Evaluation of the anti-Acanthamoeba activity of two commercial eye drops commonly used to lower eye pressure

Accepted Manuscript Evaluation of the anti-Acanthamoeba activity of two commercial eye drops commonly used to lower eye pressure Ines Sifaoui, María Reyes-Batlle, Atteneri López-Arencibia, Carolina Wagner, Olfa Chiboub, Jacqueline De Agustino Rodríguez, Pedro Rocha-Cabrera, Basilio Valladares, José E. Piñero, Jacob Lorenzo-Morales PII:

S0014-4894(17)30310-7

DOI:

10.1016/j.exppara.2017.07.012

Reference:

YEXPR 7430

To appear in:

Experimental Parasitology

Received Date: 7 June 2017 Revised Date:

28 June 2017

Accepted Date: 30 July 2017

Please cite this article as: Sifaoui, I., Reyes-Batlle, Marí., López-Arencibia, A., Wagner, C., Chiboub, O., De Agustino Rodríguez, J., Rocha-Cabrera, P., Valladares, B., Piñero, José.E., Lorenzo-Morales, J., Evaluation of the anti-Acanthamoeba activity of two commercial eye drops commonly used to lower eye pressure, Experimental Parasitology (2017), doi: 10.1016/j.exppara.2017.07.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Evaluation of the Anti-Acanthamoeba activity of two commercial eye drops

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commonly used to lower eye pressure

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Ines Sifaoui 1,2, María Reyes-Batlle 1, Atteneri López-Arencibia 1, Carolina Wagner 1,3, Olfa

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Chiboub 1,2; Jacqueline De Agustino Rodríguez 1,4 ,Pedro Rocha-Cabrera 1,4, Basilio

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Valladares 1, José E. Piñero 1, Jacob Lorenzo-Morales 1

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University Institute of Tropical Diseases and Public Health, University of La Laguna, Avda Francisco Sanchez s/n, Campus de Anchieta, 38271 la Laguna Tenerife, Canary Islands, Spain.

Laboratoire Matériaux-Molécules et Applications, IPEST, B.P 51 2070, LA Marsa, University of Carthage, Tunisia Catédra de Parasitologia, Facultad de Medicina, Escuela de Bioanalisis, Universidad Central de Venezuela, Caracas, Venezuela

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Department of Ophthalmology, Hospital Universitario de Canarias, Tenerife, Canary Islands, Spain

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Ines Sifaoui

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Corresponding author:

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E-mail: [email protected]

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Tel: +216 71 740 048

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ACCEPTED MANUSCRIPT Abstract

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Efficient treatments against Acanthamoeba Keratitis (AK), remains until the moment, as an

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issue to be solved due to the existence of a cyst stage which is highly resistant to most

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chemical and physical agents. In this study, two antiglaucoma eye drops were tested for their

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activity against Acanthamoeba. Moreover, this study was based on previous data which gave

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us evidence of a possible link between the absences of Acanthamoeba at the ocular surface in

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patients treated with beta blockers for high eye pressure both containing timolol as active

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principle.The amoebicidal activity of the tested eye drops was evaluated against four strains

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of Acanthamoeba using Alamar blue method. For the most active drug the cysticidal activity

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against A. castellanii Neff cysts and further experiments studying changes in chromatin

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condensation levels, in the permeability of the plasmatic membrane, the mitochondrial

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membrane potential and the ATP levels in the treated amoebic strains were done.

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Even though both eye drops were active against the different tested strains of Acanthamoeba,

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statistical analysis revealed that one of them (Timolol Sandoz) was the most effective one

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against all the tested strains presenting IC50s ranging from 0.529 % ± 0.206 for the CLC 16

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strain to 3.962 % ± 0.150 for the type strain Acanthamoeba castellanii Neff. Timolol Sandoz

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0.50% seems to induce amoebic cell death by damaging the amoebae at the mitochondrial

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level. Considering its effect, Timolol Sandoz 0.50% could be used in the case of contact lens

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wearers and patients with glaucoma.

