* Departments of Veterinary Clinical Medicine,
Veterinary Biosciences, and
§ Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61802; and
U.S. Food and Drug Administration, Washington, DC
Received August 31, 2000; accepted December 11, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: fumonisin; sphingosine; sphinganine; sphingolipid; cardiovascular toxicity; metabolic acidosis.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In pigs, ingestion of fumonisin B1 as culture material affects the cardiovascular system by decreasing cardiac contractility, heart rate, cardiac output, mean arterial pressure, arterial and mixed venous blood oxygen tensions, and systemic oxygen delivery by increasing mean pulmonary artery pressure, pulmonary artery wedge pressure, oxygen consumption, and oxygen extraction ratio (Constable et al., 2000; Smith et al., 1996
, 1999
). Intravenous administration of purified fumonisin B1 (1 mg/kg, daily) induces similar signs of cardiovascular dysfunction within 5 days in pigs (Smith et al., 2000
). Therefore, we were interested in determining whether intravenous administration of purified fumonisin B1 (1 mg/kg, daily) induced cardiovascular dysfunction in other species, and if so, whether the cardiovascular dysfunction was similar to that in pigs. The cardiovascular effects of fumonisin B1 were studied in milk-fed calves because of their ready availability, cost, and ease of instrumentation for cardiovascular studies. This is the first study examining the cardiovascular effects of fumonisin B1 in a species other than the pig. It was hoped the results of the study would further expand our knowledge of the mechanism of fumonisin toxicity and provide insight into the effects of fumonisin in species other than swine.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Instrumentation.
Calves were instrumented to collect blood and urine samples, and to determine heart rate (HR), cardiac output (CO), mean pulmonary-artery pressure (MPAP), pulmonary-artery wedge pressure (PAWP), central venous pressure (CVP), mean arterial pressure (MAP), and pulmonary-artery blood temperature as described previously (Smith et al., 1999; Walker et al., 1998
). Briefly, calves were anesthetized with xylazine (Astra USA, Inc., Westborough, MA) (0.1 mg/kg, I M) 8 to 12 h after feeding of milk replacement material, followed 5 to 10 min later by ketamine (Ketaset, Fort Dodge, IA) (4 mg/kg, iv). Calves were intubated orotracheally, placed in dorsal recumbency on a water-circulating heating blanket, and allowed to ventilate spontaneously, breathing 1.5% halothane in 100% oxygen to maintain anesthesia.
The right jugular furrow was prepared aseptically and the right jugular vein and carotid artery identified by surgical cut down. A 12-inch polyethylene catheter (Abbott Critical Care Systems, North Chicago, IL) (3-mm outside diameter) was placed in the right carotid artery for measurement of mean arterial blood pressure and to obtain arterial blood for analysis. A 90-cm, 7-F Swan-Ganz thermodilution catheter (Baxter Healthcare Corp, Irvine, CA) was advanced via the right jugular vein, right atrium, and right ventricle, so that the distal port was in the pulmonary artery and the proximal port in the cranial vena cava or right atrium. Correct catheter position was determined by evaluating the characteristic pressure-waveform on a strip chart recorder (Gilson Medical Electronics, Middleton, WI). Catheters were secured to the calf. The Swan-Ganz catheter was flushed every 12 h with heparinized 0.9% NaCl solution (40 IU heparin/ml) to prevent thrombosis.
Experimental protocol.
Following full recovery from instrumentation (12 to 24 h), calves were assigned randomly to 2 groups. Treated calves (n = 5) were administered purified fumonisin B1 at 1 mg/kg, iv, daily for 7 days. Fumonisin B1 was purified (>95% free acid form) as described in Smith et al., 2000, dissolved in phosphate-buffered saline (pH 7.0), and the concentration adjusted to produce an administration volume of approximately 10 ml/day. Control calves (n = 5) were administered 10 ml of isotonic saline solution (0.9% NaCl), iv, daily at the same time as the treated calves. Hemodynamic measurements were obtained at 24-h intervals (8 A.M.), immediately before feeding the milk replacement. Samples for blood gas analysis, hemoglobin concentration, plasma protein concentration (arterial), and serum sphingosine and sphinganine concentrations (mixed venous) were obtained at the same time as hemodynamic measurements. Blood for hematologic analysis was obtained at the start (baseline) and end of the study (day 7). Body weight was recorded every 24 h, immediately before feeding. At the completion of the study (day 7), each calf was euthanized with an overdose of sodium pentobarbital (60 mg/kg, iv). Serum biochemical and pathologic findings are described elsewhere (Mathur et al., 2001).
Cardiovascular measurements.
