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Inhibition of nitrosodiethylamine-induced hepatocarcinogenesis by dietary turmeric in rats
Toxicology Letters 139 (2003) 45 /54 www.elsevier.com/locate/toxlet
Short communication
Inhibition of nitrosodiethylamine-induced hepatocarcinogenesis by dietary turmeric in rats R. Thapliyal a, K.N. Naresh b, K.V.K. Rao a, G.B. Maru a,* a
Carcinogenesis Division, Cancer Research Institute, Tata Memorial Center, Parel, Mumbai 400 012, India Pathology Department, Tata Memorial Hospital, Tata Memorial Center, Parel, Mumbai 400 012, India
b
Received 1 July 2002; received in revised form 18 November 2002; accepted 20 November 2002
Abstract Turmeric, widely used in food and medicine has been shown to prevent benzo(a)pyrene [B(a)P] or dimethylbenz(a)anthracene (DMBA)-induced forestomach, skin and mammary tumors in mice and/or rats. In this study we examine the modulatory effects of turmeric on nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis in rats. Female Wistar rats were administered NDEA (200 ppm) through drinking water (5 days per week) for 4 weeks. Control and/or NDEA-treated rats received 0, 0.2, 1.0 or 5.0% turmeric diet (w/w) either before (2 weeks), during (4 weeks) and after NDEA exposure (10 weeks) or starting from 24 h after NDEA exposure for 10 weeks. NDEA-treated rats receiving 1 or 5% turmeric before, during and after carcinogen exposure showed significant decrease in number of gamma glutamyl transpeptidase (GGT) positive foci measuring /500 or /1000 mm and decrease in the incidence of NDEA-induced focal dysplasia (FD) and hepatocellularcarcinomas. Decrease in the number of GGT positive foci measuring /1000 mm was also observed in NDEA-treated rats receiving 0.2% turmeric, although no decrease in tumor incidence was noted. On the other hand, similar levels of turmeric treatment (0.2, 1 and 5%) after exposure to NDEA did not show any protective effects. The underlying mechanism(s) of chemoprevention of NDEA-induced hepatocarcinogenesis need to be explored. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nitrosodiethylamine; Rat liver; GGT /ve foci; Hepatocellular-carcinoma; Turmeric; Inhibition
1. Introduction Abbreviations: CYP 450, cytochrome P450; Fast blue BBN, diazotized 4?-amino-2?, 5?-diethoxybenzanilide; g-GMNA, gglutamyl-4-methoxy-2-naphthylamide; GGT, gamma glutamyltranspeptidase; GG, glycyl-glycine; HCC, hepatocellularcarcinoma; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone; NDEA, nitrosodiethylamine; PAHs, polycyclic aromatic hydrocarbons. * Corresponding author. Tel.: /91-22-412-3803; fax: /9122-414-6089. E-mail address: [email protected] (G.B. Maru).
One effective approach to cancer control is prevention. Identification and development of effective chemopreventive agents especially from dietary constituents that can block activation or enhance detoxification of environmental agents is an important aspect. Dietary intervention that counters/prevents/delays the harmful effects of carcinogens may then become part of our daily
0378-4274/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-4274(02)00440-X
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life-style changes. Spices are part of regular diet in certain populations and one such agent namely, turmeric with its perceived human safety following centuries of use in food and medicine is currently under investigation in several laboratories. Turmeric, the powdered rhizome of the plant Curcuma longa Linn. and curcumin(s), the yellow phenolic compounds in turmeric besides possessing antiinflammatory (Srimal and Dhawan, 1973) and anti-oxidant (Sharma, 1976) properties have also shown their efficacy in preventing chemical-induced experimental carcinogenesis in various tissues including skin (Huang et al., 1992, 1988; Limtrakul et al., 1997), colorectal (Pereira et al., 1996; Rao et al., 1995; Huang et al., 1994), oral (Huang et al., 1994), forestomach (Deshpande et al., 1997), and mammary glands (Deshpande et al., 1998; Singletary et al., 1996). The anti-initiation effects of turmeric/curcumin in experimental systems have mostly been established employing representative polycyclic aromatic hydrocarbon(s) (PAHs) which require metabolic activation. Turmeric/curcumin(s) have been shown to inhibit isozymes of CYP 450s in vitro as well as in vivo (Thapliyal and Maru, 2001; Oetari et al., 1996). Chemopreventive efficacy of turmeric in combination with other agent(s) have been reported against presumed carcinogenic metabolite of dimethylnitrosamine i.e. methyl (acetoxylmethyl)nitrosamine (Azuine and Bhide, 1994, 1992), while potential of turmeric (alone) to prevent hepatocarcinogenesis induced by nitrosamines requiring metabolic activation has not been established. Since hepatocellular carcinoma (HCC) is a common cause of cancer morbidity and mortality in Asia and Africa and its prognosis extremely poor effective measures of chemoprevention by dietary constituent like turmeric need to be studied. In this communication we report the potential chemopreventive action of turmeric against nitrosodiethylamine (NDEA), a hepatocarcinogen, by comparing the tumor incidence, number and size of gglutamyltranspeptidase (GGT) positive foci in liver and relative as well as absolute liver weights in rats receiving turmeric either before, during and after NDEA exposure or only after NDEA exposure.
2. Materials and methods 2.1. Chemicals NDEA, diazotized 4?-amino-2?, 5?-diethoxybenzanilide (Fast blue BBN), g-glutamyl-4-methoxy2-naphthylamide (g-GMNA) and glycylglycine (GG), were purchased from Sigma Chemical Co., USA. All other chemicals used were purchased locally and were of AR grade. 2.2. Animals Inbred female Wistar rats (8 /10 weeks) were obtained from the animal colony of Cancer Research Institute, Mumbai, India. They were randomly distributed into various groups and housed in cages (five per cage) and maintained under standard conditions i.e. 229/2 8C, 459/10% relative humidity and 12-h light:12-h dark cycles each day. All animals were fed powdered standard laboratory (control)/experimental diet and drinking water or 200 ppm NDEA in drinking water ad libitum. 2.3. Experimental diet(s)/water Turmeric rhizomes purchased in bulk locally were thoroughly washed with water, sun-dried and powdered in a grinding mill. It was stored at room temperature throughout the experiment. The curcumin(s) content from turmeric was analysed by preparative TLC (Roughley and Whiting, 1973). The content of curcumin(s) (% weight of turmeric powder) was 5% and proportion of components i.e. curcumin, demethoxycurcumin and bisdemethoxycurcumin was 67, 28 and 5%, respectively (Deshpande and Maru, 1995). Quantities of turmeric powder required for preparation of diet(s) with defined concentrations i.e. 0.2, 1.0 and 5.0% were weighed and added to preweighed standard laboratory diet and thoroughly mixed while adding aldehyde-free distilled ethanol (upto 5% concentration) to ensure its uniform distribution. A control diet (standard laboratory diet) was also prepared using similar concentration of aldehyde-free distilled ethanol. Ethanol from the control as well as the experi-
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mental diet(s) was allowed to evaporate completely and then the diets were used. All control and experimental diet(s) were prepared monthly in our laboratory and stored at 4 8C. Food cups were replenished with fresh diet every day. NDEA, a hepatocarcinogen, was administered in drinking water for 4 weeks (5 days a week) at a dose of 200 ppm in dark-colored water bottles to protect from direct light. NDEA in water was prepared weekly and stored in the refrigerator. Water bottles were replenished every day with fresh NDEA/plain drinking water. 2.4. Experimental design Animals were divided into eleven groups of untreated control (1), 0.2 (2), 1 (3), 5% (4) turmeric controls, NDEA-treated (5), 0.2% turmeric/ NDEA (6), 1% turmeric/NDEA (7), 5% turmeric/NDEA (8) (pretreatment groups 6 /8), NDEA/0.2% turmeric (9), NDEA/1% turmeric (10) and NDEA/5% turmeric (11) (posttreatment groups 9 /11). The experimental protocol followed is presented in Fig. 1. Groups 1 and 5 were kept on standard laboratory diet while groups 2, 3 and 4 were kept on 0.2, 1.0 and 5% turmeric diet, respectively, throughout the experiment. Groups 1, 2, 3 and 4 were kept on plain drinking water while group 5 was kept on 200 ppm NDEA 5 days a week for 4 weeks. Animals in groups 6, 7 and 8 were kept on 0.2, 1.0 and 5% turmeric diet, respectively, 2 weeks before, 4 weeks during and 10 weeks after NDEA administration i.e. till the end of experiment. Animals in groups 9,10 and 11 were kept on 0.2, 1.0 and 5% turmeric diet, respectively, 24 h after last NDEA administration for 10 weeks i.e. till the end of experiment. Body weights were recorded in the beginning and during the experimental period (once a week). Animals were anaesthetized and sacrificed at the end of the experiment. Final body and liver weights were recorded and livers were further utilized for GGT staining and histopathology. Pieces of liver (/0.5 /0.5 cm) from each liver lobe were fixed in 10% phosphate buffered formalin and embedded in paraffin. Serial sections of liver pieces so obtained were stained
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with haematoxylin and eosin. Tumor incidence was determined histologically by a surgical pathologist who was blinded from other information. The lesions were classified as no tumor, dysplasia and HCC (Bannasch and Zerban, 1990).
