Therapeutic role of olive leaves and hyperthermia on nitrosodiethylamine induced hepatocarcinogenesis in rats.
Abstract: Aim of the study

The present investigation was carried out to evaluate the effects of ethanolic olive leaves extract (OLE) alone or with whole body hyperthermia(WBH) in rat models of liver carcinogenesis, initiated by nitrosodiethylamine (NDEA) and promoted by phenobarbital(PB).


Liver cancer was induced by daily intraperitoneal injection of NDEA (200mg/kg) for three weeks. Two weeks after the last day of treatment with NDEA, PB was used with the aim to promote the carcinogenic effects for up to 6 weeks. OLE (200mg/kg) was administered intraperitoneally for 3 weeks either during or after the period of PB administration to hepatocellular carcinoma-bearing rats. After the experimental period, serum and liver samples were collected for biochemical and histopathological analysis.


On administration of the carcinogens, the levels of serum tumor markers and liver enzymes increased markedly, but were significantly lowered in the groups of OLE treatment, especially when OLE was administered during the period of PB administration. On the contrary, the albumin serum levels were decreased in the carcinogen-administered animals, which was improved up to normal levels in the OLE groups alone or with hyperthermia. The results were dependent on the stage of carcinogenesis, rather than the duration of the protective treatment. The histopathological observations of liver tissues were corresponding with the biochemical results and confirmed the difference between control and treated groups.


OLE may efficiently prevent the initiation step of Carcinogenesis and modulate the development of NDEA-induced and PB-promoted hepatocellular carcinogenesis in rats.

Keywords: Olive tree, hyperthermia, hepatocarcinogenesis, nitrosodiethylamine, oleuropein, rats.
Article Type: Report
Subject: Animal models in research (Usage)
Fever (Causes of)
Fever (Research)
Hyperthermia (Causes of)
Hyperthermia (Research)
Liver cancer (Development and progression)
Liver cancer (Control)
Liver cancer (Research)
Nitrosoamines (Health aspects)
Nitrosoamines (Research)
Olive (Usage)
Olive (Health aspects)
Olive (Chemical properties)
Olive (Research)
Authors: Abdel-Gawad, Eman I.
Hassan, Amal I.
Pub Date: 01/01/2009
Publication: Name: Experimental Medicine Publisher: Renaissance Medical Publishing Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 Renaissance Medical Publishing ISSN: 1791-9967
Issue: Date: Jan, 2009 Source Volume: 1 Source Issue: 1
Topic: Event Code: 310 Science & research
Product: Product Code: 0176000 Olives NAICS Code: 111339 Other Noncitrus Fruit Farming SIC Code: 0179 Fruits and tree nuts, not elsewhere classified
Geographic: Geographic Scope: Egypt Geographic Code: 7EGYP Egypt
Accession Number: 204482754
Full Text: Introduction

The olive leaf is the first botanical mentioned in the Bible. Through the history of civilization, the olive plant has been an important source of nutrition and was used for medical reasons. The olive leaf extract contains compounds with potent antimicrobial activities against bacteria, fungi and mycoplasma. (1) Several epidemiological studies have shown that the incidence of coronary heart diseases and certain cancers, e.g., breast and colon cancers, are rarer in the Mediterranean basin where the diet is rich in olives and olive products. (2) In addition, the olive leaves are considered a source of several antioxidants (3) and of substances with anti-inflammatory activities. (4)

There is an increasing interest in the phenolic compounds of olive by-products, due to their biological properties. Oleuropein is the most abundant biophenol in olive leaves and has been used in a number of medical treatments since its first reference in the literature (5); oleuropein prevents cardiac diseases by protecting membrane lipid oxidation acting on coronary dilation and by its antiarrythmic action (6) improves lipid metabolism and prevents obesity, protects vital enzymes and hypertensive cell death in colon cancer patients (7) and finally presents antiviral properties. (8) Recently, AIDS patients have begun to use olive leaf extract for a variety of indications, among them as a drug for the support of immune system, to relieve chronic fatigue, to boost the effects of anti-HIV medications, and to treat HIV-associated Kaposi's sarcoma and HSV infections. (3) All these effects represent the antioxidant and nutraceutical capacities of olive leaf extracts in which by-products show a synergistic effect. (10)

