J Arthropod-Borne Dis, Dec 2022, 16(4): 288–300 ME Alcigir and O Ozkan: The Evaluation of … 288 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Original Article The Evaluation of Androctonus crassicauda Antivenom against the Effects of Aegaeobuthus nigrocinctus Scorpion Venom on Autophagy, Apoptosis and Necroptosis Mehmet Eray Alcigir1, *Ozcan Ozkan2 1Department of Pathology, Kirikkale University, Faculty of Veterinary Medicine, Kirikkale, Turkey 2Department of Biology, Çankırı Karatekin University, Faculty of Science, Çankırı, Turkey *Corresponding Author: Dr Ozcan Ozkan; Email: ozcanozkan@karatekin.edu.tr (Received 27 May 2021; accepted 13 Aug 2022) Abstract Background: In this study aimed to show the role of autophagy acting as a seesaw between apoptosis and necroptosis in certain vital organs under the effects of the Aegaeobuthus nigricinctus venom and different dosages of the Androcto- nus crassicauda antivenom administration in mice. Methods: In the venom group (VG), mice (n= 6) were inoculated with 2LD50 A. nigrocinctus venom. In the antivenom administered groups (AVG), the effects of the potency of the A. crassicauda antivenom were evaluated to have a neu- tralization effect against 20LD50 of the A. nigrocinctus venom. After histopathological examination, expressions of mammalian target of rapamycin (mTOR) as an autophagy activator, receptor-interacting serine/threonine-protein kinase 3 (RIPK3) as a necroptosis activator, and caspase-3, caspase-9 as the markers of apoptotic cell death signals were eval- uated by the immunoperoxidase method in addition to DNA in-situ fragmentations by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Results: Only in VG, caspases and TUNEL expressions were found to be higher after the envenomation process in contrast to the elevated RIPK3 expressions. mTOR expressions remained almost stable in the organs. In AG, mTOR expressions were further increased in the 30LD50 and 40LD50 groups. Conclusion: There were an increased mTOR expression and stabilized caspases and TUNEL expression in these sub- groups, the RIPK3 expressions were found to be low when compared with all of the antivenom administration groups. Increasing doses of the antivenom drifts more the cells to autophagy while cell fate in organs under envenomation get- ting rid of apoptosis and necroptosis pathways. Keywords: Scorpion; Aegaeobuthus nigrocinctus venom; Androctonus crassicauda antivenom; Cell death mechanisms Introduction Venom secretions from scorpions are com- prised of a complex mixture of salts, mucopro- teins, histamine, serotonin, biogenic amines, low molecular weight peptides and high molecular weight proteins. The venom of each scorpion species has a different component profile. Low molecular weight peptides which are neurot- oxins is the most important components in scor- pion venoms, and it is also the component that is believed to be responsible for envenomation. These peptides stimulate the ion channels of cells such as sodium (Na+), potassium (K+), cal- cium (Ca2+) and chloride (Cl−) (1). Scorpion ven- oms can cause oxidative stress on cells and mi- tochondrial instability depending on overpro- duction of reactive oxygen species (ROS) (1– 5). Against this situation, autophagy machinery is activated (2, 3). Mammalian target of rapamy- cin (mTOR), a serine/threonine kinase, is like- ly to be the chief of this orchestra. It has a pivotal role in cellular metabolism, such as cell growth and proliferation (6). However, mTOR creates an inhibition in the autophagy induc- tion (7). Another reaction in response to cellu- lar damage like mitochondrial distress is necrop- tosis to be known as a regulated type of necro- Copyright © 2022 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, Dec 2022, 16(4): 288–300 ME Alcigir and O Ozkan: The Evaluation of … 289 http://jad.tums.ac.ir Published Online: Dec 31, 2022 sis. It includes the spilling of the cellular con- tents and therefore, the triggering of the chem- oattractant factors to independently develop, aris- ing from the absence of caspase activations. The mechanism is continued mainly by the ser- ine-threonine kinase receptor-interacting pro- tein (RIP) although the absolute activation is unknown (8–12). However, in programmed cell death or apoptosis, cellular contents cannot al- ways spill out in response to several damages. The cells sometimes prefer silent deaths, trig- gered by intrinsic and/or extrinsic pathways un- der the effect of chemoattractant or immune- mediated signals (12, 13). Amongst these, the cysteine-dependent aspartate driven proteases (caspases) are known to have a well-defined role in the apoptosis complex for a prolonged time. Caspase-9 