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TOXICOLOGY
Departments of Pharmacology (K.M.) and Molecular Bacteriology (H.A., Y.O.), School of Medicine, The University of Tokushima, Kuramoto, Tokushima, Japan
Received February 9, 2003; accepted April 1, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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; Ames et al.,
1993; Diplock et al.,
1998; Wang et al.,
1998). In addition to these actions, polyphenolic flavonoid
compounds have been shown to have the capacity to inhibit hepatocellular
cholesterol biosynthesis (Gebhardt,
1998). However, most studies of the actions of these phenolic
compounds have been carried out using the appropriate in vitro experimental
systems, which are considered favorable for investigating the underlying
molecular mechanisms, and hence it seems necessary to precisely characterize
and compare the actions of these phenolic compounds in the in vivo and in
vitro systems for evaluating their beneficial health effects.
Since polyphenolic flavonoid compounds have been shown to be rapidly
metabolized in the intestinal tract after ingestion
(Graefe et al., 1999;
Hollman and Katan, 1999;
Wiseman, 1999;
Gee et al., 2000), it seems
quite possible that the beneficial effects of these compounds may be
attributed fully or partly to the biological actions of their metabolites in
experimental animals and humans. Therefore, the biological activities of their
possible metabolites have been investigated in comparison with those of the
parent compounds. For example, the antioxidant activities, and inhibitory
actions on cholesterol biosynthesis, of quercetin and its possible metabolites
have been studied, and 4-methylcatechol, also called 3,4-dihydroxytoluene and
well known as a metabolite of quercetin produced in the intestinal tract after
ingestion, has been shown to inhibit both lipid peroxidation and cholesterol
biosynthesis as potently as the parent phenolic compounds in the primary
cultures of rat hepatocytes (Glässer
et al., 2002). On the other hand, the differences in the
biological actions between the parent flavonoid compounds and their
metabolites have been reported as well, and the cytotoxic effects of these
compounds are well known as such cases. Flavonoid compounds have been shown to
cause their cytotoxic effects on malignant cells in culture
(Chen et al., 1998;
Paschka et al., 1998;
Richter et al., 1999;
Russo et al., 1999;
Saeki et al., 1999;
Sergediene et al., 1999),
which proposes their protective effects against the development of cancer. In
addition, 4-methylcatechol, a metabolite of flavonoid compounds and also known
to be produced by the biodegradation of toluene in liver, has been shown to
cause the cytotoxic effect on several types of the cells in culture
(Ito et al., 1981;
Shen, 1998;
Shen et al., 2000). However,
despite the in vitro cytotoxicity, this compound has been reported to show
carcinogenic rather than carcinostatic activity in rat stomach (Hirose et al.,
1988,
1989;
Furihata et al., 1993;
Asakawa et al., 1994). In our
preliminary studies, the effect of 4-methylcatechol on the rate of tumor
growth in mice inoculated with B16-F10 melanoma cells was examined to assess
its carcinostatic action, but this compound failed to reveal any substantial
antitumor activity in these tumor-bearing animals. Thus, the in vitro
cytotoxicity of phenolic compounds is not always considered to be a positive
index of the in vivo carcinostatic action.
Recently, the cytotoxic effects of various chemicals and natural substances on malignant tumor cells in culture have been extensively studied as a primary screening for their antitumor activities, and hence it seems important and necessary to confirm the connection between the in vitro cytotoxic and in vivo antitumor activities. In this respect, it seems helpful for assessment of the antitumor activities of phenolic compounds to understand the reason for the inconsistency between the in vivo and in vitro effects of 4-methylcatechol. For this purpose, the in vitro cytotoxic effect of 4-methylcatechol on murine tumor cells was further investigated.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Cell Culture. Cells were maintained as monolayer cultures on a 60-mm culture dish in the growth medium [Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated bovine calf serum, 5% equine serum, 50 units/ml of penicillin, 50 µg/ml of streptomycin, and 50 µg/ml of gentamycin sulfate] at 37°C in a humidified incubator containing a 95% air/5% CO2 atmosphere.
Cell Growth and Viability. For the growth determination, the cells were plated on a 24-well cluster plate at a density of 2 x 104 cells/well and cultured for 24 h to allow the cells to attach to the bottom of the plastic plate. The medium was replaced with fresh growth medium and then cultured with or without 4-methylcatechol for different periods. For the determination of cell viability, the cells were plated at a density of 5 x 104 cells/well and cultured for 48 h. The medium was replaced with the serum-free medium (DMEM containing 50 units/ml of penicillin, 50 µg/ml of streptomycin, and 50 µg/ml of gentamycin sulfate, 5 µg/ml of insulin, 5 µg/ml of transferrin, and 5 ng/ml of sodium selenite), and the cells were cultured for 24 h to arrest the cell growth and then treated with various concentrations of 4-methylcatechol for 24 h in the serum-free medium.
