Life Sciences 78 (2006) 1448 – 1456

www.elsevier.com/locate/lifescie

Comparative toxicity of oleic and linoleic acid on human lymphocytes

Maria F. Cury-Boaventura a,*, Renata Gorjão a, Thaı́s Martins de Lima a,
Philip Newsholme b, Rui Curi a
a
Department of Physiology and Biophysics, Institute of Biomedical Sciences, Av. Prof. Lineu Prestes, 1524, CEP 05508-900, University of São Paulo, SP, Brazil
b
Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland

Received 4 April 2005; accepted 12 July 2005

Abstract

Commercially available lipid emulsions for parenteral nutrition are mainly composed by long chain triacylglycerol containing a high
proportion of linoleic acid (LA) or oleic acid (OA). The immunological impact of such therapy is particularly important because parenteral
diets are often administered to critically ill patients as a mechanism to supply adequate nutrition during catabolic stress conditions. The
comparative toxicity of OA and LA on human lymphocytes and the type of cell death induced by these fatty acids were determined in vitro.
Parameters of cell death were investigated by flow cytometry – cell viability, DNA fragmentation, phosphatidylserine externalization,
mitochondrial depolarization, neutral lipid accumulation and production of reactive oxygen species – and by fluorescence microscopy—
chromatin condensation. Additionally a spectrofluorometric assay was employed to determine the activities of caspase—3, 6 and 8. Evidence is
presented herein that OA is less toxic to human lymphocytes than LA. However, both fatty acids promoted apoptosis and necrosis of these
cells. The mechanism of cell death induced by OA involved activation of caspase 3 while the mechanism of death induced by LA involved
mitochondrial depolarization and ROS production. Importantly, neutral lipid accumulation may be a mechanism to protect lymphocytes against
the toxicity induced by OA. OA may offer an immunological less problematic alternative to LA with respect to fatty acid composition of
parenteral nutritional emulsions.
D 2005 Elsevier Inc. All rights reserved.