as initiator of apoptosome and caspase-3 as effector have pivotal roles after the mitochondrial cytochrome c. Caspases, fur- thermore, lead to cleaving in cellular proteins to be like in receptor-interacting serine/ threo- nine-protein kinase 1–3 (RIPK1-RIPK3) com- plex in necrosome of dying cells (14, 15). There- fore, caspases can take a role in a number of non-apoptotic molecular interaction independent- ly of apoptotic cell death (16). Up to now, scorpionism has been one of the lasting global health problems in tropical and subtropical countries (17). Therefore, it is extremely important to know about the medi- cal importance of scorpion venom (17, 18). In Turkey, the Aegaeobuthus nigrocinctus scor- pion was reported to exist in the Adıyaman, Erzincan, Gaziantep, Hatay, Kahramanmaras, Kilis and Malatya provinces of the South-east- ern Mediterranean and Eastern Anatolian re- gions. In these regions, therefore, this scorpion species may be responsible for most of the cases of scorpion stings (19, 20). Nowadays, although there are no vaccines or other effective agents against animal ven- oms, hence the serotherapy stands as the only and unique treatment option available as re- ported before (21). The Androctonus crassi- cauda monovalent antidote has been used in the treatment of all cases of scorpion stings in Turkey (22). As far as we know, currently there are a few studies about the A. nigrocinctus scorpion venom. Moreover, cell death pathways, unfor- tunately, has have not been observed on dif- ferent organs, although there have been numer- ous reports on cellular damage stemming from a few scorpion species. Therefore, the species at hand is a neglected scorpion species regard- ing studies all over the world and in Turkey (20). In this respect, there is a requirement to fill the gap over the cell death cascade. Both different cell death types have been compara- tively evaluated in different organs and the su- premacy of the antivenom over the neglected A. nigrocinctus scorpion has been shown by this study. So, the presented study is one of the first studies conducted on the A. nigrocinctus scor- pion venom. The aim of this study was to measure the neutralization efficiency of the A. crassicauda antivenom against the A. nigrocinctus venom; (a) the effect of autophagy on the seesaw role between apoptosis and necroptosis (b) and to measure the response in different cell death reactions in the various vital organs. Materials and Methods The usage of animals and their care In this study which was approved by the local ethics committee (2017/04), 30 healthy CD-1 mice of 20±2g were used in total. Until the end of the experiment, mice (n= 30) were housed in a polycarbonate mouse cage (EU Type 2) and they were maintained at 22±2 °C on a 12h light / dark cycle with free access to food and water. Venom and antivenom handling In all the experimental procedures, the A. nigrocinctus venom was collected from The Nemrut Mountain National Park, which is in the Adıyaman Province in the Southeastern An- atolia Region of Turkey, and its LD50 is 0.38 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 288–300 ME Alcigir and O Ozkan: The Evaluation of … 290 http://jad.tums.ac.ir Published Online: Dec 31, 2022 mg/kg on mice (20). The monovalent A. cras- sicauda antivenom produced in horses by the Ministry of Health was used by The General Directorate of Public Health. The potency ca- pacity (ED50) of 1mL of antivenom neutralizes 50LD50 of the venom. Experimental procedure The mice modelling system is performed simply according to two main caption: antive- nom administered envenomation group (AVG), only venom administered group (V), and the control (C) group which was not administered any agent. The Effect of the monovalent antivenom and the A. nigrocinctus venom in mice: Patho- logical examination In antivenom group, mice were classified into three sub-groups with six mice in each group (n= 18). For each antivenom group, in- dividually, 1ml of the antivenom was mixed with an equivalent volume of doses of 20LD50 (GI), 30LD50 (GII), and 40LD50 (GIII) of the A. nigrocinctus venom and incubated for 45 min at 37 °C. Then, mice in each AG were sub- cutaneously (s.c.) injected with 200µL of each of the mixtures. After injection, mice were mon- itored for abnormal reactions and signs of en- venomation for 12h. The mice in the AG were euthanized with overdose of the mixtures of ketamine and xylazine at the end of the obser- vation. The animals (n= 6) in the venom group (VG) as positive control were injected with 2 LD50 of A. nigrocinctus venom in 200 µL physiological saline solution (PSS), while the negative control group (CG) were injected with 200 µL of