1. Development of a sensitive in vitro assay to quantify the biological
activity of pro-inflammatory phorbol esters in Jatropha oil
Guillaume Pelletier & Bhaja K. Padhi & Jalal Hawari &
Geoffrey I. Sunahara & Raymond Poon
Received: 11 July 2014 /Accepted: 16 December 2014 /Published online: 15 January 2015 / Editor: T. Okamoto
# The Society for In Vitro Biology 2015
Abstract New health safety concerns may arise from the
increasing production and use of Jatropha oil, a biodiesel
feedstock that also contains toxic, pro-inflammatory, and co-
carcinogenic phorbol esters. Based on the exceptional sensi-
tivity of Madin-Darby canine kidney (MDCK) cells to the
model phorbol ester 12-O-tetradecanoylphorbol-13-acetate
(TPA), a robust bioassay was developed to quantify the bio-
logical activity of Jatropha phorbol esters directly in oil,
without sample extraction. We first verified that the character-
istic response of MDCK cells to TPA was also observed
following direct exposure to phorbol esters in Jatropha oil.
We further confirmed that similarly to TPA, Jatropha oil’s
phorbol esters can activate protein kinase C (PKC). We then
assessed the transcriptional response of MDCK cells to
Jatropha oil exposure by measuring the expression of
cyclooxygenase-2 (COX-2), a gene involved in inflammatory
processes which is strongly upregulated following PKC acti-
vation. Based on the parameterization of a TPA dose-response
curve, the transcriptional response of MDCK cells to Jatropha
oil exposure was expressed in term of TPA toxic equivalent
(TEQ), a convenient metric to report the inflammatory poten-
tial of complex mixtures. The sensitive bioassay described in
this manuscript may prove useful for risk assessment, as it
provides a quantitative method and a convenient metric to
report the inflammatory potential of phorbol esters in Jatropha
oil. This bioassay may also be adapted for the detection of
bioactive phorbol esters in other matrices.
Keywords In vitro bioassay . Jatropha oil . MDCK cell line .
Phorbol ester . Cyclooxygenase-2
Introduction
The use of biodiesel is promoted worldwide, as it can contrib-
ute to a reduction of fossil fuel dependency and greenhouse
gas emissions (Adler et al. 2007; Huo et al. 2009). Biodiesels
are generally less toxic than fossil fuels (Poon et al. 2007;
Poon et al. 2009), but potential human health hazards arising
from their increasing use also need to be properly assessed,
especially for biodiesels manufactured from nonedible or
toxic feedstock (Poon et al. 2013).
Biodiesel production from Jatropha curcas seed oil may
avoid competition with food crop and reduce pressure on arable
land, as this shrub can withstand harsh conditions and poor soils
otherwise unsuitable for agriculture (Makkar and Becker 2009;
Brittaine and Lutaladio 2010). Numerous toxins including
curcin, lectins, trypsin inhibitors, phytates, and saponins are
found in Jatropha seeds, but most of Jatropha oil’s toxicity is
attributed to the inflammatory and co-carcinogenic properties
of phorbol esters (Furstenberger et al. 1981; Hirota et al. 1988;
Makkar et al. 1998; Devappa et al. 2010b). So far, six different
phorbol esters (named C1, C2, C3, C4, C5, and C6) have been
characterized in Jatropha oil, and their bioactivity was assessed
(Haas et al. 2002; Roach et al. 2012).
Total phorbol ester concentration in Jatropha oil generally
varies from 2 to 4 mg/g. Although Jatropha oil refining and
esterification in a small-scale laboratory setting appears to
G. Pelletier (*) :B. K. Padhi :R. Poon
Hazard Identification Division, Environmental Health Science and
Research Bureau, Health Canada, Environmental Health Centre,
50 Colombine Driveway, P.L. 0803B, Tunney’s Pasture, Ottawa,
ON K1A 0L2, Canada
e-mail: guillaume.pelletier@hc-sc.gc.ca
J. Hawari :G. I. Sunahara
National Research Council of Canada, Biotechnology Research
Institute, 6100 Royalmount Avenue, Montreal, QC H4P 2R2,
Canada
J. Hawari
Department of Civil Engineering, Geology and Mining,
Polytechnique Montréal, 2500 Chemin dePolytechnique, C.P. 6079,
succ. Centre-ville, Montreal, QC H3C 3A7, Canada
In Vitro Cell.Dev.Biol.—Animal (2015) 51:644–650
DOI 10.1007/s11626-014-9861-z

2. remove or degrade phorbol esters (Haas and Mittelbach 2000;
Makkar et al. 2009; Ichihashi et al. 2011), concentrations of
phorbol esters ranging from 0.41 to 1.32 mg/g were reported
in industrially produced Jatropha biodiesel (Makkar et al.
