55, 162–170 (2000)
Copyright © 2000 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Relaxant Effects of Aflatoxins on Isolated Guinea Pig Trachea
Hanin Abdel-Haq, Maura Palmery, Maria Grazia Leone, Luciano Saso, and Bruno Silvestrini 1
Department of Pharmacology of Natural Substances and General Physiology, University of Rome La Sapienza, Rome, Italy
Received July 18, 1999; accepted December 10, 1999
Dyspnea is one of the symptoms of acute aflatoxicosis. Contrary
to expectations, we observed that naturally occurring aflatoxins
(AF) AFB 1, AFB 2, AFG 1, and AFG 2 and their major metabolites
AFM 1, AFM 2, AFP 1, AFQ 1, and AFG 2a relaxed carbachol (C)
precontracted guinea pig trachea to different degrees. The efficacies but not the potencies of AFB 1, AFB 2, AFG 1, and AFG 2 were
similar to that of the ␤-agonist, isoprenaline, whose activity was
potentiated by the AF. Their mechanism of action is not clearly
understood but several mechanistic indications were obtained with
AFB 1: 1) its effect was not influenced by the ␤-blocker, timolol,
indicating that a direct interaction with ␤ 2-adrenergic receptors
was not involved. 2) AFB 1 potentiated PGE 1 and PGE 2, two
relaxant prostaglandins, and its activity was reduced by indomethacin. 3) The cAMP level in the guinea pig trachea relaxed by AFB 1
increased, possibly due to inhibition of phosphodiesterase; direct
interaction with PG receptors; and/or interaction with A 2 adenosinic receptors, suggested by the inhibitory activity of XAC, a
specific antagonist. 4) Finally, since tetrodotoxin reduced the
relaxant activity of AFB 1, it is speculated that this mycotoxin
could stimulate inhibitory nonadrenergic, noncholinergic nerves
(i-NANC). In conclusion, the symptoms of acute aflatoxicosis do
not seem to be due to a direct activity on the tracheal muscle, but
rather, to the well-known pro-inflammatory activity of the aflatoxins, which are capable of releasing arachidonic acid from cell
membranes.
Key Words: aflatoxins; acute aflatoxicosis; guinea pig trachea;
relaxation.
Aflatoxins are a group of closely related mycotoxins
produced by Aspergillus flavus (AFB 1 and AFB 2 ), A. parasiticus (AFB 1 , AFB 2 , AFG 1 , and AFG 2 ), and other fungi
(Mclean and Dutton, 1995). Chronic intoxication with AFB 1
is associated with liver (Eaton and Gallagher, 1994; Linsell
and Peers, 1972; 1977; Saracco, 1995) and lung carcinogenesis (Dvorackova, 1976; Dvorackova et al., 1981; Dvorackova and Pichova, 1986; Hayes et al., 1984; Massey,
1995), due mainly to its biotransformation to the 8,9-epoxide derivative, which forms stable adducts with endogenous
1
To whom correspondence should be addressed at the Department of
Pharmacology of Natural Substances and General Physiology, University of
Rome La Sapienza, P. le Aldo Moro 5, 00185 Rome, Italy. Fax: ⫹39-649912480. E-mail: [email protected].
proteins and nucleic acids (Eaton and Gallagher, 1994;
Guengerich et al., 1998; Massey et al., 1995; McLean and
Dutton, 1995). In the liver, this metabolic reaction is catalyzed chiefly by mixed function mono-oxygenase enzyme
systems (cytochrome P450-dependent), while in the lung, a
co-oxidative mechanism that involves the enzymes cyclooxygenase and lipoxygenase also operates (Battista and
Marnett, 1985; Donnelly et al., 1996; Liu et al., 1990; Liu
and Massey, 1992; Massey et al., 1995).
On the other hand, certain symptoms of acute aflatoxicosis
such as lung congestion, respiratory distress, cough, dyspnea,
pulmonary edema, and alveolar damage (Brucato et al., 1986;
Clark et al., 1984; Lougheed et al., 1995; Patten, 1981) following inhalation of contaminated dusts and powders (Sorenson et al., 1981, 1984), are less clearly understood. It was
proposed that AFs cause these symptoms through a pro-inflammatory action, e.g., by releasing arachidonic acid from cell
membranes, which is then transformed into inflammatory prostaglandins (PG) and leukotrienes (Amstad and Cerutti, 1983;
Amstad et al., 1984; Levine, 1977; Liu et al., 1990; Liu and
Massey, 1992).
