Biochemical Pharmacology, Vol. 51, pp. 39-45, Copyright 0 1995 Elsevier Science Inc.

1996.

ISSN 0006-2952/96/$15.00 + 0.00 SSDI 0006-2952(95)02113-Z

ELSEVIER

Effects of Curcumin on Cytochrome P450 and Glutathione S-Transferase Activities in Rat Liver
S. Oeturi,? M. Sudibyo,? Jan N. M. Commandeur, R. Samhoedi,# Nice P. E. Vermeukn
LEIDEN/AMSTERDAMCENTER FOR DRUG RESEARCH, DEPARTMENTOF PHARMACOCHEMISTRY,DIVISION OF MOLECULAR TOXICOLOGY, VF.IJEUNIVERSITEIT,DE B~ELELAAN 1083-1081 HV AMSTERDAM, THE NETHERLANDS

ABSTRACT. The stability of curcumin, as well as the interactions between curcumin and cytochrome P45Os (P45Os) and glutathione s-transferases (GSTs) in rat liver, were studied. Curcumin is relatively unstable in phosphate buffer at pH 7.4. The stability of curcumin was strongly improved by lowering the pH or by adding glutathione (GSH), N-acetyl L-cysteine (NAC), ascorbic acid, rat liver microsomes, or rat hver cytosol. Curcumin was found to he a potent inhibitor of rat liver I450 lAl/lA2 measured as ethoxyresorufin deethylation (EROD) activity in P-naphthoflavone (PNF)-induced microsomes, a less potent inhibitor of I450 2B1/2B2, measured as pcntoxyresorufin depentylation (PROD) activity in phenobarbital (PB)-induced microsomes and a weak inhibito- of I450 2E1, measured as p-nitrophenol (PNP) hydroxylation activity in pyrazole-induced microsomes. K, values were 0.14 and 76.02 FM for the EROD- and PROD-activities, respectively, and 30 PM of curcumin inhihiced only 9% of PNP-hydroxylation activity. In ethoxyresorufin deethylation (EROD) and pentoxyresoruiin depentylation (PROD) experiments, curcumin showed a competitive type of inhibition. Curcumin was also a potent inhibitor of glutathione S-transferase (GST) activity in cytosol from liver of rats treated with phenobarbital (PB), P-naphthoflavone (PNF) and pyrazole (Pyr), when measured towards l-chloro-2,4dinitrobenzenr (CDNB) as substrate. In liver cytosol from rats treated with phenobarbital (PB), curcumin inhibited GST activity in a mixed-type manner with a K, of 5.75 PM and K, of 12.5 PM. In liver cytosol from rats treated with pyrazole (Pyr) or &naphthofIavone (PNF), curcumin demonstrated a competitive type of inhibition with K, values of 1.79 I_~Mand 2.29 PM, respectively. It is concluded that these strong inhibitory properties of curcumin towards P45Os and GSTs, in addition to its well-known antioxidant activity, may help explain the previously observed anticarcinogenic, antimutagenic, and cytoprotective effects of this important natural compound and food constituent. RIOCHEMPHARMACOL 51;1:39-45, 1996. KEY WORDS. curcumin; inhibitory potency; rat liver; cytochromes P450; glutathione S-transferases

Turmeric, tein tion kinase

a yellow

product

in the rhizomes activity [l] and

of Ctlrcuma to possess

longa proac12-

Topical to inhibit tabolites B[a]P against properties,

application to epidermal [9, lo]. which

of curcumin binding

before

B[a]P has been shown hydrocarbon tumor initiation strong antioxidant meby

L. and others activity [2-4].

containing C inhibition The involves

cm-cumin,

is reported

the covalent

of polycyclic has

anti-inflammatory

DNA and to prevent curcumin peroxidation similarities

mechanism

of the anti-inflammatory of 5-lipoxygenase,

Moreover, lipid

of curcumin

inhibition

were shown

to play a role in the protection (e.g. of paracetamol to ferulic acid, which is

lipoxygenase, and cyclooxygenase [5, 61. Curcumin also inhibits the microsomal-mediated mutagenicity of B[a]P, capsaicin, chili extract, and cigarette smoke condensate [7, 81.

