TY  - JOUR
AU  - Lukić, Ivana
AU  - Blagojević, Veljko
AU  - Minić, Rajna
AU  - Ivanović, Saša
AU  - Borozan, Sunčica
AU  - Ćupić, Vitomir
AU  - Živković, Irena
PY  - 2024
UR  - http://intor.torlakinstitut.com/handle/123456789/976
AB  - that can most closely evaluate the biological activity of the drug of interest. For testing the potency
of antivenom, these are the methods used to assess cytotoxicity. The aim of this study was to evaluate
the most commonly used cytotoxicity methods for determining the protective potency of the antivenom
Viekvin, which neutralizes Vipera ammodytes venom.
Material and methods: The selected methods are based on different biological mechanisms: MTT
assay, based on the activity of cell oxidoreductase enzymes; crystal violet staining, based on the degree
of cell adhesion; trypan blue staining, based on cell membrane permeability, and propidium iodide
staining, based on measurement of nucleic acids of dead cells. The pro-apoptotic effect of the venom
was also determined with annexin V staining.
Results: The IC50 value of V. ammodytes venom obtained by these methods was very similar, while
the EC50 values differed significantly.
Conclusions: We concluded that the choice of the method used to measure the anticytotoxic antivenom
potency depends on the immunogenicity of the venom components that cause cell death; for each
venom/antivenom pair, it is necessary to select the appropriate assay separately, and at present, none
of the standard cytotoxic methods can be universally applied to determine antivenom potency.
PB  - Termedia Publishing
T2  - Central European Journal of Immunology
T1  - Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom
EP  - 104
IS  - 2
SP  - 94
VL  - 49
DO  - 10.5114/ceji.2024.142417
ER  - 

@article{
author = "Lukić, Ivana and Blagojević, Veljko and Minić, Rajna and Ivanović, Saša and Borozan, Sunčica and Ćupić, Vitomir and Živković, Irena",
year = "2024",
abstract = "that can most closely evaluate the biological activity of the drug of interest. For testing the potency
of antivenom, these are the methods used to assess cytotoxicity. The aim of this study was to evaluate
the most commonly used cytotoxicity methods for determining the protective potency of the antivenom
Viekvin, which neutralizes Vipera ammodytes venom.
Material and methods: The selected methods are based on different biological mechanisms: MTT
assay, based on the activity of cell oxidoreductase enzymes; crystal violet staining, based on the degree
of cell adhesion; trypan blue staining, based on cell membrane permeability, and propidium iodide
staining, based on measurement of nucleic acids of dead cells. The pro-apoptotic effect of the venom
was also determined with annexin V staining.
Results: The IC50 value of V. ammodytes venom obtained by these methods was very similar, while
the EC50 values differed significantly.
Conclusions: We concluded that the choice of the method used to measure the anticytotoxic antivenom
potency depends on the immunogenicity of the venom components that cause cell death; for each
venom/antivenom pair, it is necessary to select the appropriate assay separately, and at present, none
of the standard cytotoxic methods can be universally applied to determine antivenom potency.",
publisher = "Termedia Publishing",
journal = "Central European Journal of Immunology",
title = "Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom",
pages = "104-94",
number = "2",
volume = "49",
doi = "10.5114/ceji.2024.142417"
}

Lukić, I., Blagojević, V., Minić, R., Ivanović, S., Borozan, S., Ćupić, V.,& Živković, I.. (2024). Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom. in Central European Journal of Immunology
Termedia Publishing., 49(2), 94-104.
https://doi.org/10.5114/ceji.2024.142417

Lukić I, Blagojević V, Minić R, Ivanović S, Borozan S, Ćupić V, Živković I. Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom. in Central European Journal of Immunology. 2024;49(2):94-104.
doi:10.5114/ceji.2024.142417 .

Lukić, Ivana, Blagojević, Veljko, Minić, Rajna, Ivanović, Saša, Borozan, Sunčica, Ćupić, Vitomir, Živković, Irena, "Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom" in Central European Journal of Immunology, 49, no. 2 (2024):94-104,
https://doi.org/10.5114/ceji.2024.142417 . .

