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A Review on Biological Control and Metabolism of

Aflatoxin
a a
H. N. Mishra & Chitrangada Das
a
Post Harvest Technology Centre, Department of Agricultural and Food Engineering, Indian
Institute of Technology, Kharagpur—721302, India. E-mail: hnm@agfe.iitkgp.ernet.in.
Telephone: 91-3222-83130 (Office), 91-3222-283131 (Residence). Fax: 91-3222-282244,
91-3222-255303

Version of record first published: 03 Jun 2010.

To cite this article: H. N. Mishra & Chitrangada Das (2003): A Review on Biological Control and Metabolism of Aflatoxin,
Critical Reviews in Food Science and Nutrition, 43:3, 245-264

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 Critical Reviews in Food Science and Nutrition, 43(3):245–264 (2003)

A Review on Biological Control and Metabolism of

Aflatoxin
H. N. Mishra and Chitrangada Das*
Post Harvest Technology Centre, Department of Agricultural and Food Engineering, Indian Institute of
Technology, Kharagpur — 721302, India. E-mail: hnm@agfe.iitkgp.ernet.in. Telephone: 91-3222-83130 (Office),
91-3222-283131 (Residence). Fax: 91-3222–282244, 91-3222–255303.

Referee: Dr. F. S. Chu, 16458 Denhave Court, Chino Hills, CA 91709

Downloaded by [North Carolina State University] at 22:20 01 October 2012

* Corresponding author: Dr. Chitrangada Das, NFP Division, National Metallurgical Lab, Jamshedpur, 831007, India. phone: 91-657-
2240062, email: chitrangadas@rediffmail.com

ABSTRACT: The series of events that led to the discovery of aflatoxin as a potent carcinogen, its biosynthesis,
mechanism of action, structure-function relationship provide interesting insight into the economical and techno-
logical factors involved in the development of an effective control measure for the toxin. Scientists all over the
world are making continuous efforts to explore a generalized process of detoxification, which can bring down the
toxin content in heterogeneous commodities to a threshold level. In this article biological control methods with
special emphasis on in vivo and in vitro enzymatic detoxification of aflatoxin have been reviewed. Future areas
of research involving large-scale enzymatic detoxification and modified atmosphere storage are also discussed.

I. INTRODUCTION 1961; Vander Zijden, 1962) and led to the discov-

Aflatoxins are a group of closely related het-
erocyclic compounds produced predominantly by
two filamentous fungi, Aspergillus flavus and
Aspergillus parasiticus. Recent studies have shown
that some A. nominus and A. tamarii starins are
also aflatoxin producing, of which A. nominus is
phenotypically similar to A. flavus (Kurtzman et
al, 1987; Goto et al., 1996; Goto et al., 1997). Ito
et al. (2001) isolated one more strain,
A. pseudotamarii, that can produce aflatoxin. These
fungi belong to the class Hyphomycetes, subdivi-
sion Deuteromycotina and family Aspergillaceae
(Beuchat, 1987). They contaminante a vast array
of food and agricultural commodities. Aspergillus
species are capable of growing on a variety of
substrates and under a variety of environmental
conditions. Therefore, most foods are susceptible
to aflatoxigenic fungi (Palmgren and Hayes, 1987)
at some stage of production, processing, transpor-
tation, and storage.
The outbreak of aflatoxicosis (famous as
Turkey “X” disease) in England in 1960 caused
the death of a large population of livestock (Blount,
ery of aflatoxin in groundnut meal contaminated
by A. flavus (Hesseltine, 1979). Subsequently,
aflatoxins were found in other feeds, especially
maize (Shotwell, 1977; Chakrabarty, 1981) and
cottonseed meal (Lillehoj, 1979; Sharma et al.,
1994). The economic impacts attributed to afla-
toxin are incurred directly by loss in crops, live-
stock, and dairy and indirectly by a recurring
expenditure in quality-control programs, research
and education, lower foreign exchange earnings,
and increased storage and packaging costs of vul-
nerable commodities.
The potential hazard of aflatoxins to human
health has led to worldwide monitoring programs
for the toxin in various commodities as well as
regulatory actions by nearly all countries. Levels
varying from zero tolerance to 50 ppb have been
set for total permissible aflatoxin content in foods
and feed (Patterson, 1983). While most countries,
including the U.S., have a regulatory level around
20 ppb in foods, considerably lower limits for
aflatoxin in food have been established by the
European Economic Community on January 1,
1999, that is, 2.0 ppb for aflatoxin B1 and 4.0 ppb

