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Food and Chemical Toxicology 46 (2008) 2124–2127

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Changes in spontaneous contractions of rat ileum by aflatoxin in vitro

Nevcihan Gursoy a,*, Bulent Sarac b, Nedim Durmus b, Ahmet Parlak b, Sahin Yildirim b,
Tijen Kaya b, Ihsan Bagcivan b
a
Faculty of Engineering, Department of Food Engineering, Cumhuriyet University, 58140 Sivas, Turkey
b
Department of Pharmacology, School of Medicine, Cumhuriyet University, 58140 Sivas, Turkey

Received 28 December 2007; accepted 4 February 2008

Abstract

Aflatoxins are a group of mycotoxins produced by toxigenic strains of Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius
as secondary metabolites. Most of the studies on the aflatoxins have focused mainly on their chronic toxic effects but aflatoxins have also
a lot of acute effects on the respiratory, cardiovascular and gastrointestinal systems. In this study the acute gastrointestinal effects of the
aflatoxins on rat isolated ileum and the possible mechanisms underlying contractile responses to them were investigated. Aflatoxin
increased both of the amplitude and the frequency of spontaneous contractions in a dose-dependent manner. Pretreatment with a cho-
linergic system inhibitor, atropine sulfate (23.6 nM), a specific sodium-channel blocker, tetrodotoxin (0.3 lM) and an inhibitor of ACh
release from terminal motor neurons, morphine (0.3 lM) decreased both of aflatoxin induced spontaneous contractions’ amplitude and
frequency, in contrast a nicotinic ganglionic blocker, hexamethonium chloride (55 lM) did not change the aflatoxin effect. But the
decrease of amplitude was more than the frequency in the presence of these antagonists. In conclusion, these findings of aflatoxin on
isolated rat ileum may explain their acute gastrointestinal effects in humans and animals.
Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Aflatoxin; Amplitude; Frequency; Ileum

1. Introduction tural commodities are contaminated with mycotoxins, lead-

ing to significant economic losses (Galvano et al., 2001;
Aflatoxins are a group of mycotoxins produced by toxi- Atroshi et al., 2002; Aziz and Moussa, 2002).
genic strains of Aspergillus flavus, Aspergillus parasiticus Various foods may support the growth of these micro-
and Aspergillus nomius as secondary metabolites (McLean organisms and consequently lead to contamination with
and Dutton, 1995). Aflatoxins are the collective term for four aflatoxins (Wood, 1992). The contamination may take
major naturally occurring secondary compounds (B1, B2, G1 place during growth, harvest, transportation and storage
and G2) produced from acetate unit via the polyketide path- and thus is difficult to prevent. Aflatoxins are considered
way with the contribution of methionine to methoxy-methyl by the US Food and Drug Administration (FDA) to be
group (Sweeney and Dobson, 1998; Creppy, 2002). Up until an unavoidable contaminant of foods. The occurrence of
now, approximately 400 secondary metabolites with toxi- toxigenic Aspergillus is worldwide but the problem is espe-
genic potential produced by more than 100 moulds, have cially prevalent in developing countries. Techniques for the
been reported, with the Food and Agriculture Organization elimination or inactivation of aflatoxins include physical,
(FAO) estimating that as much as 25% of the world agricul- chemical and biological methods (Park, 1993). However,
no effective, universally applicable and practical methods
*
are currently available.
Corresponding author. Tel.: +90 346 2191010x2889; fax: +90 346
2191179.
The toxic effect of mycotoxin ingestion in both humans
E-mail addresses: gursoy_gu@hotmail.com, ngursoy@cumhuriyet. and animals depends on a number of factors includ-
edu.tr (N. Gursoy). ing intake levels, duration of exposure, toxin species,

0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2008.02.005
 N. Gursoy et al. / Food and Chemical Toxicology 46 (2008) 2124–2127 2125