drug-induced

[Ill. Curcumin

has structural

also known as an alkaline degradation product [12]. Plant phenols, such as ellagic acid, ferulic * Corresponding author. t Vwting fellow, Laboratory oc Pharmacology and Toxicology, Faculty of
Pharmacy, Gadjah Mada University, Yogyakarm, Indonesia. t Faculry of Pharmacy, Gadjah Mada University, Yogyakarra, Indonesia. 5 Abheuiations-P450, cytochrome P450; GST, glutxhione s-rmnsferase; GSH, glurathione; NAC, N-aceryl L-cysreine; EROD, ethoxyresorufin deethylation; PROD, pentoxyresorufin depentylation; PNP, p-nitrophenol; PB, naphthoflavone-phenobarbital; 3NF, P-naphthoflavone; Pyt, pyrazole; CDNB, I-chloro-2,4-dinxrohenzrne; LPO, lipid peroxidation; BSA, serum albumin; B[a]P, benzo[a]pyrene. Received 31 January 1995; accsrpted 1 September 1995.

of curcumin acid, caffeic inhibitory

acid, and chlorogenic

acid, were shown

to possess

properties towards GSTs with CDNB as substrate [13]. As yet, no data are available concerning possible inducing or inhibitory properties two of the bioactivation of curcumin towards GSTs nor towards P45Os, most important enzyme systems involved in the and bioinactivation of xenobiotic compounds

U41. In this report, the effects of curcumin on P450 and GST activities were studied. The effect of curcumin on EROD and

40

S. Oetari

et al.

PROD cytosolic

and PNP hydroxylation GSTs were investigated.

activities The

of rat liver microsoactivity stability of rat liver of curcumin

PNF-microsomes (2.2 mg/mL

(0.27 mg/mL

of protein)

or PB-microsomes stock solution in

ma1 I450 and CDNB was also investigated.

GSH-conjugation

of protein),

25 PL of curcumin

methanol (80, 240, 800, and 2400 PM for IC,, determination and 0, 2.4, 8, 24, and 80 PM for the determination of K, and type of inhibition) (10 mM in DMSO). and 10 PL of ethoxyFluorescence of 530 nm and an emission Elmer Model of NADPH to the Resorufin aliquot reactions. in these and PROD or pentoxyresorufin at an excitaof 586 buffer the specof All spectrometo start wavelength was recorded 3000 fluorescence mixtures

MATERIALS Materials Curcumin acetylaceton ments rity >99%.

AND

METHODS

tion wavelength nm with a Perkin of vanillin curcumin from Aldrich and ter. A lOO+L pH dealkylation curcumin final EROD

was synthesized according Resorufin that

by condensation the synthetical

(5 mM in phosphate formation was followed solution

to [15]. H-NMR was purchased

and HPLC

measurehad a puChemie

7.8) was added

incubation

demonstrated

trofluorometrically concentration)

for 2 minutes. mixtures, was added measurements

To ensure before adding

the stability curcumin. in duplicate.

(Beerse, Belgium), ethoxyresorufin, pentoxyresorufin, GSH, CDNB, PNF, PB and Pyr from Sigma Chemical Co. (St Louis, MO), PNP from JT Baker (Deventer, from Janssen and ascorbic and BSA from Boehringer The Netherlands), (Beerse, Mannheim paranitrocatechol NADPH The stadt, Chimica Belgium), BV (Almere, (Darm-

25 PL of a GSH were done

(1 mM,

Netherlands), Germany).

acid from

E. Merck

p-Nitrophenol Hydroxylation determined sisting

Hydroxylution of PNP in pyrazole microsomes of rat liver was mixtures consus-

as described

by Koop [20]. Reaction

Animals, Pretreatment and Preparation of Subcellular Male Wistar rats (ZOO-220 were housed

of 1.68 mL of phosphate (100 mM in aquadest), (1.5 mg/mL),

buffer (0.1 M, pH 6.8), 40 yL 150 PL of a microsomal

Fractions Olac CPB, Zeist, The

of PNP pension (t = 25C on a standard The were

g, Harlan

and 25 PL of a curcumin stock solution were preincubated (80, 240, 800, and 2400 PM m methanol) started by adding 100 PL buffer pH 6.8). After 10 min, by the addition of 0.5 mL triProteins and 511 were precipi1.0 mL of the for a nm with g, 15 min), at