Cell growth and viability were determined by measuring the amount of
neutral red taken up into the cells as reported previously
(Fautz et al., 1991;
Morita et al., 1999;
Morita and Wong, 2000).
Briefly, the cells were washed with saline solution and incubated in 0.5 ml of
DMEM containing neutral red (50 µg/ml) for 2 h in a humidified incubator.
Then, the cells were washed with saline solution and extracted with acidified
ethanol solution (50% ethanol/1% acetic acid) for 20 min at room temperature
with constant gentle shaking. The amount of neutral red taken up into the
cells was spectrophotometrically determined by measuring the optical density
at 540 nm, and the cell viability was calculated as a percent of the
control.
Cell viability was also determined by measuring the amount of total
protein. The cells were cultured for 24 h as described above, washed with
saline, and then dissolved in 0.5 ml of 1 M NaOH for 20 min at room
temperature with gentle shaking. The amount of total protein in an aliquot of
the extract was determined by the method of Bradford
(Bradford, 1976) using bovine
immunoglobulin G as a standard.
Numbers of viable and dead cells were determined using a trypan blue dye exclusion method. Briefly, the cells were cultured on a 35-mm dish and exposed to 4-methylcatechol in the growth medium. Both attached and floating cells were collected by trypsinization, and an aliquot of the cells were mixed with an equal volume of trypan blue solution. The cells excluding dye (viable cells) and those taking up dye (dead cells) were counted in duplicate using a hemocytometer, and the numbers of these cells were expressed as the percent of total cell number.
Apoptotic Cell Damage. For the fluorescence cytochemical study, the cells were plated on a poly-D-lysine-coated 35-mm culture dish at a density of 1 x 104 cells/dish and treated with 4-methylcatechol for 24 h as described above. The medium was removed by aspiration, and the cells were rinsed with phosphate-buffered saline and fixed with methanol/acetic acid solution (3:1) for 1 h at room temperature. The fixed cells were incubated in fluorescence dye solution (5 µg/ml of propidium iodide and 50 µg/ml of acridine orange in phosphate-buffered saline) for 1 h at room temperature and then examined by an inverted fluorescence microscope.
For the DNA fragmentation analysis, the cells were plated on a 60-mm
culture dish at a density of 5 x 105 cells/dish and treated
with 4-methylcatechol for 24 h as described above. The cells attached at the
bottom were scraped off and collected together with unattached cells by
centrifuging at 1500g for 5 min at 4°C. The DNA was prepared from
the pelleted cells and applied to a 1.8% agarose gel containing 0.5 µg/ml
of ethidium bromide for the electrophoretic DNA analysis as described
previously (Morita et al.,
1999; Morita and Wong,
2000).
For the caspase-3 assay, the cells were plated on a 24-well cluster plate
at a density of 5 x 104 cells/well and treated with
4-methylcatechol for 6 h as described above. The medium was removed, and the
cells were rinsed with ice-cold saline and then lysed with the extraction
buffer [50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 150 mM NaCl, and 0.5% Nonidet
P-40] followed by a freeze-thaw cycle. The lysate was passed 20 to 25 times
through a blue pipetter tip and then centrifuged at 150,000 rpm
(20,000g) for 20 min at 4°C. The caspase-3 activity in the
supernatant fraction was then determined as reported previously
(LaRue et al., 2000). An
aliquot (50 µl) of the supernatant fraction was mixed with 150 µl of the
reaction buffer (20 mM HEPES, 10% glycerol, and 2 mM dithiothreitol)
containing 10 µl of 1 mM acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin in
an enzyme-linked immunosorbent assay plate microwell. The mixture was
incubated for2hat room temperature under the light-protecting conditions, the
fluorescence intensity was measured at 365 (Ex) to 450 nm (Em) using a
microplate reader, and the enzyme activity was expressed as a percent of the
control.
Hydrogen Peroxide Generation. Cells were plated on one half of a
24-well cluster plate at a density of 5 x 104 cells/well, and
the growth medium only was added to the other half. The cells were cultured as
described above and incubated with various concentrations of 4-methylcatechol
for 2 h. The medium was collected and centrifuged at 15,000 rpm
(20,000g) for 20 min to remove the floating cells and cell fragments.
