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Histochemical demonstration of dehydrogenase activity in the cuticle of cestodes
EXPERIMENTAL
PARASITOLOGY
14, 333-336 (1963)
Histochemical Activity Alvin
H.
Department
Demonstration in the Cuticle Rothmanl of Biology,
(Submitted
and
of Dehydrogenase of Cestodes Donald
Rice University,
for publication,
L. Lee2
Houston,
13 March
Texas
1963)
With the aid of nitro blue tetrazolium, the cuticle of four species of cestodes has been shown capable of reducing enzymatically isocitrate, glutamate, a-ketoglutarate, succinate, malate, and lactate. It is considered that this activity resides in the cuticular mitochondria.
Threadgold (1962) and Rothman (1963) have shown that the cuticle of cestodes contains structures which morphologically resemble the mitochondrion. With the aid of the electron microscope and modern biochemical and histological techniques, mitochondria have been shown capable of oxidizing the intermediates of the tricarboxylic acid cycle and the reduced form of the pyridine nucleotides (Ziegler and Linnane, 1958; Sedar and Rosa, 1961). Read (1952, 1953) indicated that homogenates of the tapeworm Hymenolepis diminuta oxidized malic, succinic, and glutamic acids and converted fumaric acid to malic acid. Heyneman and Voge (1960) demonstrated succinic dehydrogenase in the hymenolepid cysticercoid and Goldberg and Nolf ( 1954) demonstrated succinic dehydrogenase in the strobilate phase of Hymenolepis nana. The present report will show that the cuticle of cestodes is potentially capable of metabolic activity.
MATERIALS
AND METHODS
The species of cestodes investigated included Hymenolepis diminuta, Hymenolepis microstoma, Hymenolepis citelli, and Taenia (Hydatigera) taeniaeformis. The cysticercoid larvae of the hymenolepid species were reared in the grain beetle, Tribolium confusum, and the strobilocercus larvae of T. taeniaeformis were reared in Sprague-Dawley rats. The strobilate form of the tapeworms were obtained as follows: H. diminuta from SpragueDawley rats, H. microstoma from Swiss mice, H. citelli from golden hamsters, and T. taeniaeformis from domestic cats. Portions of the worms were either fresh frozen before sectioning or were immersed in acetone at -20°C for 15 minutes before embedding in ice and sectioning. Worms were also fixed in 10% neutral formalin for 15 minutes and 120 minutes at 5°C before embedding and sectioning. Control worms were either dipped in boiling water for 1 minute or exposed to a dry heat of 95°C for 20 minutes. Sections approximately 8 u thick were cut in an International cryostat, removed on the end of a sharp needle, and floated in the test solution. Fresh tissue was incubated for 15 minutes, and acetone-fixed or formalin-fixed tissue was incubated for 60 minutes. The sec-
1 Present address: Department of Zoology, University of Maryland. 2 Present address: Molten0 Institute of Parasitology, University of Cambridge. This investigation was supported by grants from the U.S. Public Health Service (E-427.5 and E-1384). 333
334
ROTHMAN
tions were then removed from the incubation medium by means of a sharp needle, placed in a drop of glycerine on a microscope slide, covered with a glass slip, and examined. The test solution contained, per milliliter: 2 pmoles of substrate, 1 umole MgCl?, 0.25 mg nitro blue tetrazolium (NBT), 7.5mg polyvinyl pyrrolidinone, and 4 umoles tris maleate buffer at pH 7.4. Diphosphopyridine nucleotide (DPN) was added to give a final concentration of 2 ymoles per milliliter. Malonate was tested at 2 umoles per milliliter and arsenite was tested at 4 pmoles per milliliter. The substrates included the sodium salts of citric, isocitric, succinic, glutamic, a-ketoplutaric, fumaric, and malic acids. The calcium salt of lactic acid and the reduced form of diphosphopyridine nucleotide (DPNH) were also tested as substrates.” RZSULTS
As summarized in Table I fresh-frozen section of the four species of cestodes have histochemically demonstrable NBT reducing systems. Figures 1 and 2 show that both the cuticle and the subcuticular parenchyma have activity. Negative results were obtained with isocitrate, glutamate, malate, and lactate when the DPN was absent from the incubation medium. The succinate and a-ketoglutarate systems were positive in the absence of coenzyme. Diphosphopyridine nucleotide in the absence of substrate gave negative results, whereas DPNH gave a positive result in the absence of any other electron donor. Worms fixed in acetone and worms fixed in formalin for a brief period gave the same results indicated in Table 1. Worms fixed for 120 minutes in the formalin yielded negative results except in the case of DPNH where a weak result was obtained. 3 The DPN, DPNH, and NBT mere obtained from the Sigma Chemical Company, St. Louis 18, Missouri. The substrates were obtained from the California Corporation for Biochemical Research, Los Angeles 63, Calif.