Keywords: Lymphocytes; Oleic acid; Linoleic acid; Apoptosis; Necrosis

Introduction
The incorporation of lipid emulsions in parenteral diets is
necessary to provide energy requirement to critically ill patients.
Lipid emulsions for parenteral nutrition that are commercially
available are mainly composed of long chain triglycerides
(LCT) containing a high proportion of N-6 polyunsaturated fatty
acids (PUFA) (60%), usually derived from soybean or
sunflower oil. However, lipid emulsions containing N-9
monounsaturated fatty acids (MUFA) as the main fatty acids
are also now available (Allwood, 2000).
The immunological impact of such therapy is particularly
important because parenteral diets are often administered to
critically ill patients who may be immuno-compromised
* Corresponding author. Tel.: +55 11 30917245; fax: +55 11 30917285.
E-mail address: mafecury@icb.usp.br (M.F. Cury-Boaventura).
0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2005.07.038
(Lindgren et al., 2001; Lin et al., 2002). Severe trauma and
sepsis are associated with depressed immune functions and
increase levels of putative immunosuppressive agents such as
PGE2, IL-4, and IL-10 (Ahmad et al., 1997; Lanza_Jacoby et
al., 2001). In fact, infection and injury activate the immune
system and bring about widespread metabolic changes. A
combination of enhanced lipolysis in adipose tissue and
increased hepatic lipogenesis in infected and injured indivi-
duals cause a rapid increase in plasma concentrations of free
fatty acids (Grimble, 1998; Zuijdgeest_Van_Leeuwen et al.,
2002).
Studies on the effect of fatty acids on immune response date
back over 30 years (Piette and Saugier, 1970). In vitro and in
vivo studies have shown that fatty acids modulate a number of
leukocyte functions (Arrington et al., 2001; Kelley, 2001),
including proliferation (Otton et al., 1998; Granato et al., 2000;
Furukawa et al., 2002; Cavaglieri et al., 2003), activation by
antigens (Granato et al., 2000), natural killer cell activity
 M.F. Cury-Boaventura et al. / Life Sciences 78 (2006) 1448 – 1456
(Yaqoob et al., 1998), cytokine release (Granato et al., 2000;
Furukawa et al., 2002; Hara et al., 2003; Kurita-Ochiai et al.,
2004), adhesion molecule expression (Yaqoob et al., 1998;
Kurita-Ochiai et al., 2004), mitogenic signalling cascade
activity (Yusufi et al., 2003), and cell death (Emenaker et al.,
2001; Garnacho-Montero et al., 2002; Lima et al., 2002;
Pompeia et al., 2002; Healy et al., 2003; Switzer et al., 2004).
The increased risk of infection in patients receiving
parenteral nutrition has been associated with immunosuppres-
sive effects of the specific lipid content of the formula, mainly
LA (Muller et al., 1986; Freeman et al., 1990; Sosenko et al.,
1993; Battistella et al., 1997; Anderson and Fritsche, 2002).
Therefore, current standard lipid emulsions for parenteral
nutrition based on soybean oil, rich in LA, may have
immunosuppressive effects (Soyland et al., 1993; Morlion et
al., 1996; Granato et al., 2000; Furukawa et al., 2002; Koller et
al., 2003; Lekka et al., 2004). It is therefore of interest that the
role of lipid emulsion rich in OA and free OA on immune
function still remains controversial. Some reports have shown
that OA has no effect on lymphocyte proliferation and natural
killer cell activity (Yaqoob et al., 1998; Granato et al., 2000). In
contrast, others have found decreased lymphocyte proliferation,
natural killer cell activity, cytokine release by lymphocyte after
infusion of a lipid emulsion based on olive oil (Moussa et al.,
2000), and adhesion molecule expression induced by OA in
both animal and human lymphocytes (Tsang et al., 1977;
Yaqoob et al., 1998). In the latter cases, the inhibition of
immune response observed due to OA treatment was less
pronounced than that obtained with LA.
Cell death usually occurs by two possible mechanisms:
apoptosis or necrosis. Apoptosis, also known as programmed
cell death, occurs during both physiological and pathological
processes. In opposition, necrosis is linked mostly to
pathological conditions, when cells die after exposure to
extreme physical, biological or chemical conditions (Proskur-
yakov et al., 2003). Apoptosis is characterized by changes in
cell morphology including translocation of phosphatidylserine
(PS) from the inner to the outer leaflet of the cell membrane, a
decrease in cell size, an increase in DNA fragmentation and
caspase activation (Kerr et al., 1972). Necrosis is characterized