TY  - JOUR
AU  - Ivanović, Saša R.
AU  - Borozan, Nevena
AU  - Ćupić Miladinović, Dejana
AU  - Živković, Irena
AU  - Borozan, Sunčica
PY  - 2023
UR  - http://intor.torlakinstitut.com/handle/123456789/658
AB  - Diazinon is an organophosphate pesticide (OP) that has significant potential for accidental and intentional poisoning of wildlife, domestic animals and humans. The aim of the study is to investigate the correlation between cholinesterase activity and oxidative stress parameters in liver and diaphragm by continuous monitoring as a function of time during prolonged use of diazinon. Wistar rats were treated orally with diazinon (55 mg/kg/day): 7, 14, 21 and 28 days. At the end of each period, blood, liver and diaphragm were collected to examine cholinesterase activity and enzymatic/non-enzymatic oxidative stress parameters: superoxide dismutase 1 (SOD1), catalase (CAT), thiobarbituric acid substances (TBARS), protein carbonyl groups. In all four time periods, there was a significant change in acetylcholinesterase (AChE) in erythrocytes and butyrylcholinesterase (BuChE) in blood plasma, CAT in liver and diaphragm and SOD1 in diaphragm. Parameters significantly altered during the cholinergic crisis included: cholinesterases and TBARS in liver and diaphragm and partially SOD1 in liver. Protein carbonyl groups in liver and diaphragm were significantly altered outside the cholinergic crisis. In the liver, there was a very strong negative correlation between BuChE and TBARS in all four time periods and BuChE and CAT on day 7. In the diaphragm, a very strong negative correlation was found between AChE and TBARS at days 7 and 14, and a very strong positive correlation between AChE and SOD1 at days 14, 21 and 28. A better understanding of the relationship between cholinergic overstimulation and oxidative stress may help to better assess health status in prolonged OPs intoxication.
PB  - Elsevier
T2  - Toxicology and Applied Pharmacology
T1  - The relationship between the cholinergic mechanism of toxicity and oxidative stress in rats during subacute diazinon poisoning
SP  - 116598
VL  - 473
DO  - 10.1016/j.taap.2023.116598
ER  - 

@article{
author = "Ivanović, Saša R. and Borozan, Nevena and Ćupić Miladinović, Dejana and Živković, Irena and Borozan, Sunčica",
year = "2023",
abstract = "Diazinon is an organophosphate pesticide (OP) that has significant potential for accidental and intentional poisoning of wildlife, domestic animals and humans. The aim of the study is to investigate the correlation between cholinesterase activity and oxidative stress parameters in liver and diaphragm by continuous monitoring as a function of time during prolonged use of diazinon. Wistar rats were treated orally with diazinon (55 mg/kg/day): 7, 14, 21 and 28 days. At the end of each period, blood, liver and diaphragm were collected to examine cholinesterase activity and enzymatic/non-enzymatic oxidative stress parameters: superoxide dismutase 1 (SOD1), catalase (CAT), thiobarbituric acid substances (TBARS), protein carbonyl groups. In all four time periods, there was a significant change in acetylcholinesterase (AChE) in erythrocytes and butyrylcholinesterase (BuChE) in blood plasma, CAT in liver and diaphragm and SOD1 in diaphragm. Parameters significantly altered during the cholinergic crisis included: cholinesterases and TBARS in liver and diaphragm and partially SOD1 in liver. Protein carbonyl groups in liver and diaphragm were significantly altered outside the cholinergic crisis. In the liver, there was a very strong negative correlation between BuChE and TBARS in all four time periods and BuChE and CAT on day 7. In the diaphragm, a very strong negative correlation was found between AChE and TBARS at days 7 and 14, and a very strong positive correlation between AChE and SOD1 at days 14, 21 and 28. A better understanding of the relationship between cholinergic overstimulation and oxidative stress may help to better assess health status in prolonged OPs intoxication.",
publisher = "Elsevier",
journal = "Toxicology and Applied Pharmacology",
title = "The relationship between the cholinergic mechanism of toxicity and oxidative stress in rats during subacute diazinon poisoning",
pages = "116598",
volume = "473",
doi = "10.1016/j.taap.2023.116598"
}

Ivanović, S. R., Borozan, N., Ćupić Miladinović, D., Živković, I.,& Borozan, S.. (2023). The relationship between the cholinergic mechanism of toxicity and oxidative stress in rats during subacute diazinon poisoning. in Toxicology and Applied Pharmacology
Elsevier., 473, 116598.
https://doi.org/10.1016/j.taap.2023.116598

Ivanović SR, Borozan N, Ćupić Miladinović D, Živković I, Borozan S. The relationship between the cholinergic mechanism of toxicity and oxidative stress in rats during subacute diazinon poisoning. in Toxicology and Applied Pharmacology. 2023;473:116598.
doi:10.1016/j.taap.2023.116598 .

