Ecotoxicology and Environmental Safety 74 (2011) 342–349

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Ecotoxicology and Environmental Safety

0147-65

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Subchronic effects of dipyrone on the fish species Rhamdia quelen$

J.H. Pamplona a, E.T. Oba a, T.A. da Silva b, L.P. Ramos b, W.A. Ramsdorf c, M.M. Cestari c,C.A. Oliveira Ribeiro d, A.R. Zampronio a, H.C. Silva de Assis a,n

a Departamento de Farmacologia, Universidade Federal do Parana, Caixa Postal 19031, CEP 81531-970, Curitiba-PR, Brazilb Departamento de Quımica, Universidade Federal do Parana, Caixa Postal 19031, CEP 81531-970, Curitiba-PR, Brazilc Departamento de Genetica, Universidade Federal do Parana, Caixa Postal 19031, CEP 81531-970, Curitiba-PR, Brazild Departamento de Biologia Celular, Universidade Federal do Parana, Caixa Postal 19031, CEP 81531-970, Curitiba-PR, Brazil

a r t i c l e i n f o

Article history:

Received 29 January 2010

Received in revised form

31 July 2010

Accepted 19 September 2010Available online 30 October 2010

Keywords:

Dipyrone

Biomarkers

Rhamdia quelen

Fish

13/$ - see front matter & 2010 Elsevier Inc. A

016/j.ecoenv.2010.09.010

research was partially supported by the B

nd Fundac- ~ao Araucaria). All the procedures of

nce with the Guide for the Care and Use of L

on Animal Care) and the animal protocol

ity�s ethical committee for animal experiment

esponding author. Fax: +55 41 3266 2042.

ail address: helassis@ufpr.br (H.C. Silva de As

a b s t r a c t

The use of the non-steroidal anti-inflammatory drugs such as dipyrone is so widespread that this drug and

its metabolites have been detected in effluents and surface water. This study aimed to evaluate the

potential toxic effects of dipyrone on the aquatic environment, using a native fish species, Rhamdia quelen.

Fish were exposed to three concentrations of dipyrone, 0.5, 5 and 50 mg/L, in the water for 15 days, and

hematological, biochemical, genetic and morphological biomarkers were evaluated. The glutathione

S-transferase activity decreased in the highest concentration in relation to the control group. In addition,

hematocrit, red blood cells and thrombocyte counts were decreased in all three exposed groups in relation

to the control group. The comet assay showed DNA damage at the lowest concentration of dipyrone and

significant kidney damage. Those results suggest that a constant exposure of aquatic organisms to

dipyrone presents potential toxic effects.

& 2010 Elsevier Inc. All rights reserved.

1. Introduction

Studies about the effects of pharmaceuticals on the aquaticenvironment have been increasing in the last years. Potential risksassociated with the release of pharmaceuticals into the environ-ment have become an increasingly important issue for environ-mental regulators and pharmaceutical industry (Kummerer et al.,2004). Despite the fact that some pharmaceuticals have been usedin similar quantities to many pesticides and other organic micro-pollutants, they have not been subjected to the same rigoroustreatment like them to reduce their presence in the environment(Daughton and Ternes, 1999; Bound and Voulvoulis, 2004).

Pharmaceuticals and their metabolites may be introduced to theenvironment through human excretion and disposal in wastewater(Nikolaou et al., 2007). Due to an incomplete elimination inwastewater-treatment plants, residues are found in surface anddrinking waters. In Brazil, there are few studies about the presenceof pharmaceuticals in the environment, although some authorshave already reported the presence of some of the pollutantsmentioned earlier. Stumpf et al. (1999) and Ternes et al. (1999), for

ll rights reserved.

razilian government (CNPq,

this study were carried out in

aboratory Animals (Canadian

approved by Parana Federal

ation.

sis).

example, have found estrogens, lipid regulators and anti-inflam-matory drugs as pollutants in the water.