.I_HNLJ
--LfiI.5
Control worms, which were dipped in boiling water for 1 minute before freezing, were negative for all substances tested. Worms exposed to a dry heat, before freezing, gave a barely detectable color. Malonate inhibited the response with succinate, and interestingly, when arsenite was tried with the fumarate-DPN system, a positive result was obtained with two of the tapeworm species. When arsenite was tested with the other systems of H. diminuta, an enhancement of the formazan production occurred. Arsenite had no effect on the DPN-requiring systems if DPN were absent. Also, arsenite in the no substrate-DPN system had no effect. DISCUSSION
The advantages of using NBT over other tetrazoliums is adequately discussed by Nachlas et al. (1957) and need not be considered here. The reduction of NBT in the cuticle and the parenchyma of the four species of cestodes studied, as well as the demonstrated requirement for DPN, argues for the presence of the oxidative enzyme systems. The lack of activity in the heat-treated controls supports this contention. The specificity of the electron donor was demonstrated by using reduced glutathione as a substrate. At approximately pH 10 a general bluing of the medium occurred due to formazan formation, as indicated by Pearse (1960). That the cuticle has metabolic activity is plain; it remains to be proven, however, that the enzyme activity is in the mitochondria. The work of Sedar and Rosa ( 1961) on rat tissues lends support to this possibility. Except for the histochemical demonstration of isocitric dehydrogenase, the present report agrees with the biochemical determination of dehydrogenase presented by Read (1952, 1953). Read (1953) was unable to demonstrate manometrically isocitric dehydrogenase in tapeworm homogenates. This
DEHYDHOGENASE
ACTIVITY
No substrate
with
DPN
Citrate
eb
cl
c2
-
-
-
with
DPN
Isocitrate
with
DPN
Glutamate Glutamate
with
DPN
+
with
malonate
-
-
-
-
-
-
-
with
DPN
Cl
C2
+ + +
+ + +
-
-
-
-
-
-
r -
+
-
+
+ -
-
+
-
+
-
glutathione
Reduced glutathione with DPN
e
-
-
-
with DPN
+ -
r
DPN
Lactate
Reduced
-
-
Malate
Lactate DPNH
-
c2
5
-
+ + +
r -
Fumarate with DPN with arsenite Malate
e
-
+
Succinate
Fumarate
c2
-
+ -
+
with
cl
-
Alpha ketoglutarate Succinate Fumarate
e
-
-
Isocitrate
335
CUTICLE
-
-
Citrate
CESTODE
TABLE I Activity irk the Cuticle of Cestodesa
Dehydrogenase
No substrate
OF
-
-
-
-
+ -
-
+
-
-
+
-
-
+
-
+
-
-
+
-
-
w
+
-
w
-
+ -
-
-
-
+ -
W
-
w
-
a The test solution contained, per milliliter: 2 pmoles of substrate, 1 umole MgCI,, 0.25 mg nitro blue tetrazolium (NBT), 75 mg polyvinyl pyrrolidinone, and 4 umoles tris buffer at pH 7.4. Diphosphopyriclme nucleotide (DPN) was added to give a final concentration of 2 umoles per milliliter. Malonate was tested at 2 umoles per milliliter and arsenite was tested at 4 umoles per milliliter. b e, experimental; cl, control worms immersed 1 minute in boiling water; c2, control worms heated at 95°C for 20 minutes; +, positive result; -, negative result; r, reduced result; w, barely perceptible result.