mechanisms of action, metabolism, and defense mecha- preloaded with 1–1.5 g tension. Tissues were allowed to equilibrate for
nisms (Hussein and Brasel, 2001a,b). Consumption of 30 min.
After equilibrium, at the beginning of each experiment, 80 mM KCl
mycotoxin-contaminated food or feed does however lead was added to the bath and the contraction was considered as reference
to the induction of teratogenic, carcinogenic, oestrogenic, response. Following KCl response, smooth muscle segments were allowed
neurotoxic, and immunosuppressive effect in humans to equilibrate for 30 min before addition of aflatoxin cumulatively at
and/or animals (Atroshi et al., 2002). 10 8–10 5 M doses.
Studies in various animal species have reported acute Isometric tension was recorded on a Grass model 79 E polygraph. To
ensure a maximal effect, ileum segments were incubated with aflatoxins for
aflatoxin induced effects on the respiratory, cardiovascular 2 min. All experiments were repeated on four different preparations
and gastrointestinal systems (Lougheed et al., 1995; Bortell obtained from different animals. Then a cholinergic system inhibitor
et al., 1983; Oyelami et al., 1997; Guengerich et al., 1998). atropine sulfate (23.6 nM), a nicotinic ganglionic blocker hexamethonium
In the bovine species, Cook et al. (1986) found that acute chloride (55 lM), a specific sodium-channel blocker tetrodotoxin
AFB1 intoxication alters the amplitude or frequency or (0.3 lM), and morphine (0.3 lM) which is an inhibitor of ACh release
from terminal motor neurons were added to the bath 20 min before the
both of rumen contractions in a dose-dependent manner. submaximal dose of aflatoxin (10 6 M) (Luzi et al., 2002).
Abdel-Haq et al. investigated the acute actions of four Changes in the amplitude of spontaneous contractions in the presence
common aflatoxins on isolated organs, and AFB1 and and absence of antagonists were calculated as a percentage of the con-
AFG1 relaxed guinea pig isolated tracheal tissue (Abdel- traction caused by KCl (80 mM) on ileum smooth muscle isolated from
Haq et al., 2000a) and stimulated guinea pig isolated atria rats (number of ileum segments for each antagonist = 8). The frequency
(number/min) changes of spontaneous contractions in the presence and
(Abdel-Haq et al., 2000b). They concluded that these absence of antagonists were expressed as a percentage of the initial
effects were probably mediated by inhibition of cyclic spontaneous contractions for 10 min intervals (number of ileum segments
nucleotide phosphodiesterase activity, as demonstrated for each antagonist = 8).
for AFB1 (Bonsi et al., 1999). At the end of all experiments we repeated the KCl contraction
Peraica et al. (1999) investigated the acute gastrointesti- responses which were not statistically different from the beginning of
experiment.
nal toxicity of aflatoxins in humans and described the
symptoms of vomiting, diarrhoea, pyrexia, abdominal
pain, ascites and severe gastrointestinal erosions. They 2.3. Drugs
could not explain the pharmacological mechanisms under- Aflatoxin, dimethyl sulfoxide (DMSO), atropine sulfate, hexametho-
lying how the aflatoxins cause diarrhoea and other intesti- nium chloride, tetrodotoxin and morphine were obtained from Sigma
nal effects. Luzi et al. (2002) studied acute effects of Chemical Co. (St Louis, MO, USA). Aflatoxin was dissolved in DMSO at
aflatoxins on guinea pig isolated ileum and concluded that a concentration of 2 mg/ml stock solutions and stored at 0–5 °C, all the
the aflatoxins cause acute gastrointestinal effects on iso- other substances were dissolved in deionized water and ethanol (1:1) as
1 mg/ml stock solutions; stock solutions were diluted in deionized water.
lated tissues and act indirectly by stimulating ACh release
from nerve terminals.
In this study we aimed to investigate the acute gastroin- 2.4. Statistical analysis
testinal effects of total aflatoxin on rat isolated ileum All data are expressed as mean ± SEM. Groups were compared sta-
in vitro and the possible mechanisms underlying contractile tistically using general linear models of ANOVA followed by Newman–
responses to them. Keuls test and, t-test when appropriate. Differences were considered to be
significant when p < 0.05.

2. Materials and methods

3. Results
2.1. Animals
Aflatoxin was given cumulatively at 10 8–10 5 M con-
Sixteen male Wistar albino rats each weighing approximately 280 g
were maintained in accordance with the recommendations of the Guide
centrations and the amplitude of ileum contractions was
for the Care and Use of Laboratory Animals and the experiments were increased significantly at 10 6 M and 10 5 M doses com-
approved by Cumhuriyet University—Medical Faculty, Animal Care pared with vehicle induced contraction amplitude
Committee and rats were acclimatized to a 12 h light/dark cycle at 23 °C (Fig. 1A) (p < 0.05). In the presence of a cholinergic system
with food and water available ad lib. inhibitor atropine sulfate (23.6 nM), a specific sodium-
channel blocker tetrodotoxin (0.3 lM) and morphine
2.2. Isometric measurements (0.3 lM) which is an inhibitor of ACh release from terminal
motor neurons, the amplitude of aflatoxin induced sponta-
The rats were killed by cervical dislocation and the abdomen was
opened with a midline incision of approximately 3 cm. Specimens of neous contractions decreased significantly (p < 0.05), but a
1.5 cm ileum were resected. After resection, specimens were isolated from nicotinic ganglionic blocker hexamethonium chloride
their fat and mesenteric tissue and their lumens were washed out with (55 lM) did not change the amplitude of aflatoxin induced
previously aerated (95% O2 and 5% CO2) Krebs bicarbonate solution spontaneous contractions (Fig. 1B).
(KBS) (composition in mM: NaCl, 120; KCl, 4.6; CaCl2, 2.5; Mg Cl2, 1.2;
Similarly frequency of ileum contractions was increased
107 NaHCO3, 22; NaH2PO4 and glucose 11.5). Whole full-thickness seg-
ments of ileum was placed in circular direction in a 10 ml tissue baths, significantly at 10 6 M and 10 5 M doses of aflatoxin com-
filled with pre-aerated KBS at 37 °C. The upper end of the preparations pared with vehicle induced contraction frequency (Fig. 2A)
was tied to an isometric transducer (Grass FT 03, Quincy, MA, USA) and (p < 0.05). Atropine, tetrodotoxin and morphine decreased
2126 N. Gursoy et al. / Food and Chemical Toxicology 46 (2008) 2124–2127