2009). There are currently no guidelines, regulations, or la-
beling requirements concerning the presence of phorbol esters
in biodiesel.
The 3–10 μg/g detection limit for Jatropha phorbol esters
achieved by an early high-pressure liquid chromatography
(HPLC) method proved insufficient for adequate health protec-
tion (Kumar et al. 2010). Further refinements brought detection
limit down to 26 ng/g (Devappa et al. 2013), while a sensitivity
of 2 ng/ml was claimed for a liquid chromatography-mass spec-
trometry (LC-MS/MS) method (Ichihashi et al. 2011). However,
chromatographic protocols provide no information on the poten-
cy of phorbol esters and phorbol ester derivatives. The biological
activity of Jatropha phorbol esters has been tested using snails,
artemia, daphnia, bacteria, fungi, mammalian cell lines, and
various other in vitro bioassays (Beutler et al. 1989; Becker
and Makkar 1998; Vogg et al. 1999; Demissie and Lele 2010;
Devappa et al. 2010a; Devappa et al. 2012). However, these
assays present shortcomings, as they all require phorbol esters
pre-fractionation and, depending on the specific bioassay, can be
labor-intensive, lack sensitivity, or require additional biosafety
measures.
We took advantage of the exceptional responsiveness of
M a d i n - D a r b y c a n i n e k i d n e y c e l l s t o 1 2 - O -
tetradecanoylphorbol-13-acetate (Ohuchi and Levine 1978;
Blumberg 1980) to develop a simple, robust, and sensitive
bioassay to directly quantify the biological activity of phorbol
esters in Jatropha oil. We first verified that exposure to
Jatropha oil triggered the same characteristic alterations of
MDCK cellular morphology observed following exposure to
TPA (Fey and Penman 1984). We then confirmed that simi-
larly to TPA, Jatropha phorbol esters’ effects were mediated
(at least in part) through activation of protein kinase C (Griner
and Kazanietz 2007). Finally, we selected cyclooxygenase-2
(COX-2), a well-known, highly inducible gene involved in
inflammation (Langenbach et al. 1999) to assess MDCK
transcriptional response to Jatropha phorbol esters. This tran-
scriptional response was then compared to a TPA dose-
response curve, and phorbol ester biological activity in
Jatropha oil was expressed as TPA toxic equivalent (TEQ), a
well-known approach (Van den Berg et al. 2006) that repre-
sents a convenient way to quantitatively report the pro-
inflammatory potential of Jatropha oil.
Material and Methods
Material and reagents. MDCK (NBL-2, Catalog No. CCL-34)
cell line was purchased from American Type Culture Collection
(Manassas, VA) in 2002, grown for a few passages and then
cryopreserved in liquid nitrogen. Thawed cell aliquots used in
this study were cultured for a maximum of 15 passages. Jatropha
oil was obtained from Agroils (Firenza, Italy, 50129) whereas
corn oil (Mazola brand, ACH Food Companies, Oakville, ON,
Canada) was purchased from a local grocery store. Fatty acid
profiles for these two oils were described by Poon et al. (2013).
Unless otherwise stated, other reagents were purchased from
Sigma-Aldrich Canada (Oakville, ON, Canada).
Phorbol ester quantification. Jatropha oil aliquots were dilut-
ed using minimal volume of tetrahydrofuran and methanol
before loading on a Gemini-NX C18 HPLC reverse phase
column (Phenomenex, Torrance, CA). Peak separation was
performed using acetonitrile (80 v/v %) and 0.05% of formic
acid in water (20%) at room temperature. Phorbol esters (exact
mass 710.38) were analyzed using a HPLC/UV system
(Agilent Technologies, Santa Clara, CA) connected to a photo
diode array (PDA) detector and a Bruker micro Q-TOF mass
analyzer using electrospray positive ionization mode. The
esters were identified by comparison with the reference stan-
dard chemical TPA using their protonated molecular mass [M
+ H]+, [MH-H2O]+, and other characteristic mass fragments.
Quantification was done using UV detection at 280 nm. The
results are expressed on the basis of equivalent absorption
using TPA as external standard (Makkar et al. 2009).