Cardiac output was measured by the thermodilution technique with the aid of a cardiac output computer (American Edwards Laboratories, Inc., Irvine, CA). Three to 5 ml of 5% dextrose solution (0° C) was injected rapidly into the proximal port of the Swan-Ganz catheter, and the change in pulmonary artery temperature monitored. The mean value of 3 CO determinations was used as the experimental value. Heart rate was obtained simultaneously with CO determination and stroke volume (SV) calculated as SV = CO/HR. Arterial and venous pressure measurements were obtained with the calf standing and referenced to the scapulo-humeral joint. Systemic vascular resistance (SVR) and pulmonary vascular resistance (PVR) were calculated (in units of dyne s/cm5) as SVR = CO x 80/(MAP-CVP) and PVR = CO x 80/(MPAP-PAWP). A standard base-apex electrocardiogram was obtained (PageWriter Xli, Hewlett-Packard, Boise, ID) with the calf standing.
Blood pH, PO2, PCO2, and hemoglobin concentration were measured (Ciba-Corning 288 Blood Gas System; Medfield, MA) and pH, Po2, Pco2 values corrected for pulmonary artery blood temperature. Plasma-bicarbonate concentration and base-excess values were calculated using standard equations. Systemic oxygen delivery, oxygen consumption, oxygen-extraction ratio, alveolar-arterial oxygen gradient, and physiologic shunt fraction were calculated. Systemic O2 delivery was calculated as the product of arterial O2 content and cardiac output, and was indexed to body weight. Total blood O2 content was calculated to be 1.39 ml of O2/g of hemoglobin plus dissolved O2 equal to 0.3 volume %/100 mm of Hg. Mass specific oxygen consumption (VO2) was calculated from the difference between arterial (CaO2) and mixed venous oxygen content (CvO2), multiplied by CO, and indexed to body weight: VO2 (ml O2/min.kg) = CO x (CaO2 CvO2)/body weight. Systemic O2 extraction ratio was calculated as the ratio of the arterio-venous O2 content difference to the arterial O2 content. Room air alveolar-arterial O2 gradient [P(A-a)O2] was calculated by use of the alveolar gas equation: PAO2 = PIO2 (PaCO2/R), where PIO2 is the inspired partial pressure of oxygen calculated from the barometric pressure and PAO2 is the alveolar O2 tension. The respiratory exchange ratio (R) was assumed to equal 0.8. The physiologic shunt to total blood flow ratio (Qs/Qt) was calculated by use of the shunt equation: Qs/Qt = (CiO2 CaO2)/(CiO2 CvO2), where CiO2 is the oxygen content of ideal end-pulmonary capillary blood. Plasma protein concentration ([PP]) was determined by refractometry and change in plasma volume at dayi (from baseline), calculated as: change in plasma volume from baseline = ([PPi] [PPbaseline]) x 100/[PPi].
Serum and myocardial sphingolipid analysis.
Mixed venous blood samples were collected, allowed to clot at room temperature, and the serum harvested after centrifugation (3000 x g). Serum samples were stored at 20° C and thawed immediately before determining free sphinganine and sphingosine concentrations by modification of the methods described by Riley et al. (1994b) and Yoo et al. (1996). Serum sphingolipid concentrations were determined after adding 200 ml of 10% sulfosalicylic acid to each 1-ml aliquot of serum. Samples were allowed to stand for 5 min at room temperature, centrifuged, and the supernatant discarded. The precipitate was disrupted mechanically, and the homogenates hydrolyzed and extracted with a mixture of chloroform and 0.2 M KOH in methanol at 40°C for 2 h. Sphinganine C 20 (internal standard), 100 ml 2 N NH4OH, and the chloroform mixture used for extraction hydrolysis were added to the precipitated protein. Samples were washed (Yoo et al., 1996), dried through Na2SO4 columns, evaporated to dryness under a stream of nitrogen, and derivatized with o-phthaldialdehyde (Riley et al., 1994b
). Concentrations of sphinganine, sphingosine, and sphinganine C 20 were determined by high performance liquid chromatography with fluorescence detection.
The left ventricular myocardium was obtained immediately after euthanasia, stored at 20°C, thawed, and a 50-mg (fumonisin-treated calves) or 200-mg (control calves) tissue sample homogenized in 0.05 M potassium phosphate buffer before being processed, as stated previously, for sphingolipid determination.
Hematologic analysis.
Red-blood-cell indices, white-blood-cell count, and differential and platelet counts were determined using a hemocytometer (Cell-Dyne 3500, Abbott Diagnostics, Santa Clara, CA).
Statistical analysis.
