1
Sub-chronic exposure to MeHg at low levels is associated with oxidative
stress in rats: protective effects of fish oil
Denise Grotto1; Juliana Vicentini2, José Pedro Friedmann Angeli3, Elder Francisco
Latorraca4, Patrícia Alves Pontes Monteiro4, Gustavo Rafael Mazzaron Barcelos1;
Sabrina Somacal5; Tatiana Emanuelli5; Fernando Barbosa Jr1*.
1. Departamento de Análises Clínicas, Toxicológicas e Bromatológicas. Faculdade
de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo.
2. Centro de Ciências da Saúde, Departamento de Análises Clínicas e
Toxicológicas, Universidade Federal de Santa Maria.
3. Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo.
4. Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto –
Universidade de São Paulo – Avenida Bandeirantes, 3900, 14049-900, Ribeirão
Preto, SP, Brasil
5. Centro de Ciências Rurais, Departamento de Tecnologia e Ciência dos
Alimentos, Universidade Federal de Santa Maria.
*Corresponding author:
[email protected]
Tel: +55 16 36024701
Fax: +55 16 36024725
Avenida do Café s/n, Universidade de São Paulo, Ribeirão Preto,
CEP.: 14040-903, RibeirãoPreto, São Paulo, Brasil.
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Abstract
The present study evaluates in rats the oxidative damage associated with
sub-chronic exposure to MeHg at low levels and a possible protective effect of fish
oil. Rats receiving MeHg or MeHg plus fish oil had glutathione peroxidase and
catalase activities dropped when compared to the control groups without
methylmercury exposure. On the other hand, fish oil co-administration showed a
significant DNA protective effect. No differences in Hg distribution in rat tissues
were observed after MeHg administration with or without fish oil co-adminstration.
However, histopathological analysis showed a significant leukocyte infiltration in rat
tissues (brain and heart) after MeHg exposure that is minimized in rats also treated
with fish oil. Finally, this study demonstrated an oxidative damage of MeHg
exposure even at low levels and a protective effect of fish oil administration.
However, this protection seems not to be related to the re-establishment of
antioxidant enzymes activity or Hg re-distribution in rat tissues.
Key words: methylmercury, fish oil, oxidative stress, DNA damage, protective
effects.
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1. Introduction
Several experimental and epidemiological studies have demonstrated that
exposure to methylmercury (MeHg) at low and moderate levels has been
associated with neurotoxic effects on motor functions (Dolbec et al., 2000),
damage in immune system (Moszczynski et al., 1998), kidneys (Rutowski et al.,
1998), cardiovascular system (Virtanen et al., 2007) and with genetic damage
(Ben-Ozer et al., 2000; Grotto et al., 2009a). On the other hand, some
epidemiological studies failed to identify such associations (Myers et al., 2003). A
possible reason for this disagreement is the presence of several nutritional factors
that may modify mercury toxicity (Chapman and Chan, 2000).
The process responsible for triggering the MeHg toxicity is not well
established, however it is known that MeHg, has a high affinity to thiol-containing
molecules such as reduced glutathione (GSH), cysteine, N-acetylcysteine,
metallothionein, and albumin (Clarkson, 1997), which are also the basis for MeHg
transport, binding, distribution, metabolism, and detoxification in biological systems
(Zalups, 2000). Moreover, Hg is related to changes in the activities of antioxidant
enzymes, such as superoxide dismutase (SOD) (Ariza et al., 1998) and catalase
(CAT) (Hussain, et al., 1999), besides inducing reactive oxygen species (ROS)
production, especially of H2O2 e O2•- (Lund et al., 1991), promoting oxidative
stress and lipid peroxidation.
Human exposure to MeHg occurs primarily through the consumption of fish
or seafood rich in methylmercury (Dórea et al., 2006). Fish and other seafood are
4
also source of important nutrients such as very long chain omega-3 fatty acids
polyunsaturated
fatty
acids
(Stone,
1996,
Calder
&
Yaqoob,
2009).
Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and other n-3
polyunsaturated fatty acids (PUFAS) found in fish are critical to neural
development (Marszalek and Lodish, 2005), and they are known to have anticancer (Li, 2003), anti-inflammatory (Simophoulos, 2002), and cardiovascularprotective (Carroll and Roth, 2002) effects.
Although the fundamental mechanisms of MeHg toxicity are not totally
known, there is some evidence that oxidative stress is implicated (Sarafian, 1999).
Therefore, the aims of this study were verify toxic or protective effects of MeHg and
fish oil on DNA, besides evaluating oxidative stress through GSH-Px, CAT and
SOD activities. Moreover, histopathological analysis of liver, kidney, heart and
brain were carried out.
2. Materials and Methods
2.1. Chemicals
Methylmercury
chloride,
glycine,
epinephrine,
potassium
phosphate
monobasic, potassium phosphate dibasic, hydrogen peroxide, nicotinamide
adenine dinucleotide phosphate-oxidase (NADPH), reduced glutathione (GSH),
reductase glutathione (GSHR), sodium azide, ethidium bromide, agarose, triton®
X-100 and tetramethylammonium (TMAH) 25% (w/v) in water were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Formalin, ethanol, xylene, paraffin and all the
5
other reagents used were of analytical grade. Aqueous solutions were prepared in
Milli-Q water (Millipore, Bedford, MA, USA). Fish oil capsules were purchased from
Sundown. Agarose low melting point (LMP) at 0.5% (w/v) and agarose normal
melting point (NMP) at 1.5% (w/v) were dissolved in Ca2+ and Mg2+ free phosphate
buffered saline. A clean laboratory and laminar-flow hood capable of producing
class 100 were used for preparing solutions. All the others operations were
performed in a clean room class 1000.
2.2. Animals
Male Wistar rats weighting 200 – 220 g from our Central Bioteruim
(University of São Paulo – Ribeirão Preto, Brazil) were used. The animals were
kept under a 12h light/dark cycles, maintained in an air-conditioned room at 2225°C, with free access to food and water. Animals were used according to the
guidelines of the Committee on Care and Use of Experimental Animal Resources,
University of São Paulo, Brazil (Approved protocol number: 07.1.1185.53.3).
2.3. Dose selection and experimental groups
The exposure dose of MeHg (140 µg/Kg/day) was chosen based on a recent
study of Passos et al., 2008, in which the daily ingestion of MeHg from riparian
Amazon populations that are environmentally exposed to Hg by fish ingestion was
estimated. Selected doses for fish oil were (EPA 15.50 μg/kg/day and DHA 10.30
μg/kg/day).
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Animals were assigned in four groups of eight rats in each group :(Group I)
received water by gavage; (Group II) received fish oil by gavage (Group III)
received MeHg by gavage; (Group IV) received MeHg plus fish oil by gavage. The
total treatment time was 100 days.
After the treatment, animals were euthanized by anesthesia overdose with
ketamine and xylazine and they had their blood and organs collected. Part of the
total blood was used to the comet assay and to the measure of hemoglobin after
the collection; the remaining blood was stored in heparinized microcentrifuge tubes
at -80ºC until analysis. Organs (liver, right kidney, heart and brain were stored in
10% formalin.
2.4. Comet Assay
The alkaline version of the comet assay was performed according to
guidelines proposed by Singh et al., 1988, with a slight modification (Silva et al.,
2000). After the treatment, 20 µL of heparinized periphery blood were mixed with
120 µL 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 a microscope coverslip and refrigerated for 5
min to gel. This was followed by immersion in ice-cold alkaline lysing solution
(2.5M NaCl, 10 mM Tris, 100 mM EDTA, 10% DMSO, 1% Triton X-100, final pH
10.0) for at least 1 h.