Fig. 1. The effect of cumulative aflatoxin doses (AF) on the amplitude of Fig. 2. The effect of cumulative aflatoxin doses (AF) on the frequency of
rat ileum spontaneous contractions (A) and the amplitude change of rat ileum spontaneous contractions (A) and the frequency change of
submaximal aflatoxin induced spontaneous contractions in the presence of submaximal aflatoxin induced spontaneous contractions in the presence of
atropine sulfate (ATR), hexamethonium chloride (HEXA), tetrodotoxin atropine sulfate (ATR), hexamethonium chloride (HEXA), tetrodotoxin
(TTX) and morphine (MORP) (B). Data are expressed as the (TTX) and morphine (MORP) (B). Data are expressed as the means ±
means ± SEM. * P < 0.05 denotes significant difference from control SEM. * P < 0.05 denotes significant difference from control groups.
**
groups. ** P < 0.05 denotes significant difference from all groups. P < 0.05 denotes significant difference from all groups.

the frequency of contractions induced by aflatoxin signifi-

cantly (p < 0.05) but hexamethonium did not (Fig. 2B). Aflatoxin can cause both acute and chronic toxicity in
Interestingly, the decrease at the amplitude of spontane- animals and no animals species is resistant to the acute
ous contractions by aflatoxin was more significant than the toxic effect of aflatoxin. Acute toxicity is produced by mod-
frequency in the presence of these antagonists. erate to high levels of aflatoxins. Specific, acute episodes of
disease ensue may include hemorrhage, acute liver damage,
4. Discussion edema, alteration in digestion, absorption and/or metabo-
lism of nutrients, necrosis, cirrhosis, cholinergic symptoms
Aflatoxins have received a considerable amount of and possibly death (Cook et al., 1986; CAST, 2003). The
attention due to their ability to produce both immunosup- only study investigating the acute effect of aflatoxins in
pressive and carcinogenic effect on animals and humans. humans, describes symptoms of vomiting, diarrhea,
Research on aflatoxin over the last 40 years has made it abdominal pain and severe gastrointestinal erosions (Pera-
one of the best studied fungal secondary metabolites. In ica et al., 1999). Reported mortality rates in the acute phase
spite of the large volume of research in this area, many range from 10 to 60% (Tandon et al., 1978; Chao et al.,
unanswered questions remain concerning the genetic regu- 1991; Peraica et al., 1999). More recently, the acute effects
lation of aflatoxin production and the molecular signals on gastrointestinal, respiratory, cardiovascular and central
that intimately associate the synthesis of aflatoxin with spe- nervous systems, have been reported both in humans and
cific nutritional and environmental conditions (Scheidegger animals after exposure to common aflatoxins (Hussein
and Payne, 2003). and Brasel, 2001a,b; IARC, 1993; Peraica et al., 1999).
Humans and animals can be exposed to aflatoxin by The many studies on the aflatoxins have focused mainly
consuming food products contaminated with mainly A. fla- on the chronic toxic effects because of their carcinogenicity,
vus, A. parasiticus. The fungi can colonize most food prod- hepatotoxicity and mutagenicity (Peraica et al., 1999; Hus-
ucts during processing and storage, but the pathogenic sein and Brasel, 2001a,b). According to some investiga-
ability of Aspergillus also enables it to infect crops such tions, the cause of abnormalities are immunosuppression
as corn, peanuts, cotton, and tree nuts long before harvest of organ extreme getting fat, coagulation and impairing
(Brown et al., 1999). protein structure (Steyn, 1995; Farfan-Amaya, 1999).
 N. Gursoy et al. / Food and Chemical Toxicology 46 (2008) 2124–2127 2127

In line with in vivo experiments on acute aflatoxin intox- Brown, M.P., Brown-Jenco, C.S., Payne, G.A., 1999. Genetic and
ication (Cook et al., 1986; Luzi et al., 2002), in our study molecular analysis of aflatoxin biosynthesis. Fungal Genetics and
Biology 26 (2), 81–98.
aflatoxin evoked acute effects on rat isolated ileum but dif- CAST, 2003. Mycotoxins. Risks in plant, animal, and human systems.
ferently it increased both of frequency and amplitude of Task Force Report, No. 139. Council for Agricultural Science and
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