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Comparative Biochemistry and Physiology Part C 136 (2003) 127–134
The heart of Daphnia magna: effects of four cardioactive drugs
Arturo Villegas-Navarroa, *, Esperanza Rosas-La, Jose´ L. Reyesb
a
´ Ambiental, 95 Oriente No. 1649, Col. Granjas de San Isidro, Puebla Pue C.P. 72590, Mexico
Laboratorio de Investigacion
b
´ y Estudios Avanzados, IPN, Mexico DF, Mexico
Physiology and Biophysics Department, Centro de Investigacion
Received 15 May 2003; received in revised form 29 July 2003; accepted 30 July 2003
Abstract
We used Daphnia magna bioassays to determine the LC50 and the effects on the heart of the cardioactive drugs
ouabain, verapamil, metaproterenol and metoprolol. Distinctions were made between the pharmacological and toxicological effects of these drugs and the adequacy of physicochemical characteristics of its habitat (reconstituted water). Video
microscopy and digital image processing were used to study the pharmacological effects on the heart. D. magna exhibited
the expected sensitivity to the reference toxicant sodium dodecyl sulfate with a LC50 of 15.6"4.5 mgyl. All drugs were
toxic with 48 h-LC50 of 2.03 mgyl ouabain, 7.04 mgyl verapamil, 32.45 mgyl metaproterenol and 76.21 mgyl metoprolol.
Ouabain was the most toxic and caused a positive concentration-dependent inotropic effect. Verapamil caused positive
chronotropic and inotropic effects, while metaproterenol showed positive concentration-dependent chronotropic effects at
high concentrations (10y3 and 10y4 M). Metoprolol induced a positive chronotropic effect at low concentrations (10y8,
10y7, 10y6 M) and a negative chronotropic effect at high concentration (10y4 M). Ouabain, metaproterenol and
metoprolol in D. magna caused similar effects to those produced in mammals. In contrast, verapamil caused opposite
effects. The results suggest the presence of Naq, Kq-ATPase receptors to verapamil and of non-specific adrenergic
receptors in heart of D. magna.
䊚 2003 Elsevier Inc. All rights reserved.
Keywords: Contraction strength; Daphnia magna; Heart rate; Metaproterenol; Metoprolol; Ouabain; Verapamil
1. Introduction
Pharmacology has made tremendous strides in
the sophistication of the models used to identify
and understand the mechanisms of action of agents
and in diversification of specific models to study
one determined pharmacological effect (Van der
Kloot, 1967; Clemedson and Ekwall, 1999; Guilhermino et al., 2000). Furthermore, for reasons of
concern about animal welfare, economics and the
need for greater sensitivity and further understanding of pharmacodynamics, the interest in in vitro
*Corresponding author. Tel.yfax: q52-222-2-45-77-40.
E-mail address: [email protected]
(A. Villegas-Navarro).
models has been increased (Ekwall, 1999; Keddy
et al., 1995).
One kind of in vitro assay is the use of invertebrates (Yeoman and Faragher, 2001). These
organisms have less developed cortical and sensorial systems and are characterized by fast tissue
regeneration. Daphnia magna has been used to
study drugs with cardiac action such as acetylcholine, tetraethylpyrophosphate, pilocarpine, adrenaline and rotenone. They caused negative
chronotropic effects on D. magna’s heart, while
atropine had a positive chronotropic action and
adrenaline accelerated cardiac rate only at high
concentrations (Bekker and Krijgsman, 1951).
Crozier et al. (1999) reported that nicotine slowed
1532-0456/03/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S1532-0456(03)00172-8
128
A. Villegas-Navarro et al. / Comparative Biochemistry and Physiology Part C 136 (2003) 127–134
D. magna’s heart rate, while ethanol raised it.
Postmes et al. (1989) reported that agonists and
antagonists were either inactive or lowered the
heart frequency and the negative chronotropic
effect of epinephrine could not be blocked by the
antagonist propranolol.
D. magna bioassay is recommended as advantageous over other models for assessment of aquatic toxicity (Environment Canada, 1990) but it
remains to be shown if bioassays with D. magna
are feasible and show advantages in pharmacological studies. One advantage that can be pointed
out is that D. magna are transparent (Chapman,
1976). Transparency is a useful characteristic to
obtain insights into animal physiology, as it allows
the researcher to apply optical methods to visualize
physiological functions and to measure several
¨
different parameters simultaneously (Rudiger
et
al., 1997) and non-invasive method in physiological research (Colmorgen et al., 1995).