The concentration of hydrogen peroxide in the supernatant fraction was
determined based on the method reported previously
(Nourooz-Zadeh et al., 1994)
using PeroXOquant quantitative peroxide assay kit.
Data Analysis. Results were presented as the mean ± S.E. Data were analyzed by an analysis of variance followed by Tukey's post hoc test. A p value of <0.05 was accepted as a statistically significant difference.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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To characterize the effect of 4-methylcatechol on mouse B16 melanoma cells, the cells were exposed to 100 µM of the drug for 24 h, and the numbers of viable and dead cells were determined. As shown in Fig. 2, the percentage of viable cells was slightly reduced by treatment of these cells with 4-methylcatechol (approximately 91 and 76% in the control and treated groups, respectively), whereas the percentage of dead cells in the treated group was approximately 260% higher than that in the control. Consistent with the results presented in Fig. 1, an approximately 50% reduction of neutral red uptake was also observed in the separate experiments (data not shown). Even though the discrepancy in the reduction of cell viabilities estimated by the trypan blue exclusion and neutral red uptake methods was observed, these results seemed to indicate that 4-methylcatechol might be able to induce the cytotoxic damage to B16 cells in culture.
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To further characterize the cytotoxic effect of 4-methylcatechol on mouse B16 melanoma cells, the cell growth was arrested by maintaining them in the serum-deprived culture medium for 24 h, and the effect of 4-methylcatechol on these cells was examined under the serum-free conditions. As shown in Fig. 3, the reduction of neutral red uptake was observed in a manner dependent on the concentration of 4-methylcatechol. In addition, 4-methylcatechol also caused the reduction of total protein amount, similar in extent to the reduction of dye uptake. These results indicated that the reduction of neutral red uptake was not due to the impairment of dye transport across the plasma membrane of these cells but directly reflected a decrease in the number of viable cells, providing evidence that 4-methylcatechol could exert cytocidal rather than cytostatic action on B16 cells in culture.
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To test the susceptibilities of other murine tumor cells to the cytotoxic effect of 4-methylcatechol, mouse LL/2 Lewis lung carcinoma cells, rat C6 glioma cells, and rat PC12 pheochromocytoma cells were exposed to different concentrations of the drug for 24 h under the serum-free culture conditions, and their viabilities were determined by measuring the uptake of neutral red into these cells. As shown in Fig. 4, these cells were susceptible to 4-methylcatechol, with slight differences in their susceptibilities. The cytotoxic effect of 4-methylcatechol on C6 cells and LL/2 cells was almost similar to that on B16 cells, and PC12 cells showed the slightly less but substantially similar susceptibility to the drug as compared with that of B16 cells. These results were thought to indicate that 4-methylcatechol might induce the damage to murine tumor cells, resulting in cell death through the common process to these cells.
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To investigate the manner of cell death induced by 4-methylcatechol, B16 melanoma cells were exposed to the drug, and the assessment of apoptotic cell death was then carried out using fluorescence dye staining, DNA fragmentation analysis, and caspase-3 assay. Morphological study showed that B16 cells were uniformly stained with acridine orange (green color) in the control group, and the cells with dots of condensed chromatin were observed in the treated group, whereas the cells stained with propidium iodide (red color) were not detected in both the control and treated groups (Fig. 5a). Electrophoretic analysis also showed that 4-methylcatechol induced the degradation of DNA into nucleosomic fragments at the concentration range required to reduce the cell viability (Fig. 5b). In the control, there was no fluorescent signal observed in the region of agarose gel ranging from 100 to 500 base pair(s), whereas the signal appeared in this region by exposure of the cells to 4-MC. This signal was intensified by increasing the drug concentration from 50 to 100 µM, but the signal at a higher dose (250 µM) was less intense in comparison with that observed at a lower dose (100 µM). In contrast, the fluorescence in the region ranging lower than 100 base pair(s) increased according to the drug concentration. Therefore, it seemed possible that the degradation of DNA to nucleosomic fragments might be induced by the exposure to 4-MC, and the further degradation of DNA fragments could occur in the presence of higher concentrations of the drug. Moreover, the elevation of caspase-3 activity was observed by short-term exposure of the cells to the drug (Fig. 5c). These results indicated that 4-methylcatechol reduced the viability of B16 cells as a result of inducing the apoptotic cell death under the conditions used here.
