The Heart of Daphnia Magna: Effects of Four Cardioactive Drugs

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-Lab , Jose´ L. Reyes

aLaboratorio de Investigacion´ Ambiental, 95 Oriente No. 1649, Col. Granjas de San Isidro, Puebla Pue C.P. 72590, Mexico bPhysiology and Biophysics Department, Centro de Investigacion´ y Estudios Avanzados, IPN, Mexico DF, Mexico

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 Naqq , K -ATPase receptors to verapamil and of non-specific adrenergic receptors in heart of D. magna. ᮊ 2003 Elsevier Inc. All rights reserved.

Keywords: Contraction strength; Daphnia magna; Heart rate; Metaproterenol; Metoprolol; Ouabain; Verapamil

1. Introduction models has been increased (Ekwall, 1999; Keddy et al., 1995). Pharmacology has made tremendous strides in One kind of in vitro assay is the use of inver- the sophistication of the models used to identify tebrates (Yeoman and Faragher, 2001). These and understand the mechanisms of action of agents organisms have less developed cortical and sen- and in diversification of specific models to study sorial systems and are characterized by fast tissue one determined pharmacological effect (Van der regeneration. Daphnia magna has been used to Kloot, 1967; Clemedson and Ekwall, 1999; Guil- study drugs with cardiac action such as acetylcho- hermino et al., 2000). Furthermore, for reasons of line, tetraethylpyrophosphate, pilocarpine, adrena- concern about animal welfare, economics and the line and rotenone. They caused negative need for greater sensitivity and further understand- chronotropic effects on D. magna’s heart, while ing of pharmacodynamics, the interest in in vitro atropine had a positive chronotropic action and q adrenaline accelerated cardiac rate only at high *Corresponding author. Tel.yfax: 52-222-2-45-77-40. ( ) E-mail address: [email protected] concentrations Bekker and Krijgsman, 1951 . (A. Villegas-Navarro). Crozier et al. (1999) reported that nicotine slowed

1532-0456/03/$- see front matter ᮊ 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. kept at 4 8C. Before use, the pellets were diluted Postmes et al. (1989) reported that agonists and in 50 ml of reconstituted water (Villegas-Navarro antagonists were either inactive or lowered the et al., 1997). heart frequency and the negative chronotropic effect of epinephrine could not be blocked by the 2.1. Forty eight hours-LC50 determination antagonist propranolol. D. magna bioassay is recommended as advan- Ten neonates were placed in each 150 ml con- tageous over other models for assessment of aquat- tainers containing 100 ml of the drug solution ic toxicity (Environment Canada, 1990) but it dissolved in reconstituted water. All assays and a remains to be shown if bioassays with D. magna control group were made in triplicate, except for are feasible and show advantages in pharmacolog- the sodium dodecyl sulfate (SDS, Sigma, St. Lou- ical studies. One advantage that can be pointed is, MO) tests, which were done in duplicate. A out is that D. magna are transparent (Chapman, 24-h preliminary test was carried out to determine 1976). Transparency is a useful characteristic to if the drug produced any effect on the physico- obtain insights into animal physiology, as it allows chemical characteristics of the solution and to the researcher to apply optical methods to visualize determine the concentration range to be used in physiological functions and to measure several the definitive test. The criterion for a valid bioas- different parameters simultaneously (Rudiger¨ et say was an unmoving rate of less than 10% in the al., 1997) and non-invasive method in physiolog- control group. In the definitive test, the minimum ical research (Colmorgen et al., 1995). number of dilutions was five plus the control The purpose of the present study is to evaluate group. Immobile organisms were counted after 48 the D. magna model to study drugs with action h to calculate 48 h-LC50 . upon the heart, and was chosen because of its size and because this organ can be easily observed by 2.2. SDS assay optical methodologies. SDS was used to determine the sensitivity of 2. Materials and methods the D. magna to this chemical according to recommended procedures (Lewis and Horning, 1991).

