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Changes in oxidative stress parameters and acid phosphatase activity in the pre-regressing and regressing tail of Indian jumping frog Polypedates maculatus (Anura, Rhacophoridae)
Comparative Biochemistry and Physiology Part C 130 Ž2001. 281᎐288
Changes in oxidative stress parameters and acid phosphatase activity in the pre-regressing and regressing tail of Indian jumping frog Polypedates maculatus ž Anura, Rhacophoridae/ Pravati Kumari Mahapatra, Priyambada Mohanty-Hejmadi, Gagan B.N. Chainy U Biochemistry Unit, Department of Zoology and ICABPS, Utkal Uni¨ ersity, Bhubaneswar-751004, India Received 22 March 2001; received in revised form 27 June 2001; accepted 3 July 2001
Abstract Activities of acid phosphatase Žnormal and Co 2q-sensitive., superoxide dismutase and catalase and levels of lipid peroxidation, hydrogen peroxide were compared in the tails of tadpoles of stage III, XVIII, XXI and XXIII, respectively, of the Indian Jumping frog Polypedates maculatus. It is noticed that acid phosphatase activity Žnormal and Co 2q-sensitive., and levels of lipid peroxidation and hydrogen peroxide increased during tail regression. There is also an increase in the level of superoxide dismutase and catalase in the regressing tail. A positive correlation between activity of acid phosphatase and lipid peroxidation, hydrogen peroxide and lipid peroxidation, acid phosphatase and hydrogen peroxide was noticed in the tail of tadpoles during different developmental stages, suggesting a critical interaction between reactive oxygen species and lysosomal activity during metamorphosis. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Acid phosphatase; Anuran tail during pre-regression and regression; Oxidative stress
1. Introduction In living organisms continuous generation of reactive oxygen species wROS: such as superoxide . Ž . x radicals ŽOy 2 ; hydroxyl radicals OH , etc. and Ž . hydrogen peroxide H 2 O 2 is a common phenomenon ŽSies, 1997.. The ROS, commonly referred
U
Corresponding author. Fax: q91-0674-509037r509107. E-mail address: [email protected] ŽG.B.N. Chainy..
as proxidants, are produced as a consequence of various metabolic activities of cells, and due to their unspecific and high reactive nature, they attack almost all biomolecules including membrane lipids present in their vicinity, causing lipid peroxidation, protein carbonylation and DNA strand breaks ŽHalliwell and Gutteridge, 1985.. Therefore, the cellular system is well equipped both enzymatic and non-enzymatic defences to neutralize the ROS. Superoxide dismutase dismutates superoxide radicals to hydrogen peroxide,
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which in turn is broken down by catalase and glutathione peroxidase. The major non-enzymatic antioxidants are ascorbic acid, vitamin E, uric acid and reduced glutathione. Whenever the rate of generation of the ROS exceeds its rate of neutralisation, the cells lead into oxidative stress condition. Consequently, cells exhibit several pathophysiological states including that of apoptosis ŽChandra et al., 2000.. And in general, lipid peroxidation is taken as an index of oxidative stress ŽKappus, 1985.. Recently, the regression of tails in tadpoles of anurans during metamorphosis has drawn considerable attention as an ideal model to understand the phenomenon of apoptosis. Earlier studies have indicated the crucial role of hydrogen peroxide in tail regression during spontaneous metamorphosis in several species of frogs ŽKashiwagi et al., 1999; Hanada et al., 1997.. Furthermore, using Rana japonica tadpole tail as a model system to unveil the mechanism of tail regression, Kashiwagi et al. Ž1999. observed that T4 enhances generation of NO which in turn inhibits catalase activity and results in augmentation of H 2 O 2 , thereby triggering apoptosis. An increase in the generation of superoxide radicals in the tadpole tail tissue of Rana catesbeina undergoing regression was also recorded ŽLittle and Flores, 1990.. The involvement of macrophages as effectors of cell death has also been described ŽWeber, 1968; Lohmann-Matthes et al., 1972; Nathan et al., 1980.. In anuran tail the macrophages undergo activation prior to performing functions such as phagocytosis or autolysis. There is a progressive activation of macrophages, beginning in connective tissue of tail fin and spreading later to the connective tissue of the tail stem ŽWeber, 1968; Kerr et al., 1974.. Production of hydrogen peroxide Žoxidant. by macrophages in the tail of metamorphosing Rana japonica has been demonstrated by Sasaki et al. Ž1988.. It is also evident that in most of the biological system, an increase in oxidants Žhydrogen peroxide and superoxide radicals. lead to lipid peroxidation, an index of oxidative stress ŽSevanian and Hochstein, 1985.. Apart from the ROS, the role of acid phosphatase, a lysosomal enzyme has also gained attention in the tail regression phenomenon of tadpoles during metamorphosis ŽGilbert and Frieden, 1981.. While there is ample information available in the literature on the role of ROS in regression of
tail of tadpole of anurans particularly in various species of genus Rana during metamorphosis, very little information is available on species of other genus of anurans. Furthermore, a comparative study of the role of ROS in tail regression among various species of anurans will help in understanding the physiological role of ROS in tail regression, thereby apoptosis. Since it is well known that tail regression begins in anuran tail at stage XX ŽSasaki et al., 1988. two critical stages, i.e. stage III Žhind limb bud. and stage XVIII Žwell developed hind limbs. are taken as stages prior to tail regression, and two more stages, i.e. stage XXI Žboth fore limbs emerged. and stage XXIII Žmore than half of tail regressed. are taken as stages following tail regression Žstages according to Taylor and Kollros, 1946.. The present study sets out to investigate: Ži. whether oxidative stress is generated in the tail of the tadpoles of the Indian jumping frog Polypedates maculatus ŽAnura, Rhacophoridae. prior to tail regression or during tail regression; and if so, Žii. whether there exists any correlation between oxidative stress and acid phosphatase, a lysosomal enzyme. Presence of cobalt sensitive acid phosphatase is reported for the first time in anuran tail.
