reoxygenation in semiaquatic rodents

CBB-09930; No of Pages 9 Comparative Biochemistry and Physiology, Part B xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb

F

5 6 7 8

a

9

a r t i c l e

10 11 12 13 14

Article history: Received 9 May 2015 Received in revised form 20 August 2015 Accepted 25 August 2015 Available online xxxx

15 Q3 16 17 18 19 20 21 22 23

Keywords: Adaptation Catalase Diving Glutathione Hypoxia/reoxygenation Lactate dehydrogenase Superoxide dismutase Semiaquatic rodents

Institute of Biology, Karelian Research Centre, Russian Academy of Sciences, Petrozavodsk, Russia Institute of Animal Sciences, University of Agriculture in Krakow, Krakow, Poland Institute of Veterinary Sciences, University of Agriculture in Krakow, Krakow, Poland

i n f o

a b s t r a c t

To meet the challenges presented by dive-derived hypoxia/reoxygenation transition, the aquatic mammals possess multi-level adaptations. However, the adjustments of the semiaquatic animals as modern analogs of evolutionary intermediates between ancestral terrestrial mammals and their fully aquatic descendants are still not fully elucidated. The aim of this study was to analyze the total lactate dehydrogenase (LDH) activity (in the lactate to pyruvate direction), the LDH patterns and the antioxidant defense in the tissues (heart, kidney, liver, lung, muscle, spleen) of semiaquatic rodents such as Eurasian beaver (Castor fiber), muskrat (Ondatra zibethicus) and nutria (Myocastor coypus). Samples from Wistar rat were used for comparison. Semiaquatic rodents had higher catalase activity compared to rats. The superoxide dismutase activity was higher and the catalase activity was lower in almost all tissues of muskrat than of both beaver and nutria. Comparing beaver and nutria, no significant differences in the antioxidant enzyme activities were found for the heart, kidney and liver. In beaver, most of the examined tissues (heart, kidney, lung and spleen) use lactate as preference to glucose as a substrate but in muskrat the heart, liver and skeletal muscle showed the increased LDH activity. Nutria had the unusual LDH properties that are needed to be further investigated. Our results suggest that beaver, nutria and muskrat have distinct mechanisms of adaptation to diving hypoxia/reoxygenation and support the hypothesis that semiaquatic mammals are the intermediate animals that help to define which potential selection factors and mechanical constraints may have directed the evolution of the aquatic forms. © 2015 Published by Elsevier Inc.

E

T

E

c

C

b

44 42 41

R

43

1. Introduction

46 47

The aquatic and semiaquatic lifestyles of mammals assume the existence of developed multi-level adaptations to maximize aerobic dive duration and to cope with diving hypoxia/reoxygenation (Kooyman et al., 1981; Davis and Kanatous, 1999; Zenteno-Savín et al., 2002; Davis, 2014). Most dives are within the aerobic dive limit (ADL) (Butler and Jones, 1997; Ponganis et al., 1997), which is defined as the longest dive that a mammal can make while relying principally on oxygen stored in the lungs, blood and muscles to maintain aerobic metabolism (Kooyman et al., 1981). Dives shorter in duration than the ADL showed no post-dive increase in blood lactic acid, indicating that metabolism had remained aerobic (Kooyman et al., 1981). The dive response involving bradycardia and peripheral vasoconstriction is a

54 55 56 57

U

52 53

N C O

Q4 45

50 51

24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

R

40

48 49

O

4

Svetlana Sergina a,⁎, Ekaterina Antonova a, Viktor Ilyukha a, Stanisław Łapiński b, Marcin Lis c, Piotr Niedbała b, Alexey Unzhakov a, Vladimir Belkin a

R O

3Q2

P

2

Biochemical adaptations to dive-derived hypoxia/reoxygenation in semiaquatic rodents

D

1Q1

⁎ Corresponding author at: Institute of Biology, Karelian Research Centre, Russian Academy of Sciences, Puskinskaya st., 11, Petrozavodsk, Karelia, Russia. Tel./fax: + 7 8142 76 98 10. E-mail address: [email protected] (S. Sergina).

primitive cardiovascular response to protect animals from asphyxia and to regulate blood flow in a manner that maintains aerobic metabolism at different levels of submerged exercise (Butler and Jones, 1997; Davis and Williams, 2012). Although the degree of bradycardia and peripheral vasoconstriction may vary with the dive duration or level of exertion (Davis and Williams, 2012), all organs and tissues including the heart, kidneys, and splanchnic organs experience a reduction in convective oxygen delivery resulting from both hypoxic hypoxia (decrease in oxygen supply without a decrease in blood flow) and ischemic hypoxia (the condition in which blood flow is reduced or stopped) (Butler and Jones, 1997; Davis and Kanatous, 1999; Kanatous et al., 2001). Nevertheless, pinnipeds maintain aerobic metabolism during most of freeranging dives (Kooyman et al., 1983; Davis et al., 1991; Hochachka, 1992; Butler and Jones, 1997). Depending on the availability of oxygen the tissue metabolism is adjusted by changing the activity of key enzymes of metabolic pathways. Castellini et al. (1981) showed that, on average, marine mammals do not possess significantly elevated anaerobic enzyme activities when compared with terrestrial mammals. Fuson et al. (2003) showed that the heart, liver, kidneys and gastrointestinal organs of harbor seals exhibit adaptations that promote an aerobic,

http://dx.doi.org/10.1016/j.cbpb.2015.08.012 1096-4959/© 2015 Published by Elsevier Inc.

Please cite this article as: Sergina, S., et al., Biochemical adaptations to dive-derived hypoxia/reoxygenation in semiaquatic rodents, Comp. Biochem. Physiol. B (2015), http://dx.doi.org/10.1016/j.cbpb.2015.08.012

58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77

101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143

F

O

R O

99 100

144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161

2. Materials and methods

162 Q5

2.1. Ethical procedures

163

P

97 98

All the experiments were conducted according to EU guidelines on 164 the use of animals for biochemical research (86/609/EU) with the spe- 165 cial permission of Local Ethic Committee of Institute of Biology. 166

D

95 96

Despite the high energetic costs of semiaquatic existence these animals represent modern analogues of evolutionary intermediates between ancestral terrestrial mammals and their fully aquatic descendants (Fish, 1992; Fish and Baudinette, 1999; Williams, 1999). Therefore, we suppose that semiaquatic rodents must have the similarities and differences in antioxidant capacity and biochemical parameters involved in the regulation of tissue energy demands with non-diving closely related species and with each other. The aim of this study was to analyze and compare the LDH patterns, the total LDH activity and the antioxidant defenses in some tissues (heart, kidney, liver, lung, skeletal muscle and spleen) among rodent species (semiaquatic Eurasian beaver, nutria and muskrat and non-diving rat). We used several tissues such as those containing oxygen stores (lung, muscle), those participating in dive response (heart) and other splanchnic organs (liver, kidney and spleen). The LDH activity was measured in the lactate to pyruvate (aerobic) direction. The antioxidant parameters such as GSH level and SOD and catalase activities were also measured.

2.2. Animals and tissue collection

167

The research was carried out using the facilities of the Equipment Sharing Centre of the Institute of Biology, Karelian Research Centre of Russian Academy of Sciences. Fresh tissue samples were obtained from 12 Eurasian beavers (C. fiber; 8 males and 4 females, average body mass 17.22 ± 1.27 kg), 5 nutrias (M. coypus; 2 males and 3 females, average body mass 4.35 ± 0.41 kg), 12 muskrats (O. zibethicus; 8 males and 4 females, average body mass 1.00 ± 0.06 kg) and 5 Wistar rats (R. norvegicus; males, average body mass 0.33 ± 0.03 kg). Skinned beaver and muskrat carcasses were collected from commercial trappers during the autumns of 2004–14; traplines were located on the rivers of the North-West region (Karelia), Russia. The nutrias were of the standard breed and kept at a fur farm in Poland (Permission No. 32/2010, First Local Ethical Committee on Animal Testing at the Jagiellonian University in Krakow). Nutrias were kept in brick pens with water reservoirs, under conventional feeding consisting of a clover-grass mixture with grain supplement (2/3 of soaked barley and 1/3 of wheat), and waste bread was given occasionally. The method of nutria farming providing water reservoirs contributes to the preservation of diving adaptations of the species (Katomski and Ferrante, 1974). Rats were bred in the laboratory. Nutria carcasses and rats were collected after pelting in autumns of 2007 and 2010, respectively. Samples of the heart, kidney, liver, lung, skeletal muscle (hindlimb muscle) and spleen were obtained within the first 2 h after death. No samples were analyzed from animals that had been dead for more than 4 h. All samples were immediately placed on ice for transportation to the laboratory, where samples were stored at −80 °C awaiting analyses. All samples were analyzed in triplicate. For assay of LDH properties and determination of antioxidant enzyme activities tissue samples were homogenized in 0.05 M phosphate buffer, pH 7.0, and centrifuged at 6000 ×g for 15 min. To measure GSH, tissue samples were homogenized in 0.02 M EDTA, and then centrifuged at 5000 × g for 15 min. Preliminary tests revealed no differences between sexes, hence, data for males and females were pooled in all subsequent analyses.

