Drug and Chemical Toxicology

ISSN: 0148-0545 (Print) 1525-6014 (Online) Journal homepage: http://www.tandfonline.com/loi/idct20

Genotoxic response and oxidative stress induced

by diclofenac, ibuprofen and naproxen in Daphnia
magna

Leobardo Manuel Gómez-Oliván, Marcela Galar-Martínez, Sandra García-

Medina, Analleli Valdés-Alanís, Hariz Islas-Flores & Nadia Neri-Cruz

To cite this article: Leobardo Manuel Gómez-Oliván, Marcela Galar-Martínez, Sandra García-
Medina, Analleli Valdés-Alanís, Hariz Islas-Flores & Nadia Neri-Cruz (2014) Genotoxic response
and oxidative stress induced by diclofenac, ibuprofen and naproxen in Daphnia magna, Drug
and Chemical Toxicology, 37:4, 391-399, DOI: 10.3109/01480545.2013.870191

To link to this article: http://dx.doi.org/10.3109/01480545.2013.870191

Published online: 07 Jan 2014.

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ISSN: 0148-0545 (print), 1525-6014 (electronic)

Drug Chem Toxicol, 2014; 37(4): 391–399

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2013.870191

RESEARCH ARTICLE

Genotoxic response and oxidative stress induced by diclofenac,

ibuprofen and naproxen in Daphnia magna
Leobardo Manuel Gómez-Oliván1, Marcela Galar-Martı́nez2, Sandra Garcı́a-Medina2, Analleli Valdés-Alanı́s2,
Hariz Islas-Flores1, and Nadia Neri-Cruz1
1
Laboratorio de Toxicologı́a Ambiental, Facultad de Quı́mica, Universidad Autónoma del Estado de México, Toluca, State of Mexico, Mexico and
2
Laboratorio de Toxicologı́a Acuática, Departamento de Farmacia, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico
City, Mexico

Abstract Keywords
Context: Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used Catalase, DNA damage, glutation peroxidase,
pharmaceuticals in Mexico, but there is not proper regulation on the sale, use and disposal. These lipid peroxidation, NSAID, protein carbonyl,
drugs can enter water bodies by diverse pathways, attaining significant concentrations and superoxide dismutase
inducing damage on hydrobionts. Objective: To evaluate the oxidative stress and consequent
damage to genetic material induced by DCF, IBP and NPX on Daphnia magna. Methods: The acute History
toxicity assays were performed to 48-h by nonsteroidal anti-inflammatory drugs evaluated. A
sublethal assay were done after 48 h of exposure to DCF, IBP and NPX added to water with the Received 21 June 2013
concentration equivalent to the lowest observed adverse effect level (LOAEL), 9.7 mg/L for DCF, Revised 18 September 2013
2.9 mg/L for IBP and 0.017 mg/L for NPX. The DNA damage (comet assay) was evaluated at 12, 48 Accepted 22 November 2013
and 96 h. The oxidative biomarkers were evaluated: lipid peroxidation; protein carbonyl content; Published online 23 December 2013
activity of the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase.
Results: D. magna exposed to DCF, IBP and NPX showed a significant increase (p50.05) with
respect to controls in LPX. PCC was increased in IBP exposure. SOD and CAT activity were
increased by exposure to IBP and NPX. GPX shows a significant increase with respect to control in
IBP and DCF exposure and significant decrease by NPX exposure. DNA damage was observed in
48 and 96 h. Discussion and conclusion: DCF, IBP and NPX were responsible of alterations in
biochemical biomarkers evaluated and DNA damage.

Introduction make pharmaceuticals to be evaluated for potential effects on

aquatic flora and fauna (Fent et al., 2006).
The presence of pharmaceutical chemicals in the aquatic
The occurrence pattern and the concentration of the wide
environment has long been recognized as a concern
range of pharmaceutically active compounds largely vary
(Richardson & Bowron, 1985). The primary routes for
from country to country. Nevertheless, the most frequently
pharmaceuticals into the environment are through human
detected prescription classes include non-steroidal antiin-
excretion, disposal of unused products and through agricultural
flammatory drugs (NSAIDs), antibiotics, beta-adrenergic
usage (Poynton & Vulpe, 2009). A wide range of pharmaceut-
antagonists (b-blockers) and iodinated X-ray contrast media
ical products have been detected in surface and groundwater,
(Láng & Köhidai, 2012). Many environmental analyses have
associated with wastewater disposal (Barnes et al., 2008;
been performed in various countries, which are summarized
Vulliet & Cren-Olivé, 2011; Watkinson et al., 2009).
by various reports (e.g. Richardson, 2007; Palmer et al.,
Pharmaceuticals are a class of emerging environmental
2008; Togola & Budzinski, 2008; Yu & Chu, 2009; Wang
contaminants that are extensively and increasingly being used
et al., 2011).
in human and veterinary medicine. These chemicals are
In the Valley of Mexico, substantial concentrations have
designed to have a specific mode of action and many of them
been found of pharmaceutical agents such as trimethoprim,
for some persistence in the body. These features among others
clarithromycin, clindamycin, erythromycin, metoprolol, IBP,
gemfibrozil, DCF, sulfasalazine, bezafibrate and NPX
(Siemens et al., 2008).
Address for correspondence: Dr. Leobardo Manuel Gómez-Oliván, NSAIDs are chemicals with anti-inflammatory, analgesic
Laboratorio de Toxicologı́a Ambiental, Facultad de Quı́mica, and antipyretic effects. The most common members of this
Universidad Autónoma del Estado de México, Paseo Colón intersección group of therapeutic agents are acetylsalicylic acid (ASA),
Paseo Tollocan, s/n., Col. Residencial Colón, Toluca, Estado de México, paracetamol (PAR), DCF, IBP and NPX (Katzung, 2007).
C.P. 50120 México. Tel: + (52) 7222173890. Fax: + (52) 7222173890.
E-mail: [email protected], lgolivan74 @gmail.com These pharmaceutical compounds are widely used to relieve
392 L. M. Gómez-Oliván et al. Drug Chem Toxicol, 2014; 37(4): 391–399

