livestock, the biological activities of glucosinolate hydrolysis products have generated considerable
toxicological and pharmocological interest. De- pending on glucosinolate composition and on the
prevalence of hydrolysis products, consumption of glucosinolates by mammals has been linked with
goitrogenic effects thiocyanates or with a re- duced risk of developing cancer isothiocyanates
in experimental animals [6 – 10].
Natural isothiocyanates derived from aromatic and aliphatic glucosinolates are effective chemo-
protective agents that block chemical carcinogene- sis and prevent several types of cancer in rodent
models [10]. Mechanistic studies have shown that isothiocyanates target mammalian Phase 1 and
Phase 2 drug-metabolizing enzymes and their cod- ing genes, resulting in decreased carcinogen-DNA
interactions and in increased carcinogen detoxifi- cation [8]. For example, the methionine-derived
4-methylsulfinylbutyl
isothiocyanate sul-
foraphane inhibits Phase 1 enzyme-mediated acti- vation of procarcinogens [11], induces Phase 2
detoxification enzymes such as quinone reductase QR and glutathione-S transferase in hepatoma
cells [12,13], and blocks mammary tumor forma- tion in rats [14,15]. Sulforaphane is the most pow-
erful natural inducer of chemoprotective enzymes thus far reported [12] and has become a metabolic
target of breeding strategies to enhance the anti- carcinogenic potency of cruciferous vegetables
[15,16].
The chemoprotective properties of natural isoth- iocyanates have renewed interest in glucosinolate
biosynthesis. While significant progress has been made in understanding the biochemistry and enzy-
mology of glucosinolate synthesis, very little is known about the structural and regulatory genes
involved [2]. In Arabidopsis thaliana L. Heynh., a member of the Cruciferae family and a premier
reference species for plant biology [17], 23 differ- ent glucosinolates have been identified [18]. Inter-
estingly,
4-methylsulfinylbutyl glucosinolate
glucoraphanin, precursor to sulforaphane, is the major leaf glucosinolate of ecotype Columbia
[18,19]. Here, we show that a QR bioassay in murine hepatoma cells reliably reports gluco-
raphanin content in A. thaliana. Furthermore, we have optimized the bioassay to allow for high-
throughput analysis of leaf extracts. The bioassay for the major chemopreventive glucosinolate in A.
thaliana, glucoraphanin, allows for rapid analysis of a large number of samples in an effort to dissect
glucosinolate biosynthesis by genetic and molecu- lar genetic approaches.
2. Materials and methods
2
.
1
. Chemicals NADP, FAD, 3-4,5-dimethylthiazo-2-yl-2,5-
diphenyltetrazolium bromide MTT, 2-methyl- 1,4-naphthoquinone menadione, bovine serum
albumin BSA, glucose 6-phosphate, crystal vio- let, baker’s yeast glucose-6-phosphate dehydroge-
nase, and aryl sulfatase were obtained from Sigma Chemical Co. St. Louis, MO; digitonin was ob-
tained from Boehringer Mannheim Indianapolis, IN; and acetonitrile, dimethyl sulfoxide, and
dimethyl formamide were obtained from Fisher Scientific Pittsburgh, PA. All the other chemicals
were of commercial purity or of plant cell culture grade.
2
.
2
. Plant material Arabidopsis thaliana L. Heynh. ecotype Co-
lumbia and mutant line TU1 derived from eco- type Columbia [19] were obtained from the
Biological Resource Center at Ohio State Univer- sity. Organically grown green onions Allium sa-
ti6a and apples Malus malus were obtained from a local market and were stored at − 20°C
after purchase. When Arabidopsis plants were grown to maturity, seeds were planted in vermi-
culite, and modified Hoagland’s nutrient media was subirrigated every 3 days [20]. For plant
growth under sterile conditions, seeds were surface sterilized for 10 min in 30 vv bleach contain-
ing 0.1 vv Triton X-100 and were placed on solid medium containing 8 g l
− 1
agar, 5 g l
− 1
sucrose and 2.15 g l
− 1
0.5 × Murashige-Skoog salts [21], pH 5.6. All the Arabidopsis plants were
grown in a growth chamber at 22°C under illumi- nation with fluorescent and incandescent light at
an intensity of 60 mE m
− 2
s
− 1
for 16 h daily.
2
.