1040-8398/03/$.50
© 2003 by CRC Press LLC
245
 for total aflatoxins (cited from National Peanut
Research Laboratory, Agricultural Research Labo-
ratory, USDA, CRIS no 6604-42000-006-00D).
Intensive research has been undertaken on
aflatoxin-related problems (Goldblatt, 1969, 1971;
Pons and Goldblatt 1969; Pons 1976). The search
is on for a suitable remedial measure to combat
this problem since the 1960s. In principle, there
are three possibilities to avoid harmful effects
caused by aflatoxin:
• Prevention of aflatoxin producing fungal at-
tack at preharvest stage
• Detoxification of aflatoxin-contaminated food
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and agricultural commodities
• Inhibition of absorption of aflatoxin in con-
sumed food in the digestive tract
These aspects have been reviewed thoroughly
in the following paragraphs.
II. STRUCTURE AND FUNCTION OF
AFLATOXIN MOLECULES
Aflatoxins are chemically difuro-coumarin
derivatives derived through polyketide pathway
(Heathcote and Hibbert 1976; Vederas and
Nakashima, 1980; Wantanabe, 1996). Recently,
genetic studies by Payne and Brown (1998) on
aflatoxin producing fungi has given an interesting
insight and led to the cloning of 17 genes respon-
sible for 12 enzymatic conversions in the afla-
toxin biosynthetic pathway. The physical factors
influencing the pathway have been found out, but
the exact role played by them is not yet clear. The
pathway-specific regulation occurs by a Zn(II) 2
cys 6 DNA binding protein that regulates the
transcription of all pathway genes.
Presently, 18 different types of aflatoxins have
been identified, with aflatoxin B1, B2, G1, G2, M1,
and M2 being the most common. Of these, Afla-
toxin B1 (AFB1) and G1 (AFG1) occur most fre-
quently, with AFB1 being the most potent. Physi-
cochemical and biochemical characteristics of the
AFB1 molecule reveal two important sites for
toxicological activity (Heathcote and Hibbert,
1978). The first site is the double bond in position
8,9 of the furofuran ring (Figure 1). The interac-
246
tions of aflatoxin, DNA, and proteins, which oc-
cur at this site alter the normal biochemical func-
tions of these macromolecules and lead to delete-
rious effects at the cellular level. The second
reactive group is the lactone ring in the coumarin
moeity (Lee et al., 1981). The lactone ring is
easily hydrolyzed and therefore is vulnerable to
degradation.
Monteiro et al. (1996) studied the in vitro
interaction of groundnut proteins, namely, arachin
and conarachins (I and II) with AFB1 molecule.
The addition of aflatoxin quenched the intrinsic
fluorescnec of all the three proteins. The associa-
tion constants of interaction were 3.5 ± 0.4 × 104,
4.8 ± 0.5 × 104 and 23 ± 3 × 104 M–1, respectively,
with arachin, conarachin II, and conarachin I at
26°C. There was an increase in beta structure
content of arachin and conarachin II, while an
aperiodic content of conarachin was observed.
Kinetic studies showed the reaction to be of
pseudo-first order. The modification of lysyl resi-
dues by succinylation decreased the strength of
binding with conarachins, with no significant
change in arachin.
Biological effects of aflatoxin can be subdi-
vided into its toxicity, carcinogenicity, mutage-
nicity, and teratogenicity. The effects are influ-
enced by species variation, sex, age, nutritional
status, and effect of other chemicals (Ellis et al.,
1991). In addition, the dose level and period of
exposure of the organism to the toxin are very
important. The toxicological effects of AFB1 oc-
cur after the metabolic activation of the molecule
by the microsomal mixed function oxidase sys-
tem. These enzymatic reactions involve metabo-
lism and detoxification. Metabolic activation of
AFB1 leads to the formation of reactive AFB1-
epoxide, which can lead to toxic or detoxification
pathway or both. While the epoxide exerts its
effect by interacting with DNA and some en-
zymes to alter the p-53 gene and to inhibit the
enzymatic activities, it can also conjugate with
proteins and glutathione, which is then excreted
from the body. Various factors affecting the ki-
netics of formation of adducts and detoxification
greatly affect the toxicity of aflatoxin and other
mycotoxins. Mutation is caused due to binding of
the aflatoxin molecule to DNA and the subse-
quent erroneous protein synthesis. Sarasin et al.
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FIGURE 1. Structure of some commonly occurred aflatoxin molecule.