The slides were then incubated for 20 min in ice-cold
electrophoresis solution (0.3 M NaOH, 1 mM EDTA, pH>13), followed by
electrophoresis at 25 V:300 mA (1.25 V/cm) for 25 min (22). After electrophoresis,
the slides were then neutralized (Tris 0.4 M, pH 7.5) and stained with ethidium
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bromide (20 µg/mL). One hundred cells per animal (two slides of 50 cells each)
were analyzed at 400 X using a fluorescence microscope (Nikon) with a blue (488
nm) excitation filter and yellow (515 nm) emission (barrier) filter. Quantification of
DNA breakage was achieved by Komet 4.0 software.
2.5. GSH-Px activity
Antioxidant
enzyme
glutathione
peroxidase
(GSH-Px)
activity
was
determined using GSH, GSHR, NADPH, sodium azide and H2O2. The method is
based on the oxidation of NADPH at 25°C in the presence of the reagents above
and diluted blood, and the GSH-Px activity is monitored in absorbance at 340 nm,
according to Paglia and Valentine, 1967. Dates were expressed in nmol
NADPH/min/ml erythrocytes.
2.6. CAT activity
Catalase activity was assayed by measuring the rate of decrease in
hydrogen peroxide (H2O2) 10 mM absorbance in a spectrophotometer at 240 nm
(Aebi, 1984). Total blood is diluted in PBS 50 mM; an aliquot of 20 µL is added to
1,910 µL of PBS and 70 µL of H2O2. The CAT activity is monitored by the
consumption of H2O2. Dates were expressed in κ/gHb.
Hemoglobin levels (Hb) from the rats were analyzed in blood through routine
laboratorial methods to the correction of the enzyme values.
2.7. SOD activity
8
Superoxide activity was assayed by quantifying the inhibition of superoxidedependent epinephrine self-oxidation by spectrophotometer at 480 nm (McCord
and Fridovich, 1969). Dates were expressed in USOD/mgHb.
A ratio between SOD and CAT activities (SOD/CAT) were submitted to
statistical analyses to better comprehend MeHg effects on these two antioxidant
enzymes.
2.8. Histopathological Analysis
It was carried out histopathological analysis of liver, right kidney, heart and
brain. Organs were fixed in 10% formalin and they were dehydrated in an
ascending graded series of ethanol, cleared in xylene and embedded in paraffin.
Sections of 5 µm were obtained with a standard microtome and were stained with
hematoxylin and eosin. The sections were examined by a pathologist without
knowledge of the experimental groups, according to a score in relation to. Normal
organ was given a score of 0. Organs with brand leukocitary infiltration were given
a score of +1. Organs with moderate leukocitary infiltration were given a score of
+2. Those with severe leukocitary infiltration were given a score of +3.
2.9. Determination of Mercury in tissues
Determination of Hg in kidney, liver, heart and brain was performed by using
a inductively coupled plasma mass spectrometer (ICP-MS) (ELAN DRCII,
PerkinElmer, SCIEX, Norwalk, CT, USA). Sample introduction system was
composed by quartz cyclonic spray chamber and a Meinhard® nebulizer
connected by Tygon® tubes to the ICP-MS’s peristaltic pump (set at 20 rpm). For
9
this analysis we have adopted the method proposed by Batista el at., 2009. Briefly,
10-50 mg of each tissue was weight and transferred to a conical tubes (15 mL).
Then, 1mL of 50% (v/v) TMAH solution was added to the samples, incubated at
room temperature for 12 h and the volume made up to 10mL with a solution
containing 0.5% (v/v) HNO3, 0.01% (v/v) Triton® X-100. Analytical calibration
standards were prepared daily over the range of 0–20 µg L−1 in a diluent containing
5% (v/v) TMAH, 0.5% (v/v) HNO3, 0.01% (v/v) Triton® X-100.
In order to verify the accuracy of data, certified reference materials DOLT-3,
DORM-3 and TORT-2 from National Research Council Canada (NRCC) were
analyzed. Found values were in good agreement with the certified values.
