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1“ WM“

DIETARY TOXICITY OF THE F USARI W MY COTOXIN S MONILIFORMIN AND
FUMONISIN S TO MINK (MUSIELA VISON)

By

Marsha K. Morgan

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Science

1998

COPyright by
Marsha K. Morgan
1 998

ABSTRACT

DIETARY TOXICITY 01: THE FUSARIUM MYCOTOXINS MONILIFORMIN AND
FUMONISINS TO MINK (MUSTELA WSON)

By
Marsha K. Morgan

Mycotoxins are toxic secondary metabolites produced by fungi under favorable
environmental conditions. F usarium firngi are common contaminates of grain worldwide,
and because they can produce several mycotoxins, including moniliformin and fiimonisins,
they are of concern to human and animal health. Because cereal grains are a major
component of farm mink diets, the effects of these mycotoxins on mink (Mustela vison)
were investigated. The moniliformin study was conducted to ascertain the acute,
subacute, subchronic, and reproductive efi‘ects in mink resulting from exposure to this
compound. In a preliminary trial, adult mink presented diets containing moniliforrnin
provided by F. firjikuroi culture material (M-1214) of 40 mg/kg or greater refused to eat
significant quantities of feed. Feeding adult mink diets that contained 8.1 or 17.0 mg/kg,
wet weight, moniliformin in a 30 d subchronic trial produced no significant adverse effects
on feed consumption, body weights, hematologic parameters or serum chemical values, or
notable histologic changes in their tissues. In the reproduction trial, female mink were
exposed to the same dietary concentrations of moniliformin as in the subchronic test from
two wk prior to the breeding season (February 15, 1996) until their offspring (kits) were
eight wk old (June 30, 1996). Consumption of the high-dose (17 mg/kg) diet resulted in
significant neonatal mortality and reduced kit body weights at birth and at eight wk of age.

Necropsy of eight-wk-old kits fi'om the control and high-dose groups revealed no gross or

histologic alterations in liver, lung, or heart tissues that could account for the mortality
observed in the kits exposed to moniliformin. These results indicate that long-term (105-
135 d) dietary exposure to 17 mg/kg moniliformin is not lethal to adult female mink, but
can have adverse efi‘ects on neonatal mink. In the acute trial, the i.p. LDso was determined
to be within the range of 2.2-2.8 mg/kg b.w. of moniliformin for nine-mo-old female mink.
In the subacute study, nine-mo-old female mink receiving 0.33 mg/kg b.w. of moniliformin
by i.p. injection for three d had hearts that were visibly enlarged, rounded, and dilated on
the right sides at necropsy on day six post-dosing. Electron microscopic evaluation of the
hearts from both acutely and subacutely exposed mink showed alterations occurring in the
mitochondria, myofibers, myofilaments, nuclei, and in extracellular collagen deposition.
This study showed that adult female mink are very sensitive to moniliformin, and that this
compound specifically targets and damages the ultrastructure of hearts of exposed mink.
The fumonisin study investigated the effects of dietary Fusarium moniliforme
culture material (M-1325) containing known concentrations of fumonisins B1, B2, and B3
on sphingolipids in urine and hair of mink for use as potential, non-invasive biomarkers of
exposure to firmonisins in this species. Feeding mink diets containing 86, 22, and 7 mg/kg
or 200, 42, and 12 mg/kg of fumonisins B1, B2, and B3, respectively, for two, four, or
seven (1 caused marked increases in urinary fiee sphinganine‘ (Sa) and free sphingosine
(So) concentrations, and fiee Sa/fi'ee So ratios (2 to 11-fold), compared to controls. Free
Sa and free So concentrations and Sa/So ratios in hair samples from mink fed the control
or firmonisin-treated diets for 100 d were similar and were not altered by fumonisin-
exposure. These results suggest that Sa/So ratios in urine, but not in hair, of mink can

serve as an early indicator of exposure to firmonisins in this species.

This thesis is dedicated to my savior Jesus Christ.

ACKNOWLEDGEMENTS

First, I would like to thank God for providing me with the strength and courage to
finish my PhD. program here at Michigan State University. I would also like to extend
my sincere graditude to Dr. Richard J. Aulerich for his never ending guidance, support,
and encouragement during my Doctoral degree program. Thank you for understanding
my physical limitations as well as keeping me focused on my program.

I would also like to thank my other committee members, Drs. Steven J. Bursian,
Kuo-Chuan K. Chou, Michael A. Kamrin, and Scott D. Fitzgerald for their help and
guidance throughout my program. My research was greatly assisted by Chris R. Bush,
Angelo Napolitano, Debra M. Powell, Dr. Joseph Schroeder, and Dr. James Render.
Thank you for helping me conduct and analyze my studies. A special thank you to the
Mink Farmers Research Foundation for generously funding my research at the mink farm.

Lastly, I would like to thank my parents, William and Karen Morgan, for

encouraging me to pursue all my dreams and goals in life.

TABLE OF CONTENTS

LIST OF TABLES .......................................................................... ix
LIST OF FIGURES .......................................................................... x
INTRODUCTION ............................................................................ 1
LITERATURE REVIEW .................................................................... 4
Fusarium Fungi .................................................................... 5
Monilil'ormin ....................................................................... 5
Decontamination and Detoxification .................................... 7
Animal Studies ............................................................. 7
Acute Toxicity ...................................................... 9

Subacute Toxicity .................................................. 11

In Vitro Toxicity ................................................... 12

Human Implications ....................................................... 12

Pharmacokinetic Data ..................................................... 13

Toxic Mechanism of Action .............................................. 13

Regulatory Aspects ........................................................ 15

F umonisins .......................................................................... 1 5

Decontamination and Detoxification ..................................... 16

Animal Diseases ............................................................ 21

Equine Leukoencephalomalacia (ELEM) ...................... 21

Porcine Pulmonary Edema (PPE) ............................... 22

Other Toxic Effects ............................................... 22

In Vitro Studies ............................................................. 22

Human Implications ........................................................ 23

Pharmacokinetic Data ..................................................... 23

Toxic Mechanism of Action .............................................. 24

Regulatory Aspects ........................................................ 28

Mink Sensitivity to Fusarium Mycotoxins ................................... 28

Fumonisins .................................................................. 28

Zearalenone ................................................................. 29

Deoxynivalenol (DON; Vomitoxin) ...................................... 30

T2 Toxin ..................................................................... 3O

vii

PART I . SUBCHRONIC AND REPRODUCTIVE EFFECTS IN MINK FROM
EXPOSURE TO F USARI UIVI F UJIK UROI CULTURE MATERIAL (M-1214)
CONTAINING KNOWN CONCENTRATIONS OF MONILIFORIMIN.

Abstract .............................................................................. 31
Introduction ......................................................................... 32
Materials and Methods ........................................................... 33
Subchronic Study ........................................................... 33
Reproduction Study ........................................................ 35
Statistical Analysis ......................................................... 37
Results ............................................................................... 37
Subchronic Study ........................................................... 37
Reproduction Study ....................................................... 39
Discussion ........................................................................... 42
Conclusions .......................................................................... 46

PART 11. ACUTE AND SUBACUTE EFFECTS IN MINK FROM EXPOSURE
To FUSARIUM FUJIKUROI CULTURE MATERIAL (M-1214) CONTAINING
KNOWN CONCENTRATIONS OF MONILIFORMIN.

Abstract .............................................................................. 47
Introduction ........................................................................ 48
Materials and Methods ........................................................... 49
Acute Study ................................................................. 49
Subacute Study ............................................................. 51
Statistical Analysis .......................................................... 52
Results and Discussion ............................................................ 52
Acute Study ................................................................. 52
Subacute Study ............................................................. 53
Conclusions ......................................................................... 59

PART III. DIETARY FUMONISINS DISRUPT SPHINGOLIPID METABOLISM
IN MINK AND INCREASE THE FREE SPHINGANINE TO SPHINGOSINE
RATIO IN URINE BUT NOT IN HAIR.

Abstract .............................................................................. 60
Introduction ........................................................................ 61
Materials and Methods ........................................................... 62
Results and Discussion ........................................................... 64
Conclusions ......................................................................... 67
FUTURE RESEARCH ...................................................................... 67
Moniliformin ....................................................................... 67
Fumonisins ......................................................................... 68
REFERENCES .............................................................................. 69

LIST OF TABLES

Table Page
. Chemical and Physical Parameters of Moniliforrnin ................................ 8
. Highest Moniliforrnin Concentrations Reported in Natural Samples in
Various Countries ....................................................................... 8
. Acute Toxicity of Moniliforrnin in Experimental Animals .......................... 10
. Chemical Parameters of Fumonisins B1 and B2 ...................................... l8

. Highest Concentrations Reported for F umonisins B1 and B2 in Feeds or
Grains from Several Countries ......................................................... 2O

. Reproductive Performance of Female Mink Fed Various Concentrations of
Moniliforrnin ............................................................................. 4O

. Body Weights and Survivability of Kit Mink Whelped and Nursed by Females
Fed Various Concentrations of Moniliforrnin ........................................ 41

'. Mean Sphinganine (Sa) and Sphingosine (So) Concentrations and Sa/So

Ratios for Urine of Female Mink Fed Various Concentrations of Fumonisins
for 2, 4, or 7 d ........................................................................... 65

ix

LIST OF FIGURES

Figure Page
1. Chemical Structure of Moniliforrnin ................................................... 6
2. Diagram Showing the Inhibition of Pyruvate Dehydrogenase Complex and

Ot-Ketoglutarate Complex by Moniliformin in the TCA Cycle ..................... 14
3. Chemical Structures of Fumonisins Bl, B2, and B3 .................................. 17
4. Chemical Structures of Sphinganine, Sphingosine, and Fumonisins B1 ........... 25
5. Diagram Showing Where Fumonisins Target and Inhibit Sphinganine N-

Acyltransferase and/or Sphingosine N—Acyltransferase ............................. 27
6. Electron Photomicrograph of a Section of Heart from a Control Mink

Showing Normal Ultrastructure. Mitochondria (A), Myofibers (B), and

Transverse Striations are Normal in Appearance (8040x) ....................... 56
7. Electron Photomicrograph of a Section of Heart from an Acutely Exposed

Mink Showing Histologic Lesions in the Mitochondria (A) and Myofibers

(B). Massive Vacuolation and Edema are Present in the Tissue (63 50x) ........ 57
8. Electron Photomicrograph of a Section of Heart from a Subacutely

Exposed Mink. Note the Presence Of Massive Vacuolation and Edema,

an Increase in the Volume of Mitochondria, Several Degenerating Nuclei

(A), Extracellular Collagen Bundles (B), and Disintegrating Myofibers

(Lower Right) with Cellular Debris (3 525x) ......................................... 58

INTRODUCTION

Mycotoxins are secondary toxic metabolites produced by fungi infecting cereal
grains, tree nuts, fiuits, vegetables, Oil seeds, and meats, worldwide (Jelinek et al., 1989;
Steyn, 1995). In recent years, numerous mycotoxins have been detected in animal feeds
and human foods throughout the world including the United States (Jelinek et al., 1989;
Sydenham, et al., 1991; Bosch and Mirocha, 1992; Sydenham et al., 1992; Wood, 1992;
Murphy et al., 1993; Park etal., 1996). These natural contaminants are a global concern
since many are toxic in low amounts (mg/kg) to animals and humans (Jofi‘e, 1986; Berry et
al., 1988; Smith and Henderson, 1991; Yamashita et al., 1995). Reports indicate that
some of these mycotoxins have been detected in animal feeds and human foods at or
above levels toxic to experimental animals (Thiel et al., 1992; Murphy et al., 1993).
Currently in the US, the trend is toward less regulation and research of mycotoxins in
animal feeds and human foods (Kuiper-Goodman, 1995). However, because of the
potential hazards, it would appear that the government should increase surveillance
programs and encourage research on mycotoxins to adequately protect animals and
humans from exposure to toxic levels of these compounds.

Of particular concern are the mycotoxins produced by F usarium firngi:
trichothecenes, zearalenone, firsaric acid, firsarin C, fumonisins, butenolide, gibberellins,
and moniliformin. These mycotoxins can cause a diversity of toxic effects in animals

including feed refusal, emesis, hepatocarcinogenesis, equine leukoencephalomalacia,

porcine pulmonary edema, hyperestrogenic efi‘ects, impaired fertility, cardiotoxicity etc.
(Osweiler et al.,1985; Marquardt, 1996).

Presently, the effects of moniliformin and the firmonisins in exposed experimental
animals are receiving considerable attention. Several Fusarium species are known to
produce these mycotoxins including F. moniliforme (Cole et al., 1973; Nelson, 1992;
Norred and Voss, 1994), and F. proliferatum (Nelson et al., 1993; Bullerman and Tsai,
1994; Norred and Voss, 1994). Both are Ofien detected in corn and corn products
worldwide (Logrieco et al., 1993; Murphy etal., 1993; Bacon and Nelson, 1994).
Moniliforrnin and fumonisins B1 and B2 have been found as co-contaminants with
nivalenol, deoxynivalenol, and zearalenone (Thiel et al., 1982; Jelinek et al., 1989;
Sydenham et al., 1990a; Dombrink-Kurtzman et al., 1993; Nagaraj and Wu, 1994;
Yamashita et al., 1995). Concentrations of naturally occurring moniliformin and
firmonisin B1 have been reported to be as high as 530 mg/kg and 334 mg/kg w.w. in maize
and corn screenings, respectively (Logrieco et al., 1993; Dutton, 1996).