by early loss of membrane integrity and occurs when there is
great damage to the cell membrane or when the production of
ATP ceases abruptly (Proskuryakov et al., 2003). It has been
shown that higher concentrations of certain fatty acids,
particularly PUFA, can cause cell death via apoptosis or, when
concentrations are excessive, necrosis (Lima et al., 2002).
In the present study, the comparative toxicity of OA or LA
on isolated human lymphocytes was investigated. We postu-
lated that a decrease in lymphocyte number due to apoptosis
and/or necrosis may play an important role in the impairment
of immune function induced by these fatty acids. The induction
and type of cell death was investigated by changes in
membrane integrity, DNA fragmentation, and phosphatidylser-
ine externalization using flow cytometry. The chromatin
condensation assay and determination of the proportion of
necrotic and apoptotic cells, using acridine orange/ethidium
bromide staining, were carried out through fluorescence
1449
microscopy. The possible mechanisms involved were examined
by measuring mitochondrial depolarization, neutral lipid
accumulation, ROS production and activities of caspases 3, 6
and 8.
Materials and methods
Reagents
RPMI-1640 medium, HEPES, penicillin, Trizol reagenti
and streptomycin were purchased from Invitrogen (Carlsbad,
CA, USA). Oleic acid, linoleic acid, Triton X-100, glutamine,
nile red, and trypan blue were obtained from Sigma (St. Louis,
MO, USA). Propidium iodide was from ICN Biomedicals (Costa
Mesa, CA, USA). Citrate was purchased from Merck (Frank-
furter, Darmstadt, Germany) and sodium bicarbonate from
Labsynth products (Diadema, SP, Brazil). Hoechst 33342,
acridine orange, dihydroethidium and rhodamine 123 were
obtained from Molecular Probes (Eugene, OR, USA). Ethidium
Bromide was purchased from BioRad Laboratories (Hercules,
CA, USA). AC-DEVD-AMC (caspase 3), AC-VEID-AMC
(caspase 6), AC-IETD-AFC (caspase 8) and annexin V-FITC
were from Pharmingen – BD Biosciences (San Diego, CA,
USA).
Study design
The study was approved by the Ethical Committee of the
Institute of Biomedical Sciences. The blood was obtained from
The Blood Bank of the Federal University of São Paulo. The
blood was considered as completely healthy after a routine
laboratory analysis.
Cell isolation
Human peripheral blood mononuclear cells (PBMC; a
mixture of monocytes and lymphocytes) were isolated by
centrifugation. Blood was diluted in PBS (1:1) and this
suspension was layered into Histopaque-1077 and centrifuged
(Harrier 18/80, Sanyo, London, UK), for 30 min, at 800g
and 4 -C. PBMC were collected from the interphase, lysed
with 150 mM NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA, pH
7.4, and washed twice with PBS. The PBMC were
maintained in RPMI-1640 medium to allow the adherence
of monocytes as to obtain a pure lymphocyte preparation
(about 98%).
Culture conditions
The cells were grown in RPMI-1640 medium containing
10% fetal calf serum (FCS). This medium was supplemented
with glutamine (2 mM), HEPES (20 mM), streptomycin
(10,000 Ag/mL), penicillin (10,000 UI/mL) and sodium
bicarbonate (24 mM). Cells were grown in 25-mL flasks
containing 1 –2  106 cells/mL. The cells were kept in a
humidified atmosphere at 37 -C, containing 5% CO2. Cells
were used at 2  106 cells/mL.
1450 M.F. Cury-Boaventura et al. / Life Sciences 78 (2006) 1448 – 1456
Fatty acid treatment
The cells were treated with various concentrations (25, 50,
100, and 200 AM) of OA or LA for 24 h. The fatty acids were
dissolved in ethanol. The final concentration of ethanol in the
culture medium did not exceed 0.05%. This concentration of
ethanol was not toxic to the cells as also reported by Siddiqui et
al. (2001).
Viability of the cells
Cells were centrifuged at 1000g for 15 min at 4 -C and
the pellet obtained was resuspended in 500 AL phosphate-
buffered saline (PBS). Afterwards, 50 AL of propidium iodide
solution (50 mg/mL in PBS) were added and the cells
analyzed using a FACSCalibur flow cytometer (Becton
Dickinson, San Juan, CA, USA). Propidium iodide is a
highly water-soluble fluorescent compound, which cannot
pass through intact membranes, and is generally excluded
from viable cells. It binds to DNA by intercalating between
the bases with little or no sequence preference. Fluorescence
was measured using the FL2 channel (orange-red fluores-
cence = 585/42 nm). Ten thousand events were analyzed per
experiment. Cells with propidium iodide fluorescence were