pain in acute and chronic inflammatory conditions, rheumatic (Scenedesmus spp, 3  107 cells/ml). Third to fifth brood
syndromes, degenerative disease, acute lumbar pain, tendin- juveniles524 h old and differing in age by53 h were used for
itis, bursitis, sciatica, gout, dental surgery, dysmenorrhea and testing.
migraine (Mycek et al., 2004).
NSAIDs having similar mechanisms of action have been Chemicals
studied in different species. Thus, Borgmann et al. (2007)
found that these compounds are toxic to Hyallela azteca, and Diclofenac
Cleuvers (2004) revealed they affect growth and reproduction The pharmaceutical agent DCF (CAS number 15307-86-5,
in the cladoceran Daphnia magna and algae Scenedesmus 499% purity) C14H11Cl2NO2, 296 Da, was purchased from
subspicatus. DCF affect gills and kidney of freshwater fish, Sigma-Aldrich Corporation Mexico. Stock solutions were
brown trout (Salmo trutta f. fario), suggesting possible risk to prepared by dissolving 1 g DCF in deionized water.
fish populations (Hoeger et al., 2005). Results obtained by
Matozzo et al. (2012), indicate that exposure of clams
Ibuprofen
Ruditapes philippinarum to IBP induces significant alter-
ations in the immune parameters and suggest potential IBP (CAS Number 15687-27-1, 498% purity) was purchased
immunosuppression in treated clams. NPX has been reported from Sigma-Aldrich Corporation Mexico. IBP (1 g) was
to be a potential teratogenic agent in studies using Hydra dissolved in deionized water to make stock solutions.
attenuata as a model (Quinn et al., 2008).
NSAIDs can form reactive products by oxidation Naproxen
(Masubuchi et al., 2002; Gómez-Lechón et al., 2003), being
then able to induce oxidative stress. Oxidative stress is NPX (CAS Number 22204-53-1, 98% purity) was pur-
defined as disruption of the balance between reactive oxygen chased from Sigma-Aldrich Corporation Mexico. NPX (1 g)
species (ROS) and the antioxidant systems in the body. ROS was dissolved in deionized water to make stock solutions.
are normally produced in cells as a result of metabolic
processes (Vlahogianni et al., 2007); however, they can also Toxicity assays
be formed by diverse environmental contaminants (Sinha Test systems consisted in 150 mL polyethylene containers
et al., 2007), increasing lipid peroxidation (LPX) and protein holding reconstituted water, and supplied with constant
carbonyl content (PCC) as well as changes in the activity of oxygen under a 14 h:10 h light:dark photoperiod at room
diverse antioxidant enzymes, including superoxide dismutase temperature. Intoxication systems were static and no food was
(SOD), catalase (CAT), and glutathione peroxidase (GPX) provided to specimens during exposure periods.
(Parvez & Raisuddin, 2005; Vlahogianni et al., 2007), and
may ultimately damage genetic material.
Acute toxicity test
Studies by Parolini et al 2010 have revealed the capacity of
paracetamol to induce moderate genotoxicity in Dreissena Preliminary acute toxicity tests were conducted in order to
polymorpha exposed to environmental concentrations. In calculate DCF, IBP and NPX LC50 data. All experiments
other studies biomarkers of damage (LPX and DNA damage) were performed according to the OECD standard procedure
were significantly affected in marine mussel (Mytilus spp) by (OECD, 2004) for determining the 48 h LC50 for D. magna.
DCF exposure in concentrations of 1000 mg/L at 96 h Different test concentrations for DCF (43, 54, 68, 86, 108 and
(Schmidt et al., 2011). 136 mg/L), for IBP (20.4, 25.7, 32.4, 40.7 and 51.3 mg/L), for
Daphnia magna (D. magna) is commonly used in aquatic NPX (0.025, 0.05, 0.075, 0.1, 0.15 and 0.2 mg/L), plus a DCF,
toxicity testing because of many characters that make it easy IBP and NPX-free control system were set up. Static systems
and economical to culture in the laboratory: it is relatively were used without renewal of test solution.
small, has short life cycle, high fecundity, and parthenogen- Ten neonates (524 h old) from a designated brood were
etic reproduction. placed in a 30 ml glass beaker containing 25 ml for each test
The aim of this study was to evaluate oxidative stress and concentration and control. Test organisms were not fed
genotoxicity induced on D. magna by DCF, IBP and NPX during the testing period. The temperature of test was
exposure to sublethal concentration. 20  1  C, 16 h:8 h light:dark photoperiod. The glass beaker
were loosely covered to reduce the loss of water due to
Methods evaporation and to avoid the entry of dust into the solutions.
Observations were made at 48 h, and results recorded.
Experimental organisms
Dead (immobilized) specimens were counted after 48 h.
The test organism was D. magna Straus, obtained from Oil Immobilization, which was employed as an endpoint, was
Mexican Institute and has been maintained in the considered to have happened if no movement was detected
Environmental Toxicology laboratory of the University for 15 s after gentle shaking of the test vehicle. Five
Autonomous of State of Mexico, for several generations. replicates of the assay were performed. A total of 350
The Daphnia medium consisted of CaCl2, KCl, NaHCO3 and organisms were used for DCF and NPX LC50 determination
MgSO2. The living green algae Scenedesmus spp. were used and 300 organisms for IBP LC50 determination. The 48 h
as the sole food for D. magna. Organisms were maintained at LC50 of DCF, IBP and NPX and its 95% confidence limits
25  1  C under a 14:10 (light:dark) photoperiod. D.magna (p50.05) were estimated by Probit analysis (EPA Probit
were fed once daily with 5 ml of concentrated algae Analysis Program v1.5).
DOI: 10.3109/01480545.2013.870191 Genotoxic response and oxidative stress induced by NSAID in D. magna 393