Since direct effects on the airways could also be involved, it
was decided to investigate the effect of the most common AFs
(AFB 1, AFB 2, AFG 1, and AFG 2) and their major metabolites
(AFM 1, AFM 2, AFP 1, AFQ 1, and AFG 2a), on isolated tracheal
tissue of guinea pigs; these animals are highly susceptible to
acute aflatoxicosis (Hsieh et al., 1977; Patterson, 1973;
Schoental, 1967; Wogan, 1966).
MATERIALS AND METHODS
Animals and Reagents
Male Dunkin-Hartley guinea pigs (250 –350 g) were obtained from Charles
River and acclimated for 1 week to a 12/12-h light-dark illumination cycle at
23°C, with food and water provided ad libitum.
Aflatoxin B 1 (AFB 1), carbachol chloride (C), dimethylsulphoxide (DMSO),
indomethacin (IND), isoprenaline [ISO, (S)-(⫹)-isoproterenol L-bitartrate],
theophylline (THEO), timolol (TIM), prostaglandin E 1 (PGE 1), PGE 2, histamine (HIS) and tetrodotoxin (TTX) were obtained from Sigma Chemical Co.
(St. Louis, MO, USA).
5⬘-(N-cyclopropyl)-carboxamidoadenosine (CPCA), 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine (xanthine amine
162
TRACHEA RELAXATION BY AFLATOXINS
163
FIG. 1. Relaxant effect of aflatoxins on carbachol-precontracted
trachea. (A) Tracheal strips were
maximally contracted by carbachol
and relaxed by isoprenaline (ISO) or
aflatoxin B 1 (AFB 1). (B) Relaxation
(%) of ISO (circle), AFB 1 (down triangle filled), as shown in (A), AFB 2
(down triangle open), AFG 1 (filled
square), and AFG 2 (open square)
were calculated with the formula reported in Materials and Methods.
Proper dimethylsulphoxide (DMSO)
blanks (0.5, 1.5, 2.5, 4, 6, 7.5, 10 ␮l)
were assayed (up filled triangle). Results are means ⫾ SD of 6 experiments; *p ⬍ 0.05, by ANOVA test,
indicates that the effects of AFB 1 and
AFB 2 were statistically different
from those of AFG 1 and AFG 2, respectively. (C) Using the abbreviations of AFs as symbols, pEC 50 (-log
EC 50) were plotted, calculated from
curves shown in panel (B) according
to Tallarida and Murray (1986).
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ABDEL-HAQ ET AL.
FIG. 2. Relaxant effect of aflatoxin metabolites on carbachol-precontracted trachea. (A) The major AF
metabolites were tested at the dose of
6 ⫻ 10 – 6 M (AFM 1, AFM 2, and
AFQ 1), 3.3 ⫻ 10 – 6 M (AFP 1) and
5.8 ⫻ 10 –5 M (AFG 2a), and (B) relaxation (%) was calculated with the
formula reported in Materials and
Methods and plotted using the abbreviations of AFs as symbols.
congener or XAC), and 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (RO 20 –1724) were obtained from Research Biochemicals International (Natick, MA, USA).
Relaxant Effect of Aflatoxins on Carbachol-Contracted Trachea
The animals were sacrificed by cervical dislocation and the trachea was
rapidly excised and placed in Krebs solution at room temperature. After
removal of adhering fat and connective tissue, the trachea was opened longitudinally by cutting through the cartilage opposite the smooth-muscle layer,
and cut again in transverse segments to obtain two strips. Each strip was
mounted in an organ bath containing 5 ml of Krebs solution, maintained at
37°C and gassed continuously with 95% O 2, 5% CO 2 under a tension of 0.5 g,
measured by an isotonic transducer (Basile, model 7006) and equilibrated for
30 min prior to the start of experiments. Tracheal strips were then maximally
contracted by C (5.5 ⫻ 10 –7 M) and relaxed by ISO or AFs at different
concentrations:
● A typical cumulative relaxant curve was obtained by adding ISO at 3.0 ⫻
10 –9, 8.0 ⫻ 10 –9, 1.7 ⫻ 10 – 8, 2.8 ⫻ 10 – 8, 8.3 ⫻ 10 – 8, 2.8 ⫻ 10 –7 M;
● AFB 1 , AFB 2 , AFG 1 , and AFG 2 , dissolved in DMSO (10 mg/ml), were
tested at the following cumulative doses: 3.2 ⫻ 10 – 6, 1 ⫻ 10 –5, 1.6 ⫻ 10 –5,
2.6 ⫻ 10 –5, 3.8 ⫻ 10 –5, 4.8 ⫻ 10 –5, 1.0 ⫻ 10 – 4; AF metabolites were tested at
a single dose of 6 ⫻ 10 – 6 M (AFM 1, AFM 2, and AFQ 1), 3.3 ⫻ 10 – 6 M (AFP 1),
and 5.8 ⫻ 10 –5 M (AFG 2a).
