Epigenetic ( protein ) patteren of albino rat Rattus norvegicus after treatment with homocysteine and trimethylglycine

Hyperhomocysteineamia (hHcys) is thought to be a risk factor for cardiovascular disease. The epigenetic (protein) pattern due to the effect of hHcys in kidney and liver was profiled in the supplemented developing rats. A group treated with 0.6 % Hcys for 8 weeks and trimethylglycine (Tri or Betaine) antioxidant treated group (6 g / Kg) were compared with four weeks age control group. The results indicated that, a great variety in protein pattern was obtained. Specific type of protein with M.W. of 214.29 K. d. was produced in the kidney samples treated with Trimethylglycine. Common types of protein with M.W. of 278.64, 157.07, 52.39, 39.31, 29.24, 24.49, 20.86, 20 and 18.87 K. d. were obtained in the same sample. Characteristic band with 59.58 K. d. was obtained in control the kidney after 8 weeks while three types of protein were obtained in the control liver with M.W. of 221.4, 28.25 and 18.07 K.d. The highest similarity index was between control and Trimethylglycine treated kidney group (0.76), but the lowest one was between the control and liver trimethylglycine treated group (0.38).


INTRODUCTION
Hyperhomocysteineamia (hHcys) has been indicated as an independent risk factor causing a variety of pathological changes in different cells or tissues.Homocysteine (Hcys) is produced from S-adenosylhomocysteine (SAH) through the catalysis of (SAH) hydrolase, (Li et al., 2006).Hcys is derived from the essential amino acid methionine, during normal condition, excess Hcys is converted back to methionine or broken down be excreted (Stead et al., 2000).While the remethylation process of Hcys was 5-methyltetrahydrofolate and the activity of betaine homocystein S-methyltransferase (BHMT) as the methyl donor (Finkelstein et al., 1971;Castro et al., 2002 andFinkelstein, 2007).In rat, the kidney is a major site for the removal of plasma Hcys House et. al, 1997B & 1999.According to Stead et al., 2000, the liver is a key organ of Hcys metabolism and potentially control the plasma Hcys level.Betaine was involved in Hcys metabolism as an alternative methyl donor, it is used in the treatment of homocystineuria in human as revealed by Schwab et al., 2002.Betaine is found naturally in most living organisms, it protects plants (Sakamoto and Murata, 2000), microbes (Rozwadowski et al., 1991), marine and freshwater invertebrates (Konosu and Hayashi 1975) against osmotic stress and acts as an osmolyte in mammalian tissues as concluded by Garcia-Perez and Burg, 1991 andBurg, 1997.Betaine is formed in cells as an oxidation product of choline and can be obtained externally from food, (McCue and Hanson, 1992).Several nutrients are including folate, vitamin B-12 and vitamin B-6 influence the metabolism of Hcys, (Selhub and Miller, 1992;Cuskelly et al., 2001 andHu, 2002).The objective of this study is the revealence of the protein pattern after different treatments to asses the physiological state of rat as a response to this treatment.To achieve this purpose vertical slab electrophoresis process was performed and Polyacrylamide gel electrophoresis (PAGE) was used.

MATERIAL AND METHODS
The used albino rats Rattus norvegicus (4 weeks age and 50 -60 g in weight) were adapted to the laboratory conditions for 7 days before the study.Animals were divided into 4 groups, group I was control, group II was treated with Trimethylglycine (6 g / Kg) orally for 2 months (Wilcken et al., 1985 andSchwab et al., 2002), group III received Homocysteine (Hcys) at the dose of (0.6 %) orally for 2 months, (Masse et al., 2003) and group IV received both Homocysteine and Trimethylglycine (Hycs + Tri) orally for 2 months.Animal's autopsy took place after 2 and 8 weeks.Kidney and liver organs were prepared to biochemical studies.Proteins were separated through 8% Polyacrylamide gel electrophoresis (PAGE) according to method of (Davis, 1964).Electrode and gel buffer and polyacrylamide stocke were prepared according (Laemmli, 1970).
Staining solution (commsie brilliant blue) was used for 12-18 hr and destaining by destain solution.The gel were photographed, scanned and analyzed using Gel Doc 2000 Bio Red system.

