XENOBIOTICA, 1996, VOL.26, NO.

7, 667-680

Cytotoxicity and cytoprotective activities of natural

compounds. The case of curcumin

J. N. M . COMMANDEUR" and N . P. E. VERMEULEN

Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology,
Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV
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Amsterdam, T h e Netherlands

Received 30 November 1995

Introduction
Many pharmaceutical drugs exhibit undesired side-effects in patients in addition
to their desired pharmalogical activity. These side-effects may either exist as
undesired pharmacological activities, due to non-selectivity of the drug towards
pharmacological receptors or to toxicity as a result of metabolic bioactivation to
reactive intermediates which are capable of covalent binding to cellular macro-
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molecules. Covalent binding to proteins can cause cell death by disturbing essential
biochemical processes or immunological reactions, whereas covalent binding to
DNA may initiate the process leading to cancer (Vermeulen et al. 1993).
There are several approaches to avoid the toxic side-effects by drugs, such as
withdrawal from the market, or reduction of the therapeutic dose. If no safe
alternatives are available and reduction of dose is not allowed because therapeutic
ineffectiveness will result, an alternative approach is chemoprotection. Chemo-
protection is defined as protection against drug toxicity by concomitant adminis-
tration of protective compounds. At present a large number of chemicals, mainly
thiol-compounds, have been evaluated in experimental animals as chemoprotectants
against the toxic side effects of antitumor agents (Treskens and Van der Vijgh 1993).
Mesna (sodium 2-mercaptoethanesulphonate) is now used clinically to protect
against bladder toxicity of ifosfamide. Other thiol-compounds are used in the
management of poisoning, such as N-acetylcysteine (paracetamol poisoning),
penicillamine and succimer (lead poisoning), dimercaprol (arsenic, gold, mercury
poisoning). Recently, selenium compounds, such as sodium selenite and ebselen,
have been shown to protect rat against nephrotoxicity of cisplatin (Baldew et al.
1989, 1990) and hepatotoxicity of paracetamol (Li et al. 1994).
During the past decade a large number of natural products and dietary
components have been evaluated as potential chemopreventive agents (Sharma et al.
1994). Chemoprevention is defined as the inhibition or reversal of carcinogenesis by
daily administration of specific drugs. In order to be useful as a chemopreventive
agent, a compound has to meet several criteria: (1) low cost (because of the necessity
of daily administration), (2) capability of oral administration, (3) little or no toxic
effects, (4) a high efficacy, and (5) a known mechanism of action. Mechanisms which
may explain chemopreventive action of a compound may involve amongst others :
inhibition of bioactivating enzymes, scavenging of reactive intermediates or free
radicals, induction of inactivating enzymes or antiproliferative activity (Morse and
Stoner 1993). It is clear that some of the mechanisms of chemoprevention can also

*Author for correspondence.

0049-8254/96 $12.00 01996 Taylor & Francis Ltd

 668 J . N. M . Cornrnandeur and N . P. E. Vermeulen
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Figure 1 . Proposed structures of curcumin.

be applied to prevent against drug-induced toxicities. Knowledge of the mech-

anism(s) of action of chemoprotectants is required for a rational selection of
chemoprotectant in order to achieve optimal protection against a toxic drug without
interfering with the disposition or biological activity of endogenous or other
exogenous compounds.
A class of dietary compounds which are currently intensively evaluated as
potential chemopreventive and chemoprotective agents is the class of polyphenols
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(Stoner and Mukhtar 1995). One of the members of this class, which will be the topic
of the present review, is curcumin (figure 1). Curcumin is an anti-oxidant and
colouring agent in different food products and can be regarded as a low cost
compound which can be isolated from different Curcuma species, or can be
synthesized in high yields using the Pabon reaction (Pabon 1964). As a dietary
component curcumin has been ingested orally by man for centuries. Because of its
different pharmacological activities (Ammon and Wahl 1990), curcumin containing
extracts (‘turmeric’) has been used in traditional medicine in Asia.

