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The effect of DDT on the level of Di- and triphosphopyridine nucleotides in Triatoma infestans
EXPERIMENTAL
PARASITOLOGY
The
13,
Effect of DDTl on the Level of DiTriphosphopyridine Nucleotides in Triatoma inf estans’ M.
Department
199.203 (1963)
Agosin
of Parasitology,
and
Maria
Biochemistry
(Submitted
L&a
Section,
for publication,
and
Dinamarca University
20 April
of Chile, Santiago,
Chile
1962)
1. The levels of oxidized and reduced pyridine nucleotides have been measured in normal and DDT- and DDE-treated nymphs and adult males of T. infestans. 2. The total content of DPN (DPN plus DPNH) is higher than TPN (TPN plus TPNH) in both nymphs and males. In nymphs, DPN is mainly present in the oxidized form and TPN in the reduced form, whi!e in males TPN is mainly found in the reduced form and DPN in the oxidized form. 3. DDT significantly increases the total TPN content in nymphs, but does not affect the ratio TPN/TPNH, while in males neither the total TPN level nor the ratio TPN/ TPNH are changed. 4. DDT significantly increases the total content of DPN in males after 24 hours of intoxication. DDE is without any effect on the DPN or TPN levels in nymphs and males, but the ratios DPN/DPNH are significantly increased in males.
Nymphs of T. infestans are more resistant to DDT than adult males (Agosin, Scaramelli, and Neghme, 1961). This is partially explained by the fact that DDT is metabolized at a much higher rate in nymphs than males, one of the metabolites corresponding possibly to Kelthane (Dinamarca, Agosin, and Neghme, 1962). The latter compound has been shown to be produced in vitro by a microsomal enzyme essentially requiring TPNH and molecular oxygen (Agosin, Michaeli, Miskus, Nagasawa, and Hoskins, 1961). In vivo estimation of the C140r yields t Abbreviations: DDT, 2,2-bis-(p-chlorophenyl) DDE, 2,2-bis-(p-chlorol,l,l-trichloroethane; phenyl)-l,l-dichloroethylene; Kelthane, 2,2-bis-(pchlorophenyl)-l,l,l-trichloroethanol; DPN, oxidized diphosphopyridine nucleotide; DPNH, reduced diphosphopyridine nucleotide; TPN, oxidized triphosphopyridine nucleotide ; TPNH, reduced triphosphopyridine nucleotide. 2 Supported by Grant E-2300 of the Division of Research Grants and Fellowships of the National Institutes of Health, U.S. Public Health Service.
from glucose-l-V4 and glucose-6-Cl4 in T. infestam, indicate that DDT-intoxication induces preferential oxidation of glucose carbon 1 over glucose carbon 6. This is especially pronounced in nymphs (unpublished data from this laboratory). These observations suggested that glucose oxidation via pentose phosphate pathway is stimulated by DDT. The latter metabolic pathway has been shown to occur in Triatoma infestans (Agosin, Scaramelli, and Neghme, 1961). The rate-limiting step in the operation of the pentose phosphate pathway appears to be the supply of TPN (Kinoshita, 1957). Thus, as a consequence of DDT-metabolism, TPN would be generated resulting in increased oxidation of glucose by the pentose phosphate pathway. Furthermore, the TPN levels should not be appreciably increased in the DDT-susceptible males since here DDT-hydroxylation plays a minor role as detoxifying mechanism (Dinamarca et al., 1962). The present study was undertaken in order to determine whether the 199
200
AGOSIN
AND
levels of pyridine nucleotides in nymph and male T. infestans are modified under the influence of DDT, and whether differences exist in this respect between both developmental stages of the insect. METHODS
