Effects of pharmacological interventions on emetine cardiotoxicity in isolated perfused rat hearts

ELSEVIER

Toxicology 97 (1995) 93- 104

Effects of pharmacological interventions on emetine cardiotoxicity in isolated perfused rat hearts Shujia J. Pan, Alan B. Combs* Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712-1074, USA

Received 13 April 1994, accepted 2 August 1994

Abstract

The cardiotoxicity of emetine continues to be a significant clinical problem. The purpose of this study was to investigate the effect of several mechanistic interventions, including ICRF-187, an iron-chelating agent which protects against doxorubicin toxicity, atropine, and fructose-1,6-bisphosphate (FBP) on the toxicity of emetine in our isolated, perfused rat heart model. The model includes functional, electrocardiographic, and biochemical determinations in the same preparation. Atropine and ICRF-187 had no effect on the time needed for emetine to induce ventricular asystole, while FBP significantly increased this time. Administration of 47 PM atropine, 300 pM FBP, or 1 mM FBP decreased the release of lactate dehydrogenase (LDH) into the coronary elfluent, while ICRF-187 had no effect. These pharmacological interventions variably changed the amplitude of the biphasic response of the coronary flow to emetine. Finally, FBP was very effective in slowing the rate of QRS-waveform degeneration in the perfused hearts. Emetine caused PR- and QRS-prolongation which was not altered by FBP. Keywords:

Atropine; Cardiotoxicity; Electrocardiography; Lactate dehydrogenase; Perfused rat heart

1. lntroductlon Emetine is the active alkaloid in ipecac syrup which currently is the acute treatment of choice for

many toxic oral ingestions, especially in children (Anonymous, 1982; Amitai et al., 1987). For this purpose, the drug is effective and safe. Emetine is cardiotoxic, however, when used chronically. Such

* Corresponding author.

Emetine; Fructose-1,6_bisphosphate;

dangerous use can occur when emetine is used therapeutically as a secondary drug for the treatment of amebiasis, or when ipecac is abused chronically by people with bulimia nervosa (Murphy, 1985). Our laboratory has been studying emetine cardiotoxicity for the past few years, because of its clinical relevance and scientific interest. Generally, we have combined functional, electrocardiographic, and biochemical determinations in rat hearts perfused in the non-working Langen-

0300-483x/95/$09.50 0 1995Elsevier Science Ireland Ltd. All rights reserved SSDI 0300-483X(94)02928-N

ICRF-187; In vitro;

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dorff mode (Diiring and Dehnert, 1988) to characterize emetine toxicity and to study cardiotoxicity. In the characterization of our system, we found degradation of electrocardiogram waveforms, and we reported that emetine perfusion at concentrations of 19 PM and 37 PM causes dose-dependent decreases in contractility, and biphasic changes in coronary flow (Pan and Combs, 1991,1993). In addition, we have reported that emetine causes lactate dehydrogenase (LDH) release into the coronary effluent (Pan and Combs, 1991, 1993). In contrast to the functional changes caused by emetine, which may be pharmacological in nature, the release of LDH is clearly adverse and toxicological, since LDH is a large, intracellular protein which cannot leave the cell under normal circumstances (Evans, 1991). Several mechanisms have been proposed for the cardiotoxic actions of emetine. These mechanisms include inhibition of mitochondrial respiration (Brink et al., 1969), reduction of calcium permeability (Salako, 1972), and inhibition of protein synthesis (Agarwal et al., 1983; Combs and Acosta, 1991). For the most part, these studies have not been definitive and more work is needed. We investigated various pharmacological interventions to point toward possible mechanisms for emetine’s cardiotoxicity. Because decreased AVconduction and heart block were caused by emetine (Zbinden et al., 1980), and because atropine would be expected to enhance AVconduction through its vagal-blocking action, we tested the effect of this drug in our system. The compound ICRF-187 is an iron-chelating agent which decreases the production of reactive oxygen species (Rajagopalan et al., 1988), and which protects against the toxicity of doxorubicin (Yeung et al., 1992). We tested ICRF-187 in our system to determine if emetine’s cardiotoxicity might be associated with iron-mediated oxidative stress. Fructose-1,6-bisphosphate (FBP) protects against doxorubicin toxicity in the isolated perfused rat heart (Danesi et al., 1990). It may do this by cells cardiac enhancing glycolysis within (Nuutinen et al., 1991). Emetine cardiotoxicity does not appear to be associated with decreased mitochondrial function (Appelt and Heim, 1964, 1965). Therefore, we used FBP to test whether a disturbance in glycolytic function might be im-