Disposition of curcumin
Curcumin is known to be unstable at neutral and basic pH, resulting in
degradation to ferulic acid and feruloylmethane (Tonnesen and Karlsen 1985).
Although the exact mechanism of degradation is not known yet, it appears to occur
via an oxidative mechanism because the presence of antioxidants such as ascorbic
acid, N-acetylcysteine or glutathione completely prevented the degradation of
curcumin at p H 7.4 (Oetari et al. 1996). At the conditions in the stomach ( p H 1-2)
and small intestines ( p H 6.5) curcumin is expected to be stable since between p H 1
and 7 curcumin degradation is extremely slow (Tonnesen and Karlsen 1985). Its
high lipophilicity allows curcumin to be absorbed rapidly from the gastrointestinal
tract by passive diffusion. Using radioactive curcumin it was determined that in rat
approximately 90 Yoof oral doses, ranging from 2.5 to 1000 mg/kg, is excreted by the
faeces within 48 h (Holder et al. 1978, Ravindranath and Chandrasekhara 1982).
Because only 35 yo of the given curcumin was excreted unchanged, it was concluded
that 65% of the dose was absorbed (Ravindranath and Chandrasekhara 1982).
Intraperitoneal administration of curcumin gave similar results, indicating that
curcumin is readily absorbed from the peritoneal cavity (Holder et al. 1978).
Biliary metabolites of curcumin have been identified by mass spectrometry, after
collection of the bile from rats given 50 mg/kg curcumin intravenously (Holder et
al. 1978). It was shown that 50-60 yo of the dose was excreted in bile within 5 h of
administration. T h e main biliary metabolites were identified as the glucuronides of
tetrahydrocurcumin and hexahydrocurcumin, representing 52 and 42 yo of the
 Cytotoxicity and cytoprotective activities of natural compounds 669

biliary metabolites respectively. A minor metabolite was dihyroferulic acid. These

data show that a major fraction of curcumin is reduced by endogenous reductase
systems, and subsequently glucuronidated by UDP-glucuronosyl transferases.
Following an oral dose of radiolabelled curcumin, 6.3 & 2.5 yo of the radioactivity
was excreted in the urine in 72 h and intraperitoneal administration gave approx-
imately two-fold higher urinary excretion, 11.2 & 0.7 yo of the dose (Holder et al.
1978). T h e nature of the urinary metabolites has not yet been elucidated.
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Toxicity of curcumin
In the literature different chemical structures have been attributed to curcumin
(figure 1). Because of the presence of a$-unsaturated ketone moieties in its
structure, (I, 11, figure l ) , curcumin was originally suspected to be capable of
undergoing Michael-type addition reactions with glutathione (GSH) or protein
thiols. From numerous studies in experimental animals as well in human volunteer
studies, however, it is clear that curcumin given orally at high doses does not cause
For personal use only.

apparent toxic effects. Wahlstom and Blennow (1978) did not observe toxicity in rat
after doses up to 5 g/kg. Also, a chronic study in rat, in which curcumin was
administered at approximately 750 mg/kg/day for 10 weeks, did not reveal toxicity
(Pulla Reddy and Lokesh 1994a).
Controversial data exist regarding the ulcerogenic activity of curcumin in the
stomach. Prasad et al. (1976) reported that 100 mg/kg curcumin over 6 days
produced gastric ulceration in the albino rat. Sinha et al. (1975), however, reported
an anti-ulcerogenic activity, whereas Bhatia et al. (1964) did not find any protective
action of curcumin in guinea pig against histamine-induced ulceration.
So far there has been no reason to suppose that consumption by man as a part of

