Synthesis and assessment of the relative toxicity of the oxidised derivatives of campesterol and dihydrobrassicasterol in U937 and HepG2 cells

Article abstract of DOI:10.1016/j.biochi.2012.04.019

The cytotoxic effects of the oxidised derivatives of the phytosterols, stigmasterol and β-sitosterol, have previously been shown to be similar but less potent than those of the equivalent cholesterol oxides in the U937 cell line. The objective of the present study was to compare the cytotoxic effects of the oxidised derivatives of synthetic mixtures of campesterol and dihydrobrassicasterol in both the U937 and HepG2 cell lines. The parent compounds consisted of a campesterol: dihydrobrassicasterol mix at a ratio of 2:1 (2CMP:1DHB) and a dihydrobrassicasterol:campesterol mix at a ratio of 3:1 (3DHB:1CMP). The 2CMP:1DBH oxides were more cytotoxic in the U937 cells than the 3DBH:1CMP oxides but the difference in cytotoxicity was less marked in the HepG2 cells. The order of toxicity of the individual oxidation products was found to be similar to that previously observed for cholesterol, β-sitosterol and stigmasterol oxidation products in the U937 cell line. There was an increase in apoptotic nuclei in U937 cells incubated with the 7-keto and 7β-OH derivatives of both 2CMP:1DHB and 3DHB:1CMP and also in the presence of 3DHB:1CMP-3β,5α,6β-triol and 2CMP:1DHB-5β,6β-epoxide. An additional oxidation product synthesised from 2CMP:1DHB, 5,6,22,23-diepoxycampestane, was cytotoxic but did not induce apoptosis. These results signify the importance of campesterol oxides in the overall paradigm of phytosterol oxide cytotoxicity.

Full text of DOI:10.1016/j.biochi.2012.04.019

Biochimie 95 (2013) 496e503
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Research paper
Synthesis and assessment of the relative toxicity of the oxidised
derivatives of campesterol and dihydrobrassicasterol in U937
and HepG2 cells
Yvonne O’Callaghan a, Olivia Kennya, Niamh M. O’Connell b, Anita R. Maguire c,
,
Florence O. McCarthy b, Nora M. O’Brien a
*
a School of Food and Nutritional Sciences, University College Cork, Cork, Ireland
b Department of Chemistry, Analytical and Biological Chemistry Research Facility, University College Cork, Cork, Ireland
c Department of Chemistry and School of Pharmacy, Analytical and Biological Chemistry Research Facility, University College Cork, Cork, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
The cytotoxic effects of the oxidised derivatives of the phytosterols, stigmasterol and b-sitosterol, have
Received 19 December 2011
Accepted 18 April 2012
Available online 26 April 2012
previously been shown to be similar but less potent than those of the equivalent cholesterol oxides in the
U937 cell line. The objective of the present study was to compare the cytotoxic effects of the oxidised
derivatives of synthetic mixtures of campesterol and dihydrobrassicasterol in both the U937 and HepG2
cell lines. The parent compounds consisted of a campesterol: dihydrobrassicasterol mix at a ratio of 2:1
Keywords:
Campesterol
Dihydrobrassicasterol
Oxidation product
Cell culture
(2CMP:1DHB) and
a dihydrobrassicasterol:campesterol mix at a ratio of 3:1 (3DHB:1CMP). The
2CMP:1DBH oxides were more cytotoxic in the U937 cells than the 3DBH:1CMP oxides but the difference
in cytotoxicity was less marked in the HepG2 cells. The order of toxicity of the individual oxidation
products was found to be similar to that previously observed for cholesterol,
terol oxidation products in the U937 cell line. There was an increase in apoptotic nuclei in U937 cells
incubated with the 7-keto and 7 -OH derivatives of both 2CMP:1DHB and 3DHB:1CMP and also in the
presence of 3DHB:1CMP-3 ,5 ,6 -triol and 2CMP:1DHB-5 ,6 -epoxide. An additional oxidation product
b-sitosterol and stigmas-
Cytotoxicity
b
b
a
b
b b
synthesised from 2CMP:1DHB, 5,6,22,23-diepoxycampestane, was cytotoxic but did not induce
apoptosis. These results signify the importance of campesterol oxides in the overall paradigm of
phytosterol oxide cytotoxicity.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
exist as either of two epimers, 24
or 24 -methylcholesterol (dihydrobrassicasterol) (Fig. 1). It is esti-
mated that campesterol in terrestrial plants is comprised of
approximately 65% 24 -methylcholesterol and 35% 24 -methyl-
a-methylcholesterol (campesterol)
b
Campesterol is one of the most prevalent phytosterols found in
foods, along with
b
-sitosterol and stigmasterol [1]. Although
b-
a
b
sitosterol is present in the human diet at higher concentrations, it has
been demonstrated that campesterol is more readily absorbed by rat
jejunal villus cells and brush border membranes [2] and is present at
higher concentrations in human serum, both at baseline and also
following dietary supplementation with sterol enriched spreads
[3,4]. In contrast to the heavily promoted health benefits of dietary
phytosterol supplementation, a number of groups have identified
phytosterols with adverse health effects: induction of endothelial
dysfunction and increased size of ischaemic stroke [5]; inhibition of
cell growth [6]; aggressive vascular disease in sitosterolaemic
patients [7]. The methylgroup attheC24 positionofcampesterolmay
cholesterol, however, certain species of phytoplankton have been
found to contain campesterol comprised entirely of one or other of
the two epimers [8]. The epimers are difficult to isolate and studies
investigatingthebiological activityofcampesterolhave notgenerally
distinguished between the two forms quoting instead a total cam-
pesterol content.
Similar to cholesterol, campesterol has a double bond at the C5-
6 position and therefore oxidation of campesterol may occur during
food processing and storage. Grandgirard et al. [9] demonstrated
that campesterol oxides can be absorbed in the intestine of the rat
and the 3b,5a,6b-triol derivative of campesterol has been detected
in the plasma of healthy human subjects [10]. It is also possible that
campesterol may be oxidised in vivo by both enzymatic and non-
enzymatic mechanisms [10]. The cytotoxic effects of cholesterol
oxides have been extensively reported [11] however, data relating
* Corresponding author. Tel.: þ353 21 4902884.
