Relaxant Effects of Aflatoxins on Isolated Guinea Pig Trachea
Hanin Abdel-Haq,
Maura Palmery,
Maria Grazia Leone,
Luciano Saso and
Bruno Silvestrini1
Department of Pharmacology of Natural Substances and General Physiology, University of Rome La Sapienza, Rome, Italy
Received July 18, 1999;
accepted December 10, 1999
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ABSTRACT
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Dyspnea is one of the symptoms of acute aflatoxicosis. Contrary to expectations, we observed that naturally occurring aflatoxins (AF) AFB1, AFB2, AFG1, and AFG2 and their major metabolites AFM1, AFM2, AFP1, AFQ1, and AFG2a relaxed carbachol (C) precontracted guinea pig trachea to different degrees. The efficacies but not the potencies of AFB1, AFB2, AFG1, and AFG2 were similar to that of the ß-agonist, isoprenaline, whose activity was potentiated by the AF. Their mechanism of action is not clearly understood but several mechanistic indications were obtained with AFB1: 1) its effect was not influenced by the ß-blocker, timolol, indicating that a direct interaction with ß2-adrenergic receptors was not involved. 2) AFB1 potentiated PGE1 and PGE2, two relaxant prostaglandins, and its activity was reduced by indomethacin. 3) The cAMP level in the guinea pig trachea relaxed by AFB1 increased, possibly due to inhibition of phosphodiesterase; direct interaction with PG receptors; and/or interaction with A2 adenosinic receptors, suggested by the inhibitory activity of XAC, a specific antagonist. 4) Finally, since tetrodotoxin reduced the relaxant activity of AFB1, 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.
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INTRODUCTION
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Aflatoxins are a group of closely related mycotoxins produced by Aspergillus flavus (AFB1 and AFB2), A. parasiticus (AFB1, AFB2, AFG1, and AFG2), and other fungi (Mclean and Dutton, 1995
). Chronic intoxication with AFB1 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 endogenousproteins 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 cyclo-oxygenase 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 (AFB1, AFB2, AFG1, and AFG2) and their major metabolites (AFM1, AFM2, AFP1, AFQ1, and AFG2a), 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
).
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MATERIALS AND METHODS
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Animals and Reagents
Male Dunkin-Hartley guinea pigs (250350 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 B1 (AFB1), carbachol chloride (C), dimethylsulphoxide (DMSO), indomethacin (IND), isoprenaline [ISO, (S)-(+)-isoproterenol L-bitartrate], theophylline (THEO), timolol (TIM), prostaglandin E1 (PGE1), PGE2, 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 congener or XAC), and 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (RO 201724) 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% O2, 5% CO2 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 x 107 M) and relaxed by ISO or AFs at different concentrations:
- A typical cumulative relaxant curve was obtained by adding ISO at 3.0 x 109, 8.0 x 109, 1.7 x 108, 2.8 x 108, 8.3 x 108, 2.8 x 107 M;
- AFB1, AFB2, AFG1, and AFG2, dissolved in DMSO (10 mg/ml), were tested at the following cumulative doses: 3.2 x 106, 1 x 105, 1.6 x 105, 2.6 x 105, 3.8 x 105, 4.8 x 105, 1.0 x 104; AF metabolites were tested at a single dose of 6 x 106 M (AFM1, AFM2, and AFQ1), 3.3 x 106 M (AFP1), and 5.8 x 105 M (AFG2a).
The percentage of relaxation was calculated with the standard formula E = 100 x (Tinitial-Tfinal)/(Tinitial-T0) where T was the tone of the organ, before contraction with C (T0), after contraction with C (Tinitial), and after the addition of the relaxant agent (Tfinal).
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 AFB1 (2.6 x 106 and 3.2 x 106 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 x 106 M for AFM1, AFM2, and AFQ1; 3.3 x 106 M for AFP1 and 5.5 x 105 M for AFG2a) on C-contracted tracheal strips. Then, on the partially relaxed tissues, an identical dose of ISO (1 x 108 M) was added.
Studies on the Mechanism of the Relaxant Action of Aflatoxin B1
Interaction with ß-adrenergic receptors.
Tracheal strips precontracted with C (5.5 x 107 M) were incubated for 10 min with TIM (2.3 x 107 M) and cumulative doses of ISO and AFB1 were added as described above.
Prostaglandin-related effects.
Tracheal strips were either equilibrated with IND (3 x 106 M) and subsequently contracted by a single dose of HIS (3.6 x 106) or C (5.5 x 107 M), or were allowed to develop a spontaneous tone (ST), and cumulative doses of AFB1 were added.
In another set of experiments, tracheal strips with ST were precontracted with C, incubated for 15 min with IND (1 x 106 and 2 x 106 M) and cumulative doses of AFB1 were added.
PGE1 and PGE2 were examined by testing a single dose (5.6 x 105 and 5.7 x 106 M, respectively) on C-precontracted tracheal strips with ST. In addition, C-precontracted tracheal strips were equilibrated with a nonrelaxant dose of AFB1 (1.9 x 106 M) and the same doses of PG were added.
Effect on cyclic AMP levels.
Tracheal strips with ST, precontracted by C (5.5 x 107 M), were relaxed by AFB1 (5.8 x 105, 1.4 x 104 and 2 x 104 M), THEO (3.3 x 104 M) and RO 201724 (1.44 x 104 M), rapidly removed from the bath, immersed in liquid nitrogen, weighed, equilibrated in phosphate-buffered saline containing 4 x 103 EDTA, and stored at 70°C. The samples were then homogenized, deproteinized by heating at 100°C for 10 min, centrifuged at 16,000 x 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 A2 adenosinic receptors.
Tracheal strips with ST, precontracted by C (5.5 x 107 M), were incubated with XAC (2.3 x 107 and 7 x 107 M) for 10 min and cumulative doses of AFB1 (5.1 x 106, 1 x 105, 1.6 x 105, 2.6 x 105, 3.8 x 105, 4.8 x 105, 1.0 x 104 and 3.0 x 104 M) were added.
Neuronal effects.
Tracheal strips with ST, precontracted by C (5.5 x 107 M) were incubated with TTX (1.3 x 105 and 1.9 x 105 M) for 30 min and cumulative doses of AFB1 were added.
Statistical Analysis
Calculations of pEC50 (-log EC50) 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.
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RESULTS
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Relaxant Effect of Aflatoxins on Carbachol-Contracted Trachea
Both isoprenaline (ISO) and aflatoxin (AF) B1, B2, G1, and G2, at different concentrations (pE50 = 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 (010 µl) induced only a minimal relaxation (Fig. 1B
). From the effective doses 50 (EC50) of the curves shown in Figure 1B
, the pEC50 (-logEC50) were calculated and plotted (symbols correspond to the abbreviations) in Figure 1C
.

