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Introduction
The hepatic drug metabolizing enzymes include phase I and phase II enzymes. In the phase I enzymes, cytochrome
p-450 is mainly responsible for the oxidation-reduction metabolic processing of most xenobiotics and also for the inactivation of
certain endogenous substances such as steroid hormones. Through phase I metabolism, the formed metabolites with polar
groups conjugate with glutathione (GSH) under the catalysis of glutathione
S-transferase (GST), and the formed complexes
are then excreted from the urine and bile. So, there are tight connections among
p-450, GST and GSH in the detoxification of the
body[1]. A number of compounds can affect
the p-450, GST and GSH, which results in alternations of the body
detoxification function. Our previous study demonstrated that the natural clausenamide, isolated from the leaves of Clausena
Lansium (Lour) Skeels[2], significantly protected against hepatotoxicity of
CCl4 and induced the hepatic cytochrome P-450
(P-450) in mice[3,4]. The authors further found that the synthetic nine clausena-mides and their ennatiomers could induce or
inhibit the liver p-450s and p-450-dependent enzymes in mice (data to be published). The purpose of the present paper
was to further study the effect of nine synthetic racemic mixtures of clausenamide, neoclausenamide and epineoclausenamide and
their corresponding ennatiomers (Figure 1) on liver GSH biosynthesis and GST activity in mice.
Materials and methods
Animals Male Kunming strain mice weighing
20±2 g were obtained from the Experimental Animal Center of Chinese
Academy of Medical Sciences, Beijing.
Chemicals Nine clausenamides were totally synthesized and kindly provided by Liang HUANG (Institute of Materia
Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing). The purity of each
compound was over 98%. They were suspended in 0.5%
Tween-80 before being administered to mice by gavage (ig).
Trihydro-methyl-aminomethane (Tris), sulfosalicylic acid, 3, 5-diamino-benzoic acid (DABA)
5,5-dithio-bis (2-nitrobenzoic acid (DTNB),
L-buthionine-[S, R]-sulfoximine
S-(n-butyl)-homo-cysteine sulfoximine
(L-BSO) were purchased from Sigma. All other reagents
used were of analytical grade.
Animal treatment Male mice were divided into groups of control, clausenamide, neoclausenamide,
epineoclausen-amide and their corresponding ennatiomers. The control group contained 6_10 mice, the other groups consisted of
5-8 mice each. The test compound (125 and 250 mg·10
mL-1·kg-1) was given to mice once daily for three
consecutive days. The control mice received 5% Tween-80 10 mL/kg. All mice were killed by decapitation 24 h after the last medication. The
livers were removed and weighed for preparation of cytosol. The rationale for choosing the dose of clausenamide as
well as the timing of administration was based on our study of the hepatoprotective action of clausenamides in
mice.
L-BSO was dissolved in 0.9% saline and adjusted to pH 8.5 with 0.1
mol/L NaOH before use. Mice were ip injected a dose
of 400 mg·20
mL-1·kg-1 of L-BSO. The control mice were ip injected saline 20 mL/kg.
Preparation of liver cytosol and determination of GST and
GSH The procedure for preparation of liver
cytosol[5], and the determination of
protein[6], liver cytosol GST
activity[7], and GSH
content[8], and glutathione peroxidase
(GSH-px)[9] and glutathione disulfide reductase
(GR)[10] were performed as described in related references.
Statistical analysis Data are expressed as mean±SD. Student’s
t-test was used to calculate the differences
between groups. P<0.05 was considered to be statistically significant.
Results
Effects of natural (±)clausenamide on liver GSH content and GST activity in
mice Oral administration of (±)clausena-mide 250
mg/kg to mice once daily for 3 d induced significant increases in liver cytosol GST activity and GSH content (Table
1).
Effects of synthesized clausenamide and its ennatiomers on liver cytosol GST activity and GSH content in
mice In this test, three groups of mice were treated with 125 mg/kg, 250 mg/kg of synthetic (±)clausenamide, (+)clausenamide,
(-)clausenamide once daily for 3 d, respectively. As a result, the three clausenamides showed different effects on GST and GSH
in mice. (±)Clausenamide, (+)clausenamide and
(-)clausenamide at doses of 250 mg/kg and 125 mg/kg all increased GST activity significantly. Only (+)clausenamide and
(±)clausenamide induced a remarkable increase of liver cytosol GSH. The liver GSH content of mice treated with synthetic
(+)clausenamide and natural (±)clausenamide was almost 2_3 folds of that of control mice. While (-)clausena-mide only at 250
mg/kg increased liver GSH content, and its potency to induce liver GSH is weaker than that of 125 mg/kg of (+)clausenamide.
The group of mice treated with the combined suspension of 125 mg/kg of both (+)clausenamide and (-)clausenamide, which
was mixed just before ig, also induced a significant increase of GSH content and GST activity. The potency of this mixture at
the same dosage was essentially corresponding to that of the synthesized (±)clausenamide (Table 1).
Effects of neoclausenamide and epineoclausenamide and their ennatiomers on liver cytosol GST activity and GSH in
mice Similar experiments for neoclausenamide and epineo-clausenamide and their ennatiomers were performed as clausenamide
ennatiomers. All of the six compounds induced a significant increase in GST activity, but none of the six test compounds
increased the liver cytosol GSH content (Table 2).