E-mail address: nob@ucc.ie (N.M. O’Brien).
0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2012.04.019

Y. O’Callaghan et al. / Biochimie 95 (2013) 496e503
497
shown to be cell line specific [15] therefore, the toxicity of the POPs
was also investigated in the HepG2, human hepatoma cell line.
Previous studies have demonstrated that the cholesterol oxides are
cytotoxic but do not induce apoptosis in HepG2 cells [21].
2. Materials and methods
2.1. Materials
All chemicals and cell culture reagents were obtained from
Sigma Chemical Co. (Poole, UK) unless otherwise stated. Cell lines
were obtained from the European Collection of Animal Cell
Cultures (Salisbury, UK).
Samples of campesterol enriched 2CMP:1DHB 1c and dihydro-
brassicasterol enriched 3DHB:1CMP 1d were synthesized as per
O’Connell et al. [22] and were subsequently used in the oxidations
below.
Fig. 1. Structures of cholesterol and related phytosterols.
to the biological effects of phytosterol oxides is limited [12].
Previous research conducted in our laboratory has demonstrated
that the oxidised products of b-sitosterol and stigmasterol are toxic
to cells in a similar manner but to a lesser extent than their
cholesterol oxide counterparts in the U937 cell line [13,14].
Phytosterol oxides have also demonstrated cytotoxic effects in
normal cell lines [15]. This may have significant clinical implica-
tions for individuals consuming phytosterol enriched products and
warrants further investigation.
Minor differences in the chemical structure of cholesterol oxides
can significantly impact on their biological effects. Massey and
Pownall [16] found that there was a relationship between the 3-
dimensional structure of cholesterol oxides, their membrane
biophysical properties and ultimately their ability to induce
apoptosis. A recent study conducted by Paillasse et al. [17] found
Description of synthesis of CMP:DHB mixtures.
CMP:DHB mixtures were synthesised in 6 steps starting from
stigmasterol (>95% purity, Sigma Aldrich). Stigmasterol was pro-
tected via tosylate formation, solvolysed with methanol, and cleaved
under ozonolysis conditions to yield an aldehyde. This aldehyde was
converted via Wittig reaction to the required alkene for CMP:DHB
which could then be hydrogenated and deprotected to yield the
CMP:DHB mixtures (2CMP:1DHB and 3DHB:1CMP; both conclu-
sively assigned by 1H and 13C NMR). All the CMP:DHB oxides
described in this manuscript were synthesised from these materials.
that neither cholesterol-5a,6a-epoxide or cholesterol-5b,6b-
epoxide reacted with nucleophiles in non-catalytic conditions
although chemicals bearing epoxide groups are generally potent
2.2. Synthesis of 2CMP:1DHB oxides
alkylating agents. The authors also found that cholesterol-5
epoxide reacted with nucleophiles under catalytic conditions but
cholesterol-5 ,6 -epoxide remained unreactive. Rimner et al. [18]
also examined the relationship between oxysterol structure and
their cytotoxic effects and found that the -isomers of oxysterols
(7 -hydroxycholesterol, 5 ,6 -epoxide) were up to 10-fold more
cytotoxic than their equivalent -iosmers in human arterial endo-
thelial cells. The authors stated that the stereospecific effects of the
oxysterols could have been mediated either at the level of the cell
membrane or through a putative oxysterol receptor. Carvalho et al.
[15] have also reported a relationship between the structure and
biological activity of oxysterols in a variety of both normal and
cancer cell lines.
The objective of the present study was to compare the cytotoxic
effects of a series of oxides synthesised from two parent compounds,
the first parent compound was predominantly campesterol, 2 cam-
pesterol:1 dihydrobrassiacasterol (2CMP:1DHB) 1c and the second
was predominantly dihydrobrassicasterol, 3 dihydrobrassicasterol:1
campesterol (3DHB:1CMP) 1d.The individual oxides of 2CMP:1DHB
and 3DHB:1CMP were subsequently synthesised and characterised
according to the methods previously outlined for stigmasterol oxides
a,6a-
The key oxides targeted in this study are 7-keto 4c and 4d, 7-
b-
OH 5c and 5d, -epoxide 7c and 7d, -epoxide 8c and 8d and triol
9c and 9d to form a comparative study with other phytosterol
oxides (Scheme 1) [19,23].
2CMP:1DHB 1c was converted to its corresponding acetylate 2c
using acetic anhydride and pyridine. Allylic oxidation employing
chromium trioxide forms the acetate of 7-keto-2CMP:1DHB 3c. The
acetate group is cleaved using base-catalysed hydrolysis conditions
furnishing 7-keto-2CMP:1DHB 4c. Synthesis of the other 7-position
b
a
b
b
b
b
b b
a
oxide 7-b-OH 5c was continued via stereoselective reduction of the
carbonyl group. The keto-steroid was reduced using sodium boro-
hydride in the presence of cerium chloride heptahydrate which
promotes attack of hydride from the axial position allowing
formation of the equatorial alcohol functional group [24].
Oxidation was then focused on the alkene bond between
carbons 5 and 6. Stereoselective epoxidation was achieved using
a biphasic system of potassium permanganate and copper sulphate
in order to enhance oxidation on the more hindered
Removal of the acetate group affords -epoxide 7c.
Exposure of 2CMP:1DHB 1c to mCPBA yields the
The peracid approaches from the less hindered side of the double
bond producing a mixture of epimers where the -epoxide
predominates over the
-epoxide in a ratio of 4.2:1 evident from 1H
NMR analysis (dH 2.90, d, , 6-H and dH 3.06, d, , 6-H). These
b-face [25].
b
a
-epoxide 8c.
[19]. The oxides were 2CMP:1DHB-5
,6 -epoxide 7c, 7-Keto-2CMP:1DHB 4c, 7
5c, 2CMP:1DHB -3 ,5 ,6 -triol 9c, 3DHB:1CMP-5
3DHB:1CMP-5 ,6 -epoxide 7d, 7-keto-3DHB:1CMP 4d, 7
3DHB:1CMP 5d and 3DHB:1CMP-3 ,5 ,6 -triol 9d. The oxidised
a,6
a
-epoxide 8c, 2CMP:1DHB-
-Hydroxy-2CMP:1DHB
,6 -epoxide 8d,
-Hydroxy-
a
5b
b
b
b
b
a
b
a a
a
b
b
b
b
isomers were inseparable by chromatography. Completing this
panel of oxides, synthesis of triol 9c was accomplished by acid-
b
a b
derivative of 2CMP:1DHB, 5,6,22,23-Diepoxycampestane (4-
epoxides) 12c was also investigated. Cell viability and apoptotic
nucleiweredeterminedforeachof thephytosteroloxidationproducts
catalysed ring-opening of a-epoxide 8c.
