Pergamon Food and Chemical Toxicology 35 (1997) 991-999

TJ~

The use of the Chicken Embryo Screening

Test and Brine Shrimp (Artemia salina)
Bioassays to Assess the Toxicity of
Fumonisin B1 Mycotoxin
J. J. H L Y W K A I*, M. M. B E C K 2 and L. B. B U L L E R M A N I
IDepartment of Food Science and Technology, University of Nebraska, Lincoln, NE 68583 and
2Department of Animal Science, University of Nebraska, Lincoln, NE 68583, USA

(Accepted 1 June 1997)

Ala~tract~hicken embryos and brine shrimp naulpii were utilized in short-term toxicity bioassays to
assess their :;ensitivity to the mycotoxin fumonisin Bl (FBI). Fertile chicken eggs (Cobb x) were dosed
with FBt or. day 2 of incubation by the injection of 100 #1 of aqueous solution into the air space of
each egg. Eggs were incubated with mechanical rotation until hatch, at which time mortality was
assessed. Probit transformation of the mortality data produced a linear line of best fit (P < 0.05), from
which an LI)5o of 52 #g FBl/egg, equivalent to a concentration of 1.3 gM hatched in artificial seawater
and exposed to FB~ in an optimized 96-well plate assay with a 48 hr mortality endpoint. Probit trans-
formation of the mortality data resulted in an LCso of 1.7 #M FBt, or 1.2 gg FBt/ml. Thus, at the cellu-
lar level, both bioassays appeared sensitive to FBg however, from the standpoint of use as a screening
assay, the chicken embryo bioassay is limited by the relatively high dose of FBI required per egg. It is
anticipated lhat the design and simplicity of the brine shrimp bioassay will accommodate screening for
FBI toxicity in contaminated samples. © 1997 Elsevier Science Ltd. All rights reserved

Abbreviations: DAS = diacetoxyscirpenol; DON = deoxynivalenol; FB~ = fumonisin B~; LC5o= median
lethal concentration; LD~ = median lethal dose.

Keywords: b:oassay; brine shrimp; chicken embryo; fumonisin Bx.

INTRODUCTION 1991). Epidemiologically, higher concentrations of

Fumonisin Bt (FFh) is a mycotoxin produced pri- fumonisins have been found in areas of Africa,
China and Italy, where human oesophageal cancer
marily by Fusariura moniliforme and F. proliferatum
rates are elevated and corn is the dietary staple
as the predominant fungal contaminants of corn
(Cheng et al., 1985; Franceschi et al., 1990; Rheeder
(Bacon and Nelson, 1994). As these moulds are ubi-
et al., 1992; Sydenham et al., 1990).
quitous in temperate and subtropical regions, the
Fumonisins have been detected in virtually all
extent of FBl-contaminated corn is widespread
types of processed corn products and on a world-
(Bullerman, 1996) O f a group of !1 structurally
wide basis (Doko and Visconti, 1994; Pittet et al.,
similar toxins ( B e ~ i d e n h o u t et al., 1988; Branham
1992; Stack and Eppley, 1992; Sydenham et al.,
and Plattner, 1993; Cawood et al., 1991; Seo et al.,
1991). Overall, the level of FB1 found in corn-based
1996), collectively referred to as fumonisins, FBI
foods appears to be dependent on the environmen-
occurs naturally in the greatest abundance and is,
tal conditions of the harvest year and the extent of
quantitatively, considered the most toxic (Norred
processing to which the corn has been subjected
and Voss, 1994). FBI is the causative agent of sev-
(Bullerman, 1996). Typically, minimally processed
eral acute diseases in livestock, including equine leu-
products such as corn grits and cornmeal have
coencephalomalacia (Sydenham et al., 1992; Wilson
higher detectable levels of fumonisins, than do
et al., 1992) and porcine pulmonary oedema
more extensively processed products such as canned
(Colvin and Harrison, 1992). FBt has been demon-
corn, corn chips, corn flakes and tortillas. The levels
strated experimentally to be hepatotoxic and hepa-
of fumonisin contamination found in fresh sweet-
tocarcinogenic in rats (Gelderblom et al., 1988 and
corn or canned corn tend to be lower than those
reported for field corn (Doko and Visconti, 1994;
*Author for correspondence. Trucksess et al., 1995; Bullerman, 1996).

