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Cardiovascular Effects of Fumonisins in Swine
FUNDAMENTAL AND APPLIED TOXICOLOGY ARTICLE NO.
31, 169–172 (1996)
0088
Cardiovascular Effects of Fumonisins in Swine GEOF W. SMITH,* PETER D. CONSTABLE,† CHARLES W. BACON,‡ FILMORE I. MEREDITH,‡
AND
WANDA M. HASCHEK*
Departments of *Veterinary Pathobiology and †Clinical Medicine, University of Illinois, Urbana, Illinois 61801; and ‡Mycotoxin Research Unit, Russell Research Center, USDA-ARS, Athens, Georgia 30601 Received August 14, 1995; accepted January 29, 1996
Cardiovascular Effects of Fumonisins in Swine. SMITH, G. W., CONSTABLE, P. D., BACON, C. W., MEREDITH, F. I., AND HASCHEK, W. M. (1996). Fundam. Appl. Toxicol. 31, 169–172. Fumonisins, mycotoxins produced by Fusarium moniliforme, induce hepatic damage and acute lethal pulmonary edema in swine. We examined the cardiovascular effects of short-term fumonisin exposure in anesthetized and conscious male cross-bred pigs weighing 30–36 kg. Culture material containing fumonisins at £20 mg/kg/day (fumonisin B1 and B2 backbone) was added to the feed of treated pigs (n Å 5) for 7 days, while control pigs (n Å 5) were fed a diet free of fumonisins. On Day 8, pigs were anesthetized with halothane and instrumented with Swan-Ganz catheters to facilitate hemodynamic measurements. Mean pulmonary artery pressure, central venous pressure, heart rate, cardiac output, and electrocardiographic variables were recorded and stroke volume was calculated. All measurements were repeated at least 18 hr after recovery from anesthesia. Pigs fed fumonisins had a significant increase in mean pulmonary artery pressure, accompanied by decreased heart rate, cardiac output, and mixed venous oxygen tension. The electrocardiogram was normal, and there was no evidence of pulmonary edema formation either histologically or by altered lung wet/dry weights. This study suggests that pulmonary hypertension caused by hypoxic vasoconstriction may be associated with the pulmonary edema observed in fumonisin toxicity. q 1996 Society of Toxicology
Fumonisins are a group of naturally occurring mycotoxins produced primarily by the fungus Fusarium moniliforme which frequently infests corn. These toxic fungal metabolites have been implicated in naturally occurring outbreaks of disease affecting horses and pigs known as equine leukoencephalomalacia (ELEM) (Wilson et al., 1990) and porcine pulmonary edema (PPE) (Osweiler et al., 1992). Experimentally, fumonisins cause liver damage in all species examined as well as species-specific target organ toxicity (Casteel et al., 1993; Gumprecht et al., 1995a; Haschek et al., 1992; Jaskiewicz et al., 1987; Lim et al., 1995; Osweiler et al., 1993; Ross et al., 1993; Voss et al., 1989). Fumonisins have The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged.
also been shown to be hepatocarcinogenic in rats (Gelderbloom et al., 1988) and have been linked epidemiologically with human esophageal cancer (Sydenham et al., 1991). We were intrigued by the recent finding of medial hypertrophy of the small pulmonary arteries and right ventricular hypertrophy observed in pigs fed culture material containing fumonisin B1 at a concentration of 150 to 170 ppm for up to 210 days (Casteel et al., 1994). These results suggest that pulmonary hypertension occurs in pigs that chronically ingest fumonisins and that the heart and pulmonary vasculature may be targets of fumonisin toxicity. Pulmonary intravascular macrophages are known to release arachidonic acid metabolites that activate neutrophils, alter capillary permeability, and induce pulmonary hypertension (Bertram et al., 1989). In fumonisin-treated pigs, we have observed membrane-associated ultrastructural alterations in pulmonary intravascular macrophages (PIMs) and Kupffer cells (Haschek et al., 1992), as well as in capillary endothelial cells (Gumprecht et al., 1995b). These alterations could be the direct result of fumonisin-induced membrane damage since fumonisin B1 inhibits sphingolipid biosynthesis (Wang et al., 1991). It is possible that fumonisin-induced damage to PIMs and subsequent release of vasoactive products could lead to altered pulmonary hemodynamics and gas exchange, while damage to the endothelial cells could lead to increased permeability. The combination of these events could lead to pulmonary edema with high concentrations of fumonisins. Since cardiovascular effects may play an important role in the mechanism of fumonisin toxicity in pigs, the purpose of our study was to evaluate selected cardiovascular effects following short-term feeding of fumonisins. This report provides new information about the cardiovascular toxicity of fumonisins in pigs. MATERIALS AND METHODS Animals and treatment. This protocol was approved by the institutional laboratory animal care committee. Ten 30- to 36-kg, male castrated crossbred pigs, born and raised at the University of Illinois Veterinary Research Farm, were housed individually in farrowing crates beginning 5 days prior to treatment. Pigs were fed an 18% protein, complete grower ration that was free of fumonisin B1 , fumonisin B2 (detection limit of 0.5 ppm), aflatoxin, vomitoxin, T-2 toxin, ochratoxin, and zearalenone on analysis by HPLC at the University of Illinois Laboratories of Veterinary Diagnostic Medicine.