Ivanović, Saša R., Borozan, Nevena, Ćupić Miladinović, Dejana, Živković, Irena, Borozan, Sunčica, "The relationship between the cholinergic mechanism of toxicity and oxidative stress in rats during subacute diazinon poisoning" in Toxicology and Applied Pharmacology, 473 (2023):116598,
https://doi.org/10.1016/j.taap.2023.116598 . .

TY  - GEN
AU  - Milovanović, Vladimir
AU  - Blagojević, Veljko
AU  - Lukic, Ivana
AU  - Minić, Rajna
AU  - Borozan, Sunčica
AU  - Ćupić, Vitomir
AU  - Ivanović, Saša
AU  - Živković, Irena
PY  - 2023
UR  - http://intor.torlakinstitut.com/handle/123456789/894
AB  - Introduction: In recent decades, many efforts have been made in laboratories to develop alternative methods that would replace animal tests or reduce the number of animals used. In this sense, the methods for measuring cytotoxicity have proven to be very suitable substitutes for testing toxicity. As each individual substance being tested has its own specificities, it is necessary to carefully select a specific, appropriate cytotoxic method. This is especially important if the investigated substance is complex, such as animal venom. Also, antivenoms are drugs for which it is necessary to correctly determine the protective power.Methods: This study aims to evaluate most commonly methods for evaluating citotoxiciy of Vipera ammodytes venom and protective potency of appropriate antivenom. For this purpose, methods based on different biological mechanisms: MTT enzyme assay, Crystal Violet (CV), Trypan Blue (TB) and Propidium Iodide (PI) staining, were examined on Vero cells. The proapoptotic effect of the venom was also determined with AnexinV staining.Results: The IC50 value of V. ammodytes venom evaluated by MTT, CV, TB, PI (%) methods was similar: 62.5, 55, 66, 87 g/ml, respectively, and the most suitable methods for testing the cytotoxicity of V. ammodytes venom were methods based on the measurement of living cells, which were MTT and CV staining. The same methods were used to test the anticytotoxic potency of the antivenom and each method confirmed significant anticytotoxic potential, but the EC50values obtained by those methods varied significantly.Conclusion: Choice of the method used to measure the anticytotoxic antivenom potency depends on the immunogenicity of the venom components that cause cell death.
T2  - SSRN
T1  - Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom
DO  - 10.2139/ssrn.4374567
ER  - 

@misc{
author = "Milovanović, Vladimir and Blagojević, Veljko and Lukic, Ivana and Minić, Rajna and Borozan, Sunčica and Ćupić, Vitomir and Ivanović, Saša and Živković, Irena",
year = "2023",
abstract = "Introduction: In recent decades, many efforts have been made in laboratories to develop alternative methods that would replace animal tests or reduce the number of animals used. In this sense, the methods for measuring cytotoxicity have proven to be very suitable substitutes for testing toxicity. As each individual substance being tested has its own specificities, it is necessary to carefully select a specific, appropriate cytotoxic method. This is especially important if the investigated substance is complex, such as animal venom. Also, antivenoms are drugs for which it is necessary to correctly determine the protective power.Methods: This study aims to evaluate most commonly methods for evaluating citotoxiciy of Vipera ammodytes venom and protective potency of appropriate antivenom. For this purpose, methods based on different biological mechanisms: MTT enzyme assay, Crystal Violet (CV), Trypan Blue (TB) and Propidium Iodide (PI) staining, were examined on Vero cells. The proapoptotic effect of the venom was also determined with AnexinV staining.Results: The IC50 value of V. ammodytes venom evaluated by MTT, CV, TB, PI (%) methods was similar: 62.5, 55, 66, 87 g/ml, respectively, and the most suitable methods for testing the cytotoxicity of V. ammodytes venom were methods based on the measurement of living cells, which were MTT and CV staining. The same methods were used to test the anticytotoxic potency of the antivenom and each method confirmed significant anticytotoxic potential, but the EC50values obtained by those methods varied significantly.Conclusion: Choice of the method used to measure the anticytotoxic antivenom potency depends on the immunogenicity of the venom components that cause cell death.",
journal = "SSRN",
title = "Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom",
doi = "10.2139/ssrn.4374567"
}

Milovanović, V., Blagojević, V., Lukic, I., Minić, R., Borozan, S., Ćupić, V., Ivanović, S.,& Živković, I.. (2023). Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom. in SSRN.
https://doi.org/10.2139/ssrn.4374567

Milovanović V, Blagojević V, Lukic I, Minić R, Borozan S, Ćupić V, Ivanović S, Živković I. Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom. in SSRN. 2023;.
doi:10.2139/ssrn.4374567 .

