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Effects of monocrotaline on energy metabolism in the rat liver
Toxicology Letters 182 (2008) 115–120
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Effects of monocrotaline on energy metabolism in the rat liver Fábio Erminio Mingatto a,∗ , Marcos Antonio Maioli a , Adelar Bracht b , Emy Luiza Ishii-Iwamoto b a b
Laboratório de Bioquímica, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Dracena, 17900-000 Dracena, SP, Brazil Laboratório de Metabolismo Hepático, Departamento de Bioquímica, Universidade Estadual de Maringá, 87020-900 Maringá, PR, Brazil
a r t i c l e
i n f o
Article history: Received 30 April 2008 Received in revised form 5 September 2008 Accepted 5 September 2008 Available online 16 September 2008 Keywords: Monocrotaline Dehydromonocrotaline Liver metabolism Glycogenolysis Gluconeogenesis Urea cycle
a b s t r a c t Monocrotaline (MCT) is a pyrrolizidine alkaloid present in the plants of the Crotalaria species that causes cytotoxicity and genotoxicity in animals and humans, and it is hepatically metabolized to the alkylating agent dehydromonocrotaline by cytochrome P-450. The exact cellular and molecular mechanisms of MCTinduced tissue injury remain unclear. We previously demonstrated that dehydromonocrotaline, but not monocrotaline, inhibits the activity of NADH-dehydrogenase at micromolar concentrations in isolated liver mitochondria, an effect associated with significantly reduced ATP synthesis. Impairment of energy metabolism is expected to lead to several alterations in cell metabolism. In this work, the action of different concentrations of monocrotaline (250, 500, and 750 M) on energy metabolism-linked parameters was investigated in isolated perfused rat livers. In the fed state, monocrotaline increased glycogenolysis and glycolysis, whereas in the livers of fasted rats, it decreased gluconeogenesis and urea synthesis from lalanine. These metabolic alterations were only found in livers of phenobarbital-treated rats, indicating that active metabolites including dehydromonocrotaline were responsible for the observed activity. Our findings indicate that hepatic metabolic changes may be implicated, partly at least, in the hepatotoxicity of monocrotaline in animals and humans. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Monocrotaline (MCT), an alkaloid pyrrolizidine phytotoxin, has well-documented hepatic and cardiopulmonary toxicity for animals, including ruminants, and man (Mclean, 1970; Mattocks, 1986; Huxtable, 1989; Souza et al., 1997; Schultze and Roth, 1998; Stegelmeier et al., 1999; Nobre et al., 2004a, b, 2005). Monocrotaline is frequently ingested accidentally because of food grain contamination or intentionally in the form of herbal medicine preparations (Huxtable, 1989). It was demonstrated that liver injury occurs 24 h after monocrotaline administration in rats at doses higher than 150 mg kg−1 . MCT-induced hepatic lesions are characterized by centrilobular cell necrosis, congestion and dilation of sinusoids, hemorrhage, damage to sinusoidal and central venular endothelial cells (DeLeve et al., 1999; Yee et al., 2000; Copple et al., 2002), and parenchymal cell oncosis and apoptosis (Copple et al., 2004). Monocrotaline toxicity requires cytochrome P-450-mediated bioactivation to reactive pyrrolic metabolite dehydromonocrotaline (DHM) (Butler et al., 1970; Lafranconi and Huxtable, 1984; Roth and Reindel, 1990; Wilson et al., 1992; Pan et al., 1993; Schultze and Roth, 1998). This metabolite, despite having a half-life of only a
∗ Corresponding author. Tel.: +55 18 3821 8200; fax: +55 18 3821 8208. E-mail address: [email protected] (F.E. Mingatto). 0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2008.09.004
few seconds in aqueous media (Mattocks et al., 1990), is a powerful alkylating agent that binds to cellular DNA and proteins (Petry et al., 1984; Hincks et al., 1991; Niwa et al., 1991; Wagner et al., 1993; Yan and Huxtable, 1995a; Lamé et al., 2005). The exact cellular and molecular mechanisms of MCT-induced hepatic injury remain unclear. We previously demonstrated that dehydromonocrotaline, but not monocrotaline, inhibits the activity of NADH-dehydrogenase when added at micromolar concentrations to isolated rat liver mitochondria, an effect associated with significantly reduced ATP synthesis (Mingatto et al., 2007). Because the activity of complex I is regulated by thiol groups, it was suggested that the inhibition of complex I NADH oxidase activity resulted from the oxidation of cysteine thiol groups by dehydromonocrotaline. Inhibition of mitochondrial function is likely to affect cell metabolism in several ways. In principle, increases in catabolic processes that can lead to ATP synthesis independently of oxidative phosphorylation as well as decreases in energy-dependent anabolic processes should be expected. However, this toxic effect on mitochondria has not been reproduced in the intact liver until now. It must be stressed that the conditions under which mitochondria operate within intact cells are quite different from those of the rather simple environment of a suspension of isolated organelles. Added factors include the passage of drug through plasma membrane, the presence of numerous intracellular binding sites, and
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biotransformation reactions, all of which are important for making the active drug metabolite available to the mitochondria. Furthermore, the possibility exists that the actions of monocrotaline in the intact liver are not solely the consequence of a primary action of dehydromonocrotaline on mitochondria. The present study, performed in isolated perfused rat livers, was planned to address these questions. Monocrotaline was infused into the livers, and several metabolic fluxes were measured, with special emphasis on metabolic fluxes related to energy metabolism in terms of glycogenolysis, glycolysis and oxygen consumption in the livers of fed rats, as well as gluconeogenesis and ureogenesis in the livers from fasted rats. In order to stimulate the production of monocrotaline metabolites by perfused livers, rats were previously treated with phenobarbital, a well-known inducer of hepatic cytochrome P-450 isoenzymes (Vlasuk et al., 1982), which have, in turn, been used to stimulate MCT metabolism (Reid et al., 1998). 2. Materials and methods 2.1. Chemicals The liver perfusion apparatus was built in the workshops of the University of Maringá. Monocrotaline, enzymes and coenzymes used in the enzymatic assays were purchased from Sigma–Aldrich (St. Louis, MO, USA). Phenobarbital was purchased from Rhodia Farma, Brazil. Sodium pentobarbital was a gift from Cristália, Brazil. All other reagents were of the highest commercially available grade.
2.2. Animals Male albino rats (Wistar) weighing 180–220 g were fed ad libitum with a standard laboratory diet (Nuvilab® , Colombo, Brazil). In some experimental protocols, the rats were starved for 24 h before the surgical removal of the liver. In experiments with phenobarbital induction, rats were dosed intraperitoneally (90 mg kg body weight−1 ) daily for 3 consecutive days. 2.3. Liver perfusion For the surgical procedure, fed or 24 h fasted rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg kg body weight−1 ). Hemoglobin-free, non-recirculating perfusion was performed. The surgical technique was the same as that described by Scholz and Bücher (1965). After cannulation of the portal and cava veins, the liver was positioned in a Plexiglas chamber. The perfusion fluid was Krebs/Henseleit-bicarbonate buffer (pH 7.4), saturated with a mixture of oxygen and carbon dioxide (95:5) using a membrane oxygenator with simultaneous temperature adjustment at 37 ◦ C. The flow, provided by a peristaltic pump, was between 30 and 35 ml min−1 , depending on the liver weight. Samples of the effluent perfusion fluid were collected according to the experimental protocol and analyzed for their metabolite contents. The oxygen concentration in the outflowing perfusate was monitored continuously using a Teflon-shielded platinum electrode adequately positioned in a Plexiglas chamber at the point where the perfusate exits (Scholz and Bücher, 1965). Monocrotaline (250, 500 and 750 M) or l-alanine (2.5 mM) was dissolved in the perfusion fluid. 2.4. Analytical The following compounds were assayed using standard enzymatic procedures: glucose (Bergmeyer and Bernt, 1974), l-lactate (Gutman and Wahlefeld, 1974), pyruvate (Czok and Lamprecht, 1974), urea (Bergmeyer, 1974) and ammonium (Kun and Kearney, 1974). Metabolic rates were calculated from input–output differences and
Fig. 1. Time courses of the effects of monocrotaline on glycogen catabolism and oxygen consumption in perfused livers of phenobarbital-treated fed rats. Livers from fed rats were perfused with Krebs/Henseleit-bicarbonate buffer as described in Section 2. Panel A represents the livers of control rats. Monocrotaline was infused for 10–50 min at concentrations of 250 M (panel B), 500 M (panel C) or 750 M (panel D) as indicated by the horizontal bars. Samples of the effluent perfusate were withdrawn for metabolite assays. Oxygen consumption was monitored polarographically. The results represent the mean values ± S.E.M. of four perfusion experiments. Significant differences in the time course of oxygen consumption, glucose and l-lactate production in the monocrotaline infusion series (panels B, C and D) as compared to the control series (panel A) are indicated by horizontal lines (two-way analysis of variance, with post hoc Newman–Keuls test).