then evaluated by using the Cell Quest software (Becton
Dickinson).
Staining DNA using propidium iodide
DNA fragmentation was analyzed by flow cytometry after
DNA staining with propidium iodide according to the method
described by Nicoletti et al. (1991). The presence of detergent
in the solution permeabilizes the cells, which promptly
incorporate the dye into DNA. Briefly, after the different
incubation periods, the cells were centrifuged at 1000g, for
15 min, at 4 -C. The pellet was gently resuspended in 300 AL
hypotonic solution containing 50 Ag/mL propidium iodide,
0.1% sodium citrate, and 0.1% Triton X-100. The cells were
then incubated for 2 h at 4 -C. Fluorescence was measured and
analyzed as described above.
Chromatin condensation
After treatment with the fatty acids for 24 h, cells were
pelleted, resuspended in Hoechst 33342 solution (100 Ag/
mL), and stained for 10 min in the dark to visualize the
location of DNA. Cells were examined for chromatin
condensation (Zeiss, Munchen-Hallbergmoos, Germany) us-
ing a fluorescence microscope with a filter for 365/80 nm.
The Hoechst 33342 dye has been widely used for staining the
nuclei of living cells. Hoechst dyes preferentially bind to AT
regions, making them quite selective (but not specific) for
DNA. Hoechst dye-stained cells and tissues show no
cytoplasmic staining. The Hoechst 33342 dye is commonly
used to distinguish the compact chromatin of apoptotic nuclei,
to identify replicating cells and to sort cells based on their
DNA content. Cells stained with Hoechst 33342 were then
evaluated by fluorescence microscopy using the KS 300
software (Zeiss).
Determination of the proportion of necrotic and apoptotic cells
by using acridine orange/ethidium bromide assay
After treatment with the fatty acids for 24 h, cells were
pelleted and resuspended in 25 AL PBS. Afterwards, 1 AL of
aqueous solution of acridine orange/ethidium bromide (100 Ag/
mL each) was added and the cells were examined using a
fluorescence microscope. Acridine orange intercalates into the
DNA giving it a green appearance. Thus, viable cells have a
bright green nucleus. Ethidium bromide is only taken up by
non-viable cells. This dye also intercalates into DNA, making it
appears orange. Live cells with intact membranes have a
uniform green color in their nuclei. Early apoptotic cells have
chromatin condensation with bright green colored nuclei. Late
apoptotic cells have bright orange areas of condensed
chromatin in the nucleus that distinguish them from necrotic
cells, which have a uniform orange color. The cells were
analyzed using a fluorescence microscope with a filter for 470/
40 nm (Zeiss). The cells were then classified as follows: live,
apoptotic and necrotic cells. The percentage of apoptotic and
necrotic cells was then calculated.
Phosphatidylserine (PS) externalization
PS externalization was analyzed by flow cytometry after PS
staining with annexin V according to the method described by
Vermes et al. (1995). Cells were washed twice with cold PBS
and then resuspended in 100 AL binding buffer (10 mM
HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl 2) at a
concentration of 1 106 cells/mL. 5 AL of fluorescein-
conjugated annexin V (annexin V-FITC) were added. The
cells were gently vortexed and incubated for 15 min at room
temperature (20 – 25 -C) in the dark. Afterwards, 10 AL of PI
and 400 AL of binding buffer were added and the cells were
analyzed by flow cytometry. Annexin V is a phospholipid-
binding protein that has a high affinity for PS. PI is used to
distinguish viable from non viable cells. Fluorescence of
annexin V-FITC was measured in FL1 channel (green
fluorescence = 530/30 nm) and PI in FL2 channel (orange-
red fluorescence = 585/42 nm). Cells stained with annexin V-
FITC were then evaluated as described above.
Mitochondrial transmembrane potential (MTP)
Cells were centrifuged at 1000g for 15 min at 4 -C and
the pellet obtained was resuspended in 1000 AL of phosphate-
buffered saline (PBS). Rhodamine 123 is a cell-permeable,
cationic, fluorescent dye that is readily sequestered by active
mitochondria without inducing cytotoxic effects. Uptake and
equilibration of rhodamine 123 is rapid. Therefore, rhodamine
123 allows for quick and easy detection of changes in MTP
(Darzynkiewicz et al., 1981). Rhodamine 123 (5 AM) was
added and the cells were then incubated for 15 min at 37 -C
in the dark. Afterwards, the cells were washed twice with
 M.F. Cury-Boaventura et al. / Life Sciences 78 (2006) 1448 – 1456 1451