-
the diet results in any deleterious effects. It is estimated that adults in India consume
80-200 mg curcumin per day. Dietary curcumin at 1200 mg/day ( 20 mg/kg/day)
has been reported to provide a moderate relief to patients suffering from rheumatoid
arthritis (Deodhar et al. 1980).
In in vitro studies curcumin also appeared to have a very low intrinsic toxicity.
A concentration up to 5 mM curcumin only produced minor cell death in freshly
isolated rat hepatocytes (Donatus et al. 1990). T h e non-toxicity of curcumin could
probably be explained by the fact that the diketone-moiety is, by internal hydrogen
bonding, comparable with an aromatic system (111, figure 1). Nmr studies of
curcumin solution are consistent with structure I11 (Pedersen et al. 1985). Binding
of nucleophiles, such as G S H or protein thiols, to this molecule will disturb the
highly conjugated system of double bonds of curcumin and is therefore energetically
not favourable.
Tonnesen et al. (1987) showed that the cytotoxicity of curcumin to bacteria
(Salmonella typhimurium and Escherichia coli) was greatly enhanced upon irradiation
with visible light ( > 400 nm). Curcumin was also found to be phototoxic in
mammalian cells (rat basophilic leukaemia cells) at a concentration < 1 ,UM (Dahl et
al. 1994). It has been suggested that the photochemical toxicity of curcumin may be
used for therapeutic purposes, for example in the treatment of psoriasis, bacterial
and viral diseases, and as a new tool in cancer therapy (Tonnesen et al. 1987).
Curcumin might have the advantage over psoralens in that photoactivation is by
visible light and not in the UVA region.
 670 J . N . M . Commandeur and N . P . E. Vermeulen

serum GPT
6oo 1 7-

I.U.
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control CCM + 7.72 mgkg + 38.6 mghg + 77.2 mgkg + 154.2 mgkg
Cu rcum In

Liver lipid peroxide levels

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control CCM + 7.72 mgkg + 38.6 mgkg + 77.2 mgkg + 154.2 mgkg
Curcumln

Figure 2. Dose-dependent protection of orally administered curcumin against tetrachloromethane-

induced hepatotoxicity and lipid peroxide formation (Nishigaki et al. 1992). GPT, glutamate
pyruvate transaminase, IU, international units.

Efficacy of curcumin as a chemoprotectant

T h e potential of curcumin to act as a chemoprotective agent was first
demonstrated in vitro using cultured rat hepatocytes. Kiso et al. (1983) showed that
1 mg/ml ( - 2.5 mM) curcumin protected against tetrachloromethane- (CC1,) and
D-galactosamine-induced cytotoxicity. More recently, a protective effect of low
concentrations ( < 20 ,UM) of curcumin was shown against oxidative damage caused
by the antimalarial drug primaquine to red blood cells (Tonnesen et al. 1994).
However, concentrations up to 5 mM curcumin did not protect against cytotoxicity
of paracetamol in freshly isolated hcpatocytes (Donatus et al. 1990).
T h e chemoprotective efficacy of curcumin has recently also been demonstrated
in vivo. Intraperitoneal doses of 200 mg/kg/day curcumin for 2 weeks protected
completely against liver toxicity of cyclophosphamide in mouse (Soudamini and
Kuttan 1991). Subsequent studies showed that curcumin also protects against
toxicities in different organs upon oral administration. A dose-dependence was
observed in the chemoprotection of curcumin against hepatotoxicity of tetra-
 Cytotoxicity and cytoprotective activities of natural compounds 67 1

chloromethane (CCI,), as indicated by the decreased release of glutamate pyruvate

transaminase (GPT) from the liver (figure 2) (Nishigaki et al. 1992). Doses of
39 mg/kg for 3 days significantly reduced hepatotoxicity and lipid peroxidation
caused by CCl, in rat. Curcumin mixed with feed, at 200 mg/kg/day, protected
duckling from liver damage produced by aflatoxin B, (Soni et al. 1992). Inter-
estingly, oral doses of 200 mg/kg/day for 7 days also protected against the lung
toxicity of cyclophosphamide in rat (Venkatesan and Chandrakasan 1995).
A partial decrease of paracetamol-induced hepatotoxicity and methaemo-
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globinemia was observed after 6-day pretreatment of mouse with 1.87 mg/kg/day
curcumin (Donatus 1994). However, at higher doses of curcumin (60 mg/kg/day),
curcumin potentiated these toxicities. Therefore, knowledge of the mechanism(s) by
which curcumin exerts its protective as well as potentiating activities is required
before such a compound can be used rationally as a chemoprotectant against drug-
induced toxicities.