Netherlands)

in an environmentally room, kept

at 37C for 2 min and the reaction NADPH (10 mM in phosphate the reactions were terminated chloroacetic tated the resulting Novaspec droxylation by centrifugation supernatant of II Pharmacia measurements measurement (2500 was mixed

and air humidity-60%)

controlled

laboratory diet (RMH TM-IO, Hope Farms, Woerden, Netherlands) and starved overnight before use. Animals pretreated &turn with either PNF, once by i.p. injection kg/day in arachidis by i.p. injection, fractions -80C were until oil, 0.1% (w/v) PB in the drinking in physiological

acid (0.5 M in aquadest).

of 80 mg/ water ad saline 24 to at by

with 0.1 mL 10 N NaOH

for 7 days, or Pyr, 200 mg/kg/day

4-nitrocatechol were done

once a day for 2 days. Livers were isolated Rat liver microsomal by ultracentrifugation et al. [17], respectively, concentrations [18] with prepared according and stored

LKB spectrophotometer. in duplicate.

All PNP hy-

hr after the last treatment. Lake [16] and Lundgren use. Protein of Bradford the method

and cytosolic

were determined

C$H Conjugation GST activities

of CDNB CDNB were measured of Habig spectrophotoet al. [21]. The

BSA as a standard.

towards

metrically Stability Experiments Solutions (final tween mM), of curcumin with Curcumin in phosphate 25 PM), buffer, pH 7.4 and pH 6.5 every 5 min be1 150-20 spectrophotometer. GSH (final concentration rat liver mi-

according

to the method

reaction mixtures contained either 25 PL of liver cytosol (0.20.3 mg/mL of protein) of rats pretreated with PNF, with PB, or with Pyr, 50 PL of GSH concentrations (10 mM in aquadest), of CDNB of phosphate and 10 I_LLof varying (10, 12.5, 16.5, 25, and 50 buffer (50 mM, pH studies, 5-PL samples

concentration 200-600 NAC solutions

were scanned

nm using a Hitachi of curcumin, ascorbic

mM in ethanol) 6.5, final volume

in a solution

To similar

500 PL). In inhibition

(50 PM),

acid C (25 PM),

crosomes (0.03 mg/mL protein), or cytosol tein) were added and solutions subsequently 200-600 nm every 5 min.

(0.01 mg/mL proscanned between

of solutions of curcumin in methanol were preincubated for 4 min and protected from light before GSH and CDNB were added. Methanol in the incubation mixtures (2% v/v) had no effect on the enzyme activities. The absorption differences (A abs/min) were recorded on a Philips PU 8720 UV-VIS scanning spectrophotometer at 340 nm.

7-Ethoxy-

and 7-Pentoxyresorufin

0-Dealkylation

Cytochrome P450-mediated 0-dealkylation of EROD in PNFmicrosomes of rat liver and of PROD in PB-microsomes were measured according to Burke and Mayer [19]. The reaction mixtures, prepared in a fluorimeter cuvette, contained 1.94 mL of of phosphate buffer (0.1 M, pH 7.8), 25 PL of a suspension

Type of lnhibition For the ethoxyresorufin 0-deethylation (PNF induction) and pentoxyresorufin 0-depentylation (PB induction) reactions in the final incubation mixtures, the concentrations of ethoxy-

Effects of Circumin

on Biotransformation

Enzymes

41

and pentoxyresorufin those of curcumin

were 113, 5, 2.5, 1.25, and 0.63 PM, and 0, 0.03, 0.1, 0.3, and 1.0 PM (BNF-induc-

(IC,,)

was determined inhibitor

from the plots of remaining concentration

activity

vs

the (varied)

at a fixed concentration

tion) and 0 and 3 /.tM (PB induction). In GSH conjugation reactions, the final concentrations of CDNB were 1.0, 0.5, 0.33, 0.25, and 0.20 mM, and those yM (PB induction), 10, and 20 yM (BNF induction). of curcumin 0, 7.5 and 15 and 0, 0, 3.5, and 15 PM (Pyr induction)

of the substrate.