F.E. Mingatto et al. / Toxicology Letters 182 (2008) 115–120 the total flow rates, normalized to the wet weight of the liver, and expressed as mol min−1 (gram liver wet weight)−1 . 2.5. Data analysis The data in the figures are expressed as mean ± standard error of the mean (S.E.M.). The statistical significance of the differences between parameters among the experimental groups was evaluated using two-way analysis of variance, and differences in the same experimental groups were tested by repeated-measures one-way analysis of variance (ANOVA). Significant differences among means were identified by Newman–Keuls testing. The results are given in the text as probability values (P). P ≤ 0.05 was adopted as the criterion of significance. Statistical analysis was performed using StatisticaTM or GraphPAD Software programs.
3. Results 3.1. Effects of monocrotaline on glycogen catabolism and oxygen consumption In the first experiments, the effects of monocrotaline on glycogen catabolism and oxygen consumption were investigated. Livers from fed rats were perfused with substrate-free perfusion medium in a non-recirculating mode. Under these conditions, the livers respire mainly at the expense of endogenous fatty acids, but
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they simultaneously exhibit extensive glycogenolytic and glycolytic activity (Scholz and Bücher, 1965), as indicated by glucose, l-lactate and pyruvate release. Monocrotaline was infused at 250, 500 and 750 M. It was previously demonstrated by Yan and Huxtable (1994) that, at these concentrations, perfused rat livers produce and release significant amounts of potentially toxic metabolites. When monocrotaline was infused for 40 min at 500 M in perfused livers from non-treated rats, there were no significant changes in glucose, l-lactate or pyruvate productions or in oxygen consumption (data not shown). Thus, subsequent experiments were performed with livers from phenobarbital-treated rats using three doses of monocrotaline (250, 500 and 750 M). Fig. 1 illustrates the experimental protocol and the time course of the metabolic fluxes when livers were perfused for 90 min in the absence (control) or in the presence of monocrotaline. In the control series (panel A), the rates of oxygen consumption and pyruvate production did not change during the experimental time period. However, glucose and l-lactate production progressively decreased, possibly due to the low glycogen content of livers from phenobarbital-treated rats. As shown by panels B, C and D, the introduction of 250 M, 500 M or 750 M monocrotaline at 10 min produced changes, especially in glucose and l-lactate
Fig. 2. Time course of the effects of 500 M monocrotaline on glucose, l-lactate and pyruvate production and on oxygen consumption as well as urea and ammonium production derived from l-alanine in perfused livers from phenobarbital-treated rats. Livers from 24 h fasted rats were perfused with Krebs/Henseleit-bicarbonate buffer as described in Section 2. l-Alanine (2.5 mM) was infused after 10–88 min of perfusion. Panels A and B represent the livers of the control rats. Monocrotaline (500 M) was infused at 30–70 min as indicated by the horizontal bars in the panels C and D. Samples of the effluent perfusate were withdrawn for metabolite assay. Data represent the mean values ± S.E.M. of four to six perfusion experiments. Significant differences in the time course of pyruvate production (panel C) and urea and ammonium production (panel D) in the monocrotaline infusion series as compared to the control series are indicated by horizontal lines (two-way analysis of variance, with post hoc Newman–Keuls test). *P ≤ 0.05 compared with values obtained immediately before monocrotaline infusion (panel C), according to repeated-measures ANOVA testing.
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production. Although significantly increased by all concentrations of monocrotaline, glucose and l-lactate production did not show a clear concentration-effect relationship. Glucose production, for example, was increased by 243%, 239% and 278% after infusion of 250, 500 and 750 M monocrotaline, respectively. After cessation of monocrotaline infusion, glucose production remained elevated for at least the following 20 min. The increase in l-lactate production was not as large as that of glucose production, and the stimulation kinetics were different. At 250 M, a gradual increase was observed during the entire course of monocrotaline infusion and l-lactate production stabilized at a level nearly 84% higher than that of the control series. l-Lactate production was also stimulated after the onset of 500 and 750 M monocrotaline infusion. After nearly 10 min, the rates decreased progressively and stabilized at lower values than those observed for 250 M monocrotaline; however, this level was still higher than that of the control series. Finally, slight stimulation of oxygen consumption was observed upon the infusion of 250 M, 500 M or 750 M monocrotaline, and pyruvate production was not significantly affected. 3.2. Effects of monocrotaline on gluconeogenesis, the urea cycle and oxygen consumption In order to determine the effects of monocrotaline on energydependent biosynthetic processes, gluconeogenesis, ureogenesis, oxygen consumption, ammonium level, and l-lactate and pyruvate production from l-alanine were measured in perfused livers of fasted rats. The control experiments, in which only the gluconeogenic precursor l-alanine was infused in livers of phenobarbital-treated rats, are shown in Fig. 2A and B. After a pre-perfusion period of 10 min, 2.5 mM l-alanine was infused for 80 min. As shown by Fig. 2A, the amounts of glucose, l-lactate and pyruvate were negligible prior to l-alanine infusion due to low endogenous levels of glycogen and gluconeogenic substrates. After the onset of l-alanine infusion, glucose, l-lactate and pyruvate production progressively increased. After approximately 15 min, steady-state conditions were attained for glucose production. For l-lactate and pyruvate production, on the other hand, steady-state conditions only occurred after 40 min of substrate infusion. In previous experiments, we have found that the effects of 750 M monocrotaline infusion on l-alanine metabolism closely resembled those of 500 M monocrotaline, as it was demonstrated to occurs in the livers of fed rats (Fig. 1). The actions of a selected concentration of monocrotaline (500 M) are thus illustrated by Fig. 2 (panels C and D). The introduction of monocrotaline produced a rapid and significant inhibition of glucose production (Fig. 2C). The maximum inhibition (nearly 25%) occurred at 4 min of monocrotaline infusion. After this time, however, there was a gradual recovery of glucose production such that at the terminus of monocrotaline infusion, glucose production was not statistically different from its level before monocrotaline infusion or from the control series. Fig. 2C also shows that l-lactate production was not significantly affected. Pyruvate production, on the other hand, was progressively and significantly reduced. At the end of monocrotaline infusion, pyruvate production was 42% lower than in the control series at the corresponding time point (70 min). There was no evident recovery of pyruvate production; at the end of the experimental period (90 min), it was 50% lower than in the control series. Oxygen consumption and the production of urea and ammonium by control livers are shown in Fig. 2B. In the absence of exogenous substrate, ammonium production was negligible, and the basal rate of urea production was 0.076 mol min−1 g−1 . lAlanine infusion increased oxygen consumption as well as urea and ammonium production. Steady-state levels of oxygen con-
sumption and urea production were maintained during the entire l-alanine infusion. Ammonium production, on the other hand, increased progressively during the same period. The introduction of 500 M monocrotaline significantly inhibited urea production as can be seen in Fig. 2D. Twenty-five percent inhibition was observed at the end of monocrotaline infusion when compared to the values found in the control series. Upon cessation of monocrotaline infusion, there was a gradual recovery of urea production. The rates of oxygen consumption and ammonium production were not significantly affected by monocrotaline infusion, although a tendency toward higher values was evident for oxygen consumption. After the end of monocrotaline infusion, a small but significant reduction of ammonium production was observed (Fig. 2D). 