Mechanism of chemoprotective activity of curcumin

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In order to understand the mechanism(s) by which a chemoprotectant exerts its

protective activity, it is important to know at what levels the process leading to tissue
necrosis can be influenced. A hypothetical scheme of several different processes
involved in drug-induced toxicity is shown in figure 3.
Most drugs are lipophilic compounds that undergo biotransformation to form
more water-soluble metabolites. However, biotransformation of a lipophilic drug
may also result in the formation of highly reactive intermediates which can be
deactivated enzymatically or non-enzymatically by G S H (Sipes and Gandolfi 1991).
I n the case this protective mechanism is insufficient, reactive intermediates may
covalently bind to cellular macromolecules, such as proteins, membrane phos-
pholipids and DNA. Because of their important biochemical functions, damage to
these latter macromolecules may result in biochemical disturbances. Depletion of
cellular A T P and increase of cytosolic calcium levels have been suggested as crucial
biochemical disturbances which may lead to cell damage (Commandeur and
Vermeulen 1990, Nicotera et al. 1990). Finally, it has been shown that damaged cells
can induce inflammatory processes, such as activation of macrophages or infiltrating
neutrophils. These inflammatory responses which expose the tissue to high levels of
a variety of reactive oxygen species may cause extensive tissue necrosis (Kehrer
1993).
Therefore, according to figure 3, protection by chemoprotective agents can
therefore arise from :
(a) Inhibition of the activating enzyme system (enzyme I) ;
(b) Stimulation of non-activating enzymes (enzymes IT and 111), thus leading to
lower availability of substrate for the bioactivating enzyme ;
(c) Scavenging of the reactive intermediate by chemoprotectants;
(d) Stimulation of enzymic and non-enzymic deactivation of reactive intermediates
by increasing levels of glutathione or glutathione S-transferase (enzyme IV) ;
(e) Interference with biochemical disturbances leading to cell death ; and
( f ) Anti-inflammatory activity.
T h e potential effects of curcumin on each of these levels will be subsequently
discussed.
 672 J . N . M . Commandeur and N . P. E . Vermeulen

Metabolite A

enzyme I
Reactive
intermediate
-
Deactivation
(enzpe Iv)
Metabolite C
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Metabolite D -
Repair
4
Reaction with cellular
macromolecules
enzyme V

I mitochondria
Biochemical
disturbances
I plasma membrane
endoplasmic reticulum
nucleus
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1
Damaged
cytoskeleton

NECROSIS

activation of macrophages
recruitment of neutrophiles
Figure 3. Hypothetical scheme of the different processes involved in the mechanism of drug-induced
toxicity.

Inhibition of the activating enzyme systems

T h e first indication that curcumin decreased covalent binding of drugs to
macromolecules was the fact that topical application of curcumin inhibited covalent
binding of benzo(a)pyrene to epidermal DNA (Huang et al. 1992). This inhibitory
effect was explained by inhibition of epidermal cyclo-oxygenase and lipoxygenase,
which are known to be able to bioactivate benzo(a)pyrene to electrophilic metabolites
(Huang et al. 1991). IC5,’s of curcumin towards epidermal cyclo-oxygenase and
lipoxygenase were approximately 5 and 10 ,UM respectively. More recently it was
shown that dietary curcumin at levels of approximately 15-30 mg/kg/day for 4
weeks almost completely blocked covalent binding of benzo(a)pyrene in rat liver
(Mukundan et al. 1993). Because rat liver cytochrome P450 isoenzymes are
quantitatively more important in the bioactivation of benzo(a)pyrene, blocking of
these enzyme systems is more likely. Oetari et al. (1996) recently showed that
curcumin is an extremely potent inhibitor of cytochrome P4501A1, which is indeed
the most important isoenzyme in the initial bioactivation of benzo(a)pyrene. T h e
IC,,’s of curcumin using ethoxyresorufin as a marker substrate was as low as 2 ,UM.
Curcumin was less potent in inhibiting cytochrome P4502B-isoenzymes with an
IC,, = 14 ,UM using pentoxyresorufin as a substrate. Curcumin was not able to
inhibit p-nitrophenol hydroxylation indicating that cytochrome P4502E1 is not
inhibited by curcumin. T h e fact that curcumin shows a high inhibitory effect
towards cytochrome P4501A1 may be explained by curcumin being a flat molecule
 Cytotoxicity and cytoprotective activities of natural compounds 673

