Research in Veterinary Science 89 (2010) 325–331

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Research in Veterinary Science

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Aflatoxin B1 in poultry: Toxicology, metabolism and prevention

Sumit Rawal, Ji Eun Kim, Roger Coulombe Jr. *
Graduate Program in Toxicology, Department of Veterinary Sciences, Utah State University, Logan, UT 84322-4620, USA

a r t i c l e i n f o a b s t r a c t

Article history: Aflatoxins (AF) are ubiquitous in corn-based animal feed and causes hepatotoxic and hepatocarcinogenic
Accepted 13 April 2010 effects. The most important AF in terms of toxic potency and occurrence is aflatoxin B1 (AFB1). Poultry,
especially turkeys, are extremely sensitive to the toxic and carcinogenic action of AFB1, resulting in mil-
lions of dollars in annual losses to producers due to reduced growth rate, increased susceptibility to dis-
Keywords: ease, reduced egg production and other adverse effects. The extreme sensitivity of turkeys and other
Aflatoxin B1 poultry to AFB1 is associated with efficient hepatic cytochrome P450-mediated bioactivation and defi-
Aflatoxicosis
cient detoxification by glutathione S-transferases (GST). Discerning the biochemical and molecular mech-
P450s
Metabolism
anisms of this extreme sensitivity of poultry to AFB1, will contribute in the development of novel
Bioactivation strategies to increase aflatoxin resistance. Since AFB1 is an unavoidable contaminant of corn-based poul-
Liver cancer try feed, chemoprevention strategies aimed at reducing AFB1 toxicity in poultry and in other animals have
Cytochrome P450 been the subject of numerous studies. This brief review summarizes many of the key recent findings
Glutathione S-transferase regarding the action of aflatoxins in poultry.
Chemoprevention Ó 2010 Elsevier Ltd. All rights reserved.

1. Aflatoxins causes a myriad of other effects either directly or indirectly associ-

Aflatoxins (AF) are the naturally-occurring mycotoxins, pro-
duced as secondary metabolites by the fungus Aspergillus flavus, A.
parasiticus, and A. nominus. The name ‘‘aflatoxin” is derived from
the first letter in Aspergillus, and the first three letters in flavus
(Schoental, 1967). Structurally, AFs are difurocoumarin derivatives
that fluoresce under ultraviolet light. Depending upon color of the
fluorescence, AFs are divided into aflatoxin B1 and B2 (AFB1, AFB2)
for blue, and G1 and G2 (AFG1, AFG2) for green (Hartley et al.,
1963; Dalvi, 1986) (Fig. 1). Aflatoxin M1 and M2 (AFM1, AFM2),
known as milk-AFs, are the metabolites of AFB1 and AFB2, respec-
tively (Carnaghan et al., 1963). Other metabolites of AFB1 are afla-
toxin Q1 (AFQ1) and aflatoxicol. Aflatoxins are the most
intensively researched group of mycotoxins, because of their dem-
onstrated toxic and carcinogenic effects in the susceptible labora-
tory animals and livestock and their acute toxicological and
chronic hepatocarcinogenic effects in humans. Of the known AFs,
AFB1 is the most potent, and is a classified human carcinogen (Wo-
gan et al., 1974; Wong and Hsieh, 1976; Bondy and Pestka, 2000).
2. Toxicity of aflatoxins
The AFB1 is toxic to a wide range of animal species. AFB1 is prin-
cipally an hepatotoxin and hepatocarcinogen (JECFA, 1998), but it
* Corresponding author. Tel.: +1 435 797 1598; fax: +1 435 797 1601.
E-mail address: roger@usu.edu (R. Coulombe Jr.).
ated with this toxicity: immunosuppresion, reduced growth rate,
lowered milk and egg production, reduced reproductivity, reduced
feed utilization and efficiency and anemia. AFB1 has been shown to
induce hepatocellular carcinoma in many species of animals
including fish (rainbow trout, sockeye salmon, and guppy), poultry
(turkeys, ducks, and geese), non-human primates (rhesus, cyno-
molgus, African green, and squirrel monkeys), and rodents (rats,
mice, and tree shrews) (Wogan, 1992).