The panel of 2CMP:1DHB oxides were extended to the bisep-
oxide 12c as seen in previous studies [19]. Following deprotection
of 10c [22], excess mCPBA resulted in epoxidation of both double
bonds of 11c on the side chain and in the B ring, forming the
epoxide on both upper and lower faces of the molecule (Scheme 2).
(POP) at 30 mM (12.5 mg/ml), 60 mM (25 mg/ml) and 120 mM (50 mg/ml)
in the U937 human monocytic lymphoma cell line. The U937 cell line
is commonly used as a macrophage reference model in studies
investigating the cytotoxic effects of cholesterol oxidation products
[20] and phytosterol oxidation products [13]. The cytotoxic effects of
cholesterol oxides and phytosterol oxides have previously been
This produced a mixture of diastereomers 5
,6 ,22 ,23 - 12c2, 5 ,6 ,22 ,23 - 12c3 and 5
in a ratio of 3.7:3.7:1:1 deduced from 1H NMR analysis. The 3.7:1
a
b
,6
,6
a
b
,22
,22
b
a
,23
,23
b
- 12c1,
5a
a
a
a
b
b
b
b
a- 12c4

498
Y. O’Callaghan et al. / Biochimie 95 (2013) 496e503
Scheme 1. Synthesis of campesterol and dihydrobrassicasterol oxides (c ¼ 2CMP:1DHB and d ¼ 3DHB:1CMP) (i) Ac2O, pyridine (2c ¼ 98%; 2d ¼ 96%); (ii) CrO3, dimethylpyrazole,
CH2Cl2 ꢂ20 to 5 ꢀC (3c ¼ 44%; 3d ¼ 56%); (iii) K2CO3, MeOH, H2O (4c ¼ 97%; 4d ¼ 95%); (iv) CeCl3.7H2O, NaBH4, MeOH, (5c ¼ 59%; 5d ¼ 45%); (v) KMnO4ꢂCuSO4.5H2O, t-BuOH, H2O,
CH2Cl2 (6c ¼ 32%; 6d ¼ 74%); (vi) Na2CO3, MeOH (7c ¼ 78%; 7d ¼ 77%); (vii) mCPBA, CH2Cl2 (8c ¼ 88%; 8c ¼ 49%); (viii) H2SO4, THFꢂH2O, (9c ¼ 77%; 9d ¼ 56%).
ratio was calculated from dH 2.90 (1H, bd, J 4.5, 6-H, 12c1 and 12c2)
and dH 3.07 (1H, d, J 2.2, 6-H, 12c3 and 12c4). The 1:1 ratio was
calculated from dH 2.76 (1H, dd, J 6.0, one of 23-H,12c3 or 12c4) and
dH 2.82 (1H, dd, J 6.0, one of 23-H, 12c3 or 12c4). The mixtures of
diastereomers could not be separated by column chromatography
(Fig. 4).
2.5. Cell treatment with phytosterol oxides
Cells (U937 or HepG2) were adjusted to a density of 1 ꢁ105cells/
ml in media supplemented with 2.5% (v/v) FBS. HepG2 cells were
allowed to adhere overnight prior to the addition of the oxides. The
oxides were dissolved in ethanol and were added to the cells to
a final concentration of 30, 60 and 120 mM. Equivalent quantities of
ethanol were added to control cells and samples were incubated for
24 h.
2.3. Synthesis of 3DHB:1CMP oxides
The oxidation products were directly produced as above from
a racemic sample of dihydrobrassicasterol and campesterol 1 in
2.6. Cytotoxicity, cell viability and apoptosis
a ratio of 3:1 synthesised previously. The proportion of
epoxides differs slightly from the previous 2CMP:1DHB epoxides as
follows: -epoxide 7d predominates over the -epoxide in a ratio of
7.9:1 evident from 1H NMR analysis (dH 3.06, d,
, 6-H and dH 2.90, d,
, 6-H) and -epoxide 8d predominates over the -epoxide in
a ratio of 8.0:1 evident from 1H NMR analysis (dH 2.90, d,
, 6-H and
dH 3.06, d, , 6-H). In both cases, these isomers were inseparable by
a- and b-
The cytotoxic and apoptotic effects of the oxides were assessed
as previously described in O’Callaghan et al. [14]. Briefly, the
viability of U937 cells was determined by staining the cells using
a solution containing 0.1 mM fluorescein diacetate and 0.02 mM
ethidium bromide (FDA-EtBr) which causes live cells to fluoresce
green and non-viable cells to fluoresce red. Cells were mixed (1:1)
with FDA-EtBr and incubated at 37 ꢀC for 5 min. Two hundred cells
were scored at 200x magnification on a Nikon Optiphot-2 fluo-
rescence microscope and the viable cells were expressed as
a percentage of the total cell count.
b
a
b
a
a
b
a
b
chromatography.
2.4. Cell maintenance
Human monocytic, U937 cells were grown in Royal Park
Memorial Institute (RPMI)-1640 medium supplemented with 10%
(v/v) foetal bovine serum (FBS). The human, hepatoma cell line,
HepG2, was maintained in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% (v/v) FBS and 1% non-essential
amino acids. The cells were grown at 37 ꢀC and 5% (v/v) CO2 in
a humidified incubator. Cells were cultured in the absence of
antibiotics.
U937 cell viability was determined using the MTT cell prolifer-
ation Kit (Roche, Mannheim, Germany). The MTT assay is based on
the cleavage of the yellow tetrazolium salt MTT to purple formazan
by metabolically active cells. Cells were seeded in the wells of a 96
well plate and were exposed to POP for 24 h. MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (10
was added to each of the wells and cells were incubated for 4 h
prior to the addition of 100 l solubilisation solution (10% SDS in
ml)
m
Scheme 2. Synthesis of bisepoxide of 2CMP:1DHB (i) 2.5 M H2SO4, THFꢂH2O (58%); (ii) mCPBA, CH2Cl2, 0 ꢀC (54%).