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Keywords: Acanthamoeba; Timolol; Chemotherapy; mechanism of action; mitochondria

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ACCEPTED MANUSCRIPT Introduction

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Acanthamoeba is an ubiquitous organism, which has been isolated from several habitats such

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as soil, water, air, dental unit water among others (1,2). Depending on the external conditions,

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this organism exists in two different forms: an active, infective trophozoite stage and a highly

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resistant cyst stage. Acanthamoeba is the causative agent of infections in human such as

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Acanthamoeba keratitis (AK) and Granulomatous Amoebic Encephalitis (GAE). Moreover,

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Acanthamoeba keratitis is a serious infection of the cornea that can cause blindness in some

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cases when not diagnosed and treated early (3,4). Recently, the incidence of AK cases due to

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Acanthamoeba has shown a remarkable increase worldwide which is mainly related to the

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increase of contact lens wearers (3-5).

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Generally, AK, favored by local immunosuppression, is the result of corneal trauma and

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different factors such as poor hygiene in the handling and storage of the contact lenses and

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their cases. Currently, the first line treatment regimen for AK includes biguanides [0.02%

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polyhexamethylene biguanide (PHMB) or 0.02% chlorhexidine digluconate] associated with a

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diamidine (0.1% propamidine isethionate or 0.1% hexamidine) (4,6). Even though, several

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studies have been focused on the development of novel therapeutic agents against AK, no

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fully effective drugs are available at the moment.

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Recently, Rocha-Cabrera et al., 2015 demonstrated the efficacy of the Schirmer test strips to

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detect Acanthamoeba at the ocular surface (6). The author reported that most of the patients

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that were positive for Acanthamoeba did present ocular surface pathologies such as

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staphylococcal blepharitis, seborrhoeic blepharitis and keratoconjunctivitis sicca. Moreover, it

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was also observed in this study an absence of Acanthamoeba in patients undergoing treatment

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for glaucoma or ocular hypertension (data not published). Glaucoma is a neurodegenerative

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disease considered as the second cause of blindness in the world. Furthermore, in 2020 it is

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expected that the number of people suffering this disease will reach around 79.6 million (7).

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ACCEPTED MANUSCRIPT In general, this condition is related to high intraocular pressure (IOP). Beta blockers (beta-

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adrenergic antagonists) are one of five classes of drugs used to treat glaucoma as they are able

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to decrease the intraocular pressure (8). In Spain, timolol containing eye drops such as

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Combigan and Timolol Sandoz 0.5% are drugs often prescribed to treat the IOP.

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Therefore and based on the previous data mentioned above, this two commercial eye drops

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used were tested for their activity against Acanthamoeba in the present study.

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2- MATERIAL AND METHODS

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2.1- Chemicals

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Two beta blockers drugs available for topical use against IOP and glaucoma were selected for

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analysis based on previous data. The drugs included a Timolol (Timolol Sandoz 0.50%) and

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a mix of Brimonidine tartrate and timolol (Combigan). (see Table 1 for a detailed

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composition of both eye drops).

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2.2- In vitro drug sensitivity assay

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Amoebic strain

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The amoebicidal activity of the tested eye drops was evaluated against four strains: one type

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strain from the American Type Culture Collection the Acanthamoeba castellanii Neff (ATCC

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30010), and three clinical isolates, CLC-16 and Acanthamoeba Griffini, genotype T3 and

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CLC-51, genotype T1 genotype obtained in a previous studies by Martin Navarro et al., 2008

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and González-Robles et al., 2014 (9, 10). The strains were axenically grown in PYG medium

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(0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract and 1.5% (w/v) glucose)

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containing 40 µg gentamicin ml−1 (Biochrom AG, Cultek, Granollers, Barcelona, Spain).

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In vitro effect against the trophozoite stage of Acanthamoeba.

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The anti-Acanthamoeba activities of the molecules were determined using the Alamar Blue®

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assay as previously described (9,11). Briefly, Acanthamoeba strains were seeded in duplicate

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on a 96-well microtiter plate with 50 µl from a stock solution of 104 cells ml−1. Amoebae were

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ACCEPTED MANUSCRIPT allowed to adhere for 15 min process which was checked using a Leika DMIL inverted

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microscope (Leika, Wetzlar, Germany). After that, 50 µl of serial dilutions of the fraction

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tested drugs was added to each well. Finally, the Alamar Blue Assay Reagent® (Biosource,

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Europe, Nivelles, Belgium) was placed into each well at an amount equal to 10% of the

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medium volume. Test plates containing Alamar Blue were then incubated for 120 h at 28 °C

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with a slight agitation.

