Effects of Desiccation on Metamorphic Climax in Bombina variegata: Changes in Levels and Patterns of Oxidative Stress Parameters

Simple Summary Global warming alters patterns of precipitation and drought, which are important factors in the survival of amphibian populations. Metamorphosis is affected by environmental changes; this is especially true of metamorphic climax, the crucial stage of amphibian development that is accompanied by significant morphological, physiological and behavioral adaptations necessary for the transition to a terrestrial habitat. This study investigated naturally occurring changes in the cellular oxidative status (antioxidant system and oxidative damage) of yellow-bellied toad larvae during this phase, and how exposure to exogenous factors such as desiccation affected them. Our results revealed clear changes in the antioxidant system’s (AOS) response and the levels of oxidative damage during metamorphic climax, with the highest response and damage observed at the end stage. Decreasing water levels during larval development altered the components of the AOS and increased oxidative damage, resulting in increased oxidative stress. The knowledge gained from this study could contribute to a better understanding of the oxidative stress that larvae experience during this critical stage of development, and the consequences of global warming—such as water loss—on amphibians. Abstract In this paper, we examined how the oxidative status (antioxidant system and oxidative damage) of Bombina variegata larvae changed during the metamorphic climax (Gosner stages: 42—beginning, 44—middle and 46—end) and compared the patterns and levels of oxidative stress parameters between individuals developing under constant water availability (control) and those developing under decreasing water availability (desiccation group). Our results revealed that larvae developing under decreasing water availability exhibited increased oxidative damage in the middle and end stages. This was followed by lower levels of glutathione in stages 44 and 46, as well as lower values of catalase, glutathione peroxidase, glutathione S-transferase and sulfhydryl groups in stage 46 (all in relation to control animals). Comparison between stages 42, 44 and 46 within treatments showed that individuals in the last stage demonstrated the highest intensities of lipid oxidative damage in both the control and desiccation groups. As for the parameters of the antioxidant system, control individuals displayed greater variety in response to changes induced by metamorphic climax than individuals exposed to desiccation treatment. The overall decrease in water availability during development led to increased oxidative stress and modifications in the pattern of AOS response to changes induced by metamorphic climax in larvae of B. variegata.

The concentration of total proteins was determined by Lowry et al. (1951). For the construction of the standard protein curve a series of bovine serum albumin (BSA) dilutions was used (the final concentration ranges between 100-1000 μg/mL). The procedure for protein concentration determination is based on the biuretic reaction of cupric ions (Cu 2+ ) with peptide bonds of the protein in the alkaline environment and the reaction of the phosphomolybdenum-phosphoflavolframic reagent (Folin-Ciocalteu reagent) with aromatic amino acids tyrosine and tryptophan, which are constitutive parts of measured proteins. After binding to the peptide bonds, Cu 2+ ions are reduced in the cuprous ions (Cu + ) and the Cu + -protein complex is formed. This complex further reacts with the added Folin-Ciocalte reagent to form a blue colored complex. The color intensity is proportional to the protein content and is measured spectrophotometrically at 500 nm.
The activity of SOD was determined by the adrenaline method (Misra and Fridovich, 1972), which is based on the ability of SOD to reduce spontaneous autoxidation of adrenaline in the adrenochrome in the alkaline environment. Autoxidation of adrenaline depends on the presence of O2 •-. SOD present in the sample removes O2 •-and thus inhibits the autoxidation reaction. Reduction in the rate of adrenaline autoxidation is determined spectrophotometrically at a wavelength of 480 nm. The change in the absorbance is due to the pink-colored adrenaline. Solutions: 3 x 10 -4 M adrenaline in 0.1 M HCl, carbonate buffer (0.05 M Na2CO3 + 10 -4 M EDTA) pH 10.2 adjusted with 10% HCl and 8 mM KCN. Experimental procedure: 3 ml of carbonate buffer was poured into the glass cuvette, together with an appropriate volume of the pre-adjusted adrenaline and the amount of sample that induces the adrenaline autooxidation inhibition ranging from 16.66% to 66.66%. To calculate the activity of SOD, the value of the sample absorption changes in the blank test (buffer and adjusted adrenaline) was used. The SOD activity unit is defined as the amount of enzyme which leads to 50% inhibition of adrenaline autoxidation in the linear portion of the change in absorbance per minute. The activity of SOD in the samples was expressed in units per milligram of protein (U/mg protein).