Acute and/or subacute dietary exposure to moniliformin can be toxic to several
experimental animals and livestock species, including broiler chicks (Kriek et al., 197 7;
Allen et al., 1981; Engelhardt et al., 1989; Javed et al., 1993; Ledoux et al., 1995),
ducklings (Engelhardt er al., 1989), turkey poults (Engelhardt et al., 1989), mice
(Burmeister et al., 1980), and donkeys, horses, pigs, and rabbits (Chen et al., 1990).
However, very little research has been reported on the chronic effects of moniliformin in
animals (Lew et al., 1996) and none in mink.

At least seven firmonisins have been identified and of these, fumonisins B1, B2, and

B3 are the most prevalent in field studies (Thiel etal., 1982; Marasas et al., 1984; Nelson

et al., 1993). Fumonisin B1 and/or F. monilifonne have been shown to cause porcine
pulmonary edema (PPE), equine leukoencephalomalacia (ELEM), nephrotoxicity and
hepatotoxicity in rats and have been implicated in cases of human eSOphageal cancer (Ross
et al., 1991; Osweiler et al., 1992; Voss et al., 1993; Chu and Li, 1994; Riley eta1.,
1994b). Studies with mink have shown that F. moniliforme culture material containing
fumonisins B1, B2, and B3 adversely affects liver and kidney function, hematologic and
serum chemistry values, whelping success, kit body weights at birth, and decreases the
biosynthesis of sphingolipids (Restum et al., 1995; Powell et al., 1996).

Fumonisins are structurally similar to sphingoid bases, such as sphingosine (So)
and Sphinganine (Sa), and several fumonisins (B1, B2, and hydrolyzed B1) have been
shown to block the de novo synthesis of sphingolipids through the inhibition of
sphingosine— and Sphinganine N-acyltransferases. This inhibition results in a decrease in
the biosynthesis of complex sphingolipids and an increase in free Sa and sometimes SO in
cells (Norred et al., 1991; Wang et al., 1991; Riley et al., 1993; Riley et al., 1994a,b).

Studies by Riley et al. (1994a,b) have shown that fumonisins significantly increase
the Sa/So ratio in the liver, kidneys, lungs, sera, and urine Of rats, ponies, or pigs. In a
previous study conducted with mink, it was found that firmonisins significantly increased
hepatic and renal free Sa concentrations as well as the Sa/So ratios in these tissues
(Restum er al., 1995). The results of these studies suggested that, in mink, as in rats and
pigs, the free Sa and free So in urine may serve as a non-invasive, early biomarker of
exposure to firmonisins. Hair is also a tissue that contains sphingolipids and can be easily
obtained from many animals. However, disruption of sphingolipid synthesis due to

exposure tO fumonisins has not been examined. Because cereal grains are important

components of farm mink diets and previous studies have shown mink to be sensitive to
other mycotoxins produced by F usarium firngi, such as zearalenone, deoxynivalenol, and
firmonisins (Bursian et al., 1992; Aulerich et al., 1993; Restum et al., 1995; Yang et al.,
1995; Powell etal., 1996), the following studies were conducted to investigate the effects
of moniliformin and the fumonisins B1, B2, and B3 on mink. The objectives Of the
moniliformin studies were to determine the acute, subacute, subchronic, and reproductive
toxicity of dietary moniliformin to mink, to characterize any observed clinical signs or
histopathologic lesions or alterations in the mink, and to examine the effects of the
mycotoxin on hematologic parameters, serum chemical values, reproductive performance
of female mink, and growth and viability of exposed ofi‘spring (kits). In the firmonisin
study, the objectives were to determine free Sa and free So concentrations in urine and
hair from fumonisin-treated mink and evaluate their use as potential, non-invasive

biomarkers for firmonisin exposure in this species.
LITERATURE REVIEW

“Molds (firngi) develop from spores that are found ubiquitously” around the world
and thrive on most organic material (Marquardt, 1996). As firngi grow, they can produce
toxic by-products known as mycotoxins. Most mycotoxins are polar, small molecular
weight chemicals that are simple to complex in structure (Steyn, 1995; De Nijs et al.,
1996). In the field, firngi can invade seedlings/plants and can greatly reduce the value of
infected crops and even cause mycotoxicoses, which are diseases in animals and humans
that consume feeds and foods contaminated with mycotoxins (Osweiler et al., 1985;

Nelson et al., 1993; Marquardt, 1996). Mycotoxin levels in field crops can be greatly

increased by favorable environmental conditions such as drought, insect infestation,
humidity, and moisture (Osweiler et al., 1985; Marquardt, 1996). Many of the mycotoxins
produced by F usarium fungi damage crops and greatly reduce their economic value to the
agricultural industry.
Fusarium Fungi

Sixty-one F usarium species have been identified, and the six most common species
detected in fields are moniliforme, culmorum, oxysporum, graminearum, equiseti, and
sporotrichioides (De Nijs et al., 1996). These firngi are soil-borne microorganisms that
grow throughout the world, but particularly in cool climates with high humidity (>90%)
and moisture (25%; Osweiler et al., 1985; Bird, 1994; Marquardt, 1996). Fusarium firngi
are known as “field firngi” since they infect and produce toxic metabolites in plants prior
to the harvesting of crops (Joffe, 1986; Bird, 1994; Miller, 1995; De Nijs et al., 1996).
Mycotoxins produced by F usarr'um fungi have been found in a diversity of foods and feeds
worldwide including oats, wheat, rye, bananas, corn, lettuce, rice, potatoes, watermelon,
avacados, chickpeas, and coconuts and many appear to be stable under food processing
conditions, such as pasteurization and sterilization (Jofl‘e, 1986; Bird, 1994; Miller, 1995;
De Nijs et al., 1996).
Moniliformin

In 1970, the United States experienced the “southern corn leaf blight epidemic”,
and this corn was subsequently screened for firngi (Cole et al., 1973; Lansden et al., 1974;
Ueno, 1985; Betina, 1989). Two firngi were isolated, Helminthosporium maydis and
Fusarium moniliforme (Cole et al., 1973). Moniliformin was first isolated fiom the F.

moniliforme cultures (Figure 1; Cole etal., 1973; Ueno, 1985; Gilbert, 1989; Sharman et

 

 

 

 

 

Figure 1. Chemical Structure OfMoniliformin (Thiel, 1978).

al., 1991). It is a simple, low molecular weight chemical that occurs naturally as a
potassium or sodium salt (Table 1; Cole etal., 1973; Steyn et al., 1978; Sydenham and
Thiel, 1996). Moniliformin has been reported in animal feeds and human foods at
moderate to high (530 mg/kg) concentrations throughout the world (Table 2).

Laboratory analysis of moniliformin has been difiicult due to its polar and
ionophoric nature. It can be qualitatively detected by thin layer chromatography with
fluorescence detection and quantified by ion-pair high performance liquid chromatography
(HPLC) or by I-IPLC with ultraviolet detection methods (Gilbert et al., 1986; Gilbert,
1989; Sharman et al., 1991; Hong and Jilun, 1997).

Decontamination and Detoxification

Very little research has been conducted on the stability of moniliformin in grains.
Moniliformin (potassium salt) is fairly heat stable in corn and wheat at temperatures
between 50 - 150°C (Scott and Lawrence, 1987; Gilbert, 1989). It can be reduced to
undetectable levels and detoxified (100%) in grains by using a 5% H202 spray which is
economically feasible and generates no toxic by-products (Hong and Jilun, 1997).

Animal Studies

Moniliformin is acutely and subacutely toxic to several animals and primarily
targets the hearts of birds and mammals (Kriek et al., 1977; Engelhardt et al., 1989;
Abbas et al., 1990; Lili et al., 1991; Deyu et al., 1993; Javad et al., 1993). Often, animals
exposed to increasing concentrations of moniliformin exhibit physical signs of muscular

weakness, respiratory stress, cyanosis, coma, and death (Kriek et al., 1977; Thiel, 1978).

 

Table 1. Chemical and Physical Parameters ofMoniliformin‘

 

Chemical Name

 

Chemical Formula

Molecular Weight (amu)

salt)
Melting Point
Pka 1.7

 

C4I'I203

2 320°C

3 -Hydroxycyclobut-3 -ene-1 ,2-dione

98.0 (free acid), 137 (potassium salt), 121 (sodium

 

‘Cole et al., 1973; Steyn et al., 1978; Sydenham and Thiel, 1996

 

Table 2. Highest Moniliformin Concentrations Reported from Field Samples
in Various Countries.

 

 

 

 

 

 

Country Source MON‘ Citation
(mg/kg)

Poland Wheat (damaged) 17.1 Shannan et al., 1991
Poland Oats (damaged) 38.3 Sharman et al., 1991
Poland Maize (damaged) 399 Sharman et al., 1991
Poland Maize (damaged) 530 Lew et al., 1996
Poland Corn (damaged) 425 Logrieco et al., 1993
Gambia Maize 3.2 Sharman et al., 1991
South Africa Maize 2.7 Sharman et al., 1991
Italy Maize (damaged) 200 Logrieco and Moretti, 1995
USA Corn screenings 2.8 Thiel et alg 1986

 

‘MON = Moniliformin

 

 

Acute Toxicity

As shown in Table 3, the acute toxicity (LDso) of moniliformin has been
determined in several animal species. The acute toxic efl'ects of moniliformin in animals
include heart lesions, cyanotic and enlarged hearts, gizzard and intestinal hemorrhaging,
and death. Abbas et a1. (1990) reported death in four out of five female rats (20-d-old)
orally administered 20 mg/kg b.w. (approximately 1.0 mg) of moniliformin (99% purity)
and higher concentrations (40, 60, 80, or 100 mg/kg b.w.) caused death in five out of five
rats within 16 hr. All the rats that died had hemorrhaging in the small intestines. At 10
mg/kg b.w. (0.5 mg) or less, there were no signs Of toxicity. Also, Abbas et al. (1990) fed
female rats (20 d-Old) O - 8.0 g/kg of moniliformin (99%) for 24 hr. At concentrations of
1.0 and 1.5 g/kg, one rat from each group consisting of three animals died, but no signs of
toxicity were Observed. Intestinal hemorrhaging occurred at 2.0 g/kg and higher in the
rats. At concentrations greater than 3.0 g/kg, all rats died within 16 hr and had intestinal
hemorrhaging. Cole et al. (1973) showed that cockerels orally administered over 500 ug
(12.5 mg/kg b.w.) of moniliformin died in less than two hr and had edema in the
mesenteries and minor hemorrhaging in the intestinal tract, gizzard, and proventriculus.
Animals that died after two hr did not have any lesions in any body system. Deyu et a1.
(1993) administered 29.5 mg/kg of moniliformin (purified) to 10 adult male Kunming mice
and collected the hearts of two mice at one, two, three, or 24 hr post-dosing. Upon
necropsy, the hearts from the treatment group at one, two, three, or 24 hr post-dosing
were cyanotic and enlarged compared to controls hearts which were normal in
appearance. Electron microscopy Of the hearts showed lesions in the mitochondria,

myofibers, and cytoskeleton. Mitochondrial lesions appeared first and were the most

 

Table 3. Acute Toxicity of Moniliformin in Experimental Animals.

 

 

 

 

 

 

 

Animal Age Route LDso Citation
Chick embryos 4 (1 Air sac 2.8 ug/egg Burmeister et al., 1979
Ducklings 7 d Oral 3.7 mg/kg Ueno, 1985
Cockerels l (1 Oral 4.0 mg/kg Cole et al., 1973
Chicks 1 d Intubation 5.4 mg/kg Burrneister et al., 1979
Chicks 7 wk IV. 1.4 mg/kg Allen et al., 1981
Female mice Adult I.P. 20.9 mg/kg Burrneister et al., 1979
Female mice ---- Oral 47.6 mg/kg Bunneister et al., 1979
Male mice Adult Oral 29.5 mg/kg Deyu et al., 1993
Male mice Adult I.P. 29.1 mg/kg Burrneister et al., 1979
Female rats 20 d Oral >20 mg/kg Abbas et al., 1990
Female rats Adult Oral 41.6 mg/kg Kriek et al., 1977
Male rats Adult Oral 50.0 mg/kg Kriek er al., 1977

 

10

 

predominant lesion in the exposed hearts. Nagaraj et al. (1996) showed that three-wk-
old broiler chicks injected iv. with 1 mg moniliformin/kg b.w. had bradycardia within 15
min. of injection. Three out of seven chicks died within 50 min. after injection due to
cardiac failure (Nagaraj etal., 1996).

Subacute Toxicity

In subacute studies, pathological examinations have shown heart lesions, increased
heart and liver weights, reduced body weight gains and feed consumption, and death in
moniliformin-exposed animals. Allen et al. (1981) showed that one-d-Old broiler chicks
fed 64 mg/kg of moniliformin (either purified or culture material) for 21 d had decreased
feed consumption and body weights. Also, three of the 10 chicks died at this
concentration, but no lesions or other signs of toxicity were observed in the chicks. Javed
et al. (1993) showed that one-d-old broiler chicks fed 27 or 154 mg/kg of moniliformin
for 14 d had a significant decrease in body weight gains and increased mortality compared
to controls. Kubena et al. (1997) reported that one-d-old male broiler chicks fed 100 mg
moniliformin/kg for three wk had a 29% reduction in body weight gains and an increase in
heart weights when compared to the controls. Deyu et al. (1993) orally closed 30 adult
Wistar rats with 6 mg/kg of moniliformin (purified) for 56 d, and each wk five rats were
necropsied and their hearts collected for electron microscopy. The rat hearts had
mitochondrial, myofibril, and cytoskeletal alterations and lesions. The mitochondrial
lesions appeared first and were the most predominant lesions. Ledoux et al. (1995)
showed that one-d-old broiler chicks were adversely affected afier dietary exposure to
moniliformin for 21 d. Heart weights and lesions were significantly increased at doses

between 50-200 mg/kg of moniliformin compared to the controls. Also, liver weights

ll

were significantly higher at 100-200 mg/kg and mortality was significantly higher at
concentrations greater than 200 mg/kg of moniliformin when compared to the controls.