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Animals were randomly assigned to two groups (5 pigs per group). Treated pigs were given fumonisin-containing culture material in their feed, while control pigs were untreated. The culture material was prepared using a modification of the Weibking et al. (1993) procedure, mixed with the grower ration, and fed to treated pigs at a dose of not more than 20 mg/ kg/day hydrolyzed fumonisin (3.3 mg fumonisin B1 /kg), for 7 days prior to instrumentation. This dose was based on pilot studies in which pigs fed culture material at a higher concentration of fumonisin died of pulmonary edema after 4–6 days of feeding. The daily dose was divided and fed twice a day in an attempt to simulate normal feeding. Control pigs were fed only the grower ration, on the same schedule as the treated pigs. Instrumentation. On Day 8, anesthesia was induced with an intramuscular injection of 2 mg/kg xylazine and 10 mg/kg ketamine hydrochloride, followed by mask induction with 3–5% halothane in 100% O2 . Pigs were then orotracheally intubated, placed in dorsal recumbency, and maintained at 1.5% halothane in 100% O2 . A 7-F Swan-Ganz thermodilution catheter (Baxter Healthcare Corp., Irvine, CA) was placed in the jugular vein after surgical cut-down and advanced until the proximal port was in the cranial vena cava or right atrium [for measurement of central venous pressure (CVP)] and the distal port was in the pulmonary artery [for measurement of mean, systolic, and diastolic pulmonary artery pressures (MPAP, SPAP, DPAP)]. Correct catheter position was confirmed by evaluation of pressure tracings which were monitored on a multichannel strip chart recorder (Gilson Medical Electronics, Middleton, WI). The Swan-Ganz catheter was secured to the neck and pigs were returned to their crates and allowed to recover from anesthesia for 18 hr, at which time all cardiovascular measurements were repeated. The catheters were flushed every 8 hr with heparinized saline (40 IU/ml) to prevent thrombosis. Cardiovascular measurements. Cardiac output (CO) was measured by thermodilution with the aid of a CO computer (American Edwards Laboratories Inc., Irvine, CA). Three milliliters of 5% dextrose solution (07C) was injected rapidly into the proximal port of the Swan-Ganz. The mean of five CO determinations was used as the experimental value for each animal. Heart rates (HR) were obtained simultaneously with CO determination. Stroke volume (SV) was calculated by standard methods. Mean pulmonary artery pressure, systemic pulmonary artery pressure, diastolic pulmonary artery pressure, central venous pressure, cardiac output, and heart rate were determined during anesthesia and when pigs were conscious. Routine four-limb lead ECGs were acquired on all pigs at the end of halothane anesthesia using a Hewlett–Packard M1707A electrocardiographic unit (Hewlett–Packard, Boise, ID). The PR interval, QRS duration, QT interval (mean value for six leads), and Q, R, and S wave amplitudes (Lead 2) were determined. Blood gas analysis. A mixed venous (pulmonary artery) blood sample was collected when pigs were conscious and analyzed using a Ciba Corning 288 blood gas system (Ciba Corning Diagnostics Corp., Medfield, MA). Pulmonary arterial blood pH, PO2 , and PCO2 were corrected for blood temperature and bicarbonate (HCO03 ) and base excess (BE) values calculated. Lung wet/dry weight determination and pathology. At necropsy, a section of the left apical lobe was weighed, dried at 1107C for 48 hr, and then reweighed. The wet/dry weight ratio was calculated. The lungs were also evaluated both grossly and histologically for evidence of pulmonary edema. Statistics. Variables were evaluated by independent t tests and means were considered significantly different at p õ 0.05.
RESULTS
Hemodynamics. The mean pulmonary artery pressure of fumonisin-treated pigs was significantly increased under halothane anesthesia and when conscious (Table 1). This increase was accompanied by a significant decrease in cardiac
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output and heart rate. Mean central venous pressure and stroke volume were similar in both groups. Electrocardiography. There were no significant differences in the PR interval, QRS duration, QT interval, or lead II QRS wave amplitudes. Arrhythmias (other than sinus arrhythmia) or cardiac conduction abnormalities were not observed in any pigs. Blood gas analyses. A significant decrease in pulmonary artery O2 tension was present in fumonisin-treated pigs, with no changes in pulmonary artery blood CO2 tension, pH, or HCO3 concentrations (Table 2). Base excess values tended to be lower in treated pigs (p Å 0.069). Pulmonary pathology and wet/dry weight ratios. There were no changes in the lung wet/dry weight ratios between treated and control groups. Pulmonary edema was not observed either grossly or histologically. DISCUSSION
