Oxidized derivatives of dihydrobrassicasterol: Cytotoxic and apoptotic potential in U937 and HepG2 cells

Article abstract of DOI:10.1021/jf204737e

The ability of phytosterol compounds to reduce plasma serum cholesterol levels in humans is well investigated. However, phytosterols are structurally similar to cholesterol with a double bond at the C_5-6 position and are therefore susceptible to oxidation. Much research has been carried out on the biological effects of cholesterol oxidation products (COPs) in vitro. In contrast, there is less known about phytosterol oxidation products (POPs). From previous studies, it is apparent that oxidized derivatives of the phytosterols, β-sitosterol and stigmasterol, are cytotoxic in vitro but are less potent than their COP counterparts. In the present study, the cytotoxic and apoptotic potential of oxidized derivatives of dihydrobrassicasterol (DHB) including 5α,6α-epoxyergostan-3β-ol (α-epoxide), 5β,6β-epoxyergostan-3β-ol (β-epoxide), ergost-5-en-7-on- 3β-ol (7-keto), ergost-5-ene-3β,7β-diol (7-β-OH), and ergostane-3β,5α,6β-triol (triol) were evaluated in the U937 and HepG2 cell lines. In general, 7-keto, 7-β-OH, and triol derivatives had a significant cytotoxic impact on U937 and HepG2 cells. The oxides appear to be more toxic toward U937 cells. In line with previous findings, the POPs investigated in this study were less potent than the equivalent COPs. The results add to the body of data on the toxicity of individual POPs.

Full text of DOI:10.1021/jf204737e

Article
pubs.acs.org/JAFC
Oxidized Derivatives of Dihydrobrassicasterol: Cytotoxic and
Apoptotic Potential in U937 and HepG2 Cells
Olivia Kenny,† Yvonne O’Callaghan,† Niamh M. O’Connell,‡ Florence O. McCarthy,‡
Anita R. Maguire,‡,§ and Nora M. O’Brien*,†
‡
†School of Food and Nutritional Sciences, Department of Chemistry, Analytical and Biological Chemistry Research Facility, and
§School of Pharmacy, Analytical and Biological Chemistry Research Facility, University College Cork, Cork, Ireland
ABSTRACT: The ability of phytosterol compounds to reduce plasma serum cholesterol levels in humans is well investigated.
However, phytosterols are structurally similar to cholesterol with a double bond at the C5−6 position and are therefore susceptible
to oxidation. Much research has been carried out on the biological effects of cholesterol oxidation products (COPs) in vitro. In
contrast, there is less known about phytosterol oxidation products (POPs). From previous studies, it is apparent that oxidized
derivatives of the phytosterols, β-sitosterol and stigmasterol, are cytotoxic in vitro but are less potent than their COP
counterparts. In the present study, the cytotoxic and apoptotic potential of oxidized derivatives of dihydrobrassicasterol (DHB)
including 5α,6α-epoxyergostan-3β-ol (α-epoxide), 5β,6β-epoxyergostan-3β-ol (β-epoxide), ergost-5-en-7-on-3β-ol (7-keto),
ergost-5-ene-3β,7β-diol (7-β-OH), and ergostane-3β,5α,6β-triol (triol) were evaluated in the U937 and HepG2 cell lines. In
general, 7-keto, 7-β-OH, and triol derivatives had a significant cytotoxic impact on U937 and HepG2 cells. The oxides appear to
be more toxic toward U937 cells. In line with previous findings, the POPs investigated in this study were less potent than the
equivalent COPs. The results add to the body of data on the toxicity of individual POPs.
KEYWORDS: U937, HepG2, dihydrobrassicasterol oxidation products, cytotoxicity, apoptosis
abundant followed by the epoxide derivatives. To date, it has
been difficult to separate DHB from campesterol and therefore
accurately quantify DHB oxides. Thus, the contribution of
DHB oxides to the diet is unclear but is anticipated to be low in
comparison to POPs generated from the major phytosterol, β-
sitosterol. However, the potential effects of DHB oxides may be
of more significance than β-sitosterol oxides as they are closer
in structure to the parent cholesterol oxides.
In addition to dietary intake, POPs in plasma may be derived
from phytosterols penetrating the skin via cosmetic products
and subsequently oxidized by UV light.7 The majority of
research on the biological effects of sterol oxidation products
has focused on COPs.8−10 In relation to POPs, proposed
biological effects include modulation of cholesterol metabolism
via liver X receptor (LXR), lipid lowering and antidiabetic
properties, modulation of oxidation capacity of cytochrome
P450, cytotoxicity in vitro and in vivo, modulation of
inflammation and immunity, and estrogenic and/or androgenic
activity.11 However, definitive information on the potential
toxic effects of POPs is scarce mainly due to the lack of
commercial availability of the individual oxide standards.
Existing knowledge on the cytotoxicity of POPs in vitro
generally pertains to blends of POPs rather than pure
compounds. There is some evidence that mixes of sterol
oxidation compounds act in a different way to a single purified
oxide compound.12
INTRODUCTION
■
Plant sterols or phytosterols are present in the unsaponifiable
matter of vegetable oils and fats. Phytosterols have been
incorporated into a range of cholesterol-lowering products, and
inclusion of these products in the diet has been documented to
reduce serum cholesterol levels in human clinical trials.1 For
this reason their addition to foods has increased significantly
over the past decade. Phytosterols have an unsaturated ring
structure similar to cholesterol (Figure 1). Due to the presence
of a double bond between C5 and C6, sterols can undergo
oxidative processes. Therefore, cholesterol and phytosterols
present in foods are susceptible to oxidation especially in those
foods which have been exposed to heat treatments in the
presence of oxygen or have been stored for long periods
subjected to sunlight and oxygen. Oxidation causes the
formation of cholesterol oxidation products (COPs) and
phytosterol oxidation products (POPs).2 It has been reported
that POPs are present in phytosterol-enriched products at
concentrations of approximately 0.1% (12−68 μg/g).3,4 The
recommended daily intake of phytosterol-enriched spreads is
four servings of 12 g each which could potentially equate to a
POP intake of 0.6−3.4 mg.
The content and profile of phytosterols/stanols added to
foods is determined by the ingredient source.5 In tall oil
products, β-sitosterol predominates, followed by sitostanol and
campesterol. In soybean products, β-sitosterol, stigmasterol,
and campesterol are the major components. Vegetable oils
contain mainly β-sitosterol but also contain sitostanol and
campesterol. Garcia-Llatas et al. have reviewed studies
investigating POPs in plant sterol-enriched food, and it is
evident that β-sitosterol oxides predominate.6 In general, 7-keto
and 7-OH oxide derivatives of all phytosterols are the most
Received: November 21, 2011
Revised: May 18, 2012
Accepted: May 18, 2012
Published: May 18, 2012
© 2012 American Chemical Society
5952
dx.doi.org/10.1021/jf204737e | J. Agric. Food Chem. 2012, 60, 5952−5961

Journal of Agricultural and Food Chemistry
Article
Figure 1. Structures of cholesterol and common phytosterols.
Figure 2. Structures of the DHB oxides investigated in the present study. (1) DHB; (2) DHB acetate; (3) ergost-5-en-7-on-3β-ol acetate, 7-keto
acetate; (4) ergost-5-en-7-on-3β-ol, 7-keto; (5) ergost-5-ene-3β,7β-diol, 7-β-OH; (6) 5β,6β-epoxyergostan-3β-ol acetate, β-epoxide acetate; (7)
5β,6β-epoxyergostan-3β-ol, β-epoxide; (8) 5α,6α-epoxyergostan-3β-ol, α-epoxide; (9) ergostane-3β,5α,6β-triol, triol. (i) Ac2O, pyridine (87%); (ii)
CrO3, dimethylpyrazole, CH2Cl2 −20 to 5 °C (43%); (iii) K2CO3, MeOH, H2O (95%); (iv) CeCl3·7H2O, NaBH4, MeOH, (71%); (v) KMnO4−
CuSO4·5H2O, t-BuOH, H2O, CH2Cl2 (50%); (vi) Na2CO3, MeOH (83%); (vii) mCPBA, CH2Cl2 (89%); (viii) H2SO4, THF−H2O, (74%).
Thus, in order to obtain a more comprehensive insight into
the cytotoxicity of POPs, synthesis and evaluation of single
oxides is paramount. To date, studies on the effects of
individual POPs have involved those derived from β-sitosterol
and stigmasterol.12−17 In general, the oxides of β-sitosterol were
similar to the equivalent COPs, however higher concentrations
of POPs were required to elicit comparable toxic effects.6,14−16
In relation to stigmasterol oxides, the 7-β-OH, epoxydiol, and
diepoxide derivatives were cytotoxic to the U937 cell line and
all three induce significant apoptotic cell death.17 In fact, the
diepoxide and epoxydiol derivatives of stigmasterol, which are
oxidized on the side chain, were found to be the most cytotoxic
of all the derivatives tested in the U937 cell line and are also
significantly cytotoxic in HepG2 and Caco-2 cells.17,18
Stigmasterol differs structurally from cholesterol and β-
sitosterol, in that it contains an additional double bond,
positioned at C22−23. The presence of the additional double
bond allows for the formation of oxides such as diepoxide and
epoxydiol, compounds that do not have cholesterol or β-
sitosterol equivalents.17 Given these novel findings, it is of
interest to examine other individual oxides derived from
different phytosterols.