2.5. Histochemical staining for GGT positive foci Determination of GGT positive foci was performed as described (Rutenberg et al., 1969). Freshly excised livers were cryosectioned and 8 / 10 m thick sections were mounted on slides and airdried. One square cm pieces of livers were used to make cryosections. Freshly prepared solution containing the substrate GMNA, the acceptor GG and the azo coupling dye, fast blue BBN, in 0.1 M tris buffer pH 7.4 and saline was then pipetted on to the cryosections and incubated at room temperature in dark for 30/45 min. Following incubation, the slides were rinsed in saline for 2 min and then transferred to 0.1 M cupric sulfate solution for 2 min. The slides were again rinsed in saline followed by a rinse in distilled water. The sections were air-dried, mounted in 10% glycerol and observed under a light microscope with a 10 X objective lens that had a graticule for measuring the foci size. Five fields from four sections of each liver were examined. The numbers of foci are, therefore, an average of 20 fields for each liver sample. The GGT positive foci, thus, counted were divided according to various size ranges and results have been expressed as number of foci per mm2. Cryosections of kidney served as positive control for GGT staining.
2.6. Statistical analysis The incidence of mortality and HCC was analysed by the X2 test. Differences in body, liver weights, liver to body weight ratios and number of GGT positive foci between different groups (multiple comparisons) were analysed by one-way ANOVAs with Bonferroni post-hoc test. Differences were considered statistically significant at P 5/ 0.05.
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Fig. 1. Experimental design for NDEA-induced HCC in female Wistar rats.
3. Results In groups of untreated control rats as well as those receiving 0.2, 1 and 5% turmeric through diet for 16 weeks of experimental period no mortalities were observed. However, 10 out of 22 (45%) rats receiving 200 ppm of NDEA through drinking water (5 days per week /4) died either just after NDEA treatment or within 3/4 weeks after stopping the NDEA treatment. The observed mortality in NDEA-treated group was significantly higher compared with the untreated control group (Table 1). In groups of rats receiving 0.2, 1 and 5% turmeric 2 weeks before, 4 weeks during NDEA treatment and 10 weeks after cessation of exposure to NDEA, some improvement in survival was observed when compared with NDEA-treated group although statistical significance in survival was attained only in group of rats receiving 1% turmeric diet but not in those receiving either 0.2
or 5% turmeric diet (Table 1). In groups of rats receiving 0.2 or 1 or 5% turmeric diet 24 h after last NDEA exposure for a period of 10 weeks, the mortalities were 60, 52 and 42% respectively, i.e. similar to the group exposed to NDEA alone (45% mortality) and significantly higher compared with untreated control groups (0% mortality, Table 1). Data on initial and final body weights, weight gain, liver weight and liver/body weight /100 (relative liver weight), number of GGT positive foci and focal dysplasia (FD), HCC and incidence of tumor from animals belonging to various experimental groups have been compared among survivors at the end of 16 weeks of experimental period and have been presented below (Tables 2 / 4). The initial and final body weights of untreated controls, 0.2% (group 2) or 1% (group 3) or 5% (group 4) turmeric diet referred as turmeric controls, NDEA-treated (group 5) and those receiving
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Table 1 Effect of dietary turmeric on NDEA-induced mortality in rats Experimental group No
Treatment
1 2 3 4 5
UC 0.2% TC 1% TC 5% TC NDEA
T administration prior (2 weeks), NDEA treatment (10 weeks) 6 0.2% T/NDEA 7 1% T/NDEA 8 5% T/NDEA
n
Survived (%)
11 5 12 12 22
11/11 (100) 5/5 (100) 12/12 (100) 12/12 (100) 12/22 (55)*
during (4 weeks), and after 10 17 18
7/10 (70) 15/17 (88)# 13/18 (72)
T administration after NDEA treatment (10 weeks) 9 NDEA/0.2% T 10 4/10 (40)* 10 NDEA/1% T 21 10/21 (48)* 11 NDEA/5% T 19 11/19 (58)* UC, untreated control; TC, turmeric control; NDEA, nitrosodiethylamine; *, significantly different from UC, P 5/ 0.05. #, significantly different from NDEA, P 5/0.05.
turmeric diet either before, during and after NDEA exposure (groups 6 /8) or only after NDEA exposure (groups 9 /11) were comparable.
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Similarly weight gains in animals belonging to various groups were also comparable except in those receiving 0.2% turmeric after exposure to NDEA (group 9), wherein a significant decrease in weight gain was observed (Table 2) when compared with untreated control group. The absolute and relative liver weights in animals exposed to 0.2, 1 and 5% turmeric diet (groups 2 /4) for experimental period of 16 weeks did not differ when compared with absolute and relative liver weights in untreated controls (group 1). Animals treated with 200 ppm NDEA through drinking water 5 days per week for 4 weeks had significantly increased absolute (8.279/0.65 vs. 5.939/0.25) as well as relative (4.519/0.35 vs. 3.09/0.09) liver weights at the time of sacrifice when compared with respective liver weights in untreated controls (Table 2). Turmeric administration before, during and after NDEA treatment appears to protect against NDEA-induced increase in absolute and relative liver weights although statistical significance was attained only in the group receiving 1% turmeric diet (relative liver weights 4.519/0.35 vs. 3.189/0.30) (group 7). This protection is not seen in group receiving 5% turmeric diet and treated
Table 2 Effect of dietary turmeric and/or NDEA treatment on body and liver weights of rats Experimental group
n
Initial weight@
Final weight@
Gain in weight@
Liver weight@
Liver:body weight@ /100
11 5 12 12 12
1409/7 1229/9 1449/7 1419/6 1529/6
1989/7 2009/10 2089/7 2059/6 1859/7
589/6.30 789/6.04 659/4.54 659/3.66 339/4.82
5.939/0.25 5.429/0.17 5.629/0.39 6.279/0.33 8.279/0.65*
3.09/0.09 2.729/0.05 2.689/0.13 3.049/0.13 4.519/0.35*
No Treatment 1 2 3 4 5
UC 0.2% TC 1% TC 5% TC NDEA
T administration prior (2 weeks), during (4 weeks), and after NDEA treatment (10 weeks) 6 0.2% T/NDEA 7 1519/5 1999/9 479/10.29 6.89/0.6 7 1% T/NDEA 15 1529/5 2039/5 519/4.14 6.59/0.28 8 5% T/NDEA 13 1549/6 1959/8 429/6.76 7.379/0.58
3.459/0.30 3.189/0.10# 3.809/0.30$
T administration after NDEA treatment (10 weeks) 9 10 11
NDEA/0.2% T NDEA/1% T NDEA/5% T
4 1509/7 10 1589/4 11 1519/5
1659/5 1989/13 1939/8
159/10.8% 409/10 429/8.9
7.479/0.81 7.739/0.36 9.59/0.74%
4.519/0.42% 4.069/0.3% 4.889/0.26%
Data expressed are mean9/S.E.; @, weight in gm; UC, untreated control; TC, turmeric control; NDEA, nitrosodiethylamine. Groups 2, 3, 4 and 5 compared with group 1 (UC). *, Significantly different from UC, P 5/0.05. Groups 6, 7 and 8 compared with group 1 (UC). $, Significantly different from UC, P 5/0.05. Groups 9, 10 and 11 compared with group 1 (UC). %, significantly different from UC, P 5/0.05. Groups 6, 7 and 8 compared with group 5 (NDEA). #, Significantly different from NDEA, P 5/0.05.