3
. Preparation of plant extracts Acetonitrile extracts of green onions, apples,
and rosette leaves of mature A. thaliana plants 4 weeks old were essentially prepared according to
Prochaska et al. [22]. The plant material was ho- mogenized with 2 vol. of deionized cold water in a
Waring blender for 2 min at 4°C. The suspension was lyophilized, and 400 mg of the dried powder
were extracted with 14 ml acetonitrile for 24 h at 4°C. The extract was filtered and evaporated to
dryness in a rotating evaporator B 40°C. The residue was dissolved in 100-ml acetonitrile and
was directly used for induction of Hepa 1c1c7 murine hepatoma cells. Aqueous extracts water
extracts and phosphate buffer extracts from the Arabidopsis plants grown under greenhouse and
sterile conditions were prepared from rosette and primary leaves, respectively. Using a polypropy-
lene mini-pestle, the leaf material 40 mg fresh weight was homogenized in an 1.5-ml Eppendorf
tube with either 50 ml deionized water or 50 ml extraction buffer 5 mM K
2
HPO
4
– KH
2
PO
4
, 1 mM EDTA, pH 7.6. The pestle was rinsed with
150-ml water or extraction buffer, and the ho- mogenate was vortexed for 2 h at room tempera-
ture. For organic extracts using triple solvent solution [15], leaves 200 mg fresh weight were
homogenized with 1 ml triple solvent prepared by mixing of equal volumes of dimethyl sulfoxide,
dimethyl formamide, and acetonitrile and ex- tracted at − 50°C for 1 h. All extracts were
cleared by repeated centrifugation 10 min at 16 000 × g, and the supernatant was directly used
for induction of Hepa 1c1c7 murine hepatoma cells. Plant material was extracted on at least three
occasions and analyzed separately.
2
.
4
. Induction of cultured Hepa
1
c
1
c
7
murine hepatoma cells
The Hepa 1c1c7 murine hepatoma cell line was kindly provided by Paul Talalay, The Johns Hop-
kins University School of Medicine. Mutant Hepa 1c1c7 cells, BP
r
c1 and TAOc1BP
r
c1, were ob- tained from Michael Denison, University of Cali-
fornia, Davis. All cell lines were propagated in a-minimal essential medium supplemented with
10 fetal calf serum FCS [15] in a humidified incubator in 5 CO
2
at 37°C as previously de- scribed [22,23]. To monitor inducer potency of
plant extracts, Hepa 1c1c7 murine hepatoma cells were grown in 96-well microtiter plates. Typically,
10 000 Hepa 1c1c7 cells were seeded into each well, grown for 24 h, and then induced for 24 h by
exposure to fresh culture medium containing serial dilutions of the plant extract to be assayed aceto-
nitrile, water, phosphate buffer, or triple solvent extracts. Usually, 20 ml of the extract was diluted
to 4 ml with cell culture medium, and two-fold serial dilutions were prepared in the microtiter
plate using an octapipet, one column of wells receiving the same amount of plant extract. The
final volume in each well was 200 ml, and the concentration of the extract solvent was 0.5. For
the assay of triple solvent extracts, ascorbic acid 0.5 mM final concentration and 0.003 units of
myrosinase Sigma were added to each well to achieve complete glucosinolate hydrolysis.
2
.
5
. Assay of quinone reductase acti6ity After Hepa 1c1c7 cells were exposed to plant
test extracts in culture medium for 24 h, QR activity was assayed in cell lysates, using mena-
dione, MTT, and a NADPH-generating system [23]. The cell culture medium was decanted and 50
ml of lysing solution 0.8 digitonin, 2 mM EDTA, pH 7.8 was added to each well. To facili-
tate cell lysis, microtiter plates were incubated for 10 min at 37°C and were subsequently agitated on
an orbital shaker 100 rpm for 10 min at room temperature. For measurement of QR activity, 200
ml of assay solution were added to each well using an octapipet. The QR assay solution contained 25
mM Tris – HCl, pH 7.4, 1 mM glucose-6-phos- phate, 50 mM menadione, 30 mM NADP, 5 mM
FAD, 0.07 wv bovine serum albumin BSA, 0.03 wv MTT, 0.01 vv Tween-20, and 2
units ml
− 1
of yeast glucose-6-phosphate dehydro- genase [23]. The reaction mixtures were incubated
at room temperature and reactions were termi- nated after 5 min by the addition of 50 ml of 0.1 M
HCl. A reagent blank was prepared by adding stop solution to one column of each plate before
the addition of assay solution. Absorbances were measured by scanning the microtiter plates at 595
nm. The average absorbance value of the reagent blank column n = 8 wells was subtracted from
the average absorbance value of each test column. The standard deviation of the mean column ab-
sorbance was generally B 10. Occasionally, the absorbance of a well deviated as much as 50
from the mean column absorbance, due to un- equal cell density. Cell densities were determined
either by staining with crystal violet or by mea- surement of total protein with the bicinchoninic
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
