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Chemopreventive action of mace (Myristica fragrans, Houtt) on DMBA-induced papillomagenesis in the skin of mice
Cancer Letters, (1991) 56 (1991) 59-63 Elsevier Scientific Publishers Ireland Ltd.
59
Chemopreventive action of mace (Myristica fragrans, HOW) on DMBA-induced papillomagenesis in the skin of mice L.N. Jannu, Cancer
Biology
(Received (Accepted
S.P. Hussain Laboratory,
School
and A.R. Rao of Life Sciences,
Jawaharlal
Nehru
University,
New Delhi-110
067 (India)
30 October 1990) 6 November 1990)
Summary The present paper reports the chemopreventive property of mace (aril covering the seed of Myristica fragrans) on DMBA-induced papillomagenesis in the skin of male Swiss albino mice. When a single topical application of DMBA (150 pg in 100 ~1 of acetone) was followed, 2 weeks later, by repeated applications ofcroton oil (1% in acetone, three times/week) skin papillomas appeared in 100% animals and the average tumors per tumor-bearing animal was 5.67. On the other hand, when animals receiving similar treatments were put on a diet containing 1% mace during the periinitiational phase of tumorigenesis, the skin papilloma incidence was reduced to 50% and the average tumor per tumor-bearing mouse was only 1.75. This decline in papilloma was significant (P < 0.05). Keywords: skin papillomagenesis DMBA; chemoprevention; mace
Materials
Myristica fragrans Houtt, is an evergreen, dioecious and aromatic tree which yields comCorrespondence to: Professor A.R. Rao, Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru Universi067, India.
and Methods
in mice;
Introduction
ty, New Delhi-110
mercial nutmeg and mace. Nutmeg is the kernel of the seed whereas mace is the fibrous aril covering the testa of the seed. Both these products are known to contain a fixed oil, a volatile oil and starch, and are widely used as spices and herbal medicines. In medicine it is used as a carminative, astringent and aphrodisiac and the oil of these products is used in flavouring food products, and liquors [lo]. Recently our laboratory has demonstrated that administration of mace through the diet increased the activity of glutathione S-transferase (GST) and the level of acid soluble sulfhydryl (SH) in mouse liver [4]. These findings stimulated us to study the possible chemopreventive action of mace on chemical carcinogenesis, and the present paper deals with the chemopreventive action of mace on DMBAinduced papillomagenesis in the skin of male Swiss albino mice.
Animals Random-bred, male Swiss albino mice (7-8 weeks old) obtained from our JNU Animal Facility, New Delhi, were maintained in an airconditioned room and were provided with (unless otherwise stated) standard food pellets (Hindustan Lever Ltd., India) and tap-water ad libitum. Three days before the commencement of the experiment, the dorsal skin in the interscapular area was shaven and only those
60
animals showing no hair regrowth were used for the study. Chemicals
DMBA and croton oil were procured from Sigma Chemical Co., U.S.A. Mace was purchased from the local market in New Delhi. DMBA was dissolved in acetone at a concentration of 150 pg/O. 1 ml. Croton oil was diluted in acetone to give 1% dilution. The mace was ground into a smooth powder and then mixed thoroughly with the powdered diet to give 1% experimental diet. Experimental
design
