African Journal of Biotechnology
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
|
African
Journal of Biotechnology Vol. 1 (2), pp. 46–49, December 2002 ISSN 1684-5315 © 2002 Academic Journals
1UNLV Cancer Institute/Chemistry Department, University of Nevada Las Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154-4003 2McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, WI 53706-1599
Accepted 13 November 2002 |
||||||||||||||||||||||||||||||||||||
| Abstract | |||||||||||||||||||||||||||||||||||||
|
|
Perillyl alcohol and d-limonene are naturally occurring plant compounds that exhibited anti-carcinogenic activities in mammary tumor models. The effects of these monoterpenes at the initiation stage of aflatoxin B1-induced hepatocarcinogenesis were investigated. Male F344 rats were fed Control or treatment diets throughout the study and exposed to aflatoxin for 5 days. Three days after the last aflatoxin dose, blood and liver samples were obtained. Analysis of liver samples showed that both limonene and perillyl alcohol significantly inhibited (p<0.05) aflatoxin-DNA adducts formation in hepatocytes. The monoterpenes may have potential for use as chemopreventive agent against aflatoxin-induced liver cancer. Keywords: Aflatoxin B1, hepatocarcinogenesis, monoterpenes, chemoprevention. |
|||||||||||||||||||||||||||||||||||
| Introduction | |||||||||||||||||||||||||||||||||||||
|
|
Liver cancer (primary
hepatocellular carcinoma) is a major public health hazard in the
developing countries of Africa and Asia.
The etiology of this disease implicates both environmental (diet)
and infectious (hepatitis B and C) factors.
Aflatoxin B1
(AFB1) is a major
contaminant of foods in the humid tropical regions of Africa and Asia and
has been linked, with hepatitis B and C infections, to the high incidence
of liver cancer in these regions (Montalto et
al., 2002; Sarin et
al., 2001; Wild et
al., 2000).
The poor prognosis and survival rates among liver cancer patients
indicate that prevention may offer a useful approach to reducing the
incidence of human liver cancer in high-risk areas.
Economic considerations, however, dictate that such chemopreventive
agent must be inexpensive and readily available to the general populace
especially in the developing countries. Monoterpenes (d-limonene, perillyl alcohol, etc) are naturally occurring, nontoxic and ubiquitous plant compounds. Dietary sources of monoterpenes include d-limonene in orange and citrus peel oils, caraway, dill and green leafy vegetables; perillyl alcohol in cherry and spearmint, other monoterpenes in lemongrass oil, and spearmint (Crowell, 1999). It was reported earlier that the monoterpenes, principally d-limonene and its analogs, elicited preventive (Elegbede et al., 1986; Haag et al., 1992) or therapeutic (Maltzman et al., 1989) effects against experimentally induced mammary tumors in rat cancer models. Mills et al. (1995) also reported that perillyl alcohol significantly inhibited the growth of diethylnitrosamine-induced liver tumors in rats by increasing the frequency of apoptosis. Although the mechanism of action of the monoterpenes is still not yet clearly understood, both limonene (Poon et al., 1996; Vigushin et al., 1998) and perillyl alcohol (Ripple et al., 1998, 2000) have been tested in Phase I human clinical trials.
Figure
1:
Structures of Limonene, Perillyl alcohol and Oltipraz.
In this study, the effects of the monoterpenes, d-limonene and perillyl alcohol (Figure 1), at the initiation stage of AFB1-induced liver carcinogenesis in rats were investigated. Specifically, male F344 rats were fed control or test diets [5% d-limonene, 2% perillyl alcohol, and 30mg% oltipraz] from two weeks before aflatoxin exposure to the end of the study. For aflatoxin exposure, each animal was anesthetized and intubated with [3H]-AFB1 (260 mCi/µmol; 250 µg/kg body wt) in tricaprylin (Groopman et al., 1992). Three days after the last intubation with [3H]-AFB1, animals were sacrificed and blood and liver were obtained. Twenty-four hour urine samples were also collected. Tritium-labeled aflatoxin B1 (3H-AFB1) was used to facilitate tracking of aflatoxin metabolic products. A non-therapeutic dose of the known hepatic chemopreventive agent, oltipraz, was included as positive control.