In Vitro Toxicity

Moniliformin was not mutagenic in the Ames assay, but was cytotoxic to the
following cell lines: L6 myoblasts, chicken primary cell cultures, Chinese hamster ovary
fibroblast cells, and dog kidney fibroblast cells (Vesonder et al., 1993; Wu et al., 1995a;
Reams ela1., 1996).

Human Implications

Moniliforrrrin may be one of the causative agents of Keshan Disease (Chen et al.,
1990; Lili er al., 1991; Nagaraj and Wu, 1994; Hong and Jilun, 1997). A This disease occurs
primarily in China and causes ischemic myocardial necrosis (cardiomyopathy) in humans
(Yang and Wang, 1991; Yu et al., 1995). This disease develops in humans who have low
dietary intakes of selenium and/or vitamin E which are important in the removal of fiee
radicals from the body (Chen et al., 1990; Yang and Xia, 1995; Yu et al., 1995). In
animals, moniliformin decreases glutathione peroxidase activity, and selenium is required
for activation of this enzyme; Chen et al., 1990; Yang and Wang, 1991). Therefore,
moniliformin may contribute to decreased removal of fiee radicals in heart tissue,
particularly in selenium and/or vitamin E deficient humans, and cause Keshan Disease
(Yang and Xia, 1995). Presently, this disease is only associated with consumption of
grains contaminated with moniliformin, and to date scientific evidence has not shown that

moniliformin causes Keshan Disease.

12

Pharmacokinetic Data

No information could be found in the literature on the absorption, distribution,

metabolism, storage, or elimination of moniliformin in exposed animals or humans.
Toxic Mechanism of Action

Forster (1992) showed that moniliformin can undergo molecular rearrangement
and become structurally similar to pyruvate and Ot-ketoglutarate. These substrates are
important components of the TCA cycle which generates ATP in mitochondria for energy
production in cells (Figure 2). It has been proposed that moniliformin may act as a
competitive and/or suicide inhibitor of pyruvate dehydrogenase complex and/or or-
ketoglutarate complex in this cycle (Thiel, 1978; Gathercole et al., 1986). This inhibition
would be very detrimental to muscles (cardiac and skeletal) which rely heavily on aerobic
metabolism for energy production (Reams er al., 1997). With increasing inhibition, this
reduction in ATP production in cardiac tissue would cause reduced heart performance and
contraction, and/or cardiac arrest.

Chen et al. (1990) showed that glutathione peroxidase and glutathione reductase
were competitively and non-competitively inhibited, respectively, by moniliformin in the
hearts of Wister rats in vitro. This type of inhibition would likely increase the levels of
free radicals in cells and cause lipid peroxidation of membranes and/or DNA damage in
cells (likely secondary toxic mechanism of action).

Moniliformin has also been reported to inhibit brain enzymes in vitro. Burka et al.
(1982) showed that transketolase and pyruvate dehydrogenase from the brains Of

Sprague-Dawley rats were inhibited 25% by 10'9 M moniliformin. Also, inhibition of these

13

Pyruvate

‘1’ x Pyruvate dehydrogenase complex

11
Acetyl CoA MONILIFORMIN
Citrate
Oxaloacetate or - ketoglutarate
MONILIFORMIN
T Citric Acid 11
CYCIC ‘1’ Xa - ketoglutarate
Fumarate dehydrogenase complex
\ Succinyl CoA
Succinate

Figure 2. Diagram Showing the Inhibition of Pyruvate Dehydrogenase Complex and Ot-
Ketoglutarate Complex by Moniliformin in the Citric Acid Cycle (Thiel, 197 8;
Gathercole et al., 1986).

14

two enzymes was much less for moniliformin analogs (groups added on the square
structure; Burka et al., 1982).
Regulatory Aspects
Regulatory action levels or guidelines for animals or humans have not been issued

by the Food and Drug Administration (FDA) for moniliformin (Marquardt, 1996).

Fumonisins

In 1988, two research groups independently discovered a family of mycotoxins
called the fumonisins when investigating the cause of equine leukoencephalomalacia
(ELEM) and human esophageal cancer (Bacon and Nelson, 1994; Jackson et al., 1996a).
Afier this discovery, the South Afiican Medical Research Council was first to successfully
isolate fumonisin B1 and B2 from F. moniliforme MRC 826 culture material (Bacon and
Nelson, 1994; Jackson et al., 1996a). Research on fumonisins has greatly increased in the
United States after many cases of mycotoxicosis occurred in livestock from consumption
of the 1989-1990 corn crops (Jackson et al., 1996a). These compounds were identified as
the major cause of the mycotoxicoses in exposed animals (Jackson et al., 1996a).

At least seven firmonisin analogues have been identified in the past few years: B1,
B2, B3, B4, A1, A2, and C1(Thiel etal, 1982; Marasas et al., 1984; Nelson et al., 1993;
Marasas, 1995). These mycotoxins are a closely related group of compounds that are
very polar in nature (Diaz and Boerrnans, 1994; Dutton, 1996). All seven have a long
hydroxylated hydrocarbon backbone with attached methyl groups, but differ slightly by the
attachment of amides (A series) or free amino (B series) groups on this backbone (Diaz

and Boerrnans, 1994; Dutton, 1996). The firmonisins within a series designated by letters

15

A, B, or C difi‘er by the arrangement of hydroxyl groups on the hydrocarbon backbone
(Diaz and Boerrnans, 1994). The exception is firmonisin C; which lacks the terminal
methyl group adjacent to the amine on the hydrocarbon backbone (Dutton, 1996). The A
series firmonisins are not toxic; however, the B series fumonisins are biologically active in
animals (Dutton, 1996; Jackson et al., 1996a).

Fumonisins B1, B2, and B3 are the most commonly detected members of the family
in cultures and field samples (Figure 3; Plattner et al., 1992; Bullerman and Tsai, 1994;
Scott et al., 1994; Marasas, 1995; Bucci and Howard; 1996). These compounds are fairly
low molecular weight chemicals that are complex in structure (Figure 3, Table 4;
Sydenham and Thiel, 1996). However, fumonisin B1 is ofien reported as the major
contaminant of the B series firmonisins (Plattner, et al., 1992).

Fumonisins in animal feeds and human foods can be detected by enzyme-linked
immunosorbent assay (ELIZA), gas chromatograph-mass spectrometer (GC-MS), and
high performance liquid chromatography (HPLC; Pestka et al., 1994; Rice and Ross,
1994). Pestka et al.(1994) reported that the ELIZA is a better qualitative method,
whereas GC-MS and HPLC analyses are good quantitative methods for detecting
firmonisins. Unfortunately, the quantitative methods normally involve very intensive and
time consuming procedures. Recently, Solfiizzo et al. (1997) developed a rapid reverse-
phase HPLC method with fluorimetric detection for quantifying fumonisins. This method
is more precise and accurate than previous quantitative methods (Solfiizzo et al., 1997).

Decontamination and Detoxification
Chemical, physical, biological and/or thermal treatments have been used to reduce

the amount and/or toxicity of fumonisins in corn with minimal to moderate success

16

Fumonisin B1
We
CH3 0R CH3 “2

Fumonisin B3
R=C0CH2 CH(COOH)CHZCOOH

Figure 3. Chemical Structures of Fumonisin B1, B2, B3 (Merrill et al., 1993; Nelson etal.,
1993) '

l7

 

Table 4. Chemical Parameters of Fumonisin B1 and B2 (Sydenham and Thiel, 1996).

 

Fumonisin B1

 

Chemical Name

 

1,1'-[14,15-(2-aminO-3,5, lO-tri-hydroxy-12,16-
dimethyli-cosandiyl)] di-(2,3-dihydrogen propane-1,2,3-
tricarboxylate)

 

 

 

Chemical F orrnula CgJ-IsgNOls
Molecular Weight (amu) 722
Fumonisin B2
Chemical Name 1, l'-[14,15-(2-amino-3,5-dihydroxy-12, 16-

Cherrrical Formula

Molecular Weight (amu)

 

dimethylicosandiyl)] di-(2,3-dihydrogen propane-1,2,3-
tricarboxylate)

C34H59N014

706

 

l8

 

(Norred et al., 1991; Park et al., 1992; Jackson et al., 1996b). Ammoniation of F.
moniliforme culture material reduced the amount of fumonisin B1 in corn up to 79%;
however, the toxicity of the culture material was not decreased when fed to four-mo-old-
male Sprague-Dawley rats (Norred et al., 1991; Park et al., 1992). Sydenham et al.
(1994) showed that fumonisin B1 levels in corn were reduced up to 69% by removing the
screenings and fines. Dupuy et al. (1993) found that fumonisin B1 was not significantly
destroyed after thermal processing of contaminated corn for “10 min, 38 min, 175 min, or
8 hr at 150, 125, 100, or 75°C, respectively.” Jackson et al. (1996b) reported that
temperatures greater than 173°C for one hr were necessary to reduce fumonisin B; by
90%. Scott and Lawrence, (1994) showed that fumonisins B1 and B2 in corn meal were
reduced by 60% when heated at 190°C for one hr and completely destroyed at 220 °C after
24 min. This evidence indicates that fumonisins are fairly heat stable and are not greatly
reduced by common industrial thermal and drying processes (Alberts et al., 1990; Dupuy
et al., 1993; Bullerman and Tsai, 1994).

Fumonisins Bl and B2 have been reported in many animal feeds and human foods
throughout the world. As shown in Table 5, concentrations as high as 360 mg/kg
fumonisin B, and 48.0 mg/kg B2 have been reported. These concentrations are near or
similar to firmonisin levels in feeds associated with porcine pulrnonary edema (PPE),
equine leukoencephalomalacia (ELEM), nephrotoxicity, and/or hepatotoxicity in animals
and human esophageal cancer (Marasas et al., 1988a,b; Sydenham et al., 1990; Ross et
al., 1991; Osweiler et al., 1992; Thiel et al., 1992; Merrill et al., 1993; Voss et al., 1993;
Chu and Li, 1994; Riley et al., 1994b; Restum et al., 1995; Voss et al., 1995; Dutton,

1996)

19

 

Table 5. Highest Concentrations Reported for Fumonisins B1 and B2 in Feeds and
Grains from Several Countries.

 

 

 

Country Source B1 B2 Citation
(mg/kg) (ms/ks)

Thailand Corn 18.8 1.4 Yoshizawa et al., 1996
Brazil Feed 38.5 12.0 Sydenham et al., 1992
Brazil Feed 38.5 11.8 Thiel et al., 1992
Brazil Corn 18.5 19.1 Hirooka et al., 1996
China Corn 155 --- Chu and Li, 1994
South Afiica Corn 46.9 16.3 Sydenham et al., 1991
South Afiica Corn 117 23.0 Sydenham et al., 1991
Honduras Maize 6.6 --- Julian et al., 1995
Egypt Corn meal 3.0 0.8 Sydenham etal., 1991
Italy Pufi‘ed corn 6.1 0.5 Doko and Visconti, 1994
Italy Maize 250 --.. Logrieco and Moretti, 1995
Italy Pufl‘ed corn 6.1 0.5 Bullerman and Tsai, 1994
Italy Corn grits 3.8 0.9 Bullerman and Tsai, 1994
Italy Corn (moldy) 250 --- Bottalico et al., 1995
USA Corn grits 2.5 1.1 Sydenham et al., 1991
USA Corn meal 2.8 0.9 Sydenham et al., 1991
USA Corn meal 15.6 ---- Pestka et al., 1994
USA Corn 37.9 0.9 Murphy et al., 1993
USA Corn 37 .9 12.3 Bullerman and Tsai, 1994
USA Feed 86 ~---- Park et al., 1992
USA Feed 122 23.0 Thiel et al., 1992
USA Corn 360 -- Ross et al., 1991
USA Feed 270 12.8 Thiel et al., 1992
USA Feed 330 ---- Ross et al., 1992
USA Feed 37.0 ---- Rice and Ross, 1994
USA Corn ' 239 ---- Rice and Ross, 1994
USA Corn 166 48.0 Motelin et al., 1994
USA Corn ‘ 149 44.0 Motelin et al., 1994

 

 

 

 

 

‘Screenings

20

 

Animal Diseases

F umonisins can cause several adverse effects in exposed animals, including equine
leukoencephalomalacia and porcine pulmonary edema (V oss, 1990; Osweiler etal., 1992;
Ross et al., 1991; Voss et al., 1993; Riley et al., 1994a,b).