Stress oxidative biomarkers evaluation reactive carbonyls formed (C¼O)/mg protein based on their
molar extinction coefficient of 21 000 M/cm
Sublethal toxicity assays involved adding DCF, IBP and NPX
at a concentration equivalent to the lowest observed adverse
SOD activity determination
effect level (LOAEL), 9.7 mg/L for DCF; 2.9 mg/L for IBP;
and 0.018 mg/L for NPX, to one test systems and 150 mg wet SOD activity was determined according to the Misra &
weight of D. magna was added (about 250 entire organisms Fridovich (1972) method. To 20 mL supernatant in a 1 cm
were used). The exposure period was 48 h. DCF, IBP and cuvette were added 150 mL of a carbonate buffer solution
NPX-free control system were set up for exposure time in the (50 mM sodium carbonate and 0.1 mM EDTA) pH 10.2 and
sublethal study. Static systems were used without renewal of 100 mL adrenaline (30 mM). Absorbance was read at 480 nm,
test solution. at 30 s and 5 min. SOD activity was determined by
Daphnids were maintained in dilution water at the test interpolating the data on a type curve. Results were expressed
temperature for at least 48 h prior to start the test. as IU SOD/mg protein.
The temperature of test was 20  1  C, 16 h:8 h light:dark
photoperiod, pH 7.0, hardness 150 mg/L as CaCO3. The glass CAT activity determination
beaker were loosely covered to reduce the loss of water due to CAT activity was determined according to Radi et al. (1991)
evaporation and to avoid the entry of dust into the solutions. method. To 20 mL supernatant were added 1 mL of an
After exposure time, test specimens were removed and isolation buffer solution (0.3 M saccharose, 1 mM EDTA,
suspended in 1 mL of Tris buffer solution (pH 7). The mixture 5 mM HEPES and 5 mM KH2PO4) and 0.2 mL of hydrogen
was maintained in an ice bath throughout the procedure and peroxide (20 mM). Absorbance was read at 240 nm, at 0 and
homogenized. The supernatant was centrifuged at 12 500 rpm 60 s. Results were obtained by substituting the absorbance
and 4  C for 15 min. The following biomarkers were value of each reading in the formula: catalase
evaluated: LPX and PCC to determine oxidized protein concentration ¼ (A60–A0)/MEC, where the MEC of H2O2
content; activity of the antioxidant enzymes SOD, CAT and equals 0.043 Mm/cm. Results were expressed as mM H2O2/
GPX. Total protein content was used to express the results of mg protein.
all the biomarkers employed. All tests were performed on the
supernatant except for LPX determination in which the GPX activity determination
cellular pellet was used. Five replicates of the assay were
performed. GPX activity was determined using the Paglia & Valentine
(1967) methodology. To 100 mL supernatant were added
900 mL of buffer reagent solution (5 M K2HPO4, 5 M
Lipid peroxidation determination
KH2PO4, 3.5 mM reduced glutathione, 1 mM sodium azide,
Lipid peroxidation (LPX) was measured using the thiobar- 2 U glutathione reductase and 0.12 mM NADPH pH 7.0;
bituric acid (TBA) assay according to the method described Sigma) and 200 mL of H2O2 (20 mM). Absorbance was read at
by the Büege & Aust (1978) method. The cellular pellet 340 nm at 0 and 60 s. Activity was estimated using the molar
was reconstituted with Tris-HCl buffer (pH 7.4) to attain a extinction coefficient of NADPH (6.2 mM/cm). Results were
volume of 5 mL. 1 mL of this solution was taken and expressed as mM NADPH/mg protein.
incubated at 37  C for 30 min; 2 mL of TCA-TBA reagent
(0.375% thiobarbituric acid in 15% trichloroacetic acid) was Total protein determination
added and the sample was shaken in a vortex, then placed
To 25 mL of supernatant was added 75 mL deionized water and
in a bath of boiling water for 45 min, allowed to cool and
2.5 mL Bradford’s reagent (0.05 g Coommassie Blue dye,
the precipitate removed by centrifugation at 3 000 rpm for
25 mL of 96% ethanol and 50 mL H3PO4, in 500 mL
10 min. Absorbance was read at 535 nm using a reaction
deionized water). The test tubes were shaken and allowed to
blank. Results were expressed as mM malondialdehyde/mg
rest for 5 min prior to the reading of absorbance at 595 nm and
protein using the molar extinction coefficient of 1.56  105/
interpolation on a bovine albumin curve (Bradford, 1976).
M/cm.

Genotoxic response evaluation

Protein carbonyl content determination
Comet assay
PCC was determined by the method of Levine et al. (1994)
method. Soluble proteins were obtained by centrifugation of To prepare Daphnia, a total of 150 neonates, aged less than
samples at 10 500 rpm for 30 min. To 100 mL of this 24 h, were collected 12, 24 and 96 h from the control and
supernatant was added 150 mL of 10 mM dinitrophenylhy- experimental tanks after exposure to DCF, IBP, NPX, H2O2
drazine in 2 M HCl (Sigma) prior to incubation at room (10 mM) and pooled for a Comet assay.
temperature for 1 h in the dark. Next, 500 mL of 20% DNA damage was evaluated by comet assay as proposed
trichloroacetic acid was added and the sample was allowed by Tice et al. (2000) and Lankoff et al. (2006). The
to rest for 15 min at 4  C, then centrifuged at 16 000 rpm for microscope slide containing the cells was prepared 1 h
5 min. The bud was rinsed three times in 1:1 ethanol:ethyl before the sample was obtained. First, a 200-mL layer of 1%
acetate (Baker), dissolved in 150 mL of 6 M guanidine (Sigma) normal agarose was placed on the slide, then 10 mL of a cell
pH 2.3, and incubated at 37  C for 30 min. Absorbance was suspension was mixed with 75 mL of 0.7% normal agarose and
read at 366 nm and results were expressed as nmols of 50 mL of this mixture was placed on the initial agarose layer.
394 L. M. Gómez-Oliván et al. Drug Chem Toxicol, 2014; 37(4): 391–399

To extract DNA, slides were placed in a coplin jar with according to their LC50:51 mg/L is extremely toxic, 1–10 mg/
lysis solution [2.5 M NaOH, 10 M ethylendiaminetetraacetic L is toxic, and 10–100 mg/L is hazardous for aquatic
acid (EDTA), 10 mM Tris, 10% dimethyl sulfoxide (DMSO), organisms. Based on this ranking, NPX is extremely toxic,
1% Triton, at pH 10] for 1 h at 4  C. After lysis, samples were IBP and DCF are hazardous for D. magna.
incubated with 1 mg/mL formamidopyrimidine glycosylase
(FPG) and endonuclease III (ENDO) with 40 mM 4-(2- Evaluation of oxidative stress
hydroxyethyl)-1-piperazinee ethane sulfonic acid (HEPES), Lipid peroxidation
0.1 M KCl, 0.5 mM EDTA at pH 8.0, and 0.2 mg/mL fetal
bovine serum for 45 min at 37  C. Each enzyme was LPX results are shown in Figure 1. D. magna exposed to
incubated separately. An internal control was set up for DCF, IBP and NPX showed a significant increase (p50.05)
each group and incubated without enzymes. with respect to controls of 96.3, 379.4 and 285.3%, respect-
Slides were placed in the electrophoresis chamber for ively at 48 h. The most significant effect on this biomarker
20 min with an alkaline solution (300 mM NaOH and 1 mM was presented by exposure to IBP
EDTA) at a pH of 13. Electrophoresis was performed at
300 mAmp, 25 V and pH 413 for 20 min and the process was Protein carbonyl content
stopped with a neutralization buffer (0.4 M trizma base) at a PCC is shown in Figure 2. A significant increase (p50.05) of
pH of 7.4. 3,578% with respect to controls took place at 48 h for IBP
The DNA was stained with 50 mL ethidium bromide and exposure. However, no significant increases (p40.05) of 79
examined with an epifluorescence microscope attached to an and 39.8% with respect to controls occurred at 48 h for DCF
image analyzer equipped with a program for measurement of and NPX exposure, respectively.
the cell nucleus. A total of 100 measurements per replicate
were made and % DNA damage in the tail was obtained.
Measuring was done with a Zeiss Axiophot KS400 micro-
scope equipped with epifluorescence and a 510–560 nm filter.
To identify oxidative damage to DNA induced by DCF,
IBP, NPX and H2O2 a comet assay was performed with the
addition of two enzymes involved in the initial steps of DNA
excision repair. To evaluate levels of oxidized purines, FPG
was added, and to determine the rate of oxidized pyrimidines,
endo III was used in the assay (Lankoff et al., 2006). The level
of FPG or endo III-sensitive sites was obtained from the
difference between the DI of nuclei incubated with enzymes
and the internal control.

