Benefits and Side Effects of Different Vegetable Oil
`Vectors on Apoptosis, Oxidative Stress, and P2X7 Cell
`Death Receptor Activation
`
`Toibiri Said,‘ Melody Dut0t,1 Raymond Cl9rist0n,2 jean-Louis Beaudeux,3 Chantal Martin,‘
`jean-Michel Warnet,1’4 and Patrice Rat1’4
`
`PURPOSE. Ocular side effects in patients using eye drops may be
`due to intolerance to the vector used in eye drops. Castor oil is
`the commonly used lipophilic vector but has been shown to be
`cytotoxic. Effects on cells of four oils (olive, camelina, Aleu-
`rites moluccana, maize) were compared with those of castor
`oil in human conjunctival cells.
`METHODS. Human conjunctival cells were incubated with the
`oils for 15 minutes. After a 24-hour recovery period, cells were
`tested for viability, proliferation, apoptosis (P2X7 cell death
`receptor and caspase 3 activation), intracellular redox poten-
`tial, and reactive oxygen species production. Fatty acid incor-
`poration in cell membranes was also analyzed. In vivo ocular
`irritation was assessed using the Draize test.
`
`RESULTS. Compared to the four other oils, castor oil was shown
`to induce significant necrosis and P2X7 cell death receptor and
`caspase 3 activation and to enhance intracellular reactive oxy-
`gen species production. Aleurites moluccana and camelina
`oils were not cytotoxic and increased cell membrane omega-3
`fatty acid content. None of the five tested oils showed any in
`vivo ocular irritation.
`CONCLUSIONS. The results demonstrated that castor oil exerts
`
`cytotoxic effects on conjunctival cells. This cytotoxicity could
`explain the side effects observed in some patients using eye
`drops containing castor oil as a vehicle. The lack of cytotoxic
`effects observed with the four other oils, Aleurites, camelina,
`maize, and olive, suggest that they could be chosen to replace
`castor oil in ophthalmic formulations. (Invest Opbtbalmol Vis
`Sci. 2007;48:5000 -5006) DOI:10.1 167/iovs.07-0229
`
`Topical drug administration is very often used to treat ocular
`surface and intraocular disease, providing higher local
`drug levels than systemic administration,‘ with minimal gen-
`eral side effects.2 The therapeutic efficacy of a topical formu-
`
`From the llaboratoire de Toxicologie and 3Stress Oxydant et
`Atteintes Vasculaires, Faculte des Sciences Pharmaceutiques et Bi-
`ologiques, Universite Rene Descartes Paris, Paris, France; ZINRA (Insti-
`tut National de la Recherche Agronomique), Lipides Membranaires et
`Regulation Fonctionnelle du Coeur et des Vaisseaux, UMR (Unite Mixte
`de Recherche)-INRA Physiologic, Universite Paris XI, Ch:itenay-Mala-
`bry, France; and 4INSERM (Institut National de la Sante et de la Recher-
`che Medicale), UMRS (Unite Mixte de Recherche en Sante), Institut
`Biomedical des Cordeliers, Paris, France.
`Submitted for publication February 22, 2007; revised July 2, 2007;
`accepted September 4, 2007.
`Disclosure: T. Said, None; M. Dutot, None; R. Christon, None;
`J.-L. Beaudeux, None; C. Martin, None; J.-M. Warnet, None; P. Rat,
`None
`The publication costs of this article were defrayed in part by page
`charge payment. This article must therefore be marked “advertise-
`ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`Corresponding author: Patrice Rat, Iaboratoire de toxicologie
`(INSERM U 598), Fac11lte des sciences Pharmaceutiques et Biologiques,
`Universite Rene Descartes Paris 5, 4 avenue de l’Observatoire, 75006,
`Paris, France; patrice.rat@univ-paris5.fr.
