were statistically similar when the MeHg-contaminatedfish group was compared to the group without fish and theuncontaminated-fish group. Likewise, PC did not show differencesin levels when all groups were compared. On the other hand,MDA levels in the plasma of rats receiving a contaminated-fishdiet were higher when compared to the other two groups(po0.05), indicating an increase in lipid peroxidation.

Since SBP increased at the end of the study, we also chose toevaluate the levels of plasmatic nitric oxide (NO). Table 2 showsNO levels in the three groups. In rats fed a diet containing MeHg-contaminated fish, NO levels did not differ from those of rats feduncontaminated fish or fed a diet without fish. Moreover, NOlevels were not correlated with an increase in SBP.

Comet assay results, representing DNA% in the tail, are shownin Fig. 4. There was no difference between the non-fish group andthe group receiving uncontaminated fish. In contrast, rats fedMeHg-contaminated fish demonstrated a significant increase inDNA migration when compared to the other two groups, suggest-ing a genotoxic effect resulting from MeHg dietary exposure.

Histopathological analyses are presented in Table 3. Therewere no atypical histological findings for liver, kidney, heart orbrain in either the control group or the uncontaminated fishgroup. On the other hand, rats fed a diet of MeHg-contaminatedfish displayed weak but significant leukocyte infiltration in theheart and brain in comparison with the other two groups.

Total Hg levels in blood, liver, kidney, heart and brain arepresented in Table 4. Rats fed a MeHg-contaminated fish dietshowed a significantly higher Hg concentration than the othergroups. Rats fed an uncontaminated fish diet also showed anincrease in Hg levels in blood, liver and kidney compared to thecontrol. However, these Hg levels were much lower than the Hglevels found in rats exposed to contaminated fish.

Total Se levels in blood, liver, kidney, heart and brain arepresented in Table 5. A significant increase in Se levels was foundin both groups that were fed fish compared to the group that wasnot fed fish. This increase in Se was proportional in both theMeHg-contaminated and uncontaminated fish diet and the simi-larity is due to Se levels being alike in fish from both the northand south of Brazil as shown above.

4. Discussion

There are a large number of papers evaluating the toxic effectsof MeHg exposure in fish-eating communities, but the results areconflicting (Cordier et al., 2002; Dorea et al., 2003; Lebel et al.,1998; Myers et al., 2003). It has been suggested that dietarymodulation of MeHg toxicity may occur through interactionbetween nutrients and MeHg (Chapman and Chan, 2000) or dueto the chemical form of MeHg found in fish (Harris et al., 2003).Strain et al. (2004), in a study about nutrition and neurodevelop-ment with children from Seychelles, examined fish nutrients suchas docosohexaenoic acid, Se, iodine, vitamins B6 and B12, choline,zinc and copper. The present study thus sheds new light on theeffects of a diet rich in fish contaminated with MeHg in ratsmimicking the diet of riparians living in the Amazon region.

Kehrig et al. (2008) observed Hg levels of between 0.087 and1.43 mg/g in carnivorous fish species from the Tapajos River. Thisvalue is in agreement with Hg levels found in the fish used toprepare our diet. Moreover, we determined that almost 100% ofHg in fish was in the form of MeHg. It is known that fish are asource of Se, but Se concentrations in fish can vary from oneregion to another (Combs, 2001). We found comparable Secontents in fish from different rivers in Brazil.

There was no difference in animal body weight among thegroups. It showed the animals’ acceptance of the fish-rich diet,with no growth-related effects.

There were increases in SBP at the end of the study in ratstreated with contaminated fish. In agreement with our presentfinding, our team had previously observed an increase in SBP inrats treated subchronically with low levels of CH3HgCl in aqueoussolution (Grotto et al., 2009b). Among the toxic effects of MeHg,cardiovascular dysfunctions have received increased attention inthe recent years (Guallar et al., 2002; Uchino et al., 1995).

Fig. 3. Systolic blood pressure (SBP) of rats treated for 12 weeks with diets rich in fish contaminated or uncontaminated with methylmercury (MeHg). Values are

expressed as mean7SD. *Significantly different when compared with control (po0.05).