Equine Leukoencephalomalacia (ELEM)

ELEM is a well known disease that occurs only in Equidae. Fumonisin B1 appears
to be the primary causative agent in cultures and crop samples associated with ELEM
(Ross et al., 1993; Diaz and Boerrnans, 1994; Norred and Voss, 1994; Kuiper-Goodman,
1995). Recently, Ross et al. (1994) showed that fumonisin B2 in F. proliferatum (M-
6104) produced this disease in equines as well. Toxicoses can develop one to two wk
after exposure to firmonisin-contaminated feeds (Diaz and Boerrnans, 1994). There are
two main types of ELEM: neurotoxic and hepatotoxic (Osweiler et al., 1985; Diaz and
Boerrnans, 1994; Kuiper-Goodman, 1995, Dutton, 1996). In neurotoxic ELEM, afi‘ected
animals may exhibit signs of hyperexcitability, head pressing, aimless walking, unilateral
blindness, ataxia, anorexia, depression, coma, and/or death (Osweiler et al., 1985; Diaz
and Boennans, 1994, Dutton, 1996). Histopathologic examinations usually reveal mild to
severe liquefactive necrosis in the white matter of the cerebral herrrispheres of the brain
(Osweiler er al., 1985; Diaz and Boerrnans, 1994; Dutton, 1996). In hepatotoxic ELEM,
Equidae may experience anorexia, depression, facial edema, tongue and lip paralysis,
and/or elevated icterus (Diaz and Boerrnans, 1994). This exposure usually causes mild to
severe legions and discoloration (yellow) of the livers (Diaz and Boermans, 1994; Dutton,
1996). Ross et al., (1991) reported that cases of ELEM have occurred at concentrations

of less than 1 to 126 mg/kg of firmonisin B1 in equine feeds.

21

 

Porcine Pulmon_ar_y Edema (PPE)

PPE is a disease that occurs in swine that are usually fed corn or corn screenings
contaminated with firmonisins (Colvin and Harrison, 1992; Osweiler etal., 1992; Diaz and
Boerrnans, 1994; Motelin et al., 1994; Norred and Voss, 1994). Fumonisin B1 appears to
be the causative agent in both cultures and field samples (Osweiler et al., 1992; Motelin et
al., 1994). PPE usually develops one to two wk after consuming firmonisin-contaminated
feeds (Guzman and Casteel, 1995). Physical symptoms can include weakness, anorexia,
dyspnea, cyanosis, and death (Diaz and Boerrnarrs, 1994). Exposure can cause
hydrothorax, massive lung edema, and/or death (Colvin and Harrison, 1992; Norred,
1993; Dutton, 1996). Ross et al. (1991) reported that swine developed PPE when
exposed at less than 1 to 330 mg/kg fumonisin B1 in the feed.

Other Tordc Efi‘ects

F umonisins are hepatotoxic and/or nephrotoxic to several species of animals
including rats, chicks, ducklings, lambs, mice, turkeys, pigs, and Equidae (Haschek et al.,
1992; Riley et al., 1994b; Bermudez et al., 1995;“ Bondy et al., 1995; Edrington et al.,
1995; Dutton, 1996; Ledoux et al., 1996).

In Vitro Studies

The fiimonisins are promoters and weak cancer initiators in rat liver cells and
moderately cytotoxic at high doses (>345 uM in rat hepatocytes) in mammalian cell
cultures (Gelderblom et al., 1992; Park etal., 1992; Gelderblom et al., 1993; Jackson et
al., 1996a). Gelderblom et a1. (1993) showed that cytotoxicity increases as polarity

decreases for the firmonisins (B2> B3 > B1> A2 > A1 ). Also, cytotoxicity increases with

22

the attachment of free amino groups on the firmonisin backbone (B series only).
However, the fumonisins were not mutagenic in the Ames assay.
Human Implications

Presently, fumonisins are only associated with esophageal cancer, and to date
scientific evidence has not shown that fumonisins cause this type of cancer in humans
(Norred, 1993; Bullerman and Draughon, 1994; Norred and Voss, 1994; Shepard et al.,
1996). Also, esophageal cancer has not been reported in animals exposed to fumonisins
(Norred and Voss, 1994).

Pharmacokinetic Data

Fumonisins are polar metabolites of fungi that are poorly absorbed and eliminated
fairly quickly from the body (Dutton, 1996). Prelusky et al. (1995) showed that two
Holstein cows (452-630 kg) injected i.p. with 0.1 or 0.2 mg/kg of fumonisin Bl had a very
rapid decrease in plasma levels Of the mycotoxin. Fumonisin B1 was undetectable (4.0
ng/ml) 120 rrrin. after dosing. Also, in two Holstein cows gavaged with either 1.0 or 5.0
mg/kg of firmonisin Bl, firmonisin B1 or its metabolites were not detected in the plasma up
to 14 d after exposure. Shepard et al. (1995a) showed that two male Vervet monkeys
gavaged with 6.4 mg/kg of fumonisin B1 had minimal absorption of this compound. The
plasma concentrations peaked (z 210 ng/ml) at one and a half hr after toxin administration
and then sharply declined to under 50 ng/ml afier four hr.

Prelusky et al. (1996) fed pigs up to 3.0 mg/kg l4C-fumonisin B; for 24 d and
noted accumulations Of fumonisin B1 occurring only in the liver and kidneys. Gumprecht
et al., (1995) showed that, in adult New Zealand White rabbits injected iv. with 1.25

mg/kg of firmonisin B1, the toxin affected the kidneys and livers. However, the kidneys

23

were more severely affected than the livers in four male Vervet monkeys gavaged or
injected iv. with either 6.4 mg/kg b.w or 1.7 mg/kg b.w. of 14C fumonisin B1 (Shepard et
al., 1995a). l“C fumonisin B1 accumulated at mean concentrations of 0.6 and 1.9% of
the administered doses, respectively, in the livers and kidneys of monkeys gavaged or iv.
injected. Also, the main route of elimination of the administered doses was via the feces at
64.0 and 68.1%, respectively. Shepard et al. (1992) showed that male BD IX rats injected
iv. with 7.5 mg/kg b.w. Of firmonisin B1 eliminated only 16% of firmonisin B1
(unmetabolized) in the urine after 24 hr. Also, Shepard et al. (1994) found that male
Wistar rats injected i.p. with 7.5 mg/kg b.w. offumonisin B1 had eliminated 67% of it in
the bile after 24 hr. Recently, Shepard et al. (1995b) showed that male BD IX rats mainly
eliminated both gavaged (82.0%) and i.p. injected (84.1%) firmonisin B2 (unmetabolized)
via the feces within three d.
Toxic Mechanism of Action

Sphingolipids are found in cellular membranes of all animal species (Riley et al.,
1994c). Fumonisins are structurally similar to the simplest sphingolipids, such as free
Sphinganine (Sa) and free sphingosine (So) (Figure 4; Riley et al., 1994b,c). These
sphingoid bases are produced by the condensation of an amino acid and a fatty acid and
are the precursors of complex sphingolipids (glycosphingolipids, sphingomyelin, and
ceramides) in cellular membranes (Riley et al., 1994c). The enzymes, sphingarrine N-
acyltransferase and sphingosine N-acyltranferase, commonly convert sphingoid bases into
complex sphingolipids (Riley et al., 1994c). Because firmonisins are structurally similar to

sphingoid bases, these mycotoxins target and inhibit Sphinganine N-acyltransferase

24

0H

WNW“

Sphinganine "”2
0H CHZOI'I
/\/\/\/\/\/\/\/W
Sphingosine "”2
CH3 0R CH3 01‘! m2

Fumonisin B1

R=C0CH2 CH(COOH)Cl{2COOH

Figure 4. Chemical Structures of Sphinganine, Sphingosine, and Fumonisin B1
(Jackson et al., 1996a).

25

(primary target) and/or sphingosine N-acyltransferase (Figure 5; Riley et al., 1994c). This
disrupts de novo biosynthesis of complex sphingolipids which is critically important in
regulating cell firnction (e. g. “cell-cell communication and intracellular signal
transduction” pathways) and leads to accumulation of free Sa and sometimes fi'ee So in
cells in vitro (Wang et al., 1991; Riley et al., 1994a). Also, complex sphingolipids can be
converted back into free SO which acts as a second messenger in regulating cellular
processes (e. g. turning on/ofi‘ genes or proteins; Wang et al., 1992; Riley et al., 1994a).
Therefore, fumonisins can disrupt the amount of complex sphingolipids and sphingoid
bases in cells and drastically alter cellular processes. This mechanism of action has been
postulated as the probable cause of ELEM, PPE, and hepatotoxicity in fumonisin-exposed
animals (Wang et al., 1992).

Wang et al. (1992) found that ponies fed 15 to 44 mg/kg firmonisin B1 had
reduced levels of complex sphingolipids and elevated levels of free Sa, and sometimes free
So in sera. Also, free Sa and free So levels were elevated before liver enzyme levels were
increased in sera (Wang et al., 1992). Riley et al. (1994b) showed that rats fed 15, 50,
and 150 mg/kg of firmonisin B1 had increased free Sa /So ratios in the kidneys, which was
similar to the free Sa/SO ratios in their urine. The fumonisins adversely afi‘ected the
kidneys sooner than the liver in these rats. Gumprecht et al. (1995) showed that adult
New Zealand White rabbits intravenously administered a single dose of 1.25 mg/kg b.w. of
fumonisin B1 had elevated fiee Sa/So levels in their kidneys and livers. The kidneys had
severe lesions compared to mild lesions in the livers. In a previous study with mink,
Restum et al. (1995) showed that fumonisins significantly increased fi'ee Sa levels and free

Sa/So ratios in kidney and liver, and this ratio was much higher in kidney than liver of the

26

Serine + Palmityl COA

i
3-Ketosphirrganine
l
Sphinganine
FUMONISIN S
U
i X Sphinganine N-acyltransferase
(or ceramide synthase)
N-acylsphinganine
l

Sphingomyelin <—> N-acylsphingosine <—> Glycosphingolipids

ll XSphingosine N -acyltransferase
(or ceramide synthase)

> Sphmgosrne <

 

 

 

FUMONISINS

Figure 5. Diagram Showing Where Fumonisins Target and Inhibit Sphinganine N-
Acyltransferase and/or Sphingosine N-Acyltransferase (Wang et al., 1992;
Riley et al., 1994c).

27

. exposed females. In the past, sera concentrations of free Sa and free So were viewed as a
good biomarker of fumonisin exposure. However, the above evidence suggests that urine
concentrations of free Sa and free SO may be a better biomarker of fumonisin exposure
than sera in exposed mink and other mammals (Wang et al., 1992; Riley et al., 1994b;
Restum et al., 1995).
Regulatory Aspects

According to Vainio et al. (1993), as cited by Jackson et al. (1996a), firmonisins
are listed as Group 2B carcinogens (possible human carcinogen) by the International
Agency for Research on Cancer. The FDA has set informal guidance levels for firmonisin
B, in some animal feeds (equines s 5 ppm, pigs 3 10 ppm, and beef cattle s 50 ppm
firmonisin B1), but not for human foods (Jackson et al., 1996a). These informal guidelines
are designed to help protect animals and hopefirlly humans from toxic levels of firmonisins
in animal products (Jackson er al., 1996a).
Mink Sensitivity to Fusarium Mycotoxins

Previous studies with mink have shown them to be sensitive to fumonisins, as well
as to other mycotoxins produced by F usarium fungi, such as deoxynivalenol, zearalenone,
and T2 toxin (Cameron et al., 1989; Aulerich and Bursian, 1993; Restum et al., 1995;
Powell et al., 1996).

F umonisins

Dietary firmonisins have produced several adverse efl‘ects in mink. Restum er al.
(1995) fed, nine-mo-old, pastel female mink F. moniliforme culture material (M-1325)
containing 0 or 118 mg/kg firmonisins [fumonisin B1 (89 mg/kg), B2 (21 mg/kg), B3 (8

mg/kg)] for 87 d. Several hematologic and serum chemical values were significantly

28

altered in the treated group when compared to the control. Also, free Sa and free Sa/So
ratios in liver and kidneys were statistically much greater in the treated group compared to
the control. Powell et al. (1996) fed adult, pastel, female mink 0, 115, or 254 mg
fumonisins/kg diet consisting of fumonisin B1 (86 or 200 mg/kg), B2 (22 or 42 mg/kg), and
B3 (7 or 12 mg/kg) from F. moniliforme culture material (M-1325) for 108 (1. Only 58%
of the mated females whelped in the 254 mg/kg fumonisins B1, B2, and B3 group
compared to 100% in the 115 mg/kg and control groups. There was a statistically
significant decrease in kit body weights at birth and only 23 kits were born alive in the 254
mg/kg group compared to 61 and 76 in the 115 mg/kg and control groups. All three
fumonisins were detected at very low levels (0.7 % of dietary concentration) in milk
samples collected from the high dose group.
Zearalenone

Mink are very sensitive to the estrogenic effects of zearalenone at low
concentrations in the diet (Cameron et al., 1989; Aulerich and Bursian, 1993). Cameron
et al. (1989) fed 0, 10 and 20 mg/kg of zearalenone to ovariectomized natural dark mink
for 21 d. The females had a dose-related increase in uterine weights. In addition, a
reproductive study on female mink was conducted with the same zearalenone
concentrations. The diets were fed to the females for approximately two mo. prior to
mating to untreated males. No females whelped in the 20 mg/kg group compared to
100% in the 10 mg/kg and control groups. All females in the 20 mg/kg group had
enlarged uteri. Kit mortality at three wks of age was much greater in the 10 mg/kg group
(17%) than the control group (0%). The kit sex ratio (%, maleszfemales) was quite

different in the control group (64:36) compared to the 10 mg/kg group (22:78).

29

Deoxynivalenol (DON; Vomitoxin)
Temporary feed refirsal was the only toxic efi‘ect that occurred in female mink fed
1.7 mg/kg Of deoxynivalenol in the diet for approximately six mo. (Aulerich et al., 1994).
Also, no adverse reproductive efi'ects occurred in the females or kits up to six wk of age in
this study. Mink given a choice between “clean feed” or deoxynivalenol-containing feed
(as low as 0.28 mg/kg) preferred the clean feed (Aulerich et al., 1994).
T2 toxin
Mink have been reported to be very sensitive to low concentrations of i.p. injected

T2 toxin. The LDso was within the range of 0.5 - 2.0 mg/kg b.w. for mink (Cameron et

aL,l989)

30

PART I

SUBCHRONIC AND REPRODUCTIVE EFFECTS IN MINK FROM EXPOSURE
TO F USARI UIVI F UJIK UROI CULTURE MATERIAL (M-1214) CONTAINING
KNOWN CONCENTRATIONS OF MONILIFORIMIN.