The non-steroidal anti-inflammatory drugs (NSAIDs) are amongthe most used pharmaceuticals in the world. Many of them, such asibuprofen, diclofenac, acetaminophen, salicylic acid and naproxenare often found in sewage and surface water (Gomez et al., 2007;Fent et al., 2006). Dipyrone sodium or metamizole sodium belongsto the NSAID family and is used as an analgesic, antipyretic and anti-inflammatory, albeit its anti-inflammatory efficacy has been disputed,when compared to its intense analgesic and antipyretic properties(Souza et al., 2002). Its use is controversial and has been forbidden inmany countries, such as United States, United Kingdom and Sweden,for its relation with many blood dyscrasias. Due to its strong analgesiceffect, availability as parenteral formulation and low cost, the use ofdipyrone is still on the rise in Europe and South America. Dipyrone is apro-drug, and after its oral ingestion it is rapidly hydrolyzed into itsmain metabolite, 4-methylaminoantipyrine, from which many othersare produced by enzymatic reactions (Ergun et al., 2004).

Dipyrone is one of the drugs most commonly found in hospitaleffluents (Gomez et al., 2007). The presence of its metabolites isalso reported in effluents and surface water by many authors(Wiegel et al., 2004; Feldmann et al., 2007; Gomez et al., 2008).Despite the fact that dipyrone is a widely utilized drug in Brazil andother countries in South America, there are no studies about its fatein the environment, nor about its toxic effects to organisms. Thisresearch aims at offering important data which also concernssocial health, given that dipyrone has been ranked the top-sellingtherapeutic drug in Brazil.

/ Ecotoxicology and Environmental Safety 74 (2011) 342–349 343

Many studies apply the use of biomarkers to evaluate the toxiceffects of many substances to the aquatic environment. Theglutathione S-transferases (GSTs) and catalase activities, the lipo-peroxidation, genetic damage, histology and hematology arebecoming important tools, in elucidating the effects of aquaticenvironmental contaminants.

This study aimed to evaluate the effects of dipyrone on theaquatic environment using as an animal model the fish speciesjundia or silver catfish (Rhamdia quelen), a neotropical species thatis found in the region between middle Argentina to the south ofMexico. It is a commercially relevant species for fisheries in thesouth of Brazil, due to its high-quality meat (Carneiro and Mikos,2005). This species of fish adapts well to the cold winter and has afast growth rate during the warmer months, consequently it hasbeen intensively cultured and used as a model to laboratory studies(Gomes et al., 2000).

Three concentrations of dipyrone were used for this study: 5 mg/L,the concentration closest to what is found in the environment, asreported by Gomez et al. (2007), as well as 0.5 and 50 mg/L,concentrations 10 times lower and higher than 5 mg/L, respectively.A selected group of biomarkers were used: activities of glutathioneS-transferase (GST) and catalase (CAT), lipoperoxidation (LPO),hematological parameters, the comet assay and histopathology.

2. Material and methods

2.1. Animals

R. quelen fish were purchased from Panama Pisciculture, Santa Catarina, Brazil.

Animals about 70 days old were separated into four groups of 14 individuals each in

aquaria of 100 L, kept at a controlled temperature of 28 1C and fed ad libitum with

commercial food. The project was approved by the Animal Experimentation Ethics

Committee of Federal University of Parana, under number 317.

2.2. Chemical analysis

Before starting the experiments, a kinetic study of the dipyrone degradation was

carried out in order to estimate the half-life time of this compound in water.

Dipyrone was purchased from Sigma Aldrichs. Experiments were performed in the

presence and the absence of the fish in the aquaria, with 50 mg/mL of dipyrone.

Water samples were collected at 0, 6, 12, 24, 36 and 48 h after the addition in

the aquaria and frozen at �70 1C for posterior analysis. In another experiment,

dipyrone was added to the water at 0, 24 and 48 h after the beginning of the kinetics.

The water analyses were carried out by high performance liquid chromatography

(HPLC), consisting of a Shimadzu LC10AD, an UV detector SPD-10A having a

Rheodyne injector with a 20 mL sample loop. Chromatographic separation was

achieved on a 5 mm, 150�4.6 mm, C18 column. The mobile phase was methanol

100%, the flow rate was 1.0 mL/min and the detector wavelength was set at 265 nm.

The data system provided readout of the digitally integrated area under the peaks

and determined the retention time. The quantification was made by external

standardization, based on calibration curves constructed for the monitored

compound. All analyses were performed at room temperature (21 1C). The results

were used to estimate the time of water changing in bioassay.