Milovanović, Vladimir, Blagojević, Veljko, Lukic, Ivana, Minić, Rajna, Borozan, Sunčica, Ćupić, Vitomir, Ivanović, Saša, Živković, Irena, "Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom" in SSRN (2023),
https://doi.org/10.2139/ssrn.4374567 . .

TY  - CONF
AU  - Ivanović, Saša
AU  - Ćupić, Vitomir
AU  - Živković, Irena
AU  - Milovanović, Vladimir
AU  - Ćupić Miladinović, Dejana
AU  - Borozan, Sunčica
PY  - 2023
UR  - http://intor.torlakinstitut.com/handle/123456789/845
AB  - Poskok (Vipera ammodytes) predstavlja najzastupljeniju zmiju otrovnicu u Srbiji i na Balkanu, kako po brojnosti tako i po arealu rasprostranjenosti. U poređenju sa venomima ostalih otrovnica iz familije Viperidae na Balkanu, toksičnost venoma poskoka je najveća. Neurotoksičnost sirovog venoma poskoka smo ispitivali u in vitro uslovima na osnovu kontrakcija preparata dijafragme izazvanih poljnom električnom stimulacijom i aktivnosti enzima: acetilholinesteraze (AChE) i ukupnih ATP-aza u dijafragmi.Formirano je 5 grupa pacova (n=5): kontrola (dijafragma bez venoma), dijafragma sa venomom, dijafragma sa smešom venom/antivenom („Viekvin“, Torlak, Srbija) u odnosima 1:2, 1:10 i 1:20 (m/m). Kontrakcije dijafragme su opale na 50% kontrolnih kontrakcija: pod uticajem venoma za 62,00±2,31minuta, a sa smešom venom/antivenom 1:2 za 78,50±7,51 minuta, 1:10 za 150,00±17,32 minuta, 1:20 za 307,50±2,89 minuta. Statistička razlika (p0,05) pod dejstvom venoma. Venom inhibira aktivnost ukupnih ATP-aza dijafragme za 51,81% u odnosu na kontrolu. Venom poskoka ne deluje na AChE i nikotinske receptore u neuro-mišićnoj sinapsi dijafragme, već narušava energetski metabolizam mitohondrija.
AB  - Vipera ammodytes is the most common venomous snake in Serbia and the Balkans, both in terms of numbers and area of distribution. Compared to the venoms of other poisonous snakes from the family Viperidae in the Balkans, the toxicity of the V. ammodytes venom is the highest. We investigated the neurotoxicity of crude venom in vitro using contractions of the diaphragm preparation induced by electric field stimulation and enzyme activity: acetylcholinesterase (AChE) and total ATPases in the diaphragm. Five groups of rats were formed (n=5): control (diaphragm without venom), diaphragmwith venom, diaphragm with mixture of venom/antivenom ("Viekvin", Torlak, Serbia) in the ratio 1:2, 1:10 and 1:20 (m/m). Diaphragmatic contractions decreased to 50% of control contractions: under the influence of venom for 62.00±2.31 minutes, and with venom/antivenom mixture 1:2 for 78.50±7.51 minutes, 1:10 for 150.00±17.32 minutes, 1:20 for 307.50±2.89 minutes. A statistical difference (p 0.05) under the influence of the venom. Venom inhibits the total ATPases activity of the diaphragm by 51.81% compared with control.The venom of V. ammodytes does not act on AChE and nicotinic receptors in the neuromuscular synapse of the diaphragm, but interferes with mitochondrial energy metabolism.
PB  - Belgrade : Serbian Society of Toxicology
C3  - 13th International Congress of the Serbian Society of Toxicology and 1st TOXSEE Regional Conference, belgrade, 10 - 12 May, 2023
T1  - Neurotoksično dejstvo venoma Vipera ammodytes na modelu izolovane dijafragme pacova
T1  - Neurotoxic effect of Vipera ammodytes venom on the rat isolated diaphragm model
EP  - 108
SP  - 107
UR  - https://hdl.handle.net/21.15107/rcub_intor_845
ER  - 

