Tumor Suppressor p53 Gene Mutations Pattern Induced by Repeated Fried Palm Oil in Rats

1Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt 2Department of Biochemistry, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt 2Department of Chemistry, Al Jouf University, Al-Jawf, Saudi Arabia 3Department of Physiology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt 4Laboratory of Marine Biotechnology, Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima 890-0056, Japan


Introduction
In today's society with all its modern trappings, repeated frying with vegetable oils is a widely used procedure for food preparation (Bouchon, 2009;Shila et al., 2011). Several studies reported that using vegetable oils in prolonged frying processes may be a principle or strong reason for inducing or forming mutagenic or carcinogenic diets (Hamilton et al., 1997;Coultate, 2009;Gouveia De Souza et al., 2004;Paiva-Martins and Gordon, 2005;Khalil et al., 2009).
During the frying process at temperatures of 170°-200°C, steam formed from moisture in the food being fried help volatile products rise to the surface of the frying medium and into the kitchen atmosphere, imparting a mixture of fried-flavours and off-flavours. The non-volatile compounds formed, however, gradually build up in the oil as it is being repeatedly-used for food frying operations. These non-volatiles, primarily "Polar Compounds" (PC) and to a lesser extent lipid polymers, get absorbed into fried foods and eventually end up in our body system (Wai, 2007). The nutritional consequences of ingesting deep-fried oils include a variety of symptoms ranging from allergic reactions of digestive tract, growth retardation, increase in liver and kidney weights, to other biochemical reactions.
A series of Heterocyclic Aromatic Amines (HAAs) that have been found to be mutagens/carcinogens may arise in oil of fat during prolonged frying processes (Nagao et al., 1997;Raloff, 1999). The mutagenic and possibly carcinogenic products of HAAs are metabolized and activated by enzymes of cytochrome systems-mediated N hydroxylation to a number of hydroxylated metabolites which react with the DNA to induce mutations (Davis and Snyderwine, 1995;Schut et al., 1997).
Tumor suppressor genes are normal genes that slow down cell division, repair DNA mistakes, or tell cells when to die (a process known as apoptosis or programmed cell death). When tumor suppressor genes don't work properly, cells can grow out of control, which can lead to cancer. Many different tumor suppressor genes have been found, including TP53 (p53), BRCA1, BRCA2, APC and RB1 (ACS, 2011).
The p53 tumor suppressor gene is the most apparent among these because mutations have been demonstrated in a fraction of almost every tumor type tested. Most mutation detected was a single nucleotide mismatch Single Nucleotide Polymorphism (SNP) that occurred in the p53-gene coding region. P53 mutations indicate that the sites and features of DNA base changes differ among the various tumor types and depend on carcinogen type. Thus; the molecular epidemiology of p53 mutations allows the possibility of correlating particular mutations with specific carcinogens and establishing one step in the causal pathway between exposure to carcinogens and the development of cancer. Moreover, these carcinogens leave molecular fingerprints on the p53 gene (Nadia and Marwa, 2012).
Assessing p53 status may very well be beneficial in early detection and monitoring of tumor relapse, by detecting mutant p53 DNA. Furthermore, analysis of p53 status can serve as a tool in the prediction of effective therapeutic regimens, whereas p53 itself, particularly mutant p53, may represent targets for cancer therapy (Rivlin et al., 2011). Therefore, the present study focused on monitoring p53 gene mutations pattern in liver of mature male albino rats fed on basal diet containing thermally oxidized commercial palm oil repeatedly used for frying process (FO) in a dose of 150 mL kg −1 diet for one month.

Material and Method
Material Unpackaged palm oil was used in this experiment because it is less expensive and more available to consumers, especially in rural areas in Egypt. The oil and potatoes were obtained from local market in Ismailia governorate, Egypt.

Animals
Eighteen sexually mature male Albino rats (weighing approximately 150±10 g) were obtained from Faculty of Veterinary Medicine animal house and used throughout the experiment. Rats were housed in metal cages, fed on a standard diet and water ad libitum, maintained under a 12 h light/dark cycle, at a laboratory temperature of 23±3°C. Rats were acclimatized for two weeks before starting the experiments. All rats were handled in accordance with the standard guide for the care and use of laboratory animals. Killing was performed under sodium pentobarbital anesthesia and all efforts were made to minimize suffering.

Preparation of Frying Oil
For 2 liter palm oil, pan frying was performed in an uncovered stainless steel pan fryer, where about 400 g potatoes or tamea (Reciprocal) were divided into 2 batches and fried each time at 175±5°C. The frying processes were repeated for 15 times (12.3 min each) twice daily for 8 successive days. No replenishment of oil was considered and oil was taken out at the end of the experiment, filtered and placed in a bottle in the refrigerator (4°C), then thoroughly mixed with the basal diet.