The purpose of the present study is to evaluate
the D. magna model to study drugs with action
upon the heart, and was chosen because of its size
and because this organ can be easily observed by
optical methodologies.
2. Materials and methods
D. magna was cultured for several generations
in hard-reconstituted water as previously described
(Villegas-Navarro et al., 1997). The quality criteria
applied to the culture of D. magna were those
published by Poirier et al. (1988). For the toxicity
tests, neonates of the 2nd–6th generations with
less 24 h old were used. Hard-reconstituted water
for the cultures was prepared in agreement to
Mexican Official Norm NMX-AA-087 (SCFI,
1995) and was aerated for 48 h to obtain O2
concentrations exceeding 3 mgyl (Conductronic
Model Ox25). The reconstituted water had the
following physicochemical parameters: pH 7.5–
8.5 (Corning Model 7), hardness of 160–180 mgy
l expressed as CaCO3 (Fritz and Schenk, 1969),
conductivity of 250–600 mSycm (YSI Model
520A) and temperature 20"3 8C. These are adequate conditions for the growth of this species. D.
magna were set in the container for reproduction
and growth along with 1000 ml of reconstituted
water, which was changed once a week. Chlorella
vulgaris was used to feed the D. magna (Naylor
et al., 1993) and was cultivated according to Stein
(1973), then concentrated and the pellets were
kept at 4 8C. Before use, the pellets were diluted
in 50 ml of reconstituted water (Villegas-Navarro
et al., 1997).
2.1. Forty eight hours-LC50 determination
Ten neonates were placed in each 150 ml containers containing 100 ml of the drug solution
dissolved in reconstituted water. All assays and a
control group were made in triplicate, except for
the sodium dodecyl sulfate (SDS, Sigma, St. Louis, MO) tests, which were done in duplicate. A
24-h preliminary test was carried out to determine
if the drug produced any effect on the physicochemical characteristics of the solution and to
determine the concentration range to be used in
the definitive test. The criterion for a valid bioassay was an unmoving rate of less than 10% in the
control group. In the definitive test, the minimum
number of dilutions was five plus the control
group. Immobile organisms were counted after 48
h to calculate 48 h-LC50.
2.2. SDS assay
SDS was used to determine the sensitivity of
the D. magna to this chemical according to recommended procedures (Lewis and Horning, 1991).
The LC50 for SDS was established in a 48 h
bioassay in advance to the pharmacological bioassays. The concentrations were 4, 8, 16, 32 and
64 mgyl.
2.3. Drugs
The pharmacological solutions used were: Ouabain, (")-verapamil hydrochloride, metaproterenol-hemisulfate and (")-metoprolol (q)-tartrate
(SIGMA, St. Louis, MO). They were selected by
the following criteria: (1) by their site of action;
(2) they are prototypes of their action mechanisms;
(3) they are used in clinical practice; and (4) they
are soluble in reconstituted water. Before performing bioassays the basic physicochemical parameters of the drug solutions were measured to assess
that D. magna were in a satisfactory habitat to
study their normal physiological function and to
discriminate between any abnormal function due
to physicochemical parameters and the pharmacological effect. The drug solutions were placed
under experimental conditions of light and air
environmental.
A. Villegas-Navarro et al. / Comparative Biochemistry and Physiology Part C 136 (2003) 127–134
2.4. Experimental set-up
The main component was an inverted microscope (IROSCOPE Model SI-PH), a digital video
camera (PANASONIC, Model GP-KR222), a videotape recorder VHS (SONY Model SLV-LX7S)
and an assembled computer. Image information
was stored using VHS videotape recorders and
was displayed on a monitor. One recorder stored
the original video information from the camera,
while the other stored the processed data in the
computer. The software Asymetrix and Corel Draw
in a PC slot were used for on-line image
processing.
2.5. Recording of cardiac events
D. magna of 10 days old were used. If most of
the water is withdrawn, the animals are maintained
in place by the surface tension of the remaining
fluid and unable to displace out of the microscope’s field. Then, the frequency and the type of
muscular contraction can be observed; the area of
the heart in systole and diastole served to judge
the magnitude of contraction. It could be discriminated between normal and increased force of beat
(least area ‘strong’) and contractions in dilated
position (major area ‘weak’), a criterion used by
echocardiography (Yaoita et al., 2002). Irregularity
of rhythm could be clearly discriminated. D. magna were maintained separately in 50 ml of drug
solution, in beakers at room temperature (20"3
8C), during the experiment.