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Table 3 Effect of dietary turmeric on the number of NDEA-induced GGT positive foci in rat liver Experimental group
Number of GGT positive foci@ per mm2
No
Treatment
n
100 /250 mm
5
NDEA
12
1.39/0.47
251 /500 mm
501 /1000 mm
/1000 mm
1.939/0.49
1.529/0.31
1.129/0.29
T administration prior (2 weeks), during (4 weeks), and after NDEA treatment (10 weeks) 6 0.2% T/NDEA 7 2.719/0.56 2.299/0.38 0.979/0.21 7 1% T/NDEA 13 3.489/0.66 1.239/0.25 0.179/0.09* 8 5% T/NDEA 12 6.689/1.40* 2.539/0.43 0.839/0.28
0.099/0.09* 0* 0.29/0.09*
T administration after NDEA treatment (10 weeks) 9 NDEA/0.2% T 4 3.559/1.06 10 NDEA/1% T 10 3.669/0.68 11 NDEA/5% T 11 3.629/0.8
0.959/0.56 0.49/0.16 1.079/0.39
1.959/0.45 2.269/0.35 2.369/0.38
1.29/0.2 0.829/0.25 1.079/0.23
@, Average of five fields from four sections of each liver/n , data expressed are mean9/S.E. T, turmeric; NDEA, nitrosodiethylamine. Groups 6, 7, and 8 compared with group 5 (NDEA). *, Significantly different from NDEA; P 5/0.05.
compared with untreated control (group 1) (Table 2). Gamma glutamyltranspeptidase is known to be absent in adult normal rat liver but it has been shown to be present in high levels in neoplastic liver (Hanigan and Pitot, 1985). GGT is a wellaccepted marker enzyme for hepatic neoplasia. Data on the effect of dietary turmeric on the number of various sizes of NDEA-induced GGT positive foci in rat liver are presented in Table 3.
with NDEA (group 8) as absolute and relative weights were not only similar to NDEA-treated group (group 5) but remained elevated when compared with untreated control (group 1) (Table 2). Absolute and relative liver weights in rats which received 0.2% (group 9), 1% (group 10) and 5% (group 11) turmeric diet after NDEA treatment were comparable with those in NDEA-treated group (group 5) and significantly higher when Table 4 Effect of dietary turmeric on NDEA-induced HCC incidence in rats Experimental group No
Treatment
1 2 3 4 5
UC 0.2% TC 1% TC 5% TC NDEA
n
Normal
FD
11 5 12 12 12
11 5 12 12 2
0 0 0 0 2
HCC
0 0 0 0 8$
Tumor incidence (FD/HCC) (%)
0/11 (0) 0/5 (0) 0/12 (0) 0/12 (0) 10/12 (83)*
T administration prior (2 weeks), during (4 weeks), and after NDEA treatment (10 weeks) 6 0.2% T/NDEA 7 3 3 1 4/7 (57)* 15 15 0 0% 0/15 (0) # 7 1% T/NDEA 8 5% T/NDEA 13 8 1 4 5/13 (38)* # T administration after NDEA treatment (10 weeks) 9 NDEA/0.2% T 4 0 10 NDEA/1% T 10 0 11 0 11 NDEA/5% T
0 3 1
4$ 7$ 10$
4/4 (100)* 10/10 (100)* 11/11 (100)*
UC, untreated control; TC, turmeric control; NDEA, nitrosodiethylamine. *, $, significantly different from UC, P 5/0.05; #, %, significantly different from NDEA, P 5/0.05.
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No GGT positive foci were observed in liver sections of untreated rats (group 1) and those from 0.2, 1 and 5% turmeric-treated controls (groups 2, 3 and 4) (Fig. 2a). In NDEA-treated (200 ppm, 5 days per week for 4 weeks) rats various sizes of GGT positive foci were noted and average number of foci were 1.3 (100 /250 mm), 1.93 (251 /500 mm), 1.52 (501 /1000 mm) and 1.12 (/1000 mm) per mm2 (Table 3, Fig. 2). Administration of turmeric diet before, during and after NDEA exposure reduced the number of
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NDEA-induced big sized foci (/1000 mm and 501/1000 mm) when compared with NDEA alone exposed group. A significant decrease in the number of NDEA-induced foci ( /1000 mm) was observed in livers of groups receiving 0.2 (group 6), 1% turmeric (group 7) and 5% turmeric (group 8) diet while in the 501 /1000 mm range only group receiving 1% turmeric diet (group 7) showed a significant decrease in the number of foci as compared with NDEA alone treated group. The numbers of foci ranging from 251 to 500 mm were not different between various groups while 5%
Fig. 2. GGT expression in rat liver. (a) Untreated liver, (b) /(f) NDEA-treated liver. The darkly stained areas are the GGT positive foci of various sizes. For details see text.
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turmeric diet (group 8) significantly increased the numbers of NDEA-induced foci (100 /250 mm) as compared with NDEA alone treated group. The increase in the number of NDEA-induced foci observed with 0.2 and 1% turmeric diet was not statistically significant when compared with NDEA alone (Table 3). Analysis of NDEA-induced GGT positive foci of various sizes in livers of animals wherein turmeric diet was administered 24 h after cessation of exposure to NDEA for 10 weeks also showed an increase in number of relatively smaller foci (100 / 250 mm) while number of relatively bigger foci i.e. /500 mm decreased only in group receiving 1% turmeric diet (Table 3). The increases and decreases were, however, not statistically significant in all groups (group 5 vs. groups 9, 10 and 11) examined. Effects of dietary turmeric on incidence of NDEA-induced FD and HCC are presented in Table 4, Fig. 3. As seen in the table, no malignant or benign tumors were observed in untreated control
as well as 0.2, 1 and 5% turmeric control groups. Two hundred ppm of NDEA administered through drinking water 5 days per week for 4 weeks resulted in development of HCC in eight out of 12 rats while two out of 12 animals showed FD. The incidence of NDEA-induced FD and tumors (83%) were significantly decreased in rats which received 1 (0%) or 5% (38%) turmeric diet 2 weeks before, during (4 weeks) and continued after exposure to NDEA (10 weeks) but not in group receiving 0.2% turmeric diet (57%)(Table 4). When incidence of HCC only was compared between turmeric pretreated/ NDEA (groups 6, 7 and 8) and NDEA alone treated group (group 5) a statistically significant decrease was noted only in group receiving 1% turmeric diet. On the other hand, administration of 0.2 or 1 or 5% turmeric diet 24 h after cessation of exposure to NDEA for 10 weeks did not have such protective effects and incidence of HCC in these groups was 100% (Table 4).
