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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. 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