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Phenolic compounds are generally known to show not only their antioxidative effects but also pro-oxidant actions under the in vitro assay conditions, and hence it seemed possible that 4-methylcatechol might exert its pro-oxidant action on the cells in culture. To test this possibility, the cytotoxicity of 4-methylcatechol on B16 melanoma cells was assessed in the presence of a hydroperoxide scavenger, e.g., catalase or GSH. As shown in Fig. 6, the reduction of cell viability induced by 4-methylcatechol was blocked by catalase and GSH at the concentrations of 500 units/ml and 2 mM, respectively. In addition, the generation of hydrogen peroxide during the incubation of 4-methylcatechol in the culture of B16 cells was determined to directly examine its pro-oxidant action. As shown in Fig. 7, the concentration of hydrogen peroxide in the cell culture was increasing in a concentration-dependent manner, and the substantial generation of hydrogen peroxide was also observed even by incubating the drug in the medium without the cells. These results indicated that 4-methylcatechol could be oxidized in the cell culture and exerted prooxidant action on the cells.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The viabilities of murine tumor cells were shown to be reduced by the exposure of these cells to 4-methylcatechol in a concentration-dependent manner, and the cytotoxic effect was observed to be nonselective about their original tissues and species. Further studies showed that characteristics of the apoptotic cell damage, such as chromatin condensation, DNA fragmentation, and caspase-3 activation, were observed in B16 melanoma cells treated with 4-methylcatechol (Fig. 5). Together, these observations clearly indicate that 4-methylcatechol induces the apoptotic damage to murine tumor cells in culture, resulting in the reduction of their viabilities under the in vitro experimental conditions. In general, the apoptotic cell death is considered to be mostly induced by oxidative insults, and hence it seems conceivable that the cytotoxic effect of 4-methylcatechol observed here may be the result of the oxidative damage to the cells. Then, the effect of 4-methylcatechol on the viability of B16 melanoma cells was again examined in the presence of catalase and GSH, and these hydroperoxide scavengers were shown to successfully protect the cells against the cytotoxic effect of this drug (Fig. 6). Therefore, it seems likely that the cytotoxicity of 4-methylcatechol observed here may be due to the toxic effect of hydrogen peroxide on B16 melanoma cells in culture.
Previously, quercetin and related phenolic compounds containing a catechol
moiety in their chemical structures have been shown to be oxidized, resulting
in lipid peroxidation under certain in vitro assay conditions
(Laughton et al., 1989;
Yamanaka et al., 1997;
Galati et al., 1999). Recent
studies have also shown that these compounds can be oxidized and can rapidly
generate hydrogen peroxide in commonly used cell culture media
(Long et al., 2000). Moreover,
both DOPA and dopamine have been shown to undergo oxidation to generate
hydrogen peroxide and semiquinones/quinones by interacting with commonly used
culture media (Clement et al.,
2002). Based on these previous findings, it seems reasonable to
consider that catechol compounds can exert oxidative damage to the cells as a
result of generating hydrogen peroxide in the culture medium, and hence the
generation of hydrogen peroxide from 4-methylcatechol was examined by
incubating the drug in the medium with or without B16 melanoma cells.
Considerable amounts of hydrogen peroxide were shown to be generated by
incubating 4-methylcatechol in the cell culture, and the substantial
generation of hydrogen peroxide was also observed even by incubating the drug
in the culture medium without the cells
(Fig. 7). In addition to the
generation of hydrogen peroxide observed here, it seems conceivable that,
similar to DOPA and dopamine, 4-methylcatechol may have the property of
generating semiquinones/quinones by interacting with cells and/or unidentified
constituent(s) in the culture medium. Thus, it is likely that 4-methylcatechol
can be oxidized and can generate hydrogen peroxide, and probably
semiquinones/quinones, in the cell culture, resulting in the oxidative damage
to murine tumor cells in vitro.
In summary, 4-methylcatechol is shown to induce the apoptotic damage to murine tumor cells as a result of generating hydroperoxides in the cell culture, suggesting that the cytotoxic effect of this drug may be attributed to its prooxidant action on the cells. This may be able to account for the discrepancy between the in vitro cytotoxic and the in vivo antitumor activities of 4-methylcatechol and its derivatives. Recently, as a primary screening for the antitumor activity, the cytotoxic effects of various compounds on malignant tumor cells have been studied using the in vitro culture system. However, the results presented here suggest that caution is required when the in vitro cytotoxicity is assessed to predict the carcinostatic actions of phenolic compounds in vivo.
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Footnotes |
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ABBREVIATIONS: GSH, reduced-form glutathione; DMEM, Dulbecco's modified Eagle's medium; 4-MC, 4-methylcatechol.
Address correspondence to: Dr. Kyoji Morita, Department of Pharmacology, Tokushima University School of Medicine, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. E-mail: km{at}basic.med.tokushima-u.ac.jp
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, significantly different from control (p
< 0.05).
