- Email: [email protected]
Piperine, a plant alkaloid of the piper species, enhances the bioavailability of aflatoxin B1 in rat tissues
Cancer Letters, (1991) 195 - 199 Elsevier Scientific Publishers Ireland
195 Ltd
Piperine, a plant alkaloid of the piper species, bioavailability of aflatoxin B1 in rat tissues Manju
A. Allarneh”‘, N. Srivastavaa
Saxenab”,
Gopa
Biswasa,
“Biochemistry Department. Vollabhbhai Pate/ Chest Institute. Research Laboratory. C.S.I.R., Jammu-Tawi-180 001. (India) (Received 9 August 1991) (Revision received 27 September (Accepted 30 September 1991)
Branch
0304-3835/92/$05.00 Printed and Published
of Delhi,
Delhi-110
and
007 and
Keywords: aflatoxin B,; piperine; formation; DNA binding
bRegional
Piperine (Fig. 1) is an alkaloid of black pepper (Piper nigram L.) and long pepper (Piper longum L.) which are commonly used as spices in food. Atal et al. [l] for the first time reported that piperine strongly inhibits hepatic hydroxylase (AHH) and arylhydrocarbon other cytochrome P-450 activities. Similarly the rate of glucuronidation and UDPGA content was lowered in isolated epithelial small intestine cells when incubated with piperine [2]. We undertook this study to examine whether
Biochemistry Department. University of Delhi. Delhi-
at Galveston,
Galveston.
\ 0’1, v
H
7
C=C-&C-CO-N A
A
Texas.
Fig. 1. 0 1992 Elsevier Scientific Publishers in Ireland
biotrans-
Introduction
110 007. India. Tarbial Modaress University, ‘Department of Biochemistry, P.O. Box 14 155-4838, Tehran, Iran. “Department of Biological Chemistry and Genetics, University Medical
University
Singhb
metabolism thus closely resembles the mode of action of SKF 525-A on biotransformation of foreign compounds.
Piperine is known to modify the biotransformation of drugs. The effect ofpiperine on the metabolic activation and distribution of [3H]aflatoxin BI (AFB,) in rats has been described. Piperine markedly inhibited liver microsomecatalysed t3H’jAFB1 binding to calf thymus DNA in vitro, in a dose dependent manner. Rats pretreated with piperine accumulated considerable [3H]AFBI radioactivity in plasma and in the tissues examined as compared to the controls. However, piperine had no injluence on hepatic [3H]AFBI-DNA binding in vivo, which could possibly be due to the null effect of piperine on liver cytosolic glutathione (GSH) 5-transferase activity. Piperine-treated rat liver microsomes demonstrated a tendency to enhance [3HjAFB1 binding to calf thymus DNA in vivo. The effect of piperine on AFBl
of Texas, U.S.A.
Raj”, Jaswant
the
1991)
Summary
Correspondence to: H.G. Raj, Vallabhbhai Pate1 Chest Institute.
H.G.
enhances
Ireland
Ltd
Structure
of piperine
3
196
piperine (a) influences distribution of [‘HIAFBl in rat tissues and (b) has an effect in vitro or in vivo on the metabolic activation of the carcinogen. Materials
and Methods
Piperine was prepared in the Biochemical Division, Regional Research Laboratory, Jammu, India as described earlier [ 11. The source of all other chemicals used is indicated in an earlier communication [3]. Male albino Wistar rats (175-215 g body wt.) were maintained on a commercial diet supplied by Hindustan Lever Ltd., Bombay, India. Distribution of [3H]AFBI
radioactivity
Rats were divided into piperine treated and control groups. The former were fed a single dose of piperine (50 mg/kg) in 1 ml coconut oil while the controls received the vehicle alone. After 1 h all the animals were injected i.p. with 2 &i [3H]AFB1 containing 40 pg AFB1/lOO g body wt. The animals were killed in groups of 3, from control as well as piperinetreated groups, 30 min, 1 and 2 h after [ 3H]AFB, administration. Blood was collected from heart puncture into heparinized tubes. Liver and lung were removed and homogenized in 0.25 M sucrose. Aliquots of plasma and tissue homogenate were placed in liquid scintillation vials containing tissue solubiliser supplied by Beckman Instrument Co., U.S.A. The samples were digested overnight followed by the addition of a liquid scintillation cocktail. The vials were counted in Beckman liquid scintillation counter Model 6 000 IC. on AFBI-DNA binding in uitro Liver microsomes were prepared. Microsome mediated [3H]AFB, binding to calf thymus DNA was carried out in the presence of several concentrations of piperine (0, 10, 40 and 100 PM) prepared in 0.2 ml DMSO. DNA was extracted and AFB1-DNA binding determined as described earlier [3].