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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 B1 (AFB1). (B) Relaxation (%) of ISO (circle), AFB1 (down triangle filled), as shown in (A), AFB2 (down triangle open), AFG1 (filled square), and AFG2 (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 µl) were assayed (up filled triangle). Results are means ± SD of 6 experiments; *p < 0.05, by ANOVA test, indicates that the effects of AFB1 and AFB2 were statistically different from those of AFG1 and AFG2, respectively. (C) Using the abbreviations of AFs as symbols, pEC50 (-log EC50) were plotted, calculated from curves shown in panel (B) according to Tallarida and Murray (1986).
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When the major metabolites of AFB1 and AFB2 were tested, the most active compounds appeared to be AFM1 and AFP1 (Figs. 2A and 2B
).

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FIG. 2. Relaxant effect of aflatoxin metabolites on carbachol-precontracted trachea. (A) The major AF metabolites were tested at the dose of 6 x 106 M (AFM1, AFM2, and AFQ1), 3.3 x 106 M (AFP1) and 5.8 x 105 M (AFG2a), and (B) relaxation (%) was calculated with the formula reported in Materials and Methods and plotted using the abbreviations of AFs as symbols.
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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 AFB1 at nonrelaxant doses (2.6 and 3.2 x 106 M), a potentiating effect was observed (pE50 = 7.78 ± 0.015 and 8.18 ± 0.017, respectively, Figs. 3A and 3B
).