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Subsequently, the plates were analyzed, during an interval of time between 72 and 144 h,

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with an Enspire microplate reader (PerkinElmer, Massachusetts, USA) using a test

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wavelength of 570 nm and a reference wavelength of 630 nm. Percentages of growth

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inhibition, 50% and 90% inhibitory concentrations (IC50 and IC90) were calculated by linear

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regression analysis with 95% confidence limits. All experiments were performed three times

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each in duplicate, and the mean values were also calculated.

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Cysticidal activity

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A. castellanii Neff cysts were prepared as previously described (12). Briefly, trophozoites

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were transferred from PYG medium based cultures (trophozoite medium) to Neff´s

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encystment medium (NEM; 0.1 M KCl, 8 mM MgSO4·7H2O, 0.4 mM CaCl2·2H2O, 1 mM

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NaHCO3, 20 mM ammediol [2-amino-2-methyl-1,3-propanediol; Sigma Aldrich Chemistry

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Ltd., Madrid, Spain], pH 8.8, at 25 °C) and were cultured in this medium with gently shaking

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for a week in order to obtain mature cysts. After that, mature cysts were harvested and washed

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twice using PYG medium.

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The effects of the active molecules against cysts were evaluated by incubating 104 cysts of A.

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castellanii Neff with the previously calculated IC90s of the bioactive molecules in PYG

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medium. The numbers of trophozoites, cysts, and nonviable cysts were counted with a

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Neubauer chamber at 96, 120, 144, and 168 h.

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ACCEPTED MANUSCRIPT Double-stain assay for apoptosis determination.

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A double-stain apoptosis detection kit (Hoechst 33342/PI) (GenScript, Piscataway, NJ, USA)

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and an inverted confocal microscope (Leica DMI 4000B) were used. The experiment was

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carried out by following the manufacturer’s recommendations, and thus 105 cells/well were

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incubated in a 24-well plate for 24 h with the previously calculated IC50 and IC90. The double-

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staining pattern allows the identification of three groups in a cellular population: live cells

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will show only a low level of fluorescence, apoptotic cells will show a higher level of blue

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fluorescence, and dead cells will show low-blue and high-red fluorescence (13).

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Plasma membrane permeability

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The SYTOX® Green assay was performed to detect the parasite’s membrane permeability

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alterations. Briefly, 1 × 105 trophozoïtes were washed and incubated in saline water with the

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SYTOX® Green at a final concentration of 1 µM (Molecular Probes) for 15 min in the dark.

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Subsequently the compounds were added (IC50s and IC90s). The increase in fluorescence due

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to binding of the fluorescent marker to the parasite’s DNA was measured using a

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spectrofluorimeter with excitation filter of 485 nm and emission filter at 520 nm. The reading

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was normalized by subtracting the baseline fluorescence of the untreated group (negative

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control) (13,14).

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Analysis of Mitochondrial Membrane Potential

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The collapse of an electrochemical gradient across the mitochondrial membrane during

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apoptosis was measured using a JC-1 mitochondrial membrane potential detection kit (Cell

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Technology) by flow cytometry as previously described (14, 15). After being treated with IC50

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and IC90 of the active drug for 24 h, the cells were centrifuged (1000 r.p.m. × 10 min) and

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resuspended in JC-1 buffer. 100µl of each treated culture was added to a black 96 well plate

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then 10 µl of JC-1 was added and incubated at 26 °C for 30 min. Analysis for mean green and

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red fluorescence intensity was done using flow cytometry for 30 minutes .

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ACCEPTED MANUSCRIPT Measurement of ATP

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ATP level was measured using a CellTiter-Glo® Luminescent Cell Viability Assay. The

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effect of the drug on the ATP production was evaluated by incubating (1*105) of cells/ml with

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the previously calculated IC50 and IC90 of the active drug (14,15).

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3. Results and discussion

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Acanthamoeba keratitis is a common condition that can lead to sight-threatening

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complications and blindness. While the infection rate was reported to be 1.2 per million adults

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and 0.2–1 per 10,000 contact lens wearers per year (16) rates have recently risen to more than

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seven-fold in contact lens wearers (17). Acanthamoeba keratitis is often misdiagnosed due to

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initial non-specific symptoms. At a later stage of the infection, some AK symptom could be

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developed confirming the infection such as the typical ring infiltrate and radial perineuritis.

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Nerveless, at this stage the prognosis is guarded and most cases require surgical intervention

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(4,18).

