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The metabolic fate of C14-DDT in Triatoma infestans
EXPERIMENTAL
PARASITOLOGY
The Maria
12, 61-72 (1962)
Metabolic
Fate of C14-DDT in
Luisa
Moises
Dinamarca,
Department of Parasitology,
Agosin,
Triatoma
infestud2
and Amador
Biochemistry Section, Santiago, Chile
University
Neghme
of Chile,
(Submitted for publication, 10 August 1961) (1) Nymph and male T. infestans specimens differ in their rate of absorption, excretion and metabolism of Cr4-DDT. Nymphs absorb DDT to a lesser extent than males. Furthermore, DDT is excreted at a faster rate in nymphs than males. After 96 hours of intoxication, 27.5% of the absorbed DDT is converted to a mixture of five metabolites in nymphs, while only 4.7% of the absorbed DDT is accounted for by DDT-metabolites in males after the same time of intoxication. (2) DDT is metabolized into five metabolites in both nymph and male adult T. infestans. DDE is the major DDT-metabolite produced by nymphs, while in males, metabolite No. 5 is the most important one, DDE being found only in negligible amounts. Metabolite 3 has the same chromatographic identity as Kelthane, a DDT metabolite found in Drosophib and Blattella germanica. (3) It is postulated that the production of the more polar metabolites involves oxidative processes independent of the mechanism of formation of DDE. A tentative sequence is given for the formation of DDT metabolites in T. infestans and the availability of TPNH is suggested as a common lii for both the oxidation and the dehydrochlorination processes. (4) It is considered that the differences regarding DDT penetration, excretion, and metabolism shown by nymphs as compared to males, may account at least partly for the higher tolerance to DDT exhibited by nymph T. infestam.
Previous work in this laboratory has shown that nymph and male adult T. infestans specimens differ markedly in their susceptibility towards DDT, males being much more susceptible than nymphs (Agosin, Scaramelli, and Neghme, 1961). Apparently, this 1 Supported by grant E-2300 of the Division of Research Grants and Fellowships of the National Institutes of Health, U.S. Public Health Service, and by funds of the Commission for Scientific Research of the University of Chile. 2 Abbrevidons: DDT, 2,2-bis-(p-cblorophenyl)l,l,l-trichloroethane; DDE, 2,2-bis-(p-chlorophenyl)l,l-dicbloroethylene; DDA, 2,2-bis-(p-chlorophenyl) acetic acid ; DDD, 2,2-bis-(p-chlorophenyl)-l,ldichloroethane; Kelthane, 2,2-bis-(p-chlorophenyl)l,l,l-trichloroethanol; DBP, p,p’-dichlorobenzophenone; DBH, p,p’-dichlorobenzohydrol; GSSG, oxidized glutathione ; GSH, reduced glutathione ; TPN, oxidized triphosphopyridine nucleotide; TPNH, reduced triphosphopyridine nucleotide.
difference in response to DDT is not related to enzyme content of male and nymph specimens. The latter have, in general, a lower enzyme content than males, at least in respect to glycolytic and pentose phosphate pathway enzymes (Agosin et d., 1961). The possibility that key enzymes which are inhibited by DDT in males are not affected in nymphs is supported by the fact that in males some glycolytic and pentose phosphate pathway enzymes are inhibited in vivo as well as in vitro by DDT. The corresponding nymphal enzymes are not affected (Agosin et al., 1961). These observations suggest that the hypothetical DDT-sensitive key enzyme(s) may be structurally different in male and nymph T. infestans as seems to be the case for triose phosphate dehydrogenase (Agosin, Neghme, and Scaramelli, 1960). However, other factors in addition to differ61
DINAMARCA,AGOSIN,ANDNEGHME
62
ential enzymatic inhibition may be involved in the lesser susceptibility of nymphs to DDT. These factors, such as differences in the rate of absorption and excretion of DDT, and/or different or more efficient detoxifying mechanisms in nymphs have been explored in the present studies. The results obtained indicate that DDT is absorbed less and excreted faster in nymphs than in males. Furthermore, although both nymph and males metabolize DDT to at least five metabolites, the rate of production of these metabolites is much higher in the nymph. It is hoped that these studies will contribute to our understanding of the mode of action of DDT, and the mechanism involved in the differences of susceptibility towards DDT exhibited by the various developmental stages of T. infestans. METHODS
