MSc Chemistry
(Joint degree)
Analytical science track
Literature Thesis
Comprehensive overview of In vitro assays for studying neuro-.,
hemo-. & cytotoxicity caused by snake venom
by
Mayra Menke
12934372 (UvA); 2593475 (VU)
September 2020 – January 2021
12 EC
Semester 1
Supervisor: Examiner:
Mátyás Bittenbinder (MSc) Dr. Jeroen Kool
Division of Bio-analytical Chemistry within the Department of Chemistry & Pharmaceutical
Science , Faculty of Science VU Amsterdam
Table of Contents
Abstract ...2
Abbreviations: ...2
Chapter 1. Introduction ...3
1.1 Snakebite envenoming ...3
1.2 Pathological effects ... 5 1.1.1 Cytotoxicity ...5
1.1.2 Neurotoxicity ...5
1.1.3 Hemotoxicity and cardiovascular disruptions ...
6
Chapter 2. In Vitro assays ...8
2.1 In vitro cytotoxicity ...
8
2.1.1 Colorimetric assays ...
8
2.1.2 Fluorometric and dye exclusion assays...
12
2.2 In vitro neurotoxicity ...
16
2.2.1 In vitro muscle preparations ...
16
2.2.2 Cell based assays ...
19
2.3 In vitro hemotoxicity ... 22
2.3.1 Hemorrhagic assays...
22
2.3.2 Coagulation assays ...
25
Chapter 3. Analytical techniques for snake venom analysis... 32
3.1 Effect- directed analysis and high- resolution screening ...
32
3.2 Snake venomics and high-throughput analysis ...
33
Conclusion ... 36
Acknowledgements ... 36
Abstract
This literature study provides an extensive overview of in vitro assays and analytical techniques used to study snake venoms and gain knowledge on their compositional and toxicological characteristics. Snakebites envenomation is a serious medical issue causing approximately 138,000 deaths and 400,000 cases of severe chronic morbidity annually. This important yet neglected tropical disease mainly affects communities in developing countries. Snake venoms are complex mixtures of various components, including toxins that can induce several pharmacological effects. These effects are categorized into three main types, cytotoxic, neurotoxic, and hemotoxic. Currently, intravenous administration of antivenom is the only effective treatment for neutralizing these toxicological effects. However, this treatment has some drawbacks, such as poor antibody specificity and high expenses. The low specificity is mainly due to the variability between snake venoms and their complexity. Therefore, better and affordable therapies are needed. For this, a better understanding of the mechanisms of actions, complexity, and variability of snake venom is necessary.
Abbreviations:
PLA2s (phospholipase A2s), SVSPs (snake venom serine proteases), SVMPs (snake venom
metalloproteinases), LAAO (L-amino acid oxidases), 3FTx (three-finger proteins), CRISPs (cysteine-rich secretory proteins), CVF (cobra venom factors), VEGF (vascular endothelial growth factor), nAChRs (nicotinic acetylcholine receptors), ACh (acetylcholine), AChE (acetylcholinesterase), TLE (thrombin-like enzymes), MTT Dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium), XTT (2,3-bis-(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide), WST-1 (2-(4-iodophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt), WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium, monosodium salt), LDH (lactate dehydrogenase), HTS (high-throughput screening), FRET (Fluorescence Resonance Energy Transfer), ELISA (enzyme-linked immunosorbent assays), AChBP (acetylcholine-binding protein), PT (Prothrombin Time), aPTT (activated partial thromboplastin time), TT (thrombin time), thromboelastography (TEG), OD (optical density), SVTLEs (Snake venom thrombin-like enzymes), SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), EDA (effect-directed analysis), LC (liquid chromatography), RP-HPLC (Reverse phase high performance liquid chromatography), MS (mass spectrometry), HRS (high-resolution screening), ESI (Electrospray ionization)
Chapter 1. Introduction
1.1 Snakebite envenoming
Annually, around 2.7 million people are affected by the envenomation of snakebites, and approximately 81,000 to 138,000 deaths and 400,000 survivors suffering severe morbidity are reported 1,2. Snakebite envenoming is a neglected tropical disease which is common in poor
communities of underdeveloped tropical countries in South Asia, South Asia, Sub-Saharan Africa and Latin America 1,3. The high numbers of fatalities and morbidity have a major effect on the health care
systems of these countries 2. An increasing number of encounters of humans with snakes due to
agriculture activities, scarcity and availability of good health services and lack of effective antivenom all contribute to this problem. The majority of severe cases are caused by snakes belonging to the
Viperidae family (rattlesnakes, lance-headed pit vipers, and true vipers) and the Elapidae family
(cobras, kraits, mambas, Australasian species, and sea snakes) 4. These snake families are part of a
superfamily known as the advanced snakes formerly called Colubroidae and contains more than 2500 species 5. In addition, subfamilies of rear-fanged Colubroid snakes are often harmless to humans,
however, some species are able to envenom snakebite victims 2.
Snakes use their venom for prey immobilization and digestion as well as protection against predators
2,6. The venom is injected via modified fangs that are connected via a duct to muscularized secretory
venom glands positioned on both sides of the head on the upper jaw of vipers (Figure 1a) 7,8. The
delivery system of Elapid and Colubrid snakes is similar to that of the viperids but the shape of the glands and the connection with the fangs is different 7. Moreover, the fangs of species from the
Colubrid family are positioned at the back of the mouth, hence, the name rear-fanged snakes. Venom is a complex combination of various biologically active substances, the majority being proteins and peptides which are known as toxins (Figure 1b) 9. These can be divided into enzymatic and
non-enzymatic toxins. The main difference of this division is that small molecules are able to inhibit the
enzymatic proteins 10. Furthermore, venoms also contain metal ions, salts and organic compounds
such as carbohydrates, nucleosides and lipids 7,11. Enzymes that are often found in snake venom
include phospholipase A2s (PLA2s), snake venom serine proteases (SVSPs), metalloproteinases
(SVMPs), L-amino acid oxidases (LAAO), nucleotidases (5’-nucleotidases, ATPases, phosphodiesterases and DNases) and hyaluronidases 6,12. The common non-enzymatic components include the
three-finger proteins (3FTx), cysteine-rich secretory proteins (CRISPs), kunitz peptides, C-type lectins, disintegrins, natriuretic peptides, nerve growth factors, bradykinin-potentiating peptides, cobra venom factors (CVF), proteinase inhibitors and vascular endothelial growth factor (VEGF) 5,9,10. Many
of these snake venom components cause various pathophysiological and systemic effects by binding selectively to receptors, ion channels, enzymes and other molecular targets with high affinity in the victim 11,13. The PLA
2s enzymes are arguably the most bioactive and multi-effect snake venom
components and can act synergistically with other venom components 10.
Antivenom comprised of antibodies, is currently the only effective medical treatment for snake bite envenomations 14. Intact IgG or F(ab’)2- or Fab-fragment therapies are prepared by extracting these
antibodies from serum or plasma from animals immunized with snake venom 15,16. The resulting
venom antiserum is injected into the victim’s bloodstream to neutralize the toxins, preferably before
they cause any permanent damage 10,17.Due to the wide variation in snake venom composition and
different therapeutic properties among various snake species producing a single antiserum that is effective for the venom of all snake species is impossible 18. The production of these antiserums is a
complicated and highly expensive process making it difficult for countries that lack the financial resources to purchase these medications 19. The combination of these factors leads to the limited
Therefore, it is of great importance to develop antisera that are more specific and affordable. In order to achieve this, a better understanding of the underlying molecular mechanisms following envenomation is necessary.