2.3. Bioassay

The animals were exposed to three concentrations of dipyrone sodium: 0.5, 5

and 50 mg/L, for 15 days on semi-static assay. The 5 mg/L concentration was chosen

due to the fact that it is found in the aquatic environment. The other two

concentrations were 10 times lower and higher than this basic concentration,

0,.5 and 50 mg/L, respectively. Every day (each 24 h), one-third of the aquarium

water was replaced with the same concentration of dipyrone. Control animals were

submitted to the same water change schedule without addition of dipyrone. At the

end of the exposure, the fish were anesthetized with 2% benzocaine and killed by

medullar section. The total length of each fish was measured and blood was

collected by caudal venipuncture, taken with a syringe containing ethylenediami-

netetraacetic acid (3%) for the evaluation of hematological parameters and the

comet assay. The liver was also collected and frozen at �70 1C for biochemical

analysis.

2.4. Biomarkers analysis

2.4.1. Biochemical biomarkers

Samples of liver were homogenized (10% w/v) in cold (4 1C) phosphate buffer

(K2HPO4, 0.1 M, and KH2PO4 0.1 M; pH 6.5). Homogenates were centrifuged at

10 000� g, for 20 min at 4 1C. Aliquots of the supernatant of each sample were

collected for analysis of glutathione S-transferase (GST) and catalase (CAT) activities

and lipid peroxidation (LPO).

The GST activity was measured at 340 nm using the method described by

Keen et al. (1976). The enzyme activity was calculated in mmol of CDNB conjugate

formed/min/mg protein.

The CAT activity was measured at 240 nm based on the method described by Aebi

(1984). Enzyme activity was expressed in mmol H2O2 consumed/min/mg protein.

Analysis of LPO was carried out using the ferrous oxidation-xylenol (FOX) assay

at 570 nm (Jiang et al., 1992). LPO concentration was expressed in nmol of

hydroperoxide/mg protein.

Protein concentration was determined using Bradford’s method (1976) with

bovine serum albumin as the standard.

2.4.2. Hematological biomarkers

Red blood cell (RBC) count was taken using a Neubauer chamber, hematocrit

(Ht) by the microhamatocrit method (Hine,1992) and hemoglobin concentration

(Hb) using the cyanomethemoglobin technique (Collier, 1944). According to

Carvalho (1994), the mean corpuscular volume (MCV– mm3), mean corpuscular

hemoglobin (MCH – pg cell�1) and mean corpuscular hemoglobin concentration

(MCHC – g 100 mL�1) were determined according to the following equations:

MCV¼ ðHt=RBCÞ10; MCH¼ ðHb=RBCÞ10 and MCHC¼ ðHb=HtÞ100

2.4.3. Genetical biomarker

For the comet assay, a blood sample was diluted in fetal bovine serum, stored on

ice and in the dark for 24 h and then prepared for the comet assay (Ramsdorf et al.,

2009), which was performed as described by Singh et al. (1988) with modifications by

Ferraro et al. (2004). The slides for microscopy were prepared with the cell suspension

(10 mL) in low melting point agarose (120 mL) at 37 1C followed by incubation in lysis

solution at 4 1C for 7 days. After lysis, the slides were placed in buffer NaOH (10 N) and

EDTA (200 mM), pH413 for 20 min, to effect DNA denaturation. Electrophoresis was

carried out at 25 V and 300 mA for 25 min at 4 1C and slides were neutralized for

15 min with 0.4 M Tris, pH 7.5, fixed in ethanol 95% for 5 min and stained with

ethidium bromide (0.02 mg/mL). DNA strand breaks were scored using a Leica

epifluorescence microscope at a magnification of 400� . For each fish, 100 cells

were visually analyzed according to the method described by Collins et al. (1997).

2.4.4. Histopathological biomarkers

Posterior kidney samples were preserved in ALFAC fixative solution for 16 h

(85 mL ethanol 80%, 25 mL of formaldehyde 40%, and 5 mL of acetic acid 100% for

each 100 mL of fixative solution), dehydrated in a graded series of ethanol baths,

diaphanized in xilol, and embedded in Paraplast-Plus resin (Sigmas). The sections

(5 mm) were stained with Harris hematoxylin and eosin/floxin 1% and observed

under a Zeiss-Axiophot photomicroscope.

New nephrons (NN/mm2) were determined microscopically using an eyepiece

grade. The structures were quantified in 15 fields (sorted randomly) on each slide.