D. magna was cultured for several generations The LC50 for SDS was established in a 48 h in hard-reconstituted water as previously described bioassay in advance to the pharmacological bio- (Villegas-Navarro et al., 1997). The quality criteria assays. The concentrations were 4, 8, 16, 32 and applied to the culture of D. magna were those 64 mgyl. published by Poirier et al. (1988). For the toxicity tests, neonates of the 2nd–6th generations with 2.3. Drugs less 24 h old were used. Hard-reconstituted water for the cultures was prepared in agreement to The pharmacological solutions used were: Oua- Mexican Official Norm NMX-AA-087 (SCFI, bain, (")-verapamil hydrochloride, metaproteren- ) ( ) (q) 1995 and was aerated for 48 h to obtain O2 ol-hemisulfate and " -metoprolol -tartrate concentrations exceeding 3 mgyl (Conductronic (SIGMA, St. Louis, MO). They were selected by Model Ox25). The reconstituted water had the the following criteria: (1) by their site of action; following physicochemical parameters: pH 7.5– (2) they are prototypes of their action mechanisms; 8.5 (Corning Model 7), hardness of 160–180 mgy (3) they are used in clinical practice; and (4) they ( ) l expressed as CaCO3 Fritz and Schenk, 1969 , are soluble in reconstituted water. Before perform- conductivity of 250–600 mSycm (YSI Model ing bioassays the basic physicochemical parame- 520A) and temperature 20"3 8C. These are ade- ters of the drug solutions were measured to assess quate conditions for the growth of this species. D. that D. magna were in a satisfactory habitat to magna were set in the container for reproduction study their normal physiological function and to and growth along with 1000 ml of reconstituted discriminate between any abnormal function due water, which was changed once a week. Chlorella to physicochemical parameters and the pharmaco- vulgaris was used to feed the D. magna (Naylor logical effect. The drug solutions were placed et al., 1993) and was cultivated according to Stein under experimental conditions of light and air (1973), then concentrated and the pellets were environmental. A. Villegas-Navarro et al. / Comparative Biochemistry and Physiology Part C 136 (2003) 127–134 129

2.4. Experimental set-up ed using a 10 mm rule on the microscope stage and storing that image as a file in the computer. The main component was an inverted micro- One frame was selected with Asymetrix Software scope (IROSCOPE Model SI-PH), a digital video and transported to Corel Draw up to subfile Photo camera (PANASONIC, Model GP-KR222), a vid- Paint and used to measure areas. In Photo Paint eotape recorder VHS (SONY Model SLV-LX7S) the file-ruling image was compared to Photo Paint and an assembled computer. Image information scale throughout superimposing their images (10 was stored using VHS videotape recorders and mms9.25 mm) and heart area was estimated as a was displayed on a monitor. One recorder stored ellipse. In this way readings can be made more the original video information from the camera, conveniently and with greater accuracy. while the other stored the processed data in the computer. The software Asymetrix and Corel Draw 2.8. Statistics in a PC slot were used for on-line image processing. All data were assessed for homogeneity of variance to ensure that assumptions of analysis of 2.5. Recording of cardiac events variance ANOVA were met. The data were ana- lyzed by one-way ANOVA and the Tukey’s test to D. magna of 10 days old were used. If most of narrow down which columns were significantly the water is withdrawn, the animals are maintained different from other columns (GraphPad InStat, in place by the surface tension of the remaining Windows 95). For 48 h-LC50 the Probit method fluid and unable to displace out of the micro- (Fevrier, 1987) was used. The significance level scope’s field. Then, the frequency and the type of was set at P-0.05s*; PF0.01s**; PF0.001s muscular contraction can be observed; the area of ***. The graphic results are shown normalized: the heart in systole and diastole served to judge decrease of the areas1yvycv; dispersion values the magnitude of contraction. It could be discrim- S.E.M.yvc, where cvscontrol value; vsvalue for inated between normal and increased force of beat each experimental group and S.E.M.sstandard (least area ‘strong’) and contractions in dilated error for means. position (major area ‘weak’), a criterion used by echocardiography (Yaoita et al., 2002). Irregularity 3. Results of rhythm could be clearly discriminated. D. magna were maintained separately in 50 ml of drug 48 h-LC50 using SDS was 15.6"4.5 mgyl and solution, in beakers at room temperature (20"3 did not differ significantly from the reference result 8C), during the experiment. of 14.5"4.5 mgyl (SCFI, 1995). Table 1 shows a summary of the physicochem- 2.6. Image processing ical parameters for the four drug solutions. There were no differences between physical and chemical The image processor allowed real time opera- characteristics of these four drug solutions and the tions (i.e. 25 framesys with a PAL or CCIR video recommended test conditions for D. magna, neither camera as the input source), which was essential during change of concentration nor for change of for analyzing fast heart movements. An electronic drugs. circuit on the card ensured jitter-free image cap- The ultraviolet spectrophotometric readings for tures, also from video recorders. This allowed the ouabain at 272, verapamil at 278, metaproterenol acquisition of already-processed and recorded data at 282 and metoprolol at 223 nm indicated chem- replayed on a video recorder, thus starting a further ical instability at concentrations of 10 mgyl, under processing step. In this way, the implementation light and air surrounding during 48 h (Table 2). of complex algorithms is possible, if the operation The later column shows in terms of percentage the was divided into sequentially executable parts. change in spectrophotometric reading indicating they were satisfactory for ouabain, verapamil, 2.7. Area calibration metaproterenol and unacceptable for metoprolol ()20%).