The animals were assorted into the following control and experimental groups (see also Table 1): ’ Group 1 (n = 20): A single dose of 150 pg of DMBA in 100 ~1 of acetone was applied topically over the shaven skin. Two weeks later, croton oil (100 ~1 of 1% croton oil in acetone) was applied three times/week until the end of the experiment. All these mice received normal diet. Group 2 (n = 20): Animals of this group received 1% diet for 21 days, i.e. 10 days before, the day of and 10 days following the carcinogen treatment. DMBA and croton oil treatments were given in Group 1. Group 3 (n = 20) : Mice of this group received 1% mace diet as in Group 2. Croton oil was applied as in Group 1. However, these animals were not treated with DMBA. Group 4 (n = 20): Animals of this group received 1% mace diet and DMBA treatment as in Group 2. However, they were not treated subsequently with croton oil. Group 5 (n = 20): These animals received DMBA and normal diet as in Group 1. However they did not receive either mace or croton oil treatment. Animals were weighed initially, at fortnightly intervals and at autopsy. Papillomas appearing in the shaven area of the skin were recorded at weekly intervals, and papillomas L 1 mm in diameter were included for data analysis only if they persisted for two weeks or more. Animals
were sacrificed 20 weeks after the commencement of the treatments. The x*-test was applied to evaluate the influence of the modulator on tumor incidence by analyzing the significance level of the difference between control and experimental values. Results The findings of the present investigation are depicted in Table I. Further, Fig. 1 depicts the percentage of mice with papillomas in control (Group 1) and experimental (Group 2) lots. Figure 2 depicts the number of tumors per tumor-bearing mouse documented during the observation period in control and experimental groups (Group 1 and Group 2). Figure 3 depicts the cumulative number of tumors induced during the observation period in control (Group 1) and experimental (Group 2) animals. Various treatments did not affect the body weight gain during the observation period (Table I). Spontaneous skin tumors are not encountered in our colony of Swiss albino mice. When a single application of DMBA was followed, 2 weeks later, by repeated applications of croton oil (Group 1) skin papillomas appeared in all (100%) animals and the average number of tumors per tumor-bearing animal was 5.67. Mice of Group 2 which received, in addition to DMBA and croton oil as in Group 1, mace diet instead of normal diet during the peri-initiation phase of tumorigenesis showed only 50% tumor incidence and the average papillomas per tumorbearing mouse was reduced to 1.75. Animals of Group 3 (which were first treated with only mace diet for 21 days and then received repeated topical applications of croton oil), Group 4 (which were treated with a single dose of DMBA and mace diet during the periinitiational phase of tumorigenesis) and Group 5 (which received only DMBA and normal diet) did not show any skin papilloma occurrence during the observation period. Discussion A wide spectrum of naturally occurring as well
‘P < 0.05.
1 2 3 4 5
Group no.
Nil Mace Mace Mace Nil
DMBA DMBA Nil DMBA DNBA
Croton oil Croton oil Croton oil Nil Nil
Promoter 20 20 20 20 20
Initial
Initiator
Modulator 19 220 20 20 20
Effective
papillomagenesis
Animal no.
effect of mace on DMBA-induced
Treatment
Table 1. Chemopreventive
26.85 26.2 27.5 26.5 28.0
Initial f zt zt + zt
0.6 1.05 0.5 0.8 0.25
Body weight (g) Mean * SD.
37.2 36.05 35.9 35.4 36.5
Final
in the skin of mice.
f f f f zt
2.1 3.0 1.5 1.8 0.9
19/19 10/20’ o/20 o/20 o/20
(%I
(100.0) (50.0) (00.0) (00.0) (00.0)
Mice with papillomas
5.67 1.75 0.00 0.00 0.00
Papillomas/ papillomasbearing mouse
62
‘lo-
GROUP-l:INITIATOR+PROMOTOR GROUP-2:MODIFIER+INITlATOR+ PROMOTOR
GROUP-l
INITIATOR+PROMOTOR
GROUP-2
MODIFIER+INITIATOR+ PROMOTOR
loo-
GROUP-l go-
i-
FJOln 2 ?
70-
2
60-
d g
50-
d = w
40-
2 2
30-
2
zo-
z
lo-
2
4
6
OBSERVATION
8
10 PERIOD
12
14
16
18
(weeks)
Fig. 1. The percentage of mice with papillomas in control (Group 1) and experimental (Group 2) lots. GROUP-t’INITIATOR+PROMOTOR GROUP-2 MODIFIER+INITIATOR+
2
4 6 OBSERVATION
8
IO PERIOD
12 lL (weeks1
16
18
Fig. 3. Cumulative number of papillomas induced during the observation period in control (group 1) and experimental (group 2) animals.