Abstract

This study was conducted to ascertain the subchronic and reproductive efi‘ects in
mink (Mustela vison) resulting from exposure to moniliformin, a toxic mycotoxin
produced by F usarium firngi In a preliminary trial, adult female mink presented diets that
contained targeted concentrations of moniliformin provided by F. filjikuroi culture
material (M-1214) of 40 mg/kg, or greater, refused to eat significant quantities of feed.
Feeding adult mink diets that contained 8.1 or 17.0 mg/kg, wet weight, moniliformin in a
30-d subchronic trial produced no significant adverse effects on feed consumption, body
weights, hematologic parameters or serum chemical values, or notable histologic changes
in their tissues. In the reproduction trial, female mink were exposed to the same dietary
concentrations of moniliformin as in the subchronic test from two wk prior to the breeding
season until their offspring (kits) were eight-wk-Old. Consumption Of the high-dose (17
mg/kg) diet resulted in significant neonatal mortality and reduced kit body weights at birth
and at eight wk of age. Necropsy Of eight-wk-old kits from the control and high-dose
groups revealed no gross or histologic lesions or alterations in liver, lung, or heart tissues
that could account for the mortality observed in the kits exposed to moniliformin. These

results indicate that long-term (105-135 (1) dietary exposure to F. flrjikuroi culture

31

material containing 17 mg/kg moniliformin is not lethal to adult female mink, but can have
adverse effects on neonatal mink.
Introduction

Moniliformin is a mycotoxin produced by several species of F usarium firngi that
occur on cereal grains, including F. moniliforme (Cole et al., 1973; Nelson, 1992), F.
proliferatum (Nelson et al., 1993), F. oxysporum (Abbas et al., 1990), F. subglutinans
(Logrieco et al., 1993; Lew et al., 1996), and F. fujikuror‘ (Ledoux et al., 1995). It has
been found as a co-contaminant with deoxynivalenol, zearalenone, and fusarin C which are
also produced by F usarium firngi (Thiel et al., 1982, 1986). Concentrations of naturally
occurring moniliformin as high as 425 mg/kg in corn (Logrieco et al., 1993) and 530
mg/kg w.w. in maize (Lew et al., 1996) have been reported.

Less than chronic dietary exposure to moniliformin can be toxic to broiler chicks
(Kriek etal., 1977; Allen et al., 1981; Engelhardt et al., 1989; Javed et al., 1993; Ledoux
et al., 1995), ducklings (Engelhardt et al., 1989), turkey poults (Engelhardt et al., 1989),
mice (Burmeister et al., 1980; Deyu er al., 1993), rats (Kriek et al., 1977; Abbas et al.,
1990; Deyu etal., 1993) and donkeys, horses, pigs, and rabbits (Chen et al., 1990).
However, very little research has been reported on the chronic effects of moniliformin in
animals (Lew et al., 1996).

Because cereal grains are important components of farm mink diets and previous
studies have shown mink (Mustela vison) to be sensitive to other mycotoxins produced by
F usarium firngi, such as zearalenone (Bursian et al., 1992; Yang et al., 1995),
deoxynivalenol (Aulerich et al., 1994), and fumonisins (Restum et al., 1995; Powell et al.,

1996), studies were conducted to investigate the effects of moniliformin on mink. The

32

objectives of these studies were to determine the toxicity of dietary moniliformin to mink,
to characterize any observed clinical signs or histopathologic lesions or alterations in the
mink, and to examine the efiects of the mycotoxin on hematologic parameters, serum
chemical values, reproductive performance of female mink, and growth and viability of
exposed Offspring (kits).
Materials and Methods
Subchronic Study

Twenty-four pastel, adult, female mink were divided equally into a control and
three treatment groups. Litterrnates were not placed in the same group to minimize any
confounding factors such as genetic susceptibility or resistance to moniliformin. The mink
were vaccinated as kits against canine distemper, virus enteritis, botulism, and
hemorrhagic pneumonia. They were housed individually in suspended wire mesh cages
(76 cm L x 61 cm W x 46 cm H) in an animal room at the Michigan State University
Experimental Fur Farm. The temperature in the room was maintained above 0°C by
thermostatically-controlled electric heaters. Ventilation was provided by a wall fan and
ceiling vents. The natural photoperiod was simulated by the use of a time clock. The
mink were acclimated to the room, cages, and control diet for five (1 prior to the start of
the feeding trial on January 10, 1996. Feed and drinking wateriwere provided ad libitum
throughout the study.

F. filjikuroi Nirenburg (M-1214; Fusarium Research Center, Pennsylvania State
University, University Park, PA) corn culture material was prepared as described by
Ledoux et al. (1995). The culture material, which contained approximately 10,000 mg/kg

moniliformin, was blended with “toxin free” ground corn to form a premix for

33

incorporation of the mycotoxin into the mink diets. The basal diet consisted of 34.3%
water, 23.8% commercial mink cereal, 23.8% poultry by-products, 6.3% herring meal,
6.3% eggs, 4.8% beef liver, and 0.6% corn premix. During the reproduction and lactation
periods (March through May), 4.7 IU vitamin E/kg, 625 USP vitamin A/kg, 6.25 IU
vitamin D/kg, and 8 ml com oil/kg, were added to the diets. Proximate analysis of the
basal diet, as fed, yielded 55.9% moisture, 17.1% crude protein, 8.9% fat, 4.1% ash and
1.5% crude fiber (Litchfield Analytical Services, Litchfield, MI). Ground corn without the
culture material was added to the control diet. The mixed diets were stored frozen (-5°C)
until fed to the mink. Targeted dietary concentrations of 0 (control), 10, 20, and 40
mg/kg moniliformin were selected for the 30-d subchronic feeding trial based on results of
a pilot feeding trial that indicated feed refirsal at dietary concentrations in excess of 40
mg/kg moniliformin. Samples of the culture material and the control, 10, and 20 mg/kg
moniliformin diets were collected for moniliformin analysis (Ledoux et al., 1995). The 40
mg/kg diet was not analyzed because this dietary group was dropped fiom the trial due to
reduced feed consumption. The cereal used in the diets was screened for the presence of
deoxynivalenol, zearalenone, and aflatoxins. Mink feed consumption was recorded daily
and body weights were measured weekly. The animals were observed daily for signs of
toxicity. Mink losing more than 30% of their initial body weight were removed fiom the
trial. At the end of the 30-d feeding trial, the mink were anesthetized (Ketamine HCl, 30
mg/mink) and 12 ml blood collected (via cardiac puncture) for hematologic and serum
chemical determinations (analyses by Veterinary Clinical Pathology Laboratory, Michigan

State University, East Lansing, MI).

34

Red and white blood cell counts, hemoglobin concentration, hematocrit, mean
corpuscular volume, mean corpuscular hemoglobin concentration, spun packed cell
volume, plasma total solids, and difi‘erential cell counts were determined using a
Technicon H1 system (Technicon Diagnostics Systems Division, Tarrytown, NY). The
serum chemistry analyses and calculations were performed with an Abbott Spectrum
Analyzer (Abbott Laboratories, Dallas, TX) and included calcium, chloride, iron,
magnesium, phosphorus, potassium, sodium, sodium/potassium ratio, carbon dioxide,
anion gap, total protein, albumin, globulin, albumin/globulin ratio, creatinine, alanine
aminotransferase, alkaline phosphatase, amylase, aspartate aminotransferase, creatine
kinase, total bilirubin, cholesterol, glucose, blood urea nitrogen, and osmolality.

Afier blood collection, the mink were euthanized (CO2) and necropsied. Brain,
liver, kidneys, heart, spleen, lungs, and adrenal glands were collected, weighed and a
sample of heart, liver, and lung placed in bufi‘ered 10% formalin. The fixed tissues were
processed according to routine histological procedures, embedded in paraffin, sectioned (5
um), stained with hematoxylin and eosin, and examined with the assistance of a veterinary

pathologist using a light microscope.
Reproduction Study

For the reproduction study, 36 pastel, adult, female mink were allocated to a
control or one of two treatment groups each containing 12 mink. Littermates were placed
in different dietary groups. The mink were housed individually in suspended wire cages
(as described for the subchronic study) in an open-sided shed. A wooden next box (38 cm

L x 28 cm W x 27 cm H) bedded with aspen shavings and excelsior (“wood wool”) was

35

attached to the outside of each cage. The mink were acclimated to the cages, nest boxes,
and control diet for seven d before initiation of the trial on February 15, 1996.

The control and treatment diets containing targeted concentrations of 0 (control),
10 (low-dose), or 20 (high-dose) mg/kg moniliformin (supplied by the corn culture
material) were prepared as described for the subchronic study. Feed and drinking water
were provided ad libitum.

The degree of vulvar swelling in the females was assessed prior to the breeding
season (February 28, 1996). Vulvar swelling has been correlated with mating
performance and serum estradiol concentrations in female mink (Travis et al., 1978). It
was scored on a scale from 0 to 3. A score of 0 was assigned to designate that the vulva
was pale and unswollen (anestrus). Scores from 1 to 3 were used to indicate increasing
degrees of tumefaction and a progressive color change from cream to pink to red as vulvar
swelling increased. Most female mink will not mate until they have a vulva swelling score
(VSS) of 2 or greater.

Mink body weights were recorded at weekly intervals fiom the start of the trial
until March 14, 1996, and at whelping, and three and six wk post-partum. The females
were not weighed during pregnancy because of the variation in litter size and fetal body
weights due to possible adverse effects handling may have on gestation. The females were
mated to untreated males between March 4 and 25, 1996. Mated females were given at
least two additional opportunities to mate to different males during the breeding period (a
common commercial mink breeding practice to increase conception rates). All matings
were verified by examination, with a light microscope, of vaginal aspirations taken

immediately after mating for the presence of normal appearing motile spermatozoa.

36

During the whelping period (April 15 through May 15, 1996), the nest boxes were
checked daily for kits. Gender of all kits was determined at birth, and the alive and
stillborn offspring were counted. Kit body weights were recorded at birth and at three,
six, and eight wk Of age. At three wk of age, the kits were ofi‘ered the same experimental
diet as their dams, but could continue to nurse up to weaning at six wk. Milk samples
were collected (Jones et al., 1980) for moniliformin analysis fi'om five or six females in
each dietary group at about three wk post-partum

Three kits from the control group and 12 kits from the high-dose group were
euthanized (CO2) at eight wk of age and necropsied. Samples of liver, lung, and heart
from the kits were collected and prepared for histologic examination as previously
described.

Statistical Analysis

The Sigma Stat Statistical Analysis System (1992) was used to analyze the data.
Continuous variables (body weights, organ weights, hematological parameters, serum
chemical values, and gestations) were evaluated using one-way analysis of variance.
Where the F value statistic was significant at p<0.05, treatment means were tested by
Tukey’s test. Discrete variables (kit survival and the number of alive and stillborn kits)
were analyzed using Bonferroni Chi-square test. Statements of statistical significance are
based on p<0.05.

Results
Subchronic Study
Laboratory analysis by HPLC of duplicate samples Of the M-1214 corn culture

material yielded a mean moniliformin concentration of9174 mg/kg (analysis by USDA

37

National Center for Agricultural Utilization Research, Peoria, IL). Analyses of the
control, 10, and 20 mg/kg diet samples for moniliformin yielded concentrations of <02
(detection limit), 8.1, and 17.0 mg/kg, respectively. HPLC analysis of the cereal for
aflatoxins, zearalenone, and deoxynivalenol showed no detectable concentrations
(detection limits were 0.004 mg/kg for aflatoxins B1, B2, G1, and G2; 0.2 mg/kg for
zearalenone; and 0.4 mg/kg for deoxynivalenol) of these mycotoxins (analysis by Animal
Health Diagnostic Laboratory, Michigan State University, E. Lansing, MI).

Feed consumption by mink in the 40 mg/kg group steadily decreased to about 15%
of that of the controls by day seven at which time they were removed fiom the trial. Other
than the decreased feed consumption and reduced body weights of the mink in the 40
mg/kg group, no other clinical signs were observed. There were no significant difi‘erences
in feed consumption or body weights among the mink in the control, 8.1 (a.k.a 10 mg/kg),
and 17 mg/kg (a.k.a. 20 mg/kg) groups throughout the trial.

No gross lesions or alterations were observed in the mink at necropsy and there
were no significant difi‘erences in brain, liver, kidney, heart, lung, or adrenal gland weights
(expressed as absolute weight or as percent of body weight) among the groups. The only
significant difl‘erence in hematologic values among the groups was in the leukocyte
differential cell counts. The percentage of segmented neutrophils in the high-dose group
was less than in the control and low-dose groups [66.17 i 3.32 (mean : S.E.)% vs. 79.50
i 1.49 and 79.20 i 3.64%, respectively] and the percentage of lymphocytes was greater in
the high-dose group compared to the control and low-dose groups (26.67 i 3.13% vs.
14.00 i 3.13 and 14.40 i 3.42%, respectively). The serum calcium concentration in the

high-dose group (9.88 i 0.13 mg/dl) was significantly greater than in the low-dose group

38

(9.34 i 0.15 mg/dl) but not significantly greater than in the control (9.62 i 0.13 mg/dl).
Also, the serum magnesium concentration of the mink in the low-dose group (1.82 3: 0.05
mEq/l) was significantly less than in the control (2.00 1: 0.04 mEq/l) but not significantly
different from that of the high-dose group (1.85 i 0.04 mqu 1). Histologic examination
of the hearts, livers and lungs did not reveal any consistent lesions or alterations among

these tissues from mink in the three groups.