Insect Material Laboratory reared T. infestans specimens were used throughout. They were maintained as indicated elsewhere (Agosin et al., 1961). The experimental work was done with adult males and third instar larvae (nymphs) that were starved at least one week before being used. Groups of 3.5 specimens were treated with C14-DDT. Twenty microliters of an acetone C14-DDT solution, containing 16.8 ug of DDT (2,380 c.p.m. per pg) were topically applied to the ventral abdominal region of the insects by means of a micropipette. At 6, 24, 48, 72, and 96 hours, five surviving specimens were selected for analysis. The dead nymph or male specimens were discarded. After application of C14-DDT, the insects were kept as previously described (Agosin et aZ., 1961). As a control, the amount of applied C14-DDT was determined in several experiments by extracting and measuring the radioactivity immediately after the topical application of the insecticide. Extraction
Procedures
At the above indicated time intervals, the insects selected for analysis were rinsed three times individually in about 5 ml of acetone to eliminate any external insecticide. The abdomen of the insects was gently pressed
with a forceps and the fecal matter obtained pooled with the feces present in the insects container. The rinsed insects were then homogenized in about 10 ml of chloroform in a semimicro Virtis homogenizer. About 2 ml of water was added to the homogenate which was then extracted three times each with about 20ml of chloroform in a separatory funnel. The combined chloroform layers, filtered through anhydrous sodium sulfate, were concentrated to dryness under a stream of hot air. The insect container and excreta were extracted for 48 hours with 10 ml of a solution of 80 ml acetone, 20 ml water and 1 ml 1 N sulfuric acid, according to Lindquist and Dahm (1956). The mixture was then extracted three times with chloroform, the chloroform layer filtered through anhydrous sodium sulfate, and the filtrate concentrated as above. The efficiency of the extraction procedure for both insect and fecal material was 98 to 99%. Chromatographic Procedures The dry extracts, dissolved in a minimum volume of acetone, were spotted on Whatman #l one-inch-wide filter paper strips, and chromatographically purified by the procedure of Menn, Eldefrawi, and Gordon (1960) with 100% acetonitrile. The purified extracts from the strips were made up to a volume of 1 ml with acetone and an aliquot was taken for measuring the radioactivity. The remaining extract was again concentrated to a minimum volume and chromatographed with betamethoxypropionitrile as stationary phase and iso-octane saturated with beta-methoxypropionitrile as mobile phase (Gordon, 1960; I/M system). In some special cases, 75% N,N-dimethyl-formamide in water as mobile phase, and 10% mineral oil in chloroform as stationary phase (D/M system) or isopropyl ether saturated with glutaronitrile as mobile phase and 15% glutaronitrile in acetone as stationary phase (E/G system) were also used. The dye reference system of Gordon (1960) formed by a mixture of Oil Blue N (A + B), Oil Orange 2311, and Sudan Yellow RRA was used with the I/M system. Confirming Gordon’s work (1960) the position of insecticide spots relative to that of the different dyes was constant. After develop-
METABOLIC
FATE
OF DDT IN TRIATOMA
ment, the chromatograms were scanned in a model ClOO-A Nuclear Chicago Actigraph with a thin-window D-47 Flow-Counter, with a speed of 0.75 inches/mm and a slit of l/4 inch. The metabolites were located, eluted with 100% acetonitrile and rechromatographed with the above chromatographic systems. Finally, when the metabolites were clearly separated, they were eluted with 100% acetonitrile, and the radioactivity measured on planchets at infinite thinness in a Model 6000 Nuclear Chicago Dynacon Electrometer. Standard corrections were applied in measuring the radioactivity. Non-radioactive chromatographic reference compounds were detected with Mitchell’s silver nitrate reagent (1957). Reagents 2-C14-ethane-labelled DDT obtained through the courtesy of the Communicable Diseases Center, Savannah, Georgia, was used at the beginning of this work. However, the specific activity was low for our purposes. Therefore, most of the results given here were obtained with ring-labelled-C14-DDT, prepared in the laboratory (Miskus, 1959). The dyes were a gift of Dr. H. T. Gordon. Chromatographically pure DDE, DBP, DBH, DDD, DDA, were kindly provided by R. Miskus. The rest of the reagents were of the highest purity commercially available. RESULTS