4. Discussion The results of the present study show that monocrotaline affects energy-linked hepatic metabolism in different manners, with glycogenolysis activation and gluconeogenesis/ureogenesis inhibition being the most significant effects. It is well known that liver glycogenolysis and gluconeogenesis are the major sources of circulating glucose and that impairment of these processes can have negative consequences on other organs. Hepatic glycogen is replenished postprandially during the absorptive period. As fasting progresses, the glycogen supply is depleted, and new glucose synthesis, or gluconeogenesis, becomes the predominant process contributing to glucose production (Ruderman, 1975). Gluconeogenesis is also important for ruminant metabolism since, although ruminant animals have a major need for glucose (principally during lactation), little of this need is met by glucose absorbed from the gastrointestinal tract (Lindsay, 1970). The liver is also the major site of conversion of amino-N to urea, and the urea cycle is well known as the main pathway for the removal of potentially toxic ammonium from blood of ruminants as well as other mammals (Meijer et al., 1990; Huntington and Archibeque, 1999; Watford, 2003). Activation of glycogenolysis and inhibition of gluconeogenesis and ureogenesis all occur when uncoupling agents or inhibitors of oxidative phosphorylation are infused into isolated perfused rat livers (Scholz and Bücher, 1965; Constantin et al., 1995; SalgueiroPagadigorria et al., 1996; Petrescu and Tarba, 1997). Furthermore, l-lactate production from endogenous glycogen was enhanced by monocrotaline perfusion, indicating increased glycolysis. This effect, together with the increased glycogenolysis, is an expected compensation for decreased mitochondrial ATP formation, which is an effect of dehydromonocrotaline that has recently been demonstrated in isolated mitochondria (Mingatto et al., 2007). The inhibition of energy metabolism by monocrotaline is quite clear, and the involvement of dehydromonocrotaline is highly probable, due to its inhibitory action on respiratory chain complex I (Mingatto et al., 2007). It should be stressed however, that monocrotaline is not a typical inhibitor of oxidative phosphorylation because inhibition of oxygen consumption was not observed in the perfused liver. In contrast, there was a small stimulatory effect. This apparent discrepancy may reflect a superimposition of the actions of dehydromonocrotaline and possibly other monocrotaline metabolites on mitochondrial respiration and the activation of cytochrome P-450 monooxygenases. The latter enzymes are implicated in the metabolic transformation of monocrotaline (Reid et al., 1998) and could be responsible, at least in part, for the stimulation of oxygen consumption observed during monocrotaline infusion. The finding that livers only responded to monocrotaline after induction of cytochrome P-450 by phenobarbital pre-treatment clearly shows that the observed metabolic changes were caused by monocrotaline metabolites. Although dehydromonocrotaline is
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considered to be the most toxic metabolite, we cannot exclude the possibility that other metabolites are equally responsible for some of the observed changes. The different kinetics of glycogenolysis stimulation and gluconeogenesis and ureogenesis inhibition support this possibility. While the inhibition of gluconeogenesis and ureogenesis occurred a few minutes after monocrotaline infusion, the development of glycogenolysis stimulation was relatively slow. Furthermore, 20 min after the interruption of monocrotaline treatment, glycogenolysis and pyruvate production from l-alanine were still affected in contrast with the inhibition of gluconeogenesis and ureogenesis, which recovered more quickly. Yan and Huxtable (1995b) demonstrated that the fraction of monocrotaline converted to dehydromonocrotaline in perfused rat livers after 1.0 h of recirculation varied from 4.6% at the lowest monocrotaline concentration (125 M) to 2.4% at the highest concentration (1500 M). These are equivalent to dehydromonocrotaline concentrations of approximately 5.8 to 36 M, respectively. The inhibitory action of dehydromonocrotaline in isolated mitochondria occurs at a similar range of concentrations (IC50 = 62.06 M; Mingatto et al., 2007). In the present work, monocrotaline was infused for 40 min in a non-recirculating mode, and rats were previously treated with phenobarbital. It therefore seems reasonable that dehydromonocrotaline was produced at a concentration that could potentially affect mitochondrial function. It should be considered, however, that dehydromonocrotaline has an aqueous half-life of 2.7 s, according to Mattocks et al. (1990). If the metabolic changes caused by monocrotaline infusion are due to a direct action of dehydromonocrotaline, persistent effects cannot be expected after cessation of infusion of the parent compound. On the other hand, such persistent effects would be expected of compounds that bind extensively to intracellular structures, a possibility that can be considered based on the data of Yan and Huxtable (1995b), who found that, in addition to dehydromonocrotaline, livers produced and released the pyrroles DHP (6,7-dihydro-7-hydroxy1-hydroxymethyl-5H-pyrrolizine) and GSDHP (7-glutathionyl-6,7dihydro-1-hydroxymethyl-5H-pyrrolizine) into the perfusate or bile; a considerable fraction of these pyrrolic metabolites remained bound to liver tissues. It is therefore likely that not one but various metabolites were acting in isolated perfused rat livers; those bound to cellular structures possibly caused glycogenolysis activation and inhibition of pyruvate production from l-alanine, whereas rapidly washed out metabolites including dehydromonocrotaline likely induced gluconeogenesis and ureogenesis inhibition. Clearly, further experiments are necessary to test this hypothesis. It should be also mentioned that, although Yan and Huxtable (1995b) demonstrated a concentration-dependent increase in metabolite production when monocrotaline was infused in the range of 125–1500 M, in the present work the maximal effects on glycogen catabolism as well as gluconeogenesis and ureogenesis were attained with 500 M monocrotaline. Because the action of monocrotaline depends on its metabolization, the metabolic transformation of monocrotaline in phenobarbital-treated rats likely becomes saturated at lower concentrations than in untreated rats. Animals and humans intoxicated with monocrotaline have been demonstrated to present clinical signs related to hepatic failure including anorexia, weight loss and jaundice, with concomitant histological changes characterized by fibrosis, megalocytosis and centrilobular necrosis (Brooks et al., 1970; Nobre et al., 2004a, b, 2005). In addition, liver injury from monocrotaline has been divided into acute and chronic phases with damage in the sinusoidal endothelial cells, central venular endothelial cells and hepatic parenchymal cells followed by the hepatic veno-occlusive disease or sinusoidal obstruction syndrome (Copple et al., 2003). Since an inhibition of energy metabolism may affect several processes,
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including metabolite and macromolecules synthesis, active transport systems, membrane integrity and others, it is reasonable that, the energy-linked liver metabolic changes described in the present work may contribute to monocrotaline hepatotoxicity in animals and humans. Conflict of interest None declared. Acknowledgements This work was supported by grants from Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Process number 2004/09882-7, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. The authors thank Célia Akemi Gasparetto, Luiz Saraiva Arraes, Irene Aparecida Bernardino and Aparecida Pinto Munhoz Hermoso for their technical assistance. References Bergmeyer, H.U., 1974. Determination of urea with glutamate dehydrogenase as indicator enzyme. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 1794–1801. Bergmeyer, H.U., Bernt, E., 1974. Determination of glucose with glucose oxidase and peroxidase. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 1205–1215. Brooks, S.E., Miller, C.G., McKenzie, K., Audretsch, J.J., Bras, G., 1970. Acute venoocclusive disease of the liver. Fine structure in Jamaican children. Arch. Pathol. 89, 507–520. Butler, W.H., Mattocks, A.R., Barnes, J.M., 1970. Lesions in the liver and lungs of rats given pyrrole derivatives of pyrrolizidine alkaloids. J. Pathol. 100, 169–175. Constantin, J., Ishii-Iwamoto, E.L., Suzuki-Kemmelmeier, F., Yamamoto, N.S., Bracht, A., 1995. Bivascular liver perfusion in the anterograde and retrograde modes: zonation of the response to inhibitors of oxidative phosphorylation. Cell Biochem. Funct. 13, 201–209. Copple, B.L., Banes, A., Ganey, P.E., Roth, R.A., 2002. Endothelial cell injury and fibrin deposition in rat liver after monocrotaline exposure. Toxicol. Sci. 65, 309–318. Copple, B.L., Ganey, P.E., Roth, R.A., 2003. Liver inflammation during monocrotaline hepatotoxicity. Toxicology 190, 155–169. Copple, B.L., Rondelli, C.M., Maddox, J.F., Hoglen, N.C., Ganey, P.E., Roth, R.A., 2004. Modes of cell death in rat liver after monocrotaline exposure. Toxicol. Sci. 77, 172–182. Czok, R., Lamprecht, W., 1974. Pyruvate, phosphoenolpyruvate and d-glycerate-2phosphate. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 1446–1451. DeLeve, L.D., MsCuskey, R.S., Wang, X., Hu, L., McCuskey, M.K., Epstein, R.B., Kanel, G.C., 1999. Characterization of a reproducible rat model of hepatic venoocclusive disease. Hepatology 29, 1779–1791. Gutman, J., Wahlefeld, A.W., 1974. l-(+)-Lactate determination with lactate dehydrogenase and NAD+ . In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 1464–1468. Hincks, J.R., Kim, H.-Y., Segall, H.J., Molyneux, R.J., Stermitz, F.R., Coulombe Jr., R.A., 1991. DNA cross-linking in mammalian cells by pyrrolizidine alkaloids: structure-activity relationships. Toxicol. Appl. Pharmacol. 111, 90–98. Huntington, G.B., Archibeque, S.L., 1999. Practical aspects of urea and ammonia metabolism in ruminant. Proc. Am. Soc. Anim. Sci. Available: http://www.asas.org/symposia/98-99proc.htm (accessed September 3, 2008). Huxtable, R.J., 1989. Human health implications of pyrrolizidine alkaloids and herbs containing them. In: Cheeke, P.R. (Ed.), Toxicants of Plant Origin, vol. 1. CRC Press, Boca Raton, pp. 41–86. Kun, F., Kearney, E.B., 1974. Ammonia. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 1802–1806. Lafranconi, W.M., Huxtable, R.J., 1984. Hepatic metabolism and pulmonary toxicity of monocrotaline using isolated perfused liver and lung. Biochem. Pharmacol. 33, 2479–2484. Lamé, M.W., Jones, A.D., Wilson, D.W., Segall, H.J., 2005. Monocrotaline pyrrole targets proteins with and without cysteine residues in the cytosol and membranes of human pulmonary artery endothelial cells. Proteomics 5, 4398–4413. Lindsay, D.B., 1970. Carbohydrate metabolism in ruminants. In: Phillipson, A.T. (Ed.), Physiology of Digestion and Metabolism in the Ruminant. Oriel Press, Newcastle upon Tyne, England, pp. 438–541. Mattocks, A.R., 1986. Chemistry and Toxicology of Pyrrolizidine Alkaloids. Academic Press, London, pp. 1–393. Mattocks, A.R., Legg, R.F., Jukes, R., 1990. Trapping of short-lived electrophilic metabolites of pyrrolizidine alkalois escaping from perfused rat liver. Toxicol. Lett. 54, 93–99.
