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Protective effect of aloe extract against the cytotoxicity of 1,4-
naphthoquinone in isolated rat hepatocytes involves modulations in cellular
thiol levels
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C Pharmacology & Toxicology 2002, 90, 278–284. Copyright C
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ISSN 0901-9928
Protective Effect of Aloe Extract against the Cytotoxicity of
1,4-Naphthoquinone in Isolated Rat Hepatocytes Involves
Modulations in Cellular Thiol Levels
Toshio Norikura1, David O. Kennedy1, Alexander K. Nyarko2, Akiko Kojima1 and Isao Matsui-Yuasa1
1Department of Food and Human Health Sciences, Graduate School of Human Life Science, Osaka City University,
3–3–138 Sugimoto, Sumiyoshi-ku, Osaka 558–8585, Japan, and 2)Chemical Pathology Unit, Noguchi Memorial Institute
for Medical Research, University of Ghana, P.O.Box LG 581, Legon, Accra, Ghana
(Received June 19, 2001; Accepted January 10, 2002)
Abstract: Aloe is a familiar ingredient in a wide range of health care and cosmetic products and has been reported to
possess various physiological effects, antioxidative, anticarcinogenic, antiinflammatory and laxative. Aloe has also been
reported to have an effect on liver function. The cytoprotective effect of aloe extract against 1,4-naphthoquinone-induced
hepatotoxicity was evaluated in primary cultured rat hepatocytes. After exposure to 1,4-naphthoquinone (100 mM), a
decrease in cell viability measured as ±60% lactate dehydrogenase depletion was induced. Cellular glutathione (GSH)
and protein-SH levels were also significantly decreased in a time-dependent manner. However addition of aloe extract
resulted in a dose-dependent improvement of these effects. This cytoprotective effect of aloe could be attributed to its
inhibition of GSH and protein-SH depletions. The effect of the aloe extracts were also dose-dependent. Addition of
diethyl maleate (1 mM), a cellular glutathione-depleting agent, to hepatocytes treated with both 1,4-naphthoquinone and
aloe extract, induced depletion of GSH, but did not affect protein-SH or lactate dehydrogenase. These results suggest that
the 1,4-naphthoquinone-induced toxicity in rat hepatocytes was inhibited by aloe extract, and that this protective effect
was due to the maintenance of cellular thiols, especially protein-SH.
Aloes have been used therapeutically since Roman times chemical reactivities and mechanisms of cell killing by indi-
and perhaps long before (Morton 1961). It is now a familiar vidual quinones differ (Henry & Wallace 1996). Quinonoid
ingredient in a wide range of health care and cosmetic prod- compounds can exist in various redox states and participate
ucts. Aloes have been reported to possess various physio- in electron transport processes and photosynthesis and also
logical effects, such as antioxidative (Malterud et al. 1990; act as environmental pollutants in atmospheric particulate
Lee et al. 2000), anticarcinogenic (Hyung & Byung 1997; matter and cigarette smoke, thus they are of general toxico-
Corsi et al. 1998), anti-inflammatory (Udupa et al. 1994; logical relevance (O’Brien 1991).
Hutter et al. 1996) and laxative (Schorkhuber et al. 1988; Thiol groups play a complex role in biological systems
Ishii et al. 1998). Administration of aloe has also been re- and, in particular, glutathione (GSH) exhibits coenzymatic,
ported to have an effect on liver function. For example, regulatory, protective, and reparative roles. GSH levels of
after oral administration of ethanol to rats, aloe caused a tissues decrease in response to oxidative stress caused by
faster elimination of ethanol in blood. Other studies have radiation, chemical compounds, drugs, hyperoxia, and isch-
shown that this rapid elimination effect of aloe is due to aemia/reperfusion (Ishikawa & Sies 1984; Curello et al.
the supply of nicotinamide dinucleotide and a protection of 1985). Similarly, free sulfydryl groups in proteins are highly
alcohol dehydrogenase in liver (Sakai et al. 1989). Adminis- reactive functional groups in biological systems that partici-
tration of aloe to normal mice induced metallothionein, pate in several different reactions, such as alkylation, aryl-
which acts as radical scavenger in liver (Sato et al. 1990). ation, oxidation, thiol-disulfide exchange, etc. Therefore,
Quinones are a widely distributed class of compounds modification of protein thiol groups can result in severe
that occur naturally in many biological systems. The cyto- functional damage, including loss of enzyme activity (Or-
toxicity of quinones has been shown in several studies (Di- renius 1995).