168 169

T

93 94

C

91 92

E

89 90

R

87 88

R

85 86

O

84

C

82 83

N

80 81

fat-based metabolism under hypoxic conditions but can provide ATP anaerobically if required. One of the most important enzyme systems that can cause a shift in metabolism toward the glycolytic or aerobic pathway is the lactate dehydrogenase (LDH) system (Hochachka and Somero, 2002; Philp et al., 2005). Random combinations of two different LDH subunits designated H (heart-type) and M (muscle-type) yield five isoenzymes: LDH1 (H4), LDH-2 (H3M1), LDH-3 (H2M2), LDH-4 (H1M3) and LDH-5 (M4). The M and H subunits of the enzyme evolved in order to fulfill certain definitive metabolic roles. The M isomer is more abundant in glycolytic skeletal muscles and in anaerobic environments leading to the production of lactate from pyruvate. The H isomer predominates in the myocardium and functions primarily in aerobic conditions by converting lactate to pyruvate. Consequently, the amount of H subunits reflects the aerobic capacity of the tissue, while preponderance of M subunits means that the tissue functions under relatively anaerobic conditions. Although generally accepted that the predominant isoenzyme of the mammal skeletal muscle is LDH-5 (M4) (Hochachka and Somero, 2002), Blix and From (1971) have demonstrated that the skeletal muscle of diving seal has more H subunits than the same tissue of sheep that is probably connected with higher oxygen stores in seal skeletal muscle. Shoubridge et al. (1976) found that LDH-5 (M4), which seems to be predominant in the tissues of high anaerobic capacity, was the main isoenzyme in the heart and brain tissues of cetaceans indicating that this is a biochemical adaptation to hypoxia during prolonged dives. When a dive exceeding an animal's ADL is required, there is an additional advantage for aerobic tissues to produce the ATP by glycolysis. Messelt and Blix (1976) also demonstrated that anaerobic ATP production may be of considerable importance even in the brain and heart tissues that was shown in semiaquatic Eurasian beaver during prolonged dives. The first breath after a dive restores the oxygenated blood flow to all tissues, known as “reperfusion” (Elsner et al., 1998); nevertheless, an increase in reactive oxygen species (ROS) generation and, consequently, a potential oxidative stress can occur (Fridovich, 1998; Halliwell and Gutteridge, 1999). To prevent or attenuate the deleterious effects promoted by ROS, cells possess a set of enzymatic and low-molecular antioxidants, which maintain the intracellular ROS at proper levels. Endogenous antioxidants include antioxidant enzymes, i.e. superoxide dismutases (SODs), catalyzing O− 2 • dismutation into H2O2, catalase and glutathione peroxidases removing H2O2, as well as low-molecular ubiquitous glutathione (GSH), tocopherols, carotenoids and others, removing or transforming ROS into less toxic metabolites (Halliwell and Gutteridge, 1999). Studies on the adaptations of marine mammals to hypoxia/reoxygenation associated with diving suggest that an enhanced antioxidant capacity allows them to avoid oxidative damage (Elsner et al., 1998; Zenteno-Savín et al., 2002; Vázquez-Medina et al., 2006, 2007). However, along with numerous studies on marine diving mammals there is a lack of investigations concerning speciesdependent features of antioxidant status as well as of regulation of tissue energy demands in freshwater semiaquatic mammals including such rodents as Eurasian beaver, nutria and muskrat. According to modern taxonomy of Rodentia (Romanenko et al., 2012), the Eurasian beaver (Castor fiber L., 1758) (Castoridae) belongs to suborder Sciuromorpha. It is the largest rodent in the Northern hemisphere that can perform dives lasting up to 15 min without asphyxiation (Graf et al., 2012). The muskrat (Ondatra zibethicus L., 1766) (Cricetidae, Myomorpha) which is common from the tropics to the Arctic has the maximum dive time estimated of 9–12 min (Scholander, 1940). The nutria (Myocastor coypus Molina, 1782) (Myocastoridae, Hystricomorpha) is native to southern South America and is commonly bred in captivity. Wild nutria is able to submerge for periods exceeding 10 min (Katomski and Ferrante, 1974). The Wistar rat (Rattus norvegicus Berkenhout, 1769) (Muridae, Myomorpha) which is a terrestrial rodent species with a maximal forced dive underwater endurance of 2 min (Scholander, 1940) was used for comparison.

U

78 79

S. Sergina et al. / Comparative Biochemistry and Physiology, Part B xxx (2015) xxx–xxx

E

2

Please cite this article as: Sergina, S., et al., Biochemical adaptations to dive-derived hypoxia/reoxygenation in semiaquatic rodents, Comp. Biochem. Physiol. B (2015), http://dx.doi.org/10.1016/j.cbpb.2015.08.012

170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202

S. Sergina et al. / Comparative Biochemistry and Physiology, Part B xxx (2015) xxx–xxx

3

2.3. Lactate dehydrogenase properties

3. Results

255

204 205

2.3.1. Total lactate dehydrogenase (EC 1.1.1.27) activity Total LDH activity in tissues was quantified by measuring the lactate-dependent rate of reduction of NAD+ at 340 nm (Karlsson et al., 1974). One unit of the enzyme was defined as the amount that catalyzes the reduction of 1 μmol of NAD+ per minute. The results were expressed in μmol per minute per g wet tissue weight.

3.1. Lactate dehydrogenase properties

256

3.1.1. Total lactate dehydrogenase activity Results of the interspecies comparison of total LDH activity (in the lactate to pyruvate direction) in tissues are presented in Fig. 1. Unfortunately we did not measure the tissue activities of LDH in the pyruvate reductase direction and therefore we could not assess the tissue anaerobic capacities in the species. Both liver and skeletal muscle LDH activities were the highest in muskrat among species. The LDH activity was higher in the heart, liver and skeletal muscle of muskrat than of beaver. The LDH activity was higher in the lung and spleen of beaver than of muskrat. The LDH activity was the lowest in the heart, kidney, lung and spleen of nutria as compared to the other examined species. No significant differences in the LDH activity were found between beaver and rat for the heart, kidney and spleen; whereas the LDH activity was higher in the liver, lung and skeletal muscle of rat than of beaver.

257 258

3.1.2. Lactate dehydrogenase pattern Results of the LDH assay are summarized in Table 1. Relative to other organs, mammalian liver and muscle are well adapted for glycolysis, containing high amount of LDH-5 isoenzyme, heart and kidney — for aerobic ATP production, containing more LDH-1, and main metabolic pathways in the lung and spleen might be more aerobic or anaerobic, depending on the species (Hochachka and Somero, 2002). The heart LDH pattern of rodents was characterized by prevalence of aerobic metabolism due to high contents of the aerobic LDH-1 and LDH2 isoenzymes and, respectively, of H-subunits. But if heart LDH pattern in beaver and rat contained all of the five isoenzymes, there was a reduction in the anaerobic LDH-5 isoenzyme in the heart of both muskrat and nutria, and also in LDH-4 isoenzyme in the case of nutria. The heart H:M ratio was the lowest in rat among species. The kidneys of beaver, muskrat and rat also contained large amounts of aerobic LDH-1 and LDH-2 isoenzymes and, respectively, the high percentage of H subunits. The LDH pattern in nutria kidney was an exception with nearly equal contents of all isoenzymes, the total H:M ratio being 52.49:47.51. Muskrat kidney was characterized by the highest amount of H-subunits and the reduction of LDH-5 isoenzyme in its LDH pattern. Liver LDH pattern showed similarity in all examined species with the prevalence of anaerobic LDH-5 isoenzyme over others and higher

272 273

2.4. Antioxidant enzyme activities

220

2.4.1. Superoxide dismutase (EC 1.15.1.1) The total SOD activity was measured by the adrenochrome method based on the spontaneous autoxidation of epinephrine with the formation of product with an absorbance peak at 480 nm (Misra and Fridovich, 1972). This reaction depends on the presence of superoxide anions and is specifically inhibited by SOD. The amount of enzyme that caused 50% inhibition of epinephrine autoxidation is defined as 1 unit (U). SOD activity was expressed as U per mg protein after normalization with estimated total protein in milligrams in the respective tissues.