Reproduction Study

Body weights of the adult female mink in the three groups were not significantly
difi‘erent fiom the initiation of the trial through whelping. However, the body weights of
the adult females in the low-dose group were significantly greater than the controls at
three wk (1092 i 27.0 g, vs. 991 j; 28.4 g), and at six wk post-partum (923 i 25.0 g, vs.
814 i 29.3 g).

Vulvar swelling scores for the females were similar among the groups and all the
females on the trial were mated, except for one in the low-dose group that died prior to
the breeding season. Necropsy findings (Michigan State University Health Diagnostic
Laboratory) indicated that pyelonephritis and urolithiasis were the probable causes of
death.

The reproductive performance of the mink is summarized in Table 6. Breeding
performance, gestation, and kit sex ratios were not significantly different among the
groups. There was, however, a significant difi‘erence in the number of alive versus stillborn
kits in the high-dose group compared to the control and low-dose groups (Table 6). Kit

body weights and survival from birth through eight wk of age are shown in Table 7.

39

 

Table 6. Reproductive Performance of Female Mink Fed Various Concentrations

 

 

 

of Moniliformin.
Dietary group (mg/kg moniliformin)
Parameter
Control Low-dose High-dose
(<0.2) (8.1) (17.0)

No. females 12 1 1' 12
No. females whelping
per no. mated 10/12 11/11 10/12
Mean no. verified
matings per female
per attempted 2.8/5.3 3.0/5.0 2.8/5.1
matings°
Mean gestation (d)°"' 48.1 i 0.72 47.3 3: 0.68 48.0 i 0.72
No. kits whelped

Alive 62 51 34°

Dead 4 4 24
Mean litter size per
female whelping

Total kits 6.6 5.0 5.8

Live kits 6.2 4.6 3.4
Kit sex ratio (%
males: females) 45:55 49:51 48:52

 

 

 

 

 

3One female died prior to mating
bMatings verified by presence of “normal”, motile sperm in vaginal aspirations taken

after copulation
cMean : SE.

°Based on date of final mating

°Significantly different (p<0.05) from control and low-dose groups

 

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41

Although the kits in the high-dose group weighed significantly less than controls at birth
and low-dose kits at three wk, there were no significant differences in kit body weights
among the groups at six wk of age. However, the kits in the high-dose group weighed
significantly less than those in the control and low-dose groups at eight wk, and kit
mortality increased markedly between six and eight wk in the high-dose group. Histologic
examination of the liver, heart and lungs from eight-wk-old kits in the control and high-
dose groups, however, revealed no lesions or alterations in the tissues that could account
for the mortality. No free moniliformin by HPLC analysis could be detected in the
samples of mink milk and recoveries fiom spiked milk samples were very low
(approximately 20%).
Discussion

In the subchronic study, mink fed the diet containing a targeted concentration of
40 mg/kg moniliformin had an 85% decrease in feed consumption within seven d
compared to controls. In contrast to the mink, feed intake by rats provided diets that
contained 750 or 1000 mg/kg purified moniliformin decreased by 16% and dietary
concentrations of 450 mg/kg moniliformin, from F. oxysporum MT 6 rice culture material,
resulted in an average reduction in feed consumption Of only 11% compared to control
values (Abbas et al., 1990). In broiler chicks, significantly (p<0.05) lower feed intakes
were not Observed with diets that contained less than 100 mg/kg moniliformin provided by
F. firjikuroi culture material (Ledoux et al., 1995). Thus, compared to rats and chicks,
mink appear to be considerably less tolerant of dietary moniliformin. It was not
determined whether the sensitivity of mink to dietary moniliformin was due to decreased

palatability of the diet or to a toxic response. However, the efi‘ect of monilifomrin on

42

mink feed consumption precluded the use of higher dietary concentrations of mycotoxin in
this study which may account for the absence of other toxic effects in the adult mink that
have been reported for some other species.

The statistically significant differences observed in the hematologic values and
serum chemical concentrations between the groups of adult mink in the subchronic study
may not be biologically relevant, as the percentages of segmented neutrophils and
lymphocytes, and serum calcium and magnesium concentrations observed in this study
were within the ranges reported for “normal” mink. Mean segmented neutrophil and
lymphocyte values for “normal” adult female mink were reported to be 72.0 i 1.72
(S.E.)% and 15.2 i 1.34%, respectively, by Asher etal., (1976) and, according to Kubin
and Mason (1948), can range from 44-81 and 14-51%, respectively. Serum calcium
concentrations reported for normal mink were 10.66 (mean) (Asher et al., 1976), 11.02 i
0.70 (SD) (Rotenberg and Jorgensen, 1971), and 9.47 i 0.39 (SD) mg/dl (Weiss et al.,
1994). In a recent study using the same strain of adult mink and a similar diet as fed in
this study, serum calcium and magnesium concentrations of the control animals were 9.37
i 0.58 (SE) mg/dl and 2.20 i 0.11 mEq/l, respectively (Powell et al., 1996). Serum
magnesium concentrations for normal, adult, female mink fed a conventional (“wet”) diet
were reported to be 2.63 i 0.37 (SD) mEq/l by Rotenberg and Jorgensen (1971).

NO studies were found in the literature that examined the efl‘ects of moniliformin
on mammalian reproduction and offspring survival. Thus, no comparisons of the effects
observed in the mink in this study with those in other species could be made.

The breeding performance, conception rates, and duration of gestations of the

mink fed moniliformin were comparable to the controls. Mean litter size in all groups,

43

based on total kits whelped (Table 6), was equivalent to or greater than the normal
average of five kits reported for mink by Sundquist et al., (1989). However, over 41% of
the kits whelped by females in the high-dose group were stillborn or died within 24 hr
post-partum compared to 6.1 and 7.3% mortality for the control and low-dose groups,
respectively (Table 6). The mortality in the latter two groups is comparable to the 7.2%
mortality reported for the first 24 hr for kits whelped on commercial mink farms (Venge,
1973). Normal kit mortality fi'om birth through weaning (six wk) for mink was reported
to be 17% (Venge, 1973), and 20% (Schneider and Hunter 1993). These “normal” six wk
kit mortality rates are greater than the 13.6 and 12.7% observed in the control and low-
dose groups, respectively, but considerably less than the 51.7% mortality observed in the
high-dose group. The 72.4% overall mortality fi'om birth to eight wk in the high-dose
group kits was notably greater than the normal mortality of 18-20% (V enge, 1973) and
21% (Schneider and Hunter, 1993) reported for mink fiom birth to pelting (approximately
seven mo) on commercial mink farms.

The high percentage of kit deaths within the first 24 hr in the high-dose group
would suggest that there was placental transfer of moniliformin fi'om the dam to the fetus.
The lesser (5.1%) mortality from 24 hr to six wk of age suggests that moniliformin was
not transferred to kits via the milk. However, this could not be confirmed analytically.
The marked increase in kit mortality between six and eight wk of age in the high-dose
group (Table 7) may reflect the exposure of the kits to a lethal concentration of
moniliformin via the feed following weaning and indicates that young mink are more

sensitive to moniliformin than adult females.

Mink kits usually begin to consume “solid” feed at about three wk of age.
Therefore, their nourishment for the first three wk is provided entirely by the dams. The
non-significant difi‘erence in kit body weights between the kits in the control and high-dose
groups at three wk (Table 7) may be misleading if one considers that the average litter size
per lactating female was 6.3, 4.4, and 3.9 for the control, low and high-dose groups,
respectively. On average, the dams in the control group were nursing 2.4 more kits than
dams in the high-dose group but still had heavier kits. The difi‘erences in average litter
size may also account to some extent for the lower body weights of the dams in the
control group compared to the low-dose group at three and six wk post-partum.

When exposed to increasing concentrations of moniliformin, animals have shown
muscular weakness, respiratory stress, cyanosis, coma, and death (Kriek et al., 1977;
Thiel, 1978). Orally administered monilifornrin caused severe myocardial lesions and
degeneration and necrosis of the myocardium in rats, mice, chicks, poults and ducklings
(Thiel, 1978; Rearns et al., 1997), intestinal and thymic hemorrhages in rats (Abbas et al.,
1990), and ascites, hydropericardium and myocardial pallor in broiler chicks, turkey
poults, and ducklings (Engelhardt et al., 1989). According to Thiel (1978), the toxic
mechanism of moniliformin may involve the selective inhibition of mitochondrial pyruvate
and or-ketoglutarate oxidations. Serum pyruvate concentrations in chicks and poults have
been reported to be directly proportional to the concentration of moniliformin in their diets
(Reams et al., 1997). Perhaps the toxic effect in the mink kits exposed to moniliformin
was associated with the ionophore nature of this mycotoxin which could alter biochemical

events detrimental to normal development. Nevertheless, the absence of lesions or

45

alterations at necropsy and on histological examination of tissues of the kit mink from the
high-dose group is unclear and was the subject of Part II of this thesis.

The results of this study indicated that dietary exposure of up to 17 mg/kg
moniliformin (provided by F. firjikuroi culture material M-1214), from prior to breeding
through weaning (about four mo) was not lethal to adult female mink, but caused
significant neonatal mortality and reduced kit body weights. The toxic effects observed in
the kits appeared to be due to placental transfer of moniliformin, or its metabolites, and
consumption Of moniliformin-treated feed by the kits.

Conclusions

The study clearly showed that kit mink are more sensitive to moniliformin than

adult females and that the use of moniliformin-contaminated ingredients in mink diets

could have adverse effects on reproduction and kit survival.

46

PART H

ACUTE AND SUBACUTE EFFECTS IN MINK FROM EXPOSURE
TO MONILIFORMIN EXTRACTED FROM FUSARIUM
FUJIKUROI CULTURE MATERIAL (NI-1214)

Abstract

This study was conducted to investigate the acute and subacute effects in mink
exposed to F usarium firjikuroi culture material containing 9,174 mg/kg of moniliformin.
In the acute study, nine-mo-Old pastel female mink were i.p. injected with either 0.5, 1.0,
1.5, 2.0, or 2.5 ml ofa solution containing 1.1 mg moniliformin/kg (1 ml = 1.1 mg
moniliformin/ml) to ascertain a LDso. The i.p. LDso was determined to be within the range
of 2.2-2.8 mg/kg b.w. of moniliformin for these mink. The cardiac tissue of mink acutely
exposed at all doses to moniliformin appeared normal after staining with hematoxylin and
eosin or trichrome. However, electron microscopy of right ventricular free wall of the
hearts from these mink showed alterations in the mitochondria, myofibers, myofilaments,
sarcoplasrnic reticula, nuclei, and in extracellular collagen deposition. In the subacute
study, eight previously exposed female mink from the acute trial were i.p. injected daily
with 0.3 ml/kg b.w. (0.33 mg/kg b.w.) of the moniliformin solution for three consecutive d
and observed for a total of six d. Statistically significant difi‘erences were observed in the
spun packed cell volumes, red blood cell counts, hematocrit values, hemoglobin
concentrations, serum chloride and albumin concentrations, and amylase activities between
the control and moniliformin-treated mink, but were not considered biologically significant

47

because the values were within the ranges reported for normal mink. The hearts of the
monilifonnin-exposed mink were the only grossly afi‘ected organ. They appeared
enlarged, rounded, and dilated on the right side. Sections of brain, heart, kidney, and liver
appeared normal after being stained with hematoxylin and eosin. Transmission electron
microscopy of the right ventricular free wall of the hearts showed alterations occurring in
the mitochondria, myofibers, myofilaments, sarcoplasmic reticula, nuclei, and in
extracellular collagen deposition in exposed mink. Subacutely exposed mink had greater
ultrastructural damage occurring in the myocardial tissue than acutely exposed mink. This
evidence clearly showed that moniliformin is toxic to mink at relatively low doses, and that
this mycotoxin specifically targets and damages the ultrastructure of the hearts of exposed
female mink.

Introduction

Moniliformin is a mycotoxin produced by several F usarium fungi, including F.
moniliforme, F. proliferatum, and F. firjr'kuroi, that under favorable conditions can infect
grains and vegetables. Concentrations of naturally occurring moniliformin have been
reported as high as 530 mg/kg in maize and 425 mg/kg w.w. in corn (Logrieco et al.,
1993; De Nijs et al., 1996; Lew et al., 1996).

This mycotoxin is acutely toxic to most animals at relatively low dietary
concentrations, and the young of a species are more sensitive than adults (Cole et al.,
1973; Kriek etal., 1977; Burrneister et al., 1979, Ueno, 1985; Abbas et al., 1990; Deyu et
al., 1993; Morgan et al., 1998). Studies have shown that moniliformin primarily targets

the hearts of avians and mammals. Animals exposed to increasing concentrations of

48

moniliformin generally experienced muscular weakness, respiratory stress, cyanosis, coma,
and death (Kriek et al., 1977; Thiel, 1978).

In a previous study conducted in our laboratory (Part I), a high incidence of
mortality was observed in young kit mink (Mustela vison) exposed to moniliformin during
gestation and lactation (via their dams receiving 17 mg/kg of moniliformin in the diet), and
consumption of their dam’s diet following weaning (Morgan et al., 1998). However,
necropsy of the eight-wk-old kits exposed to the moniliformin failed to reveal any gross or
histologic lesions that could account for the mortality.