Monte et al. 1984; O’Brien 1991), and provides a suitable We have previously shown that a green tea extract pro-
experimental model for cytotoxicity studies. However, the tected against liver injury induced by 1,4-naphthoquinone
(Miyagawa et al. 1997) and taurine protected liver injury
caused by carbon tetrachloride (Wu et al. 1999) due to
Author for correspondence: Isao Matsui-Yuasa, Department of maintenance of protein thiol levels. In this study, we exam-
Food and Human Health Sciences, Graduate School of Human Life
Science, Osaka City University, 3–3–138 Sugimoto, Sumiyoshi-ku, ined the effect of aloe extract on liver injury induced by 1,4-
Osaka 558–8585, Japan (fax π81 6 6605 2810, e-mail yuasa/life. naphthoquinone in primary cultured rat hepatocytes and
osaka-cu.ac.jp). assessed the involvement of cellular thiol levels. Primary
ALOE PROTECTS AGAINST 1,4-NAPHTHOQUINONE CYTOTOXICITY 279
cultured rat hepatocytes represent a good in vitro experi-
mental model that preserves its differentiated character-
istics.
Materials and Methods
Materials. Aloe powder was purchased from Maruho Pharmaceut-
icals (Osaka, Japan). This powder was a mixture of Aloe ferox
Miller, Aloe africana Miller and Aloe spicata Baker. Collagenase,
sodium pyruvate, 5-sulfosalicylic acid dihydrate, 5,5’-dithio-bis-2-
nitrobenzoic acid were obtained from WAKO Pure Chemical Indus-
tries, Ltd (Osaka, Japan). NADH and GSH (reduced forms) were
purchased from Sigma-Aldrich Fine Chemicals (Tokyo, Japan). 1,4-
naphthoquinone was obtained from Tokyo Kasei Co. (Tokyo,
Japan).
Extraction. Aloe powder (1 g) was mixed with twenty parts of 50%
ethanol for 24 hr. This was centrifuged at 11,400¿g for 10 min.
Ethanol in the supernatant was evaporated and the remaining resi-
due was freeze-dried. To this residue was added 10 parts of water
followed by mixing for 3 hr, and subsequent centrifugation at
11,400¿g for 10 min. The supernatant was removed for freeze-dry- Fig. 2. Effect of aloe extract on 1,4-naphthoquinone (NQ)-induced
ing to yield the 50% ethanol fraction. The 50% ethanol fraction cytotoxicity. Isolated hepatocytes were incubated with or without
was suspended in water and further extracted with chloroform. The NQ (100 mM), and further treated with the 50% ethanol fraction of
residual water fraction was extracted with ethyl acetate followed by aloe (50% EtOH) for 2 hr. Lactate dehydrogenase (LDH) activity
butanol, in that order. Each of the fractions was evaporated to dry- in the suspension buffer was measured, as described in Materials
ness in vacuo (fig. 1). and Methods, to monitor the degree of cell injury. Results show
mean∫S.D. of three different determinations. *P,0.05, **P,0.01,
Hepatocyte preparation and culture. The Animal Research Commit- compared with NQ-treated cultures.
tee of the Osaka City University approved the protocol for this
experiment, and care of the animals was in accordance with the
standards of this institution (Guide for Animal Experimentation, 1,4-naphthoquinone (100 mM) or 3 ml carbon tetrachloride/2 ml me-
Osaka City University). Hepatocytes were isolated from male dium. Carbon tetrachloride was dissolved in dimethyl sulfoxide (3:5
Sprague-Dawley rats weighing 200–250 g by collagenase perfusion by volume) and this solution was dissolved in Hank’s buffer. The
(Moldeus et al. 1993). The viability of the isolated hepatocytes was incubation buffer was changed to Hank’s buffer because this did
over 90% as determined by 0.2% trypan blue exclusion. The cells not interfere with other factors such as amino acids in this experi-
were plated in 35 mm plastic dishes at a density of 2.5¿105 cells/ml ment. The diethyl maleate concentration used was on the basis of
in 2 ml Williams’ Medium E supplemented with 10% foetal bovine literature (Wu et al. 1999) and also from pilot experiments. The
serum, 0.1 mM insulin and 1 mM dexamethasone, and were cultured suspension buffer was used for assay of lactate dehydrogenase activ-
in humidified atmosphere of 5% CO2 and 95% air at 37æ overnight. ity, and the cells were used for assay of cell viability, GSH and
The medium was replaced the next day with Hank’s buffer with or protein-SH concentrations.