214 215 216

221 222 223 224 225 226 227 228 229 230

237

2.5. Total protein assay

238 239 240 241

Results for antioxidant enzymes' activities were standardized to total soluble protein content in tissue homogenates. Total tissue protein content was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard.

242

2.6. Glutathione measurement

243 244 245

The GSH content was determined using the Ellman method in the presence of 5,5′-dithiobis-(2-nitrobenzoic acid) (Sedlak and Lindsay, 1968). The results were expressed in mmol GSH per 100 g of wet tissue.

246

2.7. Treatment of data

247

All the collected and calculated numerical data were transformed into SI units and processed statistically as mean ± standard error of the mean. Statistical analysis was performed using the nonparametric Mann–Whitney U-test. Differences between samples were considered to be significant when the p value was less than 0.05. The statistical analyses were performed using Sigma-Stat 2.03 (SPSS Science Software Ltd., USA), while figures were prepared using Grapher 7.0 (Golden Software Inc., USA).

248 249 250 251 252 253 254

E

R

R N C O

234 235

U

233

C

236

2.4.2. Catalase (EC 1.11.1.6) The catalase activity was evaluated by measuring the decrease in H2O2 concentration at 240 nm (Bears and Sizes, 1952). One enzyme unit (IU) is defined as the amount of catalase capable of transforming 1.0 μmol of H2O2 for a minute. Catalase activity was expressed as IU per mg protein after normalization with estimated total protein in milligrams in the respective tissues.

231 232

O

219

212 213

T

217 218

2.3.2. Lactate dehydrogenase pattern Separation of the LDH isoenzymes was performed by Wieme's method of horizontal electrophoresis on agar gel plates (Wieme, 1959), using a PEPh-3 apparatus (Russia). The LDH isoenzyme ratios were estimated quantitatively after histochemical staining of samples by scanning electropherograms on a Chromoscan-200 microdensitometer. The percentage of M subunits was calculated as: M = LDH5 + 0.75 · LDH-4 + 0.5 · LDH-3 + 0.25 · LDH-2, and the percentage of H subunits was calculated as: H = 100% − M.

R O

210 211

P

209

D

208

E

206 207

F

203

Fig. 1. The activity of LDH in the rodents' tissues. Results (in μmol/min/g wet tissue) are expressed as mean ± SEM. * significant difference from beaver, ◊ from nutria, ♦ from muskrat in the same tissue. *, ◊, ♦p b 0.05; **, ◊◊, ♦♦p b 0.01.

Please cite this article as: Sergina, S., et al., Biochemical adaptations to dive-derived hypoxia/reoxygenation in semiaquatic rodents, Comp. Biochem. Physiol. B (2015), http://dx.doi.org/10.1016/j.cbpb.2015.08.012

259 260 261 262 263 264 265 266 267 268 269 270 271

274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295

4

LDH isozymes, %

t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28

Beaver (n = 3–11)

Nutria (n = 4–5)

Muskrat (n = 9–12)

Rat (n = 5)

Heart Kidney Liver Lungs Skel. muscle Spleen Heart Kidney Liver Lungs Skel. muscle Spleen Heart Kidney Liver Lungs Skel. muscle Spleen Heart Kidney Liver Lungs Skel. muscle Spleen

Subunits, %

LDH-1 (HHHH)

LDH-2 (HHHM)

LDH-3 (HHMM)

LDH-4 (HMMM)

LDH-5 (MMMM)

H

M

51.87 ± 2.93 56.70 ± 5.24 1.62 ± 0.74 18.29 ± 4.35 2.76 ± 0.78 16.06 ± 5.29 61.94 ± 3.01 27.04 ± 2.03** 0 24.95 ± 3.67 9.43 ± 1.10* 3.96 ± 0.63 53.34 ± 1.23◊ 70.57 ± 3.23*◊◊ 0 4.19 ± 1.52**◊◊ 2.20 ± 1.53◊ 4.04 ± 0.52* 42.59 ± 1.50*◊♦♦ 42.30 ± 2.59◊♦♦ 0.77 ± 0.25 4.86 ± 0.24**◊ 1.25 ± 0.63◊ 2.32 ± 0.22**♦

39.52 ± 1.19 28.36 ± 2.72 5.90 ± 2.71 36.12 ± 2.04 17.79 ± 2.51 40.45 ± 6.66 33.18 ± 1.83* 16.40 ± 0.37* 0 34.77 ± 1.98 14.05 ± 1.33 23.88 ± 0.85* 36.59 ± 1.44 26.55 ± 2.48◊◊ 0 9.48 ± 1.33***◊◊ 7.40 ± 4.34 13.58 ± 1.23***◊◊ 36.31 ± 1.01 34.00 ± 0.98◊ 2.42 ± 0.31 14.27 ± 1.85**◊ 1.16 ± 0.68*◊ 12.25 ± 0.69**◊

7.34 ± 2.42 13.15 ± 2.89 17.77 ± 4.46 38.54 ± 4.45 22.73 ± 0.40 30.23 ± 6.64 4.58 ± 1.30 14.97 ± 1.33 0.57 ± 0.57* 27.87 ± 1.78 4.32 ± 1.32* 37.17 ± 0.54 10.07 ± 1.58 2.78 ± 0.95**◊◊ 2.66 ± 1.85* 32.88 ± 4.00 9.26 ± 3.92 34.66 ± 2.16 18.83 ± 1.35*◊♦ 15.66 ± 1.75♦♦ 12.43 ± 3.57◊♦ 15.09 ± 1.11**◊♦♦ 7.03 ± 3.71* 32.48 ± 0.93◊

1.18 ± 0.47 1.56 ± 0.50 6.31 ± 0.77 5.62 ± 2.00 13.61 ± 1.53 11.16 ± 3.42 0.30 ± 0.30 22.67 ± 1.63** 10.19 ± 2.63 9.63 ± 3.08 4.79 ± 2.09* 27.65 ± 1.01** 0 0.10 ± 0.10**◊◊◊ 21.40 ± 2.48*◊ 17.37 ± 2.57** 13.06 ± 2.27 24.13 ± 1.36** 1.99 ± 0.86 6.53 ± 1.61*◊♦♦♦ 3.29 ± 0.28*◊♦♦ 27.97 ± 2.29**◊♦ 5.00 ± 2.16*♦ 11.34 ± 0.96◊♦♦

0.09 ± 0.06 0.23 ± 0.23 68.39 ± 7.54 1.43 ± 0.93 43.10 ± 2.38 2.10 ± 0.94 0 18.92 ± 0.74** 89.24 ± 3.18 2.78 ± 0.80* 67.41 ± 1.91* 7.34 ± 0.63* 0 0 75.94 ± 3.71 36.08 ± 4.10***◊◊ 68.07 ± 8.90 23.59 ± 2.98***◊◊ 0.29 ± 0.19 1.50 ± 0.41*◊ 81.08 ± 2.92 37.81 ± 3.48**◊ 85.55 ± 7.01* 41.60 ± 1.49**◊♦♦

85.48 84.93 16.51 66.05 30.87 64.31 89.19 52.49** 2.83 67.37 23.32* 47.37** 85.82◊ 91.90*◊◊ 6.68 32.08***◊◊ 15.65 37.59***◊ 79.73*◊♦♦ 77.27*◊♦♦ 9.63◊ 30.10**◊ 6.89*◊ 30.59**◊

14.52 15.07 83.49 33.95 69.13 35.69 10.81 47.51** 97.17 32.63 76.68* 52.63** 14.18◊ 8.10*◊◊ 93.32 67.92***◊◊ 84.35 62.41***◊ 20.27*◊♦♦ 22.73*◊♦♦ 90.37◊ 69.90**◊ 93.11*◊ 69.41**◊

F

Tissue

O

Species

t1:4

R O

t1:3

Table 1 The LDH spectra and the percentages of H and M subunits in the rodents' tissues.

P

t1:1 t1:2

S. Sergina et al. / Comparative Biochemistry and Physiology, Part B xxx (2015) xxx–xxx

LDH = lactate dehydrogenase, skel. muscle = skeletal muscle. Values are means ± SEM. * significant difference from beaver, ◊ from nutria, ♦ from muskrat in the same tissue. *, ◊, ♦p b

t1:30

0.05; **, ◊◊, ♦♦p b 0.01; ***, ◊◊◊, ♦♦♦p b 0.001.