Rate of Absorption and Excretion and Metabolites
of DDT
The rate of absorption of DDT as well as the rate of excretion of DDT and metabolites was found to be different in nymph as compared to male T. infestans (Fig. 1). Six hours after DDT application, only about 11% of the applied C14-DDT was absorbed in nymphs, while 27% was found in males. At 72 hours the percentage of absorbed DDT was fairly similar in male and nymphal specimens, but while the amount of C14-DDT absorbed at 96 hours was the same as at 72 hours in nymphs, it continued to increase almost linearly in males (Fig. 1). In experiments not shown in Fig. 1, the level of the absorbed radioactivity remained constant up to at least 480 hours in nymphs. On the other hand, the
63
absorbed radioactivity continued increasing up to 120 hours in males, at which time 100% of the insects were dead. The excretion of DDT and metabolites in nymphs increased rapidly to a fairly constant level, a steadystate between internal and excreted radioactivity being reached at about 72 hours after application of the insecticide (Fig. 1). This steady-state was maintained up to 480 hours. In males, the excretion of radioactivity was always below 5% of the applied dose, a steady-state between internal and excreted radioactivity not being reached at any moment. The rate of absorption and excretion of DDT and metabolites in nymphs appears to be fairly similar to what has been reported for the Madeira roach (Lindquist and Dahm, 1956) while in males the overall rate of absorption and excretion can be compared with the rate observed for the American cockroach (Robbins and Dahm, 1955). Figure 2 shows the plotting of absorbed radioactivity, that is internal plus excreted radioactivity, and the radioactivity of internal and excreted metabolites plus the excreted unchanged Cl4-DDT. It is evident from the data that over 37% of the absorbed DDT is either metabolized or excreted in nymphs, while only slightly over 9% is metabolized or excreted in male specimens. This marked difference may partly account for the higher susceptibility of males to DDT. A similar situation has been reported for the Madeiraroach as compared with the American-roach (Robbins and Dahm, 1955). Chromatographic Separation of Metabolites The paper chromatography of internal extracts of C14-DDT treated T. infestans nymphs revealed five metabolites. The characteristic movement of these metabolites in the I/M chromatographic system is shown in Fig. 3. Figure 3 shows an R, scale for the four dyes used, and a reference for the known pure compounds together with the relative movement of the five metabolites. As can be seen, metabolites 1, 2, and 3 are more polar in this system than DDT, while metabolites 4 and 5 are less polar. Figure 4
64
DINAMARCA,
AGOSIN, AND NEGHME
::
80
2 2 :: ? 2 4
60
2
07
2 t - 40
40
ABSORBED
% INTERNAL
RADIOACTIVITY 2 85 E 20
20
INTEdNAL
RAOIOAC7IVI7Y
2 z! 0 0
24
48
hours
72
96
0
24
FIG. 1. Rate of absorption and excretion treated with ring-labelled-C14-DDT.
40
hours
of intoxication of radioactivity
in nymph
72
of intoxication
and male
T. infestans topically
NYMPH SPECIMENS
Unabsorbed
"
L.
DD 1
.(I
hours of intoxication FIG. 2. The relationship of internal time intervals in nymph and male T.
‘nabsorbed
72
96
24
DDT
4a
7.2
96
hours of intoxico tion unchanged DDT versus metabolized or excreted infestans specimens treated with C14-DDT.
DDT
at various
METABOLIC FATE OF DDT IN TRIATOMA
l.O--
q
0.9 -o.a--
T 10
O Oil Blue 8
Q9 0.8
0.7-Q6--
0.7
0
m
m
Oil Blue A
0.5--
!3
t
0
!xl
OilOrangt
El:
pJ
0
El
m
0.2-O.l--
0.5
1 0.4
0.4-0.3--
0.6
Sudah Yellow
q
0.3
Q2
0.1
1
FIG. 3. Chromatographic solubilities of dyes, reference compounds and T. infestans metabolites in the I/M system. Standards: l,DDA; 2,DBH; 3,Kelthane ; 4,DDD ; 5,DBP ; 6,DDT; t,DDE.