2. Materials and methods 2.1. Chemicals Chemicals used in this study were of analytical grade. Thiobarbituric acid ŽTBA., bovine serum albumin ŽBSA. and mercaptoethanol were obtained from Sigma Chemical Co.; USA, sodium dodecyl sulfate ŽSDS., nicotinamide adenine disodium salt ŽNADH., horse radish peroxidase ŽHRP. and p-nitrophenyl phosphate were obtained from Sisco Research Laboratory, Mumbai, India; Sephadex G-25 was obtained from Pharmacia Biotech, Sweden; Folin᎐Ciocalteus reagent was obtained from Qualigens Fine Chemicals, Glindia Ltd., Mumbai, India. All other chemicals were of the highest purified grade available. 2.2. Tadpoles Foam nests containing eggs of Polypedates maculatus were collected from nature during the monsoon period ŽJuly᎐September, year
P. Kumari Mahapatra et al. r Comparati¨ e Biochemistry and Physiology Part C 130 (2001) 281᎐288
1998᎐2000.. The hatchlings were reared in the laboratory following standard procedure ŽMohanty-Hejmadi et al., 1992.. They were fed with boiled Amaranthus green and boiled egg ad libitum. Four stages of tadpoles, i.e. stage III Žhind limb bud stage., stage XVIII Žwell developed hind limbs., stage XXI Žboth fore limbs emerged. and stage XXIII Žmore than half of the tail regressed. of Taylor and Kollros Ž1946. were selected for the experiment. The tadpoles were anaesthetized with MS 222 prior to tail amputation through the base of the tail. For each assay, a pool comprising of five tadpoles were taken. 2.3. In¨ estigation of oxidati¨ e stress parameters To investigate oxidative stress in the tail during different developmental stages, the level of lipid peroxidation Žan index of oxidative stress . and hydrogen peroxide Žan oxidant or Reactive Oxygen Species., and the activities of superoxide dismutase and catalase Žboth enzymatic antioxidants. were estimated. To evaluate the lysosomal enzymes, the activity of acid phosphatase along with cobalt sensitive acid phosphatase were determined. 2.3.1. Lipid peroxidation The tissue lipid peroxidation ŽLPX. was assayed as thiobarbituric acid reacting substance ŽTBARS. by thiobarbituric acid ŽTBA. assay of Ohkawa et al. Ž1979.. A 10% Žwrv. homogenate was prepared with 1.5% KCl in an ice-cooled hand mortar. The homogenate was centrifuged at 4⬚C for 5 min at 1000 = g. The supernatant was taken for the assay. The assay mixture contained 0.4 ml sample, 0.1 ml of 8.1% SDS, 0.75 ml of 20% acetic acid ŽpH adjusted to 3.5 with NaOH. and 0.75 ml of 0.8% aqueous solution of TBA. The mixture was heated in a water bath for 60 min at 95⬚C using glass balls as condensers. The tubes were cooled in running tap water, centrifuged at 1000 = g for 10 min at room temperature and absorbance of the supernatant was read at 532 nm. Protein content of the sample was assayed by the method of Lowry et al. Ž1951.. The concentration of TBARS was calculated from extinction coefficient of 1.56 = 10 5 My1 cmy1 ŽWills, 1969.. The amount of TBARS was expressed as nmol malonaldehyde ŽMDA. formedr mg protein.
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2.3.2. Hydrogen peroxide The level of hydrogen peroxide ŽH 2 O 2 . was measured by the method of Pick and Keisari Ž1981. based on the H 2 O 2-mediated and horse radish peroxidase ŽHRPO.-dependent oxidation of phenol red. A 10% homogenate Žwrv. was prepared with 50 mM phosphate buffer ŽpH 7.4., centrifuged for 10 min at 1000 = g at 4⬚C. The supernatant was taken for the assay. The assay mixture contained 0.28 mM phenol red, 1 unit of HRPO and 500 g protein in 50 mM phosphate buffer ŽpH 7.4., in the final volume of 2.0 ml. Absorbance was measured at 610 nm. The level of hydrogen peroxide was expressed as nmol H 2 O 2rmg protein. 2.3.3. Superoxide dismutase The method of Paoletti and Macoli Ž1990. was followed to measure superoxide dismutase ŽSOD.. A 10% Žwrv. homogenate was prepared with 50 mM phosphate buffer ŽpH 7.4. and centrifuged at 10 000 = g for 20 min at 4⬚C. The supernatant was passed through Sephadex G-25 column Ž1 ml supernatant through 5 ml Sephadex column. with 50 mM phosphate buffer ŽpH 7.4.. The second 2 ml was taken for the assay in the above fraction. Assay mixture contained: NADH 7.5 mM; EDTArMnCl 2 100 mMr50 mM; mercaptoethanol 28 mM; 12.5 g protein; and phosphate buffer 60 mM ŽpH 7.4. to make the final volume 3 ml. Absorbance was taken at 340 nm. The reading was observed at the 8th and 16th min after addition of mercaptoethanol. The level of SOD was expressed as unitsrmg protein w1 unit of SODs Ždifference in OD of blankrdifference in OD of sample. y 1x. 2.3.4. Catalase Catalase activity was measured according to the method of Aebi Ž1974.. A 10% Žwrv. homogenate was prepared with 50 mM phosphate buffer ŽpH 7.0.. The homogenate was centrifuged at 10 000 = g for 10 min at 4⬚C. The supernatant was used for the assay. The assay mixture contained: 0.1 ml Ž60 mM. H 2 O 2 ; 150 g protein; 50 mM phosphate buffer ŽpH 7.0. to make the final volume 3 ml. The reaction was initiated by adding 0.1 ml H 2 O 2 Ž60 mM.. The absorbance was taken at 240 nm and reading was noted at a 60-s interval. Catalase activity was expressed as pmolrmg proteinrmin w1 mol sy1 s 1 katal Žkat.x.
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2.3.5. Acid phosphatase Acid phosphatase ŽACP. activity was determined according to the method of Guha et al. Ž1974. with p-nitrophenyl phosphate as substrate. A 10% homogenate Žwrv. was made with 0.25 M sucrose. The homogenate was treated with Triton X-100 Žfinal conc. 0.1%, vrv. for 15 min in ice. The homogenate was centrifuged at 4⬚C for 10 min at 8000 = g. The assay mixture contained 100 g protein in a total volume of 0.6 ml of 0.1 M acetate buffer ŽpH 5.0.. After preincubation of 5 min at room temperature, 0.3 ml of p-nitrophenyl phosphate Ž1 mM dissolved in 0.1 M acetate buffer, pH 5.0. was added to it. The incubation was carried out at 37⬚C for 30 min. The reaction was stopped by adding 2.1 ml of 0.5 M NaOH solution. Absorbance was measured at 410 nm. The standard was prepared using various concentrations of p-nitrophenol. In order to study the effect of cobalt acetate on acid phosphatase activity, the supernatant was pre-incubated with acetate buffer containing cobalt acetate Ž10 mM final concentration . for 5 min at room temperature. The enzyme activity was expressed as mol p-nitrophenol ŽpNP. formedrmg proteinrmin at 37⬚C. 2.4. Statistics Statistical analysis was followed by Gomez and Gomez Ž1984.. The ANOVA test to find out significance difference between means was calculated by Duncan’s multiple range test. The same superscripts over the bars in Figs. 1᎐5 represent the data which are not significantly different.