Species susceptibility to various acute toxic manifestations, as
measured by TD50, is likewise variable (Gold et al., 1984; Wogan,
1992). While Fisher rats are highly sensitive (TD50: 1.3 mg/kg/body
weight/day), Swiss mouse are highly resistant (TD50: >5300 mg/kg/
body weight/day). Rhesus and cynomolgus monkeys dosed for an
average of 3.3 and 14 years, respectively, yielded a TD50 value of
156 and 848 mg/kg/body weight/day, respectively.
A wide variation exists in species susceptibility to AFB1 hepato-
carcinogenesis. Fish and poultry, known to be extremely sensitive
to AFB1, responded to doses as low as 15–30 lg/kg. Rats responded
at levels of 15–1000 lg/kg, whereas mice showed no effects to lev-
els as high as 150,000 lg/kg (Wogan, 1992). In rainbow trout, die-
tary AFB1 concentrations of 20 lg/kg resulted in a liver tumor
incidence of 62% (Bailey et al., 1988).
Non-human primates show a wide variability in AFB1 suscepti-
bility to hepatic tumors (Adamson, 1989). While squirrel monkeys
developed liver cancer when fed AFB1 at 2000 lg/kg for 13 months,
much higher doses were required over a longer period of time to
induce low incidence of liver carcinoma in rhesus, cynomolgus
and African green monkeys (99–1225 mg/animal administered

0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.rvsc.2010.04.011
326 S. Rawal et al. / Research in Veterinary Science 89 (2010) 325–331
Fig. 1. Chemical structures of aflatoxin B1, B2, G1 and G2.
p.o. over periods of 48–179 months). However, AFB1-induced tu-
mors in extrahepatic tissues in the latter species.
In humans, acute aflatoxicosis is manifested by vomiting,
abdominal pain, pulmonary edema, coma, convulsions, and death
with cerebral edema and fatty involvement of the liver, kidney,
and heart (Strosnider et al., 2006). The occurrence of acute aflatox-
icosis was evidenced by the severe outbreak in Kenya in 2004
(Probst et al., 2007). Epidemiological studies have consistently
demonstrated that AFB1 is a liver carcinogen in humans (Van Rens-
burg et al., 1985; Groopman et al., 1988). Studies conducted in
Swaziland and Guangxi, China has linked AFB1 exposure to devel-
opment of liver cancer in humans (Peers et al., 1987; Yeh et al.,
1989). The International Agency for Research on Cancer has con-
cluded that there is sufficient evidence for the carcinogenicity of
AFB1 in humans and hence placed this mycotoxin under group I.
Aflatoxin B1 is a ‘‘pro-carcinogen” in that enzymatic bioactiva-
tion is a prerequisite for carcinogenic (and toxic) activity (Garner
et al., 1972). Accordingly, elucidation of the mechanisms of AFB1
metabolism has been the focus of intense research over the years.
AFB1 is metabolized by hepatic microsomal cytochrome P450s
(P450) to the reactive, electrophilic exo-AFB1-8,9-epoxide (AFBO)
which binds to DNA and other critical cellular macromolecules
(Ball and Coulombe, 1991; Wogan, 1992; Coulombe, 1993; Galla-
gher et al., 1996; Guengerich et al., 1996). The AFBO is highly
unstable, and it reacts with the DNA to form N7 guanine adducts
by intercalation of AFBO between base pairs (Iyer et al., 1994).
There are a number of urinary and serum biomarkers that have
been validated to accurately predict AFB1 cancer risk in humans.