93-104

plicated in emetine cardiotoxicity. The results of these mechanistic interventions are reported in this manuscript. 2. Methods 2. I. Chemicals Emetine dihydrochloride, atropine sulfate, ICRF-187, fructose- 1,bbisphosphate (FBP) and all other reagents were obtained *from Sigma Chemical Company (St Louis, MO). 2.2. Isolated perfused heart preparations Male Sprague-Dawley rats (200-325 g, Indianapolis, IN) were anesthetized with sodium pentobarbital(80 mgikg) via i.p. injection. Sodium heparin (150 units) was injected into the posterior vena cava after the chest of the rat was opened. The heart was rapidly removed, washed and cleaned with icecold Krebs-Henseleit bicarbonate buffer. Then, the heart was mounted through the aorta to a non-working heart Langendorff apparatus at a constant perfusion pressure of 80 cmHz0 above the aorta. The perfusate flow rate into the apparatus was 23 ml/min of Krebs-Henseleit bicarbonate buffer at 37°C. The Krebs-Henseleit bicarbonate buffer was composed of (in mM) 115 NaCl, 25 NaHCO,, 10 glucose, 5.9 KCl, 1.18 MgC12.6Hz0, 1.23 NaH#O,+, 1.2 NazSOd and 2.5 CaC&, and it was maintained at pH 7.4 by continuous gassing with 95% 02-5% CO*. 2.3. Deiermination of parameters All hearts were equilibrated by perfusion with Krebs-Henseleit buffer for 30 min after the heart was attached to the apparatus. In order to monitor contractile performance, a 21 G needle was inserted vertically into the left ventricle. This needle was attached to a Statham transducer connected to a Beckman Dynagraph Model R611 recorder. The onset time for ventricular asystole was defined as the first time period during which the ventricle quit beating for more than four seconds. Electrocardiography was monitored by an Macintosha-based EKG device (Combs et al., 1992) in which the active lead was attached to the 21 G intraventricular needle and the grounded lead was attached to the aortic cannula. The PR-interval, the QRSduration, and the &T-segment duration were

S.J. Pan er al. / Toxicology 97 (1995) 93-104

measured as defined by Budden et al. (1981). At the given time points, the coronary flow rate was measured, and the etlluent was collected for lactate dehydrogenase (LDH) analysis. 2.4. LDH assay The LDH activity was determined by measuring the rate of NADH change at 30°C. The standard unit of LDH activity is defined as 1 pm01 NADH oxidized by LDH during 1 min. The reaction was initiated by the addition of assay mixture and the absorbance was measured by spectrophotometer at 340 nm. The assay mixture contained 100 mM triethanolamine HCl buffer (pH 7.6), 0.15 mM NADH, 1 mM EDTA, and 1.5 mM pyruvate (Kehrer et al., 1988). 2.5. Pharmacological intervention studies A constant rate Harvard infusion pump was used to add drugs to the perfusion solution. Injection was made through an injection port located just before the heart. After the 30-min equilibration with Krebs-Henseleit, the treatment groups started to receive emetine for 10 min given by a constant infusion syringe pump. The time for the start of emetine injection was set as zero for purposes of data presentation and comparison. Atropine sulfate was infused at concentrations of 1 PM or 47 PM, starting 5 min before emetine perfusion and continuing through the lo-min period of emetine perfusion. The higher concentration was chosen from a report in which atropine protected against the cardiodepressive effect of a phthalate plasticizer on the human myocardium (Barry et al., 1990), and the lower concentration was chosen to be within the range of those reported to cause anticholinergic effects in isolated, perfused hearts (Fenton and Dobson, 1985). The ICRF-187 perfusion was started 10 min before emetine and was given at a concentration of 25 PM. This time and concentration was adopted from Rajagopalan et al. (1988). Because the early literature indicated some controversy about whether perfused FBP would be taken up into the cell (Bemardini et al., 1988; Stames et al., 1992), we started perfusion with FBP 20 min before emetine was started, and we continued this perfusion until the end of each experiment. The later literature more clearly shows that FBP is taken up

