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Oxidative stress during vitamin A-induced abnormal tail regeneration in the tadpoles of Polypedates maculatus
Comparative Biochemistry and Physiology Part B 131 (2002) 403–410
Oxidative stress during vitamin A-induced abnormal tail regeneration in the tadpoles of Polypedates maculatus Pravati Kumari Mahapatraa,*, Priyambada Mohanty-Hejmadia, Gagan B.N. Chainyb a
Developmental Biology Laboratory, Department of Zoology, Utkal University, Bhubaneswar-751004, India b Biochemistry Unit, Department of Zoology, Utkal University, Bhubaneswar-751004, India Received 3 August 2001; received in revised form 12 November 2001; accepted 22 November 2001
Abstract Vitamin A and its derivatives inhibit normal tail regeneration in amphibians. The most remarkable effect is the development of limbs at the cut end of the tail in anurans. Prior to ectopic limb development, there is an abnormal tail regeneration in the treated tadpoles. The purpose of the present study was to compare oxidative stress condition in the regenerated tail of normal and vitamin A (10I Uyml, 72 h) treated tadpoles. The present findings show a hyper-oxidative stress condition in the regenerated tail of the vitamin A-treated tadpoles of the Indian jumping frog, Polypedates maculatus (Anura: Rhacophoridae). 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Anuran; Tail amputation; Normal tail regeneration; Abnormal tail regeneration; Homeotic transformation; Ectopic limb; Oxidative stress; Vitamin A
1. Introduction Normal tail regeneration in anurans is inhibited by vitamin A and its derivatives, the retinoids (Niazi and Saxena, 1968; Mahapatra and MohantyHejmadi, 1994; Mahapatra et al., 2001a; Muller et al., 1996; Scadding, 1987). Retinoids also have a multiple effect on regenerating anuran limbs, ranging from proximo-distal duplication, antero-posterior duplication, hypomorphic or phocomelic limb to complete inhibition of limb regeneration (Bryant and Gardiner, 1992; Mahapatra and MohantyHejmadi, 1994; Mahapatra et al., 2001b; Scadding, 1996). The most striking effect of vitamin A is the induction of ectopic limb development (homeotic transformation of tail to limb) at the cut end of the tail in anurans (Das and Mohanty-Hejmadi, 1999; Maden, 1993; Mahapatra and MohantyHejmadi, 1994; Mahapatra et al., 2001b; Mohanty*Corresponding author. E-mail address: mahap [email protected] (P.K. Mahapatra).
Hejmadi et al., 1992; Muller et al., 1994, 1996). However, the precise mechanism of homeotic transformation of tail to limbs has not yet been properly understood. It is known that oxygenderived free radicals or oxidants, products of oxygen metabolism, are one of the causative factors underlying development and differentiation (Allen, 1991). Oxygen-derived free radicals are also very important mediators of cell injury and death (Joseph and Knight, 1995), and the cellular environment becomes more pro-oxidizing during differentiation (Sies, 1997). Cells contain antioxidant defenses that respond to variations in cellular oxidant production. The imbalance between oxidants and antioxidants is termed oxidative stress (Sies, 1997). Evidence is present that implicates oxidants as a factor that can stimulate alteration in gene expression (Allen, 1991; Schulze-Osthoff and Baeuerle, 1998) and the appearance of new tissue is preceded by the transcription of tissue-specific genes and the concomitant suppression of tran-
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P.K. Mahapatra et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 403–410
scription of genes that are specific to pleuripotent stem cells (Allen, 1991). During homeotic transformation of tail into limbs, the possible cause has been explained earlier as the change in positional value of the tail blastema cells to body flank (Bryant and Gardiner, 1992) and activation of limb-specific homeotic genes (Bryant and Gardiner, 1992; Maden, 1993; Mahapatra, 1994; Mahapatra and Mohanty-Hejmadi, 1994; MohantyHejmadi et al., 1992; Muller et al., 1996). Histological studies of the abnormal tail have shown a massive amount of notochord in the regenerated tail (Das and Mohanty-Hejmadi, 1999; Maden, 1993; Mahapatra et al., 2001b; Muller et al., 1996) along with a thickened notochordal sheath, thick nerve cord and scattered muscle (mesenchymal) cells. The aim of the present investigation was to study the oxidative stress conditions during abnormal and normal tail regeneration in the tadpoles of the Indian jumping frog, P. maculatus (Anura, Rhacophoridae). The species was selected because of its prolific reaction to vitamin A treatment, as reported earlier (Mahapatra and Mohanty-Hejmadi, 1994). In the present study, lipid peroxidation has been investigated as an index of oxidative stress. The level of hydrogen peroxide (an oxidant) has also been investigated. Furthermore, the activity of two enzymatic antioxidants, related to normal development of anurans, i.e. superoxide dismutase (SOD) and catalase, and the level of reduced glutathione (GSH), a non-enzymatic antioxidant expressed during regeneration and cell division has been analyzed. 2. Materials and methods 2.1. Chemicals Chemicals used in this study were of analytical grade. Thiobarbituric acid (TBA), bovine serum albumin (BSA), 5,59-dithiobis-2-nitrobenzoic acid (DTNB) and mercaptoethanol were obtained from Sigma Chemical Co.; USA, sodium dodecyl sulf (SDS), nicotinamide adenine disodium salt (NADH), horseradish peroxidase (HRP) and reduced glutathione (GSH) were obtained from Sisco Research Laboratory, Mumbai, India; Sephadex G-25 was obtained from Pharmacica 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 P. maculatus were collected from nature during the monsoon period (July–September, 1998–2000). The hatchlings were reared in the laboratory following standard procedure (Mohanty-Hejmadi et al., 1992). They were freely fed with boiled Amaranths greens and boiled egg. 2.3. Tail amputation and treatment Limb-bud stage tadpoles were taken for the experiment. Tadpoles were anaesthetized with MS 222 prior to tail amputation through the middle of the tail. Following amputation, the experimental tadpoles were treated with vitamin A palmitate (Arovit, Roche) 10 IUyml for 72 h (the optimal treatment condition), then transferred to conditioned water. The control tadpoles were reared in conditioned water following amputation. For both the treated and control group of amputated tadpoles, parallel non-amputated, i.e. vitamin A treatment without tail amputation and control without tail amputation, tadpoles were reared. 