The objectives of this study were to ascertain the target organ(s) of moniliformin
toxicosis in mink, characterize any changes in hematologic parameters and serum
chemistry values associated with exposure to moniliformin, describe any lesions and/or
alterations observed in tissues of moniliformin-treated mink, and determine a LDso for
moniliformin in mink.

Materials and Methods
Acute Study

Initially, attempts were made to determine an oral LD3o for moniliformin in nine-
mo-old or adult female mink by gavage. Although, mink were found to be very sensitive
to moniliformin, dosing by gavage caused vomiting, even at non-lethal concentrations.
Since an accurate measure of the dosages retained could not be determined with gavage,
i.p. injection was selected for administering moniliformin in firture tests.

Ten, nine-mo-old pastel female mink were injected i.p. with various concentrations
of moniliformin extracted from F. firjikuroi culture material containing 9,174 mg/kg

moniliformin. Since moniliformin is highly soluble in water, a solution containing 1.1 mg

49

moniliformin/ml was prepared daily by stirring 10 g culture material/80 ml distilled H20 in
a glass container for one hr. The solution was filtered through cheese cloth, and no. 1
filter paper via a vacuum pump, followed by filtration through a sterile Millex-GS 0.22 um
filter unit using a 22 G-l needle into an autoclaved glass vial fitted with a rubber septum.
Aqueous extractions of the same culture material yielded a mean recovery for
moniliformin of 93% (n=2) by HPLC analysis (G. Rottinghaus, University of Missouri,
personal communication). The acute toxicity of moniliformin was assessed using two,
nine-mo-old, pastel, female mink per dose group. The mink in each group were
administered (i.p.) either 0.6, 1.1, 1.7, 2.2, or 2.8 mg moniliformin/kg b.w. (0.5, 1.0, 1.5,
2.0, or 2.5 ml/kg b.w.), respectively, of the extract containing 1.1 mg moniliformin/ml.
Following dosing, the mink were placed into individual 51 cm L x 51cm W x 30 cm H
wire cages and observed for signs of toxicity for 48 hr. Mink that died (n=2) were
necropsied with the assistance of a veterinary pathologist and samples of brain, heart,
liver, and kidney were fixed in 10% formalin. The fixed tissues were processed according
to routine histological procedures, embedded in parafiin, sectioned (5-6 um), stained with
hematoxylin and eosin or trichrome (cardiac tissue only; Prophet et al., 1992) and
examined by a veterinary pathologist using a light microscope. Samples of the fixed right
ventricular free wall of the hearts were also processed for electron microscopy. They
were embedded in Epon-Araldite resin (Polysciences, Warrington, PA), sectioned (<1
um), and stained with uranyl acetate and lead citrate. The ultrathin sections were then
examined by a trained technician (Center for Electron Optics, Michigan State University,
East Lansing, MI) using a transmission electron microscope (Philips 301; Philips

Electronic Instruments, Mahwah, NJ).

50

Subacute Study

Ten, nine-mo-old pastel female mink were used to achieve the other objectives of
the study (possible target organs, any changes in hematologic parameters and serum
chemical values, and observable lesions and/or alterations in the tissues of moniliforrnin-
treated mink). The mink were divided into a treatment group consisting of the eight dosed
mink that survived the acute trial, two d prior, and a control group of two untreated mink.
Treatment mink were i.p. injected with 0.3 ml/kg b.w. Of moniliforrrrin solution (0.33 mg
moniliformin/kg b.w.; prepared as above), daily for three consecutive d and observed for
signs of toxicity for a total of six d. The mink were housed as previously described and
provided feed and water ad Iibitum throughout the study. On day six of the trial, the mink
were anesthetized (Ketamine HCl, 30 mg/mink) and five ml of blood were collected (via
the jugular vein) for hematologic and serum chemical determinations (analyses by the
Veterinary Clinical Pathology Laboratory, Michigan State University, East Lansing, MI).

Red and white blood cell counts, hemoglobin concentration, hematocrit, mean
corpuscular volume, mean corpuscular hemoglobin concentration, spun packed cell
volume, plasma total solids, and difi‘erential cell counts were determined using a
Technicon H1 system (Technicon Diagnostics Systems Division, Tarrytown, NY). The
serum chemistry analyses and calculations were performed with an Abbott Spectrum
Analyzer (Abbott Laboratories, Dallas, TX) and included calcium, chloride, iron,
magnesium, phosphorus, potassium, sodium, sodium/potassium ratio, carbon dioxide,
anion gap, total protein, albumin, globulin, albumin/ globulin ratio, creatinine, alanine
aminotransferase, alkaline phosphatase, amylase, aspartate aminotransferase, creatine

kinase, total bilirubin, cholesterol, glucose, blood urea nitrogen, and osmolality. After

51

blood collection, the mink were euthanized (CO2) and necropsied. Brain, liver, kidneys,
heart, spleen, and lungs were grossly examined, and a sample of each were placed in

bufi‘ered 10% formalin. The fixed tissues were processed and stained as described above.
Statistical Analysis

Sigma Plot Scientific Graphing System (1992) was used to statistically analyze the
data. The Student’ 5 t-test was used to determine difi‘erences in hematologic parameters
and serum chemical values between the control and moniliformin-treated groups.

Statements of statistical significance were based on p<0.05.

Results and Discussion
Acute Study

Intraperitoneal injection of 1.1 mg/kg b.w. or more of the moniliformin caused
vomiting and inactivity in the mink within 30 and 60 min after exposure, respectively. To
my knowledge, no previous studies have reported emesis occurring in animals exposed to
moniliformin, however, other undetected chemicals in the culture material could have
caused the Observed emesis in the mink. The Fusarium mycotoxins, deoxynivalenol and
T2 toxin, have also produced vomiting in several exposed animals, including mink.
However, the mechanism of action is currently unknown (Vesonder et al., 1976; Betina,
1989; Aulerich and Bursian, 1993). One mink exposed to 0.6 mg/kg b.w. of the
moniliformin extract was noticeably inactive within 60 rrrin. while the other mink in the
group appeared normal throughout the observation period. NO other signs of toxicity
were observed in the mink dosed with 0.6 mg/kg b.w. of moniliformin. After three hr, the
mink administered 1.1 mg/kg b.w., or less, of moniliformin were alert and active.

52

However, mink exposed to 1.7 mg/kg b.w., or more, Of the compound remained lethargic.
Both mink dosed with 2.8 mg/kg b.w. of moniliformin died within 24 hr. The acute LD3o
was determined to be within the range of 2.2-2.8 mg/kg b.w. of moniliformin for nine-mo-
Old female mink. Burrneister et al. (1979) reported LD30 values for i.p. injected adult male
and female mice to be about 10 times greater at 29.1 and 20.9 mg/kg b.w. of moniliformin,
respectively. Gavaged adult mice and rats have been reported to have similar or higher
LDso values (29.5 - 50 mg moniliformin/kg b.w.) than i.p. injected animals (Kriek et al.,
1977; Burrneister et al., 1979; Deyu er al., 1993). Nevertheless, these findings indicate
that moniliformin administered by either route is highly toxic to exposed mink and other
animals.

In the two expired mink, there were no histologic alterations observed in the
sections of brain, heart, liver, or kidney after staining the tissues with hematoxylin and
.esoin, and/or trichrome. Alterations in cardiac tissue observed with electron microscopy
are presented and discussed in the subacute section.

Subacute Study

The mink that survived the acute trial and were dosed (i.p.) with 0.33 mg
moniliformin/kg b.w. for three consecutive d did not vomit or show any signs of toxicity
during the six d observation period. Analysis of the serum chemistry values showed
statistically significant difi‘erences between the control and moniliformin-treated rrrink in
chloride (122.6 i 3.2 mmol/l, mean t SE, control group vs. 118.2 i 0.4 mmol/l, treated
group), albumin concentrations (3.05 :l: 0.05 g/dl, control group vs. 3.59 d: 0.08g/dl,
treated group), and amylase activities (106.5 3: 14.5 U/l, control group vs. 77.3 i 4.89 U/l,
treated group). Several hematologic values were significantly higher in the treatment

53

group than the control group. The mean (i SE). spun packed cell volume, red blood cell
count, hemoglobin concentration, and hematocrit value for the treatment group were
54.25 a 0.75 %, 8.98 :l: 0.23 x 10°/ul, 16.75 :1: 0.14 g/dl, and 57.77 i 0.70 %, respectively.
The greater hematologic values noted for the treated mink compared to the controls might
be due to hemoconcentration caused by dehydration, although water consumption by the
mink was not monitored. These values for the control animals were 41.50 :1: 1.50 %, 7.05
i 0.55 x 10°/ul, 12.45 i 0.25 g/dl, and 43.70 i 0.80 %, respectively. Since the control
group contained only two animals, these statistically significant blood values were also
compared to values for control animals from other mink studies conducted in our
laboratory and normal values reported for female mink in the literature (Fletch and
Karstad, 1972; Restum et al., 1995; Powell et al., 1996). It was concluded that these
statistical differences in serum and hematologic values may not be biologically significant,
as they were comparable to control values for mink of the same strain fed a similar diet in
previous studies and were within the ranges reported for normal mink. Kubena et al.
(1997) reported that male broiler chicks fed 100 mg moniliformin/kg diet for 21 d had
increased alanine arninotransferase and alkaline phosphatase activities, and serum calcium
and creatinine concentrations compared to the controls. Ledoux et al. (1995) showed that
male broiler chicks fed 200 mg moniliformin/kg diet for 21 d had decreases in mean
corpuscular volume and hemoglobin concentration compared to the controls.

No gross lesions or alterations were observed in the mink at necropsy in any organ
except for the hearts. The hearts appeared rounded, enlarged, and dilated (right sides,
only) in the treated mink. Sections of liver, brain, kidney, and heart tissue stained with
hematoxylin and eosin appeared normal. Trichrome staining showed fibrosis in the

54

connective tissue of the heart in both a treated (total subacute exposure = 2.8 mg/kg b.w.
of moniliformin) and control mink. An increase in fibrotic connective tissue can be
indicative of myocardial injury and chronic inflammation (Cheville, 1983; Haschek and
Rousseaux, 1991).

Electron rrricroscopy of the ventricular tissue from the moniliformin-treated mink
showed histologic alterations occurring in the myofibers, mitochondria, nuclei,
myofilaments, sarcoplasmic reticula, and in extracellular collagen deposition. Figure 6 is
an electron photomicrograph of a cardiomyocyte fiom a control animal. Numerous
mitochondria, circular in shape, are distributed among the closely spaced myofibers of the
myocyte. The sarcomeres have Z- and M-lines that appear as distinct dark and light lines,
respectively, with defined edges. Figure 7 is an electron photomicrograph showing a
cardiomyocyte of a mink acutely exposed to moniliformin. Extensive vacuolation and
edema of the myocyte are evident. The mitochondria are degenerated, swollen, and
irregular in appearance. The Z- and M-lines are thickened and irregular in width. These
myocardial alterations are characteristic of cellular injury (Cheville, 1983; Haschek and
Rousseaux, 1991). Deyu et a1. (1993) showed that adult, male mice acutely exposed to
29.5 mg moniliformin/kg b.w. had similar alterations occurring in the mitochondria,
myofibers, and nuclei in the hearts. In a subacutely exposed mink (total exposure = 2.8
mg moniliformin/kg b.w.), these lesions were even more severe (Figure 8). The
myofibers and mitochondria exhibit marked degrees of degeneration, disintegration, and
necrosis. Massive vacuolation and edema of the myocyte are evident. Mitochondrial
volume is greatly increased, and the Z-lines are thickened and irregular in width.

Increased numbers of nuclei are present within the myocyte which is characteristic of

55

 

Figure 6. Electron Photomicrograph of a Section of Heart from a Control Mink Showing
Normal Ultrastructure. Mitochondria (A), Myofibers (B), and Transverse Striations are
Normal in Appearance (8040x).

56

 

Figure 7. Electron Photomicrograph of a Section of Heart from an Acutely Exposed Mink
Showing Histological Lesions in the Mitochondria (A) and Myofibers (B). Massive
Vacuolation and Edema are Present in the Tissue (63 50x).

57

 

Figure 8. Electron Photomicrograph of a Section of Heart fi'om a Subacutely Exposed
Mink. Note the Presence of Massive Vacuolation and Edema, an Increase in the Volume
of Mitochondria, Several Degenerating Nuclei (A), Extracellular Collagen Bundles (B),
and Disintegrating Myofibers (Lower Right) with Cellular Debris (3 525x).

58

inflammation and regeneration (Cheville, 1983; Haschek and Rousseaux, 1991). The
presence of extracellular bundles of collagen can be indicative of chronic inflammation.
Engelhardt et al. (1989) showed that chicks, ducklings, and turkey poults exposed to
12.5%, 25 %, or 50% of a corn culture material that contained 1.15 g moniliformin/kg
culture material (fermented feed) diet for 14 d had similar degenerative and necrotic
changes occurring in the myofibers and nuclei in the hearts. Ledoux et al. (1995) reported
that male broiler chicks fed 50-3 00 mg moniliformin/kg diet for 21 d had myocardial
lesions and an increase in nuclei that were abnormal in appearance. Reams et al. (1997)
showed that turkey poults and broiler chicks fed F. firjilmroi culture material containing
160-330 mg/kg of moniliformin for up to 28 d had an increase in the size and number of
mitochondria and abnormal nuclei in heart tissue.
Conclusions

This study showed that adult female mink were approximately 10 times more
sensitive than laboratory rats and mice to acute i.p. exposure to moniliformin. The LD3o
was determined to be within the range of 2.2-2.8 mg moniliformin/kg b.w. for nine-mo-old
female mink. The mycotoxin was also shown to specifically target and damages the

ultrastructure of the hearts of acutely and subacutely exposed mink.