Fig. 3. NDEA-induced HCC in female Wistar rats (H and E staining). (a) normal/40, (b) FD/40, (c) HCC/400.
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4. Discussion Turmeric and/or curcumin have been shown to prevent carcinogen-induced tumors of many organs. However, its effects on hepatocarcinogenicity have not been addressed until recently. The major aim of the study was to investigate the efficacy of dietary turmeric in inhibiting NDEAinduced hepatocarcinogenesis in rats. The results of the present study and the NDEAinduced hepatocarcinogenesis model established in Wistar rats in our laboratory (Fernandes and Rao, 1994) have comparable tumor incidence and tumor type but differ with respect to significantly higher NDEA-induced toxicity/mortality observed in the present investigation. Higher mortality may possibly be due to (a) susceptibility of female than male rats to NDEA toxicity and/or (b) relatively high dose of carcinogen received per kg body weight because of lower (average) body weight in the present study. The results from the experiment on chemopreventive effects of various doses of dietary turmeric demonstrate that administration of 1 or 5% turmeric diet 2 weeks before, 4 weeks during and 10 weeks after NDEA exposure significantly decreased the incidence of HCC and FD taken together as compared with NDEA-treated group. It may be noted that clear cut dose response has not been observed under the experimental conditions employed and this may probably be due to relatively narrow optimal/effective dose range of turmeric and it may not have been appropriately covered (e.g. 0.5, 1, 1.5 and 2%) in dose range used in our experiment (0.2, 1 and 5%). Our results of turmeric-mediated chemopreventive effects in Wistar rats are in agreement with a recent study (Chuang et al., 2000) wherein curcumin containing diet has been shown to inhibit NDEA-induced hepatocarcinogenesis in male C3H/HeN mice, a strain most susceptible to NDEA-induced hepatocarcinogenesis. It was also interesting to note that dietary turmeric-mediated protection against NDEA-induced hepatocarcinogensis was not observed when same levels of dietary turmeric were administered 24 h after last day of exposure to NDEA for 10 weeks (postinitiation period). Reduction in NDEA-induced
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increases in absolute and relative liver weights as well as decrease in number of NDEA-induced bigger foci due to exposure to dietary turmeric indicate growth inhibition/inhibition of cell proliferation which may ultimately attenuate or delay the development of cancer. Although the exact mechanism(s) of turmericmediated prevention of NDEA-induced HCC are not known, inhibitory effects of turmeric on PAHinduced carcinogenesis have been attributed to (i) inhibition of phase I enzymes (CYP 1A1/1A2) and (ii) induction of phase II (glutathione S -transferase) enzymes. Similarly several reports of inhibitory effects of cruciferous vegetables-derived isothiocyanates on nitrosamine-induced lung adenocarcinomas (Chung et al., 1996; Morse et al., 1989; Wattenberg, 1987) have also been attributed to modulation of phase I (CYP 2B1) and phase II enzymes (Yang et al., 1994; Guo et al., 1993, 1992). A significant reduction in tumor incidence when turmeric was administered during initiation phase as opposed to the post-initiation phase is suggestive of a similar mechanism at play in this experiment. This suggestion is in agreement with earlier reports of antiinitiating effects of turmeric/ curcumin in vitro (Deshpande and Maru, 1995; Goud et al., 1993) as well as in vivo (Deshpande et al., 1998, 1997; Huang et al., 1992). Further mechanistic studies on dietary turmeric need to be pursued employing various molecular markers.
Acknowledgements We thank Dr N. Hasgekar for advise and help in preparing the cryosections of liver and L. Motiwale for providing useful information on NDEAinduced rat liver carcinogenesis.
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forestomach and oral cancer models. J. Ethnopharmacol. 44, 211 /217. Bannasch, P., Zerban, H., 1990. Tumours of the liver. In: Turusov, V., Mohr, U. (Eds.), Pathology of Tumours in Lab Animals, vol. 1 */Tumours of the Rat, IARC Scientific Publication no. 99, IARC, France, pp 199 /240. Chuang, S.E., Kuo, M.L., Hsu, C.H., Chen, C.R., Lin, J.K., Lai, G.M., Hsieh, C.Y., Cheng, A.L., 2000. Curcumin containing diet inhibits diethylnitrosamine-induced murine hepatocarcinogenesis. Carcinogenesis 21, 331 /335. Chung, F.L., Kelloff, G., Steele, V., Pittman, B., Zang, E., Jiao, D., Rigotty, J., Choi, C.-I., Rivenson, A., 1996. Chemopreventive efficacy of arylalkyl isothiocyanates and N-acetylcysteine for lung tumorigenesis in Fischer rats. Cancer Res. 56, 772 /778. Deshpande, S.S., Maru, G.B., 1995. Effects of curcumin on the formation of benzo(a)pyrene-derived DNA adducts in vitro. Cancer Lett. 96, 71 /80. Deshpande, S.S., Ingle, A.D., Maru, G.B., 1997. Inhibitory effects of curcumin-free aqueous turmeric extract on benzo(a)pyrene-induced forestomach papillomas in mice. Cancer Lett. 118, 79 /85. Deshpande, S.S., Ingle, A.D., Maru, G.B., 1998. Chemopreventive efficacy of curcumin-free aqueous turmeric extract on 7,12 dimethylbenz(a)anthracene-induced rat mammary tumorigenesis. Cancer Lett. 123, 35 /40. Fernandes, C., Rao, K.V.K., 1994. Dose related promoter effect of metanil yellow on the development of hepatic preneoplastic lesions induced by N-nitrosodiethylamine in rats. Indian J. Med. Res. 100, 140 /149. Goud, V.K., Polasa, K., Krishnaswamy, K., 1993. Effects of turmeric on xenobiotic metabolising enzymes. Plant Foods Hum. Nutr. 44, 87 /92. Guo, Z., Smith, T.J., Wang, E., Sadrieh, N., Ma, Q., Thomas, P.E., Yang, C.S., 1992. Effects of phenethylisothiocyanate, a carcinogenesis inhibitor, on xenobiotic-metabolising enzymes and nitrosamine metabolism in rats. Carcinogenesis 13, 2205 /2210. Guo, Z., Smith, T.J., Wang, E., Eklind, K.I., Chung, F.-L., Yang, C.S., 1993. Structure activity relationships of arylalkyl isothiocyanates for the inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolism and the modulation of xenobiotic-metabolising enzymes in rats and mice. Carcinogenesis 14, 1167 /1173. Hanigan, M.H., Pitot, H.C., 1985. Gamma-glutamyl transpeptidase */its role in hepatocarcinogenesis. Carcinogenesis 6, 165 /172. Huang, M.T., Smart, R.C., Wong, C.Q., Conney, A.H., 1988. Inhibitory effect of curcumin, chlorogenic acid, caffeic acid and ferulic acid on tumor promotion in mouse skin by 12-Otetradecanoyl phorbol-13-acetate. Cancer Res. 48, 5941 / 5946. Huang, M.T., Wang, Z.Y., Georgiadis, C.A., Laskin, J.D., Conney, A.H., 1992. Inhibitory effects of curcumin on