Figure 1. A) Schematic illustration of the venom system. The produced venom is stored in the venom glands and secreted by the action of a compressor muscle, when a bite occurs. Then the duct delivers the venom to the fangs that inject it into the tissues of a snakebite victim 2,7.B) Schematic overview of the snake venom composition 5.
The multifunctional toxins PLA2s, SVMPs, SVSPs, 3FTXs are the most important targets to investigate
for the “next generation” snakebite therapeutics due to the major pathologies that they produce in snakebite victims 9. This can be done by using in vitro assays to study the toxicities generated, as well
as by analytical techniques for the identification, characterization and quantification of these venom components.
Most snake venom-related research published focusses on venom composition and pathophysiological effects of various toxin classes. Meanwhile, there no papers that give a comprehensive overview of the in vitro assays that exist for studying the many pathologies. An overview of this would be a useful starting point for developing treatments against snakebite. This literature study provides an extensive overview of the in vitro assays used to study the different toxicity effects (neurotoxicity, hemotoxicity, and cytotoxicity) caused by snake venom. The focus will also be on the various analytical methods used for venom profiling and how these can be used in combination with the in vitro assays.
1.2 Pathological effects
The main pathologies and toxicity caused by snakebite envenomation include tissue necrosis, local swelling, blistering and apoptosis (cytotoxicity), hemorrhage and coagulopathy (hemotoxicity) and neuromuscular paralysis (neurotoxicity) and in some cases myotoxicity (Figure 2) 15,20,21. Isolated
enzymes from snake venom predominantly target cell membranes, the blood coagulation cascade and vascular walls 20. The variability of these clinical symptoms is directly related to the diversity of toxic
venom components; such variation occurs on various levels including inter- and intra-species, intersubspecies, intergenus and interfamily 21,22. These variations are influenced by environmental
conditions, geographical location, age, gender and feeding habits 3,23.
1.1.1 Cytotoxicity
The characterization of pathological activities of snake venom cytotoxins is mainly based on studies of cytotoxins extracted from cobra venom 13.
Most cytotoxins are non-enzymatic amphipathic short polypeptides belonging to the three-finger toxin family 2,13. These toxins are mainly found in the venom of Elapid snakes, especially that of cobras,
whose venom is made up of 40-70% of these toxins 24,25. Other cytotoxins include PLA
2s, LAAOs, and
CRISPs 26. The actions of cytotoxins lead to necrotic cell death and alter the function and structure of
cell membranes resulting in serious damage 24. They do this by forming cation channels in cell
membranes, which leads to increased levels of Na+ and Ca2+ ions in the cytosol causing cell death and
calcium intoxication, disruption of the ionic balance, and cytolysis 10. Cytotoxins have a high affinity
for binding to anionic lipids in cell membranes 25. This interaction with negatively charged lipids can
cause cytotoxin dimerization, ultimately leading to oligomerization, an important part of membrane pore formation 13.
A few cytotoxins that can depolarize cardiac myocytes and cause heart arrest are also known as cardiotoxins 27. The cytotoxic effects produced by the cardiotoxins in heart cells are usually caused by
the opening of voltage-gated Ca2+ channels, resulting in background K+ channels being blocked and the
generation of new abnormal ion-conducting pathways 28. It is believed that these toxins interact with
target proteins present in membranes of cardiac muscle cells. Other cell types affected by cytotoxins include lymphocytes, spleen cells, different tumor cells, and erythrocytes 13.
Disruption of cell membranes by PLA2s is a secondary process of their catalytic activities on membrane
phospholipids, indicating that venom PLA2s possess cytotoxicity. These toxins are abundantly present
in both Elapidae and Viperidae snakes. However, only PLA2s of vipers show cytotoxic effects 26. Snake
venom LAAOs have also shown to cause cell death due to H2O2 generation during the deamination of
L-amino acid to α-keto acid 29. The produced H
2O2 is a reactive oxygen species that is very toxic and is
able to act on proteins, plasma cell membranes, and nucleic acids 30.
1.1.2 Neurotoxicity
Many of the neurotoxins are ion channel and membrane receptor blockers that interfere with the cholinergic transmission of the peripheral nervous system, where the main target is the skeletal neuromuscular junction 3,5. This may cause acute neuromuscular paralysis, which could lead to
respiratory failure by affecting the muscles that support breathing 31. These effects are mainly
observed in elapid envenomations but also in some species of Viperidae such as rattlesnakes and Russell’s viper 3.
Based on their site of action, neurotoxins are classified as pre-or postsynaptic, also known as - and -neurotoxins, respectively 32. The major neurotoxic components present in Elapid venoms are 3FTxs
proteins, which are - neurotoxins that competitively bind with high affinity to muscle () nicotinic acetylcholine receptors (nAChRs) positioned on the membrane of postsynaptic neurons 33,34. This
hardly irreversible interaction subsequently disrupts neuromuscular transmission. Fasciculins, a class of 3FTx present in the venom of mambas, can interfere with the hydrolysis of acetylcholine (ACh) by inhibiting acetylcholinesterase (AChE) 35. The degradation of ACh results in the termination of
neurotransmission at cholinergic synapses, which allows the neuron to go back to its resting state after activation 3,36. Mamba venoms also contain dendrotoxins and fasciculins that block certain types
of voltage-gated potassium channels on nerve endings and synaptic AChE, respectively 35,37. The
inhibition of these ion channels results in the accumulation of ACh at the neuromuscular junction, which produces longer action potentials causing hyperexcitability of neurons that lead to convulsions and direct cardiotoxicity resulting in death 38. The blocking of synaptic AChE by fasciculins leads to
fasciculations, also known as muscle twitches 3,35.
Other pathways that lead to neurotoxicity include the presynaptic actions of -neurotoxic PLA2s that
cause depletion of ACh synaptic vesicles and irreversible damage to motor nerve terminals due to phospholipid hydrolysis 3. Secondary contributing mechanisms of these enzymatic proteins include
interactions with muscle nAChRs and voltage-gated potassium channels 35. Much more is known about
the mechanism of action of - neurotoxins, compared to those of presynaptic PLA2 -neurotoxins 13.
However, it seems that hydrolysis of phospholipid bilayer of neurons is essential for generating presynaptic neurotoxicity 39. Therefore, a better understanding of the activities of PLA
2 neurotoxins
involved in presynaptic toxicity is necessary for the development of therapies to prevent neurodegeneration 40. This deterioration is due to the accumulation of lysophospholipids and fatty
acid levels in plasma membranes of the synaptic cleft, presumably caused by the action of endogenous lipases at the nerve terminals 13,40.
1.1.3 Hemotoxicity and cardiovascular disruptions
Haemotoxicity is the most common toxic effect observed following snake bite envenoming, especially those snakes belonging to the Viperidae family 15. While this type of toxicity is mainly inflicted by
viperid snakes, particularly pit vipers and true vipers, some rear-fanged Colubroid species, Australian elapids, and African spitting cobras are also capable of causing hemotoxicity 15,17,41,42. Venom toxins
cause haemotoxic effects by directly inducing hemorrhage and disturbing the regulation of blood pressure, clotting factors, and platelets. The most recognized hemotoxins are the zinc-dependent SVMPs, which are abundant in viperid snake venoms and can be divided into three classes based on their domain structure(i.e., P-I, P-II, and P-III) 26,43,44. These toxins are well known for rupturing capillary
vessels and predominantly responsible for hemorrhages; hence they are also called hemorrhagins 20,43.