70
cold PBS and incubated for 30 min at 30 -C in the dark. *
60

fragmentation (%)
Fluorescence of rhodamine 123 was determined using the

cells with DNA

50
FL1 channel (green fluorescence = 530/30 nm). *
40 oleic acid
Production of ROS 30 linoleic acid
20

Dihydroethidium was used for the flow cytometric 10

measurement of superoxide production (Rothe and Valet, 0
1990). Dihydroethidium is rapidly oxidized to ethidium (a red 0 25 50 100 200

fluorescent compound) by H2O2 (in the presence of perox- Concentration (µM)

idase) and superoxide. Ethidium is trapped in the nucleus by Fig. 2. Effects of oleic or linoleic acid on DNA fragmentation. The
intercalating into DNA, leading to an increase of ethidium percentage of cells with DNA fragmentation after treatment for 24 h with
fluorescence. The cells were stained with dihydroethidium 25, 50, 100 and 200 AM of oleic or linoleic acid is shown. Cells were
(100 AM) by incubating for 30 min at room temperature in stained with a buffer containing citrate, Triton X-100 and propidium iodide
and analyzed by flow cytometry. The indicated percentages correspond to
the dark and analyzed in a FACSCalibur flow cytometer
the fraction of cells with subdiploid DNA. Fluorescence was measured in
(Becton Dickinson). Fluorescence was measured using filter channel FL2 (585/42 nm). The values are presented as mean T S.E.M. of five
FL-3 (670 nm). Histograms of 10 000 events were analyzed determinations from two experiments. *P < 0.05, for comparison between the
per experiment. Cells with dihydroethidium fluorescence were treatment with OA or LA vs. control (in the absence of fatty acids = 0 AM
then evaluated by using Cell Quest software (Becton concentration).
Dickinson).
at a 5-min intervals by using a spectrofluorometer (Molecular
Caspase activity Devides Spectra MAX Gemini XS, CA, USA) (Andersson et
al., 2000).
Cells were centrifuged at 1000g for 15 min at 4 -C and
the pellet obtained was resuspended in 200 L of lyse buffer
containing 10% sucrose, 0.1% CHAPS, 100 mM HEPES, pH
7.4, 10 mg/mL leupeptin, 10 mg/mL aprotinin, 200 mM
PMSF, and 10 mM dithiothreitol. The cells were incubated
for 30 min at 4 -C and centrifuged at 12 000g for 30 min at
4 -C. The supernatant was collected and protein was
determined by Bradford (1976). Caspase protease activity
was determined by incubating the lysate (20 Ag of total
protein) with 50 AM of the fluorogenic substrates, AC-
DEVD-AMC (caspase 3), AC-VEID-AMC (caspase 6) or
AC-IETD-AFC (caspase 8) in the buffer (10% sucrose, 100
mM HEPES, pH 7.4, 10 mg/mL leupeptin, 10 mg/mL
aprotinin, 200 mM PMSF, and 10 mM dithiothreitol). The
caspase activity was assessed by measuring fluorescence of
Asp-7-amino-4-trifluoromethyl coumarin released for 30 min

