Chemopreventive Activity of Turmeric Essential Oil and Possible Mechanisms of Action

Many human cancers are caused by chemical carcinogens, such as polycyclic aromatic hydrocarbons, heterocyclic amines, aromatic amines etc present in our environment. Continued exposure of these substances to human cells leads to genomic instability, including repair deficiency and accumulation of genetic alteration (Ames, 1983). Mutation is a major factor in carcinogenesis and incidence of cancer may be reduced by decreasing the rate of mutations induced by various chemical mutagens. Many carcinogens are activated through Phase I (cytochrome p450) enzymes present in endoplasmic reticulum of the liver cells. Induction of Phase II detoxification enzymes, such as glutathione S-transferase and UDP-glucuronyl transferase, is another mechanisms of protection against carcinogenesis (Piengchai et al., 2011). Modulating these enzymes may reduce cancer incidence. Spices which are widely used as food ingredient exhibits different pharmacological properties. Essential oils from spices are volatile compounds produced as secondary metabolites. The essential oil from Curcuma longa rhizome is a complex mixture obtained by steam distillation. A total number of 12 components of the essential oil are identified by GC-MS analysis and the principal compounds include ar-turmerone (61%),


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2002). The chemopreventive efficacy of TEO has been reported against submucous fibrosis in humans (Deepa et al., 2010) and was found to reduce benzo[a]pyrene induced DNA damage in vitro in oral mucosa cells (Hastak et al., 1997). TEO prevented the growth of Aspergillus flavus species and significantly reduced aflatoxin B1 production which is a potent liver carcinogen (Ferreira et al., 2013). It could also improve the bioavailability of curcumin after oral administration in humans (Antony et al., 2008).
Food and drug administration (FDA) has accepted TEO as a food additive and is Generally Recognized As Safe (GRAS). It is safe in rats (NOAEL) up to an oral dose of 500 mg/kg body weight (Chapter 3).
The protective effects of TEO as a chemopreventive agent against NDEA induced hepatocellularcarcinoma in male Wistar rats and two-stage mouse skin papilloma development induced by DMBA as initiator and croton oil as promoter is also being reported in this study. In order to assess the anticarcinogenic efficacy of TEO we have also analyzed 3-methyl cholanthrene (3-MC) induced sarcoma in mice. To determine the possible mechanism of action of TEO we have evaluated the inhibition of different cytochrome P450 enzymes (Phase I enzymes) by TEO in vitro and in vivo. Moreover, we have studied the levels of the drug metabolizing enzymes, glutathione-S-transferase (GST) and UDP-glucuronyl transferase after TEO administration in rats.

Effect of TEO on N-nitrosodiethylamine induced hepatocarcinogenes is in rats
Six groups of male Wistar rats, each group consisting of 8 animals were used for this study.

Group I : Untreated
Group II : Control receiving (NDEA alone).
Group III : Vehicle control (NDEA and paraffin oil).
Group IV : NDEA + 20 mg/kg b. wt. TEO Group VI : NDEA + 100 mg/kg b. wt. TEO Group VI : NDEA + 500 mg/kg b. wt. TEO Animals were treated with 0.02% NDEA (2.5 ml/animal/dose) for 20 weeks (6 days/week) and thereafter observed for 9 more weeks. At the end of 29 th week, all the animals were sacrificed by ether anaesthesia. Liver were excised and washed with ice cold saline. A small piece of liver was kept in 10% formalin for histopathological analysis.

Morphology and weight of the liver
Liver from each animal was washed in ice-cold saline (0.9%) and observed for tumour nodules and other morphological abnormalities. Weight of each liver was recorded and expressed in relation to the body weight.

Biochemical analysis of serum
Blood was collected by direct heart puncture in non-EDTA tubes and serum was separated after centrifugation at 5000 rpm for 10 minutes and used for the following investigations. Gamma-glutamyl transferase (γ-GT) activity was assayed by the method of Tate and Meister (Tate et al., 1975). Total bilirubin was determined by Jendrassikdiazotized sulphanilic acid method (Garber et al., 1981). Alkaline phosphatase (ALP) was estimated by p-nitrophenyl phosphate (PNPP) hydrolysis (McComb et al., 1979) and alanine amino transferase (ALT) as well as aspartate aminotransferase (AST) by kinetic method using commercially available kits (Span Diagnostics, India).