In the present study, we report on the synthesis and toxicity
of a range of individual oxides of DHB (1) (Figure 2). DHB
(1) is a 24-epimer of campesterol. At the C24 position, the
methyl group is on the alpha-face in DHB and the beta-face in
campesterol (Figure 1). DHB occurs naturally in specific
Chlorella species19 and can be biosynthesized from ergosterol.20
The significance of this key phytosterol becomes clear when
one considers that DHB and campesterol are usually quoted as
a “campesterol fraction” in analysis of phytosterols in foods
(primarily due to the fact that the analysis is done by GC or 1H
NMR and these isomers are only separated by chiral GC or 13C
NMR) and that this campesterol fraction is made up of 2:1
5953
dx.doi.org/10.1021/jf204737e | J. Agric. Food Chem. 2012, 60, 5952−5961

Journal of Agricultural and Food Chemistry
Article
campesterol:DHB.21 Therefore, DHB content of phytosterol
sources can be as high as 10%. The significance of the novel
DHB oxides targeted in this study can be related to their
potential to answer a fundamental question at the heart of
dietary phytosterol supplementation. Once these benchmark
compounds have been fully characterized via biological
evaluation, key toxic POPs can be identified and hence avoided
by assiduous use of phytosterol mixtures in formulation or
storage regimens.
(multiplet). Thin layer chromatography (TLC) was carried out on
precoated silica gel plates (Merck 60 PF254). Visualization was
achieved by UV light (254 nm), vanillin or potassium permanganate
staining. Column chromatography was carried out using Kieselgel 60,
0.040−0.063 nm (Merck). Specific rotations were recorded on a
Perkin-Elmer 341 polarimeter at 20 °C in the solvents indicated. The
sodium D line (589 nm) was used. Abbreviations used: mCPBA, 3-
chloroperbenzoic acid; 4-DMAP, 4-(dimethylamino)pyridine; DMSO,
dimethyl sulfoxide.
DHB Acetate (2). Acetic anhydride (1.037 mL, 11.00 mM) and
pyridine (0.885 mL, 11.00 mM) were combined, and DHB (1) (2.200
g, 5.50 mM) in CH2Cl2 (50 mL) was added slowly via addition funnel.
The reaction mixture was refluxed for 24 h. The reaction mixture was
stirred with a saturated aqueous solution of NaHCO3 (40 mL) for 30
min. The organic layer was extracted with CH2Cl2 (3 × 30 mL). The
combined organic layers were washed with 2 M HCl (2 × 30 mL),
water (30 mL), and brine (10 mL), dried, filtered, and concentrated
under reduced pressure to give a white solid. Purification by flash
chromatography on silica gel [hexane:ethyl acetate (85:15)] yielded
the title compound (2) as a white solid (2.119 g, 87%): mp 135−137
°C; [α]2D0 −55.45° (c 1.000 in CHCl3); νmax(KBr)/cm−1 2961, 2938,
2905, 2822, 1731, 1466, 1441, 1367; δH (300 MHz, CDCl3) 0.68 (3H,
s, 18-CH3), 0.77−2.00 [40H, m containing 0.77−0.93 (12H,
overlapping 4 × d, J 6.8, 21-CH3, 26-CH3, 27-CH3 and 28-CH3),
1.02 (3H, s, 19-CH3)], 2.03 (3H, s, COCH3), 2.32 (2H, bd, J 7.8),
4.55−4.66 (1H, m, 3α-H), 5.37 (1H, bd, J 4.2, 6-H); δC (75 MHz,
CDCl3) 11.9 (CH3), 15.5 (CH3), 17.6 (CH3), 18.9 (CH3), 19.3
(CH3), 20.5 (CH3), 21.1 (CH2), 21.4 (CH3), 24.3 (CH2), 27.8 (CH2),
28.2 (CH2), 30.6 (CH2), 31.5 (CH), 31.9 (CH), 31.92 (CH2), 33.7
(CH2), 36.2 (CH), 36.6 (quaternary C), 37.0 (CH2), 38.1 (CH2), 39.1
(CH), 39.7 (CH2), 42.3 (quaternary C), 50.1 (CH), 56.0 (CH), 56.7
(CH), 74.0 (CH), 122.7 (CH), 139.7 (quaternary C), 170.5 (CO);
m/z (ESI+) 383 [M + H − AcOH]+ (20%), 149 (50), 116 (100);
HRMS calc for C28H47 [M + H − AcOH]+ 383.3678, found 383.3676.
The following peaks were distinguishable for the minor campesterol
component at a level <5%: 15.4 (CH3), 18.3 (CH3), 18.7 (CH3), 20.2
(CH3), 30.3 (CH2), 32.4 (CH), 35.9 (CH), 38.9 (CH), 56.1 (CH).
Ergost-5-en-7-on-3β-ol Acetate (7-Keto Acetate) (3). Chro-
mium trioxide (4.751 g, 47.55 mM) was suspended in dry
dichloromethane (150 mL) and stirred for 30 min at −25 °C.
Dimethylpyrazole (4.571 g, 47.55 mM) was added in one portion and
the reaction mixture stirred for 30 min at −20 °C. DHB acetate (2)
(1.400 g, 3.17 mM) in CH2Cl2 (50 mL) was added and the mixture
stirred at −20 °C allowing it to warm to 5 °C over 2.5 h. Ethyl acetate
(500 mL) was then added and the brown suspension filtered through
Celite. The filtrate was concentrated under reduced pressure to give a
brown residue. This residue was purified by chromatography on silica
gel using hexane−ethyl acetate (95:5), yielding 7-keto acetate (3)
(0.620 g, 43%) as a white solid: mp 179−181 °C; [α]2D0 −105.40° (c
1.000 in CHCl3); νmax(KBr)/cm−1 2957, 2871, 1729, 1672, 1466,
1376; δH (300 MHz, CDCl3) 0.68 (3H, s, 18-CH3), 0.77−2.03 [36H,
m, containing 0.77−0.94 (12H, overlapping 4 × d, J 6.8, 21-CH3, 26-
CH3, 27-CH3 and 28-CH3), 1.21 (3H, s, 19-CH3)], 2.05 (3H, s,
COCH3), 2.23 (1H, t, J 10.6), 2.35−2.59 (3H, m), 4.66−4.77 (1H, m,
3α-H), 5.70 (1H, bd, J 1.5, 6-H); δC (75 MHz, CDCl3) 12.0 (CH3),
15.5 (CH3), 17.3 (CH3), 17.6 (CH3), 19.1 (CH3), 20.5 (CH3), 21.2
(CH2), 21.3 (CH3), 26.3 (CH2), 27.4 (CH2), 28.5 (CH2), 30.6 (CH2),
31.5 (CH), 33.7 (CH2), 36.0 (CH2), 36.1 (CH), 37.8 (CH2), 38.3
(quaternary C), 38.7 (CH2), 39.1 (CH), 43.1 (quaternary C), 45.4
(CH), 49.8 (CH), 49.9 (CH), 54.6 (CH), 72.2 (CH), 126.7 (CH),
163.8 (quaternary C), 170.3 (CO), 201.9 (CO); m/z (ESI+) 457
[M + H]+ (3%), 397 (35), 149 (40), 116 (100); HRMS calc for
C30H48O3 [M + H]+ 457.3682, found 457.3693. The following peaks
were distinguishable for the minor campesterol component at a level
<5%: 15.4 (CH3), 18.3 (CH3), 18.9 (CH3), 20.2 (CH3), 28.6 (CH2),
30.3 (CH2), 32.4 (CH), 35.8 (CH), 38.8 (CH), 54.8 (CH).
Thus, the objective of this study was to synthesize DHB and
characterize a range of oxidized derivatives of DHB including
5α,6α-epoxyergostan-3β-ol (α-epoxide, 8), 5β,6β-epoxyergo-
stan-3β-ol (β-epoxide, 7), ergost-5-en-7-on-3β-ol (7-keto, 4),
ergost-5-ene-3β,7β-diol (7-β-OH, 5), and ergostane-3β,5α,6β-
triol (triol, 9) (Figure 2). The U937 cell line is commonly used
as a macrophage reference model in studies investigating the
cytotoxic effects of COPs and POPs.15,22 Previous studies have
demonstrated that the COPs are cytotoxic but do not induce
apoptosis in HepG2 cells.23 Thus, in this study the cytotoxicity
and apoptotic potential of each oxidized derivative was assessed
in the U937, monocytic blood cell line and the HepG2, hepatic
cell line.
MATERIALS AND METHODS
Materials. Cell lines were obtained from the European Collection
of Animal Cell Cultures (Sailsbury, U.K.).
■
Chemicals. All chemicals were obtained from Sigma Chemical Co.
(Poole, U.K.) unless otherwise stated. Information on the purity of
COPs was obtained from Sigma
Cell Maintenance. Human monocytic U937 cells were grown in
suspension in RPMI-1640 medium supplemented with 10% (v/v) fetal
bovine serum (FBS). Human hepatoma HepG2 cells were maintained
in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v)
FBS and 1% nonessential 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. Exponentially growing cells were used in all
experiments.
Description of Synthesis of DHB (1). Synthetic DHB was
obtained in >95% purity (with the remaining <5% being campesterol)
via a recently described method.24 Briefly, DHB (1) was synthesized in
6 steps starting from stigmasterol (>95% purity). Stigmasterol was
protected via tosylate formation, solvolyzed with methanol, and
cleaved under ozonolysis conditions to yield an aldehyde. This
aldehyde was converted via Wittig reaction to the required alkene for
DHB, which could then be hydrogenated and deprotected to yield
DHB in >95% purity (as assigned by 1H and 13C NMR). All the DHB
oxides described in this manuscript were synthesized from this
material.