tumor initiation by benzo(a)pyrene and 7,12 dimethylbenz(a)anthracene. Carcinogenesis 13, 2183 /2186. Huang, M.T., Lou, Y.R., Ma, W., Newmark, H.L., Reuhl, K.R., Conney, A.H., 1994. Inhibitory effects of dietary curcumin on forestomach, duodenal and colon carcinogenesis in mice. Cancer Res. 54, 5841 /5847. Limtrakul, P., Lipigorngoson, S., Namwong, O., Apisariyakul, A., Dunn, F.W., 1997. Inhibitory effect of dietary curcumin on skin carcinogenesis in mice. Cancer Lett. 116, 197 /203. Morse, M.A., Eklind, K.I., Amin, S.G., Hecht, S.S., Chung, F.L., 1989. Effects of alkyl chain length on the initiation of 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung neoplasia in A/J mice. Carcinogenesis 10, 1757 /1759. Oetari, S., Sudibyo, M., Commandeur, J.N., Samhordi, R., Vermeulen, N.P., 1996. Effects of curcumin on cytochrome P450 and GST activities in rat liver. Biochem. Pharmacol. 51, 39 /45. Pereira, M.A., Grubbs, C.J., Barnes, L.H., Hong, L., Olson, G.R., Eto, I., Juliana, M., Whitaker, L.M., Kelloff, G.J., Steele, V.E., Lubet, R.A., 1996. Effects of the phytochemicals, curcumin and quercetin, upon azoxymethane-induced colon cancer and 7,12-dimethylbenz(a)anthracene-induced mammary cancer in rats. Carcinogenesis 17, 1305 /1311. Rao, C.V., Rivenson, A., Simi, B., Reddy, B.S., 1995. Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound. Cancer Res. 55, 259 /266. Roughley, P.J., Whiting, A.D., 1973. Experiments in the biosynthesis of curcumin. J. Chem. Soc. 20, 2379 /2388. Rutenberg, A.M., Kim, H., Fischbein, J.W., Hanker, J.S., Wasserkrug, H.L., Seligman, A.M., 1969. Histochemical and ultrastructural demonstration of g-glutamyl transpeptidase activity. J. Histochem. Cytochem. 17, 517 /525. Sharma, O.P., 1976. Antioxidant activity of curcumin and related compounds. Biochem. Pharmacol. 25, 1811 /1812. Singletary, K., MacDonald, C., Waltig, B., Fischer, C., 1996. Inhibition of dimethylbenz(a)anthracene (DMBA)-induced mammary tumorigenesis and DMBA-DNA adduct formation by curcumin. Cancer Lett. 103, 137 /141. Srimal, R.C., Dhawan, B.N., 1973. Pharmacology of diferuloylmethane (curcumin), a non-steroidal anti-inflammatory agent. J. Pharm. Pharmacol. 25, 447 /452. Thapliyal, R., Maru, G.B., 2001. Inhibition of cytochrome P450 isozymes by curcumins in vitro and in vivo. Food Chem. Toxicol. 39, 541 /547. Wattenberg, L.W., 1987. Inhibitory effects of benzylisothiocyanates administered shortly before diethylnitrosamine or benzo(a)pyrene on pulmonary and stomach neoplasia in A/J mice. Carcinogenesis 8, 1971 /1973. Yang, C.S., Smith, T.J., Hong, J.-Y., 1994. Cytochrome P450 enzymes as targets for chemoprevention against chemical carcinogenesis and toxicity: opportunities and limitations. Cancer Res. 54 (Suppl), 1982s /1986s.
Short communication
Inhibition of nitrosodiethylamine-induced hepatocarcinogenesis by dietary turmeric in rats R. Thapliyal a, K.N. Naresh b, K.V.K. Rao a, G.B. Maru a,* a
Carcinogenesis Division, Cancer Research Institute, Tata Memorial Center, Parel, Mumbai 400 012, India Pathology Department, Tata Memorial Hospital, Tata Memorial Center, Parel, Mumbai 400 012, India
b
Received 1 July 2002; received in revised form 18 November 2002; accepted 20 November 2002
Abstract Turmeric, widely used in food and medicine has been shown to prevent benzo(a)pyrene [B(a)P] or dimethylbenz(a)anthracene (DMBA)-induced forestomach, skin and mammary tumors in mice and/or rats. In this study we examine the modulatory effects of turmeric on nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis in rats. Female Wistar rats were administered NDEA (200 ppm) through drinking water (5 days per week) for 4 weeks. Control and/or NDEA-treated rats received 0, 0.2, 1.0 or 5.0% turmeric diet (w/w) either before (2 weeks), during (4 weeks) and after NDEA exposure (10 weeks) or starting from 24 h after NDEA exposure for 10 weeks. NDEA-treated rats receiving 1 or 5% turmeric before, during and after carcinogen exposure showed significant decrease in number of gamma glutamyl transpeptidase (GGT) positive foci measuring /500 or /1000 mm and decrease in the incidence of NDEA-induced focal dysplasia (FD) and hepatocellularcarcinomas. Decrease in the number of GGT positive foci measuring /1000 mm was also observed in NDEA-treated rats receiving 0.2% turmeric, although no decrease in tumor incidence was noted. On the other hand, similar levels of turmeric treatment (0.2, 1 and 5%) after exposure to NDEA did not show any protective effects. The underlying mechanism(s) of chemoprevention of NDEA-induced hepatocarcinogenesis need to be explored. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nitrosodiethylamine; Rat liver; GGT /ve foci; Hepatocellular-carcinoma; Turmeric; Inhibition
1. Introduction Abbreviations: CYP 450, cytochrome P450; Fast blue BBN, diazotized 4?-amino-2?, 5?-diethoxybenzanilide; g-GMNA, gglutamyl-4-methoxy-2-naphthylamide; GGT, gamma glutamyltranspeptidase; GG, glycyl-glycine; HCC, hepatocellularcarcinoma; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone; NDEA, nitrosodiethylamine; PAHs, polycyclic aromatic hydrocarbons. * Corresponding author. Tel.: /91-22-412-3803; fax: /9122-414-6089. E-mail address: [email protected] (G.B. Maru).
One effective approach to cancer control is prevention. Identification and development of effective chemopreventive agents especially from dietary constituents that can block activation or enhance detoxification of environmental agents is an important aspect. Dietary intervention that counters/prevents/delays the harmful effects of carcinogens may then become part of our daily
0378-4274/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-4274(02)00440-X
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life-style changes. Spices are part of regular diet in certain populations and one such agent namely, turmeric with its perceived human safety following centuries of use in food and medicine is currently under investigation in several laboratories. Turmeric, the powdered rhizome of the plant Curcuma longa Linn. and curcumin(s), the yellow phenolic compounds in turmeric besides possessing antiinflammatory (Srimal and Dhawan, 1973) and anti-oxidant (Sharma, 1976) properties have also shown their efficacy in preventing chemical-induced experimental carcinogenesis in various tissues including skin (Huang et al., 1992, 1988; Limtrakul et al., 1997), colorectal (Pereira et al., 1996; Rao et al., 1995; Huang et al., 1994), oral (Huang et al., 1994), forestomach (Deshpande et al., 1997), and mammary glands (Deshpande et al., 1998; Singletary et al., 1996). The anti-initiation effects of turmeric/curcumin in experimental systems have mostly been established employing representative polycyclic aromatic hydrocarbon(s) (PAHs) which require metabolic activation. Turmeric/curcumin(s) have been shown to inhibit isozymes of CYP 450s in vitro as well as in vivo (Thapliyal and Maru, 2001; Oetari et al., 1996). Chemopreventive efficacy of turmeric in combination with other agent(s) have been reported against presumed carcinogenic metabolite of dimethylnitrosamine i.e. methyl (acetoxylmethyl)nitrosamine (Azuine and Bhide, 1994, 1992), while potential of turmeric (alone) to prevent hepatocarcinogenesis induced by nitrosamines requiring metabolic activation has not been established. Since hepatocellular carcinoma (HCC) is a common cause of cancer morbidity and mortality in Asia and Africa and its prognosis extremely poor effective measures of chemoprevention by dietary constituent like turmeric need to be studied. In this communication we report the potential chemopreventive action of turmeric against nitrosodiethylamine (NDEA), a hepatocarcinogen, by comparing the tumor incidence, number and size of gglutamyltranspeptidase (GGT) positive foci in liver and relative as well as absolute liver weights in rats receiving turmeric either before, during and after NDEA exposure or only after NDEA exposure.