Other hemotoxins include SVSPs and PLA2s that primarily target the coagulation cascade in which they
act as procoagulants or anticoagulants and may also affect platelet aggregation 10,20. In addition,
LAAOs, C-type lectins-like, and 5’-nucleotidases also exhibit some haemotoxicity by either inhibiting or inducing platelet aggregation 45. Hydrogen peroxide (H
2O2) generated from the enzymatic activities
of LAAOs also interferes with interactions between blood coagulation factors 46.
The pathological pathway of hemorrhage induced by hemorrhagic toxins involves the direct damage to blood vessels causing extravasation, which combined with various hemostatic effects, may lead to massive blood loss resulting in hemodynamic instability and cardiovascular shock 47,48. Profuse
bleeding is thus caused by the synergistic action of hemostatic interferences (e.g., action of SVSPs) and microvessel disturbances (e.g., action of SVMPs) 15. However, hemorrhagins can also cause
bleeding without the presence of changes in the hemostatic system. The induced local and systemic bleeding by SVMPs can affect different organs such as the brain, heart, liver, and lungs 20.
Furthermore, these enzymes can also cause tissue necrosis, swelling, and blisters (Figure 2). The P-II and III classes of SVMPs are mostly hemorrhagic by digesting vessel wall components, while the P-Is are fibrinogenolytic 15. Several SVSPs have shown kininogenase, thrombin-like, and fibrino(geno)lytic
activities similar to thrombin; hence they are often referred to as thrombin-like enzymes (TLE) 41. Many
TLEs proteolytically cleave fibrinogen into fibrinopeptides resulting in the formation of clots by polymerization of fibrin monomers 15. In addition, TLEs are unable to activate factor XIII to cross-link
the polymers, making the formed fibrin thrombi very unstable and therefore easily dissolved by plasmin. The constant production and destruction of clots result in depletion of circulating fibrinogen, making the blood uncoagulable 41.
Chapter 2. In Vitro assays
One of the first in vitro bioassay techniques developed for studying toxicity of compounds on various tissues were cell toxicity assays 49. These assays can be a promising alternative to in vivo testing for
determining toxicity of snake venoms.
2.1 In vitro cytotoxicity
Membrane integrity is the main feature utilized for detection, whether in vitro cultured eukaryotic cells are dead or alive 50. Cytotoxicity is mainly predicted by cell viability assays based on various cell
functions, including cell membrane permeability, nucleotide uptake activity, cell adhesion, enzyme
activity, adenosine triphosphate (ATP) production, and coenzyme production 51. Non-viable or dead
cells are permeable to molecules that are otherwise excluded by living cells due to the loss of their membrane integrity 50. On the other hand, cytosolic proteins and molecules can also leak out of the
cell because of disturbances in the membrane that is supposed to keep them in the cell 52. The
movement of these molecules in and out of the cell can be measured by various assays.
Several types of cell viability tests exist and can be broadly classified as (a) colorimetric assays, (b) dye exclusion assays, (c) luminometric assays, (d) flow cytometric assays, and (e) fluorometric assays 53.
The simplest techniques are the dye exclusion assays that use various dyes such as trypan blue, eosin,
congo red, and erythrosine B that are permeable to dead cells 51. Colorimetric assays measure the
leakage of a biomarker usually an enzyme to determine the metabolic activity of the cells 50,51. A
spectrophotometer is used for colorimetric measurements. The most utilized colorimetric assays include MTT Dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium), XTT (2,3-bis-(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide), WST-1 (2-(4-iodophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt), WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium, monosodium salt), lactate dehydrogenase (LDH), sulforhodamine B (SRB), neutral red uptake (NRU), and crystal violet stain (CVS) assays 53. Fluorometric assays use fluorescent dyes such as resazurin (alamarBlue) and
5-carboxyfluorescein diacetate acetoxymethyl ester (5-CFDA-AM) and can be carried out with a fluorometer, fluorescence microplate reader, fluorescence microscope, or flow cytometer 51,53. Other
methods include luminometric assays such as the ATP assay and real-time viability assay that generate a stable and persistent glow- type signal after the addition of a reagent 53.
2.1.1 Colorimetric assays
MTS assay
MTS belongs to the newer class of tetrazolium reagents that are negatively charged and are analogs of MTT 51. The MTS cell proliferation assay is based on the reduction of this tetrazolium salt by
mitochondrial activity of viable cells generating colored formazan products that are soluble in cell culture medium 54,55. This assay is easy to use, reliable, inexpensive and gives a rapid indication of
toxicity 53. One study compared the in vitro cytotoxic effects of venom from Acanthophis spp. and and
Naja spp. snakes, which belong to the Elapidae family 56. The species studied included A. antarcticus,
A. praelongus, A. rugosus and A. sp. Seram (Acanthophis spp) and N. nigricollis, N. haje, and N. mossambica (Naja spp.) snakes.
The effect of these venoms on cell viability was determined using a rat aorta smooth muscle cell line, A7r5 and rat skeletal muscle cell line (L6), as many snakes target skeletal muscles. For this study the MTS assay was used to measure cell proliferation.
The A7r5 and L6 cells in 96- well micro-titer tissue culture plate are first incubated for 24 hours with the venom stock solutions at 37 C with 5% CO256. The cells are then washed with PBS, the culture
media is refreshed and MTS is added to the solution of each well, and further incubated for 3 hours
56. Following incubation, the absorbance is recorded with a fusion α plate reader at 492 nm. Usually
for the detection, electron-acceptor redox intermediates such as PMS (phenazine methyl sulfate) or PES (phenazine ethyl sulfate) are used in combination with MTS 55. These intermediates are not added
in this study. The number of cells present in the culture is directly proportional to the amount of
formazan produced by dehydrogenase enzymes 54.
The resulting IC50 values showed that the cytotoxicity level varied amongst both Acanthophis spp. and
Naja spp. venoms (Table 1) 56. The rank of potency was as followed: A. antarticus ≥ A. rugosus ≥ A.
praelongus N A. sp. Seram and N. mossambica >N. nigricollis > N. haje. The Acanthophis spp were
remarkably less cytotoxic compared to those of the Naja spp. snakes. Generally, the N. nigricollis and
N. mossambica venoms are mainly cytotoxic; hence, they show a higher potency compared to N. haje
venom that is mostly neurotoxic 56. Thus, this ranking also shows consistency with the observed clinical
effects observed in these snake venoms. Various parameters, such as cell type and incubation time, can influence the absorption level of tetrazolium reduction tests, including the MTS assay 54,55. This is
also observed in the current study where the N. nigricollis and N. mossambica, show a significantly higher potency in the L6 cells, which indicates that skeletal muscle cell lines might be more sensitive to cytotoxins and thus may be the most suitable for analyzing cytotoxic snake venoms. Moreover, in both cell lines the venom of N. mossambica (spitting cobra) snake was more potent than that of N.
nigricollis (spitting cobra), which is in contrast to a previous study that found N. nigricollis to be more
cytotoxic among the Naja species 56,57. This may also reflect the differences in the cell lines used in
these assays 56.
Table 1. IC50 values for Acanthophis spp. (n=3) and Naja spp. (n=4) venoms 56.
The other colorimetric assays were all utilized for evaluating the cytotoxicity of Macrovipera lebetina
snake venom in HEK-293 cells 58.