Y. O’Callaghan et al. / Biochimie 95 (2013) 496e503
499
Table 1
0.01M HCl). The absorbance was determined at 570 nm using
platereader (Spectrafluorplus, Tecan) and the data were
expressed as a percentage of the control.
Percent viable U937 cells following exposure to 3DHB:1CMP or 2CMP:1DHB oxides
at concentrations of 30 M, 60 M or 120 M for 24 h, as determined by the fluo-
rescein diacetate (FDA/EtBr) assay.
a
m
m
m
The Neutral Red Uptake assay was used to test the viability of
HepG2 cells exposed to POP for 24 h. Following incubation, the cells
were washed with KrebseRinger buffer (115 mM NaCl, 4.7 mM KCl,
1.2 mM KH2PO4, 1.2 mM MgSO4e7H2O, 24 mM NaHCO3, 20 mM
Hepes, 1.1 mM glucose, 1.28 mM CaCl2e6H2O) and the neutral red
3DHB:1CMP (d)
mean ꢃ se
2CMP:1DHB (c)
mean ꢃ se
Control
95.3 ꢃ 0.3
87.5 ꢃ 4.3
89.4 ꢃ 3.0
79.2 ꢃ 2.9
84.7 ꢃ 5.9
92.3 ꢃ 1.6
85.8 ꢃ 3.4
90.3 ꢃ 3.1
77.3 ꢃ 5.5
30.2 ꢃ 2.8*
88.5 ꢃ 4.6
69.0 ꢃ 8.8*
38.5 ꢃ 8.5*
62.9 ꢃ 2.2*
34.0 ꢃ 12.0*
11.5 ꢃ 3.5*
97.5 ꢃ 0.4
95.6 ꢃ 0.9
96.0 ꢃ 1.5
88.7 ꢃ 2.0
96.0 ꢃ 0.6
94.6 ꢃ 0.4
62.3 ꢃ 5.4*
95.0 ꢃ 1.2
82.8 ꢃ 3.6
68.8 ꢃ 1.4*
63.0 ꢃ 3.1*
36.2 ꢃ 1.6*
27.3 ꢃ 2.2*
63.2 ꢃ 4.4*
45.5 ꢃ 2.8*
32.6 ꢃ 3.1*
30
60
m
m
M
M
a
a
-epoxide (8)
-epoxide (8)
dye (50 mg/ml KrebseRinger) was added. Cells were then incubated
120
m
M
a
-epoxide (8)
for 2 h at 37 ꢀC and 5% (v/v) CO2. Cells were subsequently lysed by
adding 1% glacial acetic acid and the absorbance was determined at
a wavelength of 540 nm using a platereader (Spectrafluorplus,
Tecan).
30
60
m
m
M
M
b
b
-epoxide (7)
-epoxide (7)
120 mM b-epoxide (7)
30
60
m
m
M 7-keto (4)
M 7-keto (4)
120 mM 7-keto (4)
30
60
m
m
M 7
M 7
b
b
-OH (5)
-OH (5)
2.7. Apoptosis
120 mM 7b-OH (5)
To measure apoptosis, U937 cells were incubated with POP for
24 h. Cells were harvested by centrifugation and the morphology of
30
60
m
m
M 3,5,6-triol (9)
M 3,5,6-triol (9)
120 mM 3,5,6-triol (9)
the cell nuclei was examined following staining with 200
ml
Hoechst 33342 (0.008 mM). Stained samples were placed on
a microscope slide and examined under UV light on the Nikon
Optiphot-2 fluorescence microscope. A total of 300 cells per sample
were counted and the percentage of fragmented and condensed
nuclei was calculated.
The effect of compounds 8d, 8c, 7d, 7c, 4d, 4c, 5d, 5c, 9d and 9c on U937 cell viability
at concentrations of 30 M, 60 M and 120 M was evaluated following a 24 h
m
m
m
incubation by the fluorscein diacetate:ethidium bromide (FDA:EtBr) staining
method. The control represents the percentage viability in untreated cells. U937
cells were mixed 1:1 with FDA:EtBr stain. A total of 200 cells were scored per sample
and the percent viable cells were determined. Data represent the mean of four
independent experiments ꢃ standard error of the mean. *P < 0.05, indicates that the
value obtained for samples treated with either 3DHB:1CMP or 2CMP:1DHB differed
significantly from their respective control, ANOVA followed by Dunnetts.
The DNA fragmentation assay was carried out to confirm the
presence of apoptotic nuclei in the samples. Briefly, 2 ꢁ 106 cells
were harvested. The pellets were lysed and 10 ml RNase A (1 mg/ml)
and 10
ml Proteinase K (20 mg/ml) were added. Samples were
loaded to the wells of a 1.5% (w/v) agarose gel and electrophoresis
was carried out (110 V, 3 h). The gel was visualised under UV light
on a transilluminator following staining with ethidium bromide
and was photographed using the Genegenius Bioimaging system.
cytotoxic at all three concentrations of 7
at the two higher concentrations (60
eOHe3DHB:1CMP 5d following a 24 h incubation. The triol
derivative was the most cytotoxic of all the derivatives as previ-
ously demonstrated for cholesterol, -sitosterol and stigmasterol.
b
eOHe2CMP:1DHB 5c and
mM
and 120 M) of
m
7b
2.8. Statistical analysis
b
The triol derivatives of both 3DHB:1CMP 9d and 2CMP:1DHB 9c
significantly reduced cell viability at all three concentrations. To
summarise, the order of toxicity of the 2CMP:1DHB and
3DHB:1CMP oxides was similar to that previously demonstrated for
All data points are the mean and standard error values of at least
three independent experiments. Data were analysed by ANOVA
followed by Dunnett’s test. The software employed for statistical
analysis was Graphpad Prism, version 4.00 for windows (Graphpad
software, San Diego, California, USA).