Effect of piperine pretreatment on the microsome-dependent AFBI-DNA binding Male albino Wistar rats were used in all the experiments. Rats received a single dose of piperine (50 mg/kg) orally, in 1.0 ml coconut oil. Control animals received coconut oil alone. All the rats were killed 1 h after treatment. Microsomes and cytosols of rat liver were prepared. Binding of [3H]AFB1 to calf thymus DNA catalysed by microsomes was quantitated by the method described earlier [31. Results Pretreatment of rats with piperine resulted in significant accumulation of [ 3H]AFB1 radioactivity in plasma and tissues (Fig. 2). Piperine was found to interfere with rat liver microsome-catalysed AFBl metabolism resulting in
Effect of piperine
I
2
3
Time in hours
Fig. 2. Effect of piperine on distribution of 13H]AFB, in the rat. Experimental details described under Materials and Methods. Control: - piperine. Treated: + piperine. Values are the average of 3 animals with less than 5% variation.
197
traperitoneally (Table I). Piperine administration had no effect on rat liver cytosolic GSH S-transferase activity while microsomes showed a tendency to stimulate AFBr-DNA binding in vitro (Table I). Discussion
I
I
50
100
PIPERINE
Inhibition of drug metabolism is specifically defined as the interference in the metabolism of one xenobiotic by another at the enzymic site [5]. Piperine conforms to this definition according to the reports of Atal et al. [l]. The inhibitory potency of the agent is often examined by in vitro oxidative metabolism [6]. Keeping in tune with this, piperine proved a good inhibitor of microsome mediated AFBr epoxidation (Fig. 3). Progressive inhibition of drug metabolism leads to elevated levels of the drug in the plasma and tissues [5]. Similarly piperine markedly enhances bioavailability of AFBr in rat tissues (Fig. 2). Pretreatment with piperine prolonged hexobarbital sleeping time and zoxazolamine paralysis time in mice dosed with piperine [l]. Recently, piperine administration to rats was shown to induce potentiation of pentobarbitone sleeping time by enhancement of barbiturate uptake in the brain and to reduce the rate of degradation of the narcotic in the liver [7]. Oxidative metabolism of AFBi is described in Fig. 4. Formation of AFBr-2,3-oxide is the major pathway for oxidative degradation [8]. Although piperine can
( IJM 1
Fig. 3. Inhibition of liver microsome-catalysed AFB,DNA binding in vitro by piperine. Various concentrations of piperine 0, 10, 40 and 100 PM were prepared in 0.2 ml DMSO and included in the liver microsome-mediated binding [“H)AFB, to calf thymus DNA as described earlier [3]. Values are the average of 3 analyses with less than 5% variation.
diminished binding of AFBr to calf thymus DNA (Fig. 3). The inhibition was found to be concentration dependent. However, the hepatic AFBr-DNA binding in vivo remained unaltered in rats injected with piperine inTable 1. Effect of oral administration GSH S-transferase activity. Treatment
[3H]AFB,-DNA (pmol/mg in vivo
Control Piperine
of piperine
21.66 25.68
Values are the mean f S.E.M. “Habig et al. [13]. bP > 0.05
DNA)
•t 2.86 zt 1.41b
of 4 animals.
on hepatic
AFBr-DNA
binding in vivo and in vitro and cytosolic
(pmol/mg DNA per 30 min) in vitro
GST activitya (nmol CDNB) conjugated/ mg protein)
107 * 8.0 122 f 10.ob
1453 f 40 1501 f 51b
binding
198
Fig. 4.