2.5. Data analysis

One-way analysis of variance (ANOVA) was used, followed by the Tukey’s post-

hoc test for multiple comparisons to analyze biochemical and hematological data,

new nephron counts and total length of fish. The data of comet assay and catalase

activity were analyzed using the Kruskal–Wallis test. All data were statistically

analyzed using GraphPad Prism v5.00.288 (GraphPad Software, Inc.). All tests were

regarded as statistically significant when po0.05.

3. Results

3.1. Chemical analysis

The important goal of the chemical analysis was to allow thevisualization of the hydrolysis profile, to guide the aquaria waterchange process and exposition of dipyrone during the bioassay. Thekinetics of dipyrone in aquaria water in the absence of fish showeda decrease of 47.3% in the first 24 h and of 56.24% in 48 h (Fig. 1).With a population of one fish weighing 30 g/L of water in theaquarium, the decrease in the concentration of dipyrone in 24 hwas of 72.94% and of 76.98% in 48 h, when an equilibrium seemed

Fig. 1. Dipyrone sodium kinetics in filtered water in the absence (A) and in the presence (B and C) of fish in the water. In (B) and (C) the population of approximately 30 g of fish

per liter of water was used. In (C), addition of the same initial concentration of dipyrone was done each 24 h with the change of one-third of the water. Points show the

mean7SEM of samples collected from different aquaria at different time points.

/ Ecotoxicology and Environmental Safety 74 (2011) 342–349344

to be established (Fig. 1). The results of the experiment with additionof dipyrone each 24 h are shown in Fig. 1. After the addition in the24th hour, the total concentration of dipyrone increased by 17% inrelation to the initial quantity, possibly due to the remainingconcentration of dipyrone after the equilibrium. After reintroducingthe initial concentration in the 48th hour, there was an increase indipyrone of approximately 13% in relation to the 24th hour, onceagain because of remaining dipyrone in the water. In the HPLCanalysis, a second peak besides dipyrone was also observed in theall samples, with an average retention time of between 3 and4 min. This peak is probably represented by the hydrolysis to mainmetabolite, 4- methylaminoantipyrine. This compound was notquantified but, following a kinetic pattern, its concentrationincreases as the concentration of dipyrone diminishes.

Fig. 2. Specific GST activity in the liver of R. quelen after its exposure to dipyrone.

Animals were exposed to the indicated dipyrone concentration for 15 days. After

this period, GST activity was measured in the liver. Control group was kept in filtered

water without dipyrone. Bars show the mean7SEM of GST activity (mmol of

conjugated CDNB/min/mg protein) of fishes. *po0.05.

3.2. Biomarkers

The GST activity of the group exposed to 50 mg/L of dipyronedecreased when compared to control group while no significantdifferences were observed at lower dipyrone concentrations(Fig. 2). On the other hand, no changes in CAT activity wereobserved after dipyrone exposure when compared to the controlgroup (Fig. 3). Likewise, no difference was observed in LPO levels inthe exposed groups when compared to control (Fig. 4).

The cell types observed in R. quelen peripheral blood were red bloodcells (mature erythrocytes), white blood cells (leukocytes, among themlymphocytes, monocytes and neutrophils) and thrombocytes. Inabsolute numbers, mature erythrocytes and thrombocytes decreasedin all exposed groups after 15 days of exposure, while no differenceswere seen for leukocytes (Table 1). In the same way, hematocrit (Ht)decreased in all three groups in relation to control (Table 2) but nodifferences were seen in the other hematological parameters.

The comet assay results evidence a statistically significant DNAdamage in the group exposed to the lowest dipyrone sodiumconcentration (Fig. 5).

Important histopathological findings were observed in poster-ior kidneys of individuals from tested group when compared withthe control one. Kidney showed a normal distribution of glomeruli(capillaries and Bowman’s space) and tubules (Fig. 6A) in oppositeto the exposed groups. Individuals exposed to 0.5 mg/L of dipyrone,presented important lesions as large necrosis areas and enlarge-ment of Bowman’s space (Fig. 6B). In addition, a significant increaseof new nephron development was observed (Fig. 7).

When exposed to 5 mg/L of dipyrone more severe damageswere observed in the renal parenchyma such as a continuousBowmann’s space dilatation, necrosis and the presence of differ-entiated tissue areas not observed at the lowest concentration(Fig. 6C and D).