`
`5000
`
`lation depends on both its composition and the physicochem-
`ical properties of the vehicle. Use of an appropriate vehicle is
`critical to increase the optimal efficacy of the pharmacologi-
`cally active drug.3 Most commercialized eye drops are pre-
`pared in aqueous form, although most active components are
`lipophilic. Another drawback of the hydrophilic formulations
`is the fast elimination of the eye drop by tears, reducing
`contact duration between the drug and the ocular tissue.
`The use of a lipophilic vehicle in eye drops increases solu-
`bility and pharmacologic effects of the drug. An improvement
`of the cell delivery of drugs using vector oil is based on the
`modulation of membrane fluidity that directly depends on its
`fatty acid composition. Castor oil, which mainly contains rici-
`noleic acid (90% of total fatty acid content),4 is one of the
`lipophilic vehicles used in cyclosporine eye drops.2’5‘(’ How-
`ever,
`it presents both a low-stability and an epithelial and
`conjunctival toxicity7 as well as systemic adverse effects such
`as purgative effects, hypersensitivity, nephrotoxicity, and neu-
`rotoxicity.4’8 Since castor oil is presumed to be responsible for
`cytotoxic effects in the eye, its replacement by another li-
`pophflic vector could result in better tolerance of the drops.
`Omega-3 fatty acids are important in the structure and
`function of the visual system.9 ac-Linolenic acid (ALA) is the
`precursor of the long-chain omega-3 polyunsaturated fatty ac-
`ids (n-3 PUFA)—mainly, docosahexaenoic acid (DHA) and ei-
`cosapentaenoic acid (EPA)1°—found in fish oils and in nerve
`tissue cell membranes. Several vegetable oils exhibit a high
`content of omega-3 fatty acids. For instance, Aleurites m0luc-
`cana and camelina oils are rich sources of linolenic acid (36%-
`40%).”‘12 Long-chain omega-3 fatty acids are rapidly incorpo-
`rated into cell membrane phospholipids, thereby resulting in
`changes in receptor functions and alteration in cell signaling
`mechanisms and membrane-bound enzymes.” On the other
`hand, incorporation of ac-linolenic acid or other n-3 series fatty
`acids into the diet results in marked changes in cell membrane
`composition as well as in arachidonic acid metabolism.” Ara-
`chidonic acid, a long-chain omega-6 polyunsaturated fatty acid
`(n-6 PUFA) abundant in membrane phospholipids, is the first
`step of the inflammatory process after metabolism by cyclooxy-
`genases and lipoxygenases, which are present in the conjunc-
`tiva.15‘16 The omega-3 fatty acids contained in vegetable oils,
`such as camelina oil, could thus replace arachidonic acid in cell
`membranes and decrease inflammation. Therefore, it is of in-
`terest to develop lipophilic vectors that would be free of
`negative side effects and contain omega-3 fatty acids.
`In this study, we evaluated the in vitro and in vivo toxicity
`of four vegetable oils to validate their use as vehicles of li-
`pophflic drugs in eye drops. They were chosen because of
`their biological properties and fatty acid composition“‘17’18:
`Two of them are rich in omega-3 fatty acids (Aleurites and
`camelina) and two are free of omega-3 (maize and olive). The
`olive oil was highly refined (neutral, denatured, and free of
`antioxidants) and was chosen as the negative control; castor oil
`was tested to confirm its cytotoxic effects in our cell model.
`Cell assays for necrosis, apoptosis, intracellular redox status,
`
`Investigative Ophthalmology & Visual Science, November 2007, Vol. 48, No. 1 1
`Copyright © Association for Research in Vision and Ophthalmology
`
`ALL 2003
`ARGENTUM PHARMACEUTICALS V. ALLERGAN
`IPRZO16-01232
`
`1
`
`ALL 2003
`ARGENTUM PHARMACEUTICALS V. ALLERGAN
`IPR2016-01232
`
`

`
`IOVS, November 2007, Vol. 48, No. 11
`
`Cytotoxicity Evaluation of Different Vector Oils
`
`5001
`
`and antioxidant properties were performed on human con-
`junctival cells. In addition, we analyzed the incorporation of
`oils (i.e., of their constitutive fatty acids) in cultured conjunc-
`tival epithelial cell membranes. In vivo ocular irritation was
`evaluated by using the Draize test.