0278-6915/97/$19.00 + 0.00 © 1997 Elsevier Science Ltd. All rights reserved. Printed in Great Britain
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992 J. J. Hlywka et al.
Although some decomposition of FBI can be
induced by various chemical and physical treat-
ments (Dupuy et al., 1993; Jackson et al., 1996;
Sydenham et al., 1994), a correlated decrease in as-
sociated toxicity cannot be assumed. Hendrich et al.
(1993) observed that apparent decreases in the
fumonisin content of corn following nixtamilization
processing may be offset by the formation of
equally toxic hydrolyzed decomposition products.
The thermal processing of aqueous solutions of FB1
at various pHs resulted in identified decomposition
products of hydrolyzed and partially hydrolyzed
FBI (Jackson et al., 1996). The analytical determi-
nation of FBI by liquid chromatography typically
involves derivatization of its primary amine group
(Rice et al., 1995). Modification of FBI, such that
this functional group is removed or effectively
blocked (Murphy et al., 1996), may result in novel
FB) decomposition products that are not detected
by standard derivatization procedures.
In order to attest that toxicity is lost on modifi-
cation of the parent toxin through processing, bio-
logical activity must be evaluated. Published
bioassays for evaluating the toxicity of FBt include
the use of mammalian and plant cells (Shier et al.,
1991; Wang et al., 1991; Abbas et al., 1993; Hanelt
et al., 1994), as well as chicken embryos (Javed
et al., 1993; Bacon et al., 1995), although the sensi-
tivity of each assay and practicality of use varies
greatly. In an attempt to develop an appropriate
biological screening assay for FBl-contaminated
samples, the responses of two short-term bioassay
systems to pure FBI toxin were examined. The
objective of this study was to assay the acute tox-
icity of FB~ using the chicken embryo screening test
and brine shrimp (Artemia salina) bioassays.
MATERIALS AND METHODS
Chicken embryo screening test bioassay
The chicken embryo screening test bioassay pro-
cedure followed the method recommended by
Prelusky et al. (1987). Fertile chicken eggs (Cobb x,
Tyson Foods, Inc., Springdale, AR) were candled
(Ultraviolet Products, Inc., San Gabriel, CA) prior
to incubation to remove those that were cracked or
otherwise defective. The eggs were incubated at
37.5°C and 55% relative humidity (Natureform
inc., Jacksonville, FL) for approximately 19days
with mechanical turning every 12 hr. The eggs were
then transferred to hatching trays at 37.5°C and
85% relative humidity until hatch at approximately
21 days. On day 2 of incubation, eggs were dosed
with sterile aqueous solutions of FBI prepared from
a stock solution of 10 mg/ml in H20. The FBI used
in this study was of 95-98% purity and was cour-
tesy of Dr Robert Eppley (US FDA, Washington,
DC). Prior to treatment with FB~, the eggs were
candled to select only those which appeared fertile.
To dose the eggs, a small hole (approximately
1 mm diameter) was drilled over the air space of
each egg, using a Dremel ~ tool (Dremel
Manufacturing Co., Racine, WI, USA) equipped
with a l-ram dental drill bit. A total volume of
100 #1 FBI solution was injected into the air space
over the developing chicken embryo using a
Hamilton '~ syringe (Hamilton Co., Reno, NV). The
hole was covered with transparent tape and the egg
returned to the incubator. Negative and positive
controls of 100/~1 sterile water and 100/~1, 0.05/zg
deoxynivalenol//~l (DON) (Sigma Chemical Co., St
Louis, MO), respectively, were also included.
Natural mortality was observed by the incubation
of untreated eggs. One replication of this exper-
iment consisted of 20 eggs per treatment or control,
with a total of four replications performed. Eggs
were candled at 3-4-day intervals to record chick
development, or lack thereof. The chicks were
allowed to develop until hatch (approximately
21 days), at which time mortality was assessed and
any gross abnormalities according to Hamilton
(1952) were noted.
Brine shrimp ( Artemia salina) bioassay
The brine shrimp (A. salina) bioassay procedure
followed the microwell plate method proposed by
Solis et al. (1993) with some modifications. Briefly,
microlitre volumes of FB~ solutions prepared in
acetonitrile-water (70:30, v/v) were transferred to
the wells of a 96-well plate (Costar, Cambridge,
MA) and allowed to air-dry. The amount of FBI
applied to each well was calculated based on a final
well volume of 200 #1. Negative and positive con-
trols of acetonitrile-water (70:30, v/v) and the tri-
chothecene mycotoxin, diacetoxyscirpenol (DAS)
(Applied Science Laboratories, State College, PA),
respectively, were also included. Brine shrimp eggs
(100mg, Novalek, Inc., Hayward, CA) were
hydrated and hatched in 100 ml artificial seawater
(6.5 g Instant Ocean, Mentor, OH + 6 mg yeast/
liter deionized H20) under fluorescent illumination
at 27~C for 24hr in an orbital incubator
(Gallenkamp, Leicestershire, UK) at 140 rpm. After
24 hr, the shrimps were transferred to fresh artificial
seawater using a 10-ml wide mouth pipette and a
light source to attract the nauplii to one side of the
hatching flask. The volume was adjusted to attain a
density of approximately 200 shrimps/ml, and
100/~1 of the hatching solution were transferred to a
FB~-treated microwell plate containing 100/al artifi-
cial seawater per well. Thus, each well contained a
final volume of 200/~1 and 10-20 brine shrimps.
The plates were wrapped in Parafilm* (American
National Can, Greenwich, CT) to prevent moisture
loss and incubated for up to 48 hr at 27°C.
Following incubation, plates were examined under
a binocular microscope (Nikon Optiphot, Nikon,
JA) at x40 magnification and the number of dead
shrimps recorded. The mortality endpoint of this
bioassay was defined as the absence of controlled
forward motion during 10 sec of observation
(Barahona-Gomariz et al., 1994; Eppley, 1974). The
total number of shrimps per well was enumerated
 Bioassays for the toxicity assessment of fumonisin Bt 993