Hyperthermia has been used as a cancer treatment in which body tissues are exposed to high temperatures. High temperatures can damage and kill cancer cells, usually with minimal injury to normal tissues. It has been proposed that with killing cancer cells and damaging proteins and structures within the cells, hyperthermia may shrink tumors (11) Preliminary data suggest that heat may be especially destructive to two types of tumor cells: those that are making DNA in preparation for division and those that are acidic and poorly oxygenated. These cell types tend to be resistant to radiation. It has been suggested by proponents that heat also appears to be make cells more sensitive to radiation by preventing radiation-damaged cells from repairing themselves. (12) When used as an adjunct to chemotherapy, hyperthermia may potentiate the effects of some chemotherapeutic agents. (13)

Primary liver cancer has been classified as the fifth most common cause of cancer and the fourth most cause of cancer mortality in the world. (14) The major risk factors of this disease include N--nitrosodiethylamine (NDEA) which is one of the most important environmental hepatocarcinogens that has been reported to generate free radicals to exert its carcinogenic effects. (15) It has been widely used in the field of experimental hepatocarcinogenesis. (16) Foodstuffs such as milk and meat products, salted fish, alcoholic beverages and a few varieties of vegetables are the principal sources of nitroso-compounds. (16) In rats, NDEA has been shown to be metabolized to its active ethyl radical metabolite, and the reactive product interacts with nuclear enzymes involved in DNA repair/replication causing mutation, which would lead to carcinogenesis. (17) Phenobarbital (PB) is known as a model tumor which enhances hepatocarcinogenesis in rodents if administered subsequent to initiation by a liver carcinogen such as nitrosodiethylamine. (18)

The present study aims to carry out a systematic investigation of the therapeutic influences of ethanolic OLE alone or with WBH on NDEA-induced hepatocarcinogenesis by analyzing serum [alpha]-FP, [alpha]-2M and TNF-[alpha] levels along with biomarkers of liver function as endpoints of chemotherapy.

Materials and Methods


Nitrosodiethylamine (NDEA) and Phenobarbital (PB) were purchased from Sigma-Aldrich Chemicals Co (St. Louis, USA). All other chemicals were of analytical grade.

Preparation of olive leaf extract

The olive leaves were collected from a piece of land at Anshas and the extraction procedure was done in the department of biochemistry, Faculty of Agriculture, Banha University. The leaf samples were air-dried at room temperature and blended to a mesh size of 1 mm. The blended samples (1kg) in 4 litres of 70% ethanol for 48 hours, filtered and concentrated to dryness using rotary evaporator. The ethanolic extract was kept in the refrigerator until usage. (19)

Experimental induction of hepatocarcinogenesis

Nitrosodiethylamine (NDEA) initiated and phenobarbital (PB) promoted rat hepatoma. NDEA was injected intraperitoneally to rats in a single daily dose of 200 mg/ kg b.w. dissolved in saline solution for 3 weeks (initiation period). After two weeks of the last NDEA injection, the carcinogenic effect was promoted by phenobarbital (0.05% PB Sigma Chem. Co., St Louis, MO, USA) (20) through intraperitoneal injection at a dose of 500 mg/kg b.w./day for 6 weeks (promotion period).

Treatments schedule

Forty-eight adult albino male rats weighing 140-150 gr were used in this study. They were made to acclimatize to laboratory conditions for 10 days and were fed a commercial pelleted rat chow and water ad libtum. The animals were grouped into three main groups:

Group I: (control group) rats served as control.

Group II: (carcinogenic group) rats were received intraperitoneal injection of NDEA for three weeks (initiation period). After two weeks of initiation period, the carcinogenic effect was promoted by Phenobarbital injection for 6 subsequent weeks (promotion period).

Group III: (Treated group) rats were treated with OLE alone (200mg/kg b.w.) or with WBH for three subsequent weeks alone or with hyperthermia exposure twice weekly at 41.50 [degrees]C for half an hour during the treatment period. The relative temperature of the rats was recorded using a thermocouple (Cole Parmer type T. thermocouple thermometer) connected with a rectal probe, which was inserted 4 cm beyond the anal sphincter. The animals in this group were sub-divided into two subgroups according to the time of treatment.

The first subgroup received the treatment during the last 3 weeks of promotion period. The second subgroup received the treatment after the end of promotion period. Blood samples were collected from orbital venous plexus of the experimental animals after 24 hours of the last dose of OLE and serum was separated for different biochemical analyses.

