At high radiation doses, significant effects can occur in exposed individuals within a short time of exposure, and in severe cases this can lead to early death. At low radiation doses, the principal concern is the risk of radiation-induced cancer in exposed individuals and hereditary disease in their descendants. The risks of these late effects have been quantified and this provides the basis for recommendations on limits for exposure. Free radicals have been implicated in the pathogenesis of many degenerative disorders, including atherosclerosis, cancer and diabetes [1]. When cells are exposed to ionizing radiation the standard physical effects between radiation and the atoms or molecules of the cells occur first and the possible biological damage to cell functions follows later [2]. The biological effects of radiation result mainly from damage to the DNA, which is the most critical target within the cell; however, there are also other sites in the cell that, when damaged, may lead to cell death when directly ionizing radiation are absorbed in biological material [3]. Radiation interacts with other molecules and atoms (mainly water, since about 80% of a cell is composed of water) within the cell to produce free radicals, which can, through diffusion in the cell, damage the critical target within the cell. In interactions of radiation with water, short lived yet extremely reactive free radicals such as H₂O⁺ (water ion) and OH• (hydroxyl radical) are produced. The free radicals in turn can cause damage to the target within the cell. The free radicals that break the chemical bonds and produce chemical changes that lead to biological damage are highly reactive molecules because they have an unpaired valence electron. Free radicals were needed to produce energy and various substances that the body requires. If there is excessive free radical formation, damage to cells and tissues can occur [4]. The formation of a large number of free radicals stimulates the formation of more free radicals, leading to even more damage. Antioxidants are substances that may protect cells from the damage caused by unstable molecules known as free radicals. Free radical damage may lead to cancer. Antioxidants interact with and stabilize free radicals and may prevent some of the damage free radicals might otherwise cause [5]. Reactive oxygen species are involved in several disorders. The harmful action of the free radicals however is blocked by antioxidant substances which scavenge the free radicals and detoxify the organism. Current research into free radicals has confirmed that foods rich in antioxidants play an essential role in the prevention of cardiovascular diseases and cancers [6]. Radiation exposure alters the balance of endogenous defense system through generation of ROS resulting in an imbalance in prooxidant, antioxidant status in the cell [7,8]. Oxidative stress leads to both DNA damage and cell death [9]. Appropriate antioxidant intervention seems to inhibit or reduce free radical toxicity and offer protection against radiation damage. A number of dietary antioxidants have been reported to decrease free radical attack on biomolecules [10]. Lycopene is a dietary carotenoid synthesized by plants and microorganisms. It occurs primarily in red fruits, vegetables, tomatoes, watermelon, pink grape fruit, apricots, pink guava and papaya [11,12]. Tomato (Lycopersicon esculentum) is one of the most popular natural antioxidant that had a potent effect on scavenging free radicals comes from different toxins. The regular intake of tomatoes or its products has been associated with a reduced risk of chronic diseases and these effects have been mainly attributed to Lycopene [13,14]. Tomato Lycopene content varies considerably, reflecting the influence of variety (generally genetic factors), maturity, and both agronomic and environmental conditions during growing. Lycopene has been under considerable investigation for its antioxidant benefits in treating various chronic human diseases like cancer, liver diseases, osteoporosis and diabetes [15]. Tomato extract, partially protected against acute liver injury due to chemically-induced oxidant stress [16]. Lycopene found to suppress liver cancer remarkable 50% hepatocellular carcinoma (liver cancer) suppression in those participants

who consumed a daily combination of natural tomato extract [17]. Lycopene exhibited local anti inflammatory activity and also attenuated liver injury induced by I/R. Lycopene administration might be useful in the pharmacological modulation of inflammatory events [18]. Lycopene supplementation significantly reduced radiotherapy-induced oxidative liver injury [19]. Our goal of this study is to ameliorate damage induced by radiation using natural antioxidant that are available to most of the people, like Lycopene that found in tomato, which is a relatively new carotenoid known to play an important role in human health and disease.

The present study was done on healthy adult male albino rats in the weight range from (150-200 gm), selected from an inbred group housed in specially designed cages and maintained under standard conditions of light and temperature All animals were cared for according to the Guiding Principle in the Care and Use of Animals. They were divided into groups each of 10 rats on the basis of initial weight and kept in individual cages.

Group 1: considered normal Control.

Group 2: considered irradiated animals exposed to Fractionated doses of gamma radiation, (1/2Gy) every two days for one month.

Group 3: considered control animals delivered Lycopene alone orally at a dose of (5 mg/kg per day) for Two months Group 4: considered irradiated animals treated with Lycopene for 2 months. Fractionated doses were delivered simultaneously with Lycopene for only one month.