Statistical design
For the acute toxicity assay (DCF, IBP and NPX LC50) the
Figure 1. Lipid peroxidation in D. magna after exposure to 2.9 mg/L of
Probit analysis was carried out and significance was assessed IBP, 0.018 mg/L of NPX and 9.7 mg/L of DCF for 48 h. Values are the
by the degree of overlap of 95% LC (EPA Analysis Program mean of three replicates SE. *Significantly different from control
v1.5). The X2 linear adjustment test was not significant at values, ANOVA and Tukey–Kramer (p50.05). The results are referred
to milligrams protein (mg PT).
p50.05.
For the sublethal toxicity assay statistical evaluation of the
results obtained in the oxidative stress assay was made by
applying to a one-way analysis of variance (ANOVA) and
differences between means were compared using the Tukey–
Kramer multiple comparisons test, with p set at 50.05. For
this purpose we used the package, SPSS (ver.10: SPSS Inc.,
Chicago, IL).
Kruskal–Wallis nonparametric ANOVA and Dunn’s mul-
tiple comparisons test were applied to DNA damage results,
with p set at 50.05.

Results
Acute toxicity
In this study, the LC50 of DCF was 96.6 mg/L (84.2–112.7),
IBP was 24.9 mg/L (14.4–31.4) and NPX was 0.174 mg/L Figure 2. Proteyn Carbonyl Content in D. magna after exposure to
2.9 mg/L of IBP, 0.018 mg/L of NPX and 9.7 mg/L of DCF for 48 h.
(0.149–0.222) in D. magna at 48 h of exposure. EU guidelines
Values are the mean of three replicates SE. *Significantly different
(93-67-EEC) laid down by the Commission of the European from control values, ANOVA and Tukey–Kramer (p50.05). The results
Communities (1996) classify substances into different types are referred to milligrams protein (mg PT).
DOI: 10.3109/01480545.2013.870191 Genotoxic response and oxidative stress induced by NSAID in D. magna 395

Figure 3. Superoxide dismutase (SOD) activity in D. magna after Figure 5. Glutathione peroxidase (GPX) activity in D. magna after
exposure to 2.9 mg/L of IBP, 0.018 mg/L of NPX and 9.7 mg/L of DCF exposure to 2.9 mg/L of IBP, 0.018 mg/L of NPX and 9.7 mg/L of DCF
for 48 h. Values are the mean of three replicates SE. *Significantly for 48 h. Values are the mean of three replicates SE. *Significantly
different from control values, ANOVA and Tukey–Kramer (p50.05). different from control values, ANOVA and Tukey–Kramer (p50.05).
The results are referred to milligrams protein (mg PT). The results are referred to milligrams protein (mg PT).

Comet assay
As shown in Figure 6, DCF, IBP and NPX exposure at 48 and
96 h increased significantly (p50.05) with respect to control
in the median DNA (%) in the tail without adding enzymes.
To estimate the magnitude of oxidized base lesions after
exposure, cells were treated with endonucleases. When FPG
and ENDO were added, observed a significant increase
(p50.05) with respect to control at 48 and 96 h by IBP and
NPX exposure, while DCF didn’t show significant increase
(p40.05) with respect to control when add ENDO enzyme.
However when add FPG enzyme at 48 and 96 h, all exposures
increased significantly (p50.05) with respect to control the %
DNA in the tail (oxidation of purine bases). The exposure of
Figure 4. Catalase (CAT) activity in D. magna after exposure to 2.9 mg/ these drugs at 12 h didn’t show a difference with respect to
L of IBP, 0.018 mg/L of NPX and 9.7 mg/L of DCF for 48 h. Values are control. Further when organisms were exposed to a concen-
the mean of three replicates SE. *Significantly different from control
values, ANOVA and Tukey–Kramer (p50.05). The results are referred tration of H2O2 10 mM (positive control) was observed a
to milligrams protein (mg PT). significant increase over the control (p50.05) in all exposure
times without adding enzyme and with ENDO and FPG
enzymes, this increase was time dependent.
SOD activity
SOD activity (Figure 3) showed a significant increase with
Discussion
respect to controls (p50.05) at 48 h by IBP and NPX The LC50 data of DCF, IBP and NPX in D. magna found in
exposure of 434.7 and 171.8%, respectively. However a this study may be clarified by the fact that these NSAID acts
significant decrease (p50.05) with respect to control of by blocking the enzyme cyclooxygenase. It is responsible for
95.5% occurred by DCF exposure. catalyzing arachidonic acid degradation in prostaglandin
production (Cha et al., 2006; Fortier et al., 2008). These
CAT activity eicosanoids act as autocrine and paracrine messengers and in
invertebrate species such as D. magna as important mediators
CAT activity (Figure 4) increased significantly (p50.05) with
during reproduction and in the immune system (Deridovich &
respect to control in IBP and NPX at 48 h exposure times,
Reunova, 1993; Fortier et al., 2008; Stanley, 2000).
showing values of 2,250 and 521.7%, respectively. A not
Prostaglandins are also involved in neural transmission and
significant increase (p40.05) with respect to control occurred
the transport of ions across cell membranes (Arkhipova et al.,
by DCF exposure.
2005). The end result of these actions in the present study
may have been inhibition of neural transmission evidenced
GPX activity
by daphnid immobilization.
Figure 5 shows GPX activity in D. magna. Significant DCF, IBP and NPX has been identified and quantified
increases (p50.05) of 1578 and 962.3% with respect to in diverse water bodies (Aga, 2008; Siemens et al., 2008).
control were observed at 48 h by IBP and NPX exposure, DCF is biodegradable by microorganisms or photodegradable
respectively. However, GPX activity decreased with respect to into the following metabolites: 5,40 -dihydroxy-diclofenac,
control by 75.9% at 48 h (p50.05). 3-dihydroxy-diclofenac, 40 -dihydroxymethyl-diclofenac,
396 L. M. Gómez-Oliván et al. Drug Chem Toxicol, 2014; 37(4): 391–399

Figure 6. Determination of DNA damage by the comet assay in D. magna after exposure to 2.9 mg/L of IBP, 0.018 mg/L of NPX, 9.7 mg/L of DCF and
10 mM of H202 (hydrogen peroxide) for 48 h. Each value is the mean of three replicates SEM. *, significantly different from the corresponding
control. Kruskal-Wallis nonparametric ANOVA and Dunn’s test (p50.05).