RESULTS The primary properties and cytosolic hydroxylation

AND

DISCUSSION the inhibitory P45Os and PNP of

aim of this study was to investigate towards EROD P450 microsomal GSTs. and PROD activities of GST

of curcumin reactions

cytochrome

Analysis of Data Apparent equations: K, constants were calculated inhibition - K,,,); using the following

dealkylation

were chosen

for study of the inhibiand GSH conjugation

tion

of differential

(i) for competitive

CDNB

for the inhibition

activities.

K, = K,,JI/Kj,*

Solubility and Stability of Curcumin The experimental conditions were carefully considered be-

(ii) for noncompetitive

inhibition, - V,,,,,*); inhibition, - V,,,*K,,J

cause of the low solubility K, = V,,,*W(V,,, and (iii) for mixed-competitive min at higher experiments concentrations sured K, = Vmax* &,,[4/W,,,&,,* and K,, = where buffers. 1). The pH values in aqueous

and suspected (Tonnesen

instability

of curcu[12]). Most at

and Karlsen of curcumin nm)

solutions

were, therefore, (200-600

performed in phosphate rapidly

~50 /,tM. The

stability

was mea(Fig.

spectrophotometrically At pH 7.4, curcumin absorbance

(25 PM) degraded 10 min the remaining appeared was colorless,

at 426 nm decreased

to approximately absorbance at 210 nm that

50% after 5 min, and after

V,,,*WW,,, - Vmax*)r

was only about

10%. Two new absorptions

and 262 nm. The final solution

indicating

a yellow conjugated system no longer existed in the degradation products of curcumin. Phosphate buffer pH 6.5 resulted in K,,,, V,,.,, Km*, V,,,ax* and in the presence of the inhibitor (Cai in 50% resulting of inhibitor et al. [22]. inhibition significantly the spectral curcumin instability GSH increased pattern solution (Fig. stability of curcumin. No changes in the occurred up to 30 min after preparing buffer prevented

were obtained The

in the absence

1). In phosphate was completely

pH 7.4, the by adding

(1). [I] is the concentration concentration

of curcumin

of inhibitor

(1 mM), NAC

(50 PM), ascorbic

acid (25 PM), and also

Abs .

Abs.

,c

so--

0,5
,

080

200

&
300 400 w
cur.pH. 7.40

\ \

500 1 (nm)

cur.pH.

6.50

FIG. I. Overlay of UV-VIS spectra of a 25 pM solution of curcumin in 50 mM phosphate buffer pH 7.4 (right) and pH 6.5 (left) every 5 min (200-600 nm; upper line t = 0).

42

S. Oetari

et ai.

by adding cytosolic Apparently, strongly containing

protein fractions increased

from microsomal (0.01 mg/mL) by the of curcumin presence

(0.03 mg protein/ml) in aqueous of thioland solutions non

and is

presence centrations, induced

of curcumin, the

the K,,, value was increased With K,,, values were The average activity plots increasing of both when K, values

from 0.99 to curcumin PNFand compared of curcumin respec1). to conPBto

of rat liver (data not shown). thiol-

10.05 PM in PNF-microsomes. apparent liver microsomes of curcumin.

the stability antioxidants.

increased

the controls PROD inhibited by 36 up by 13 values of GSH the acthat as tively

were 0.14 PM for the EROD Effects on P450 Activities activity (Table At concentrations of 1,3, 10, and 30 PM, curcumin the EROD activity in PNF-induced liver microsomes to 93%, the PROD microsomes PROD (1 mM) inhibitory tivities curcumin EROD measured only activity in PB-induced up to 88%, and the PNP hydroxylation were calculated activities in the effects activity

and 76.02 PM for the microsomes, indicated (Table a competi-

in PNF- and PB-induced 1). Lineweaver-Burk for both application activities

tive type of inhibition Recently, inhibit against dermis topical the formation tumorigenic of female CD-l

P450 activities of curcumin adducts et al., [lo]).