100

80 *#
Viability (%)

60 oleic acid
40 * linoleic acid

20

0
0 25 50 100 200

Concentration (µM)

Fig. 1. Effects of oleic or linoleic acid on viability of human lymphocytes.

Lymphocytes were treated with ethanol (control), oleic or linoleic acid at 25,
50, 100 and 200 AM for 24 h. The values are presented as mean T S.E.M. of
four determinations from three experiments. *P < 0.05, for comparison
between the treatments with OA or LA vs. control (in the absence of
fatty acids = 0 AM concentration). #P < 0.05, for comparison between LA and
OA.
Fig. 3. Effects of oleic or linoleic acid on chromatin condensation. Human
lymphocytes were stained with Hoechst 33342 and incubated for 10 min at
room temperature in the dark to visualize DNA after treatment for 24 h with
100 and 200 AM of oleic or linoleic acid. Cells were examined by fluorescence
microscopy for determination of chromatin condensation using a 365/80 nm
filter.
1452 M.F. Cury-Boaventura et al. / Life Sciences 78 (2006) 1448 – 1456

16
*

cells with phosphatidylserine

14 * *

externalization (%)
12

10

0
control oleic acid oleic acid oleic acid linoleic linoleic linoleic
50 µM 100 µM 200 µM acid 50 µM acid 100 µM acid 200 µM

Fig. 4. Effects of oleic or linoleic acid on phosphatidylserine externalization. The percentage of cells with phosphatidylserine externalization after treatment for 24
h with oleic (100 AM) or linoleic acid (50 and 100 AM) is shown. Cells were resuspended in binding buffer, stained with annexin-FITC and incubated for 10 min at
37 -C in the dark. Afterwards, cells were stained with propidium iodide. Fluorescence was measured using filter FL1 (530/30 nm) and filter FL3 (670 nm). The
values are presented as mean T S.E.M. of four determinations from three experiments. *P < 0.05, for comparison between the treatment with OA or LA vs. control (in
the absence of fatty acids = 0 AM concentration).

Measurement of intracellular neutral lipids using the Tukey – Kramer’s test (INStat –Graph Pad Software,
Inc., San Diego, CA, USA).
Cells were pelleted as described. Nile red (0.1 Ag/mL), a The level of significance was set for P < 0.05.
selective fluorescent stain for intracellular lipid droplets
(Healy et al., 2003), was added and fluorescence was Results
determined using the FL1 channel (green fluorescence = 530/
30 nm). Cells stained with nile red fluorescence were Loss of cell membrane integrity
evaluated as describe above.
No significant effect of fatty acids on membrane integrity
Statistic analysis was observed up to 100 AM. However, at 200 AM loss in
membrane occurred, LA inducing a more pronounced effect
Results are presented as means T S.E.M. of 4 – 5 determina- (Fig. 1).
tions from 2 to 3 experiments. Comparisons with control and
between OA and LA treatments were performed by analysis of DNA fragmentation
variance (ANOVA). Significant differences were analyzed by
Apoptotic nuclei are distinguished by hypodiploid DNA
content compared with the diploid DNA content of normal cells.
2 The percentage of human lymphocytes with hypodiploid DNA
cells with mitochondrial

1.8
depolarization (%)

1.6
1.4 * 60 *
1.2
50
ROS production
(arbitrary units)