Macrovipera lebetina (M. lebetina) is one of the most venomous
snakes found in Iran with acute renal failure (ARF) as one of the main effects observed 58,59. It is
presumed that cytotoxic effects of the venom on the kidney play a major role in ARF pathogenesis 58.
Necrosis and apoptosis are some of the effects identified in cells undergoing renal failure. Therefore, cells from human embryonic kidney 293 cell line were used to measure the cytotoxicity with MTT, LDH and neutral red uptake assays.
IC50 concentration (μg/mL) A7r5 L6 A. antarcticus 47.7 ± 2.5 67.1 ± 6.4 A. rugosus 51.0 ± 6.9 81.5 ± 17.0 A. praelongus 59.2 ± 5.6 116.6 ± 28.8 A. sp. Seram 107.4 ± 16.7 188.1 ± 17.7 N. mossambica 7.9 ± 0.9 3.1 ± 0.4 N. nigricollis 10.0 ± 1.0 7.2 ± 0.6 N. haje 13.6 ± 1.1 24.5 ± 2.0
In all three experiments, the HEK-293 cells are seeded in 96-well plates at 20,000 cells per well and incubated for 3 and 24 hours to attach. After incubation the cells are treated with a concentration series of 1, 5, 10, 20, 40, 80 μg/mL crude venom and incubated for another 3 to 24 hours.
MTT assay
The MTT tetrazolium assay was the first homogeneous cell viability assay developed for 96-well plates suitable for high-throughput screening (HTS) and is considered the golden standard 51,60. This method
is similar to the previously discussed MTS assay where the tetrazolium salt is converted into colored formazan crystals by dehydrogenases inside mitochondria of living cells 60. However, the formed
crystals in the MTT assay are insoluble and must first be dissolved before measuring absorbance 55.
The level of absorbance for this method is determined at 570 nm using a spectroscopic multiplate reader. Following the exposure of HEK-293 cells to the crude venom and incubation, 20 μL of MTT (5 mg / mL) is added to each well and incubated for an additional 3 hours 58. The culture medium
consisting of MTT is then taken out of the wells and 100 μL of dimethyl sulfoxide solvent (DMSO) is added to dissolve the formazan crystals. An ELISA plate reader is used to measure the absorbance at 570 nm.
Neutral red uptake assay (NRU)
The NRU assay is used for the quantification of viable cells 51. Living cells are able to incorporate and
bind to the supravital dye neutral red. This dye is a weak cationic substance that passes the cell
membrane through nonionic passive diffusion and accumulates in lysosomes 61. It binds to anionic
and/or phosphate groups of the lysosomal matrix via electrostatic hydrophobic interactions. The dye is then extracted from the viable cells with an acidified ethanol solution and the absorbance of the solubilized dye is measured with a spectrophotometer. After the 24 hours of incubation of the venom-exposed cells, the medium of the wells are changed with a new one consisting of 40 μg /mL NR dye 58.
Followed by an additional 3 hours of incubation, the neutral red medium was removed, and the cells were washed with a PBS buffer. Finally, the extraction solution containing 50% from ethanol 96%, deionized water 49% and glacial acetic acid 1% is added to each well. Then the optical density (OD) of the dye extract is recorded at 540 nm with a microplate reader.
LDH assay
LDH cytotoxicity assays are mostly used for the measurement of necrotic cell death 62. Necrosis is a
type of cell death with morphological features, including swelling of cells and organelles and rapid
rupture of the cell membrane 63. The breakdown of the plasma membrane can cause cell contents,
such as cytoplasmic enzymes, including LDH, to leak into the extracellular environment 51. In this assay
the yellow tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) is used to detect the release of LDH into the cell culture medium 53. When LDH catalyzes the oxidation
of lactate to pyruvate, reduced nicotinamide adenine dinucleotide (NADH) is generated. The newly formed NADH then reduces the yellow tetrazolium salt to a red water-soluble dye of which the absorbance can be recorded at 490 nm. The amount of formazan measured represents the total LDH activity and is directly related to the amount of damaged cells 51. This LDH assay was used to
characterize of the mechanism of M. lebetina venom cytotoxicity by studying the LDH activity in the HEK-293 cells 58.
The measurements obtained with both the MTT and NRU assays showed only cytotoxic effects of the venom on the cells exposed for 24 hours, indicating that the effect of the venom is time-dependent
58. A dose-dependent relationship was also observed after 24 hours of exposure. In the culture media
of the HEK293 cells exposed to venom concentrations higher than 20 µg/ml, an increase in LDH activity was measured by the LDH assay. However, this was not statistically significant and thus suggesting that the cytotoxic effects of the M. lebetina venom are of apoptotic nature rather than necrotic. The LDH assays are able to discriminate between necrosis and apoptosis 64.
By also conducting a separate apoptosis assay, one can confirm whether the induction of this process is one of the mechanisms responsible for the cytotoxic effects caused by snake venoms 65.
Such assays include measuring caspase activity fluorometrically or colorimetrically, flow cytometric assays (e.g., annexin V staining), TUNEL assay and other staining assays such as the dual ethidium bromide and acridine orange (EB/AO) staining assay 51,66–68.
Assays used to determine the cytotoxicity of crude snake venoms can also be correlated to the activity of specific components in those venoms. An example of such a study shows that L-amino acid oxidase isolated from the venom of Bothrops atrox can induce caspase-mediated apoptosis 69. The main
characteristics of apoptosis include DNA fragmentation, chromatin condensation and caspases activation 70.
In this study, the MTT assay was used to measure cytotoxicity and, for apoptosis detection, the Annexin V staining flow-through cytometric assay, and cellular caspases 9 and 3 activity measurements were performed 69. Apoptosis was quantified with fluorescence microscopy. In
addition, the LAAO activity was recorded with a spectrophotometric assay using L-leucine as substrate. Murine and human cell lines (PC12, B16F10 and HL-60 and Jurkat) derived from cancer cells were utilized for these experiments 69,71–74.The MTT assay was performed with all four cell lines and for caspase activity and flow cytometric analysis the HL-60 cells were utilized 69.
L-amino acid oxidase assay
This is a spectrophotometric microplate assay based on a horseradish peroxidase (HRP)-linked reaction with o-phenylenediamine (OPD) as a H2O2 probe 75. Generated H2O2 is detected by the HRP
catalyzed oxidation of the H2O2 sensitive probes.One unit of LAAO activity was defined as the amount
of enzyme needed to produce 1 µmol of H2O2 per minute, under the specified conditions. In this study
L-leucine was used as a substrate that was oxidatively deaminated catalyzed by LAAO, producing H2O269. The reduction of the formed H2O2 by OPD resulted in a colored product that was measured at
490 nm.