Third instar larvae (nymphs) and adult male T. infestans were obtained and handled as previously described (Agosin, Scaramelli, and Neghme, 1961; Dinamarca et al., 1962). DDT and DDE, a nontoxic DDT analogue, dissolved in acetone were topically applied at the dose of 300 ug per specimen to the ventral abdominal region of the insects by means of a micropipette. Control groups treated only with acetone yielded identical values with nontreated insects. Twenty-four and fortyeight hours after application of the insecticide, extracts for the estimation of pyridine nucleotides were prepared as follows: The insects were cut into small pieces and blotted on filter paper to eliminate any blood. They were then rapidly weighed into a glass Virtis homogenizing cup and homogenized with 5 volumes of either 0.1 N perchloric acid or 0.1 N sodium carbonate at high speed during 30 seconds and at low speed for 30 additional seconds. During the homogenization, the Virtis homogenizing cup was immersed in crushed ice. After centrifuging the homogenate at 14,000 X g for 60 minutes at 2”C, the perchloric acid supernatant fluid was adjusted to pH 7.0 with KOH and the insoluble perchlorate removed by centrifugation in the cold. The sodium carbonate supernatant fluid was adjusted to pH 7.8 with HCl and then filtered through Whatman No. 1 filter paper. When the concentration of the pyridine nucleotides was too low for the methods of assay used, several corresponding extracts were pooled and lyophilized. The lyophilized material was resuspended in an appropriate amount of HZ0 and an aliquot was taken for assay. DPN and DPNH were estimated spectrophotometrically with alcohol dehydrogenase in the presence of ethanol and
DINAMARCA
acetaldehyde, TPN with glucose-6-phosphate dehydrogenase in the presence of glucose-6phosphate, and TPNH with glutathione reductase in the presence of oxidized glutathione, as described by Ciotti and Kaplan (1957). The methods of extraction were checked by determining the recovery of added coenzymes which were added in known amounts to insect preparations during the extraction. Recoveries were always over 96%. Particular consideration was given to the presence of blood which interferes markedly with the recovery of TPNH. The results given are derived from a minimum of 5 duplicate determinations. Glutathione reductase was prepared from dry peas (Kaplan, Colowick, and Neufeld, 1953). Alcohol and glucose-6-phosphate dehydrogenases, as well as glucose-6-phosphate, oxidized glutathione, DPN, DPNH, TPN, TPNH, were obtained commercially.” RESULTS As shown in Table I the level of total DPN (DPN f DPNH) is much higher than the level of total TPN (TPN +TPNH) in both nymphs and males. As in mammalian tissues (Glock and McLean, 1955) most of the DPN in nymphs ( 77% ) is in the oxidized form, while about 60% of the TPN is in the reduced form. The reverse situation occurs in males, where about 685% of the DPN is in the reduced form, while over 60% of the TPN is in the oxidized form. Statistical analysis at a 5% level indicates that the exposure of nymphs to either DDT or DDE during 24 and 48 hours does not affect the total level of DPN, or the ratio DPN/ DPNH. On the other hand, the level of total DPN is significantly increased in males after 24 hours of DDT-intoxication, although the level reverts to normal after 48 hours of intoxication. Furthermore, the DPN/DPNH ratio, which has a value of 0.5 in normal males, is significantly increased to 9.8 at 24 hours and 3 Sigma Chemical
Co., St. Louis, MO., U.S..4.
-
24 48 24 48
Conditions
Normal
DDT DDT DDE DDE
44.9 38.0 36.8 34.1 34.3
% f f ? ?
DPN
4.6 1.6 3.2 6.3 7.9
13.6 16.7 14.3 14.7 13.2
2 -c C f k
DPNH
1.5 0.7 3.7 0.6 3.6
58.5 56.2 59.7 48.8 47.5
k k k & ?
TPN
5.4 4.5 & 1.1 6.9 11.6 zk 0.0 6.6 11.3 k 0.5 3.8 4.6 f. 0.0 4.8 4.2 k 0.0
DPNb + DPNH
TABLE
I
Level
TPNH
TPNb +
Nucleotide
6.6k0.7 11.3 & 10.9 k 4.8 k 4.7 3z 0.4 0.3 0.3 1.1
11.1~1.3 22.9 k 22.2 k 9.4 -r8.9 f 0.4 1.7 1.0 1.5
All values in pg/gm/fresh
TPNH
on the Pyridine
Nymphs
and DDE
a The figures after the k sign correspond to the standard error of the mean. b -4verages of the single experiments.