95

into the heart and that it serves as a substrate for glycolysis (Nuutinen et al., 1991; Tavazzi et al., 1992). The initial FBP concentration we tested was chosen arbitrarily to be 100 PM. Since this concentration did not have an effect on any of our measured parameters, higher concentrations of 300 PM and 1 mM were tested. These concentrations were the same, or less, than the perfusion concentrations previously reported (Stames et al., 1992). It has been reported that FBP has calcium chelating actions. FBP at a conecntration of 1 mM has a calcium chelation equivalent of 0.2 mM (Hassinen et al., 1991). We tested whether removal of this concentration of calcium would protect against LDH release, and whether addition of this concentration of calcium would prevent FBP’s effect upon LDH release. Because our data indicated that atropine and FBP prevented LDH release from our emetineperfused hearts, we investigated whether this might be an artifact caused by direct inhibition of LDH in the coronary etlluents by these drug interventions. In these studies, either 47 PM atropine or 1 mM FBP was added, after the fact, to the coronary effluents collected from each of three hearts perfused with 37 pM emetine. The LDH activity was analyzed as described above. 2.6. Statistical analysis All data are expressed as means * S.E. Analysis of statistical significance was performed using SPSS software (SPSS for the Macintosh’s’, SPSS Inc., Chicago, IL). A significant difference was considered to be a probability of less than 0.05. One-way ANOVA, the Kruskal-Wallis one-way nonparametric ANOVA, the Student-NewmanKeuls (SNK) post hoc test, and Student’s t-test were used to compare the means of the various groups. 3. Results 3.1. Effect of interventions on time neededfor ventricular asystole

As reported previously (Pan and Combs, 1991, 1993), 37 PM emetine perfusion quickly causes ventricular asystole within 2 min. The effects of atropine, ICRF- 187, and FBP upon the time need-

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Table 1 Effect of emetine perfusion and drug pretreatments on time to ventricular asystole Perfusion condition?

Emetine 37 CM alone (7) Atropine 1 CM + Emetine 37 PM (4) Atropine 47 gM + Emetine 37 PM (6) ICRF-187 + Emetine 37 &I (3) Fructose- 1&bisphosphate 100pM + Emetine 37 CM (5) Fructose- 1&bisphosphate 300 PM + Emetine 37 pA4 (3) Fructose-l$bisphosphate 1 mM + Emetine 37 CM (7)

Time to ventricular asystoleb 0.90 * 0.04 1.09 +z 0.18 1.10 f 0.06 1.30 f 0.50

-“; 2.4 $ z 1

2.o 1.6

E2

1.2

s

0.6

3

0.4

f

0.0

0.98 * 0.05 2.08

l

0.32’

>4.46 > 1.52+

sValues in parentheses are number of hearts per group. bResuhs are in min (mean l SE.). ‘This fructose-1,6-bisphosphate group differs significantly from the emetine alone group (P s 0.05, one-way ANOVA, followed by the Student-Neuman-Ketds post hoc test). It also differs from the atropine, ICRF-187, and low dose fructose-l ,6bisphosphate treatment groups which do not differ significantly from the emetine alone group. dThe value shown includes two IO-nun readings from hearts that were protected to the extent that emetine did not cause ventricular asystole during the 10&n emetine perfusion period. CThisgroup differs significantIy from the emetine alone group (P s 0.05, Kruskal-Wallis one-way, nonparametric ANOVA), but not from the group that received 300 PM fructose-1,6bisphosphate.

ed for ventricular asystole to occur are shown in Table 1. Neither the two concentrations of atropine, nor the ICRF-187 significantly changed the time for ventricular asystole. On the other hand, the two higher concentrations of FBP significantly prolonged the time for asystole to occur, and in a couple of instances, prevented asystole completely. 3.2. Effects of interventions on LDH release from emetine treated hearts These data are shown in Figs. l-3. Perfusion with 37 pM emetine caused a very significant loss of LDH from within the cells. This loss was changed only slightly by ICRF-187 (Fig. 1). Atropine caused a dose dependent decrease in the release of LDH from emetine-treated hearts (Fig. 2). Both