microsomes IC,,

was reported

in Pyr-induced

of B[a]P-DNA mice (Huang the levels

and to protect in the epiet Mukundan adducts

by 0.5 up to 9%, respectively. to be 2 yM for the EROD (Table 1). The presence mixtures incubation of curcumin inhibitor activity,

of B[a]P and DMBA

and 14 PM for the or absence influence and EROD demonstrate measured

al. [23] found that 0.03% curcumin significantly liver mechanism carcinogenesis ides as a result reduced of action involved of rats. Soudamini and Kuttan of curcumin scavenging

in the diet for 4 weeks also in the that the [24] suggested as inhibitor of peroxides capacity. The

did not

of B[a]P-DNA

on the PROD of P450 lAl/lA2 inhibitor

(data not shown). is a potent activity, as PROD a much

The results clearly weaker

of chemical and superoxcarcinogen

of I450 2B1/2B2 inhibi-

of its antioxidant

and causes no significant

tion of I450 2El measured as PNP Ferulic acid (one of the degradation showed induced no significant inhibition rat liver microsomes not shown).

hydroxylation activity. products of curcumin) activities of 1,3, with in PNF10, and

B[a]P, however, requires dihydrodiol-9,10-epoxide, bind to DNA the present potent EROD study,

oxidative bioactivation to B[a]P-7,8the ultimate carcinogen known to by P450 1Al lAl/lA2, [26]. In as that curcumin is an extremely measured (Fig. 2). Our reactivity because P450 lAl/lA2. of azoxymethby curcumin explanation was AOM seems [I I], curof its strong

[25]. B[a]P is bioactivated we found inhibitor of P450

on EROD

at concentrations

competitive activity that,

30 FM (data

in PNF-induced

microsomes

In liver microsomes

from rats pretreated and PROD respectively. in liver inhibited,

PNF and PB, were 33.9 of miof PBwas obsignif1). In the

sults suggest cumin and specific ane

apart from its antioxidant activity that colon modulation in the present towards

the V,,, values of the EROD and 1.3 nmol/min/mg protein, curcumin, pretreated crosomes served. icantly these I450 activities only rats were In both when

activities microsomes inhibition (Table

could also act as an anticarcinogen inhibitory

In the presence but in liver

Rao et al. [27] suggested (AOM)-induced through because, mediated metabolism unlikely

the inhibition

weakly

carcinogenesis However, this

of PNF-pretreated cases, the V,,, compared

rats a strong to the controls

of P450 2El-dependent

values did not increase

by curcumin.

study, we found that curcumin

TABLE 1. Enzyme kinetic parameters of the effects of curcumin from differentially induced rats Curcumin

on P450-dependent

EROD

and PROD

activities in microsomal rat liver fractions

added

Microsomes

(PM)

V max (nmol/min/mg)

Type of inhibition

PNF-microsomes/EROD
activity 0.00 0.03 0.10 0.30 1 .oo PB-microsomes/PROD activity 0.00 1.00 3.00 10.00 1.27 1.10 0.94 1.05 6.41 6.52 6.66 6.95 % inhibition 0.5 4 8 9
two experiments. Mkxed, mixed type of inhtbition; competitwe, competitive type of inhibitmn.

33.96 31.45 32.37 37.06 36.38

0.99 1.19 1.60 2.94 10.05 0.15 0.16 0.15 0.11 Competitive Competitive Competitive Competitive

76<0 76.02 75.10

14

Competitive Competitive Competitive

Pyr-microsomes/PNP hydroxylation activity

1 3 10 30
The values of V,_ K,, K,, and K: are the average from

nd

nd

Effects of Circumin

on Biotransformation

Enzymes

43

Q
l n

CURCOpM CURC 0.03 PM

CURC CURC 0.1 PM 0.3 PM

.
n

CURC.1$v4

-1

0 l/S &Id)

FIG. 2. Lineweaver-Burk plot showing the inhibition of cytochrome P450-mediated ER0D activity in liver microsomes from PNF-induced rats, by curcumin at concentrations of 0, 0.03, 0.1, 0.3, and 1 FM.

showed

no significant

inhibition

hydroxylation ble 1).