Hours of intoxication
Efiect of DDT
14.7k0.9 56.3 k 33.4 & 40.4 C 24.2 f
tissue
DPN
4.6 3.8 1.6 1.9
of Nymph
44.4 2 4.6 13.8 k 0.7 48.0 C 2.1 8.4 k 1.1 38.6 k 2.0 8.4 k 0.6
11.0 f 0.8 7.6 k 0.2 14.4 f 2.1
9.3LO.8 9.2e1.5
TPN
46.1k5.1 62.3e4.4
DPNH
DPNb +
Males
infestan
31.4k1.7 6.0&1.1
DPNH
and Male Triatoma
2.8 2 0.5 5.6 21 1.4 6.9 -e 0.8
6.120.0 6.6-r-1.3
TPNH
16.6 C 1.0 14.0 k 1.37 15.3 2 1.2
15.4eO.6 15X21.9
TPNH
TPNb+
E 5 r
8
0 M” s
z
3
z 2 s
E
::
!2 H E
& ct
: z 2 4
202
AGOSIN AND DINAMARCA
3.3 at 48 hours of intoxication. DDE does not change the level of total DPN in males. However, the ratio DPN/DPNH is significantly altered to 5.1 at 24 hours and 1.5 at 48 hours of intoxication, as compared with the ratio of 0.5 observed in normal males. When the levels of TPN are considered, it is seen that the opposite situation exists as compared to DPN. The level of total TPN is significantly increased by DDT after 24 or 48 hours of intoxication in nymphs, although the ratio TPN/TPNH is not significantly altered from the statistical standpoint. In males, only the ratio TPN/TPNH increases from 1.5 in normal specimens to over 5.0 in intoxicated ones, but the level of total DPN is not appreciably changed. Finally, DDE does not affect the total level of TPN as the ratio TPN/TPNH either in nymphs or males. DISCUSSION
Our results are consistent with the hypothesis that the detoxication of DDT by a mechanism requiring TPNH (Agosin, Michaeli, Miskus, Nagasawa, and Hoskins, 1961) should produce the accumulation of TPN (Table I.) This phenomenon is observed only in nymphs where DDT detoxication is more active (Dinamarca et al., 1962). The net increase in the total level of TPN corresponds to a net synthesis from DPN, since the increases of both TPN and TPNH parallel each other without a significant change of the ratio TPN/TPNH. However, it should be pointed out that so far DPN kinase has not been studied in this organism. On the other hand, there is apparently a net synthesis of total DPN in males after 24 hours of DDT-intoxication, which cannot expressed simply in terms of an increase of the oxidized DPN at the expense of the reduced form, but no net synthesis of total TPN can be observed (Table I). DDE, a non-toxic analogue, does not affect the total level of DPN and TPN as the ratio DPN/DPNH and TPN/TPNH in nymphs,
suggesting that here DDE is not metabolized, at least not by hydroxylating mechanisms. DDE is also without effect on the total level of TPN in males. However, although the total level of DPN is not significantly changed in males, the relative proportion of the reduced and oxidized form of DPN is strikingly modified in favor of the accumulation of the oxidized form (Table I). Several glycolytic enzymes are inhibited by both DDT and DDE in males, but some of these enzymes are not affected in nymphs, for example triosephosphate dehydrogenase (Agosin, Scaramelli, and Neghme, 1961) . This observation may be an explanation for the increase in total DPN observed in males under the influence of DDT (Table I). The probability of this assumption being correct is enhanced by the fact that the increase is mainly of the oxidized form of DPN. Our results also support the idea that the increased glucose oxidation via pentose phosphate pathway induced by DDT in nymphs (unpublished data of the authors) may be due to the increased generation of TPN. This view is also supported by the observation that pentose phosphate pathway enzymes show a lesser degree of inhibition by DDT in nymphs than males (Agosin, Scaramelli, and Neghme, 1961). It is hoped that these results throw some light on the mechanisms underlying the marked differences in susceptibility towards DDT shown by nymphs and males of T. infestans. REFERENCES M., MICHAELI, D., MISKUS, R., NAGASAWA. S., AND HOSKINS, W. M. 1961. A new DDTmetabolizing Enzyme in the German Cockroach. Journal of Economical Entomology 54, 340-342.