Synthesis of POPs: General Procedures. Melting points were
measured on a Uni-Melt Thomas-Hoover capillary melting point
apparatus and are uncorrected. Low resolution mass spectra were
recorded on a Waters Micromass Quattro Micro mass spectrometer
(instrument number QAA1202) in electrospray ionization (ESI)
positive and negative modes and a Waters Micromass LCT Premier
(instrument number KD160) was used for high resolution
acquisitions. Infrared (IR) spectra were recorded as potassium
bromide (KBr) disks on a Perkin-Elmer FT-IR Paragon 1000 or a
1
Spectrum One FT-IR spectrophotometer. H NMR (300 MHz) and
13C NMR (75 MHz) were recorded on a Bruker Avance 300 NMR
spectrometer unless otherwise stated. All spectra were recorded at 20
°C in deuterated chloroform (CDCl3) with tetramethylsilane (TMS)
as an internal standard unless otherwise stated. Chemical shifts (δH
and δC) are reported in parts per million (ppm), relative to TMS, and
coupling constants are expressed in hertz (Hz). Splitting patterns in
1H NMR spectra are designated as s (singlet), br s (broad singlet), d
(doublet), dd (doublet of doublets), ddd (doublet of doublet of
doublets), dt (doublet of triplets), t (triplet), q (quartet), and m
Ergost-5-en-7-on-3β-ol (7-Keto) (4). A suspension of 7-keto
acetate (3) (0.550 g, 1.21 mM) in methanol (75 mL) was stirred at rt
for 5 min. Potassium carbonate (0.183 g, 1.33 mM) in water (15 mL)
was added to the suspension, and the mixture was stirred at rt for 24 h.
5954
dx.doi.org/10.1021/jf204737e | J. Agric. Food Chem. 2012, 60, 5952−5961

Journal of Agricultural and Food Chemistry
Article
The reaction mixture was then partitioned between water (200 mL)
and ethyl acetate (100 mL). The organic layer was washed with water
(2 × 200 mL) and saturated aqueous sodium chloride (2 × 200 mL)
and dried over magnesium sulfate, and the solution was then
concentrated under reduced pressure to yield title compound (4) as
a white solid (0.476 g, 95%): mp 152−154 °C; [α]2D0 −96.15° (c 1.000
in CHCl3); νmax(KBr)/cm−1 3526, 3338, 2955, 2869, 1673, 1655,
1462, 1375; δH (300 MHz, CDCl3) 0.68 (3H, s, 18-CH3), 0.77−2.05
[37H, m, containing 0.77−0.94 (12H, overlapping 4 × d, J 6.8, 21-
CH3, 26-CH3, 27-CH3 and 28-CH3), 1.20 (3H, s, 19-CH3)], 2.24 (1H,
t, J 11.2), 2.36−2.54 (3H, m), 3.62−3.71 (1H, m, 3α-H), 5.69 (1H, s,
6-H); δC (75 MHz, CDCl3) 12.0 (CH3), 15.5 (CH3), 17.3 (CH3), 17.6
(CH3), 19.1 (CH3), 20.5 (CH3), 21.2 (CH2), 26.3 (CH2), 28.5 (CH2),
30.6 (CH2), 31.1 (CH2), 31.5 (CH), 33.8 (CH2), 36.1 (CH), 36.4
(CH2), 38.3 (quaternary C), 38.7 (CH2), 39.1 (CH), 41.8 (CH2), 43.1
(quaternary C), 45.4 (CH), 49.9 (2 × CH), 54.6 (CH), 70.4 (CH),
126.0 (CH), 165.6 (quaternary C), 202.6 (CO); m/z (ESI+) 415
[M + H]+ (60%), 149 (40), 116 (100); HRMS calc for C28H47O2 [M
+ H]+ 415.3576, found 415.3575. The following peaks were
distinguishable for the minor campesterol component at a level
<5%: 15.4 (CH3), 18.3 (CH3), 18.9 (CH3), 20.2 (CH3), 28.6 (CH2),
30.3 (CH2), 32.4 (CH), 35.8 (CH), 38.8 (CH), 54.8 (CH).
Ergost-5-ene-3β,7β-diol (7-β-OH) (5). A suspension of 7-keto
(4) (0.340 g, 0.82 mM) and cerium chloride heptahydrate (0.459 g,
1.23 mM) in methanol (15 mL) was stirred at rt for 10 min. Sodium
borohydride (0.034 g, 0.90 mM) was added to the suspension and the
mixture stirred at rt for 24 h. The reaction was worked up by
partitioning between water (50 mL) and ethyl acetate (50 mL). The
organic layer was washed with water (2 × 20 mL) and saturated
aqueous sodium chloride (2 × 20 mL) and dried over magnesium
sulfate, and the solution was then concentrated under reduced
pressure to yield the crude product as a white solid. This was purified
by chromatography on silica gel eluting with 60% ethyl acetate−
hexane, yielding a white solid (5) (0.244 g, 71%): mp 164−166 °C;
[α]D20 −10.20° (c 1.000 in CHCl3); νmax(KBr)/cm−1 3381, 2957, 2869,
1670, 1465, 1383; δH (300 MHz, CDCl3) 0.69 (3H, s, 18-CH3), 0.77−
1.63 [35H, m, containing 0.77−0.94 (12H, overlapping 4 × d, J 6.8,
21-CH3, 26-CH3, 27-CH3 and 28-CH3), 1.05 (3H, s, 19-CH3)], 1.76−
1.93 (4H, m), 2.02 (1H, dt, J 12.6, 3.3), 2.20−2.36 (2H, m), 3.48−
3.59 (1H, m, 3α-H), 3.84 (1H, d, J 7.8, 7-H), 5.28 (1H, s, 6-H); δC (75
MHz, CDCl3) 11.9 (CH3), 15.5 (CH3), 17.6 (CH3), 19.0 (CH3), 19.2
(CH3), 20.6 (CH3), 21.1 (CH2), 26.4 (CH2), 28.5 (CH2), 30.6 (CH2),
31.47 (CH), 31.53 (CH2), 33.8 (CH2), 36.2 (CH), 36.5 (quaternary
C), 37.0 (CH2), 39.1 (CH), 39.6 (CH2), 40.9 (CH), 41.7 (CH2), 42.9
(quaternary C), 48.3 (CH), 55.3 (CH), 56.0 (CH), 71.4 (CH), 73.3
(CH), 125.5 (CH), 143.5 (quaternary C); m/z (ESI+) 399 [M + H −
H2O]+ (10%), 381 (70), 149 (20), 116 (100); HRMS calc for
C28H47O [M + H − H2O]+ 399.3627, found 399.3610. The following
peaks were distinguishable for the minor campesterol component at a
level <5%: 15.3 (CH3), 18.2 (CH3), 18.7 (CH3), 20.2 (CH3), 30.2
(CH2), 32.4 (CH), 35.8 (CH), 38.8 (CH), 55.4 (CH).
(12H, overlapping 4 × d, J 6.8, 21-CH3, 26-CH3, 27-CH3 and 28-
CH3), 1.00 (3H, s, 19-CH3), 2.03 (3H, s, COCH3)], 3.07 (1H, bd, J
2.1, 6-H), 4.72−4.82 (1H, m, 3α-H); δC (75 MHz, CDCl3) 11.8
(CH3), 15.5 (CH3), 17.0 (CH3), 17.6 (CH3), 18.9 (CH3), 20.5 (CH3),
21.3 (CH3), 21.9 (CH2), 24.2 (CH2), 27.2 (CH2), 28.1 (CH2), 29.7
(CH), 30.5 (CH2), 31.5 (CH), 32.5 (CH2), 33.7 (CH2), 35.0
(quaternary C), 36.1 (CH), 36.7 (CH2), 38.0 (CH2), 39.1 (CH), 39.8
(CH2), 42.3 (quaternary C), 51.0 (CH), 56.0 (CH), 56.2 (CH), 62.5
(quaternary C), 63.6 (CH), 71.4 (CH), 170.5 (CO); m/z (ESI+)
459 [M + H]+ (8%), 441 (10), 399 (8), 149 (40), 116 (100); HRMS
calc for C30H51O3 [M + H]+ 459.3838, found 459.3820. The following
peaks were distinguishable for the minor campesterol component at a
level <5%: 18.3 (CH3), 18.7 (CH3), 20.2 (CH3), 30.3 (CH2), 32.1
(CH2), 35.8 (CH), 38.8 (CH).
5β,6β-Epoxyergostan-3β-ol (β-Epoxide) (7). A suspension of
the β-epoxide acetate (6) (0.250 g, 0.55 mM) in methanol (50 mL)
was stirred at rt for 5 min. Sodium carbonate (0.116 g, 1.09 mM) was
added, and the mixture was stirred at rt for 16 h. The reaction mixture
was then concentrated under reduced pressure to yield the crude
product as a white solid. This product was purified by chromatography
on silica gel using hexane−ethyl acetate (90:10) to give the epoxide
(7) as a white solid (0.190 g, 83%). 1H NMR analysis (δH 3.06, d, β, 6-
H and δH 2.90, d, α, 6-H) showed this to be a mixture of the β- and α-
epoxides in a ratio of 6.1:1 which could not be separated by
chromatography: mp 132−134 °C; [α]2D0 −7.10° (c 1.000 in CHCl3);
νmax(KBr)/cm−1 3436, 2957, 2869, 1466, 1377; δH (300 MHz, CDCl3)
0.60−2.11 [46H, m, containing 0.61 (3H, s, 18-CH3), 0.76−0.90
(12H, overlapping 4 × d, J 6.8, 21-CH3, 26-CH3, 27-CH3 and 28-
CH3), 0.99 (3H, s, 19-CH3)], 3.06 (1H, bd, J 2.1, 6-H), 3.63−3.73
(1H, m, 3α-H); δC (75 MHz, CDCl3) 11.8 (CH3), 15.5 (CH3), 17.1
(CH3), 17.6 (CH3), 18.9 (CH3), 20.5 (CH3), 22.0 (CH2), 24.2 (CH2),
28.1 (CH2), 29.8 (CH), 30.6 (CH2), 31.0 (CH2), 31.4 (CH), 32.6
(CH2), 33.7 (CH2), 34.9 (quaternary C), 36.1 (CH), 37.3 (CH2), 39.0
(CH), 39.8 (CH2), 42.2 (CH2), 42.3 (quaternary C), 51.3 (CH), 56.0
(CH), 56.2 (CH), 63.0 (quaternary C), 63.8 (CH), 69.3 (CH); m/z
(ESI+) 417 [M + H]+ (6%), 399 (46), 381 (20), 149 (20), 116 (100);
HRMS calc for C28H49O2 [M + H]+ 417.3733, found 417.3730. The
following peaks were distinguishable for the minor campesterol
component at a level <5%: 18.3 (CH3), 18.7 (CH3), 20.2 (CH3), 35.8
(CH), 38.8 (CH).