2.4. Estimation of oxidative stress The original tail tips of limb-bud stage tadpoles were analyzed as the original group. Regenerated tails of 5, 10, 15 and 20 days post-amputated tadpoles of both the control and experimental groups were taken for analysis as the control and treated group, respectively (Table 1). Five days post-amputated tadpoles were selected to study oxidative stress during the earlier stage of regeneration, when the regenerated tail blastema was distinct. The 20 days post-amputated tadpoles were analyzed because bulbular mass, a prerequisite for ectopic limb development, was visible by this period. The 10 and 15 days post-amputated tadpoles were taken as the intermediate stages. Tail of parallel non-amputated tadpoles of both the control and vitamin A-treated tadpoles were also taken after 5, 10, 15 and 20 days of the experiment. For each assay, a pool comprising of 50 tadpoles was taken. 2.4.1. Lipid peroxidation The tissue lipid peroxidation (LPX) was assayed as thiobarbituric acid reacting substance (TBARS) by the thiobarbituric acid (TBA) assay of Ohkawa
P.K. Mahapatra et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 403–410 Table 1 Changes in oxidative stress parameters of the regenerated tail of control (C) and vitamin A-treated (T) tadpoles of P. maculatus Oxidative stress parameters
Group
LPXa
C T
H 2O 2 c
Days following tail amputation 5
10
15
20
1.16b 1.61
1.08 1.91
0.8 1.16
0.63 1.07
C T
1.87 3.85
1.88 5.37
1.82 2.04
1.14 1.27
SODd
C T
14.37 9.06
8.1 9.38
6.9 4.19
0.91 0.9
CATe
C T
1 1.6
1.21 1.96
1.3 1.81
1.09 2.12
GSHf
C T
2.11 2.11
1.4 1.67
1.15 1.29
1.15 1.18
a Level of lipid peroxidation (nmol MDA formedymg protein). b Values in folds relative to original; C, control group; T, treated group. c Level of H2O2 (nmolymg protein). d Activity of SOD (units of SODymg protein). e Activity of Catalase (pmolymg proteinymin). f Level of Glutathione (mmygm tissue).
et al. (1979). A 10% (wyv) homogenate was prepared with 1.5% KCl in an ice-cooled hand mortar. The homogenate was centrifuged at 4 8C for 5 min at 1000=g. The supernatant was taken for the assay. The assay mixture contained a 0.4ml sample, 0.1 ml of 8.1% SDS, 0.75 ml of 20% acetic acid (pH 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 8C, using glass balls as condensers. The tubes were cooled in running tap water, centrifuged at 1000=g for 10 min at room temperature and the absorbence of the supernatant was read at 532 nm. The protein content of the sample was assayed by the method of Lowry et al. (1951). The concentration of TBARS was calculated from the extinction coefficient of 1.56=105 My1 cmy1 (Wills, 1969). The amount of TBARS was expressed as nmol malonaldehyde (MDA) formedymg protein. 2.4.2. Hydrogen peroxide The level of hydrogen peroxide (H2O2) was measured by the method of Pick and Keisari (1981), based on the H2O2-mediated and horseradish peroxidase (HRPO)-dependent oxidation of phenol red. A 10% homogenate (wyv) was pre-
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pared with 50 mM phosphate buffer, pH 7.4, centrifuged for 10 min at 1000=g at 4 8C. The supernatant was taken for the assay. The assay mixture contained 0.28 mM phenol red, 1 unit of HRPO and 500 mg protein in 50 mM phosphate buffer, pH 7.4, in the final volume of 2.0 ml. Absorbence was measured at 610 nm. The level of hydrogen peroxide was expressed as nmol H2O2 ymg protein. 2.4.3. Superoxide dismutase (SOD) Superoxide dismutase activity was measured according to the method of Paoletti and Macoli (1990). A 10% (wyv) homogenate was prepared with 50 mM phosphate buffer, pH 7.4 and centrifuged at 10 000=g for 20 min at 4 8C. The supernatant was passed through a Sephadex G-25 column (1 ml supernatant through 5 ml Sephadex column) with 50 mM phosphate buffer, pH 7.4. The second 2 ml was taken for the assay in the above fraction. The assay mixture contained NADH 7.5 mM, EDTAyMnCl2 100y50 mM, mercaptoethanol 28 mM, 12.5 mg protein and phosphate buffer 60 mM, pH 7.4 to make the final volume 3 ml. The absorbence was taken at 340 nm and readings were noted at the 8th and 16th min after addition of mercaptoethanol. The activity of SOD was expressed as unitsymg protein w1 unit of SODs(difference in OD of blankydifference in OD of sample)y1x. 2.4.4. Catalase Catalase activity was measured according to the method of Aebi (1974). A 10% (wyv) homogenate was prepared with 50 mM phosphate buffer, pH 7.0. The homogenate was centrifuged at 10 0005=g for 10 min at 4 8C. The supernatant was used for the assay. The assay mixture contained 0.1 ml (60 mM) H2O2, 150 mg protein, 50 mM phosphate buffer, pH 7.0 to make the final volume 3 ml. The reaction was initiated by adding 0.1 ml H2O2 (60 mm) and absorbence was noted at 240 nm at a 60-s interval. Catalase activity was expressed as pmolymg proteinymin w1 mol secy1s1 katal (kat)x. 2.4.5. Reduced glutathione (GSH) The assay of GSH was done following the method of Ellman (1959). A 10% (wyv) homogenate was prepared with 5% metaphosphoric acid. The homogenate was kept at room temperature for 15 min, centrifuged for 30 min at 1000=g at room
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Fig. 1. Lipid peroxidation (nmol MDA formedymg protein) in the original and regenerated tail of the control and vitamin A (10I Uyml)-treated tadpoles following 5, 10, 15 and 20 days of post-amputation, respectively in the tadpoles of P. maculatus.