AGOSIN,
M., SCARAMELLI, N., AND NEGHME, A. 1961. Intermediary Carbohydrate Metabolism of Triatoma infestam (insecta; Hemiptera). I.Glycolytic and Pentose Phosphate Pathway Enzymes and the Effect of DDT. Comparative Biochemistry and Physiology 2, 143.159.
.4cosrw,
CIOTTI,
M.
M.,
AND KAPLAN,
N. 0.
1957.
In
S. P.
EFFECT
OF
DDT
ON
TRIATOMA
Colowick and N. 0. Kaplan, Methods in Enzymology, Vol. III. Academic Press, Inc. New York, p. 890. DINAMARCA, M. L., AGOSIN, M., AND NECHME, A., 1962. The Metabolic Fate of Cr4-DDT in Triatoma infestans. Experimental Parasitology 12, 61-72. GLOCK, G. E., AND MCLEAN, P., 1955. Levels of Oxidized and Reduced Diphosphopyridine Nucleotide and Triphosphopyridine Nudeotide in
PYRIDINE
NUCLEOTIDE
LEVEL
203
Animal Tissues. Biochemical Journal 61, 388390. KAPLAN, N. O., COLOWICK, S. P., NEUFELD, E. F. 1953. Pyridine Nucleotide Transhydrogenase. III. Animal Tissue Transhydrogenases. Journal of Biological Chemistry 206, 1-15. KINOSKITA, J. H. 1957. The Stimulation of Phosphogluconate oxidation pathway by Pyruvate in Bovine Cornea1 Epithelium. Journal of BioEogical Chemistry 228, 247-253.
PARASITOLOGY
The
13,
Effect of DDTl on the Level of DiTriphosphopyridine Nucleotides in Triatoma inf estans’ M.
Department
199.203 (1963)
Agosin
of Parasitology,
and
Maria
Biochemistry
(Submitted
L&a
Section,
for publication,
and
Dinamarca University
20 April
of Chile, Santiago,
Chile
1962)
1. The levels of oxidized and reduced pyridine nucleotides have been measured in normal and DDT- and DDE-treated nymphs and adult males of T. infestans. 2. The total content of DPN (DPN plus DPNH) is higher than TPN (TPN plus TPNH) in both nymphs and males. In nymphs, DPN is mainly present in the oxidized form and TPN in the reduced form, whi!e in males TPN is mainly found in the reduced form and DPN in the oxidized form. 3. DDT significantly increases the total TPN content in nymphs, but does not affect the ratio TPN/TPNH, while in males neither the total TPN level nor the ratio TPN/ TPNH are changed. 4. DDT significantly increases the total content of DPN in males after 24 hours of intoxication. DDE is without any effect on the DPN or TPN levels in nymphs and males, but the ratios DPN/DPNH are significantly increased in males.
Nymphs of T. infestans are more resistant to DDT than adult males (Agosin, Scaramelli, and Neghme, 1961). This is partially explained by the fact that DDT is metabolized at a much higher rate in nymphs than males, one of the metabolites corresponding possibly to Kelthane (Dinamarca, Agosin, and Neghme, 1962). The latter compound has been shown to be produced in vitro by a microsomal enzyme essentially requiring TPNH and molecular oxygen (Agosin, Michaeli, Miskus, Nagasawa, and Hoskins, 1961). In vivo estimation of the C140r yields t Abbreviations: DDT, 2,2-bis-(p-chlorophenyl) DDE, 2,2-bis-(p-chlorol,l,l-trichloroethane; phenyl)-l,l-dichloroethylene; Kelthane, 2,2-bis-(pchlorophenyl)-l,l,l-trichloroethanol; DPN, oxidized diphosphopyridine nucleotide; DPNH, reduced diphosphopyridine nucleotide; TPN, oxidized triphosphopyridine nucleotide ; TPNH, reduced triphosphopyridine nucleotide. 2 Supported by Grant E-2300 of the Division of Research Grants and Fellowships of the National Institutes of Health, U.S. Public Health Service.