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FIG. 3. Potentiating effect of aflatoxin B1 on isoprenaline-induced relaxation of carbachol-precontracted trachea. (A) Carbachol-precontracted tracheal strips were equilibrated for 5 min with a nonrelaxant dose of aflatoxin B1 (AF B1) (3.2 x 106 M), cumulative doses of isoprenaline (ISO, 3 x 109, 1.1x108, 2.8 x 108, 5.5 x 108, 1.4 x 107, 4.2 x 107 M) were added, and (B) relaxation (%) was calculated with the formula reported in Materials and Methods: (circle), ISO; (triangle), ISO + AFB1 (2.6 x 106 M); (square), ISO + AFB1 (3.2 x 106 M). Results are means ± SD of 6 experiments; *p < 0.05, **p < 0.01, and ***p < 0.001, by ANOVA test.
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The relaxant effect of ISO (1 x 108 M), on C-precontracted trachea strips was increased by a previous partial relaxation by the AF metabolites AFM1, AFM2, AFP1, AFQ1, and AFG2a (Figs. 4A and 4B
). In Figure 4B
, the potentiating effect of AFs on ISO corresponded to the difference EISO after AF EISO alone: this apparently complicated plot, in which the effect EISO 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 (r2 = 0.55) was found.

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FIG. 4. Potentiating effect of aflatoxin metabolites on isoprenaline-induced relaxation of carbachol-precontracted trachea. (A) Relaxant effect of aflatoxin (AF) metabolites (AFM1, AFM2 and AFQ1, 6 x 106 M; AFP1, 3.3 x 106 M; AFG2a, 5.5 x 105 M) on carbachol-precontracted tracheal strips; then, on the partially relaxed tissues, an identical dose of isoprenaline (ISO) (1 x 108 M) was added. The activity of ISO administered after the AF (EISO after AF) was higher than that of ISO alone (EISO 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 EISO after AF EISO 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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Studies on the Mechanism of the Relaxant Action of Aflatoxin B1
Interaction with ß-adrenergic receptors.
The relaxant effect of ISO, but not that of AFB1, on C-precontracted trachea was antagonized by the ß-blocker, timolol (TIM) (Fig. 5
).

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FIG. 5. Interaction with ß-adrenergic receptors. Tracheal strips precontracted with carbachol (5.5 x 107 M) and cumulative doses of isoprenaline (ISO) and aflatoxin B1 (AFB1) were added following incubation for 10 min with ISO (open circle) and AFB1 (open square), and without ISO (filled circle) and AFB1 (filled square) to timolol (2.3 x 107 M). Relaxation (%) was calculated with the formula reported in Materials and Methods. Results are means ± SD of 6 experiments.
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Prostaglandin-related effects.
The potency, but not the efficacy, of the relaxant effect of AFB1 was higher on spontaneously contracted trachea preparations (pE50 = 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 (pE50 = 4.65 ± 0.015 and 4.39 ± 0.24, respectively, Fig. 6A
).

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FIG. 6. Prostaglandin-related effects. (A) Tracheal strips were either equilibrated with indomethacin (3 x 106 M) and subsequently contracted by a single dose of histamine (3.6 x 106 M, [triangle]) or carbachol (5.5 x 107 M, [circle]), or were allowed to develop a spontaneous tone (diamond), and cumulative doses of aflatoxin B1 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 x 106 and 2 x 106 M), and cumulative doses of aflatoxin B1 were added: filled circle, AFB1; triangle, AFB1 + IND (1 x 106 M); open circle, AFB1 + IND (2 x 106 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 x 105 and 5.7 x 106 M, respectively) of prostaglandins (PG), E1 and E2, were tested on carbachol-precontracted tracheal strips with spontaneous tone in the presence or absence of a nonrelaxant dose of aflatoxin B1 (1.9 x 106 M). (D) Data are means ± SD of 6 experiments; **p < 0.01 by Student's t-test.
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Incubation of C-precontracted trachea with low doses of IND (1 x 106 and 2 x 106 M) reduced the relaxant effect of AFB1 (Fig. 6B
). AFB1, at a non-active concentration (1.9 x 106 M), potentiated the relaxant activity of PGE1 and PGE2 on C-precontracted trachea (Figs. 6C and 6D
).
Effect on cyclic AMP levels.
The levels of cAMP in C-precontracted trachea relaxed by AFB1, theophylline (THEO) and RO 201724 were increased (Fig. 7
). In particular, the effect of AFB1, at the highest concentration tested (2.0 x 104 M), was not significantly lower than that of RO 201724 (1.44 x 104 M) and THEO (3.3 x 104 M) (Fig. 7
).