The activity of CAT was determined by the method described by Claiborne (1984). The method is based on monitoring the decomposition rate of H2O2 to H2O and O2 under the action of CAT. Reduction of the absorbance due to the consumption of H2O2 was detected spectrophotometrically at a wavelength of 240 nm. Experimental procedure: The H2O2 solution in phosphate buffer is adjusted so that the blank sample absorption at a wavelength of 240 nm is between 0.525 and 0.550. In a quartz cuvette, 1.5 ml of the adjusted solution of H2O2 in phosphate buffer was poured, and then the amount of sample which leads to a mean change in the absorbance in the range of 0.03 to 0.06 was added. In the sample starts the process of H2O2 decomposition reaction due to the presence of CAT. The reduction of the absorbance was monitored spectrophotometrically at 240 nm every 30 seconds for 3 minutes at a temperature of 25 °C. To calculate the CAT activity, a molar extinction coefficient for H2O2 (43.6 M-1 cm-1), at a wavelength of 240 nm, was used. CAT activity unit is determined as the number of millimoles of H2O2 reduced per minute (mmol H2O2/min). The activity of enzymes in the tested samples is expressed in units per milligram of protein (U/mg protein).
The GSH-Px activity was measured by the method developed by Tamura et al. (1982). The principle of the method is based on the coupled activity of GSH-Px (catalyses the oxidation of GSH in GSSG with the reduction of organic hydroperoxides) and GR (enables the reduction of GSSG in GSH with the oxidation of NADPH as a coenzyme). The organic peroxide tert-butyl hydroperoxide is added to the reaction mixture, with NADPH and GR. The activity of the GSH-Px enzyme was detected by spectrophotometric monitoring of NADPH oxidation into NADP + . Into quartz cuvette were poured: 1.6 mL of H2O (or less volume depending on the amount of added sample), 0.3 mL of 1mM GSH, 0.6 mL of 0.2 mM NADPH, 0,1 mL of 1mM NaN3 (which inactivated CAT), 0.1 mL of 1 mM EDTA, 0.3 mL 0.5M phosphate buffer (pH 7.0), 0.1 mL of 0.03 M tert-butyl hydroperoxide, adequate amount of sample and 5μL GR. Decrease of the absorbance was monitored spectrophotometrically at 340 nm every 30 seconds for 3 minutes at a temperature of 25 °C. The activity of GSH-Px was determined according to the blank, using a molar extinction coefficient of 6.22 x 103 M -1 cm -1 for NADPH at 340 nm. The GSH-Px enzyme activity unit was defined as the number of oxidized nanomoles of NADPH per minute (nmol NADPH/min), and the activity of GSH-Px in the tested tissues is expressed in units per milligram of protein (U/mg protein).
To determine the activity of GR in the tested samples the method according to Glatzle et al. (1974) was used. This method is established on the ability of GR to catalyze the GSSG reduction into GSH with the oxidation of coenzyme NADPH to NADP + . In the reaction mixture, the GSSG and NADPH are added, and GR activity is measured spectrophotometrically by reducing the NADPH concentration. Experimental procedure: In a quartz cuvette 0.6 mL 0.5 M phosphate buffer (pH 7.4), 0.1 mL 2 mM GSSG, 0.1 mL 0.5 mM EDTA, 2 mL H2O (or a smaller volume depending on the volume of the sample) and 0.1 mL 0.1 mM NADPH were added, and at the end adequate amount of the sample. The absorbance was measured at a wavelength of 340 nm every 30 seconds for 3 minutes at a temperature of 25 °C. To calculate the GR activity, a molar extinction coefficient for NADPH at 340 nm of 6.22 x 10 3 M -1 cm -1 was used. The unit of activity of the enzyme GR is defined as the number of nanomoles oxidized NADPH per minute (nmol NADPH/min). The activity of this enzyme in the tested samples is presented in units per milligram of protein (U/mg protein).