Natural antioxidants

Lycopene Extract Liquid Alcohol Free was purchased from Sigma Chemical Co. and used at dose of (5 mg/kg per day).

Radiation facility

Fractionated dose of gamma irradiation was performed at the National Centre for Radiation Research and Technology, Atomic Energy Authority (NCRRT), Cairo, Egypt, using Cesium -137 in a Gamma cell40 Irradiator (Atomic Energy of Canada Limited, Canada). Animals were exposed to fractionated dose of γ-radiation, 0.5 Gy every 2 days, at a dose rate of 0.61 Gy min-1 for one month

Light microscopy

Histological changes of liver by light microscopy were studied by using paraffin method as described earlier [20]. Liver tissue was cut into pieces of desired size and fixed into Bouin’s fixative (saturated picric acid 75 ml + conc. Formalin 25ml + glacial acetic acid 5 ml) for 24-48 hrs. Tissues were removed from fixative solution and washed thoroughly with distilled water for few hours so as to remove extra Bouin’s fixative. After fixation, samples were washed with 70% alcohol to remove excess of picric acid from the tissues and dehydrated in graded series of ethanol. Liver biopsies were dehydrated in ascending grades of molten paraplast 58-62C. Three μm thick histological sections were cut and stained with Hematoxylin and eosin and examined under bright field light microscope.

Transmission electron microscopy

Liver biopsy were removed from the animals instantaneously and killed by decapitation, sliced into one mm pieces in a drop of 3 % glutareldehyde. Tissue was then immersed in fresh ice cold fixative for two hours and then in 0.1 M cacodylate buffer for next 4 h. The tissues was then rinsed briefly in buffer and post osmicated in 1% osmic acid for one to two hours. Liver was dehydrated in an ascending series of alcohol, followed by propylene oxide and finally embedded in resin that was polymerized at 600°C. Subsequently the blocks were prepared in araldite and 1 m sections were cut with a glass knife on LKB-2000S, ultra microtome mounted on glass slides and stained with buffered toluidine blue. Appropriate areas were selected with the light microscope. Finally, ultra thin sections of selected blocks were cut with a diamond knife, picked up on copper grids and stained with uranil acetate and lead citrate for final viewing.

Light microscopy

Light microscopic study declared that animals delivered accumulated dose of 0.5 gray gamma radiation every two days for one month up to 7.50 gray which is sub lethal dose caused severe changes in the liver cells, Some sections showed that the cell come to be Pre cancerous stage on the other hand the supplementations of Lycopene showed a trend toward lower incidence of hepatic pathological changes induced by radiation. This effect did not reach statistical significance amelioration but it come to the level of helping cells to regenerate and overcome pathological changes. This can be explained in several examined sections from the different groups of the experiment. Examined sections of (Group 1) which was normal control showed normal pattern of cellular structures (Figure 1), meanwhile those examined from group 2 which was exposed to gamma radiation showed sever disorganization cytoplasmic disintegration and gradual vacuolization, area of necroses, pyknosis, fatty change and inflammation (Figure 2 and 3). Atrophy, hemorrhage, apoptosis, pyknosis and hyperplasia are common feature found in (Figure 4 and 5). Examined sections from (Group 3) which delivered Lycopene alone had no remarkable changes (Figure 6). A remarkable restoration in cell structure were recognized in (Group 4) which were irradiated and treated with Lycopene , (Figure 7a and 7b) showed healthy well designed cellular structure with normal central rounded nucleuses, and homogeneous cytoplasm.

Figure 1: Light micrograph of a section from normal rat liver (Group1) showing radial organization of hepatocytes and sinusoids (S) around the central vein (CV).

Figure 2: light micrograph of a section from rat liver exposed to accumulated dose of gamma radiation (Group 2) presented gradual vacuolization (arrows), cytoplasmic disintegration, pyknotic nuclei (PK) hepatic necrosis (Ne), inflammation and fatty accumulation.

Figure 3: Light micrograph of a section rat liver exposed to accumulated dose of gamma radiation (Group2) presented, Area of necrosis (Ne).

Figure 4: Light micrograph of a section of rat liver exposed to accumulated dose of gamma radiation (Group 2) presented hemorrhage (HR) , globular red hyaline (Rh), Atrophy (AT) and Hyperplasia (Hp).

Figure 5: Light micrograph of a section rat liver exposed to accumulated dose of gamma radiation (Group 2) presented Pyknosis (PK) and congestion of the portal track (PT).

Figure 6: Light micrograph of a section of rat liver cell given Lycopene alone (5 mg/kg per day) (Group 3) showing no restricted cellular changes.

Figure 7: a,b: Light micrograph of a section of gamma irradiated rat liver given Lycopene (5 mg/kg per day) (Group 4) showing great amelioration and recovery of the hepatic cells . Lipid and fatty features are still recorded.