65
shows a radiogram of nymph internal extracts 6 hours after application of C14-DDT. When the radioactivity between 0.0 and 0.45 RF was eluted with acetonitrile and rechromatographed with the I/M system, metabolites 2 and 3 were detected, while the radioactivity between 0.45 and 0.72 RF was found to be only DDT (Fig. 5). Metabolite 5 of Fig. 4 is apparently a single one in the I/M system. A radiogram of nymphal internal extracts 48 hours after application of DDT is shown in Fig. 6. Rechromatography of the region between 0.0 and 0.38 R,s shows the presence of three metabolites: number 1, which did not move from the origin; number 2, which almost coincides with the Sudan yellow spot, and number 3 between the Sudan yellow and Oil orange spots. (Fig. 7). Rechromatography of the region between 0.38 and 0.94 Rts showed the presence of another DDT metabolite, metabolite number 4 (Fig. 8). All these metabolites were detected at 24, 48, 72, and 96 hours. However metabolite 5 was not found after 24 hours of intoxication (Table I). Metabolite 5 was from the beginning very distinctive in males, as shown in Fig. 9. Metabolites 2 and 3 were also detected but in
Solvent front
I
I
1.0
.9
8 I
.8
I
I
.7
.6
.s
I
I
I
.4
.3
I
-2
I
.I
I
W
Rf
FIG. 4. Radiogram of nymph internal extracts after 6 hours of topical application of ring-labelled-WDDT. Chromatographic system, I/M. The dotted line corresponds to the background radioactivity.
66
DINAMARCA,
AGOSIN,
AND
NEGHM’R
Solvent front
Origin I oil
BlueB
0
1.0
.9
.8
Oil Blue A
Oil
Sudan Yellow
(
.7
.6
.C
.S
.3
.2
.I
0.0
5 FIG. 5. Radiogram of the eluate between 0.0 and 0.45 R,s obtained from the chromatogram Chromatographic system, I/M. The dotted line corresponds to the background radioactivity.
Solvent front
Origin
\ Oil Orange
1.0
II .9
I
I
.8
.7
Sudan Yellow
I
I .6
.S
of Fig. 4.
.4
.3
.2
.l
I .O
Rf FIG. 6.
DDT.
Radiogram of nymph internal extracts after 48 hours of topical application Chromatographic system, I/M. The dotted line corresponds to the background
of ring-labelled-C14radioactivity.
METABOLIC
FATE
OF DDT IN
67
TRIATOMA
Solvent front
1.0
.9
.8
.7
.6
.5
4
.3
.2
.I
QO
FIG. 7. Radiogram of the eluate between 0.0 and 0.38 R,s obtained from the chromatogram Chromatographic system, I/M. The dotted line corresponds to the background radioactivity.
of Fig. 6.
Solvent Sudan Yellow
1.0
.9
.8
.7
33
.!i
I .4
I .3
.2
.i
Origia
OD
% FIG. 8. Same as Fig. 7, but the eluate was obtained from 0.38 to 0.94 R,s from chromatogram The dotted line corresponds to the background radioactivity.
of F.ig. 6.