-n-

Controls

-e-

Em&he 37 PM

+--

ICRF 25 PM + Emetine 37 FM

_ 0

10

Emetine infusion ICRF infusion 20 30 Time (min)

40

50

Fig. 1. Effects of ICRF-187 perfusion on lactate dehydrogenase (LDH) release (upper chart) and on coronary flow (lower chart) induced by 10 min perfusion with 37 PM emetine. Perfusion with ICRF-187 was started 10 mitt before emetine and was continued throughout the 10 min of emetine perfusion. The ICRF-187 concentration was 1 CM (n = 3). Control hearts received only Krebs-Henseleit bicarbonate buffer (n = 4) and the circles represent hearts that received 37 FM emetine for IO mitt starting at time = 0 (n = 7). One-way ANOVA was performed at the 2 min and 16 min time points which corresponded to the maximum and minimum values of the biphasic response of the coronary flow to emetine. Extrapolated control values were. used for these comparisons. At 2 min (marked *), the controls differ significantly from the other two values which do not differ from each other. At 16 min (marked 1), each value differs significantly from each of the others.

doses of FBP were very effective in preventing LDH release in the rat hearts (Fig. 3). The cumulated amounts of LDH released over the first 40 min following the start of emetine perfusion are shown in Fig. 4. The high concentration of atropine and both doses of FBP reduced the amount

S. J. Pan

et al. / Toxicology

of LDH released almost to control levels. The ICRF-187 and the low concentration of atropine provided only slight protection from emetineinduced LDH release.

s3 a .f

97 (1995)

7

93-104

91

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2

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1.2 0.6

5

0.0

0.4

-10

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0

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30

40

-e-

Emetine 37 PM

--+

Atropine 1 PM + Emetine 37 FM

Emetine infusion Atropine infusion O-10

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Atropine 47 NM + Emetine 37 UM

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20

30

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Time (min) Fig. 2. Effects of a&opine perfusion on lactate dehydrogenase (LDH) release (upper chart) and on coronary flow (lower chart) induced by 10 min perfusion with 37 CM emetine. Atropine perfusion was started 5 min before emetine and was continued throughout the 10 min of emetioe perfusion. The atropine concentrations were 1 CM (n = 4) and 47 PM (n = 6). Control hearts received only the Krebs-Henseleit bicarbonate buffer (n = 4), and the circles represent hearts that received 37 PM emetine for 10 mio starting at time = 0 (n = 7). One-way ANOVA was performed at the 2 mitt and 16 min time points which corresponded to the maximum and minimum values of the biphasic response of the coronary flow to emetine. Extrapolated control values were used for these comparisons. At 2 min, the emetine alone and high dose atropine groups (each marked with *) differ from the control group, but not from each other, and the low dose atropine group does not differ from any of the other groups. At 16 min (marked 1), the control group differs from all of the other groups. IO addition, the two atropine-treated groups differ from the other groups, but not from each other.

0

10 20 Time (min)

30

40

50

Fig. 3. Effects of fructose-l&bisphosphate (FBP) perfusion on lactate dehydrogenase (LDH) release (upper chart) and on coronary flow (lower chart) induced by 10 mitt perfusion with 37 $4 emetine. Perfusion with FBP was started 20 min before emetine and was continued through the 10 min of emetine perfusion and until the end of etlluent collection. The FBP concentrations were 300 CM (n = 3) and I mM (n = 7). Control hearts received only the Krebs-Heoseleit bicarbonate buffer (n = 4), and the circles represent hearts that received 37 pM emetine for 10 mm starting at time = 0 (n = 7). One-way ANOVA was performed at the 2 mio and 16 min time points which corresponded to the maximum and minimum values of the biphasic response of the coronary flow to emetine. Extrapolated control values were used for these comparisons. At 2 min (marked I), the emetine alone group differs from all of the other groups which do not differ from each other. At 16 min (marked ). the control group ditfers from all of the other groups which do not differ from each other.