These include urinary aflatoxin-N7-guanine and AFM1 (Gan et al.,
1988; Groopman et al., 1992; Groopman and Kensler, 1999). Uri-
nary excretion of the mercapturic acid (AFB-NAC), a product of glu-
tathione (GSH) adduction of AFB1 mediated by glutathione-S-
transferases (GSTs), has also been used in field studies (Wang
et al., 1999). Serum AFB-albumin adducts, which are positively
associated with hepatocellular carcinoma in humans (Wang
et al., 1996) have found wide use in epidemiologic studies (Wild
and Turner, 2001). Analysis of serum adducts indicates a positive
correlation between dietary AFB1 exposure and serum AFB-albu-
min adducts (Gan et al., 1988; Wild et al., 1992).
3. Aflatoxin B1 toxicity in poultry
Poultry, especially turkeys are extremely sensitive to the toxic
effects of AFB1 (Carnaghan et al., 1966; Arafa et al., 1981; Giamb-
rone et al., 1985; Huff et al., 1986; Kubena et al., 1995; Klein
et al., 2000). Extreme sensitivity of turkeys to AFB1 was first and
graphically demonstrated by association with ‘Turkey X Disease’
which caused widespread deaths of turkeys and other poultry
throughout Europe in the 1960s (Stevens et al., 1960). The disease
was shown to be caused by AFB1 contaminated feed (Smith, 1960).
It was later reported that the contamination with AFs came from
Brazilian peanut meal (Blount, 1961). Among different poultry spe-
cies, turkeys are shown to be the most susceptible to AFs, quails are
intermediate, while chickens are considered relatively resistant
(Arafa et al., 1981; Lozano and Diaz, 2006). Although direct com-
parisons have not been conducted, wild turkeys appear to be less
susceptible to AFB1 than their commercial counterparts (Quist
et al., 2000).
Aflatoxin at 0.7 mg/kg reduced the growth rate of turkey poults,
but had no effect in quails and chickens (Arafa et al., 1981). A diet
containing 400 mg/kg AFB1 severely affected body and relative li-
ver weights in turkeys, while chickens showed no effect at this die-
tary concentration (Leeson et al., 1995). A study examining the
effects of AFB1 on the development of liver lesions in poults, re-
ports ducks as more susceptible than turkeys and chickens (Coker,
1979). In that study, ducks developed hepatic lesions by dietary
exposure to 30 lg/kg, turkeys at 300 lg/kg, while chickens re-
sponded to 500 lg/kg. Another study evaluated the effects of
AFB1 on the development of cytopathology in the tracheal culture
in day-old turkeys, Japanese quails, chicken and ducks (Colwell
et al., 1973). While cultures derived from ducks developed pathol-
ogy at 6 lg/kg AFB1, the levels needed for equivalent pathology
were as high as 100 lg/kg for those from chickens. Tracheal cul-
tures from turkeys and quails responded to 28 and 47 lg/kg of
AFB1, respectively.
4. Economic Impacts to the poultry industry due to aflatoxins
Turkeys are an important international food commodity. The
United States accounts for roughly one-half of the world’s turkey
production at approximately 7.30 billion pounds live weight, with
an estimated value of nearly US$ 3 billion (National Agricultural
Statistics Service, USDA). The per capita consumption of turkeys
in the United States is approximately 18 pounds, and turkeys are
now the fourth major food and protein source, behind chicken, beef
and pork, respectively (National Turkey Federation).
Aflatoxins result in economic losses to poultry industry from
reductions in growth rate, hatchability, feed efficiency and immu-
nity towards diseases (Richard et al., 1986; Coulombe, 1993).