Pathway
for cellular biotransformation
of
AFB,.
inhibit microsome mediated AFBr epoxidation in vitro (Fig. 3) it is unlikely that piperine can be effective in vivo as measured by AFBi-DNA binding (Table I). It is known that not all compounds that are effective in vitro can act as inhibitors of drug metabolism in vivo [6]. The enhancement of AFBr epoxide, if any, in vivo is effectively conjugated by cytosolic GSH S-transferases [9]. It is pertinent to cite the effect of phenobarbital in vivo, where inhibition of hepatic AFBi-DNA binding is observed inspite of increased cytochrome P-450 content as the cellular GSH Stransferase activity is markedly induced [lo]. Since piperine administration does not affect the catalytic activity of cellular GSH S-transferase AFBr-DNA binding is unaltered (Table I) in vivo. Piperine-like SKF 525-A [ll, 121 is known to inhibit cellular UDP-glucuronyl transferase [2]. It is possible that enhanced [3H]AFB1 levels in the plasma and tissues of rats can be due to suppression of glucuronidation of various hydroxy metabolites of AFBi (Fig. 4) in vivo by piperine.
Acknowledgements Financial Department Delhi.
support was provided by the of Science and Technology, New
References Atal, C.K.,
Dubey, R.K. and Singh, J. (1984)
basis of enhanced
Biochemical
drug availability of piperine:
evidence
that piperine is a potent inhibitor of drug metabolism. Pharmacol.
Exp. Therap.,
Singh, J.. Dubey, mediated
R.K.
232,
J.
258 - 262.
and Atal. C.K.
inhibition of glucuronidation
(1986)
Piperine-
activity in isolated
epithelial cells of the guinea pig small intestine: evidence that piperine lowers the endogenous content. J. Pharmacol.
UDP-glucuronic
Exp. Therap.,
236,
Allameh. A., Saxena,
M. and Raj. N.C.
effects
hydroxyanisole
of butylated
(1988) on
acid
893-899. Differential
metabolism
aflatoxin B, in vitro by liver and lung microsomes.
of
Cancer
Lett., 40. 49-57. Daoud. A.H.
and Irving, C.C.
(1977)
Methylation of DNA
in rat liver and intestine by dimethylnitrosamine methylnitrosourea. Mannering, Handbook Anders,
Chem.-Biol.
G.J.’ (1971).
and N-
167, 135 - 143.
Inhibition of drug metabolism.
Exp. Pharmacol.,
M.W.
Interact.,
and Mannering.
28, 452 - 476. G.J.
(1966).
Inhibition of
199
drug metabolism. I. Kinetics of the inhibition of ethyl morphine by SKF 525-A type compounds. Mol. Pharmacol., 2, 319-327. 7
8
9
Majumdar. A.M., Dhuley, J.N.. Deshmukh, V.K., Raman, P.H.. Thorat. S.L. and Naik. S.R. (1990) Effect of piperine on pentobarbitone induced hypnosis in rat. Indian J. Exp. Biol.. 28. 486-487. Swenson, D.H.. Miller, E.C. and Miller, J.A. (1974) Aflatoxin Bt-2,3-oxide: evidence for its formation in viva and by human liver microsomes in vitro. Biochem. Biophys. Res. Commun.. 60, 1036- 1043. H.G., Santhanam, K., Gupta, R.P. and Raj, Venkitasubramanian, T.A. (1975) Oxidative metabolism of aflatoxin Bj: observations on the formation of epoxideglutathione conjugate, Cbem. Biol. Interactions. 11. 301-305.
10
11
Lotlikar, P.D., Raj., H.G. and Bohm et al. (1989) A mechanism of inhibition of aflatoxin Br-DNA binding in the liver by phenobarbital treatment of rats. Cancer Res., 49, 951- 957, Neumann, N.R., Miya. T.S., Bousquet, W.F. (1963) Mechanism of development of tolerance to SKF 525-A in the rat. Proc. Sot. Exp. Biol. Med., 114. 141- 145.
12
Hargreeves. T. (1967) The effect of SKF 525-A on uridine 5’-pyrophosphate-glucuronyl transferase. Biochem. Pharmacol.. 16. 1481- 1488.