2.6. Image processing
The image processor allowed real time operations (i.e. 25 framesys with a PAL or CCIR video
camera as the input source), which was essential
for analyzing fast heart movements. An electronic
circuit on the card ensured jitter-free image captures, also from video recorders. This allowed the
acquisition of already-processed and recorded data
replayed on a video recorder, thus starting a further
processing step. In this way, the implementation
of complex algorithms is possible, if the operation
was divided into sequentially executable parts.
2.7. Area calibration
To measure areas an inverted microscope was
used (100=, eyepiece and objective) and calibrat-
129
ed using a 10 mm rule on the microscope stage
and storing that image as a file in the computer.
One frame was selected with Asymetrix Software
and transported to Corel Draw up to subfile Photo
Paint and used to measure areas. In Photo Paint
the file-ruling image was compared to Photo Paint
scale throughout superimposing their images (10
mms9.25 mm) and heart area was estimated as a
ellipse. In this way readings can be made more
conveniently and with greater accuracy.
2.8. Statistics
All data were assessed for homogeneity of
variance to ensure that assumptions of analysis of
variance ANOVA were met. The data were analyzed by one-way ANOVA and the Tukey’s test to
narrow down which columns were significantly
different from other columns (GraphPad InStat,
Windows 95). For 48 h-LC50 the Probit method
(Fevrier, 1987) was used. The significance level
was set at P-0.05s*; PF0.01s**; PF0.001s
***. The graphic results are shown normalized:
decrease of the areas1yvycv; dispersion values
S.E.M.yvc, where cvscontrol value; vsvalue for
each experimental group and S.E.M.sstandard
error for means.
3. Results
48 h-LC50 using SDS was 15.6"4.5 mgyl and
did not differ significantly from the reference result
of 14.5"4.5 mgyl (SCFI, 1995).
Table 1 shows a summary of the physicochemical parameters for the four drug solutions. There
were no differences between physical and chemical
characteristics of these four drug solutions and the
recommended test conditions for D. magna, neither
during change of concentration nor for change of
drugs.
The ultraviolet spectrophotometric readings for
ouabain at 272, verapamil at 278, metaproterenol
at 282 and metoprolol at 223 nm indicated chemical instability at concentrations of 10 mgyl, under
light and air surrounding during 48 h (Table 2).
The later column shows in terms of percentage the
change in spectrophotometric reading indicating
they were satisfactory for ouabain, verapamil,
metaproterenol and unacceptable for metoprolol
()20%).
Table 3 shows the 48 h-LC50 values for D.
magna, based on nominal concentrations and the
A. Villegas-Navarro et al. / Comparative Biochemistry and Physiology Part C 136 (2003) 127–134
130
Table 1
Initial physicochemical properties of test drug solutions (10 mgyl)
Parameters
Ouabain
Verapamil
Metaproterenol
Metoprolol
pH
Dissolved O2 (mgyl)
Total hardness
(mgyl as CaCO3)
Conductivity (mSycm)
Temperature (8C)
7.6
8.4
184
7.7
8.8
182
7.5
9.2
174
7.7
8.8
180
513
23
516
23
516
23
510
23
Table 2
Ultraviolet spectrophotometric readings (absorbance) at several time intervals
Drug (10 mgyl)
0h
24 h
48 h
Change%
Ouabain
Verapamil
Metaproterenol
Metoprolol
0.070
0.112
0.066
0.030
0.075
0.116
0.074
0.035
0.080
0.118
0.074
0.040
14.3
5.3
12.0
33.3
Table 3
Forty-eight hours-LC50 value"95% confidence limits for cardiac drugs tested against D. magna and LD50 i.v. in rats
Drug
48 h-LC50"C.L.95%
(mgyl)
LD50 (i.v.)a
(mgykg)
Ouabain
Verapamil
Metaproterenol
Metoprolol
2.0"0.2
7.0"0.3
32.4"4.2
76.2"5.7
14.0
16.0
42.0b
90.0
a
b
(Merck Index, 1989).
Orally in rats.
Fig. 1. Concentration–response curve for ouabain. D. magna
were exposed for 2 h to ouabain from 10y9 to 10y5 M. Effects
were observed individually under microscope and recorded for
quantitative analyses. Mean"S.E.M. are shown normalizing.