59

PART III

EFFECTS OF DIETARY FUMONISIN S ON SPHINGOLIPID
CONCENTRATIONS IN THE URINE AND HAIR OF MINK

Abstract

This study was conducted to investigate the effects of dietary Fusarium
moniliforme culture material (M-1325) containing known concentrations of fumonisins
B1, B2, and B3 on sphingolipids in urine and hair of mink (Mustela vison) for use as
potential, non-invasive biomarkers of exposure to fumonisins in this species. Feeding
mink diets containing 86, 22, and 7 mg/kg or 200, 42, and 12 mg/kg offumonisins B1, B2,
and B3, respectively, for two, four, or seven (1 yielded marked increases in urinary free
Sphinganine (Sa) and fi'ee sphingosine (So) concentrations, and fi'ee Sa/free So ratios (2 to
11-fold), compared to controls. Free Sa and fiee So concentrations and Sa/So ratios in
hair samples from mink fed the control or firmonisin-treated diets for 100 d were similar
and were not apparently altered by exposure to these mycotoxins. These results suggest
that Sa/So ratios in urine, but not in hair of mink can serve as an early indicator of

exposure to fumonisins in this species.

60

Introduction

Fumonisins are mycotoxins produced by several F usarr'um fungi, including
F usarr‘um moniliforme. At least seven fumonisins have been identified (Nelson et al.,
1993) and Of these, fumonisins B1, B2 and B3 are the most prevalent and are commonly
found on corn, sorghum, and other grains throughout the world (Thiel et al., 1982;
Marasas et al., 1984). These mycotoxins are toxic to animals and affect a variety of
organs (Merrill et al., 1993; Riley et al., 1994b; Restum er al., 1995; Voss et al., 1995).
Fumonisin B1 and/or F. moniliforme have been shown to cause porcine pulmonary edema
(Osweiler et al., 1992), equine leukoencephalomalacia (Ross et al., 1991), nephrotoxicity
and hepatotoxicity in rats (Voss et al., 1993; Riley et al., 1994a) and have been implicated
in cases of human esophageal cancer (Chu and Li, 1994). Studies with mink have shown
that F. moniliforme culture material containing firmonisins B1, B2 and B3 adversely afi'ects
liver and kidney function, hematologic and serum chemistry values, whelping success, kit
body weights at birth, and biosynthesis of sphingolipids (Restum et al., 1995; Powell et
aL,1996)

Fumonisins are structurally similar to sphingoid bases, such as sphingosine (So)
and sphinganine (Sa), and several fumonsins (B1, B2, and hydrolyzed B1) have been shown
to block the de novo synthesis of sphingolipids through the inhibition of sphingosine- and
sphinganine N-acyltransferase. This inhibition can cause an accumulation of free Sa and
fiee So (Wang et al., 1991, 1992; Riley et al., 1994b,c). Studies by Riley et al. (1994b,c)
have shown that firmonisins significantly increase the Sa/So ratio in the liver, kidneys,
lungs, sera and urine of rats, ponies, and pigs. A previous study conducted with mink

showed that firmonisins significantly increased hepatic and renal fi'ee Sa concentrations as

61

well as the Sa/So ratios in these tissues (Restum et al., 1995). The results of these studies
suggested that, in mink, as in rats and pigs, urine may serve as a non-invasive, early
biomarker of exposure to firmonisins.

Hair is also a tissue that contains sphingolipids and can be easily obtained from
many animals. However, sphingolipid disruption within hair due to exposure to
fumonisins has not been examined. Therefore, the Objectives of this preliminary study
were to determine free Sa and free So concentrations in urine and hair of mink fed F.
moniliforme culture material containing firmonisins and to assess the use of Sa/So ratios
for urine and hair as potential, non-invasive biomarkers for firmonisin exposure in this
species.

Materials and Methods

This research was part of a previous investigation to determine the efi‘ects of
subacute and chronic exposure to firmonisins in mink. The following is a brief description
of the materials and methods that pertain to this phase of the study. A more detailed
description of the study is presented by Powell et a1. (1996).

Thirty-six, adult, pastel female mink were housed individually in wire mesh cages
in an animal room at the Michigan State University Experimental Fur Farm on February
13, 1995. The rrrink were acclimated to the facilities for 7 (1. They were then randomly
allocated into three groups and provided a basal mink diet supplemented with either 0
(control), 1.45 (low-dose, 115 mg/kg of firmonisins B r, B2, and B3) or 2.90% (high-dose,
254 mg/kg of firmonisins B1, B2, and B3) F. moniliforme culture material (M-1325,
Fusarium Research Center, Pennsylvania State University, University Park, PA) known to

contain 10,197 mg/kg of fumonisins B1, B2 and B3.

62

The mink diets were prepared by blending an appropriate quantity of the culture
material with ground corn to yield a 10 kg premix which was then mixed with the other
dietary ingredients. Ten kg of ground corn without culture material was added to the
control diet. The mink diet consisted of 21 .2% commercial mink cereal, 21.2% poultry
by-products, 17.0% ocean fish scrap, 6.4% beef liver, 5.9% eggs, 2.9% ground corn,
25.4% water, 0.02 mg d-biotin/kg, 4.7 IU vitamin E/kg, 625 USP vitamin A/kg, and 62.5
IU vitamin D/kg, as fed. Proximate analysis Of the diet (“as-f ” basis) yielded 65.8%
moisture, 5.5% fat, 13.1% crude protein, 0.9% crude fiber, and 3.5% ash (Litchfield
Analytical Service, Litchfield, MI). Samples of the culture material and diets were
analyzed by HPLC/fluorescence for fumonisins B1, B2 and B3 at the National Veterinary
Service Laboratory, Ames, IA The mink were fed the control and fumonisin-treated diets
ad Iibitum for 100 d (February 20 to May 31).

Urine samples were collected fiom nine mink in the control group and from two to
five treated mink on days two, four, and seven of fumonisin exposure. In an attempt to
obtain fresh, uncontaminated urine samples, the mink were caught and held over a beaker
for a few sec. The process of being restrained in mid-air frequently induced urination.

Hair samples, which consisted of straight guard hairs, intermediate guard hairs, and
underfirr, were collected, with electric clippers, from the dorsal- surface of the back of 12
control and 10 high-dose mink at the end of the feeding trial (May 31). The hair and urine
samples were stored frozen (-6.7°C) until analyzed.

Sphingoid bases were extracted and mass measurements were conducted as
described by Merrill et al. (1988). Briefly, C20 sphinganine was added as an internal

standard, and the long-chain bases were extracted with chloroform and methanol and then

63

treated with base to remove interfering glycerolipids. After preparation of the o-
phthaldehyde derivatives, the long-chain bases were separated by reverse-phase HPLC
(Shimadzu Scientific Instruments, Inc., Wood Dale, 1]..) using a C13 column and a mobile
phase of methanol, 5 mM potassium phosphate, pH 7.0 (91 :9 v/v).

The data were statistically analyzed using the Sigma Stat Statistical Analysis
System (1992). Comparisons were made by one-way analysis of variance. Where the F
value was significant at p<0.05, treatment means were tested by Dunnett’s test.

Results and Discussion

The F. monilifonne culture material used in the study contained 7300, 2200, and
697 mg/kg firmonisins Bl, B2 and B3, respectively. Analysis of the mink diets yielded
fumonisin Br, B2 and B3 concentrations of <05 mg/kg (detection limit) for the control
diet, 86, 22, and 7 mg/kg for the low-dose diet, and 200, 42, and 12 mg/kg for the high-
dose diet, respectively. No clinical signs of toxicity were observed in the mink during the
trial.

The mean free Sa and free So concentrations and Sa/So ratios in urine fiom mink
fed the control diet or diets that contained the culture material are shown in Table 8. The
urinary free Sa and free So concentrations Of individual mink within groups were variable
and the only statistically significant (p<0.05) difference in the Sa/So ratios between the
controls and firmonisin-treated mink was in the low-dose mink exposed for seven d.
However, since the mean Sa/So ratios for the urine of mink fed the low-dose and high-
dose diets for 2, 4 or 7 d were all 2 to 11-fold greater than the ratio for the control mink,
they are probably biologically significant. Riley et al. (1994b) reported significantly

elevated Sa/So ratios in urine of rats fed 50 or 150 mg/kg dietary firmonisin B1 for 4 wk.

64

 

Table 8. Mean Sphinganine (Sa) and Sphingosine (So) Concentrations and Sa/So
Ratios for Urine of Female Mink Fed Various Concentrations of
Fumonisins for 2, 4, or 7 D.

 

 

Dietary treatment Treatment Sa 80

(mg/kg filmonisins) period (d) No. (pmoI/ml) (pmol/ml) Sa/So

0 (control) - 9 57.0:2110‘ 250.6:1088' 0.17:0.22'

115 (low-dose) 2 2 67.8:4476 2059:2308 0.82:0.47
4 3 149.5:365 5374:1885 0.36:0.38
7 3 1254.8:365 4104:1885 1.86:0.38"

254 (high-dose) 2 4 216.3:316 5353:1632 0.44:0.33
4 2 1222.4:447 8368:2308 1.65:0.47
7 5 319.6:2831 5019:1460 0.89:0.30

 

 

 

 

 

 

 

' Standard error of the mean

" Significantly different from control (p<0.05).

 

65

 

These Sa/So ratios ranged from about 2.5 (females) to 8 (males) for rats fed 50 mg/kg
firmonisin B1 and about 6 (males) to >10 (females) for those fed 150 mg/kg firmonisin Br,
whereas control Sa/SO ratios were <1.

The notably higher Sa/SO ratios observed by d two in both the low- and high-dose
groups compared to the control shows the rapid effect of fumonisins on urinary
sphingolipids in mink. Wang et al. (1992) reported increased free Sa/SO ratios in pony
sera within one d following consumption of fumonisins. In a previous trial with mink fed
diets that contained 0 (control) or 118 mg/kg fumonisins B1 + B2 + B3 (provided by the
same F. moniliforme culture material as in this study) for 87 (1, mean kidney Sa/So ratios
of 0081028 (x _+_ SE, control) and 3.75:0.28 (fumonisin-treated) were reported (Restum
et al., 1995).

The hair samples collected for sphingolipid analysis were fiom mink fed the control
and high-dose diets fi'om February 20 through May 31. This period of exposure includes
the early stages of the spring molt, which in adult female mink begins around April 10 and
is completed by mid July (Bassett and Llewellyn, 1949; Allain and Martinet, 1985). Thus,
some new growth hair, produced during the treatment period, as well as some hair fi'om
the previous (fall) molt were present in the samples. The mean free Sa and free SO
concentrations and Sa/So ratios for the hair samples of the control and treated mink were
similar (79 pmol/g, 147 pmng, and 0.54, for the control and 74 pmol/g, 135 pmol/g, and
0.55 for the high-dose group, respectively) and appeared to be unaltered by exposure to
the culture material containing firmonisins.

Riley et al. (1994c) suggested that a serum Sa/SO ratio greater than twice the

average ratio for control animals should be considered suspect and that three times the

66

average as highly suspect of firmonisin toxicosis. Ifthese suggested guidelines for sera
are applied to the Sa/So ratios for urine and hair of mink obtained in this study, the results
indicate that Sa/So ratios in urine, but not in hair can serve as an early indicator of possible
exposure to fumonisins in mink. However, confirmation of firmonisin toxicosis should be
based on analysis for the presence of fumonisins in the diet.
Conclusions

Urine, but not hair, can be used as a non-invasive biomarker of fumonisin exposure
in adult female mink and as an early indicator of fumonisin toxicity than the standard
biomarker, sera, in mammals.

FUTURE RESEARCH

Moniliformin

Acute and subacute studies have been conducted on the toxic effects of
moniliformin in several experimental animals (Kriek et al., 1977; Allen et al., 1981;
Engelhardt et al., 1989; Abbas et al., 1990; Chen et al., 1990; Deyu et al., 1993; Javed et
al., 1993; Ledoux et al., 1995). These studies have reported that moniliformin was highly
toxic to these animals at relatively low concentrations. More studies are needed on. other
possible adverse (e. g. immunological, reproductive, chronic, carcinogenic) effects of this
mycotoxin in exposed animals. Electron microscopic evaluation has also indicated that
moniliformin targets and damages the ultrastructure of the hearts and livers of exposed
birds and mammals (Deyu et al., 1993; Ledoux et al., 1995; Reams et al., 1997). More
research is necessary to determine if monilifomrin targets and damages the organ(s) of

other exposed animals.

67

Since this mycotoxin is highly toxic to experimental animals and has been detected
at high levels in grains, the United States should screen for the presence of moniliformin
in animal feeds and human foods. These measures would help better protect animals and
humans from possible toxic exposure levels of moniliformin in their diets.

Fumonisins

The B series firmonisins have been reported to cause nephrotoxicity (Riley et al.,
1994b), hepatotoxicity (V oss er al., 1993), ELEM (Ross et al., 1991), and PPE (Osweiler
et al., 1992) in exposed animals. The mechanism(s) of action leading to these toxic effects
is currently unknown. More research is greatly needed to determine how fumonisins
produce these toxic efl‘ects and diseases in animals.

Urine is considered as an early biomarker of firmonisin exposure in mammals (rats
and mink) (Riley et al., 1994b). This fluid could possibly be used as a non-invasive
biomarker of filmonisin toxicosis in other animal species.

Presently, the United States has implemented advisory levels for fumonisins B1 and
B2 in some animal feeds, but not human foods (Jackson et al., 1996a). More studies are
required to determine if these advisory levels are adequately protecting exposed animals

and humans.

68

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