247
 (1977) revealed that a DNA repair mechanism
occurs in human cells after treatment with acti-
vated AFB1. Aflatoxins, being potent protein syn-
thesis inhibitors, impair differentiation in sensi-
tive primordial cells (Moss and Smith, 1985) and
lead to a teratogenic effect on certain animals.
The structure-activity relationship in toxicity and
carcinogenicity of aflatoxins and analogues were
studied by Wogan et al. (1972).
Aflatoxins may be considered biosynthetic
inhibitors both in vivo and in vitro, with large
doses causing the total inhibition of biochemical
processes and lower doses affecting different meta-
bolic pathways. They inhibit O2 uptake in whole
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tissues by acting on adenosine triphosphatase
enzyme of electron transport chain resulting in
the decreased production of ATP (Morceau and
Moss, 1979). Aflatoxin also reduces hepatic gly-
cogen level, probably by inhibiting glycogenesis
or depression of glucose transport to liver cells or
acceleration of glycogenolysis (Moss and Smith,
1985).
Aflatoxin binds strongly to DNA and RNA.
At the molecular level aflatoxins impart their ef-
fect either interfering with DNA replication or
transcription of messenger RNA into protein.
Clifford and Rees (1967) revealed that the admin-
istration of a single dose of aflatoxin (7 mg/kg
body weight) resulted in slow development of
periportal necrosis. Hepatic enzymes were released
into the serum after 48 h of poisoning. This was
followed by rise in serum phosphatase activity
and bilirubin concentration. Shortly after this study
it was revealed (Clifford and Rees, 1976a and b) that
the biochemical changes underlying the develop-
ment of liver necrosis in the rat after administra-
tion of AFB1 were initiated by the toxin interact-
ing with DNA. This interaction prevented the
RNA polymerase transcribing the DNA and in-
hibited the formation of m-RNA. The failure of
mRNA formation resulted in an inhibition in pro-
tein synthesis that they considered to be the cause
of the liver necrosis. They compared interactions
of AFB1, AFG1, and AFG2 with DNA. Raney et al.
(1993) studied the binding of AFB1 to DNA and
DNA adduction by corresponding epoxide. Their
results demonstrated that duplex structure favors
adduct formation. Adduct yields were compared
for A, B, and Z form of DNA helices. About 12
248
times less adduct was produced from the A form
helix when compared with the B form, while no
adduct was produced from a Z-form duplex. They
concluded that reaction of AFB1–8,9 epoxide
with DNA proceeds via an intercalated transi-
tion state complex only with the B form of the
double helix.
Afltoxin binds with lysine component of se-
rum albumin resulting in the formation of lysine-
AFB1 (Sabbioni, 1990). Ch’ih et al. (1993) inves-
tigated the nuclear translocation of AFB1-protein
complex. The in vitro binding of 3[H] AFB1 to
various proteins was studied by equilibrium di-
alysis. It was reported that at 23°C, 3[H] AFB1
binding activity (mMol/ Mole) decreased as fol-
lows: pyruvate kinase> albumin-NLS> albumin>
carbonic anhydrase> RNase> histone. The nuclear
translocation and activation of AFB1 and AFB1
protein complexes were investigated using iso-
lated rat liver nuclei in the presence of an ATP
and NADPH regenerating system.
Acute and chronic effects of aflatoxins in farm
and laboratory animals as well as human beings are
well documented. Dietary intake of aflatoxin leads
to the disease aflatoxicosis. The risk posed by afla-
toxin depends on the level and type of aflatoxin in
diet, the strain of animal, and its nutritional status.
Repeated ingestion of aflatoxin causes bile duct
proliferation, hepatic necrosis, osteosclerosis of
bone, childhood cirrhosis, immune suppression,
and hepatic veno-occlusive lesions. The reported
outbreaks of aflatoxicosis in men were due to the
consumption of staple foods such as maize and not
due to the consumption of groundnut. Bhat (1989)
reported that groundnut meal contaminated with
aflatoxin caused Indian childhood cirrohsis and
liver cancer.
III. PREVENTION OF AFLATOXIN AT
THE PREHARVEST STAGE
The level of aflatoxin that cause regulatory
concern is continually being lowered and it is
difficult to produce commodities free of these
exceedingly small amounts of aflatoxin. One most
rational and economic approach is to control afla-
toxin problem is preharvest elimination of afla-
toxin. This can be achieved in the following ways.
 A. Ideal Agronomic Practices
Prevention of aflatoxigenic fungal attack is
possible by using healthy seeds, proper irrigation,
rotation of crops, harvesting after full maturity,
drying the harvested crops within time and store
them under proper atmosphere, preventing insect
or other damages to the crop (Santha et al., 1993).
Proper planning of planting, irrigation, harvesting
are to be done (Jones et al., 1981). The recom-
mendations of Food and Agricultural Organiza-
tion (1979) and World Health Organization (1988)
provide some useful guidelines in this regard.
This level of primary prevention is the most im-
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portant and effective plan for reducing fungal
growth and mycotoxin production. Some research-
ers also tried to employ artificial intelligence and
neural network approach to predict and estimate
the aflatoxin contamination at preharvest stage
and peanut maturity (Tollner et al., 1998;
Handerson et al., 1999; Parmer et al., 1997).
B. Breeding Resistant Varieties
A number of researchers have been working
on A. flavus-resistant or -tolerant varieties of corn
(Windstrom and Wilson, 1984; Zuber et al., 1978)
and peanuts (Mixon, 1981; Rao and Tulpule,
1967). This is done by the identification of resis-
tant germplasm and movement into production
lines of the crops. Lillehoj and Zuber (1975) in
several laboratory studies and field work showed
varietal differences in corn with respect to resis-
tance to A. flavus and aflatoxin production. Davis
et al. (1984) performed extensive field studies to
aflatoxin formation by A. flavus. However, the
resistance to invasion of toxigenic fungi has been
attributed to several biochemical, environmental,
and physical factors. Uncontrollable factors could
bring the failure in the utilization of selected fun-
gal-resistant variety.
C. Use of Genetic Engineering
Control of aflatoxin is possible by targeting
mechanisms governing aflatoxin biosynthesis
(Yu et al., 2000; Chang et al., 2000). This can be
achieved with the identification of genes and en-
zymes responsible for aflatoxin synthesis and the
transfer of genes that encode resistant factors in-
hibiting toxin synthesis by genetic engineering of
commercial corn, cotton, and peanut varieties
(Cary et al., 2000), which may lead to the forma-
tion of resistant varieties to mild attack or inhibit
toxin production.
To prevent the contamination of food with
aflatoxigenic fungi is the most suitable approach
for avoiding potential hazards. However, preven-
tion is not always possible under certain agro-
nomic and storage practices, particularly where
environmental conditions provide a congenial at-
mosphere for the growth of fungi and toxin pro-
duction. In this context detoxification is another
option for commodities already contaminated with
toxic fungal metabolites.
IV. DETOXIFICATION OF AFLATOXINS
Some physical methods adopted for the re-
moval of aflatoxin from groundnut meal are widely
accepted, but certain nutrients are destroyed in
the process. A variety of chemicals have been
screened for their ability to react with aflatoxins.
Of which methanol, oxidizing agents, ammonia,
etc. are notable (Samarajeewa et al., 1990). How-
ever, their applicability in foods is restricted by
safety problems that may arise due to chemical
residues. The use of microorganisms for degrada-
tion of aflatoxin in food or feed appears attractive
but may have certain disadvantages, in that the
organism would not only utilize the food for its
growth, but may themselves release undesirable
compounds. The commonly used physical, chemi-
cal, and biological methods of detoxification of
aflatoxin are shown in Table 1. The isolation of
enzyme or enzyme system that can degrade afla-
toxin resulting in a nontoxic or less toxic product(s)
retaining the nutritive value and appearance of the
sample seems to be an effective approach.
Any detoxification procedure should fulfill
the following requirements (Bata and Lasztity,
1999).
• The mycotoxin should be destroyed or trans-
formed to nontoxic compounds.
249
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TABLE 1