2. Materials and methods 2.1. Chemicals NDEA, diazotized 4?-amino-2?, 5?-diethoxybenzanilide (Fast blue BBN), g-glutamyl-4-methoxy2-naphthylamide (g-GMNA) and glycylglycine (GG), were purchased from Sigma Chemical Co., USA. All other chemicals used were purchased locally and were of AR grade. 2.2. Animals Inbred female Wistar rats (8 /10 weeks) were obtained from the animal colony of Cancer Research Institute, Mumbai, India. They were randomly distributed into various groups and housed in cages (five per cage) and maintained under standard conditions i.e. 229/2 8C, 459/10% relative humidity and 12-h light:12-h dark cycles each day. All animals were fed powdered standard laboratory (control)/experimental diet and drinking water or 200 ppm NDEA in drinking water ad libitum. 2.3. Experimental diet(s)/water Turmeric rhizomes purchased in bulk locally were thoroughly washed with water, sun-dried and powdered in a grinding mill. It was stored at room temperature throughout the experiment. The curcumin(s) content from turmeric was analysed by preparative TLC (Roughley and Whiting, 1973). The content of curcumin(s) (% weight of turmeric powder) was 5% and proportion of components i.e. curcumin, demethoxycurcumin and bisdemethoxycurcumin was 67, 28 and 5%, respectively (Deshpande and Maru, 1995). Quantities of turmeric powder required for preparation of diet(s) with defined concentrations i.e. 0.2, 1.0 and 5.0% were weighed and added to preweighed standard laboratory diet and thoroughly mixed while adding aldehyde-free distilled ethanol (upto 5% concentration) to ensure its uniform distribution. A control diet (standard laboratory diet) was also prepared using similar concentration of aldehyde-free distilled ethanol. Ethanol from the control as well as the experi-
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mental diet(s) was allowed to evaporate completely and then the diets were used. All control and experimental diet(s) were prepared monthly in our laboratory and stored at 4 8C. Food cups were replenished with fresh diet every day. NDEA, a hepatocarcinogen, was administered in drinking water for 4 weeks (5 days a week) at a dose of 200 ppm in dark-colored water bottles to protect from direct light. NDEA in water was prepared weekly and stored in the refrigerator. Water bottles were replenished every day with fresh NDEA/plain drinking water. 2.4. Experimental design Animals were divided into eleven groups of untreated control (1), 0.2 (2), 1 (3), 5% (4) turmeric controls, NDEA-treated (5), 0.2% turmeric/ NDEA (6), 1% turmeric/NDEA (7), 5% turmeric/NDEA (8) (pretreatment groups 6 /8), NDEA/0.2% turmeric (9), NDEA/1% turmeric (10) and NDEA/5% turmeric (11) (posttreatment groups 9 /11). The experimental protocol followed is presented in Fig. 1. Groups 1 and 5 were kept on standard laboratory diet while groups 2, 3 and 4 were kept on 0.2, 1.0 and 5% turmeric diet, respectively, throughout the experiment. Groups 1, 2, 3 and 4 were kept on plain drinking water while group 5 was kept on 200 ppm NDEA 5 days a week for 4 weeks. Animals in groups 6, 7 and 8 were kept on 0.2, 1.0 and 5% turmeric diet, respectively, 2 weeks before, 4 weeks during and 10 weeks after NDEA administration i.e. till the end of experiment. Animals in groups 9,10 and 11 were kept on 0.2, 1.0 and 5% turmeric diet, respectively, 24 h after last NDEA administration for 10 weeks i.e. till the end of experiment. Body weights were recorded in the beginning and during the experimental period (once a week). Animals were anaesthetized and sacrificed at the end of the experiment. Final body and liver weights were recorded and livers were further utilized for GGT staining and histopathology. Pieces of liver (/0.5 /0.5 cm) from each liver lobe were fixed in 10% phosphate buffered formalin and embedded in paraffin. Serial sections of liver pieces so obtained were stained
47
with haematoxylin and eosin. Tumor incidence was determined histologically by a surgical pathologist who was blinded from other information. The lesions were classified as no tumor, dysplasia and HCC (Bannasch and Zerban, 1990).
2.5. Histochemical staining for GGT positive foci Determination of GGT positive foci was performed as described (Rutenberg et al., 1969). Freshly excised livers were cryosectioned and 8 / 10 m thick sections were mounted on slides and airdried. One square cm pieces of livers were used to make cryosections. Freshly prepared solution containing the substrate GMNA, the acceptor GG and the azo coupling dye, fast blue BBN, in 0.1 M tris buffer pH 7.4 and saline was then pipetted on to the cryosections and incubated at room temperature in dark for 30/45 min. Following incubation, the slides were rinsed in saline for 2 min and then transferred to 0.1 M cupric sulfate solution for 2 min. The slides were again rinsed in saline followed by a rinse in distilled water. The sections were air-dried, mounted in 10% glycerol and observed under a light microscope with a 10 X objective lens that had a graticule for measuring the foci size. Five fields from four sections of each liver were examined. The numbers of foci are, therefore, an average of 20 fields for each liver sample. The GGT positive foci, thus, counted were divided according to various size ranges and results have been expressed as number of foci per mm2. Cryosections of kidney served as positive control for GGT staining.
2.6. Statistical analysis The incidence of mortality and HCC was analysed by the X2 test. Differences in body, liver weights, liver to body weight ratios and number of GGT positive foci between different groups (multiple comparisons) were analysed by one-way ANOVAs with Bonferroni post-hoc test. Differences were considered statistically significant at P 5/ 0.05.
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Fig. 1. Experimental design for NDEA-induced HCC in female Wistar rats.