cholesterol, b-sitosterol and stigmasterol in the U937 cell line. In all
cases higher concentrations of the phytosterol oxides were
required in order to induce similar levels of toxicity to those
induced by cholesterol oxides. Therefore, the introduction of an
3. Results and discussion
ethyl group as in b-sitosterol and stigmasterol or a methyl group as
3.1. Toxicity of oxides in U937 cells
in campesterol and 22-dihydrobrassicasterol at the C24 position on
the sterol molecule reduced the cytotoxicity of these compounds in
U937 cells compared to their equivalent COPs. It has previously
been established that oxidised phytosterols are approximately
three-fold less cytotoxic than their equivalent cholesterol oxides in
U937 cells [13,26]. Carvalho et al. [15] also found that cholesterol
The FDA-EtBr assay was used to compare the toxicity of the
oxidised derivatives of 2CMP:1DHB 1c and 3DHB:1CMP 1d in the
U937 cell line (Table 1) following a 24 h incubation period. The a-
epoxide derivatives of both 2CMP:1DHB 8c and 3DHB:1CMP 8d
were not cytotoxic in the U937 cell line at the concentrations
oxides were more cytotoxic than 7
lines, however the level of cytotoxicity induced by
eOHesitosterol was almost equal to that of cholesterol oxides in
normal cells derived from the eye (ARPE-19) and the skin (BJ). The
authors suggested that phytosterol oxides may therefore be as
harmful as cholesterol oxides in vivo. In the present study, the
2CMP:1DHB oxides were significantly more cytotoxic (P < 0.05) at
lower concentrations than the 3DHB:1CMP oxides.
The FDA-EtBr staining assay is a membrane integrity assay and
the MTTassay determines cell viability by measuring mitochondrial
activity and can be a more sensitive measure of cytotoxicity. All of
the oxidised derivatives of 2CMP:1DHB 4-9c significantly (P < 0.05)
reduced viability of U937 cells as determined by the MTT assay
(Table 2), the observed reduction in cell viability was dose depen-
dent. U937 cell viability as determined by MTT was less affected by
the addition of the oxides of 3DHB:1CMP 4-9d in comparison with
beOHesitosterol in cancer cell
investigated in the present study. Similarly, the
tives of cholesterol, sitosterol and stigmasterol have been shown
not to be cytotoxic in the U937 cell line [10,11]. -Epoxide deriva-
tives of 3DHB:1CMP 7d were not toxic, following 24 h incubation,
as has previously been shown for the -epoxide derivative of
stigmasterol but cell viability was significantly reduced (P < 0.05)
a-epoxide deriva-
7b
b
b
to 62.3%, at the highest concentration (120
m
M) of 2CMP:1DHB-
b
-
-
epoxide 7c. The -epoxide derivatives of both cholesterol and
b
b
sitosterol have also been shown to cause a significant reduction in
U937 cell viability [13]. The 7-keto derivative of both 3DHB:1CMP
4d and 2CMP:1DHB 4c induced a significant (P < 0.05) reduction in
cell viability at the 120
significantly cytotoxic at the 120
7-keto derivative of stigmasterol was not cytotoxic in the U937 cell
line [14]. The 7 -OH derivative was significantly (P < 0.05)
m
M concentration. 7-Keto-b-sitosterol was
m
M concentration [13] while the
b

500
Y. O’Callaghan et al. / Biochimie 95 (2013) 496e503
Table 2
Table 3
Cell viability in U937 cells following exposure to 3DHB:1CMP or 2CMP:1DHB oxides
Viability in HepG2 cells following exposure to 3DHB:1CMP or 2CMP:1DHB oxides at
concentrations of 30 M, 60 M or 120 M for 24 h expressed as percent control, as
determined by the neutral red uptake assay.
at concentrations of 30
assay.
m
M, 60
mM or 120
m
M for 24 h, as determined by the MTT
m
m
m
3DHB:1CMP (d)
mean ꢃ se
2CMP:1DHB (c)
mean ꢃ se
3DHB:1CMP (d)
mean ꢃ se
2CMP:1DHB(c)
mean ꢃ se
30
60
m
m
M
M
a
a
-epoxide (8)
-epoxide (8)
0.96 ꢃ 0.09
0.93 ꢃ 0.07
0.52 ꢃ 0.05*
0.71 ꢃ 0.07
0.68 ꢃ 0.05*
0.66 ꢃ 0.04*
0.80 ꢃ 0.06
0.60 ꢃ 0.07*
0.12 ꢃ 0.03*
0.92 ꢃ 0.14
0.55 ꢃ 0.08*
0.20 ꢃ 0.04*
0.49 ꢃ 0.11*
0.15 ꢃ 0.03*
0.05 ꢃ 0.01*
0.74 ꢃ 0.03*
0.67 ꢃ 0.05*
0.35 ꢃ 0.06*
0.86 ꢃ 0.04*
0.81 ꢃ 0.04*
0.35 ꢃ 0.01*
0.55 ꢃ 0.02*
0.11 ꢃ 0.00*
0.04 ꢃ 0.01*
0.14 ꢃ 0.07*
0.02 ꢃ 0.02*
0.00 ꢃ 0.01*
0.25 ꢃ 0.04*
0.04 ꢃ 0.02*
0.03 ꢃ 0.02*
30
60
120
30
60
m
m
M
M
a
a
-epoxide (8)
-epoxide (8)
97.1 ꢃ 5.3
99.3 ꢃ 5.8
99.4 ꢃ 4.2
105.8 ꢃ 4.4
100.3 ꢃ 4.2
87.5 ꢃ 4.9
92.7 ꢃ 3.6
82.6 ꢃ 4.3*
65.7 ꢃ 3.4*
94.6 ꢃ 3.0
88.4 ꢃ 1.6
80.7 ꢃ 1.7*
79.2 ꢃ 5.4*
69.3 ꢃ 4.1*
45.0 ꢃ 4.2*
97.9 ꢃ 4.1
90.9 ꢃ 4.0
77.9 ꢃ 1.9*
87.5 ꢃ 2.0
92.0 ꢃ 1.3
89.5 ꢃ 4.8
79.8 ꢃ 4.2*
80.0 ꢃ 10.1*
78.3 ꢃ 4.8*
77.0 ꢃ 3.0*
95.6 ꢃ 4.8
45.8 ꢃ 4.3*
52.3 ꢃ 7.4*
48.3 ꢃ 5.5*
41.1 ꢃ 5.0*
120
m
M
a-epoxide (8)
m
M
a
-epoxide (8)
30
60
m
m
M
M
b
b
-epoxide (7)
-epoxide (7)
m
m
M
M
b
b
-epoxide (7)
-epoxide (7)
120
m
M
b-epoxide (7)
120 mM b-epoxide (7)
30
60
120 mM 7-keto (4)
30
60
120 mM 7b-OH (5)
30
60
30
60
m
m
M 7-keto (4)
M 7-keto (4)
m
M 7-keto (4)
mM 7-keto (4)
120
m
M 7-keto (4)
30
60
m
m
M 7
M 7
b
b
-OH (5)
-OH (5)
m
m
M 7
M 7
b
b
-OH (5)
-OH (5)
120
m
M 7b
-OH (5)
30
60
m
m
M 3,5,6-triol (9)
M 3,5,6-triol (9)
m
M 3,5,6-triol (9)
mM 3,5,6-triol (9)
120
m
M 3,5,6-triol (9)
120 mM 3,5,6-triol (9)
The effect of compounds 8d, 8c, 7d, 7c, 4d, 4c, 5d, 5c, 9d and 9c on U937 cell viability
at concentrations of 30 M, 60 M and 120 M was evaluated following a 24 h
The effect of compounds 8d, 8c, 7d, 7c, 4d, 4c, 5d, 5c, 9d and 9c on HepG2 cell
viability at concentrations of 30 M, 60 M and 120 M was evaluated following
m
m
m
m
m
m
incubation by the MTT assay. Absorbance of the samples was determined at 570 nm