Effect of TEO on antioxidant enzymes in the liver
Liver homogenate (25%) was prepared in Tris-HCl buffer (0.1 M, pH-7.4), centrifuged at 1000 rpm for 10 minutes at 4 o C to remove the cell debris. The supernatant was used for assessing the activity of GSH by its reaction with 5,5-dithiobis-2nitrosobenzoic acid (Moron et al., 1979). Glutathione peroxidase (GPx) activity was checked from the degradation of H 2 O 2 in the presence of GSH (Hafeman et al., 1974).

Histopathological analysis
A portion of the liver was cut, washed in PBS and fixed in 10% neutral buffered formalin (Chapter 2, Section 2.2.6).

Activity of TEO on DMBA induced two-stage skin papilloma formation in mice
Male Balb/c mice were used for the DMBA induced skin papilloma study.
DMBA, croton oil and TEO was applied on the shaved dorsal side (2 cm diameter) of the mice at least two days before the application of chemicals. Animals having no hair growth after 2 days were selected for the experiment. Balb/C mice were divided in to eight groups and each group having 8 animals. in all groups were watched for food intake as well as any apparent toxicity such as weight loss or mortality during the entire period of the study. Skin papilloma formation was recorded weekly and the tumor growth greater than 1 mm in diameter was included in the cumulative total if they persisted for 2 weeks or more. Formation of the onset papilloma and number of papilloma per cage in various groups were recorded every week.

Effect of TEO against 3-methylcholanthrene (3-MC) induced sarcoma development in mice
Balb/C mice weighing 20-25 g was divided into 5 groups and each group consisting of 15 animals. Homogenate was initially centrifuged at 14000 g for 20 min in a cold centrifuge (Remi) and supernatant was then further centrifuged at 10, 5000 g for 1 h in an ultracentrifuge C Where 'C' is the optical density of control without essential oil; 'T' is the optical density with essential oil.

Evaluation of the effect of TEO on aniline hydroxylase
Inhibition of aniline hydroxylase activity (an indicator of CYP2E1 activity) was measured by the method of Mazel (1971).

Principle
Aniline hydroxylase catalyses the hydroxylation of aniline to p-aminophenol in the presence of NADPH. The activity of aniline hydroxylase was determined by measuring (630 nm) the blue color formed when p-aminophenol reacts with phenol.

Procedure
The total volume of the reaction mixture is 1.5 ml containing the microsomal fraction (1 -1.5 mg protein) phosphate buffer (150 mM, pH 7.4), MgCl 2 (5 mM), aniline (32 mM in ethanol) and various concentration of TEO (50, 100 and 200 μg/ml) was incubated at 37 o C for 5 minutes. NADPH (0.33 mM in buffer) was added to the mixture and incubation contained for 2 hours at 37 o C. After incubation 500μl of 20% TCA was added to the mixture and was centrifuged at 3000 rpm for 10 minutes. Supernatant (1.5ml) was mixed with 750μl of 10% Na 2 CO 3 , 1.5ml of phenol (2% in 0.2M NaOH) and mixture was incubated at 37 o C for 30 minutes. The absorbance was measured at 630 nm against the phosphate buffer. The percentage of inhibition was calculated.

Principle
Aminopyrene was dealkylated by microsomal enzymes to form 4aminoantipyrene and formaldehyde. Formaldehyde so formed was measured by condensation with Nash reagent. The colour development and absorbance was measured at 412 nm.

Procedure
Total volume of the reaction mixture was 1.5 ml containing microsomal fraction (1-1.5 mg protein), phosphate buffer (150 mM, pH 7.4), MgCl 5 (5 mM), aminopyrene (320 mM), semicarbazide hydrochloride (120 mM) and various concentrations of TEO (50, 100 and 200 µg/ml) were incubated at 37ºC for 5 min. NADPH (33.3 mM in buffer) was added to the mixture and incubation continued for 2 h at 37ºC. After incubation, reaction was stopped by the addition of a mixture at 10% ZnSO 4 (500 μl) and saturated Ba (OH) 2 solution (500 μl), the mixture was centrifuged at 3000 rpm for 10 min, the supernatant was mixed with 500 μl Nash reagent (Nash reagent is a mixture of ammonium acetate (30 g) and acetyl acetone (400 μl) in a total volume of 100 ml distilled water). The tubes were then placed in a water bath at 60ºC for 30 minutes for colour development and absorbance was measured at 412 nm against distilled water. The percentage of inhibition was calculated.

