L-amino acid oxidase from Bothrops atrox snake venom triggers autophagy, apoptosis and necrosis in normal human keratinocytes

Abstract

Snake venom L-amino acid oxidases (LAAOs) are flavoproteins, which perform diverse biological activities in the victim such as edema, myotoxicity and cytotoxicity, contributing to the development of clinical symptoms of envenomation. LAAO cytotoxicity has been described, but the temporal cascade of events leading to cell death has not been explored so far. This study evaluates the involvement of LAAO in dermonecrosis in mice and its cytotoxic effects in normal human keratinocytes, the major cell type in the epidermis, a tissue that undergoes extensive necrosis at the snakebite site. Pharmacological inhibition by the antioxidant NAC (N-acetyl cysteine) prevented B. atrox venom-induced necrosis. Consistent with the potential role of oxidative stress in wounding, treatment with purified LAAO decreased keratinocyte viability with an Effective Concentration (EC₅₀) of 5.1 μg/mL. Cytotoxicity caused by LAAO was mediated by H₂O₂ and treated cells underwent autophagy, followed by apoptosis and necrosis. LAAO induced morphological alterations that precede cell death. Our results show the chronological events leading to cell death and the temporal resolution from autophagy, apoptosis and necrosis as distinct mechanisms triggered by LAAO. Fluorescently-labelled LAAO was efficiently and rapidly internalized by keratinocytes, suggesting that catalysis of intracellular substrates may contribute to LAAO toxicity. A better understanding of LAAO cytotoxicity and its mechanism of action will help to identify potential therapeutic strategies to ameliorate localized snake envenomation symptoms.

Figure 1

Dermonecrosis inhibition by NAC. Mice gastrocnemius muscle was injected with 100 μL of PBS (negative control), B. atrox venom (150 µg/animal), B. atrox venom (150 µg/animal) + NAC (1:50 w/w) or NAC. After 72 h animals were euthanized and the tissue injury was evaluated.

Figure 2

Isolation and characterization of L-amino acid oxidase from B. atrox. B. atrox venom was fractionated using (a) Molecular exclusion in Sephadex S-200 and (b) Ion exchange chromatography in DEAE-Sepharose. (c) The enzymatic active fraction was selected and applied to a HiTrap Heparin Hp in HPLC Shimadzu. In all chromatographic steps fractions were monitored at 280 nm. (d) Schematic diagram of LAAO purification. (e) LAAO with (+) and without (−) β-mercaptoethanol (β-met) was applied to a 12% polyacrylamide gel and protein was stained with Coomassie Blue (cropped image). Full-length gel is presented in Supplementary Fig. S2. (f) For 2D electrophoresis, LAAO (30 μg) was applied to a pH 4–7 IPG strip and electrophoresis was carried out on 12% acrylamide gel. (g) Temperature and activation effects on LAAO activity. LAAO aliquots were kept at −80 °C, −20 °C or 4 °C for 5 days. Their activity was tested with (black bars) or without (white bars) pre-activation with acetate buffer pH 5.0 for 30 min at 37 °C. Absorbance was determined at 490 nm and LAAO activity was expressed as ΔA492 nm/min/mg).

Figure 3

LAAO cytotoxicity against keratinocytes. (a) Concentration-response of LAAO cytotoxicity. Cells were incubated with different concentration of LAAO (0.6–40 μg/mL) and after 24 hours cell viability was analyzed using Alamar Blue reagent. (b) EC₅₀ curve (concentration capable of reducing cell viability to 50%) using Graph Pad Prism software. (c) Catalase effect (100 μg/mL) in the cytotoxicity trigged by LAAO (0, 0.25, 0.5, 1 and 2EC₅₀). Cells were incubated with LAAO, with or without catalase and after 24 hours cell viability was analyzed. Values represent means of three independent assays and error bars indicate standard error of the means (SEM). (d) Morphological alteration in LAAO treated keratinocytes. Following incubation with different LAAO concentrations, cells were fixed and phase contrast images were acquired. Black arrows indicate colony edges retractions and white arrows show pyknotic nuclei. *p ≤ 0.05, ***p ≤ 0.001. Scale bar = 100 μm.

Figure 4

Morphological analysis of cells treated with LAAO. Keratinocytes were treated with LAAO (2EC₅₀) and imaged using phase contrast microscopy for 6 hours. Non-treated cells were used as control. Black arrows: cytoplasmic vesicles; red arrows: cell retraction; black arrows head: pyknotic nuclei. Images were acquired in a widefield timelapse microscopy. Scale bar = 50 μm.

Figure 5

LAAO triggers autophagy in keratinocytes. (a) Cells transfected with LC3-GFP and treated with 2EC₅₀ of LAAO for 1.5; 3 e 6 hours. Non-treated cells were used as negative control and starved cells (treated with EBSS for 30 min) were considered positive control. Cells were incubated with DAPI for nuclei staining and images were acquired on a fluorescence microscope. Scale bar = 10 μm. (b) Average number of LC3 puncta per transfected cell. Values represent means of three independent assays and error bars indicate standard error of the means (SEM). *p ≤ 0.05.

Figure 6

Apoptosis pathway (6, 12 and 24 hours) evaluation after LAAO treatment. Keratinocytes were treated with LAAO (2EC₅₀) for different time points and results were obtained using flow cytometry. (a) Dot plots showing cells labeled with Annexin-V and/or PI. Non-treated cells were used as negative control and cells treated with 1 μM Staurosporine or Triton X-100 0,1% were used as positive control for apoptosis and necrosis, respectively. (b–e) Graphs show quantifications of the proportion of (b) Viable cells. (c) Apoptotic cells (labelled with Annexin-V). (d) Late apoptotic/necrotic cells (labelled with Annexin-V and Propidium Iodide (PI)). (e) Necrotic cells (labelled with PI). Values represent means of three independent assays and error bars indicate standard error of the means (SEM). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 7

Mitochondrial membrane potential (12 e 24 hours) after LAAO treatment. Keratinocytes were treated with LAAO (2EC₅₀) of different time points. (a) Dot plots show cells labeled with JC-1 reagent. Non-treated cells were used as negative control and cells treated with 50 μM FCCP were used as positive control. (b,c) Graphs show quantifications of the proportion of (b) Depolarized mitochondrial membrane. (c) Polarized mitochondrial membrane. Values represent means of three independent assays and error bars indicate standard error of the means (SEM). *p ≤ 0.05.

Figure 8

LAAO treatment induces necrosis from 12 hours onwards. Cells were treated with LAAO (2EC₅₀) for 6, 12 e 24 h. Cells treated with Triton X-100 0.1% were used as positive control and non-treated cells as negative control. (a) Cells were stained with DAPI (nuclei) and Sytox Green. Scale bar = 50 μm. (b) Quantification of percentage of necrotic cells using FIJI software. Values represent means of three independent assays and error bars indicate standard error of the means (SEM). *p ≤ 0.05, ***p ≤ 0.001.

Figure 9

LAAO internalization by keratinocytes. LAAO was labeled with Alexa-555 as manufactor’s instructions and incubated with cells for 1.5 hours. The same procedure was performed for BSA, used control. Untreated cells were used as negative control. Z-profile of 20 pixels wide (white shadow) is projected in Z (shows the height of the cells). Images were acquired in a widefield microscopy. Scale bar = 20 μm.