Calculations and data analysis
The similarity coefficient and genetic distance were calculated according to (Nei and Li,1979).As following :-S.I = 2Nxy/ (Nx + Ny) G. d = 1 -S.I Where: S = similarity value, Nx and Ny are the number of bands in individuals x and y, N is the number of shared bands.The value produced by this index ranges from zero, representing no bands sharing, to (1), representing complete identity, while G. d is the genetic distance value.

RESULTS
There is no different between protein of treated and control samples as a native technique.Sodium deudosyle sulphate polyacrylamide gel electrophoresis (SDS PAGE) showed highly variation in kidney protein pattern.The tabulated data in table (1A) and fig.
No characteristic bands for the control, while the bands no. 3, 8, 9, 17 and 19 with Rf value 0.091, 0.30, 0.33, 0.65 and 0.69 and M.W. 214.29, 69.5, 66.5, 34 and 31.75 K.d. were characteristic bands for the Tri treated animals.Band no.18 appeared only in Hcys and Tri + Hcys treated animals with Rf value 0.66.Densitometric scanning of electrophoresis showed that, the 7 th band in Tri + Hcys treated group had the highest density than the others.The quantitative mutation was recorded according to band % related to control samples.The highest quantitative mutation was recorded in band no. 15 in Tri + Hcys samples with M.W. 39.75 K.d.equal 42.6 times of control.In table (1B) and fig.
(1), the total number of bands in treated animals after 8 weeks were 14, 22 and 22 for Tri, Hcys and Tri + Hcys respectively.Bands no. 1, 4, Epigenetic patteren of albino rat after treatment with Homocysteine and Trimethylglycine.169 11, 15, 19, 21, 24, 25 and 26 were common bands in all treated animal and control.Bands no. 10 and 16 with M.W. 59.583 and 35.25 K.d.specific to the control.Band no. 5 with M.W. 110 K.d.specific to Tri treated animals and recorded high denisty, while band no. 2 with M.W. 242.86 K.d specific to Tri + Hcys treated animals.There was a quantitative mutation recorded in band no.11 in Hcys treated group with M.W. 52.66 K.d.reached to 11.5 times if compared to control.The similarity index (S.I.) and genetic distance (G.D.) at the epigenetic level were recorded between control and other treated animals, table (1C).The highest value of 1 was found between Tri + Hcys and Hcys group, while the lowest value was 0.54 found between Tri and control group.The highest G.d. value of 0.46 was found between control and Tri group, while the lowest value of 0 was found between Hcys and Tri + Hcys group.From data in table (1D), the highest S.I. of 0.95 was found between Tri + Hcys and Hcys group, while the lowest value of 0.54 was found between Tri + Hcys and control group.The highest G.d. value of 0.46 was found between control and Tri + Hcys group, while the lowest value of 0.24 was found between control and Tri group.(2), summarized the data collected from liver electrophoresis of all groups.In table (2A), six types of protein from each group under study were produced except for control, it was 11 bands.There were 4 common bands were no.3, 8, 12 and 13 with average M.W. 138, 44.22, 25 and 17.49 K.d for control group.In control group , there were 6 characteristic bands, were appeared their no.were 4, 5, 6, 9, 10 and 11, while band no 7 with M.W. 50 K.d was disappeared.Band no. 1 was a common band between Hcys and Tri + Hcys treated groups with Rf value 0.049 for both.Also band no. 2 was a common band between control group and Tri treated group with Rf value 0.053 for both.Densitometric scanning of electrophoresis showed that bands no. 3 and 8 in all three treared groups had higher density than others, table (2A).It is good to mention that, protein with M.W. 17.077 in control samples has a small band % (0.3) compared to Tri samples (14.8), i.e 49.3 times as compared with control samples.
In table (2B), the total number of protein types in control group bands, Tri treated group, Hcys treated group and Tri + Hcys treated group were 11, 14, 12 and 12 respectively.Bands no. 3, 5, 6, 7, 10, 11, 12 and 15 with average M.W. 140, 85.32, 75.02, 56.43, 44.06, 36.43, 32.52 and 23.29 K.d were common bands for all 4 groups.There was one characteristic band for control group, it was no. 2 with M.W. 221.43 K.d.The rest treated groups agree with each other in all bands, except Tri treated group which contained 2 characteristic bands, their number were 1 and 9 with M.W 235.71 and 49.474 K.d respectively.Densitomeric scanning of electrophorogram showed that band no. 12 in Hcys treated group and band no. 15 in both Tri treated group and Tri + Hcys treated group had higher density than other bands.The highest quantitative mutation recorded in band no. 10 in Hcys samples, it equal 6.7 times if compared to control samples.