To measure areas an inverted microscope was Table 3 shows the 48 h-LC50 values for D. used (100=, eyepiece and objective) and calibrat- magna, based on nominal concentrations and the 130 A. Villegas-Navarro et al. / Comparative Biochemistry and Physiology Part C 136 (2003) 127–134

Table 1 Initial physicochemical properties of test drug solutions (10 mgyl)

Parameters Ouabain Verapamil Metaproterenol Metoprolol pH 7.6 7.7 7.5 7.7 ( ) Dissolved O2 mgyl 8.4 8.8 9.2 8.8 Total hardness 184 182 174 180 ( ) mgyl as CaCO3 Conductivity (mSycm) 513 516 516 510 Temperature (8C) 23 23 23 23

Table 2 LD in rats collected from different sources in the ( ) 50 Ultraviolet spectrophotometric readings absorbance at sev- literature (Merck Index, 1989). The increasing eral time intervals order of toxicity for four drugs is the same for Drug (10 mgyl) 0h 24 h 48 h Change% both species. Ouabain 0.070 0.075 0.080 14.3 Fig. 1 shows the average effects on six hearts Verapamil 0.112 0.116 0.118 5.3 of ouabain on area values during systole. Ouabain Metaproterenol 0.066 0.074 0.074 12.0 produced a positive inotropic effect in a dose- Metoprolol 0.030 0.035 0.040 33.3 dependent manner and no effect was observed on diastole and heart rate. Systole of D. magna was y6 y5 Table 3 sensitive to ouabain at 10 and 10 M and its

Forty-eight hours-LC50 value"95% confidence limits for car- response was significant. No irregularities were diac drugs tested against D. magna and LD50 i.v. in rats observed in heartbeat after 2 h immersions at these

( )a high concentrations and no diastolic arrest occurred Drug 48 h-LC50"C.L.95% LD50 i.v. (mgyl)(mgykg) after 24 h in reconstituted water. On diastole, ouabain produced less area, but was not Ouabain 2.0"0.2 14.0 Verapamil 7.0"0.3 16.0 significative. Metaproterenol 32.4"4.2 42.0b Verapamil caused dose-dependent acceleration Metoprolol 76.2"5.7 90.0 in D. magna heart, from 10y7 to 10y5 M; at y4 a (Merck Index, 1989). 10 M it caused slowing (Fig. 2a), so that the b Orally in rats. frequency decreased by approximately 9% as an adverse reaction since it increased in a time- dependent 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 pro- Fig. 1. Concentration–response curve for ouabain. D. magna were exposed for 2 h to ouabain from 10y9 to 10y5 M. Effects gressive slowing, so that the frequency decreased y4 were observed individually under microscope and recorded for significantly 32% at 10 M (Fig. 4a). Systole y quantitative analyses. Mean"S.E.M. are shown normalizing. was not changed, whereas diastole at 108 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 statisti- cally 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 h- ( ) LC50 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 2h.