without aloe extract, diethyl maleate (1.0 mM), and incubated with
Cell viability was measured by the Neutral Red assay as described
previously (Zhang et al. 1990). Neutral Red stock solution (0.4%
Neutral Red in water) was diluted 1:80 in phosphate-buffered saline.
Hepatocytes were incubated with the Neutral Red solution for 2 hr
at 37æ to allow the lysosomes of viable cells to take up the dye. The
Table 1.
Effect of ethyl acetate fraction of aloe (EA) on carbon tetrachloride
(CCl4)-induced cytotoxicity. EA (100 mg/ml) was used to assay pre-
vention of cell damage induced of CCl4 by lactate dehydrogenase
(LDH) leakage and cell viability by Neutral Red assay. Isolated
hepatocytes were incubated with or without 3 ml CCl4/2 ml medium
and further treated with the EA for 2 hr. Control and CCl4 cultures
contain dimethyl sulfoxide (final concentration was 0.05%) and me-
dium. Results show mean∫S.D. of three different determinations.
Data not sharing common alphabet are significantly different.
***P∞0.01.
Released LDH Cell viability
(U/L) (%)
Control 30.0∫7.8a 100.0∫4.1 a
CCl4 463.7∫99.5b 35.1∫2.0b
a a
Fig. 1. Method of extraction of aloe fractions. CCl4πEA 83.3∫48.6 100.5∫2.1
280 TOSHIO NORIKURA ET AL.
with 0.5 ml 2% 5-sulfosalicylic acid and briefly sonicated and centri-
fuged at 11,400¿g for 10 min. The supernatant obtained was used
for the assay of intracellular GSH concentration and the cell pellet
was used for the determination of protein-SH. Briefly, the pellet was
suspended in 1.0 ml 0.5 M Tris-HCl, pH 7.6, and sonicated. 5,5ø-
dithio-bis-2-nitrobenzoic acid (at a final concentration of 100 mM)
was then added and after 20 min. absorbance was measured at 412
nm. Data were expressed as nmols SH/5¿105 cells, calculated on
the basis of a GSH calibration curve.
Statistical analysis. Data are expressed as mean∫S.D. The signifi-
cance of difference in assay values was evaluated with ANOVA fol-
lowed by Bonferroni/Dunn test. A p value of less than 0.05 was
considered significant.
Results
Effect of aloe extract on 1,4-naphthoquinone-induced cyto-
toxicity.
Isolated hepatocytes incubated with 100 mM 1,4-naphtho-
quinone induced a release of lactate dehydrogenase. Ad-
dition of 50% ethanol fraction of aloe, however, prevented
this in a dose-dependent manner (fig. 2). The 50% ethanol
Fig. 3. Effect of various fractions of aloe extract on 1,4-naphthoqui-
none (NQ)-induced cytotoxicity. 50% ethanol fraction (50% EtOH),
butanol fraction (Butanol), water fraction (Water) and ascorbic acid
were dissolved in water and chloroform fraction (Chloroform),
ethyl acetate fraction (EA) and a-tocopherol were dissolved in di-
methyl sulfoxide (DMSO). Concentration of DMSO in medium was
0.05%. Each fraction and antioxidants (100 mg/ml) were assayed to
determine which aloe fraction prevented cell damage induced by
NQ (100 mM) by lactate dehydrogenase (LDH) leakage and cell
viability by Neutral Red assay. Cell culture treatment was as in Ma-
terials and Methods. Results show mean∫S.D. of four different de-
terminations. *P,0.05, **P,0.01
Neutral Red solution was then removed and the cultures were
washed rapidly (in less than 2.5 min.) with a mixture of 1% form-
aldehyde-1% calcium chloride. A mixture of 1% acetic acid-50%
ethanol was added to the cells to extract the Neutral Red from
hepatocytes at room temperature for 30 min. Each sample was then
measured at 540 nm with a spectrophotometer (Beckman DU-640).