2.10. Statistical Analysis
Data from DNA damage and oxidative stress were reported as mean 
standard deviation (SD). Results from histopathological analysis were expressed
as a leukocitary infiltration score. Differences among the treatments were
evaluated by Kruskal-Wallis test, followed by Duncan pos hoc. Spearman
regression analysis was used. P values equal to or less than 0.05 were considered
significant. Data were analyzed with Statistica 6.0 software system (Statsoft Inc.,
2001).
3. Results
10
The results of comet assay are showed in Figure 1. Fish oil group (7.02 
1.55 µm) was not genotoxic, since the mean had not significant difference in tail
length compared to the control group (5.85  1.32 µm). On the other hand, MeHg
exposed rats (Group III) had 3x fold increased in tail length (18.53  4.8 µm)
compared to control group (p<0.001). Fish oil co-administration (13.6  4.1 µm)
showed increased when compared to control (p<0.05), however this group also
showed a significant diminish in DNA damage when compared to MeHg group
(p<0.05), about 30% of protection.
Regarding the antioxidant enzymes results, they are showed in Table 1. The
group treated only with fish oil did not present changes in its antioxidant capacity
when compared to control group. On the other hand, group treated with MeHg
presented a drop in GSH-Px and CAT activities but did not modify SOD activity
when compared to control and fish oil treatment. Co-administration of fish oil with
MeHg did not provide protective effects against MeHg-induced enzymes inhibition,
since GSH-Px activity in MeHg + fish oil did not differ from MeHg group. CAT
activity in MeHg + fish oil group was also not different from MeHg group. We have
observed a significant increase in the SOD/CAT ratio in the group of rats treated
with MeHg compared to control and fish oil, but SOD/CAT ratio in MeHg + fish oil
group was not different or MeHg group or control group.
Histopathological analyses are presented in Table 2 and the results are
expressed in percentage of rats that presenting different scores of leukocitary
infiltration. Figure 2 and Figure 3 represented some examples of the analyses in
brain and heart. Fish oil group was not statistically different from control group
11
(related to what??). Rats that received MeHg presented significant leukocyte
infiltration in liver, kidney, heart and brain compared to control and fish oil groups
(p<0.01). Fish oil showed to be anti-inflammatory since leukocyte infiltration
significantly dropped in all tissues when it was co-administrated with MeHg.
Results of Hg distribution in liver, kidney, heart and brain of control group
and fish oil group were, respectively: 0.018  0.017 and 0.026  0.024 µg/g; 0.004
 0.001 and 0.004  0.002 µg/g; 0.007  0.004 and 0.004  0.001 µg/g; 0.002 
0.001 and 0.004  0.001 µg/g, showing no statically difference. Moreover, data
about total Hg distribution in MeHg treated group and MeHg associated with fish oil
are presented in Figure 4. There was a significantly difference among MeHg
treatment, MeHg+fish oil and control (p<0.001), however there was no difference
between MeHg group and MeHg+fish oil. It demonstrates that fish oil did not
modify Hg distribution.
4. Discussion
Since intoxication from contaminated fish consumption in Minamata, Japan,
in the late 1950s (Weiss, 1996), MeHg has been a public health problem.
Although many populations have been exposed to similar doses of MeHg
through the consumption of fish and seafood, some of them have experienced
subsequent adverse effects (Lebel et al., 1998; Salonen et al., 2000), whereas
others have not (Grandjean et al., 1995; Myers et al., 2003). This different finding
12
may be explained by differences in the diet components and/or fish nutrients that
might act as potential modifiers of MeHg toxicity.