following the addition of 100/zi acetonitrile to each
well and the percent mortality calculated. A total of
six replicates per dose were performed on each
plate.
Data analyses
The sensitivity of each bioassay procedure to FBI
was determined through the generation of dose-
response curves aad the calculation of LCs0 values
by probit analysis (Hubert, 1984). Percentage mortal-
ities were adjusted relative to the natural or control
percentage mortalities, following Abbott's formula,
p = pr--C/l - C, where Pi denotes the observed non-
zero dose mortality rate and C represents the natu-
ral mortality rate of the control (Hubert, 1984).
RESULTS
Chicken embryo screening test bioassay
Preliminary studies using a dose range of 0.5 to
250/ag FBl/egg produced a sigmoidal dose-response
curve with no mortality at 0.5/~g/egg and 100%
mortality at 175 or greater /zg/egg. To determine
the LDso, five (lose levels ranging from 25 to
125/~g FBt/egg were selected for subsequent repli-
cated dose-resporse experiments. The mean percen-
tage mortalities far FBrtreated eggs, adjusted for
the percent mortality of H20-treated control eggs,
is shown in Fig. I. The mean mortality rate in eggs
dosed with 100~! sterile H20 was 22.2 + 20.7%.
This was comparable with the results of Prelusky
et al. (1987) and was consistent with the observed
natural mortality rate of untreated eggs, which
averaged 19.2 _+ 8.3%. These results indicate that
the carrier solvent of H20 and method of toxin
delivery by injection over the air space did not
affect chick mortality. The adjusted mortality rate
for those embryos dosed with 5 #g of the trichothe-
cene mycotoxin DON as a positive control was
26.7 _+ 14.0%. Prelusky et al. (1987) reported LDso
values of 5.2 to 17.8 #g DON/egg which were
dependent upon the day of incubation when trea-
ted.
Probit transformation of the mortality data for
FBl-treated eggs produced a linear line of best fit
(P < 0.05), from which an LDs0 of 52/zg FBl/egg
was calculated (Fig. 2). Assuming an average egg
weight of 60 g, with 12% egg weight as shell and an
egg density of 0.97 g/ml (data not shown); this
LDs0 equates to an FBt concentration of 1.3 #M.
In general, FB~ was lethal to chicken embryos
very early in their development (e.g. day 3-4 of in-
cubation), regardless of the dose level. At least 80%
of all embryos that died in FBl-treated eggs did so
within 1-2 days of treatment. There was no signifi-
cant occurrence of any particular type of malfor-
mation in embryos that died at later stages of
development. Malformations that were observed
within all treated and untreated eggs included
splayed feet, deformed legs, crossed beaks and/or
enlargement of the posterior of the skull. The over-
all basal malformation rates of the dead in shell
embryos of untreated eggs and those dosed with
100#l H20 were 13.3 and 18.8%, respectively.
Similar malformation rates of 18.4 and 16.1% were
observed among the mortalities of eggs dosed with