The percentage of relaxation was calculated with the standard formula E ⫽
100 ⫻ (T initial-T final)/(T initial-T 0) where T was the tone of the organ, before
contraction with C (T 0), after contraction with C (T initial), and after the addition
of the relaxant agent (T final).
TRACHEA RELAXATION BY AFLATOXINS
165
FIG. 3. Potentiating effect of aflatoxin B 1 on isoprenaline-induced relaxation of carbachol-precontracted trachea. (A) Carbachol-precontracted tracheal
strips were equilibrated for 5 min with a nonrelaxant dose of aflatoxin B 1 (AF
B 1) (3.2 ⫻ 10 – 6 M), cumulative doses of isoprenaline (ISO, 3 ⫻ 10 –9,
1.1⫻10 – 8, 2.8 ⫻ 10 – 8, 5.5 ⫻ 10 – 8, 1.4 ⫻ 10 –7, 4.2 ⫻ 10 –7 M) were added, and
(B) relaxation (%) was calculated with the formula reported in Materials and
Methods: (circle), ISO; (triangle), ISO ⫹ AFB 1 (2.6 ⫻ 10 – 6 M); (square),
ISO ⫹ AFB 1 (3.2 ⫻ 10 – 6 M). Results are means ⫾ SD of 6 experiments; *p ⬍
0.05, **p ⬍ 0.01, and ***p ⬍ 0.001, by ANOVA test.
Potentiating Effect of Aflatoxins on Isoprenaline-Induced Relaxation
of Carbachol Precontracted Trachea
C-precontracted tracheal strips were equilibrated for 5 min with nonrelaxant
doses of AFB 1 (2.6 ⫻ 10 – 6 and 3.2 ⫻ 10 – 6 M) and cumulative doses of ISO
were added as described above.
The relaxant effect of AF metabolites was evaluated by testing a single dose
(6 ⫻ 10 – 6 M for AFM 1, AFM 2, and AFQ 1; 3.3 ⫻ 10 – 6 M for AFP 1 and 5.5 ⫻
10 –5 M for AFG 2a) on C-contracted tracheal strips. Then, on the partially
relaxed tissues, an identical dose of ISO (1 ⫻ 10 – 8 M) was added.
Studies on the Mechanism of the Relaxant Action of Aflatoxin B 1
Interaction with ␤-adrenergic receptors. Tracheal strips precontracted
with C (5.5 ⫻ 10 –7 M) were incubated for 10 min with TIM (2.3 ⫻ 10 –7 M) and
cumulative doses of ISO and AFB 1 were added as described above.
Prostaglandin-related effects. Tracheal strips were either equilibrated
with IND (3 ⫻ 10 – 6 M) and subsequently contracted by a single dose of HIS
(3.6 ⫻ 10 – 6) or C (5.5 ⫻ 10 –7 M), or were allowed to develop a spontaneous
tone (ST), and cumulative doses of AFB 1 were added.
In another set of experiments, tracheal strips with ST were precontracted
FIG. 4. Potentiating effect of aflatoxin metabolites on isoprenaline-induced relaxation of carbachol-precontracted trachea. (A) Relaxant effect of
aflatoxin (AF) metabolites (AFM 1, AFM 2 and AFQ 1, 6 ⫻ 10 – 6 M; AFP 1, 3.3 ⫻
10 – 6 M; AFG 2a, 5.5 ⫻ 10 –5 M) on carbachol-precontracted tracheal strips; then,
on the partially relaxed tissues, an identical dose of isoprenaline (ISO) (1 ⫻
10 – 8 M) was added. The activity of ISO administered after the AF (E ISO after AF)
was higher than that of ISO alone (E ISO alone), indicating a potentiating effect.