Fig. 1. Endogenous level of TBARS Žnmol MDA formedrmg protein. at different developmental stages in the tail of Polypedates maculatus. Data are expressed as mean " S.D. of five observations ŽS.D., standard deviation.. Same superscripts over the bar represent data that are not significantly different.
to XXIII of tadpoles, there was no statistical difference in levels of hydrogen peroxide between stages XVIII and XXI ŽFig. 2..
3. Results 3.1. Lipid peroxidation In the present study, lipid peroxidation level was recorded to be the lowest in the stage XVIII tadpoles, which gradually increased in XXI and XXIII stages of tadpoles ŽFig. 1.. However, lipid peroxidation in stage III was observed to be higher than in stage XVIII. 3.2. Hydrogen peroxide Although the level of H 2 O 2 increased gradually in regressing tails of tadpoles from stage III
Fig. 2. The level of hydrogen peroxide Žnmol H 2 O 2 rmg protein. at different developmental stages in the tail of Polypedates maculatus. Data are expressed as mean " S.D. of five observations. Same superscripts over the bar represent data that are not significantly different.
P. Kumari Mahapatra et al. r Comparati¨ e Biochemistry and Physiology Part C 130 (2001) 281᎐288
Fig. 3. The level of superoxide dismutase Žunits of SODrmg protein. at different developmental stages in the tail of Polypedates maculatus. Data are expressed as mean " S.D. of five observations. Same superscripts over the bar represent data that are not significantly different.
3.3. Antioxidant Enzymes Superoxide dismutase ŽSOD. activity was the lowest in stage III tadpoles ŽFig. 3.. The activity
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Fig. 5. The activity of normal acid phosphatase Žmol pNP formedrmg proteinrmin. and Co 2q-sensitive acid phosphatase Žmol Co 2q-sensitive pNP formedrmg proteinrmin. at different developmental stages in the tail of Polypedates maculatus. Data are expressed as mean " S.D. of five observations. Same superscripts over the bar represent data that are not significantly different.
increased before the on set of metamorphosis Žstage XVIII.. A further increase in the enzyme activity was recorded as tail regression proceeds. In contrast to SOD, the activity of catalase ŽFig. 4. was higher in stage III tadpoles, which then decreased in stage XVIII and XXI, and then increased in stage XXIII. 3.4. Acid phosphatase In the present study the activity of acid phosphatases Žnormal. was the lowest in stage III tadpoles ŽFig. 5. which increased gradually in tadpoles of stages XVIII, XXI and XXIII, respectively. In contrast to the normal acid phosphatase, the activity of Co 2q-sensitive acid phosphatase was higher in stage III, decreased in stage XVIII and increased gradually thereafter till stage XXIII ŽFig. 5..
Fig. 4. Catalase activity Žpmolrmg proteinrmin. at different developmental stages in the tail of Polypedates maculatus. Data are expressed as mean " S.D. of five observations. Same superscripts over the bar represent data that are not significantly different.
4. Discussion Increase in the level of lipid peroxidation in the regressing tail of Polypedates maculatus during
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spontaneous metamorphosis is not surprising, and is in good agreement with the results of earlier finding in other species of anurans. It is reported that hydrogen peroxide production by macrophages occurred in the tails of metamorphosing Rana japonica tadpoles ŽSasaki et al., 1988.. Similarly, superoxide production is also elevated in tadpole tissue of Rana catesbeiana Žat stage XVIII. during metamorphosis, along with rise in -glucuronidase activity ŽLittle and Flores, 1990.. The higher level of lipid peroxidation at stage III of tadpole may be associated with a comparatively higher level of cell division of the growing tail. We have observed high level of lipid peroxidation in the regenerating tail of Polypedates maculatus Žunpublished data.. The level of hydrogen peroxide, an oxidant, is noticed to be low during earlier stage Žstage III. of metamorphosis and is gradually increased as tail regression proceeds. The pattern of hydrogen peroxide level in tails of different stages of tadpole is similar to that of superoxide dismutase. Superoxide dismutase is an enzyme which dismutates superoxide radicals to hydrogen peroxide. In contrast to our results, Kashiwagi et al. Ž1999. observed that when tadpole tail of Rana japonica in culture were exposed to thyroxin, hydrogen peroxide could not be detected either in culture media or in tail homogenates. The difference between results obtained by Kashiwagi et al. Ž1999. and us may be due to different experimental conditions and selection of a different species. It is well known that regression of tail in amphibian tadpole begins at stage XX ŽSasaki et al., 1988.. It is evident from our study that lipid peroxidation is detectable by stage XVIII. Since LPX is the end point of ROS reaction, it is possible that production of the ROS in tail Žeither by macrophages or by cells of tail tissue in response to some stimuli. is initiated earlier than stage XVIII. We have detected both LPX and H 2 O 2 in stage III tadpoles. Although the origin . of reactive oxygen species ŽOy 2 , H 2 O 2 and their role in the process of tissue regression of this species is not clear, their role in programmed cell death in regressing tadpole tail is suggested ŽSasaki et al., 1988.. A decrease in catalase activity in the regressing tail was noted by Hanada et al. Ž1997. and Kashiwagi et al. Ž1999.. Our result corroborates their findings. However, in contrast to their findings, we have observed an increase in the activity of
