Anaerobiosis and the toxicity of cyanide in turtles

Comp. Biochem. Physiol., 1968, Vol. 24, pp. 543 to 548. Pergamon Press. Printed in Great Britain

ANAEROBIOSIS AND T H E T O X I C I T Y OF CYANIDE IN T U R T L E S D. BELLAMY and J. A. PETERSEN* Department of Zoology, The University, Sheffield 10

(Received 27 ffuly 1967) A b s t r a c t - - 1 . Aquatic and terrestrial chelonians from the Amazon basin sur-

vived several hours in an atmosphere of nitrogen. The major change in tissue composition was an increase in plasma lactic acid. 2. Injections of potassium cyanide were fatal at doses which only partly inhibited respiration. From changes in the composition of plasma and brain it appeared that there was an impairment in the homeostatic mechanisms for glucose, lactic acid, sodium and potassium. 3. The results are discussed in relation to the control of metabolism in anoxia. INTRODUCTION IT IS generally acknowledged that many invertebrates can survive from hours to days in the absence of oxygen (Slater, 1928), but resistance to anoxia is not usually considered to be an important characteristic of mature vertebrates. However, nearly a hundred years ago it was reported that amphibians could survive in an atmosphere of nitrogen for several hours (Pfliiger, 1875; Aubert, 1881). Later, these observations were extended to include turtles where the survival times were measured in terms of days (Johlin & Moreland, 1934). Although chelonians are outstanding in their ability to withstand anoxia (BeUdn, 1963) the available evidence suggests that the ability to derive a large proportion of energy from anaerobic reactions may be a fairly widespread adaptation of lower vertebrates to special environmental conditions (e.g. Blazka, 1958). Potassium cyanide is a potent inhibitor of aerobic oxidative metabolism and its toxicity in mammals has been ascribed solely to an action on electron transport. The long-term survival of chelonians in nitrogen suggested the possibility that cyanide could be tested in these animals for physiological effects apart from the inhibition of respiration. Also a comparison of the effects of anoxia with those of cyanide administration might throw light on the role of oxygen tension in the control of anaerobic metabolism in turtles. METHODS

Animals Experimental work was carried out aboard the R.V. Alpha Helix at the confluence of the Rio Negro and Rio Branco, Amazonas, Brazil. Turtles were obtained * Present address: Department of Physiology, Caixa Postal 6868, S~o Paulo, Brazil. 54-3

544

D. BELLAMY AND J. A. PETERSEN

locally and kept in running water drawn from the Rio Negro (temperature, 29°C; pO2, 60-70 mm Hg).

Glucose, urea and lactic acid in plasma Because the experiments were carried out in the field it was convenient to make use of analytical kits available from commercial sources. For each method blood was collected in a heparinized beaker after decapitation. Glucose. A specific coupled enzyme system of glucose oxidase and peroxidase was used (Glucostat, Worthington Biochemical Corp. New Jersey, U.S.A.). Urea. After the addition of urease, ammonia derived from urea was measured with Nessler's reagent (Kit No. 14, Sigma Chemical Co. Ltd., New York). Lactic acid. Lactic dehydrogenase was used to give a quantitative reduction of flavine adenine dinucleotide (Test combination 15972; Boehringer Corp.).

Sodium and potassium Inorganic ions were extracted from pieces of brain tissue by shaking for 2 hr at 37°C with 0-1 N HNO3 (1 ml/10 mg wet wt. tissue). The extract was analysed with a flame photometer, using appropriate interference standards.

Oxygen uptake The respiration of intact animals was determined by placing them in a thickwalled plastic box containing about 4 1. of water and 1 1. of air at 30°C. Samples of air and water were taken at intervals and the oxygen tension measured with an electrode system (Beckman Instrument Co.). At the beginning of the experiments the oxygen tension in air and water was between 100 and 150 mm Hg.