following a 4 h incubation with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide). The results are expressed relative to the control value
(1.00). Data represent the mean of four independent experiments ꢃ standard error
of the mean. *P < 0.05, indicates that the value obtained for samples treated with
either 3DHB:1CMP or 2CMP:1DHB differed significantly from their respective
control, ANOVA followed by Dunnetts.
a 24 h incubation by the neutral red uptake assay. Absorbance of the samples was
determined at 540 nm following a 2 h incubation with neutral red dye. The results
are expressed as a percentage of the control value (100%). Data represent the mean
of four independent experiments ꢃ standard error of the mean. *P < 0.05, indicates
that the value obtained for samples treated with either 3DHB:1CMP or 2CMP:1DHB
differed significantly from their respective control, ANOVA followed by Dunnetts.
Koschutnig et al. [28] found that 7
cytotoxic in HepG2 cells and cell viability was 50% at 120
keto- -sitosterol. Again, the triol derivatives 9c, 9d were the most
cytotoxic of all the derivatives tested, reducing cell viability to less
than 50% at 120 M 3DHB:1CMP-triol 9d and at both the 60 M and
120 M concentrations of 2CMP:1DHB-triol 9c. The difference in
b
eOHe
b
-sitosterol was not
the 2CMP:1DHB oxides. The
8d caused a significant (P < 0.05) reduction in cell viability at the
120 M concentration only, the -epoxide 7d, 7-keto 4d and 7 -OH
5d derivatives significantly (P < 0.05) reduced cell viability at the
two higher concentrations (60 M and 120 M) and the triol 9d
derivative significantly (P < 0.05) reduced cell viability at all
concentrations employed in the present study. The data obtained
by the MTT assay demonstrated that the oxidised derivatives of
2CMP:1DHB 4-9c were more cytotoxic than the oxidised deriva-
tives of 3DHB:1CMP 4-9d in the U937 cell line.
a-epoxide derivative of 3DHB:1CMP
mM 7-
b
m
b
b
m
m
m
m
m
toxicity between the derivatives of 3DHB:1CMP and 2CMP:1DHB
was not as evident in the HepG2 cell line as in the U937 cell line.
3.3. Apoptosis induced by oxides
The ability of a compound to induce apoptosis in cancer cell
lines may be used as an indication of its potential as a chemother-
apeutic agent. Certain novel oxides of stigmasterol (5,6,22,23-
diepoxystigmastone) have previously demonstrated an ability to
induce cell death which was almost exclusively apoptotic at certain
concentrations [14]. One of the objectives of the present study was
to differentiate between the apoptogenicity of the oxidised deriv-
3.2. Toxicity of oxides in HepG2 cells
Studies have demonstrated that the effects of oxysterols are cell
line specific [15,27]. Therefore, we also investigated the toxicity of
the 2CMP:1DHB and 3DHB:1CMP oxides in the HepG2, human
hepatoma cell line, using the neutral red uptake assay. The order of
toxicity of the 2CMP:1DHB and 3DHB:1CMP oxides were found to
be similar in the HepG2 and U937 cell lines (Table 3). However, the
atives of 3DHB:1CMP and 2CMP:1DHB. The
both 3DHB:1CMP 8d and 2CMP:1DHB 8c did not induce apoptosis
(Table 4). The -epoxide derivative of 2CMP:1DHB 7c caused
a significant increase (P < 0.05) in apoptotic nuclei to greater than
7-fold of the untreated control, at the 120 M concentration. There
a-epoxide derivative of
b
-epoxide 7c, 7d derivative was not significantly (P < 0.05) cyto-
toxic in the HepG2 cell line (Table 3). The -epoxide derivative of
2CMP:1DHB 8c was toxic at the 120 M concentration only. Ryan
et al. [13] demonstrated that - and - epoxysitosterol were not
b
a
m
b
m
a
was also a significant decrease in cell viability at this concentration
to 62.3% therefore, apoptotic cell death accounted for approxi-
mately 50% of total cell death. The 7-keto derivatives 4c, 4d also
cytotoxic in the HepG2 cell line. 7-Keto-2CMP:1DHB 4c signifi-
cantly (P < 0.05) reduced cell viability at all three concentrations
(Table 3) however, no dose response was observed as the level of
increased apoptotic nuclei relative to control samples. The 7
derivatives 5c, 5d increased apoptotic nuclei significantly (P < 0.05)
at the 60 M and 120 M concentration of 7 eOHe3DHB:1CMP 5d
and at all three concentrations of 7 eOHe2CMP:1DHB 5c. The triol
b-OH
cytotoxicity did not increase at concentrations above 30
cell viability was significantly (P < 0.05) decreased in the presence
of 60 M and 120 M 7-keto-3DHB:1CMP 4d and a dose response
was observed for this compound. 7 eOHe2CMP:1DHB 5c signifi-
cantly (P < 0.05) decreased HepG2 cell viability at the 30
concentration and to less than 50% of the control level at the
120 M concentration. 7 eOHe3DHB:1CMP 5d was only signifi-
cantly cytotoxic at the highest concentration (120 M) in the
HepG2 cell line. 7-Keto-and 7 eOHe -sitosterol have previously
been shown to be more toxic in HepG2 cells, reducing viability to
7.1% and 4.6% of the control, respectively, at 120 M [13] however,
mM. HepG2
m
m
b
m
m
b
b
derivatives of 2CMP:1DHB 9c did not significantly (P < 0.05)
increase the apoptotic nuclei in U937 cells. It has previously been
observed that the predominant mode of cell death induced by the
mM
m
b
triol derivative of b-sitosterol is necrosis [13]. The ability of the
m
oxides to induce cell death by apoptosis was confirmed by the
appearance of a ladder like pattern, representing cleavage of the
DNA to fragments of 200 base pairs, in an agarose gel (Figs. 2 and 3).