The significant DNA damage observed after MeHg treatment is in
agreement with results obtained by other studies (Ariza et al., 1998; Grotto et al.,
2009a), which showed Hg is a reactive metal that bind to DNA, leading to
alterations in its structure, even at low levels as presented in our study. Jin et al.,
2008, in a study carried out with rats exposed to MeHg, had also demonstrated
DNA damage measuring 8-hydroxydeoxyguanosine in liver and kidneys. In a
recent and important review, Crespo-López et al., 2009, presented a scheme about
the four main mechanisms that lead to Hg genotoxicity: production of ROS, which
react direct or indirectly on DNA; direct action on DNA, forming Hg species-DNA
adducts; inhibition of the DNA repair systems, and inhibition of mitotic spindle formation
and chromosome segregation (action over the microtubules). In relation to fish oil, this
compound did not present genotoxicity and it showed to be protectors against DNA
damage. The association MeHg+ fish oil reduced DNA injury by about 35%. It is in
according to Kikugawa et al., 2003, who showed that polyunsaturated fatty acids decreased
the DNA damage caused by ADP/Fe(II) in rat liver cells.
In relation to GSH-Px activity, a Se-dependent enzyme, fish oil group did not
present significant difference to control, according to other findings (Erdogan et al.,
2004; Iraz et al., 2005). Chautan et al., 1990, observed that plasma GSH-PX
remained constant among different diets constituent from fat (among them fish oil),
suggesting that Se status of rats was not altered by the diets. Moreover, it was
found that GSH-Px had its activity significantly decreased in MeHg treated group
when compared to control group. Its decrease in GSH-Px activity could be
13
explained by a direct enzyme inhibition by MeHg, such as occurred with CAT
inhibition (Abdel-Hamid et al., 2001), or by overproduction of free radical from
MeHg (Huang et al. 1996; Clarkson, 1997). Our results are in accord to
Vijayalakshmi and Sood, 1994, who found GSH-Px activity diminished in mice
treated with MeHg, but the treatment was 7 days. Regarding the associated
treatment, we observed that fish oil combined to MeHg did not ameliorate GSH-Px
activity, showing that fish oil did not present antioxidant effect on MeHg oxidative
induction.
CAT activity in fish oil treated animals did not show any statistical
differences compared to the control rats. It is in according to Ibrahim et al., 1999,
that found mice renal CAT activity not significantly affected by dietary fat. Contrary
to our findings, Iraz et al., 2005, found a raise in CAT activity after fish oil
administration to rats, suggesting an increase in antioxidant capacity even no
oxidative stimulus. Nevertheless we support the proposal that under physiological
conditions, antioxidant enzymes appear to be in balance, as the results presented
in GSH-Px. Contrasting, MeHg treatment decreased CAT activity when compared
to control and fish oil treatment. This result is in according with other studies with
mice (Hussain et al 1999) and rats (Grotto et al., 2009b) exposed to MeHg and
with riparians from the Brazilian Amazon exposed to MeHg (Grotto et al., 2010)
and male workers exposed to Hg from fluorescent lamp (Abdel-Hamid et al., 2001).
However when fish oil administration was associated with MeHg exposure, CAT
activity was not statistically different to MeHg treatment, showing fish oil
administration was not able to attenuate the oxidant effect induced by MeHg
exposure.
14
Respecting to SOD activity, fish oil treated rats did not present significative
difference from control, corroborating to other study with mice fat fed (Ibrahim et
al., 1999). Besides, no difference was found in MeHg group or in MeHg + fish oil
group compared to control group. Contrary to our results, Ariza el at., 1998,
observed an increase in SOD activity, but it was in cell culture exposed to Hg2+,
and Abdel-Hamid et la., 2001 found an inhibition of SOD activity in male workers
exposed to Hg from fluorescent lamp plant. These controversial results may be
explained by the difference in doses of exposure, differences in chemical form of
mercury and mainly because the group studied (rats, cells and humans).
Thus, to better understand MeHg oxidant effects, we calculate the ratio
between SOD and CAT activities, whereas SOD converts superoxide anion (O2-)
in hydrogen peroxide (H2O2) and CAT converts H2O2 in water (Halliwell and
Guteridge, 1999). We observed an enlarged SOD/CAT ratio in rats treated with
MeHg compared with the control group. High levels of O2- increase SOD activity,
since it is the main SOD allosteric activator (Halliwell and Gutteridge, 1999). CAT
activity may be inhibited by O2- (Kono and Fridovich, 1982; Shimizu et al., 1984).