original tail. The oxidative stress of the parallel non-amputated tadpole tails (both control and treated) were also compared with the original tail and no significant difference was observed. Therefore, comparative data have been represented for the regenerated tail of the control and treated tadpoles. In the regenerated tail of 5 days postamputated (dpa) tadpoles of the control group, lipid peroxidation (LPX) was higher than that of the original tadpoles (Fig. 1). A gradual decrease in the level was noticed in the regenerating tail following 10, 15 and 20 days of amputation in the control group. On the other hand, a gradual elevation in LPX was noticed in regenerating tails of the vitamin A-treated group up to 10 days and thereafter a reduction in its level was recorded in the 15 and 20 dpa tadpoles (Fig. 1). However, the level of LPX was always higher in the treated group than the respective control tadpoles. A similar trend was noticed in the level of hydrogen peroxide (Fig. 2). The level of H2O2 was remarkably higher in the treated tadpoles than the respective control tadpoles up to 10 days of post-amputation. There was a fall in the level and by the 20th day the level declined further and was
temperature. The supernatant was used for the assay. The assay mixture contained 0.5 ml supernatant, 2.5 ml DTNB (5,59-dithiobis-2-nitrobenzoic acid) 0.3 mM. The mixture was incubated for 25 min at room temperature and centrifuged for 5 min at 4500=g. The absorbence was taken at 412 nm. The level of GSH was expressed as mmygm tissue. 2.5. Statistics Statistical analysis was followed by the method of Gomez and Gomez (1984). The ANOVA test to find out the significant 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 (control and treated to be considered separately). 3. Results The regenerated tail of the control and treated tadpoles were analyzed and compared with the
Fig. 2. Level of H2O2 (nmolymg protein) in the original and regenerated tail of the control and vitamin A (10I Uyml)-treated tadpoles following 5, 10, 15 and 20 days of post-amputation, respectively in the tadpoles of P. maculatus.
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higher throughout and was always higher than the respective control. The maximum level was recorded in 20 dpa tadpoles of the treated group. The level of reduced glutathione (GSH) in control and the vitamin A-treated group was the highest in 5 dpa tadpoles. Thereafter, a gradual fall in the level was noticed in both the groups (Fig. 5). A comparative account of the changes in oxidative stress parameters in the regenerated tail of the control and treated tadpoles has been summarized in Table 1. 4. Discussion
Fig. 3. Superoxide dismutase (units of SODymg protein) in the original and regenerated tail of the control and vitamin A (10I Uyml) -treated tadpoles following 5, 10, 15 and 20 days of post-amputation, respectively in the tadpoles of P. maculatus.
close to the original tail value. For both lipid peroxidation and H2O2, the highest level was observed in the regenerated tail of the experimental tadpoles 10 days after amputation (Fig. 1 and Fig. 2). In comparison to the control group, a lesser elevation in the activity of SOD was recorded in regenerated tails of 5 dpa tadpoles of the treated group (Fig. 3). In the treated group, activity remained unaltered in 10 dpa tadpoles, thereafter a decrease was observed in the 15-dpa tadpole. In the control group, there was a fall in activity in 10 and 15 dpa tadpoles. An interesting observation was that, for both the treated and experimental tadpoles, the activity of SOD was close to the original tail by the 20th day of amputation (Fig. 3). No change in catalase activity in the regenerating tail of 5 dpa tadpoles of the control group was noted in comparison to the original group (Fig. 4). There was an increase in activity, which remained unchanged, from 10 to 20 dpa tadpoles. In the treated group, the catalase activity remained
We have observed a higher level of lipid peroxidation and H2O2 in 10 dpa tadpoles of the treated group (Fig. 1 and Fig. 2). The level is always more in the treated group than the respective control group (Table 1). H2O2 has been proposed to act as a second messenger molecule, affecting the proliferation and differentiation of cells (Schulze-Osthoff and Fiers, 1991). Histolog-
Fig. 4. Catalase activity (pmolymg proteinymin) in the original and regenerated tail of the control and vitamin A (10I Uyml)treated tadpoles following 5, 10, 15 and 20 days of post-amputation, respectively in the tadpoles of P. maculatus.