Effect of TEO on various isoforms of cytochrome P450 enzymes in vivo
Wistar rats (120-150 g) were divided into the following groups consists of six animals per group. Different doses of TEO were administered once daily for 15 days orally, and phenobarbitone (60 mg/kg body weight, i.p./day) was started on day 12 and continued for 4 days. The rats were sacrificed 24 h after the last dose of phenobarbitone. The livers of all the animals were excised quickly and washed thoroughly in ice-cold saline and kept at -70°C. Liver homogenate (25%) was prepared in cold phosphate buffer (pH 7.4, 0.1M).
Homogenate was initially centrifuged at 14000 g for 20 min in a cold centrifuge (Remi) and supernatant was then further centrifuged at 10,5000 g for 1 h in an ultracentrifuge

Assessment of the effect of TEO on phase II enzymes in vivo.
Five groups of (120-150 g) Wistar rats, with each group consisting of 6 animals were divided into the following groups.
UDP glucuronic acid (30 mM) was dissolved in dist. water.

Procedure
The incubation mixture, containing 0.5ml buffer (0.2 ml Triton X-100, 0.05 ml MgCl 2 , 0.05 ml p-nitro phenol, 0.18 ml water and 0.1ml enzyme was incubated at 37 o C for 2 minutes. Then 0.1ml of UDP glucuronic acid was added. Then 0.1ml of aliquot of this mixture was arrested at 0, 10 and 15 minutes with TCA and centrifuged. Supernatant (1 ml) and NaOH (0.25ml) was added and read at 450 nm using a photochemical colorimeter. The activity of UDP glucuronyl transferase was expressed as n moles/min/mg protein.

Glutathione-S-transferase
The glutathione-S-transferase was estimated by the methods of Habig et al.

Statistical analysis
The values are expressed as mean ± SD. The statistical significance was compared between control and experimental groups by one way analysis of variance (ANOVA) followed by appropriate post hoc test (Dunnet multiple comparison test) using Graph pad in Stat software (version 3.05). Data of essential oil treated animals were compared with control animals.

Morphology and liver weight
Morphology of NDEA alone treated rats liver showed numerous tumour nodules on the surface with variable shapes, colour and normal morphology of liver was completely lost in most of the animals. Irregular protuberances were seen in most of the liver of NDEA treated animals ( Fig. 7.1. A, B, C). Liver of animals treated with TEO (20 and 100 mg/kg body weight) showed fewer incidences of tumour nodules and retained its normal morphology of the liver with small necrotic masses seen in some animals. In animals treated with 500 mg/kg body weight TEO normal morphology of liver was seen and did not show any significant tumour incidence. Liver weight was increased in both control (NDEA alone) and vehicle control (NDEA+paraffin oil) groups. TEO treated groups (20, 100 and 500 mg/kg body weight) showed decreased liver weight when compared to control groups (Table 7.1). The present study revealed that TEO significantly restored NDEA induced altered liver weight and morphology in a concentration dependent manner.

Biochemical analysis of serum
The levels of bilirubin, AST, ALT and ALP were drastically elevated in serum of control and vehicle control animals. TEO treated groups (100 and 500 mg/kg body weight) showed significant (p<0.001) decrease in these hepatic parameters when compared with control group. The elevated level of γ-GT as seen a marker of cell proliferation was decreased significantly (p<0.001) by TEO administration (Table 7.2).

Effect of TEO on antioxidant enzymes in the liver
NDEA administration suppressed the GSH, GPx and GST level in liver tissue, which was significantly (p<0.001) increased by the TEO administration in a concentration dependent manner (Table 7.3).