Assay to measure caspase-3 and caspase-9 activities
Apoptotic caspases are cysteine proteases that induce apoptosis by cleaving the peptide bond following an aspartic acid in their various protein substrates 76. The caspase family is made up of 14
members divided into three different subfamilies, including apoptosis activators, executioners, and inflammatory mediators 77. Caspases can be activated by either a death receptor- mediated pathway
or a mitochondrion-mediated pathway (i.e. a caspase-9-dependent pathway) 77,78. Moreover, these
enzymes can activate each other and thereby accelerate the process of cell death 70. The cleavage site
varies by subfamily, but caspases within a subfamily may prefer the same cleavage sequence 79. The
active form of caspase-3 is able to cleave the enzyme poly (ADP-ribose) polymerase (PARP) 80. PARP
appears to play a key role in DNA repair and proteolytic cleavage of this enzyme prevents it from being recruited to sites of DNA damage. Mapping of the cleavage site of PARP showed that caspase- 3 prefers to cleave the tetrapeptide (Asp-Glu-Val-Asp) DEVD sequence 81. The caspase-3 activity assay is
based on the cleavage of a synthetic tetrapeptide DEVD labeled with either a fluorescent molecule, 7-amino-4-trifluoromethyl coumarin (AFC), or a colorimetric molecule, p-nitroanilide (pNA) 80. In the
study of the Bothrops atrox LAAO the colorimetric type was utilized for measuring the absorbance of the released chromophore pNA after cleavage of the Ac-DEVD- pNA substrate with a spectrophotometer at 405 nm 69,80. The same principle was used for assessing the activity of
caspase-9 but with the cleavage sequence LEHD, which is the preferred site of this enzyme. Therefore, the Ac-LEHD-pNA peptide was utilized 69.The level of activity can be assessed by comparing the measured absorbance of an uninduced control sample with an apoptotic one 80.
Flow cytometry analysis
Flow cytometry is based on the detection of cells passing by a laser in a liquid flow 51. Cell components
labelled with a fluorescent compound are excited by the laser and subsequently emit light that can be detected. Flow cytometry machines are thus able to quantitatively analyze each cell. The most common flow cytometric assays include mitochondria assays, membrane permeability (e.g., nucleic acid and inclusion and exclusion dyes), and membrane asymmetry (e.g., annexin V and F2N12S staining assays). For this study, the asymmetry and exclusion dye assay with annexin V and propidium iodide (PI), respectively, were used 69. In the early stages of apoptosis, the cell membrane architecture
undergoes some changes, including phospholipids being loosely packed and phosphatidylserines (PS)
emerging on the outer surface of the membrane 81. In addition, during these changes the membrane
integrity stays intact 51. Annexin V is a fluorescent protein that interacts with the PS exposed
phospholipid membrane in the presence of calcium ions 82. The exclusion dye propidium iodide (PI) is
often used in combination with annexin V to make sure that only cells with a compromised membrane integrity are stained 81. In this way apoptotic cells with an intact cell membrane that are only stained
by annexin V can be distinguished from necrotic cells that have both stains. Exclusion dyes are fluorescent molecules that are not excluded by leaky membrane cells as is the case with necrosis 51.
High catalytic activity of B. atrox LAAO was observed against the L-Leu. The results of the MTT assay showed a dose-dependent cytotoxic effect of B. atrox LAAO on the HL-60 cellswith an IC50 of
approximately 50 μg/mL 69. In addition, the B. atrox LAAO was also able to induce apoptosis in the
other cell lines.The microscopic analysis revealed the highest percentage of apoptotic cells was found in the HL-60 cell line. The flow cytometry analysis used to confirm the results acquired from the microscopy experiment showed both apoptotic (annexin V + /propidium iodide −) and necrotic (annexin V +/propidium iodide +) HL-60 cells. The activity of both caspases increased in a dose-dependent manner. Caspase-3 activity indicated that induction of apoptosis by B. atrox LAAO was responsible for the reduction of viable cells. In addition, the caspase-9 activity suggested that a mitochondrial pathway triggered the caspase cascade 69,77.
2.1.2 Fluorometric and dye exclusion assays
The Alamar Blue (resazurin) assay was used to measure the cytotoxicity of venom from 5 Brazilian
Bothrops spp. snake species including B. jararaca, B. alternatus, B. jararacussu, B. neuwiedi, and B. moojeni and was then related to activities of enzymes including LAAO 83. The venom of these snake
species are constituents of an antigenic mixture used to produce Brazilian antivenom. In this study a MGSO-3 cell line derived from primary human breast cancer was utilized.
Alamar Blue assay
This fluorometric assay is based on the irreversible reduction of the cell-permeable and nearly non-fluorescent molecule resazurin (blue color) by viable cells with an active metabolism to resorufin, a highly fluorescent pink compound 55,84. Resorufin is extracted from the living cells into the medium
resulting in a color change of the medium that can be measured with microplate fluorometer with a filter using a range 530-570 nm for excitation and 580- 620 nm emission 51. This method is more
sensitive compared to the classic tetrazolium reduction assays because it mainly measures fluorescence instead of absorbance. Moreover, Alamar Blue also avoids many incompatibility issues observed in tetrazolium assays 85. In addition, resazurin is not only water soluble, but also non-toxic
and stable in culture medium, allowing for continuous cell monitoring 85. Furthermore, various Alamar
Blue cell viability and proliferation kits are commercially available that can also be readily used in high throughput applications 51,55,84.
The MGSO-3 cells are plated in 96-well microtiter plate and incubated for 24 hours at 37 C.
Afterwards, 20 µg/mL of each venom in decreasing concentrations (0.31-20 µg/mL) is added to each well. Then 10% of Alamar Blue in DMEM is added following a 24 hour incubation. The fluoresence is measured after 3 hours with a Synergy 2 (Biotek) fluorimeter at 540 nm of excitation and 590 nm of emission. The results for this assay showed a concentration-dependent inhibition of cell viability on these cultured cancer cells. The cytotoxic dose (CD50) or the amount of venom needed to kill half of
cells was used to determine the cytotoxic activity of the venoms. The cytotoxicity level varied between
the species where B. neuwiedi, B. jararacussu, and B. moojeni with the lowest CD50 value were the
most toxic while B. jararaca B. alternatus with the highest CD50 value were the least toxic (Table 2).
The results obtained from the Alamar Blue assay were than correlated to the activity of LAAO enzymes
83. To measure this activity, a L-amino acid oxidases assay was used and has the same principle of the
previously described LAAO assay in the section of colorimetric assays of the present literature
overview.One unit of LAAO activity is defined as the amount of enzyme needed to produce 1 µmol of
H2O2 per minute, under the specified conditions. A significant correlation between the LAAO activity
and cytotoxicity was observed (Table 2). These results were then compared with data from experiments performed in a previous study that included the determination of venom-induced necrotizing activities 83,86. In addition, other in vitro experiments showed a dose-dependent
relationship between the exposure of cells to snake venom LAAO and necrosis they induced 83. The
combined results indicate that at least for Bothrops spp. venoms, the in vitro assays determining LAAO activity can replace in vivo necrosis assays.
Table 2. CD50 values and LAAO activity of the venoms from Bothrops spp snake species 83.