@conference{
author = "Ivanović, Saša and Ćupić, Vitomir and Živković, Irena and Milovanović, Vladimir and Ćupić Miladinović, Dejana and Borozan, Sunčica",
year = "2023",
abstract = "Poskok (Vipera ammodytes) predstavlja najzastupljeniju zmiju otrovnicu u Srbiji i na Balkanu, kako po brojnosti tako i po arealu rasprostranjenosti. U poređenju sa venomima ostalih otrovnica iz familije Viperidae na Balkanu, toksičnost venoma poskoka je najveća. Neurotoksičnost sirovog venoma poskoka smo ispitivali u in vitro uslovima na osnovu kontrakcija preparata dijafragme izazvanih poljnom električnom stimulacijom i aktivnosti enzima: acetilholinesteraze (AChE) i ukupnih ATP-aza u dijafragmi.Formirano je 5 grupa pacova (n=5): kontrola (dijafragma bez venoma), dijafragma sa venomom, dijafragma sa smešom venom/antivenom („Viekvin“, Torlak, Srbija) u odnosima 1:2, 1:10 i 1:20 (m/m). Kontrakcije dijafragme su opale na 50% kontrolnih kontrakcija: pod uticajem venoma za 62,00±2,31minuta, a sa smešom venom/antivenom 1:2 za 78,50±7,51 minuta, 1:10 za 150,00±17,32 minuta, 1:20 za 307,50±2,89 minuta. Statistička razlika (p0,05) pod dejstvom venoma. Venom inhibira aktivnost ukupnih ATP-aza dijafragme za 51,81% u odnosu na kontrolu. Venom poskoka ne deluje na AChE i nikotinske receptore u neuro-mišićnoj sinapsi dijafragme, već narušava energetski metabolizam mitohondrija., Vipera ammodytes is the most common venomous snake in Serbia and the Balkans, both in terms of numbers and area of distribution. Compared to the venoms of other poisonous snakes from the family Viperidae in the Balkans, the toxicity of the V. ammodytes venom is the highest. We investigated the neurotoxicity of crude venom in vitro using contractions of the diaphragm preparation induced by electric field stimulation and enzyme activity: acetylcholinesterase (AChE) and total ATPases in the diaphragm. Five groups of rats were formed (n=5): control (diaphragm without venom), diaphragmwith venom, diaphragm with mixture of venom/antivenom ("Viekvin", Torlak, Serbia) in the ratio 1:2, 1:10 and 1:20 (m/m). Diaphragmatic contractions decreased to 50% of control contractions: under the influence of venom for 62.00±2.31 minutes, and with venom/antivenom mixture 1:2 for 78.50±7.51 minutes, 1:10 for 150.00±17.32 minutes, 1:20 for 307.50±2.89 minutes. A statistical difference (p 0.05) under the influence of the venom. Venom inhibits the total ATPases activity of the diaphragm by 51.81% compared with control.The venom of V. ammodytes does not act on AChE and nicotinic receptors in the neuromuscular synapse of the diaphragm, but interferes with mitochondrial energy metabolism.",
publisher = "Belgrade : Serbian Society of Toxicology",
journal = "13th International Congress of the Serbian Society of Toxicology and 1st TOXSEE Regional Conference, belgrade, 10 - 12 May, 2023",
title = "Neurotoksično dejstvo venoma Vipera ammodytes na modelu izolovane dijafragme pacova, Neurotoxic effect of Vipera ammodytes venom on the rat isolated diaphragm model",
pages = "108-107",
url = "https://hdl.handle.net/21.15107/rcub_intor_845"
}

Ivanović, S., Ćupić, V., Živković, I., Milovanović, V., Ćupić Miladinović, D.,& Borozan, S.. (2023). Neurotoksično dejstvo venoma Vipera ammodytes na modelu izolovane dijafragme pacova. in 13th International Congress of the Serbian Society of Toxicology and 1st TOXSEE Regional Conference, belgrade, 10 - 12 May, 2023
Belgrade : Serbian Society of Toxicology., 107-108.
https://hdl.handle.net/21.15107/rcub_intor_845

Ivanović S, Ćupić V, Živković I, Milovanović V, Ćupić Miladinović D, Borozan S. Neurotoksično dejstvo venoma Vipera ammodytes na modelu izolovane dijafragme pacova. in 13th International Congress of the Serbian Society of Toxicology and 1st TOXSEE Regional Conference, belgrade, 10 - 12 May, 2023. 2023;:107-108.
https://hdl.handle.net/21.15107/rcub_intor_845 .

Ivanović, Saša, Ćupić, Vitomir, Živković, Irena, Milovanović, Vladimir, Ćupić Miladinović, Dejana, Borozan, Sunčica, "Neurotoksično dejstvo venoma Vipera ammodytes na modelu izolovane dijafragme pacova" in 13th International Congress of the Serbian Society of Toxicology and 1st TOXSEE Regional Conference, belgrade, 10 - 12 May, 2023 (2023):107-108,
https://hdl.handle.net/21.15107/rcub_intor_845 .