The major findings of this study were that short-term fumonisin exposure induces pulmonary hypertension, mixed venous blood hypoxemia, and a decrease in heart rate and cardiac output in growing swine. The lack of an increased wet/dry weight ratio and histologic evidence of pulmonary edema indicates that the fumonisin levels fed in this study were below the threshold level for the induction of pulmonary edema, which has an acute onset 3 to 6 days after initiation of feeding and is a terminal event. Our results suggest possible mechanisms for fumonisin toxicity in swine. Pulmonary hypertension is a common hemodynamic feature of acute lung injury and, if severe enough, can cause pulmonary edema. Pulmonary edema results from an increase in lung interstitial fluid which can be due to either increased pulmonary venous pressure (usually left-sided heart failure) or increased endothelial permeability. The increase in interstitial fluid adversely affects pulmonary gas exchange and may be associated with pulmonary and systemic arterial hypoxemia and pulmonary hypertension. The exact cause of pulmonary hypertension observed in this study is unknown, but may be due to direct damage to the pulmonary vascular endothelium and/or smooth muscle resulting in pulmonary vasoconstriction. Although we failed to observe any evidence of lung injury or interstitial edema by light microscopy in this study, preliminary results from another ultrastructural study indicate that fumonisins alter pulmonary vascular endothelial cells, characterized by duplication and disorganization of the Golgi apparatus and/or smooth endoplasmic reticulum (Gumprecht et al., 1995b). This damage could be due to fumonisin-induced inhibition of sphingolipid biosynthesis with accumulation of sphinganine and could lead to increased endothelial permeability and interstitial edema. Endotoxemia is a common cause of pulmonary hypertension, mixed venous hypoxemia, and decreased cardiac output (Olson et al., 1992). Based on our recent findings that fumonisin-
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FUMONISINS ALTER CARDIOPULMONARY FUNCTION
TABLE 1 Effects of Feeding Fumonisin-Containing Culture Material on Selected Cardiovascular Parameters in Pigs Anesthetized
Conscious
Control CO (liters/min) HR (bpm) SV (ml) MPAP (mm Hg) SPAP (mm Hg) DPAP (mm Hg) CVP (mm Hg)
5.21 107 48.7 21 42 15 4.4
{ { { { { { {
Treated
0.27 2 2.6 5 8 2 0.5
3.77 90 42.0 45 64 25 4.0
{ { { { { { {
Control
0.23* 6* 1.8 5* 8* 2* 0.7
7.09 114 64.1 17 39 11 1.8
{ { { { { { {
0.37 5 4.5 4 3 7 0.7
Treated 5.20 81 65.7 45 62 33 1.7
{ { { { { { {
0.70* 4* 11.3 4* 5* 4* 0.3
* Mean values are significantly different from control pigs by unpaired t test at p õ 0.05; mean { SEM; n Å 5 for control and treated groups.
treated pigs have reduced ability to clear bacteria by the lung and liver (Smith et al., in press), it is logical to assume that fumonisin treatment would predispose to endotoxemia. However we did not see any histological or ultrastructural alterations indicative of endotoxemia, nor did we observe a rise in rectal temperature which is usually associated with endotoxemia. We did not observe an increase in plasma TNF or IL-6 concentrations during the development of fumonisin-induced pulmonary edema in pigs (unpublished data), which further refutes the idea of an endotoxemic-induced pulmonary hypertension associated with fumonisin toxicity. Pulmonary hypertension has been linked to nodular hyperplasia of the liver in humans (Portmann et al., 1993; Robalino and Moodie, 1991). Fumonisins cause hepatic injury in all species and nodular hyperplasia of the liver has been reported to occur with chronic ingestion of fumonisins in pigs (Casteel et al., 1993). As Casteel et al. pointed out, it is tempting to hypothesize that the events seen in humans could be related to those seen in fumonisin-induced pulmonary hypertension (Casteel et al., 1994). In contrast to our findings in pigs, virtually all human patients with pulmonary hypertension and nodular hyperplasia of the liver had ECG abnormalities with normal heart rates and cardiac outputs (Portmann et al., 1993; Robalino and Moodie, 1991). However, the possibility that
TABLE 2 Effects of Feeding Fumonisin-Containing Culture Material on Pulmonary Arterial Blood Gas Analyses in Pigs Control pH PCO2 (mm Hg) PO2 tension (mm Hg) HCO03 (mEq/liter) Base excess (mEq/liter)
7.34 57.4 45.4 31.4 5.5
{ { { { {
0.02 2.8 3.2 0.4 0.6
Treated 7.28 59.6 33.9 28.6 3.2
{ { { { {
0.02 4.1 2.8* 1.9 1.9
* Values are significantly different from controls by unpaired t test at p õ 0.05; mean { SEM; n Å 5 for control and treated groups.