PRoMoToR/r-G-
OBSERVATION
PERIOD
(weeks)
Fig. 2. Tumor burden (the average number of tumors per tumor-bearing mouse) documented in control (group 1) and experimental (group 2) animals.
as synthetic substances have been shown to cause inhibition of chemical carcinogenesis in different animal model systems and some of these chemopreventive agents are on trial in human situations [ 1,11,121.The present investigation demonstrates the chemopreventive potential of mace on DMBA-induced skin tumorigenesis in male Swiss albino mice. The findings depict that there is reduction not only in tumor incidence but also in cumulative tumor frequency and tumor burden following exposure of DMBAtreated mice to mace diet during the periinitiational phase alone. Earlier our laboratory demonstrated that the administration of the mace of Myristicafragrans in the diet at the concentration of 1% and 2% for 10 days would enhance the activity of GST in the liver of mice significantly [4]. In addition this study revealed that the mace diet would augment the level of acid soluble SH groups in the liver of mice. Since
63
GST enzyme system is responsible for catalyzing the conjugation of xenobiotics with glutathione and bringing about cellular detoxication [6,9] it is possible that mace by increasing the GST activity could potentially decrease the carcinogenic effect of DMBA. Then, since mace is also reported by us to enhance acid soluble nonprotein thiols which are known to offer protection against cellular damage possibly through free radical scavenging mechanism, this pathway may also be operating in inhibiting DMBA-induced tumorigenesis in the skin. Myristica fragrans has been reported to interfere with either the synthesis or the action of prostaglandins [3,5]. At several extrahepatic sites, co-oxidation of xenobiotic chemicals including polycyclic aromatic hydrocarbons (PAH) is known to depend upon prostaglandin synthetase [2,7,8]. If there is any involvement of prostaglandin synthetase co-oxidation of PAH in activation process in the epithelial cells of the skin, the suppression of this pathway by mace also would conceivably have inhibited tumorigenesis in the present study. References Boone, C.W., Kelloff, G.J. and Malone, W.E. (1990) Identification of Candidate Cancer Chemopreventive Agents and Their Evaluation in Animal Models and Human Clinical Trials: A Review. Cancer Res., 50, 2-9. Craven, P .A., Se Robertis, S.R. and Fox, J. W. (1983) Fatty
acid-dependent
3
4
5
6
7
8
9
10 11 12
benzo(a)pyrene
oxidation in colonic mucosal
microsomes: evidence for distinct metabolic pathway. Cancer Res., 43, 35-40. Fawell, N.N. and Thompson, G. (1973) nutmeg for diarrhoea of medullary carttnoma of thyroid. New Eng. J. Med., 289, 108-109. Kumari, M.V.R. and Rao, A.R. (1989) Effects of mace (Myristica fiagrans, Houtt. ) on cytosolic glutathione Stransferase activity and acid soluble sulfhydryl level in mouse liver. Cancer Lett., 46, 87-91. Misra, V., Misra, R.N. and Linger, W.G. (1978) Role of nutmeg in inhibiting prostaglandin biosynthesis. Indian J. Med. Res., 67, 482-484. Newmark, H.L. (1987) Plant phenolics as inhibitors of mutational and precarcinogenic events. Can. J. Physiol. Pharmacol., 65, 461-466. Silvarajah. K., Lasker. J. M. and Eling, Y.E. (1981) Prostaglandin synthetase-dependent cooxidation of ( = ) benzo(a)pyrene 7,8-dihydrodiol by human lung and other mammalian tissues. Cancer Res., 41, 1834- 1839. Silvarajah, K., Jones, K.G., Fouts, J.R., Devareaux. T., Shinley, J.E. and Eling, T.E. (1983) Prostaglandin synthatase and cytochrome P-450-dependent metabolism of benzo(a)pyrene-7,8-dihydrodiol by enriched populations of rat Clara cells and alveolar type II cells. Cancer Res., 43, 2632-2636. Sparnins, V.L., Venegas, F.L. and Wattenberg, L.W. (1982) Glutathione S-transferase activity enhancement by compounds inhibiting chemical carcinogenesis by dietary constituents. J. Natl. Cancer Inst., 68, 493-496. The Wealth of India: Raw Materials (1962) Council of Scientific and Industrial Research, New Delhi, VI, 474-479. Wattenberg, L. W (1985) Chemoprevention of cancer. Cancer Res., 45, l-8. Williams, G.W. (1984) Modulation of Chemical carcinogenesis by xenobiotics. Fund. Appl. Toxicol., 4, 325-344.