An increase in SOD activity and a decrease in CAT activity may result in high
levels of H2O2, which facilitates the production of hydroxyl radical (OH•), the most
powerful oxidant molecule (Halliwell, 2006). Our findings are in agreement with Ali
et al., 1992, that observed MeHg raising O2- production. So, SOD/CAT ratio could
explain the oxidative damage caused by MeHg exposure. Fish oil was not able to
return the increased SOD/CAT ratio to control value, showing no antioxidant effect.
15
Although knowing the protector effects of fish oil (Carrol and Roth, 2002;
Simophoulos, 2002), there are many researches that present fish oil as oxidative
stress inductor (Vossen et al., 1995; Ibrahim et al., 1999; Farina et al., 2003), like
in this study, generating some discussion about it.
Polyunsaturated fatty acids are very susceptible to oxidation, leading to
increases in peroxidative tissues damage (Haliwell and Chirico, 1993). Increased
intake of fish oil is associated with increased susceptibility of membranes to
oxidation and an increased requirement for antioxidants (Saito and Nakatsugawa,
1994). Ibrahim et al., 1999, in a study with mice fed with different oils, showed prooxidant effects of high fish oil intake through the formation of lipid peroxidation
products, and suggested that dietary fats play an important role in determining
cellular susceptibility to oxidative stress.
On the other hand, Simopoulos, 2002, in a very important article, reviewed
about omega-3 fatty acids and their anti-inflammatory effects. The researcher
reported that the EPA and DHA from fish lead to a diminish in prostaglandin E2
metabolites production; a decrease in thromboxane A2 (an effective platelet
aggregator and vasoconstrictor); a reduction in leukotriene B4 formation (an
inducer of leukocyte chemotaxis and adherence), among others good evidences
(Simopoulos, 2002)
Considering the above findings, in our histopathological analyses we
observed that fish oil treatment was able to protect liver, kidney, heart and brain of
the damage induced to MeHg exposure. There was not significantly difference
between organs from rats treated with fish oil compared to control, however
significantly leukocyte infiltration was found in all tissues from rats treated with
16
MeHg (p<0.01). Augusti et al., 2008, found significantly renal tubular necrosis, but
in their study rats were exposed to HgCl2 at 5 mg/Kg, a much higher dose than the
dose used in our study.
And the most important, we observed a significantly
protection in MeHg + fish oil treatment compared to MeHg, showing that fish oil
reduce inflammatory process since the leukocyte infiltration decreased in
associated treatment.
On the other hand, we observed a significantly difference in Hg distribution
in MeHg group and MeHg+fish oil group compared to the control and fish oil
groups in liver, kidney, heart and brain. It means that fish oil reduces inflammatory
process caused by MeHg but it is not followed by the decrease in Hg distribution.
Thus, our findings have demonstrated the genotoxic effects of MeHg even at
low levels of exposure, besides oxidative effects under antioxidant enzymatic
system. Fish oil minimized MeHg genotoxic effects, but it had not effect over
MeHg-mediated oxidative stress, showing that fish oil is not protective by this way.
The mechanism by which fish oil proved to be protective was even the antiinflammatory mechanism, corroborating with Simopoulos, 2002. This fact may
explain the leukocyte infiltration absent in tissues of rats treated with fish oil
showed in our study. Moreover, fish oil treatment was able to overturn the toxic
effects induced by MeHg treatment.
Acknowledgements
The authors would like to thank the financial support of the São Paulo State
Foundation for Scientific Research (FAPESP, Brazil) and the Brazilian National
17
Council for Scientific and Technologic Development (CNPq) and the Coordination
of improvement of higher level students (CAPES) by the fellowships.
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Captions for figures
Figure 1.
Cometa
Figura 2. corte histopatologico 1
Figure 3. Corte histopatológico 2
Figura 4. Total Hg distribution in different tissues. Means with same letter
did not present statistical difference, p< 0.05.