Data were presented as mean ± SD. Non normally distributed variables were log transformed or ranked before statistical analyses were performed. Two-way analysis of variance (group, time) with repeated measures on one factor (time) was used for comparison. Appropriate Bonferroni-adjusted post-tests were conducted whenever the F test was significant. Within-group comparisons were to the baseline value. Between-group comparisons for each variable were made at each time interval. A statistical software package (SAS, release 6.12, SAS Institute, Inc., Cary, NC) was used for analysis. A p value of <0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Blood gas analysis.
Arterial pH was decreased in fumonisin-treated calves on days 6 and 7, and arterial-plasma bicarbonate concentrations and base excess were decreased in treated calves on days 5 to 7, with decreased arterial Pco2 on day 7 (Fig. 2). This indicates development of metabolic acidosis in treated calves, with partial respiratory compensation.
|
Serum and myocardial sphingolipid analysis.
The baseline serum sphinganine concentration was 0.010 ± 0.007 µmol/l, the baseline serum sphingosine concentration was 0.021 ± 0.025 µmol/l, and the baseline serum sphinganine to sphingosine ratio was 0.50 ± 0.23. Serum sphinganine concentrations were increased in treated calves by day 3, and then appeared to plateau from days 5 to 7 (Fig. 3). Serum sphingosine concentrations were unchanged in treated calves (Fig. 4
), although they were numerically 4 to 5 times higher than baseline values at the end of the study. Serum sphinganine and sphingosine concentrations tended to decrease in control calves over time.
|
|
The left ventricular sphinganine concentration in control calves was 0.75 ± 0.60 µmol/kg wet weight of tissue, the sphingosine concentration was 1.90 ± 1.00 µmol/kg, and the sphinganine to sphingosine ratio was 0.40 ± 0.13. Left ventricular sphinganine concentration (67.5 ± 126.0 µmol/kg), sphingosine concentration (18.5 ± 21.5 µmol/kg), and sphinganine to sphingosine ratio (2.86 ± 1.80) were markedly increased in fumonisin-treated calves.
Hematologic analysis.
Blood hemoglobin concentration was increased transiently in treated calves on day 4 (8.8 ± 2.4) and day 5 (9.0 ± 2.6), but had returned to baseline value (7.8 ± 1.9) by day 7. There was no change in erythrocyte count and indices, total and differential leukocyte count, or platelet count in treated or control calves (data not shown).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There are alternative reasons for the absence of cardiovascular toxicity in calves; they include species differences in the conversion rate of sphinganine to sphinganine-1-phosphate and sphingosine to sphingosine-1-phosphate, differences in the number or responsiveness of receptors to sphinganine and sphingosine (as well as their metabolites), alternative pathways for sphinganine and sphingosine metabolism, and differences in the metabolic pathways for complex sphingolipids and ceramide. Obviously, much more work is required to characterize the reason for the variation in species susceptibility to fumonisin.
In healthy milk-fed calves, normal ranges for serum sphinganine and sphingosine concentration were similar to those reported in adult cattle (Prelusky et al., 1995), pigs (Smith et al., 1999
), horses (Goel et al., 1996
; Wang et al., 1992; ), and rats (Riley et al., 1994a
), but lower than those reported for vervet monkeys (Shephard et al., 1996
).
Although fumonisin B1 did not induce cardiovascular depression in milk-fed calves, intravenous fumonisin administration did induce metabolic acidosis. As there were no changes in cardiac output, arterial PO2, blood hemoglobin concentration, oxygen delivery, and oxygen consumption in treated calves, metabolic acidosis was attributed to renal failure secondary to proximal tubular damage (Mathur et al., 2001).
In conclusion, the results of the present study support findings that cattle are more resistant to the toxic effects of fumonisins than horses and pigs (Osweiler et al, 1993; Prelusky et al., 1995
; Richard et al., 1996
) and indicate that calves are more resistant to fumonisin B1 cardiovascular toxicity than pigs. The mechanism for this resistance remains to be determined.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
1 To whom correspondence should be addressed at the University of Illinois at Urbana-Champaign, Department of Veterinary Clinical Medicine, 1008 W. Hazelwood Dr., Urbana, IL 61802. E-mail: p-constable{at}uiuc.edu.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Constable, P. D., Smith, G. W., Rottinghaus, G. E., and Haschek, W. M. (2000). Ingestion of fumonisin B1-containing culture material decreases cardiac contractility and mechanical efficiency in swine. Toxicol. Appl. Pharmacol. 162, 151160.[ISI][Medline]
Edrington, T. S., Kamps-Holtzapple, C. A., Harvey, R. B., Kubena, L. F., Elissalde, M. H., and Rottinghaus, G. E. (1995). Acute hepatic and renal toxicity in lambs dosed with fumonisin-containing culture material. J. Anim. Sci. 73, 508515.
