acid reagent [15]. The potency of QR induction by plant extracts is expressed as unit g
− 1
fresh or dry weight. One unit of QR inducer activity is defined
as the amount of plant material required to double the specific QR activity in a microtiter well con-
taining 200-ml cell culture medium.
2
.
6
. HPLC analysis of desulfoglucosinolates Glucosinolates were extracted from A. thaliana
leaves 50 mg fresh weight by boiling in water 1 ml [19]. After washing the leaves with water 1
ml, the combined extract was applied to a DEAE- Sephadex A-25 40 mg column pyridine acetate
form. The glucosinolates were converted into their desulfo analogs by overnight treatment with
100 ml 0.1 1.4 U aryl sulfatase, and the desul- foglucosinolates were eluted with 1 ml water [18].
HPLC of desulfo-glucosinolates was carried out using a Shimadzu VP Liquid Chromatograph.
Samples 100 ml were separated at ambient tem- perature on a Waters Spherisorb C18 column
150 × 4.6 mm i.d., 5-mm particle size, using the following methanol gradient in water at a flow
rate of 1.0 ml min
− 1
: 3 5 min, 3 – 21 6 – 20 min. Desulfoglucosinolates were detected at 226
and 280 nm. Sinigrin allyl glucosinolate was used as a standard. Farnham et al. [24] reported that
the relative
integrated absorbance
areas for
equimolar concentrations of glucoraphanin and sinigrin at 226 nm are identical.
3. Results
3
.
1
. Induction of quinone reductase in murine hepatoma cells by Arabidopsis leaf extracts
Methylsulfinylalkyl isothiocyanates are potent and specific inducers of mammalian Phase 2
detoxification enzymes such as QR or glutathione- S transferase [7]. Since methylsulfinylalkyl glucosi-
nolates
are synthesized
in A.
thaliana and
glucoraphanin is the most abundant leaf glucosi- nolate of ecotype Columbia [17,18], we tested the
potency of Columbia leaf extracts in inducing QR activity in cultured Hepa 1c1c7 murine hepatoma
cells. As indicated by the colorimetric QR bioassay, rosette leaves of A. thaliana contain
readily detectable levels of Phase 2 enzyme induc- ers Figs. 1 and 2A. We used green onion and
apple extracts as controls for the bioassay, which were reported to contain high and negligible levels
of QR inducers, respectively [22]. As shown in Fig. 2, at the highest concentration tested acetonitrile
extract derived from 8 mg dry weight ml
− 1
cell culture medium, green onion extract induced
murine specific QR activity 3.5-fold, whereas the effect of apple extract was marginal less than 20
induction. The potency of green onions is calcu- lated at 2250 U g
− 1
dry weight, while apple extracts are considered inactive Fig. 2B and C.
Interestingly, the potency of acetonitrile extract of mature leaves of A. thaliana, calculated at 38 500
U g
− 1
dry weight, exceeded that of green onion extract by more than one order of magnitude Fig.
2A. Less than 0.15 mg dry weight ml
− 1
medium were required for A. thaliana extract to double the
specific QR activity of Hepa 1c1c7 cells, as op- posed to 2.3 mg dry weight ml
− 1
medium of green onion extract. Maximal, about 9-fold, induction of
specific QR activity by A. thaliana extract was achieved at a concentration of 4 mg dry weight
ml
− 1
medium. Examination of detectable cytotox- icity showed that A. thaliana extract at 2 mg dry
weight ml
− 1
medium reduced cell density by 20. However, the cytotoxic effect was negligible at and
below 1 mg dry weight ml
− 1
medium Fig. 1BFig. 2A.
Fig. 1. Colorimetric assay of QR activity A and of cell density B. Hepa 1c1c7 murine hepatoma cells were grown in
96-well microtiter plates and induced with serial two-fold dilutions of Columbia leaf extract only one half of each assay
plate is shown. Each column of wells received the same dilution of a single plant extract. A more intense color
denotes higher QR activity and higher cell density.
Fig. 2. Induction of QR activity in murine Hepa1c1c7 cells by acetonitrile extract of A. thaliana leaves A, green onion B,
and apple C. Shown are, expressed as ratios of treated cells to control cells, total QR activities open circles, cell densities
triangles, and specific QR activities closed circles. Repre- sentative data of one experiment out of three independent
plant extractions are shown.
thaliana. The acetonitrile extraction method is lengthy and requires more leaf material than a
developing seedling can provide. We tested three procedures to rapidly extract isothiocyanates or
glucosinolates from rosette leaves of A. thaliana plants. During tissue homogenization, myrosinase-
catalyzed hydrolysis of glucosinolates to isothio- cyanates is favored at neutral pH, whereas nitrile
formation dominates at acidic conditions and re- quires ferrous ions [3]. We compared QR inducer
potencies of buffered 5 mM K
2
HPO
4
KH
2
PO
4
, 1 mM EDTA, pH 7.6 and non-buffered H
2
O leaf extracts. As shown in Table 1A, the QR inducer
potency of phosphate buffer extracts was as about twice as high as the potency of water extracts,
which suggests preferential formation of isothio- cyanates during leaf extraction with phosphate
buffer.