250
Some Commonly Used Physical, Chemical, and Biological Methods of Aflatoxin
 • Fungal spores and mycelia should be destroyed
so that new toxins are not formed.
• The food or feed material should retain its
nutritive value and remain palatable.
• The physical properties of raw material should
not change significantly.
• The detoxification process should be economi-
cally feasible, that is, the cost of detoxification
should be less than the value of the treated
commodity.
The detoxification treatments of AFB1 should
be aimed either at removing the double bond of
the terminal furan ring or in opening the lactone
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ring. Once the lactone ring is opened, further
reactions can occur to alter the binding properties
of terminal furan ring to DNA and proteins
(Basappa and Santha, 1996). Such structural
changes can be induced through supply of energy
in a form absorbable by AFB1 or by using any
chemical to block or remove the active sites. Bio-
logical or enzymatic treatments also modify the
structure such that to result some less toxic or
nontoxic compounds. Probable mechanisms of
reactions are shown in Figure 2. In the following
paragraphs biological control methods of afla-
toxin, in vivo metabolism of aflatoxin by enzymes
in animal system, and the possible control of
aflatoxin by dietary modification are discussed in
detail.
V. CONTROL BY MICROORGANISMS
Many microorganisms, including bacteria,
actinomycetes, yeasts, molds, and algae, can de-
grade aflatoxins. In the following paragraphs the
control of aflatoxin by some important bacteria
and fungi and their mode of are discussed.
A. Enzymatic Degradation or Adhesion
of Aflatoxin by Bacteria
Ciegler et al. (1966)a and b screened approxi-
mately 1000 microorganisms for their ability to
either destroy or transform AFB1 and AFG1. Only
one of the bacteria assayed, Flavobacterium
aurantiacum – NRRL B-184, removed AFB1 from
solution irreversibly. Temperature and pH influ-
enced the uptake of the toxin by the cells. A high
population of cells (1011 cells/ml) permanently
removed greater percentages of the aflatoxin from
solutions than did lower populations. Heat-inacti-
vated cells removed some amount of aflatoxin
that could be recovered by water wash (Line and
Brackett, 1995). Toxin-contaminated milk, oil,
peanut butter, groundnuts, and corn were com-
pletely detoxified and contaminated soybeans were
partially detoxified. Duckling assay showed that
detoxification of aflatoxin solution by
F. aurantiacum — NRRL B-184 was complete,
with no new toxic compounds being formed
(Ciegler et al., 1966a). Later, Hao et al. (1988,
1989) supported the efficiency of this organism in
removing AFB1 from peanut milk.
Recent investigation in this field concentrate
on the study of possible mechanisms of degrada-
tion, that is, whether this bacterium actually de-
grades the aflatoxin or the disappearance of the
toxin resulted from adsorption to the cells. Line et
al. (1994) used 14C labeled AFB1 and exposed to
F. aurantiacum to trace and detect the radiola-
beled product with a scintillation counter. The
CO2, AFB1, and water-soluble degradation prod-
ucts and the adsorbed toxin by the cells were
measured. The rate of removal of AFB1 by live
cells were much higher compared with dead cells,
but both these cell types adsorbed some amount
of AFB1. Also, the release of labeled CO2 only by
the living cells shows that some amount of AFB1
is metabolized by flabacteria cells. D’Souza and
Brackett (1998) investigated the role of trace metal
ions (Cu2+, Mn2+, Zn2+, and Co2+) in an effort to
understand the enzymatic system involved in AFB1
degradation by F. aurantiacum. The effect of di-
valent chelators (D’Souza and Brackett, 2000)
and the effect of seryl and sulfhydryl group in-
hibitors were also studied. Copper and Zinc ions
inhibit the degradation of aflatoxin. These effects
show the influence of the enzyme system in the
degradation process (D’Souza and Brackett,
2001). Crude protein extracts (800 g of total pro-
tein/ ml) from F. aurantiacum was tested for its
ability to degrade AFB1 in aqeous solution and
was able to remove 74.1% of the toxin, whereas
only 5.5% of AFB1 was removed in heat-inacti-
vated crude protein. DNase-I treated crude pro-
251
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252
FIGURE 2. Probable mechanism of degradation of Aflatoxin B1.
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FIGURE 3. (a) Biologically reduced AFB1 by T. pyriformis W. (Robertson et al., 1970) Rhizopus sp. (Nakazato et
al, 1990). (b) Hydroxy derivative of AFB1 by Lactobacillus delbrueckii (Maing et al., 1973).