3. Results In groups of untreated control rats as well as those receiving 0.2, 1 and 5% turmeric through diet for 16 weeks of experimental period no mortalities were observed. However, 10 out of 22 (45%) rats receiving 200 ppm of NDEA through drinking water (5 days per week /4) died either just after NDEA treatment or within 3/4 weeks after stopping the NDEA treatment. The observed mortality in NDEA-treated group was significantly higher compared with the untreated control group (Table 1). In groups of rats receiving 0.2, 1 and 5% turmeric 2 weeks before, 4 weeks during NDEA treatment and 10 weeks after cessation of exposure to NDEA, some improvement in survival was observed when compared with NDEA-treated group although statistical significance in survival was attained only in group of rats receiving 1% turmeric diet but not in those receiving either 0.2
or 5% turmeric diet (Table 1). In groups of rats receiving 0.2 or 1 or 5% turmeric diet 24 h after last NDEA exposure for a period of 10 weeks, the mortalities were 60, 52 and 42% respectively, i.e. similar to the group exposed to NDEA alone (45% mortality) and significantly higher compared with untreated control groups (0% mortality, Table 1). Data on initial and final body weights, weight gain, liver weight and liver/body weight /100 (relative liver weight), number of GGT positive foci and focal dysplasia (FD), HCC and incidence of tumor from animals belonging to various experimental groups have been compared among survivors at the end of 16 weeks of experimental period and have been presented below (Tables 2 / 4). The initial and final body weights of untreated controls, 0.2% (group 2) or 1% (group 3) or 5% (group 4) turmeric diet referred as turmeric controls, NDEA-treated (group 5) and those receiving
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Table 1 Effect of dietary turmeric on NDEA-induced mortality in rats Experimental group No
Treatment
1 2 3 4 5
UC 0.2% TC 1% TC 5% TC NDEA
T administration prior (2 weeks), NDEA treatment (10 weeks) 6 0.2% T/NDEA 7 1% T/NDEA 8 5% T/NDEA
n
Survived (%)
11 5 12 12 22
11/11 (100) 5/5 (100) 12/12 (100) 12/12 (100) 12/22 (55)*
during (4 weeks), and after 10 17 18
7/10 (70) 15/17 (88)# 13/18 (72)
T administration after NDEA treatment (10 weeks) 9 NDEA/0.2% T 10 4/10 (40)* 10 NDEA/1% T 21 10/21 (48)* 11 NDEA/5% T 19 11/19 (58)* UC, untreated control; TC, turmeric control; NDEA, nitrosodiethylamine; *, significantly different from UC, P 5/ 0.05. #, significantly different from NDEA, P 5/0.05.
turmeric diet either before, during and after NDEA exposure (groups 6 /8) or only after NDEA exposure (groups 9 /11) were comparable.
49
Similarly weight gains in animals belonging to various groups were also comparable except in those receiving 0.2% turmeric after exposure to NDEA (group 9), wherein a significant decrease in weight gain was observed (Table 2) when compared with untreated control group. The absolute and relative liver weights in animals exposed to 0.2, 1 and 5% turmeric diet (groups 2 /4) for experimental period of 16 weeks did not differ when compared with absolute and relative liver weights in untreated controls (group 1). Animals treated with 200 ppm NDEA through drinking water 5 days per week for 4 weeks had significantly increased absolute (8.279/0.65 vs. 5.939/0.25) as well as relative (4.519/0.35 vs. 3.09/0.09) liver weights at the time of sacrifice when compared with respective liver weights in untreated controls (Table 2). Turmeric administration before, during and after NDEA treatment appears to protect against NDEA-induced increase in absolute and relative liver weights although statistical significance was attained only in the group receiving 1% turmeric diet (relative liver weights 4.519/0.35 vs. 3.189/0.30) (group 7). This protection is not seen in group receiving 5% turmeric diet and treated
Table 2 Effect of dietary turmeric and/or NDEA treatment on body and liver weights of rats Experimental group
n
Initial weight@
Final weight@
Gain in weight@
Liver weight@
Liver:body weight@ /100
11 5 12 12 12
1409/7 1229/9 1449/7 1419/6 1529/6
1989/7 2009/10 2089/7 2059/6 1859/7
589/6.30 789/6.04 659/4.54 659/3.66 339/4.82
5.939/0.25 5.429/0.17 5.629/0.39 6.279/0.33 8.279/0.65*
3.09/0.09 2.729/0.05 2.689/0.13 3.049/0.13 4.519/0.35*
No Treatment 1 2 3 4 5
UC 0.2% TC 1% TC 5% TC NDEA
T administration prior (2 weeks), during (4 weeks), and after NDEA treatment (10 weeks) 6 0.2% T/NDEA 7 1519/5 1999/9 479/10.29 6.89/0.6 7 1% T/NDEA 15 1529/5 2039/5 519/4.14 6.59/0.28 8 5% T/NDEA 13 1549/6 1959/8 429/6.76 7.379/0.58
3.459/0.30 3.189/0.10# 3.809/0.30$
T administration after NDEA treatment (10 weeks) 9 10 11
NDEA/0.2% T NDEA/1% T NDEA/5% T
4 1509/7 10 1589/4 11 1519/5
1659/5 1989/13 1939/8
159/10.8% 409/10 429/8.9
7.479/0.81 7.739/0.36 9.59/0.74%
4.519/0.42% 4.069/0.3% 4.889/0.26%
Data expressed are mean9/S.E.; @, weight in gm; UC, untreated control; TC, turmeric control; NDEA, nitrosodiethylamine. Groups 2, 3, 4 and 5 compared with group 1 (UC). *, Significantly different from UC, P 5/0.05. Groups 6, 7 and 8 compared with group 1 (UC). $, Significantly different from UC, P 5/0.05. Groups 9, 10 and 11 compared with group 1 (UC). %, significantly different from UC, P 5/0.05. Groups 6, 7 and 8 compared with group 5 (NDEA). #, Significantly different from NDEA, P 5/0.05.
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Table 3 Effect of dietary turmeric on the number of NDEA-induced GGT positive foci in rat liver Experimental group
Number of GGT positive foci@ per mm2
No
Treatment
n
100 /250 mm
5
NDEA
12
1.39/0.47
251 /500 mm
501 /1000 mm
/1000 mm
1.939/0.49
1.529/0.31
1.129/0.29
T administration prior (2 weeks), during (4 weeks), and after NDEA treatment (10 weeks) 6 0.2% T/NDEA 7 2.719/0.56 2.299/0.38 0.979/0.21 7 1% T/NDEA 13 3.489/0.66 1.239/0.25 0.179/0.09* 8 5% T/NDEA 12 6.689/1.40* 2.539/0.43 0.839/0.28
0.099/0.09* 0* 0.29/0.09*
T administration after NDEA treatment (10 weeks) 9 NDEA/0.2% T 4 3.559/1.06 10 NDEA/1% T 10 3.669/0.68 11 NDEA/5% T 11 3.629/0.8
0.959/0.56 0.49/0.16 1.079/0.39
1.959/0.45 2.269/0.35 2.369/0.38
1.29/0.2 0.829/0.25 1.079/0.23
@, Average of five fields from four sections of each liver/n , data expressed are mean9/S.E. T, turmeric; NDEA, nitrosodiethylamine. Groups 6, 7, and 8 compared with group 5 (NDEA). *, Significantly different from NDEA; P 5/0.05.
compared with untreated control (group 1) (Table 2). Gamma glutamyltranspeptidase is known to be absent in adult normal rat liver but it has been shown to be present in high levels in neoplastic liver (Hanigan and Pitot, 1985). GGT is a wellaccepted marker enzyme for hepatic neoplasia. Data on the effect of dietary turmeric on the number of various sizes of NDEA-induced GGT positive foci in rat liver are presented in Table 3.
with NDEA (group 8) as absolute and relative weights were not only similar to NDEA-treated group (group 5) but remained elevated when compared with untreated control (group 1) (Table 2). Absolute and relative liver weights in rats which received 0.2% (group 9), 1% (group 10) and 5% (group 11) turmeric diet after NDEA treatment were comparable with those in NDEA-treated group (group 5) and significantly higher when Table 4 Effect of dietary turmeric on NDEA-induced HCC incidence in rats Experimental group No
Treatment
1 2 3 4 5
UC 0.2% TC 1% TC 5% TC NDEA
n
Normal
FD
11 5 12 12 12
11 5 12 12 2
0 0 0 0 2
HCC
0 0 0 0 8$
Tumor incidence (FD/HCC) (%)
0/11 (0) 0/5 (0) 0/12 (0) 0/12 (0) 10/12 (83)*
T administration prior (2 weeks), during (4 weeks), and after NDEA treatment (10 weeks) 6 0.2% T/NDEA 7 3 3 1 4/7 (57)* 15 15 0 0% 0/15 (0) # 7 1% T/NDEA 8 5% T/NDEA 13 8 1 4 5/13 (38)* # T administration after NDEA treatment (10 weeks) 9 NDEA/0.2% T 4 0 10 NDEA/1% T 10 0 11 0 11 NDEA/5% T
0 3 1
4$ 7$ 10$
4/4 (100)* 10/10 (100)* 11/11 (100)*
UC, untreated control; TC, turmeric control; NDEA, nitrosodiethylamine. *, $, significantly different from UC, P 5/0.05; #, %, significantly different from NDEA, P 5/0.05.