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Fig. 5. Level of Glutathione (mmygm tissue) in the original and regenerated tail of the control and vitamin A (10I Uyml) -treated tadpoles following 5, 10, 15 and 20 days of post-amputation, respectively in the tadpoles of P. maculatus.
ical studies have revealed that in the treated tadpoles, de-differentiations of normal tail tissue do not occur during regeneration. In contrast, it leads to development of an abnormal bulbular mass, mainly containing a huge mass of notochordal cells and scattered mesenchymal cells (Das and Mohanty-Hejmadi, 1999; Maden, 1993; Mahapatra et al., 2001b; Muller et al., 1996). A positive relation between the high level of H2O2 and abnormal tail regeneration in the treated group is suggested. A comparatively high activity of SOD (Fig. 3) was observed in 5 dpa tadpoles of the control group. An increase in the level of SOD has been observed during development in several amphibians (Barja De Quiroga and Gutierrez, 1984). It has been explained that increases in SOD activity associated with development are partly due to enzyme induction, it is oxidation associated with the increase in SOD rather than the antioxidant properties of the enzyme that seem to play a role in stimulating changes in gene expression (Allen
and Balin, 1989; Allen, 1991). The level of LPX and H2O2 in 5 dpa tadpoles of the control group is comparatively less than that of the treated tadpoles and the level of SOD is more in the control group at the same time. Our finding support the view of Allen and Balin (1989), that high levels of SOD are not the antioxidant properties of SOD but of cell differentiation, as a normallooking tail regenerates in the control tadpole by the 5th day of amputation. The activity of catalase always remained higher in all the experimental tadpoles than the respective controls (Fig. 4). An increase in catalase activity has been observed in amphibians during development (Gil et al., 1987). It has been observed in frog muscle that increased catalase activity minimizes cellular oxidative stress by lowering the concentration of H2O2 (Hermis-Lima and Story, 1998). Rohrdanz and Kahl (1998) have reported that H2O2 induces the expression of catalase in cultured primary rat hepatocytes. In the present findings, an increase in catalase activity in the treated group is suggested to be due to an elevation in the level of H2O2 in the earlier stages, i.e. 5 and 10 dpa tadpoles (Fig. 2). It is known that higher catalase activity degrades H2O2 (Barja De Quiroga, 1992). We have observed a fall in the level of H2O2 in 15 and 20 dpa tadpoles (Fig. 2). This fall in H2O2 is suggested to be due to the higher activity of catalase during this period. It has been explained earlier that the hypersensitivity towards oxidative stress is retained by the expression of catalase (Schmidt et al., 1995). In our finding, a comparatively higher activity of catalase for a longer period in the treated group shows a hyper-oxidative stress condition in the regenerated tail of treated tadpoles. It is evident in amphibians that GSH concentration increases during the mitotic phase of regeneration and decreases during redifferentiation of regenerating tissue. Decrease in GSH is seen in cells exhibiting a decline in mitotic activity and is considered as an important factor that governs the onset of mitosis (Allen, 1991; Sohal et al., 1988). In our observation, there is a sharp rise in the level of GSH in 5 dpa tadpoles of both the control and treated tadpoles (Fig. 5). This rise is suggested to be due to the high level of cell division during this period. The decline in the level in 10–20 dpa tadpoles is suggested to be due to a slowing down in the rate of cell division.
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The remarkable observations of the present study include higher level of LPX (an index of oxidative stress) and H2O2 (an oxidant) in the 10 dpa tadpoles of the treated group (Fig. 2) and higher catalase activity in the treated tadpoles than the control group (Fig. 4). The above finding establishes a hyper-oxidative stress condition in the abnormally regenerated tail of the treated tadpoles in comparison to the respective, normally regenerated tail. Acknowledgments PKM thanks the Council of Scientific and Industrial Research, Council of Scientific Research, Government of India for a Senior Research Associateship (Scientist’s Pool Scheme), pool no 13 (7338-A), 1998. We also thank the University Grants Commission (SAP) for financial support. References Aebi, H., 1974. Catalase. In: Bergmeyer, H.U. (Ed.), Methods in enzymatic analysis, Vol. 2. Academic Press, New York, pp. 673–678. Allen, G.R., Balin, A.K., 1989. Oxidative influence on development and differentiation: an over view of a free radical theory of development. J. Free Rad. Biol. Med. 6, 631–661. Allen, G.R., 1991. Oxygen reactive species and Antioxidant responses during development: the metabolic paradox of cellular differentiation. Proc. Soc. Exp. Biol. Med. 196, 117–129. Barja De Quiroga, G., Gutierrez, P., 1984. Superoxide dismutase during development of two amphibian species and its role in hyperoxia tolerance. Mol. Physiol. 6, 221–232. Barja De Quiroga, G., 1992. Oxygen radicals, a failure or a success of evolution? Free Rad. Res. Comms. 18, 63–70. Bryant, S.V., Gardiner, D.M., 1992. Retinoic acid, local cell– cell interaction and pattern formation in vertebrate limbs. Dev. Biol. 152, 1–25. Das, P., Mohanty-Hejmadi, P., 1999. Histological effect of vitamin A on the tail amputated tadpoles of Polypedates maculatus with special reference to homeotic transformation. Cell Tissue Organs 164, 90–101. Ellman, G.L., 1959. Tissue sulfgydryl group. Arch. Biochem. Biophys. 82, 70–77. Gil, P., Alonso-Bedate, M., Barja De Quiroga, G., 1987. Different levels of hyperoxia reversibly induce catalase activity in amphibian tadpoles. Free Rad. Biol. Med. 3, 137–146. Gomez, K.A., Gomez, A.A., 1984. Statistical procedure for agricultural research. 2nd Wiley Interscience Publication. Hermis-Lima, M., Story, K.B., 1998. Role of antioxidant defense in the tolerance of severe dehydration by anurans. The case of the leopard frog Rana pipiens. Molecular and Cellular Biochemistry 189, 79–89.