Electron microscopic investigation

Fine structure examination of Group1 which was normal control showed normal pattern of cellular structures like healthy nucleus, homogeneous cytoplasm with well developed mitochondria, rough endoplasmic reticulum and glycogen particles (Figure 8 and 9). Fine structure examination of liver cells from (Group 2) reveled that was exposed to accumulated dose of gamma radiation revealed that exposure to accumulated dose caused an obvious injuries to the cell fine structure. The cytoplasm was ill defined and loses its normal differentiation with necrotic areas and Lipid vacuoles which was many and varied in shape (lipoma) (Figure 11 and 12). Also, there were a conspicuous disorganized features found in nucleus including morphological alterations, irregular and dilated envelope. The heterochromatin was altered. An obvious clumping of heterochromatin which is peripherally and randomly distributed within the nucleus was detected. Amorphous polymorphic mitochondria were found to be malformed, in some hepatocytes; the outer mitochondrial membrane appeared to be degenerated (Figure 10). Rough endoplasmic reticulum was seen as elongated rupture and fragmented with detachment of ribosome. Collagen fiber was also increased as a feature of liver fibroses (Figure 13). Liver sections of Lycopene delivered animals (Group 3) showed no remarkable changes (Figure 14 and 15). Electron micrograph of a liver cell from (Group 4) which was irradiated and treated by Lycopene showing that most of the cells had an ameliorant sign of the cell organoids, the cytoplasm is homogeneous and granulated with well designed strands of rough endoplasmic reticulum (RER) with ribosome. The nucleus retains its normality, semi-rounded and containing permanent, heterochromatin tightly packed form of DNA (Figure 16 and 17). The ER was well organized with ribosomes (Figure 18). On the other hand further examined sections revealed that lipid droplets still recognized in the cell but not as in the treated sections some sections shows that the cytoplasm still vacuolated (Figures 19 and 20).

Figure 8: An electron micrograph of a section of normal control liver rat (Group1) showing normal structure illustrated in healthy nucleus (N), homogeneous cytoplasm (Cy) with well developed mitochondria (M) and rough endoplasmic reticulum (RER).

Figure 9: An electron micrograph of a section of normal control rat liver (Group1) showing parallel strands of rough endoplasmic reticulum (RER) with ribosome. Rosette form of glycogen particles (white stars).

Figure 10: An electron micrograph of a section of irradiated rat liver (Group 2) showing markedly altered morphology. The nucleus showed irregular nucleus (N) and reduced number of mitochondria (M), destructive (RER), lipid droplets (LD).

Figure 11: An electron micrograph of a section of irradiated rat liver (Group 2). Note, lipoma can be restricted in a big lipid droplet (BLD).

Figure 12: An electron micrograph of a section of irradiated rat liver (Group 2) showing hemorrhage (HR), increased amount of collagen fiber (CF) and malformated mitochondria (M).

Figure 13: An electron micrograph of a section of irradiated rat liver (Group2) showing several striking alterations of mitochondrial ultra structure.

Figure 14: An electron micrograph of a section of rat liver given Lycopene alone (5 mg/kg per day) (Group 3) for Two months showing no cellular changes, the Mitochondria (M) and the rough endoplasmic reticulum (RER) showed no cellular changes.

Figure 15: An electron micrograph of a section of rat liver given Lycopene alone (5 mg/kg per day) (Group3) for Two months, Note classic cell structure with healthy nucleus (N), and endoplasmic reticulum with ribosome attached (RER).

Figure 16: An electron micrograph of a section of liver from Group 4 which was treated with Lycopene and radiation. A significant regression in the rate of the degenerative changes was found in (Group 3).

Figure 17: An electron micrograph of a section of liver from Group 4 which was treated with Lycopene and radiation showing cytoplasm with normal patches of smooth and rough endoplasmic reticulum and ribosome’s inside. The nucleus (N) looks more healthy with euchromatin.

Figure 18: An electron micrograph of a section of liver from Group 4 which was treated with Lycopene and radiation showing nearly normal structure.

Figure 19: An electron micrograph of a section of liver from Group 4 which was treated with Lycopene and radiation Note that lipid droplets still found in abundant (Lp).

Figure 20: Higher magnification of Figure 19. Note increased amount of lipid droplets.