Experimental Design
The animals were divided randomly into three groups, 6 animals each, namely: Group 1, animals were orally administered by distilled water and considered as Control (CO). Group 2, was fed basal diet containing fresh pure palm oil (NO) that was not used in frying process. Group 3, was fed the basal diet containing thermally oxidized palm oil repeatedly used for frying process (FO) with a dose of 150 mL kg −1 diet (Ahmed and Sharma, 1997). All rat groups were fed the corresponding previously mentioned diets for one month.
At the end of the experiment, all animals were fasted for 12 h then killed under sodium pentobarbital anesthesia. Liver tissue samples were taken, snap frozen in liquid nitrogen at -196°C, kept at -80°C for genetic alteration studies.

Total RNA Extraction
Total RNA was extracted from liver samples using Quick Gene RNA tissue kit S II (RT-S2) (Life Sciences Advanced Technologies Inc.) according to the manufacture's protocol. Total RNA concentration and purity were determined spectrophotometrically as described by Sambrook and Russel (2001) and A260/A280 ratio was between 1.7 and 1.9.

Reverse Transcriptase-Assisted Polymerase Chain Reaction
Reverse transcription of total RNA was performed with Superscript II reverse transcriptase (Gibco BRL, USA) to generate 3' -RACE-Ready first strand cDNA using a SMART TM RACE cDNA amplification kit (Clontech, USA) according to the manufacture's protocol. Oligonucleotide Primers and PCR Amplification of p53 Gene Two degenerate primers, one sense (F) and one antisense (R) were designed by web-based software Primer3Plus (Untergasser et al., 2007)

PCR and Agarose Gel Electrophoresis
PCR reactions contained 3' -RACE-Ready cDNA (2.5 µL), 20 µM each of degenerate primers, 25 µL of SapphireAmp Fast PCR Master Mix (Takara, Japan) in a final volume of 50 µL. The cycling conditions were as follows: 35 cycles of denaturation at 94°C for 30 sec, annealing at 68°C for 30 sec and extension at 72°C for 3 min. PCR products were analyzed on 1% agarose gels. The DNA band of expected size was excised with a scalpel, purified using GFX PCR DNA and a gel band purification kit (GE Health Care, UK), the PCR products obtained were cloned into PT7BlueT-vector (Novagen, USA). Purified plasmids were directly sequenced by dye terminator cycle sequencing using an ABI PRISM dye terminator cycle sequencing kit (PE Biosystemes, USA) and an applied Biosystems 3130×L DNA sequencer.

Phylogenetic Analysis
The resulted nucleotide sequences were aligned with the corresponding functional p53 sequences available in GenBank. DNA sequences with the following Genbank accession numbers were retrieved from the database and used in the phylogenetic analysis: NM_ 030989.3 (Rat p53), AB020317 (Mouse p53), AF307851 (Human p53), sequence alignment was performed by the CLUSTAL W method using Laser gene Megalign program (Ver 5.52,2003, DNASTAR Inc).

Partial Nucleotide Sequence Analysis of P53 cDNA
A nucleotide sequence of 799 bp of the Open Reading Frame (ORF) of p53 cDNA was amplified using the sense and antisense primers in Control (CO), Normal Oil (NO) and Fired Oil (FO) groups respectively ( Fig. 1-3). The amplified region corresponding to the p53 cDNA gene codon regions 104-370 (exons 4-9). Phylogenetic analysis (Fig. 4) clearly shows that the resulted sequences are closely related to functional rat p53, Rattus norvegicus, tumor protein p53 (Tp53) sequence.  Table 2 showing the percent identities of the resulted sequences with the sequence of p53 cDNA in other species. P53 cDNA sequence from the control group and group fed on normal oil shows the highest identity (100%) with rat p53, followed by 91.7% with mouse p53 then ended by 85.4% with human p53. The percent identity between p53 cDNA sequence in control group and group fed on repeated fried oil was 91.6%.

Detection of p53 Sequence Mutations
Sequence alignment of control, Normal oil and fried oil groups p53 sequences by the CLUSTAL W method using Laser gene Megalign program (Ver 5.52,2003, DNASTAR Inc) (Fig. 5) shows presence of fifty eight base-pair substitutions mutations. A total of twelve base pair substitutions arose at A:T base pairs (five A:T→T:A, five A:T →G:C and two A:T → C:G). However, there were a total of fourteen base pair substitutions arose at G:C base pairs (three G:C→ T:A, eight G:C → A:T and three G:C → C:G). At T: A base pairs, there were a total of eighteen base pair substitutions include (fourteen T: A→ C:G and four T:A→ G:C). Finally, A total of fourteen base pair substitutions arose at C: G base pairs (eight C: G→T: A, four C: G→G:C and two C:G→A:T). Of the fifity eight substitutional mutations recorded, there were thirty one silent (same sense) mutations and twenty seven missense mutations which causing the substitution of amino acids (Fig. 6).