3.3. Effects of perfusate calcium concentrations on LDH released by emetine from control and FBPtreated hearts

Decreasing perfused calcium to a 2.3-mM concentration did not prevent emetine-induced LDH

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t Emetine 37pM+

Interventions

Fig. 4. Total LDH released from isolated, perfused hearts. The total amount of LDH released between the start of emetine perfusion and 40 mitt after the start of emetine is shown, and is expressed as total LDH units released/g dry heart weight (left axis), or as percentage of total cardiac LDH (right axis). This graph summa&es the data shownin the first three figures and statistical analysis was performed on these aggregate data (ANOVA followed by SNK post hoc analysis). The group marked with * (emetine alone, n = 7) differs from ah of the other groups. The groups marked with * (control, n = 4; 47 phi atropine, n = 6; 300 pM FBP, n = 3; and 1 mM FBP, n = 6) do not differ from each other. The two groups marked * * * (low dose atropine, n = 6; ICRF-187, n = 3) do not differ from each other, but each does differ from groups marked * and * * .

release. Increasing the perfused calciwn concentration to 2.7 mM did not prevent the protective effect of 1 mM FBP. 3.4. Direct effects of ah-opine and FBP on LDH activity in coronary effluent To determine whether the protective effect of atropine and FBP upon LDH released by emetine perfusion might be an artifact of direct inhibition of LDH by these drugs, we added atropine or FBP to coronary eBbtents after their collection, but before LDH analysis. The results indicated that there are no significant differences among the control groups, the atropine groups, and the FBP groups. 3.5. Effects of interventions on rate of coronary flow These data are shown in the lower graphs in Figs. l-3. The hearts were very variable in their coronary flow rates. For this reason, the coronary flow data were normalized to facilitate comparison in Figs. l-3. In the case of atropine and FBP, the

coronary flow rates were normalized so that the flow rates at the beginning of emetine perfusion were lOO?&In contrast to atropine and FBP, in which pretreatment did not have a significant effect on coronary flow, ICRF-187, itself, increased the coronary flow. Therefore, the coronary flows in hearts treated with ICRF-187 were normalized to the start of ICRF-187, so that the coronary flows at 10 min before emetine were set at lOO?/. Emetine, itself, caused the typical biphasic response previously reported (Pan and Combs, 1993). There was a rapid early increase in flow that peaked about 2 min after the start of emetine. Following that came a gradual fall in coronary flow rate to below control values. This decreased coronary flow rate persisted for at least 20 min after the discontinuation of emetine. The coronary flow data for ICRF-187 are shown in Fig. 1. The ICRF-187 group had decreased flows starting after the addition of emetine. The decrease somewhat paralleled, but was greater than that caused by emetine. In the

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S.J. Pan et al. /Toxicology 97 (1995) 93-104 Seconds following start of emetine perfusion

t-TG-G+

-4++b-+P +45sec (bigeminy)

t Heart

1

Heart 2

Heart 3

Fig. 5. Effect of perfusion with 37 CM emetine on the EKG in three isolated, perfused hearts. Records from three hearts are shown. The vertical axis, proceeding from top to bottom, indicates time in seconds after the start of emetine perfusion (a zero time recording was not obtained for Heart 3, but the -5-min recording was not significantly different from the +20-s recording). The bottom recording for each heart represents the last coherent, organized heart beat before ventricular fibrillation.

case of hearts pretreated with atropine, the initial phase of increased flow was essentially unchanged. Both doses of atropine enhanced the depression of flow during the second phase. In contrast to ICRF-187 and atropine, FBP blocked the initial phase of increased coronary flow, although the second phase of decreased coronary flow was not changed. 3.6. Effects of emetine and FBP upon the EKG in isolated, perfused rat hearts The sequence of EKG changes in three rat

hearts given 37 @I emetine is shown in Fig. 5, and

the effect of pretreatment with 1 mM FBP upon the emetine-induced EKG changes is shown in Fig. 6. In the emetine-treated hearts, the control recordings exhibit typical PR-intervals and the typical complexity of the QRS-waveforms. As emetine began to be perfused, however, the PRintervals became prolonged, the magnitude of the QRS-waveforms became less, and these waveforms became more and more simplified, or debased. Ventricular fibrillation occurred in less than 1 min in all three of these hearts. The Pwaveforms changed very little during this time period. The recordings from three hearts