296

314

content of M- than of H-subunits. Aerobic LDH-1 and LDH-2 isoenzymes were completely lacking in the livers of nutria and muskrat. The lung LDH patterns of both beaver and nutria were similar in isoenzyme distribution and contained high amounts of LDH-1, LDH-2 and LDH-3, respectively, and more H- than M-subunits. On the contrary the percentage of M-subunits was higher than of H-subunits in the lung LDH pattern of both muskrat and rat. Muskrat lung contained large amounts of LDH-3 and LDH-5 isoenzymes, but rat lung contained large amounts of LDH-4 and LDH-5 isoenzymes. The anaerobic LDH-5 isoenzyme prevailed over others, and, respectively, the content of H- was higher than of M-subunits in the skeletal muscle of all examined species. No significant differences in LDH pattern were found between beaver and muskrat and also between beaver and muskrat for skeletal muscle. The percentage of M-subunits was higher in the skeletal muscle of rat than of both beaver and nutria. Beaver spleen contained the highest amount of LDH-2, the lowest amount of LDH-5 isoenzyme and, respectively, the highest H:M ratio as compared to the spleens of the other examined species. The content of anaerobic LDH-5 isoenzyme was the highest in rat spleen.

315

3.2. Antioxidant enzyme activities

316

3.2.1. Superoxide dismutase Results on the SOD activity are presented in Fig. 2. The SOD activity was not measured in rat tissues and in nutria lung. No significant differences in the SOD activity were found between beaver and nutria for the heart, kidney, liver and spleen. But in skeletal muscle the SOD activity was higher in beaver than in nutria. The SOD activity was higher in the heart, kidney, skeletal muscle and spleen of muskrat than of nutria. The SOD activity was higher in the kidney, liver and spleen of muskrat than of beaver. The SOD activity was higher in beaver lung than in muskrat. No significant differences in the SOD activity were found between beaver and muskrat for the heart and skeletal muscle.

308 309 310 311 312 313

317 318 319 320 321 322 323 324 325 326 327 328 329

341

E

3.3. Glutathione

T

C

E

R

307

R

305 306

O

303 304

3.2.2. Catalase Results on the catalase activity are presented in Fig. 3.

331 332 333 334 335 336 337 338 339 340

Results were summarized in Fig. 4. The interspecies comparison 342 showed the lowest GSH levels in the kidney, liver and lung of nutria. 343 The GSH level was lower in all examined tissues of nutria than of both 344

C

301 302

330

N

299 300

No significant differences in the catalase activity were found among species for the kidney. The catalase activity was higher in the heart, liver and skeletal muscle of beaver than of muskrat. No significant differences in the catalase activity were found between beaver and muskrat for the kidney, lung and spleen, and between beaver and nutria for the heart, kidney and liver. The catalase activity was higher in almost all nutria tissues analyzed, besides the heart and kidney, as compared to the same tissues of muskrat. The interspecies comparison showed the highest catalase activity in the skeletal muscle of beaver and in the lung and spleen of nutria, and the lowest catalase activity in the heart, lung, skeletal muscle and spleen of rat.

U

297 298

D

t1:29

Fig. 2. The activity of SOD in the rodents' tissues. Results (in U/mg protein) are expressed as mean ± SEM. * significant difference from beaver, ◊ from nutria, ♦ from muskrat in the same tissue. *, ◊, ♦p b 0.05; **, ◊◊, ♦♦p b 0.01. SOD activity was not measured in nutria lung and in rat tissues.

Please cite this article as: Sergina, S., et al., Biochemical adaptations to dive-derived hypoxia/reoxygenation in semiaquatic rodents, Comp. Biochem. Physiol. B (2015), http://dx.doi.org/10.1016/j.cbpb.2015.08.012

S. Sergina et al. / Comparative Biochemistry and Physiology, Part B xxx (2015) xxx–xxx

5

O

F

10 min) (Katomski and Ferrante, 1974) and muskrat (9–12 min) (Scholander, 1940). Physiological recordings during natural and simulated diving indicate that both rat and muskrat possess the same basic physiological responses to underwater submersion that occur in marine animals (McCulloch, 2012). But in contrast to rats, muskrats show an intensification of bradycardia during forced dives compared with voluntary dives (McCulloch, 2012). Due to the variability and the differences of the responses compared with voluntary diving, it is suggested that the use of forced submergence in naïve rats not trained to dive should be avoided when investigating the hemodynamic responses to diving (Panneton et al., 2010). Our results showed that, in rat, similar to diving rodents, the heart had the highest total LDH activity and the liver had the highest catalase activity and GSH level. According to the tissue LDH isoenzyme patterns and H:M ratios the rat's heart and kidney had mainly aerobic metabolism, that is similar to other examined species and the other organs were characterized by mainly anaerobic ATP production, and in the lung and spleen this was similar to muskrat but in contrast to beaver and nutria. Previously Blix and From (1971) have demonstrated that the skeletal muscle of the diving seal has more H-subunits than the same sheep tissue that is probably connected with higher oxygen stores in the skeletal muscle of seal than that of sheep. Similarly, in our study the skeletal muscle of both beaver and nutria contained more H-subunits as compared to the same rat tissue. Previously it was shown (Castellini et al., 1981) that the tissue activities of LDH (in the pyruvate to lactate direction) and pyruvate kinase are very similar in marine and terrestrial mammals with only a few exceptions. Elevated marine mammal liver and kidney LDH activities could play an important role in postdive gluconeogenesis and in restoring circulating levels of glucose. In our study the liver and skeletal muscle of rat had the higher aerobic capacities as compared to the same tissues of beaver, and the lower aerobic capacities as compared to the same muskrat tissues. Diving mammals generally have constitutively higher antioxidant capacity than nondiving ones to avoid oxidative injuries caused by overproduction of ROS during repetitive cycles of hypoxia/reoxygenation (Elsner et al., 1998; Zenteno-Savín et al., 2002; Vázquez-Medina et al., 2006, 2007). Moreover, the tissue capacity to produce O− 2 • is higher in seal than in pig heart, kidney and skeletal muscle under basal conditions, and in response to an oxidant-generating system (xanthine + xanthine oxidase) (Zenteno-Savín et al., 2002). We did not measure the SOD activity in rat tissues, so our conclusions are based only on the GSH level and catalase activity. No differences in the GSH content were found between rat and beaver tissues, whereas the GSH level was higher in the heart and liver but was lower in the lung and muscle of rat than of muskrat. Comparing ringed seals to pigs, the concentrations of GSH are 20-, 6-, 2- and 3-fold higher in the heart, skeletal muscle, kidneys and lungs, respectively (VázquezMedina et al., 2007). Our results showed that in rat, among the other species, the catalase activity was the lowest in the heart, lung, skeletal muscle and spleen. Similarly, the SOD activity was higher and the catalase activity was lower in the heart of rat compared to muskrat (Galantsev et al., 1994) and the catalase activity was lower in the liver of pig as compared to seal (Vázquez-Medina et al., 2006). Our data may indicate the important role of catalase as a regulator of oxygen-sensing and redox signaling which are dependent on ROS, particularly on H2O2, and are essential for mediating the physiological and pathophysiological responses to hypoxia (Bunn and Poyton, 1996; Brunelle et al., 2005). The second messenger, H2O2, can potentially mediate the adaptive response to oxidative stress during apnea in seals since Nrf2, the redox sensitive transcription factor that regulates antioxidant gene expression, translocates into the nucleus in response to increased intracellular H2O2 production and xanthine oxidase produces mainly H2O2 (Kelley et al., 2010).

4. Discussion

353

4.1. Comparison of non-diving (rat) and semiaquatic rodents

354

The transition from terrestrial to aquatic or semiaquatic lifestyle of mammals was accompanied by numerous morphological modifications, physiological traits and biochemical adjustments. As revealed in our study the similarities and differences in the LDH properties and antioxidant levels among rodents may be connected with the intermediate position of semiaquatic mammals between terrestrial and aquatic animals (Fish and Baudinette, 1999). In this study the non-diving rat was used for comparison. This species has a maximal forced underwater endurance of 2 min (Scholander, 1940), which is much less than the dive durations of beaver (up to 15 min) (Graf et al., 2012), nutria (about

360 361 362 363 Q6

T C

E

R

359

R

357 358

N C O

355 356

U

349 350

P

352

347 348

E

351

muskrat and rat. The GSH level was higher in the heart and liver of rat than of muskrat. The GSH level was higher in the lung and skeletal muscle of muskrat than of rat. No significant differences in GSH level were found between beaver and muskrat, and also between beaver and rat for any of the tissues analyzed. No significant differences in GSH level were found between beaver and nutria for the heart, skeletal muscle and spleen.

D

345 346

R O

Fig. 3. The activity of catalase in the rodents' tissues. Results (in IU/mg protein) are expressed as mean ± SEM. * significant difference from beaver, ◊ from nutria, ♦ from muskrat in the same tissue. *, ◊, ♦p b 0.05; **, ◊◊, ♦♦p b 0.01.