b
b
m
The oxidised derivatives of
b-sitosterol have previously

Y. O’Callaghan et al. / Biochimie 95 (2013) 496e503
501
Table 4
Apoptotic nuclei in U937 cells following exposure to 3DHB:1CMP or 2CMP:1DHB
oxides at concentrations of 30
staining with Hoechst 33342.
m
M, 60
m
M or 120
m
M for 24 h, as determined by
3DHB:1CMP (d)
mean ꢃ se
2CMP:1DHB (c)
mean ꢃ se
Control
1.00 ꢃ 0.08
1.48 ꢃ 0.20
1.85 ꢃ 0.30
2.33 ꢃ 0.16
1.54 ꢃ 0.40
1.65 ꢃ 0.22
1.93 ꢃ 0.30
2.12 ꢃ 0.37
3.47 ꢃ 0.58*
3.53 ꢃ 0.42*
1.97 ꢃ 0.34
2.62 ꢃ 0.24*
3.15 ꢃ 0.36*
2.05 ꢃ 0.12
2.65 ꢃ 0.50*
3.55 ꢃ 0.12*
1.00 ꢃ 0.08
0.86 ꢃ 0.32
1.59 ꢃ 0.23
2.77 ꢃ 0.45
1.50 ꢃ 0.27
2.23 ꢃ 0.27
7.23 ꢃ 0.91*
3.04 ꢃ 0.36
3.95 ꢃ 0.64
4.86 ꢃ 1.73*
5.86 ꢃ 2.00*
6.54 ꢃ 1.86*
6.82 ꢃ 0.18*
2.91 ꢃ 0.36
4.45 ꢃ 0.27
3.23 ꢃ 0.73
30
60
m
m
M
M
a
a
-epoxide (8)
-epoxide (8)
120
m
M
a
-epoxide (8)
30
60
m
m
M
M
b
b
-epoxide (7)
-epoxide (7)
120 mM b-epoxide (7)
30
60
m
m
M 7-keto (4)
M 7-keto (4)
120 mM 7-keto (4)
30
60
m
m
M 7
M 7
b
b
-OH (5)
-OH (5)
120 mM 7b-OH (5)
30
60
m
m
M 3,5,6-triol (9)
M 3,5,6-triol (9)
120 mM 3,5,6-triol (9)
The effect of compounds 8d, 8c, 7d, 7c, 4d, 4c, 5d, 5c, 9d and 9c on the quantity of
apoptotic nuclei in U937 cells exposed to 30 M, 60 M and 120 M of the
m
m
m
compounds for 24 h was evaluated. U937 cells were harvested and nuclei were
stained with Hoechst 33342. A total of 300 nuclei were scored per sample and the
percent condensed/fragmented nuclei was determined. Data are expressed as fold
increase in condensed/fragmented nuclei relative to control cells. The control cells
represent untreated cells and were 2.2% apoptotic. Data represent the mean of four
independent experiments ꢃ standard error of the mean. *P < 0.05, indicates that the
value obtained for samples treated with either 3DHB:1CMP or 2CMP:1DHB differed
significantly from their respective control, ANOVA followed by Dunnetts.
Fig. 3. DNA fragmentation: U937 cells were incubated with 60
m
M campesterol oxides
for 24 h. Cells were harvested, lysed and incubated with proteinase K and RNAse A.
Samples were loaded to the wells of an agarose gel and a current of 110 V was applied
for 3 h. Apoptotic cells form a ladder like pattern representative of the cleavage of DNA
to 200 base pair units which is the hallmark of apoptosis. Lanes: MW: molecular
weight marker (200 bp); Control: untreated cells,
(8c); 2CMP:1DHB-5 ,6 -epoxide (7c), 7-K: 7-Keto-2CMP:1DHB (4c), 7-OH:
Hydroxy-2CMP:1DHB 5c, Triol: 2CMP:1DHB -3 ,5 ,6 -triol (9c).
a-E: 2CMP:1DHB-5a,6a-epoxide
b
b
7b-
b
a b
demonstrated higher levels of apoptotic cell death [13] than the
oxides investigated in the present study. Four of the five oxides
investigated induced apoptosis, the highest level of apoptosis was
a 20 fold increase in apoptotic nuclei, relative to the control sample.
7b
-OH Stigmasterol was the only one of the five oxides (
a-epoxide,
found in U937 cells exposed to 7-keto-b-sitosterol which caused
b
-epoxide, 7-keto, 7 -OH, triol) of stigmasterol which induced
b
apoptosis in U937 cells causing a 10-fold increase at 60 mM [14].