1
0.8 40
0.6 30
0.4
0.2 20
0 10
control oleic acid linoleic acid
200 µM 200 µM 0
control oleic acid linoleic acid
Fig. 5. Effects of oleic or linoleic acid on mitochondrial depolarization. The 200 µM 200 µM
percentage of lymphocytes with depolarized mitochondria membrane after
treatment for 24 h with oleic or linoleic acid at 200 AM is indicated. Cells Fig. 6. Effects of oleic or linoleic acid on ROS production. ROS production
were stained with rhodamine 123 and incubated for 15 min at 37 -C in the by human lymphocytes after treatment for 24 h with oleic or linoleic acid at
dark. Afterwards, cells were washed and incubated for a period of 30 min at 200 AM is presented as arbitrary units. Cells were stained with
30 -C in the dark. Fluorescence was measured using filter FL1 (530/30 nm). dihydroethidium (100 AM) and incubated for 30 min at 37 -C in the dark.
Histograms of ten thousands events are shown (logarithmic scale). The Fluorescence was measured using filter FL3 (670 nm). Histograms of ten
values are presented as mean T S.E.M. of eleven experiments. *P < 0.01, for thousands events are shown (logarithmic scale). The values are presented as
comparison between the treatment with LA vs. control (in the absence of mean T S.E.M. of eleven experiments. *P < 0.05, for comparison between the
fatty acids = 0 AM concentration). #P < 0.01, for comparison between LA and treatment with OA or LA vs. control (in the absence of fatty acids = 0 AM
OA. concentration).
 M.F. Cury-Boaventura et al. / Life Sciences 78 (2006) 1448 – 1456 1453

30
(fluorescence unit/min)
determined by acridine orange/bromide ethidium assay. Treat-
* ment with OA resulted in detection of 15% cells in apoptosis
Caspase 3 activity

25
and 8% in necrosis, whereas treatment with LA showed 23% of
20
cells in apoptosis and 54% in necrosis. The control cells had
15 2% in apoptosis and 1% in necrosis. LA precipitated
10 lymphocyte death mainly by causing necrosis at 200 AM (data
not shown).
5

0 Phosphatidylserine externalization
control oleic acid
100 µM
Fig. 7. Effects of oleic or linoleic acid on caspase 3 activity. The activity of caspase
3 after treatment for 24 h with 100 AM oleic or linoleic acid is shown. Caspase
protease activity was determined by incubating the lysate (20 Ag of total protein)
with 50 AM, the AC-DEVD-AMC fluorogenic substrate, in the lyse buffer. Caspase
activity was assessed by measuring fluorescent Asp-7-amino-4-trifluoromethyl
coumarin released for 30 min at 5-min intervals using a spectrofluorometer. The
values are presented as mean T S.E.M. of four determinations from three
experiments. *P < 0.05, for comparison between the treatment with OA or LA
vs. control (in the absence of fatty acids = 0 AM concentration).
was only significantly increased at 200 AM. DNA fragmentation
was slightly more pronounced by treatment with LA (Fig. 2).
linoleic acid
100 µM OA raised the proportion of cells with phophatidylserine
externalization at 100 AM concentration but had no effect at
50 and 200 AM. LA led to phosphatidylserine externalization
at 50 and 100 AM but this effect was not observed at 200 AM
(Fig. 4).
Mitochondrial transmembrane potential
The treatment with OA had no effect on mitochondrial
depolarization. On the other hand, the treatment with 200 AM
LA significantly raised ( P < 0.01) the proportion of cells with
depolarized mitochondria (50%) (Fig. 5).

Chromatin condensation ROS production

Under fluorescence microscopy, nuclei of untreated cells The treatment with OA for 24 h did not significantly affect
were observed as blue and round, characteristic of viable cells. ROS production. However, the treatment with LA (200 AM)
Treatment of the cells with OA and LA (200 AM) for 24 raised lymphocyte ROS production by 2-fold (Fig. 6).
h resulted in nuclear fragmentation and chromatin condensa-
tion, which are characteristic of apoptosis. The fluorescence Caspase activity
intensity of chromatin condensation after treatment of the cells
with the fatty acids for 24 h tended to be slightly higher for LA OA and LA at 50 AM did not affect caspase 3 activity
(19%) as compared to OA (11%) (Fig. 3). (data not shown). Cells treated with OA for 24 h had an
increase in caspase 3 activity at 100 AM (by 32%) (Fig. 7)
Determination of the proportion of necrotic or apoptotic cells but had no effect on caspases 6 and 8 activity (data not
shown). In contrast, the treatment with LA at 100 AM had no
The proportion of viable, apoptotic or necrotic lymphocytes significant effects on caspases 3 (Fig. 7), 6 and 8 activity
after treatment with OA and LA (200 AM) for 24 h was (data not shown).