6 24 48 72 96
Hours of intoxication
0.0 0.0 0.0 0.7 0.9
1
0.6 1.6 1.7 0.7 0.9
2
3
0.0 0.0 0.4 0.5 0.3
4
5
3.4 3.3 2.3 3.7 1.2
metaholites
0.5 1.0 0.5 0.7 0.2
Internal
95.5 94.1 95.1 94.4 97.4
DDT
Males
Rate of Production
0.0 0.0 0.0 0.4 0.5
1 0.0 0.0 0.0 0.6 0.4
2
TABLE I C14-DDT-Metabolites
Internal
0.0 0.0 0.0 0.2 0.3 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0
100.0 100.0 100.0 98.8 98.8
0.0 4.1 1.7 0.8 0.5
0.1 1.9 4.0 5.9 1.8
0.1 5.4 8.2 14.5 8.1
0.0 8.1 4.0 4.6 13.1
4
metabolites
in Male and Nymph
3 4 5 DDT 1 2 3 Percentage of the absorbed radioactivity
metaholites
and Excreted
Excreted
of Internal
0.3 0.3 0.0 0.0 0.0
5
99.5 80.2 82.1 74.2 76.5
DDT
Nymphs
0.0 0.0 0.0 0.0 0.0
1
0.0 0.0 0.0 0.0 0.0
3
Excreted
0.0 1.6 2.8 4.7 2.4
2
T. infestans Specimens
0.0 14.3 14.0 6.3 1.6
4
0.0 0.0 0.0 0.0 0.0
5
metabolites
100.0 84.1 83.2 89.0 96.0
DDT
Ori Solvent 1 front
I l.0
I .9
I .a
I
1
.7
.6
I
.s
I
1
.4
.3
L
gi I n
I
.2
.l
0.0
FIG. 9. Radiogram of male internal extracts after 6 hours of topical application of ring-labelled W-
DDT.
Chromatographic system, I/M.
The dotted line corresponds to the background radioactivity.
l-
Solvent front
Origin
Oil
Blue B
1sNoQ
Oil Blue A
udan Yellow
00
I
e3ti .----,---
----B---m-
1
1.0
e--w
I 1
.9
4
.7
-------
I
I
.6
.s
I
.4
--
I
.3
--
I I
.2
--_
*
.l
01)
Rf FIG. 10. Radiogram of the eluate between 0.0 and 0.4 RF obtained from a chromatogram of male internal extracts after 72 hours of topical application of ring-labelled Cl4-DDT. Chromatographic system, I/M. The dotted line corresponds to the background radioactivity.
70
DINAMARCA,
AGOSIN,
small amounts at 6 hours. A typical radiochromatogram is shown in Fig. 10 which corresponds to rechromatography of the radioactivity found between 0.0 and 0.4 Rfs in chromatograms of internal male extracts after 72 hours of Cl*-DDT application. Metabolite 4 was present in very small amounts and was detected only after 48 hours of intoxication, while metabolite 1 was found only at 72 hours of intoxication (Table I). The chromatographic resolution of fecal extracts showed the presence of metabolites 2 and 4 during all intoxication periods in nymphs, while in males only metabolites 1, 2, and 3 were detected in feces after 72 hours of DDT application. As shown in Fig. 3 metabolite 4 has the same chromatographic mobility as DDE. When metabolite 4 obtained from internal and external nymph extracts as well as internal male extracts was eluted from paper chromatograms and rechromatographed in the D/M and the E/G system, its position corresponded clearly to DDE. Furthermore, co-chromatography of metabolite 4 with non-radioactive DDE in the I/M system showed that the radioactivity coincides with the spot produced by DDE when treated with Mitchell’s reagent (1957). These observations indicate that metabolite 4 found in T. infestans can be identified as DDE. The remaining four metabolites have as yet not been identified. However metabolites 1 and 2 correspond closely from a chromatographic point of view to metabolites 1 and 2 described by Hoskins et al. (1958) in Blattella germanica. Metabolite 1 and 2 of T. infestans apparently do not correspond to DDA (Fig. 3). However metabolite 2 has the same solubility characteristics as DBH in the I/M system. Further chromatographic studies of metabolite 2 will be made in order to check chromatographic identity with DBH. Metabolite 3 possesses a chromatographic identity with metabolite 4, as indicated by Hoskins (1958), and has the same mobility as Kelthane, as seen in Fig. 3. Kelthane has been reported as a major DDT-metabolite in Drosophila (Tsukamoto, 1959). We do not have any clue as to the identity of metabolite 5 as yet.