According to a report by Council for Agricultural Science and Tech-
nology, losses due to AFs to the United States poultry industry ex-
ceeded $143 million annually (CAST, 1989). A recent study
reported annual crop losses of $932 million due to mycotoxin con-
tamination and additional losses of $466 million in efforts to pre-
vent or reduce contamination (CAST, 2003). Although AFs are
found in most of the feed ingredients; corn, peanut meal, cotton-
seed meal, and sorghum appears to be at greatest risk for introduc-
ing AFs in turkey diets (Pons and Goldblatt, 1965; Brekke et al.,
1977; Winn and Lane, 1978; Hill et al., 1983). Crops contaminated
with AFs are a worldwide problem and approximately 25% of
world’s food supply is contaminated with mycotoxins annually
(CAST, 1989). Conditions that favor contamination by mycotoxins
include excessive moisture both in field and post harvest storage,
high humidity, temperature extremes, drought stress, and insect
damage to crops (Coulombe, 1993). Aflatoxins are deleterious to
poultry and their contamination in feed is practically unavoidable
(Coulombe et al., 2005). The United States Food and Drug Adminis-
tration regulates the amount of AFB1 in poultry feed. Current action
 S. Rawal et al. / Research in Veterinary Science 89 (2010) 325–331
level for corn and peanut products is 100 ppb and for cottonseed
meal is 300 ppb.
Consumption of AFB1 contaminated feed results in poor perfor-
mance, decreased body and organ weights, immunosuppression,
morbidity, and mortality in turkeys (Kubena et al., 1990, 1991;
Coulombe, 1993). Aflatoxins significantly affect feed consumption,
total plasmatic proteins and cholesterol levels of turkeys (Rauber
et al., 2007). Furthermore, AFs lead to irreversible liver damage
as indicated by decreased liver-to-body weight ratios, liver enzyme
alterations, altered blood coagulation patterns, and histologic
changes like hepatocellular necrosis and biliary hyperplasia in tur-
keys (Quist et al., 2000; Klein et al., 2002b).
There are numerous reports that feed-borne AFB1 contamina-
tion has a profound and negative impact on feed efficiency, which
significantly reduces productivity in the poultry industry. For
example, dietary AFs (2.5 mg/kg) significantly reduced the feed in-
take by 9–11% of poultry among all age groups (7–280 days old)
(Pandey and Chauhan, 2007). Rauber et al. (2007) reported signif-
icant reductions in the feed consumption due to low dietary AF
concentrations (100, 200, 500 and 1000 ppb). Another study also
reports significant reductions in the dietary feed intake and feed
efficiency due to aflatoxins (1–3 mg/kg in the diet) (Ehrich et al.,
1986). Dietary exposure of broiler hens to AF (10 mg/kg) resulted
in embryonic mortality and lowered the immunity in the progeny
chicks (Qureshi et al., 1998). Embryonic exposure with AFs resulted
in long-term depression of the immune function in chicks (Neldon-
Ortiz and Qureshi, 1992). As in other species, the liver is the most
severely affected organ in poultry, primary consequences being
hepatotoxicity and carcinogenicity (Klein et al., 2000).
5. AFB1 metabolism in poultry: role of cytochrome P450s
Cytochrome P450s are mixed function oxidases that catalyze the
biotransformation of a wide variety of xenobiotics. They are a
superfamily of hemoproteins that aid in the oxidation of various
substrates such as steroids, eicosanoids, pharmaceuticals, pesti-
cides, pollutants, and carcinogens (Parikh et al., 1997). Cytochrome
P450s play an important role in the formation of carcinogenic and
mutagenic electrophilic intermediates from naturally-occurring
dietary compounds (Guengerich et al., 1996). As mentioned above,
AFB1 is not toxic per se, but requires metabolic conversion to the
reactive and electrophilic exo-AFBO by P450s to exert its toxicity
(Ball and Coulombe, 1991; Coulombe, 1993; Gallagher et al.,
1996; Guengerich et al., 1996). This electrophilic metabolite reacts
with cellular nucleophiles and can induce mutations by alkylating
DNA, principally at the N7 position of guanine forming the 8,9-dihy-
dro-8-(N7-guanyl)-9-hydroxy-AFB1 (Lin et al., 1977). In addition
AFBO can bind to proteins and other critical cellular nucleophiles.