120
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Mclean, E.K., 1970. The toxic actions of pyrrolizidine (Senecio) alkaloids. Pharmacol. Rev. 22, 429–483. Meijer, A.J., Lamers, W.H., Chamuleau, R.A., 1990. Nitrogen metabolism and ornithine cycle function. Physiol. Rev. 76, 701–748. Mingatto, F.E., Dorta, D.J., Santos, A.B., Carvalho, I., Silva, C.H.T.P., Silva, V.B., Uyemura, S.A., Santos, A.C., Curti, C., 2007. Dehydromonocrotaline inhibits mitochondrial complex I. A potential mechanism accounting for hepatotoxicity of monocrotaline. Toxicon 50, 724–730. Niwa, H., Ogawa, T., Yamada, K., 1991. Alkylation of nucleoside by dehydromonocrotaline, the putative toxic metabolic of the carcinogenic alkaloid monocrotaline. Tetrahedron Lett. 32, 927–930. Nobre, V.M.T., Riet-Correa, F., Barbosa Filho, J.M., Dantas, A.F.M., Tabosa, I.M., Vasconcelos, J.S., 2004a. Poisoning by Crotalaria retusa (Fabaceae) in Equidae in the semiarid region of Paraíba. Pesquisa Vet. Brasil. 24 (3), 132–143. Nobre, V.M.T., Riet-Correa, F., Dantas, A.F.M., Tabosa, I.M., Medeiros, R.M.T., Barbosa Filho, J.M., 2004b. Intoxication by Crotalaria retusa in ruminants and eqüidae in the state of Paraíba, northeastern Brazil. In: Acamovic, T., et al. (Eds.), Poisonous Plant and Related Toxins. CAB International, Glasgow, UK, pp. 275–278. Nobre, V.M.T., Dantas, A.F.M., Riet-Correa, F., Barbosa Filho, J.M., Tabosa, I.M., Vasconcelos, J.S., 2005. Acute intoxication by Crotalaria retusa in sheep. Toxicon 45, 347–352. Pan, L.C., Wilson, D.W., Lame, M.W., Jones, A.D., Segall, H.J., 1993. COR pulmonale is caused by monocrotaline and dehydromonocrotaline but not by glutathione or cysteine conjugates of dihydropyrrolizine. Toxicol. Appl. Pharmacol. 118, 87–97. Petrescu, I., Tarba, C., 1997. Uncoupling effects of diclofenac and aspirin in the perfused liver and isolated hepatic mitochondria of rat. Biochim. Biophys. Acta 1318, 385–394. Petry, T.W., Bowden, G.T., Huxtable, R.J., Sipes, I.G., 1984. Characterization of hepatic DNA damage induced in rats by the pyrrolizidine alkaloid monocrotaline. Cancer Res. 44, 1505–1509. Reid, M.J., Lamé, M.W., Morin, D., Wilson, D.W., Segall, H.J., 1998. Involvement of cytochrome P450 3A in the metabolism and covalent binding of 14 Cmonocrotaline in rat liver microsomes. J. Biochem. Mol. Toxicol. 12, 157–166. Roth, R.A., Reindel, J.F., 1990. Lung vascular injury from monocrotaline pyrrole, a putative hepatic metabolite. In: Witner, C.M., et al. (Eds.), Advances in Experimental Medicine & Biology, Biological Reactive Intermediates IV. Plenum Press, New York, pp. 477–487. Ruderman, N.B., 1975. Muscle amino acid metabolism and gluconeogenesis. Ann. Rev. Med. 26, 245–258.
Salgueiro-Pagadigorria, C.L., Constantin, J., Bracht, A., Nascimento, E.A., IshiiIwamoto, E.L., 1996. Effects of the nonsteroidal anti-inflammatory drug piroxicam on energy metabolism in the perfused rat liver. Comp. Biochem. Physiol. 113C, 93–98. Scholz, R., Bücher, T., 1965. Hemoglobin-free perfusion of rat liver. In: Chance, B., Estabrook, R.W., Williamson, J.R. (Eds.), Control of Energy Metabolism. Academic Press, New York, pp. 393–414. Schultze, A.E., Roth, R.A., 1998. Chronic pulmonary hypertension-the monocrotaline model and involvement of the hemostatic system. J. Toxicol. Environ. Health B: Crit. Rev. 1, 271–346. Souza, A.C., Hatayde, M.R., Bechara, G.H., 1997. Pathological aspects of poisoning by Crotalaria spectabilis (Fabaceae) seeds in swine. Pesquisa Vet. Brasil. 17, 12–18. Stegelmeier, B.L., Edgar, J.A., Colegate, S.M., Gardner, D.R., Scloch, T.K., Coulombe, R.A., Molyneux, R.J., 1999. Pyrrolizidine alkaloid plants, metabolism and toxicity. J. Nat. Toxins 8, 95–116. Vlasuk, G.P., Ghrayeb, J., Ryan, D.E., Reik, L., Thomas, P.E., Levin, W., Walz Jr., F.G., 1982. Multiplicity strain differences, and topology of phenobarbital-induced cytochromes P450 in rat liver microsomes. Biochemistry 21, 789–798. Wagner, J.G., Petry, T.W., Roth, R.A., 1993. Characterization of monocrotaline pyrroleinduced DNA cross-linking in pulmonary artery endothelium. Am. J. Physiol. Lung Cell. Mol. Physiol. 264, L517–L522. Watford, M., 2003. The urea cycle: teaching intermediary metabolism in a physiological setting. Biochem. Mol. Biol. Educ. 31, 289–297. Wilson, D.W., Segall, H.J., Pan, L.C., Lame, M.W., Estep, J.E., Morin, D., 1992. Mechanisms and pathology of monocrotaline pulmonary toxicity. Crit. Rev. Toxicol. 22, 307–325. Yan, C.C., Huxtable, R.J., 1994. Quantitation of the hepatic release of metabolites of the pyrrolizidine alkaloid, monocrotaline. Toxicol. Appl. Pharmacol. 127, 58–63. Yan, C.C., Huxtable, R.J., 1995a. The effect of the pyrrolizidine alkaloids, monocrotaline and trichodesmine, on tissue pyrrole binding and glutathione metabolism in the rat. Toxicon 33, 627–634. Yan, C.C., Huxtable, R.J., 1995b. The relationship between the concentration of the pyrrolizidine alkaloid monocrotaline and the pattern of metabolites released from the isolated liver. Toxicol. Appl. Pharmacol. 130, 1–8. Yee, S.B., Kinser, S., Hill, D.A., Barton, C.C., Hotchkiss, J.A., Harkema, J.R., Ganey, P.E., Roth, R.A., 2000. Synergistic hepatotoxicity from coexposure to bacterial endotoxin and the pyrrolizidine alkaloid monocrotaline. Toxicol. Appl. Pharmacol. 166, 173–185.