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S.J. Pan et al. /Toxicology 97 (1995) 93-104

pretreated with FBP before the emetine are shown in Fig. 6. Generally, the changes are similar to those described in Fig. 5, but the rate at which the adverse changes occur is much, much slower, and

the time for ventricular fibrillation, if it occurred at all, was much greater. The heart from the rat designated as ‘Rat 6’ went through a phase of complete heart block. In this heart, the atria and ven-

Seconda following start of emetine perfusion

I-

1. ..... “..2

1

O-f+-+--

;;::s ;:::::-:, .+ +2 4 30

--W---d-

-+--&--+-/ -b---J* .G-_ 1

P-wave f QRS

P-wave

Complete block with ventricular tachycardia

-+---df-----

? Heart 4

Heart 5

Heart 6

Fig. 6. Effect of perfusion with FBP on the action of 37 PM emetine on the EKG in three isolated, perfiwd hearts. Records from three hearts are shown. The vertical axis, proceeding from top to bottom, indicates the time in s after the start of emetine perfusion.

S.J.Pan er a/./Toxicology97 (1995) 93-104

-e-

37wLM Emetine

-s-

101

1 mM FBP + 37 FM Emetine

400

Ventricular Rate

1

z8 0.06 g 0.05 z ,m J 0.04 a

2004J’ (-in, 0

20

40

60

60 100 120 140

0.03 t-20. 0

20

40

60

60 100 120 140

SaT-Duration z 8 0.03 : ‘3 !! 0.02 z o.o2c/.‘, (-:2& 0

20

Time

40

60

60 100 120 140

After Start of Emetine(sec)

0.01 c/.p, (-;20”,0

20

40

60

60 100 120 140

Time AfterStartof Emetine(sec)

Fig. 7. Electrocardiographic parameters. Hearts perfused with 37 pM emetine are designated by squares (n = 3). Hearts perfused with FBP and then 37 PM emetine are indicated by circles (n = 3). Data represent means f SE. The horizontal axes represent time in seconds, in which emetine perfusion was started at time = 0. The upper left graph shows the heart rates in beats per min on the vertical axis. The upper right graph shows the PR-intervals, the lower left graph shows the QRSdurations and the lower right graph shows the SaTdurations. The vertical axes in the latter three graphs represent duration of the indicated intervals in seconds. The 37 &4 emetine values marked + differ significantly (P s 0.05) from the FBP values at the same time points.

tricles each beat independently, and the ventricular rate was greater than the atria1 rate. The averaged EKG data are shown in Fig. 7. Until the moment of ventricular fibrillation, the ventricular rates did not change significantly in the hearts given emetine alone. The administration of FBP by itself caused the rate to be significantly slower than in the hearts treated with emetine alone. This difference was not modified by the addition of emetine to the FBP-perfused hearts. The PR-intervals for the emetine-alone hearts and the FBP-treated hearts became more and more prolonged, and the two groups were almost identical throughout the period that the former group kept beating. There was greater and greater PRprolongation, a process which continued in the FBP-treated hearts long after the emetine-alone ventricles had stopped beating. The QRSdurations also were similar between the two

groups of hearts. The QRS-intervals became more prolonged as time went on, and this process continued in the FBP-treated hearts after the emetinealone ventricles had stopped functioning. The FBP administration caused the SarT-intervals to be significantly greater than in the emetine-alone hearts. The intervals became shorter with the perfusion of emetine, but the difference between emetine-alone and FBP-treated animals was maintained until ventricular fibrillation in the emetine alone group. The SarT-values in the FBP-treated hearts became very highly variable during the 60-100-s time period. 4. DIselc4sIon As Table 1 shows, atropine and ICRF-187 had no effect on the time to asystole, and infusion with FBP prolonged the time needed for emetine to