3.4. Cytotoxic effects of 5,6,22,23-diepoxycampestane
The bisepoxide derivative of 2CMP:1DHB (5,6,22,23-
diepoxycampestane 12c) was found to the most cytotoxic of all
the compounds investigated in the present study (Table 5) reducing
cell viability to 11.7% at the 30
presence of an additional epoxide group on the side chain greatly
increased the toxicity of the compound in comparison to the
epoxide, -epoxide derivatives of campesterol. However, the level
mM concentration therefore, the
a
-
b
of apoptosis was low and it must be concluded that cell death
induced by this compound was largely necrotic. A similar derivative
of stigmasterol (5,6,22,23-diepoxystigmastane) was less cytotoxic
than 5,6,22,23-diepoxycampestane but induced cell death almost
exclusively by apoptosis at the 30
The concentrations of phytosterol oxide employed in the
present study (30 M, 60 M and 120 M) were selected to allow
a comparison with cholesterol oxides which have typically been
investigated at concentrations ranging from 10 M to 150 mM
mM concentration [14].
m
m
m
m
[19,20,27]. In general to induce a similar level of toxicity the
concentrations of phytosterol oxides required were approximately
three-fold higher than their cholesterol oxide counterpart. The
difference in the cytotoxicity of the various phytosterol and
cholesterol oxides (a-epoxide, b-epoxide, 7-keto, 7b-OH, triol) may
be caused by differences in their uptake and metabolism by cells.
Few studies have investigated the uptake of phytosterol oxides in
Fig. 2. DNA fragmentation: U937 cells were incubated with 60
mM dihydro-
brassicasterol oxides for 24 h. Cells were harvested, lysed and incubated with
proteinase K and RNAse A. Samples were loaded to the wells of an agarose gel and
a current of 110 V was applied for 3 h. Apoptotic cells form a ladder like pattern
representative of the cleavage of DNA to 200 base pair units which is the hallmark of
cells, however it has been found that cholesterol-5,6
a-epoxide,
apoptosis. Lanes: MW: molecular weight marker (200 bp); Control: untreated cells,
E: 3DHB:1CMP-5 ,6 -epoxide (8d); -E: 3DHB:1CMP-5 ,6 -epoxide (7d), 7-K: 7-keto-
-Hydroxy-3DHB:1CMP (5d), Triol: DHB:1CMP-3 ,5 ,6
a-
cholesterol-5,6 -epoxide and ,5 ,6 -cholestane-triol are all
b
3b a b
a
a
b
b b
taken up at a similar rate by rabbit aortic endothelial cells [29].
Cholesterol oxides with similar structures have demonstrated
3DHB:1CMP (4d), 7-OH: 7
triol (9d).
b
b a b-

502
Y. O’Callaghan et al. / Biochimie 95 (2013) 496e503
Fig. 4. Structures of the mixture of bisepoxides of 2CMP:1DHB in 12c
different metabolisms [17]. Cholesterol-5,6
modulator of Liver-X-Receptors (LXR) [30] and bis 5,6
didiepoxycholesterol is a selective LXR modulator [31]. The 3
sulphated form of cholesterol-5,6 -epoxide is an antagonist of LXR
[32] whereas the 3 sulphated form of cholesterol-5,6 -epoxide is
inactive. Both cholesterol-5,6 -epoxide and cholesterol-5,6
epoxide can be converted to 3 ,5 ,6 -cholestane triol by action of
a
-epoxide is a direct
4. Conclusion
a-24(S),25-
a
b
-
Elucidation of the potential detrimental health effects of
phytosterol oxidation products has hitherto been hampered by the
lack of standard compounds. The present study which investigates
the cytotoxic effects of campesterol oxides and dihydro-
brassicasterol oxides in U937 and HepG2 cells adds to previous
a
b
b
a
b-
b
a b
the enzyme cholesterol epoxide hydrolase (ChEH) which may be
active in tumour cells [33]. ChEH has demonstrated a preference to
studies which reported the cytotoxic effects of b-sitosterol oxides
and stigmasterol oxides [13,14]. Cholesterol oxides have greater
cytotoxicity than phytosterol oxides in both the U937 and HepG2
cell lines. In relation to the phytosterol oxides, the oxidised deriv-
hydrate Cholesterol-5,6
[34] which may account for the greater cytotoxicity of cholesterol-
5,6 -epoxide in U937 cells. To date, there has been no investigation
b-epoxide over cholesterol-5,6a-epoxide
b
atives of b-sitosterol were the most cytotoxic and the most
into the interaction of ChEH and phytosterol oxides.
apoptotic, followed by the campesterol (67%) oxides, stigmasterol
oxides and dihydrobrassicasterol (75%) oxides, although it is
possible by enhancing the purity of campesterol, the toxicity of the
oxides may be increased. These results signify the importance of
campesterol oxides in the overall paradigm of phytosterol oxide
cytotoxicity.
Compounds which induce apoptosis can provide targeted anti-
cancer therapies as the apoptotic program is altered in tumour cells
[35] therefore phytosterol oxides such as 5,6,22,23-diepoxyst
igmastane may have the potential to be developed as chemothera-
peutic agents. Cholesterol oxides have demonstrated selective toxicity
to cancer cells and are relatively non-toxic in normal cells, however
certain phytosterol oxides have demonstrated cytotoxic effects in
normal cells [15] therefore it requires further study to elucidate the
potential benefits of these compounds as anticancer therapies.
Acknowledgements
Funding for this research was provided under the National
Development Plan, through the Food Institutional Research
Measure, administered by the Department of Agriculture, Food and
Fisheries, Ireland.
Table 5
Cytotoxicity in U937 and HepG2 cells and apoptotic nuclei in U937 cells following
exposure to 5,6,22,23-Diepoxycampestane (Bisepoxide 12c) at concentrations of
30
m
M, 60
m
M or 120
mM for 24 h.
Appendix A. Supplementary material
Viability
(U937 cells)
Viability
Apoptosis
(HepG2 cells)
mean ꢃ se
(U937 cells)
mean ꢃ se
mean ꢃ se
Supplementary material related to this article can be found
online at doi:10.1016/j.biochi.2012.04.019.
30
60
m
m
M Bisepoxide (12c)
M Bisepoxide (12c)
11.7 ꢃ 5.2*
1.3 ꢃ 1.8*
0.0 ꢃ 0.0*
48.4 ꢃ 8.2*
0.0 ꢃ 5.6*
0.0 ꢃ 5.4*
4.45 ꢃ 0.91*
0.59 ꢃ 0.27
0.41 ꢃ 0.14
120 mM Bisepoxide (12c)
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m
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