Cytotoxicity effects of di- and tri-hydroxybenzaldehydes as a chemopreventive potential agent on tumor cells

Article abstract of DOI:10.1016/S0300-483X(01)00346-8

As part of our earlier search for new compounds with improved biological activities including antioxidant, anti-inflammatory, and tumor growth inhibition activities, we synthesized 2,4,5-trihydroxybenzaldehyde (2,4,5-THBA) from commercially available Sesa

Full text of DOI:10.1016/S0300-483X(01)00346-8

Toxicology 161 (2001) 179–187
www.elsevier.com/locate/toxicol
Cytotoxicity effects of di- and tri-hydroxybenzaldehydes as a
chemopreventive potential agent on tumor cells
Tsui-Hwa Tseng a, Yen-Min Tsheng a, Yean-Jang Lee b,*
a Institute of Biochemistry, Chung Shan Medical and Dental College, Taichung, Taiwan
b Department of Chemistry, National Changhua Uni6ersity of Education, No. 1, Ching Der Road, Changhua 50058, Taiwan
Received 30 November 2000; received in revised form 19 December 2000; accepted 22 January 2001
Abstract
As part of our earlier search for new compounds with improved biological activities including antioxidant,
anti-inflammatory, and tumor growth inhibition activities, we synthesized 2,4,5-trihydroxybenzaldehyde (2,4,5-
THBA) from commercially available Sesamol. First we examined the free radical-quenching capacity of 2,4,5-THBA,
3,4-dihydroxybenzaldehyde (3,4-DHBA), and 2,5-dihydroxybenzaldehyde (2,5-DHBA) in vitro by 1,1-diphenyl-1,2-
picryhydrazylradical (DPPH) test. The antioxidant bioactivity was also evaluated using the model of t-butyl
hydroperoxide (t-BHP)-induced cytotoxicity in rat primary hepatocytes. Finally, three chemicals were tested by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay on human nasopharynx carcinoma cells
(KB cells), human hepatoblastoma (HepG2), and human leukemia HL-60 cells. The 2,4,5-THBA shows significant
cytotoxicity on HL-60 cells. The results suggest that 2,4,5-THBA may be a potential chemopreventor or chemother-
apy agent against HL-60 cells. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Trihydroxybenzaldehyde (THBA); DPPH; Antioxidant; MTT; Lipid peroxidation; HL-60 cells
stituents, possess chemopreventive activities in
tongue, liver, and large bowel carcinogenesis
(Tanaka et al., 1993a,b, 1994a,b; Kawamori et al.,
1994; Hirose et al., 1995). For example, protocate-
chuic acid (PCA) seems to be a promising com-
pound regarded as a candidate group for cancer
preventive agents (Mori et al., 1997). Further-
more, we have isolated and investigated Hibiscus
protocatechuic acid (PCA) from Hibiscus sabdar-
iffa L. For instance, PCA showed against oxida-
tive damage induced by t-butyl hydroperoxide
(t-BHP) in rat primary hepatocytes (Tseng et al.,
1996), and inhibitory effect on tumor promotion
1. Introduction
Chemoprevention is a rapidly expanding field,
and also a hopeful and promising one in the
management of cancer (Wattenberg 1985;
Tanaka, 1992; Szarka et al., 1994; Ip et al., 1994),
especially urinary bladder cancer (Boone and
Wattenberg, 1994; Meyskens et al., 1994). In ad-
dition, Mori et al. (1997) have reported phenolic
compounds, widely distributed as plant con-
* Corresponding author. Fax: 886-4-7211190.
E-mail address: leeyi@cc.ncue.edu.tw (Y.-J. Lee).
0300-483X/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0300-483X(01)00346-8

180
T.-H. Tseng et al. / Toxicology 161 (2001) 179–187
Table 1
Thus, PCA has anticarcinogenic potential and
may play a role in chemoprevention. Since several
compounds that possess antioxidant or anti-infl-
ammatory activity are able to inhibit 12-O-te-
Inhibition of free radical (DPPH) by 2,5-dihydroxybenzalde-
hyde(2,5-DHBA), 3,4-dihydroxybenzaldehyde (3,4-DHBA)
and 2,4,5-trihydroxybenzaldehyde (2,4,5-THBA)
Treatment
Concentration
(mM)
% of DPPH
bleachinga
tradecanoylphorbol-13-acetate
(TPA)-induced
tumor promotion in mouse skin (Wang et al.,
1995; Huang et al., 1992a,b; Chen et al., 1993;
Kozumbo et al., 1983), it is expected that natural
phenolic antioxidants, such as catechol, resor-
cinol, and pyrogallol analogues, may have some
modifying effects on the tumor promotion of
carcinogenesis or inhibitory effects of tumor cells.