Biochemical investigations

Serum was collected for analyses of the following parameters: a-fetoprotein (AFP), [alpha]-2 macroglobulin ([alpha]-2M) and tumor necrosis factor (TNF-[alpha]) for rats were done by ELISA according to the techniques of Watanabe et al (21),Hoyer et al (22) and Whittle et al (23), respectively. Serum gamma glutamyltrans-ferase ([gamma]-GT) was determined using a kinetic photometric method according to Szasz and Persijn. (24) Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin activities were assayed colorimetrically according to the method described by Breuer (25) and Dumas & Biggs. (26)

Histopathological investigation

Autopsy samples were taken from the liver sacrificed rats in different experimental groups then fixed in 10% formalin saline solution for twelve hours. Serial dilutions of alcohol (methyl, ethyl and absolute ethyl) were used. Specimen were cleared in xylene embedded in paraffin at 56 degree in hot air oven for twenty four hours. Paraffin bees wax tissue blocks were prepared for sectioning at 4 micron thickness by slidge microtome. The obtained tissue sections were collected on glass slides, deparaffinized and stained by hematoxylin and eosin stain (27) for histopathological examination through the light microscope.

Statistical analysis

Our data were expressed as the mean [+ or -] S.E. Means were compared by one--way analysis of variance (ANOVA) followed by Duncan test, which was used to identify differences between groups. A value of P< 0.05 and 0.01 were accepted as significant.


Biochemical results

NDEA significantly increased AFP, [alpha]-2M and TNF--[alpha] level compared to control level. OLE treatment significantly lowered AFP and TNF--[alpha] level, which approached to normal values. However, OLE co-administered significantly inhibits AFP [alpha]-2M and TNF-[alpha] level to a great extent when administered during promotion period more than after promotion period in NDEA-treated group. Exposure to whole body hyperthermia has a slightly effect on OLE efficiency in decreasing AFP [alpha]-2M and TNF-[alpha] levels of rats treated during promotion period only. The serum AFP [alpha]-2M and TNF-[alpha] activity of animals treated with OLE alone or with hyperthermia were not significantly different from each other (Table 1).

In table 2, the serum activities of [gamma]-GT, AST and ALT in NDEA- group were significantly elevated and albumin activity was significantly decreased when compared to normal group. OLE co-administered to animals during promotion period restored [gamma]-GT, AST and ALT levels more than after promotion period when compared to NDEA group. The animals treated with OLE showed significant increase in albumin activity when compared to NDEA group. Exposure to whole body hyperthermia has no synergistic effect on the OLE as a treatment either during or after promotion period of tested parameters shown in table 2.

Histopathological Findings

Examination of liver sections of different groups illustrated that: liver tissue of normal group showed hepatic lobules with normal architecture (Figure 1). Liver tissues of the NDEA-group showed sever degeneration and disorganization of the histological pattern of the hepatocytes (d) with inflammatory cells infiltration (m) in the portal area (Fig.2). Some cells possessed pyknotic nuclei, and numerous Kupffer cells (Fig 3). According to the histopathological findings, the figures indicating the early stage of carcinogenesis in the hepatocytes which is called dysplastic activity associated with degenerative changes as well as inflammatory section. Liver tissue from the OLE-treated rats during promotion period showed inflammatory cells infiltration (m) and Kupffer cells proliferation (k) in between the degenerated hepatocytes(d) (Fig.4). Section of rat liver exposed to hyperthermia plus OLE treated during promotion period showed Kupffer cells (k) with fewer inflammatory cells infiltration (m) in between the hepatocytes (Fig. 5). Liver section of rats treated with OLE alone (Fig.6) or with whole body hyperthermia(WBH) (Fig.7) after promotion period showed congestion in central vein (cv), Kupffer cells proliferation (k) and inflammatory cells infiltration (m) in between the degenerated hepatocytes.









It is well known that liver cancer is one of the most important cancers in the world, resulting in more than 1 million patients and over 260,000 deaths per year. (28) Therefore, the chemoprevention and treatment of liver cancer is very important. AFP, [alpha]-2M, TNF-[alpha], ALT, AST and a-GT are valuable references, widely used in animal studies to diagnose and observe the development of hepat ocarcinogenesis. (21,29,30,31) In the present study, the values of previously mentioned parameters showed sharp increase in NDEA-group as compared with that of the normal control group (Tables 1 & 2).