|
||||||||||||||||||||||||||||||||||||
| Results and Discussion | |||||||||||||||||||||||||||||||||||||
|
|
During the period of intubation with aflatoxin, rats in control, d-limonene and oltipraz groups lost 7, 8 and 4% of their body weights respectively while those on perillyl alcohol diets gained 5%. Livers of rats on the experimental diets appeared grossly normal. Average liver weights (g) for the respective groups were 5.6±1.29 for control, 8.0±1.76 for d-limonene, 9.8±0.78 for perillyl alcohol, and 9.6±0.94 for oltipraz. Animals on the treatment diets had significantly (p<0.05) larger livers than the Control animals. The effect of dietary monoterpenes against AFB1-induced initiation of liver cancer was assessed as the ability of the monoterpenes to inhibit the formation of [3H] AFB1-DNA adducts in liver cells. The levels of AFB1-DNA adduct formed in the livers of animals fed different diets are shown in Table I. Animals fed perillyl alcohol or d-limonene diets had significantly (p<0.01) lower reduction (52% and 46%, respectively) in the level of AFB1-DNA adducts compared to the control group. Oltipraz, which was used at a non-therapeutic dose, elicited a 22% reduction in adduct formation compared to the control. This reduction in DNA adduct formation with oltipraz was consistent with earlier reports (Bolton et al., 1993; Groopman et al., 1994).
Table 1: Inhibition of hepatic AFB1-DNA adduct formation in rats by dietary monoterpenes.
Liver DNA was extracted and
quantitated by the method of Sambrook et al. (1989). Aliquots of DNA extracts were counted for [3H]
radioactivity in a Beckman LS 6000 IC Scintillation Counter. Values
represent Mean ± SEM, (n=6). aValues
are significantly different from the Control (p< 0.01)
Urinary excretion of aflatoxin products has been used as a measure of exposure to the carcinogen. In this study we measured aflatoxin metabolic products excreted in the urine as [3H] aflatoxin equivalents (nmoles/mg creatinine). Three days after the last AFB1 intubation, the presence of labeled AFB1 products in the blood and urine of exposed animals was measured. There were no significant differences in blood levels of aflatoxin products between the control and monoterpene groups. Rats in the monoterpene-fed groups had lower urinary aflatoxin B1 compared to the control group (Table 2).
Table 2:
Distribution of [3H] aflatoxin metabolites in blood and urine
of rats intubated with aflatoxin B1 and fed different
monoterpene diets.
Rats were fed respective diets
throughout the study. Seventy-two
hours after the last aflatoxin dose, 24-hr urine and blood samples were
obtained. Serum albumin was extracted and quantified (Chapot and Wild, 1991)
and urinary creatinine determined (Procedure No 555, Sigma Diagnostics, St
Louis, MO). Aliquots of each sample were counted for total [3H]
activity. Values are Mean ± SD aValues
are significantly different from the Control (p<0.05) bValues
are significantly different from perillyl alcohol (p<0.05)
Incorporation of the monoterpenes in the diet of male F344 rats during the period of exposure to aflatoxin B1 resulted in significant (p<0.01) reduction in the formation of aflatoxin-DNA adducts in the liver (Table I). The cumulative reduction in the formation of hepatic aflatoxin-DNA adducts were perillyl alcohol (52%), d-limonene (46%), and oltipraz (22%). Both d-limonene and perillyl alcohol significantly (p<0.01) lowered AFB-DNA adduct formation than in the Control group. In earlier studies, Bolton et al. (1993) and Groopman et al. (1994) showed that transient intervention with oltipraz caused about 25% cumulative reduction in levels of hepatic aflatoxin-DNA adducts. According to these authors, later analysis showed that oltipraz reduced the hepatic glutathione S-transferase placental form-positive foci by 54% and 72%, an indication that reduced cumulative adduct formation underestimated the potency of oltipraz (Bolton et al., 1993). By inference, the cumulative effects of d-limonene and perillyl alcohol against aflatoxin-induced liver carcinogenesis may be better than