Cell culture and exposure protocols. MDCK cells were
grown according to ATCC’s protocol in Eagle’s minimum
essential medium with Earle’s balanced salt solution (ATCC
Catalog No. 30–2003) supplemented with 10% fetal bovine
serum (ATCC Catalog No. 30–2020) and 1% penicillin-
streptomycin solution (Catalog No. 15140–122, Life
Technologies, Burlington, ON, Canada) in a humidified incu-
bator at 37 o
C and 5% CO2. MDCK cells seeded at 70,000
cells/cm2
were allowed to attach to the bottom of a petri dish
and to grow for 24 h. The cell culture media were then
removed, and the test substance was added in fresh culture
media. TPAwas dissolved in dimethyl sulfoxide (DMSO) and
added to culture media at 0, 0.003, 0.015, 0.075, 0.375, 1.875,
and 9.375 nM, keeping final DMSO concentration constant at
0.1% (v/v). Jatropha and corn oils were emulsified directly in
cell culture media by five ultrasonic bursts of 5 s, using an
ultrasonic processor equipped with a microtip (Cole Parmer,
Vernon Hills, IL). Although lactate dehydrogenase assay
(Roche Diagnostics, Laval, QC, Canada) suggested that
MDCK cells can easily withstand up to 6 μl/ml oil exposures
(data not shown), oil emulsions above 1.5 μl/ml proved un-
stable as an oily phase quickly reformed. In order to avoid
such dispersion issue, oil exposure was limited to 1.5 μl/ml.
At this exposure level, corn oil did not affect transcriptional
response of MDCK cells to TPA (data not shown). MDCK
cells were therefore exposed to 0, 0.0015, 0.015, 0.15, and
1.5 μl/ml Jatropha oil, keeping oil volume constant at 1.5 μl/
ASSESSMENT OF JATROPHA OIL’S BIOACTIVITY 645

3. ml cell culture media across control and treatment groups
using corn oil. Cells were exposed to TPA or Jatropha oil for
24 h. For the assessment of protein kinase C (PKC) activity,
MDCK cells were harvested after 2 h of exposure. For the
qualitative assessment of cellular morphology, cells were
seeded at a lower density (approximately 30,000 cells/cm2
),
stained with Sigma-Aldrich’s modified Giemsa stain accord-
ing to the manufacturer’s protocol, and observed under light
microscopy.
Immunoblot analysis. MDCK cells were rinsed twice with
ice-cold phosphate-buffered saline (PBS) and lysed directly
in sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) sample loading buffer (62.5 mM Tris pH 6.8, 3%
w/v SDS, 10% v/v glycerol, 5% v/v β-mercaptoethanol).
Samples were then boiled for 5 min, and the supernatant was
collected after centrifugation. Sample protein concentrations
were assessed using RC-DC protein assay kit (Bio-Rad
Laboratories, Mississauga, ON, Canada) according to the man-
ufacturer’s instructions. Equal amounts of protein (10 μg) were
loaded on a 12% polyacrylamide gel, electrophoresed, and
transferred to Immobilon PVDF (EMD Millipore, Billerica,
MA). Membranes were blocked for 1 h at room temperature
with Tris-buffered saline containing 0.1% Tween-20 (TBST)
supplemented with 5% bovine serum albumin (BSA).
Incubation with rabbit polyclonal antibodies against phosphor-
ylated myristoylated alanine-rich C kinase substrate (phospho-
MARCKS [Ser152/156], Novus Biochemicals, Littleton, CO)
was performed overnight at 4 o
C at 1:500 dilution in TBST
plus 1% BSA. Membranes were then washed four times for
10 min at room temperature in TBST, before incubation for 1 h
at room temperature with peroxidase-conjugated donkey anti-
rabbit IgG antibody (Jackson Immunoresearch Laboratories,
West Grove, PA) at 1:10,000 dilution in TBST. Membranes
were washed four times for 5 min in TBST, incubated in
Immun-Star WesternC reagent (Bio-Rad) for 5 min. Images
were acquired on a ChemiDoc XRS+ system and bands quan-
tified using Image Lab software (Bio-Rad). Six samples per
treatment group were used. A reference sample created by
mixing an equal amount of all the control samples was loaded
on all gels. Each polyacrylamide gel contained one sample
from all four treatment groups loaded in duplicate, and each
gel was also run in duplicate. The intensity of each sample was
normalized against the reference sample present on the same
gel, and the four normalized values generated from each
individual sample were averaged.