DISCUSSION
Nine new types of protein with average M. W. 106.11, 93.33, 76.43, 50.13, 46.4, 44.6, 27.82, 23.39 and 22.35 K.d were produced in the treated groups, as in table (1A) and three new types of protein with average M.W. 34.5, 33.33 and 22.35 K.d were produced in treated groups, as in table (1B), which mean the activation of some genes to produce these types of protein (Holdane, 1937).
There were six types of protein with M. W. 85, 69.09, 54.848, 35.83, 33.05 and 28.25 K.d, as showed in table 2A, and three types of protein with M. W. 221.43, 28.25 and 18.07 K.d, (table 2B), were disappeared from all treated groups, this mean the inhibition of their genes to produce these types of protein.While there was one protein type with M.W. 50 K.d, (table 2A), and four types of protein with average M. W. 126.19, 50.90, 29.16 and 14.16 K.d were appeared in treated groups, which indicated that the activation of their genes to produce these types of protein.hHcys is very dangerous for the body and toxic for endothelium , it enhanced vascular smooth muscle cell proliferation, (Satta et al., 2006).In the present study the protein electrophoresis revealed differences between control and the treated groups and also between treated groups themselves.This is due to the production or the disappearance of different types of proteins.Finkelstein et al., 1971;Breska and Garrow 1999;Castro et al., 2002 andYap 2005, indicated that, the Betaine -homocysteine -S -methyltransferase 1 (BHMT 1) protein, with M.W. 44.9 K.d and the Betaine -homocysteine -S -methyltransferase 2 (BHMT 2) protein with M.W. 39.9 K.d were involved in the regulation of Hcys metabolism.Betaine and Hcys were converted to dimethylglycine and methionine respectively.This reaction is also required for the irreversible oxidation of choline.These two proteins were existed already in this study with M.W. 44.6 K.d in all treated groups, which represented to (BHMT1) protein.Also the common protein with M.W. 39.31 K.d, represented the BHMT2 protein, the two types of protein BHMT1 and BHMT2 were important in regulation of Hcys metabolism.The protein with M.W. 44.6 K.d which presented in Hcys group and Tri + Hcys group (band no 14) refered to the presence of BMHT1 protein and absent from Tri group indicates that, betaine supplementation decreased the plasma Hcys concentration as resulted by Brouwer et al., 2000;Tangerman et al., 2000 andSchwab et al., 2002.From other obtained data, the protein of BHMT1 was represented in all treated groups with average M.W. 44.2 K.d and average M.W. 44.06 K.d in all treated groups.Finkelstein 2007, indicated that, all tissues posses the methionine cycle with methyltetrahydrofolate as the methyl donor, but only kidney, liver, pancrease, intestine and brain also contain the transsulfuration pathway.Also, Treberg and Driedzic (2007), suggested that, the kidney and liver appear to be the major sites of betaine synthesis.
Finally, it would be concluded that the protein pattern is changed in each of kidney and liver and quantitative mutation recorded in all tables leading to change in homocysteine pattern which causes different behavioral pattern in each of control and the treated groups.

Table (
1A) : Molecular weight (M.W.), amxount (%) and rate of flow (RF) for kidney of Rattus norvegicus fractionated protein for the treated and control groups after 2 weeks.
Table (1B): Molecular weight (M.W.), amount (%) and rate of flow (RF) of kidney fractionated protein for the treated and control groups of Rattus norvegicus after 8 weeks.Sahar Abd El-Razik Mosallam 170

Table (
2A) : Molecular weight (M.W.), amount (%) and rate of flow (RF) of liver fractionated protein for the treated and control groups of Rattus norvegicus after 2 weeks.Molecular weight (M.W.), amount (%) and rate of flow (RF) of liver fractionated protein for the treated and control groups of Rattus norvegicus after 8 weeks.From table (2C), the highest S. I. recorded between Tri + Hcys group and Hcys group, it reached one, while the smallest value of 0.44 was found between Hcys & Tri + Hcys and control group.Data from table (2D) revealed that, the smallest G.d. value of 0.05 was found between Hcys and Tri + Hcys group.