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In vitro drug sensitivity assay

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Both drugs were screened for their activity against the trophozoites stage of different

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Acanthamoeba strain. From the figure 1, it could be observed that the amoebicidal activity

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was based on a dependent-dose application. The IC50 and IC90

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appropriate and comparable data to give as previously described (9,11). The results are

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illustrated in Table 2.

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The analysis of variance by Multifactor ANOVA, illustrated that the biological activity was

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strain dependent with p = 0.0001 < 0.001. In fact, the CLC51 was the most sensitive strain to

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both drugs with an IC50 ranged 1.243 % ± 0.103 for TS and 2.715 % ± 0.007 for Combigan.

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With 3.125% of Timolol Sandoz a few trophozoïtes of CLC51 were observed as shown in

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figure 2. Meanwhile, A. Neff and A. griffini were the most resistant strains to both drugs. On

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the other hand, the effect of drug type was statistically significant on the activity since the p =

at

/96 h were chosen as the

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ACCEPTED MANUSCRIPT 0.0000 < 0.001, the Timolol Sandoz was the most effective drugs against all the strains with

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the IC50 ranged from 0.529 % ± 0.206 for the CLC 16 until 3.962 % ± 0.150 for A. Neff.

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Figure 3 showed that up to 120h the excystation did not occur with all the tested drugs. In

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fact, 4,5 % of the Timolol Sandoz could inhibit 50% of the initial inoculums in cysts,

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meanwhile, at 40% the same drug was able to inhibit 90% of the cysts.

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Timolol treated cells stained positive in the double-stain assay. When double staining was

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performed, the tested drug at both concentration IC50 and IC90 caused nuclei staining with

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Hoechst, indicating the presence of condensed chromatin (Fig. 4). Moreover, the differences

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between the three cell populations were clear, and thus live cells were detected under

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fluorescence microscopy, as they showed faint-blue nuclei, whereas cells displaying PCD

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presented bright-blue nuclei due to chromatin condensation. Meanwhile, the nuclei of cells

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treated with the IC90 showed a double stain blue and red demonstrating a late stage of PCD.

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Timolol Sandoz caused plasma membrane permeability in treated cells. As shown in

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Figure 5.D, amoebae treated with both concentrations IC50 and IC90 of the tested drug induced

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cellular membrane damage after 30 min of treatment. Nevertheless, cellular membrane

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disruption and the cell integrity was checked and confirmed using fluorescence microscopy in

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treated cells (Fig. 5 A to C).

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Timolol Sandoz induced mitochondrial malfunction. As it can be observed in figure 6,

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histograms of the mitochondrial potential fluorescence demonstrated that the treatment with

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the IC50 and IC90, decreased the membrane potential (∆Ψm) of A.Neff by 28%, 75%,

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respectively.

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generation in 24h. We found that both concentrations produced a pronounced decrease in the

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total ATP level (Figure 7). In fact, cells treated with the IC90 generated a percentage of ATP

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less than 80% of the untreated cells.

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The mitochondrial damage has been confirmed with the measure of ATP

ACCEPTED MANUSCRIPT Glaucoma constitutes the second-leading cause of blindness in the world. Furthermore, in

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2020 the number of people affected by this illness could exceed the 79 million (7).

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Keratoplasty in eyes with glaucoma are high-risk procedures and glaucoma has to be

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monitored more efficiently pre- and postoperatively. Combigan and Timolol Sandoz are

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beta blockers drugs used against IOP and Glaucoma. Both drugs present an amoebicidal

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activity against trophozoite and cyst, although Timolol Sandoz exhibits a stronger activity

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against the four strains. Furthermore, this drug contains 0.01% of preservative agent the

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benzalkonium chloride. Sunada et al., (2014) have reported the effectiveness of this

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preservative on several Acanthamoeba strains by altering membrane permeability and the

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formation of pseudocyst (19). Even though, Benzalkonium chloride has the highest

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effectiveness among the preservative agents, its toxicity to the human cornea needs to be

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considered (19). During our study, the amount of Benzalkonium chloride used in well is less

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than 0.001%. Niszl & Markus, (1998) reported that eye drop containing 0.004% of BAK are

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not efficient to kill Acanthameoba cysts (20). Several reports confirmed the important role of

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phospholipases in Acanthamoeba virulence (4, 21). The timolol maleate, Timolol Sandoz’s

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active ingredient, is a beta blocker that could inhibit the production of phospholipase secreted

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by Acanthamoeba as reported by Hänel et al, (1995). In fact, those authors demonstrated that

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the inhibition of Candida albicans by blocker-like structures was mainly due to the decrease

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of phospholipase secretion (22).