`
`MATERIALS AND METHODS
`
`Reagents
`Castor, maize, and olive oils were purchased from Sigma-Aldrich (St.
`Louis, M0) at the highest purity grade available. Camelina oil was
`purchased from Phytocos (Poitiers, France); and Aleurites moluccamz
`oil was purchased from Plant Extract laboratory (Antanarivo, Madagas-
`car).
`
`Ocular Cytotoxicity Assessment on a
`Conjunctival Cell Line
`Wong Kilboume— derived human conjunctival epithelial cells (WKD,
`ECACC 93120839) Were cultured under standard conditions (moist
`atmosphere of 5% CO2 at 37°C) in Dulbecco’s minimum essential
`medium (DMEM; Eurobio, Les Ulis, France) supplemented with 10%
`fetal bovine serum (FBS, Eurobio), 2 mM 1-glutamine, 50 IU/mL peni-
`cillin, and 50 IU/mL streptomycin (Eurobio). The culture medium was
`changed every 3 days; confluent cultures were removed by trypsin
`incubation. The cells were counted, seeded into 96-well microplates
`(Costar; VWR, Fontenay sous Bois, France), and kept at 37°C for 24
`hours. The different undiluted oils, including olive oil as the control,
`were incubated for 15 minutes (30 minutes for the fatty acid incorpo-
`ration test), and tests were performed after a 24-hour recovery period
`in culture medium. Olive oil was chosen as the control.
`Fluorometry was performed with a microplate cold light cytoflu-
`orometer” (Fluorolite 1000; Dynex, Cergy Pontoise, France). All fluo-
`rescent dyes were added to living cells, since this method allows
`detection of the fluorescent signal directly from the microplate.
`
`Cell Viability
`
`Membrane Integrity. Membrane integrity, which is closely
`correlated with cell viability, was evaluated with neutral red (Fluka,
`Buchs, Switzerland), by fluorometric detection (Aexcitation [ex.] =
`535 run; Aemission [em.] = 600 nm). Neutral red was used at a
`concentration of 50 (g/mL, according to Borenfreund and Puerner2°;
`200 ML per well of medium containing neutral red was added to living
`cells, and the microplate was incubated for 3 hours at 37°C in moist
`
`atmosphere With 5% C02. The neutral red fluorescence was then
`measured.
`
`Redox Status. The Alamar blue assay was used to measure redox
`status variations.2"22 Alamar blue penetrates cells passively and
`thereby is reduced. The oxidized nonfluorescent dye becomes a re-
`duced fluorescent dye. A solution at 0.1 mg/mL in PBS is diluted (1/1 1)
`in culture medium containing 2.5% FBS. Cells were incubated at 37°C
`for 7 hours, and fluorescence detection (Aex. = 535 um; Aem. = 600
`nm) was then measured.
`Cell Proliferation. Cell proliferation was evaluated by the in-
`corporation of BrdU in DNA during replication. The BrdU kit (Cell
`Proliferation ELISA, BrdU) was provided by Roche (Meylan, France).
`
`Apoptosis
`
`P2X7 Cell Death Receptor Activation: the YO-PRO-1
`Test. YO-PRO-1 , a DNA probe (lnvitrogen-Molecular Probes, PoortGe-
`bouw, The Netherlands), penetrates apoptotic cells only through the
`P2X7 receptor. After incubation with the different oils, a 2-p.M YO-
`PRO-1 solution in phosphate-buffered saline was applied (200 ML per
`well), and the microplate was placed for 10 minutes in the dark at
`room temperature. The fluorescence signal was scanned by using a
`cytofluorometer with a small band pass and precise wave lengths for
`YO-PRO-1 fluorescence detection (Aex. = 491 nm; Aem. = 509 nm).
`This test was simultaneously performed with the neutral red test.