CD50 concentration (μg/mL) LAAO activity (U/mg/min) MGSO-3 B. neuwiedi 4.07 B. jararacussu 4.24 B. moojeni 4.66 B. jararaca 9.96 B. alternatu 12.42 9.91 9.16 9.24 3.92 3.28
Fluorescent based assays are also commonly used to measure enzymatic activities including those of snake venom PLA2s and serine proteases 25,87,88. The proteolytic activity of the 5 Brazilian Bothrops spp
snakes was determined with a Fluorescence Resonance Energy Transfer (FRET) assay 83. FRET assay
This technique is based on the non-radiative energy transfer froman excited molecular fluorophore
(the donor) to another fluorophore (the acceptor a process that is distant-dependent 89. The donor
and acceptor are coupled via dipole-dipole interactions 90. Peptides with specific amino acid residues
that mimic the cleavage site in the target protein are used for determining protease activity 91. On one
end of the peptide a fluorescent donor group is attached and on the other end a quenching group (Figure 3). The energy transfer from the donor to the acceptor in the intact molecule leads to a decrease in fluorescence. Cleavage of the peptide by the protease separates the fluorophore and quencher which results in a large increase of fluorescence from the donor that can be detected. With FRET peptides used as enzyme substrates one is able to quantitatively measure enzymatic activities. The fluorophore abz (ortho-amino benzoic acid) and the quenching group EDDnp (2,4-dinitrophenyl ethylenediamine) are often used as donor/acceptor pair in FRET peptides. This is a perfect pair because the emission spectrum of abz perfectly overlaps with the absorption spectrum of EDDnp, which is necessary for the energy transfer to occur 90,91. Furthermore, it allows for a high efficiency of
The substrate Abz-FLPRSFRQ-EDDnp containing the canonical arginine (Arg) at the P1 position and suitable for cleavage by SVSP was used to measure the hydrolytic activity of serine proteases. For this, 1µg of each venom and 47 mM of substrates are added to MGSO-3 cells 83. The hydrolysis is monitored
at 340 nm of excitation and 440 nm of emission following incubation of 30 min at 37°C in a microplate reader. The highest activities were observed in B. jararacussu and B. moojeni venoms.
Figure 3. A schematic overview of the FRET assay using peptides as enzyme substrates. In the intact peptide, the FRET between the donor(fluorophore) and acceptor (non-fluorescent quencher) results in low fluorescence. Cleavage of the peptide separates the fluorophore from the quencher, increasing fluorescence 92.
Experiments used in a study that evaluated the effects of a Naja naja kaouthia cobra venom cytotoxin free of PLA2 on model membranes included the trypan blue assay and a fluorometric assay for
measuring PLA2 activity 25. Trypan Blue stain assay
Trypan blue stain assay is one of the most straightforward methods used for the quantification of viable cells.93 Living cells with an intact plasma membrane prevent the uptake of Trypan Blue, a large
negatively charged molecule 94. Therefore, viable cells appear colorless by light microscopy, while
dead cells which have compromised membranes are immediately stained blue after exposure to this dye. For determining the cytotoxic effect of the cobra venom cytotoxin, Jurkat cells derived from human T cell leukemia and normal human lymphocytes (NHL) are incubated with defined concentrations of the cytotoxin with or without 10-9 M PLA
2 for 30 minutes at 37°C 25. The dye can
have toxic effects on mammalian cells when incubated for too long 53. Therefore, cells should be
counted 3- 5 minutes following mixing with Trypan Blue 51. Often a hemocytometer, which is a
counting chamber device, is used to determine the amount of viable or dead cells by light microscopy
53,95. The cytotoxicity is calculated as percentage of untreated control cells (Table 3) 93.
The results showed that the cytotoxin was less effective on the normal human lymphocytes at the concentrations studied 25. Around 6% of these cells died as a consequence of the cytotoxin activity. A
higher activity of the cytotoxin at a concentration of 10-5 M was observed on Jurkat cells, killing
approximately 80% of them (Table 3). The addition of PLA2 significantly reduced the cell viability of
both cell types incubated with the cytotoxin. About 92% of normal lymphocytes and 98% of Jurkat cells died after treatment with PLA2in combination with 10-5 M of the toxin. These results indicate that
cytotoxin might be able to discriminate between malignant and normal lymphocytes. Furthermore, they also suggest that it acts synergistically with PLA2 to induce cytotoxicity in both the Jurkat cells and
Table 3. Cytotoxicity induced by the cytotoxin (CT) on normal human lymphocytes and Jurkat cells in the presence or absence of 10-9 M PLA2 25. The results in the table are expressed as percent viable cells after the indicated treatments.
CT concentration (M) Cell untreated with PLA2 Cell treated with PLA2
NHL Jurkat NHL Jurkat 0 100* 100 95.4 ± 2.8 98.7 ± 3.4 10-10 98.1 ± 3.0 91.4 ± 2.5 - - 10-9 99.3 ± 5.5 86.5 ± 2.0 - - 10-8 98.3 ± 2.3 74.3 ± 4.5 62.0 ± 5.6 59.5 ± 3.4 10-7 99.0± 3.6 46.2 ± 4.9 34.9 ± 5.1 26.5 ± 4.1 10-6 96.7 ± 3.1 35.3 ± 2.0 18.2 ± 3.5 9.1 ± 3.0 10-5 94.0 ± 4.0 21.5 ± 3.1 8.0 ± 3.0 2.6 ± 2.0 * Percent viable cells is based on control cell samples not incubated with CT or PLA2 25. The data are based on the means of
three preparations ± the standard deviation.
The Trypan Blue staining method is quick and easy, but it has some drawbacks 50. The limitations
include the analysts' subjective interpretation of what a dead cell is or colored debris. It is possible that cells with a compromised membrane can become completely viable again by repairing themselves, which is another problem because the viability of the cells is measured indirectly from the cell membrane integrity 93. In addition, Trypan Blue staining assay is not able to discriminate
between healthy cells and living cells that are losing cell functions 53. Hence, it is not sensitive enough
to use for in vitro cytotoxicity testing. Furthermore, this technique is not suitable for high throughput screening because it requires manual counting of the cells 94. An alternative approach for more precise
analysis of cell viability is to use flow cytometry with Trypan Blue for determining dye exclusion 93. PLA2 activity assay
The PLA2 activity was also measured in the study of the cobra venom cytotoxin using a continuous
fluorescent displacement assay 25. This method is based on the interaction of a fluorescent probe
DAUDA (11-(dansylamino)undecanoic acid) with a rat liver fatty-acid-binding protein (FABP) that leads to a high level of fluorescent enhancement 96. The hydrophobic DAUDA probe is almost
non-fluorescent in buffer but shows increased fluorescence when present in a non-polar phase. This is the case for phospholipid micelles in which fluorescence increases and will be a function of the micelle concentration. The DAUDA probe has a high affinity for rat liver FABP; hence, it preferentially binds to this protein in a mixture with phospholipid micelles. Long-chain fatty acids are released as a result of hydrolysis of phospholipids by PLA2. These long-chain fatty acids then displace the DAUDA probe by
binding with an equal or higher affinity to FABP, which leads to a decrease of fluorescence. The rate of fatty acid release and phospholipid hydrolysis by PLA2 is parallel to the rate at which fluorescence
is reduced. The PLA2 activity can be determined as a result of this continuous displacement and
reduction of fluorescence. For the cobra venom cytotoxin study, an assay cocktail is prepared consisting of DAUDA and phosphatidylcholine in methanol mixed with an assay buffer (0.1 M Tris-HCl,
pH 7.8, 0.1 M NaCl, 0.1 mM CaCl2, 0.02 mM Triton X-100) 25. Then 0.01 mg of cobra venom fractions
obtained from chromatography analysis are mixed with 2 mL of the assay mixture and incubated at 37°C for 30 minutes in a fluorimeter cell. Lipid hydrolysis is stopped by the addition of 20 mM EDTA, and then 30 µg of rat liver FABP is added. The DAUDA fluorescence is measured at 500 nm after excitation of the solution with a pulsed laser at 350 nm. This assay is a very sensitive method capable of detecting free fatty acid concentrations as low as 0.1 nM. All six fractions, except the first one hydrolyzed phosphatidylcholine, and the highest PLA2 activity was observed in fraction F2.