253
 tein extracts degraded 80.5% of AFB1 in solution,
suggesting that removal of of aflatoxin by F.
aurantiacum is not due to nonspecific binding
with the bacterium’s genomic DNA. Proteainase-
K-treated crude protein extracts degraded 34.5%
AFB1, which shows the possible involvement of
enzyme (Smiley and Droughon, 2000). Efficient
degradation occurred at pH 7.
B. Binding of Aflatoxin by Some
Probiotic Bacterial Strains
Karunaratne (1990) showed that Lactobacillus
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acidophilus, L. bulgaricus, and L. planatarum could
be used to either prevent mold growth or to de-
grade aflatoxin. The effect of AFB1 adhesion capa-
bility of Lactobacillus rhamnosus strain GG was
investigated by Kankaanpaa and Tuomola (2000)
using a caco-2 adhesion model. The removal of
AFB1 by the strain reduced its adhesion capability
from 30 to 5%. It was therefore concluded by them
that aflatoxins may influence the adhesion proper-
ties of probiotic bacteria able to sequester them,
and subsequently it may reduce the accumulation
of AFB1 in the intestine via increased aflatoxin–
bacteria complex. Nezami et al. (2000) studied the
adhesion capability of L. rhamnosus strain GG and
LC 705 and Propionibactrium freudenrichhi ssp.
shermani JS with aflatoxin. The complexes formed
in vitro with AFB1 and lactobacilli strains were
stable for 1 h. The binding efficiency of AFB1 at
late exponential and early stationary phase by these
bacteria by pronase E, lipase, m-periodate were
consistent; the effect of urea suggested hydropho-
bic interaction and the effect of NaCl and CaCl2
proved electrostatic interaction, while a pH profile
suggested that the interaction may involve hydro-
gen bonding as well. Viable, heat-killed, and acid-
killed bacteria responded in a similar manner
(Haskard et al., 2000). Bifidobacterium also can
bind to AFB1 efficiently (Peltonen et al., 2000).
C. Biocompetitive Inhibition of Aflatoxin
by Bacteria
In 1995, H. K. Chourasia published his experi-
mental results on kernel infection and aflatoxin
254
production in peanut (Arachis hypogaea L) by
A. flavus in the presence of geocarposporic bacte-
ria. He hypothesized that geocarposporic bacteria
would be ideal for protecting the developing ground-
nut pods against aflatoxigenic fungi. A. flavus was
grown on groundnut extract agar and on viable
groundnut kernels, either in pure culture or in dual
culture with either of Bacillus megaterium,
B. laterosporus, Cellulomonas cartae, F. odoratum,
Phyllobacterium rubiacearum, Pseudomonas
aurofaciens, and Xanthomonas maltophila. Afla-
toxin production by A. flavus, its growth, and inter-
actions with other microorganisms were studied,
and it was concluded that F. odoratum showed
inhibition in aflatoxin biosynthesis.
D. Control by Fungi
Detroy and Hesseltine (1969) studied the
conversion of AFB1 to aflatoxicol by steroid
metabolizing fungi. However, the process was
very slow and incomplete (60% detoxification).
Tetrahymena pyriformis W decreased the con-
centration of 2 µg AFB1/ml by 67% in 48 h with
production of a biologically reduced AFB 1
(Robertson et al., 1970) as shown in Figure 3.
The carbonyl in cyclopentane ring of AFB1 was
reduced to a hydroxyl. The toxicity and mutage-
nicity of this treated product were not deter-
mined. Hamid and Smith (1987) showed that
molds capable of producing aflatoxin could also
degrade them. The production of aflatoxin by
A. flavus and A. parasiticus reached a maximum
and then decreased. Aflatoxin-degradative ac-
tivity was demonstrated in 6- to 12-day-old
mycelium and cell-free extracts of A. flavus. The
addition of cycloheximide, SKF 525-A, or
metyrapone to cultures of A. flavus prevented
the subsequent degradation of the toxin. In cell-
free extracts, degradation was inhibited by SKF
525-A, metyrapone, and cytochrome-c but not
by KCN. In all the cell-free extracts aflatoxin
degradation was enhanced by NADPH and
NaIO4. They concluded the probable involve-
ment of cytochrome P-450 mono-oxygenases in
the aflatoxin-degradative activity of A. flavus.
Cole et al. (1972) investigated the conversion of
AFB1 to isomeric hydroxy compounds by Rhizo-
 pus species (Figure 3). Two new fluorescent com-
pounds thus produced were characterized by them
using 14C-labeled AFB1 as stereoisomers of hy-
droxylated AFB1. Tsubouchi et al. (1980) stud-
ied degradation of AFB1 by Aspergillus niger.
Interconversion of AFB1 and aflatoxicol by sev-
eral fungi was studied by Nakazato et al (1990).
They screened four fungal strains viz, A. niger,
Eurotium herbariorum, a Rhizopus sp. and
nonaflatoxigenic A. flavus that could convert AFB1 to
aflatoxicol. They concluded that the interconversion
occurred due to intracellular enzymes of A. flavus and
Rhizopus sp. The greenhouse experiment performed
by Chourasia and Sinha (1994) revealed the potential
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of biological control of aflatoxin in developing peanut
by atoxigenic strains of A. flavus. They inoculated
both the strains in root region of 1 to 2 weeks old
peanut plants in preharvest stage. Of the seven
atoxigenic strains tested, six showed reduction in A.
flavus contamination.
Overall, it can be concluded from the above
studies that very few microorganisms can effec-
tively degrade aflatoxin. In the process they may
release certain more toxic compounds. So the use
of these organisms to detoxify aflatoxin in com-
mercial scale needs to be investigated further.
VI. METABOLISM OF AFLATOXIN
Some important aspects of metabolic and en-
zymatic detoxification of aflatoxin are summa-
rized in Table 2.
A. Metabolism by Hepatic Enzymes
Neal and Colley (1978) performed some
HPLC studies on the metabolism of aflatoxins by
rat liver microsomal preparations. The metabo-
lites found in reverse phase column chromatogra-
phy were AFM1, AFQ1, and a compound that co-
chromatographed along with a degradation product
of AFB1 2,3–dihydrodiol. The time course of
metabolism of AFB1 by microsomal preparations
isolated from control and phenobarbitone–pre-
treated rats was examined. The rate and extent of
metabolism were greater with microsomal prepa-
rations from the latter.
B. Nuclear Metabolism
The nuclear biotransformation of AFB1 in
vitro was observed by Yoshizawa et al. (1981)
with regard to inducer specificity, pH dependency,
time course, kinetics, inhibitor sensitivity, and
nuclear localization, and these data compared with
those from the microsomal transformation of
AFB1. Like microsome, the nucleus was also ca-
pable of metabolizing AFB1 into AFM1, AFQ1,
and two unidentified compounds in presence of
fortified NADPH-generating system.
C. Rat Blood Components
Chang et al. (1985) developed a modified
procedure for preparing alginate gel to immobi-
lize rat erythrocytes. It showed high enzyme ac-
tivity for the reduction of AFB1 to aflatoxicol.
The half life of such preparation was 10 days and
could be used repeatedly at 37°C in batch mode
without a substantial loss in enzyme activity.
Hemolysis of immobilized enzymes was not ob-
served after prolonged storage at 4˚C. Compared
with the free animal cells, immobilized rat eryth-
rocytes were temperature resistant.
Dirr and Schabort (1986) revealed the mecha-
nism of transport of AFB1 in rat blood plasma
binding of AFB1 to albumin in vivo and in vitro and
performed spectroflurimetric studies of the nature
of interaction of these macromolecules to AFB1. In
1987 they characterized the AFB1 binding site in
rat albumin. A fluorescence enhancement method
was used to investigate the noncovalent interaction
between AFB1 and rat albumin. Dissociation con-
stant was calculated to be 20 µM at 20°C.
D. Cytochrome p-450 and Glutathione-S-
Transferase
Lotlikar et al. (1989) investigated the mecha-
nism of phenobarbital pretreatment of rats on both
hepatic AFB1-DNA binding and AFB1-glutathione
conjugation. In rats fed with 0.1 % PB for 1 week
liver weight and microsomal protein content was
increased to 27 and 38%, respectively. They con-
cluded that the induced cytosolic glutathione-S-
255
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TABLE 2