1(;'0

80

60
o
E
40

20

I ) I t

25 50 75 1 O0 125

FB 1 (p.g/egg)
Fig. 1. Mo=lality response of chicken embryos dosed with 25-125/tg FB1/egg, delivered in 100/Jl sterile
H20 on day 2 of incubation. Data are the mean of four replicates corrected for control mortality in
H20-treated eggs; 4- standard deviations are indicated by capped vertical bars when larger than symbols
at each point.
994 J.J. Hlywka et al.

.o
P
Q.

0.g5

i i ! i i

25 50 75 100 125
FB 1 (l~g/egg)
Fig. 2. Probit transformation of mortality data from chicken embryos dosed with 25-125/~g FB~/egg,
estimating an LDs0 of 52/~g FBgegg. A chi-square goodness-of-fit test was significant at P < 0.05; the
data represents four replicates of 20 eggs per dose.

25 and 50 #g FBt, respectively. Malformation rates of lethal toxicity at a higher dose. In those chicks
of less than 5% observed among the dead embryos of that did hatch from FBrtreated eggs, there were no
eggs treated with 75 ttg FBI or more would suggest indications of abnormalities or compromised health.
the likelihood of an earlier or more immediate onset Histopathological effects were not determined.

80

60

E
40

20

0 . . . . . . . . i . . . . . . . i . . . . . . . .

1 10 1~ 1~0

FB 1 (I~M)
Fig. 3. Mortality response of 24-hr-old brine shrimps (Artemia salina) exposed to 1.4-346 #M (1-
250/~g/ml) FB~ for periods of 24, 36 and 48 hr, respectively. Data are the mean of six replicates; + stan-
dard deviations are indicated by capped vertical bars when larger than symbols at each point.
Mortality rates in untreated control wells (0/~M FB~) were 9.2 + 10.2, 9.5 :t: 10.2 and 10.8 + 10.7% for
exposure periods of 24, 36 and 48 hr, respectively.
 Bioassays for the toxicity assessment of fumonisin B~ 995

10(]

80

,_z, 60
to

E
4Cl

20

0 . . . . . . i . . . . . .

0.1 1 10

FB 1 (pM)
Fig. 4. Mortality response of 24-hr-old brine shrimps (Artemia salina) following 48 hr exposure to 0.3-
7/aM (0.2-5 .,ag/ml) FBI. Data points represent the means and standard errors of three experiments each
with six replicates. Percentage mortalities were adjusted to account for the background mortality of
9.5 + 2.2% for brine shrimps in untreated control wells.