(B) The effect of ISO alone, different AF metabolites, and the potentiating
effect of each AF on ISO (the difference of E ISO after AF – E ISO alone) were plotted.
The entity of the direct relaxant effect and the potentiating effect were different
for each AF, indicating two distinct mechanisms of action. An inverse linear
correlation was found between these two properties of AFs (C).
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ABDEL-HAQ ET AL.
with C, incubated for 15 min with IND (1 ⫻ 10 – 6 and 2 ⫻ 10 – 6 M) and
cumulative doses of AFB 1 were added.
PGE 1 and PGE 2 were examined by testing a single dose (5.6 ⫻ 10 –5 and
5.7 ⫻ 10 – 6 M, respectively) on C-precontracted tracheal strips with ST. In
addition, C-precontracted tracheal strips were equilibrated with a nonrelaxant
dose of AFB 1 (1.9 ⫻ 10 – 6 M) and the same doses of PG were added.
Effect on cyclic AMP levels. Tracheal strips with ST, precontracted by C
(5.5 ⫻ 10 –7 M), were relaxed by AFB 1 (5.8 ⫻ 10 –5, 1.4 ⫻ 10 – 4 and 2 ⫻ 10 – 4
M), THEO (3.3 ⫻ 10 – 4 M) and RO 20 –1724 (1.44 ⫻ 10 – 4 M), rapidly removed
from the bath, immersed in liquid nitrogen, weighed, equilibrated in phosphate-buffered saline containing 4 ⫻ 10 –3 EDTA, and stored at –70°C. The
samples were then homogenized, deproteinized by heating at 100°C for 10
min, centrifuged at 16,000 ⫻ g for 5 min, and analyzed using an assay system
from Amersham Pharmacia Biotech (Uppsala Sweden, Code TRK-432), according to the manufacturer’s directions (available online at http://www.
apbiotech.com).
Interaction with A 2 adenosinic receptors. Tracheal strips with ST, precontracted by C (5.5 ⫻ 10 –7 M), were incubated with XAC (2.3 ⫻ 10 –7 and 7 ⫻
10 –7 M) for 10 min and cumulative doses of AFB 1 (5.1 ⫻ 10 – 6, 1 ⫻ 10 –5, 1.6 ⫻
10 –5, 2.6 ⫻ 10 –5, 3.8 ⫻ 10 –5, 4.8 ⫻ 10 –5, 1.0 ⫻ 10 – 4 and 3.0 ⫻ 10 – 4 M) were
added.
Neuronal effects. Tracheal strips with ST, precontracted by C (5.5 ⫻ 10 –7
M) were incubated with TTX (1.3 ⫻ 10 –5 and 1.9 ⫻ 10 –5 M) for 30 min and
cumulative doses of AFB 1 were added.
FIG. 5. Interaction with ␤-adrenergic receptors. Tracheal strips precontracted with carbachol (5.5 ⫻ 10 –7 M) and cumulative doses of isoprenaline
(ISO) and aflatoxin B 1 (AFB 1) were added following incubation for 10 min
with ISO (open circle) and AFB 1 (open square), and without ISO (filled circle)
and AFB 1 (filled square) to timolol (2.3 ⫻ 10 –7 M). Relaxation (%) was
calculated with the formula reported in Materials and Methods. Results are
means ⫾ SD of 6 experiments.
Statistical Analysis
Calculations of pEC 50 (-log EC 50) were performed according to Tallarida
and Murray (1986). Results were expressed as means ⫾ SD. Statistical
significance of differences between groups was determined by the Student’s
t-test and ANOVA, using the software Sigma-Stat (SPSS Inc., Chicago, IL).
Values of p ⬍ 0.05 indicate significant differences.