Discussion
As social economic and cultural conditions have changed over the twentieth century, significant alteration occurred in eating habits, including eating outside home and especially in fast food restaurants, which lead to increased consumption of oils used in deep fat frying (Weisburger, 2002).
Carcinogenesis is a complex process characterized by the cumulative activation of various oncogenes and the inactivation of suppressor genes. About 30-40% of human hepatocarcinomas and 20-60% of rat experimental tumors demonstrated mutations of p53 gene (Vancutsem et al., 1994;Masui et al., 1997;De Miglio et al., 2001). Interestingly, the spectrum of p53 mutations can provide clues to the etiology and molecular pathogenesis of neoplasia. The frequency and type of mutation can also be considered as a molecular dosimeter of carcinogen exposure and thus can provide information for cancer risk assessment. Chemically induced rat liver cancer proceeds through multiple, distinct initiation-promotion-progression stages and mutation of the suppressor p53 gene has been found in relatively early pre-neoplastic lesions in the rat liver. Therefore, mutant p53 molecules have been thought to have some unique properties that are important in carcinogenesis in rats (Haas and Pitot, 1998).
In this study, the analyses of p53 gene mutations induced by repeated fried oil in rat liver were confined to exons 4-9, which include the highly conserved domains of the gene and are the sites at which mutations in human and experimental liver tumors have been most frequently detected (Hollstein et al., 1991;Smith et al., 1993;Vancutsem et al., 1994). The findings of this study show that p53 mutations are a frequent event in the liver of rats fed on repeated fried oil. Our results are in an agreement with that of Mariam et al. (2010) which indicated that fried oil food has a mutagenic effect on the genomic materials of male mice. This mutagenic effect may be due to the formation of oxidative compounds of lipidperoxid or Heterocyclic Aromatic Amines (HAAs), that have been found to be mutagenic or carcinogenic (Hamilton et al., 1997;Starvic et al., 1997;Raloff, 1999;Coultate, 2009;Bou et al., 2008). Lipidperoxid compounds may be associated with generation of free radicals causing DNA fragmentation or DNA mutation (Lampe, 1999;Coultate, 2009;Nicolle et al., 2003;Bou et al., 2008).
The second major finding of this study is that the majority of mutations were missense mutations 12 of which arose at A:T base pairs, 14 at G:C, 18 at T:A and 14 of which were C:G base pairs transversions (Fig. 5). These data in rats clearly indicate a specific mutation spectrum in liver associated with repeated fried oil. The vast majority of cancer-associated mutations inTP53 are missense mutations. The great majority of these missense mutations are clustered within the central most conserved region of p53 that spans the DNA-binding domain and among these area small number (approximately six) of ''hot spot'' residues that occur with unusually high frequency (Harris and Hollstein, 1993;Cho et al., 1994;Petitjean et al., 2007).
In most cases, the p53 gene is mutated; giving rise to a stable mutant protein whose accumulation is regarded as a hallmark of cancer cells. Mutant p53 proteins not only lose their tumor suppressive activities but often gain additional oncogenic functions that endow cells with growth and survival advantages. Interestingly, mutations in the p53 gene were shown to occur at different phases of the multistep process of malignant transformation, thus contributing differentially to tumor initiation, promotion, aggressiveness and metastasis (Rivlin et al., 2011).

Conclusion
In conclusion, Assessing p53 status may very well be beneficial in early detection and monitoring of tumor relapse, by detecting mutant p53 DNA. Furthermore, analysis of p53 status can serve as a tool in the prediction of effective therapeutic regimens, whereas p53 itself, particularly mutant p53, may represent targets for cancer therapy (Rivlin et al., 2011).

Funding Information
This research received no grant from any funding agency in the public, commercial or not-for-profit sectors.

Author's Contributions
Abeer A.I. Hassanin: Designed the research plan and organized the study, have made a substantial contribution to the concept and design, acquisition of data or analysis and interpretation of data, approved the version to be published.
Abeer G.A. Hassan: Designed the research plan and organized the study have made a substantial contribution to the concept and design, acquisition of data or analysis and interpretation of data, coordinated the mouse work.
Saadia, A. Ali: Have made a substantial contribution to the concept and design, acquisition of data or analysis and interpretation of data.
Takao Itakura: Drafted the article or revised it critically for important intellectual content.

Ethics
All rats were handled in accordance with the standard guide for the care and use of laboratory animals. Killing was performed under sodium pentobarbital anesthesia and all efforts were made to minimize suffering.