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S.J. Pm et al. I Toxicology 97 (1995) 93-104

cause ventricular asystole. This action of FBP and the effect that FBP had on LDH release (Fig. 3, described below) were our first indications that this glycolysis intermediate might have protective actions against emetine cardiotoxicity. We have reported previously that emetine causes the loss of toxicologically significant amounts of LDH from within perfused hearts. In this paper, we demonstrate that atropine and FBP can protect against LDH loss. In the case of atropine, the lower concentration was used previously for its anticholinergic effects (Fenton and Dobson, 1985). We feel that the protection provided by the 47 PM atropine may not be related to its antimuscarinic effects. On the other hand, we feel that the dose-dependent reduction in LDH release caused by FBP administration may be a very specific biochemical protective effect of this compound. This concept will be developed more fully later in this discussion. As an alternative to it having a specific biochemical effect, it could be speculated that the protective effect of FBP on LDH release might occur because of FBP’s calcium chelating action. The concentration of calcium in our standard Krebs-Henseleit perfusion buffer was 2.5 mM. Based upon the values given by Hassinen et al. (1991), the concentration of calcium chelated by 1 mM FBP is 0.2 mM. If changes in this concentration of calcium are responsible for FBP’s effect on LDH release, then perfusion with 2.3 mM calcium (2.5 mM minus the 0.2 mM chelated) should protect against LDH release, by itself, and perfusion with 2.7 mM calcium in the presence of 1 mM FBP (2.5 mM plus the 0.2 mM chelated by the included FBP) should not protect against LDH release. Neither of these predictions turned out to be true. Therefore, the protective effect of FBP is unlikely to be caused by chelation-caused changes in external calcium. Another possible mechanism for the protective action of high dose atropine and FBP is that these drugs might directly inhibit LDH activity in the coronary emuent, and that the decrease in measured activity is an artifact of such inhibition. This possibility is ruled out because direct addition of these drugs to aliquots taken from coronary effluents of hearts perfused with emetine-alone did

not change the LDH activities in comparison with aliquots in which neither drug was added. The effects of emetine on the coronary flow are not understood. It has been shown that the coronary flow is inversely proportional to levels of myocardial ATP (Starnes et al., 1985). The beginning of the decrease in coronary flow of the second phase is concomitant with ventricular asystole. At this time, large scale ATP consumption would cease and the coronary flow rate would decrease in response. The enhanced decreases in coronary flow caused by pretreatment with ICRF-187 and atropine also are not understood. Treatment with FBP did not enhance the depressive phase, and it obtunded the increased flow phase. The latter observation provides the third indication that FBP can counter effects of emetine in our perfused hearts. The mechanism for protection against doxorubicin-induced cardiotoxicity by ICRF- 187 is thought to involve iron chelation which results in protection against oxidative stress (Rajagopalan et al., 1988). It is active in vivo (Speyer et al., 1988) and in vitro in the isolated, perfused heart preparation (Rajagopalan et al., 1988). In as much as ICRF- 187 proved only slight protection against emetine-induced LDH release, we feel that emetine’s cardiotoxicity may be different in mechanism from that of doxorubicin and it does not involve iron-mediated oxidative stress. The electrocardiographic records provide the fourth instance in which FBP changes the effect of emetine in our perfused hearts. In the hearts given emetine alone, the PR-intervals and QRS-intervals become more prolonged with continuing emetine perfusion, and the QRS waveforms become more simplified and abnormal. The PR- and QRSprolongations may be related to the quinidine-like action of emetine reported by De Hemptinne (1965). On the other hand, the degeneration of the waveforms may be related to an effect of emetine on myocardial energetics. Treatment with FBP prolonged the time needed for the QRS waveform degeneration to occur, but the prolongation of the PR- and QRS-durations continued unabated. In fact, in one of the hearts shown in Fig. 7 (Heart 6), complete heart block occurred without ventricular asystole. We feel that these effects on the PR- and