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Effects of monocrotaline on energy metabolism in the rat liver Fábio Erminio Mingatto a,∗ , Marcos Antonio Maioli a , Adelar Bracht b , Emy Luiza Ishii-Iwamoto b a b
Laboratório de Bioquímica, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Dracena, 17900-000 Dracena, SP, Brazil Laboratório de Metabolismo Hepático, Departamento de Bioquímica, Universidade Estadual de Maringá, 87020-900 Maringá, PR, Brazil
a r t i c l e
i n f o
Article history: Received 30 April 2008 Received in revised form 5 September 2008 Accepted 5 September 2008 Available online 16 September 2008 Keywords: Monocrotaline Dehydromonocrotaline Liver metabolism Glycogenolysis Gluconeogenesis Urea cycle
a b s t r a c t Monocrotaline (MCT) is a pyrrolizidine alkaloid present in the plants of the Crotalaria species that causes cytotoxicity and genotoxicity in animals and humans, and it is hepatically metabolized to the alkylating agent dehydromonocrotaline by cytochrome P-450. The exact cellular and molecular mechanisms of MCTinduced tissue injury remain unclear. We previously demonstrated that dehydromonocrotaline, but not monocrotaline, inhibits the activity of NADH-dehydrogenase at micromolar concentrations in isolated liver mitochondria, an effect associated with significantly reduced ATP synthesis. Impairment of energy metabolism is expected to lead to several alterations in cell metabolism. In this work, the action of different concentrations of monocrotaline (250, 500, and 750 M) on energy metabolism-linked parameters was investigated in isolated perfused rat livers. In the fed state, monocrotaline increased glycogenolysis and glycolysis, whereas in the livers of fasted rats, it decreased gluconeogenesis and urea synthesis from lalanine. These metabolic alterations were only found in livers of phenobarbital-treated rats, indicating that active metabolites including dehydromonocrotaline were responsible for the observed activity. Our findings indicate that hepatic metabolic changes may be implicated, partly at least, in the hepatotoxicity of monocrotaline in animals and humans. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Monocrotaline (MCT), an alkaloid pyrrolizidine phytotoxin, has well-documented hepatic and cardiopulmonary toxicity for animals, including ruminants, and man (Mclean, 1970; Mattocks, 1986; Huxtable, 1989; Souza et al., 1997; Schultze and Roth, 1998; Stegelmeier et al., 1999; Nobre et al., 2004a, b, 2005). Monocrotaline is frequently ingested accidentally because of food grain contamination or intentionally in the form of herbal medicine preparations (Huxtable, 1989). It was demonstrated that liver injury occurs 24 h after monocrotaline administration in rats at doses higher than 150 mg kg−1 . MCT-induced hepatic lesions are characterized by centrilobular cell necrosis, congestion and dilation of sinusoids, hemorrhage, damage to sinusoidal and central venular endothelial cells (DeLeve et al., 1999; Yee et al., 2000; Copple et al., 2002), and parenchymal cell oncosis and apoptosis (Copple et al., 2004). Monocrotaline toxicity requires cytochrome P-450-mediated bioactivation to reactive pyrrolic metabolite dehydromonocrotaline (DHM) (Butler et al., 1970; Lafranconi and Huxtable, 1984; Roth and Reindel, 1990; Wilson et al., 1992; Pan et al., 1993; Schultze and Roth, 1998). This metabolite, despite having a half-life of only a
∗ Corresponding author. Tel.: +55 18 3821 8200; fax: +55 18 3821 8208. E-mail address: [email protected] (F.E. Mingatto). 0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2008.09.004
few seconds in aqueous media (Mattocks et al., 1990), is a powerful alkylating agent that binds to cellular DNA and proteins (Petry et al., 1984; Hincks et al., 1991; Niwa et al., 1991; Wagner et al., 1993; Yan and Huxtable, 1995a; Lamé et al., 2005). The exact cellular and molecular mechanisms of MCT-induced hepatic injury remain unclear. We previously demonstrated that dehydromonocrotaline, but not monocrotaline, inhibits the activity of NADH-dehydrogenase when added at micromolar concentrations to isolated rat liver mitochondria, an effect associated with significantly reduced ATP synthesis (Mingatto et al., 2007). Because the activity of complex I is regulated by thiol groups, it was suggested that the inhibition of complex I NADH oxidase activity resulted from the oxidation of cysteine thiol groups by dehydromonocrotaline. Inhibition of mitochondrial function is likely to affect cell metabolism in several ways. In principle, increases in catabolic processes that can lead to ATP synthesis independently of oxidative phosphorylation as well as decreases in energy-dependent anabolic processes should be expected. However, this toxic effect on mitochondria has not been reproduced in the intact liver until now. It must be stressed that the conditions under which mitochondria operate within intact cells are quite different from those of the rather simple environment of a suspension of isolated organelles. Added factors include the passage of drug through plasma membrane, the presence of numerous intracellular binding sites, and
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biotransformation reactions, all of which are important for making the active drug metabolite available to the mitochondria. Furthermore, the possibility exists that the actions of monocrotaline in the intact liver are not solely the consequence of a primary action of dehydromonocrotaline on mitochondria. The present study, performed in isolated perfused rat livers, was planned to address these questions. Monocrotaline was infused into the livers, and several metabolic fluxes were measured, with special emphasis on metabolic fluxes related to energy metabolism in terms of glycogenolysis, glycolysis and oxygen consumption in the livers of fed rats, as well as gluconeogenesis and ureogenesis in the livers from fasted rats. In order to stimulate the production of monocrotaline metabolites by perfused livers, rats were previously treated with phenobarbital, a well-known inducer of hepatic cytochrome P-450 isoenzymes (Vlasuk et al., 1982), which have, in turn, been used to stimulate MCT metabolism (Reid et al., 1998). 2. Materials and methods 2.1. Chemicals The liver perfusion apparatus was built in the workshops of the University of Maringá. Monocrotaline, enzymes and coenzymes used in the enzymatic assays were purchased from Sigma–Aldrich (St. Louis, MO, USA). Phenobarbital was purchased from Rhodia Farma, Brazil. Sodium pentobarbital was a gift from Cristália, Brazil. All other reagents were of the highest commercially available grade.
2.2. Animals Male albino rats (Wistar) weighing 180–220 g were fed ad libitum with a standard laboratory diet (Nuvilab® , Colombo, Brazil). In some experimental protocols, the rats were starved for 24 h before the surgical removal of the liver. In experiments with phenobarbital induction, rats were dosed intraperitoneally (90 mg kg body weight−1 ) daily for 3 consecutive days. 2.3. Liver perfusion For the surgical procedure, fed or 24 h fasted rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg kg body weight−1 ). Hemoglobin-free, non-recirculating perfusion was performed. The surgical technique was the same as that described by Scholz and Bücher (1965). After cannulation of the portal and cava veins, the liver was positioned in a Plexiglas chamber. The perfusion fluid was Krebs/Henseleit-bicarbonate buffer (pH 7.4), saturated with a mixture of oxygen and carbon dioxide (95:5) using a membrane oxygenator with simultaneous temperature adjustment at 37 ◦ C. The flow, provided by a peristaltic pump, was between 30 and 35 ml min−1 , depending on the liver weight. Samples of the effluent perfusion fluid were collected according to the experimental protocol and analyzed for their metabolite contents. The oxygen concentration in the outflowing perfusate was monitored continuously using a Teflon-shielded platinum electrode adequately positioned in a Plexiglas chamber at the point where the perfusate exits (Scholz and Bücher, 1965). Monocrotaline (250, 500 and 750 M) or l-alanine (2.5 mM) was dissolved in the perfusion fluid. 2.4. Analytical The following compounds were assayed using standard enzymatic procedures: glucose (Bergmeyer and Bernt, 1974), l-lactate (Gutman and Wahlefeld, 1974), pyruvate (Czok and Lamprecht, 1974), urea (Bergmeyer, 1974) and ammonium (Kun and Kearney, 1974). Metabolic rates were calculated from input–output differences and
Fig. 1. Time courses of the effects of monocrotaline on glycogen catabolism and oxygen consumption in perfused livers of phenobarbital-treated fed rats. Livers from fed rats were perfused with Krebs/Henseleit-bicarbonate buffer as described in Section 2. Panel A represents the livers of control rats. Monocrotaline was infused for 10–50 min at concentrations of 250 M (panel B), 500 M (panel C) or 750 M (panel D) as indicated by the horizontal bars. Samples of the effluent perfusate were withdrawn for metabolite assays. Oxygen consumption was monitored polarographically. The results represent the mean values ± S.E.M. of four perfusion experiments. Significant differences in the time course of oxygen consumption, glucose and l-lactate production in the monocrotaline infusion series (panels B, C and D) as compared to the control series (panel A) are indicated by horizontal lines (two-way analysis of variance, with post hoc Newman–Keuls test).