Gene expression analysis. Total RNA was isolated and puri-
fied using RNeasy Kit (Qiagen, Toronto, ON, Canada) ac-
cording to the manufacturer’s instructions. RNA quality and
quantity were determined using a 2100 Bioanalyzer (Agilent
Technologies) and a Nanodrop 1000 spectrometer (Thermo
Scientific, Waltham, MA). Five micrograms of total RNAwas
used for first-strand complementary DNA (cDNA) synthesis,
using Superscript III reverse transcriptase (Life Technologies)
according to the manufacturer’s protocol. The resulting first-
strand cDNA was diluted ten times to 200 μl to be used as
templates for qPCR analyses. Given the fact that exposure to
phorbol esters alters cellular morphology (Fey and Penman
1984) and beta-actin expression (Gerstenfeld et al. 1985),
hypoxanthine phosphoribosyltransferase 1 (HPRT1) and glyc-
eraldehyde 3-phosphate dehydrogenase (GAPDH) were se-
lected as housekeeping genes to monitor the relative expres-
sion of COX-2. Information on the primers used for the
amplification of those genes is provided in Table 1.
Quantitative PCR was performed with an iCycler iQ5 Real-
Time Detection System (Bio-Rad) using SYBR-Green I dye
(Qiagen) in a reaction volume of 25 μl containing 5 μl of the
diluted cDNA synthesis reaction and a primer concentration
of 0.4 μM. The RT-qPCR reaction mix was denatured at 95 o
C
for 3 min and then submitted to 45 amplification cycles (10-s
denaturation at 95 o
C, 45-s annealing at 57 o
C, and 30-s
extension at 72 o
C). Two qPCR technical replicates for each
sample were performed and the threshold cycle values aver-
aged. Primer specificity was assessed by endpoint PCR on
agarose gel and by melt curve analysis. PCR amplification
efficiency was calculated for every plate, based on the regres-
sion analysis of a row of serially diluted samples. Plates
presenting amplification efficiency greater than 90% were
used for analysis. Four samples per treatment group were
used. Assessment of relative gene expression was performed
using two housekeeping genes as described by Vandesompele
et al. (2002).
Statistical analysis. Dataset normality and homogeneity of
variance were first assessed by Shapiro-Wilk and Levene
tests, and treatment groups significantly different from the
control group were then identified by one-way ANOVA
followed by Dunnett post hoc test. Data that did not satisfy
ANOVA homogeneity and homoscedasticity assumptions
were log-transformed, and if these assumptions were still not
met, data were then analyzed using Kruskal-Wallis ANOVA
followed by Mann-Whitney U test. Data analyses were per-
formed using SigmaPlot 11.0 software (Systat Software,
Chicago, IL), and differences between treatment groups were
considered statistically significant for p<0.05.
Results
Owing to their acute sensitivity, MDCK cells have often been
used to study the effects of TPA on cellular functions (Daniel
et al. 1999). Figure 1 clearly shows that direct exposure to
Jatropha oil triggered the typical deformation of MDCK epi-
thelial polygonal geometry and the appearance of extensive
646 PELLETIER ET AL.

4. neurite-like processes observed following exposure to TPA
(Fey and Penman 1984).
Based on the molecular mechanisms underlying the bio-
logical effects of TPA (Fig. 2), we then assessed the phos-
phorylation state of myristoylated alanine-rich C kinase sub-
strate (MARCKS), a substrate and surrogate biomarker of
PKC activity. MDCK cell exposure to 9.375 nM TPA resulted
in a 3.5-fold increase of phosphorylated MARCKS signal. A
very similar increase was observed following exposure to
1.5 μl Jatropha oil/ml, while exposure to 1.5 μl corn oil/ml
had essentially no effect (Fig. 3).
COX-2 is among the first genes induced following expo-
sure to TPA (Coyne et al. 1990; Sciorra and Daniel 1996).
Significant induction of COX-2 gene expression which was
observed following exposure to 0.375 nM TPA appeared to
reach a plateau around 1.875 nM TPA (Fig. 4). Light micros-
copy observation of MDCK cells also confirmed that the
thresholds for COX-2 gene induction and alteration of
MDCK cellular morphology were approximately similar.
Although MDCK cells can tolerate up to 6 μl/ml of vege-
table oil (data not shown), exposure to Jatropha oil was limited
to 1.5 μl/ml in the concentration-response curve presented in
Fig. 5. Exposures to Jatropha oil at 0.15 and 1.5 μl/ml of
resulted in statistically significant 3.4- and 17.3-fold induc-
tions of COX-2 gene expression. These observations clearly
show that direct exposure to raw Jatropha oil can induce COX-
2 gene expression in MDCK cells.