2.2 In vitro neurotoxicity
Neurotoxicity is mainly caused by neuromuscular block and inhibition of neuromuscular transmission
3. The main venom components responsible for these effects are the neurotoxic PLA
2s on a
pre-synaptic level and 3FTxs on a post-pre-synaptic level 97. The main targets of these toxins include pre- and
post-synaptic nAChRs, voltage-gated calcium channels, potassium channels and SNARE (Soluble N-ethylmaleimide-sensitive-factor Attachment Receptor) proteins. In vitro neurotoxicity induced by snake venom toxins is analyzed using neuromuscular preparations from avian, amphibian and
mammalian species 98. The most often used are the mouse phrenic diaphragm and the chick biventer
cervicis preparations 99. Others include frog rectus abdominis muscle preparation guinea pig isolated
ileum preparation 100. In addition, neurotoxicity is also determined with enzyme-linked
immunosorbent assays (ELISA) and assays using cultured cells that are able to express certain receptors such as the nAChR.
2.2.1 In vitro muscle preparations
The chick biventer cervicis nerve–muscle and mouse phrenic nerve–diaphragm preparations were used in a study for the comparison of in vitro neurotoxicity three Australian snakes 101. These snakes
include Hoplocephalus stephensi, Austrelaps superbus and Notechis scutatus.
Chick biventer cervicis nerve–muscle preparation
The in vitro assay of chick biventer cervicis nerve–muscle preparation is used to discriminate between
neuromuscular blocking agents (NMBAs) that cause depolarization and those that do not 102. Both
decrease the contractions induced by the stimulation of nerves; however, the depolarizing substances also cause muscle contraction. This is reflected by a decrease in twitch amplitude (due to blockade of post-synaptic muscle nAChRs) and fade of the twitch height responses on repetitive stimulation (due to blockade of pre-synaptic neuronal nAChRs) 3. The depolarizing NMBAs only reduce the twitch
amplitude. Moreover, this assay is also able to determine both pre-and post-junctional neurotoxicity
100. Innervated biventer cervicis muscle is removed from the back of the neck of 4 – 14 day old chicks 103. Thereafter, the isolated muscle is bathed in a physiological salt solution (34°C), bubbled with 95%
O2 and 5% CO2, consisting of (in mmol/L): NaCl 118.4; NaHCO3 25; glucose 11; KCl 4.7; MgSO4 1.2;
KH2PO4 1.2; CaCl2 2.5 100,101,103. Then the preparation is attached to a ring electrode and lever
transducer around the upper tendon where the nerve supplying the muscle is located (Figure 4) 102.
Stimulation is applied by the electrodes, either directly to the muscle or indirectly via the nerve, then
the venom is added to the bath medium 100. Muscle contractions and contractures are recorded via
the transducer 104. The chick biventer cervicis nerve - muscle preparation also allows to distinguish
between myotoxic and neurotoxic effects caused by a toxin or snake venom 98. Myotoxicity inhibits
both direct and indirect twitches, while neurotoxicity only reduces indirect twitches. In addition, this preparation can also respond to both electrical stimuli and exogenous agonist. This makes it possible to discriminate between prejunctional and post- junctional effects of venoms or toxins. Pure presynaptic neurotoxins would terminate indirect evoked twitches without interfering with the responses to cholinoceptor agonists such as ACh and carbachol. Postsynaptic neurotoxins would inhibit both responses to cholinoceptor agonists and to indirect stimulation, however, responses induced by high K+ concentrations would not be affected.
In the study of the three Australian snakes, the motor nerve was stimulated to evoke twitches at a
voltage greater than that required to produce a maximal twitch (0.1 Hz, 0.2 msec) 101. Followed by the
addition of venom at concentrations of 3 µg/mL and 10 µg/mL. Before and after the addition of venom, the submaximal responses to ACh (1 mmol / L, 30 s), carbachol (20 µmol / L, 60 s) and KCl (40 mmol / L, 30 s) without nerve stimulation are determined in order to test if neurotoxic activities occur 101,103.
At these concentrations ACh, carbachol and KCl generate contractures similar in amplitude to the control twitch height 103. The time it takes to block the amplitude of indirectly evoked twitched by 90%
(t90) is calculated to compare the venom potencies 101,103.
Figure 4. Illustration of electrode assembly 102. The stimulus can be directly applied to the muscle or indirectly via the muscle
nerve. The muscle contractions and contractures are then recorded via the transducer.
Twitches in the chick biventer cervicis preparation were blocked by all three venoms at 3 µg/mL and 10 µg/mL in a time-dependent manner 101. The venom of N. scutatus had the highest potency and that
of A. superbus the lowest (Figure 5; Table 4). The t90 values of the H. stephensi and A. superbus venoms
at 3 µg/mL were significantly different from those at 10 µg/mL, indicating that inhibition is concentration- dependent. However, the N. scutatus venom t90 value at 3 μg/mL was not significantly
different compared to that at 10 μg/mL, indicating a near maximum inhibition at 3 µg /L in this muscle preparation.
Figure 5. Effect of Notechis scutatus ( ), Hoplocephalus stephensi ( ) and Austrelaps superbus ( ) venoms at (a) 3 µg/mL and (b) 10 µg/mL on indirect twitches of the chick isolated biventer cervicis nerve–muscle preparation 101.
Contractile responses to exogenous ACh (1mmol/L) and carbachol (20µmol/L) were terminated by all three venoms at a concentration of 10 µg/mL 101. However, they had no significant effect on
contractures induced by KCl (40 mmol/L) that is 90 ± 13, 87 ± 4 and 100 ± 8% of initial response for N.
scutatus, H. stephensi and A. superbus, respectively.
Table 4. Time (min) it takes to generate 90% inhibition of indirect twitches 101.
Chick biventer cervicis
3 µg/mL 10 µg/mL
Notechis scutatus 38 ± 6 (8) 22 ± 2 (12) Hoplocephalus stephensi 47 ± 7 (8) 20 ± 2 (11) Austrelaps superbus 89 ± 10 (7) 26 ± 3 (11)
The data is based on the mean ± SEM of the number of experiments given in parentheses.
Mouse phrenic nerve–diaphragm preparation
The left phrenic nerve of a rat or mouse is removed from within the thoracic cavity together with the rib connected to the diaphragm 100. The preparation is then bathed in the same salt solution as
previously described for the chick biventer preparation and mounted with the nerve attached to an electrode and the muscle to a transducer. The same conditions and procedure are used as the chick biventer assay except for the temperature, which is kept at 37°C 100,101. The phrenic nerve is stimulated
at a voltage higher than that which produces a maximal twitch (0.1 Hz, 0.2 msec) 101. The indirect
twitches in the mouse diaphragm preparation were in inhibited by all three venoms at 3 µg/mL and 10 µg/mL, with the N. scutatus venom having the highest potency and A. superbus the lowest (Figure 6; Table 5). Just as in the chick biventer preparation, the t90 values of the H. stephensi and A. superbus
venoms at 3 µg/mL were significantly different from those at 10 µg/mL, indicating that inhibition is concentration- dependent. Also, the t90 of the N. scutatus venom at 3 µg/mL was not significantly
different from the one at 10 µg/mL.
Figure 6. Effect of Notechis scutatus ( ), Hoplocephalus stephensi ( ) and Austrelaps superbus ( ) venoms at (a) 3 µg /mL (n = 4–7) and (b) 10 µg/mL (n=5) on indirect twitches of the mouse phrenic nerve– diaphragm preparation 101.
The results from both experiments indicate that all three venoms induce neurotoxicity via post-synaptic action 101. This is based on the fact that they were able to block indirect twitches in both
neuromuscular preparations and selectively inhibited exogenous nicotinic receptor agonists.