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FIG. 7. Effect on cyclic AMP levels. cAMP was measured in tracheal strips with spontaneous tone, precontracted by carbachol (5.5 x 107 M), and relaxed by aflatoxin B1 (AF B1), theophylline (THEO) and RO 201724. Values are referred to control samples.
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Interaction with A2 adenosinic receptors.
Partial blockade of A2 adenosinic receptors by XAC (2.3 x 107 and 7.0 x 107 M) reduced the activity of AFB1 on C-precontracted trachea (Figs. 8A and 8B
).

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FIG. 8. Interaction with A2 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 AFB1 were added. (B) Relaxation (%) was calculated with the formula reported in Materials and Methods: circle, AFB1; triangle, AFB1 + XAC (2.3 x 107 M); square, AFB1 + XAC (7 x 107 M). Results are means ± SD of 6 experiments; *p < 0.05, **p < 0.01, and ***p < 0.001, by ANOVA test.
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Neuronal effects.
Tetrodotoxin (TTX), at inactive doses (1.3 x 105 and 1.9 x 105 M), reduced the relaxant effect of AFB1 on C-precontracted trachea (Fig. 9
).

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FIG. 9. Neuronal effects. Tracheal strips with spontaneous tone, precontracted by carbachol (5.5 x 107 M) were incubated with tetrodotoxin (TTX, 1.3 x 105 and 1.9 x 105 M) for 10 min and cumulative doses of AFB1 were added. Relaxation (%) was calculated with the formula reported in Materials and Methods: circle, AFB1; square, AFB1 + TTX (1.3 x 105 M); triangle, AFB1 + TTX (1.9 x 105 M). Results are means ± SD of 6 experiments; *p < 0.05, **p < 0.01 and, ***p < 0.001, by ANOVA test.
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DISCUSSION
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Acute aflatoxicosis following inhalation of contaminated powders is characterized by several pathological symptoms, including dyspnea (Brucato et al., 1986
; Clark et al., 1984
). Contrary to expectations, naturally occurring aflatoxins AFB1, AFB2, AFG1, and AFG2 and their major metabolites (AFM1, AFM2, AFP1, AFQ1, and AFG2a) possessed relaxant effects on carbachol-precontracted guinea pig trachea (Figs. 1 and 2
). The efficacy, but not the potency, of AFB1, AFB2, AFG1, and AFG2 was similar to that of the ß-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:
- The activity of AFB1 was not affected by the ß-blocker, timolol (TIM) (Fig. 5
), indicating that a direct interaction with ß2-adrenergic receptors was not involved.
- The relaxant effect of AFB1 was lower in the presence of indomethacin (IND) (Figs. 6A and 6B
), a well known inhibitor 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 PGD2, PGF2
, and thromboxane A2 (Campbell and Halushka, 1996
; Johnston et al., 1995
; Lindèn et al., 1991
; Raeburn et al., 1987
). Other PGs such as PGE1, PGE1, and PGI2 generally relax the tracheal muscle, but may also contract it under certain experimental conditions (Ndukwu et al., 1997
). Since IND, at low doses (1 x 106 and 2 x 106 M), reduced the relaxant activity of AFB1 on C-precontracted trachea (Fig. 6B
), and since AFB1 was able to potentiate exogenous PGE1 and PGE2 (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 AFB1 (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 AFB1 (2 x 104 M) was comparable with that of a specific inhibitor of PDE-IV (RO 201724 at 1. 44 x 104 M) and with theophylline (THEO at 3.3 x 104 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 EP2 or IP subtypes. (3) Interaction with A2 adenosinic receptors (reviewed by Collis 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 AFB1 on C-precontracted trachea (Fig. 8
).
- Finally, the reduction of the relaxant activity of AFB1 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
).
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 AFB1 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 A2 adenosinic receptors, and, presumably, to stimulate the i-NANC system.
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ACKNOWLEDGMENTS
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This research was partly supported by grants from the Noopolis Foundation and the Sovena Foundation of Italy.
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NOTES
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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: +39-6-49912480. E-mail: silvestrini{at}uniroma1.it. 
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