CI CI
I I
CI-c-CI
I
* CI-c.
I
CI CI
TEORACHLOROMETHANE

Y3 x3
0

0NH
* ob
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OH 0
PARACETAMOL (NAPOI)

CYCLOPHOSPHAMIDE
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AFLATOXIN 61 8,g-epoxide

Figure 4. Cytochrome P450-dependent bioactivation of tetrachloromethane (CCl,), paracetamol,

cyclophosphamide and aflatoxin B, to toxic reactive intermediates.

with conjugated aromatic systems (structure 111, figure 1). Cytochrome P4501A1 is
generally known to be specialized in the biotransformation of planar polycyclic
aromatic hydrocarbons (Guengerich and Shimada 1991).
The hepatotoxicities of CCl,, cyclophosphamide, paracetamol and aflatoxin B,
are all dependent on bioactivation by different cytochrome P450 isoenzymes (figure
4). CC1, is known to be bioactivated mainly by cytochrome P4502E1 (Guengerich
1991). The fact that curcumin does not inhibit this isoenzyme rules out the
possibility that the observed protection of curcumin against CC1, toxicity can be
explained by blockade of bioactivation. Cyclophosphamide is known to be
bioactivated primarily by cytochrome P4502C isoenzymes (Clarke and Waxman
1989). However, as yet the inhibition of the latter class of isoenzymes by curcumin
has not yet been studied.
Paracetamol can be bioactivated by different P450 isoenzymes, such as l A l , l A 2 ,
2E1 and 3A4 (figure 5 ) (Patten et al. 1993, Snawder et al. 1994). Enzyme kinetic
analysis and antibody studies have revealed that in rodents and human cytochrome
P4502E1 plays a major role in the bioactivation of paracetamol (Patten et al. 1993,
Snawder et al. 1994). Cytochrome P4501A2 contributes to a significant extent at
higher doses of paracetamol. Low doses (1.87 mg/kg/day for 6 days) of curcumin to
mouse decreased the excretion of the mercapturate of paracetamol in urine by 27 %,
showing that partial protection against hepatotoxicity is due to inhibition of
bioactivation rather than to increased G S H conjugation (Donatus 1994). The very
poor inhibition of cytochrome P4502E1 by curcumin (Oetari et al. 1996), explains
 674 J . N . M . Commandeur and N . P . E. Vermeulen

UDPGT A
b-Glucuronide OH

CYP IAl
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CYP 2E1
CYP 1A2
CYP 3A4

6' 0
2
GSH
-__)

OH
S-NAC
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NAPQI

Figure 5 . Biotransformation of paracetamol to non-toxic metabolites, glucuronide and sulphate

conjugates, and the toxic intermediate N-acetyl-p-benzoquinoneimine (NAPQI). U D P G T ,
UDP-glucuronosyl transferase; ST, sulphotransferase; NAC, N-acetylcysteine.

its relatively small inhibitory effect on the excretion of the mercapturate of

paracetamol.
Curcumin inhibits colon tumorigenesis induced by azoxymethane in rat (Rao et
al. 1995). It was postulated that this might be explained by inhibition of metabolic
activation of azoxymethane by cytochrome P4502E1 to methylazoxymethanol and
methylazoxyformamide. However, the observation that a relatively high con-
centration of curcumin (30 ,UM) inhibits rat liver P4502E1 activity by < 10 yo seems
to rule out this explanation (Oetari et al. 1996).