Brine s h r i m p ( A r t e m i a salina) bioassay
Initial dose-response experiments indicated that
brine shrimps' sensitivity to FBI was dependent on
the dose, the age or the growth stage of the shrimps
at the time of exposure, as well as the duration of
exposure. It was observed that brine shrimps
exposed to a fixed concentration of FBI at 24 hr
old were much mere sensitive to the toxic effects of
FBI than were brine shrimps exposed at 48 hr old.
For example, 48-hr-old old brine shrimps were re-
sistant to levels of 300/~g FBl/ml for a period of
24 hr, whereas the same dose and duration of ex-
posure caused approximately 80% mortality in
brine shrimps exposed at 24 hr old. For the pur-
poses of this study, the age of the brine shrimps
was reported relative to initial hydration of the
eggs. Further experiments indicated that the exten-
sion of the duration of exposure of brine shrimps to
FBt, from 24 to 36 to 48hr, resulted in an
increased sensitivity or mortality response (Fig. 3).
Brine shrimps were not sensitive to residues of the
acetonitrile-water carrier solvent and mortality
rates within solvent-only treated control wells were
less than 10%. The use of 2/~g DAS/ml as a posi-
tive control resulted in mortality rates of 95-100%
following 24-48 hr of exposure. Harwig and Scott
(1971) and Muller and Lepom (1985) reported 50%
mortality in brine shrimps exposed to 0.47 and
0.55/ag DAS/ml, respectively.
To optimize sensitivity and to better accommo-
date FBI levels typically encountered in contami-
nated corn samples, the 48-hr bioassay using 24-hr-
old brine shrimps was selected for use as the routine
procedure. For subsequent bioassay tests, the dose
range was extended to lower concentrations of F B b
ranging from approximately 0.3 to 7/aM, or 0.2 to
5#g/ml. The cumulative dose-response curve of
three experiments is shown in Fig. 4. Independent
probit transformations of each curve resulted in sig-
nificant linear relationships (P < 0.05), from which
an average LCs0 value of 1.7 _+0.1 /tM FBI was cal-
culated. This LC50 translates into a dose level of
1.2 +0.1 #g FBl/ml, or approximately 0.25/~g FBI/
microwell.
H P L C analyses of the artificial seawater medium
within the treated wells of the microwell plates, fol-
lowing the methods of Rice et al. (1995), confirmed