59
Chemopreventive action of mace (Myristica fragrans, HOW) on DMBA-induced papillomagenesis in the skin of mice L.N. Jannu, Cancer
Biology
(Received (Accepted
S.P. Hussain Laboratory,
School
and A.R. Rao of Life Sciences,
Jawaharlal
Nehru
University,
New Delhi-110
067 (India)
30 October 1990) 6 November 1990)
Summary The present paper reports the chemopreventive property of mace (aril covering the seed of Myristica fragrans) on DMBA-induced papillomagenesis in the skin of male Swiss albino mice. When a single topical application of DMBA (150 pg in 100 ~1 of acetone) was followed, 2 weeks later, by repeated applications ofcroton oil (1% in acetone, three times/week) skin papillomas appeared in 100% animals and the average tumors per tumor-bearing animal was 5.67. On the other hand, when animals receiving similar treatments were put on a diet containing 1% mace during the periinitiational phase of tumorigenesis, the skin papilloma incidence was reduced to 50% and the average tumor per tumor-bearing mouse was only 1.75. This decline in papilloma was significant (P < 0.05). Keywords: skin papillomagenesis DMBA; chemoprevention; mace
Materials
Myristica fragrans Houtt, is an evergreen, dioecious and aromatic tree which yields comCorrespondence to: Professor A.R. Rao, Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru Universi067, India.
and Methods
in mice;
Introduction
ty, New Delhi-110
mercial nutmeg and mace. Nutmeg is the kernel of the seed whereas mace is the fibrous aril covering the testa of the seed. Both these products are known to contain a fixed oil, a volatile oil and starch, and are widely used as spices and herbal medicines. In medicine it is used as a carminative, astringent and aphrodisiac and the oil of these products is used in flavouring food products, and liquors [lo]. Recently our laboratory has demonstrated that administration of mace through the diet increased the activity of glutathione S-transferase (GST) and the level of acid soluble sulfhydryl (SH) in mouse liver [4]. These findings stimulated us to study the possible chemopreventive action of mace on chemical carcinogenesis, and the present paper deals with the chemopreventive action of mace on DMBAinduced papillomagenesis in the skin of male Swiss albino mice.
Animals Random-bred, male Swiss albino mice (7-8 weeks old) obtained from our JNU Animal Facility, New Delhi, were maintained in an airconditioned room and were provided with (unless otherwise stated) standard food pellets (Hindustan Lever Ltd., India) and tap-water ad libitum. Three days before the commencement of the experiment, the dorsal skin in the interscapular area was shaven and only those
60
animals showing no hair regrowth were used for the study. Chemicals
DMBA and croton oil were procured from Sigma Chemical Co., U.S.A. Mace was purchased from the local market in New Delhi. DMBA was dissolved in acetone at a concentration of 150 pg/O. 1 ml. Croton oil was diluted in acetone to give 1% dilution. The mace was ground into a smooth powder and then mixed thoroughly with the powdered diet to give 1% experimental diet. Experimental
design
The animals were assorted into the following control and experimental groups (see also Table 1): ’ Group 1 (n = 20): A single dose of 150 pg of DMBA in 100 ~1 of acetone was applied topically over the shaven skin. Two weeks later, croton oil (100 ~1 of 1% croton oil in acetone) was applied three times/week until the end of the experiment. All these mice received normal diet. Group 2 (n = 20): Animals of this group received 1% diet for 21 days, i.e. 10 days before, the day of and 10 days following the carcinogen treatment. DMBA and croton oil treatments were given in Group 1. Group 3 (n = 20) : Mice of this group received 1% mace diet