RESULTS
Relaxant Effect of Aflatoxins on Carbachol-Contracted
Trachea
Both isoprenaline (ISO) and aflatoxin (AF) B 1, B 2, G 1, and
G 2, at different concentrations (pE 50 ⫽ 7.61 ⫾ 0.03, 4.39 ⫾
0.24, 4.37 ⫾ 0.24, 4.26 ⫾ 0.21 and 4.28 ⫾ 0.27, respectively),
induced a dose-dependent relaxation of guinea pig isolated
trachea, maximally precontracted by carbachol (C) (Figs. 1A,
1B, and 1C): the efficacies of these AFs were similar to that of
ISO but their potency was much lower (Fig. 1B). Dimethylsulphoxide (DMSO) blanks (0 –10 ␮l) induced only a minimal
relaxation (Fig. 1B). From the effective doses 50 (EC 50) of the
curves shown in Figure 1B, the pEC 50 (-logEC 50) were calculated and plotted (symbols correspond to the abbreviations) in
Figure 1C.
When the major metabolites of AFB 1 and AFB 2 were tested,
the most active compounds appeared to be AFM 1 and AFP 1
(Figs. 2A and 2B).
Potentiating Effect of Aflatoxins on Isoprenaline-Induced
Relaxation of Carbachol Precontracted Trachea
When increasing amounts of ISO were added to C-precontracted trachea in the presence of AFB 1 at nonrelaxant doses
(2.6 and 3.2 ⫻ 10 – 6 M), a potentiating effect was observed
(pE 50 ⫽ 7.78 ⫾ 0.015 and 8.18 ⫾ 0.017, respectively, Figs. 3A
and 3B).
The relaxant effect of ISO (1 ⫻ 10 – 8 M), on C-precontracted
trachea strips was increased by a previous partial relaxation by
the AF metabolites AFM 1, AFM 2, AFP 1, AFQ 1, and AFG 2a
(Figs. 4A and 4B). In Figure 4B, the potentiating effect of AFs
on ISO corresponded to the difference E ISO after AF – E ISO alone: this
apparently complicated plot, in which the effect E ISO after AF was
arbitrarily divided into two different stacks (ISO and AF potentiating effect), has the advantage of showing that the direct
relaxant effect and the potentiating effect on ISO appear to be
different properties of AFs. In fact, when they were plotted,
one vs. the other in Figure 4C, an inverse linear correlation
(r 2 ⫽ 0.55) was found.
Studies on the Mechanism of the Relaxant Action
of Aflatoxin B 1
Interaction with ␤-adrenergic receptors. The relaxant effect of ISO, but not that of AFB 1, on C-precontracted trachea
was antagonized by the ␤-blocker, timolol (TIM) (Fig. 5).
Prostaglandin-related effects. The potency, but not the
efficacy, of the relaxant effect of AFB 1 was higher on spontaneously contracted trachea preparations (pE 50 ⫽ 5.08 ⫾ 0.04,
Fig. 6A) when compared with those without spontaneous tone
(ST), due to pre-incubation with indomethacin (IND), and
contracted by either histamine (HIS) or C (pE 50 ⫽ 4.65 ⫾
0.015 and 4.39 ⫾ 0.24, respectively, Fig. 6A).
Incubation of C-precontracted trachea with low doses of
IND (1 ⫻ 10 – 6 and 2 ⫻ 10 – 6 M) reduced the relaxant effect of
AFB 1 (Fig. 6B). AFB 1, at a non-active concentration (1.9 ⫻
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TRACHEA RELAXATION BY AFLATOXINS
FIG. 6. Prostaglandin-related effects. (A) Tracheal strips were either equilibrated with indomethacin (3 ⫻ 10 – 6 M) and subsequently contracted by a single
dose of histamine (3.6 ⫻ 10 – 6 M, [triangle]) or carbachol (5.5 ⫻ 10 –7 M, [circle]), or were allowed to develop a spontaneous tone (diamond), and cumulative
doses of aflatoxin B 1 were added. Relaxation (%) was calculated with the formula reported in Materials and Method. Results are means ⫾ SD of 6 experiments.