Fig. 4. The GSH content in the rodents' tissues. Results (in mmol/100 g wet tissue) are expressed as mean ± SEM. * significant difference from beaver, ◊ from nutria, ♦ from muskrat in the same tissue. *, ◊, ♦p b 0.05; **, ◊◊, ♦♦p b 0.01.

Please cite this article as: Sergina, S., et al., Biochemical adaptations to dive-derived hypoxia/reoxygenation in semiaquatic rodents, Comp. Biochem. Physiol. B (2015), http://dx.doi.org/10.1016/j.cbpb.2015.08.012

364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 Q7 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 Q8 419 420 421 422 423 424 425 426 427 428 429

S. Sergina et al. / Comparative Biochemistry and Physiology, Part B xxx (2015) xxx–xxx

450 451 452

453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492

4.2.1. Eurasian beaver In beaver, the liver had the highest catalase activity, connected with the detoxifying role of the organ, and the heart had the highest total LDH and SOD activities, reflected high aerobic capacity of this tissue. The GSH contents were not different between organs indicating the high rate of the GSH maintenance. The heart, kidney, lung and spleen of Eurasian beaver possessed the ability for mainly aerobic metabolism as a result of the prevalence of Hsubunits over M-subunits, but the liver and muscle were characterized as glycolytic tissues. This is in contrast to the other examined species having only two of six examined organs with mainly aerobic ATP production. Our results indicate that lactate can be used in preference to glucose as a substrate in at least four of six examined beaver tissues. Similar findings for the lung of Weddel seal were detected by Murphy et al. (1980), who showed that the dominant fate of lactate appears to be oxidation. In such a role the lung may be assisted by the heart which may also be effective in reducing lactate accumulation in central blood during diving and at early stages of recovery. Lung volume in beaver is similar to the values in terrestrial mammals but provide at least half of the oxygen delivered to the well-circulated compartment during a maximum physiological dive of about 5 min (Clausen and Ersland, 1968). Previously (Messelt and Blix, 1976; Shoubridge et al., 1976), the presence of high amount of LDH-5, which seems to be predominant in the tissues of high anaerobic capacity, in such aerobic tissues as the heart and brain of cetaceans and Eurasian beaver was found. In our study in contrast to muskrat and nutria, beaver had all of the five LDH isoenzymes in its heart and kidney patterns, although no differences in heart H:M ratio were found between beaver and both muskrat and nutria. Moreover, the total LDH activity and, respectively, the aerobic capacity were lower in the heart of beaver compared to muskrat and it was nearly equal in the kidney of these species. The muscles that are rich in hypoxia-tolerant enzyme systems (Blix and From, 1971; Hochachka and Somero, 2002; Philp et al., 2005) are most likely the main sources of the postdive rise in arterial lactate (Scholander, 1940). This lactate accumulation is the price paid for extended diving capacity and must be eliminated by the liver before the animal can dive again. In our study total LDH activity was the lowest in beaver liver among species indicating the lowest level of lactate elimination in this organ.

518 519

F

4.2.2. Nutria Being the only member of the family Myocastoridae, the nutria belongs to the suborder Hystricomorpha (Romanenko et al., 2012), a group of rodents with some unusual biochemical characteristics. Hystricomorph insulins differ from those of other mammals in that they do not bind zinc and have very little biological activity with respect to glucose metabolism: insulin exhibits only 1–10% of biological activity in comparison to other mammals (King and Kahn, 1981). Nevertheless, blood glucose concentration values in this group of rodents after fasting and in the glucose tolerance test were within the expected range for mammals (Opazo et al., 2004). This indicates that hystricomorphs have compensatory mechanisms that may permit the maintenance of standard values of plasma glucose. In our study, within the nutria, the skeletal muscle had the highest total LDH activity and the liver, as the organ with high metabolic rate, had the highest antioxidant enzyme activities. The heart and lung of nutria possessed the ability for mainly aerobic metabolism as a result of the prevalence of H-subunits over M-subunits and the high contents of aerobic isoenzymes in its patterns. The higher H:M ratio was found in the heart of nutria than of muskrat and rat. According to LDH isoenzyme pattern, nutria was characterized by mainly aerobic metabolism in its lung that was similar to beaver but in contrast to muskrat and rat. Similarly with beaver, the lactate is used in preference to glucose as a substrate in the lung of nutria. It is confirmed by the study of Katomski and Ferrante (1974) who concluded that nutria's respiratory system can tolerate the physiological consequences of diving which include high levels of carbon dioxide and lactic acid in the blood. Also, the LDH activity was the lowest in the lung of nutria among species. Similarly with the other examined species, nutria liver and skeletal muscle are dependent on anaerobic metabolism for energy production. Although the liver H:M ratios were similar among examined semiaquatic rodents, nutria liver as well as the same muskrat tissue possessed the more pronounced ability to anaerobic ATP production than beaver and rat livers. Both kidney and spleen of nutria contained nearly equal amounts of H- and M-subunits, which reflect the importance of both aerobic and anaerobic ATP productions for functions of these organs. This type of lactate metabolism is not common to all rodents. The advantage of possessing the nearly equal percentages of H- and M-subunits in the kidney and spleen is the ability to produce lactate and to consume it

T

448 449

C

446 447

E

444 445

R

442 443

R

440 441

O

438 439

C

436 437

N

435

U

433 434

O

The evolution of increased aquatic habits is accomplished by elevated body oxygen stores and adaptations that promote their economical use (Mirceta et al., 2013). The most ancient invaders of the marine environment (cetaceans) along with the intermediate group (pinnipeds) rely primarily on intrinsic oxygen stores mostly bound in the blood to hemoglobin and in the muscle to myoglobin (Kooyman et al., 1981; Mirceta et al., 2013; Davis, 2014), while the semiaquatic mammals retained the use of their lungs as oxygen stores (Kooyman and Ponganis, 1998), with the increased use of the muscle oxygen reserves. Diving rodents along with shallow-diving otariid seals, small cetaceans, sea otters and sirenians have lower myoglobin content than deepdiving animals but higher myoglobin content than most terrestrial species (Lenfant et al., 1970; Snyder and Binkley, 1985). Previously Mirceta et al. (2013) showed the convergent evolution of high muscle oxygen storage capacities in both beaver and muskrat along with such proficient divers as otaroids, phocids and cetaceans. As revealed in our study, the similarities and differences in LDH properties and antioxidant levels among diving rodents reflect different evolutionary ways of adaptation to diving hypoxia/reoxygenation in the closely related species. Our findings are therefore species-dependent and tissue-specific, since each organ system is distinct in its physiological and biochemical contributions to whole body metabolism during and after a dive.

493 494

R O

431 432

The spleen serves primarily as a blood depot, but it is also capable of temporarily sequestering red blood cells that are expelled into the hepatic sinus by contraction of the splenic capsular smooth muscle associated with the dive response (Cabanac et al., 1997). However, in small cetaceans the spleen is relatively small and probably does not serve a blood storage role (Cowan and Smith, 1999). Similarly to the majority of rodents, but unlike mice or seals which are characterized by storage type spleens (Eurell, 2003; Cesta, 2006), beavers have defensive type spleens (Dolka et al., 2014). An absence of blood-storing properties in beaver spleens could reflect the beavers' adaptation to an aquatic mode of life and their reluctance to remain on land for longer periods of time (Dolka et al., 2014). In contrast to the spleens of nutria, muskrat and rat, this organ of Eurasian beaver possessed the ability for mainly aerobic metabolism, as a result of the prevalence of H-subunits over M-subunits and higher LDH activity as compared to nutria and muskrat. No differences in GSH content were found between beaver and both muskrat and rat for any of the examined tissues. It may indicate that the redox state of cells is strongly regulated and not affected by hypoxia. Beaver and nutria had similar SOD activities in almost all tissues. Nevertheless, as compared to the muskrat, the beaver had lower SOD activity in the kidney, liver and spleen, and higher catalase activity in the heart, liver and muscle. Moreover, the heart and muscle catalase activities were the highest in beaver among species. These findings might be related with the important role of H2O2 in oxygen sensing and redox signaling, mediated by H2O2 (Brunelle et al., 2005).