Recently we synthesized and studied the con-
stituents of plants of the Cinnamomum SP. of the
family Lauraceae, which are used as an antide-
pressant in Asiatic folk medicine. Carpacin
showed anticarcinogenic potency for exhibiting
the effects of GST-inducing activity and inhibition
on benzo[a]pyrene induced unscheduled DNA
synthesis in rat primary hepatocytes (Tseng et al.,
2000b). Moreover, according to the report (Ogata
et al., 1997), an electron donor and/or bulky
groups at the ortho- or para-position of the phe-
nol were required for inhibition of lipid oxidation.
Furthermore, some investigations highlight an ad-
ditional role of polyphenolic components of
higher plants that may act as antioxidants or via
other mechanisms contributing to the anticarcino-
genic or cardioprotective actions (Ho, 1992;
Huang and Ferrano, 1992; Newmark, 1992). To
find a causal relationship between the antioxidant
and antitumor activity, we synthesized the pheno-
Control(DMSO) 0.2%
–
Quercetin
10
2
5
10
50
2
96.4690.14*b
24.7790.22*
54.8690.22*
97.3490.14*
96.4690.18*
33.6290.08*
70.7990.14*
97.3490.07*
97.3490.18*
19.4690.08*
48.6790.16*
89.3890.32*
96.4690.18*
2,5-DHBA
2,5-DHBA
2,5-DHBA
2,5-DHBA
3,4-DHBA
3,4-DHBA
3,4-DHBA
3,4-DHBA
2,4,5-THBA
2,4,5-THBA
2,4,5-THBA
2,4,5-THBA
5
10
50
2
5
10
50
a % of DPPH (1,1-diphenyl-2-picrylhydrazyl radical) bleach-
ing=[(absorbance of control group−absorbance of the agent
pretreated group)/(absorbance of control group)]×100%.
b Values are the average of triplicate determinations. *PB
0.05, compared with control.
in mouse skin (Tseng et al., 1998). The mechanism
studies have demonstrated PCA can induce apop-
tosis in human leukemia HL-60 cells, and that this
process was associated with the reduction of RB
phosphorylation and Bcl-2 expression (Tseng et
al., 2000a).
In our previous studies, PCA exhibited strong
antioxidant and antitumor promotion effects.
Table 2
Microculture tetrazolium assay (MTT) of 2,5-dihydroxybenzaldehyde (2,5-DHBA), 3,4-dihydroxybenzaldehydeand (3,4-DHBA)
2,4,5-trihydroxybenzaldehyde (2,4,5-THBA) at various concentrations in primary rat hepatocytes
Treatmenta
Concentration (mM)
Optical density (OD) mean9S.D.
% of absorbanceb
Control(DMSO)
2,5-DHBA
2,5-DHBA
3,4-DHBA
3,4-DHBA
0.2%
100
500
100
500
0.6590.05
0.6890.08
0.2090.02
0.7090.12
0.6990.10
0.7090.16
0.9490.12
100.00
104.87
30.76*
108.00
106.15
107.22
144.61*
2,4,5-THBA
2,4,5-THBA
100
500
a Primary rat hepatocytes were treated with 2,5-DHBA, 3,4-DHBA and 2,4,5,-THBA at various concentrations for 24 h.
b % of absorbance=(OD of test /OD of control)×100%. Values are the average of triplicate determinations. *PB0.05, compared
with control.