256
Metabolic and Enzymatic Detoxification of Aflatoxin
 transferases after PB treatment of rats played a
significant role in inhibiting hepatic AFB1-DNA
binding and hepatic carcinogenesis, presumably
by inactivation of the AFB1-epoxide. Some role
also might be for the inactivation of AFB1 by
cytochrome P-450 mediated hydroxylation for the
inhibition of hepatic AFB1-DNA linking. Hayes
et al. (1991) revealed that the toxicity of AFB1
was a result of it being metabolized to AFB1-8,9
epoxide, a reaction catalyzed in the rat by cyto-
chrome P-450 isozymes. The same enzymes were
also responsible for the conversion of AFB1 into
less toxic metabolites, AFQ1, AFM1, AFP1. Once
formed, this epoxide did not necessarily produce
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genotoxic/cytotoxic damages, because it could also
be inactivated through the formation of AFB1
dihydrodiol catalyzed by epoxide hydrolase or
through formation of an AFB1-GST conjugate by
GST. The latter is the major biliary metabolite
produced in rat liver. They schemed a purification
process for 2 ethoxyquin alpha class glutathione-S-
transferases that possessed 25-fold greater activity
to AFB1-8,9 epoxide than that exhibited by the
other GSTs. They identified Yc2 subunit of GST,
which had high catalytic activity toward AFB1.
Daniels and Massey (1992) investigated the role
of polycyclic aromatic hydrocarbon-inducible forms
of cytochrome-P 450 in the pulmonary and hepatic
microsomal activation (3[H] AFB1-DNA binding)
and detoxification (3[H] AFM1 and 3[H] AFQ1 for-
mation) of 3[H] AFB1. They determined Km and
Vmax of each reaction using different combinations
of alpha and beta napthoflavone and in the presence
or absence of NADPH-generating systems. Their
results indicated an important role for the 1A sub-
class of P-450 isozymes in the biotransformation of
AFB1 to AFM1 in rabbit lung and liver and a minor
role in AFB1 activation in liver.
E. Tissue-Specific Bioinactivation of
Aflatoxin
Schlemper et al. (1991) performed in vitro stud-
ies with rat liver parenchymal, Kupffer and endothe-
lial cells isolated from male Sprague–Dawley rats to
investigate cell-specific bioactivation of AFB1, DNA
binding, and adduct formation. In the mutagenicity
studies using homogenates of all three separated
liver cell populations (co-incubated with NADP+
and Glucose–6–phosphate as cofactors). Parenchy-
mal, Kupffer and endothelial cells were able to ac-
tivate AFB1 to epoxide. The incorporation of 3[H]
AFB1, HPLC analysis and DNA hydrolyzates of all
three cell types showed the major adduct to be 8,9–
dihydro-8–(N7–guanyl)–9–hydroxy-AFB1.
F. Aldehyde Reductase
Ellis et al. (1993) isolated an ethoxyquin-
inducible aldehyde reductase from rat liver that
metabolizes AFB1. It belongs to the aldo-keto-
reductase subfamily. They suggested that protec-
tion of liver against the toxic and carcinogenic
effects of AFB1 can be achieved by induction of
detoxification enzymes by chemoprotectors such
as phenolic antioxidant ethoxyquin. They cloned
and sequenced c-DNA encoding aldehyde reduc-
tase (AFB1-AR), expressed in Escherichia coli.
The purification of an recombinant enzyme re-
vealed that it was capable of converting the pro-
tein binding dialdehyde form of AFB1-dihydrodiol
to the nonbinding dialcohol metabolite. AFB1-
AR represents the only carcinogen-metabolizing
aldehyde reductase identified so far that is in-
duced by a chemoprotector.
G. Recombinant Yeast Cells
Pelkonen et al. (1994) studied the ability of
three highly homologous mouse liver CYP 2A
enzymes to activate AFB1 by expressing them in
recombinant yeast cells. The reconstituted
monooxygenase complex with CYP 2A 5 puri-
fied from yeast microsomes produced AFB1-ep-
oxide at a rate of 17.2 nMol/min/nMol P-450 in
presence of 50 µM AFB1, while CYP 2A 4 had
about 10% and P-4507 alpha had only 1.5% of
this activity. They studied the effect of coumarin,
an inhibitor of CYP 2A 4 and performed kinetic
study as well. Their data demonstrated that highly
homologous mouse CYP 2A 4 enzymes activate
AFB1 in a different manner, and recombinant yeast
cells expressing mammalian CYP enzymes are a
useful and inexpensive system to test the role of
different enzymes in AFB1 toxicity.
257
 H. Metabolsim by Human c-DNA
The kinetics of oxidation by human c-DNA-
expressed human liver microsomal cytochrome
P-450 1A-2 and 3A-4 was studied by Gallagher et
al (1996). AFB1-oxide formation by cDNA–ex-
pressed human CYP1A-2 followed Michaelis–
Menten kinetics (Km = 41 µM, Vmax = 2.63 nMol/
min/nMol P-450). The formation of AFB1-oxide
(activated product) in cDNA-expressed human
CYP 3A 4 microsomes was sigmoid and consis-
tent with the kinetics of substrate activation.
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I. Role of Peroxidase Group of Enzymes