30 -hydroxymethyl-diclofenac, 40 -hydroxy-diclofenac and The hydroxylated metabolites of NSAIDs can be oxidized

50 -hydroxy-diclofenac (Deng et al., 2003). The latter two to intermediates of a reactive quinone imine that reacts with
are oxidized to intermediates of benzoquinone imine, com- protein nucleophilic groups to form adducts. There is also
pounds that are highly toxic to aquatic organisms (Oviedo- formation of NSAIDs cationic radicals to nitroxides and
Gómez et al., 2010). quinolone imines, both of which are associated with the redox
IBP is rapidly degraded under aerobic conditions, with cycle (Hoeger et al., 2008). These findings could contribute to
removal rates up to 90% (Ternes, 1998). The main photo- PCC increase by IBP, DCF and NPX exposure.
degradation metabolites are arylcarboxyl IBP; hydroxy-IBP y Asensio et al. (2007) say that NSAID-induced inhibition of
el carboxy-IBP (Carballa et al., 2004; Méndez-Arriaga G6PD elicits damage by oxidation of protein thiols. This may
et al., 2008). not be the only process of protein carbonyl production:
NPX photodegradation products include 4-isobutylaceto- formation of free radicals during biotransformation of this
phenone, 1-(6-methoxy-2-naphthyl) ethanol and 2-acetyl-6- pharmaceutical may be one of the sources of oxidation of this
methoxynaphthalene (Miranda et al., 1991). biomolecule.
Besides the abiotic transformations already mentioned, It is also well known that when NSAIDs are ingested they
the biotransformation NSAIDs may undergo after entering enters in contact with the vasculature where they acetylates
the body must be considered. It is known that in humans the the enzyme COX2 present in endothelium or circulating
biotransformation of NSAID is mediated by the CYP2C9, leukocytes to produce 15-epi-lipoxin A4, which promotes
CYP2C8, CYP2C18, CYP2C19 and CYP2B6 (Tang, 2003). nitric oxide (NO) synthesis mediated by endothelial (eNOS)
The invertebrate Daphnia is the first crustacean to have its and inducible (iNOS) nitric oxide synthase (Paul-Clark et al.,
genome sequenced. This genus is known to have cytochrome 2004). When the superoxide anion and NO bind they may
P450 (CYP) and the CYP2, CYP3 and CYP4 families form a reactive nitrogen specie (peroxynitrite) through a
(Feyereisen, 2006). CYP2 subfamily is specifically the diffusion-limited reaction (Hule & Padmaja, 1993). The
responsible for the NSAID biotransformation to hydroxylated oxidant agent peroxynitrite is known to induce protein
metabolites. oxidation and nitration in absence of GSH, eliciting
In the present study, LPX showed a tendency to increase in mitochondrial dysfunction and eventually leading to
DCF, IBP and NPX exposure in D. magna. This increase may irreversible damage and severe loss of cellular ATP
be due to the fact that CYP produces an oxygenated (Jaeschke et al., 2003).
intermediate – the oxycytochrome P450 complex [P450 The antioxidant defense system is crucial in the neutral-
(Fe3þ) O 2 ] – during NSAIDs biotransformation, with conse- ization of generated ROS by pharmaceuticals redox reactivity
quent release of superoxide anion (O2 ) by uncoupling of this (Regoli et al., 2002). This system is mediated by a cascade of
reaction (Doi et al., 2002). antioxidant enzymes which scavenge ROS to less toxic
Since NSAIDs affect the mitochondrion and consequently compounds. SOD leads this mechanism converting the highly
oxidative phosphorylation, increased ROS production, par- reactive superoxide anion (O2 ) into H2O2. In IBP and NPX
ticularly of O2 , may occur and as a result, an increase in exposure SOD activity is enhaced significantly over the 48 h,
LPX (Salgueiro-Pagadigorria et al., 2004). the activation of this antioxidant enzyme favors the evidence
Direct damage to proteins or the chemical alteration of of the prooxidant action of IBP and NPX. As a catalyst of
their amino acids as a result of oxidative stress may SOD activity by product H2O2 to form water and oxygen,
increase PCC (Parvez & Raisuddin, 2005) and therefore CAT action is enabled by the high enhacement of SOD
oxidized protein content (Shacter, 2000). Protein oxidation activity over 48 h or by the possible increment of H2O2
results in loss of sulfhydryl groups and rearrangement formation in the cells due to the increase of ROS linked
of amino acid resonance, altering their function and to arachidonic acid metabolism via lipoxygenase (LOX)
therefore the integrity of the organism (Parvez & pathway instead of COX pathway blockage by NSAIDs
Raisuddin, 2005). (Ardaillou et al., 1987).
DOI: 10.3109/01480545.2013.870191 Genotoxic response and oxidative stress induced by NSAID in D. magna 397