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No GGT positive foci were observed in liver sections of untreated rats (group 1) and those from 0.2, 1 and 5% turmeric-treated controls (groups 2, 3 and 4) (Fig. 2a). In NDEA-treated (200 ppm, 5 days per week for 4 weeks) rats various sizes of GGT positive foci were noted and average number of foci were 1.3 (100 /250 mm), 1.93 (251 /500 mm), 1.52 (501 /1000 mm) and 1.12 (/1000 mm) per mm2 (Table 3, Fig. 2). Administration of turmeric diet before, during and after NDEA exposure reduced the number of
51
NDEA-induced big sized foci (/1000 mm and 501/1000 mm) when compared with NDEA alone exposed group. A significant decrease in the number of NDEA-induced foci ( /1000 mm) was observed in livers of groups receiving 0.2 (group 6), 1% turmeric (group 7) and 5% turmeric (group 8) diet while in the 501 /1000 mm range only group receiving 1% turmeric diet (group 7) showed a significant decrease in the number of foci as compared with NDEA alone treated group. The numbers of foci ranging from 251 to 500 mm were not different between various groups while 5%
Fig. 2. GGT expression in rat liver. (a) Untreated liver, (b) /(f) NDEA-treated liver. The darkly stained areas are the GGT positive foci of various sizes. For details see text.
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turmeric diet (group 8) significantly increased the numbers of NDEA-induced foci (100 /250 mm) as compared with NDEA alone treated group. The increase in the number of NDEA-induced foci observed with 0.2 and 1% turmeric diet was not statistically significant when compared with NDEA alone (Table 3). Analysis of NDEA-induced GGT positive foci of various sizes in livers of animals wherein turmeric diet was administered 24 h after cessation of exposure to NDEA for 10 weeks also showed an increase in number of relatively smaller foci (100 / 250 mm) while number of relatively bigger foci i.e. /500 mm decreased only in group receiving 1% turmeric diet (Table 3). The increases and decreases were, however, not statistically significant in all groups (group 5 vs. groups 9, 10 and 11) examined. Effects of dietary turmeric on incidence of NDEA-induced FD and HCC are presented in Table 4, Fig. 3. As seen in the table, no malignant or benign tumors were observed in untreated control
as well as 0.2, 1 and 5% turmeric control groups. Two hundred ppm of NDEA administered through drinking water 5 days per week for 4 weeks resulted in development of HCC in eight out of 12 rats while two out of 12 animals showed FD. The incidence of NDEA-induced FD and tumors (83%) were significantly decreased in rats which received 1 (0%) or 5% (38%) turmeric diet 2 weeks before, during (4 weeks) and continued after exposure to NDEA (10 weeks) but not in group receiving 0.2% turmeric diet (57%)(Table 4). When incidence of HCC only was compared between turmeric pretreated/ NDEA (groups 6, 7 and 8) and NDEA alone treated group (group 5) a statistically significant decrease was noted only in group receiving 1% turmeric diet. On the other hand, administration of 0.2 or 1 or 5% turmeric diet 24 h after cessation of exposure to NDEA for 10 weeks did not have such protective effects and incidence of HCC in these groups was 100% (Table 4).
Fig. 3. NDEA-induced HCC in female Wistar rats (H and E staining). (a) normal/40, (b) FD/40, (c) HCC/400.
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4. Discussion Turmeric and/or curcumin have been shown to prevent carcinogen-induced tumors of many organs. However, its effects on hepatocarcinogenicity have not been addressed until recently. The major aim of the study was to investigate the efficacy of dietary turmeric in inhibiting NDEAinduced hepatocarcinogenesis in rats. The results of the present study and the NDEAinduced hepatocarcinogenesis model established in Wistar rats in our laboratory (Fernandes and Rao, 1994) have comparable tumor incidence and tumor type but differ with respect to significantly higher NDEA-induced toxicity/mortality observed in the present investigation. Higher mortality may possibly be due to (a) susceptibility of female than male rats to NDEA toxicity and/or (b) relatively high dose of carcinogen received per kg body weight because of lower (average) body weight in the present study. The results from the experiment on chemopreventive effects of various doses of dietary turmeric demonstrate that administration of 1 or 5% turmeric diet 2 weeks before, 4 weeks during and 10 weeks after NDEA exposure significantly decreased the incidence of HCC and FD taken together as compared with NDEA-treated group. It may be noted that clear cut dose response has not been observed under the experimental conditions employed and this may probably be due to relatively narrow optimal/effective dose range of turmeric and it may not have been appropriately covered (e.g. 0.5, 1, 1.5 and 2%) in dose range used in our experiment (0.2, 1 and 5%). Our results of turmeric-mediated chemopreventive effects in Wistar rats are in agreement with a recent study (Chuang et al., 2000) wherein curcumin containing diet has been shown to inhibit NDEA-induced hepatocarcinogenesis in male C3H/HeN mice, a strain most susceptible to NDEA-induced hepatocarcinogenesis. It was also interesting to note that dietary turmeric-mediated protection against NDEA-induced hepatocarcinogensis was not observed when same levels of dietary turmeric were administered 24 h after last day of exposure to NDEA for 10 weeks (postinitiation period). Reduction in NDEA-induced
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increases in absolute and relative liver weights as well as decrease in number of NDEA-induced bigger foci due to exposure to dietary turmeric indicate growth inhibition/inhibition of cell proliferation which may ultimately attenuate or delay the development of cancer. Although the exact mechanism(s) of turmericmediated prevention of NDEA-induced HCC are not known, inhibitory effects of turmeric on PAHinduced carcinogenesis have been attributed to (i) inhibition of phase I enzymes (CYP 1A1/1A2) and (ii) induction of phase II (glutathione S -transferase) enzymes. Similarly several reports of inhibitory effects of cruciferous vegetables-derived isothiocyanates on nitrosamine-induced lung adenocarcinomas (Chung et al., 1996; Morse et al., 1989; Wattenberg, 1987) have also been attributed to modulation of phase I (CYP 2B1) and phase II enzymes (Yang et al., 1994; Guo et al., 1993, 1992). A significant reduction in tumor incidence when turmeric was administered during initiation phase as opposed to the post-initiation phase is suggestive of a similar mechanism at play in this experiment. This suggestion is in agreement with earlier reports of antiinitiating effects of turmeric/ curcumin in vitro (Deshpande and Maru, 1995; Goud et al., 1993) as well as in vivo (Deshpande et al., 1998, 1997; Huang et al., 1992). Further mechanistic studies on dietary turmeric need to be pursued employing various molecular markers.
Acknowledgements We thank Dr N. Hasgekar for advise and help in preparing the cryosections of liver and L. Motiwale for providing useful information on NDEAinduced rat liver carcinogenesis.
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