Table I. The comparative toxicity of fumonisin B~ mycotoxinin a variety of short-term bioassay systems
Assay Endpoint (FBI) Reference
Chicken embryo 21 days 1.3 ~M=
Brine shrimp 48 hr 1.7/aM=
M'FI': turkey lymphocyte 48 hr 7 pMb Dombrink-Kurtzman
et al. (1994)
72 hr 2 ~UM
b
Madin-Darby canine kidney cell line 4 days 3.6-54 ~UMt' Shier et aL (1991); Abbas et al. (1993)
H4TG rat hepatoma cell line 4 days 5 . 6 - 1 8 ~M b Shier et aL (1991); Abbas et al. (1993)
"Endpoint measure refers to concentration which is lethal to 50oVoof population.
bEndpoint measure refers to concentration which causes a 50% inhibition of cell proliferation.
996 J.J. Hlywka et al.
that the air-dried FBI residues were dissolved at the
desired concentrations.
DISCUSSION
The chicken embryo and brine shrimp bioassays
have been used extensively to evaluate the toxicity
of mycotoxins (Panigrahi, 1993). Although often ill-
defined in terms of mechanisms of toxicity, these
bioassays have none the less allowed for the rapid
and economical screening of a variety of samples
for biological activity. The results of this study indi-
cate that the chicken embryo and brine shrimp
bioassays are sensitive tools for measuring the tox-
icity of FBI.
The toxicity of FBI to chicken embryos was first
reported by Javed et al. (1993). These authors con-
ducted an extensive histopathological examination
of the affected embryos, although their results do
differ somewhat from those reported here and else-
where (Bacon et al., 1995). Javed et al. (1993) exam-
ined three doses of FBI, ranging from 32 to
3204/~g/egg, and reported mortality results that
were linearly dose dependent. At a dose level of
3204 #g FBl/egg, Javed et al. (1993) observed mean
mortality rates of 90 and 100%, dependent on the
time of treatment. This dose level was approxi-
mately 25 times greater than the highest dose of
125 pg/egg used in this study, which resulted in a
mean mortality rate of 94%. A subsequent study by
Bacon et al. (1995) determined the toxicity of FBI
in the presence or absence of fusaric acid. When
administered as an aqueous solution, Bacon et al.
(1995) found FBI to have no effect on chick mor-
tality below 10 pg/egg and greater than 90% mor-
tality at lOOttg/egg. Bacon et al. (1995) did not
report an LDso for FBI using the chicken embryo
assay; however, the dose range used for their study
was similar to that of this study and mean mortality
rates do concur.
As in this current study, Javed et al. (1993) admi-
nistered FB~ in aqueous solutions over the air space
of fertile eggs. Prelusky et al. (1987) reported that
the administration of toxin by injection over the air
space minimized variations in toxin diffusion and
distribution. In the study by Bacon et al. (1995),
aqueous solutions of FB~ were injected into the
yolk of each egg. Prelusky et al. (1987) found that
the dosing of eggs through yolk injection increased
the overall lethality, regardless of the carrier solvent
used. Furthermore, the reproducibility of this
method of administration has been questioned with
regards to the location of toxin deposition relative
to the developing embryo, as well as the concen-
tration of toxin to which the embryo is exposed
(Walker, 1967). Although some conflicting data was
presented by Bacon et al. (1995), it was demon-
strated that the co-occurrence of mycotoxins within
a contaminated sample could have a synergistic
effect on toxicity. Bacon et al. (1995) found that
low concentrations of fusaric acid could potentiate
the toxicity of FB1 to chicken embryos. Similarly,
the co-occurrence of other metabolites or toxins
within a sample may negate or antagonize the tox-
icity of a known mycotoxin. F u s a r i u m moulds are
known to produce more than 100 secondary metab-
olites, including a variety of mycotoxins (Jarvis,
1989). Contrary to the absence of malformations
observed in this study, or reported by Bacon et al.
(1995), Javed et al. (1993) reported on the occur-
rence of enlarged beaks, elongated necks and hy-
drocephalus in FB~-exposed embryos. As the purity
of the FBI used by Javed et al. (1993) was 90% (M.
Dombrink-Kurtzman, personal communication,
1995), it could be surmised that other compounds
may have inhibited the toxicity of FBI and/or con-
tributed to the embryonic malformations observed.
Cagampang (1994) observed similar malformations,
notably the enlargement of the posterior of the
skull, in chicken embryos exposed to culture ma-
terial extracts from F. m o n i l i f o r m e , where mortality
was not entirely dependent on the FB~ content of
the extract.
The toxicity of FBt to brine shrimps has only
recently been reported (Jim6nez et al., 1997).
Jimenez et al. (1997) approximated an LDs0 of
60gg FB~/ml within a 24-well plate, 24-hr brine
shrimp bioassay. In comparison, an LDso of ap-
proximately 1.2/zgFBl/ml was determined in the
present study. Although preliminary experiments
following the method of Solis et al. (1993) indicated
that the brine shrimps were quite resistant to any
toxic manifestations, the bioassay was easily modi-
fied to optimize sensitivity and respond to lower
levels of FBI.
Mixtures of acetonitrile and water are frequently
used in the extraction of FBI and other fumonisins
from corn and food product samples (Rice et al.,
1995). As the brine shrimps were not adversely
affected by the acetonitrile-water 70:30 (v/v) solvent
residue, it is anticipated that this bioassay design