Effects on SST

activity

of P450 2El measured as PNP in pyrazole-induced microsomes (Ta-

Activities

On the CDNB-GSH conjugation activity in liver cytosolic fractions from PB-induced rats, concentrations between 3 and 12 PM of curcumin gave from 35 up to 56% of inhibition, respectively. In Pyr-induced liver cytosolic fractions, curcumin concentrations between 5 and 15 PM gave from 38 up to 56% inhibition of the CDNB GSH conjugation activity. In PNFinduced rat liver cytosol, curcumin concentrations between 9 and 30 PM gave from 39 up to 58% inhibition of the CDNB GSH conjugation activity. The respective IC,, values were calculated to be 10 PM (PB-induction), 12 PM (Pyr-induction), and 21 PM (PNF-induction) (Table 2). In liver cytosol from rats pretreated with PNF, PB, or Pyr, the apparent V,,, values for the GST activity towards CDNB as substrate were 1.24, 4.14, and 1.32 pmol/min/mg protein, respectively. In the presencm: of curcumin, the V,,, values of the CDNB-conjugation activities of GST decreased in the case of cytosol from rats pretreated with PB and Pyr (Table 2). In cytosol of PNF-pretreated rats, however, V,,, values did not decrease significantly in the presence of curcumin when

compared to controls. In all three types of cytosols, the apparent K,,, values increased upon addition of curcumin (Table 2). In cytosol of rats pretreated with PB, the type of inhibition was mixed, and in the case of Pyr-cytosol the type of inhibition was competitive at the lower concentrations of curcumin but mixed at higher concentrations (Table 2). Lineweaver-Burk plots indicated a competitive type of inhibition in rat liver cytosol pretreated with PNF (Table 2, Fig. 3). Plant phenols, such as ellagic acid, ferrulic acid, caffeic acid, and chlorogenic acid, have been reported to be in vitro inhibitors of GST activity from rat liver (Das et al., [ 131). However, Susan et al. [28] found that a semi chronically administered high oral dose of curcumin given to mice, daily for 15 days, increased hepatic GST activity towards CDNB by 1.8-fold compared to control animals. Moreover, Nijhoff et al. [29] recently reported that dietary curcumin significantly increased GST activity in the small intestine, but not in the liver of rats. Mathews et al. [30] found that curcumin interacts with GSH spontaneously and enzymatically in the presence of GSTs from a mouse liver postmichondrial fraction. In our study, we did not see a reaction between GSH and curcumin, neither in the presence nor in the absence of GSTs from cytosolic liver fractions of differentially induced rats. We found that the GST activity measured with CDNB as substrate was strongly inhibited by curcumin. Given the strong inhibition of GST activity in differentially induced rats (approx. 60%) and the approximately equal distribution of both class alpha (subunits 1 and 2) and class mu isoenzymes (subunits 3 and 4), as well as the absence of class pi-isoenzymes (Foliot and Beaune [31]), curcumin is not expected to demonstrate a high GST-isoenzyme selectivity. Based on the present data on liver fractions of rats pretreated with PNF, curcumin appeared to show an extremely potent inhibitory effect towards P450 lAl/lA2 (K, = 0.14 PM) and a 20-fold 1 ower, but still potent, inhibitory effect on GST activity towards CDNB (K, = 2.29 PM). In liver fractions of rats pretreated with PB, however, a reversed ratio of inhibitory effects of curcumin on P450 2B1/2B2 and GST was observed, albeit at a 50-lOO-fold higher concentration level (K, = 76 PM for P450 2B1/2B2 versus K, = 6 PM for GST). These strong, differential, and (iso)enzyme-selective inhibitory ef-

TABLE 2. Enzyme kinetic parameters of the effects of curcumin on GST activities towards CDNB in cytosol from diierentially induced rats Kind of cytosol Curcumin (PM) added V max (pmol/min/mg) Type of inhibition

6%

&

($)

:g,

PNF-cytosol

PB-cytosol

Pyr-cytosol

0 10 20 0 7.5 15 0 3.5 15
v,,,. K,,,, K,, and K: arc the average from two

1.24 1.60 1.53 4.14 2.59 2.22 1.32 1.30 1.09

experiments. Mlxed, mixed type

0.21 1.14 1.26 0.39 0.56 0.64 0.10 0.28 0.44

of Inhibition;

2.29 4.06 5.75 7.18 1.79 3.29

compenrive,

21 12.50 17.33

competitive competitive mixed mixed competitive mixed

10

71.28
competitive type of inhibition.