the PSS venom. Macroscopical and Histopathological Exam- inations Autopsy procedures were performed imme- diately on dead mice in VG and the animals (n= 6) in CG and AVG groups for the exam- ination of macroscopic changes and histochem- ical analysis after the animals were euthanized. Briefly, the peritoneal cavities of the mice were opened, and tissue samples were collected from the livers, kidneys, lungs, hearts, and brains of the mice in each group and were immediately placed in 10% v/v formalin solution. After em- bedding in paraffin, the sections at 4µm-thick- nesses were taken. They were placed in slides and were stained with hematoxylin and eosin (H and E) for microscopic examination. Immunohistochemical analysis Detection of mTOR, RIPK3, caspase-3 and caspase-9 expressions The Strep Avidin-Biotin Complex Peroxi- dase (strep ABC-P) method was applied fol- lowing the manual instructions described in the kit (Peroxidase Detection System, RE7110-K, Leica, Novocastra). The sections at 4µm-thick- ness were passed through xylol and alcohol se- ries (5min for each), and then de-paraffinized and rehydrated. The sections were boiled in citrate buffer (pH 6.0) at 160 ºC for 15min to reveal the antigenic determinants (Bioptica, It- aly). To eliminate endogenous peroxidase activ- ity, the tissues were kept in 3% hydrogen-per- oxide (H2O2)-methanol solution for 15min. Non- specific protein activity was prevented with the use of blocking serum (Novocastra, Leica). In- cubation with primary antibodies (mTOR-1:250 dilution Gene Tex, GTX48628, RIPK3-1:200 Antibodiesonline.com, ABIN2792102, caspa- se-3 LS-B22845, LSBio, USA, 1:100 dilution, anti-caspase-9 ADI-AAP109, EnzoLife Scienc- es, USA, 1:150) was left overnight at +4 °C. Bi- otin-linked antibody and streptavidin-linked an- tibody were dripped onto tissue sections and in- cubated at 37 ºC for 15min. Thereafter, they were rinsed twice for 5min, using PBS at the end of each phase, except in the protein blocking phase. For the control sections, PBS was used instead of primary antibody as the negative control. Dia- minobenzidine (DAB) was used as chromogen, while Gill’s hematoxylin was used as ground staining. The slices were fixated using Entel- lan® which is a non-aqueous mounting medium. Detection of DNA in-situ fragmentation The terminal deoxynucleotidyl transferase http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 288–300 ME Alcigir and O Ozkan: The Evaluation of … 291 http://jad.tums.ac.ir Published Online: Dec 31, 2022 mediated nick end labeling (TUNEL) staining assay method was applied according to the kit procedure (In situ Cell Detection Kit, Roche, USA, Cat no: 11684795910). For control sec- tions, a labeling solution without terminal trans- ferase was dripped onto the slices. Evaluation of results All histopathological and immunoexpres- sion results were illustrated in an Olympus BX51 and photographed with an Olympus DF 25 camera attachment. Mean scores were per- formed semiquantitatively counting 10 high power field at 400 magnifications for each col- lected organs in all groups. Histopathological scoring was found to be as follows; negative (-): 0–10%, mild (+): 10–30%, moderate (++): 30–70%, strong (+++): 70–100%. Statistical analysis Immunoexpressions were evaluated using a Two-way ANOVA test to compare the vari- ability in reactions among the groups. The post- hoc Bonferroni test was used in multivariate comparisons. The data was analyzed using the Graphpad (8.0 version) software. A value of p< 0.05 was accepted as statistically significant. Results Histopathological findings The Venom group and Control groups Regarding the results of the negative con- trol group; the organs aforementioned were not affected by any degeneration or necrotic changes. There was also no inflammatory re- action at all. The only hyperemic changes were present in the liver and kidney vessels at some of the cases (Fig. 1). Findings of the positive control as VG in liver suggests that there was hyperemia in central and portal ves- sels. Acute cell swelling to vacuolar degen- eration were ended in karyolysis and cyto- plasmic shrinkage in hepatocytes. In the kid- ney: hyperemic capillaries and glomerulus were present. Acute cell swellings as well as vacuolar degeneration were evaded in particu- larly cortical tubules. In the spleen, follicular hyperplasia in lymphoid follicle as well as haemorrhage was observed. In the lungs, hy- peremic capillary vessels and neutrophil ex- travasation were considered. In the heart, hy- peremic capillaries, parenchyma degeneration as well as inflammatory cell infiltration were again found (Fig. 1). Antivenom group (AVG) In the liver, the degeneration associated