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vasoactive substances normally metabolized or produced by the injured liver could be shunted from the splanchnic circulation to the pulmonary vascular bed, causing vasoconstriction, should be considered. Electrocardiographic abnormalities were not found in pigs in our study. These results are in agreement with other studies (Casteel et al., 1994; Haschek et al., 1992) and suggest the absence of a marked cardiac conductance abnormality or cardiac arrhythmias (other than bradycardia) in pigs fed fumonisin. The fumonisin-induced reduction in heart rate observed in this study has not been previously reported. One potential cause of bradycardia is stimulation of bronchopulmonary C fibers, which represent approximately 80–90% of the afferent vagal innervation of the lung (Coleridge and Coleridge, 1991). Selective stimulation of these fibers by lung injury or increased interstitial pressure results in systemic hypotension, bradycardia, withdrawal of sympathetic tone, decreased cardiac output and contractility, and apnea followed by rapid, shallow breathing (Allen et al., 1994; Roberts et al., 1986). Pigs in the terminal stages of fumonisin toxicosis exhibit a similar respiratory pattern. Bradycardia can also result from activation of carotid chemoreceptors and activation of carotid and aortic baroreceptors (Shepherd, 1981). Experimentally, activation of carotid chemoreceptors was accompanied by bradycardia, decreased cardiac output, vasoconstriction, and increased systemic arterial pressure and sympathetic tone (Daly and Scott, 1963), whereas activation of carotid and aortic baroreceptors induces a reflex bradycardia, decreased cardiac output, vasodilation, decreased systemic arterial pressure, and withdrawal of sympathetic tone (Shepherd, 1981). Finally, bradycardia could also result from a direct negative chronotropic effect on cardiac pacemaker cells. Such an effect has been demonstrated in vitro upon treatment of frog atrial myocytes with fumonisin (Sauviant et al., 1991). In summary, we found that fumonisins induce pulmonary hypertension, mixed venous hypoxemia, and a decrease in heart rate and cardiac output in swine. These findings have not been previously reported. The most likely cause for pul-
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monary hypertension was considered to be alveolar hypoxemia secondary to lung and endothelial injury, whereas the most likely reason for the bradycardia and decreased cardiac output is currently unknown. This study also confirms previous findings which suggest that the cardiovascular system is a primary target for acute fumonisin toxicity (Casteel et al., 1994). Further studies examining the cardiovascular effects of fumonisins are warranted to help define the mechanism of pulmonary edema in pigs and to determine the potential toxicity of fumonisins to humans. ACKNOWLEDGMENTS We thank Dr. C. W. Lim for technical help and Dr. L. Gumprecht for pathology support. This study was supported by Hatch Animal Health Funds and Illinois Pork Producers Association.
REFERENCES Allen, D. A., Schertel, E. R., Weisbrode, S. E., and Myerowitz, F. D. (1994). Acute lung injury isolated to an in situ lung preparation causes sustained reflex cardiovascular depression in dogs. J. Appl. Physiol. 77, 1850–1857. Bertram, T. A., Overby, L. H., Brody, A. R., and Eling, T. E. (1989). Comparison of arachidonic acid metabolism by pulmonary intravascular macrophages and alveolar macrophages exposed to particulate and soluble stimuli. Lab. Invest. 61, 457–466. Casteel, S. W., Turk, J. R., Cowart, R. P., and Rottinghaus, G. E. (1993). Chronic toxicity of fumonisin in weanling pigs. J. Vet. Diagn. Invest. 5, 413–417. Casteel, S. W., Turk, J. R., and Rottinghaus, G. E. (1994). Chronic effects of dietary fumonisin on the heart and pulmonary vasculature of swine. Fundam. Appl. Toxicol. 23, 518–524. Coleridge, H. M., and Coleridge, J. C. G. (1991). Afferent innervation of lungs, airways, and pulmonary Artery. In Reflex Control of the Circulation (I. H. Zucker and J. P. Gilmore, Eds.), pp. 579–608. CRC Press, Boca Raton, FL. Daly, M. de B., and Scott, M. J. (1963). The cardiovascular responses to stimulation of the carotid chemoreceptors in the dog. J. Physiol. 165, 179–197. Gelderblom, W. C. A., Jaskiewicz, K., Marasas, W. F. O., Thiel, P. G., Horak, R. M., Vleggaar, R., and Kriek, N. P. J. (1988). Fumonisins— Novel mycotoxins with cancer-promoting activity produced by Fusarium moniliforme. Appl. Environ. Microbiol. 54, 1806–1811. Gumprecht, L. A., Marcucci, A., Vesonder, R. F., Peterson, R. E., Scott, J. R., Riley, R. T., Showker, J. L., Beasley, V. R., and Haschek, W. M. (1995a). Effects of intravenous fumonisin B1 in rabbits: Nephrotoxicity and sphingolipid alterations. Nat. Toxins 3, 395–403. Gumprecht, L. A., Parker, H. M., Tumbleson, M. E., Beasley, V. R., and Haschek, W. M. (1995b). Fumonisin-induced ultrastructural and sphingolipid alterations in lungs of orally dosed pigs. Toxicol. Pathol. 23, 769 [Abstract]. Haschek, W. M., Motelin, G., Ness, D. K., Harlin, K. S., Hall, W. F., Vesonder, R. F., Peterson, R. E., and Beasley, V. R. (1992). Characterization of fumonisin toxicity in orally and intravenously dosed swine. Mycopathologia 117, 83–96. Jaskiewicz, K., Marasas, W. F. O., and Taljaard, J. J. F. (1987). Hepatitis