/ Ecotoxicology and Environmental Safety 74 (2011) 342–349 345

4. Discussion

In this study, it was observed that many evaluated parameterswere affected by exposure to dipyrone. The comet assay showedDNA damage, which could be caused by N-nitrosodimethylamine(NDMA), an N-nitroso compound (NOC). These kinds of compounds

Fig. 3. Specific catalase activity in the liver of R. quelen after its exposure to

dipyrone. Animals were exposed to the indicated dipyrone concentration for 15

days. After this period, CAT activity was measured in the liver. Control group was

kept in filtered water without dipyrone. Results are expressed as median and 10–90

percentiles of CAT activity (mmol of degraded H2O2/min/mg protein) of fish.

Kruskall–Wallis test.

Fig. 4. LPO content in the liver of R. quelen after its exposure to dipyrone. Animals

were exposed to the indicated dipyrone concentration for 15 days. After this period,

hydroperoxide concentration was measured. Control group was kept in filtered

water without dipyrone. Bars show the mean7SEM of hydroperoxides rate (nmol of

hydroperoxides/mg protein) of fish.

Table 1Blood cell counts of Rhamdia quelen (absolute numbers) relative to dipyrone exposure

Dipyrone sodium concentration (mg/L

Control 0.5

RBC (106 mL�1) 2.52 (0.16) 1.7

Thrombocytes (104 mL�1) 3.8 (0.38) 1.

WBC (104 mL�1) 5.6 (0.9) 5.

Mean7SEM (parenthesis), n¼14.

**po0.01 in relation to control.

***po0.001 in relation to control.

are genotoxic, and there are evidences that dipyrone may betransformed into NDMA (Brambilla and Martelli, 2007). Dipyronesodium is considered by many authors a DNA-damaging drug(Alexander et al., 2001). As reviewed by Brambilla and Martelli(2007) and Brambilla and Martelli (2009), it is one of the manydrugs that can go through endogenous transformation into thegenotoxic and carcinogenic N-nitrosodimethylamine (NDMA), anN-nitroso compound (NOC).

Glutathione is one of the chemicals that can act as an inhibitor ofnitrosation. Electrophilic species generated by promutagenic NOChave been shown to be conjugated with reduced glutathione(Brambilla and Martelli, 2007).To quantify GSH levels might beimportant in further studies with this compound.

Dipyrone was considered mutagenic to various Salmonella

lineage, and induced an enhanced sister-chromatid exchange inmouse bone marrow cells (Giri and Mukhopadhyay, 1998).Arkhipchuk et al. (2004) evaluated the same drug as toxic andgenotoxic for three organisms, belonging to three different sys-tematic groups: Allium cepa, Ceriodaphnia affinis, Hydra attenuata

and Carassius auratus gibelio.In this study, all exposed groups showed diminished hematocrit

and red blood cell counts (Tables 1 and 2). The fact that all the threegroups exposed had their hematocrits and RBC counts diminishedcould suggest an onset of anemia-like situation. Clauss et al. (2008)describe three types of anemia: hemorrhagic (blood loss), hemo-lytic (erythrocyte destruction), and hypoplastic (poor erythropoi-esis). One of the most common causes of anemias is the disruptionor destruction of hematopoietic tissues (poor erythropoiesis). Infish, hemorrhagic anemia might be caused by trauma, parasitism,etc, and is usually accompanied by a loss of iron; hypoplasticanemias are often associated with inflammatory diseases, toxinsand diseases with disruption or destruction of hematopoietictissues. Hemolytic anemia may be associated with environmentaltoxins, selected nutritional deficiencies and other factors.

Diseases are normally related to hemogram alterations inhuman beings and fish. For this reason, hematological profiles ofdifferent fish have been studied. Hematological research in fish isstill important to the comprehension of many ecological aspects(Tavares-Dias et al., 2000).

Alterations in the function and structure of the blood cells orcoagulation mechanisms can be considered hematologic disorders;some important of them can be a result of fish diseases. The primaryones are anemia, leukopenia, leukocytosis and thrombocytopenia.Hematocrit values less than 20% in fish are usually associated withanemia (Clauss et al., 2008).