with acute cell swelling and vacuolar degen- eration was not found in hepatocytes at every field as being in VG. In the kidney, acute cell swelling and vacuolar degeneration in cortical tubules were present although there were not any findings in the medullary region of the kidneys. In the spleen, follicular hyperplasia in lymphoid follicles, intrafollicular hemor- rhage as well as the presence of megakaryo- cytes was observed in some of the cases. In the heart, individual parenchyma degeneration with shrinkage pink cytoplasm in some cardi- omyocytes were observed. The findings were not observed in all the cases (Fig. 1). Immunohistochemical findings mTOR expressions Expressions were localized in the mem- brane and cytoplasm of cells. In VG, the ex- pressions were scattered diffusively from the central to peri-central region of the lobules of the liver, on the periphery of lymphoid folli- cles of the spleen, on the cortical tubule epi- theliums of the kidney and cardiomyocytes in the heart. Any statistical differences were not found between these groups (p> 0.05). In the control group, there were no expressions (p< 0.05 between this and other groups). In all the antivenom subgroups (AVG) in- cluding from GI to GIII, the expressions were increased when compared to previous sub- groups in envenomed mice organs. The ex- pressions were localized in the periphery of lobules of the liver, in the periphery of the http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 288–300 ME Alcigir and O Ozkan: The Evaluation of … 292 http://jad.tums.ac.ir Published Online: Dec 31, 2022 lymphoid follicles of the spleen, on the corti- cal tubule epitheliums of the kidney, and car- diomyocytes in the heart. Any statistical dif- ferences were not found between these groups (p> 0.05). However, there was a meaningful statistical difference in comparison between the envenomed and antivenom administration groups (p< 0.05). In the control group, no ex- pressions were found (p> 0.05 between this and other groups). Caspase-3 and caspase-9 expressions These expressions were localized in the membrane and cytoplasms of the cells. In VG, both expressions were found at high degrees regarding the aforementioned cellular locali- zation which stated in mTOR sections. How- ever, when compared between caspase-3 and caspase-9 expressions, there was not any sta- tistical difference between subgroups (p> 0.05). In comparison of two caspases, both had the same distribution with no statistical difference (p< 0.05). In the control group, there were no expressions (p< 0.05 between this and other groups). In GI to GIII, both expressions had similar characteristics in terms of localization, the expressions were found at lower degree of positivities (p< 0.05). A meaningful statistical difference was found between envenomed and antivenom administration groups (p< 0.05). In the Control group, there were no expressions (p< 0.05 between this and other groups). TUNEL reactions Expressions were the same with the previ- ous ones. In VG, the expressions were at high levels. They were found at the same localiza- tion in all of the organs as mentioned in pre- vious markers. In the control group, there were no expressions (p< 0.05 between this and other groups). The expressions were found at the same localization in all of the organs as mentioned in previous markers. However, the positivity degrees were lower in GII and GIII of AVG when compared to that of GI (p< 0.05). When these expressions were compared to the en- venomed groups, such positivities were more elevated and the distribution of positivities were more evaded in tissues in antivenom groups (p< 0.05). In the control group, there were no expressions (p< 0.05 between this and other groups). RIPK3 expressions In VG, expressions were seen in the cyto- plasms of cells. The expressions had the same localizations as being previous markers. The distribution of positivities were stronger and more prevalent in the tissues when compared to that of GI in AVG. Among groups, there was a statistical difference (p< 0.05). In the control group, there were no expressions (p< 0.05) between the previous two groups. The expressions were decreased in all of the antivenom subgroups when compared to that of all envenomed groups (p< 0.05). The distribution of positivities were the same as the previous ones. Nevertheless, the number of positive cells decreased particularly in the GII and GIII of AVG. There was a statistical difference with the comparison of GI (p< 0.05). In the control group, there were no ex- pressions (p< 0.05 between this and other groups). All the expressions according to vital organs were shown in Figure 2, 3 and Fig. 4. Statistical evaluations of tabular data were presented in Table 1. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 288–300 ME Alcigir and O Ozkan: The Evaluation of … 293 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Fig. 1. Description of the degeneration in the liver, kidney, and heart in the envenomed groups (VG), and antivenom groups (AG1 to 3), no findings to report in the control group (CG), x400, Hematoxylin-Eosin (H and E) staining Fig. 2. Immunoexpressions of mTOR, caspases, DNA in situ fragmentation and RIPK3 in the experimental group (A) and the antivenom groups (B) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 288–300 ME Alcigir and O Ozkan: The Evaluation of … 294 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Fig. 3. Immunoexporessions in the organs of the envenomed animals, Diaminobenzidine (DAB) chromogen, ABC-P, x200 Fig. 4. Immunoexporessions in organs of the antivenom administered animals, Diaminobenzidine (DAB) chromogen, ABC-P, x200 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 288–300 ME Alcigir and O Ozkan: The Evaluation of … 295 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Discussion Cells may be exposed to numerous dan- gerous factors during their lifetime. In this case, cells, due to continuous damage may be drifted to death by using self-activation of specific molecular pathways (23). These pos- sible molecular mechanisms and some trig- gering linkages are a controversial matter for a long time whether if this is a real self- suicide or only a biological result or reaction against lethal factors. Therefore, several ter- minologies have been produced meeting those mechanisms (24, 25). Cell death mechanisms can be diversified within different categories including apoptosis, necroptosis, autophagy, piroptosis etc. In particular, first three mecha- nism are related to formerly known necrosis. Serine-threonine kinase receptor-interacting protein (RIP) and kinase (RIPK), activate sig- naling pathways under cellular distress by ex- cessive free radical accumulation. On the other hand mTOR which is taken as a role regar- ding the PI3K/Akt/mTOR signaling pathway, in terms of creating a downstream in auto- phagic activation. Caspases, cysteine-dependent aspartate driven proteases, are taken into role within necroptosis. Caspases, have a role of cleaving in cellular proteins in necrosome. Herein, the key role of caspase-9 is associated to be an activator for apoptosis and a deacti- vator for autophagy. Downregulation of cas- pases, reactivated mTOR change autophagic mechanism in order to provide autolysosomal activity. By negative feedback, increased auto- somal vesicles can reverse autophagy. If free radical (i.e ROS activity) related-damages are excessive and being out of controling mech- anism, RIP activation, namely necroptosis, is generated because mitochondrial distress is triggered. As seen herein, each mechanism is closely related to each other. These mecha- nisms can be easily developed under cellular distress to be like in scorpion envenomation (thanks to toxin contents) (8–12). In the light of the current knowledge, au- tophagy is known to provide cellular homeo- stasis by inhibiting catabolic products and gene- rating nutritional substance and some mole- cular precursors for cells; namely, a cell sur- vival mechanism (25). However, more recent- ly, the subject of autophagy machinery or self- eating has been given emphasis (24). Against the cellular damage resourced from toxication, some damages happen in cells due to intoxication. Many toxins and metabolites, cause cytotoxicity by their effects on vital organs such as the heart, kidney, spleen, brain, and skin. In addition to the cellular alterations, hem- orrhage and disseminated intravascular coag- ulation can develop as a result of cytotoxici- ties (5, 26–29). The toxic effects primarily start in the mitochondria by increasing oxida- tive stress and over time, it begins to affect all organs. As a result of cellular alterations, cells can be drifted by many mechanisms into death. Several factors can determine the fate of the cell. These mechanisms include autophagy, programmed cell death or apoptosis and necrop- tosis which results in necrosis (5, 29, 30). To begin with programmed cell death, it is reported that the execution of cell death plays an orchestrate role between the mentioned pro- cesses. Apoptosis triggers cellular membrane receptor facilitating to emit death signals to cytosol. In some events, cells under oxidative stress can release cytochrome c from the mito- chondria (4, 5, 31). In such situations, the apop- tosis cascade begins to develop autonomously by the activation of Cysteinyl-aspartases or