FIG. 1. Section of H. citelli strobila showing formazan deposits in base of cuticle and in parenchyma. The light band between the dark area of the cuticle and the parenchyma is the subcuticle. FIG. 2. Section of T. taeniaefovmis strobila showing formazan deposits in base of cuticle and in parenchyma. The light band between the dark area of the cuticle and the parenchyma is the subcuticle.
336
ROTHMAN
point will bear further examination by alternative procedures. Pearse (1960) indicates that neither aconitase nor fumarase has been demonstrated histochemically. The present study supports this observation except for the unexpected response effected by arsenite on the fumarateDPN system. Since it was anticipated that arsenite would elicit just the reverse effect, the finding that arsenite also enhances the oxidation of all the substrates (except citrate) tested with H. diminuta certainly warrants further investigation. Aronson and Pharmakis (1962) report a similar effect of potassium cyanide for succinic dehydrogenase. An electron donor is important to this arsenite enhancement, for in the absence of one, DPN with arsenite has no noticeable effect. An uncoupling of phosphorylation by arsenite (Fletcher and Sanadi, 1962) could account for the increased transport of electrons, yielding more formazan. The brief formalin fixation and brief acetone fixation facilitated considerably the handling of the tissue and the interpretation of the results (cf. Pearse, 1960; Walker and Seligman, 196 1) . REFERENCES AROSSON, i%‘., AND PHARMAKIS, T. 1962. Enhancement of neotetrazolium staining for succinic dehydrogenase activity with cyanide. Stain Technology 37, 321. FLETCHER, M. J., AND SAPTADI,D. R. 1962. On the mechanism of oxidative phosphorylation. III. Effects of arsenite and 2,3-dimercaptopropanol on coupled phosphorylation in heart mitochondria. Archives of Biochemistry and Biophysics 96, 139-142. GOLDBERG, E.,
AND NOLF, L.
0.
1954.
Succinic
AND
LEE
dehydrogenase activity in the cestode Hymenolepis nana. Experimental Parasitology 3, 275-284. HEYNEMAN, D., AND VOGE, M. 1960. Succinic dehydrogenase activity in cysticercoids of Hymenolepis (Cestoda: Hymenolepididae) measured by the tetrazolium technique. Experimental Parasitology 9, 14-17. NACNLAS, M. M., Tsou, K. C., SOUZA, E., CHENG, C. S., AND SELIGMAN, A. M. 1957. Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. Journal of Histochemistry and Cytochemistry 5, 420.436. PEARSE, A. G. E. 1960. “Histochemistry: Theoretical and Applied,” 998 pp. Little, Brown and Company, Boston, Massachusetts. READ, C. P. 1952. Contributions to cestode enzymology. I. The cytochrome system and succinic dehydrogenase in Hymenolepis diminuta. Experimental Parasitology 1, 353-362. READ, C. P. 1953. Contributions to cestode enzymology. II. Some anaerobic dehydrogenases in Hymenolepis diminuta. Experimental Parasitology 2, 341-347. ROTHMAN, A. H. 1963. Electron microscope studies of tapeworms. The surface structures of Hymewolepis diminuta. Transactions of the American Microscopical Society 82, 22.30. SEDAR,.4. W., AND ROSA, C. G. 1961. Cytochemical demonstration of the succinic dehydrogenase system with the electron microscope, using nitro blue tetrazolium. Ultrastructure Research 5, 226243. THREADGOLD, L. T. 1962. An electron microscope study of the tegument and associated structures of Dipylidium caninum. Quarterly Journal of Microscopical Science 103, 135-140. WALKER, D. G., AND SELIGMAN, A. M. 1961. Formalin fixation in the cytochemical demonstration of succinic dehydrogenase of mitochondria. Journal of Biophysical and Biochemical Cytology 9, 415-428. ZIEGLER, D. M., AND LINPSANE, A. W. 1958. Studies on the electron transport system. XIII. Mitochondrial structure and dehydrogenase activity in isolated mitochondria. Biochimica et Biophysica Acta 30, 53-63.