Turkey liver P450s are especially efficient toward AFB1 bioacti-
vation compared to other poultry species thus far examined. Tur-
key liver microsomes bioactivated AFs 1.8 and 3.5 times more
than liver microsomes from quail and chicken, respectively (Loz-
ano and Diaz, 2006). When comparing livers obtained from 9, 45
and 61 day-old turkeys, microsomes from younger were more ac-
tive toward AFB1 bioactivation than that from older birds (Klein
et al., 2002a).
In turkey liver, AFB1 metabolism is mediated by homologues to
human CYP1A2 and CYP3A4 (Klein et al., 2000, 2003; Yip and Cou-
lombe, 2006). Initial studies reported that concentrations of AFB1
which are likely to be achieved in the liver following ingestion of
‘‘real-world” concentrations of AFB1 are bioactivated to AFBO pri-
marily by CYP1A2, whereas much higher concentrations are cata-
lyzed by CYP3A4 (Gallagher et al., 1996; Kelly et al., 1997; Van
Vleet et al., 2002). CYP1A homologues also metabolize AFB1 to pro-
duce the detoxified metabolite AFM1, whereas CYP3A enzymes
327
produce another detoxified metabolite, aflatoxin Q1 (AFQ1) (Camp-
bell and Hayes, 1976; Guengerich et al., 1996) (Fig. 2).
Although both CYP1A and 3A isoforms oxidize AFB1, there are
conflicting reports on their relative importance (Shimada and
Guengerich, 1989; Ramsdell and Eaton, 1990; Gallagher et al.,
1994, 1996). A recent study demonstrated a dominant contribution
of CYP3A4 homologues in AFBO production. Aflatoxin B1 metabo-
lism studies in human liver microsomal preparations indicate a
predominant role for CYP3A4 and that its expression level was
an important determinant of the AFB1 disposition in human liver
(Kamdem et al., 2006). Specific CYP3A4 inhibitors like troleando-
mycin have been shown to inhibit AFBO production (Gallagher
et al., 1994), while inducers of CYP3A4 activity such as 3-methyl-
cholanthrene and rifampicin, increase AFB1 metabolism in cultured
human hepatocytes (Langouet et al., 1995).
Mammalian P450s are well described, but limited information
is available on P450s in poultry, especially in the context of AFB1
metabolism. Chicken P450s CYP1A5 (Gilday et al., 1996) and
CYP3A37 (Ourlin et al., 2000) have been described. Our laboratory
recently cloned, expressed and characterized CYP1A5 from turkey
liver, the first functional protein amplified from turkeys (Yip and
Coulombe, 2006). CYP1A5 is predicted to be 528 amino acid with
94.7% sequence identity to chicken CYP1A5. Like its human homo-
logue, the Escherichia coli-expressed CYP1A5 efficiently bioactivat-
ed AFB1 to AFBO, and in addition, produced AFM1. The CYP1A
family identity of CYP1A5 was confirmed using specific inhibitors
of catalytic activities, and through genetic mapping studies where
the structure of the turkey gene was shown to be equivalent to that
of the human CYP1A genes with seven exons of 38, 858, 127, 90,
124, 87 and 307 bp, respectively, and six introns. A single nucleo-
tide polymorphism (SNP) in the 30 UTR (untranslated region) was
used to assign CYP1A5 to turkey linkage group M16 (equivalent
to chicken chromosome 10) (Reed et al., 2007).
The genetic structure of a CYP3A4 homologue, CYP3A37, which
along with CYP1A5 also epoxidates AFB1 in turkey liver was re-
cently elucidated. Similar to human CYP3A4, CYP3A37 gene is or-
ganized into 13 exons (124, 94, 53, 100, 114, 89, 149, 128, 73,
161, 227, 163 and 475 bases in length) and 12 introns (Rawal
et al., 2009). Studies to determine the metabolic characteristics of
the turkey CYP3A37 gene product, as well as its importance rela-
tive to CYP1A5 in AFB1 bioactivation in turkey liver, are currently
underway.
