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International Journal of Advanced Science and Engineering TechnologyOn line: ISSN 2225-9686; Print ISSN 2225-9678Vol.2, No.2 (2012) pp(120-127)www.ijaset.com Effect Of Pleurotus Ostreatus On Hyperglycemia, DNA Damage
And Chromosomes Aberrations
Department of Biochemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203 Jeddah 21589, Saudi Arabia, ABSTRACT
Some Pleurotus ostreatus (mushrooms) have shown tendency to be effective for control of blood glucose and One of the main health problems with high and
also for DNA damage and chromosomes aberrations without markedly increased complications is diabetes. Despite
any side-effects. The present work demonstrates that several projects with preventative strategies and
mushrooms have promising role in the domain of repairing armories of medication, the arrangement of diabetes
DNA damage and chromosomes aberrations. The published remains grossly unsatisfactory. Thus, it is vital to
clinical and / or academic data are, however, still not identify unfamiliar drugs or novel nutraceuticals for
sufficient enough to show how mushroom reduces treating and preventing diabetes without side effects.
hypoglycemic effects to be use as an authoritative drug or The present study deals with scientific information on
medical food (nutraceuticals). So, much scientific attention mushrooms with regards to its potential use as anti-
should be paid to the mushrooms that have effective medical diabetic active food. In addition to the anti-
functions and in particular curative functions of diabetes hyperglycemic action of mushrooms, the present study
mellitus. Although chemical and biochemical hypoglycemic presents its effect on DNA damage, chromosome
agents, e.g. insulin [1], troglitzone, repaglinide, tolbutamide aberrations and sperm alternations in streptozotocin-
and rosigitazone are the backbone of classical-treatment of induced diabetic rats. These animals have been treated,
diabetes and are so efficient in controlling diabetes, they for 30 days, with amaryl (as control treatment) (0.03
have hurtful side effects and fail to significantly alter the mg/kg b wt/dl), low-dose mushroom (100 mg/kg b wt/dl)
course of diabetic complications [2]. Mushrooms have been and high-dose mushroom (200 mg/kg .b. wt/dl). The
defined [3] as a macro fungus with special fruiting-bodies glucose level GL of streptozotocin-induced diabetic
that could be epigeous and large enough to be seen and to be animals has been markedly improved by mushroom
picked simply by naked hands [3]. Mushrooms constitute treatment; for example GL has decreases from 167.6
more than twenty thousand known species which are widely mg/dl down to 116.0 mg/dl for treatment with high-dose
distributed all-over the globe; however, only about two mushroom and 128.9 mg/dl for treatment with low-dose
thousands (10%) species are known and could be explored.
mushroom, comparing with amaryl treatment that
Huge quantity of mushrooms species are unexplored and decreases GL down to 92.6 mg/dl. But, the experimental
even undiscovered which would possibly be of certain results show that treatment with mushroom is better
medical interest [4]. For example, tremella-fuciformis (snow than treatment with amaryl in case of genetic changes
fungus) is commonly used in Chinese sweet-cuisine and it is (DNA fragmentation, disappear of some base pairs and
tasteless. Despite its unordinary nature, Tremella-fuciformis chromosome aberrations.
is highly valued for several medical benefits [5]. From its So, it is proposed that more close scientific attention be
fruiting bodies, snow fungus show an important dose paid to precede more research of functional mushrooms
dependent hypoglycemic activity in normal mice and for preventive and curative treatments for diabetes.
significant medical performance in streptozotocin inducediabetic mice [6]. The anti-diabetic activity of submerged Keywords -
culture of snow fungus, exo-polysaccharides has been Mushroom, Diabetes, Hyperglycemia, DNA
reported [7]. Their data have suggested that exo- chromosome
aberrations
polysaccharides exhibits major hypoglycemic effect and alternations, streptozotocin-induced diabetic rats
even improve the medical activity of insulin [8]. This meansthat snow fungus has potential oral hypoglycemic effect andcan be used as medical-food. Moreover, the administration INTRODUCTION
of mushroom in drinking-water and the diet has highlyopposed the hyper glycemia of streptozotocin diabetic mice [9]. Mushroom with “immune modulating” polysaccharides ethanol (95 percent). By vacuum distillation, the residue has can be used as medical food, it can be used as drug in been filtered then centered to a dry mass which has been limited conditions and it can be used as nutraceutical used as mushroom extract. Low-level of this extract (100 (health-promoting food). Hokama et al [10] have reported mg / kg.b.wt) and high-level concentrations (200 mg / that many medical plants are an important source of strong kg.b.wt) have been used as it will be seen in the antioxidants and have free radical scavenging activity [11].