T.-H. Tseng et al. / Toxicology 161 (2001) 179–187
181
Table 3
Effect of 2,5-dihydroxybenzaldehyde (2,5-DHBA), 3,4-dihydroxybenzaldehyde (3,4-DHBA) and 2,4,5-trihydroxybenzaldehyde
(2,4,5-THBA) on tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity in the primary rat hepatocytes
Treatmenta
Concentration (mM)
LDHb (mU)/106 cells
ALT (mU)/106 cells
AST (mU)/106 cells
Control
t-BHP
0.2%
1500
10
100
10
100
10
100
137.03916.26
284.0292.45*
225.07926.46
150.07916.73c
293.67914.85
221.86913.15c
257.23926.32
177.64919.32c
102.8892.78
118.9796.43
95.92914.59c
68.5990.92c
103.9696.49c
97.5398.85c
85.2092.26c
74.48914.49c
153.26920.92
280.27912.49*
235.79937.71
134.5098.24c
258.30910.46c
255.62926.85
217.57920.22c
175.77921.29c
2,5-DHBA
2,5-DHBA
3,4-DHBA
3,4-DHBA
2,4,5-THBA
2,4,5-THBA
a Primary rat hepatocytes were pretreated by 2,5-DHBA, 3,4-DHBA and 2,4,5-THBA for 30 minutes, then t-BHP were added and
incubated for 30 min.
b Values are the average of triplicate determinations.*PB0.05, compared with control. LDH: Lactate dehydrogenase; ALT,
alanine aminotransferase; AST, aspartate aminotransferase. cPB0.05, compared with positive control.
Table 4
Effect of 2,5-dihydroxybenzaldehyde (2,5-DHBA), 3,4- dihydroxybenzaldehyde (3,4-DHBA) and 2,4,5-trihydroxybenzaldehyde
(2,4,5-THBA) on tert-butylhydroperoxide (t-BHP)-induced lipid peroxidation in primary rat hepatocytes
Treatmenta
Concentration (mM)
MDA formationb (n mole/mg protein)
% of inhibition
Control
t-BHP
0.2%
1500
10
100
10
100
10
100
29.7895.86
289.96994.90*
68.2095.45c
5.6090.90c
–
–
2,5-DHBA
2,5-DHBA
3,4-DHBA
3,4-DHBA
2,4,5-THBA
2,4,5-THBA
85.23
109.28
57.18
98.16
39.29
106.87
141.2196.18c
34.5699.07c
187.72961.12
11.9096.00c
a Primary rat hepatocytes were pretreated with 2,5-DHBA, 3,4-DHBA, and 2,4,5-THBA at various concentrations for 30 min,
then t-BHP was added for 30 min.Values are the average of triplicate determinations. *PB0.05, compared with control. cPB0.05,
compared with positive control. MDA, malondialdehyde.
b % of inhibition=[(value of positive control−value of test)/(value of positive control−value of negative control)]×100%.
Table 5
Microculture tetrazolium assay (MTT) of 2,5-dihydroxybenzaldehyde (2,5-DHBA), 3,4-dihydroxybenzaldehyde (3,4-DHBA) and
2,4,5-trihydroxybenzaldehyde (2,4,5-THBA) at various concentrations in KB cells
Treatmenta
Concentration (mM)
Optical density (OD) mean9S.D.
% of absorbanceb
Control(DMSO)
2,5-DHBA
2,5-DHBA
3,4-DHBA
3,4-DHBA
0.2%
10
100
10
100
10
100
1.4290.06
1.3290.09
0.9390.01
1.4090.06
1.1990.08
1.5490.08
1.4490.04
100.00
92.95*
65.49*
98.59
83.80
108.45*
101.40
2,4,5-THBA
2,4,5-THBA
a KB cells were treated with 2,5-DHBA, 3,4-DHBA and 2,4,5-THBA at variousconcentrations for 3 days.
b % of absorbance=(OD of test /OD of control)×100%. Values are the average of triplicate determinations. *PB0.05, compared
with control.

182
T.-H. Tseng et al. / Toxicology 161 (2001) 179–187
lic structure, 2,4,5-trihydroxybenzaldehyde (2,4,5-
THBA), and investigated its biological property.
Recently much attention has been focused on
the protective biochemical function of naturally
occurring antioxidants in biological systems, and
on the mechanisms of their action. Phenolic com-
pounds were considered to play an important role
for the prevention of oxidative damage in living
systems (Block, 1992; Hertog and Feskens, 1993).
The commercial availability of 1,2,4-trihydroxy-
Table 6
Microculture tetrazolium assay (MTT) of 2,5-dihydroxybenzaldehyde (2,5-DHBA), 3,4-dihydroxybenzaldehyde (3,4-DHBA) and
2,4,5-trihydroxybenzaldehyde (2,4,5-THBA) at various concentrations in HepG2 cells
Treatmenta
Concentration (mM)
Optical density (OD) mean9S.D.