`Caspase 3 Activation. To assess apoptosis, caspase 3 activity
`was evaluated by using a caspase 3 kit assay with rhodainine 1 10-DEVD
`fluorogenic substrate (lnvitrogen-Molecular Probes).
`
`Reactive Oxygen Species Production
`Intracellular reactive oxygen species (ROS) production (mainly H202)
`was detected with the 2',7'-dichlorofluorescein diacetate fluoroprobe
`(DCFH-DA; lnvitrogen-Molecular Probes).23 This probe is a nonfluores-
`cent compound currently used in flow cytometry and adapted for use
`in the microplate assay.“ Once incorporated into the cells, the probe
`is cleaved by endogenous esterases and can no longer exit the cell. The
`de-esterified product becomes a fluorescent compound 2',7'-dichlo-
`rofluorescein after its oxidation by intracellular ROS. Fluorescent signal
`detected ().ex.: 490 rim; ).em.: 535 nm) is proportional to ROS pro-
`duction.
`
`Fatty Acid Incorporation in Membrane
`The cells were centrifuged for 5 minutes at 1000g. The cell pellets
`were resuspended in 1 mL of 0.1 M sucrose-10 mM Tris buffer (pH 7.4)
`
`250
`
`200
`
`150
`
`100
`
`50
`
`"/2Fluorescence
`
`FIGURE 1. Membrane integrity eval-
`uation using the neutral red test after
`incubation with different oils for 15
`minutes followed by a 24-hour recov-
`ery period. Results are expressed as a
`percentage of control. Castor oil de-
`creased cell viability. *P < 0.001.
`
`I Neutral Red
`
`OHVB
`
`Came-Iina
`
`Aleurites
`
`Maize
`
`Castor
`
`2
`
`

`
`5002
`
`Said et al.
`
`IOVS, November 2007, Vol. 48, No. 11
`
`140
`
`120
`
`100
`
`so
`
`60
`
`40
`
`20
`
`o
`
`abslolive
`
`I Brdu
`
`FIGURE 2. Cell proliferation evalua-
`tion with BrdU incorporation after
`incubation with different oils for 15
`minutes followed by a 24-hour recov-
`ery period. Castor oil inhibited cell
`proliferation. *P < 0.001.
`
`Olive
`
`Camelina
`
`Aleurites
`
`Maize
`
`ca5t°r
`
`at 4°C. They were then lysed after five freeze—thaw cycles and a cold
`sonication for 30 to 60 seconds. Cell membranes were isolated by
`several ultracentrifugations. Total lipids were extracted, and mem-
`brane fatty acids were analyzed and quantified by high-pressure liquid
`chromatography, as previously described.25’2(’
`
`In Vivo Assessment of Ocular Irritation:
`Draize Test
`
`All procedures in this study were performed in compliance with the
`ARVO Statement for the Use of Animals in Ophthalmic and Visual
`Research. Animal care and experimentation complied with the rules of
`European Council Guidelines:
`license for experimental studies on
`living animals. Eight-week-old male New-Zealand rabbits (Cegav, Saint
`Mars d’Egrenne, France) were placed in an individual cage and kept in
`standard laboratory conditions (at 20°C, 12-hour light— dark cycle), fed
`ad libitum on the standard laboratory diet with free access to water.
`Ocular irritation was evaluated by a modified Draize test.27 Briefly,
`0.1 mL of nondiluted (100%) oil was instilled into the conjunctival sac,
`
`and the upper and lower lids were gently held together for 10 seconds.
`The opposite eye of each animal served as the untreated control.
`Ocular responses (conjunctiva, iris, cornea) were scored at 1, 24, 48,
`72, and 96 hours, and at 7 days. A fluorescein stain was used to confirm
`corneal effects visible by examination at the 24-hour observation and at
`each subsequent observation period until the cornea failed to exhibit
`uptake of fluorescein. Irritation was scored according to the method of
`Draize et al.27 A score of less than 15 was considered to show that the
`oil was a nonirritant.