In the same way, all the exposed groups presented diminishedthrombocyte counts. These cells are mainly involved in clottingresponse and might still have other functions in fish: besideshemostasis and homeostasis, the decrease of the predisposition toinfections, as they are, together with leukocytes named organicdefense blood cells (Tavares-Dias and Moraes, 2004). Thrombocy-topenia, thus, could have critical effects on fish, because they are

and control group.

)

5 50

4 (0.09)*** 1.69 (0.1)*** 1.77 (0.1)***

6 (0.3)*** 1.6 (0.2)*** 2.0 (0.4)**

3 (0.8) 4.2 (0.6) 5.0 (0.8)

Table 2Hematological variables and indices of Rhamdia quelen relative to dipyrone exposure and control group.

Dipyrone sodium concentration (mg/L)

Control 0.5 5 50

Hematocrit (%) 35.50 (1.72) 28.69 (1.09)** 30.05 (0.89)* 28.64 (1.16)**

Hemoglobin (g/100 mL) 4.91 (0.22) 4.33 (0.35) 3.95 (0.42) 4.38 (0.31)

RBC (106 mL�1) 2.2 (0.16) 1.74 (0.09)*** 1.69 (0.1)*** 1.77 (0.1)***

MCV (mL) 144.2 (6.96) 158.9 (9.27) 164.9 (17.01) 149.7 (15.28)

MCH (r g cell�1) 20.05 (1.08) 25.35 (1.96) 22.81 (1.51) 25.38 (2.06)

MCHC (g/100 mL) 14.05 (0.64) 16.38 (1.64) 13.79 (1.28) 14.89 (0.91)

Mean7SEM (parenthesis), n¼14.

*po0.05 in relation to control.

**po0.01 in relation to control.

***po0.001 in relation to control.

Fig. 5. Score of genetic damage in blood cells. Results are expressed as median and

10–90 percentiles. *indicates significant differences among the groups (po0.05),

Kruskall–Wallis test.

/ Ecotoxicology and Environmental Safety 74 (2011) 342–349346

responsible not only for this pathway but also for controlling fluidloss from surface wounds and for their participation on defensemechanisms. High levels of glucocorticoids and vitamin K defi-ciency are normally involved in clotting alterations (Campbell andEllis, 2007). Rios et al. (2005) found thrombocytopenia in Hoplias

malabaricus as a response from starvation.There was no dose–response profile in the hematological para-

meters that showed alterations. Those data could probably haveestablished and offer important information as well, as the con-centration studied might be enough to cause hematological damage.

There is little information about the hematological effects of thedipyrone in fishes. In humans, the same drug and others wereresponsible for many blood dyscrasias, such as aplastic and hemo-lytic anemia, agranulocytosis and thrombocytopenia (Bottiger andBottiger, 1981; Ribera et al., 1981; Sansone et al.,1984).

The histology of the posterior kidney of R. quelen showed tissuedamages in all groups exposed to dipyrone related to concentra-tions. The presence of necrosis areas represent severe and perma-nent lesions and the changes in the renal parenchyma as tissuedifferentiation show lesions that could be the beginning ofneoplastic events on exposure to 5 and 50 mg/L of dipyrone. Inaddition, the Bowman’s space enlargement suggests a circulatoryeffect that in short time can induce the kidney function failure.Necrosis of the blood vessels and changes in the renal parenchyma,which may have hematopoietic activity, have already been shown(Cooley et al., 2000).These findings are in accordance with thehematological results, corroborating the idea that the pharmaceu-tical evaluated affects the hematological system. Other researchers

verified the same depletion of the hematopoietic system, followedby uranium and TNP-470 (an analog of fumagillin) exposition(Cooley et al., 2000; Morris et al., 2003).

The vacuolization of the capillaries in the glomerulus and anincrease in the new nephron development were findings observed inindividuals exposed to the lowest concentration (0.5 mg/L). Thepresence of new nephrons can be related with the injuries recoverydue to a process of renal regeneration (Cormier et al., 1995; Saliceet al., 2001; Reimschuessel et al., 1996; Reimschuessel, 2001), notobserved in the higher concentrations, where more severe damageswere found. The other changes have been previously observed,followed by the exposure of some contaminants as mercury, anti-biotics and solvents, known by their capability to cause necrosis andcellular vacuolization (Reimschuessel, 2001). According to the mor-phological findings presented in the current study, clearly dipyronepresents serious renal injuries that must be better investigated.