LD50 in rats collected from different sources in the
literature (Merck Index, 1989). The increasing
order of toxicity for four drugs is the same for
both species.
Fig. 1 shows the average effects on six hearts
of ouabain on area values during systole. Ouabain
produced a positive inotropic effect in a dosedependent manner and no effect was observed on
diastole and heart rate. Systole of D. magna was
sensitive to ouabain at 10y6 and 10y5 M and its
response was significant. No irregularities were
observed in heartbeat after 2 h immersions at these
high concentrations and no diastolic arrest occurred
after 24 h in reconstituted water. On diastole,
ouabain produced less area, but was not
significative.
Verapamil caused dose-dependent acceleration
in D. magna heart, from 10y7 to 10y5 M; at
10y4 M it caused slowing (Fig. 2a), so that the
frequency decreased by approximately 9% as an
adverse reaction since it increased in a timedependent manner (Rozman and Doull, 2001).
Verapamil caused increments in the amplitude of
systolic contraction (reduced area) at low concentrations reaching a plateau at 10y5 M (Fig. 2b).
Contrarily, in diastole it induced a negative inotropic effect at 10y7 and 10y6 M and this effect was
reversed to control values at 10y5 and 10y4 M
(Fig. 2c).
The average heart rate increased 5% and 20%
from the control heart rate when D. magna were
treated with metaproterenol, 10y6 M and 10y4 M,
respectively (Fig. 3a). Metaproterenol had no
activity at any dose upon systole and diastole.
Metoprolol induced a positive chronotropic
effect only at the lowest concentration (10y8 M),
while high concentrations caused a gradually progressive slowing, so that the frequency decreased
significantly 32% at 10y4 M (Fig. 4a). Systole
was not changed, whereas diastole at 10y8 M
A. Villegas-Navarro et al. / Comparative Biochemistry and Physiology Part C 136 (2003) 127–134
131
the conformity with the procedure, as well as the
validity of the results, thereupon, these results can
be validly compared with other studies on drugs
sensitivity (ISO, 1982). The 48 h-LC50 value for
SDS obtained in this work did not differ statistically from previous reports (Villegas-Navarro et
al., 1999).
Environmental conditions used in the experiments had no significant influence on D. magna
heart activity, since only minor differences were
observed due to daily methodological variation
(Table 1). The conditions used in the experiments
can, therefore, be considered satisfactory to the
organisms.
Ultraviolet spectrophotometric readings support
a minor chemical instability of aqueous solutions
for ouabain, verapamil and metaproterenol
(-14.3%) and larger for metoprolol (s33.3%),
under the conditions of light and air exposition
during 48 h, the time required to define the 48 hLC50 (Table 2). Color and transparency characteristics showed no changes in the drug solutions
indicating that the LC50 were not significantly
affected by the unstability of the chemicals. This
chemical instability is time-dependent since it
increases with time. The OECD (1995) guideline
indicates that the concentration of test substances
must remain within 80% of the nominal concentration. The chemical instability was negligible on
our pharmacological studies since they took only
2 h.
Fig. 2. Concentration–response curve for verapamil. D. magna
were exposed for 2 h to verapamil from 10y7 to 10y4 M.
Mean"S.E.M. are shown normalizing for systole and diastole.
induced a reduced area that was not observed at
higher doses (Fig. 4b).
4. Discussion
According to ISO description the SDS standard
toxic is used to verify the sensitivity of D. magna,
Fig. 3. Concentration–response curve for metaproterenol. D.
magna were exposed for 2 h to metaproterenol from 10y6 to
10y3 M. Mean"S.E.M. are shown.
132
A. Villegas-Navarro et al. / Comparative Biochemistry and Physiology Part C 136 (2003) 127–134
Fig. 4. Concentration–response curve for metoprolol. D. magna were exposed for 2 h to metoprolol from 10y8 to 10y4 M. Mean"S.E.M.
are shown normalizing for diastole.
As expected from data obtained in mammals,
the four drugs tested were toxic to D. magna.
Ouabain was the most toxic and metoprolol was
the least toxic. The resemblance in the sequences
of lethality by the four drugs for D. magna and
rats seems interesting (Table 3). This resemblance
is relative but meaningful, since it can indicate
similarity in toxicological sensitivity and mechanisms of action (vide supra). Rozman and Doull
(2001) give evidences of the role of time as a
quantifiable variable of toxicity and just as D.
magna and rat experiments were done under different experimental conditions of time and routes
of administration, further speculations might be
hazardous.