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in vervet monkeys caused by Fusarium moniliforme. J. Comp. Pathol. 97, 281–291. Lim, C. W., Parker, H. M., Vesonder, R. F., and Haschek, W. M. (1996). Intravenous fumonisin B1 induces cell proliferation and apoptosis in the rat. Nat. Toxins 4, 33–41. Olson, N. C., Dodam, J. R., and Kruse-Elliot, K. (1992). Endotoxemia and gram-negative bacteremia in swine: Chemical mediators and therapeutic considerations. J. Am. Vet. Med. Assoc. 200, 1884–1893. Osweiler, G. D., Ross, P. F., Wilson, T. M., Nelson, T. M., Witte, S. T., Carson, T. L., Rice, L. G., and Nelson, H. A. (1992). Characterization of an epizootic of pulmonary edema in swine associated with fumonisins in corn screenings. J. Vet. Diagn. Invest. 4, 53–59. Osweiler, G. D., Kehrli, M. E., Stabel, J. R., Thurston, J. R., Ross, P. F., and Wilson, T. M. (1993). Effects of fumonisin-contaminated corn screenings on growth and health of feeder calves. J. Anim. Sci. 71, 459–466. Portmann, B., Stewart, S., Higenbottam, T. W., Clayton, P. T., Lloyd, J. K., and Williams, R. (1993). Nodular transformation of the liver associated with portal and pulmonary arterial hypertension. Gastroenterology 104, 616–621. Robalino, B. D., and Moodie, D. S. (1991). Association between primary pulmonary hypertension and portal hypertension: Analysis of its pathophysiology and clinical, laboratory and hemodynamic manifestations. J. Am. Coll. Cardiol. 17, 492–498. Roberts, A. M., Bhattacharya, H. D., Schultz, H. D., Coleridge, H. M., and Coleridge, J. C. G. (1986). Stimulation of pulmonary vagal afferent Cfibers by lung edema in dogs. Circ. Res. 58, 512–522. Ross, P. F., Ledet, A. E., Owens, D. L., Rice, L. G., Nelson, H. A., Osweiler, G. D., and Wilson, T. M. (1993). Experimental equine leukoencephalomalacia, toxic hepatosis, and encephalopathy caused by corn naturally contaminated with fumonisins. J. Vet. Diagn. Invest. 5, 69–74. Sauviant, M-P., Laurent, D., Kohler, F., and Pellegrin, F. (1991). Fumonisin, a toxin from the fungus Fusarium moniliforme sheld, blocks both the calcium current and the mechanical activity in frog atrial muscle. Toxicon 29, 1025–1031. Shepherd, J. T. (1981). The lungs as receptors sites for cardiovascular regulation. Circulation 63, 1–10. Smith, G. W., Constable, P. D., Smith, A. R., Bacon, C. W., Meredith, F. I., Wollenberg, G. K., and Haschek, W. M. (1996). Effects of feeding fumonisin on the pulmonary clearance of particulates and bacteria in swine. Am. J. Vet. Res., in press. Staub, N. C. (1994). Pulmonary intravascular macrophages. Annu. Rev. Physiol. 56, 47–67. Sydenham, E. W., Thiel, P. G., Marasas, W. F. O., Shepherd, G. S., Van Schalkwyk, D. J., and Koch, K. R. (1991). Natural occurrence of some Fusarium mycotoxins in corn from low and high esophageal cancer prevalence areas of the Transkei, Southern Africa. J. Agric. Food Chem. 39, 2014–2018. Voss, K. A., Norred, W. P., Plattner, R. D., and Bacon, C. W. (1989). Hepatotoxicity and renal toxicity in rats of corn samples associated with field cases of equine leukoencephalomalacia. Food Chem. Toxicol. 27, 89–96. Wang, E., Norred, W. P., Bacon, C. W., Riley, R. T., and Merrill, A. H., Jr. (1991). Inhibition of sphingosine biosynthesis by fumonisins. J. Biol. Chem. 266, 14486–14490. Weibking, T. S., Ledoux, D. R., Bermudez, A. J., Thurk, J. R., Rottinghaus, G. F., Wang, E., and Merrill, A. H., Jr. (1993). Effects of feeding Fusarium moniliforme culture material, containing known levels of fumonisin B1 , on the young broiler chick. Poultry Sci. 72, 456–466. Wilson, T. M., Ross, P. F., Rice, L. G., Osweiler, G. D., Nelson, H. A., Owens, D. L., Plattner, R. D., Reggiardo, C., Noon, T. H., and Pickrell, J. W. (1990). Fumonisin B1 levels associated with an epizootic of equine leukoencephalomalacia. J. Vet. Diagn. Invest. 2, 213–221.
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31, 169–172 (1996)
0088
Cardiovascular Effects of Fumonisins in Swine GEOF W. SMITH,* PETER D. CONSTABLE,† CHARLES W. BACON,‡ FILMORE I. MEREDITH,‡
AND
WANDA M. HASCHEK*
Departments of *Veterinary Pathobiology and †Clinical Medicine, University of Illinois, Urbana, Illinois 61801; and ‡Mycotoxin Research Unit, Russell Research Center, USDA-ARS, Athens, Georgia 30601 Received August 14, 1995; accepted January 29, 1996
Cardiovascular Effects of Fumonisins in Swine. SMITH, G. W., CONSTABLE, P. D., BACON, C. W., MEREDITH, F. I., AND HASCHEK, W. M. (1996). Fundam. Appl. Toxicol. 31, 169–172. Fumonisins, mycotoxins produced by Fusarium moniliforme, induce hepatic damage and acute lethal pulmonary edema in swine. We examined the cardiovascular effects of short-term fumonisin exposure in anesthetized and conscious male cross-bred pigs weighing 30–36 kg. Culture material containing fumonisins at £20 mg/kg/day (fumonisin B1 and B2 backbone) was added to the feed of treated pigs (n Å 5) for 7 days, while control pigs (n Å 5) were fed a diet free of fumonisins. On Day 8, pigs were anesthetized with halothane and instrumented with Swan-Ganz catheters to facilitate hemodynamic measurements. Mean pulmonary artery pressure, central venous pressure, heart rate, cardiac output, and electrocardiographic variables were recorded and stroke volume was calculated. All measurements were repeated at least 18 hr after recovery from anesthesia. Pigs fed fumonisins had a significant increase in mean pulmonary artery pressure, accompanied by decreased heart rate, cardiac output, and mixed venous oxygen tension. The electrocardiogram was normal, and there was no evidence of pulmonary edema formation either histologically or by altered lung wet/dry weights. This study suggests that pulmonary hypertension caused by hypoxic vasoconstriction may be associated with the pulmonary edema observed in fumonisin toxicity. q 1996 Society of Toxicology
Fumonisins are a group of naturally occurring mycotoxins produced primarily by the fungus Fusarium moniliforme which frequently infests corn. These toxic fungal metabolites have been implicated in naturally occurring outbreaks of disease affecting horses and pigs known as equine leukoencephalomalacia (ELEM) (Wilson et al., 1990) and porcine pulmonary edema (PPE) (Osweiler et al., 1992). Experimentally, fumonisins cause liver damage in all species examined as well as species-specific target organ toxicity (Casteel et al., 1993; Gumprecht et al., 1995a; Haschek et al., 1992; Jaskiewicz et al., 1987; Lim et al., 1995; Osweiler et al., 1993; Ross et al., 1993; Voss et al., 1989). Fumonisins have The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged.