The total phorbol ester concentration measured by HPLC
in the Jatropha oil tested was estimated at 1.23 mg/g, a slightly
lower value than usually reported (Devappa et al. 2011).
Based on the parameterization of the TPA concentration-
response curve (Fig. 4), the 17.3-fold induction of COX-2
gene expression observed following exposure to 1.5 μl/ml
Jatropha oil (Fig. 5) corresponds to a final concentration of
0.53 nM TPA in cell culture media. On an undiluted basis, this
represents 352 nM TPA in Jatropha oil and on a mass basis
(using a Jatropha oil density of 0.92 g/ml and a TPA molecular
weight of 617 g/mol) to a TPA TEQ of about 0.24 μg/g.
Discussion
A large number of irritant diterpene esters based on tigliane,
daphnane, or ingenane core are synthesized by J. curcas and
other members of the Euphorbiaceae family (Devappa et al.
2010b). The biological activities and potencies of these diter-
pene esters are highly dependent on their structures (Wang
et al. 2003), and in addition to the inflammatory and co-
carcinogenic properties of TPA (a tigliane ester isolated from
Croton tiglium), they can also present anti-hypertensive, anti-
retroviral, analgesic, anti-bacterial, anti-leukemic, anti-inflam-
matory, and anti-tumorigenic activities (Devappa et al.
2010b). Consequently, we first confirmed that the acute sen-
sitivity of MDCK cells to TPA (Ohuchi and Levine 1978;
Blumberg 1980) is also observed following exposure to
Jatropha oil phorbol esters.
Direct exposure of MDCK cells to Jatropha oil unequivo-
cally triggered the typical alteration of cellular morphology
observed following exposure to TPA (Fig. 1). This observa-
tion confirmed that MDCK cells can respond to the presence
of Jatropha phorbol esters presenting only a small fraction of
TPA’s activity (Beutler et al. 1989). Incidentally, this experi-
ment also demonstrated that direct exposure to vegetable oil
did not mute MDCK cellular response to phorbol esters.
Although it is possible to directly measure and quantify the
alteration of cellular morphology resulting from exposure to
phorbol esters (Penman and Fey 1986), this approach is time-
consuming, labor-intensive, prone to artifacts, and not easily
amenable to high-throughput applications.
Table 1 Primer sequences used for qRT-PCR amplification of the COX-2, GAPDH, and HPRT1 genes
Gene Accession Primer sequence Location Amplicon size (bp)
PTGS2/COX-2 NM_001003354 F-GTTCATTCCTGATCCCCAAG Exon 6 186
R-TTGAAAAGGCGCAGTTTATG Exon 7
HPRT1 NM_001003357 F-TGACACTGGGAAAACAATGCAGACT Exon 6 110
R-AGCCAACACTTCGAGGGGTCCT Exon 7
GAPDH NM_001003142 F-AAGGTCATCCCTGAGCTGAA Exon 7 192
R-GACCACCTGGTCCTCAGTGT Exon 9
Figure 1 Illustration of the
effects of Jatropha oil on MDCK
cellular morphology at 0.15 and
1.5 μl/ml and comparison with
exposure to 9.375 nM TPA.
ASSESSMENT OF JATROPHA OIL’S BIOACTIVITY 647

5. The exceptional sensitivity of MDCK cells to TPA was
described well before the elucidation of the molecular mech-
anisms involved (Ohuchi and Levine 1978; Blumberg 1980;
Nishizuka 1984). As indicated in Fig. 2, TPA directly binds
and activates PKC (Griner and Kazanietz 2007). As a first step
toward the development or an in vitro bioassay, we confirmed
that the effects of Jatropha oil are mediated at least in part
through PKC activation (Fig. 3). The very similar 3.5-fold
increase in the phospho-MARCKS signal observed following
exposure to 9.375 nM TPA and to the much less potent 1.5 μl/
ml Jatropha oil (see Figs. 4 and 5) suggests that activation of
PKC had already plateaued.
Interestingly, all the steps leading to the production of
arachidonic acid (Fig. 2) are independent of mRNA and
protein synthesis, but the production of prostaglandin is de-
pendent on COX-2 mRNA transcription and protein synthesis
(Beaudry et al. 1985; Coyne et al. 1990). The high inducibility
and dynamic range of COX-2 expression make this gene an
ideal candidate biomarker to assess the presence and biolog-
ical activity of phorbol esters in Jatropha oil. As illustrated in
Fig. 4, induction of COX-2 gene expression can be observed at
subnanomolar TPA concentrations and presents a typical sig-
moid shape spanning two orders of magnitude.