P

4.2. Interspecies comparison of semiaquatic rodents

D

430

E

6

Please cite this article as: Sergina, S., et al., Biochemical adaptations to dive-derived hypoxia/reoxygenation in semiaquatic rodents, Comp. Biochem. Physiol. B (2015), http://dx.doi.org/10.1016/j.cbpb.2015.08.012

495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517

520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557

S. Sergina et al. / Comparative Biochemistry and Physiology, Part B xxx (2015) xxx–xxx

578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 Q9 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622

4.2.3. Muskrat Muskrat is a semiaquatic rodent with small body size which is normally accompanied by a high mass-specific metabolic rate. The species serves as usual experimental animals for studying adaptations that attend a diving mode of existence in mammals (Snyder and Binkley, 1985; MacArthur, 1992). Within the muskrat, the heart had the highest total LDH activities, reflecting high aerobic capacity of this tissue. The liver had the highest antioxidant enzymes' activities, and the kidney had the highest GSH level. Similar to rat tissues, the heart and kidney of muskrat were characterized by mainly aerobic metabolism, and the other organs — by anaerobic ATP production. The aerobic capacity of muskrat heart was higher than that of the same beaver and nutria tissues. Similarly, Snyder and Binkley (1985) suggested that muskrat tolerates submersion due to adaptations primarily associated with aerobic, rather than anaerobic, metabolism by virtue of their size-related high metabolic rate. Although field observations indicate that 86.5% of muskrat dives are within the ADL (MacArthur, 1992), its heart is adapted to hypoxic conditions, and one of the adaptations is a high potential for glycolysis (McKean, 1984). The isolated heart of the muskrat is better able to deal with hypoxia than the heart of the non-dividing guinea pig and rabbit (McKean and Landon, 1982). Moreover, the glycogen concentrations and pyruvate kinase activities in the heart, brain, and gastrocnemius muscle of muskrats are similar to those obtained from terrestrial animals (Snyder and Binkley, 1985). The liver, lung, skeletal muscle and spleen of muskrat are characterized by mainly anaerobic ATP production in various extents, depending on the tissue. However, while the liver and muscle LDH isoenzyme patterns contained high amounts of LDH-5 isoenzymes, the lung and spleen LDH patterns are LDH-3, LDH-4 and LDH-5. Being the shallow diver, muskrat should rely primarily on oxygen stores in the lung rather than in the blood and muscles (Kooyman and Ponganis, 1998), but the lung volumes in muskrat are similar to those expected for a terrestrial mammal of comparable body mass (Tenney and Remmers, 1963; Snyder and Binkley, 1985). Moreover, in contrast to the lungs of both beaver and nutria, but similar to those of rat, this organ of muskrat contained more M- than H-subunits. In contrast, the lung of Weddell seal takes up lactate and the circulating glucose either is not taken out of the blood or is released in very small amounts from the lung (Hochachka et al., 1977). The liver and skeletal muscle of muskrat had the highest aerobic capacities (assessed by total LDH activity) among the other examined species. This is consistent with the important role of the liver in gluconeogenesis and in restoring circulating glucose levels after a dive

661

To accommodate their amphibious habits the semiaquatic mammals possess the similarities and differences in physiological and biochemical traits with terrestrial and aquatic mammals. As revealed in our study species-dependent and tissue-specific features of LDH properties and antioxidant levels among rodents reflect different evolutionary ways of adaptation to diving hypoxia/reoxygenation in closely related species. Beavers have longer evolutionary diving history compared to muskrat and some beaver's parameters allow to suggest that beavers are more like aquatic mammals and birds than like terrestrial or even semiaquatic mammals (Allers and Culik, 1997; Mirceta et al., 2013). Data on nutria is lacking but McKean (1982) suggested earlier that nutrias possess physiological adaptations to diving similar to the adaptations in beavers. We suggest that semiaquatic mammals are adapted to diving hypoxia at the expense of the multiple LDH forms (isoenzymes) and also at the expense of alteration of the enzyme activity considering that LDH has proven to be an excellent model system for addressing questions about enzymatic adaptation (Hochachka and Somero, 2002). Based on LDH isoenzyme pattern and total LDH activity (in the lactate to pyruvate direction), the enhanced utilization of circulating lactate loads as the adaptation for recovery from a diving period is achieved through either involving most of the examined tissues (heart, kidney, lung and spleen) in this process as in the case of beaver, or increasing LDH activity in tissues (heart, liver and skeletal muscle) as in the case of muskrat. The nutria, representative of Hystricomorphs, had the unusual LDH properties

662 663

O

F

5. Conclusions

R O

577

P

575 576

623 624

D

573 574

(Castellini et al., 1981). For skeletal muscle the differences may be partially secondary to differences in locomotory effort, muscle oxidative capacity and fiber type distribution (Kooyman and Ponganis, 1998; Elsner, 1999). Differences in muscle aerobic capacities between species also reflect the differences in types of locomotion. For example, in comparison with the shallow/shorter-duration divers, the lower muscle aerobic capacity in the deep/long divers reflects the energy-conserving mode of locomotion, which enables them to perform longer and deeper dives (Kanatous et al., 2001). Moreover, muskrat has the higher cost of transport (2.179 J/Nm) as compared to Canadian beaver (Castor canadensis) (0.360 J/Nm) and the aquatic animals (0.026–0.410 J/Nm) (Allers and Culik, 1997; Fish, 2000). This value, cost of transport, is defined as the metabolic energy required to transport a unit of mass to a unit of distance and is calculated by dividing the mass-specific metabolic rate by the swimming velocity (Fish, 1992). The minimum cost of transport is the most efficient and is considered to occur at the velocity in which the animal can cover the largest distance for the smallest energy cost. Some cardiovascular effects can be explained by the interaction between NO• and superoxide anion during a dive ending to less NO• availability (Theunissen et al., 2013). It is known that superoxide anion can spontaneously react with NO• generating peroxynitrite (ONOO−) (Beckman et al., 1990) to diminish the vasodilatation process or be converted by SOD into oxygen and H2O2 (McCord and Fridovich, 1969). The absence of NO• may be an adaptive strategy to avoid ONOO− and HO• formation after an event of ischemia/reperfusion that can potentially increase superoxide production (Beckman et al., 1990). Hence, SOD may act as a regulator of cardiovascular effects in muskrat. In our study SOD activity was higher, but catalase activity was lower in almost all tissues of muskrat than of beaver and nutria. Additionally, Cantú-Medellín et al. (2011) suggested that the higher SOD activity in shallow/shortduration diver (bottlenose dolphin) tissues compared with deep divers (Kogia spp.) tissues could lead to higher production of H2O2, which can in turn lead to the cascade reactions of lipid peroxidation (Fridovich, 1998). ROS (particularly, H2O2) and the oxidation of lipid molecules, in turn, act as signaling mechanisms, via the Keap1–NRF2 pathway, activating the antioxidant genes in order to protect the tissues against the potential oxidative stress associated with dive-induced ischemia/reperfusion (Cantú-Medellín et al., 2011).

E

571 572

T

569 570

C

567 568

E

565 566

R

564

R

562 563

N C O

560 561

in the same extent. In this sense, nutria might be unique in the metabolism of its kidney and spleen. Another feature of nutria was the lowest aerobic capacity of the heart, kidney, lung and spleen as compared to the same tissues of examined species. At the same time, GSH content was the lowest in almost all tissues of nutria as compared to the other examined species. Previously Lance and Elsey (1983) showed that nutria has extremely low selenium content in its muscle and lower erythrocyte selenium-dependent glutathione-peroxidase activity than rat, rabbit, chicken, quail, turtle, snake and bullfrog. It is well known that GSH acts as a co-factor for glutathione-peroxidase. Therefore, our results suggest that nutria could use the other antioxidants, which were not analyzed in this study, to protect their tissues against the ROS. The SOD activity was lower in the heart, kidney, skeletal muscle and spleen and the catalase activity was higher in almost all tissues of nutria as compared to muskrat. No significant differences in antioxidant enzyme activities were found between beaver and nutria for the heart, kidney and liver. Earlier (McCean, 1982) it was revealed that nutrias possess physiological adaptations to diving similar to the adaptations of beavers.