Tissue homogenization at low temperature with equal volumes of dimethyl sulfoxide, dimethyl for-
mamide, and acetonitrile triple solvent inacti- vates myrosinases and quantitatively extracts
glucosinolates [15]. Using triple solvent extraction and a myrosinase-supplemented QR assay, Fahey
et al. [15] showed that nearly all the inducer activity of cruciferous plants is derived from glu-
cosinolates. When applied to A. thaliana leaves, the QR inducer potency of triple solvent extracts
was comparable to the potency of phosphate buffer extracts see Table 1A, indicating that glu-
cosinolates are readily hydrolyzed by endogenous myrosinase activities during tissue homogenization
in aqueous buffers. Leaf extraction with phos- phate buffer was the method of choice in subse-
quent experiments because of its simplicity and effectiveness in extracting QR inducers. The plant-
to-plant variation in QR inducer potencies among different samples of Columbia leaf extract was
between 5 and 10 see Table 1.
3
.
3
. Test of mutant Hepa
1
c
1
c
7
cells defecti6e in Phase
1
enzyme expression Phase 2 detoxification enzymes are transcrip-
tionally activated by two classes of inducers i monofunctional inducers that directly and specifi-
cally activate Phase 2 genes; and ii bifunctional inducers that sequentially activate Phase 1 genes
and, after subsequent chemical modification by Phase 1 enzymes, genes encoding Phase 2 enyzmes
[8]. Induction of QR in wild type Hepa 1c1c7
Table 1 Potency of QR induction of A. thaliana ecotype Columbia
leaf extracts, inducer potencies are given per gram fresh weight as the mean 9 S.D. n = 3
Extraction solvent Murine cell line
Inducer potency A Rosette lea6es of
4
-week-old soil-grown mature plants 6250 550
Hepa 1c1c7 H
2
O Hepa 1c1c7
11 150 850 Phosphate buffer
Hepa 1c1c7 Triple solvent
12 500 900 B Primary lea6es of
2
-week-old agar-grown seedlings Hepa 1c1c7
Phosphate buffer 16 650 1150
12 500 1050 BPrc1
Phosphate buffer TAOc1BPrc1
14 300 1100 Phosphate buffer
3
.
2
. Comparison of extraction methods Next, we evaluated extraction methods better
suited to screening large populations of A.
murine hepatoma cells is responsive to both mono- and bifunctional inducers. The mutant cell
lines BP
r
c1 and TAOc1BP
r
c1, both derived from Hepa 1c1c7 cells, are defective in the expression of
the Phase 1 enzyme, cytochrome P
1
-450 [25]. Thus, QR induction in response to bifunctional inducers,
but not monofunctional inducers, is impaired in both mutant cell lines [25].
To assess the contribution of bifunctional induc- ers to the total QR inducer potency of leaf extract
and thus the ability to preferentially detect mono- functional inducers such as sulforaphane, we com-
pared QR induction in wild type and mutant Hepa 1c1c7 cells. As shown in Table 1B, the QR inducer
potency of leaf extract obtained from 2-week-old Columbia seedlings grown on agar plates was
similar in TAOc1BP
r
c1 cells and in BP
r
c1 cells. However, the QR inducer potency of the same
extract was 15 and 30 higher in wild type Hepa 1c1c7 cells than in TAOc1BP
r
c1 and BP
r
c1 cells, respectively. The difference in inducer potency of
the same sample between wild type and mutant murine hepatoma cells indicates presence of bi-
functional inducers in Columbia leaf extract, which amount to about 20 of the total QR
inducer potency Table 1B. In order to minimize detection of bifunctional quinone reductase induc-
ers, the TAOc1BP
r
c1 cell line was used in subse- quent experiments.
3
.
4
. Quinone reductase inducer potencies of mutant and wild type Arabidopsis strains
Finally, we tested the feasibility of the QR assay to identify plants with altered glucosinolate profi-
les. We compared QR inducer potencies of wild type leaf extracts with extracts of mutant line TU1
ecotype Columbia. The TU1 line was identified in a mutant screen by HPLC for plants with
altered glucosinolate metabolism [19]. The single recessive mutation gsm
1
-
1
affects synthesis of glucoraphanin, which was reported to accumulate
in TU1 leaves to less than 2 of wild type [19]. Reexamination of glucosinolate profiles of both
accessions by HPLC analysis showed that the leaves of Columbia wild type plants contained
glucoraphanin at a concentration of 400 nmol g
− 1
fresh weight, whereas levels of glucoraphanin in leaves of TU1 plants were measured at 7 nmol g
− 1
fresh weight data not shown. Importantly, the difference in glucoraphanin content between wild
type and TU1 leaf extracts correlated with a large difference
in QR
inducer potencies
in TAOc1BP
r
c1 cells, which was readily visualized by the colorimetric QR assay Fig. 3. The QR in-
ducer potency of TU1 plants 2500 U g
− 1
fresh weight was less than 20 of wild type plants
14 300 U g
− 1
fresh weight and likely reflects the reduced concentration of glucoraphanin in TU1
leaves.
4. Discussion