AFP, a tumor-associated fetal protein, has long been employed as a serum fetal tumor marker to monitor disease progression. (29) The significant increase observed of serum AFP and a-2macroglobulin in NDEA-group is in agreement with previous authers (29,30,31,32) who stated that [alpha]-FP and [alpha]-2M together might be linked to the rat hepatocarcinogenesis from initial stage to tumor progression even in conditions, which are undetectable, by established cytochemical markers and seems to be linked with their tumorgenicity. Smorenburg et al (30) and Sukata et al (32) also confirmed that the increase in serum [alpha]-2macroglobulin during hepatocarcinogenesis was not a result of secretion by the host liver but in response to inflammatory injury, since, [alpha]-2macroglobulin is captured by cytokines rather than proteases, forming complexes which could be observed in stromal cells, sinusoidal cells, or hepatocytes.

Increased serum TNF-[alpha] level observed in the present study might be attributed to induction of TNF-[alpha] as a result of hepatocarcinogenesis processes. Since, TNF-[alpha] is one of the earliest events in hepatic inflammation, triggering a cascade of other cytokines that co-operate to kill hepatocytes, recruit inflammatory cells and initiate a wound healing response that includes fibrogenesis. (33) The present results were supported by the findings of Yuan et al (34) who reported that TNF-[alpha] participated in the activity process of liver disease. Also, Zang et al (35) reported that TNF-[alpha] is an important mediator in the pathogenesis of liver necrosis and significantly correlated with the elevated serum ALT and insignificantly correlated with AFP.

The ubiquity of elevated [gamma]-GT levels in many rodent and human hepatic and extrahepatic carcinomas have led to the hypothesis that [gamma]-GT provides a growth advantage to focal cells during carcinogenesis. (36) A plausible hypothesis is that the advantage may be due to the role of [gamma]-GT in the transport of GSH constituents, leading to increase in cellular GSH. The latter is required for proliferation and resistance. (37)

Serum transaminases are sensitive indicators of hepatic injury. Several reports have shown an increase in the activities of AST and ALT in response to NDEA administration. (38,39,40,41) It has been reported that NDEA is transported through blood and it causes hepatic injury. (38) Consequently, elevated activities of ALT and AST observed in the current study in response to NDEA administration could be a common sign of impaired liver function and subsequent leakage of these enzymes into circulation. (41)

Enhanced liver cell death and impaired regeneration are indeed features of most liver disorders. Low albumin is a sign of poor health and a predictor of a bad outcome. Thus, significant decrease in albumin level observed in NDEA-group was indicative of poor liver function and inability to fight infections. (41)

Administration of OLE to NDEA-treated rats showed restoration of tumor markers activities and serum liver enzymes towards normal and these effects was more pronounced in animals injected with OLE during promotion period rather than animals injected after promotion period. This infers that therapeutic effect of OLE depends on the stage of carcinogenesis, rather than duration of treatment. Such findings could be attributed to the biological activities of OLE which mainly derived from its contents of mixture of polyphenolic compounds. (19) Among these phenolic compounds, oleuropein and hydroxytyrosol, both of which are bioavailable, strengthening the immune system (10) and inhibite oxidation stress. (42,43) Furthermore, polyphenols exert antiproliferative and an anti-angiogenic effect that are particularly pronounced in tumor cells. (44) OLE also affects macrophage function and modulates the inflammatory response, two effects that may also contribute to activity against infectious agents. (45,46)

It is well known that endogenous and exogenous cancer risk factors generate oxygen free radicals species in vivo and the free radicals scavenging is one of the known mechanisms by which antioxidants inhibit lipid oxidation (38). In this respect, olive flavonoids, phenols and oleuropeosides have been shown to possess important antioxidant and free radicals scavenging activity towards these radicals (19,42) which is principally based on the redox power properties of their phenolic hydroxyl groups (42) and the structural relationships between different parts of their chemical structure. (42)

Whole-body hyperthermia is a new and safe therapy method of cancer treatment, increasing the activity of the immune system and the effectivity of chemotherapy. (47) The use of hyperthermia in the treatment of malignancy depends on the differential between tumor and normal cells to the application of this energy source. (48) Temperature applied in the range between 41[degrees]C and 42[degrees]C were lethal to tumor cells, whilst not damaging normal cells. (49,50)

Although the mentioned advantages of whole body hyperthermia in cancer treatment, the present study revealed adverse effect of hyperthermia exposure for half hours on animals treated with OLE after promotion period indicating that treatment with hyperthermia depends on the stage of carcinogenesis processes related to exposure time.

Our findings were further supported by the histopathological examination of liver section, which illustrated that liver tissue of NDEA-treated rats showed drastic changes in the morphology of the liver cells, manifest as nuclear pleomorphism, intranuclear vacuoles, cellular infiltration, pyknotic nuclei and numerous Kupffer cells. Whereas the OLE-treated rats showed more or less normal hepatic lobular architecture. Accordingly, we presented evidence that OLE substantially improved the liver cell metabolic indices, as well as its synthetic capacity, and further treated its malignant transformation.