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Joseph, A., Knight, M.D., 1995. Diseases related to oxygenderived free radicals. Ann. Clin. Lab. Sci. 25, 111–121. 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. Maden, M., 1993. The homeotic transformation of tail into limbs in Rana temporaria by retinoids. Dev. Biol. 159, 379–391. Mahapatra, P., 1994. Regeneration in anurans of Bhubaneswar. Ph.D. Thesis, Utkal University. Orissa, India. Mahapatra, P.K., Mohany-Hejmadi, P., 1994. Vitamin A: mediated Homeotic Transformation of Tail to limbs, limb suppression and Abnormal Tail Regeneration in the Indian Jumping frog. Polypedates maculatus. Dev. Growth and Differ. 36, 307–317. Mahapatra, P.K., Mohanty-Hejmadi, P., Dutta, S.K., 2001a. Polymelia in Bufo (Anura: Bufonidae). Current Science. (II) 80, 1447–1451. Mahapatra, Pravati Kumari, Das, Pragnya., Mohanty-Hejmadii, Priyambada., 2001b Homeotic transformation in Anurans. J. Zool. Soc. India (in press). 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. Muller, G., Striecher, J., Wurm, R., 1994. Structure and pattern in retinoid-induced ectopic limbs of anuran tadpoles. Proceedings of the second world congress of Herpetology. Adelaide, Australia. pp 176–177. Muller, G., Striecher, J., Muller, R., 1996. Homeotic duplicate of the pelvic body segment in regenerating tadpole tails induced by retinoic acid. Dev. Genes. Evol. 206, 344–348. Niazi, I.A., Saxena, S., 1968. Inhibitory and Modifying influence of the excess of vitamin A on tail regeneration in Bufo tadpoles. Expereintia 24, 852–853. 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 NAD(P)H oxidation. In: Packer, L., Glazer, A.N. (Eds.), Methods in Enzymology, Vol. 186. Academic Press, San Diego. Pick, E., Keisari, Y., 1981. Superoxide anions and hydrogen peroxide production by chemically elicited peritoneal macrophage: induction by multiple non-phagocytic stimuli. Cell. Immunol. 59, 301–318. Rohrdanz, E., Kahl, R., 1998. Alteration of antioxidant enzyme expression in response to hydrogen peroxide. Free Rad. Biol. Med. 24, 7–38. Scadding, S.R., 1987. Vitamin A inhibits amphibian tail regeneration. Can. J. Zool. 65, 457–459. Scadding, S.R., 1996. Treatment of axolotls with retinoids for limb regeneration studies. Int. J. Dev. Biol. 40, 909–910. Schmidt, N., Kerstin, A.P., Cerutti, P., Baeuerle, P.A., 1995. The role of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kB. Chem. Biol. 2, 13–22. Schulze-Osthoff, K., Fiers, W., 1991. Oxygen radicals as second messengers. Trends Cell Biol. 1, 150.
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Oxidative stress during vitamin A-induced abnormal tail regeneration in the tadpoles of Polypedates maculatus Pravati Kumari Mahapatraa,*, Priyambada Mohanty-Hejmadia, Gagan B.N. Chainyb a
Developmental Biology Laboratory, Department of Zoology, Utkal University, Bhubaneswar-751004, India b Biochemistry Unit, Department of Zoology, Utkal University, Bhubaneswar-751004, India Received 3 August 2001; received in revised form 12 November 2001; accepted 22 November 2001
Abstract Vitamin A and its derivatives inhibit normal tail regeneration in amphibians. The most remarkable effect is the development of limbs at the cut end of the tail in anurans. Prior to ectopic limb development, there is an abnormal tail regeneration in the treated tadpoles. The purpose of the present study was to compare oxidative stress condition in the regenerated tail of normal and vitamin A (10I Uyml, 72 h) treated tadpoles. The present findings show a hyper-oxidative stress condition in the regenerated tail of the vitamin A-treated tadpoles of the Indian jumping frog, Polypedates maculatus (Anura: Rhacophoridae). 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Anuran; Tail amputation; Normal tail regeneration; Abnormal tail regeneration; Homeotic transformation; Ectopic limb; Oxidative stress; Vitamin A
1. Introduction Normal tail regeneration in anurans is inhibited by vitamin A and its derivatives, the retinoids (Niazi and Saxena, 1968; Mahapatra and MohantyHejmadi, 1994; Mahapatra et al., 2001a; Muller et al., 1996; Scadding, 1987). Retinoids also have a multiple effect on regenerating anuran limbs, ranging from proximo-distal duplication, antero-posterior duplication, hypomorphic or phocomelic limb to complete inhibition of limb regeneration (Bryant and Gardiner, 1992; Mahapatra and MohantyHejmadi, 1994; Mahapatra et al., 2001b; Scadding, 1996). The most striking effect of vitamin A is the induction of ectopic limb development (homeotic transformation of tail to limb) at the cut end of the tail in anurans (Das and Mohanty-Hejmadi, 1999; Maden, 1993; Mahapatra and MohantyHejmadi, 1994; Mahapatra et al., 2001b; Mohanty*Corresponding author. E-mail address: mahap [email protected] (P.K. Mahapatra).