The GST activity showed a decrease in the highest concentrationof dipyrone in these experiments, but no changes were observed atthe CAT activity and LPO.

Decrease in GST activity is normally observed through sometoxicants’ exposure to environment, such as polychlorinateddibenzo-p-dioxins, pesticides or polycyclic aromatic hydrocarbons,and in some fish species in polluted sites (Van der Oost et al., 2003).The decrease in phase I enzymes and the increase in phase IIenzymes suggest an adaptive response to a toxic environment(Stegeman et al., 1992). Therefore, in the highest concentration ofdipyrone, not only was the GST activity diminished but the tissuedamages were more accentuated and the new nephron productionwas absent. Those findings show that at this concentration, theorganism might have already lost its capability of protecting itselffrom the damage caused by dipyrone. In addition, as only thecontrol group had some inflammatory responses, one can hypothe-size that the exposed groups may have alterations on theirimmunological system, as they have also presented necrosis.

The process of lipid peroxidation is considered the major causeof cell injury and death, to which many pathological conditions anddiseases are connected with. The lipid peroxidation may occur incell membrane or in many organelles, leading to enzyme, mem-brane and DNA degradation (Hermes-Lima, 2004).

Despite the morphological damages and DNA lesions no increaseon the oxidative stress was observed, at least with the employedbiomarkers. It is important to have in mind that different moleculesare differently affected by oxidative stress. So, we suggest that, othermechanisms acting on the cell redox system could be involved, toprotect the cell membrane against lipid peroxidation. Furtherstudies using other biomarkers in order to quantify free radicalsor enzymes involved in their balance are necessary.

With fish, increases in hepatic CAT activity were only observed insome experiments that involved polycyclic aromatic hydrocarbons

Fig. 6. Histological cross-section of the trunk kidney in R. quelen after its exposure to dipyrone. (A) Control group. White arrow shows the glomeruli and the Bowman’s space is

observed in detail (black arrow). Bar¼100 and 20 mm. (B) Group exposed to 0.5 mg/L of dipyrone. Observe a large necrotic area (white arrows) and an enlargement of Bowman’s

space in detail (black arrow). Bar¼100 and 20 mm. (C and D) A differentiated tissue is shown in individuals exposed to 5 mg/L of dipyrone (arrows). Bar¼100 mm. (E) The white

arrows show damaged glomeruli in individuals exposed to 50 mg/L of dipyrone and the black arrow shows the enlargement of Bowman’s space. Bar¼20 mm. (F) Necrotic area

in individuals exposed to 50 mg/L (white arrow) and in detail a differentiated tissue is observed around the blood vessel (black arrow). Bar¼100 mm.

/ Ecotoxicology and Environmental Safety 74 (2011) 342–349 347

(PAHs) contaminated sediments and polychlorinated biphenyls(PCBs), but most laboratory studies could not demonstrate anysignificant alterations. Even though, more responses were observedin the field (Van der Oost et al., 2003). Vinagre et al. (2003)considered an adaptive response that augmented antioxidantcompetence toward peroxyl radical against cyanotoxin microcystinand no oxidative damages were observed, in terms of lipidperoxidation.

Studies must consider that lipid peroxidation and otherdamages are modulated by antioxidant systems and metallothio-neins (Valavanidis et al., 2006). For example, evaluations of GPOXmay elucidate some questions, as it reduces a variety of peroxidesby employing GSH as a cofactor. The principal peroxidase in fish iscytosolic and is considered to play an important role in themembrane protection from LPO (Van der Oost et al., 2003). Besides,fishes appear to have lower basal activities of SOD and CAT whereashigher basal activities of GPX (Wdzieczak et al., 1982).