13
Habig, W.H., Pabst. M.J.. and Jakoby. W.B. (1974) Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem.. 249, 7130 - 7139.
195 Ltd
Piperine, a plant alkaloid of the piper species, bioavailability of aflatoxin B1 in rat tissues Manju
A. Allarneh”‘, N. Srivastavaa
Saxenab”,
Gopa
Biswasa,
“Biochemistry Department. Vollabhbhai Pate/ Chest Institute. Research Laboratory. C.S.I.R., Jammu-Tawi-180 001. (India) (Received 9 August 1991) (Revision received 27 September (Accepted 30 September 1991)
Branch
0304-3835/92/$05.00 Printed and Published
of Delhi,
Delhi-110
and
007 and
Keywords: aflatoxin B,; piperine; formation; DNA binding
bRegional
Piperine (Fig. 1) is an alkaloid of black pepper (Piper nigram L.) and long pepper (Piper longum L.) which are commonly used as spices in food. Atal et al. [l] for the first time reported that piperine strongly inhibits hepatic hydroxylase (AHH) and arylhydrocarbon other cytochrome P-450 activities. Similarly the rate of glucuronidation and UDPGA content was lowered in isolated epithelial small intestine cells when incubated with piperine [2]. We undertook this study to examine whether
Biochemistry Department. University of Delhi. Delhi-
at Galveston,
Galveston.
\ 0’1, v
H
7
C=C-&C-CO-N A
A
Texas.
Fig. 1. 0 1992 Elsevier Scientific Publishers in Ireland
biotrans-
Introduction
110 007. India. Tarbial Modaress University, ‘Department of Biochemistry, P.O. Box 14 155-4838, Tehran, Iran. “Department of Biological Chemistry and Genetics, University Medical
University
Singhb
metabolism thus closely resembles the mode of action of SKF 525-A on biotransformation of foreign compounds.
Piperine is known to modify the biotransformation of drugs. The effect ofpiperine on the metabolic activation and distribution of [3H]aflatoxin BI (AFB,) in rats has been described. Piperine markedly inhibited liver microsomecatalysed t3H’jAFB1 binding to calf thymus DNA in vitro, in a dose dependent manner. Rats pretreated with piperine accumulated considerable [3H]AFBI radioactivity in plasma and in the tissues examined as compared to the controls. However, piperine had no injluence on hepatic [3H]AFBI-DNA binding in vivo, which could possibly be due to the null effect of piperine on liver cytosolic glutathione (GSH) 5-transferase activity. Piperine-treated rat liver microsomes demonstrated a tendency to enhance [3HjAFB1 binding to calf thymus DNA in vivo. The effect of piperine on AFBl
of Texas, U.S.A.
Raj”, Jaswant
the
1991)
Summary
Correspondence to: H.G. Raj, Vallabhbhai Pate1 Chest Institute.
H.G.
enhances
Ireland
Ltd
Structure
of piperine
3
196
piperine (a) influences distribution of [‘HIAFBl in rat tissues and (b) has an effect in vitro or in vivo on the metabolic activation of the carcinogen. Materials
and Methods
Piperine was prepared in the Biochemical Division, Regional Research Laboratory, Jammu, India as described earlier [ 11. The source of all other chemicals used is indicated in an earlier communication [3]. Male albino Wistar rats (175-215 g body wt.) were maintained on a commercial diet supplied by Hindustan Lever Ltd., Bombay, India. Distribution of [3H]AFBI
radioactivity
Rats were divided into piperine treated and control groups. The former were fed a single dose of piperine (50 mg/kg) in 1 ml coconut oil while the controls received the vehicle alone. After 1 h all the animals were injected i.p. with 2 &i [3H]AFB1 containing 40 pg AFB1/lOO g body wt. The animals were killed in groups of 3, from control as well as piperinetreated groups, 30 min, 1 and 2 h after [ 3H]AFB, administration. Blood was collected from heart puncture into heparinized tubes. Liver and lung were removed and homogenized in 0.25 M sucrose. Aliquots of plasma and tissue homogenate were placed in liquid scintillation vials containing tissue solubiliser supplied by Beckman Instrument Co., U.S.A. The samples were digested overnight followed by the addition of a liquid scintillation cocktail. The vials were counted in Beckman liquid scintillation counter Model 6 000 IC. on AFBI-DNA binding in uitro Liver microsomes were prepared. Microsome mediated [3H]AFB, binding to calf thymus DNA was carried out in the presence of several concentrations of piperine (0, 10, 40 and 100 PM) prepared in 0.2 ml DMSO. DNA was extracted and AFB1-DNA binding determined as described earlier [3].