Stimulation of non-activating enzymes

A chemoprotectant may also decrease the formation of reactive intermediates
indirectly by stimulating non-bioactivating enzyme systems by enzyme induction.
Rat fed a diet containing 5510% turmeric for 4 weeks demonstrated increased
activities of UDP-glucuronosyl transferase and glutathione S-transferase ; no
significant differences were seen in the activity of cytochrome P4501A1 (Goud et al.
1993).
Paracetamol is metabolized mainly by glucuronidation and sulphation to non-
toxic conjugates (figure 5). Only a small fraction is bioactivated by cytochrome P450
to the reactive intermediate N-acetyl-p-benzoquinoneimine(NAPQI) (Vermeulen
et al. 1992). At a repeated low dose (1.87 mg/kg for 6 days), curcumin significantly
protected against paracetamol (150 mg/kg) hepatotoxicity in mouse. T h e fact that
excretion of sulphate and glucuronide of paracetamol was not affected, whereas
excretion of mercapturate was decreased, suggests that protection was due to a
decreased bioactivation rather than an increased sulphation and/or glucuronidation
(Donatus 1994). At a high dose of curcumin (60 mg/kg/day for 6 days), however,
paracetamol-induced hepatotoxicity was strongly potentiated. Urinary analysis of
 Cytotoxicity and cytoprotective activities of natural compounds 675

paracetamol metabolites revealed a complete inhibition of glucuronidation and

partial (62 yo)reduction of sulphation by curcumin (Donatus 1994). This may be
explained by curcumin itself being metabolized by these phase I1 enzymes (see
above), thus resulting in competitive inhibition at the enzyme level or resulting in
cofactor depletion (Holder 1978). T h e blockade of phase I1 enzymes by high doses
of curcumin resulted in a seven-fold increase in urinary excretion of unchanged
paracetamol (Donatus 1994). Because free paracetamol concentrations in the liver
will also be increased, whereas the major enzyme active in the bioactivation of
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paracetamol, cytochrome P4502ER1, is not inhibited (Oetari et al. 1996), an

increased rate of bioactivation of paracetamol can be anticipated. A net increase in
the bioactivation of paracetamol by high doses of curcumin is indeed reflected by a
two-fold increase in the excretion of the mercapturic acid of paracetamol (Donatus
1994).

Scavenging of reactive intermediates by curcumin

As yet no information is available about the ability of curcumin to scavenge
electrophilic reactive intermediates formed by metabolic activation of drugs.
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However, toxicity of a number of drugs has been shown to be the result of the
production of reactive oxygen species (Kehrer 1993). Redox cycling of compounds,
such as quinones, nitro-compounds and bipyridylium compounds, may lead to the
production of substantial amounts of superoxide anion radicals (0;-).Superoxide
anion radicals are not highly reactive species themselves but they can be converted
to hydrogen peroxide and hydroxyl radicals (Kehrer 1993). Recently, the in-
activation of nitric oxide by superoxide anion radicals, leading to vasoconstriction
(Nakajima et al. 1994), and formation of highly reactive peroxynitrate radicals
(Koppenol et al. 1992), were also suggested to play a role in the toxicity of reactive
oxygen species.
Curcumin appears to be a potent scavenger of oxygen free radicals, such as
superoxide anion radicals (Kunchandi and Rao 1990), hydroxyl radicals (Pulla
Reddy and Lokesh 1994b) and nitrogen dioxide radicals (Unnikrishnan and Rao
1995). Its antioxidant activity is further shown by its capacity to inhibit lipid
peroxidation in rat brain homogenates (Sreejayan and Rao 1993) and rat liver
microsomes (Pulla Reddy and Likesh 1992), and by its ability to inhibit peroxidation
of arachidonic acid (Ammon et al. 1993) by iron/ascorbate.

Stimulation of enzymic and non-enzymic deactivation of reactive intermediates

Reactive intermediates formed by bioactivation of lipophilic drugs can be
deactivated enzymatically or non-enzymatically by conjugation to G S H . Treatment
of mice for 15 days with curcumin (orally, 250 mg/kg/day) resulted in a 50%
increase in activity of glutathione S-transferase (GST) activity in the liver,
presumably due to enzyme induction (Matthews and Rao 1992). GST activities in
lung, stomach, small intestine and kidney were unchanged. Concentrations of G S H
in the organs studied were not changed.
A single dose of curcumin to the male Swiss mouse at 1 g/kg, in contrast,
resulted in a 20 yo decrease in hepatic GST activity, whereas the GSH concentration
in the liver was increased by 75% (Nagabhushan and Bhide 1992). These
contradictory results may be explained by a direct inhibitory action towards G S T
due to the higher dose administered, in contrast with inductive effects at low doses
chronically administered.
 676 J . N . M . Commandeur and N . P . E . Vermeulen

Very recently, the effect of curcumin on GST activity was investigated in cytosol
of the induced rat (Oetari et al. 1996). From these experiments it appeared that
curcumin indeed is able to inhibit GST activity with 1-chloro-2,4-dinitrobenzene as
substrate: IC,,’s ranged from 10 to 20 PM dependent on the type of induction.