Table 2Biomarkers of oxidative stress: glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), aminolevulinate

dehydratase (ALA-D), proteins carbonyls (PC) in blood or plasma of rats after MeHg exposure through diet and nitric oxide (NO) levels in plasma. The animals (male Wistar

rats; 8/group) were treated with commercial diet (Nuvital Nutrientes S/As) in control group; diet containing MeHg-uncontaminated fishes and diet containing MeHg-

contaminated fishes during a period of 100 days. Results are expressed as mean7SD.

Groups GSH1 GSH-Px2 CAT3 SOD4 ALA-D5 PC6 MDA7 NO8

Control 1.170.3a 3878a 294731a 2.370.4a 1974a 1.670.3a 1873a 43 715a

MeHg-uncontaminated fish 1.070.2a 3576a 283724a 2.270.2a 1873a 1.370.6a 2274a 40713a

MeHg-contaminated fish 0.970.2a 4076a 298740a 2.170.3a 2074a 1.670.4a 2674b 35715a

a,bMeans within the same column with the same letter do not differ statistically (p40.05); means with different letters are statistically different (po0.05).

1 mmol/mL blood.2 nmol NADPH/min/g Hb.3 k/g Hb.4 USOD/mg Hb.5 (UI).6 nmol/mg protein.7 mM.8 mmol/L; Hb—hemoglobin.

Fig. 4. Induction of DNA migration, represented by percentage of DNA in tail, in

peripheral blood of rats after exposure to a diet containing uncontaminated fish or

fish contaminated with MeHg. Rats were treated over 100 days; a, b Means with

the same letter do not differ statistically (p40.05); means with different letters

are statistically different (po0.05).

D. Grotto et al. / Environmental Research 111 (2011) 1074–10821078

In an epidemiological study with 251 riparians living alongTapajos River and exposed to MeHg through fish consumption,relatively low blood pressure was observed and just 8% of thesubjects presented hypertension. However a significant dose–effect relation between Hg exposure and blood pressure was

found (Fillion et al., 2006). Fillion’s results support our findingsconcerning blood pressure and MeHg exposure through fishconsumption since the SBP level of rats exposed to contaminatedfish increased at the end of the treatment. These results alsosuggest an association between an increase in SBP and MeHgexposure time.

Several in vivo and vitro studies with methylmercury (dis-solved salt in aqueous medium) show the oxidative damagecaused by this compound. Mercury and its compounds have agreat affinity for –SH groups, attaching to thiol-containing mole-cules such as GSH (Clarkson, 1997) or molecules involved inantioxidant cellular defense (Chen et al., 2005; Perottoni et al.,2004). Indeed in the present study GSH levels were similar in allthree groups of rats. This suggests that the form in which MeHg ispresent or linked to fish would not be as toxic as MeHg in anaqueous solution. Along the same lines, some antioxidantenzymes – GSH-Px, CAT and SOD – were analyzed and no effectsfrom their activities were observed when comparing rats fed withMeHg-contaminated fish and the other groups.

ALA-D is a thiol-containing enzyme and its inhibition hasproved to be a useful index of oxidative stress (da Silva et al.,2007; Perottoni et al., 2004). In the present study, rats fed a dietcontaining MeHg-contaminated fish did not present any altera-tion in ALA-D activity when compared to other groups.

Protein carbonyl is an important biomarker for evaluating thedamage induced by reactive oxygen species (ROS) in proteins(Stadtman and Levine, 2003). As with ALA-D activity, no proteinoxidative damage was induced by the diet rich in conta-minated fish.

D. Grotto et al. / Environmental Research 111 (2011) 1074–1082 1079

These findings are in disagreement with studies exposing animalsto MeHg aqueous solutions (Farina et al., 2004; Valentini et al., 2010)and suggest to us that the form of MeHg present in fish or some fishcomponents would reduce the effects expected from MeHgexposure.