6. AFB1 metabolism in poultry: the role of glutathione S-
transferases
In humans and most animals, the principal route of AFB1 detox-
ification appears to be through conjugation with endogenous GSH,
a reaction catalyzed by GSTs. Glutathione S-transferases
(E.C.2.5.1.18), a family of multifunctional dimeric proteins, are
important phase II biotransformation enzymes involved in cellular
detoxification and excretion of a variety of xenobiotic substances
(Eaton and Bammler, 1999; Frova, 2006; Kim et al., 2010). Most
cytosolic GSTs exist as dimeric subunits of 23–30 kDa with an
average length of 199–244 amino acids (Hayes and Pulford, 1995).
Avian GSTs comprise a complex isoenzyme system that has re-
ceived much less attention (Yeung and Gidari, 1980) which is unfor-
tunate given the critical role this enzyme plays in AFB1
susceptibility and resistance. Five groups of GST subunits (desig-
nated CL1 to CL5) have been identified in the cytosolic fraction of
Leghorn chick livers according to electrophoretic mobility on SDS/
PAGE (Chang et al., 1990). Expressed Sequence Tag (EST) databases
identified a (Chang et al., 1990, 1992; Liu et al., 1993), l (Liu and
Tam, 1991; Sun et al., 1998), s (Hsiao et al., 1995) and r (Thomson
et al., 1998) classes from cDNA sequences of the domestic chicken.
328 S. Rawal et al. / Research in Veterinary Science 89 (2010) 325–331
Fig. 2. The extreme sensitivity of turkeys to AFB1 is associated with efficient AFB1 epoxidation catalyzed by CYP1A5 and CYP3A37, coupled with deficient GST detoxification.
The hydroxylated metabolites, AFM1 and AFQ1 are formed by CYP1A5 and CYP3A37, respectively.
Full length cDNA of a-class GSTs was isolated from chicken livers
and heterologously expressed in baculovirus and E. coli systems.
GST-specific substrates such as 1-chloro-2,4-dinitrobenzene
(CDNB), ethacrynic acid (ECA), trans-4-phenyl-e-buten-2-one
(tPBO) and cumene hydroperoxide (CHP) were used for functional
characterization (Chang et al., 1992; Liu et al., 1997). Nine a-class
isozymes with distinctive molecular masses were affinity purified
from chicken livers and partially cloned and characterized (Hsieh
et al., 1999); clustering of chicken ESTs accessioned in GenBank
suggests expression of six separate a-class transcripts. The nomen-
clature of chicken a-class GSTs was recently re-named (GenBank
accession nos.) as cGSTA1 (NM_001001777), cGSTA2
(NM_001001776), cGSTA3 (NM_204818), cGSTA-CL3 (M38219)
based on subunit nomenclature proposed by (Mannervik et al.,
1992). Sun et al. (1998) newly reported that structure and enzyme
specific activities of avian m-class GST, cGSTM1-1 which has the
highest epoxidase activity among rGSTM3-3, rGSTM1-1 and
hGSTM2-2.
There exists substantial amount of evidence that the conjuga-
tion of the epoxide by GSH is the major, rate-limiting determinant
in species susceptibility to AFB1, regardless of the extent of P450-
mediated bioactivation (Hayes et al., 1991). For example, quail
and rat are much more sensitive to AFB toxicity and carcinogenic-
ity than the more resistant mouse, yet all these animals exhibit
high rates of P450-mediated AFBO formation. The relatively high
rate of glutathione conjugation by a constitutive a-class, GSTa3,
appears to be the critical resistance factor in mice (Hayes et al.,
1992). Resistance in the rat to AFB1-induced hepatocarcinogenesis
can by enhanced by induction of a similar GST in liver which is
constitutively expressed at low levels (Stresser et al., 1994). In
non-human primates, evidence has been presented that GSTs in
the l-class, rather than a, are most efficient in conjugation of AFBO
(Wang et al., 2000). Deficient or a complete lack of a functional GST
with affinity toward AFBO appears to be a major reason that poul-
try are extremely susceptible to AFB1 (Klein et al., 2000).
