Isolation and Pharmacological Characterisation of Pre-Synaptic Neurotoxins from Thai and Javanese Russell's Viper (Daboia siamensis) Venoms

Abstract

The widespread geographical distribution of Russell's vipers (Daboia spp.) is associated with marked variations in the clinical outcomes of envenoming by species from different countries. This is likely to be due to differences in the quantity and potency of key toxins and, potentially, the presence or absence of some toxins in venoms across the geographical spectrum. In this study, we aimed to isolate and pharmacologically characterise the major neurotoxic components of D. siamensis venoms from Thailand and Java (Indonesia) and explore the efficacy of antivenom and a PLA₂ inhibitor, Varespladib, against the neuromuscular activity. These data will provide insights into the link between venom components and likely clinical outcomes, as well as potential treatment strategies. Venoms were fractionated using RP-HPLC and the in vitro activity of isolated toxins assessed using the chick biventer cervicis nerve-muscle preparation. Two major PLA₂ fractions (i.e., fractions 8 and 10) were isolated from each venom. Fraction 8 from both venoms produced pre-synaptic neurotoxicity and myotoxicity, whereas fraction 10 from both venoms was weakly neurotoxic. The removal of the two fractions from each venom abolished the in vitro neurotoxicity, and partially abolished myotoxicity, of the whole venom. A combination of the two fractions from each venom produced neurotoxic activity that was equivalent to the respective whole venom (10 µg/mL), but the myotoxic effects were not additive. The in vitro neurotoxicity of fraction 8 (100 nM) from each venom was prevented by the pre-administration of Thai Russell's viper monovalent antivenom (2× recommended concentration) or preincubation with Varespladib (100 nM). Additionally, the neurotoxicity produced by a combination of the two fractions was partially reversed by the addition of Varespladib (100-300 nM) 60 min after the fractions. The present study demonstrates that the in vitro skeletal muscle effects of Thai and Javanese D. siamensis venoms are primarily due to key PLA₂ toxins in each venom.

Keywords: Russell’s viper; neuromuscular junction; neurotoxin; phospholipase A2; snake venom.

Figure 1

Effect of Javanese (JRV) or Thai (TRV) D. siamensis venoms (3–10 µg/mL) on (a,c) indirect twitches and (b,d) contractile responses to acetylcholine (ACh), carbachol (CCh), and potassium chloride (KCl) in the chick biventer cervicis nerve-muscle preparation. Effect of Javanese (JRV) or Thai (TRV) D. siamensis venoms (10–30 µg/mL) on (e,g) direct twitches or (f,h) baseline tension in the chick biventer cervicis nerve-muscle preparation. Data presented as mean ± SEM; * p < 0.05, significantly different from vehicle control (Control); one-way ANOVA followed by Bonferroni multiple comparison post hoc test, n = 3–5.

Figure 1

Effect of Javanese (JRV) or Thai (TRV) D. siamensis venoms (3–10 µg/mL) on (a,c) indirect twitches and (b,d) contractile responses to acetylcholine (ACh), carbachol (CCh), and potassium chloride (KCl) in the chick biventer cervicis nerve-muscle preparation. Effect of Javanese (JRV) or Thai (TRV) D. siamensis venoms (10–30 µg/mL) on (e,g) direct twitches or (f,h) baseline tension in the chick biventer cervicis nerve-muscle preparation. Data presented as mean ± SEM; * p < 0.05, significantly different from vehicle control (Control); one-way ANOVA followed by Bonferroni multiple comparison post hoc test, n = 3–5.

Figure 2

HPLC chromatograms of (a) Javanese and (b) Thai D. siamensis venoms using a Jupiter C18 semi-preparative column. Chromatograms of isolated fractions 8 and 10 from (c,e) Javanese and (d,f) Thai D. siamensis venoms.

Figure 3

Intact mass protein analysis LC-ESI-MS chromatogram of (upper panel) fraction 8 and (lower panel) fraction 10 from Javanese D. siamensis venom.

Figure 4

Intact mass protein analysis LC-ESI-MS chromatogram of (upper panel) fraction 8 and (lower panel) fraction 10 from Thai D. siamensis venom.