F.E. Mingatto et al. / Toxicology Letters 182 (2008) 115–120 the total flow rates, normalized to the wet weight of the liver, and expressed as mol min−1 (gram liver wet weight)−1 . 2.5. Data analysis The data in the figures are expressed as mean ± standard error of the mean (S.E.M.). The statistical significance of the differences between parameters among the experimental groups was evaluated using two-way analysis of variance, and differences in the same experimental groups were tested by repeated-measures one-way analysis of variance (ANOVA). Significant differences among means were identified by Newman–Keuls testing. The results are given in the text as probability values (P). P ≤ 0.05 was adopted as the criterion of significance. Statistical analysis was performed using StatisticaTM or GraphPAD Software programs.
3. Results 3.1. Effects of monocrotaline on glycogen catabolism and oxygen consumption In the first experiments, the effects of monocrotaline on glycogen catabolism and oxygen consumption were investigated. Livers from fed rats were perfused with substrate-free perfusion medium in a non-recirculating mode. Under these conditions, the livers respire mainly at the expense of endogenous fatty acids, but
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they simultaneously exhibit extensive glycogenolytic and glycolytic activity (Scholz and Bücher, 1965), as indicated by glucose, l-lactate and pyruvate release. Monocrotaline was infused at 250, 500 and 750 M. It was previously demonstrated by Yan and Huxtable (1994) that, at these concentrations, perfused rat livers produce and release significant amounts of potentially toxic metabolites. When monocrotaline was infused for 40 min at 500 M in perfused livers from non-treated rats, there were no significant changes in glucose, l-lactate or pyruvate productions or in oxygen consumption (data not shown). Thus, subsequent experiments were performed with livers from phenobarbital-treated rats using three doses of monocrotaline (250, 500 and 750 M). Fig. 1 illustrates the experimental protocol and the time course of the metabolic fluxes when livers were perfused for 90 min in the absence (control) or in the presence of monocrotaline. In the control series (panel A), the rates of oxygen consumption and pyruvate production did not change during the experimental time period. However, glucose and l-lactate production progressively decreased, possibly due to the low glycogen content of livers from phenobarbital-treated rats. As shown by panels B, C and D, the introduction of 250 M, 500 M or 750 M monocrotaline at 10 min produced changes, especially in glucose and l-lactate
Fig. 2. Time course of the effects of 500 M monocrotaline on glucose, l-lactate and pyruvate production and on oxygen consumption as well as urea and ammonium production derived from l-alanine in perfused livers from phenobarbital-treated rats. Livers from 24 h fasted rats were perfused with Krebs/Henseleit-bicarbonate buffer as described in Section 2. l-Alanine (2.5 mM) was infused after 10–88 min of perfusion. Panels A and B represent the livers of the control rats. Monocrotaline (500 M) was infused at 30–70 min as indicated by the horizontal bars in the panels C and D. Samples of the effluent perfusate were withdrawn for metabolite assay. Data represent the mean values ± S.E.M. of four to six perfusion experiments. Significant differences in the time course of pyruvate production (panel C) and urea and ammonium production (panel D) in the monocrotaline infusion series as compared to the control series are indicated by horizontal lines (two-way analysis of variance, with post hoc Newman–Keuls test). *P ≤ 0.05 compared with values obtained immediately before monocrotaline infusion (panel C), according to repeated-measures ANOVA testing.
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production. Although significantly increased by all concentrations of monocrotaline, glucose and l-lactate production did not show a clear concentration-effect relationship. Glucose production, for example, was increased by 243%, 239% and 278% after infusion of 250, 500 and 750 M monocrotaline, respectively. After cessation of monocrotaline infusion, glucose production remained elevated for at least the following 20 min. The increase in l-lactate production was not as large as that of glucose production, and the stimulation kinetics were different. At 250 M, a gradual increase was observed during the entire course of monocrotaline infusion and l-lactate production stabilized at a level nearly 84% higher than that of the control series. l-Lactate production was also stimulated after the onset of 500 and 750 M monocrotaline infusion. After nearly 10 min, the rates decreased progressively and stabilized at lower values than those observed for 250 M monocrotaline; however, this level was still higher than that of the control series. Finally, slight stimulation of oxygen consumption was observed upon the infusion of 250 M, 500 M or 750 M monocrotaline, and pyruvate production was not significantly affected. 3.2. Effects of monocrotaline on gluconeogenesis, the urea cycle and oxygen consumption In order to determine the effects of monocrotaline on energydependent biosynthetic processes, gluconeogenesis, ureogenesis, oxygen consumption, ammonium level, and l-lactate and pyruvate production from l-alanine were measured in perfused livers of fasted rats. The control experiments, in which only the gluconeogenic precursor l-alanine was infused in livers of phenobarbital-treated rats, are shown in Fig. 2A and B. After a pre-perfusion period of 10 min, 2.5 mM l-alanine was infused for 80 min. As shown by Fig. 2A, the amounts of glucose, l-lactate and pyruvate were negligible prior to l-alanine infusion due to low endogenous levels of glycogen and gluconeogenic substrates. After the onset of l-alanine infusion, glucose, l-lactate and pyruvate production progressively increased. After approximately 15 min, steady-state conditions were attained for glucose production. For l-lactate and pyruvate production, on the other hand, steady-state conditions only occurred after 40 min of substrate infusion. In previous experiments, we have found that the effects of 750 M monocrotaline infusion on l-alanine metabolism closely resembled those of 500 M monocrotaline, as it was demonstrated to occurs in the livers of fed rats (Fig. 1). The actions of a selected concentration of monocrotaline (500 M) are thus illustrated by Fig. 2 (panels C and D). The introduction of monocrotaline produced a rapid and significant inhibition of glucose production (Fig. 2C). The maximum inhibition (nearly 25%) occurred at 4 min of monocrotaline infusion. After this time, however, there was a gradual recovery of glucose production such that at the terminus of monocrotaline infusion, glucose production was not statistically different from its level before monocrotaline infusion or from the control series. Fig. 2C also shows that l-lactate production was not significantly affected. Pyruvate production, on the other hand, was progressively and significantly reduced. At the end of monocrotaline infusion, pyruvate production was 42% lower than in the control series at the corresponding time point (70 min). There was no evident recovery of pyruvate production; at the end of the experimental period (90 min), it was 50% lower than in the control series. Oxygen consumption and the production of urea and ammonium by control livers are shown in Fig. 2B. In the absence of exogenous substrate, ammonium production was negligible, and the basal rate of urea production was 0.076 mol min−1 g−1 . lAlanine infusion increased oxygen consumption as well as urea and ammonium production. Steady-state levels of oxygen con-
sumption and urea production were maintained during the entire l-alanine infusion. Ammonium production, on the other hand, increased progressively during the same period. The introduction of 500 M monocrotaline significantly inhibited urea production as can be seen in Fig. 2D. Twenty-five percent inhibition was observed at the end of monocrotaline infusion when compared to the values found in the control series. Upon cessation of monocrotaline infusion, there was a gradual recovery of urea production. The rates of oxygen consumption and ammonium production were not significantly affected by monocrotaline infusion, although a tendency toward higher values was evident for oxygen consumption. After the end of monocrotaline infusion, a small but significant reduction of ammonium production was observed (Fig. 2D). 