also been shown to be hepatocarcinogenic in rats (Gelderbloom et al., 1988) and have been linked epidemiologically with human esophageal cancer (Sydenham et al., 1991). We were intrigued by the recent finding of medial hypertrophy of the small pulmonary arteries and right ventricular hypertrophy observed in pigs fed culture material containing fumonisin B1 at a concentration of 150 to 170 ppm for up to 210 days (Casteel et al., 1994). These results suggest that pulmonary hypertension occurs in pigs that chronically ingest fumonisins and that the heart and pulmonary vasculature may be targets of fumonisin toxicity. Pulmonary intravascular macrophages are known to release arachidonic acid metabolites that activate neutrophils, alter capillary permeability, and induce pulmonary hypertension (Bertram et al., 1989). In fumonisin-treated pigs, we have observed membrane-associated ultrastructural alterations in pulmonary intravascular macrophages (PIMs) and Kupffer cells (Haschek et al., 1992), as well as in capillary endothelial cells (Gumprecht et al., 1995b). These alterations could be the direct result of fumonisin-induced membrane damage since fumonisin B1 inhibits sphingolipid biosynthesis (Wang et al., 1991). It is possible that fumonisin-induced damage to PIMs and subsequent release of vasoactive products could lead to altered pulmonary hemodynamics and gas exchange, while damage to the endothelial cells could lead to increased permeability. The combination of these events could lead to pulmonary edema with high concentrations of fumonisins. Since cardiovascular effects may play an important role in the mechanism of fumonisin toxicity in pigs, the purpose of our study was to evaluate selected cardiovascular effects following short-term feeding of fumonisins. This report provides new information about the cardiovascular toxicity of fumonisins in pigs. MATERIALS AND METHODS Animals and treatment. This protocol was approved by the institutional laboratory animal care committee. Ten 30- to 36-kg, male castrated crossbred pigs, born and raised at the University of Illinois Veterinary Research Farm, were housed individually in farrowing crates beginning 5 days prior to treatment. Pigs were fed an 18% protein, complete grower ration that was free of fumonisin B1 , fumonisin B2 (detection limit of 0.5 ppm), aflatoxin, vomitoxin, T-2 toxin, ochratoxin, and zearalenone on analysis by HPLC at the University of Illinois Laboratories of Veterinary Diagnostic Medicine.
169
AID
FAAT 2146
/
6k0c$$$241
05-10-96 08:35:41
0272-0590/96 $18.00 Copyright q 1996 by the Society of Toxicology. All rights of reproduction in any form reserved.