Harris et al. (2003) investigated the chemical identity of Hg infish tissues through X-ray absorption spectroscopy. The authorsobserved that the spectrum of fish tissues closely resembled thespectrum of MeHg–cysteine. They also showed that MeHg–cysteine is much less toxic than CH3HgCl. Day-old zebrafish

Table 3Histopathology of liver, kidney, heart and brain of rats (male Wistar rats; 8/group)

fed with commercial diet (Nuvital Nutrientes S/As) in control group; diet

containing MeHg-uncontaminated fishes and diet containing MeHg-contaminated

fishes during a period of 100 days. Results are expressed by number of rats in each

leukocyte infiltration score (score 0¼normal organ; scoreþ1¼brand leukocyte

infiltration; scoreþ2¼moderate leukocyte infiltration; scoreþ3¼severe leuko-

cyte infiltration.

Groups Number of rats per score

0 þ1 þ2 þ3

Control group

Livera 8

Kidneya 8

Hearta 8

Braina 8

MeHg-uncontaminated fish

Livera 8

Kidneya 7 1

Hearta 8

Braina 8

MeHg-contaminated fish

Livera 8

Kidneya 7 1

Heartb 5 3

Brainb 5 3

a,bMeans with the same letter do not differ statistically (p40.05); means with

different letters are statistically different (po0.05).

Table 4Determination of total mercury (Hg) in blood, liver, kidney, heart and brain using Induct

8/group) were treated with commercial diet (Nuvital Nutrientes S/As) in contr

MeHg-contaminated fishes for a period of 100 days. Results are expressed as mean7S

Groups Blood1 Liver2

Control 473a 1575a

MeHg-uncontaminated fish 43710b 2875b

MeHg-contaminated fish 1310740c 870730c

a,b,cMeans within the same column with the same letter do not differ statistically (p4

1 mg/L.2 ng/g.

Table 5Determination of total selenium (Se) in blood, liver, kidney, heart and brain using an Indu

8/group) were treated with commercial diet (Nuvital Nutrientes S/As) in control group; d

fishes for a period of 100 days. Results are expressed as mean7SD.

Groups Blood1 Liver2

Control 0.670.1a 0.970.1a

MeHg-uncontaminated fish 1.270.4b 1.870.2b

MeHg-contaminated fish 1.470.2b 1.970.2b

a,bMeans within the same column with the same letter do not differ statistically (p40

1 mg/L.2 mg/g.

larvae tolerated concentrations of MeHg–cysteine 20 times higherthan those of CH3HgCl (George et al., 2008; Harris et al., 2003).

Selenium is an important nutrient present in fish (Lima et al.,2005) and is known to counteract MeHg toxicity effects (Ralstonand Raymond, 2010; Grotto et al., 2009a; Su et al., 2008; Yonedaand Suzuki, 1997a, 1997b). Among this essential element’s otherimportant biological and biochemical functions it acts as anantioxidant since GSH-Px is dependent on Se for its normaloperation (Hamilton, 2004). Thus, Se in fish could be counter-acting oxidative damage from MeHg exposure (Peterson et al.,2009).

Fish is also a source of vitamin E (Afonso et al., 2008; Seriniet al., 2010). Beyrouty and Chan (2006) observed improvement inthe survivability of rat pups exposed to a high concentration ofMeHg during gestation when Se and vitamin E were co-adminis-tered in pregnancy.

In two studies in 2008 and 2010, diets containing very lowlevels of fish were administered to mice (Bourdineaud et al., 2008,2011). In the study of 2008, diets were prepared with differentfish concentrations: 0, 0.1, 1.0 and 7.5% of the total diet, repre-senting 0, 5, 62 and 520 ng MeHg/g fish, respectively, andmimicking the diet of Wayana Amerindians. Mice were fed for 1month. The authors evaluated gene expression for mitochondrialmetabolism, oxidative stress, the detoxification process andapoptosis. They observed variations in SOD2 (mitochondrial)expression in hippocampus and kidneys in 520 ng MeHg/g fish,in liver in 62 and 520 ng MeHg/g fish, and found no difference inmuscle when compared to the control. In SOD3 (extracellular)expression, the authors observed variations in hippocampus andkidneys at 62 ng MeHg/g fish, in liver at 62 and 520 ng MeHg/g fish,and in muscle at 520 ng MeHg/g fish (Bourdineaud et al., 2008). Inthe other study in 2010, Bourdineaud et al. (2011) fed mice a dietcontaining fish contaminated with MeHg (1.1570.15, 2.370.1and 35.7570.15 ng Hg/g food pellets) and mimicking the fishconsumption of Western populations (1.25% of the total diet).The authors observed no variations in SOD expression whencomparing the diet with contaminated fish and a control group.However, altered behavior and an increased anxiety level wereobserved in fish-exposed animals (Bourdineaud et al., 2011). In