in the Detoxification of Aflatoxin
Doyle and Marth (1979) revealed that
A. parasiticus is capable of producing peroxidase
and degrade AFB1. They also showed a direct
correlation between the amount of peroxidase
produced and AFB1 degraded. Ellis et al. (1991)
concluded that the biological degradation reac-
tions might occur through enzymatic activity, and
these enzymes produced products or byproducts
that react with aflatoxins. Peroxidase was specu-
lated to be one such enzyme since it catalyzes the
decomposition of hydroperoxides to produce free
radicals, which may then react with aflatoxin.
Furthermore, certain peroxidases produce hy-
pochlorite and singlet oxygen in the presence of
hydrogen peroxide and chloride ion (Allen, 1975).
According to Singh and Smith (1991), the high-
temperature metabolism of A. flavus may lead to
microsomal peroxidase-dependent detoxification
of aflatoxins.
Horseradish peroxidase (HRP) being a well-
characterized, thermostable enzyme and having
been reported to detoxify some xenobiotic com-
pounds, a study was undertaken with HRP as the
detoxifying enzyme in an in vitro assay system
(Das and Mishra, 2000a; Das and Mishra, 2000b).
The plant source of this enzyme is most cheap and
convenient to use. AFB1–HRP reaction in phos-
phate buffer was studied with respect to varied
substrate and enzyme concentration, pH depen-
dency, time course, cofactor specificity, kinetics,
and inhibitor sensitivity. The optimum enzymatic
reaction occurred in 50 mM phosphate buffer at
258
45°C temperature, pH 6.0, incubation time 45
min, 5 M H2O2, and normal pressure using 4 U of
HRP per mM of AFB1 in liquid medium, resulting
77.1% detoxification. In infected groundnut meal
(GNM) optimum reaction occurred at 8 U of HRP
used per mM of AFB1 for a 1-day incubation
period and 20 mM hydrogen peroxide. Moisture
content of the reaction mixture was maintained
between 12 to 15%. Calcium propionate 0.3 g/
100 g of GNM was used as fungistat. The reaction
resulted in 69% detoxification. Kinetic and ther-
modynamic parameters of AFB1–HRP reaction
were determined. Toxicity and mutagenicity of
the treated commodity were tested by animal feed-
ing test and Ames test, respectively. The experi-
mental results showed the detoxified product could
safely be used by living organisms.
J. Factors Affecting the Cellular
Metabolism of Aflatoxin
Chemoprotectors are the main factors that
influence the detoxification of aflatoxin metabo-
lism in vivo. The phenobarbitone pretreated rats
have an increased efficiency of afltoxin metabo-
lism. The formation of AFQ1 and aflatoxicol was
enhanced by 4- to 5-fold by phenobarbitone pre-
treatment. This result was later supported by
Shephard et al. (1984). AFB1 metabolism by he-
patic microsomes from polychlorinated biphenyls
(PCB) and polybrominated biphenyls (PBB)
treated rats resulted in 16- to 30-fold increase,
respectively, in AFM1 level. Some nuclear com-
ponents leading toward detoxification, as discussed
earlier, need a NADP + generating system.
Mc Lellan et al. (1994) investigated the regula-
tion of AFB1-metabolizing aldehyde reductase and
glutathione–S-transferase by chemoprotectors. In
the earlier investigation they showed Yc2 subunit
of GST had high activity toward AFB1–8,9 ep-
oxide. To allow an assessment of whether the
increased expression of GST Yc2 represents a
general adaptive mechanism that is invoked by
chemoprotectors and carcinogens, they examined
the effects of butylated hydroxy-anisole (BHA),
butylated hydroxy-toluene (BHT), phenobarbital
(PB), etc. on Yc2 subunit expression and AFB1-
AR production. They found a high amount of
 alpha class GST, including Yc2 in various rat
tissues. The highest level of GSH conjugating
activity to AFB1–epoxide was found in epididy-
mis. The highest level of AFB1-AR was found in
the kidney followed by moderate levels in liver
and testis. Other extra-hepatic organs such as heart,
lung, spleen, adrenal gland, epididymis, vas def-
erence, seminal vesicles, and ovary contain much
lower levels of AFB1-AR.
Although there have been many earlier re-
searches on physical, chemical, and biological
detoxification of aflatoxin, it is clear from above
discussion that the present trend is to use enzyme
as the mode of detoxification. However, most of
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the work on enzymatic detoxification was per-
formed in vivo and the conditions are shown in
Table 3. Those dealt mainly with conventional
xenobiotic-degrading enzymes and needed
chemoprotectors as inducer. If those are used in
vitro, they need a NADPH-generating system.
From the accumulated results on cellular interac-
tions and metabolism of aflatoxin, it can be con-
cluded that AFB1 must be converted to its reactive