as in Group 2. Croton oil was applied as in Group 1. However, these animals were not treated with DMBA. Group 4 (n = 20): Animals of this group received 1% mace diet and DMBA treatment as in Group 2. However, they were not treated subsequently with croton oil. Group 5 (n = 20): These animals received DMBA and normal diet as in Group 1. However they did not receive either mace or croton oil treatment. Animals were weighed initially, at fortnightly intervals and at autopsy. Papillomas appearing in the shaven area of the skin were recorded at weekly intervals, and papillomas L 1 mm in diameter were included for data analysis only if they persisted for two weeks or more. Animals
were sacrificed 20 weeks after the commencement of the treatments. The x*-test was applied to evaluate the influence of the modulator on tumor incidence by analyzing the significance level of the difference between control and experimental values. Results The findings of the present investigation are depicted in Table I. Further, Fig. 1 depicts the percentage of mice with papillomas in control (Group 1) and experimental (Group 2) lots. Figure 2 depicts the number of tumors per tumor-bearing mouse documented during the observation period in control and experimental groups (Group 1 and Group 2). Figure 3 depicts the cumulative number of tumors induced during the observation period in control (Group 1) and experimental (Group 2) animals. Various treatments did not affect the body weight gain during the observation period (Table I). Spontaneous skin tumors are not encountered in our colony of Swiss albino mice. When a single application of DMBA was followed, 2 weeks later, by repeated applications of croton oil (Group 1) skin papillomas appeared in all (100%) animals and the average number of tumors per tumor-bearing animal was 5.67. Mice of Group 2 which received, in addition to DMBA and croton oil as in Group 1, mace diet instead of normal diet during the peri-initiation phase of tumorigenesis showed only 50% tumor incidence and the average papillomas per tumorbearing mouse was reduced to 1.75. Animals of Group 3 (which were first treated with only mace diet for 21 days and then received repeated topical applications of croton oil), Group 4 (which were treated with a single dose of DMBA and mace diet during the periinitiational phase of tumorigenesis) and Group 5 (which received only DMBA and normal diet) did not show any skin papilloma occurrence during the observation period. Discussion A wide spectrum of naturally occurring as well
‘P < 0.05.
1 2 3 4 5
Group no.
Nil Mace Mace Mace Nil
DMBA DMBA Nil DMBA DNBA
Croton oil Croton oil Croton oil Nil Nil
Promoter 20 20 20 20 20
Initial
Initiator
Modulator 19 220 20 20 20
Effective
papillomagenesis
Animal no.
effect of mace on DMBA-induced
Treatment
Table 1. Chemopreventive
26.85 26.2 27.5 26.5 28.0
Initial f zt zt + zt
0.6 1.05 0.5 0.8 0.25
Body weight (g) Mean * SD.
37.2 36.05 35.9 35.4 36.5
Final
in the skin of mice.
f f f f zt
2.1 3.0 1.5 1.8 0.9
19/19 10/20’ o/20 o/20 o/20
(%I
(100.0) (50.0) (00.0) (00.0) (00.0)
Mice with papillomas
5.67 1.75 0.00 0.00 0.00
Papillomas/ papillomasbearing mouse
62
‘lo-
GROUP-l:INITIATOR+PROMOTOR GROUP-2:MODIFIER+INITlATOR+ PROMOTOR
GROUP-l
INITIATOR+PROMOTOR
GROUP-2
MODIFIER+INITIATOR+ PROMOTOR
loo-
GROUP-l go-
i-
FJOln 2 ?
70-
2
60-
d g
50-
d = w
40-
2 2
30-
2
zo-
z
lo-
2
4
6
OBSERVATION
8
10 PERIOD
12
14
16
18
(weeks)
Fig. 1. The percentage of mice with papillomas in control (Group 1) and experimental (Group 2) lots. GROUP-t’INITIATOR+PROMOTOR GROUP-2 MODIFIER+INITIATOR+
2
4 6 OBSERVATION
8
IO PERIOD
12 lL (weeks1
16
18
Fig. 3. Cumulative number of papillomas induced during the observation period in control (group 1) and experimental (group 2) animals.