4. Discussion The results of the present study show that monocrotaline affects energy-linked hepatic metabolism in different manners, with glycogenolysis activation and gluconeogenesis/ureogenesis inhibition being the most significant effects. It is well known that liver glycogenolysis and gluconeogenesis are the major sources of circulating glucose and that impairment of these processes can have negative consequences on other organs. Hepatic glycogen is replenished postprandially during the absorptive period. As fasting progresses, the glycogen supply is depleted, and new glucose synthesis, or gluconeogenesis, becomes the predominant process contributing to glucose production (Ruderman, 1975). Gluconeogenesis is also important for ruminant metabolism since, although ruminant animals have a major need for glucose (principally during lactation), little of this need is met by glucose absorbed from the gastrointestinal tract (Lindsay, 1970). The liver is also the major site of conversion of amino-N to urea, and the urea cycle is well known as the main pathway for the removal of potentially toxic ammonium from blood of ruminants as well as other mammals (Meijer et al., 1990; Huntington and Archibeque, 1999; Watford, 2003). Activation of glycogenolysis and inhibition of gluconeogenesis and ureogenesis all occur when uncoupling agents or inhibitors of oxidative phosphorylation are infused into isolated perfused rat livers (Scholz and Bücher, 1965; Constantin et al., 1995; SalgueiroPagadigorria et al., 1996; Petrescu and Tarba, 1997). Furthermore, l-lactate production from endogenous glycogen was enhanced by monocrotaline perfusion, indicating increased glycolysis. This effect, together with the increased glycogenolysis, is an expected compensation for decreased mitochondrial ATP formation, which is an effect of dehydromonocrotaline that has recently been demonstrated in isolated mitochondria (Mingatto et al., 2007). The inhibition of energy metabolism by monocrotaline is quite clear, and the involvement of dehydromonocrotaline is highly probable, due to its inhibitory action on respiratory chain complex I (Mingatto et al., 2007). It should be stressed however, that monocrotaline is not a typical inhibitor of oxidative phosphorylation because inhibition of oxygen consumption was not observed in the perfused liver. In contrast, there was a small stimulatory effect. This apparent discrepancy may reflect a superimposition of the actions of dehydromonocrotaline and possibly other monocrotaline metabolites on mitochondrial respiration and the activation of cytochrome P-450 monooxygenases. The latter enzymes are implicated in the metabolic transformation of monocrotaline (Reid et al., 1998) and could be responsible, at least in part, for the stimulation of oxygen consumption observed during monocrotaline infusion. The finding that livers only responded to monocrotaline after induction of cytochrome P-450 by phenobarbital pre-treatment clearly shows that the observed metabolic changes were caused by monocrotaline metabolites. Although dehydromonocrotaline is
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considered to be the most toxic metabolite, we cannot exclude the possibility that other metabolites are equally responsible for some of the observed changes. The different kinetics of glycogenolysis stimulation and gluconeogenesis and ureogenesis inhibition support this possibility. While the inhibition of gluconeogenesis and ureogenesis occurred a few minutes after monocrotaline infusion, the development of glycogenolysis stimulation was relatively slow. Furthermore, 20 min after the interruption of monocrotaline treatment, glycogenolysis and pyruvate production from l-alanine were still affected in contrast with the inhibition of gluconeogenesis and ureogenesis, which recovered more quickly. Yan and Huxtable (1995b) demonstrated that the fraction of monocrotaline converted to dehydromonocrotaline in perfused rat livers after 1.0 h of recirculation varied from 4.6% at the lowest monocrotaline concentration (125 M) to 2.4% at the highest concentration (1500 M). These are equivalent to dehydromonocrotaline concentrations of approximately 5.8 to 36 M, respectively. The inhibitory action of dehydromonocrotaline in isolated mitochondria occurs at a similar range of concentrations (IC50 = 62.06 M; Mingatto et al., 2007). In the present work, monocrotaline was infused for 40 min in a non-recirculating mode, and rats were previously treated with phenobarbital. It therefore seems reasonable that dehydromonocrotaline was produced at a concentration that could potentially affect mitochondrial function. It should be considered, however, that dehydromonocrotaline has an aqueous half-life of 2.7 s, according to Mattocks et al. (1990). If the metabolic changes caused by monocrotaline infusion are due to a direct action of dehydromonocrotaline, persistent effects cannot be expected after cessation of infusion of the parent compound. On the other hand, such persistent effects would be expected of compounds that bind extensively to intracellular structures, a possibility that can be considered based on the data of Yan and Huxtable (1995b), who found that, in addition to dehydromonocrotaline, livers produced and released the pyrroles DHP (6,7-dihydro-7-hydroxy1-hydroxymethyl-5H-pyrrolizine) and GSDHP (7-glutathionyl-6,7dihydro-1-hydroxymethyl-5H-pyrrolizine) into the perfusate or bile; a considerable fraction of these pyrrolic metabolites remained bound to liver tissues. It is therefore likely that not one but various metabolites were acting in isolated perfused rat livers; those bound to cellular structures possibly caused glycogenolysis activation and inhibition of pyruvate production from l-alanine, whereas rapidly washed out metabolites including dehydromonocrotaline likely induced gluconeogenesis and ureogenesis inhibition. Clearly, further experiments are necessary to test this hypothesis. It should be also mentioned that, although Yan and Huxtable (1995b) demonstrated a concentration-dependent increase in metabolite production when monocrotaline was infused in the range of 125–1500 M, in the present work the maximal effects on glycogen catabolism as well as gluconeogenesis and ureogenesis were attained with 500 M monocrotaline. Because the action of monocrotaline depends on its metabolization, the metabolic transformation of monocrotaline in phenobarbital-treated rats likely becomes saturated at lower concentrations than in untreated rats. Animals and humans intoxicated with monocrotaline have been demonstrated to present clinical signs related to hepatic failure including anorexia, weight loss and jaundice, with concomitant histological changes characterized by fibrosis, megalocytosis and centrilobular necrosis (Brooks et al., 1970; Nobre et al., 2004a, b, 2005). In addition, liver injury from monocrotaline has been divided into acute and chronic phases with damage in the sinusoidal endothelial cells, central venular endothelial cells and hepatic parenchymal cells followed by the hepatic veno-occlusive disease or sinusoidal obstruction syndrome (Copple et al., 2003). Since an inhibition of energy metabolism may affect several processes,
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including metabolite and macromolecules synthesis, active transport systems, membrane integrity and others, it is reasonable that, the energy-linked liver metabolic changes described in the present work may contribute to monocrotaline hepatotoxicity in animals and humans. Conflict of interest None declared. Acknowledgements This work was supported by grants from Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Process number 2004/09882-7, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. The authors thank Célia Akemi Gasparetto, Luiz Saraiva Arraes, Irene Aparecida Bernardino and Aparecida Pinto Munhoz Hermoso for their technical assistance. References Bergmeyer, H.U., 1974. Determination of urea with glutamate dehydrogenase as indicator enzyme. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 1794–1801. Bergmeyer, H.U., Bernt, E., 1974. Determination of glucose with glucose oxidase and peroxidase. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 1205–1215. Brooks, S.E., Miller, C.G., McKenzie, K., Audretsch, J.J., Bras, G., 1970. Acute venoocclusive disease of the liver. Fine structure in Jamaican children. Arch. Pathol. 89, 507–520. Butler, W.H., Mattocks, A.R., Barnes, J.M., 1970. Lesions in the liver and lungs of rats given pyrrole derivatives of pyrrolizidine alkaloids. J. Pathol. 100, 169–175. Constantin, J., Ishii-Iwamoto, E.L., Suzuki-Kemmelmeier, F., Yamamoto, N.S., Bracht, A., 1995. Bivascular liver perfusion in the anterograde and retrograde modes: zonation of the response to inhibitors of oxidative phosphorylation. Cell Biochem. Funct. 13, 201–209. Copple, B.L., Banes, A., Ganey, P.E., Roth, R.A., 2002. Endothelial cell injury and fibrin deposition in rat liver after monocrotaline exposure. Toxicol. Sci. 65, 309–318. Copple, B.L., Ganey, P.E., Roth, R.A., 2003. Liver inflammation during monocrotaline hepatotoxicity. Toxicology 190, 155–169. Copple, B.L., Rondelli, C.M., Maddox, J.F., Hoglen, N.C., Ganey, P.E., Roth, R.A., 2004. Modes of cell death in rat liver after monocrotaline exposure. Toxicol. Sci. 77, 172–182. Czok, R., Lamprecht, W., 1974. Pyruvate, phosphoenolpyruvate and d-glycerate-2phosphate. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 1446–1451. DeLeve, L.D., MsCuskey, R.S., Wang, X., Hu, L., McCuskey, M.K., Epstein, R.B., Kanel, G.C., 1999. Characterization of a reproducible rat model of hepatic venoocclusive disease. Hepatology 29, 1779–1791. Gutman, J., Wahlefeld, A.W., 1974. l-(+)-Lactate determination with lactate dehydrogenase and NAD+ . In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 1464–1468. Hincks, J.R., Kim, H.-Y., Segall, H.J., Molyneux, R.J., Stermitz, F.R., Coulombe Jr., R.A., 1991. DNA cross-linking in mammalian cells by pyrrolizidine alkaloids: structure-activity relationships. Toxicol. Appl. Pharmacol. 111, 90–98. Huntington, G.B., Archibeque, S.L., 1999. Practical aspects of urea and ammonia metabolism in ruminant. Proc. Am. Soc. Anim. Sci. Available: http://www.asas.org/symposia/98-99proc.htm (accessed September 3, 2008). Huxtable, R.J., 1989. Human health implications of pyrrolizidine alkaloids and herbs containing them. In: Cheeke, P.R. (Ed.), Toxicants of Plant Origin, vol. 1. CRC Press, Boca Raton, pp. 41–86. Kun, F., Kearney, E.B., 1974. Ammonia. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 1802–1806. Lafranconi, W.M., Huxtable, R.J., 1984. Hepatic metabolism and pulmonary toxicity of monocrotaline using isolated perfused liver and lung. Biochem. Pharmacol. 33, 2479–2484. Lamé, M.W., Jones, A.D., Wilson, D.W., Segall, H.J., 2005. Monocrotaline pyrrole targets proteins with and without cysteine residues in the cytosol and membranes of human pulmonary artery endothelial cells. Proteomics 5, 4398–4413. Lindsay, D.B., 1970. Carbohydrate metabolism in ruminants. In: Phillipson, A.T. (Ed.), Physiology of Digestion and Metabolism in the Ruminant. Oriel Press, Newcastle upon Tyne, England, pp. 438–541. Mattocks, A.R., 1986. Chemistry and Toxicology of Pyrrolizidine Alkaloids. Academic Press, London, pp. 1–393. Mattocks, A.R., Legg, R.F., Jukes, R., 1990. Trapping of short-lived electrophilic metabolites of pyrrolizidine alkalois escaping from perfused rat liver. Toxicol. Lett. 54, 93–99.