PARASITOLOGY
14, 333-336 (1963)
Histochemical Activity Alvin
H.
Department
Demonstration in the Cuticle Rothmanl of Biology,
(Submitted
and
of Dehydrogenase of Cestodes Donald
Rice University,
for publication,
L. Lee2
Houston,
13 March
Texas
1963)
With the aid of nitro blue tetrazolium, the cuticle of four species of cestodes has been shown capable of reducing enzymatically isocitrate, glutamate, a-ketoglutarate, succinate, malate, and lactate. It is considered that this activity resides in the cuticular mitochondria.
Threadgold (1962) and Rothman (1963) have shown that the cuticle of cestodes contains structures which morphologically resemble the mitochondrion. With the aid of the electron microscope and modern biochemical and histological techniques, mitochondria have been shown capable of oxidizing the intermediates of the tricarboxylic acid cycle and the reduced form of the pyridine nucleotides (Ziegler and Linnane, 1958; Sedar and Rosa, 1961). Read (1952, 1953) indicated that homogenates of the tapeworm Hymenolepis diminuta oxidized malic, succinic, and glutamic acids and converted fumaric acid to malic acid. Heyneman and Voge (1960) demonstrated succinic dehydrogenase in the hymenolepid cysticercoid and Goldberg and Nolf ( 1954) demonstrated succinic dehydrogenase in the strobilate phase of Hymenolepis nana. The present report will show that the cuticle of cestodes is potentially capable of metabolic activity.
MATERIALS
AND METHODS
The species of cestodes investigated included Hymenolepis diminuta, Hymenolepis microstoma, Hymenolepis citelli, and Taenia (Hydatigera) taeniaeformis. The cysticercoid larvae of the hymenolepid species were reared in the grain beetle, Tribolium confusum, and the strobilocercus larvae of T. taeniaeformis were reared in Sprague-Dawley rats. The strobilate form of the tapeworms were obtained as follows: H. diminuta from SpragueDawley rats, H. microstoma from Swiss mice, H. citelli from golden hamsters, and T. taeniaeformis from domestic cats. Portions of the worms were either fresh frozen before sectioning or were immersed in acetone at -20°C for 15 minutes before embedding in ice and sectioning. Worms were also fixed in 10% neutral formalin for 15 minutes and 120 minutes at 5°C before embedding and sectioning. Control worms were either dipped in boiling water for 1 minute or exposed to a dry heat of 95°C for 20 minutes. Sections approximately 8 u thick were cut in an International cryostat, removed on the end of a sharp needle, and floated in the test solution. Fresh tissue was incubated for 15 minutes, and acetone-fixed or formalin-fixed tissue was incubated for 60 minutes. The sec-
1 Present address: Department of Zoology, University of Maryland. 2 Present address: Molten0 Institute of Parasitology, University of Cambridge. This investigation was supported by grants from the U.S. Public Health Service (E-427.5 and E-1384). 333
334
ROTHMAN
tions were then removed from the incubation medium by means of a sharp needle, placed in a drop of glycerine on a microscope slide, covered with a glass slip, and examined. The test solution contained, per milliliter: 2 pmoles of substrate, 1 umole MgCl?, 0.25 mg nitro blue tetrazolium (NBT), 7.5mg polyvinyl pyrrolidinone, and 4 umoles tris maleate buffer at pH 7.4. Diphosphopyridine nucleotide (DPN) was added to give a final concentration of 2 ymoles per milliliter. Malonate was tested at 2 umoles per milliliter and arsenite was tested at 4 pmoles per milliliter. The substrates included the sodium salts of citric, isocitric, succinic, glutamic, a-ketoplutaric, fumaric, and malic acids. The calcium salt of lactic acid and the reduced form of diphosphopyridine nucleotide (DPNH) were also tested as substrates.” RZSULTS
As summarized in Table I fresh-frozen section of the four species of cestodes have histochemically demonstrable NBT reducing systems. Figures 1 and 2 show that both the cuticle and the subcuticular parenchyma have activity. Negative results were obtained with isocitrate, glutamate, malate, and lactate when the DPN was absent from the incubation medium. The succinate and a-ketoglutarate systems were positive in the absence of coenzyme. Diphosphopyridine nucleotide in the absence of substrate gave negative results, whereas DPNH gave a positive result in the absence of any other electron donor. Worms fixed in acetone and worms fixed in formalin for a brief period gave the same results indicated in Table 1. Worms fixed for 120 minutes in the formalin yielded negative results except in the case of DPNH where a weak result was obtained. 3 The DPN, DPNH, and NBT mere obtained from the Sigma Chemical Company, St. Louis 18, Missouri. The substrates were obtained from the California Corporation for Biochemical Research, Los Angeles 63, Calif.