Reduced SOD activity by DCF exposure, result from the 1000 mg/L at 96 h (Schmidt et al., 2011). In contrast,
fact that several DCF metabolites bind to proteins and inhibit Parolini et al. (2011) mentioned that DCF environmentally
their activity. Likewise, NSAIDs – including DCF among relevant concentrations (0.3, 1 and 2 nM) were not able to
others – affect the mitochondrion and consequently oxidative produce any significant DNA fragmentation on zebra mussel
phosphorylation, thus potentially increasing ROS production. Dreissena polymorpha hemocytes even after 96 h exposure.
This is particularly true of the O2* anion (Asensio et al., Parolini et al. (2009) tested genotoxicity of three drugs:
2007) which, added to the ROS produced by natural processes DCF, PAR and IBP at various concentrations (0.2 mM, 2.0 mM
in the D. magna, can potentially block SOD activity. and 4 mM) obtaining as a result that all the drugs tested were
Both CAT and GPX activities were also significantly able to significantly induce primary DNA damage in
increased in D. magna by IBP, NPX and DCF exposure. hemocytes of D. polymorpha.
Bagnyukova et al. (2006) state that LPX products appear to be The results observed by Parolini et al. (2009), Rocco et al.
involved in the up-regulation of several antioxidant enzymes. (2010) and by Schmidt et al. (2011) are consistent with the
Thus, LPX increases in the present study may also explain the results obtained in this study. IBP and DCF induce DNA
observed increased activity of antioxidant enzymes. On the damage in different concentrations (from ng/L to mg/L).
other hand, CAT activity was decreased by NPX exposure. On the other hand, NSAID metabolites – particularly
This result indicates this enzyme is unable to offset ROS- NSAID acyl gluconic – may be involved in adduct formation
induced damage. with DNA. The acyl glucuronides of carboxylic acids are
The comet assay allows detecting single-stranded breaks in highly reactive molecules which attack the nucleophilic
DNA, adducting formation, and DNA-DNA and protein-DNA centers of biomolecules; this is the case of DNA.
bindings (Ali et al., 2008). To evaluate more specific the Asensio et al. (2007) state that NSAIDs induce inhibition
DNA damage we introduced two enzymes, ENDO that detect of G6PD due to formation of NSAID-aryl-CoA or NSAID-
the oxidation of pyrimidine bases and FPG which detects the CoA complexes, and as a result ribose-5-phosphate synthesis
majority product of the oxidation of purine bases, the 8 - is interrupted and therefore also nucleic acid synthesis.
oxoguanine, (8-oxodG) and other alterations purine (Collins, Reactive metabolites of DCF, IBP and NPX may interact
2008). Damage to the bases in DNA in the form of 8-oxodG directly with DNA through a covalent bond, eliciting single
is a prominent indicator of oxidative stress and has been and double-strand breaks evidenced in the comet assay. As
well characterized as premutagenic injury in mammalian well as, this damage may be indirectly produced since these
cells (Bruner et al., 2000, Miller et al., 2000; Shibutani compounds are not only capable of interacting with DNA,
et al., 1991). but can also attach to the proteins in charge of DNA synthesis
DNA damage may have serious consequences such as and repair.
mutations and carcinogenic changes, and may even produce The NSAIDs such as DCF and paracetamol may induce
cell mortality. In the present study, damage to genetic material ROS formation (Gómez-Oliván et al., 2012; Islas-Flores et al.,
was determined by the comet assay and DCF, IBP, NPX and 2013; Oviedo-Gómez et al., 2010). ROS and reactive nitrogen
H2O2 were found to increase the % DNA in the tail in cells of species (RNS) are involved in DNA damage processes not
D. magna. only because they are able to interact directly with it, but also
Akcha et al. (2008) mentioned that H2O2 is a typical DNA since they affect signal transduction, cellular proliferation and
damage-inducing agent, this is a good model system to study intracellular communication. For this motive, both enzymes
the effects of oxidative stress. In this study this agent was used ENDO and FPG, which permit detection of oxidized purines
as typical genotoxic, when D. magna were exposed to 10 mM and pyrimidines by the modified comet assay, were used to
of H2O2 was observed a significant increase over the control determine if DNA damage was due to oxidation of genetic
in all exposure times without adding enzyme and with ENDO material. FPG recognizes unsaturated rings of oxidized
and FPG enzymes, this increase was time dependent. purines including 2,6-diamino-4-hydroxy-5-(N-methyl) for-
Parolini et al. (2010), indicated that paracetamol induce mamidopyrimidine and 7,8-dihydro-8-oxo-20 deoxyguanosine
moderate genotoxicity in bivalves (Dreissena polymorpha) (8-oxodG), while ENDO removes saturated rings or frag-
exposed to environmental concentrations 1–10 nM. They ments of oxidized pyrimidines (Bruner et al., 2000; Collins,
mention that DNA damage was probably due both the 2008; Miller et al., 2000).
increase in oxidative stress and/or to a direct interaction During the biotransformation of NSAID, ROS were
between its metabolite NAPQI (N-acetyl-p-benzoquinonei- generated (Gómez-Lechón et al., 2003; Masubuchi et al.,
mine) with DNA. 2002) that may affect the deoxyribose backbone and produce
In another study by Rocco et al. (2010) observed that in ruptures chain due to abstraction of a hydrogen sugar and
zebrafish exposed to IBP at the concentration of 66.40 ng/L phosphodiester bond rupture (Gregus & Klassen, 2001).
had a statistically significant DNA loss after 5 and 28 days of Our results shown, that IBP induced an increase in
exposure in 28.1 and 34.3%, respectively. DNA oxidation in both bases, indicating the existence
DNA damage was significantly affected in marine mussel of damage to DNA by oxidation. Based on these results,
(Mytilus spp) by DCF exposure in concentrations of it may be supposed that some of the DNA damage
1000 mg/L at 96 h (Schmidt et al., 2011). They mention evidenced by the comet assay may also be due to oxidative
found also LPX induction by DCF exposure after 96 h. stress. ROS may therefore be responsible for the genotoxic
In other studies biomarkers of damage (LPX and DNA activity of IBP. DCF and NPX only indicate DNA damage
damage) were significantly affected in marine mussel by oxidation of purine bases, but not by pyrimidine bases
(Mytilus spp) by DCF exposure in concentrations of oxidation.
398 L. M. Gómez-Oliván et al. Drug Chem Toxicol, 2014; 37(4): 391–399