Commercial modern turkeys appear to be deficient in this
detoxification mechanism which is a factor for their extreme sen-
sitivity to AFB1 (Klein et al., 2000). Our laboratory has recently
amplified, sequenced and mapped the a-class GST gene cluster
from turkey liver Kim et al. (2010). The a-class GST cluster in tur-
keys consist of six forms (GenBank accession nos.) –tGSTA1.1
(GQ228399), tGSTA1.2 (GQ228400), tGSTA1.3 (GQ228401), tGSTA2
(GQ228402), tGSTA3 (GQ228403) and tGSTA4 (GQ228404). The
conserved GST domains and four a-class signature motifs in turkey
GSTs (with the exception of tGSTA1.1 which lacked one motif) con-
firmed class identity of these hepatic alpha-class GSTs in the tur-
key. These tGSTA genes were heterologously expressed in E. coli
system and functionally characterized (Kim et al., in preparation).
Although tGSTA1.1 lacked one motif, six recombinant a-class GST
enzymes were shown to exhibit glutathione conjugating activities
toward 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitro-
benzene (DCNB), ethacrynic acid (ECA) and cumene hydroperoxide
(CHP), and unlike the hepatic forms, catalytic activity towards
AFBO indicating that some form of regulatory control has silenced
this gene in turkey liver.
7. Chemoprevention of aflatoxicosis in poultry
The National Cancer Institute defines chemoprevention as the
use of naturally-occurring or synthetic agents to reduce the risk
of, or delay the development or recurrence of, cancer. Given that
AFB1 contamination in the feed is nearly universal, and therefore
practically unavoidable (Coulombe et al., 2005), chemoprevention
strategies aimed at reducing AFB1 toxicity in poultry and in other
animals have been the subject of numerous studies (Klein et al.,
2002b, 2003; Guarisco et al., 2008a,b). Several chemopreventives
have been evaluated in poultry for reducing symptoms of aflatox-
icosis. Indeed, because of their sensitivity, poultry have been used
as models for discovery of novel AFB1 chemopreventives.
 S. Rawal et al. / Research in Veterinary Science 89 (2010) 325–331
Clay-based inorganic adsorbents, which prevent absorption of
AFB1 into general circulation, hence reducing bioavailability, have
been extensively studied. In broiler chicks, adsorbents like calcium
montmorillonite clay (0.5%, 0.25%, and 0.125% in diet), and Zeolites
(1% in diet), bind to AFB1 preventing their absorption have shown
to be protective against high level AFB1 exposure of 5 and 2.5 mg/
kg, respectively (Miazzo et al., 2000; Pimpukdee et al., 2004). Hy-
drated sodium calcium aluminosilicate (HSCAS), fed at dietary lev-
els, ranging from 0.25% to 1%, has been shown to diminish the
deleterious effects of AFs (up to 5 mg/kg in diet) in broiler chicks
(Kubena et al., 1993, 1998; Ledoux et al., 1999). The adsorbent clin-
optilolite (15 g/kg) provided moderate amelioration of AF associ-
ated liver toxicity (hydropic degeneration and biliary
hyperplasia) in broiler chicks (Ortatatli et al., 2005).
Detoxification of the contaminated feed with microorganisms
has been tested as a strategy to reduce the AF-associated toxicity.