catalase in stage XXIII tadpoles. This difference is suggested to be due to different experimental conditions and selection of a different species. A sharp increase in acid phosphatase activity is observed in the anuran tail during regression ŽWeber, 1968.. The increase is suggested to be due to progressive release of enzyme from preformed lysosomes of the tail tissue in response to the hormone thyroxine or released by the phagocytic macrophages which accumulate in large numbers in the regressing tail ŽWeber, 1968; Gilbert and Frieden, 1981.. Presence of multiple forms of acid phosphatase ŽACP. according to their inhibition by several inhibitors molecular weight activation by various agents, particularly divalent metal ions, has been reported for animal tissues ŽVanha-Pertulla, 1971.. A Co 2q-sensitive acid phosphatase in the soluble fraction of testicular homogenate of mammals has been reported, whose activity increases during proliferation of spermatogenic cells and decreases during suppression of spermatogenesis in different experimental conditions ŽChainy et al., 1995.. To the best of our knowledge, there is no report available on Co 2q-sensitive ACP in the tails of frogs. This is the first time we report the presence of Co 2q-sensitive ACP in the tail and its change during tail regression in Polypedates maculatus. The high level of Co 2q-sensitive ACP in stage III may be associated with cell proliferation since similar increase in activity has been observed during spermatogenesis in mammals ŽChainy et al., 1995.. It will be of interest to find out the origin and significance of Co 2q-sensitive ACP during tail regression and its regulation and expression by oxidative stress state. In addition, in the present study a positive correlation between: acid phosphatase and lipid peroxidation; hydrogen peroxide and lipid peroxidation; and acid phosphatase and hydrogen peroxide, was observed in the tail tissue during development ŽFig. 6.. The correlation between normal acid phosphatase and lipid peroxidation showed: r s 0.81, t s 6.06 and y s 0.01q 0.17x, respectively. Correlation between Co 2q-sensitive acid phosphatase and lipid peroxidation showed: r s 0.94, t s 12.73 and y s 0.27q 0.22 x, respectively. Similarly, the values of r s 0.72, t s 4.4 and y s 1.26 q 4.82 x, respectively, are the correlation between hydrogen peroxide and lipid peroxidation. For acid phosphatase and hydrogen peroxide correlation showed: r s 0.93, t s 10.83 and y s
P. Kumari Mahapatra et al. r Comparati¨ e Biochemistry and Physiology Part C 130 (2001) 281᎐288
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Fig. 6. Correlation between TBARS Žnmol MDA formedrmg protein. and Ža. activity of acid phosphatase Žmol pNP formedrmg proteinrmin., Žb. activity of Co 2q-sensitive acid phosphatase Žmol Co 2q-sensitive pNP formedrmg proteinrmin., and Žc. the level of hydrogen peroxide Žnmol H 2 O 2 rmg protein.. Žd. Correlation between level of hydrogen peroxide Žnmol H 2 O 2 rmg protein. and activity of acid phosphatase Žmol pNP formedrmg proteinrmin. at different developmental stages ŽIII, XVIII, XXI and XXIII. in the tail of Polypedates maculatus.
0.05q 0.03 x, respectively. In all the cases, r ) P at 0.001 showed the correlation to be significant. The most interesting finding of the present investigation is the identical pattern of changes in the level of LPX, activity of catalase and Co 2qsensitive acid phosphatase ŽFigs. 1, 4 and 5.. At stage III the value of the above oxidative stress parameters is higher than stage XVIII which increases in stage XXI and maximum is observed in
stage XXIII. Another identical pattern of changes was observed in the level of hydrogen peroxide, activity of SOD and normal acid phosphatase ŽFigs. 2, 3 and 5.. From our finding it is reasonable to conclude that oxidative stress is generated prior to tail regression Žstage III, XVIII. at a lower rate which increases significantly in the regressed tail Žstage XXIII.. Furthermore, the correlation between oxidative stress and acid
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phosphatase, suggests existence of a positive correlation between oxidative stress and lysosomal activity in the pre-regressing and regressing tail of Polypedates maculatus. However, it will be of interest to unveil the specific signalling pathways through which oxidative stress triggers tail regression in the species.
Acknowledgements PKM thanks the Council of Scientific and Industrial Research, Government of India for a Senior Research Associateship ŽScientist’s Pool Scheme., pool no 13Ž7338-A. year 1998. We also thank the University Grants Commission ŽSAP. and DBT, Government of India for financial support. References Aebi, H., 1974. Catalase. In: Bergmeyer, H.U. ŽEd.., Methods in Enzymatic Analysis, Vol. 2. Academic Press, New York, pp. 673᎐678. Chainy, G.B.N., Swain, C., Sahoo, A., Mohanty, K.C., 1995. Effects of Estradiol, testosterone and Bromocriptine on rat testicular acid phosphatase activity. Biochem. Arch. 11, 111᎐114. Chandra, J., Samali, A., Orrenius, S., 2000. Triggering and modulation of apoptosis by oxidative stress. Free Radic. Biol. Med. 29 Ž3r4., 323᎐333. Gilbert, L.I., Frieden, E., 1981. Metamorphosis: A Problem in Developmental Biology, 2nd edition. Plenum Press, New York, pp. 139᎐176. Gomez, K.A., Gomez, A.A., 1984. Statistical Procedures for Agricultural Research, 2nd edition. WileyInterscience Publication. Guha, K., Karkkainen, R.R., Vanha-Perttula, T., 1974. Testicular acid phosphatase of the mouse. Med. Biol. 57, 52᎐57. Halliwell, B., Gutteridge, J.M.C., 1985. Free Radicals in Biology and Medicine. Clarendon Press, Oxford. Hanada, H., Kashiwagi, A., Takehara, Y. et al., 1997. Do reactive oxygen species underline the mechanism of apoptosis in the tadpole tail? Free Radic. Biol. Med. 23 Ž2., 294᎐301. Kappus, H., 1985. Lipid peroxidation, mechanism, analysis, enzymology and biological relevance. In: Sies, H. ŽEd.., Oxidative Stress. Academic Press, London, pp. 237᎐310. Kashiwagi, A., Handa, H., Yabuki, M. et al., 1999. Thyroxine enhancement and their role in reactive oxygen species in tadpole tail apoptosis. Free Radic. Biol. Med. 26 Ž7r8., 1001᎐1009.