Ouabain, metaproterenol and metoprolol caused
similar effects on heart of D. magna to those
observed on humans. Ouabain exerted positive
systolic inotropic effects. This similarity in effects,
plus the fact that D. magna heart is myogenic
(Bekker and Krijgsman, 1951) and the presence
of Naq, Kq-ATPase in crustacean heart (Siebers
et al., 1982), suggests a mechanism of action
similar to those in mammals heart at least for
ouabain.
Verapamil exerted positive inotropic and chronotropic effects, exactly the opposite effects caused
´
´
in humans (Fernandez-Gonzalez
et al., 2000). The
response was significant at 10y7 M. This peculiar
effect was reverted at high concentrations, with
detriment of heart rate, an habitual component in
intoxicated mammals (Tanen et al., 2000). The
specific response of D. magna heart to low con-
centration suggests the presence of a verapamil
receptor. Invertebrate Ca2q channel a1 subunits
have been isolated and cloned and there are reports
that crustaceans are insensitive to organic Ca2q
channel blockers, but sensitive to inorganic Ca2q
channel blocker, such as Sr2q (Jeziorski et al.,
1998), suggesting the presence of Ca2q channels
in D. magna heart. These data support the suggestion that D. magna heart might have verapamil
receptors that are different functionally from receptors of mammal’s heart.
Metaproterenol was either ineffective or accelerated the cardiac frequency only at high concentrations (10y4 and 10y3 M). These results are in
agreement with those of Bekker and Krijgsman
(1951) and Postmes et al. (1989) who used adrenaline andyor its agonists and antagonists, respectively. However, the usual response of the heart of
other arthropods to adrenaline is acceleration at
low doses and metaproterenol at a concentration
lower than 10y5 M, increased canine heart rate by
10% and the cardiac output also increased significantly (Casthely et al., 1985). Therefore, D. magna
heart behaves different from other arthropods heart
and response was similar to mammal’s heart at
high doses only.
Metoprolol antagonist caused significant acceleration with increased amplitude of contraction at
10y8 M and subsequently significant decrease
when given at high concentration (10y3 M); similar effect was elicited in rats (Yaoita et al., 2002).
Between these two extremes it had no significant
effects. Mosby’s (1999) reported that metoprolol
A. Villegas-Navarro et al. / Comparative Biochemistry and Physiology Part C 136 (2003) 127–134
tartrate induced reduction in heart rate, cardiac
output and systolic blood pressure in most patients
and had not intrinsic sympathomimetic activity in
humans, but as this work showed, in D. magna’s
heart it had sympathomimetic activity at 10y8 M.
It is difficult to suggest a satisfactory explanation for the action of metaproterenol and metoprolol. The heart of most mammalian is accelerated
by adrenaline, an effect obtained on the heart of
D. magna only with high concentration of adrenaline, metaproterenol and metoprolol (antagonist
action). It is unlikely that the accelerating action
of metaproterenol or the delaying action of metoprolol are of physiological importance, since they
occurred only at high concentrations, therefore, it
might occur that these drug actions were not
mediated through specific adrenoceptors.
It is clear that D. magna’s heart is no similar to
the heart of mammals, arthropods and other crustaceans. However, D. magna has other advantages
for pharmacological studies, not reported until
now, such as the similarity of the response at
mammal heart for ouabain, which can be exploited
for the best understanding of his mechanism of
action; its peculiar response to verapamil, possess
unique characteristics, which seem to make this
particular animal singularly suit the specific scientific purpose to study its calcium channel and
the similarity of response for agonist and antagonist adrenergic at high doses. Moreover, D. magna
bioassay have several practical advantages, such
as: simplicity to culture them in laboratory, short
life cycle, discrete growth, transparency, easy in
handling and low cost of maintenance. This invertebrate have, important unresolved pharmacological characteristics that must help in the
understanding of the mechanisms of action of the
drugs here studied and other important ones for
the man.
Acknowledgments
This work was supported by grants from the
CONACYT. The authors thank Dr Vicente Her´
nandez
for critical remarks, Dr P Wesche for
critical comments on the manuscript and Dr Mario
Del Valle, for technical assistance.
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