Interference with biochemical disturbances leading to cell death

Damage to biomacromolecules by covalent binding of electrophiles or by proton
abstraction by radicals can lead to loss of the biological activity of these
macromolecules (Nelson and Pearson 1990). If the functioning of the macro-
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molecules is essential to the cell, loss of their activity may ultimately lead to cell
death. Increase in intracellular calcium concentration as well as depletion of A T P in
the cell have been suggested to play an important role in cell death (Commandeur
and Vermeulen 1990; Nicotera et al. 1990).
An increased cytosolic calcium concentration may result from damage to the
plasma membrane due to lipid peroxidation, inactivation of different calcium
carriers in the plasma membrane and/or release of calcium from intracellular
calcium pools (for instance, due to damage to the endoplasmic reticulum) (Nicotera
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et al. 1990). A sustained increased cytosolic calcium concentration may activate

proteases, phospholipase and endonucleases which may break down critical cellular
constituents, such as cytoskeleton, plasma membranes and D N A (Nicotera et al.
1990).
A depletion of A T P may be the result of oxygen depletion (due to ischaemia or
hypoxia) or by mitochondrial disfunctioning (Commandeur and Vermeulen 1990).
Covalent binding to proteins involved in the citric acid cycle or in the respiration
chain may cause a decrease of the mitochondrial membrane potential, which is the
driving force in the production of ATP. A T P depletion will result in the disfunction
of energy-consuming processes such as excretion of calcium by Ca-ATPases,
resulting in increase of cytosolic calcium. Defective A T P production may also be the
result of an increased cytosolic calcium concentration : mitochondria will stop A T P
production if cytosolic calcium concentration is increased, and the mitochondrial
membrane potential will be the driving force for uptake of calcium in the
mitochondria.
T h e biochemical disturbances described above can result in an increased
production of reactive oxygen species in the cell which may produce fatal cell
damage (Kehrer 1993). Cellular xanthine dehydrogenase can be converted to
xanthine oxidase by proteases which are activated by increased calcium (Waud and
Rajagopalan 1976). Xanthine oxidase can directly reduce molecular oxygen to
superoxide anion radicals and hydrogen peroxide (Parks and Granger 1986, Greene
and Paller 1994). Also, overloading of mitochondria with calcium has been shown to
result in leakage of superoxide anion radicals and hydrogen peroxide from the
respiration chain (Van de Water et al. 1994).
As mentioned in above, curcumin has been shown to be a good scavenger of
reactive oxygen species. Therefore, part of the cytoprotective effect of curcumin
may also be ascribed to these relatively late steps in the process leading to cell death.
CCl,, cyclophosphamide and paracetamol are known to be activated by
cytochrome P450 isoenzymes to carbon radical species (CC1;) or electrophilic
reactive intermediates (acrolein and N-acetyl-p-benzoquinonimine)(figure 4). All
three compounds have been shown to cause lipid peroxidation in their target organs.
Protection against the respective toxicities by antioxidants points to involvement of
reactive oxygen species in the process leading to tissue damage. Because these
 Cytotoxicity and cytoprotective activities of natural compounds 677

compounds are known not to produce oxygen radicals during their bioactivation, it
is more likely that these radicals are formed as a result of the disturbance of
subsequent biochemical processes, as described above. Pretreatment of animals with
curcumin has been shown to decrease the extent of lipid peroxidation in the livers of
CCl; treated rats (figure 2) and in lungs of the cyclophosphamide-treated rat.
Curcumin may be effective in protection against reactive oxygen species directly, by
scavenging reactive oxygen species as described above, or indirectly by inhibition of
xanthine oxidase. Curcumin was shown to inhibit xanthine oxidase in vitro;
however, its IC,, was > 200 ,UM, which may question its relevance in vivo (Lin and
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Shih 1994).
An indication that curcumin may block intracellular reactive oxygen production,
instead of blocking inflammatory processes as described below, is the fact that
curcumin very efficiently blocks paracetamol-induced lipid peroxidation in isolated
rat hepatocytes (Donatus et al. 1990). As mentioned earlier, activation of para-
cetamol does not lead to oxygen radicals and lipid peroxidation is more likely to be
the result of cell damage (Van der Straat et al. 1987).