Figure 5

The effects of Javanese D. siamensis fraction 8 (100 nM–3 µM) on (a) indirect twitches or (b) contractile responses to exogenous ACh, CCh, or KCl or (c) direct twitches or (d) baseline tension of the chick biventer cervicis nerve-muscle preparation. Effects of Javanese D. siamensis fraction 10 (1–3 µM) on (e) indirect twitches or (f) contractile responses to exogenous ACh, CCh, or KCl in the chick biventer cervicis nerve-muscle preparation. Data presented as the mean ± SEM, * p < 0.05, significantly different from vehicle control (Control) at 180 min; # p < 0.05, significantly different from same fraction at a lower concentration; one-way ANOVA followed by Bonferroni multiple comparison post hoc test; * p < 0.05, significantly different from pre-venom response; student’s paired t-test, n = 4.

Figure 6

The effects of Thai D. siamensis fraction 8 (100 nM–1 µM) on (a) indirect twitches or (b) contractile responses to exogenous ACh, CCh, or KCl or (c) direct twitches or (d) baseline tension. Effects of fraction 10 (1–3 µM) on (e) indirect twitches or (f) contractile responses to exogenous ACh, CCh, or KCl in the chick biventer cervicis nerve-muscle preparation. Data presented as the mean ± SEM, * p < 0.05, significantly different from vehicle control (Control) at 180 min; # p < 0.05, significantly different from the same fraction at a lower concentration; one-way ANOVA followed by Bonferroni multiple comparison post hoc test; * p < 0.05, significantly different from pre-venom response; student’s paired t-test, n = 3–6.

Figure 7

Comparison of the effect of whole Javanese D. siamensis venom (10 µg/mL), venom devoid of fractions 8 and 10 (10 µg/mL), and fractions 8 and 10 combined (at a 1:1 ratio; 100 nM) on (a) indirect twitches, (b) contractile responses to exogenous ACh, CCh, or KCl, (c) direct twitches, or (d) baseline tension of the chick biventer cervicis nerve-muscle preparation. Data presented as the mean ± SEM, * p < 0.05, significantly different from vehicle control (Control) at 180 min; # p < 0.05, significantly different from whole venom at 180 min; one-way ANOVA followed by Bonferroni multiple comparison post hoc test, n = 4–12.

Figure 8

Comparison of the effect of whole Thai D. siamensis venom (10 µg/mL), venom devoid of fractions 8 and 10 (10 µg/mL), and fractions 8 and 10 combined (at a 1:1 ratio; 100 nM) on (a) indirect twitches, (b) contractile responses to exogenous ACh, CCh, or KCl, (c) direct twitches, or (d) baseline tension of the chick biventer cervicis nerve-muscle preparation. Data presented as the mean ± SEM, * p < 0.05, significantly different from vehicle control (Control) at 180 min; # p < 0.05, significantly different from whole venom at 180 min; one-way ANOVA followed by Bonferroni multiple comparison post hoc test, n = 4–12.

Figure 9

The effect of (a) Javanese D. siamensis venom fraction 8 (100 nM) or (b) Thai D. siamensis venom fraction 8 (100 nM) in the absence and presence of Varespladib (100 nM) or Thai Russell’s viper monovalent antivenom (2× recommended concentration) on (a,c) indirect twitches or (b,d) contractile responses to exogenous ACh, CCh, or KCl, in the chick biventer cervicis nerve-muscle preparation. Data presented as the mean ± SEM, * p < 0.05, significantly different from vehicle control (Control) at 180 min; one-way ANOVA followed by Bonferroni multiple comparison post hoc test; for agonist responses, n = 4–10.

Figure 10

The effects of Varespladib (100–300 nM) pre-incubated with the combination of fractions 8 and 10 from (a) Javanese D. siamensis or (c) Thai D. siamensis venoms; or Varespladib (300 nM) added 60 min after the combination of fractions 8 and 10 from (b) Javanese D. siamensis or (d) Thai D. siamensis venoms in the indirectly stimulated chick biventer cervicis nerve-muscle preparation. Data presented as the mean ± SEM, * p < 0.05, significantly different from vehicle control (Control) at 180 (a,c) or 240 min (b,d); # p < 0.05, significantly different from F8/F10 alone at 180 min (a,c) or 240 min (b,d); one-way ANOVA followed by Bonferroni multiple comparison post hoc test, n = 4–6.