.I_HNLJ
--LfiI.5
Control worms, which were dipped in boiling water for 1 minute before freezing, were negative for all substances tested. Worms exposed to a dry heat, before freezing, gave a barely detectable color. Malonate inhibited the response with succinate, and interestingly, when arsenite was tried with the fumarate-DPN system, a positive result was obtained with two of the tapeworm species. When arsenite was tested with the other systems of H. diminuta, an enhancement of the formazan production occurred. Arsenite had no effect on the DPN-requiring systems if DPN were absent. Also, arsenite in the no substrate-DPN system had no effect. DISCUSSION
The advantages of using NBT over other tetrazoliums is adequately discussed by Nachlas et al. (1957) and need not be considered here. The reduction of NBT in the cuticle and the parenchyma of the four species of cestodes studied, as well as the demonstrated requirement for DPN, argues for the presence of the oxidative enzyme systems. The lack of activity in the heat-treated controls supports this contention. The specificity of the electron donor was demonstrated by using reduced glutathione as a substrate. At approximately pH 10 a general bluing of the medium occurred due to formazan formation, as indicated by Pearse (1960). That the cuticle has metabolic activity is plain; it remains to be proven, however, that the enzyme activity is in the mitochondria. The work of Sedar and Rosa ( 1961) on rat tissues lends support to this possibility. Except for the histochemical demonstration of isocitric dehydrogenase, the present report agrees with the biochemical determination of dehydrogenase presented by Read (1952, 1953). Read (1953) was unable to demonstrate manometrically isocitric dehydrogenase in tapeworm homogenates. This
DEHYDHOGENASE
ACTIVITY
No substrate
with
DPN
Citrate
eb
cl
c2
-
-
-
with
DPN
Isocitrate
with
DPN
Glutamate Glutamate
with
DPN
+
with
malonate
-
-
-
-
-
-
-
with
DPN
Cl
C2
+ + +
+ + +
-
-
-
-
-
-
r -
+
-
+
+ -
-
+
-
+
-
glutathione
Reduced glutathione with DPN
e
-
-
-
with DPN
+ -
r
DPN
Lactate
Reduced
-
-
Malate
Lactate DPNH
-
c2
5
-
+ + +
r -
Fumarate with DPN with arsenite Malate
e
-
+
Succinate
Fumarate
c2
-
+ -
+
with
cl
-
Alpha ketoglutarate Succinate Fumarate
e
-
-
Isocitrate
335
CUTICLE
-
-
Citrate
CESTODE
TABLE I Activity irk the Cuticle of Cestodesa
Dehydrogenase
No substrate
OF
-
-
-
-
+ -
-
+
-
-
+
-
-
+
-
+
-
-
+
-
-
w
+
-
w
-
+ -
-
-
-
+ -
W
-
w
-
a The test solution contained, per milliliter: 2 pmoles of substrate, 1 umole MgCI,, 0.25 mg nitro blue tetrazolium (NBT), 75 mg polyvinyl pyrrolidinone, and 4 umoles tris buffer at pH 7.4. Diphosphopyriclme nucleotide (DPN) was added to give a final concentration of 2 umoles per milliliter. Malonate was tested at 2 umoles per milliliter and arsenite was tested at 4 umoles per milliliter. b e, experimental; cl, control worms immersed 1 minute in boiling water; c2, control worms heated at 95°C for 20 minutes; +, positive result; -, negative result; r, reduced result; w, barely perceptible result.