Effect of L-BSO on the increase of liver GSH content induced by (+)clausenamide in
mice L-BSO is a known specific inhibitor of
g-glutamyl cysteine synthetase (g-GCS), a limiting enzyme for the biosynthesis of GSH. To investigate whether
the increasing effect of (+)clausenamide on the liver GSH is caused by stimulating biosynthesis of GSH or not,
L-BSO was used as a tool to inhibit the g-GCS activity. Mice were administered 125 mg/kg of (+)clausenamide once daily for
different days. As a result, the liver GSH content
increased to the higher level at the second day, and did not
increase further at the 3rd day (data not shown). So, in the
L-BSO experiment, mice were ig administered with 125 mg/kg of (+)clausenamide, and simultaneously injected ip 400
mg/kg of L-BSO once daily for 2 d. Another two groups of mice were treated with (+)clausenamide 125 mg/kg or
L-BSO 400 mg/kg alone. The mice were killed on d 3. A piece of the liver and the left renal were removed for GSH determination
as shown in Table 3. L-BSO itself showed no significant effect on the liver GSH content, while it blocked
(+)clausenamide-induced increase of liver GSH content by 49%. (+)Clausenamide showed no increasing effect on renal GSH
content. Whereas L-BSO markedly reduced the renal GSH level. The above results suggest that the increase of liver GSH
content by (+)clausenamide is the result of stimulating GSH biosynthesis.
Effect of (+)clausenamide on the activity of liver GSH peroxidase (GSH-px) and glutathione reductase (GR) in
mice Both GSH-px and GR are involved in the redox cycle of GSH and GSSG. This experiment was to investigate whether the increase
of liver GSH content by (+)clausenamide is also related to GSHpx and GR, except for the
g-GCS. Mice were treated with 125 mg/kg of (+)clausenamide once daily for
2 d, the GSHpx and GR activities in the post-mitochondrial fraction of the livers were determined.
The (+)clausenamide significantly increased GR activity, although it did not induce a significant increase of GSHpx activity
(Table 4).
Discussion
The hepatic phase II drug metabolizing enzymes is also a very important part of the major intrinsic detoxicating
enzymes in the body, in which GSH conjugated with the formed polar metabolites under the catalysis of GST and the formed
complexes were then excreted from the urine and bile. So,
p-450, GST, and GSH are strongly connected in the detoxification
of the body[11_14]. Glutathione is very important in the maintenance of human
health[15]. From the present study, it may be
seen that the nine clausenamide ennatiomers exerted different inducing effects on liver
p-450, GST and GSH. The different effects depended upon the variation of chirac structures of racemic mixture and its corresponding ennatiomers of clausenamide
(5), neoclausenamide (6) and neoclausena-mide (16) as shown below (Figure 2).
The structural variation between clausenamide and epineoclausenamide as well as between neoclausenamide and
epineoclausenamide is at C5 and C6 with different configurations, respectively, which results in alternations of their action on
the liver p-450, GST, and GSH. In the aspect of liver GST and GSH, all of the nine test compounds were capable of inducing
liver GST activity, but their effect on the liver GSH content was completely different. Only clausenamide and its two
ennatiomers induced significant increases in liver GSH. In particular, (+)clausenamide was the most active. However, neither
neoclausenamide and epineoclausenamide nor their ennatiomers induced an increase of liver GSH content. It appears that
the cis-configuration of C4 and C5 in clausenamide is a determining structural factor for inducing an increase in liver GSH
content, 3R4S5S6R configuration of (+)clausenamide is superior to its 3S4R5R6S configuration of (-)clausenamide in
inducing liver GSH. It is known that the hepatic GSH biosynthesis is modulated by
multifactors[16], including: (1) the available
precursors for GSH synthesis, especially L-cysteine
(L-CysH); (2) the activity of g-glutamylcysteine synthetase
(g-GCS) and GSH-synthetase; (3) the rate of redox cycle of GSH and GSSG; and (4) the amount and rate of transportation of GSH and its
precursors such as L-CysH and g-glutamylcysteine
(g-GC) into and out from hepatocytes. g-GCS is the rate-limiting enzyme
in liver GSH biosynthesis. L-BSO, a specific inhibitor of
g-GCS, could markedly block the increase of liver GSH induced by
(+)clausenamide, indicating that the increasing effect of (+)clausenamide on liver GSH is mainly due to enhancing the activity of
g-GCS. However, both GSHpx and GR are involved in the redox cycle of GSH and GSSG. It is interesting to investigate
whether the increase of liver GSH content by (+)clausenamide is also related to GSH-px and GR beside the
g-GCS. The results showed that (+)clausena-mide significantly increased GR activity in the post-mitochondrial fraction of livers, but it did not
induce significant increase of GSH-px activity. The increase of GR by (+)-clau-senamide helps the hepatocytes to reduce
more GSSG to GSH, and thereby may partially increase the liver GSH content. The site of stimulatory action of (+)clausenamide
on liver GSH biosynthesis is illustrated as follows (Figure 3).
In summary, the effects of nine clausenamide ennatiomers on the liver GST and GSH varied with their spatial structures.
The C4 and C5 at cis-configuration of the clausenamide can be used particularly for determining structural factors that induce
liver GSH biosynthesis. Through this study we understand more of the structure-activity relationship of nine clausenamide
ennatiomers on liver GST and GSH. The findings of the present study also provide an example of the importance of different
influences of chirac chemicals on the body function. To answer how (+)clausenamide inhibited
g-GCS or increased GR
requires further study at a molecular level.
Acknowledgement
The authors are indebted to Prof Liang HUANG for her help in the preparation of the manuscript.
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