90
*
80
neutral lipid accumulation

70
(arbitrary units)

60

50 *

40
* * *#
30

20

10

0
control oleic acid oleic acid oleic acid linoleic linoleic linoleic
50 µM 100 µM 200 µM acid 50 µM acid 100 µM acid 200 µM

Fig. 8. Effects of oleic or linoleic acid on neutral lipid accumulation. Lipid accumulation after treatment for 24 h with 100 and 200 AM oleic or linoleic acid is shown
as arbitrary units. Cells were stained with nile red and the fluorescence of the latter was measured in FL1 (530/30 nm). The values are presented as mean T S.E.M. of
four determinations from three experiments. *P < 0.05, for comparison between the treatment with OA or LA vs. control (in the absence of fatty acids = 0 AM
concentration). #P < 0.05, for comparison between LA and OA.
1454 M.F. Cury-Boaventura et al. / Life Sciences 78 (2006) 1448 – 1456
Measurement of intracellular neutral lipids
Cells treated with OA and LA for 24 h showed increased
accumulation of intracellular neutral lipids. Treatment with
OA at 50, 100 and 200 AM led to a more pronounced
increase in neutral lipids (by 1.8-, 2.5- and 5-fold, respec-
tively), as compared to LA (by 1.4-, 1.7-, and 2-fold,
respectively) (Fig. 8).
Discussion
There is increasing evidence that unsaturated free fatty acids
can cause cell death by promoting apoptotic signaling. Several
cell types such as vascular smooth muscle cells (Pilane and
Labelle, 2004), neurons (Ulloth et al., 2003), pancreatic cancer
cells (Beeharry et al., 2004), colon cancer cell lines (Avivi-
Green et al., 2002), colorectal neoplasic cells (Llor et al.,
2003), cardiomyocyte (Ghosh et al., 2004), thymocytes
(Garnacho-Montero et al., 2002), murine splenic T lympho-
cytes (Switzer et al., 2003), and leukemia cells (Lima et al.,
2002; Pompeia et al., 2003) show morphological features of
apoptosis and necrosis after exposure to fatty acids.
Previous studies reported that PUFA (such as arachidonic,
docosahexanoic and eicosapentaenoic acid) cause a more
pronounced effect on differentiated neutrophil like HL-60 cell
death than monounsaturated or saturated fatty acids (Healy et
al., 2003). Arachidonic acid, a PUFA N-6, was more toxic to
Jurkat cells than OA (Lima et al., 2002). It has been shown that
arachidonic acid was involved in the regulation of expression
of important regulatory genes (20.5% of 89 genes), whereas
OA did not markedly affects gene expression in Jurkat cells
(2.4% of 89 genes) (Verlengia et al., 2003a,b). Recently we
have found that LA was more potent than OA on Jurkat and
Raji cell death (Cury-Boaventura et al., 2004, 2005). The
findings reported herein confirm that LA was clearly a more
potent inducer of cell death than OA in human peripheral blood
lymphocytes.
The type of cell death induced by LA and OA was then
investigated. The features of programmed cell death include
cell shrinkage, extensive nuclear fragmentation, chromatin
condensation and phosphatidylserine externalization. Our
findings indicate both fatty acids provoked loss of membrane
integrity, DNA fragmentation, chromatin condensation, and
phosphatidylserine externalization. These findings are indica-
tive that both OA and LA promoted apoptosis and necrosis.
The extent and type of cell death varied according to the
concentration of the fatty acids and the period of exposure.
Therefore, signs of apoptosis such as phosphatidylserine
externalization may be difficult to be observed when a higher
proportion of cells is in necrosis as observed at 200 AM of fatty
acids.
A significant proportion of cells died by necrosis after
treatment with a high concentration of fatty acid as indicated by
acridine orange/ethidium bromide assay. Epithelial, malignant,
pancreatic cancer and leukemic cells also show morphological
features of apoptosis when dying after exposure to PUFA
during 24 h, or via ‘‘secondary necrosis or late apoptosis’’, a
feature of end-stage apoptosis, after 72 h (Hawkins et al.,
1998). Secondary necrosis occurs when apoptotic cells cannot
maintain ATP production or control oxidative stress (Schei-
nichen et al., 2003). In this study, evidence is presented that
fatty acids induced apoptosis and subsequently secondary
necrosis. In vivo apoptotic cells will be cleared by macro-
phages before secondary necrosis can occur.
A common feature of apoptosis is mitochondrial depolar-
ization. We observed herein that only LA caused significant
mitochondrial depolarization. Fatty acids can induce the
opening of the permeability transition pore but also can lead
to direct mitochondrial uncoupling resulting in mitochondrial
depolarization (Korge et al., 2003). Depolarized mitochondria
may contribute to the apoptotic process, by causing depressed
rates of ATP synthesis, and failure to maintain adequate ATP
levels (Healy et al., 2002).
Depolarized and uncoupled mitochondria may increase
reactive oxygen species generation. Reactive oxygen species
are postulated to induce cell death by promoting the leakage
of pro-apoptotic agents from the mitochondria via the opening
of the permeability transition pore, damage on the inner
membrane due to lipid peroxidation or by damage to DNA,
which in turn triggers p53-mediated changes in the mito-
chondria. During apoptosis, there is a decrease in the levels of
reduced glutathione in the mitochondria due to leakage or
extrusion, which also diminishes the antioxidant capacity of
the cells. There is evidence that oxidative stress may be
involved in the triggering of cell death by the fatty acids
(Heimli et al., 2001; Pompeia et al., 2002, 2003; Luongo et
al., 2003). In the present study only LA increased ROS
production in human lymphocytes. Therefore, oxidative stress
may be involved in the triggering of cell death, induced by
this fatty acid. OA had no effect on ROS production.
Previous study from our laboratory have shown that OA
presents a stimulating effect on expression of genes related to
oxidative stress: superoxide dismutase, glutathione reductase,
and glutathione S-transferase in a B-lymphocyte cell lines
(Verlengia et al., 2003b).
The permeabilization of the mitochondria results in the
release of cytochrome c and other proapoptotic proteins to
cytosol, which then trigger the process of apoptosis (Dussmann
et al., 2003). Cytochrome c forms a complex with Apaf-1 and
caspase 9; the formation of this apoptosome complex leads to
activation of the cascade of caspases including caspase 3,
executing apoptosis in cells (Mihara et al., 2003). OA activated
caspase 3 (but had no effect on upstream) and mitochondrial-
independent caspases 6 and 8, suggesting that OA activates
apoptosis via changes in mitochondrial function. LA had no
significant effect on caspase activities.
Treatment with OA and LA also led to an increase of neutral
lipid accumulation in human lymphocytes. Accumulation of
excess fatty acids in triacylglycerol pools has been postulated
to divert these molecules from pathways that lead to toxic
effects and may thus serve as buffer against lipotoxicity
(Listenberger et al., 2003). OA promoted higher levels of lipid
accumulation in human lymphocytes in support of previous
studies utilizing Jurkat and Raji cell lines (Cury-Boaventura et
 M.F. Cury-Boaventura et al. / Life Sciences 78 (2006) 1448 – 1456
al., 2004, 2005). Thus, OA may be less toxic due to the higher
rate of its incorporation into neutral lipid stores.
Conclusion
The results presented herein demonstrate that OA and LA
induce apoptosis and necrosis in human lymphocytes. The
mechanism of LA to induced apoptosis involved changes in
mitochondrial transmembrane potential and ROS production.
The mechanism of OA induced apoptosis involved increased
caspase 3 activity. OA induced neutral lipid accumulation may
be cytoprotective. The cytotoxic concentrations of LA and OA
are close to those found in human plasma 49– 216 AM and
108 –282 AM, respectively (Rogiers, 1981; Shimomura et al.,
1986; Puttmann et al., 1993), which suggests that modulation
of lymphocyte cell death by fatty acids may play a role in the
regulation of immune function. The administration of paren-
teral lipid emulsions can raise the plasma levels of free fatty
acids to much higher values. The findings reported herein
support the proposition that OA is less toxic to human
lymphocytes than LA and this should be considered when
selecting appropriate nutritional lipid emulsions for parenteral
nutrition formulation.
Acknowledgments
The authors are indebted to the technical assistance of J.R.
Mendonça, G de Souza, and E.P. Portiolli. This study has been
supported by FAPESP, CNPq and CAPES.
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