5α,6α-Epoxyergostan-3β-ol (α-Epoxide) (8). A solution of
mCPBA (70%, 0.518 g, 2.10 mM) in dichloromethane (30 mL) was
added dropwise to a stirred solution of ice-cold DHB (1) (0.700 g,
1.75 mM) in dichloromethane (90 mL). The resulting mixture was
stirred at rt for 2 h. The reaction mixture was then washed with 10%
aqueous sodium hydrogen sulfite solution (2 × 50 mL), 5% aqueous
sodium thiosulfate solution (2 × 50 mL), saturated aqueous sodium
bicarbonate (2 × 100 mL), and aqueous sodium chloride (2 × 150
mL). The dichloromethane extracts were then dried over magnesium
sulfate and concentrated under reduced pressure to yield the crude
product as a white solid. This product was purified by chromatography
on silica gel using hexane−ethyl acetate (80:20) to give the epoxide
(8) as a white solid (0.648 g, 89%). 1H NMR analysis (δH 2.90, d, α, 6-
H and δH 3.06, d, β, 6-H) showed this to be a mixture of the α- and β-
epoxides in a ratio of 6.6:1 which could not be separated by
chromatography: mp 144−146 °C; [α]2D0 −41.10° (c 1.000 in CHCl3);
νmax(KBr)/cm−1 3435, 2931, 2870 1631, 1467, 1377; δH (300 MHz,
CDCl3) 0.61 (3H, s, 18-CH3), 0.64−2.11 [43H, m, containing 0.76−
0.90 (12H, overlapping 4 × d, J 6.8, 21-CH3, 26-CH3, 27-CH3 and 28-
CH3), 1.05 (3H, s, 19-CH3)], 2.90 (1H, bd, J 4.2, 6-H), 3.84−3.93
(1H, m, 3α-H); δC (75 MHz, CDCl3) 11.8 (CH3), 15.4 (CH3), 15.9
(CH3), 17.5 (CH3), 18.8 (CH3), 20.5 (CH3), 20.6 (CH2), 24.0 (CH2),
28.0 (CH2), 28.7 (CH2), 29.8 (CH), 30.5 (CH2), 30.9 (CH2), 31.4
(CH), 32.4 (CH2), 33.6 (CH2), 34.8 (quaternary C), 36.1 (CH), 39.0
(CH), 39.3 (CH2), 39.7 (CH2), 42.3 (quaternary C), 42.5 (CH), 55.7
(CH), 56.8 (CH), 59.3 (CH), 65.9 (quaternary C), 68.4 (CH); m/z
(ESI+) 417 [M + H]+ (6%), 400 (10), 399 (30), 149 (40), 116 (100);
HRMS calc for C28H49O2 [M + H]+ 417.3733, found 417.3743. The
following peaks were distinguishable for the minor campesterol
5β,6β-Epoxyergostan-3β-ol Acetate (β-Epoxide Acetate) (6).
Copper sulfate pentahydrate (3.164 g, 12.67 mM) and potassium
permanganate (7.489 g, 47.40 mM) were ground together into a fine
powder with a mortar and pestle to which water (1.4 mL) was added.
The resulting paste was transferred to a flask containing DHB acetate
(2) (0.700 g, 1.58 mM) in dichloromethane (20 mL). t-Butanol (0.84
mL) was added, and the reaction mixture was refluxed for 15 min
before cooling to rt. The reaction mixture was then stirred for a further
16 h at rt. The reaction mixture was then filtered through a silica gel
plug column eluting with dichloromethane. The product rich layer was
then dried over magnesium sulfate and concentrated under reduced
pressure to give a white solid. Purification by flash chromatography on
silica gel [hexane:ethyl acetate (92.5:7.5)] yielded the title compound
(6) as a white solid (0.360 g, 50%). 1H NMR analysis (δH 3.07, d, β, 6-
H and δH 2.89, d, α, 6-H) showed this to be a mixture of the β- and α-
epoxides in a ratio of 5.3:1 which could not be separated by
chromatography: mp 130−132 °C; [α]2D0 −13.75° (c 1.000 in CHCl3);
νmax(KBr)/cm−1 2957, 2869, 1731, 1468, 1376; δH (300 MHz, CDCl3)
0.61−2.15 [48H, m, containing 0.64 (3H, s, 18-CH3), 0.76−0.91
5955
dx.doi.org/10.1021/jf204737e | J. Agric. Food Chem. 2012, 60, 5952−5961

Journal of Agricultural and Food Chemistry
Article
component at a level <5%: 18.2 (CH3), 18.6 (CH3), 20.2 (CH3), 30.2
(CH2), 35.8 (CH), 38.7 (CH).
of viable uninjured cells. The medium was removed, and cells were
washed with Krebs buffer. The cells were subsequently treated with 1
mL of 1% glacial acetic acid/50% ethanol to burst open the cells and
release the dye. After agitation for a few minutes, the absorbance of
each sample was read on a plate reader at a wavelength of 540 nm.
Morphological Analysis of Cell Nuclei. Nuclear morphology of
control, COPs-treated, and POPs-treated cells was assessed by
fluorescence microscopy after staining with Hoechst 33342. The
Hoechst method was conducted in U937 cells only. Approximately 4
× 105 cells were centrifuged at 2000 rpm for 10 min to form a pellet.
Hoechst 33342 stain (5 μg/mL) was added, and the samples were
incubated at 37 °C and 5% (v/v) CO2 for 30 min. Stained samples
were placed on a microscope slide and examined under UV light
(330−380 nm) using a Nikon fluorescence microscope (400×
magnification). A total of 300 cells per sample were investigated,
and the percentage of fragmented and condensed nuclei was
calculated. Apoptotic cells were characterized by nuclear condensation
of chromatin, nuclear fragmentation, and blebbing of the nucleus.
DNA Fragmentation. Detection of small DNA fragments was
conducted as previously described.26 DNA fragmentation was carried
out in U937 cells only. Briefly, 2 × 106 cells were harvested, the pellets
were lysed, RNase A (0.25 mg/mL) was added, and the samples were
incubated at 50 °C for 1 h. Proteinase K (5 mg/mL) was added, and
the samples were incubated again at 50 °C for a further 1 h before
being loaded into the wells of a 1.5% (w/v) agarose gel. A 100−1500
bp DNA standard (Promega; Medical Supply Co. Ltd., Dublin,
Ireland) was used to assess the DNA fragmentation. Electrophoresis
was carried out on agarose gel prepared in TBE buffer (0.45 M Tris,
0.45 M boric acid and 2 mM EDTA, pH 8) at 3 V/cm. DNA was
visualized under UV light on a transilluminator (312 nm) after
ethidium bromide staining and photographed using an image analysis
system.
Caspase-3 and Caspase-7 Activity. The caspase-Glo 3/7 assay
(Promega, Ireland) is a homogeneous, luminescent assay that
measures total activity of both caspase-3 and caspase-7. These
members of the cysteine aspartic acid-specific protease (caspase)
family play key effector roles in apoptosis in mammalian cells. The
assay provides a luminogenic caspase-3/7 substrate, which results in
cell lysis, followed by caspase cleavage of the substrate and generation
of a “glow-type” luminescent signal, produced by luciferase.
Luminescence is proportional to the amount of caspase activity
present. Briefly, U937 and HepG2 cells were seeded in the wells of a
96 well plate (HepG2 cells were allowed to adhere for 24 h) and
exposed to COPs and POPs for 24 h. The Caspase-Glo reagent was
added to the cells at a volume of 1:1, and the cells were incubated for a
further 3 h. The luminescence of the samples was measured on a
Tecan Spectrafluor Plus plate reader, and the data were expressed as
fold increase relative to an untreated control sample.
Ergostane-3β,5α,6β-triol (Triol) (9). To a solution of α-epoxide
(8) (α:β, 6.6:1, 0.470 g, 1.16 mM) in acetone (30 mL) and water (6
mL) was added 20 drops of concentrated sulfuric acid. The reaction
mixture was stirred at rt for 2.5 h and was then concentrated. Ethyl
acetate (30 mL) was added to the residue, which was then washed
with water (2 × 30 mL) and aqueous sodium chloride (30 mL) and
dried over magnesium sulfate. The solvent was then evaporated under
reduced pressure to yield the crude product as a white solid. This
product was purified by chromatography on silica gel using hexane−
ethyl acetate (30:70) to give the triol (9) as a white solid (0.363 g,
74%): mp 248−250 °C; [α]2D0 −4.9° (c 1.000 in MeOH/H2O);
νmax(KBr)/cm−1 3433, 2956, 2870, 1466, 1384; δH (300 MHz,
DMSO-d6) 0.62 (3H, s, 18-CH3), 0.75- 1.92 [42H, m containing
0.74−0.89 (12H, m, 21-CH3, 26-CH3, 27-CH3 and 28-CH3), 1.02
(3H, s, 19-CH3)], 3.30 (1H, s, OH), 3.63 (1H, s, 6-H), 3.75−3.85
(1H, m, 3α-H), 4.17 (1H, d, J 5.7, OH), 4.39 (1H, d, J 4.2, OH); δC
(150 MHz, CDCl3) 11.9 (CH3), 15.4 (CH3), 16.3 (CH3), 17.5 (CH3),
18.7 (CH3), 20.4 (CH3), 20.7 (CH2), 23.9 (CH2), 27.8 (CH2), 30.0
(CH), 30.1 (CH2), 30.8 (CH), 31.1 (CH2), 32.0 (CH2), 33.2 (CH2),
34.5 (CH2), 35.7 (CH), 37.8 (quaternary C), 38.4 (CH), 39.8 (CH2),
40.9 (CH2), 42.2 (quaternary C), 44.5 (CH), 55.6 (CH), 55.8 (CH),
65.8 (CH), 74.1 (CH), 74.3 (quaternary C); m/z (ESI+) 399 [M + H
− 2H2O]+ 10%, 381 (4), 149 (40), 116 (100); HRMS calc for
C28H47O [M + H − 2H2O]+ 399.3627, found 399.3646. The following
peaks were distinguishable for the minor campesterol component at a
level <5%: 15.3 (CH3), 18.1 (CH3), 18.5 (CH3), 20.0 (CH3), 29.8
(CH2), 31.8 (CH), 33.1 (CH2), 35.3 (CH), 38.1 (CH).