Even many medical properties have been reported forreduction of blood glucose [6] and inhabitation of plateletaggregation [10]. In general, many physiological processesof hyperglycemia produce “oxygen-center” free radicals and other reactive oxygen species [12]. These reactive oxygenspecies can easily crush the effect of protective enzymes Kits of glucose oxidase peroxidase diagnostic (antioxidant defense) and can cause high damage and even enzyme and streptozotocin have been obtained from Sigma lethal cellular defects if membrane lipids, cellular proteins (USA, MO St. louis). Glimepiride tablets (amaryl), obtained DNA and enzymes will be oxides [13]. As it is well known, from local pharmacies, have been ground to fine powder the damage of nuclear component (or any part of its host) which dissolved in pure distilled-water. For continuous leads to diabetes mediated stress [14]. Consequently, the thirty days, it has been orally administrated at 0.03 mg / physical defects of cells will be stimulated to cause different kg.b.wt. This last value is equivalent to an acceptable dose singes of neurological deficiencies, carcinogenesis aging, for human (4 mg / kg) [8]. Here, standard treatment with heart disorders, and so on, while the germinal cell damage glimepiride tablets has been used for comparison with will yield infertility [14, 15]. In addition, chromosomes aberrations, DNA damages and sperm abnormalities are themain signs in hyperglaycemic rates [14- 17]. Natural and artificial antioxidant agents stimulate the different oxidationprocesses due to hyperglycemia. However, the use of For 24 hours, the experimental animals have been artificial antioxidant results in potential risk, for example fasted, then injected with 65 mg / kg bodu (single dose) of laws of some countries prohibits their use [13, 18]. Thus, freshly prepared streptozotocin (dissolved in citrate buffer medical plants such as mushroom are considered as safer PH4.5) to create diabetes [24]. The blood samples have been antioxidants [11, 19, and 20]. Mushrooms, from these collected via retro-orbital venous plexus and serum glucose medical plants, strongly reduce the platelet aggregation [10], level have been estimated (just prior to killing the animals at reduce the blood-cholesterol concentrations [21] and prevent the end of the experiment) by enzymatic glucose oxidase induced liver per oxidation [22, 23]. Several researches have per-oxidase (GOD PAP) diagnostic kit [14, 25], the diabetes shown the medical importance of mushroom, however, no has been confirmed after 48 hours. The animals have been much information are concerning to their anti hyper- fasted for three hours then blood has been collected from glycemic agent. Thus, the aim of this work is to study the different effects of mushroom on streptozotocin-induceddiabetic rats. In particular, chromosome aberrations, sperm abnormalities and DNA damage are closely studied.
25 male rats have been selected at random then divided into 5 groups each one has 5 experimental animals: 2. Methodology,
1- Control group, C-group (non-diabetic) 2- diabetic group Materials
3- A- group which is low- and high- level amaryl treateddiabetic animals 4- mushroom treated diabetic animals, and 5- MH- groupwhich is high-level mushroom treated diabetic animals. ML- Male albino rats of about 150- 160 g have been housed and MH- groups have been orally given 100 and 200 mg/ under standard laboratory conditions and maintained on 12 kg.b.wt/ dl, respectively. After 30 days, at the experiment hours light. These rats have been provided with water and end, the rats have been sacrificed by cervical-dislocation to study molecular-genetic, cytogenetic and sperm studies.