ively Coupled Plasma Mass Spectrometer (ICP-MS). The animals (male Wistar rats;

ol group; diet containing MeHg-uncontaminated fishes and diet containing

D.

Kidney Heart2 Brain2

1075a 674a 471a

173743b 1375a 572a

8907 47c 9073b 10072b

0.05); means with different letters are statistically different (po0.05).

ctively Coupled Plasma Mass Spectrometer (ICP-MS). The animals (male Wistar rats;

iet containing MeHg-uncontaminated fishes and diet containing MeHg-contaminated

Kidney2 Heart2 Brain2

1.570.2a 0.3270.04a 0.1470.01a

2.270.3b 0.6770.12b 0.2370.02b

2.270.4b 0.6870.12b 0.2470.04b

.05); means with different letters are statistically different (po0.05).

D. Grotto et al. / Environmental Research 111 (2011) 1074–10821080

the present study, no change in SOD or other antioxidant enzymesactivities was found.

MDA is one of the best known secondary products of lipidperoxidation induced by ROS, and can damage vascular endothe-lium and promote aggregation, inflammatory cell adhesion andvasoconstriction (Esterbauer et al., 1991; Shaw et al., 2005).The only alteration observed in oxidative stress biomarkersinvolved lipoperoxides in plasma, represented by MDA levels.Our results show that animals fed contaminated fish had higherMDA levels than those on a diet without uncontaminated fish.

Different studies have observed increased MDA levels in ratsexposed to aqueous solutions containing 100 mg/kg/day ofCH3HgCl (Grotto et al., 2009b) and 2 mg/kg CH3HgCl (Farinaet al., 2004). More interestingly, rats fed with Chinese ricecontaminated with Hg had increased MDA levels. However,Hg was present in this rice sample as inorganic mercury (Ji andLiu, 2007).

In addition. our data show a positive correlation between risesin SBP and MDA levels (r¼0.66), suggesting that MeHg in fishcould be producing ROS which would attack the vascularendothelium and promote an increase in SBP after long-termexposure.

Endothelial NO plays an important role as a regulator of thecardiovascular system and it influences vascular homeostasisthrough basal vasodilator tone maintenance, platelet aggregation,reduction of leukocyte adhesion to the endothelium and modula-tion of smooth muscle proliferation (Yetik-Anacak and Catravas,2006). Thus, modifications in NO production could be related tothe hypertension induced by MeHg-contaminated fish. However,we did not observe differences in NO levels among rats fed withMeHg-contaminated fish, rats fed with uncontaminated fish andthe control group so a rise in SBP was not associated with NOlevels. Production of NO in cultured human umbilical vascularendothelial cells was shown to be inhibited by CH3HgCl(Kishimoto et al., 1995) and NO levels also decreased in ratstreated with a solution containing CH3HgCl (Grotto et al., 2009b).

The comet assay in peripheral blood cells is a practical tool fordetecting genotoxic effects. The % of DNA in the tail was significantlyhigher in rats fed a diet containing MeHg-contaminated fish. Thus,even in fish that consumed nutrients that counteract exposure toMeHg and MeHg present in fish in a less toxic form (MeHg–cysteine), the diet containing fish contaminated with MeHg inducedgenotoxic effects. Four molecular mechanisms related to the geno-toxicity of Hg compounds are presented in a recent review byCrespo-Lopez et al. (2009). According to these authors: (1) Hg maygenerate ROS, which can react directly with DNA or, indirectly,induce changes in proteins of microtubules and DNA repairenzymes; (2) Hg may act directly on DNA, forming Hg species–DNA adducts; (3) Hg may also affect DNA repair mechanisms and(4) Hg may act on microtubules, avoiding mitotic spindle formationand chromosome segregation.