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Animals were randomly assigned to two groups (5 pigs per group). Treated pigs were given fumonisin-containing culture material in their feed, while control pigs were untreated. The culture material was prepared using a modification of the Weibking et al. (1993) procedure, mixed with the grower ration, and fed to treated pigs at a dose of not more than 20 mg/ kg/day hydrolyzed fumonisin (3.3 mg fumonisin B1 /kg), for 7 days prior to instrumentation. This dose was based on pilot studies in which pigs fed culture material at a higher concentration of fumonisin died of pulmonary edema after 4–6 days of feeding. The daily dose was divided and fed twice a day in an attempt to simulate normal feeding. Control pigs were fed only the grower ration, on the same schedule as the treated pigs. Instrumentation. On Day 8, anesthesia was induced with an intramuscular injection of 2 mg/kg xylazine and 10 mg/kg ketamine hydrochloride, followed by mask induction with 3–5% halothane in 100% O2 . Pigs were then orotracheally intubated, placed in dorsal recumbency, and maintained at 1.5% halothane in 100% O2 . A 7-F Swan-Ganz thermodilution catheter (Baxter Healthcare Corp., Irvine, CA) was placed in the jugular vein after surgical cut-down and advanced until the proximal port was in the cranial vena cava or right atrium [for measurement of central venous pressure (CVP)] and the distal port was in the pulmonary artery [for measurement of mean, systolic, and diastolic pulmonary artery pressures (MPAP, SPAP, DPAP)]. Correct catheter position was confirmed by evaluation of pressure tracings which were monitored on a multichannel strip chart recorder (Gilson Medical Electronics, Middleton, WI). The Swan-Ganz catheter was secured to the neck and pigs were returned to their crates and allowed to recover from anesthesia for 18 hr, at which time all cardiovascular measurements were repeated. The catheters were flushed every 8 hr with heparinized saline (40 IU/ml) to prevent thrombosis. Cardiovascular measurements. Cardiac output (CO) was measured by thermodilution with the aid of a CO computer (American Edwards Laboratories Inc., Irvine, CA). Three milliliters of 5% dextrose solution (07C) was injected rapidly into the proximal port of the Swan-Ganz. The mean of five CO determinations was used as the experimental value for each animal. Heart rates (HR) were obtained simultaneously with CO determination. Stroke volume (SV) was calculated by standard methods. Mean pulmonary artery pressure, systemic pulmonary artery pressure, diastolic pulmonary artery pressure, central venous pressure, cardiac output, and heart rate were determined during anesthesia and when pigs were conscious. Routine four-limb lead ECGs were acquired on all pigs at the end of halothane anesthesia using a Hewlett–Packard M1707A electrocardiographic unit (Hewlett–Packard, Boise, ID). The PR interval, QRS duration, QT interval (mean value for six leads), and Q, R, and S wave amplitudes (Lead 2) were determined. Blood gas analysis. A mixed venous (pulmonary artery) blood sample was collected when pigs were conscious and analyzed using a Ciba Corning 288 blood gas system (Ciba Corning Diagnostics Corp., Medfield, MA). Pulmonary arterial blood pH, PO2 , and PCO2 were corrected for blood temperature and bicarbonate (HCO03 ) and base excess (BE) values calculated. Lung wet/dry weight determination and pathology. At necropsy, a section of the left apical lobe was weighed, dried at 1107C for 48 hr, and then reweighed. The wet/dry weight ratio was calculated. The lungs were also evaluated both grossly and histologically for evidence of pulmonary edema. Statistics. Variables were evaluated by independent t tests and means were considered significantly different at p õ 0.05.
RESULTS
Hemodynamics. The mean pulmonary artery pressure of fumonisin-treated pigs was significantly increased under halothane anesthesia and when conscious (Table 1). This increase was accompanied by a significant decrease in cardiac
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output and heart rate. Mean central venous pressure and stroke volume were similar in both groups. Electrocardiography. There were no significant differences in the PR interval, QRS duration, QT interval, or lead II QRS wave amplitudes. Arrhythmias (other than sinus arrhythmia) or cardiac conduction abnormalities were not observed in any pigs. Blood gas analyses. A significant decrease in pulmonary artery O2 tension was present in fumonisin-treated pigs, with no changes in pulmonary artery blood CO2 tension, pH, or HCO3 concentrations (Table 2). Base excess values tended to be lower in treated pigs (p Å 0.069). Pulmonary pathology and wet/dry weight ratios. There were no changes in the lung wet/dry weight ratios between treated and control groups. Pulmonary edema was not observed either grossly or histologically. DISCUSSION
The major findings of this study were that short-term fumonisin exposure induces pulmonary hypertension, mixed venous blood hypoxemia, and a decrease in heart rate and cardiac output in growing swine. The lack of an increased wet/dry weight ratio and histologic evidence of pulmonary edema indicates that the fumonisin levels fed in this study were below the threshold level for the induction of pulmonary edema, which has an acute onset 3 to 6 days after initiation of feeding and is a terminal event. Our results suggest possible mechanisms for fumonisin toxicity in swine. Pulmonary hypertension is a common hemodynamic feature of acute lung injury and, if severe enough, can cause pulmonary edema. Pulmonary edema results from an increase in lung interstitial fluid which can be due to either increased pulmonary venous pressure (usually left-sided heart failure) or increased endothelial permeability. The increase in interstitial fluid adversely affects pulmonary gas exchange and may be associated with pulmonary and systemic arterial hypoxemia and pulmonary hypertension. The exact cause of pulmonary hypertension observed in this study is unknown, but may be due to direct damage to the pulmonary vascular endothelium and/or smooth muscle resulting in pulmonary vasoconstriction. Although we failed to observe any evidence of lung injury or interstitial edema by light microscopy in this study, preliminary results from another ultrastructural study indicate that fumonisins alter pulmonary vascular endothelial cells, characterized by duplication and disorganization of the Golgi apparatus and/or smooth endoplasmic reticulum (Gumprecht et al., 1995b). This damage could be due to fumonisin-induced inhibition of sphingolipid biosynthesis with accumulation of sphinganine and could lead to increased endothelial permeability and interstitial edema. Endotoxemia is a common cause of pulmonary hypertension, mixed venous hypoxemia, and decreased cardiac output (Olson et al., 1992). Based on our recent findings that fumonisin-
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TABLE 1 Effects of Feeding Fumonisin-Containing Culture Material on Selected Cardiovascular Parameters in Pigs Anesthetized
Conscious
Control CO (liters/min) HR (bpm) SV (ml) MPAP (mm Hg) SPAP (mm Hg) DPAP (mm Hg) CVP (mm Hg)
5.21 107 48.7 21 42 15 4.4
{ { { { { { {
Treated
0.27 2 2.6 5 8 2 0.5
3.77 90 42.0 45 64 25 4.0
{ { { { { { {
Control
0.23* 6* 1.8 5* 8* 2* 0.7
7.09 114 64.1 17 39 11 1.8
{ { { { { { {
0.37 5 4.5 4 3 7 0.7
Treated 5.20 81 65.7 45 62 33 1.7
{ { { { { { {
0.70* 4* 11.3 4* 5* 4* 0.3
* Mean values are significantly different from control pigs by unpaired t test at p õ 0.05; mean { SEM; n Å 5 for control and treated groups.