Mushroom stem-bodies have been purchased from local market in dried form. 5 g of these dried mushroomparts have been powdered and extracted with 100 ml of Immediately, after sacrificing the animals their suspended in 0.5 ml of lysis buffer as it has been described livers have been collected and their tissues have been lysed by Gibb et al [26] and the fragmented-DNA parts have been in 0.5 ml of lysis buffer containing: 1 mM EDTA, 10 mM tris- HCl (pH 8), (0.2%) triton X 100 centerfuged at 10000rpm for 20 minute at 4ºC. The pellets have been re- 3 = Streptozotocin-induced diabetic rats treated high dose of mushroom4 = Streptozotocin-induced diabetic rats treated low dose of According to Sharma’s methodology [27], genomic DNA has been segregated from the animal’s liver and its 5 = Streptozotocin-induced diabetic rats and non-treated concentration has been estimated using a spectrometer of260 nm and 280 nm absorbance, respectively. The totality ofisolated DNA has been test by electrophoresis in 0.8% agar Cultures have been incubated, for one hour at 37 < T < 38ºC rose gel using DNA molecular weight marker (Eurblio, Paris and the cells have been centrifuged for ten minute at 1000 France). ISSR-analysis has been performed using three r.p.m., then re-suspended in pre-warmed hypotonic solution different primer that are procedure from integrated DNA at 37 ºC for 20 minute. The samples have been centrifuged technologies Inc (USA, CA, San Diego), based on core and fixed in cold “3:1 methanol-glacial acetic acid”. Each repeats anchored at the 3’ or 5’ end as it is shown in table 1.
sample has been washed several times fixative and slides Amplification reactions for ISSR-analysis have been used in have been performed as shown elsewhere by Preston et al final volume 25 l containing 10xPCR buffer (50mM KCl, [30]. Chromosome analyses have been carried out in 50 10mM MgCl (pH9.0, 2mM 2dNTPs, 10mM primer, 50 ng DNA and 0.5 Taq Polymerase Promega, USA). ISSR hasbeen performed using Zietkiewicz et al [29] methodology [28] (Zietkiewicz et al 1994). PCR products have beenanalyzed using 1.2% agar-rose gel electrophoresis and visualized with 10 ug/ul ethidium bromide staining. The epididimus excised and minced in about 8 ml of sizes of fragments have been calculated based on a DNA physiological saline solution, dispersed and filtered to ladder of 100 to 2000 bp (MBI, Fermentas).
exclude large tissue fragments. Spots have been preparedafter staining the sperm with aqueous-Eosin Y. At least 3000 sperms per group have been assessed formorphological abnormalities which have been evaluated For the above mentioned five animal groups; animals femurs have been removed and the bone marrowcells have been aspirated from both femurs of each animal as it has been described elsewhere [29].
SPSS-software has been used to carry out the statistical analysis and data have been analyzed using one way analysis variance; then Duncan’s-post-hot test has been used for comparison between different treatments in the same sex. The values are shown as “mean-value ± S.D.” and differences have been only considered if P < 0.05.
Table 1 Elementary sequences used for ISSR amplification Fig 1: Effect of Mushroom treatment on blood glucose levels in diabetic rats after 30 treatment-days: 1 = Normal rats2 = Streptozotocin-induced diabetic rats treated with amaryl Blood glucose level decreases in diabetic animals Blood glucose level decreases in diabetic rats when treated with mushroom (ML- and MH groups).
Blood glucose level increases in diabetic rats than Effect of Mushroom treatment on blood glucose levels (mg/dl) in diabetic rats after thirty treatment-days Normal Group-A Group-HM Group-LM Group-STZ 64.6±2.1 92.6±1.5 116±2.6 129.8±2.1 167.6±4.2 Group-A = Group of diabetic (streptozotocin) rats treated Group-STZ = Group of normal (non-diabetic rats) treated (streptozotocin) rats treated with high dose of mushroom, Group-LM = Group of diabetic (streptozotocin) rats treatedwith low dose of mushroom Effect of different treatments on the rates of DNA fragmentation in streptozotocin-induced diabetic rats after thirty 7.16±0.1 17.33±0.5 13.1±.36 13.9±.41 21.0±.6 The experimental data shown in table 3 show that: The rate of DNA fragmentation (for diabeticanimals) increases comparing with those of The rate of DNA fragmentation (for diabetic animals treated with amaryl) decreases comparing The rate of DNA fragmentation (for diabetic comparing with those of controlling group.
Curiously, the data of high-dose and low-dose ofmushroom treatment are nearly similar and no net Fig 2: Effect of treatments with amaryl and mushroom onthe rates of DNA fragmentation in streptozotocin-induceddiabetic rats after thirty treatment-days: 1 = Normal rats2 = Streptozotocin-induced diabetic rats treated with amaryl As it has been mentioned, to detect the mushroom 3 = Streptozotocin-induced diabetic rats treated high dose of treatment efficiency, three ISSR primers are used: HB10, HB12 and HB14. The present experimental results show that 4 = Streptozotocin-induced diabetic rats treated low dose of An equal four fragments which have difference of 5 = Streptozotocin-induced diabetic rats (non-treat rats) 150 bp fragment between data of control group andA-group (Amaryle treated group).
The groups of animals treated with mushroomdisplay four amplified fragments while 610 bp fragments have been disappeared comparing with control, amaryl-treated and diabetic groups.
concentration have lacked 230 bp fragments which are exist in each of control- and HM-groups.
Six fragments in the control group and HM- group,whereas diabetic animals, amaryl samples and a Effect of different treatments on the frequency of chromosome aberrations in streptozotocin-induced diabetic rats after thirty Normal Group-A Group-HM Group-LM Group-STZ 0.6±0.22 1.43±0.13 0.6±0.44 1.19±.31 2.2±.16 0.0±0.00 4.13±0.32 2.3±0.34 2.2±0.31 7.9±.47 0.4±0.00 2.22±0.24 1.2±0.31 1.2±0.37 3.2±.49 The groups of animals treated with mushroom withcontrol, amaryl-treated and diabetic groups.
display four amplified fragments while 610 bp fragments for A-group. Also, samples treated with Eight fragments in the control group, while this low and high concentrations of mushroom revealed primer revealed 4 and 5 fragments in each diabetic an equal 7 fragments which are different from the animals and amaryl samples respectively.
control samples in only one fragment of 290 bp for The groups of animals treated with mushroom differed from the control samples in 490, 290, 250 The obtained data evidently observed that 490, 290, and 50 bp fragments for diabetic samples and 50 bp and 50 bp fragments which exist in mushroom group, they disappeared in diabetic rats. Also, the show, also, that hyperglycemic rats have important increase 490, 250 and 50 bp fragments are absent in A- of chromosome aberrations compared to normal group group but they are present in HM- and LM-groups.
which is in agreement with reference [17]. The sperm The 290, 250 and 50 bp fragments (present in HM- abnormalities in streptozotocin diabetic rats, of the present and LM-groups) disappear in diabetic group.
data, have significantly increased than the normal group (un-diabetic group) which is in fair agreement with Rabbani et al [14].
DNA fragmentation, sperm abnormalities and loss of some base pair of DNA (Genetic alternations) is due to the effect chromosome aberrations are significantly (P < 0.05) of hyperglycemia [16]; several authors have reported that increases in D-group (diabetic animals) than the control the overproduction of reactive oxygen species ROS which animals (table 4). While amaryl-treated diabetic animals and are side product of hyperglycemia attack the cell-membrane, mushroom-treated diabetic group decrease of most nucleus and genetic parts leading to possible protein and frequencies of structural and numerical chromosome DNA modifications [38, 39] which leads, consequently, to aberrations than the diabetic animals (D-group).
sperm abnormalities and chromosome aberrations [14, 16 It is worth noting that the differences between amaryl- and 40]. Thus, the DNA fragmentation and chromosome treated diabetic animals and mushroom-treated diabetic derangements, in the present data, could be a result of ROS.
animals are significant (P < 0.05) and the diabetic rats In addition, several damage path ways by the ROS such as treated with low or high concentrations of mushroom have accelerated formation of advanced glycation end production, low frequencies of most structural and numerical polyol pathway, hexosamine pathway, protein kinase or chromosome aberrations than the diabetic animals.
lipid per-oxidation [14, 15 and 41].
Discussion
Conclusions
Mushroom has proved that it is an efficient exporter of main The present study shows that the treatment with mushroom amino acids which contain antioxidant vitalities and many could reduce the high blood glucose level but not as medicinal properties [23, 33 and 34]. In particular, it efficient as amaryl. However, treatment with mushroom has contains levostatin which is a cholesterol-lowering drug been more effective for decreasing the genetic alternations used for correcting hypercholesterolemia [23, 33]. In the and sperm abnormalities in diabetic conditions than amaryl present study, the medicinal-role of mushroom for anti- hyperglycemic drug, repair of DNA damage, chromosomeaberrations and sperm abnormalities. The blood glucose References
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