Incubations in vitro Slices of brain cortex were incubated at 30°C in a balanced salt medium (NaCI, 8.0g; Na2HPO4, 0.2g; KC1, 0-2g; MgC12.6H20 , 0.1 g; CaC12, 0.1 g; H,O, 1000ml), containing 5 m M glucose and UC 14 acetate (30me/m-mole; 0.1/~c/ml). Radioactive carbon dioxide was absorbed with filter paper soaked in hyamine hydroxide and the radioactivity measured with a scintillation counter after immersion of the paper in a standard toluene-based scintillation fluid (about 15 per cent counting efficiency). RESULTS (a) Nitrogen These experiments were carried out with animals in a chamber flushed with nitrogen. The atmospheric pO~ was checked at intervals and varied between 4 and 10 mm Hg. With animals kept in a sealed chamber containing air it was found that the oxygen uptake ceased when the pO~ had dropped below 10 mm Hg. Both terrestrial (Testudo tabulata) and aquatic (Podocnemys sp.) turtles rapidly developed muscular weakness which affected locomotion. However, the animals were still capable of rapid withdrawal movements of limbs and head. Six hours'

ANAEROBIOSISAND THE TOXICITYOF CYANIDEIN TURTLES

545

exposure to low oxygen tensions was not toxic for Podocnemys but individual Testudo varied in their response. Most animals survived this treatment and their reactions could not be distinguished from those of Podocnemys. Others died within 3-6 hr. T h e toxicity of nitrogen in Testudo was not related to the size of the animals. T h r e e hours in nitrogen resulted in an increase in plasma potassium (22 per cent) and lactic acid (about 70 per cent). T h e r e was no significant change in blood sodium, glucose and urea (Table 1). Brain sodium and potassium were little affected in either Testudo or Podocnemys (maximum variation of about 10 per cent; Table 2). W h e n animals that had spent 3 hr in nitrogen were returned to air the oxygen uptake increased by about 30 per cent compared with the pre-anaerobic rate. TABLE 1--EFFECT OF ANOXIAON PLASMACOMPOSITIONIN Podocnemys sextuberculata Plasma constituents

Normal Anoxic

Sodium (mN)

Potassium (mN)

Glucose (mM)

Urea (mM)

Lactic acid (mM)

124"5 ± 5"1 122'2 ± 5"5

3"91 +0"02 5"00*+ 0.04

1-97 +_0"15 2-08 +-0-19

6"71 +0-53 7"04 + 0"85

3"11 +0"07 5"30* _+0-17

* P < 0"01 compared with normal. Animals were placed in a chamber containing water to a depth of about 1 mm. Nitrogen was passed through the chamber for 3 hr (pO~ about 5 mm Hg). Animals were killed by decapitation and blood collected in a heparinized syringe. Each figure is a mean + S.E. for six animals. TABLE 2--EFFECT OF ANOXIAON BRAINSODIUM AND POTASSIUMIN P. sextuberculata and

Testudo tabulata P. sextuberculata

Normal Anoxic

T. tabulata

Sodium (mN)

Potassium (naN)

Water (%)

Sodium (mN)

Potassium (mN)

Water (%)

39"6 + 0"4 44"5*+0"6

61"5 + 0"5 53"3*+0"7

82"3+_0"7 83"3+0"5

45"3 + 0"2 40"1"+0"3

56"6 + 0"7 55"2"+1'0

84-1+ 1'0 83.6+0-6

* P < 0'01 compared with normal. Animals were treated as described in Table 1 for 4 hr. Figures are mean _+S.E. for six animals. (b) Potassium cyanide in vivo Various amounts of 0.1 M K C N (volume calculated as a percentage of body weight) were injected into the body cavity of animals kept in air (Table 3). Potassium cyanide affected muscular tone but the response was more pronounced

546

D. BELLAMYAND J. A. PETERSEN

compared with nitrogen (abolished withdrawal movements). A dose of 0-25 per cent and above was toxic for both Testudo and Podocnemys; the animals died between 5 and 12 hr after treatment. Oxygen uptake was usually inhibited at doses greater than 0.1 per cent. In P. sextuberculata this inhibitory effect increased with dose and was maximal at 0.5 per cent (90 per cent inhibition). Toxicity could not be related to the inhibition of respiration. In this connexion, one specimen of Testudo died without any change in oxygen uptake. T A B L E 3 - - E F F E C T OF POTASSIUM CYANIDE ON PLASMA AND BRAIN IN P.