Hejmadi et al., 1992; Muller et al., 1994, 1996). However, the precise mechanism of homeotic transformation of tail to limbs has not yet been properly understood. It is known that oxygenderived free radicals or oxidants, products of oxygen metabolism, are one of the causative factors underlying development and differentiation (Allen, 1991). Oxygen-derived free radicals are also very important mediators of cell injury and death (Joseph and Knight, 1995), and the cellular environment becomes more pro-oxidizing during differentiation (Sies, 1997). Cells contain antioxidant defenses that respond to variations in cellular oxidant production. The imbalance between oxidants and antioxidants is termed oxidative stress (Sies, 1997). Evidence is present that implicates oxidants as a factor that can stimulate alteration in gene expression (Allen, 1991; Schulze-Osthoff and Baeuerle, 1998) and the appearance of new tissue is preceded by the transcription of tissue-specific genes and the concomitant suppression of tran-
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scription of genes that are specific to pleuripotent stem cells (Allen, 1991). During homeotic transformation of tail into limbs, the possible cause has been explained earlier as the change in positional value of the tail blastema cells to body flank (Bryant and Gardiner, 1992) and activation of limb-specific homeotic genes (Bryant and Gardiner, 1992; Maden, 1993; Mahapatra, 1994; Mahapatra and Mohanty-Hejmadi, 1994; MohantyHejmadi et al., 1992; Muller et al., 1996). Histological studies of the abnormal tail have shown a massive amount of notochord in the regenerated tail (Das and Mohanty-Hejmadi, 1999; Maden, 1993; Mahapatra et al., 2001b; Muller et al., 1996) along with a thickened notochordal sheath, thick nerve cord and scattered muscle (mesenchymal) cells. The aim of the present investigation was to study the oxidative stress conditions during abnormal and normal tail regeneration in the tadpoles of the Indian jumping frog, P. maculatus (Anura, Rhacophoridae). The species was selected because of its prolific reaction to vitamin A treatment, as reported earlier (Mahapatra and Mohanty-Hejmadi, 1994). In the present study, lipid peroxidation has been investigated as an index of oxidative stress. The level of hydrogen peroxide (an oxidant) has also been investigated. Furthermore, the activity of two enzymatic antioxidants, related to normal development of anurans, i.e. superoxide dismutase (SOD) and catalase, and the level of reduced glutathione (GSH), a non-enzymatic antioxidant expressed during regeneration and cell division has been analyzed. 2. Materials and methods 2.1. Chemicals Chemicals used in this study were of analytical grade. Thiobarbituric acid (TBA), bovine serum albumin (BSA), 5,59-dithiobis-2-nitrobenzoic acid (DTNB) and mercaptoethanol were obtained from Sigma Chemical Co.; USA, sodium dodecyl sulf (SDS), nicotinamide adenine disodium salt (NADH), horseradish peroxidase (HRP) and reduced glutathione (GSH) were obtained from Sisco Research Laboratory, Mumbai, India; Sephadex G-25 was obtained from Pharmacica 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 P. maculatus were collected from nature during the monsoon period (July–September, 1998–2000). The hatchlings were reared in the laboratory following standard procedure (Mohanty-Hejmadi et al., 1992). They were freely fed with boiled Amaranths greens and boiled egg. 2.3. Tail amputation and treatment Limb-bud stage tadpoles were taken for the experiment. Tadpoles were anaesthetized with MS 222 prior to tail amputation through the middle of the tail. Following amputation, the experimental tadpoles were treated with vitamin A palmitate (Arovit, Roche) 10 IUyml for 72 h (the optimal treatment condition), then transferred to conditioned water. The control tadpoles were reared in conditioned water following amputation. For both the treated and control group of amputated tadpoles, parallel non-amputated, i.e. vitamin A treatment without tail amputation and control without tail amputation, tadpoles were reared. 2.4. Estimation of oxidative stress The original tail tips of limb-bud stage tadpoles were analyzed as the original group. Regenerated tails of 5, 10, 15 and 20 days post-amputated tadpoles of both the control and experimental groups were taken for analysis as the control and treated group, respectively (Table 1). Five days post-amputated tadpoles were selected to study oxidative stress during the earlier stage of regeneration, when the regenerated tail blastema was distinct. The 20 days post-amputated tadpoles were analyzed because bulbular mass, a prerequisite for ectopic limb development, was visible by this period. The 10 and 15 days post-amputated tadpoles were taken as the intermediate stages. Tail of parallel non-amputated tadpoles of both the control and vitamin A-treated tadpoles were also taken after 5, 10, 15 and 20 days of the experiment. For each assay, a pool comprising of 50 tadpoles was taken. 2.4.1. Lipid peroxidation The tissue lipid peroxidation (LPX) was assayed as thiobarbituric acid reacting substance (TBARS) by the thiobarbituric acid (TBA) assay of Ohkawa
P.K. Mahapatra et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 403–410 Table 1 Changes in oxidative stress parameters of the regenerated tail of control (C) and vitamin A-treated (T) tadpoles of P. maculatus Oxidative stress parameters
Group
LPXa
C T
H 2O 2 c
Days following tail amputation 5
10
15
20
1.16b 1.61
1.08 1.91
0.8 1.16
0.63 1.07
C T
1.87 3.85
1.88 5.37
1.82 2.04
1.14 1.27
SODd
C T
14.37 9.06
8.1 9.38
6.9 4.19
0.91 0.9
CATe
C T
1 1.6
1.21 1.96
1.3 1.81
1.09 2.12
GSHf
C T
2.11 2.11
1.4 1.67
1.15 1.29
1.15 1.18
a Level of lipid peroxidation (nmol MDA formedymg protein). b Values in folds relative to original; C, control group; T, treated group. c Level of H2O2 (nmolymg protein). d Activity of SOD (units of SODymg protein). e Activity of Catalase (pmolymg proteinymin). f Level of Glutathione (mmygm tissue).