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Programmed Cell Death or apoptosis has been described in unicellular protists and in

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multicellular

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morphological characteristics namely the chromosomal condensation, nuclear DNA

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fragmentation, cell shrinkage, loss of mitochondrial membrane potential, the formation of

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apoptotic bodies, and the externalization of phosphatidylserine (23, 24).

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organisms.

This

process

is

generally

characterized

by

distinct

ACCEPTED MANUSCRIPT Apoptosis has been already described in Acanthamoeba (T4) by Feng et al., (2009) (25). The

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authors observed that the PCD occurred 6 h after infection with Salmonella Typhimurium.

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Furthermore, the death pathway was confirmed by detection of phosphatidylserine

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externalization and chromatin condensation in A. rhysodes. Recently, evidence of apoptosis in

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Acanthamoeba species has been described by Nakisah et al., (2012) (26) and Martin- Navarro

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et al., (2015) (13). Both reports distinguish between early apoptotic cells, late apoptotic cells,

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and necrotic cells by observing chromatin condensation. In our study, the Timolol Sandoz at

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IC50 induced chromatin condensation confirmed by the blue fluorescence. While treating with

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the IC90 the cells were double stain, (Red and blue) indicating late apoptosis.

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The possible action of the tested drug on the cell membrane was studied using the fluorescent

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probe SYTOX Green. Our data indicated that the membrane damage occurs immediately after

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the contact with the tested molecules. Even though, the timolol could permeabilize the

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cytoplasmic membrane, the microscopic observation confirmed maintenance of the cell’s

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shape. Due to its low weight, the timolol maleate could get inside the cells without necrosis

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effects (27).

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In case of malfunction of the mitochondria and especially its membrane potential the cells

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stop synthesizing ATP, leading to Programmed Cell Death (28). In the present study, The

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Timolol Sandoz produced a pronounced decrease in the mitochondrial potential and therefore

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in the total ATP level. It’s likely that those drugs could induce apoptosis in Acanthamoeba

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cells through the intrinsic pathway.

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Conclusion

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In conclusion, the obtained results suggest that Timolol Sandoz possess an interesting

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amoebicidal activity. Considering the observed effects on the mitochondria function by

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decreasing both the mitochondrial potential and the ATP content, this drug seem to induce

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apoptosis via the mitochondria pathway. Although, further studies are necessary in order to

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ACCEPTED MANUSCRIPT validate this hypothesis by studying characteristic signals of this pathway such as ROS

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generation and DNA fragmentation. Finally, therapy using Timolol Sandoz 0.50% could be

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considered in the case of contact lens wearers and patients with glaucoma to prevent

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Acanthamoeba keratitis. Nevertheless, further in vitro and in vivo studies should be

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developed.

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Acknowledgments

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This work was supported by the grants RICET (project no. RD06/0021/0005 of the

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programme of Redes Temáticas de Investigación Cooperativa, FIS), Spanish Ministry of

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Health, Madrid, Spain and the Project FIS PI10/01298 “Protozoosis emergentes por amebas

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de vida libre: aislamiento y caracterización molecular, identificación de cepas transportadoras

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de otros agentes patógenos y búsqueda de quimioterapias efectivas and PI13/00490

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“Protozoosis Emergentes por Amebas de Vida Libre: Aislamiento, Caracterización, Nuevas

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Aproximaciones Terapéuticas y Traslación Clínica de los Resultados” from the Instituto de

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Salud Carlos III. IS and ALA were funded by the Agustín de Betancourt Programme. JLM

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was funded by Ayudas Proyectos Puente de la Universidad de La Laguna 2017.

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ACCEPTED MANUSCRIPT 27. Hsieh YJ, Wu CC, Chang CJ, Yu JS. Subcellular localization of Photofrin determines the

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ACCEPTED MANUSCRIPT Figure Captions

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Figure 1. Effects of (a) Timolol Sandoz 0.50% and (b) Combigan against different

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strains of Acanthamoeba strains.