Plasma

sextuberculata

Brain

Time (hr)

Sodium (mN)

Potassium (mN)

Glucose (raM)

Sodium (m-moles/kg)

Potassium (m-moles/kg)

0 1 3 6

123 124 113 118

2"45 6"00 4"50 3"30

1"45 2-39 2-59 3-05

41"0 40"5 45"1 47"7

56"7 50"8 67-2 59"6

Animals were injected with 0"1 M KCN into the body cavity (0.25 per cent of body wt.) and kept in air for 6 hr. Each figure is a mean for three animals. T h e r e was a 50 per cent inhibition of respiration in P. sextuberculata given 0.25 per cent of 0.1 M K C N . Blood potassium and glucose increased by a factor of two. Brain potassium decreased during the first 3 hr (10 per cent), then increased up to 7 hr (20 per cent). Most animals died between 7 and 8 hr after treatment. Changes in the composition of plasma were more marked in cyanide-treated animals compared with those kept in nitrogen (Table 4). TABLE A

C O M P A R I S O N OF THE EFFECTS OF ANOXIA AND POTASSIUM CYANIDE

Plasma constituent Treatment Air Anoxic Cyanide

Sodium (raN)

Potassium (mN)

Lactic acid (mM)

126"1 +7"1 130.7+6-5 127"3+4"1

3"21 +0"19 3"91 +0"21 5"77*+0"23

2.43 +0"49 3-77 +0"31 6.41"+0.33

* P < 0"01 compared with anoxic animals.

P. sextuberculata was maintained in an atmosphere of nitrogen for 6 hr and composition of plasma compared with air-exposed animals and those treated with 0.1 M KCN (0"25 per cent of body wt.). Each figure is the mean + S.E. for six animals.

Experiments in vitro T h e addition of K C N to brain slices from Testudo and Podocnemys inhibited the oxidation of C 14 acetate (60 per cent inhibition at 10 -5 M). Cyanide up to

ANAEROBIOSIS AND THE TOXICITY OF CYANIDE IN TURTLES

547

1"4 mM did not affect tissue sodium and potassium ('Fable 5). Most species showed an increase in glucose metabolism with cyanide (20-40 per cent with 10 -6 M). The addition of iodoacetate (1 mM) resulted in the equilibration of tissue sodium and potassium with the incubation medium in 2 hr (Table 5). Incubation in an atmosphere of nitrogen did not affect tissue potassium but resulted i n a slight gain of sodium (15 per cent). TABLE

5--EFFECT

O F P O T A S S I U M C Y A N I D E A N D I O D O A C E T A T E O N SLICES O F B R A I N CORTEX

Additions Nil KCN 0.01 mM 0"10 mM 1"4 mM Iodoacetate 1 " 0 mM

C1402 (10 -s c/min)

Sodium (m-moles/kg)

Potassium (m-moles/kg)