Kerr, J.F.R., Harmon, B., Searle., J., 1974. An electron microscope study of cell deletion in the anuran tadpole tail during spontaneous metamorphosis with special reference to apoptosis of striated muscle fibers. J. Cell Sci. 14, 571᎐577. Little, G.H., Flores, A., 1990. Changes in the role of production of superoxide anion in tail of Rana catesbeiana tadpoles during spontaneous and triiodothyronine-induced metamorphosis. Comp. Biochem. Physiol. 96B Ž20., 351᎐358. Lohmann-Matthes, M.L., Schipper, H., Fischer, H., 1972. Macrophage-mediated cytitoxicity against allogenic target cells in vitro. Eur. J. Immunol. 2, 45᎐49. Lowry, D.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin᎐ phenol reagent. J. Biol. Chem. 193, 265᎐275. Mohanty-Hejmadi, P., Dutta, S.K., Mahapatra, P., 1992. Limbs generated at site of tail amputation in marbled balloon frog after vitamin A treatment. Nature 355, 352᎐353. Nathan, C.F., Murray, H.W., Cohn, Z.A., 1980. The macrophage as an effector cell. N. Engl. J. Med. 303, 622᎐626. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxidation in animal tissue by thiobarbituric acid reaction. Anal. Biochem. 95, 351᎐358. Paoletti, F., Macoli, A., 1990. Determination of Superoxide Dismutase Activity by purely chemical system based on NADŽP.H oxidation. In: Packer, L., Glazer, A.N. ŽEds.., Methods in Enzymology, Vol. 186. Academic Press, San Diego, pp. 209᎐220. Pick, E., Keisari, Y., 1981. Superoxide anion and hydrogen peroxide production by chemically elicited peritoneal macrophage: induction by multiple non phagocytic stimuli. Cell. Immunol. 59, 301᎐318. Sasaki, K., Kinoshita, T., Takahama, H., Wananabe, K., 1988. Cytochemical studies of hydrogen peroxide production in the tadpole tail of Rana Japonica during metamorphic climax. Histochem. J. 20, 99᎐107. Sies, H., 1997. Oxidative stress: oxidants and antioxidants. Exp. Physiol. 82, 291᎐295. Sevanian, A., Hochstein, P., 1985. Mechanism and consequences of lipid peroxidation in biological system. Annu. Rev. Nutr. 5, 365᎐390. Taylor, A.C., Kollros, J.J., 1946. Stages in normal development of Rana pipiens larvae. Anat. Rec. 94, 7᎐24. Vanha-Pertulla, T., 1971. Chromatographic fractionation and characterization of rat testicular acid phosphatase. Biochim. Biophys. Acta 227, 390᎐401. Weber, R., 1968. The Biochemistry of Animal Development, 2nd edition. Academic Press, New York. Wills, E.D., 1969. Lipid peroxide formation in microsomes: general consideration. Biochem. J. 113, 315᎐324.
Changes in oxidative stress parameters and acid phosphatase activity in the pre-regressing and regressing tail of Indian jumping frog Polypedates maculatus ž Anura, Rhacophoridae/ Pravati Kumari Mahapatra, Priyambada Mohanty-Hejmadi, Gagan B.N. Chainy U Biochemistry Unit, Department of Zoology and ICABPS, Utkal Uni¨ ersity, Bhubaneswar-751004, India Received 22 March 2001; received in revised form 27 June 2001; accepted 3 July 2001
Abstract Activities of acid phosphatase Žnormal and Co 2q-sensitive., superoxide dismutase and catalase and levels of lipid peroxidation, hydrogen peroxide were compared in the tails of tadpoles of stage III, XVIII, XXI and XXIII, respectively, of the Indian Jumping frog Polypedates maculatus. It is noticed that acid phosphatase activity Žnormal and Co 2q-sensitive., and levels of lipid peroxidation and hydrogen peroxide increased during tail regression. There is also an increase in the level of superoxide dismutase and catalase in the regressing tail. A positive correlation between activity of acid phosphatase and lipid peroxidation, hydrogen peroxide and lipid peroxidation, acid phosphatase and hydrogen peroxide was noticed in the tail of tadpoles during different developmental stages, suggesting a critical interaction between reactive oxygen species and lysosomal activity during metamorphosis. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Acid phosphatase; Anuran tail during pre-regression and regression; Oxidative stress
1. Introduction In living organisms continuous generation of reactive oxygen species wROS: such as superoxide . Ž . x radicals ŽOy 2 ; hydroxyl radicals OH , etc. and Ž . hydrogen peroxide H 2 O 2 is a common phenomenon ŽSies, 1997.. The ROS, commonly referred
U
Corresponding author. Fax: q91-0674-509037r509107. E-mail address: [email protected] ŽG.B.N. Chainy..
as proxidants, are produced as a consequence of various metabolic activities of cells, and due to their unspecific and high reactive nature, they attack almost all biomolecules including membrane lipids present in their vicinity, causing lipid peroxidation, protein carbonylation and DNA strand breaks ŽHalliwell and Gutteridge, 1985.. Therefore, the cellular system is well equipped both enzymatic and non-enzymatic defences to neutralize the ROS. Superoxide dismutase dismutates superoxide radicals to hydrogen peroxide,
1532-0456r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 1 . 0 0 2 3 8 - 1
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which in turn is broken down by catalase and glutathione peroxidase. The major non-enzymatic antioxidants are ascorbic acid, vitamin E, uric acid and reduced glutathione. Whenever the rate of generation of the ROS exceeds its rate of neutralisation, the cells lead into oxidative stress condition. Consequently, cells exhibit several pathophysiological states including that of apoptosis ŽChandra et al., 2000.. And in general, lipid peroxidation is taken as an index of oxidative stress ŽKappus, 1985.. Recently, the regression of tails in tadpoles of anurans during metamorphosis has drawn considerable attention as an ideal model to understand the phenomenon of apoptosis. Earlier studies have indicated the crucial role of hydrogen peroxide in tail regression during spontaneous metamorphosis in several species of frogs ŽKashiwagi et al., 1999; Hanada et al., 1997.. Furthermore, using Rana japonica tadpole tail as a model system to unveil the mechanism of tail regression, Kashiwagi et al. Ž1999. observed that T4 enhances generation of NO which in turn inhibits catalase activity and results in augmentation of H 2 O 2 , thereby triggering apoptosis. An increase in the generation of superoxide radicals in the tadpole tail tissue of Rana catesbeina undergoing regression was also recorded ŽLittle and Flores, 1990.. The involvement of macrophages as effectors of cell death has also been described ŽWeber, 1968; Lohmann-Matthes et al., 1972; Nathan et al., 1980.. In anuran tail the macrophages undergo activation prior to performing functions such as phagocytosis or autolysis. There is a progressive activation of macrophages, beginning in connective tissue of tail fin and spreading later to the connective tissue of the tail stem ŽWeber, 1968; Kerr et al., 1974.. Production of hydrogen peroxide Žoxidant. by macrophages in the tail of metamorphosing Rana japonica has been demonstrated by Sasaki et al. Ž1988.. It is also evident that in most of the biological system, an increase in oxidants Žhydrogen peroxide and superoxide radicals. lead to lipid peroxidation, an index of oxidative stress ŽSevanian and Hochstein, 1985.. Apart from the ROS, the role of acid phosphatase, a lysosomal enzyme has also gained attention in the tail regression phenomenon of tadpoles during metamorphosis ŽGilbert and Frieden, 1981.. While there is ample information available in the literature on the role of ROS in regression of
tail of tadpole of anurans particularly in various species of genus Rana during metamorphosis, very little information is available on species of other genus of anurans. Furthermore, a comparative study of the role of ROS in tail regression among various species of anurans will help in understanding the physiological role of ROS in tail regression, thereby apoptosis. Since it is well known that tail regression begins in anuran tail at stage XX ŽSasaki et al., 1988. two critical stages, i.e. stage III Žhind limb bud. and stage XVIII Žwell developed hind limbs. are taken as stages prior to tail regression, and two more stages, i.e. stage XXI Žboth fore limbs emerged. and stage XXIII Žmore than half of tail regressed. are taken as stages following tail regression Žstages according to Taylor and Kollros, 1946.. The present study sets out to investigate: Ži. whether oxidative stress is generated in the tail of the tadpoles of the Indian jumping frog Polypedates maculatus ŽAnura, Rhacophoridae. prior to tail regression or during tail regression; and if so, Žii. whether there exists any correlation between oxidative stress and acid phosphatase, a lysosomal enzyme. Presence of cobalt sensitive acid phosphatase is reported for the first time in anuran tail.