from glucose-l-V4 and glucose-6-Cl4 in T. infestam, indicate that DDT-intoxication induces preferential oxidation of glucose carbon 1 over glucose carbon 6. This is especially pronounced in nymphs (unpublished data from this laboratory). These observations suggested that glucose oxidation via pentose phosphate pathway is stimulated by DDT. The latter metabolic pathway has been shown to occur in Triatoma infestans (Agosin, Scaramelli, and Neghme, 1961). The rate-limiting step in the operation of the pentose phosphate pathway appears to be the supply of TPN (Kinoshita, 1957). Thus, as a consequence of DDT-metabolism, TPN would be generated resulting in increased oxidation of glucose by the pentose phosphate pathway. Furthermore, the TPN levels should not be appreciably increased in the DDT-susceptible males since here DDT-hydroxylation plays a minor role as detoxifying mechanism (Dinamarca et al., 1962). The present study was undertaken in order to determine whether the 199
200
AGOSIN
AND
levels of pyridine nucleotides in nymph and male T. infestans are modified under the influence of DDT, and whether differences exist in this respect between both developmental stages of the insect. METHODS
Third instar larvae (nymphs) and adult male T. infestans were obtained and handled as previously described (Agosin, Scaramelli, and Neghme, 1961; Dinamarca et al., 1962). DDT and DDE, a nontoxic DDT analogue, dissolved in acetone were topically applied at the dose of 300 ug per specimen to the ventral abdominal region of the insects by means of a micropipette. Control groups treated only with acetone yielded identical values with nontreated insects. Twenty-four and fortyeight hours after application of the insecticide, extracts for the estimation of pyridine nucleotides were prepared as follows: The insects were cut into small pieces and blotted on filter paper to eliminate any blood. They were then rapidly weighed into a glass Virtis homogenizing cup and homogenized with 5 volumes of either 0.1 N perchloric acid or 0.1 N sodium carbonate at high speed during 30 seconds and at low speed for 30 additional seconds. During the homogenization, the Virtis homogenizing cup was immersed in crushed ice. After centrifuging the homogenate at 14,000 X g for 60 minutes at 2”C, the perchloric acid supernatant fluid was adjusted to pH 7.0 with KOH and the insoluble perchlorate removed by centrifugation in the cold. The sodium carbonate supernatant fluid was adjusted to pH 7.8 with HCl and then filtered through Whatman No. 1 filter paper. When the concentration of the pyridine nucleotides was too low for the methods of assay used, several corresponding extracts were pooled and lyophilized. The lyophilized material was resuspended in an appropriate amount of HZ0 and an aliquot was taken for assay. DPN and DPNH were estimated spectrophotometrically with alcohol dehydrogenase in the presence of ethanol and
DINAMARCA
acetaldehyde, TPN with glucose-6-phosphate dehydrogenase in the presence of glucose-6phosphate, and TPNH with glutathione reductase in the presence of oxidized glutathione, as described by Ciotti and Kaplan (1957). The methods of extraction were checked by determining the recovery of added coenzymes which were added in known amounts to insect preparations during the extraction. Recoveries were always over 96%. Particular consideration was given to the presence of blood which interferes markedly with the recovery of TPNH. The results given are derived from a minimum of 5 duplicate determinations. Glutathione reductase was prepared from dry peas (Kaplan, Colowick, and Neufeld, 1953). Alcohol and glucose-6-phosphate dehydrogenases, as well as glucose-6-phosphate, oxidized glutathione, DPN, DPNH, TPN, TPNH, were obtained commercially.” RESULTS As shown in Table I the level of total DPN (DPN f DPNH) is much higher than the level of total TPN (TPN +TPNH) in both nymphs and males. As in mammalian tissues (Glock and McLean, 1955) most of the DPN in nymphs ( 77% ) is in the oxidized form, while about 60% of the TPN is in the reduced form. The reverse situation occurs in males, where about 685% of the DPN is in the reduced form, while over 60% of the TPN is in the oxidized form. Statistical analysis at a 5% level indicates that the exposure of nymphs to either DDT or DDE during 24 and 48 hours does not affect the total level of DPN, or the ratio DPN/ DPNH. On the other hand, the level of total DPN is significantly increased in males after 24 hours of DDT-intoxication, although the level reverts to normal after 48 hours of intoxication. Furthermore, the DPN/DPNH ratio, which has a value of 0.5 in normal males, is significantly increased to 9.8 at 24 hours and 3 Sigma Chemical
Co., St. Louis, MO., U.S..4.