AND
NEGHME
Quantitative
Aspects of DDT-Metabolites Production
It is evident from Table I that metabolite 4, i.e., DDE, represents, from a quantitative standpoint, the most important DDT-metabolite in nymphs. Almost 21% of the absorbed radioactivity is found as DDE in the first 24 hours of intoxication. Over 14% of the produced DDE is found in the excreta. At 48 and 72 hours the rate of DDE production decreases in nymphs, only about 11% of the absorbed radioactivity being found as DDE. At 96 hours there is apparently an accumulation of DDE in nymphs, since of the 18% of radioactivity found as DDE, 13% is not excreted. Concomitant to the decrease in DDE production, a fairly rapid increase in the formation of metabolites 2 and 3 is observed; of these, only metabolite 2 is partially excreted after 24 hours. Over 25% of the absorbed radioactivity after 72 hours of intoxication is found as metabolites 2 and 3. Metabolites 1 and 5 are not excreted in nymphs. Metabolite 5 is found only during the first 24 hours of intoxication and metabolite 1 after increasing to 4.1% at 24 hours, decreases at 96 hours to a low 0.5%. Among the metabolites produced by male T. infestam, only number 5 accumulates to a significant extent. Small amounts of DDE are found after 48 hours of intoxication only in internal extracts. Very small amounts of metabolites 1, 2, and 3 are also produced and they appear in the excreta only after 72 hours of intoxication (Table I). Metabolite 5 is produced at a fairly constant rate, but none is excreted (Table I). DISCUSSION
The present studies clearly indicate that at least two mechanisms are involved in the higher resistance towards DDT exhibited by nymph as compared to male T. infestans. The first is a permeability factor, and the second, a detoxication process. It is evident that DDT penetrates at a lower rate in nymphs than in males (Fig. 1). Therefore it is safe to assume that the higher resistance shown by nymphs can be partially attributed to failure of some of the applied DDT to penetrate the nymphal cuticle. Furthermore, DDT is excreted at a faster rate in nymph than in male T. infestans.
METABOLIC
FATE
OF DDT IN
This explanation is supported by several observations by other workers; thus, the larvae of Tragoderma granarium is resistant to DDT because of a lack of penetration of the insecticide through the cuticle (Pradhan et al., 1952). Sternburg and Kearns (1958) found that Argyrotaenia velutinana absorbs DDT at an extremely low rate, which together with an efficient detoxifying mechanism, makes them resistant to DDT. Detoxication of DDT is also an important factor in the higher resistance of nymphs to DDT. Apparently the ability of nymphs to convert DDT to DDE to a much larger extent than males renders them less susceptible to DDT. Conversion of DDT to DDE has been implied as the mechanism of resistance to this insecticide by the house-fly (Perry and Hoskins, 1950). The enzyme involved in this conversion has been purified to a great extent (Lipke and Kearns, 1959) and although it is also found in susceptible house-fly strains, the amounts there are very low (Lipke and Kearns, 1959). Whether the same situation occurs in male T. infestans as compared to nymphs, is at present uncertain. There is the possibility that the smaller in vivo conversion of DDT to DDE exhibited by males might not be due to a smaller enzyme content, but to an inhibition by DDT of ancillary enzymatic reactions. It is known that reduced glutathione is required by DDT-dehydrochlorinase for activity (Lipke and Kearns, 1959). Inhibition of reactions supplying reduced glutathione would result in a lower activity of DDT-dehydrochlorinase. However, Moorefield (1958) reported that in a susceptible house fly strain, the larvae contained considerable DDT-dehydrochlorinase, but the adults lacked it. Whether conversion of DDT to the more polar metabolites (Fig. 3 and Table I) represents a protective mechanism, is difficult to establish with the available data. Similar metabolites have been found in a fairly susceptible strain of Blattella germanica (Hoskins et al., 1958). In this case, no DDE was detected and it would appear consequently that these polar metabolites would not be related to resistance. Metabolite 3 of T. infestans seems to correspond closely to metabolite 4 of Hoskins et al. (1958) and to the
71
TRIATOMA
Kelthane produced by Drosophila (Tsukamoto, 1959). Its in vitro production by a microsomal preparation of Blattella germanica has been shown by Agosin et al. (1961a). The system required TPNH, molecular oxygen, nicotinamide, and magnesium ions. The in V&O availability of TPNH could be a limiting factor for the production of Kelthane. Supporting this possibility, unpublished observations in this laboratory have shown that the pentose phosphate pathway is stimulated by DDT to a greater extent in nymph than in male T. infestans. Since this pathway is an important source of TPNH, this could explain why metabolite 3 and the other polar metabolites are produced in greater amounts in nymphs (Table I). Available evidence suggests that the mechanism of production of DDE is independent of the pathways involved in the formation of metabolites more polar than DDT. DDE is produced by a dehydrochlorinating enzyme (Lipke and Kearns, 1959), while the polar metabolites apparently are produced by oxidative processes involving hydroxylations (Agosin et al., 1961a). Assuming that the oxidative processes leading to the formation of polar metabolites of male and nymph T. infestans specimens are qualitatively similar, one is tempted to postulate, on the basis of the results shown in Table I, the metabolic sequence of Fig. 11. A common link for both
r
.