Effect of piperine pretreatment on the microsome-dependent AFBI-DNA binding Male albino Wistar rats were used in all the experiments. Rats received a single dose of piperine (50 mg/kg) orally, in 1.0 ml coconut oil. Control animals received coconut oil alone. All the rats were killed 1 h after treatment. Microsomes and cytosols of rat liver were prepared. Binding of [3H]AFB1 to calf thymus DNA catalysed by microsomes was quantitated by the method described earlier [31. Results Pretreatment of rats with piperine resulted in significant accumulation of [ 3H]AFB1 radioactivity in plasma and tissues (Fig. 2). Piperine was found to interfere with rat liver microsome-catalysed AFBl metabolism resulting in
Effect of piperine
I
2
3
Time in hours
Fig. 2. Effect of piperine on distribution of 13H]AFB, in the rat. Experimental details described under Materials and Methods. Control: - piperine. Treated: + piperine. Values are the average of 3 animals with less than 5% variation.
197
traperitoneally (Table I). Piperine administration had no effect on rat liver cytosolic GSH S-transferase activity while microsomes showed a tendency to stimulate AFBr-DNA binding in vitro (Table I). Discussion
I
I
50
100
PIPERINE
Inhibition of drug metabolism is specifically defined as the interference in the metabolism of one xenobiotic by another at the enzymic site [5]. Piperine conforms to this definition according to the reports of Atal et al. [l]. The inhibitory potency of the agent is often examined by in vitro oxidative metabolism [6]. Keeping in tune with this, piperine proved a good inhibitor of microsome mediated AFBr epoxidation (Fig. 3). Progressive inhibition of drug metabolism leads to elevated levels of the drug in the plasma and tissues [5]. Similarly piperine markedly enhances bioavailability of AFBr in rat tissues (Fig. 2). Pretreatment with piperine prolonged hexobarbital sleeping time and zoxazolamine paralysis time in mice dosed with piperine [l]. Recently, piperine administration to rats was shown to induce potentiation of pentobarbitone sleeping time by enhancement of barbiturate uptake in the brain and to reduce the rate of degradation of the narcotic in the liver [7]. Oxidative metabolism of AFBi is described in Fig. 4. Formation of AFBr-2,3-oxide is the major pathway for oxidative degradation [8]. Although piperine can
( IJM 1
Fig. 3. Inhibition of liver microsome-catalysed AFB,DNA binding in vitro by piperine. Various concentrations of piperine 0, 10, 40 and 100 PM were prepared in 0.2 ml DMSO and included in the liver microsome-mediated binding [“H)AFB, to calf thymus DNA as described earlier [3]. Values are the average of 3 analyses with less than 5% variation.
diminished binding of AFBr to calf thymus DNA (Fig. 3). The inhibition was found to be concentration dependent. However, the hepatic AFBr-DNA binding in vivo remained unaltered in rats injected with piperine inTable 1. Effect of oral administration GSH S-transferase activity. Treatment
[3H]AFB,-DNA (pmol/mg in vivo
Control Piperine
of piperine
21.66 25.68
Values are the mean f S.E.M. “Habig et al. [13]. bP > 0.05
DNA)
•t 2.86 zt 1.41b
of 4 animals.
on hepatic
AFBr-DNA
binding in vivo and in vitro and cytosolic
(pmol/mg DNA per 30 min) in vitro
GST activitya (nmol CDNB) conjugated/ mg protein)
107 * 8.0 122 f 10.ob
1453 f 40 1501 f 51b
binding
198
Fig. 4.