will accommodate sample screening. Prior (1979)
concluded that the brine shrimp bioassay was
unsuitable as a biological screening system for
mycotoxin contamination of animal feed because of
an unacceptable number of false positives found in
their study of aflatoxin Bt, ochratoxin A, and T-2
toxin contamination of cereal feeds. Contributing
factors to the incidence of high brine shrimp mor-
tality in apparent mycotoxin-free samples could
have been the occurrence of other mycotoxins not
detected or analytically determined at the time of
study, as well as the presence of non-polar com-
pounds that may have been extracted from the feed
and were inherently toxic to the brine shrimps
(Curtis et al., 1974). The extent of these endogenous
toxic compounds extracted from a cereal sample
may be limited during the extraction of the more
polar fumonisins; however, this must be determined
in future studies. Although this bioassay cannot
replace quantitative chemical analyses, modifi-
cations to the bioassay procedure have been incor-
porated, such that this bioassay should provide an
 Bioassays for the toxicity assessment of fumonisin Bi
effective means of measuring FBm-related biological
activity in experimental samples.
At an effective dose level, the LCso values of 1.3
and 1.7 ~M FBI obtained for the chicken embryo
screening test and brine shrimp bioassays, respect-
ively, were very similar. This result would appear to
support Vesel~/et al. (1982), who found a significant
correlation in the ability of these two assays to rank
mycotoxins based on toxicity. A comparison of the
LCso values determined in these studies with the
published results of other reported bioassays for
assessing FB~ toxicity (Table 1), indicates that the
chicken embryo :rod brine shrimp bioassays are
indeed sensitive tools for measuring the biological
activity of FBt. The comparative bioassays listed in
Table I all involve cell culture techniques. While
these assay types may result in a better understand-
ing of the mechanism of toxicity, they appear to be
no more effective at indicating FB~ toxicity than the
measure of general cellular toxicity provided by the
brine shrimp and <:hicken embryo bioassays.
While the mechanism of action of the fumonisins
has not been full'./ elucidated, Wang et al. (1991)
determined an in vitro biological mode of action of
FB~ to be the disruption of sphingolipid synthesis.
FBI exhibits a stntctural similarity to the sphingoid
bases, sphingosine and sphinganine, and has been
demonstrated to specifically inhibit the enzyme
sphinganine N-acyltransferase (Riley et al., 1994;
Schroeder et al., 1994; Wang et al., 1991). Alteration
of sphingolipid metabolism by FBI may affect the
critical cellular functions of differentiation, growth,
and transformatio:a (Merrill, 1991), ultimately pro-
gressing to the disease conditions, including carcino-
genicity, associate,:l with this toxin (Riley et al.,
1994). Bacon et al. (1995) speculated on a similar
mechanism of toxicity, namely a disruption of
sphingolipid metabolism, within the brain of devel-
oping chicken embryos; however, sphingolipid levels
were not examined. Results observed in the chicken
embryo assay of this study were similar to those of
the brine shrimp bioassay, which is considered a
general toxicity assay, not specific for any particular
physiological action (Meyer et al., 1982). In com-
parison to mammalian systems, brine shrimps do
not possess the necessary cytochrome P-450
enzymes required for the metabolic activation or
detoxification of certain chemicals (Soils et al.,
1993) and they exhibit differences in purine metab-
olism (Van Denbos and Finamore, 1974). Brine
shrimp bioassays have been successfully used to
detect biologically active compounds that inhibit
protein synthesis within mammalian cells (Solis
et al., 1993), as well as those that affect RNA poly-
merase (Birndoff et al., 1975) and ATPase (Ewing
et al., 1974) systems. Gelderblom et al. (1993)
demonstrated that the intact FB~ molecule was
necessary to exert a biological effect, with one
requirement of toxicity being an intact amine func-
tional group (Kraus et al., 1992; Murphy et al.,
1996).
997
Thus, at the cellular level, the toxicity of FBt was
found to be comparable between the chicken
embryo and brine shrimp bioassays; however, quan-
titatively, the chicken embryo bioassay requires
more toxin. The relatively high dose of FBI
required per egg would preclude its use as a screen-
ing tool for FBt-contaminated samples. Conversely,
the brine shrimp bioassay is a less time-consuming
and more cost-efficient bioassay that should accom-
modate the screening of FBt-contaminated samples
and may have potential for use as a means of moni-
toring detoxification.
Acknowledgements--The assistance of Mr Lyle Robeson
in conducting the chicken embryo bioassays is gratefully
acknowledged. Published as paper no. !1835 Journal
Series, Agricultural Research Division, University of
Nebraska, Lincoln, NE 68583. Research was conducted
under Project 16-056. This material is based on work sup-
ported by the Cooperative State Research, Education, and
Extension Service, US Department of Agriculture, under
Agreement no. 95-37201-2343.
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Shier W. T. (1993) Biological activities of fumonisins,
mycotoxins from Fusarium moniliforme, in jimsonweed
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