U

558 559

7

Please cite this article as: Sergina, S., et al., Biochemical adaptations to dive-derived hypoxia/reoxygenation in semiaquatic rodents, Comp. Biochem. Physiol. B (2015), http://dx.doi.org/10.1016/j.cbpb.2015.08.012

625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660

664 665 666 667 668 669 670 671 672 Q10 673 674 675 676 677 678 679 680 681 682 683 684 685 686

S. Sergina et al. / Comparative Biochemistry and Physiology, Part B xxx (2015) xxx–xxx

P

R O

O

F

capacities. Shallow/short vs. deep/long divers. J. Comp. Biochem. Physiol. A. 158, 438–443. http://dx.doi.org/10.1016/j.cbpa.2010.11.029. Castellini, M.A., Somero, G.N., Kooyman, G.L., 1981. Glycolytic enzyme activities in tissues of marine and terrestrial mammals. Physiol. Zool. 54, 242–252. Cesta, M.F., 2006. Normal structure, function, and histology of the spleen. Toxicol. Pathol. 34, 455–465. Clausen, G., Ersland, A., 1968. The respiratory properties of the blood of two diving rodents, the beaver and the water vole. Respir. Physiol. 5, 350–359. Cowan, D.F., Smith, T.L., 1999. Morphology of the lymphoid organs of the bottlenose dolphin, Tursiops truncatus. J. Anat. 194, 505–517. Davis, R.W., 2014. A review of the multi-level adaptations for maximizing aerobic dive duration in marine mammals: from biochemistry to behavior. J. Comp. Physiol. B. 184, 23–53. http://dx.doi.org/10.1007/s00360-013-0782-z. Davis, R.W., Kanatous, S.B., 1999. Convective oxygen transport and tissue oxygen consumption in Weddell seals during aerobic dives. J. Exp. Biol. 202, 1091–1113. Davis, R.W., Williams, T.M., 2012. The dive response is exercise modulated to maximize aerobic dive duration. J. Comp. Physiol. A. 198, 583–591. http://dx.doi.org/10.1007/ s00359-012-0731-4. Davis, R.W., Castellini, M.A., Williams, T.M., Kooyman, G.L., 1991. Fuel homeostasis in the harbor seal during submerged swimming. J. Comp. Physiol. B. 160, 627–635. Dolka, I., Giżejewska, A., Giżejewski, Z., Kluciński, W., Kołodziejska, J., 2014. Histological evaluation of selected organs of the Eurasian beavers (Castor fiber) inhabiting Poland. Anat. Histol. Embryol. 2014, 1–13. http://dx.doi.org/10.1111/ahe.12150. Elsner, R., 1999. Living in water, solutions to physiological problems. In: Reynolds III, J.E., Rommel, S.A. (Eds.), Biology of Marine Mammals. Smithsonian Institution Press, Washington, D.C., pp. 73–116. Elsner, R., Øyasæter, S., Almaas, R., Saugstad, O.D., 1998. Diving seals, ischemia–reperfusion and oxygen radicals. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 119 (4), 975–980. Eurell, J.A.C., 2003. Spleen. In: Cann, C.C. (Ed.), Veterinary Histology. Quick Look Series in Veterinary Medicine. New Media, Teton, pp. 42–44. Fish, F.E., 1992. Aquatic locomotion. In: Tomasi, T.E., Horton, T.H. (Eds.), Mammalian Energetics: Interdisciplinary Views of Metabolism and Reproduction. Cornell University Press, Ithaca, N.Y, pp. 34–63. Fish, F.E., 2000. Biomechanics and energetics in aquatic and semiaquatic mammals: platypus to whale. Physiol. Biochem. Zool. 73, 683–698. Fish, F.E., Baudinette, R.V., 1999. Energetics of locomotion by the Australian water rat (Hydromys chrysogaster): comparison of swimming and running in a semiaquatic mammal. J. Exp. Biol. 202, 353–363. Fridovich, I., 1998. Oxygen toxicity: a radical explanation. J. Exp. Biol. 201, 1203–1209. Fuson, A.L., Cowan, D.F., Kanatous, S.B., Polasek, L.K., Davis, R.W., 2003. Adaptations to diving hypoxia in the heart, kidneys and splanchnic organs of harbor seals (Phoca vitulina). J. Exp. Biol. 206, 4139–4154. http://dx.doi.org/10.1242/jeb.00654. Galantsev, V.P., Kamardina, T.A., Kovalenko, R.I., Molchanov, A.A., Perepelitsa, V.A., Ianvareva, I.N., Vilkova, V.A., Putilina, F.E., 1994. Cardiovascular system reactions and bioenergy metabolism in relation to adaptation to apnea. Fiziol. Zh. Im. I. M. Sechenova 80 (9), 117–123 (In Russian). Graf, P.M., Wilson, R.P., Cohen Sanchez, L.G., Hackländer, K., Rosell, F., 2012. Diving behaviour of the Eurasian beaver (Castor fiber). Book of Abstracts. 6th International Beaver Symposium. Croatia, September 2012, p. 13. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine. Oxford University Press, Oxford (888 pp.). Hochachka, P.W., 1992. Metabolic biochemistry and the making of a mesopelagic mammal. Experientia 48, 570–575. Hochachka, P.W., Somero, G.N., 2002. Biochemical Adaptation: Mechanism and Process in Physiological Evolution. Oxford University Press, Oxford (466 pp.). Hochachka, P.W., Liggins, G.C., Qvist, J., Schneider, R., Snider, M.Y., Wonders, T.P., 1977. Pulmonary metabolism during diving: conditioning the blood for the brain. Science 198, 831–834. Kanatous, S.B., Elsner, R., Mathieu-Costello, O., 2001. Muscle capillary supply in harbor seals. J. Appl. Physiol. 90, 1919–1926. Karlsson, J., Frith, K., Sjödin, B., Gollnick, P.D., Saltin, B., 1974. Distribution of LDH-isozymes in human skeletal muscle. Scand. J. Clin. Lab. Invest. 33, 307–312. Katomski, P.A., Ferrante, F.L., 1974. Catecholamine content and histology of the adrenal glands of the nutria (Myocastor coypus). Comp. Biochem. Physiol. A Physiol. 48, 539–546. Kelley, E.E., Khoo, N.K.H., Hundley, N.J., Malik, U.Z., Freeman, B.A., Tarpey, M.M., 2010. Hydrogen peroxide is the major oxidant product of xanthine oxidase. Free Radic. Biol. Med. 48 (4), 493–498. http://dx.doi.org/10.1016/j.freeradbiomed.2009.11.012. King, G.L., Kahn, C.R., 1981. Non-parallel evolution of metabolic and growth-promoting functions of insulin. Nature 292, 644–646. Kooyman, G.L., Ponganis, P.J., 1998. The physiological basis of diving to depth: birds and mammals. Annu. Rev. Physiol. 60, 19–32. Kooyman, G.L., Castellini, M.A., Davis, R.W., 1981. Physiology of diving in marine mammals. Annu. Rev. Physiol. 43, 343–356. Kooyman, G.L., Castellini, M.A., Davis, R.W., Maue, R.A., 1983. Aerobic diving limits of immature Weddell seals. J. Comp. Physiol. B. 151, 171–174. Lance, V., Elsey, R., 1983. Selenium and glutathione peroxidase activity in blood of the nutria (Myocastor coypus): comparison with guinea-pig, rat, rabbit and some nonmammalian vertebrates. J. Comp. Biochem. Physiol. B. 75 (4), 563–566. Lenfant, C., Johansen, K., Torrance, J.D., 1970. Gas transport and oxygen storage capacity in some pinnipeds and the sea otter. Respir. Physiol. 9, 277–286. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randan, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193 (1), 265–275. MacArthur, R.A., 1992. Foraging range and aerobic endurance of muskrats diving under ice. J. Mammal. 73, 565–569.

C

T

that are needed to be further investigated. Across species, nutria had the lowest LDH activities in the heart, kidney, lung and spleen. Along with 689 that, nutria heart and lung are characterized by mainly aerobic metabo690 lism, liver and muscle — by glycolytic pathway of energy production, 691 and kidney and spleen of nutria contain nearly equal amounts of H692 and M-subunits reflecting the importance of both aerobic and anaerobic 693 ATP productions for functions of these organs. 694 The more ancient invaders of aquatic environment than diving ro695 dents possess higher (than terrestrial animals) antioxidant capacity 696 Q11 (Elsner et al., 1998; Zenteno-Savín et al., 2002; Vázquez-Medina et al., 697 2006, 2007), which not only provide the defense against potentially 698 harmful effects of ROS, producing in excess during repetitive cycles of 699 hypoxia/reoxygenation, but also can act as regulators of oxygen sensing 700 and redox signaling involved in response to hypoxia (Bunn and Poyton, 701 1996; Brunelle et al., 2005). Similarly, semiaquatic rodents have higher 702 catalase activity in the tissues compared to non-diving rat. Across semi703 aquatic species, beaver had the highest catalase activities in its heart and 704 muscle, and nutria had the lowest GSH content in almost all tissues. 705 Comparison of beaver and nutria demonstrated no significant differ706 ences in the antioxidant enzyme activities in the heart, kidney and 707 liver. The SOD activity was higher, but catalase activity was lower in al708 most all tissues of muskrat than of both beaver and nutria. Our results 709 showed that catalase plays the greater role than SOD in oxygen sensing 710 and redox signaling in beaver and nutria tissues while SOD — in muskrat 711 tissues acting also as a regulator of cardiovascular effects during a dive. 712 No differences in GSH content in all tissues were found between beaver 713 and both muskrat and rat, indicating strong regulation of cell redox 714 state irrespective of hypoxia. 715 Our results suggest that semiaquatic mammals have distinct mecha716 nisms of adaptation to dive-associated hypoxia/reoxygenation and con717 firmed the hypothesis that semiaquatic mammals are the intermediate 718 animals that help to reveal which potential selection factors and me719 chanical constraints may have directed the evolution of more derived 720 aquatic forms (Fish, 1992; Williams, 1999).