12

The valuesof

44

S. Oetari

et al.

D l

CURCO~M
CURC 10pM CURC 20pM

z
-5 -4 -3 -2 -i 0 1 ] 2 (wM) 3 4 5 6 l/[CDNB

FIG. 3. Lineweaver-Burk CDNB GSH-conjugation

plot showing the inhibition of GST-mediated activity in liver cytosol from

PNF-induced

ratsby

curcumin

at concentrations

of 0.1 and 20 pM.

fects of curcumin important between and other cause at relatively the

on cytochrome low concentrations and

P450 and GSTs of curcumin the bioactivation may be strongly that curcumin

may have bethe balance of drugs influenced is relatively of curproof

physiological

and toxicological

consequences,

bioinactivation

xenobiotic

chemicals shown

[14, 261. In summary, unstable cumin GSH,

we have

in phosphate can be improved NAC, ascorbic

buffer

at pH 7.4. The

stability

by lowering

the pH and by adding and cytosolic potent inhibitor

acid, or microsomal is an extremely

teins of rat liver. Curcumin P450 lAl/lA2, a slightly 2B2, and a weak inhibitor also a potent with inhibitor pretreated selective bition observed effects curcumin, dogenous

less potent inhibitor of I450 2Bl/ of P450 2El. However, curcumin is

of GSTs in liver cytosol from rats isoenzymePB, Py r and BNF. The observed properties might as well as the GST-inhihelp explain previously properties of and cytoprotective P450and enof curcumin Given

P450 inhibition properties of curcumin. anticarcinogenic, this compound compounds.

antimutagenic, the strong

inhibitory

may also be useful in studying of exogenous

and GST-dependent

biotransformations

References
1. Liu JY, Lin SJ, Lin JK, Inhibitory effect of curcumin on protein kinase C activity induced by 12-O-tetradecanoyl-phorbol-13_acetate in NIH 3T3 cell. Carcinogenesis 14: 857-861, 1993. 2. Mukhopadhyay A, Basu N, Chatak N and Gujral PK, Antiinflammatory and irritant activity of curcumin analogues in rat. Agents Actions 213: 508-515, 1982. 3. Rao ST, Basu N, and Siddiqui HH, Anti-inflammatory activity of curcumin analogues. Indian .I Med Res 75: 574-578, 1982. of diferuloyl methane 4. Srimal RC and Dhawan BN, Pharmacology (curcumin), a non steroidal anti-inflammatory agent. J Pharm Phannacal 114: 464-468, 1973. 5. Ammon HPT, Safayhi H, Mact T and Sabieraj J, Mechanism of anti-inflammatory actions of curcumin and boswellic acids. J Ethnopharmacol38: 113-l 19, 1993. 6. Aznam Nurfina, The synthesis of some symmetrical curcumin derivatives and the study of their anti-inflammatory activities as well as structure-activity relationships, Ph.D. Thesis, Gadjah Mada University, Yogyakarta, Indonesia, 1994.