Ionizing radiation is known to induce oxidative stress through generation of ROS resulting in an imbalance in prooxidant [22]. When radiation is absorbed by a living cell, the primary damage is caused by ionization and excitation of the atoms and molecules of that cell. These interactions occur with any type of matter, whether living or nonliving; but in a living cell, the ionized or excited atoms and molecules may be highly reactive chemically. Under these circumstances, secondary reactions will occur, resulting in changes in cellular structure, damage to essential constituents, and observable biological injury [23]. In general, free radicals are very short lived, with half-lives in milli, microor nanoseconds. Details about some of the biologically important reactive species are presented as Table 1. The present results showed much cellular destruction. Light microscopic observations showed that there were sever pathological changes. The cytoplasm was disorganized. Hyperplasia and pyknotic nuclei were common this may be due to that radiation exposure alters the balance of endogenous defense system [24]. Radiation is a well-known inducer of free radicals caused to chromosomal damages.

Table 1: Reactive oxygen species of biological interest.

The use of antioxidants may help to decreasing of the genotoxicity created by radiation and may inhibit mutagenesis and carcinogenesis [25]. Exposure to Gamma-ray irradiation inducing organelle dysfunction induced cytochrome P4502E1 (CYP2E1) in the liver, which might be associated with mitochondrial damage [26]. On the other hand further studies showed that there pathological ultra structure changes were found in liver sections from animals exposed to sublethal dose of gamma radiation illustrated in degeneration of the cytoplasm, the mitochondrian were mal formatted and shrinked with destruction of its cristae. The cristae were disappeared and reduced to fin pouch –like structure this depletion may be due to that radiation affect mitochondrial protein complexes. Pyknosis and disorganization of the endoplasmic reticulum was also recognized [27]. These finding confirm our results which revealed an obvious injuries to the cell fine structure. The cytoplasm was ill defined and lost its normal structure. Necrotic areas, conspicuous disorganization of the nucleus including morphological alterations, irregular and dilated envelope were also detected. This is may be due to ionizing radiation is known to induce oxidative stress through generation of ROS resulting in an imbalance in prooxidant, antioxidant status in the cells [28]. The degree of protection against gamma radiation injuries depends on many complex factors such as dosage, when the antioxidant is taken, and the, bio availability, retention and radical-quenching profiles of the particular antioxidant in the particular organ or cell involved. Factors of importance include water and fat solubility of the antioxidant, whether it can penetrate into cell mitochondria [29]. Lycopene belongs to the class of compounds known as carotenoids and can be ingested by people as a components of certain foods, most notably tomatoes), as well as via supplements [30]. Lycopene has been found to inhibit proliferation of several types of cancer cells, including those of breast, lung, and endometrial [31]. Epidemiological studies have suggested that regular consumption of tomatoes, the most common source of lycopene in human diets is associated with a reduced risk of certain forms of cancer and other diseases. This postulated protective effect of Lycopene is thought to be due to the various antioxidant properties of this compound [32]. The present study aimed to evaluate the radio protective effect of Lycopene, a naturally occurring dietary carotenoid on γ-irradiation-induced cellular changes. Oxidative stress is an important contributor to the risk of chronic diseases that damages proteins, lipids and nucleic acids. Because of the lipid component in the membrane, lipid peroxidation is reported to be particularly susceptible to radiation damage [33]. Antioxidants scavenge free radicals, otherwise known as reactive oxygen species (ROS), and prevent the damage they can cause. Free radicals have been associated with pathogenesis of various disorders and diseases such as cancer, cardiovascular disease, osteoporosis, diabetes, and cataracts. In one study, Lycopene significantly restored the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione reductase (GR); reduced glutathione (GSH); and decreased levels of the lipid peroxide malondialdehyde (MDA) in hypertensive patients [34]. In another study, Lycopene was found to have a favorable effect in reducing MDA levels and increasing GSH levels in coronary artery disease in postmenopausal women [35]. Our results showed that treatment with Lycopene offers protection atgainst Gama irradiation induced cellular damage, great amelioration in the structure of the cellular organoids. The cytoplasm retained its granulation. The ER gained its normal pattern with restoration of ribosomes. The nucleus, mitochondria get its normality [36,37]. Pretreatment with Lycopene offers protection to normal lymphocytes against γ-radiation-induced cellular damage due to free reaction inside the cell [34]. Antioxidants act as scavengers by neutralizing the damage caused by free radicals to our cells and tissues. They have been found to be effective against oxidation by atmospheric oxygen (auto oxidation) [38]. Other interesting finding in this study was that lipid droplets still found in Lycopene treated animals in a considerable averages compared to gamma irradiated rats [39]. This is may be due to the antioxidant sparing action of Lycopene in decreasing in lipid peroxidation and improved antioxidant status preventing the damage to the cell [34].

Lycopene may have a considerable therapeutic potential as an antioxidant. A significant regression in the rate of the degenerative cellular changes found, in both light and electron microscope examination can be outlined. On the other hand lipid droplets still found in abundant this finding suggest that tomato but may not be used as a hypo lipidaemic.