Cell Treatment with Phytosterol Oxides. U937 cells or HepG2
cells at a density of 2 × 105 cells/mL were supplemented with reduced
serum media (2.5% (v/v) FBS), before compound addition. DHB
oxide derivatives were dissolved in ethanol and added to the cells at a
final concentration of 30, 60, and 120 μM. For comparison, cells were
also incubated with 30 μM of all available COPs, cholesterol-5α,6α-
epoxide (α-epoxide), cholesterol-5β,6β-epoxide (β-epoxide), 7-keto-
cholesterol (7-keto), and 7-β-OH-cholesterol (7-β-OH). Each
compound was dissolved in ethanol, and the final concentration in
media did not exceed 0.4% (v/v). Equivalent quantities of ethanol
were added to control cells, and samples were incubated for 24 h at 37
°C and 5% (v/v) CO2.
Cell Viability Using MTT Assay. U937 cells were seeded in the
wells of a 96 well plate and were exposed to COPs and POPs for 24 h.
According to the method previously outlined by Phelan et al.,25 10 μL
of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide] was added to each of the samples and they were incubated
for a further 4 h prior to the addition of 100 μL of solubilization
solution (10% SDS in 0.01 M HCl). The plates were returned to the
incubator overnight. The absorbance of each of the samples was
determined at 570 nm with a reference wavelength of 690 nm using a
Tecan Spectrafluor Plus plate reader. Data are expressed as percentage
viability relative to the control ethanol treated sample.
Cell Viability Using FDA/EtBr Assay or Neutral Red Uptake
Assay. The viability of U937 cells was assessed after 24 h treatment
with POPs and COPs by the fluorescein diacetate/ethidium bromide
assay. Briefly, cells were mixed 1:1 (v/v) with a solution of fluorescein
diacetate and ethidium bromide and incubated at 37 °C for 5 min
before being layered onto a microscope slide. Under these conditions,
viable cells fluoresce green, while nonviable cells fluoresce red. Samples
were examined at 200× magnification on a Nikon fluorescence
microscope using blue light (450−490 nm). Two hundred cells were
scored for each condition, and cell viability was expressed as the
percentage of viable (green) cells.
Statistical Analysis. All data points are the mean and standard
error values of at least three independent experiments. Data were
analyzed by ANOVA followed by Dunnett’s test. The software
employed for statistical analysis was Prism (Hearne Scientific Software,
Dublin, Ireland).
RESULTS AND DISCUSSION
■
The benchmark compounds synthesized in this study are the 7-
keto (4), 7-β-OH (5), β-epoxide (7), α-epoxide (8), and triol
(9) of DHB (Figure 2) to form a comparative study of
phytosterol oxides with previous studies.27,28 The oxidation
products were produced directly from a sample of DHB (1) of
>95% purity synthesized previously.24 The various oxidation
products formed center around the B-ring at the 5-, 6-, and 7-
positions in line with previous studies. DHB (1) was converted
to its corresponding acetate (2) using standard acetylation
conditions. Allylic oxidation employing chromium trioxide
forms the acetate of 7-keto DHB (3). The acetate group is
consequently cleaved using base-catalyzed hydrolysis conditions
furnishing 7-keto DHB (4). The 7-keto steroid (4) was
The viability of HepG2 cells was measured after 24 h using the
Neutral Red uptake (NRU) assay. The NRU assay is a more suitable
method for measuring cell viability in this adherent cell line. After 24 h
incubation with the test compounds, medium was removed and the
cells were washed with Krebs buffer solution. Neutral Red dye (50 μg/
mL) was added to each well of the 24 well plates and incubated for 2 h
at 37 °C and 5% (v/v) CO2 to allow uptake of the dye into lysosomes
5956
dx.doi.org/10.1021/jf204737e | J. Agric. Food Chem. 2012, 60, 5952−5961

Journal of Agricultural and Food Chemistry
Article
stereoselectively reduced using sodium borohydride in the
presence of cerium chloride heptahydrate. It has been reported
that attack of a cerium ion on the carbonyl oxygen forms a
cerium−steroid complex promoting attack of borohydride at
the axial position allowing formation of the equatorial alcohol
functional group.29
Table 1. Cell Viability in U937 Cells Following Exposure for
24 h to 30, 60, or 120 μM DHB Oxides (95% Purity)
a
b
cell viability
compound
mean
SE
Cholesterol Oxides
61.6**
58.4**
64.1**
49.4**
Phytosterol Oxides
103.7
Stereoselective epoxidation was achieved using a biphasic
system of potassium permanganate and copper sulfate in order
to enhance oxidation on the more hindered β-face.30 Removal
of the acetate group affords β-epoxide (7). The epoxidation
produced a mixture of epimers inseparable by chromatography
where the β-epoxide predominates over the α-epoxide in a ratio
of 6.1:1 evident from 1H NMR analysis (δH 3.06, d, β 6-H and
δH 2.90, d, α 6-H). Exposure of DHB (1) to mCPBA yields the
α-epoxide (8). The peracid approaches from the less hindered
side of the double bond and produces a mixture of epimers
where the α-epoxide predominates over the β-epoxide in a ratio
of 6.6:1 evident from 1H NMR analysis (δH 2.90, d, α, 6-H and
δH 3.06, d, β, 6-H). To complete the panel of oxides, synthesis
of triol (9) was accomplished. Acid-catalyzed ring-opening of α-
epoxide (8) provided a direct route to the desired triol (9).
Once the panel of DHB oxides had been constructed, we
proceeded to biological evaluation of these significant pure
POPs.
COPs were added to U937 cells at a concentration of 30 μM,
and POPs were added at concentrations of 30 μM, 60 μM, and
120 μM. The concentration of COPs in human plasma has
been shown to be as high as 37 μM following the ingestion of a
test meal of spray-dried powdered eggs.31 The concentrations
of POPs employed in the present study were selected to allow a
comparison with COPs which have typically been investigated
at concentrations ranging from 10 μM to 150 μM.22,32 In
general, to induce a similar level of toxicity the concentrations
of POPs required were approximately 3-fold higher than their
COP counterparts.
30 μM α-epoxide
30 μM β-epoxide
30 μM 7-keto
2.5
9.9
9.3
30 μM 7-β-OH
10.4
30 μM α-epoxide
60 μM α-epoxide
120 μM α-epoxide
30 μM β-epoxide
60 μM β-epoxide
120 μM β-epoxide
30 μM 7-keto
3.3
8.5
3.3
4.4
3.7
2.0
3.5
8.1
7.3
14.6
8.6
6.0
13.2
5.2
1.7
92.2
94.9
81.2
72.9*
48. 9**
95.6
60 μM 7-keto
76.2
120 μM 7-keto
30 μM 7-β-OH
60 μM 7-β-OH
120 μM 7-β-OH
30 μM triol
40.4**
117.1
39.3**
19.8**
44.1**
19.8**
8.2**
60 μM triol
120 μM triol
a
The derivatives investigated were α-epoxide (8), β-epoxide (7), 7-
keto (4), 7-β-OH (5), and triol (9). Cells were also treated with 30
μM of the corresponding cholesterol oxide derivatives. All compounds
were dissolved in ethanol, and equivalent quantities of ethanol
b
(EtOH) were added to control cells. Viability was assessed using the
MTT assay, percentage value relative to ethanol treated cells. Values
are means with their standard error of at least three independent
experiments. Means values were significantly different from the control
ethanol group (ANOVA followed by the Dunnett’s test): * P < 0.05,
** P < 0.01.
When compared to control ethanol treated cells, the COPs
investigated (30 μM of α-epoxide, β-epoxide, 7-keto, and 7-β-
OH) significantly reduced U937 viability as assessed by the
MTT assay (Table 1). When each of the five oxidized
derivatives of DHB were added to cells, β-epoxide at 60 and
120 μM (7), 7-keto at 120 μM (4), 7-β-OH at 60 and 120 μM
(5), and all three concentrations of triol oxides (9) significantly
reduced viability relative to control cells (Table 1). In an earlier
study, a similar trend was observed for oxides of stigmasterol in
U937 cells, whereby the α-epoxide and β-epoxide were mildly
cytotoxic, 7-keto showed a higher degree of toxicity, while triol
(22R,23R) and 7-β-OH were the most cytotoxic.17 In relation
to oxides of β-sitosterol, 7-α-OH sitosterol was the most
cytotoxic to HepG2 cells as assessed by the MTT assay
followed by 7-keto, while 7-β-OH was mildly cytotoxic.16 The
authors suggest that toxicity of POPs is cell line specific as has
previously been observed for COPs.23 In U937 cells, three of
the four COPs tested had a significant impact on cell viability
(β-epoxide, 7-keto, and 7-β-OH; Table 2) assessed using the
FDA/EtBr assay. These results are in line with previous
reported data.15,23,26 Ryan et al. also assessed 3β,5α,6β-
cholestane-triol (cholesterol triol) at 30 μM and found U937
cell viability decreased to approximately 13% using the FDA-
EtBr assay and viability decreased to approximately 11% in
HepG2 cells when assessed by the NRU assay.15 When the
oxides of DHB were investigated, 7-keto at 120 μM (4), 7-β-
OH at 60 and 120 μM (5), and triol derivatives at all
concentrations (dose-dependent) (9) caused significant cell
death (P < 0.01) with the FDA-EtBr assay (Table 2). Similar
findings were reported for the 7-β-OH and triol derivatives of
β-sitosterol and stigmasterol. However, the 7-keto derivative of
β-sitosterol, but not stigmasterol, decreased U937 cell
viability.15,17 In the present study, the α- and β-epoxide
derivatives of DHB (8, 7) did not reduce cell viability in either
cell line (Table 2). Previously, we reported the noncytotoxic
effects in U937 cells of α- and β-epoxide derivatives of
stigmasterol and β-sitosterol.15,17
U937 cell viability values were lower with the MTT assay
(Table 1) when compared to the FDA-EtBr assay (Table 2).