Lactate dehydrogenase assay. Lactate dehydrogenase activity in the
suspension buffer was measured as described previously (Bergmeyer
et al. 1965) to monitor the degree of cell injury. The assay solution
contained 0.6 mM sodium pyruvate, 0.18 mM NADH and a suit-
able volume of the enzyme solution at 25æ in a total volume of
3.15 ml. The initial rate of NADH loss, measured as a reduction in
absorbance at 340 nm, was used as an indication of lactate dehydro- Fig. 4. Dose-dependent cytoprotective effect of the ethyl acetate
genase activity. Under this assay condition, the loss of NADH was fraction of aloe (EA) against 1,4-naphthoquinone (NQ), induced
linear with respect to time and enzyme concentration over the range cytotoxicity. EA was used to assay prevention of cell damage in-
of enzyme activity monitored. duced by NQ (100 mM). Cell culture treatment was as in legend
of fig. 3. Control and ‘‘0’’ concentration cultures contain dimethyl
Cellular GSH and protein-SH assay. Cellular GSH and protein-SH sulfoxide (final concentration was 0.05%) and medium. Results
concentrations were measured by the method described by Beutler show mean∫S.D. of four different determinations. *P,0.01, com-
et al. (1963). After removing the medium, cells (5¿105) were treated pared with NQ treated cultures.
ALOE PROTECTS AGAINST 1,4-NAPHTHOQUINONE CYTOTOXICITY 281
nificantly inhibited by the addition of ethyl acetate fraction
of aloe (100 mg/ml) (table 1).
Effect of aloe extract on 1,4-naphthoquinone-induced GSH
and protein-SH depletion.
GSH and protein-SH levels were measured to examine the
effect of aloe extract on the cytotoxicity induced by 1,4-
naphthoquinone. As shown in figs. 5 and 6, the addition of
1,4-naphthoquinone to hepatocytes resulted in an extensive
loss of GSH and protein-SH. Both losses were inhibited by
the addition of the 50% ethanol and ethyl acetate fractions
of aloe in a dose-dependent manner. In order to examine
the relationship between cell injury and cellular thiols, we
measured the time course of 1,4-naphthoquinone-induced
lactate dehydrogenase leakage and loss of GSH and protein-
SH. As shown in fig. 7, the loss of GSH and protein-SH
began before a significant leakage of lactate dehydrogenase
was observed. The addition of 50% ethanol fraction of aloe
inhibited the loss of GSH and protein-SH at all times meas-
ured.
Fig. 5. Effect of 50% ethanol fraction of aloe (50% EtOH) on 1,4-
naphthoquinone (NQ)-induced GSH and protein-SH depletion.
GSH and protein-SH levels were measured to examine the effect of
50% EtOH on the cytotoxicity induced by NQ (100 mM). Cellular
GSH and protein-SH concentrations were measured as described in
Materials and Methods. Data were expressed as nmols SH/5¿105
cells, calculated on the basis of a GSH calibration curve. Results
show mean∫S.D. of five different determinations. *P,0.05,
**P,0.01, compared with NQ-treated cultures.
fraction was further extracted with chloroform, ethyl ace-
tate and butanol, and each fraction assayed to determine
which aloe fraction prevented the cell damage induced by
1,4-naphthoquinone. All fractions showed significant cyto-
protective effect but a much stronger effect close to the con-
trol value was observed with the 50% ethanol and ethyl ace-
tate fractions (fig. 3). The majority of and the dose-depend-
ent nature of the cytoprotective effect observed with the
50% ethanol fraction was retained with the ethyl acetate
fraction (fig. 4). Aloe extracts were more effective than as-
corbic acid or a-tocopherol (fig. 3).