% of absorbanceb
Control(DMSO)
2,5-DHBA
2,5-DHBA
3,4-DHBA
3,4-DHBA
0.2%
10
100
10
100
10
100
0.9090.02
0.8990.02
0.5990.01
0.8790.02
0.5190.01
0.8890.03
0.7190.02
100.00
98.88
65.55*
96.66
56.66**
97.77
78.88*
2,4,5-THBA
2,4,5-THBA
a HepG2 cells were treated with 2,5-DHBA, 3,4-DHBA and 2,4,5-THBA at various concentrations for 3 days.
b % of absorbance=(OD of test/OD of control)×100%. Values are the average of triplicate determinations. **PB0.001 and
*PB0.05, compared with control.
Table 7
Microculture tetrazolium assay (MTT) of 2,5-dihydroxybenzaldehyde (2,5-DHBA), 3,4-dihydroxybenzaldehyde (3,4-DHBA) and
2,4,5-trihydroxybenzaldehyde (2,4,5-THBA) at various concentrations in HL-60 cells
Treatmenta
Concentration (mM)
Optical density (OD) mean9S.D.
% of absorbanceb
Control (DMSO)
2,5-DHBA
2,5-DHBA
3,4-DHBA
3,4-DHBA
0.2%
10
100
10
100
10
100
0.5790.03
0.3190.05
0.0390.01
0.3890.02
0.0490.01
0.0790.01
0.0390.01
100.00
54.38*
5.26*
66.66*
7.01*
2,4,5-THBA
2,4,5-THBA
12.28**
5.26*
a HL-60 cells were treated with 2,5-DHBA, 3,4-DHBA and 2,4,5-THBA at various concentrations for 3 days.
b % of absorbance=(OD of test/OD of control)×100%. Values are the average of triplicate determinations. **PB0.001 and
*PB0.05, compared with control.
Table 8
Microculture tetrazolium assay (MTT) of 2,4,5-trihydroxybenzaldehyde (2,4,5-THBA) at various concentrations in HL-60 cells
Treatmenta
Concentration (mM)
Optical density (OD) mean9S.D.
% of absorbanceb
Control (DMSO)
2,4,5-THBA
2,4,5-THBA
2,4,5-THBA
2,4,5-THBA
0.2%
2
5
10
20
0.7590.02
0.7090.01
0.6790.02
0.5990.01
0.2290.05
100.00
93.33*
89.33*
78.66**
29.33**
a HL-60 cells were treated with 2,4,5-THBA at various concentrations for 24 h.
b % of absorbance=(OD of test/OD of control)×100%. Values are the average of triplicate determinations. **PB0.001 and
*PB0.05 compared with control. The IC50 of 2,4,5-THBA is 15.8 mM.

T.-H. Tseng et al. / Toxicology 161 (2001) 179–187
183
benzene prompted us to prepare THBA from this
2.2. Synthesis of 2,4,5-trihydroxybenzaldehyde
(2,4,5-THBA)
compound. Starting from commercially available
trihydroxybenzene, methylation gave trimethoxy-
benzene in high yield (92%). However, bromination
of trimethoxybenzene presents a problem because
of side isomer products and poor yield. To over-
come this technical difficulty, we have followed the
procedures of total synthesis of Carpacin. Starting
from commercially available sesamol, methylation
obtained anisol in 98% yield and bromination of
anisol in chloroform with bromide provided aryl
bromide in 92% yield. The sequence reactions were
employed by lithiation of aryl bromide with n-
BuLi, and quenching with dimethylformamide
(DMF) to afford the desired aldehyde in 90% yield.
Finally, the deprotected reaction was treated with
AlCl3 in chloroform under refluxing condition to
achieve the THBA product in 56% yield.
In the present work, we first examined the free
radical-quenching capacity of 2,4,5-THBA, 2,5-di-
hydroxybenzaldehyde (2,5-DHBA), and 3,4-dihy-
droxybenzaldehyde (3,4-DHBA) in vitro by DPPH
(1,1-diphenyl-1,2-picryhydrazylradical) test. The
antioxidant bioactivity was further evaluated using
the model of t-BHP-induced cytotoxicity in rat
primary hepatocytes. Finally, the inhibitory effects
of THBA, 2,5-DHBA, and 3,4-DHBA on human
leukemia HL-60 cells and other tumor cells are also
discussed.