Effect of TEO on DMBA induced two-stage papilloma development in mice
Papilloma development in mice was started 6 th week in control and vehicle control group. Onset of papilloma was found to be significantly delayed or prevented by TEO treatment. In TEO (10 and 25%) treated groups, formation of papilloma were started in the 12 th week after DMBA application. DMBA alone, croton oil alone and DMBA + croton oil + 50% TEO treated groups did not produce any papilloma on mice.
At the end of 20 th week the average number of papilloma per mouse was 7 ± 1.06 and 6.86 ± 1.04 in control and vehicle control group respectively and this number was reduced to 1.2 ± 0.5 and 1 ± 0.3 by topical application of TEO 10% and 25% respectively ( Fig. 7.2). Number of animals which developed papilloma developed was 2 out of 8 animals and 5 out of 8 animals in 25% and 10% TEO treated groups (Table 7.4).

Effect of TEO against 3-methyl cholanthrene (3-MC) induced carcinogenesis in mice
Sarcoma developed at the site of injection i.e at the subcutaneous region of the neck in mice. The animals in the control and vehicle control groups (3-MC alone) started to develop fibrosarcoma by 8 th week and 100% animals developed sarcoma at 15 th week ( Fig. 7.4). TEO treated groups (20 and 100 mg/kg body weight) started to develop sarcoma only at 10 th week and 500 mg/kg body weight at 12 th week (Table 7.5). TEO elevated the survival rate of the animals harboring sarcoma. All the animals in control and vehicle control animals died by the end of 17 th week. In the case of TEO treated group (500 mg/kg body weight) 6 out of 15 animals and in 100 and 20 mg/kg body weight TEO treated group 5 out of 15 animals were found to be alive by the end of 17 th week ( Fig. 7.5). These results indicated that TEO could significantly inhibit and delay the development of the 3-MC induced sarcoma in mice.  Data are expressed as the mean ± standard deviation (n=8 per group).
Data are expressed as the mean ± standard deviation (n=8 per group).
Data are expressed as the mean ± standard deviation (n=8 per group).
(P value) a p<0.001. b p<0.01.    Data are expressed as the mean ± standard deviation (n=6 per group).
(P value) a P<0.001. Data are expressed as the mean ± standard deviation (n=6 per group).

Effect of TEO on Phase I enzymes in vivo
Cytochrome P450 enzymes (Phase I enzymes) were found to be significantly increased following phenobarbitone administration. Significant inhibition (P<0.001) of Phase I enzymes was observed after TEO administration as seen from the hepatic MROD (CYPIA2), PROD (CYP2B1/2) and EROD (CYP1A1) levels (Table 7.7). Oral administration of TEO significantly inhibited MROD levels in a concentration dependent manner and maximum inhibitory effect was seen at 1000 mg/kg body weight (95.9%).
TEO significantly decreased the level of EROD and PROD to 91.8 and 91.5% respectively. These results indicated that oral administration of TEO inhibited the phenobarbitone induced elevation of MROD, PROD and EROD levels.

Effect of TEO on phase II enzymes.
The level of glutathione-S-transferase in the liver was found to be significantly increased (P<0.001) after oral administration of TEO. Similarly TEO administration was found to increase the level of UDP-glucuronyl transferase in a concentration dependent manner (Table 7.8).

Discussion
Chemical analysis of essential oil from C. longa indicate the presence of 13 compounds of which ar-turmerone (61.79%) and curlone (12.48%) are found to be the most prominent. Ar-turmerone present in TEO has been reported to have significant pharmacological and anticancer potential (Hislop, 1990;Sugimura, 1970 andAmes, 1983). It is already reported that ar-turmerone (12.66%) and curlone (6.82%) exhibited  et al., 1997;Bansal et al., 2005). GSH can effectively scavenge free radicals of oxygen species through nonenzymatic and enzymatic process by conjugation with GPx and GST (Ramakrishnan et al., 2006;Zeng et al., 2008 andLie, 2002). GST is located in cytosol and plays an important role in detoxification and excretion of xenobiotics (Rao et al., 2006). We have found that simultaneous administration of TEO significantly increased NDEA induced lowering of antioxidant enzymes, GSH, GPx and GST. Our previous study revealed that oral administration of TEO increased the antioxidant enzymes in normal mice (Chapter 4, section 4.3.1 and 2). The elevation of these antioxidant enzymes and GSH in vivo may be one of the reasons for protective activity of TEO against NDEA induced hepatocarcinogenesis. Histological observations of liver tissues also support the potential of TEO against hepatocellular carcinoma.