The FDA-EtBr staining assay is a membrane integrity assay, and
the MTT assay determines cell viability by measuring
mitochondrial activity and can be a more sensitive measure of
cytotoxicity.17 Therefore, although a similar trend was observed
for both viability assays, in that the relative toxicities of each of
the compounds were similar, there was a more evident decrease
in cell viability as measured by MTT. Cell viability following 24
h exposure to COPs and POPs was assessed in the HepG2 cell
line using the NRU assay (Table 2). The NRU assay has
previously been employed to assess viability in adherent cell
lines.15 Thus, we carried out this assay in HepG2 cell lines to
allow us to compare our viability results after exposure to DHB
oxides to previously published data investigating oxides
generated from other phytosterol sources. Neutral Red is a
weak, cationic, water-soluble dye that is only taken up by viable
cells. In this study, the β-epoxide derivative of cholesterol
5957
dx.doi.org/10.1021/jf204737e | J. Agric. Food Chem. 2012, 60, 5952−5961

Journal of Agricultural and Food Chemistry
Article
and β-epoxide were 10-fold more cytotoxic than their α-isomers
in human arterial endothelial cells.34 The difference in the
cytotoxicity of the various COPs and POPs may be caused by
differences in their uptake and metabolism by cells. There are
limited studies investigating the uptake of POPs in cells,
however it has been found that α-epoxide, β-epoxide, and triol
oxides of cholesterol are all taken up at a similar rate by rabbit
aortic endothelial cells.35 COPs with similar structures have
demonstrated different metabolisms:36 α-epoxide is a direct
modulator of Liver-X-Receptors (LXR),37 and bis-5,6α-24-
(S),25-epoxycholesterol is a selective LXRα modulator.38
Additionally, the 3β-sulfated form of α-epoxide cholesterol is
an antagonist of LXR, whereas the β-epoxide equivalent is
inactive.39
The morphology of U937 cells was examined under UV light
following staining with Hoechst 33342 to help identify
apoptotic cells. Nuclei, which were condensed or fragmented,
were identified as apoptotic cells. Additionally, cells experienc-
ing bulging of the membrane or “blebbing” were also defined as
apoptotic. In control cells, apoptosis was approximately 11%
(Table 3). This level appears high for control cells, but we
included cells undergoing “blebbing” in our assessment of
apoptosis. To confirm apoptosis in U937 cells, we also
Table 2. Percentage of Viable Cells Following Exposure for
24 h to 30, 60, or 120 μM of DHB Oxides (95% Purity)
a
b
cell viability
U937 cells
mean
95.3
HepG2 cells
mean
compound
SE
0.3
SE
0.0
control (EtOH)
100.0
Cholesterol Oxides
30 μM α-epoxide
30 μM β-epoxide
30 μM 7-keto
89.1
2.4
2.8
1.7
2.9
95.8
3.6
4.8
77.6**
77.7**
78.9**
73.3**
95.5
4.4
30 μM 7-β-OH
85.4
10.9
Phytosterol Oxides
30 μM α-epoxide
60 μM α-epoxide
120 μM α-epoxide
30 μM β-epoxide
60 μM β-epoxide
120 μM β-epoxide
30 μM 7-keto
90.1
1.1
3.7
2.6
2.9
4.0
2.3
3.7
3.8
5.7
4.0
7.7
1.7
2.8
7.2
1.9
100.1
98.2
2.0
2.6
3.1
3.3
3.9
2.4
2.8
2.9
2.8
3.6
3.7
3.4
3.6
2.8
2.1
86.5
89.4
103.6
100.3
99.9
93.0
89.5
85.7
97.1
89.5
93.7
60 μM 7-keto
86.9
93.4
120 μM 7-keto
30 μM 7-β-OH
60 μM 7-β-OH
120 μM 7-β-OH
30 μM triol
44.3**
85.5
89.5
93.8
64.6**
34.0**
72.7**
44.2**
7.0**
88.8
81.7**
75.9**
63.4**
45.6**
Table 3. Percentage of Condensed and Fragmented Nuclei
in U937 Cells Following Exposure for 24 h to 30, 60, or 120
a
60 μM triol
μM DHB Oxides (95% Purity)
120 μM triol
a
The derivatives investigated were α-epoxide (8), β-epoxide (7), 7-
b
% apoptosis
keto (4), 7-β-OH (5), and triol (9). Cells were also treated with 30
μM of the corresponding cholesterol oxide derivatives. All compounds
were dissolved in ethanol, and equivalent quantities of ethanol
(EtOH) were added to control cells. Viability of the U937 cells was
assessed by the fluorescein diacetate/ethidium bromide assay; viability
of the HepG2 cells was assessed via the Neutral Red uptake assay.
compound
mean
SE
1.0
control (EtOH)
11.3
Cholesterol Oxides
30 μM α-epoxide
30 μM β-epoxide
30 μM 7-keto
21.3*
28.1**
32.5**
27.5**
1.5
3.2
2.7
2.6
b
Values are means with their standard error of at least three
independent experiments. Means values were significantly different
from the control ethanol group (ANOVA followed by the Dunnett’s
test): * P < 0.05, ** P < 0.01.
30 μM 7-β-OH
Phytosterol Oxides
18.6
30 μM α-epoxide
60 μM α-epoxide
120 μM α-epoxide
30 μM β-epoxide
60 μM β-epoxide
120 μM β-epoxide
30 μM 7-keto
1.7
1.6
2.6
4.2
1.3
2.9
4.2
3.1
5.4
2.0
1.9
4.4
3.7
1.2
5.1
18.8
21.7
significantly reduced cell viability to 73%. None of the other
cholesterol derivatives significantly reduced HepG2 cell
viability. In an earlier study, O’Callaghan et al. demonstrated
that 7-β-OH cholesterol is cytotoxic to HepG2 cells.26 In the
present study, the 7-β-OH cholesterol did decrease cell viability
(Table 2) but it did not reach statistical significance. Regarding
the POPs, 7-β-OH derivatives of DHB at 120 μM (5) and all
concentrations of triol (9) significantly reduced cell viability in
HepG2 cells. Ryan et al. studied the viability of HepG2 and
Caco-2 cells exposed to oxides of β-sitosterol (via the NRU
assay).15 They reported that the triol, 7-keto, and 7-β-OH
derivatives were cytotoxic to HepG2 cells and Caco-2 cells,
while α- and β-epoxide derivatives were noncytotoxic.
Koschutnig et al. assessed the viability of HepG2 cells after
exposure to single and mixed oxides of β-sitosterol using the
trypan blue exclusion assay.16 Cell viability showed a similar
trend: 7-α-OH sitosterol had the greatest effect on viability
followed by 7-keto sitosterol and 7-β-OH sitosterol.
18.6
20.4
20.3
20.4
60 μM 7-keto
31.5**
37.1**
21.4
120 μM 7-keto
30 μM 7-β-OH
60 μM 7-β-OH
120 μM 7-β-OH
30 μM triol
23.6*
27.7**
18.3
60 μM triol
19.3
120 μM triol
18.9
a
The derivatives investigated were α-epoxide (8), β-epoxide (7), 7-
keto (4), 7-β-OH (5), and triol (9). Cells were also treated with 30
μM of the corresponding cholesterol oxide derivatives. All compounds
were dissolved in ethanol, and equivalent quantities of ethanol
(EtOH) were added to control cells. The morphology of the nuclei
was assessed using the Hoechst 33342 stain and the number of
apoptotic nuclei expressed as a percentage of the total number.
A number of studies have investigated the relationship
between the structure of COPs and their biological effects.
Generally, triol derivatives have been found to be the most
cytotoxic to cells in culture, followed by 7-β-OH, β-epoxide, 7-
keto, and finally α-epoxide.33 It has been shown that 7-β-OH
b
Values are means with their standard error of at least three
independent experiments. Means values were significantly different
from the control ethanol group (ANOVA followed by the Dunnett’s
test): * P < 0.05, ** P < 0.01.
5958
dx.doi.org/10.1021/jf204737e | J. Agric. Food Chem. 2012, 60, 5952−5961

Journal of Agricultural and Food Chemistry
Article
measured caspase activities and DNA fragmentation (see
below). All COPs significantly increased apoptosis in U937
cells relative to control as assessed by Hoechst staining (Table
3). For the POPs, 7-keto (4) and 7-β-OH (5) at 60 μM and
120 μM appeared to induce apoptosis in U937 cells (Table 3).