Protection against toxicity due to anti-inj2ammatory activity

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It has been shown during the past decade that inflammatory processes may also
play an important role in the process leading to tissue necrosis. Laskin et al. (1986)
showed that paracetamol was much more toxic to hepatocytes when they were
incubated in the presence of Kupfer cells, the resident macrophages in the liver. It
was speculated that hepatocytes damaged by paracetamol release factors which
activate Kupfer cells. T h e activated Kupfer cells subsequently release cytotoxic
cytokines and reactive oxygen species, which further damage the hepatocytes
leading to cell death (Laskin 1986, Laskin et al. 1995).
More recently, the involvement of Kupfer cells in hepatotoxicity was also
demonstrated in vivo. Pretreatment of rat with gadolinium chloride, which
selectively kills Kupfer cells, was shown to protect against the hepatotoxicity of CC1,
(Edwards et al. 1993), paracetamol (Laskin et al. 1995) and ally1 alcohol (Przybocki
et al. 1992). Ally1 alcohol is known to be bioactivated to acrolein, which is the same
reactive intermediate responsible for cyclophosphamide toxicity (figure 4).There-
fore, it can be speculated that the protective activity of curcumin towards CCl,,
paracetamol and cyclophosphamide may in part be attributed as well to blockade of
the inflammatory responses caused by these drugs. Although curcumin has not yet
been tested with activated Kupfer cells, it has been shown to be an extremely potent
inhibitor of the production of reactive oxygen species by activated rat peritoneal
macrophages (Joe and Lokesh 1994). At 10 ,UM, curcumin completely inhibited the
production of superoxide anions, hydrogen peroxide and nitrite radicals.
Other mechanisms underlying the anti-inflammatory effects of curcumin have
been associated with inhibition of cyclo-oxygenase, lipoxygenase and prostaglandin
synthesis. Very recently it was shown that curcumin also inhibits production of the
pleiotropic cytokine tumour necrosis factor-a (TNF-a) by a human monocytic
macrophage cell line (Man-Ying Chan 1995).

Conclusions
Curcumin appears to be a promising chemoprotective agent which is capable of
protecting against several drug-induced toxicities in liver and lung of rodents.
Investigations on the mechanisms of protection by curcumin have shown that this
compound can act at different levels in the process leading to tissue necrosis. Potent
 678 J. N . M . Commandeur and N . P. E. Vermeulen

inhibition of cytochrome P4501A1 may explain its protective activity against

benzo(a)pyrene-induced carcinogenicity. Because both at early, at intermediate and
at late phases of process leading to tissue necrosis, reactive oxygen species appear to
be involved, the anti-oxidant activity of curcumin may also play an important role
in its chemoprotective ability. Finally, the anti-inflammatory effect of curcumin
may contribute to its protection against drug-induced toxicities.
T h e fact that curcumin appears to block the toxicity process at different levels,
makes this drug a candidate as a more or less generally applicable chemoprotectant.
However, the toxicity of drugs which depend on glucuronidation for their
Xenobiotica Downloaded from informahealthcare.com by McGill University on 09/26/12

elimination or inactivation may be potentiated. Curcumin itself is biotransformed

by glucuronidation (Holder et al. 1978). Thus competitive inhibition of this route or
cofactor depletion may drive the biotransformation of drugs towards activating
pathways as has been observed with paracetamol and chloramphenicol.
Natural compounds such as curcumin may be useful for chemoprevention as
well as chemoprotection purposes. These compounds may also function as lead
compounds for the design of more effective and/or more selective chemo-
preventive/chemoprotective agents.
For personal use only.

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