-
24 48 24 48
Conditions
Normal
DDT DDT DDE DDE
44.9 38.0 36.8 34.1 34.3
% f f ? ?
DPN
4.6 1.6 3.2 6.3 7.9
13.6 16.7 14.3 14.7 13.2
2 -c C f k
DPNH
1.5 0.7 3.7 0.6 3.6
58.5 56.2 59.7 48.8 47.5
k k k & ?
TPN
5.4 4.5 & 1.1 6.9 11.6 zk 0.0 6.6 11.3 k 0.5 3.8 4.6 f. 0.0 4.8 4.2 k 0.0
DPNb + DPNH
TABLE
I
Level
TPNH
TPNb +
Nucleotide
6.6k0.7 11.3 & 10.9 k 4.8 k 4.7 3z 0.4 0.3 0.3 1.1
11.1~1.3 22.9 k 22.2 k 9.4 -r8.9 f 0.4 1.7 1.0 1.5
All values in pg/gm/fresh
TPNH
on the Pyridine
Nymphs
and DDE
a The figures after the k sign correspond to the standard error of the mean. b -4verages of the single experiments.
Hours of intoxication
Efiect of DDT
14.7k0.9 56.3 k 33.4 & 40.4 C 24.2 f
tissue
DPN
4.6 3.8 1.6 1.9
of Nymph
44.4 2 4.6 13.8 k 0.7 48.0 C 2.1 8.4 k 1.1 38.6 k 2.0 8.4 k 0.6
11.0 f 0.8 7.6 k 0.2 14.4 f 2.1
9.3LO.8 9.2e1.5
TPN
46.1k5.1 62.3e4.4
DPNH
DPNb +
Males
infestan
31.4k1.7 6.0&1.1
DPNH
and Male Triatoma
2.8 2 0.5 5.6 21 1.4 6.9 -e 0.8
6.120.0 6.6-r-1.3
TPNH
16.6 C 1.0 14.0 k 1.37 15.3 2 1.2
15.4eO.6 15X21.9
TPNH
TPNb+
E 5 r
8
0 M” s
z
3
z 2 s
E
::
!2 H E
& ct
: z 2 4
202
AGOSIN AND DINAMARCA
3.3 at 48 hours of intoxication. DDE does not change the level of total DPN in males. However, the ratio DPN/DPNH is significantly altered to 5.1 at 24 hours and 1.5 at 48 hours of intoxication, as compared with the ratio of 0.5 observed in normal males. When the levels of TPN are considered, it is seen that the opposite situation exists as compared to DPN. The level of total TPN is significantly increased by DDT after 24 or 48 hours of intoxication in nymphs, although the ratio TPN/TPNH is not significantly altered from the statistical standpoint. In males, only the ratio TPN/TPNH increases from 1.5 in normal specimens to over 5.0 in intoxicated ones, but the level of total DPN is not appreciably changed. Finally, DDE does not affect the total level of TPN as the ratio TPN/TPNH either in nymphs or males. DISCUSSION
Our results are consistent with the hypothesis that the detoxication of DDT by a mechanism requiring TPNH (Agosin, Michaeli, Miskus, Nagasawa, and Hoskins, 1961) should produce the accumulation of TPN (Table I.) This phenomenon is observed only in nymphs where DDT detoxication is more active (Dinamarca et al., 1962). The net increase in the total level of TPN corresponds to a net synthesis from DPN, since the increases of both TPN and TPNH parallel each other without a significant change of the ratio TPN/TPNH. However, it should be pointed out that so far DPN kinase has not been studied in this organism. On the other hand, there is apparently a net synthesis of total DPN in males after 24 hours of DDT-intoxication, which cannot expressed simply in terms of an increase of the oxidized DPN at the expense of the reduced form, but no net synthesis of total TPN can be observed (Table I). DDE, a non-toxic analogue, does not affect the total level of DPN and TPN as the ratio DPN/DPNH and TPN/TPNH in nymphs,