caspase family of proteases (32). Among the caspases family, caspase-9 is known to have an essential role for the mitochondrial signal- ing pathways. Apoptotic cascades continue by the activation of caspase-3 (33). Another idea on cell death is related to autophagy. It takes critical responsibilities on cell death as well as many roles including tissue development, dif- ferentiation and homoeostasis and taking un- der control of health and aging in a healthy organism. However, the role of housekeeping http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 288–300 ME Alcigir and O Ozkan: The Evaluation of … 296 http://jad.tums.ac.ir Published Online: Dec 31, 2022 and checking the vital functions of cells whether they behave normally is among the evitable responsibilities (34, 35). In our study, we observed that the caspase-3 and caspase-9 expressions in VG, were high. In AVG, GI to GIII, both expressions had similarly and lower caspases expression of VG. There is statistical significance between VG and AVG. But there is no meaningful difference between both caspa- ses’ level. The autophagy mechanism provides this by regulating some proteins to send signals. mTOR or rapamycin, phosphoinositide 3-kinase (PI3K), GTPases, calcium and elements of pro- tein synthesis machinery are included among them (36). Some regulatory factors control mTOR activity in cells according to whether the cell posing a threat or not. The decreased PI3K activations generally show parallel sit- uation and function mTOR signaling via Akt- mediated phosphorylation. Therefore, PI3K/Akt/ mTOR pathway signaling cascade leads to a decreasing activity in the autophagy mecha- nism (37). On the contrary, the induction of autophagy may lead to cell survival even the cell under stress conditions by oxidative stress. In addition to, it has been stated that autoph- agy might prevent cells from undergoing apop- tosis (38). Therefore, for the autophagy mech- anism, it has a pro-survival effect to antago- nize apoptosis. In this respect, the current study results are consistent with these data. On the other hand, programmed necrotic cell death or necroptosis is triggered by serine/ threonine kinases receptor-interacting protein 3 or RIPK3 activation, binding to RIPK1 after the regula- tion by caspases and ubiquitination. Thus, these enzymes facilitate to loss of the cellular car- bohydrate deposits and to increase glutamine metabolism (39). In our study, we observed that mTOR in VG were expressed in the liver, spleen, kidney and heart although there was no statistical difference between remained groups. On the other side, the present study deter mined that venom exposure triggers increased RIPK3 activity. However, we observed that of all the dosages of antivenom administration, mTOR expression continues to increase under toxin stress. Therefore, we concluded that mTOR and RIPK3 have adverse effects when each dose of antivenom was administered. In our study, the RIPK3 expressions of VG were much stronger and more prevalent in the tis- sues when compared to that of GI in AVG. RIPK3 expressions at both remained groups and control groups were similar and did not give meaningful statistical results because the ex- pressions were decreased in particularly GII and GIII of AVG. We believe that the sole envenomation proven increase in RIPK3, i.e., necroptosis. So, this situation shows that the hypothesis makes the current study right on envenomation-necroptosis interaction. On the other hand, mTOR expressions, i.e., autopha- gy, were decreased in some organs of VG in spite of increasing in AVG. Decreasing mTOR show that autophagy can be increased in some organs. But other organs were not affected from triggering autophagy as being like in control group. So, these results prove partly the hy- pothesis regarding the vice-versa effect between RIPK and mTOR activities. Cells’ fate in the way of surviving and drifting to death can be triggered at the same way in some vital organs against venom-associated cellular distress. Accordingly, we found that cellular death pathways were triggered by high apoptosis and necroptosis as well as low autophagic ac- tivity which resulted in cellular DNA breaks. TUNEL reactions have proven such kind of DNA breaks. In VG, the TUNEL reactions were at high levels. However, these reactions in AVG were lower than that of the VG. When compared within the AVG, the reactions were found lower in GII and GIII than GI. These results showed that antivenom co-administra- tion in the envenomed group inhibit cellular death mechanism. So, DNA breaks can be stopped thanks to diminished cellular death and possibly ROS-related cellular stress. Another important point in our