(B) In another set of experiments, tracheal strips with spontaneous tone were precontracted with carbachol, incubated for 15 min with indomethacin (IND, 1 ⫻
10 – 6 and 2 ⫻ 10 – 6 M), and cumulative doses of aflatoxin B 1 were added: filled circle, AFB 1; triangle, AFB 1 ⫹ IND (1 ⫻ 10 – 6 M); open circle, AFB 1 ⫹ IND
(2 ⫻ 10 – 6 M). Results are means ⫾ SD of 6 experiments; *p ⬍ 0.05, **p ⬍ 0.01 and ***p ⬍ 0.001, by ANOVA test. (C) Single doses (5.6 ⫻ 10 –5 and 5.7 ⫻
10 – 6 M, respectively) of prostaglandins (PG), E 1 and E 2, were tested on carbachol-precontracted tracheal strips with spontaneous tone in the presence or absence
of a nonrelaxant dose of aflatoxin B 1 (1.9 ⫻ 10 – 6 M). (D) Data are means ⫾ SD of 6 experiments; **p ⬍ 0.01 by Student’s t-test.
10 – 6 M), potentiated the relaxant activity of PGE 1 and PGE 2 on
C-precontracted trachea (Figs. 6C and 6D).
Effect on cyclic AMP levels. The levels of cAMP in Cprecontracted trachea relaxed by AFB 1, theophylline (THEO)
and RO 20 –1724 were increased (Fig. 7). In particular, the
effect of AFB 1, at the highest concentration tested (2.0 ⫻ 10 – 4
M), was not significantly lower than that of RO 20 –1724
(1.44 ⫻ 10 – 4 M) and THEO (3.3 ⫻ 10 – 4 M) (Fig. 7).
Interaction with A 2 adenosinic receptors. Partial blockade
of A 2 adenosinic receptors by XAC (2.3 ⫻ 10 –7 and 7.0 ⫻ 10 –7
M) reduced the activity of AFB 1 on C-precontracted trachea
(Figs. 8A and 8B).
Neuronal effects. Tetrodotoxin (TTX), at inactive doses
(1.3 ⫻ 10 –5 and 1.9 ⫻ 10 –5 M), reduced the relaxant effect of
AFB 1 on C-precontracted trachea (Fig. 9).
DISCUSSION
Acute aflatoxicosis following inhalation of contaminated
powders is characterized by several pathological symptoms,
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ABDEL-HAQ ET AL.
including dyspnea (Brucato et al., 1986; Clark et al., 1984).
Contrary to expectations, naturally occurring aflatoxins AFB 1,
AFB 2, AFG 1, and AFG 2 and their major metabolites (AFM 1,
AFM 2, AFP 1, AFQ 1, and AFG 2a) possessed relaxant effects on
carbachol-precontracted guinea pig trachea (Figs. 1 and 2). The
efficacy, but not the potency, of AFB 1, AFB 2, AFG 1, and AFG 2
was similar to that of the ␤-agonist isoprenaline (ISO) (Fig. 1B).
The AF examined were also capable of potentiating the
relaxant effect of ISO on C-precontracted trachea (Figs. 3 and
4). Interestingly, the direct relaxant effect of the metabolites
were inversely correlated with their potentiating effect, indicating that different mechanisms could be involved (Fig. 4C).
While attempting to elucidate the mechanisms responsible for
these phenomena, it was found that:
FIG. 7. Effect on cyclic AMP levels. cAMP was measured in tracheal
strips with spontaneous tone, precontracted by carbachol (5.5 ⫻ 10 –7 M), and
relaxed by aflatoxin B 1 (AF B 1), theophylline (THEO) and RO 20 –1724.
Values are referred to control samples.
FIG. 8. Interaction with A 2 adenosinic receptors. (A) Tracheal
strips with spontaneous tone precontracted by carbachol, were incubated
with a xanthine amine congener
(XAC) for 10 min and cumulative
doses of AFB 1 were added. (B) Relaxation (%) was calculated with the
formula reported in Materials and
Methods: circle, AFB 1; triangle,
AFB 1 ⫹ XAC (2.3 ⫻ 10 –7 M);
square, AFB 1 ⫹ XAC (7 ⫻ 10 –7 M).
Results are means ⫾ SD of 6 experiments; *p ⬍ 0.05, **p ⬍ 0.01, and
***p ⬍ 0.001, by ANOVA test.
● The activity of AFB 1 was not affected by the ␤-blocker,
timolol (TIM) (Fig. 5), indicating that a direct interaction with
␤ 2-adrenergic receptors was not involved.