An association between hair Hg levels and the impairment oflymphocyte proliferation measured as a mitotic index (MI) wasdescribed in a riparian Amazon population (Amorim et al., 2000).In vitro and in vivo studies with MeHg aqueous solutions have alsodemonstrated, respectively, an increase in the mitotic indexand DNA damage after Hg exposure (Crespo-Lopez et al., 2009;Grotto et al., 2011).

A weak but significant leukocyte infiltration was found in heartand brain of rats fed a MeHg-contaminated fish diet. In a comparisonof these findings and our group’s previous findings in rats treatedwith a solution containing MeHg (Grotto et al., 2011) leukocyte heartand brain infiltration in the MeHg-contaminated fish group wasslight and much lower than leukocyte infiltration in rats treated withCH3HgCl in aqueous solution. However, this slight result is significantand could be related to the increase in SBP.

The evaluation of total Hg levels in blood, liver, kidney, heartand brain showed that rats receiving a diet containing fishcontaminated with MeHg had a significant increase in Hg levelsin all tissues analyzed when compared to rats on a diet withoutfish or with uncontaminated fish. Rats that were fed uncontami-nated fish showed a slight increase in Hg levels in blood, liver andkidney, since the MeHg concentration in fish from the southregion (Parana River) was much lower than that of fish from theTapajos River (Amazon area).

Since Se levels in fish from the two areas were very similar, Selevels in blood, liver, kidney, heart and brain of rats receivingeither contaminated or uncontaminated fish were comparable.

Several groups of researchers propose different mechanisms forthe protective effects of Se against Hg toxicity. One mechanism is theapparent formation in the bloodstream of a Hg–Se–Sel P complexbetween Hg and Selenoprotein P (Yoneda and Suzuki, 1997a, 1997b).Additionally, Ralston and Raymond (2010) published a manuscriptreviewing the main chemical forms of Se, such as selenomethionineand selenocysteine, besides refreshing the main physiological func-tions of this element. Thus the Se protection could be considered inour study, since rats on a diet rich in contaminated fish did notpresent changes in oxidative stress biomarkers and NO levels, andhypertension was observed only after the 11th week of the treat-ment. On the other hand Se did not modify Hg distribution amongthe tissues.

5. Conclusion

A number of papers demonstrate several effects after MeHgexposure in experimental models. However, in most of themanimals were exposed to aqueous solutions containing MeHg,which does not by any means represent exposure conditions infish-eating communities. Thus, for the first time an experimentalstudy with rats has been performed to evaluate the effects of adiet rich in fish from the Amazon region and contaminated withMeHg in an approximation of the exposure conditions of fish-eating Amazon communities. Minimal effects were observedcompared to those observed in rats treated with aqueous solu-tions containing MeHg. There was a small increase in systolicblood pressure after the 11th week of treatment, suggestinghypertension as an effect promoted by MeHg after long-termexposure to contaminated fish. Furthermore, the same diet rich incontaminated fish induced slight lipid peroxidation and geno-toxicity, although it did not alter any of the other oxidative stressbiomarkers under investigation.

Our results support the proposition that the chemical form ofmercury found in fish (MeHg–cysteine) could be less toxic thanMeHgCl aqueous solutions frequently and incorrectly used inexperimental models or other fish components such as seleniumand vitamin E could be minimizing the effects of this MeHg–cysteine exposure. Finally, our findings have many implications,mainly for populations exposed to MeHg through fish consump-tion such as that of the Amazon basin, and can be used as areference for further epidemiological studies in this area.

Acknowledgments

The authors would like to acknowledge the financial support ofthe S~ao Paulo State Foundation for Scientific Research (FAPESP–projects 2007/05221-4; 2007/04538-4; 2009/11102-3; 2011/07416-2; 2011/07498-9) and thank the Brazilian National Council forScientific and Technological Development (CNPq–project 473418/2006-1) and the Foundation for the Coordination of Improvement ofHigher Education Personnel (CAPES) for fellowships.

D. Grotto et al. / Environmental Research 111 (2011) 1074–1082 1081

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