Goel, S., Schumacher, J., Lenz, S. D., and Kemppainen, B. W. (1996). Effects of Fusarium moniliforme isolates on tissue and serum sphingolipid concentrations in horses. Vet. Human Toxicol. 38, 265270.[ISI][Medline]
Gumprecht, L. A., Marcucci, A., Weigel, R. M., Vesonder, R. F., Riley, R. T., Shwoker, J. L., Beasley, V. R., and Haschek, W. M. (1995). Effects of intravenous fumonisin B1 in rabbits: Nephrotoxicity and sphingolipid alterations. Nat. Toxins 3, 395403.[Medline]
Mathur, S., Constable, P. D., Eppley, R. M., Waggoner, A. L., Tumbleson, M. E., and Haschek, W. M. (2001) Fumonisin B1 is hepatotoxic and nephrotoxic in milk-fed calves. Toxicol. Sci. 60, 385396.
Osweiler, G. D., Kehrli, M. E., Stable, 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, 459466.
Osweiler, G. D., Ross, P. F., Wilson, T. M., Nelson, P. E., 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 fumonisin in corn screenings. J. Vet. Diagn. Invest. 4, 5359.[ISI][Medline]
Prelusky, D. B., Savard, M. E., and Trenholm, H. L. (1995). Pilot study on the plasma pharmacokinetics of fumonisin B1 in cows following a single dose by oral gavage or intravenous administration. Nat. Toxins 3, 389394.[Medline]
Richard, J. L., Meerdink, G., Maragos, C. M., Tumbleson, M., Bordson, G., Rice, L. G., and Ross, P. F. (1996). Absence of detectable fumonisins in the milk of cows fed Fusarium proliferatum (Matsushima) Nirenberg culture material (1996). Mycopathologia 133, 123126.[ISI][Medline]
Riley, R. T., Hinton, D. M., Chamberlain, W. J., Bacon, C. W., Wang, E., Merrill, A. H., Jr., and Voss, K. A. (1994a). Dietary fumonisin B1 induces disruption of sphingolipid metabolism in Sprague-Dawley rats: A new mechanism of nephrotoxicity. Nutr. Pharm. Toxicol. 124, 594603.
Riley, R. T., Nyeon-Hyoung, L. S., Hwan-Soo, Y., Norred, W. P., Chamberlain, W. P., Wang, E., Merrill, A. H., Jr., Motelin, G., Beasley, V. R., and Haschek, W. H. (1993). Alteration of tissue and serum sphinganine to sphingosine ratio: An early biomarker to exposure to fumonisin-containing feeds in pigs. Toxicol. Appl. Pharmacol. 118, 105112.[ISI][Medline]
Riley, R. T., Wang, E., and Merrill, A. H., Jr. (1994b). Liquid chromatographic determination of sphinganine and sphingosine ratio as a biomarker for consumption of fumonisins. J. AOAC Int. 77, 533540.[ISI]
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, 6974.[ISI][Medline]
Shephard, G. S., van der Westhuizen, L., Thiel, P. G., Gelderblom, W., Marasas, W. F., and van Schalkwyk, D. J. (1996). Disruption of sphingolipid metabolism in non-human primates consuming diets of fumonisin containing Fusarium moniliforme culture material. Toxicon 34, 527534.[ISI][Medline]
Smith G. W., Constable, P. D., Eppley, R. M., Tumbleson, M. E., Gumprecht, L. A., and Haschek W. M. (2000). Purified fumonisin B1 decreases cardiovascular function but does not alter pulmonary capillary permeability in swine. Toxicol. Sci. 56, 240249.
Smith, G. W., Constable, P. D., and Haschek, W. M. (1996). Cardiovascular responses to short-term fumonisin exposure in swine. Fundam. Appl. Toxicol. 33, 140148.[ISI][Medline]
Smith, G. W., Constable, P. D., Tumbleson, M. E., Rottinghaus, G. E., and Haschek, W. M. (1999). Sequence of cardiovascular changes leading to pulmonary edema in swine fed culture material containing fumonisin. Am. J. Vet. Res. 60, 12921300.[ISI][Medline]
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, 8996.[ISI][Medline]
Walker, P. G., Constable, P. D., Morin, D. E., Drackley, J. K., Foreman, J. H., and Thurmon, J. C. (1998). A reliable, practical, and economical protocol for inducing diarrhea and severe dehydration in the neonatal calf. Can. J. Vet. Res. 62, 205213.[ISI][Medline]
Yoo, H. S., Norred, W. P., and Riley, R. T. (1996). A rapid method for quantifying free sphingoid bases and complex sphingolipids in microgram amounts of cells following exposure to fumonisin B1. Toxicol. in Vitro 10, 7784.[ISI]