currently assessed. The mechanism of action of many chemopreventive agents involves alteration of the metabolic fate of carcinogens by modulating the activities of either, or both, the phase I and/or phase II drug metabolizing enzymes. Induction of phase II detoxification enzymes enhances carcinogen inactivation by facilitating the clearance of activated metabolites through conjugation with glutathione (Coles et al., 1985; Kensler et al., 1987) and/or glucuronides (Elegbede et al., 1993). The mechanism of action of the synthetic anticarcinogen oltipraz is by the induction of glutathione S-transferases which facilitate the conjugation of glutathione to aflatoxin-8,9-oxide and its ultimate elimination in the urine (Sparfel et al., 2002). It was reported earlier that the monoterpenes, specifically limonene and a hydroxylated analog, induced phase II hepatic drug metabolizing enzymes (Elegbede et al., 1993) while limonene also decreased the formation of 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adducts in the liver, lung, kidney and spleen of rats intubated with DMBA (Maltzman et al., 1991). DMBA is an inducer of carcinogenesis. In a randomized, double-blind, placebo-controlled chemoprevention trial in China, Egner et al. (2001) reported that chlorophyllin, a mixture of semisynthetic, water-soluble derivatives of chlorophyll that is used as food colorant and over-the-counter medicine, inhibited aflatoxin adducts in urine samples collected 3 months into the study. It was therefore suggested that chlorophyllin may represent practical means to prevent the development of hepatocellular carcinoma (Egner et al., 2001). Limonene and perillyl alcohol are naturally occurring compounds that are abundant in the peel oil of oranges, lemons, limes, and other plant products that are readily available in the tropics. In this study, we showed that perillyl alcohol and d-limonene significantly reduced the formation of aflatoxin-DNA adducts in the livers of F344 rats exposed to aflatoxin B1 for 5 days. Mills et al. (1995) reported that perillyl alcohol inhibited the growth of diethylnitrosamine-induced liver tumors in rats by enhancing tumor cell loss through apoptosis. Our results suggested that perillyl alcohol and d-limonene offered protection against aflatoxin-induced liver tumor formation at initiation by reducing aflatoxin-DNA adducts formation. This finding, taken together with the low toxicity, ready availability and low cost suggests the importance of further studies to evaluate the efficacy of these monoterpenes as potential chemopreventive agents against aflatoxin-induced liver cancer in humans. The monoterpenes appear to have potential as chemopreventive agents against aflatoxin B1-induced hepatocarcinogenesis in humans. ACKNOWLEDGEMENTS We thank Dr. Gary Kelloff of the NCI for providing us with oltipraz. We also thank Wendy Kennan for technical assistance. This work was supported by PHS NIH grant CA 38128; JAE was supported by a grant (ACS International Cancer Research Fellowship) from the International Union Against Cancer (UICC), Switzerland.
|
||||||||||||||||||||||||||||||||||||
| References | |||||||||||||||||||||||||||||||||||||
|
|
Bolton MG, Munoz A, Jacobson LP, Groopman JD, Maxuitenko YY, Roebuck BD, Kensler TW (1993). Transient intervention with oltipraz protects against aflatoxin-induced hepatic tumorigenesis. Cancer Res. 53:3499-3504. [PubMed] Chapot B, Wild CP (1991). ELISA for quantification of aflatoxin-albumin adducts and their application to human exposure assessment. Technol. Diagnostic Pathol. 