Conclusions Doi H, Iwasaki H, Masubuchi Y, et al. (2002). Chemiluminescence

associated with the oxidative metabolism of salicylic acid in rat liver
In conclusion, DCF, IBP and NPX induces oxidative stress at microsomes. Chem Biol Int 140:109–119.
48 h. Increased reactive oxygen species may be associated Fent K, Weston AA, Caminada D. (2006). Ecotoxicology of human
pharmaceuticals. Aquat Toxicol 76:122–159.
with increased in genotoxic effect on D. magna at 48 and 96 h Feyereisen R. (2006). Evolution of insect P450. Biochem Soc Trans 34:
by DCF, IBP and NPX. The set of biomarkers used in the 1252–1255.
present study is therefore a reliable early marker for toxicity Fortier MA, Krishnaswamy K, Danyod G, et al. (2008). A postgenomic
assessment. integrated view of prostaglandins in reproduction: implications for
other body systems. J Physiol Pharmacol 59:65–89.
Gómez-Lechón M, Ponsoda X, O’Connor E, et al. (2003). Diclofenac
Declaration of interest induces in hepatocytes by alteration of mitocondrial function and
generation of ROS. Biochem Pharmacol 66:2155–2167.
The authors report no declarations of interest. Gómez-Oliván LM, Neri-Cruz N, Galar-Martı́nez M, et al. (2012).
Assessing the Oxidative Stress Induced by Paracetamol Spiked in
References Artificial Sediment on Hyalella azteca. Water Air Soil Poll 223:
5097–5104.
Ali D, Nagpure NS, Kumar S, et al. (2008). Genotoxicity assessment of Gregus Z, Klassen C. (2001). Manual de toxicologı́a. México DF: Mc
acute exposure of chlorpyrifos to freswater fish Channa punctatus Graw Hill, 461–462.
(Bloch) using micronucleus assay and alkaline single cell gel Hoeger B, Kollner B, Dietrich DR, Hitzfeld B. (2005). Water-borne
electrophoresis. Chemosphere 71:1823–1831. diclofenac affects kidney and gill integrity and selected immune
Aga D. (2008). Fate of pharmaceuticals in the environment and in water parameters in brown trout (Salmo trutta f. fario). Aquat Toxicol 75:
treatment systems. United States of America: CRC Press, 391.
53–64.
Akcha F, Arzul G, Rousseau S, Bardouil M. (2008). Comet assay in
Hoeger B, Dietrich D, Schmid D, Hartmann A. (2008). Distribution of
phytoplankton as biomarker of genotoxic effects of environmental
intraperitoneally injected diclofenac in brown trout (Salmo trutt f.
pollution. Mar Environ Res 66:59–61.
fario). Ecotox Environ Saf 71:412–418.
Ardaillou R, Baud L, Sraer J. (1987). Role of arachidonic acid
Hule RE, Padmaja S. (1993). The reaction rate of nitric oxide with
metabolites and reactive oxygen species in glomerular immune-
superoxide. Free Rad Res Commun 18:195–199.
inflammatory process. Springer Semin Immunopathol 9:371–385.
Islas-Flores H, Gómez-Oliván LM, Galar-Martı́nez M, et al.
Arkhipova OV, Grishin SN, Sitdikova GF, Zefirov AL. (2005).
(2013). Diclofenac-induced oxidative stress in brain, liver, gill
Presynaptic effects of arachidonic acid and prostaglandin E2 in the
and blood of common carp (Cyprinus carpio). Ecotox Environ
frog neuromuscular synapse. Ross Fiziol Zh Im I M Sechenova 91:
Saf 92:32–38.
268–276.
Jaeschke H, Knight TR, Bajt ML. (2003). The role of oxidant stress and
Asensio C, Levoin N, Guillaume C, et al. (2007). Irreversible inhibition
of glucose-6-phosphate dehydrogenase by the coenzyme A conjugate reactive nitrogen species in acetaminophen hepatotoxicity. Toxicol
of ketoprofen: a key to oxidative stress induced by non-steroidal anti- Lett 144:279–288.
inflammatory drugs? Biochem Pharmacol 73:405–416. Katzung B. (2007). Farmacologı́a Básica y Clı́nica. 10a edición. Ed.
Bagnyukova TV, Chahrak OI, Lushchak VI. (2006). Coordinated México: Manual Moderno.
response of goldfish antioxidant defenses to environmental stress. Lankoff A, Banasik A, Duma A, et al. (2006). A comet assay study
Aquat Toxicol 78:325–331. reveals that aluminium induces DNA damage and inhibits the repair of
Barnes KK, Kolpin DW, Furlong ET, et al. (2008). A national radiation-induced lesions in human peripheral blood lymphocytes.
reconnaissance of pharmaceuticals and other organic wastewater Toxicol Lett 161:27–36.
contaminants in the United States —I) groundwater. Sci Total Environ Láng J, Köhidai L. (2012). Effects of the aquatic contaminant human
402:192–200. pharmaceuticals and their mixtures on the proliferation and migratory
Borgmann U, Bennie DT, Ball AL, Palabrica V. (2007). Effect of a responses of the bioindicator freshwater ciliate Tetrahymena.
mixture of seven pharmaceuticals on Hyalella azteca over multiple Chemosphere 89:592–601.
generations. Chemosphere 66:1278–1283. Levine RL, Williams JA, Stadman ER, Shacter E. (1994). Carbonyl
Bradford M. (1976). A rapid an sensitive method for quantitation of assays for determination of oxidatively modified proteins. Meth
microgram quantities of protein utilizing the principle of protein dye- Enzymol 233:346–357.
binding. Anal Biochem 72:248–254. Masubuchi Y, Nakayama S, Horie T. (2002). Role of mitochondrial
Bruner SD, Norman DP, Verdine GL. (2000). Structural basis for permeability transition in diclofenac-induced hepatotoxicity in rats.
recognition and repair of the endogenous mutagen 8-oxoguanine in Hepatology 35:544–551.
DNA. Nature 403:859–866. Matozzo V, Rova S, Marin MG. (2012). The nonsteroidal anti-
Büege JA, Aust SD. (1978). Microsomal lipid peroxidation. Methods inflammatory drug, ibuprofen, affects the immune parameters in the
Enzymol 52:302–310. clam Ruditapes philippinarum. Mar Environ Res 79:116–121.
Carballa M, Omil F, Lema JM, et al. (2004). Behavior of pharmaceut- Méndez-Arriaga F, Torres-Palma RA, Pétrier C, et al. (2008). Ultrasonic
icals, cosmetics and hormones in a sewage treatment plant. Water Res treatment of water contaminated with ibuprofen. Water Res 42:
38:2918–2926. 4243–4248.
Cha YI, Solnica-Krezel L, DuBois RN. (2006). Fishing for prostanoids: Miller H, Prasad R, Wilson SH, Johnson F, Grollman AP. (2000).
deciphering the developmental functions of cyclooxygenase-derived 8-oxodGTP incorporation by DNA polymerase beta is modified by
prostaglandins. Dev Biol 289:263–272. active-site residue Asn279. Biochemistry 39:1029–1033.
Cleuvers M. (2004). Mixture toxicity of the anti-inflammatory drugs Miranda MA, Morera I, Vargas F, et al. (1991). In vitro assessment of the
diclofenac, ibuprofen, naproxen, and acetylsalicylic acid. Ecotoxicol phototoxicity of anti-inflammatory 2-arylpropionic acids. Toxicol In
Environ Saf 59:309–315. Vitro 5:451–455.
Collins A. (2008). The comet assay for DNA damage and repair. Mol Misra HP, Fridovich I. (1972). The role of superoxide anion in the