Effect of aloe extract on carbon tetrachloride-induced cyto-
toxicity. Fig. 6. Effect of ethyl acetate fraction of aloe (EA) on 1,4-naphtho-
quinone (NQ)-induced GSH and protein-SH depletion. GSH and
To examine the cytoprotective effect of aloe extracts against protein-SH levels were measured to examine EA on the cytotoxicity
other hepatotoxin-induced hepatocyte injury, we used car- induced by NQ (100 mM). Data were expressed as nmols SH/5¿105
bon tetrachloride as a cytotoxic agent. Carbon tetrachloride cells, calculated on the basis of a GSH calibration curve. Control
and ‘‘0’’ concentration contain dimethyl sulfoxide (final concen-
(3 ml/2 ml) induced hepatocyte injury in isolated rat hepato- tration was 0.05%) and medium. Results show mean∫S.D. of four
cytes as shown by lactate dehydrogenase leakage and a loss different determinations. *P,0.05, **P,0.01, compared with NQ-
of cell viability. However this hepatocytes injury was sig- treated cultures.
282 TOSHIO NORIKURA ET AL.
fraction of aloe, it seems that the effect of ethyl acetate
fraction of aloe is related to protein-SH modulations.
Discussion
The results obtained in the present study demonstrate for
the first time that aloe extract had a cytoprotective effect
on 1,4-naphthoquinone-induced hepatotoxicity in a cellular
thiol-dependent way. We also demonstrated that the ethyl
acetate fraction of aloe showed a most profound cytopro-
tective effect in a dose-dependent manner. This protective
effect of aloe extract was also shown on carbon tetra-
chloride-induced cytotoxicity. However, the identities of the
effective compounds in the ethyl acetate fraction of aloe
extract are still not clear.
Fig. 7. Kinetics of the effect of 50% ethanol fraction of aloe (50%
EtOH) on 1,4-naphthoquinone (NQ)-induced lactate dehydrogen-
ase (LDH) leakage and loss of GSH and protein-SH. LDH leakage,
GSH and protein-SH levels were measured to examine the time
course effect of 50% EtOH (100 mg/ml) on the cytotoxicity induced
by NQ (100 mM). LDH, cellular GSH and protein-SH concen-
trations were measured as described in Materials and Methods. Re-
sults show mean∫S.D. of four different determinations. **P,0.01,
compared with NQ-treated cultures.
Relationship between cell injury and cellular thiols.
In order to examine in detail the relationship between lac-
tate dehydrogenase leakage and cellular thiols, we used di-
ethyl maleate, a cellular thiol-depleting agent, in the follow-
ing studies (fig. 8). It was observed that the ethyl acetate
fraction of aloe reversed the increased lactate dehydrogen-
ase release induced by 1,4-naphthoquinone, and these rever-
sals were associated with both protein-SH and GSH. How-
ever, when treating these cultures with diethyl maleate, the Fig. 8. Relationship between cell injury and cellular thiols. In order
to examine in detail the relationship between lactate dehydrogenase
reversal effect of ethyl acetate fraction of aloe was not (LDH) leakage and cellular thiols, we used diethyl maleate (DEM,
quenched, but GSH levels, rather than protein-SH levels, 1 mM), a cellular thiols depleting agent, in an attempt to find the
decreased significantly. Diethyl maleate was used in an effect of depleting thiol groups in cultures after the ethyl acetate
attempt to find the effect of depleting thiol groups in cul- fraction of aloe (EA, 100 mg/ml) had reversed the thiol levels de-
pleted by 1,4-naphthoquinone (NQ, 100 mM). Control, DEM and
tures after the aloe extract had reversed the thiol levels de- NQ cultures contain dimethyl sulfoxide (final concentration was
pleted by 1,4-naphthoquinone. Since the reversal in lactate 0.05%). Results show mean∫S.D. of four different determinations.
dehydrogenase leakage was observed in the ethyl acetate **P,0.01.
ALOE PROTECTS AGAINST 1,4-NAPHTHOQUINONE CYTOTOXICITY 283
Furthermore, leakage of lactate dehydrogenase and de- Acknowledgements
pletion of GSH and protein-SH induced by 1,4-naphtho- This study was conducted with funds from the Ministry
quinone were inhibited by addition of aloe extract. We have of Education, Science, Culture and Sports, Japan, which
previously shown that a green tea extract protected against are gratefully acknowledged.
liver injury induced by 1,4-naphthoquinone due to mainten-
ance of cellular protein-SH levels (Miyagawa et al. 1997).
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