The triol derivatives (9), while very cytotoxic (Tables 1 and 2),
were nonapoptotic and therefore we assume were killing the
cells by necrosis. Necrosis is unprogrammed cell death, where
dying cells are not engulfed by phagocytes but instead fragment
into many pieces of debris, leading to disorganized and
detrimental breakdown of the cell. Necrosis was not quantified
in the present study.
Table 4. Caspase Activity Following Exposure for 24 h to 30,
60, or 120 μM DHB Oxides (95% Purity)
a
b
caspase act. (fold increase rel to control ethanol)
U937 cells
mean
Cholesterol Oxides
HepG2 cells
compound
SE
mean
SE
30 μM α-epoxide
30 μM β-epoxide
30 μM 7-keto
1.2
0.1
0.4
0.2
0.2
1.1
1.3
1.0
1.2
0.1
0.2
0.1
0.2
2.0*
1.9*
1.4
30 μM 7-β-OH
Phytosterol Oxides
It is evident that, in U937 cells exposed to 60 μM 7-keto
oxide, the degree of apoptosis appears to be greater than the
degree of cell death: 13% cell death (FDA/EtBr assay) with
31.5% apoptosis. FDA/EtBr is a marker of cell integrity;
therefore it is plausible that cell integrity was not dramatically
affected but cells were still able to enter the early stages of
apoptosis. This effect was not evident at 120 μM. It is apparent
that, in general, the α-epoxide (8) and β-epoxide (7) of DHB,
as well as the corresponding derivatives of β-sitosterol and
stigmasterol, are nonapoptotic.15,17 7-Keto-stigmasterol did not
induce apoptosis in U937 cells;17 however apoptosis did appear
to be induced in the present study after exposure to high
concentrations: 60 and 120 μM of the 7-keto oxide (4). The 7-
β-OH derivatives of β-sitosterol and stigmasterol15,17 as well as
the oxide of DHB (5) appear to be apoptotic as measured by
Hoechst staining.
Caspase activity was significantly increased after U937 cells
were exposed to 30 μM of the COPs, β-epoxide and 7-keto
(Table 4). In relation to the DHB oxides, caspase activity in
U937 cells was significantly increased following exposure to 60
and 120 μM 7-β-OH (5). Similarly, the oxides of stigmasterol
including 7-β-OH increased caspase activity in U937 cells.17
Apoptotic potential of POPs in the U937 cell line was verified
by DNA laddering (Figure 3). It is evident that the 7-keto oxide
did show an increase in apoptosis with the Hoechst method but
did not increase caspase activity in U937 cells. Morphological
changes may be evident indicating apoptosis is occurring, but
caspase activity may be unaffected as the compound may be
inducing cell death by caspase-independent mechanisms.
Prunet et al. demonstrated that 7-keto and 7-β-OH cholesterol
can induce cell death by both caspase-dependent and
-independent pathways.40 Therefore, their equivalent DHB
oxides may have the potential to induce cell death through
caspase-independent pathways.
30 μM α-epoxide
60 μM α-epoxide
120 μM α-epoxide
30 μM β-epoxide
60 μM β-epoxide
120 μM β-epoxide
30 μM 7-keto
1.0
1.1
1.5
0.9
1.3
1.8
1.0
1.6
1.5
1.3
2.3*
3.0**
1.1
1.3
1.6
0.1
0.1
0.3
0.1
0.2
0.5
0.0
0.3
0.2
0.1
0.3
0.5
0.2
0.2
0.5
1.1
0.1
0.1
0.1
0.0
0.1
0.1
0.1
0.0
0.1
0.0
0.1
0.2
0.1
0.2
0.4
1.1
1.0
0.9
1.0
1.1
0.9
60 μM 7-keto
0.9
120 μM 7-keto
30 μM 7-β-OH
60 μM 7-β-OH
120 μM 7-β-OH
30 μM triol
1.0
0.9
1.2
1.2
1.5
60 μM triol
2.5**
4.0**
120 μM triol
a
The derivatives investigated were α-epoxide (8), β-epoxide (7), 7-
keto (4), 7-β-OH (5), and triol (9). Cells were also treated with 30
μM of the corresponding cholesterol oxide derivatives. All compounds
were dissolved in ethanol, and equivalent quantities of ethanol
(EtOH) were added to control cells. Caspase activity was expressed as
b
fold increase relative to control ethanol cells. Caspase activity is total
caspase activity (caspase 3 and caspase 7) expressed as a percentage of
ethanol control. Values are means with their standard error of at least
three independent experiments. Mean values were significantly
different from the control ethanol group (ANOVA followed by the
Dunnett’s test): * P < 0.05, ** P < 0.01.
Unlike macrophage cell lines such as U937 cells, previous
studies have demonstrated that COPs are cytotoxic but do not
induce apoptosis in HepG2 cells.15,23 Therefore, only caspase
activity was assessed in the HepG2 cell line (Table 4) following
exposure to COPs and POPs. COPs had no impact on caspase
activity in this cell line. Of the DHB oxides, only triol (60 and
120 μM) significantly increased caspase activity in HepG2 cells.
Biasi et al. investigated the apoptotic potential of a mix of COPs
in Caco-2 cells.9 It was apparent that COPs were proapoptotic
in differentiated cells but not in the undifferentiated state.
Exposure of differentiated Caco-2 cells to COPs resulted in
significantly stronger caspase activity than that of unoxidized
cholesterol. Koschutnig et al. assessed the apoptotic potential of
single and mixed oxides of β-sitosterol in the HepG2 cell line,
and only 7-keto sitosterol appeared to have apoptotic
properties.16 In contrast, oxidation products of β-sitosterol
Figure 3. DNA fragmentation in U937 cells exposed to 120 μM DHB
oxides for 24 h. Lane 1: molecular weight marker (200 bp). Lane 2:
control, ethanol treated cells. Lane 3: α-epoxide (8). Lane 4: β-epoxide
(7). Lane 5: 7-keto (4). Lane 6: 7-β-OH (5). Lane 7: triol (9).
investigated by Ryan et al. were nonapoptotic in HepG2 and
Caco-2 cell lines.15
This report discloses for the first time the synthesis and
cytotoxicity of pure oxides of the significant phytosterol DHB
and as such gives invaluable information on the toxicity profile
5959
dx.doi.org/10.1021/jf204737e | J. Agric. Food Chem. 2012, 60, 5952−5961

Journal of Agricultural and Food Chemistry
Article
(13) Adcox, C.; Boyd, L.; Oehrl, L.; Allen, J.; Fenner, G. Comparative
effects of phytosterol oxides and cholesterol oxides in cultured
macrophage-derived cell lines. J. Agric. Food Chem. 2001, 49, 2090−
2095.
(14) Roussi, S.; Winter, A.; Gosse, F.; Werner, D.; Zhang, X.;
Marchioni, E.; Geoffroy, P.; Miesch, M.; Raul, F. Different apoptotic
mechanisms are involved in the antiproliferative effects of 7-β-
hydroxysitosterol and 7-β-hydroxycholesterol in human colon cancer
cells. Cell Death Differ. 2005, 12, 128−135.
(15) Ryan, E.; Chopra, J.; McCarthy, F.; Maguire, A. R.; O’Brien, N.
M. Qualitative and quantitative comparison of the cytotoxic and
apoptotic potential of phytosterol oxidation products with their
corresponding cholesterol oxidation products. Br. J. Nutr. 2005, 94,
443−451.
(16) Koschutnig, K.; Heikkinen, S.; Kemmo, S.; Lampi, A. M.;
Piironen, V.; Wagner, K. H. Cytotoxic and apoptotic effects of single
and mixed oxides of beta-sitosterol on HepG2 cells. Toxicol. In Vitro
2009, 23, 755−762.
of an important component in the diet. Certain oxidized
derivatives of DHB are cytotoxic in U937 and HepG2 cells.
Overall, they appear to be more cytotoxic to U937 cells.
Toxicity differed with each individual oxide investigated: 7-keto,
7-β-OH, and triol derivatives show significant cytotoxic effects,
and of these derivatives, 7-β-OH appears to have the most
apoptotic potential. In contrast, α- and β-epoxide derivatives
are relatively noncytotoxic and nonapoptotic. All the oxidized
derivatives investigated in this study have a similar cytotoxic
potential in U937 cells to previously reported POPs equivalent
(β-sitosterol and stigmasterol oxide). Results generated in this
study add to the data on the toxicity profile of individual POPs
and help determine the hierarchy of toxicity of POPs.
AUTHOR INFORMATION
■
Corresponding Author
(17) O’Callaghan, Y. C.; Foley, D. A.; O’Connell, N. M.; McCarthy,
F. O.; Maguire, A. R.; O’Brien, N. M. Cytotoxic and apoptotic effects
of the oxidised derivatives of stigmasterol in the U937 human
monocytic cell line. J. Agric. Food Chem. 2010, 58, 10793−10798.
(18) Misharin, A. Y.; Mehtiev, A. R.; Morozevich, G. E.; Tkachevb, Y.
V.; Timofeevb, V. P. Synthesis and cytotoxicity evaluation of 22,23-
oxygenated stigmastane derivatives. Bioorg. Med. Chem. 2008, 16,
1460−1473.
*School of Food and Nutritional Sciences, University College
Cork, Cork, Ireland. Phone: +353 21 4902884. Fax: +353 21
4270244. E-mail: nob@ucc.ie.
Notes
The authors declare no competing financial interest.
REFERENCES
(19) Patterson, G. W.; Krauss, R. W. Sterols of Chlorella. I. The
naturally occurring sterols of Chlorella vulgaris, C. ellipsoidea, and C.
saccharophila. Plant Cell Physiol. 1965, 6, 211−220.