Different concentrations of dipyrone were used during theHPLC kinetics evaluations and during the bioassay. The bioassay

concentrations were not measured analytically, due to the fact thatwe have chosen a concentration reported by some authors already,mentioned to be found in the aquatic environment, which is muchlower than the ones allowed by the employed analytical methods.We do not suppose that, analytically, different results would havebeen obtained. Due to our analytical method, an advantage wouldbe that, working with higher concentrations, there would be fewerrestrictions concerning to the detection of small differencesbetween dipyrone concentrations, as well as to observe theappearance and the accumulation of some by-product whoseresponse to our analytical method is unknown to us. The analysisof the kinetics of dipyrone sodium by HPLC shows that, due to theinstability of dipyrone in aqueous solutions, it suffers a spontaneoushydrolysis rapidly. Nevertheless, it is possible to notice that, withthe animals in the aquaria, the disappearance of the compound is48.65% higher than it is in the absence of the animals. We cansuppose that it is due to the fact that the fish are absorbing thecompound. In all experiments, at the end of 48 h, equilibriumoccurs (Fig. 1A–C). In the experiment with reintroduction of the

Fig. 7. New nephron counts. Results express as new nephrons/mm2. Bars show the

mean7SEM. *indicates significant difference (po0.05) on comparison to other

groups.

/ Ecotoxicology and Environmental Safety 74 (2011) 342–349348

concentration of dipyrone every 24 h, there was an increase of 17%in the initial concentration, which can be due to the sum of theremaining dipyrone in the equilibrium with the amount addedafterwards. In 48 h, there was an increase of about 13% in relation tothe 24th hour, once again because of the remaining dipyrone in thewater. Upon the addition of the same initial concentration (50 mg/mL) 24 h later, the same rate was proportionally eliminated. Theconcentration increased in 24 and 48 h. It is probably explained bythe short time of the analysis: the elimination of the remainingcompound was not observed because of the speed of the eliminationrate. However, it seems that with the further addition of dipyroneat every 24 h allowed us to keep a relatively constant level ofthe compound in the water (with less than 20% change) for theexposition during 15 days. Even 1000 times lower than theconcentration applied in the kinetic study (50 mg/mL), the con-centrations of dipyrone utilized during the bioassay, concentrationsnormally found in the environment, were harmful to the animals.

The HPLC revealed a second compound, which is probably aresult of the dipyrone hydrolysis. The post-hydrolysis dipyrone by-products are also reported by many authors to be responsible fortoxic effects. Nevertheless, we could not classify them, neitherquantitatively nor qualitatively, the main metabolite showed at ourchromatographic system, because we could not establish a patternfor this probable substance. However, according to Ergun et al.(2004), in a typical chromatogram of the hydrolysis of dipyrone, thesecond peak is possibly the main dipyrone metabolite, 4-methy-laminoantipyrine. This is in agreement with our observations.Considering that the separation method of choice was a reversephase liquid chromatography, in which the longer the retentiontime, the more hydrophobic is the substance, that the loss of itshighly polar sulphonic group by dipyrone would thus diminish itspolarity originating a metabolite with a longer retention time.

Thus, being disposed to the aquatic environment, often and at aconstant speed, dipyrone and its hydrolysis by-products areprobably absorbed by aquatic organisms. For instance, Feldmannet al. (2007) found residues of dipyrone and its metabolitesaminoantipyrin, 4-acetylaminoantipyrine and 4-formilaminoanti-pyrine in hospital effluents, municipal sewers and a sewagetreatment plant in Berlin. Wiegel et al. (2004) found disprovemetabolites in the river Elbe, Germany. Dipyrone sodium waspresent in a sewage treatment plant on the Mediterranean coast(Gomez et al., 2007). Besides, we observed in the analysis devel-oped in this study the presence of a degraded compound, possiblyeven more toxic and reactive. Some authors prove the increasing

toxicity of dipyrone sodium degradation by-products (Gomez et al.,2008).

The findings at this research show that the constant dipyronerelease to the environment have toxic effects to aquatic organisms.These effects are mostly related to DNA damage and might reach anultrastructural level, as the damage presented at the histopatho-logical study corroborated the hypothesis that dipyrone originproducts are even more toxic. Those metabolites are also toxic forthe posterior kidney with important consequences to excretorysystems in fish or other similar systems in vertebrates (Evans,1993; Silva and Martinez, 2007). Further studies with a morecomplete investigation must be stimulated to study the toxiceffects of dipyrone to other systems in vertebrates.

5. Conclusion

The findings showed that the constant dipyrone release to theenvironment is able to cause toxic effects to aquatic organisms.These effects are mostly related to DNA damage and, might reachan ultrastructural level.

Acknowledgments

This research was partially supported by the CAPES, CNPq andFundac- ~ao Araucaria, Brazil.

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