Fumonisin B1 Increases Serum Sphinganine Concentration but Does Not Alter Serum Sphingosine Concentration or Induce Cardiovascular Changes in Milk-Fed Calves

Sheerin Mathur*, Peter D. Constable*,1, Robert M. Eppley{dagger}, Mike E. Tumbleson{ddagger}, Geoffrey W. Smith*, William J. Tranquilli*, Dawn E. Morin* and Wanda M. Haschek§

* Departments of Veterinary Clinical Medicine, {ddagger} Veterinary Biosciences, and § Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61802; and {dagger} U.S. Food and Drug Administration, Washington, DC

Received August 31, 2000; accepted December 11, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fumonisin B1 is the most toxic and commonly occurring form of a group of mycotoxins that alter sphingolipid biosynthesis and induce leukoencephalomalacia in horses and pulmonary edema in pigs. Purified fumonisin B1 (1 mg/kg, iv, daily) increased serum sphinganine and sphingosine concentrations and decreased cardiovascular function in pigs within 5 days. We therefore examined whether the same dosage schedule of fumonisin B1 produced a similar effect in calves. Ten milk-fed male Holstein calves were instrumented to obtain blood and cardiovascular measurements. Treated calves (n = 5) were administered purified fumonisin B1 at 1 mg/kg, iv, daily for 7 days and controls (n = 5) were administered 10 ml 0.9% NaCl, iv, daily. Each calf was euthanized on day 7. In treated calves, serum sphinganine concentration increased from day 3 onward (day 7, 0.237 ± 0.388 µmol/l; baseline, 0.010 ± 0.007 µmol/l; mean ± SD), whereas, serum sphingosine concentration was unchanged (day 7, 0.044 ± 0.065 µmol/l; baseline, 0.021 ± 0.025 µmol/l). Heart rate, cardiac output, stroke volume, mean arterial pressure, mean pulmonary artery pressure, pulmonary artery wedge pressure, central venous pressure, plasma volume, base-apex electrocardiogram, arterial Po2, and systemic oxygen delivery were unchanged in treated and control calves. Fumonisin-treated calves developed metabolic acidosis (arterial blood pH, 7.27 ± 0.11; base excess, –9.1 ± 7.6 mEq/l), but all survived for 7 days. We conclude that calves are more resistant to fumonisin B1 cardiovascular toxicity than pigs.

Key Words: fumonisin; sphingosine; sphinganine; sphingolipid; cardiovascular toxicity; metabolic acidosis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fumonisins are a group of naturally occurring mycotoxins produced primarily by 2 fungi, Fusarium verticillioides (F. moniliforme) and F. proliferatum, which are found in corn. Fumonisin ingestion leads to altered sphingolipid biosynthesis and dose-dependent increases in serum and tissue sphinganine and sphingosine concentrations (Riley et al., 1993Go), and has been implicated in field cases of equine leukoencephalomalacia (Ross et al., 1993Go) and porcine pulmonary edema (Haschek et al., 1992; Osweiler et al., 1992Go). Experimentally, fumonisin causes liver damage in all species studied to date, and also has been found to have species-specific target-organ toxicity, organs such as brain in horses (Ross et al., 1993Go), heart in pigs (Casteel et al., 1994Go; Constable et al., 2000Go; Smith et al., 1999Go, 2000Go), and kidney in sheep (Edrington et al., 1995Go), rabbits (Gumprecht et al., 1995Go), and rats (Voss et al., 1989Go). The reason for the species-specific target organ toxicity remains enigmatic.

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., 2000Go; Smith et al., 1996Go, 1999Go). Intravenous administration of purified fumonisin B1 (1 mg/kg, daily) induces similar signs of cardiovascular dysfunction within 5 days in pigs (Smith et al., 2000Go). 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Our institutional committee on the care and use of laboratory animals approved this study. Ten healthy male Holstein calves, colostrum-fed and aged between 7 and 14 days (weight 43 ± 7 kg, mean ± SD), were obtained from local sources. The calves were housed at an ambient temperature of approximately 21°C in individual calf pens, and were fed a high-quality milk replacement (milk origin protein, crude protein >20%, crude fat >20%, crude fiber <0.15%) at 10% of their body weight/day, divided into 2 feedings at approximately 12-h intervals, for the duration of the study. Calves had access to supplemental water at all times.

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., 1999Go; Walker et al., 1998Go). 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., 2001Go).

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., 1996Go), dried through Na2SO4 columns, evaporated to dryness under a stream of nitrogen, and derivatized with o-phthaldialdehyde (Riley et al., 1994bGo). 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cardiovascular parameters.
Heart rate, cardiac output, mean arterial pressure, and mean pulmonary-artery pressure were unchanged in treated and control calves (Fig. 1Go). Stroke volume, pulmonary-artery wedge pressure, central venous pressure, systemic vascular resistance, pulmonary vascular resistance, and plasma volume were also unchanged in treated and control calves (data not shown).



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FIG. 1. Effect of intravenous administration of fumonisin B1 (fumonisin, n = 5) or isotonic saline (control, n = 5) on selected cardiovascular parameters. MAP = mean arterial pressure, MPAP = mean pulmonary arterial pressure.