M and Bhide SV, Non-mutagenicity of curcumin 7. Nagabhushan and its anti-mutagenic action versus chili and capsaicin. Nun Cancer 8: 201-210, 1986. 8. Nagabhushan M, Amonkar A] and Bhide SV, In vitro antimutagenicity of curcumin against environmental mutagens. Food Chem Toxic01 25, 545-547, 1987. 9. Huang MT, Lysz T, Ferraro T, Abidi TF, Laskin FA, and Conney AH, Inhibitory effects of curcumin on in vitro lipooxygenase and cyclooxygenase activities in mouse epidermis. Cant Res 5 1, 813819, 1991. 10. Huang MT, Wang ZY, Georgiadis CA, Laskin JD and Coney AH, Inhibitory effects of curcumin on tumor initiation by benzo[a]pyrene and 7,12-dimethyl-benz[a]anthracene. Carcinogenesis 13: 2183-2186, 1992. 11. Donatus IA, Sardjokog Vermeulen NPE, Cytotoxicity and cytoprotective activities of curcumin. Biochem Pharm~col 39( 12): 1869-1875, 1990. 12. Tonnesen HH and Karlsen J, Studies of curcumin and curcuminoids. V. alkaline degradation of curcumin. 2 Lebensm Unters Forsch 180: 132-134, 1985. 13. Das M, Bickers DR and Mukhtar H, Plant phenols as in vitro inhibitors of glutathione S-transferase(s). Biochem Biophys Res Comm 120(2): 427-433, 1984. NPE, Donne-Op den Kelder GM and Commandeur 14. Vermeulen JNM, Formation of and protection against toxic reactive intermediates. In: Proceedings XIIth International Symposium on Medicinal Chemistry, Basel, 13-18 September 1992, (Eds. Testa B, Kyburz E, Fuhrer W and Giger R) pp. 573-593. Verlag Helvetica Chimica Acta, Basel, and Verlag Chemie, Weinheim, 1993. 15. Pabon HJJ, A synthesis of curcumin and related compounds, Recueii 83: 379-386, 1964. 16. Lake BG, Preparation and characterisation of microsomal fractions for studies on xenobiotic metabolism. In: Biochemical Toxicology (Eds. Snell K and Mullock B), pp. 183-215. lrl Press, Oxford-Washington DC., 1987. B, Meijer J and Depierre JW, Characterization of the 17. Lundgren induction of cytosolic and microsomal epoxide hydrolases by 2-ethylhexanoic acid in mouse liver. Drug Memb Disp 15: l14121, 1987. 18. Bradford MM, A rapid and sensitive method for the quatitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976. 19. Burke MD and Mayer RT, Ethoxyresorufin: Direct fluorometric assay of a microsomal 0-dealkylation which is preferentially inducible by 3-methyl-cholanthrene, Drug Metab Disp 2: 583-588, 1974. 20. Koop DR, Hydroxylation of p-nitrophenol by rabbit ethanol inducible cytochrome P-450 isoenzymes. MO& Phurmacol29: 399404, 1986. 21. Habig WH, Pabst MJ and Jakoby WB, Glutathione S-transferases, the first enzymatic step in mercapturic acid formation. J Biol Chem 249(22): 7130-7139, 1974. 22. Cai P, Bennet D, Nair RV, Ceska 0, Smith MJA and Giovanni JD, Inhibition and inactivation of murine hepatic ethoxy and penthoxyresorufin 0-dealkylase by naturally occurring coumarins. Chem Res Toxicol 6: 872-879, 1993. 23. Mukundan MA, Chacko MC, Annapurna VP and Krishnaswamy K, Effect of turmeric and curcumin on BP-DNA adducts. Carcinogenesis 14: 193496, 1993. KK and Kuttan R, Inhibition of chemical carcino24. Soudamini genesis by curcumin. J Ethnopharmacol 27: 227-233, 1989. 25. loannides C and Parke DV, Induction of cytochrome P450-IA as an indicator of potential chemical carcinogenesis. Drug Memb Rev 25: 485-501, 1993. 26. Guengerich FP, Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. Chem Res Toxic01 4: 391407, 1991.

Effects of Circumin

on Biotransformation

Enzymes

45

27. Rao CV, Sims B and Redd*, BS, Inhibition by dietary curcumin of azoxymethane-induced crnithine decarboxylase, tyrosine protein kinase, arachidonic acid metabolism and aberrant crypt foci formation in the rat colon. Carcinogen&s 11: 2219-2225 28. Susan M and Rao MNA, Induction of glutathione S-transferase activity by curcumin in mice. Arzneim Drug Res 42(U)(7): 962964, 1992. 29. Nijhoff WA, Groen GM, Peters WHM, Induction of rat hepatic

and intestinal glutathione S-transferases and glutathione by dretary naturally occurring anti-carcinogens. Int _JOncol 3: 11311139, 1993. 30. Mathews S and Rao MNA, Interaction of curcumin with glutathrone. Int J Pharm 76: 257-259, 1991. 3 1. Foliot A and Beaune P, Effects of microsomal enzyme inducers on glutathione S-transferase isoenzymes in livers of rats and hamsters. Biochem Pharmacol 48(2): 293-300, 1994.