2. Materials and methods 2.1. Chemicals Chemicals used in this study were of analytical grade. Thiobarbituric acid ŽTBA., bovine serum albumin ŽBSA. and mercaptoethanol were obtained from Sigma Chemical Co.; USA, sodium dodecyl sulfate ŽSDS., nicotinamide adenine disodium salt ŽNADH., horse radish peroxidase ŽHRP. and p-nitrophenyl phosphate were obtained from Sisco Research Laboratory, Mumbai, India; Sephadex G-25 was obtained from Pharmacia Biotech, Sweden; Folin᎐Ciocalteus reagent was obtained from Qualigens Fine Chemicals, Glindia Ltd., Mumbai, India. All other chemicals were of the highest purified grade available. 2.2. Tadpoles Foam nests containing eggs of Polypedates maculatus were collected from nature during the monsoon period ŽJuly᎐September, year
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1998᎐2000.. The hatchlings were reared in the laboratory following standard procedure ŽMohanty-Hejmadi et al., 1992.. They were fed with boiled Amaranthus green and boiled egg ad libitum. Four stages of tadpoles, i.e. stage III Žhind limb bud stage., stage XVIII Žwell developed hind limbs., stage XXI Žboth fore limbs emerged. and stage XXIII Žmore than half of the tail regressed. of Taylor and Kollros Ž1946. were selected for the experiment. The tadpoles were anaesthetized with MS 222 prior to tail amputation through the base of the tail. For each assay, a pool comprising of five tadpoles were taken. 2.3. In¨ estigation of oxidati¨ e stress parameters To investigate oxidative stress in the tail during different developmental stages, the level of lipid peroxidation Žan index of oxidative stress . and hydrogen peroxide Žan oxidant or Reactive Oxygen Species., and the activities of superoxide dismutase and catalase Žboth enzymatic antioxidants. were estimated. To evaluate the lysosomal enzymes, the activity of acid phosphatase along with cobalt sensitive acid phosphatase were determined. 2.3.1. Lipid peroxidation The tissue lipid peroxidation ŽLPX. was assayed as thiobarbituric acid reacting substance ŽTBARS. by thiobarbituric acid ŽTBA. assay of Ohkawa et al. Ž1979.. A 10% Žwrv. homogenate was prepared with 1.5% KCl in an ice-cooled hand mortar. The homogenate was centrifuged at 4⬚C for 5 min at 1000 = g. The supernatant was taken for the assay. The assay mixture contained 0.4 ml sample, 0.1 ml of 8.1% SDS, 0.75 ml of 20% acetic acid ŽpH adjusted to 3.5 with NaOH. and 0.75 ml of 0.8% aqueous solution of TBA. The mixture was heated in a water bath for 60 min at 95⬚C using glass balls as condensers. The tubes were cooled in running tap water, centrifuged at 1000 = g for 10 min at room temperature and absorbance of the supernatant was read at 532 nm. Protein content of the sample was assayed by the method of Lowry et al. Ž1951.. The concentration of TBARS was calculated from extinction coefficient of 1.56 = 10 5 My1 cmy1 ŽWills, 1969.. The amount of TBARS was expressed as nmol malonaldehyde ŽMDA. formedr mg protein.
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2.3.2. Hydrogen peroxide The level of hydrogen peroxide ŽH 2 O 2 . was measured by the method of Pick and Keisari Ž1981. based on the H 2 O 2-mediated and horse radish peroxidase ŽHRPO.-dependent oxidation of phenol red. A 10% homogenate Žwrv. was prepared with 50 mM phosphate buffer ŽpH 7.4., centrifuged for 10 min at 1000 = g at 4⬚C. The supernatant was taken for the assay. The assay mixture contained 0.28 mM phenol red, 1 unit of HRPO and 500 g protein in 50 mM phosphate buffer ŽpH 7.4., in the final volume of 2.0 ml. Absorbance was measured at 610 nm. The level of hydrogen peroxide was expressed as nmol H 2 O 2rmg protein. 2.3.3. Superoxide dismutase The method of Paoletti and Macoli Ž1990. was followed to measure superoxide dismutase ŽSOD.. A 10% Žwrv. homogenate was prepared with 50 mM phosphate buffer ŽpH 7.4. and centrifuged at 10 000 = g for 20 min at 4⬚C. The supernatant was passed through Sephadex G-25 column Ž1 ml supernatant through 5 ml Sephadex column. with 50 mM phosphate buffer ŽpH 7.4.. The second 2 ml was taken for the assay in the above fraction. Assay mixture contained: NADH 7.5 mM; EDTArMnCl 2 100 mMr50 mM; mercaptoethanol 28 mM; 12.5 g protein; and phosphate buffer 60 mM ŽpH 7.4. to make the final volume 3 ml. Absorbance was taken at 340 nm. The reading was observed at the 8th and 16th min after addition of mercaptoethanol. The level of SOD was expressed as unitsrmg protein w1 unit of SODs Ždifference in OD of blankrdifference in OD of sample. y 1x. 2.3.4. Catalase Catalase activity was measured according to the method of Aebi Ž1974.. A 10% Žwrv. homogenate was prepared with 50 mM phosphate buffer ŽpH 7.0.. The homogenate was centrifuged at 10 000 = g for 10 min at 4⬚C. The supernatant was used for the assay. The assay mixture contained: 0.1 ml Ž60 mM. H 2 O 2 ; 150 g protein; 50 mM phosphate buffer ŽpH 7.0. to make the final volume 3 ml. The reaction was initiated by adding 0.1 ml H 2 O 2 Ž60 mM.. The absorbance was taken at 240 nm and reading was noted at a 60-s interval. Catalase activity was expressed as pmolrmg proteinrmin w1 mol sy1 s 1 katal Žkat.x.