`
`Statistical Analysis
`Results were obtained in arbitrary fluorescence units and expressed as
`a percentage of control values. For cell experiments, oils were tested
`in six wells, and each experiment was performed in triplicate. Mean
`values for each concentration were analyzed by one-way ANOVA
`followed by the Dunnett test.28*29 All statistical analyses were per-
`formed with commercial software (Sigma Stat 2.0; SPSS, Chicago, USA).
`P < 0.05 was considered statistically significant.
`
`5.0
`
`415
`
`-10
`
`35
`
`3.0
`
`25
`
`20%fluorescence
`
`1.5
`
`1D
`
`05
`
`0.0
`
`Icaspases 3Iredox status
`
`FIGURE 3. Caspase 3 activity evalu-
`ated by the rhodamine 1 10-DEVD flu-
`orogenic substrate after incubation
`with different oils for 15 minutes fol-
`lowed by a 24-hour recovery period.
`Results are expressed as a percent-
`age of the control. Castor oil acti-
`vated caspase 3. *P < 0.001.
`
`3
`
`

`
`IOVS, November 2007, Vol. 48, No. 11
`
`Cytotoxicity Evaluation of Different Vector Oils
`
`5003
`
`
`
`3.0
`
`2.5 <
`
`2.0 -
`
`1.54
`
`1.0
`
`0.5 -
`
`%fluorescence
`
`I yopro-1:‘ redox status
`
`FIGURE 4. P2X7 cell death receptor
`activation evaluated with the YO-
`
`PRO—1 test after incubation With dif
`ferent oils for 15 minutes followed
`
`OD
`
`'::::$:.::;°::::.‘::::?.‘;.“::$:::
`control. Castor oil activated P2X7
`cell death receptor. *P < 0.001.
`
`O
`
`<\«<°
`0
`.36‘
`C-'
`
`9‘
`\¢v
`Y.
`
`.-,-°
`‘1~
`
`‘D
`0
`
`RESULTS
`
`Cell Viability, Apoptosis, and Oxidative Stress
`
`Cell viability results, as evaluated with the neutral red assay
`(Fig. 1), indicated that maize, Aleurites moluccana, and cam-
`elina oils did not induce any cellular necrosis when compared
`with the control olive oil. Although an increase in neutral red
`incorporation was observed for the three oils when compared
`with olive oil, no proliferative effect was detected by the
`specific proliferation BrdU assay (Fig. 2). On the other hand,
`the neutral red and BrdU assays indicate that castor oil signif-
`icantly decreased cell viability (P < 0.001). In the same Way,
`assays for apoptosis revealed that maize, Aleurites moluccana,
`and camelina oils did not activate the PZX7 cell death receptor
`and caspase 3 (Fig. 3), whereas castor oil significantly induced
`apoptosis through both PZX7 cell death receptor activation
`(+200%, P < 0.001; Fig. 4) and caspase 3 activation (+301%,
`P < 0.001; Fig. 3).
`
`Finally, When compared with control olive oil, maize, Aleu-
`rites moluccana, and camelina oils did not alter intracellular
`redoX status, whereas castor oil significantly decreased intra-
`cellular redoX potential (—42.3%; Fig. 5). Assays with the
`H202-sensitive DCF fluorogenic probe confirmed that castor
`oil, but not other oils, significantly increased intracellular ROS
`production (+1400%; Fig. 6) that was surely responsible for
`the alteration of global redoX status measured by the Alamar
`blue assay.
`
`Fatty Acid Incorporation in Membranes
`
`When compared with olive oil, cells incubated with camelina
`or Aleurites oils significantly increased the incorporation rate
`of on-linolenic acid in their membranes, and concomitantly, a
`decrease in the membrane arachidonic acid content was ob-
`served (Table 1). Because of the strong necrotic effect of castor
`oil, the modification in membrane fatty acid composition of
`
`110
`
`IEIU
`
`90
`
`ED
`
`fl-1
`U 7n
`5u
`an
`I”
`ED EIU
`
`E "
`
`:
`3‘
`
`I Redox status
`
`4n
`
`30
`29
`
`‘”
`
`Olive
`
`camelina
`
`Aleurites
`
`Maize
`
`Castor
`
`FIGURE 5. Redox status evaluation
`using the Alamar blue test after incu—
`bation with different oils for 15 min—
`utes followed by a 24-hour recovery
`period. Results are expressed as a
`percentage of the control. Castor oil
`altered intracellular
`redox status.