2-42

42.2

58.3

1"04 0"41 0"23

42"0 44"9 40"6

60.0 57.2 59"8

--

133

4"6

Slices of cerebral cortex from P. sextubemulata were incubated for 2 hr in a balanced salt solution containing glucose and Cx4 a c e t a t e a s described in the text. Each figure is a mean for two experiments. DISCUSSION Terrestrial and aquatic turtles found in the Rio Negro area survived for several hours under a p02 at which aerobic respiration was inhibited. This condition had little effect on the steady-state concentrations of a number of body constituents. The main question raised by this study concerns the mechanisms by which homeostasis is maintained. Previous work on turtle tissues in vitro has shown that under anaerobic conditions glycolysis can maintain some energy-requiring processes close to the aerobic level (Reeves, 1963; Brodsky & Schilb, 1966). These findings were borne out in the present work in that sodium and potassium in blood and brain were little affected by anoxia. Further, the concentration gradients for sodium and potassium in brain cortex were maintained in vitro when oxidative metabolism was inhibited. As judged by the relatively small rise in blood lactic acid and the low oxygen debt, the metabolic rate was considerably reduced in nitrogen. Consequently, a number of energy-consuming reactions must have been inhibited. It is possible that the regulation of energy demands was brought about automatically by enzyme feedback, but it is more likely that endocrine responses form an important part of the metabolic adaptation to anoxia. In this connexion, the experiments with cyanide injection suggest that a 50 per cent inhibition of aerobic energy production can be fatal (cf. Robin et al., 1964). The abnormally large rise in blood lactate which accompanied this effect indicated that glycolysis was proceeding faster than in anoxic animals. All these findings point to the low pO 2 being an important releaser of metabolic control during periods of anoxia. This conclusion rests on

548

D. BELLAMYAND J. A. PETERSEN

the assumption that the only action of cyanide was to inhibit terminal electron transport. T h e fact that one cyanide-treated animal died, and others showed symptoms of cyanide poisoning without a fall in respiration, raises the possibility that the metabolic changes were not due entirely to an inhibition of oxidation. Here a great difficulty concerns the definition of basal metabolic rate and the effect of cyanide on the activity of the animal. T h e smallest injection of cyanide that was toxic in chelonians was about fifty times greater than the toxic dose for mammals. Also, in contrast to mammals, several hours elapsed before the turtles died. T h e sensitivity of oxidative metabolism of brain slices was similar to that reported for tissues from higher vertebrates, and the relatively low toxicity of K C N in chelonians is probably related to a lesser dependence on oxidative aerobic energy-producing reactions and to differences in the mobilization and excretion of the injected compound. It is difficult to assess the biological advantages which arise from the ability of tropical chelonians to survive under anaerobic conditions. In the case of aquatic species, the survival value might be related to the diving habit (Root, 1949). In terrestrial forms the tolerance to anoxia may relate to difficulties in ventilating the lungs at times when the head and limbs are withdrawn into the shell. Acknowledgements--This work was carried out aboard the R.V. Alpha Helix as part of Research Programme B of the University of California Amazon Expedition, 1967. The investigations were made possible by funds from the National Science Foundation of America. REFERENCES AUBERT H. (1881) Ueber den Einfluss der Temperatur auf die Kohlensiiureausscheidung

und die Lebensf~ihigkeit der Fr6sche in sauerstoffloser Luft. Pfliigers Arch. ges. Physiol. 26, 293-323. BELKIN D. A. (1963) Anoxia: Tolerance in reptiles. Science 139, 492-493. BLAZKa P. (1958) The anaerobic metabolism of fish. Physiol. Zool. 31, 117-128. BRODSr:YW. A. & SCrIILBT. P. (1966) Ionic mechanisms for sodium and chloride transport across turtle bladders. Am. ft. Physiol. 210, 987-996. JOHLIN J. M. & MORELAND F. B. (1934) Studies of the blood picture of the turtle after complete anoxia, ft. biol. Chem. 103, 107-114. PFLOaER E. (1875) Beitr~ige zur Lehre yon der Respiration--I. Ueber die physiologische Verbrennung in den lebendigen Organismen. Pfliigers Arch. ges. Physiol. 10, 251-367. REEVES R. B. (1963) Energy cost of work in aerobic and anaerobic turtle heart muscle. Am. ft. Physiol. 205, 17-22. ROBIN E. D., V~San~RJ. W., MtrmgAOCHH. V. & MILLENJ. E. (1964) Prolonged anaerobiosis in a vertebrate: anaerobic metabolism in the freshwater turtle, ft. gen. comp. Physiol. 63, 287-297. ROOT R. W. (1949) Aquatic respiration in the musk turtle. Physiol. Zool. 22, 172-178. SLATERW. K. (1928) Anaerobic life in animals. Biol. Rev. 3, 303-328.