The activity of the phase II biotransformation enzyme -GST was measured by the method described by Habig et al. (1974). The principle of the method is based on the ability of the GST to catalyse the reaction of binding 1-chloro-2,4-dinitrobenzene (CDNB) to the sulfhydryl group of cysteine that is a part of the tripeptide GSH, thereby forming a CDNB-GSH conjugate. The rate of absorption due to the formation of CDNB-GSH conjugate is directly comparative to the activity of the GST in the sample. Experimental procedure: 2 ml of H2O poured into the quartz cuvette (or a smaller volume depending on the amount of sample) were followed by 0.1 mL 25 mM CDNB in 95% ethanol, 0.6 mL of 0.5 M phosphate buffer (pH 6.5), 0.3 mL of 20 mM GSH and adequate amount of sample. The absorbance was monitored spectrophotometrically at a wavelength of 340 nm every 30 seconds for 3 minutes at a temperature of 25 °C. The GST activity was determined according to the blank, using the molar extinction coefficient for the CDNB-GSH conjugate at 340 nm of 9.6 x 10 3 M -1 cm -1 . The absorbance is detected spectrophotometrically at a wavelength of 340 nm. The unit of activity of the enzyme phase II biotransformation GST is expressed as the number of nanomoles of the CDNB-GSH conjugate formed per minute (nmol CDNB-GSH/min). GST activity is given in units per milligram of protein (U/mg protein).
The method described by Griffith (1980) was used to measure the concentration of total GSH in the test samples. This method is based on a cyclic enzymatic process: 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) oxidizes GSH, whereby GSSG and 2-nitro-5-thiobenzoic acid (TNB) are formed, and then the GR enzyme reduces GSSG in GSH with NADPH coenzyme oxidation. The formation rate of the yellow colored TNB compound is monitored and is proportional to the concentration of total GSH in the sample. Experimental procedure: 0.5 mL tissue sonicates and 0.25 mL of 10% sulfosalicylic acid (for protein precipitation in the sample) were poured in microcentrifuge tubes. After centrifugation for 10 minutes at 5000 rpm, the obtained supernatant was used to determine the concentration of GSH. 0.1 mL 6 mM DTNB, sample, 0.7 mL 0.3 mM NADPH, H2O to 1 mL of the reaction mixture and 5 μL of GR were poured in a quartz cuvette. For the standards, instead of the sample, the appropriate volumes of standard solutions with the known concentration of GSH were added. An increase of the absorbance is monitored spectrophotometrically at a wavelength of 412 nm every 30 seconds for 3 minutes at a temperature of 25 °C. The absorbance is determined spectrophotometrically at 412 nm. The concentration of total GSH in the sample is expressed in nanomoles per gram of tissue (nmol of GSH/g tissue).
The concentration of free -SH groups in the tested samples was measured by the method of Ellman (1959). DTNB oxidizes free -SH groups present in the sample, whereby mixed disulfides and yellow colored TNB are formed. Experimental procedure: In the cuvette 0.5 ml of sample was added, 0.5 mL of 0.1 M phosphate buffer (pH 7.3) and 0.2 mL of DTNB. After incubation for 10 minutes at room temperature, the absorbance at a wavelength of 412 nm was read. To calculate the concentration of free SH groups, a molar extinction coefficient of 14150 M -1 cm -1 was used. The concentration of free -SH groups in the sample is determined according to the blank and proportion of formed concentration of TNB. The absorbance is measured spectrophotometrically at 412 nm wavelength. The concentration of free -SH groups in the sample is expressed in micromoles of -SH group per gram of tissue (μmol SH/g tissue).
Thiobarbituric acid reactive substances (TBARS) concentrations as a marker of lipid peroxidation process (LPO)-oxidative damage were estimated according to the method of Rehncrona et al. (1980). The content of TBARS formed spontaneously was measured upon treating the samples with cold thiobarbituric acid reagent (10% trichloroacetic acid, 0.6% thiobarbituric acid) and subsequent heating at 100°C.