1H NMR spectra were taken in DMSO unless
otherwise indicated. 1H NMR spectra was obtained
using a Varian MERCURY-400 spectrometer from
National Chung-Hsing University, Taichung. Ele-
mental analyses were performed on a Heracus
CHN-OS Rapid spectrometer in the Taichung
Instrumentation
Center,
National
Science
Council. DMSO (2.50 ppm) was used as an internal
standard for silyl compounds. Tetramethylsilane
was employed as an internal standard for
other compounds. Solvents were freshly
distilled from phosphorus pentoxide or CaH2, and
tetrahydrofuran was distilled from sodium
diphenyl ketyl, prior to use. All the reactions were
carried out under nitrogen atmosphere otherwise
stated. Silica gel (silica gel 60, 230–400
mesh, Merck) was used for chromatography.
Organic extracts were dried over anhydrous
MgSO4.
2,4,5-THBA was synthesized by our developed
procedure (Tseng et al., 2000a). To a solution of
2-methoxy-4,5-methylenedioxybenzaldehyde (400
mg, 2.2 mmol) in 50 ml CHCl3 (anhydrous) was
introduced into AlCl3 solution (0.89 g, 6.6 mmol)
at room temperature, and then heated to reflux for
1 h. The solution color was changed from colorless
to brown. After cooling down, the resulting solu-
tion was added 50 ml 2 N HCl and extracted with
ether twice. The organic layer was concentrated in
vacuo, and the residue was subjected to column
chromatography (ether:CH2Cl2:ethyl acetate 1:1:1)
2. Methods
2.1. Materials
1
to provide 2,4,5-THBA (206 mg, 56%); H NMR
2,5-Dihydroxybenzaldehyde (2,5-DHBA), 3,4-
dihydroxybenzaldehyde (3,4-DHBA), DPPH (1,1-
diphenyl-1,2-picryhydrazylradical), collagenase,
3 - (4,5 - dimethylthiazol - 2 - yl) - 2,5 - diphenyltetra-
zolium bromide (MTT), thiobarbituric acid, kits
for lactate dehydrogenase (LDH), aspartate amino-
trasferase (AST) and alanine aminotransferase
(ALT) were purchased from Sigma Chemical Co.,
St Louis, MO, USA. Solvents (E. Merck Co.,
Darmstadt, Germany) and medium for cell culture
(GIBCO BRL, Grand Island, NY, USA) other
reagents or dishes (Nunc, Denmark) were from
commercially available sources.
(400 MHz, DMSO) l 6.36 (s, 1 H), 6.98 (s, 1 H),
8.91 (s, 1 H), 9.87 (s, 1 H), 10.39 (s, 2 H); C NMR
(400 MHz, DMSO) l 104.6, 115.5, 115.8, 140.6,
156.1, 158.1, 191.6; Anal. Calcd for C7H6O4: C,
54.55; H, 3.92; O, 41.52; Found: C, 54.73; H, 3.93;
O, 41.46.
2.3. Determination of free radical-quenching
capacity
The free radical-quenching capacity of agents
was tested by a method of bleaching stable DPPH
(Ursini et al., 1994). A reaction mixture contain-

184
T.-H. Tseng et al. / Toxicology 161 (2001) 179–187
ing methanol (3 ml), DPPH (10 mM, 30 ml) and
agents (final concentration: 2, 5, 10 and 50 mM)
was allowed to stand at room temperature for 30
min before mixing with redistilled water (1 ml)
and toluene (3 ml). The solution was then cen-
trifuged, and the absorbence of the upper phase
was read at 517 nm against a blank without
agents, and processed alike.
medium was removed, and the activities of lactate
dehydrogenase (LDH), alanine aminotransferase
(ALT) or aspartate aminotransferase (AST) were
analyzed following the procedures provided by
the commercial kits (Sigma Chemical Co.). The
cytotoxicity of hepatocytes is expressed in terms
of the activity of LDH, ALT or AST released
from the treated cells.
2.4. Preparation of hepatocytes
2.7. Lipid peroxidation assay
Male Sprague–Dawley rats (250–300 g)
(Taichung Veterans General Hospital Animal
Center, Taiwan) were used for the experiments.