suggesting that here DDE is not metabolized, at least not by hydroxylating mechanisms. DDE is also without effect on the total level of TPN in males. However, although the total level of DPN is not significantly changed in males, the relative proportion of the reduced and oxidized form of DPN is strikingly modified in favor of the accumulation of the oxidized form (Table I). Several glycolytic enzymes are inhibited by both DDT and DDE in males, but some of these enzymes are not affected in nymphs, for example triosephosphate dehydrogenase (Agosin, Scaramelli, and Neghme, 1961) . This observation may be an explanation for the increase in total DPN observed in males under the influence of DDT (Table I). The probability of this assumption being correct is enhanced by the fact that the increase is mainly of the oxidized form of DPN. Our results also support the idea that the increased glucose oxidation via pentose phosphate pathway induced by DDT in nymphs (unpublished data of the authors) may be due to the increased generation of TPN. This view is also supported by the observation that pentose phosphate pathway enzymes show a lesser degree of inhibition by DDT in nymphs than males (Agosin, Scaramelli, and Neghme, 1961). It is hoped that these results throw some light on the mechanisms underlying the marked differences in susceptibility towards DDT shown by nymphs and males of T. infestans. REFERENCES M., MICHAELI, D., MISKUS, R., NAGASAWA. S., AND HOSKINS, W. M. 1961. A new DDTmetabolizing Enzyme in the German Cockroach. Journal of Economical Entomology 54, 340-342.
AGOSIN,
M., SCARAMELLI, N., AND NEGHME, A. 1961. Intermediary Carbohydrate Metabolism of Triatoma infestam (insecta; Hemiptera). I.Glycolytic and Pentose Phosphate Pathway Enzymes and the Effect of DDT. Comparative Biochemistry and Physiology 2, 143.159.
.4cosrw,
CIOTTI,
M.
M.,
AND KAPLAN,
N. 0.
1957.
In
S. P.
EFFECT
OF
DDT
ON
TRIATOMA
Colowick and N. 0. Kaplan, Methods in Enzymology, Vol. III. Academic Press, Inc. New York, p. 890. DINAMARCA, M. L., AGOSIN, M., AND NECHME, A., 1962. The Metabolic Fate of Cr4-DDT in Triatoma infestans. Experimental Parasitology 12, 61-72. GLOCK, G. E., AND MCLEAN, P., 1955. Levels of Oxidized and Reduced Diphosphopyridine Nucleotide and Triphosphopyridine Nudeotide in
PYRIDINE
NUCLEOTIDE
LEVEL
203
Animal Tissues. Biochemical Journal 61, 388390. KAPLAN, N. O., COLOWICK, S. P., NEUFELD, E. F. 1953. Pyridine Nucleotide Transhydrogenase. III. Animal Tissue Transhydrogenases. Journal of Biological Chemistry 206, 1-15. KINOSKITA, J. H. 1957. The Stimulation of Phosphogluconate oxidation pathway by Pyruvate in Bovine Cornea1 Epithelium. Journal of BioEogical Chemistry 228, 247-253.