Fig. 2. Concentration–response curve for verapamil. D. magna were exposed for2htoverapamil 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 Fig. 3. Concentration–response curve for metaproterenol. D. According to ISO description the SDS standard magna were exposed for 2 h to metaproterenol from 10y6 to toxic is used to verify the sensitivity of D. magna, 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 for2htometoprolol from 10y8 to 10y4 M. Mean"S.E.M. are shown normalizing for diastole.

As expected from data obtained in mammals, centration suggests the presence of a verapamil 2q the four drugs tested were toxic to D. magna. receptor. Invertebrate Ca channel a1 subunits Ouabain was the most toxic and metoprolol was have been isolated and cloned and there are reports the least toxic. The resemblance in the sequences that crustaceans are insensitive to organic Ca2q of lethality by the four drugs for D. magna and channel blockers, but sensitive to inorganic Ca2q rats seems interesting (Table 3). This resemblance channel blocker, such as Sr2q (Jeziorski et al., is relative but meaningful, since it can indicate 1998), suggesting the presence of Ca2q channels similarity in toxicological sensitivity and mecha- in D. magna heart. These data support the sugges- nisms of action (vide supra). Rozman and Doull tion that D. magna heart might have verapamil (2001) give evidences of the role of time as a receptors that are different functionally from recep- quantifiable variable of toxicity and just as D. tors of mammal’s heart. magna and rat experiments were done under dif- Metaproterenol was either ineffective or accel- ferent experimental conditions of time and routes erated the cardiac frequency only at high concen- of administration, further speculations might be trations (10y4 and 10y3 M). These results are in hazardous. agreement with those of Bekker and Krijgsman Ouabain, metaproterenol and metoprolol caused (1951) and Postmes et al. (1989) who used adren- similar effects on heart of D. magna to those aline andyor its agonists and antagonists, respec- observed on humans. Ouabain exerted positive tively. However, the usual response of the heart of systolic inotropic effects. This similarity in effects, other arthropods to adrenaline is acceleration at plus the fact that D. magna heart is myogenic low doses and metaproterenol at a concentration (Bekker and Krijgsman, 1951) and the presence lower than 10y5 M, increased canine heart rate by of Naqq , K -ATPase in crustacean heart (Siebers 10% and the cardiac output also increased signifi- et al., 1982), suggests a mechanism of action cantly (Casthely et al., 1985). Therefore, D. magna similar to those in mammals heart at least for heart behaves different from other arthropods heart ouabain. and response was similar to mammal’s heart at Verapamil exerted positive inotropic and chron- high doses only. otropic effects, exactly the opposite effects caused Metoprolol antagonist caused significant accel- in humans (Fernandez-Gonzalez´´ et al., 2000). The eration with increased amplitude of contraction at response was significant at 10y7 M. This peculiar 10y8 M and subsequently significant decrease effect was reverted at high concentrations, with when given at high concentration (10y3 M); sim- detriment of heart rate, an habitual component in ilar effect was elicited in rats (Yaoita et al., 2002). intoxicated mammals (Tanen et al., 2000). The Between these two extremes it had no significant specific response of D. magna heart to low con- effects. Mosby’s (1999) reported that metoprolol A. Villegas-Navarro et al. / Comparative Biochemistry and Physiology Part C 136 (2003) 127–134 133 tartrate induced reduction in heart rate, cardiac Casthely, P.A., Cottrell, J.E., Urquhart, P.A., 1985. Comparison output and systolic blood pressure in most patients of metaproterenol isoetharine and salbutamol in the relief of metacholine-induced bronchospasm in dogs. Can. and had not intrinsic sympathomimetic activity in Anaesth. Soc. J. 32, 112–118. humans, but as this work showed, in D. magna’s Chapman, G., 1976. 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