Biotechnol 26:249–261. autoxidation of epinephrine and a simple assay for superoxide
Commission of the European Communities (1996). Technical Guidance dismutase. J Biol Chem 247:3170–3175.
Document in Support of Commission Directive 93/67/EEC on Risk Mycek MJ, Harvey RA, Champe PC. (2004). Farmacologı́a. México DF:
Assessment for New Notified Substances and Commission Regulation Mc Graw Hill, 486.
(EC) No. 1488/94 on Risk Assessment. Luxembourg: Office for OECD. (2004). OECD Guidelines for the testing of Chemicals/Section 2:
Official Publications of the European Communities. Effects on biotic systems. Test No 202: Daphnia sp. Acute immo-
Deng A, Himmelsbach M, Zhu QZ, et al. (2003). Residue analysis of the bilisation test. OECD Publishing, 23 Nov 2004.
pharmaceutical diclofenac in different water types using ELISA and Oviedo-Gómez DGC, Galar-Martı́nez M, Garcı́a-Medina S, et al.
GC–MS. Environ Sci Tech 37:3422–3429. (2010). Diclofenac-enriched artificial sediment induces oxida-
Deridovich II, Reunova OV. (1993). Prostaglandins: reproduction control tive stress in Hyalella azteca. Environ Toxicol Pharmacol 29:
in bivalve mollusks. Comp Biochem Physiol A 104:23–27. 39–43.
DOI: 10.3109/01480545.2013.870191 Genotoxic response and oxidative stress induced by NSAID in D. magna 399
Paglia DE, Valentine WN. (1967). Studies on the quantitative and (Mytilus spp) and their comparison with standardized toxicity tests.
qualitative characterization of erythrocyte glutathione peroxidase. Mar Poll Bull 62:1389–1395.
J Lab Clin Med 70:158–169. Shibutani S, Takeshita M, Grollman AP. (1991). Insertion of specific
Palmer PM, Wilson LR, O’Keefe P, et al. (2008). Sources of bases during DNA synthesis past the oxidation-damaged base
pharmaceutical pollution in the New York City watershed. Sci Total 8-oxodG. Nature 349:431–434.
Environ 394:90–102. Siemens J, Huschek G, Siebe C, Kaupenjohann M. (2008).
Parvez S, Raisuddin S. (2005). Protein carbonyls: novel biomarkers of Concentrations and mobility of human pharmaceuticals in the
exposure to oxidative stress-inducing pesticides in freshwater fish world’s largest wastewater irrigation system, Mexico City-Mezquital
Channa punctata (Bloch). Environ Toxicol Pharmacol 20:112–117. Valley. Water Res 42:2124–2134.
Parolini M, Binelli A, Cogni D, et al. (2009). An in vitro biomarker Sinha S, Mallick S, Misra RK, et al. (2007). Uptake and translocation of
approach for the evaluation of the ecotoxicity of non-steroidal anti- metals in Spinacia oleracea L. grown on tannery sludge-amended and
inflammatory drugs (NSAIDs). Toxicol In Vitro 23:935–942. contaminated soils: effect on lipid peroxidation, morpho-anatomical
Parolini M, Binelli A, Cogni D, Provoini A. (2010). Multi-biomarker changes and antioxidants. Chemosphere 67:176–187.
approach for the evaluation of the cyto-genotoxicity of paracetamol on Shacter E. (2000). Quantification and significance of protein oxidation in
the zebra mussel (Dreissena polymorpha). Chemosphere 79:489–498. biological samples. Drug Metab Rev 32:307–326.
Parolini M, Binelli A, Provoini A. (2011). Assessment of the potential Stanley DW. (2000). Eicosanoids in invertebrate signal transduction
cyto-genotoxicity of the nonesteroidal anti-inflammatory drug systems. New Jersey: Princeton University Press.
(NSAID) diclofenac on the zebra mussel (Dreissena polymorpha). Tang W. (2003). The metabolism of diclofenac enzymology and
Water Air Soil Poll 217:589–601. toxicology perspectives. Curr Drug Met 4:319–329.
Paul-Clark MJ, van Cao T, Moradi-Bidhendi N, et al. (2004). 15-epi- Ternes TA. (1998). Occurrence of drugs in German sewage treatment
lipoxin A4-mediated induction of nitric oxide explains how aspirin plants and rivers. Water Res 32:3245–3260.
inhibits acute inflammation. J Exp Med 200:69–78. Tice R, Anderson D, Burlinson D, et al. (2000). The single cell gel/comet
Poynton HC, Vulpe CD. (2009). Ecotoxicogenomics: emerging technol- assay; guidelines for in vitro and vivo genetic toxicology testing.
ogies for emerging contaminants. J Am Water Res Assoc 45:83–95. Environ Mol Mut 35:206–221.
Quinn B, Gagné F, Blaise C. (2008). The effects of pharmaceuticals on Togola A, Budzinski H. (2008). Multi-residue analysis of pharmaceutical
the regeneration of the cnidarians, Hydra attenuata. Sci Total Environ compounds in aqueous samples. J Chromatogr 1177:150–158.
402:62–69. Vlahogianni T, Dassenakis M, Scoullos MJ, Valavanidis A. (2007).
Radi R, Turrens JF, Chang LY, et al. (1991). Detection of catalase in rat Integrated use of biomarkers (superoxide dismutase, catalase and lipid
heart mitochondria. J Biol Chem 26:22028–22034. peroxidation) in mussels Mytilus galloprovincialis for assessing heavy
Regoli F, Gorbi S, Frenzilli G, et al. (2002). Oxidative stress in metals’ pollution in coastal areas from the Saronikos Gulf of Greece.
ecotoxicology: from the analysis of individual antioxidants to a more Mar Pollut Bull 54:1361–1371.
integrated approach. Mar Environ Res 54:419–423. Vulliet E, Cren-Olivé C. (2011). Screening of pharmaceuticals and
Richardson SD. (2007). Water analysis: emerging contaminants and hormones at the regional scale, in surface and groundwaters intended
current issues. Anal Chem 79:4295–4324. to human consumption. Environ Poll 159:2929–2934.
Richardson ML, Bowron JM. (1985). The fate of pharmaceutical Wang C, Shi H, Adams CD, et al. (2011). Investigation of pharmaceut-
chemicals in the aquatic environment. J Pharm Pharmacol 37:1–12. icals in Missouri natural and drinking water using high performance
Rocco L, Frenzilli G, Fusco D, et al. (2010). Evaluation of zebrafish liquid chromatography-tandem mass spectrometry. Water Res 45:
DNA integrity after exposure to pharmacological agents present in 1818–1828.
aquatic environments. Ecotox Environ Saf 73:1530–1536. Watkinson AJ, Murby EJ, Kolpin DW, Costanzo SD. (2009). The
Salgueiro-Pagadigorria C, Kelmer-Bracht A, Bracht A, Ishii-Iwamoto E. occurrence of antibiotics in an urban watershed: from wastewater to
(2004). Naproxen affects Ca2þ fluxes in mitochondria, microsomes drinking water. Sci Total Environ 407:2711–2723.
and plasma membrane vesicles. Chem Biol Interact 147:49–63. Yu CP, Chu KH. (2009). Occurrence of pharmaceuticals and personal
Schmidt W, O’Rourke K, Hernan R, Brian Q. (2011). Effects of the care products along the West Prong Little Pigeon River in east
pharmaceuticals gembribozil and diclofenac on the marine mussel Tennessee, USA. Chemosphere 75:1281–1286.