D

687 688

E

8

Acknowledgments

722 723

728 729

The authors are extremely grateful to our colleagues F.V. Fyodorov, K.F. Tirronen, D.V. Panchenko, E.A. Khizhkin and A.V. Morozov (Institute of Biology, Karelian Research Centre of RAS, Petrozavodsk, Russia) for providing the animals and the assistance for this study. The study was carried out under state order (project no. 0221-2014-0001) and it was partially funded by the President of the Russian Federation grant for Leading Scientific School # 1410.2014.4 and partially of the grant for Russian–Polish Interacademic Cooperation.

730

References

731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752

Allers, D., Culik, B.M., 1997. Energy requirements of beavers (Castor canadensis) swimming underwater. Physiol. Zool. 70, 456–463. Bears, R.F., Sizes, I.N., 1952. A spectral method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195 (1), 133–140. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., Freeman, B.A., 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. U. S. A. 87 (4), 16–20. Blix, A.S., From, S.H.J., 1971. Lactate dehydrogenase in diving animals — a comparative study with special reference to the eider (Somateria mollissima). Comp. Biochem. Physiol. 40B, 579–584. Brunelle, J.K., Bell, E.L., Quesada, N.M., Vercauteren, K., Tiranti, V., Zeviani, M., Scarpulla, R.C., Chandel, N.S., 2005. Oxygen sensing requires mitochondrial reactive oxygen species but not oxidative phosphorylation. Cell Metab. 1 (6), 409–414. http://dx.doi.org/ 10.1016/j.cmet.2005.05.002. Bunn, H.F., Poyton, R.O., 1996. Oxygen sensing and molecular adaptation to hypoxia. Physiol. Rev. 76 (3), 839–885. Butler, P.J., Jones, D.R., 1997. Physiology of diving of birds and mammals. Physiol. Rev. 77 (3), 837–899. Cabanac, A., Folkow, L.P., Blix, A.S., 1997. Volume capacity and contraction control of the seal spleen. J. Appl. Physiol. 82 (6), 1989–1994. Cantú-Medellín, N., Byrd, B., Hohn, A., Vázquez-Medina, J.P., Zenteno-Savín, T., 2011. Differential antioxidant protection in tissues from marine mammals with distinct diving

R

R

O

C

N

726 727

U

724 725

E

721

Please cite this article as: Sergina, S., et al., Biochemical adaptations to dive-derived hypoxia/reoxygenation in semiaquatic rodents, Comp. Biochem. Physiol. B (2015), http://dx.doi.org/10.1016/j.cbpb.2015.08.012

753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838

S. Sergina et al. / Comparative Biochemistry and Physiology, Part B xxx (2015) xxx–xxx

Romanenko, S.A., Perelman, P.L., Trifonov, V.A., Graphodatsky, A.S., 2012. Chromosomal evolution in Rodentia. Heredity 108, 4–16. http://dx.doi.org/10.1038/hdy.2011.110. Scholander, P.F., 1940. Experimental investigations on the respiratory function in diving animals and birds. Hvalradets Skrifter 22 pp. 1–131. Sedlak, J., Lindsay, R.H., 1968. Estimation of total, protein-bound and non-protein sulfhydryl groups in tissue with Ellman's reagent. Anal. Biochem. 25, 192–205. Shoubridge, E.A., Carscadden, J.E., Leggett, W.C., 1976. LDH isozyme patterns in cetaceans: evidence for a biochemical adaptation to diving. Comp. Biochem. Physiol. 53B, 357–359. Snyder, G.K., Binkley, E.L., 1985. Oxygen transport, tissue glycogen stores, and tissue pyruvate kinase activity in muskrats. Can. J. Zool. 63, 1440–1444. Tenney, S.M., Remmers, J.E., 1963. Comparative quantitative morphology of the mammalian lung: diffusing area. Nature 197, 54–56. Theunissen, S., Guerrero, F., Sponsiello, N., Cialoni, D., Pieri, M., Germonpré, P., Obeid, G., Tillmans, F., Papadopoulou, V., Hemelryck, W., Marroni, A., De Bels, D., Balestra, C., 2013. Nitric oxide-related endothelial changes in breath-hold and scuba divers. Undersea Hyperb. Med. 40 (2), 135–144. Vázquez-Medina, J.P., Zenteno-Savín, T., Elsner, R., 2006. Antioxidant enzymes in ringed seal tissues: potential protection against dive-associated ischemia/reperfusion. Comp. Biochem. Physiol. C 142, 198–204. http://dx.doi.org/10.1016/j.cbpc.2005.09. 004. Vázquez-Medina, J.P., Zenteno-Savín, T., Elsner, R., 2007. Glutathione protection against dive-associated ischemia/reperfusion in ringed seal tissues. J. Exp. Mar. Biol. Ecol. 345, 110–118. http://dx.doi.org/10.1016/j.jembe.2007.02.003. Wieme, R., 1959. Studies on Agar-gel Electrophoresis (Brussels). Williams, T.M., 1999. The evolution of cost efficient swimming in marine mammals: limits to energetic optimization. Philos. Trans. R. SOC. Lond. B. Biol. Sci. 353, 1–9. Zenteno-Savín, T., Clayton-Hernandez, E., Elsner, R., 2002. Diving seals: are they a model for coping with oxidative stress? Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 133 (4), 527–536.

N C O

R

R

E

C

T

E

D

P

R O

O

F

McCord, J.M., Fridovich, I., 1969. Superoxide dismutase. J. Biol. Chem. 244 (22), 6049–6055. McCulloch, P.F., 2012. Animal models for investigating the central control of the mammalian diving response. Front. Physiol. 3, 1–16. http://dx.doi.org/10.3389/fphys.2012.00169. McKean, T.A., 1982. Cardiovascular adjustments to laboratory diving in beavers and nutria. Am. J. Physiol. 242 (5), R434–R440. McKean, T.A., 1984. Response of isolated muskrat and guinea pig hearts to hypoxia. Physiol. Zool. 57, 557–562. McKean, T., Landon, R., 1982. Comparison of the response of muskrat, rabbit, and guinea pig heart muscle to hypoxia. Am. J. Physiol. 243 (3), R245–R250. Messelt, E.B., Blix, A.S., 1976. The LDH of the frequently asphyxiated beaver (Castor fiber). Comp. Biochem. Physiol. 53B, 77–80. Mirceta, S., Signore, A.V., Burns, J.M., Cossins, A.R., Campbell, K.L., Berenbrink, M., 2013. Evolution of mammalian diving capacity traced by myoglobin net surface charge. Science 340, 1234192. http://dx.doi.org/10.1126/science.1234192. Misra, H.P., Fridovich, F., 1972. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247 (10), 3170–3175. Murphy, B., Zapol, W.M., Hochachka, P.W., 1980. Metabolic activities of heart, lung and brain during dividing and recovery in the Weddell seal. J. Appl. Physiol. 48, 596–605. Opazo, J.C., Soto-Gamboa, M., Bozinovic, F., 2004. Blood glucose concentration in caviomorph rodents. J. Comp. Biochem. Physiol. A. 137 (1), 57–64. http://dx.doi. org/10.1016/j.cbpb.2003.09.007. Panneton, W.M., Gan, Q., Juric, R., 2010. The rat: a laboratory model for studies of the diving response. J. Appl. Physiol. 108 (4), 811–820. http://dx.doi.org/10.1152/ japplphysiol.00600.2009. Philp, A., Macdonald, L.A., Watt, P.W., 2005. Lactate — a signal coordinating cell and systemic function. J. Exp. Biol. 208, 4561–4575. Ponganis, P.J., Kooyman, G.L., Baranov, E.A., Thorson, P.H., Stewart, B.S., 1997. The aerobic submersion limit of Baikal seals, Phoca sibirica. Can. J. Zool. 75, 1323–1327.

U

839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868

9

Please cite this article as: Sergina, S., et al., Biochemical adaptations to dive-derived hypoxia/reoxygenation in semiaquatic rodents, Comp. Biochem. Physiol. B (2015), http://dx.doi.org/10.1016/j.cbpb.2015.08.012

869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 Q12 894 895 896 897 898