FIG. 1. Section of H. citelli strobila showing formazan deposits in base of cuticle and in parenchyma. The light band between the dark area of the cuticle and the parenchyma is the subcuticle. FIG. 2. Section of T. taeniaefovmis strobila showing formazan deposits in base of cuticle and in parenchyma. The light band between the dark area of the cuticle and the parenchyma is the subcuticle.
336
ROTHMAN
point will bear further examination by alternative procedures. Pearse (1960) indicates that neither aconitase nor fumarase has been demonstrated histochemically. The present study supports this observation except for the unexpected response effected by arsenite on the fumarateDPN system. Since it was anticipated that arsenite would elicit just the reverse effect, the finding that arsenite also enhances the oxidation of all the substrates (except citrate) tested with H. diminuta certainly warrants further investigation. Aronson and Pharmakis (1962) report a similar effect of potassium cyanide for succinic dehydrogenase. An electron donor is important to this arsenite enhancement, for in the absence of one, DPN with arsenite has no noticeable effect. An uncoupling of phosphorylation by arsenite (Fletcher and Sanadi, 1962) could account for the increased transport of electrons, yielding more formazan. The brief formalin fixation and brief acetone fixation facilitated considerably the handling of the tissue and the interpretation of the results (cf. Pearse, 1960; Walker and Seligman, 196 1) . REFERENCES AROSSON, i%‘., AND PHARMAKIS, T. 1962. Enhancement of neotetrazolium staining for succinic dehydrogenase activity with cyanide. Stain Technology 37, 321. FLETCHER, M. J., AND SAPTADI,D. R. 1962. On the mechanism of oxidative phosphorylation. III. Effects of arsenite and 2,3-dimercaptopropanol on coupled phosphorylation in heart mitochondria. Archives of Biochemistry and Biophysics 96, 139-142. GOLDBERG, E.,
AND NOLF, L.
0.
1954.
Succinic
AND
LEE
dehydrogenase activity in the cestode Hymenolepis nana. Experimental Parasitology 3, 275-284. HEYNEMAN, D., AND VOGE, M. 1960. Succinic dehydrogenase activity in cysticercoids of Hymenolepis (Cestoda: Hymenolepididae) measured by the tetrazolium technique. Experimental Parasitology 9, 14-17. NACNLAS, M. M., Tsou, K. C., SOUZA, E., CHENG, C. S., AND SELIGMAN, A. M. 1957. Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. Journal of Histochemistry and Cytochemistry 5, 420.436. PEARSE, A. G. E. 1960. “Histochemistry: Theoretical and Applied,” 998 pp. Little, Brown and Company, Boston, Massachusetts. READ, C. P. 1952. Contributions to cestode enzymology. I. The cytochrome system and succinic dehydrogenase in Hymenolepis diminuta. Experimental Parasitology 1, 353-362. READ, C. P. 1953. Contributions to cestode enzymology. II. Some anaerobic dehydrogenases in Hymenolepis diminuta. Experimental Parasitology 2, 341-347. ROTHMAN, A. H. 1963. Electron microscope studies of tapeworms. The surface structures of Hymewolepis diminuta. Transactions of the American Microscopical Society 82, 22.30. SEDAR,.4. W., AND ROSA, C. G. 1961. Cytochemical demonstration of the succinic dehydrogenase system with the electron microscope, using nitro blue tetrazolium. Ultrastructure Research 5, 226243. THREADGOLD, L. T. 1962. An electron microscope study of the tegument and associated structures of Dipylidium caninum. Quarterly Journal of Microscopical Science 103, 135-140. WALKER, D. G., AND SELIGMAN, A. M. 1961. Formalin fixation in the cytochemical demonstration of succinic dehydrogenase of mitochondria. Journal of Biophysical and Biochemical Cytology 9, 415-428. ZIEGLER, D. M., AND LINPSANE, A. W. 1958. Studies on the electron transport system. XIII. Mitochondrial structure and dehydrogenase activity in isolated mitochondria. Biochimica et Biophysica Acta 30, 53-63.