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2.3.5. Acid phosphatase Acid phosphatase ŽACP. activity was determined according to the method of Guha et al. Ž1974. with p-nitrophenyl phosphate as substrate. A 10% homogenate Žwrv. was made with 0.25 M sucrose. The homogenate was treated with Triton X-100 Žfinal conc. 0.1%, vrv. for 15 min in ice. The homogenate was centrifuged at 4⬚C for 10 min at 8000 = g. The assay mixture contained 100 g protein in a total volume of 0.6 ml of 0.1 M acetate buffer ŽpH 5.0.. After preincubation of 5 min at room temperature, 0.3 ml of p-nitrophenyl phosphate Ž1 mM dissolved in 0.1 M acetate buffer, pH 5.0. was added to it. The incubation was carried out at 37⬚C for 30 min. The reaction was stopped by adding 2.1 ml of 0.5 M NaOH solution. Absorbance was measured at 410 nm. The standard was prepared using various concentrations of p-nitrophenol. In order to study the effect of cobalt acetate on acid phosphatase activity, the supernatant was pre-incubated with acetate buffer containing cobalt acetate Ž10 mM final concentration . for 5 min at room temperature. The enzyme activity was expressed as mol p-nitrophenol ŽpNP. formedrmg proteinrmin at 37⬚C. 2.4. Statistics Statistical analysis was followed by Gomez and Gomez Ž1984.. The ANOVA test to find out significance difference between means was calculated by Duncan’s multiple range test. The same superscripts over the bars in Figs. 1᎐5 represent the data which are not significantly different.
Fig. 1. Endogenous level of TBARS Žnmol MDA formedrmg protein. at different developmental stages in the tail of Polypedates maculatus. Data are expressed as mean " S.D. of five observations ŽS.D., standard deviation.. Same superscripts over the bar represent data that are not significantly different.
to XXIII of tadpoles, there was no statistical difference in levels of hydrogen peroxide between stages XVIII and XXI ŽFig. 2..
3. Results 3.1. Lipid peroxidation In the present study, lipid peroxidation level was recorded to be the lowest in the stage XVIII tadpoles, which gradually increased in XXI and XXIII stages of tadpoles ŽFig. 1.. However, lipid peroxidation in stage III was observed to be higher than in stage XVIII. 3.2. Hydrogen peroxide Although the level of H 2 O 2 increased gradually in regressing tails of tadpoles from stage III
Fig. 2. The level of hydrogen peroxide Žnmol H 2 O 2 rmg protein. at different developmental stages in the tail of Polypedates maculatus. Data are expressed as mean " S.D. of five observations. Same superscripts over the bar represent data that are not significantly different.
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Fig. 3. The level of superoxide dismutase Žunits of SODrmg protein. at different developmental stages in the tail of Polypedates maculatus. Data are expressed as mean " S.D. of five observations. Same superscripts over the bar represent data that are not significantly different.
3.3. Antioxidant Enzymes Superoxide dismutase ŽSOD. activity was the lowest in stage III tadpoles ŽFig. 3.. The activity
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Fig. 5. The activity of normal acid phosphatase Žmol pNP formedrmg proteinrmin. and Co 2q-sensitive acid phosphatase Žmol Co 2q-sensitive pNP formedrmg proteinrmin. at different developmental stages in the tail of Polypedates maculatus. Data are expressed as mean " S.D. of five observations. Same superscripts over the bar represent data that are not significantly different.
increased before the on set of metamorphosis Žstage XVIII.. A further increase in the enzyme activity was recorded as tail regression proceeds. In contrast to SOD, the activity of catalase ŽFig. 4. was higher in stage III tadpoles, which then decreased in stage XVIII and XXI, and then increased in stage XXIII. 3.4. Acid phosphatase In the present study the activity of acid phosphatases Žnormal. was the lowest in stage III tadpoles ŽFig. 5. which increased gradually in tadpoles of stages XVIII, XXI and XXIII, respectively. In contrast to the normal acid phosphatase, the activity of Co 2q-sensitive acid phosphatase was higher in stage III, decreased in stage XVIII and increased gradually thereafter till stage XXIII ŽFig. 5..
Fig. 4. Catalase activity Žpmolrmg proteinrmin. at different developmental stages in the tail of Polypedates maculatus. Data are expressed as mean " S.D. of five observations. Same superscripts over the bar represent data that are not significantly different.
4. Discussion Increase in the level of lipid peroxidation in the regressing tail of Polypedates maculatus during
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spontaneous metamorphosis is not surprising, and is in good agreement with the results of earlier finding in other species of anurans. It is reported that hydrogen peroxide production by macrophages occurred in the tails of metamorphosing Rana japonica tadpoles ŽSasaki et al., 1988.. Similarly, superoxide production is also elevated in tadpole tissue of Rana catesbeiana Žat stage XVIII. during metamorphosis, along with rise in -glucuronidase activity ŽLittle and Flores, 1990.. The higher level of lipid peroxidation at stage III of tadpole may be associated with a comparatively higher level of cell division of the growing tail. We have observed high level of lipid peroxidation in the regenerating tail of Polypedates maculatus Žunpublished data.. The level of hydrogen peroxide, an oxidant, is noticed to be low during earlier stage Žstage III. of metamorphosis and is gradually increased as tail regression proceeds. The pattern of hydrogen peroxide level in tails of different stages of tadpole is similar to that of superoxide dismutase. Superoxide dismutase is an enzyme which dismutates superoxide radicals to hydrogen peroxide. In contrast to our results, Kashiwagi et al. Ž1999. observed that when tadpole tail of Rana japonica in culture were exposed to thyroxin, hydrogen peroxide could not be detected either in culture media or in tail homogenates. The difference between results obtained by Kashiwagi et al. Ž1999. and us may be due to different experimental conditions and selection of a different species. It is well known that regression of tail in amphibian tadpole begins at stage XX ŽSasaki et al., 1988.. It is evident from our study that lipid peroxidation is detectable by stage XVIII. Since LPX is the end point of ROS reaction, it is possible that production of the ROS in tail Žeither by macrophages or by cells of tail tissue in response to some stimuli. is initiated earlier than stage XVIII. We have detected both LPX and H 2 O 2 in stage III tadpoles. Although the origin . of reactive oxygen species ŽOy 2 , H 2 O 2 and their role in the process of tissue regression of this species is not clear, their role in programmed cell death in regressing tadpole tail is suggested ŽSasaki et al., 1988.. A decrease in catalase activity in the regressing tail was noted by Hanada et al. Ž1997. and Kashiwagi et al. Ž1999.. Our result corroborates their findings. However, in contrast to their findings, we have observed an increase in the activity of