● The relaxant effect of AFB 1 was lower in the presence of
indomethacin (IND) (Figs. 6A and 6B), a well known inhibitor
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TRACHEA RELAXATION BY AFLATOXINS
and Hourani, 1993; Losinski and Alexander, 1995; Ongini and
Fredholm, 1996; Poulsen and Quinn, 1998), suggested by the
ability of the antagonist, XAC (Jacobson, 1986; Ukena et al.,
1986), to reduce the relaxant activity of AFB 1 on C-precontracted trachea (Fig. 8).
● Finally, the reduction of the relaxant activity of AFB 1 on
C-precontracted trachea in the presence of tetrodotoxin (TTX)
(Fig. 9), indicated that this mycotoxin could also interfere with
neuronal mechanisms, e.g., stimulate inhibitory nonadrenergic,
noncholinergic nerves (i-NANC) (Rhoden and Barnes, 1990).
FIG. 9. Neuronal effects. Tracheal strips with spontaneous tone, precontracted by carbachol (5.5 ⫻ 10 –7 M) were incubated with tetrodotoxin (TTX,
1.3 ⫻ 10 –5 and 1.9 ⫻ 10 –5 M) for 10 min and cumulative doses of AFB 1 were
added. Relaxation (%) was calculated with the formula reported in Materials
and Methods: circle, AFB 1; square, AFB 1 ⫹ TTX (1.3 ⫻ 10 –5 M); triangle,
AFB 1 ⫹ TTX (1.9 ⫻ 10 –5 M). Results are means ⫾ SD of 6 experiments; *p ⬍
0.05, **p ⬍ 0.01 and, ***p ⬍ 0.001, by ANOVA test.
In conclusion, acute symptoms of aflatoxicosis, such as
dyspnea, do not appear to be due to a direct activity on the
tracheal muscle but, probably, to the well-known pro-inflammatory activity of AFs, which are capable of releasing arachidonic acid from cell membranes, hence stimulating the synthesis of leukotrienes and prostaglandins (Amstad and Cerutti,
1983; Amstad et al., 1984; Levine, 1977; Liu et al., 1990; Liu
and Massey, 1992). While attempting to explain the relaxant
activity of AFB 1 on the tracheal smooth muscle, however, we
observed properties of this mycotoxin that need to be studied in
further detail, such as its ability to potentiate the activity of
PGEs, to interact with A 2 adenosinic receptors, and, presumably, to stimulate the i-NANC system.
ACKNOWLEDGMENTS
of cyclo-oxygenase, the enzyme responsible for the synthesis
of prostaglandins (PG) and thromboxanes. IND has previously
been used to abolish the spontaneous tone (ST) of the guinea
pig trachea (Ndukwu et al., 1997; Orehek, et al., 1975) which
is mainly due to cyclo-oxygenase products such as PGD 2,
PGF 2␣, and thromboxane A 2 (Campbell and Halushka, 1996;
Johnston et al., 1995; Lindèn et al., 1991; Raeburn et al.,
1987). Other PGs such as PGE 1, PGE 1, and PGI 2 generally
relax the tracheal muscle, but may also contract it under certain
experimental conditions (Ndukwu et al., 1997). Since IND, at
low doses (1 ⫻ 10 – 6 and 2 ⫻ 10 – 6 M), reduced the relaxant
activity of AFB 1 on C-precontracted trachea (Fig. 6B), and
since AFB 1 was able to potentiate exogenous PGE 1 and PGE 2
(Figs 6C, D), it is speculated that AFs could act by either
facilitating the relaxant PGs or by interfering with the contracting ones.
● The level of cyclic AMP was increased in C-precontracted
trachea preparations relaxed by AFB 1 (Fig. 7). This could be
due to different mechanisms, including: (1)The possible inhibition of phosphodiesterase (PDE) suggested by the observation that the effect of AFB 1 (2 ⫻ 10 – 4 M) was comparable with
that of a specific inhibitor of PDE-IV (RO 20 –1724 at 1. 44 ⫻
10 – 4 M) and with theophylline (THEO at 3.3 ⫻ 10 – 4 M) (Fig.
7). Evidence for this activity of AF exist in the literature (Bonsi
et al., 1999; Hoult and Paya, 1996; Prasanna et al., 1975). (2)
Interaction with PG receptors, e.g., the EP 2 or IP subtypes. (3)
Interaction with A 2 adenosinic receptors (reviewed by Collis
This research was partly supported by grants from the Noopolis Foundation
and the Sovena Foundation of Italy.
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