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Figure 2: Effect of the Timolol Sandoz 0.50% against CLC51 observed by inverted

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microscopy (x20). a. Timolol Sandoz at 50% absence of trophozoites or cysts. b. Timolol

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Sandoz at 3.125% few trophozoites were observed. c. Negative control

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Figure 3: Effects of Timolol Sandoz against cysts were evaluated by incubating 104 cysts of

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A. castellanii Neff with the previously calculated IC50 and IC90 values of the selected drugs in

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PYG medium, and cells were counted with a Neubauer chamber at between 24 and 120 h.

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Number of cysts that reverted to trophozoites in PYG medium after incubation with the drugs.

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Number of nonviable cysts when cysts were incubated with the previously calculated IC50 and

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IC90 in PYG medium.

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Figure 4: Hoechst staining is different in control cells, where uniformly faint-blue nuclei are

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observed, and in treated cells, where the nuclei are bright blue. (A to C) Overlay images:

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control (24 h) (A), Timolol Sandoz IC50 (24 h) (B), Timolol Sandoz IC90 (24 h). (D to F)

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Hoechst channel: control (24 h) (D), Timolol Sandoz IC50 (24 h) (E), Timolol Sandoz IC90 (24

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h) (F). (G to I) Propidium iodine channel: control (24 h) (G), Timolol Sandoz IC50 (24 h) (H),

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Timolol Sandoz IC90 (24 h) (I). (Magnification of 64x).

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Figure 5: Permeabilization of the cellular membrane. Fluorescence from the Sytox green

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nucleic acid stain can be observed when cells were treated with the different treatments after 2

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h. (A) Control; (B) Timolol Sandoz at IC50; (C) Timolol Sandoz at IC90. (D) Differences

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between Negative control and the drug-treated cells were apparent when fluorescence of the

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cells was measured. (Magnification of 64x)

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ACCEPTED MANUSCRIPT Figure 6: Mitochondrial membrane potential (∆ψm) Showing change in the Ratio of

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fluorescence intensity at 590/530 nm after the 24 hours of treatment with the IC50 and IC90 of

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the tested drug. Results are representing in percentage relative to the negative control.

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Figure 7: The effect of both molecules on the ATP content, using CellTiter-Glo®

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Luminescent Cell Viability Assay. Results are representing in percentage relative to the

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negative control. Cells were treated by the IC50 and IC90 concentration for 24 hours.

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ACCEPTED MANUSCRIPT Table 1. Detailed composition of the tested drugs Combigan 

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Brimonidina taratrato 2mg/ml

Timolol 5 mg/ml

Timolol 5 mg/ml

benzalkonium chloride 0.01%

benzalkonium chloride 0.005%

Disodium phosphate dodecahydrate

Sodium phosphate monobasic monohydrate

Sodium phosphate dihydrate

Fosfato sodico dibasico heptahidratado

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Timolol Sandoz 0.5%

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Combigan

Timolol Sandoz 0.5

BT (mg/ml)

T (mg/ml)

%

T (mg/ml)

IC50

5.305 ± 0.153

0.106 ±0.004

0.265 ± 0.009

3.962 ± 0.150

0.198 ±0.008

IC90

11.378 ±1.085

0.227± 0.022

0.568 ± 0.054

IC50

5.187 ± 0.274

0.104 ± 0.007

0.260 ± 0.018

IC90

9.555 ± 0.091

0.191 ± 0.001

0.478 ± 0.004

IC50

2.715 ± 0.007

0.054 ± 0.002

0.136 ± 0.007

IC90

4.853 ± 0.000

0.097 ± 0.000

0.243 ± 0.003

2.917 ± 0.013

0.146 ± 0.000

IC50

5.063 ±0.291

0.101± 0.006

0.253 ± 0.015

0.529 ± 0.206

0.188 ± 0.005

IC90

9.941 ± 0.154

0.199 ± 0.003

5.708 ± 0.154

0.446 ±0.001

A. Neff

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8.384 ± 0.185

0.420 ± 0.054

3.628 ± 0.274

0.182 ± 0.014

7.604 ± 0.091

0.380 ± 0.004

1.243 ± 0.103

0.062 ± 0.005

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0.497 ± 0.008

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- The anti-amoebic activity of two beta blockers eye drops was evaluated - Timolol Sandoz was the most effective drugs against all the Acanthamoeba strains - Timolol Sandoz decrease the mitochondrial membrane potential to less than 75%

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- The PCD was induced by the collapse of the mitochondrial membrane potential