catalase in stage XXIII tadpoles. This difference is suggested to be due to different experimental conditions and selection of a different species. A sharp increase in acid phosphatase activity is observed in the anuran tail during regression ŽWeber, 1968.. The increase is suggested to be due to progressive release of enzyme from preformed lysosomes of the tail tissue in response to the hormone thyroxine or released by the phagocytic macrophages which accumulate in large numbers in the regressing tail ŽWeber, 1968; Gilbert and Frieden, 1981.. Presence of multiple forms of acid phosphatase ŽACP. according to their inhibition by several inhibitors molecular weight activation by various agents, particularly divalent metal ions, has been reported for animal tissues ŽVanha-Pertulla, 1971.. A Co 2q-sensitive acid phosphatase in the soluble fraction of testicular homogenate of mammals has been reported, whose activity increases during proliferation of spermatogenic cells and decreases during suppression of spermatogenesis in different experimental conditions ŽChainy et al., 1995.. To the best of our knowledge, there is no report available on Co 2q-sensitive ACP in the tails of frogs. This is the first time we report the presence of Co 2q-sensitive ACP in the tail and its change during tail regression in Polypedates maculatus. The high level of Co 2q-sensitive ACP in stage III may be associated with cell proliferation since similar increase in activity has been observed during spermatogenesis in mammals ŽChainy et al., 1995.. It will be of interest to find out the origin and significance of Co 2q-sensitive ACP during tail regression and its regulation and expression by oxidative stress state. In addition, in the present study a positive correlation between: acid phosphatase and lipid peroxidation; hydrogen peroxide and lipid peroxidation; and acid phosphatase and hydrogen peroxide, was observed in the tail tissue during development ŽFig. 6.. The correlation between normal acid phosphatase and lipid peroxidation showed: r s 0.81, t s 6.06 and y s 0.01q 0.17x, respectively. Correlation between Co 2q-sensitive acid phosphatase and lipid peroxidation showed: r s 0.94, t s 12.73 and y s 0.27q 0.22 x, respectively. Similarly, the values of r s 0.72, t s 4.4 and y s 1.26 q 4.82 x, respectively, are the correlation between hydrogen peroxide and lipid peroxidation. For acid phosphatase and hydrogen peroxide correlation showed: r s 0.93, t s 10.83 and y s
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Fig. 6. Correlation between TBARS Žnmol MDA formedrmg protein. and Ža. activity of acid phosphatase Žmol pNP formedrmg proteinrmin., Žb. activity of Co 2q-sensitive acid phosphatase Žmol Co 2q-sensitive pNP formedrmg proteinrmin., and Žc. the level of hydrogen peroxide Žnmol H 2 O 2 rmg protein.. Žd. Correlation between level of hydrogen peroxide Žnmol H 2 O 2 rmg protein. and activity of acid phosphatase Žmol pNP formedrmg proteinrmin. at different developmental stages ŽIII, XVIII, XXI and XXIII. in the tail of Polypedates maculatus.
0.05q 0.03 x, respectively. In all the cases, r ) P at 0.001 showed the correlation to be significant. The most interesting finding of the present investigation is the identical pattern of changes in the level of LPX, activity of catalase and Co 2qsensitive acid phosphatase ŽFigs. 1, 4 and 5.. At stage III the value of the above oxidative stress parameters is higher than stage XVIII which increases in stage XXI and maximum is observed in
stage XXIII. Another identical pattern of changes was observed in the level of hydrogen peroxide, activity of SOD and normal acid phosphatase ŽFigs. 2, 3 and 5.. From our finding it is reasonable to conclude that oxidative stress is generated prior to tail regression Žstage III, XVIII. at a lower rate which increases significantly in the regressed tail Žstage XXIII.. Furthermore, the correlation between oxidative stress and acid
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phosphatase, suggests existence of a positive correlation between oxidative stress and lysosomal activity in the pre-regressing and regressing tail of Polypedates maculatus. However, it will be of interest to unveil the specific signalling pathways through which oxidative stress triggers tail regression in the species.
Acknowledgements PKM thanks the Council of Scientific and Industrial Research, Government of India for a Senior Research Associateship ŽScientist’s Pool Scheme., pool no 13Ž7338-A. year 1998. We also thank the University Grants Commission ŽSAP. and DBT, Government of India for financial support. References Aebi, H., 1974. Catalase. In: Bergmeyer, H.U. ŽEd.., Methods in Enzymatic Analysis, Vol. 2. Academic Press, New York, pp. 673᎐678. Chainy, G.B.N., Swain, C., Sahoo, A., Mohanty, K.C., 1995. Effects of Estradiol, testosterone and Bromocriptine on rat testicular acid phosphatase activity. Biochem. Arch. 11, 111᎐114. Chandra, J., Samali, A., Orrenius, S., 2000. Triggering and modulation of apoptosis by oxidative stress. Free Radic. Biol. Med. 29 Ž3r4., 323᎐333. Gilbert, L.I., Frieden, E., 1981. Metamorphosis: A Problem in Developmental Biology, 2nd edition. Plenum Press, New York, pp. 139᎐176. Gomez, K.A., Gomez, A.A., 1984. Statistical Procedures for Agricultural Research, 2nd edition. WileyInterscience Publication. Guha, K., Karkkainen, R.R., Vanha-Perttula, T., 1974. Testicular acid phosphatase of the mouse. Med. Biol. 57, 52᎐57. Halliwell, B., Gutteridge, J.M.C., 1985. Free Radicals in Biology and Medicine. Clarendon Press, Oxford. Hanada, H., Kashiwagi, A., Takehara, Y. et al., 1997. Do reactive oxygen species underline the mechanism of apoptosis in the tadpole tail? Free Radic. Biol. Med. 23 Ž2., 294᎐301. Kappus, H., 1985. Lipid peroxidation, mechanism, analysis, enzymology and biological relevance. In: Sies, H. ŽEd.., Oxidative Stress. Academic Press, London, pp. 237᎐310. Kashiwagi, A., Handa, H., Yabuki, M. et al., 1999. Thyroxine enhancement and their role in reactive oxygen species in tadpole tail apoptosis. Free Radic. Biol. Med. 26 Ž7r8., 1001᎐1009.
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