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QRS-durations are pharmacologic in nature, rather than toxicologic, and the data show that if ventricular fibrillation is prevented, the PR- and QRS-prolongation processes continue. The heart rate is dependent upon the functioning of atria1 pacemakers. Perfusion with FBP decreases the heart rate in comparison with hearts not getting the FBP pretreatment, but subsequent emetine administration does not change the heart rate in either group. The primary time-dependent event during the SaT-segment is the rate of ventricular repolarization. At first observation, it may seem that ventricular repolarization occurs more quickly as more emetine is perfused into these hearts. However, the decrease in the &T-segment caused by emetine may be in part artifactual because this time period, as usually measured in animals (Budden et al., 1981), also contains a component of ventricular depolarization. It is interesting that FBP, a glycolytic intermediate product, counters several of the toxic actions of emetine. One possible explanation follows from the recent observations that the production of ATP in cardiac tissue is compartmentalized (Gudbjamason et al., 1970; Weiss and Hiltbrand, 1985; Lopaschuk et al., 1992). This means that the ATP necessary for contraction is produced almost solely by the mitochondria, and the ATP needed for plasma membrane function and integrity is produced almost completely by glycolysis. It has been established that FBP does enter into the cardiac cell and that it can act as a glycolytic substrate after entry (Stames et al., 1992; Tavazzi et al., 1992). It is a substrate which can greatly increase the efficiency of glycolysis because it is already phosphorylated and does not require ATP for its utilization, as is the case when glucose is the glycolytic substrate. The decrease in QRS waveform amplitude and complexity caused by emetine provides evidence that cardiac plasma membrane function is being disturbed. The prolonged ventricular beating that pretreatment with FBP allows to occur, even in the face of the decreased atrialto-ventricular and ventricular conduction velocities, indicates that FBP is acting to preserve membrane function. Finally, the greatest argument that FBP preserves membrane function is that FBP prevents the efflux of LDH into the coronary efflu-

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ent. Because the ATP needed for membrane function and integrity comes from glycolysis, and because FBP, an efficient glycolytic substrate, is so effective in preserving membrane function and integrity, we speculate that the cardiotoxic action of emetine in our isolated, perfused hearts may be due to inhibition of glycolysis. Additional studies are in progress to investigate this possibility. Acknowledgments A.B. Combs is the Bergen-Brunswig Centennial Fellow in Pharmacy at the University of Texas at Austin. S.J. Pan was a recipient of an NIH Predoctoral Training Grant Position at the University of Texas, College of Pharmacy. References Agarwal, S., Dube, P. and Sagar, P. (1983) In vivo distribution and action of emetine on protein synthesis in hamster tissues. Indian J. Exp. Biol. 21, 353-354. Amitai, Y., Mitchell, A.A., McGuigan, M.A. and Lovejoy, F.H., Jr. (1987) Ipecac-induced emesis and reduction of plasma concentrations of drugs following accidental overdose in children. Pediatrics 80, 346-367. Anonymous (1982) Syrup of ipecac still number one choice. Am. Phar. NS21, 46. Appelt, G.D. and Heim, H.C. (1964) Effect of chronic poisoning by emetine on oxidative process in rat heart: I. J. Pharm. Sci. 53, 1080-1083. Appelt, G.D. and Heim, H.C. (1965) Effect of chronic poisoning by emetine on oxidative process in rat heart: II. J. Pharm. Sci. 54, 1621-1625. Barry, Y.A., Labow, R.S., Keon, W.J. and Tocchi, M. (1990) Atropine inhibition of the cardiodepressive effect of mono(2-ethylhexyl)phthalate on human myocardium. Toxicol. Appl. Pharmacol. 106 48-52. Bemardini, N., Danesi, R., Bemardini, M.C. and Del Tacca, M. (1988) Fructose-I$-diphosphate reduces acute ECG changes due to doxorubicin in isolated rat heart. Experientia 44, 1000-1002. Brink, A.J., Kotze, J.C.N., Muller, S.P. and Lochner, A. (1969). The effect of emetine on metabolism and contractihty of the isolated rat heart. J. Pharmacol. Exp. Ther. 165, 251-256. Budden, R., Buschmann, G. and Kiihl, U.G. (1981). The rat ECG in acute pharmacology and toxicology. In: Budden, R., Detweiler, D.K. and Zbinden, G. (Eds), The Rat Electrocardiogram in Pharmacology and Toxicology, Pergamon Press, Oxford, pp. 41-81. Combs, A.B. and Acosta, D. (1991) Toxic mechanisms of the heart: a review. Toxicol. Pathol. 18, 583-596.

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