8,9-epoxide form to exert its effects, and this
epoxide in turn reacts with cellular macromolecules
(McLellan and Dutton, 1995). Cytochrome P-450
enzymes catalyzed hydroxylation of AFB1 to AFQ1
and AFM1 and demethylation to AFP1. Conjuga-
tion of AFB1 to GST and its subsequent excretion
was regarded as an important detoxification path-
way in animals. Aldehyde reductase is another
promising enzyme that also leads to the detoxifica-
tion of aflatoxin. Horseradish peroxidase has the
potential to be used to detoxify aflatoxin in vitro.
VII. DIETARY MODIFICATION AS A
SOLUTION TO THE AFLATOXIN
PROBLEM
Chen et al. (1995) showed that dietary restric-
tion reduced the metabolic activation of AFB1 in
rats. This reduction might be attributed to the
decrease of cytochrome P-450-mediated AFB1-
epoxidation and/or increase in the detoxification
of AFB1 catalyzed by hepatic GST and other
phase II detoxification enzymes. Galvano et al.
(2001) proposed dietary modifications as a help-
ful tool to overcome aflatoxin-induced diseases.
They studied the effect of antioxidant compounds
(selenium, vitamins, provitamins), food compo-
nents (phenolic compounds, coumarin, chloro-
phyll, and its derivatives, fructose, aspartane),
medicinal herbs and plant extracts, and mineral
and biological binding agents (hydrated sodium
calcium alumino silicate, bentonites, zeolites, ac-
tivated carbons, bacteria and yeast). Compounds
having antioxidant properties have the ability to
act as superoxide anion scavengers. Some veg-
etables also showed interesting efficiency. Some
herbs and medicinal plants provide protection
against AFB1. Activated carbons, hydrated so-
dium calcium aluminosilicate (HSCAS), and some
bacteria seemed to effectively bind the toxin.
Another simple but very effective approach was
to use HSCAS clay in the diet that act as an
aflatoxin enterosorbent that tightly and selectively
binds these poisons in the gastrointestinal tract of
animals, decreasing their bioavailability and as-
sociated toxicities (Phillips, 1999). Further stud-
ies to know the molecular mechanisms of action
have shown that the dicarbonyl system of afla-
toxin is essential for tight binding by HSCAS.
Evidence suggests that aflatoxins may react at
multiple sites on the clay particle especially
interlayer regions, edges, and basal surfaces.
However, there may be certain risk factors of the
inclusion in diet before properly tested. Coumarin,
a natural food constituent, offers chemoprotection
against AFB1 (Goeger et al., 1998).
VIII. CONCLUSION AND FUTURE SCOPE
OF WORK
Degradation by selective microorganisms pre-
sents the possibility of detoxification of aflatoxin
under mild conditions without using harmful
chemicals and without significant losses in nutri-
tive value. However, acceptability and palatabil-
ity of the food and feed is yet to be confirmed.
The explosion in commercial and synthetic appli-
cations of enzymes has stimulated much interest
in enhancing functionality and stability. Catalyti-
cally self-sufficient cytochrome P-450 reductase
fusion proteins and E. coli and baculovirus
coexpression constructs can be engineered
(Guenrich and Parikh, 1997) and employed for
259
 the successful metabolism of aflatoxin. Janssen
and Schanstra (1994) reviewed protein engineer-
ing for environmental application. New insight
was obtained into the structure and catalytic
mechanism of enzymes that convert environmen-
tal pollutants and toxins. Recent advances in this
field have made it possible to use this information
for improving catalytic performance of engineered
enzymes to achieve increased stability and ex-
panded substrate range. De Santis and Jones (1999)
performed the covalent chemical modification of
enzymes for enhanced functionality. They con-
verted hydrolase into peroxidase. The difficulty
they faced was the lack of chemo- or regio-speci-
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ficity of chemically modified enzymes that yielded
heterogeneous and irreproducible products. AFB1,
being insoluble in water and soluble in organic
solvents, limits its detoxification in an aqueous
environment in which the enzymes are generally
active. Khmelnitsky and Rich (1999) reviewed
biocatalysis in nonaqeous solvents that may over-
come this limitation. However, dietary strategies
may be the cheapest and easiest acceptable way to
overcome aflatoxin problem. Apart from this,
research on modified atmosphere storage of detoxi-
fied commodity should also be taken care of.
Immobilized HRP enzyme may hold promise if
used to detoxify the toxin in liquid commodities.
ACKNOWLEDGMENT
The Council of Scientific and Industrial Re-
search, New Delhi, India, financially assisted this
work and the same is thankfully acknowledged.
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