We are grateful to Dr. Adel M. Bakeer Kholoussy, Professor of Pathology, Faculty of Veterinary Medicine, Cairo University for his help in the examination of the histopathological slides and for his valuable comments.


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Eman I. Abdel-Gawad and Amal I. Hassan

Radioisotopes Department, Atomic Energy Authority, Cairo, Egypt

Corresponding author:

Amal I. Hassan, Radioisotopes Department, Atomic Energy Authority, Malaeb El- Gamma Street, P.O. 12311, Dokki, Giza, Egypt E-mail:

HIV:           Human Immunodefiency Virus
HSV:           Herpes simplex virus
DNA:           Deoxyribonucleic acid
NDEA:          Nitrosodiethylamine
PB:            Phenobarbital
AFP:           Alpha-fetoprotein
[alpha]-2M:    Alph-2 macroglobulin
TNF[alpha]:    Tumor necrosis factor alpha
bw.:           Body weight
OLE:           Olive leaves extract
[gamma]-G.T.:  Gamma glutamyltransferase
AST:           Aspartate aminotransferase
ALT:           Alanine aminotransferase
ANOVA:         Analysis of variance
GSH:           Glutathione
ELISA:         Enzyme-linked immunosorbent assay

Table 1. Effect of olive leaf extracts on DNEA-induced alteration in
serum levels of some tumor markers.

 Groups               Control                    NDEA-
Parameters             group                     group

AFP              2.77ab [+ or -] 0.31      95.09c [+ or -] 5.21
[alpha]-2M     114.56ab [+ or -] 3.31     200.87c [+ or -] 4.78
TNF-[alpha]     41.72ab [+ or -] 1.05     126.62c [+ or -] 2.97

                               OLE-treated group
                            (During promotion period)

  Groups               OLE                     OLE+WBH

AFP              4.64b [+ or -] 0.50        3.71a [+ or -] 0.21
[alpha]-2M     130.23ab [+ or -] 2.22     122.11ab [+ or -] 3.73
TNF-[alpha]     59.35b [+ or -] 1.28       55.74b [+ or -] 1.69

                             OLE-treated group
  Group                  (After promotion period)
                         OLE                     OLE+WBH

AFP              7.86d [+ or -] 0.40        8.10d [+ or -] 0.28
[alpha]-2M     150.10d [+ or -] 0.89      155.12d [+ or -] 2.49
TNF-[alpha]     73.01d [+ or -] 1.85       80.47d [+ or -] 1.56

Values represent means [+ or -] S.E.

Values with same superscript in the raw are not statistically

Table 2. Effect of olive leaf extracts on DNEA-induced alteration in
some biochemical indices of rats.

      Group                 Control                   NDEA-
   Parameters                group                    group

[gamma]-GT (U/L)      5.22a [+ or -] 1.92      11.48b [+ or -] 0.51
AST (U/L)            40.26a [+ or -] 1.80     111.75b [+ or -] 2.6
ALT (U/L)            35.0 a  [+ or -] 0.9      97.84b [+ or -] 2.4
Albumin(g/dl)         4.41a [+ or -] 0.16       2.13b [+ or -] 0.16

      Group                        OLE-treated group
   Parameters                  (During promotion period)

                              OLE                    OLE+WBH

[gamma]-GT (U/L)      5.47a [+ or -] 0.16      5.60 a  [+ or -] 0.16
AST (U/L)            53.61a [+ or -] 1.91     57.65a [+ or -] 2.62
ALT (U/L)            52.08c [+ or -] 1.2      59.35c [+ or -] 0.69
Albumin(g/dl)         4.73 a [+ or -] 0.16     5.39 a [+ or -] 0.09

      Group                        OLE-treated group
   Parameters                  (After promotion period)

                              OLE                    OLE+WBH

[gamma]-GT (U/L)      6.13a [+ or -] 0.09      7.35d [+ or -] 0.25
AST (U/L)            71.94c [+ or -] 1.11     81.51d [+ or -] 1.17
ALT (U/L)            64.71d [+ or -] 1.73     74.03d [+ or -] 1.04
Albumin(g/dl)         3.14b [+ or -] 0.22      2.93c [+ or -] 0.13

Values represent means [+ or -] S.E.

Values with same superscript in the raw are not statistically
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