et al. (1979). A 10% (wyv) homogenate was prepared with 1.5% KCl in an ice-cooled hand mortar. The homogenate was centrifuged at 4 8C for 5 min at 1000=g. The supernatant was taken for the assay. The assay mixture contained a 0.4ml sample, 0.1 ml of 8.1% SDS, 0.75 ml of 20% acetic acid (pH 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 8C, using glass balls as condensers. The tubes were cooled in running tap water, centrifuged at 1000=g for 10 min at room temperature and the absorbence of the supernatant was read at 532 nm. The protein content of the sample was assayed by the method of Lowry et al. (1951). The concentration of TBARS was calculated from the extinction coefficient of 1.56=105 My1 cmy1 (Wills, 1969). The amount of TBARS was expressed as nmol malonaldehyde (MDA) formedymg protein. 2.4.2. Hydrogen peroxide The level of hydrogen peroxide (H2O2) was measured by the method of Pick and Keisari (1981), based on the H2O2-mediated and horseradish peroxidase (HRPO)-dependent oxidation of phenol red. A 10% homogenate (wyv) was pre-
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pared with 50 mM phosphate buffer, pH 7.4, centrifuged for 10 min at 1000=g at 4 8C. The supernatant was taken for the assay. The assay mixture contained 0.28 mM phenol red, 1 unit of HRPO and 500 mg protein in 50 mM phosphate buffer, pH 7.4, in the final volume of 2.0 ml. Absorbence was measured at 610 nm. The level of hydrogen peroxide was expressed as nmol H2O2 ymg protein. 2.4.3. Superoxide dismutase (SOD) Superoxide dismutase activity was measured according to the method of Paoletti and Macoli (1990). A 10% (wyv) homogenate was prepared with 50 mM phosphate buffer, pH 7.4 and centrifuged at 10 000=g for 20 min at 4 8C. The supernatant was passed through a Sephadex G-25 column (1 ml supernatant through 5 ml Sephadex column) with 50 mM phosphate buffer, pH 7.4. The second 2 ml was taken for the assay in the above fraction. The assay mixture contained NADH 7.5 mM, EDTAyMnCl2 100y50 mM, mercaptoethanol 28 mM, 12.5 mg protein and phosphate buffer 60 mM, pH 7.4 to make the final volume 3 ml. The absorbence was taken at 340 nm and readings were noted at the 8th and 16th min after addition of mercaptoethanol. The activity of SOD was expressed as unitsymg protein w1 unit of SODs(difference in OD of blankydifference in OD of sample)y1x. 2.4.4. Catalase Catalase activity was measured according to the method of Aebi (1974). A 10% (wyv) homogenate was prepared with 50 mM phosphate buffer, pH 7.0. The homogenate was centrifuged at 10 0005=g for 10 min at 4 8C. The supernatant was used for the assay. The assay mixture contained 0.1 ml (60 mM) H2O2, 150 mg protein, 50 mM phosphate buffer, pH 7.0 to make the final volume 3 ml. The reaction was initiated by adding 0.1 ml H2O2 (60 mm) and absorbence was noted at 240 nm at a 60-s interval. Catalase activity was expressed as pmolymg proteinymin w1 mol secy1s1 katal (kat)x. 2.4.5. Reduced glutathione (GSH) The assay of GSH was done following the method of Ellman (1959). A 10% (wyv) homogenate was prepared with 5% metaphosphoric acid. The homogenate was kept at room temperature for 15 min, centrifuged for 30 min at 1000=g at room
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Fig. 1. Lipid peroxidation (nmol MDA formedymg protein) in the original and regenerated tail of the control and vitamin A (10I Uyml)-treated tadpoles following 5, 10, 15 and 20 days of post-amputation, respectively in the tadpoles of P. maculatus.
original tail. The oxidative stress of the parallel non-amputated tadpole tails (both control and treated) were also compared with the original tail and no significant difference was observed. Therefore, comparative data have been represented for the regenerated tail of the control and treated tadpoles. In the regenerated tail of 5 days postamputated (dpa) tadpoles of the control group, lipid peroxidation (LPX) was higher than that of the original tadpoles (Fig. 1). A gradual decrease in the level was noticed in the regenerating tail following 10, 15 and 20 days of amputation in the control group. On the other hand, a gradual elevation in LPX was noticed in regenerating tails of the vitamin A-treated group up to 10 days and thereafter a reduction in its level was recorded in the 15 and 20 dpa tadpoles (Fig. 1). However, the level of LPX was always higher in the treated group than the respective control tadpoles. A similar trend was noticed in the level of hydrogen peroxide (Fig. 2). The level of H2O2 was remarkably higher in the treated tadpoles than the respective control tadpoles up to 10 days of post-amputation. There was a fall in the level and by the 20th day the level declined further and was
temperature. The supernatant was used for the assay. The assay mixture contained 0.5 ml supernatant, 2.5 ml DTNB (5,59-dithiobis-2-nitrobenzoic acid) 0.3 mM. The mixture was incubated for 25 min at room temperature and centrifuged for 5 min at 4500=g. The absorbence was taken at 412 nm. The level of GSH was expressed as mmygm tissue. 2.5. Statistics Statistical analysis was followed by the method of Gomez and Gomez (1984). The ANOVA test to find out the significant 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 (control and treated to be considered separately). 3. Results The regenerated tail of the control and treated tadpoles were analyzed and compared with the
Fig. 2. Level of H2O2 (nmolymg protein) in the original and regenerated tail of the control and vitamin A (10I Uyml)-treated tadpoles following 5, 10, 15 and 20 days of post-amputation, respectively in the tadpoles of P. maculatus.