Pathway
for cellular biotransformation
of
AFB,.
inhibit microsome mediated AFBr epoxidation in vitro (Fig. 3) it is unlikely that piperine can be effective in vivo as measured by AFBi-DNA binding (Table I). It is known that not all compounds that are effective in vitro can act as inhibitors of drug metabolism in vivo [6]. The enhancement of AFBr epoxide, if any, in vivo is effectively conjugated by cytosolic GSH S-transferases [9]. It is pertinent to cite the effect of phenobarbital in vivo, where inhibition of hepatic AFBi-DNA binding is observed inspite of increased cytochrome P-450 content as the cellular GSH Stransferase activity is markedly induced [lo]. Since piperine administration does not affect the catalytic activity of cellular GSH S-transferase AFBr-DNA binding is unaltered (Table I) in vivo. Piperine-like SKF 525-A [ll, 121 is known to inhibit cellular UDP-glucuronyl transferase [2]. It is possible that enhanced [3H]AFB1 levels in the plasma and tissues of rats can be due to suppression of glucuronidation of various hydroxy metabolites of AFBi (Fig. 4) in vivo by piperine.
Acknowledgements Financial Department Delhi.
support was provided by the of Science and Technology, New
References Atal, C.K.,
Dubey, R.K. and Singh, J. (1984)
basis of enhanced
Biochemical
drug availability of piperine:
evidence
that piperine is a potent inhibitor of drug metabolism. Pharmacol.
Exp. Therap.,
Singh, J.. Dubey, mediated
R.K.
232,
J.
258 - 262.
and Atal. C.K.
inhibition of glucuronidation
(1986)
Piperine-
activity in isolated
epithelial cells of the guinea pig small intestine: evidence that piperine lowers the endogenous content. J. Pharmacol.
UDP-glucuronic
Exp. Therap.,
236,
Allameh. A., Saxena,
M. and Raj. N.C.
effects
hydroxyanisole
of butylated
(1988) on
acid
893-899. Differential
metabolism
aflatoxin B, in vitro by liver and lung microsomes.
of
Cancer
Lett., 40. 49-57. Daoud. A.H.
and Irving, C.C.
(1977)
Methylation of DNA
in rat liver and intestine by dimethylnitrosamine methylnitrosourea. Mannering, Handbook Anders,
Chem.-Biol.
G.J.’ (1971).
and N-
167, 135 - 143.
Inhibition of drug metabolism.
Exp. Pharmacol.,
M.W.
Interact.,
and Mannering.
28, 452 - 476. G.J.
(1966).
Inhibition of
199
drug metabolism. I. Kinetics of the inhibition of ethyl morphine by SKF 525-A type compounds. Mol. Pharmacol., 2, 319-327. 7
8
9
Majumdar. A.M., Dhuley, J.N.. Deshmukh, V.K., Raman, P.H.. Thorat. S.L. and Naik. S.R. (1990) Effect of piperine on pentobarbitone induced hypnosis in rat. Indian J. Exp. Biol.. 28. 486-487. Swenson, D.H.. Miller, E.C. and Miller, J.A. (1974) Aflatoxin Bt-2,3-oxide: evidence for its formation in viva and by human liver microsomes in vitro. Biochem. Biophys. Res. Commun.. 60, 1036- 1043. H.G., Santhanam, K., Gupta, R.P. and Raj, Venkitasubramanian, T.A. (1975) Oxidative metabolism of aflatoxin Bj: observations on the formation of epoxideglutathione conjugate, Cbem. Biol. Interactions. 11. 301-305.
10
11
Lotlikar, P.D., Raj., H.G. and Bohm et al. (1989) A mechanism of inhibition of aflatoxin Br-DNA binding in the liver by phenobarbital treatment of rats. Cancer Res., 49, 951- 957, Neumann, N.R., Miya. T.S., Bousquet, W.F. (1963) Mechanism of development of tolerance to SKF 525-A in the rat. Proc. Sot. Exp. Biol. Med., 114. 141- 145.
12
Hargreeves. T. (1967) The effect of SKF 525-A on uridine 5’-pyrophosphate-glucuronyl transferase. Biochem. Pharmacol.. 16. 1481- 1488.
13
Habig, W.H., Pabst. M.J.. and Jakoby. W.B. (1974) Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem.. 249, 7130 - 7139.