2:135-155. Coles B, Meyer DJ, Ketterer B, Stanton CA, Garner RC (1985). Studies on the detoxication of microsomally-activated aflatoxin B1 by glutathione and glutathione transferases in vitro. Carcinogenesis 6:693-697. [PubMed] Crowell PL (1999). Prevention and therapy of cancer by dietary monoterpenes. J. Nutr. 129:775S-778S. [PubMed] Egner PA, Wang JB, Zhu YR, Zhang BC, Wu Y, Zhang QN, Qian GS, Kuang SY, Gange SJ, Jacobson LP, Helzlsouer KJ, Bailey GS, Groopman JD, Kensler TW (2001). Chlorophyllin intervention reduces aflatoxin-DNA adducts in individuals at high risk for liver cancer. Proc. Natl. Acad. Sci. USA 98:14601-14606. [PubMed] Elegbede JA, Elson CE, Tanner MA, Qureshi A, Gould MN (1986). Regression of rat primary mammary tumors following dietary d-limonene. J. Natl. Cancer Inst. 76:323-325. [PubMed] Elegbede JA, Maltzman TH, Elson CE, Gould MN (1993). Effects of anticarcinogenic monoterpenes on phase II hepatic metabolizing enzymes. Carcinogenesis 14:1221-1223. [PubMed] Groopman JD, Dematos P, Egner PA, Love-Hunt A, Kensler TW (1992). Molecular dosimetry of urinary aflatoxin-N7-guanine and serum aflatoxin-albumin adducts predicts chemoprotection by 1,2-dithiole-3-thione in rats. Carcinogenesis 13:101-106. [PubMed] Groopman JD, Wogan GN, Roebuck BD, Kensler TW (1994). Molecular biomarkers for aflatoxins and their application to human cancer prevention. Cancer Res. 54:1907s-1911s. [PubMed] Haag JD, Lindstrom MJ, Gould MN (1992). Limonene-induced regression of mammary carcinomas. Cancer Res. 52:4021-4026. [PubMed] Kensler TW, Egner PA, Dolan PM, Groopman JD, Roebuck BD (1987). Mechanism of protection against aflatoxin tumorigenicity in rats fed 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) and related 1,2-dithiol-3-thiones and 1,2-dithiol-3-ones. Cancer Res. 47:4271-4277. [PubMed] Maltzman TH, Christou M, Gould MN, Jefcoate CR (1991). Effects of monoterpenoids on in vivo DMBA-DNA adduct formation and on phase I hepatic metabolizing enzymes. Carcinogenesis 12:2081-2087. [PubMed] Maltzman TH, Hurt LM, Elson CE, Gould MN (1989). The prevention of nitrosomethylurea-induced mammary tumors by d-limonene and orange peel oil, Carcinogenesis 10:781-783. [PubMed] Mills JJ, Chari RS, Boyer IJ, Gould MN, Jirtle RL (1995). Induction of apoptosis in liver tumors by the monoterpene perillyl alcohol. Cancer Res. 55:979-983. [PubMed] Montalto G, Cervello M, Giannitrapani L, Dantona F, Terranova A, Castagnetta LA (2002). Epidemiology, risk factors, and natural history of hepatocellular carcinoma. Ann. NY Acad. Sci. 963:13-20. [PubMed] Poon GK, Vigushin D, Griggs LJ, Rowlands MG, Coombes RC, Jarman M (1996). Identification and characterization of limonene metabolites in patients with advanced cancer by liquid chromatography/mass spectrometry. Drug Metab. Dispos. 24:565-571. [PubMed] Ripple GH, Gould MN, Arzoomanian RZ, Alberti D, Feierabend C, Simon K, Binger K, Tutsch KD, Pomplun M, Wahamaki A, Marnocha R, Wilding G, Bailey HH (2000). Phase I clinical and pharmacokinetic study of perillyl alcohol administered four times a day. Clin. Cancer Res. 6:390-396. [PubMed] Ripple GH, Gould MN, Stewart JA, Tutsch KD, Arzoomanian RZ, Alberti D, Feierabend C, Pomplun M, Marnocha R, Wilding G, Bailey HH (1998). Phase I clinical trial of perillyl alcohol administered daily. Clin. Cancer Res. 4:1159-1164. [PubMed] Roebuck BD, Liu YL, Rogers AE, Groopman JD, Kensler TW (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short-term molecular dosimetry. Cancer Res. 51:5501-5506. [PubMed] Sambrook J, Fritsch EF, Maniatis T (1989). Analysis and cloning of eukaryotic genomic DNA. In: Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Lab. Press, 2nd Ed., Plainview, NY pp. 9.1-9.62. Sarin SK, Thakur V, Guptan RC, Saigal S, Malhotra V, Thyagarajan SP, Das BC (2001). Profile of hepatocellular carcinoma in India: An insight into the possible etiologic associations. J. Gastroenterol. Hepatol. 16:666-673. [PubMed] Sparfel L, Langouet S, Fautrel A, Salles B, Guillouzo A (2002). Investigations on the effects of oltipraz on the nucleotide excision repair in the liver. Biochem. Pharmacol. 63:745-749. [PubMed] Vigushin DM, Poon GK, Boddy A, Jarman M, Coombes RC (1998). Phase I and pharmacokinetic study of d-limonene in patients with advanced cancer. Cancer Chemother. Pharmacol. 42:111-117. [PubMed] Wild CP, Yin F, Turner PC, Chemin I, Chapot B, Mendy M, Whittle H, Kirk GD, Hall AJ (2000). Environmental and genetic determinants of aflatoxin-albumin adducts in the Gambia. Int. J. Cancer 86:1-7. [PubMed] |
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |