|Year : 2009 | Volume
| Issue : 2 | Page : 72-79
Modulation of Phosphatase Levels in Mice Liver by Genistein Treatment against Radiation Exposure
Ajay Gaur1, AL Bhatia2
1 L.B.S. College of Pharmacy, Jaipur-302004, Rajasthan, India
2 Radiation Biology Laboratory, Center for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur-302004, Rajasthan, India
|Date of Web Publication||2-Jan-2010|
L.B.S. College of Pharmacy, Jaipur-302004, Rajasthan
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Genistein is a soya isoflavone, which is found naturally in legumes, such as soybeans and chickpeas. The intraperitoneal administration of optimum dose (200 mg/kg body weight) of Genistein before 24 hrs and 15 minutes of irradiation (8 Gy at a dose rate of 1.02 Gy/min) increased the acid phosphatase (by 34.7 ± 12.39%) and decreased the alkaline phosphatase (by 49.59 ± 13.82%) in experimental group as compared to control group in liver of Swiss albino mice. Statistically analyzed survival data produced a dose reduction factor (DRF) = 1.24. The results indicate that Genistein against radiation effect may pave way to the formulation of medicine in radiotherapy for normal tissue and possible against radiomimetic drug induced toxicity. Present study also establishes the fact that Genistein may be used as a radioprotector before and after radiation exposure. Hence the possibility of its use as a radioprotectant and radiotherapeutic drug in accidental conditions or nuclear war conditions cannot be ruled out.
Keywords: Genistein, Tyrosine kinase inhibitor, Radiation, Liver, Oxidative stress, acid and Alkaline phosphatases
|How to cite this article:|
Gaur A, Bhatia A L. Modulation of Phosphatase Levels in Mice Liver by Genistein Treatment against Radiation Exposure. Phcog Res 2009;1:72-9
|How to cite this URL:|
Gaur A, Bhatia A L. Modulation of Phosphatase Levels in Mice Liver by Genistein Treatment against Radiation Exposure. Phcog Res [serial online] 2009 [cited 2020 Aug 11];1:72-9. Available from: http://www.phcogres.com/text.asp?2009/1/2/72/58135
| Introduction|| |
At present there is hardly any aspect of human welfare in which radiation does not play an important role. Radiations have cytotoxic and immunosuppressive effects. Hence, preventive methods to protect not only human but also animals and plants are necessary. Therefore, radioprotectors for use prior to exposure has been identified as one of the highest priority areas for research. Recently Interest has been generated to develop potential drugs of plant origin which can quench the reactive energy of free radicals and eliminate oxygen and are capable of modifying radiation responses with minimum side effects especially during the radiotherapy where the necessity of protection of normal tissue occurs. Plants products appear to have an advantage over synthetic products in terms of low/no toxicity at effective dose  .
Radiation brings about several biochemical alterations in the affected tissues by influencing the metabolic processes occurring in them. Radiation induced cellular degranulation of tissue damage was shown, as an increase in acid phosphatase activity was reported in animals ,,, . Some scientists reported an increased activity of acid phosphatase in mammals after sublethal doses of gamma radiation  . The enhanced activity of acid phosphatase found in the liver could be due to either a direct effect of irradiation on the lysosomal membrane or some indirect effect such as liberation of thyroid hormone, they proposed that radiation can cause the formation of lipid peroxides in lysosomes presumably due to direct oxidation of unsaturated fatty acids of the lysosomal membrane by free radical formed. This may be one of the reasons for the rupture of lysosomal membrane in the gamma irradiated mice liver. Acid phosphatase is localized in cellular lysosomes and its activity may be changed following to whole-body irradiation. An increased acid phosphatase level may also be attributed to an elevated Golgi activity and peroxidation of lysosomal membranes after irradiation causing lysis of membrane and oozing out of enzyme  .
Scientists reported a dose dependent fall in alkaline phosphatase activity in rats exposed to lethal doses of radiations ,, . Similarly, some also reported deterioration in alkaline phosphatase activity in male mice after irradiation with 5 Gy gamma rays , .
Genistein a tyrosine kinase inhibitor, compete with the ATP binding site of the catalytic domain of several oncogenic tyrosine kinases. This is possible because of structural similarities between ATP and the TKIs. Kinases use ATP as a source of phosphate, but if a TKI binds to the enzyme instead of ATP, then the kinase cannot phosphorylate proteins and signaling halts. Genistein inhibits protein tyrosine kinase, which is involved in phosphorylation of tyrosyl residues of membrane-bound receptors leading to signal transduction, and it inhibits topoisomerase II, which participates in DNA replication, transcription and repair. By blocking the activities of PTK, topoisomerase II and matrix metalloprotein and by down-regulating the expression of about 11 genes, including that of vascular endothelial growth factor, Genistein can arrest cell growth and proliferation, cell cycle at G2/M, invasion and angiogenesis. Genistein can alter the expression of gangliosides and other carbohydrate antigens to facilitate their immune recognition ,, .
Liver is selected as a testing organ because some scientists reported it as highly radiosensitive organ  . The present study has been carried out in order to check ameliorating capacity of Genestein against radiation with respect to protein and DNA content of mice liver.
| Materials and Methods|| |
Swiss albino mice (Mus musculus) obtained from All India Institute of Medical Sciences (AIIMS), New Delhi and kept at controlled condition of temperature (25 ± 2 °C) and light (light : dark, 12 : 12 hrs). They were provided standard mice feed (procured from Hindustan Uniliver ltd. Mumbai) and water ad libitium. For experimentation, healthy male mice of 6-8 weeks old with an average body weight of 22 ± 3 gm were selected from inbred colony.
Genistein: Genistein was obtained as gift sample from Mr. M. Maniar (Palm Pharmaceuticals, Inc., USA). Genistein was manufactured by L.C. Laboratories, 165 New Boston St. Woburn, MA01801 USA.
Genistein solution: Genistein was dissolved in dimethyl sulfoxide and then prepared different concentration solutions so that the volume administered intraperitoneally was 0.5 ml.
Mode of administration: Mice were administered intraperitoneally optimum dose (200 mg/kg body weight) of Genistein before 24 hrs and 15 minutes of irradition.
Biochemical Assays: Five autopsies were performed by mean of cervical dislocation of 6 mice from each group at each post irradiation interval (1 st , 3 rd , 7 th , 15 th and 30 th ) were selected for the biochemical studies. Liver was removed at each autopsy interval from the sacrificed animal of each group and placed on a piece of filter paper to remove excess of moisture. At least six observations were taken. Spectrophotometer was used to measure the optical density. Acid phosphatase activity was estimated in serum by King's method  , whereas alkaline phosphatase activity was estimated by Kind and King's method  , using commercially accessible kits (Span diagnostics Ltd.). The values are expressed as mean ± S.D. The difference between various groups was analyzed by Student's t-test.
| Experimental Protocol|| |
The experiment has been conducted in following 4 phases:
PHASE-I: Drug Tolerance Study
Mice were divided into six groups, each containing ten mice. First group of mice did not receive any treatment, second group were administered intraperitoneally dimethyl sulfoxide, as a vehicle before 24 hrs and 15 minutes of study time and other four groups of mice were administered intraperitoneally different doses 100, 200, 300 and 400 mg/kg body weight of Genistein before 24 hrs and 15 minutes of study time. All six groups were kept under normal conditions and then observed for 30 day for any sign of morbidity, mortality, body weight change and behavioral toxicity.
PHASE-II: Optimum Dose Selection
Mice were divided into five groups, each containing ten mice. First group of mice were administered intraperitoneally dimethyl sulfoxide, as a vehicle before 24 hrs and 15 minutes of irradiation and other four groups were administered intraperitoneally different doses 100, 200, 300 and 400 mg/kg body weight of Genistein before 24 hrs and 15 minutes of irradition. Finally, all the animals of five groups were exposed to 10 Gy of gamma radiation. Radiation sickness, mortality, behavioural toxicity and morbidity were observed for 30 days after irradiation. The dose of Genistein which show highest percentage of survival of mice against radiation has been selected as optimum dose for further experiment.
PHASE-III: LD 50/30 and Dose Reduction Factor
The protective action of any radio protective agent may be represented as a Dose Reduction Factor (DRF) and DRF can calculated as follows:
The DRF of Genistein was calculated by the aforementioned formula, by exposing a large number of Swiss albino mice to different doses of gamma rays in the presence or absence of Genistein. DRF of Genistein was calculated and for this mice were divided into two groups control and experimental, each containing 30 male Swiss albino mice.
Control Group: In this group, three subgroups (10 mice in each group) were made and then all mice were administered intraperitoneally dimethyl sulfoxide, as a vehicle before 24 hrs and 15 minutes of irradiation, equivalent to the optimum dose of Genistein. Now these three subgroups of mice were exposed to 6, 8, 10 Gy of gamma radiation and then observed for 30 days. Mortality and body weight were recorded every day.
Experimental Group: Mice of this group were administered intraperitoneally optimum dose (200 mg/kg body weight) of Genistein before 24 hrs and 15 minutes of irradition and then divided into 3 subgroups and then exposed to 6, 8, 10 Gy of gamma radiation.
PHASE-IV: Genistein against Radiation Damage
Mice were divided into following five groups:
Mice of this group were not received any treatment and kept under normal conditions.
Group-II Genistein Treated
Mice of this group were administered intraperitoneally optimum dose (200 mg/kg body weight) of Genistein before 24 hrs and 15 minutes of study time.
Mice of this group were administered intraperitoneally dimethyl sulfoxide as a vehicle before 24 hrs and 15 minutes of irradiation, equivalent to the optimum dose of Genistein.
Group-IV Experiment-1 or or G+IR
Mice of this group were administered intraperitoneally optimum dose (200 mg/kg body weight) of Genistein before 24 hrs and 15 minutes of irradiation.
Group-V Experiment-2 or or IR+G
This group of mice was first exposed to gamma radiation and then intraperitoneally administered optimum dose (200 mg/kg body weight) of Genistein after 15 minutes and 24 hrs of irradiation.
Mice of above treated group were observed from the day of treatment till their autopsy time with respect to body weight changes, sickness, general activity, mobility and other visible abnormalities. Mice were killed by cervical dislocation at various intervals ranging between 1-30 day and whole liver was removed and processed for biochemical estimation of acid and alkaline phosphatases.
| Results|| |
The intraperitoneal administration of Genistein did not caused any toxic effect on mice and Genistein treatment offers better survivability of mice. All irradiated mice without Genistein treatment have shown 100% mortality within 11 days. However, maximum survival of mice (30%, even beyond 30 days) has been recorded in the 200 mg/kg body weight dose of Genistein. On the basis of this survivability experiment, 200 mg/kg body weight dose of Genistein was found as the optimum dose and this was selected for further investigation against 8 Gy of gamma radiation [Table 1] and [Figure 1]. The LD 50/30 values for control group and for pre-irradiation administration of Genistein (G+IR) group were computed as 7.25 Gy and 9 Gy, respectively. The dose reduction factor has been 1.24 [Table 2] and [Figure 2].
Genistein vs. Normal: Lower activities of acid phosphatase were recorded in Genistein treated group as compared to those of normal (by 10.63%), though not significant [Table 3] and [Figure 3].
Control vs. Normal: A sharp increase in acid phosphatase activity in control group was noticed till 7 th day (by 246.12%) followed by a decreasing trend upto 30`" day. A statistically highly significant increase (p < 0.001) by 202.16%, 214.28%, 246.12%, 184.48% and 132.22% in acid phosphatase activities in control group has been noticed on 1 st, 3rd, 7th, 15th and 30th post-irradiation days, respectively, as compared to those of normal groups. An average increase in acid phosphatase activity of control group was approximately 195.85 ± 16.8345% (±SD) [Table 3] and [Figure 3].
Experimental-1 (G+IR) vs. Control: In Experimental-1 group, a sharp increase in acid phosphatase activity was recorded upto 7th day and then a decline observed till 30th day. As compared to those of control group, a statistically significant recovery (p < 0.001) by 24.01, 23.87%, 29.85%, 50.3 1 % and 45.46% in acid phosphatase activity in Experimental-1 group was recorded on 1st, 3rd, 7th, 5th and 30 th post-irradiation days, respectively. An average recovery in acid phosphatase activity of Experimental-1 group was approximately 34.7 ± 12.3949% (±SD). While comparing with those of normal, a highly significant increase (p < 0.001) in activity of acid phosphatase of Experimental-1 group was noticed on 1 st and 3 rd post-irradiation days which recovered and attained almost near normal value on 30 th day [Table 3] and [Figure 3].
Experimental-2 (IR+G) vs. Control: In Experimental-2 group, a sharp increase in acid phosphatase activity was recorded upto 7 th day which is followed by a decline till 30 th day. A statistical significant recovery (p < 0.05) by 21.56%, 18.19%, 26.15%, 40.61%, and 35.41% in acid phosphatase activities of Experimental-2 group were recorded on 1 st, 3 rd, 7 th, 15 th and 30 th post-irradiation days, respectively, as compared to those of control groups. From control, an average recovery in acid phosphatase activity of Experimental-2 group was approximately 28.38 ± 9.4116% (±SD). While comparing with those of normal, though a highly significant level (p < 0.001) in activity of acid phosphatase of Experimental-2 group was noticed on 1 st and 7 th post-irradiation days, but this followed by a significant decline (p < 0.05) upto 15 th day which attained almost near normal level on 30 th day. In Experimental-2 group, an insignificant increased level of acid phosphatase has been noticed on all post-irradiation days in comparison to those of Experimental-1 group [Table 3] and [Figure 3].
Genistein vs. Normal:
Genistein administration of mice did not produce any appreciable difference in alkaline phosphatase (by 1.61%) [Table 4] and [Figure 4].
Control vs. Normal: A sharp decline in control group was noticed upto 7 th day followed by a slight recovery by 30 th day. Statistically highly significant decreases (p < 0.001) by 49.13%, 59.62%, 61.19%, 47.22% and 31.3% in activities of alkaline phosphatase of control group were recorded on 1 st , 3 rd , 7 th , 15 th and 30 th post- irradiation days, respectively, as compared to those of normal groups. An average decrease in alkaline phosphatase activity of control group was approximately 49.69 ± 11.9943% (±SD) [Table 4] and [Figure 4]
Experimental-1 (G+IR) vs. Control: In Experimental-1 groups, a gradual decline in activities of alkaline phosphatase by 7 th day were recorded which was followed by a recovery on 30 th day. Statistically a highly significant recovery by 43.61%, 69.31%, 58.27%, 40.94%, and 35.83% in activities of alkaline phosphatase in Experimental-1 group was recorded on 1 st, 3 rd , 7 th , 15 th and 30th post-irradiation days, respectively, as compared to those of control groups. An average recovery in alkaline phosphatase activity of Experimental-1 group was approximately 49.59 ± 13.8215% (±SD). While comparing with those of normal, though a highly significant decreased level at p < 0.001 in activity of alkaline phosphatase of Experimental-1 group by 7 th day was noticed, however, it was maintained upto 15 th post-irradiation days which recovered on the later interval and attained almost near normal value by 30 th day [Table 4] and [Figure 4].
Experimental-2 (IR+G) vs. Control: A decrease in activity of alkaline phosphatase of Experimental-2 group was recorded by 7 th day and which was followed by a slight recovery by 30 th day. A statistical significant recovery of alkaline phosphatase of Experimental-2 group being maximum (by 67.19% on 3 rd day) has been recorded which is followed by a lesser recovery by 55.92%, 36.13% and 31.83% on 7 th , 15 th and 30 th post-irradiation days, respectively, as compared to those of control group. From control, an average recovery in alkaline phosphatase activity of Experimental-2 group was approximately 45.44 ± 15.3417% (±SD). While comparing with those of normal, a highly significant decrease (p < 0.001) in activity of alkaline phosphatase of Experimental-2 group was noticed on 7 th day, which was maintained upto 15 th post-irradiation day and later attained almost near normal value by 30 th day. In Experimental-1 group, an insignificant increase in activities of alkaline phosphatase occurred on all post-irradiation days as compared to Experimental-2 group [Table 4] and [Figure 4].
| Discussion|| |
An increasing value of acid phosphates enzyme was observed in present study on 7 th day after irradiation then declined progressively, but it could not be restored to normal even till the end of study. Pretreated irradiated animals exhibited a decline in acid phosphatase continuously (i.e. great recovery) from 7 th day to last autopsy interval (30 th day) just similar the trend that of control group. However, in case of Experimental-1 (pre-irradiation treated) group more protection was observed (an overall average recovery by 34.7 ± 12.394 in terms of low acid phosphatase activity, in comparison to that those of control group. In Experimental-2 group (post irradiation treated), the activity of enzyme was similar to Experimental-1 group (an average recovery by 28.38 ± 9.41 from the control). This indicates that Genistein administration is also protective against radiation damage even when given after radiation and prior to radiation too.
Several researchers reported a radiation induced cellular degradation of tissue damage. Acid phosphatase is localized in cellular lysosomes and its activity may be changed following to whole-body irradiation. The enhanced activity of acid phosphatase found in the liver could be ascribed either to a direct effect of irradiation on the lysosomal membrane or some indirect effect such as liberation of thyroid hormone. It was proposed that radiation can cause the formation of lipid peroxides in lysosomes presumably due to direct oxidation of unsaturated fatty acids of the lysosomal membrane by free radical formed. This may be one of the reasons for the rupture of lysosomal membrane in the gamma irradiated mice liver. An increased acid phosphatase level may also be attributed to an elevated Golgi activity in addition to peroxidation of lysosomal membranes after irradiation causing lysis of membrane and oozing out of enzyme  .
It is well known that radiation increases the permeability of membranes of several cellular organelles and hence an increase in serum acid phosphatase activity was seen after irradiation. A rise in acid phosphatase activity can be attributed to gastrointestinal syndrome however; further rise can be assigned to other factors like hematopoitic injury.
These findings are in close agreement with the present investigation where the acid phosphatase level was also found to be elevated after irradiation (in control group) by an average approximately 195.85 ± 16.83%. Genistein administration produced a significant recovery in acid phosphatase level in Experimental-1 group and in Experimental-2 group by approximately 34.7 ± 12.3949% and 28.38 ± 9.4116%, respectively from control.
Whole-body gamma-irradiation of 8 Gy dose revealed a decrease in the serum alkaline phosphatase activity by an average approximately 49.69 ± 11.99% (in control group). Pre-irradiation administration and post-irradiation administration of Genistein recovered the level of alkaline phosphatase significantly by an average approximately 49.59 ± 13.82% and 45.44 ± 15.34%, respectively, as compared to those of control group, though the level of alkaline phosphatase could not reach the normal value at last autopsy interval. This result further corroborates the therapeutic action of Genistein against radiation damage. Concomitantly, it also proves to be prophylactic as well as preventive against radiation action.
Scientists suggested that lesions are produced in the membrane lipids, possibly by peroxides, due to irradiation leading to the activation of latent acid hydrolase, which could result in the digestion of the membrane itself with the consequent activation and release of the other lysosomal enzymes. Although, the activation of lysosomal enzymes in tissues with interphase death is well documented, information on lysosomal enzymes in liver, kidneys and brain are meagre and often contradictory. It is known that lysosome from different cell types or even from the same tissue varies greatly in their susceptibility to damage by irradiation. An increase in lysosomal enzymes has also been reported at sublethal doses of radiation. The release of enzymes from lysosome may be due to activation of pre-existing latent enzymes or due to synthesis of new lysosome as a consequence of radiation , .
Researchers observed that alkaline phosphatase activity decreased after whole body irradiation to the mice, whereas acid phosphatase activity increased. Alkaline phosphatase plays an important role in maintenance of cellular permeability and acts as monophosphoesters. Damage to cell membrane caused by radiation may be the reason for declined activity of alkaline phosphatase. Post irradiated damage to liver can be another reason attributing to increased level of acid and alkaline phosphatase. Radiation induced stress also accounts for an increased activity of these enzymes. Alkaline phosphatase, a brush border enzyme splits various phosphate esters in an alkaline medium and mediate membrane transport. Thus acid phosphatase helps in early recovery from radiation damage by removing debris and alkaline phosphatase helps in stabilizing the membrane. One of the causes of radiation damage is due to lipid peroxidation. This alters the lysosomal membrane permeability resulting into release of hydrolytic enzymes. So, an increase in acid phosphatase was noticed after radiation treatment. The alkaline phosphatase activity is associated with membrane permeability and on account of membrane damage and depletion of hepatocytes after irradiation the enzyme activity was decreased , .
| Conclusion|| |
Man is exposed to a number of toxic substances in the environment including radiation as well as to toxic metabolites and ROS generated within the body. From the present study it is obvious that Genistein prevent the toxic effects of ROS, there is likelihood that Genistein may exert an antiradiation influence in the body. So, it would further pave way to the formulation of medicine against radiation and toxicity induced during radiotherapy. Owing to this property, the Genistein known for its functional properties can be further extended to exploit its possible application for various health benefits as nutraceuticals and food ingredient in radiotherapy to protect the normal tissue. Genistein, a potent protein tyrosine kinase inhibitor maintained the normal levels of acid phosphatase, alkaline phosphotase and other biochemical parameters against the oxidative stress produced by radiation in normal tissue of mice. The results indicate that Genistein against radiation effect may pave way to the formulation of medicine in radiotherapy for normal tissue and possible against radiomimetic drug induced toxicity.
| Significance of Findings|| |
Present study established the fact that Genistein may be used as a radioprotector before and after radiation exposure. Hence the possibility of using Genistein as a radioprotectant and radiotherapeutic drug in accidental conditions or nuclear war conditions cannot be ruled out.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]