PRoMoToR/r-G-
OBSERVATION
PERIOD
(weeks)
Fig. 2. Tumor burden (the average number of tumors per tumor-bearing mouse) documented in control (group 1) and experimental (group 2) animals.
as synthetic substances have been shown to cause inhibition of chemical carcinogenesis in different animal model systems and some of these chemopreventive agents are on trial in human situations [ 1,11,121.The present investigation demonstrates the chemopreventive potential of mace on DMBA-induced skin tumorigenesis in male Swiss albino mice. The findings depict that there is reduction not only in tumor incidence but also in cumulative tumor frequency and tumor burden following exposure of DMBAtreated mice to mace diet during the periinitiational phase alone. Earlier our laboratory demonstrated that the administration of the mace of Myristicafragrans in the diet at the concentration of 1% and 2% for 10 days would enhance the activity of GST in the liver of mice significantly [4]. In addition this study revealed that the mace diet would augment the level of acid soluble SH groups in the liver of mice. Since
63
GST enzyme system is responsible for catalyzing the conjugation of xenobiotics with glutathione and bringing about cellular detoxication [6,9] it is possible that mace by increasing the GST activity could potentially decrease the carcinogenic effect of DMBA. Then, since mace is also reported by us to enhance acid soluble nonprotein thiols which are known to offer protection against cellular damage possibly through free radical scavenging mechanism, this pathway may also be operating in inhibiting DMBA-induced tumorigenesis in the skin. Myristica fragrans has been reported to interfere with either the synthesis or the action of prostaglandins [3,5]. At several extrahepatic sites, co-oxidation of xenobiotic chemicals including polycyclic aromatic hydrocarbons (PAH) is known to depend upon prostaglandin synthetase [2,7,8]. If there is any involvement of prostaglandin synthetase co-oxidation of PAH in activation process in the epithelial cells of the skin, the suppression of this pathway by mace also would conceivably have inhibited tumorigenesis in the present study. References Boone, C.W., Kelloff, G.J. and Malone, W.E. (1990) Identification of Candidate Cancer Chemopreventive Agents and Their Evaluation in Animal Models and Human Clinical Trials: A Review. Cancer Res., 50, 2-9. Craven, P .A., Se Robertis, S.R. and Fox, J. W. (1983) Fatty
acid-dependent
3
4
5
6
7
8
9
10 11 12
benzo(a)pyrene
oxidation in colonic mucosal
microsomes: evidence for distinct metabolic pathway. Cancer Res., 43, 35-40. Fawell, N.N. and Thompson, G. (1973) nutmeg for diarrhoea of medullary carttnoma of thyroid. New Eng. J. Med., 289, 108-109. Kumari, M.V.R. and Rao, A.R. (1989) Effects of mace (Myristica fiagrans, Houtt. ) on cytosolic glutathione Stransferase activity and acid soluble sulfhydryl level in mouse liver. Cancer Lett., 46, 87-91. Misra, V., Misra, R.N. and Linger, W.G. (1978) Role of nutmeg in inhibiting prostaglandin biosynthesis. Indian J. Med. Res., 67, 482-484. Newmark, H.L. (1987) Plant phenolics as inhibitors of mutational and precarcinogenic events. Can. J. Physiol. Pharmacol., 65, 461-466. Silvarajah. K., Lasker. J. M. and Eling, Y.E. (1981) Prostaglandin synthetase-dependent cooxidation of ( = ) benzo(a)pyrene 7,8-dihydrodiol by human lung and other mammalian tissues. Cancer Res., 41, 1834- 1839. Silvarajah, K., Jones, K.G., Fouts, J.R., Devareaux. T., Shinley, J.E. and Eling, T.E. (1983) Prostaglandin synthatase and cytochrome P-450-dependent metabolism of benzo(a)pyrene-7,8-dihydrodiol by enriched populations of rat Clara cells and alveolar type II cells. Cancer Res., 43, 2632-2636. Sparnins, V.L., Venegas, F.L. and Wattenberg, L.W. (1982) Glutathione S-transferase activity enhancement by compounds inhibiting chemical carcinogenesis by dietary constituents. J. Natl. Cancer Inst., 68, 493-496. The Wealth of India: Raw Materials (1962) Council of Scientific and Industrial Research, New Delhi, VI, 474-479. Wattenberg, L. W (1985) Chemoprevention of cancer. Cancer Res., 45, l-8. Williams, G.W. (1984) Modulation of Chemical carcinogenesis by xenobiotics. Fund. Appl. Toxicol., 4, 325-344.