In a recent study, the detoxification potential of Nocardia coryne-
bacteroides (NC), as a feed additive, was evaluated in day-old Ross
308 chicks; NC significantly reduced the AF associated lesion
severity in liver, duodenum, and kidneys (Tejada-Castaneda et al.,
2008). Another report indicates the protective effect of silymarin,
a potent hepatoprotective in humans, against aflatoxicosis in broi-
ler chicks. Although the mechanism of protection from Silymarin is
uncertain, reports suggest that it acts as an antioxidant and cell
membrane stabilizer and promotes the cellular synthesis of macro-
molecules (Tedesco et al., 2004). Dietary administration of a silym-
arin-phospholipid complex (600 mg/kg of body weight) resulted in
significant improvement in weight gain and feed intake in broiler
chicks fed AFB1 (0.8 mg/kg) in diet (Tedesco et al., 2004). In addi-
tion, water soluble vitamins are protective against aflatoxicosis in
quail chicks (Wilson et al., 1975, 1978).
The food antioxidant butylated hydroxytolouene (BHT) has
been shown in numerous studies to reduce symptoms of aflatoxi-
cosis in mammalian and avian models. In rodent models, BHT has
been shown to reduce the development of cancer by either, induc-
tion of phase II enzymes like GSTs or inhibition of phase I enzymes
like P450s (Hocman, 1988; Singletary, 1990). Weight loss in chick-
ens due to AFB1 (3000 lg/kg in diet) was shown to be dramatically
improved by dietary BHT at a level of (0.39%) (Larsen et al., 1985).
Likewise, dietary BHT protects against aflatoxicosis in turkeys. A
diet containing 4000 mg/kg BHT provided significant protection
against AFB1-induced weight loss, hepatocellular necrosis, biliary
hyperplasia and changes in serum enzymes indicative of hepato-
toxicity, in turkeys (Klein et al., 2002b). Mechanism studies later
revealed that while BHT induced GST expression and activity, none
of the GSTs so induced had affinity toward AFBO (Klein et al.,
2003). The protective properties of BHT appeared to be due to an
inhibitory effect on the activity of P450 1A enzymes, suggesting re-
duced bioactivation of AFB1 to the AFBO (Klein et al., 2003). Further
studies revealed that dietary BHT reduced AFBO formation, as well
as other CYP1A5-mediated activities (Guarisco et al., 2008b).
Importantly, BHT inhibited conversion of AFB to AFBO, exhibiting
Michaelis–Menton competitive inhibition kinetics (Ki = 0.81 lM).
Inhibition of AFBO formation was found in the microsomes pre-
pared from the birds fed 4000 mg/kg BHT, as well as in the micro-
somes treated with BHT in vitro. Moreover, BHT (4000 mg/kg in
diet for 10 days) caused significant reductions in AFB1 bioavailabil-
ity, AFB1–DNA adduct formation in the liver, and AFB1 residues in
tissues in turkeys (Guarisco et al., 2008a).
A number of recent studies have demonstrated that probiotic
bacteria offers protection against AFB1 in humans and animals.
The mechanism of protection is through binding of AFB1 by cell
wall constituents of probiotic bacteria, such as Lactobacillus
rhamnosus. Because of its use in various dairy products including
yogurt, L. rhamnosus is considered a safe and effective chemopre-
ventive. Lactobacillus rhamnosus strain GG and LC-705, were found
329
to be most efficient in binding a range of mycotoxins including AFs
(El-Nezami et al., 1998, 2002a,b; Haskard et al., 2001; Peltonen
et al., 2001). In addition, L. rhamnosus reduced AFB1 transport,
metabolism, and toxicity in cultured Caco-2 cells (Gratz et al.,
2007). Furthermore, L. rhamnosus reduced AFB1 bioavailability in
rats, thereby decreasing its toxicity (Gratz et al., 2006). Thus, there
exists substantial evidence of the protective role of probiotics in
preventing aflatoxicosis.
Acknowledgements
The authors gratefully acknowledge support of competitive
grant 2002-35204-12294, from the USDA-NIFA, by competitive
grant 2007-35205-17880 from the USDA-NIFA Animal Genome
program, and the Utah and Agricultural Experiment Station.
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