`*P < 0.001.
`
`4
`
`

`
`5004
`
`Said et al.
`
`IOVS, November 2007, Vol. 48, No. 11
`
`%fluorescence
`
`I H2CFDAIredox status
`
`FIGURE 6. ROS production evalu-
`ated by DCF-DA test after incubation
`with different oils for 15 minutes fol-
`lowed by a 24-hour recovery period.
`Results are expressed in DCF/NR flu-
`orescence ratio. Castor oil increased
`ROS production. *P < 0.001.
`
`cells maintained for 30 minutes in the presence of this oil was
`impossible to determine in our experimental conditions.
`
`In Vivo Assessment of Ocular Irritation:
`Draize Test
`
`As indicated in Table 2, none of the five tested oils induced any
`noticeable in vivo ocular irritation. For each oil, the score
`remained S2, whereas positivity for this test corresponds to a
`score of :15 or higher.
`
`DISCUSSION
`
`The results of our study demonstrate that castor oil exhibited
`significant ir1 vitro cell toxicity in comparison with olive oil.
`Other tested oils (i.e., camelina, Aleurites moluccama, and
`maize oils) did not exhibit such deleterious effects, since there
`was no difference in the necrotic and apoptotic processes or
`the alteration of ROS production between these oils and olive
`oil.
`
`When it occurs, eye drop intolerance can result from either
`the active component or the vehicle. As an example, benzal-
`konium chloride, a well-known preservative, is known to in-
`duce apoptosis in certain ocular cells; recent published studies
`from our laboratory demonstrated that benzalkonium chloride
`activates P2X7 cell death receptor, leading to fluoroquinolone
`eye drop intolerance.3° Available cyclosporine eye drops con-
`
`TABLE 1. Fatty Acid Incorporation in Conjunctival Cell Membranes
`after Incubation with Different Oils
`
`Fatty Acid
`
`Control Olive Maize Camelina Aleurites
`
`Arachidonic acid
`20:4 (116)
`an-Linolenic acid
`1823 (11-3)
`
`6.75
`
`0.60
`
`4.62
`
`0.60
`
`4.45
`
`2.82
`
`4.09
`
`7.20
`
`1.65
`
`1 3-52
`
`Incubation lasted 30 minutes followed by a 24-hour recovery
`period. The different oils modified cell membrane fatty acid composi-
`tion.
`
`tain maize oil or castor oil as the vehiclef“ Reports indicate
`that ophthalmic formulations containing castor oil are more
`cytotoxic than those containing maize oil.” Our present re-
`sults showed that castor oil induced necrosis, apoptosis (P2X7
`receptor and caspase 3 activations), and intracellular H202
`overproduction in an in vitro model of conjunctival cells. As a
`hypothesis, the cytotoxic effects of castor oil could be due to
`its high ricinoleic acid content.
`Indeed, high concentrations of ricinoleic acid (20.1 mM)
`were shown to be cytotoxic in isolated hamster intestinal
`epithelial cells.“ The lack of in vivo toxicity (Draize test) may
`be due to the single application of castor oil, which is not
`sufficient to induce a toxic effect and ocular irritation. As a
`
`further study, in vivo assays testing repeated applications of
`castor oil (as well as ricinoleic acid) might show toxicity and
`explain clinical observations. Indeed, some patients who use
`eye drops containing castor oil exhibit discomfort and ocular
`irritation after long-term treatment. Use of nontoxic lipophilic
`eye drop vehicles, instead of castor oil, could suppress such
`adverse effects. The other tested oils presented better in vitro
`tolerance than castor oil and could be used in ophthalmic
`formulations instead of castor oil. Maize oil is not cytotoxic and
`is already used in cyclosporine eye drop preparations at the
`French Ophthalmologic National Center (Hopital des Quinze-
`Vingts, Paris, France). Our in vitro results confirm the tolerance
`of this oil and suggest that long-term treatment of patients
`would be safe.