The most convenient and straightforward way to express the
potential toxicity of a poorly characterized mixture of chemicals
sharing a common molecular mechanism of action is to use the
toxic equivalency (TEQ) concept (Murk et al. 1997). In this
approach, the biological activity of a given mixture is expressed
as the concentration of a well-known compound (in this case
TPA) required to achieve the same outcome. Based on the
0.24 μg/g TPA-TEQ determined for Jatropha oil and on the
phorbol ester concentration of 1.23 mg/g determined chromato-
graphically, the biological activity of Jatropha phorbol esters
therefore represents approximately 1/5000 of TPA’s potency.
This biological activity is significantly lower than suggested by
phorbol dibutyrate receptor displacement assays where Jatropha
phorbol ester potency was estimated at 1/500 of TPA-containing
croton extract (Beutler et al. 1989). However, it is worth noting
Figure 2 Simplified representation of the molecular pathways involved
in the production of inflammatory prostaglandins following exposure to
TPA. Dark gray arrows indicate mRNA and protein synthesis-
independent events, while the light gray arrow is dependent on COX-2
gene transcription and protein synthesis.
Figure 3 Exposure to Jatropha oil activates protein kinase C in vitro. The
relative phospho-MARCKS immunoblot signal of unexposed (control)
MDCK cells is presented along with the signal from cells exposed to
corn oil, Jatropha oil (1.5 μl/ml), and TPA (9.375 nM). Errors bars
represent standard deviation. *p<0.05, statistically significant difference
from control group values (n=6). Inset: illustration of a typical
immunoblot where the 87-kDa phospho-MARCKS signal (indicated by
an arrow) is located above a nonspecific band cross-reacting with the
antibody (indicated by a short line).
Figure 4 Alteration of MDCK cellular morphology and induction of COX-
2 gene expression following exposure to TPA occur approximately at the
same concentration. Errors bars represent standard deviation. *p<0.05,
statistically significant difference from the control group (n=4).
Figure 5 Induction of COX-2 gene expression in MDCK cells following
exposure to Jatropha oil. Errors bars represent standard deviation. *p<
0.05, statistically significant difference from the control group (n=4).
648 PELLETIER ET AL.

6. that while these phorbol dibutyrate displacement comparisons
were performed on a mass basis, our estimation of Jatropha
phorbol ester potency was based on absorption at 280 nm. In
light of a recent report claiming that the absorption at 280 nm of
the most abundant phorbol ester in Jatropha oil is about 40 times
greater than TPA on a mass basis (Roach et al. 2012), our results
may underestimate the potency of Jatropha oil phorbol esters.
In spite of the relatively weak potency of phorbol esters
present in Jatropha oil, we were able to detect a significant
induction of COX-2 gene expression following direct exposure
of MDCK cells to 0.15 μl/ml Jatropha oil. Hence, in addition to
routine testing, the described bioassay may prove useful for
many other applications such as selection of J. curcas strains
presenting lower toxicity, assessment of phorbol ester extraction
or deactivation procedures, detection of Jatropha oil blended in
other feedstock, or detection of pro-inflammatory phorbol esters
from other sources. Although the ability to quantify the biolog-
ical activity of phorbol esters directly in vegetable oil without
pre-concentration significantly improves the convenience and
throughput of this assay, pre-fractionation and purification steps
may also be added in order to measure more dilute samples or
assess phorbol ester biological activity in other matrices. Of
course, the development of a stable MDCK reporter cell line
would further improve the convenience and throughput of this
bioassay, providing that the exceptional sensitivity of the parental
cell line can be retained.
Conclusions
With the increasing production and use of Jatropha oil and the
development of economically viable methods to detoxify the
remaining protein-rich seed cake for animal feed (Brittaine
and Lutaladio 2010; Makkar et al. 2012), regulations and
guidelines on phorbol ester levels in various matrices (poten-
tially expressed as TPA TEQ) will need to be developed. The
sensitive assay described in this manuscript may prove useful,
as it provides a quantitative method easily amenable to high-
throughput applications and a convenient metric to report the
inflammatory potential of phorbol esters in Jatropha-based
products.
Acknowledgments The authors wish to thank Sheila Masson and
Annamaria Halasz for technical assistance and Drs. Phil Shwed and
Subramanian Karthikeyan for critical review of this manuscript. This
work was supported by the Program of Energy Research and Develop-
ment (PERD project C24.001) from Natural Resources Canada (NRcan).
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