Hepatocytes were prepared by the method of
two-stage collagenase perfusion (Bonney et al.,
1974) and cultured in Williams’ E medium supple-
mented with antibiotic mixture of penicillin, strep-
tomycin and neomycin (1%), glutamine (1%), and
fetal calf serum (FCS) (10%), and gassed with
O2/CO2 (95%/5%). Cells were plated out at a
density of 1×106 cells/60 mm dish, and treated
with chemicals as indicated in the following assay
3 h after attachment.
Hepatocytes were pretreated with agents, and
then with t-BHP as described above. The lipid
peroxidation product-malondialdehyde (MDA)
was assayed according to an improved thiobarbi-
turic acid fluorometric method at 553 nm with
excitation at 515 nm using 1,1,3,3-te-
tramethoxypropane as the standard (Yagi, 1987).
The protein concentration was determined using a
standard commercial kit (Bio-Rad Lab. Ltd) with
bovine serum albumin (BSA) as a standard. The
results were expressed as MDA formation per mg
protein.
2.8. Cell line and cell culture
2.5. Microculture tetrazolium (MTT) assay
Human leukemia cells (HL-60), and human
nasopharynx carcinoma cells (KB cells) were
maintained in RPMI 1640 and human hepato-
blastoma (HepG2) cells in Dulbecco’s modified
Eagle medium (DMEM) supplemented with 10%
fetal bovine serum (FBS), 100 U/ml of penicillin,
The range of non-toxic dose levels of agents
was established using the MTT assay with some
modification (Alley et al., 1988). Briefly, the pri-
mary cultured rat hepatocytes at a density of
5×104 cells per dish were treated with agents at
various concentrations of 100–500 mM. After a
24-h incubation, medium was replaced by the one
containing 100 ml MTT (5 mg/ml) with phosphate
buffered saline (PBS) washing in between. The
cells were incubated for another 4 h, then the blue
crystals that are the metabolized product of MTT
were extracted by isopropanol. Absorbence at 563
nm was determined and used for the measurement
of the proportion of surviving cells.
100 mg/ml of streptomycin, and 2 mM
L-glu-
tamine. Cell lines were subcultured on day 3 and
were diluted 1 day before each experiment.
2.9. Statistical analysis
The results are reported as mean9S.D. from
individual determinations. Statistical differences
were analyzed according to Student’s t-test; sig-
nificant differences were established at PB0.05.
2.6. Cytotoxicity assay of hepatocytes
After pretreatment with agents for 0.5 h at the
final concentration of 0.01–0.1 mM, hepatocytes
were incubated with t-BHP (1.5 mM) for 0.5 h
(Tseng et al., 1996). Then, 1 ml of the culture
3. Results
2,4,5-THBA, 2,5-DHBA and 3,4-DHBA com-
pounds are phenolic compounds, and their an-

T.-H. Tseng et al. / Toxicology 161 (2001) 179–187
185
tioxidative effects were first determined in this
study. The result summarized in Table 1 showed
that 2,4,5-THBA, 2,5-DHBA, and 3,4-DHBA
were able to quench the DPPH free radicals in a
concentration-dependent manner. 2,4,5-THBA,
2,5-DHBA, and 3,4-DHBA at the concentration
of 5.1, 4.7, and 4.1 mM, respectively, quenched by
50% free radicals. In addition, 3,4-DHBA exhib-
ited the strongest antioxidant capacity in vitro at
5 mM by 71%. This assay indicates that 3,4- and
2,5-DHBA function as a radical inhibitor, bleach-
ing 97% of DPPH at 10 mM. We further evaluated
the range of non-toxic dose levels of 2,4,5-THBA
and DHBA analogues by MTT assay. The MTT
assay showed that 2,4,5-THBA and 3,4-DHBA,
up to 500 mM concentration, exhibited no toxic
effect to the primary culture of hepatocytes, but
2,5-DHBA showed significant cytotoxicity 31% at
500 mM (Table 2). Therefore, 10 and 100 mM
2,4,5-THBA and DHBA analogues were used for
the following cell culture experiments.
