An isoflavone from Dipteryx alata Vogel is active against the in vitro neuromuscular paralysis of Bothrops jararacussu snake venom and bothropstoxin I, and prevents venom-induced myonecrosis

Abstract

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 μg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 μg/mL) caused irreversible paralysis. Preincubation of TM (200 μg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

Figure 1

Chemical structure of 7,8,3'-trihydroxy-4'-methoxyisoflavone isolated from D. alata Vogel [32].

Figure 2

Twitch tension responses of indirectly stimulated PND incubated with 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM, 200 μg/mL), B. jararacussu (Bjssu) venom (40 μg/mL) or a mixture of TM (200 µg/mL) and venom (40 μg/mL). The TM + venom mixture was preincubated at 37 °C for 30 min prior to testing. The points are the mean ± SEM of the number of experiments indicated in the figure. * p < 0.05 for TM + venom compared to venom alone.

Figure 3

Neuromuscular responses of indirectly stimulated PND incubated with BthTX-I (20 µg/mL) in the absence (A) and presence (B,C) of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM, 200 µg/mL). In (B), TM (200 µg/mL) was preincubated with BthTX-I (20 µg/mL) at 37 °C for 30 min before addition to the bath, whereas in (C) TM (200 µg/mL) was added separately (at *) 10 min after the addition of BthTX-I (20 µg/mL). Arrows show the moment of sample addition to the bath. Bar = tension of 5 g/cm; W = wash.

Figure 4

Histological analysis of muscle damage in PND incubated with or without 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM). Panel (A) shows a representative trace of PND incubated with TM (n = 4). The arrow indicates the addition of TM. Bar = tension of 5 g/cm. W = wash. Panels B and C show cross-sections of diaphragm muscle incubated with Tyrode solution alone (negative control; n = 6) (B) or TM alone (n = 4) (C). Note the normal appearance of the fibers (polygonal aspect and peripheral nuclei) in both panels. Bar = 50 μm in B and C.

Figure 5

Neuromuscular responses of indirectly stimulated PND incubated with B. jararacussu venom (40 μg/mL) for 120 min (A). Notice the irreversible blockade. Venom was added at the arrow. Bar = tension of 5 g/cm. W = wash. Panel (B)—Cross-section of diaphragm muscle incubated with venom alone (40 μg/mL, n = 6). Note the edema and intense myonecrosis (muscle fiber atrophy, hyaline aspect, sarcolemmal disruption and myofibril lysis). Panel (C)—Cross-section of diaphragm muscle incubated with a mixture of TM (200 μg/mL) + venom (40 μg/mL). Note the normal appearance of the fibers (polygonal aspect and peripheral nuclei), but with occasional edema. Bar = 50 μm for B and C.