`
`Omega-3 fatty acids are critical for membrane functions and
`were shown to exhibit anti-inflammatory effects.“ In response
`to proinflammatory stimuli, enhanced catalytic activity of phos-
`pholipase A2 degrades membrane phospholipids to form free
`arachidonic acid that is converted into prostaglandins and
`other eicosanoids through activation of the oxidative enzymes
`cyclooxygenases and lipoxygenases. All these enzymes are
`found in ocular conjunctiva.“ '57 Replacement of arachidonic
`acid by DHA and EPA in cell membranes reduces the inflam-
`matory process related to proinflammatory prostaglandin syn-
`thesis, since products of the catalytic action of phospholipase
`A2, cyclooxygenases, and lipoxygenases more likely result in
`
`5
`
`

`
`IOVS, November 2007, Vol. 48, No. 11
`
`Cytotoxicity Evaluation of Different Vector Oils
`
`5005
`
`TABLE 2. In Vivo Ocular Irritation Score
`
`Oils
`
`1 h
`
`24 h
`
`48 h
`
`72 h
`
`96 h
`
`7 d
`
`Total
`
`Comparison
`
`Observations
`
`Olive
`Castor
`Maize
`Aleurites moluccana
`Camelina
`
`0
`2
`1
`0
`0
`
`0
`0
`0
`0
`0
`
`0
`0
`0
`0
`0
`
`0
`0
`0
`0
`0
`
`0
`0
`0
`0
`0
`
`0
`0
`0
`0
`0
`
`0
`2
`1
`0
`0
`
`< 1 5
`< 1 5
`< 1 5
`< 1 5
`< 1 5
`
`N0 irritant
`N0 irritant
`N0 irritant
`N0 irritant
`N0 irritant
`
`Undiluted oils (100 %) were instilled into the conjunctival sac of rabbits. None of the tested oils was an irritant.
`
`the formation of anti-inflammatory38 and proresolving than
`proinflammatory products,59*4" especially in the retina.“ As
`another beneficial effect of the membrane enrichment
`in
`
`omega-3, modification of membrane fatty acids is responsible
`for quantitative and/or qualitative changes in lipid raft domain
`activity. Lipid rafts consist of lateral assemblies of cholesterol
`and sphingolipids in the outer exoplasmic leaflet connected to
`phospholipids and cholesterol in the inner cytoplasmic leaflet
`of the membrane bilayer. Components of the death-inducing
`signaling complex have been shown to be present in the raft
`fraction.“
`Both Aleurites moluccana and camelina oils have a high
`content of omega-3 fatty acids (35% and 40% of total fatty acids,
`respectively), which may precipitate a better tolerance and
`efficacy of the drug, improving cell membrane fluidity, and
`drug incorporation. These oils should be considered as inter-
`esting candidates for alternative lipophilic vehicles in eye drop
`formulations, since our data showed that they did not exhibit
`in vitro and in vivo toxic effects and contributed to beneficial
`changes in cell membrane fatty acid composition (i.e., deple-
`tion in arachidonic acid and a substantial enrichment of cell
`membranes in ALA; Table 1).
`
`CONCLUSION
`
`In our in vitro experimental conditions, castor oil induced
`necrosis and apoptosis and enhanced oxidative stress, which
`could contribute to the side effects of eye drops containing this
`oil as a vehicle. Aleurites moluccana and camelina oils did not
`exhibit such cell deleterious effects and may exert beneficial
`actions on both inflammatory process and drug incorporation
`by decreasing membrane content in arachidonic acid on behalf
`of omega-3 acids. The innocuous effects of these oils allow
`their potential use as novel vehicles for eye drops that require
`lipophilic vectors.
`
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