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F.E. Mingatto et al. / Toxicology Letters 182 (2008) 115–120
Mclean, E.K., 1970. The toxic actions of pyrrolizidine (Senecio) alkaloids. Pharmacol. Rev. 22, 429–483. Meijer, A.J., Lamers, W.H., Chamuleau, R.A., 1990. Nitrogen metabolism and ornithine cycle function. Physiol. Rev. 76, 701–748. Mingatto, F.E., Dorta, D.J., Santos, A.B., Carvalho, I., Silva, C.H.T.P., Silva, V.B., Uyemura, S.A., Santos, A.C., Curti, C., 2007. Dehydromonocrotaline inhibits mitochondrial complex I. A potential mechanism accounting for hepatotoxicity of monocrotaline. Toxicon 50, 724–730. Niwa, H., Ogawa, T., Yamada, K., 1991. Alkylation of nucleoside by dehydromonocrotaline, the putative toxic metabolic of the carcinogenic alkaloid monocrotaline. Tetrahedron Lett. 32, 927–930. Nobre, V.M.T., Riet-Correa, F., Barbosa Filho, J.M., Dantas, A.F.M., Tabosa, I.M., Vasconcelos, J.S., 2004a. Poisoning by Crotalaria retusa (Fabaceae) in Equidae in the semiarid region of Paraíba. Pesquisa Vet. Brasil. 24 (3), 132–143. Nobre, V.M.T., Riet-Correa, F., Dantas, A.F.M., Tabosa, I.M., Medeiros, R.M.T., Barbosa Filho, J.M., 2004b. Intoxication by Crotalaria retusa in ruminants and eqüidae in the state of Paraíba, northeastern Brazil. In: Acamovic, T., et al. (Eds.), Poisonous Plant and Related Toxins. CAB International, Glasgow, UK, pp. 275–278. Nobre, V.M.T., Dantas, A.F.M., Riet-Correa, F., Barbosa Filho, J.M., Tabosa, I.M., Vasconcelos, J.S., 2005. Acute intoxication by Crotalaria retusa in sheep. Toxicon 45, 347–352. Pan, L.C., Wilson, D.W., Lame, M.W., Jones, A.D., Segall, H.J., 1993. COR pulmonale is caused by monocrotaline and dehydromonocrotaline but not by glutathione or cysteine conjugates of dihydropyrrolizine. Toxicol. Appl. Pharmacol. 118, 87–97. Petrescu, I., Tarba, C., 1997. Uncoupling effects of diclofenac and aspirin in the perfused liver and isolated hepatic mitochondria of rat. Biochim. Biophys. Acta 1318, 385–394. Petry, T.W., Bowden, G.T., Huxtable, R.J., Sipes, I.G., 1984. Characterization of hepatic DNA damage induced in rats by the pyrrolizidine alkaloid monocrotaline. Cancer Res. 44, 1505–1509. Reid, M.J., Lamé, M.W., Morin, D., Wilson, D.W., Segall, H.J., 1998. Involvement of cytochrome P450 3A in the metabolism and covalent binding of 14 Cmonocrotaline in rat liver microsomes. J. Biochem. Mol. Toxicol. 12, 157–166. Roth, R.A., Reindel, J.F., 1990. Lung vascular injury from monocrotaline pyrrole, a putative hepatic metabolite. In: Witner, C.M., et al. (Eds.), Advances in Experimental Medicine & Biology, Biological Reactive Intermediates IV. Plenum Press, New York, pp. 477–487. Ruderman, N.B., 1975. Muscle amino acid metabolism and gluconeogenesis. Ann. Rev. Med. 26, 245–258.
Salgueiro-Pagadigorria, C.L., Constantin, J., Bracht, A., Nascimento, E.A., IshiiIwamoto, E.L., 1996. Effects of the nonsteroidal anti-inflammatory drug piroxicam on energy metabolism in the perfused rat liver. Comp. Biochem. Physiol. 113C, 93–98. Scholz, R., Bücher, T., 1965. Hemoglobin-free perfusion of rat liver. In: Chance, B., Estabrook, R.W., Williamson, J.R. (Eds.), Control of Energy Metabolism. Academic Press, New York, pp. 393–414. Schultze, A.E., Roth, R.A., 1998. Chronic pulmonary hypertension-the monocrotaline model and involvement of the hemostatic system. J. Toxicol. Environ. Health B: Crit. Rev. 1, 271–346. Souza, A.C., Hatayde, M.R., Bechara, G.H., 1997. Pathological aspects of poisoning by Crotalaria spectabilis (Fabaceae) seeds in swine. Pesquisa Vet. Brasil. 17, 12–18. Stegelmeier, B.L., Edgar, J.A., Colegate, S.M., Gardner, D.R., Scloch, T.K., Coulombe, R.A., Molyneux, R.J., 1999. Pyrrolizidine alkaloid plants, metabolism and toxicity. J. Nat. Toxins 8, 95–116. Vlasuk, G.P., Ghrayeb, J., Ryan, D.E., Reik, L., Thomas, P.E., Levin, W., Walz Jr., F.G., 1982. Multiplicity strain differences, and topology of phenobarbital-induced cytochromes P450 in rat liver microsomes. Biochemistry 21, 789–798. Wagner, J.G., Petry, T.W., Roth, R.A., 1993. Characterization of monocrotaline pyrroleinduced DNA cross-linking in pulmonary artery endothelium. Am. J. Physiol. Lung Cell. Mol. Physiol. 264, L517–L522. Watford, M., 2003. The urea cycle: teaching intermediary metabolism in a physiological setting. Biochem. Mol. Biol. Educ. 31, 289–297. Wilson, D.W., Segall, H.J., Pan, L.C., Lame, M.W., Estep, J.E., Morin, D., 1992. Mechanisms and pathology of monocrotaline pulmonary toxicity. Crit. Rev. Toxicol. 22, 307–325. Yan, C.C., Huxtable, R.J., 1994. Quantitation of the hepatic release of metabolites of the pyrrolizidine alkaloid, monocrotaline. Toxicol. Appl. Pharmacol. 127, 58–63. Yan, C.C., Huxtable, R.J., 1995a. The effect of the pyrrolizidine alkaloids, monocrotaline and trichodesmine, on tissue pyrrole binding and glutathione metabolism in the rat. Toxicon 33, 627–634. Yan, C.C., Huxtable, R.J., 1995b. The relationship between the concentration of the pyrrolizidine alkaloid monocrotaline and the pattern of metabolites released from the isolated liver. Toxicol. Appl. Pharmacol. 130, 1–8. Yee, S.B., Kinser, S., Hill, D.A., Barton, C.C., Hotchkiss, J.A., Harkema, J.R., Ganey, P.E., Roth, R.A., 2000. Synergistic hepatotoxicity from coexposure to bacterial endotoxin and the pyrrolizidine alkaloid monocrotaline. Toxicol. Appl. Pharmacol. 166, 173–185.