Table 5.Time (min) it takes to generate 90% inhibition of indirect twitches 101.
Mouse diaphragm
3 µg/mL 10 µg/mL
Notechis scutatus 29 ± 4 (9) 21 ± 3 (5) Hoplocephalus stephensi 75 ± 8 (5) 25 ± 4 (5) Austrelaps superbus 110 ± 8 (7) 45 ± 7 (11)
The data is based on the mean ± SEM of the number of experiments given in parentheses.
2.2.2 Cell based assays
As previously mentioned, neurotoxicity can also be analyzed with bioassays using cultured cells that express certain targets. This includes the use of SH-SY5Y cells in a FLIPR-based calcium flux assay conducted in a study analyzing neurotoxicity of snake venoms utilizing online microfluidic acetylcholine-binding protein (AChBP) profiling 105. These cells are derived from a human
neuroblastoma cell line that expresses endogenous 7- nAChRs 106. Bioactivity of the venoms is first
measured with the AChBP bioassay followed by the purification of the active components. The purified compounds are then examined with the functional calcium flux assay.
AChBP fluorescent enhancement binding assay
AChBP has a similar extracellular ligand binding domain as nAChRs, especially the 7- nAChR 107.
Hence, it is often used as an nAChR mimicking protein of nAChR ligands for neurotoxicity profiling of
venoms targeting nAChRs 105. The 7- nAChR plays a key role in neurotransmission by transforming
neurotransmitter interactions into electrical membrane depolarization and has a high Ca2+
permeability 105,108. In this assay, AChBP is used as the receptor and DAHBA
((E)-3-(3-(4-diethylamino-2-hydroxybenzylidene)- 3,4,5,6-tetrahydropyridin-2-yl) pyridine) is the fluorescent tracer ligand 109. This method is based on the binding competition between ligands and
DAHBA (Figure 7) 110. The DAHBA molecule shows enhanced fluorescence in the binding site of AChBP
and when displaced by the binding of other ligand the fluorescence decreases.
Venoms of 47 snakes were first separated with nano-liquid chromatography (LC). Then the effluent containing potential ligands is infused together with AChBP and DAHBA in a microfluidic chip comprising the AChBP assay. Fluorescence was monitored with a LED-based confocal fluorescence detection (CFD) system 105,109. Out of all the snake venoms analyzed, 15 of them contained high affinity
bioactive components 105.
FLIPR-based intracellular calcium assay
Intracellular calcium signaling is a very important event for neurotransmission 111. Depolarization of
neurons triggers the opening of voltage-gated calcium channels causing an influx of calcium ions. Increasing calcium levels in the cell triggers other signaling mechanisms that lead to the release of ACh in the synaptic cleft 3. Disruption of this calcium signaling pathway is an important phenomenon in
neurotoxicity 106. This dysregulation can be measured with calcium flux assays that use calcium-binding
fluorescent probes. These probes include fluo-3, fluo-4, fura-2, indo-1, fluo-8 and others 112–114. These
membrane permeable fluorescent Ca2+ indicator dyes usually have an ester moiety that gets
hydrolyzed by esterases, trapping the active probe in the cell 112,113. Fura-2 and indo-1 are ratiometric
probes that have two excitation and emission wavelengths, respectively 114. Binding of such probes to
Ca2+ leads to a shift in wavelength and allows for accurate quantification of Ca2+ concentrations 112.
While fluo-3 and fluo-4 are better suited for measuring dynamic changes in Ca2+ measurements rather
than quantifying 114. Compared to fura-2 and indo-1, these probes are excited by visible light (~ 488
nm) and binding to Ca2+ increases fluorescence at a single emission wavelength near 510 nm.
Fluorescent imaging plate readers (FLIPR) and flow cytometry analysis are usually used for the detection 115,116. The FLIPR instrument
contains a
CCD camera imaging system that simultaneouslyrecords the signals from each well 117. It is typically used in a high-throughput screening setup and is
able to measure the Ca2+ flux in real-time 113,116.
First cultured SH-SY5Y cells are incubated for two days at 37C and 5% CO2 105. The assay buffer
reagents included 11.5 mM glucose, 1.4 mM MgCl2, 140 mM NaCl, 5.9 mM KCl, 1.2 mM NaH2PO4, 1.8
mM CaCl2, 5 mM NaHCO3, and 10 mM HEPES (pH 7.4) 105. The fluo-4 calcium dye and an 7 agonist
( N-(5-chloro-2,4-dimethoxyphenyl)-N’ (5-methyl-3- isoxazolyl)-urea) are added to the cells and then
incubated for 30 min at 37C and 5% CO2. Then fluorescence is measured on excitation wavelengths
of 470-495nm and emission wavelengths of 515-575 nm. In addition, antagonists are measured by using the agonist activation mode in the FLIPR setup. For this choline is used to get an agonist signal and tubocurarine is used as antagonist control. Analysis of purified toxins from the D. russelli and A.
s. scutatus. snakes with the calcium-flux cell-based assays showed that they were able to inhibit
7-nAChR.
Other methods used to assess bioactivity of possible neurotoxins present in snake venom include radioligand displacement assays 109. This type of assay was among the different experiments
conducted in a study in which bioactive compounds from venom proteomes were rapidly screened and purified 118. The radioligand binding assay was used for testing the affinity of crude venom and
purified peptides for 7-nAChR.
Radioligand displacement assay
This assay is based on the ability of an unlabeled test compound to inhibit or compete the binding of a radioligand to a receptor of interest 119. Thus, the radioligand is “displaced” by the nonradioactive
ligand. Cells containing the receptor are incubated with a known amount of a suitable radioligand and range of concentrations of the unlabeled ligands 120. An important part of such a receptor binding
assay is the separation of free radioligand from bound radioligand 119. A filter assay is usually used for
A scintillation liquid is often used to measure the radioactivity. This fluid contains scintillation proximity assay (SPA) beads to which the bound radioligand binds 120. Radiant energy is transferred
from the radioligand to the scintillant in the beads, resulting in the emission of light from the surface of the beads 121. The emitted light is then counted with a 96-well plate reader.
The binding assay was performed with membranes of SH-SY5Y cells in buffer (PBS, 20 mM Tris, pH 7.4/0.05% Tween) expressing 7- nAChR 118. The radioligand used for this experiment is 3
H-methyllycaconitine (3H-MLA) at a final concentration of 2.0 nM. A concentration series of 10-7 – 10-14
M crude N. mossambica mossambica venom and two purified cytotoxins (1 and 2) are added to a mixture containing the receptor and radioligand. Membrane fragments containing bound radioligands were trapped on Unifilter-96 GF / C filters pre-rinsed with 0.3% polyethylenimine. The pre-rinsing step is done to limit nonspecific binding to the filter 118. In addition, the filter containing the
radioligand-receptor complex is also washed with ice cold 50 mM Tris-HCl buffer (pH 7.4) to further reduce nonspecific binding 118. The next step is the addition of 25 L/well scintillation liquid to the dried
filters. A Wallac 1450 MicroBeta liquid scintillation counter is used to measure radioactivity with a 300 min delay time. Both the crude venom and cytotoxin 1 showed low affinity binding
(Figure 8A,B).
Figure 8. Displacement of 3H-MLA by the crude N. Mossambica mossambica venom (A) and the purified cytotoxins (B) on the human7- nAChR 118.