study is the antivenom or immunotherapeutic usage, how http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 288–300 ME Alcigir and O Ozkan: The Evaluation of … 297 http://jad.tums.ac.ir Published Online: Dec 31, 2022 it can affect cellular damage or can reverse the adverse effect in such envenomation. It has been reported that the improvement of the immu- notherapeutic treatment in such envenomation events require a better knowledge of the phar- macological actions of the scorpion venom and of the mechanism of its in vivo neutralization by the antivenom. Selection of the proper an- tivenom dose has a vital effect on immediate and durable intervention with regards to sting events by complete neutralization of the tox- ins. As such, the cellular and vascular or other damages can be better prevented by the effec- tive diffusion of antivenom to all the organs (40). In the current study, decreasing mTOR and RIPK3 expressions as well as relatively in- creasing caspases and DNA in-situ fragments in all the antivenom subgroups show us that necroptosis decreases, and cell death increases as a result of the autophagy mechanism con- trolling cell survival in the way of preventing apoptosis. By evaluating the results of the cur- rent study (a) there is a close relationship be- tween autophagy-apoptosis and necroptosis. (b) In mice, the A. nigricinctus venom and the monovalent antivenom administration can be a useful model for coming to a better under- standing of the potential harmful effects over cells in various vital organs such as the liver, kidneys, spleen, heart, and lungs. (c) The mon- ovalent antivenom against high LD value of the venom may reverse the potential necrop- totic effects on cells due to envenomation. (d) Caspase signals apart from apoptotic cell death can also aid in decreasing mTOR expressions in envenomed animals. Thus, combined expres- sions may trigger the activation of autophagy. (e) RIPK3 may solely change the fate of cells in the course of necroptosis. Conclusion Envenomation by scorpion toxin cause a cellular damage in several organs. Excessive free radicals disturb de facto cellular membran, cytoplasmic organels and nuclear structure. In this situation is known that cells are drifted to degeneration or necrosis. However, by this ex- perimental study, we show there are definitive mechanisms which is related to each other. According to exposure degree of toxin and serum support, cell fate can be easily changed under free radical distress. These mechanisms can be turned appear as apoptosis, necroptosis and autophagy. We obtained sustainable infor- mation from this experimental that autophagic mechanism shows parallel to decreased of ven- om capacity and increased serum support. At the same time, we understood that this condi- tion gets the irreversible necroptosis capacity decreased. By this, we have also seen that apop- totic mechanism is more effective in initial phase of envenomation. But we have conclud- ed that apoptotic signals get less effective in presence of higher serum support. In this sit- uation, cell makes a decision living or death after such mechanisms are run in cell at the same time. The obtained results can facilitate in order to understand the relations amongst different cell death mechanisms as well as re- versal of the monovalent antivenom effectiv- ity on organ damage. At the same time, we inferred from result of this study that serum support the more earlier time is early getting started and suitable dose is selected, the less organ damages are developed. However, the results should be confirmed by correlating with other markers taking place in apoptotic, necroptotic and autophagic cascades. Acknowledgements The authors declare that they do not have any conflict of interest with any researchers. The authors express their sincere thanks to the Ministry of Health in Ankara, Turkey for kindly providing the A. crassicauda spesific anti-venom. The study has not been funded by any cooperation. The researchers performed the study by own facilities. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 288–300 ME Alcigir and O Ozkan: The Evaluation of … 298 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Ethical considerations The animal care and all of the experimental protocols were performed in accordance with the guidelines defined by the local ethical co- mmittee in Experimental Animal Research Comitee, Health Ministry (2017-E330341). Conflict of interest statement The authors declare no conflict of interest with any researchers. References 1. Zhang X, Zhang X (2016) Scorpion venoms in gastric cancer. Oncol Lett. 12: 3683– 3686. 2. 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