Adding 2,4,5-THBA and DHBA analogues to
the primary cultured hepatocytes effectively pro-
tected the cells from the cytotoxicity induced by
t-BHP as expressed by the leakage of LDH, ALT
and AST (Table 3). The pretreatment by three
agents significantly decreased the LDH, ALT, and
AST leakage of hepatocytes induced by t-BHP
(1.5 mM; for 30 min) at the concentration of
10–100 mM (PB0.05). In addition, lipid peroxi-
dation has been recognized as a potential mecha-
nism of cell injury. The MDA concentration, an
index of lipid peroxidation, was increased in the
hepatocytes treated with 1.5-mM t-BHP for 30
min alone. Pretreatment by 2,4,5-THBA and
DHBA compounds (10 and 100 mM) significantly
decrease the formation of MDA (PB0.05) (Table
4). The data indicated that the treatment of three
agents resulted in an effective inhibition of the
t-BHP-induced lipid peroxidation. At 10 mM
2,4,5-THBA, 3,4-DHBA, and 2,5-DHBA, the for-
mation of MDA was inhibited by 39, 57, and
85%, respectively.
nificant cytotoxicity on HL-60 cells only, which
were treated with different amounts of three
agents for 3 days. Table 7 showed that these three
agents presented a concentration-dependent in-
hibitory effect on HL-60 cells. Based on the MTT
assay resulted 2,4,5-THBA is the best in inducing
the cytotoxicity on the HL-60 cells. Further exam-
ination of 2,4,5-THBA in 2-20 mM to HL-60 cell,
the MTT assay resulted in Table 8. Treatment
with 2,4,5-THBA (2, 5, 10, and 20 mM) on HL-60
cells for 24 h, the cell survival of HL-60 was 93,
89, 79, and 29%, respectively.
4. Discussion
Recently we have investigated the antioxidant
and anti-tumor activity of caffeic acid phenethyl
ester (CAPE) analogues, and the result indicated
that CAPE may be acting as potential chemother-
apic agent against oral cancer (Chen et al., 1999;
Lee et al., 2000). Similarly, phenolic 2,4,5-THBA
and DHBA analogues might be expected to show
antioxidant and anti-tumor activity. To evaluate
them as chemopreventive or chemotherapy
agents, we started to test the free radical-quench-
ing capacity of 2,4,5-THBA and DHBA ana-
logues. For the determination of the free
radical-quenching capacity of 2,4,5-THBA and
DBHA analogues, the bleaching of DPPH in vitro
was primarily measured. These observations
demonstrated that 2,4,5-THBA and DHBA ana-
logues exhibited antioxidant bioactivity in the in
vitro test. Furthermore, we examined antioxidant
activity in primary hepatocytes under non-toxic
concentrations. The results showed that three
agents significantly decreased the LDH, ALT, and
AST leakage from hepatocytes induced by t-BHP.
After our evaluation, 2,4,5-THBA and DHBA
analogues exhibited an antioxidant effect against
the t-BHP-induced cytotoxicity probably via their
ability of quenching free radicals and decreasing
MDA formation, but the exact mechanism of
which will still need to be defined. However, it
implies that 2,4,5-THBA and 2,5-DHBA are able
to protect hepatocytes from the oxidative damage
caused by t-BHP. In our previous studies, PCA
showed strong antioxidant and antitumor promo-
Finally, in our investigation the cytotoxicities of
three agents on KB, Hep G2 and HL-60 were
studied by MTT assay. The MTT results were
shown in Table 5, Table 6, and Table 7. Treat-
ment with 10–100 mM concentration showed sig-

186
T.-H. Tseng et al. / Toxicology 161 (2001) 179–187
tion effects. We continued examining the cytotox-
icities of 2,4,5-THBA and DHBA analogues to
KB, Hep G2, and HL-60 cells. Fortunately, based
upon the MTT assay, 2,4,5-THBA is the strongest
for induce the cytotoxicity on the HL-60 cells. It
is worth noting that the treatment with 2 mM
2,4,5-THBA also exhibited significantly inhibitory
effect by 7% (PB0.05). These observations sug-
gest that 2,4,5-THBA may act as a potential
chemopreventor or chemotherapy for preferential
cytotoxicity on HL-60 cells. These observations
are consistent with our previous studies of PCA
which possess antioxidant is able to inhibit the
growth of tumor cells. Furthermore, Wick and
FitzGerald (1987) have reported that the antitu-
mor effects of biologic reducing agents related to
trometry facility Department of Chung-Hsing
University, for providing high-resolution NMR
spectra.
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