■
(1) Gupta, A. K.; Savopoulos, C. G.; Ahuja, J.; Hatzitolios, A. I. Role
of phytosterols in lipid-lowering: current perspectives. Q. J. Med. 2011,
104, 301−308.
(20) Thompson, M. J.; Cohen, C. F.; Lancaster, S. M. Brassicasterol
and 22, 23-dihydrobrassicasterol from i-ergosterol via -ergosterol.
Steroids 1965, 5, 745−752.
(21) Phillips, K. M.; Ruggio, D. M.; Toivo, J. I.; Swank, M. A.;
Simpkins, A. H. Free and esterified sterol composition of edible oils
and fats. J. Food Compos. Anal. 2002, 15, 123−142.
(22) Lizard, G.; Gueldry, S.; Sordet, O.; Monier, S.; Athias, A.;
Miguet, C.; Bessede, G.; Lemaire, S.; Solary, E.; Gambert, P.
Glutathione is implied in the control of 7-ketocholesterol-induced
apoptosis, which is associated with radical oxygen species production.
FASEB J. 1998, 15, 1651−1663.
(23) O’Callaghan, Y. C.; Woods, J. A.; O’Brien, N. M. Characteristics
of 7β-hydroxycholesterol-induced cell death in a human monocytic
blood cell line, U937 and a human hepatoma cell line, HepG2. Toxicol.
In Vitro 2002, 16, 245−251.
(24) O’Connell, N.; O’Callaghan, Y. C.; O’Brien, N. M.; Maguire, A.
R.; McCarthy, F. O. Synthetic routes to campesterol and
dihydrobrassicasterol: a first reported synthesis of dihydrobrassicaster-
ol. Tetrahedron 2012, 68, 4995−5004.
(25) Phelan, M.; Aherne, S. A.; Wong, A.; O’Brien, N. M. Bioactive
properties of wood knot extracts on cultured human cells. J. Med. Food
2009, 12, 1245−1251.
(2) Lea, L. J.; Hepburn, P. A.; Wolfreys, A. M.; Baldrick, P. Safety
evaluation of phytosterol esters. Part 8. Lack of genotoxicity and
subchronic toxicity with phytosterol oxides. Food Chem. Toxicol. 2004,
42, 771−783.
(3) Grandgirard, A.; Martine, L.; Joffre, C.; Juaneda, P.; Berdeaux, O.
Gas chromatographic separation and mass spectrometric identification
of mixtures of oxyphytosterol and oxycholesterol derivatives −
application to a phytosterol-enriched food. J. Chromatogr., A 2004,
1040, 239−250.
(4) Johnsson, L.; Dutta, P. C. Determination of phytosterol oxides in
some food products by using an optimized transesterification method.
Food Chem. 2006, 97, 606−613.
(5) Gonzal
L.; Barbera,
́
ez-Larena, M.; García-Llatas, G.; Vidal, M.; San
R.; Lagarda, M. Stability of plants sterols in ingredients
́
chez-Siles,
́
used in functional foods. J. Agric. Food Chem. 2011, 59, 3624−3631.
(6) García-Llatas, G.; Rodríguez-Estrada, M. T. Current and new
insights on phytosterol oxides in plant sterol-enriched food. Chem.
Phys. Lipids 2011, 164, 607−624.
(7) Tonello, A.; Poli, G. Serum phytosterols not only from dietary
intake. Br. J. Nutr. 2006, 96, 791−792.
́
(8) Wielkoszynski, T.; Gawron, K.; Strzelczyk, J.; Bodzek, P.;
(26) O’Callaghan, Y. C.; Woods, J. A.; O’Brien, N. M. Oxysterol-
induced cell death in U937 and HepG2 cells at reduced and normal
serum concentrations. Eur. J. Nutr. 1999, 38, 255−262.
(27) McCarthy, F. O.; Chopra, J.; Ford, A.; Hogan, S. A.; Kerry, J. P.;
O’Brien, N. M.; Ryan, E.; Maguire, A. R. Synthesis, isolation and
characterisation of β-sitosterol and β-sitosterol oxide derivatives. Org.
Biomol. Chem. 2005, 3, 3059−3065.
Zalewska-Ziob, M.; Trapp, G.; Srebniak, M.; Wiczkowski, A. Cellular
toxicity of oxycholesterols. Bioessays 2006, 28, 387−398.
(9) Biasi, F.; Mascia, C.; Astegiano, M.; Chiarpotto, E.; Nano, M.;
Vizio, B.; Leonarduzzi, G.; Poli, G. Pro-oxidant and proapoptotic
effects of cholesterol oxidation products on human colonic epithelial
cells: a potential mechanism of inflammatory bowel disease
progression. Free Radical Biol. Med. 2009, 47, 1731−1741.
(28) Foley, D. A.; O’Callaghan, Y.; O’Brien, N. M.; McCarthy, F. O.;
Maguire, A. R. Synthesis and characterisation of stigmasterol oxidation
products. J. Agric. Food Chem. 2010, 58, 1165−1173.
(10) Lordan, S.; Mackrill, J. J.; O’Brien, N. M. Oxysterols and
mechanisms of apoptotic signaling: implications in the pathology of
degenerative diseases. J. Nutr. Biochem. 2009, 20, 321−336.
̌
̌
(29) St'astna, E.; Cerny, I.; Pouzar, V.; Chodounska, H. Stereo-
́
́
́
(11) Hovenkamp, E.; Demonty, I.; Plat, J.; Lutjohann, D.; Mensink,
̈
selectivity of sodium borohydride reduction of saturated steroidal
ketones utilizing conditions of Luche reduction. Steroids 2010, 75,
721−725.
R. P.; Trautwein, E. A. Biological effects of oxidized phytosterols: a
review of the current knowledge (review). Prog. Lipid Res. 2008, 47,
37−49.
́
(30) Salvador, J. A. R.; Sae Melo, M. L.; Campos Neves, A. S.
(12) Maguire, L.; Konoplyannikov, M.; Ford, A.; Maguire, A. R.;
O’Brien, N. M. Comparison of the cytotoxic effects of beta-sitosterol
oxides and a cholesterol oxide, 7-beta-hydroxycholesterol, in cultured
mammalian cells. Br. J. Nutr. 2003, 90, 767−775.
Oxidations with potassium permanganatemetal sulphates and
nitrates. β-Selective epoxidation of Δ5-unsaturated steroids. Tetrahe-
dron Lett. 1996, 37, 687−690.
5960
dx.doi.org/10.1021/jf204737e | J. Agric. Food Chem. 2012, 60, 5952−5961

Journal of Agricultural and Food Chemistry
Article
(31) Emanuel, H. A.; Hassel, C. A.; Addis, P. B.; Bergman, S. D.;
Zavoral, J. H. Plasma cholesterol oxidation products (or sterols) in
human subjects fed a meal rich in oxysterols. J. Food Sci. 1991, 56,
843−847.
̀
(32) Lizard, G.; Monier, S.; Cordelet, C.; Gesquiere, L.; Deckert, V.;
Gueldry, S.; Lagrost, L.; Gambert, P. Characterisation and comparison
of the mode of cell death, apoptosis versus necrosis, induced by 7beta-
hydroxycholesterol and 7-ketocholesterol in the cells of the vascular
wall. Arterioscler., Thromb., Vasc. Biol. 1999, 19, 1190−1200.
́
(33) Carvalho, J. F.; Silva, M. M.; Moreira, J. N.; Simoes, S.; Sa e
̃
Melo, M. L. Sterols as anticancer agents: synthesis of ring-B
oxygenated steroids, cytotoxic profile and comprehensive SAR analysis.
J. Med. Chem. 2010, 53, 7632−7638.
(34) Rimner, A.; Al Makdessi, S.; Sweidan, H.; Wischhusen, J.;
Rabenstein, B.; Shatat, K.; Mayer, P.; Spyridopoulos, I. Relevance and
mechanism of oxysterol stereospecificity in coronary artery disease.
Free Radical Biol. Med. 2005, 38, 535−544.
(35) Sevanian, A.; Berliner, J.; Peterson, H. Uptake, metabolism, and
cytotoxicity of isomeric cholesterol-5,6-epoxides in rabbit aortic
endothelial cells. J. Lipid Res. 1991, 32, 147−155.
(36) Paillasse, M. R.; Saffon, N.; Gornitzka, H.; Silvente-Poirot, S.;
Poirot, M.; de Medina., P. Surprising “un” reactivity of cholesterol-5,6-
epoxides towards nucleophiles. J. Lipid Res. 2012, 53, 718−725.
(37) Berrodin, T. J.; Shen, Q.; Quinet, E. M.; Yudt, M. R.; Freedman,
L. P.; Nagpal., S. Identification of 5α, 6α-epoxycholesterol as a novel
modulator of liver X receptor activity. Mol. Pharmacol. 2010, 78,
1046−1058.
(38) Janowski, B. A.; Grogan, M. J.; Jones, S. A.; Wisely, G. B.;
Kliewer, S. A.; Corey, E. J.; Mangelsdorf., D. J. Structural requirements
of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 266−271.
(39) Song, C.; Hiipakka, R. A.; Liao, S. Auto-oxidized cholesterol
sulfates are antagonistic ligands of liver X receptors: implications for
the development and treatment of atherosclerosis. Steroids 2001, 66,
473−479.
́ ́ ́
(40) Prunet, C.; Lemaire-Ewing, S.; Menetrier, F.; Neel, D.; Lizard,
G. Activation of caspase-3-dependent and -independent pathways
during 7-ketocholesterol- and 7β-hydroxycholesterol-induced cell
death: a morphological and biochemical study. J. Biochem. Mol.
Toxicol. 2005, 19, 311−326.
5961
dx.doi.org/10.1021/jf204737e | J. Agric. Food Chem. 2012, 60, 5952−5961