treated pigs have reduced ability to clear bacteria by the lung and liver (Smith et al., in press), it is logical to assume that fumonisin treatment would predispose to endotoxemia. However we did not see any histological or ultrastructural alterations indicative of endotoxemia, nor did we observe a rise in rectal temperature which is usually associated with endotoxemia. We did not observe an increase in plasma TNF or IL-6 concentrations during the development of fumonisin-induced pulmonary edema in pigs (unpublished data), which further refutes the idea of an endotoxemic-induced pulmonary hypertension associated with fumonisin toxicity. Pulmonary hypertension has been linked to nodular hyperplasia of the liver in humans (Portmann et al., 1993; Robalino and Moodie, 1991). Fumonisins cause hepatic injury in all species and nodular hyperplasia of the liver has been reported to occur with chronic ingestion of fumonisins in pigs (Casteel et al., 1993). As Casteel et al. pointed out, it is tempting to hypothesize that the events seen in humans could be related to those seen in fumonisin-induced pulmonary hypertension (Casteel et al., 1994). In contrast to our findings in pigs, virtually all human patients with pulmonary hypertension and nodular hyperplasia of the liver had ECG abnormalities with normal heart rates and cardiac outputs (Portmann et al., 1993; Robalino and Moodie, 1991). However, the possibility that
TABLE 2 Effects of Feeding Fumonisin-Containing Culture Material on Pulmonary Arterial Blood Gas Analyses in Pigs Control pH PCO2 (mm Hg) PO2 tension (mm Hg) HCO03 (mEq/liter) Base excess (mEq/liter)
7.34 57.4 45.4 31.4 5.5
{ { { { {
0.02 2.8 3.2 0.4 0.6
Treated 7.28 59.6 33.9 28.6 3.2
{ { { { {
0.02 4.1 2.8* 1.9 1.9
* Values are significantly different from controls by unpaired t test at p õ 0.05; mean { SEM; n Å 5 for control and treated groups.
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vasoactive substances normally metabolized or produced by the injured liver could be shunted from the splanchnic circulation to the pulmonary vascular bed, causing vasoconstriction, should be considered. Electrocardiographic abnormalities were not found in pigs in our study. These results are in agreement with other studies (Casteel et al., 1994; Haschek et al., 1992) and suggest the absence of a marked cardiac conductance abnormality or cardiac arrhythmias (other than bradycardia) in pigs fed fumonisin. The fumonisin-induced reduction in heart rate observed in this study has not been previously reported. One potential cause of bradycardia is stimulation of bronchopulmonary C fibers, which represent approximately 80–90% of the afferent vagal innervation of the lung (Coleridge and Coleridge, 1991). Selective stimulation of these fibers by lung injury or increased interstitial pressure results in systemic hypotension, bradycardia, withdrawal of sympathetic tone, decreased cardiac output and contractility, and apnea followed by rapid, shallow breathing (Allen et al., 1994; Roberts et al., 1986). Pigs in the terminal stages of fumonisin toxicosis exhibit a similar respiratory pattern. Bradycardia can also result from activation of carotid chemoreceptors and activation of carotid and aortic baroreceptors (Shepherd, 1981). Experimentally, activation of carotid chemoreceptors was accompanied by bradycardia, decreased cardiac output, vasoconstriction, and increased systemic arterial pressure and sympathetic tone (Daly and Scott, 1963), whereas activation of carotid and aortic baroreceptors induces a reflex bradycardia, decreased cardiac output, vasodilation, decreased systemic arterial pressure, and withdrawal of sympathetic tone (Shepherd, 1981). Finally, bradycardia could also result from a direct negative chronotropic effect on cardiac pacemaker cells. Such an effect has been demonstrated in vitro upon treatment of frog atrial myocytes with fumonisin (Sauviant et al., 1991). In summary, we found that fumonisins induce pulmonary hypertension, mixed venous hypoxemia, and a decrease in heart rate and cardiac output in swine. These findings have not been previously reported. The most likely cause for pul-
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monary hypertension was considered to be alveolar hypoxemia secondary to lung and endothelial injury, whereas the most likely reason for the bradycardia and decreased cardiac output is currently unknown. This study also confirms previous findings which suggest that the cardiovascular system is a primary target for acute fumonisin toxicity (Casteel et al., 1994). Further studies examining the cardiovascular effects of fumonisins are warranted to help define the mechanism of pulmonary edema in pigs and to determine the potential toxicity of fumonisins to humans. ACKNOWLEDGMENTS We thank Dr. C. W. Lim for technical help and Dr. L. Gumprecht for pathology support. This study was supported by Hatch Animal Health Funds and Illinois Pork Producers Association.
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