 
PR interval, QRS duration, and QT interval of the base-apex electrocardiogram were unchanged in treated and control calves (data not shown). Cardiac arrhythmias, other than sinus arrhythmia, were not observed in treated or control calves.

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. 2Go). This indicates development of metabolic acidosis in treated calves, with partial respiratory compensation.



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FIG. 2. Effect of intravenous administration of fumonisin B1 (fumonisin, n = 5) or isotonic saline (control, n = 5) on selected acid-base parameters; *p < 0.05, compared with baseline value {dagger}p < 0.05, compared with control value at the same time.

 
Arterial Po2 and oxygen delivery remained constant in treated and control calves (data not shown). For treated and control calves on day 7, no differences were observed for oxygen consumption (treated, 8.2 ± 2.0 ml O2/min/kg; control, 9.7 ± 4.3 ml O2/min/kg), oxygen extraction ratio (treated, 0.49 ± 0.07; control, 0.49 ± 0.16), P(A–a)O2 (treated, 34 ± 15 mm Hg; control, 21 ± 10 mm Hg), or physiologic shunt to blood flow ratio (treated, 10 ± 6%; control, 9 ± 5%).

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. 3Go). Serum sphingosine concentrations were unchanged in treated calves (Fig. 4Go), 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.



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FIG. 3. Effect of intravenous administration of fumonisin B1 (fumonisin, n = 5) or isotonic saline (control, n = 5) on serum sphinganine concentration. *p < 0.05, compared with baseline value, {dagger}p < 0.05, compared with control value at the same time.

 


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FIG. 4. Effect of intravenous administration of fumonisin B1 (fumonisin, n = 5) or isotonic saline (control, n = 5) on serum sphingosine concentration. *p < 0.05, compared with baseline value.

 
Serum sphinganine to sphingosine ratio progressively increased from day 3 onwards in treated calves, to 5.01 ± 2.81 by day 7. Serum sphinganine to sphingosine ratio at day 7, in control calves, was similar to the baseline value.

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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The major findings in this study were that intravenous administration of purified fumonisin B1 (1 mg/kg, daily for 7 days) in milk-fed calves increased serum sphinganine and myocardial sphinganine and sphingosine concentrations, but did not significantly change serum sphingosine concentration or induce cardiovascular changes. In contrast, we recently reported that the same dosage regimen of purified fumonisin B1 increased both serum and myocardial sphinganine and sphingosine concentrations in pigs, and decreased cardiovascular function by day 5 of administration (Smith et al., 2000Go). The absence of fumonisin-induced cardiovascular effects in milk-fed calves may be related to the animals` much lower serum sphinganine (0.24 µmol/l) and sphingosine concentrations (<0.05 µmol/l) following fumonisin administration. This result contrasts with plasma sphinganine and sphingosine concentrations of approximately 0.9 µmol/l and 0.4 µmol/l, respectively, in pigs with fumonisin-induced cardiovascular dysfunction (Smith et al., 2000Go), and plasma sphinganine and sphingosine concentrations >=2.2 µmol/L and >=1.0 µmol/L, respectively, in pigs dying from fumonisin-induced cardiovascular dysfunction (Smith et al., 1999Go). Also in contrast, milk-fed calves had higher myocardial sphinganine (68 µmol/kg) and sphingosine (19 µmol/kg) concentrations following fumonisin administration, relative to myocardial sphinganine and sphingosine concentrations of 8–12 µmol/kg and 5–7 µmol/kg, respectively, in pigs with fumonisin-induced cardiovascular dysfunction (Smith et al., 1996Go, 2000Go), and myocardial sphinganine and sphingosine concentrations of approximately 19 µmol/kg and 12 µmol/kg, respectively, in pigs dying from fumonisin-induced cardiovascular dysfunction (Smith et al., 1999Go). The physiological importance of changes in extracellular sphingolipid concentration relative to changes in tissue sphingolipid concentration is currently unclear.

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., 1995Go), pigs (Smith et al., 1999Go), horses (Goel et al., 1996Go; Wang et al., 1992; ), and rats (Riley et al., 1994aGo), but lower than those reported for vervet monkeys (Shephard et al., 1996Go).

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., 2001Go).

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, 1993Go; Prelusky et al., 1995Go; Richard et al., 1996Go) and indicate that calves are more resistant to fumonisin B1 cardiovascular toxicity than pigs. The mechanism for this resistance remains to be determined.


    ACKNOWLEDGMENTS
 
This article fulfilled part of the requirements for the MS degree for S.M. We gratefully acknowledge the assistance of Ms. Amy Waggoner in completing the serum sphingolipid analyses. This study was supported by funding from the U.S. Department of Agriculture Cooperative Regional Project NC129: Fusarium mycotoxins in cereal grains. G.W.S was supported by an American Heart Association Fellowship Award (9804717X).


    NOTES
 
A portion of the results in this article were reported as an Abstract at the Society of Toxicology-2000 Annual Meeting in Philadelphia (abstract #1622).

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. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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