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higher throughout and was always higher than the respective control. The maximum level was recorded in 20 dpa tadpoles of the treated group. The level of reduced glutathione (GSH) in control and the vitamin A-treated group was the highest in 5 dpa tadpoles. Thereafter, a gradual fall in the level was noticed in both the groups (Fig. 5). A comparative account of the changes in oxidative stress parameters in the regenerated tail of the control and treated tadpoles has been summarized in Table 1. 4. Discussion
Fig. 3. Superoxide dismutase (units of SODymg protein) in the original and regenerated tail of the control and vitamin A (10I Uyml) -treated tadpoles following 5, 10, 15 and 20 days of post-amputation, respectively in the tadpoles of P. maculatus.
close to the original tail value. For both lipid peroxidation and H2O2, the highest level was observed in the regenerated tail of the experimental tadpoles 10 days after amputation (Fig. 1 and Fig. 2). In comparison to the control group, a lesser elevation in the activity of SOD was recorded in regenerated tails of 5 dpa tadpoles of the treated group (Fig. 3). In the treated group, activity remained unaltered in 10 dpa tadpoles, thereafter a decrease was observed in the 15-dpa tadpole. In the control group, there was a fall in activity in 10 and 15 dpa tadpoles. An interesting observation was that, for both the treated and experimental tadpoles, the activity of SOD was close to the original tail by the 20th day of amputation (Fig. 3). No change in catalase activity in the regenerating tail of 5 dpa tadpoles of the control group was noted in comparison to the original group (Fig. 4). There was an increase in activity, which remained unchanged, from 10 to 20 dpa tadpoles. In the treated group, the catalase activity remained
We have observed a higher level of lipid peroxidation and H2O2 in 10 dpa tadpoles of the treated group (Fig. 1 and Fig. 2). The level is always more in the treated group than the respective control group (Table 1). H2O2 has been proposed to act as a second messenger molecule, affecting the proliferation and differentiation of cells (Schulze-Osthoff and Fiers, 1991). Histolog-
Fig. 4. Catalase activity (pmolymg proteinymin) in the original and regenerated tail of the control and vitamin A (10I Uyml)treated tadpoles following 5, 10, 15 and 20 days of post-amputation, respectively in the tadpoles of P. maculatus.
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Fig. 5. Level of Glutathione (mmygm tissue) in the original and regenerated tail of the control and vitamin A (10I Uyml) -treated tadpoles following 5, 10, 15 and 20 days of post-amputation, respectively in the tadpoles of P. maculatus.
ical studies have revealed that in the treated tadpoles, de-differentiations of normal tail tissue do not occur during regeneration. In contrast, it leads to development of an abnormal bulbular mass, mainly containing a huge mass of notochordal cells and scattered mesenchymal cells (Das and Mohanty-Hejmadi, 1999; Maden, 1993; Mahapatra et al., 2001b; Muller et al., 1996). A positive relation between the high level of H2O2 and abnormal tail regeneration in the treated group is suggested. A comparatively high activity of SOD (Fig. 3) was observed in 5 dpa tadpoles of the control group. An increase in the level of SOD has been observed during development in several amphibians (Barja De Quiroga and Gutierrez, 1984). It has been explained that increases in SOD activity associated with development are partly due to enzyme induction, it is oxidation associated with the increase in SOD rather than the antioxidant properties of the enzyme that seem to play a role in stimulating changes in gene expression (Allen
and Balin, 1989; Allen, 1991). The level of LPX and H2O2 in 5 dpa tadpoles of the control group is comparatively less than that of the treated tadpoles and the level of SOD is more in the control group at the same time. Our finding support the view of Allen and Balin (1989), that high levels of SOD are not the antioxidant properties of SOD but of cell differentiation, as a normallooking tail regenerates in the control tadpole by the 5th day of amputation. The activity of catalase always remained higher in all the experimental tadpoles than the respective controls (Fig. 4). An increase in catalase activity has been observed in amphibians during development (Gil et al., 1987). It has been observed in frog muscle that increased catalase activity minimizes cellular oxidative stress by lowering the concentration of H2O2 (Hermis-Lima and Story, 1998). Rohrdanz and Kahl (1998) have reported that H2O2 induces the expression of catalase in cultured primary rat hepatocytes. In the present findings, an increase in catalase activity in the treated group is suggested to be due to an elevation in the level of H2O2 in the earlier stages, i.e. 5 and 10 dpa tadpoles (Fig. 2). It is known that higher catalase activity degrades H2O2 (Barja De Quiroga, 1992). We have observed a fall in the level of H2O2 in 15 and 20 dpa tadpoles (Fig. 2). This fall in H2O2 is suggested to be due to the higher activity of catalase during this period. It has been explained earlier that the hypersensitivity towards oxidative stress is retained by the expression of catalase (Schmidt et al., 1995). In our finding, a comparatively higher activity of catalase for a longer period in the treated group shows a hyper-oxidative stress condition in the regenerated tail of treated tadpoles. It is evident in amphibians that GSH concentration increases during the mitotic phase of regeneration and decreases during redifferentiation of regenerating tissue. Decrease in GSH is seen in cells exhibiting a decline in mitotic activity and is considered as an important factor that governs the onset of mitosis (Allen, 1991; Sohal et al., 1988). In our observation, there is a sharp rise in the level of GSH in 5 dpa tadpoles of both the control and treated tadpoles (Fig. 5). This rise is suggested to be due to the high level of cell division during this period. The decline in the level in 10–20 dpa tadpoles is suggested to be due to a slowing down in the rate of cell division.
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