FIG. 11. Postulated sequence for the production of DDT-metabolites in 2’. infestens and the relationship with normal metabolic reactions of the insect.
metabolic pathways would be TPNH, which is required for DDT-hydroxylation (Agosin et al., 1961a) and for the production of reduced glutathione probably through glutathione-reductase. TPNH would be produced mainly by the reactions of the pentose phos-
72
DINAMARCA,
AGOSIN,
phate pathway which have been previously demonstrated in T. infestans (Agosin et al., 1961). Once the various metabolites are properly identified, attempts to isolate the enzyme(s) responsible for their production would be undertaken, and the validity of the metabolic sequence of Fig. 11 could be tested. REFERENCES ACOSIN, M., NECHME, A., AND SCARAMELLI, N. 1960. Non-identity of triosephosphate dehydrogenase of adult male and nymph development stages of Triutomu infestans. Neture 186, 1052-1053. AGOSIN, M., SCARAIMELLI,N., AND NEGHME, A. 1961. Intermediary carbohydrate metabolism of Tubatoma infestans. I. Glycolytic and pentose phosphate pathway enzymes and the effect of DDT. Comparative Biochemistry and Physiology 2, 143-159. AGOSIN, M., MICHAELI, D., MISXUS, R., NAGASAWA,S., AND HOSKINS, W. M. 1961a. A new DDT-metabolizing enzyme in the German Cockroach. Journal of Economic Entomology 64, 340-342. GORDON, H. T. 1960. Personal communication. HOSKINS, W. M., MISKUS, R., AND ELDEFRAWI, M. E. 1958. The biochemistry of DDT resistance in insects. Seminar on the susceptibility of insects to insecticides. Panama, Pan-American Health Orga&ation 239-253. LINDQUIST, M., AND DAECM, P. A. 1956. Metabolism of radioactive DDT by the American Roach and European Corn Borer. Joud of Ecolzomic Entomology 49, 579-584. LIPKE, H., AND KEARNS, C. W. 1959. DDT dehydrochlorinase. 1. Isolation, chemical properties, and spectrophotometric assay. Journal of Biological Chemistry 234, 2123-2128.
AND
NEGHME
MENN, J., ELDEFRAWI, M. E., AND GORDON, H. T. 1960. Prechromatographic purification of insecticides from insect tissue extracts. Journal of Agricultural and Food Chemistry 8, 41-42. MISKUS, R. 1959. Personal communication. MITCHELL, L. C. 1957. Note on the detection of chlorinated organic pesticides in the paper chromatogram. JournaZ of the Association of Oficiol Agricultural Ch,emists 40, 891-2. MOOREFIELD, H. 1958. The origin of DDT resistance in the house fly. Contributions of the Boyce Thompson Institute 19, 403-409. PERRY, A. S., AND HOSKINS, W. M. 1950. The detoxification of DDT by resistant house flies and inhibition of this process by piperonyl cyclonene. Science 111, 600-601. PRADHAN, S., NAIR, M., AND KRISCHMASWANMI, S. 1952. Lipoid solubility as a factor in the toxicity of contact insecticides. Nature 170, 619-620. ROBBINS, W. E., AND DAHM, P. A. 1955. Absorption and excretion, distribution and metabolism of C14-labelled DDT by the American Cockroach. Journal of Agricultural and Food Chemists 3, 500-508. STERNBURG,J., AND KEARNS, C. W. 1958. Metabolic fate of DDT when applied to certain naturally tolerant insects. Journal of Economic Entomology 46, 497-508. TSUKILMOTO, M. 1959. Metabolic fate of DDT in Drosophila melanoguster. I. Identification of a non DDE metabolite. Botyu-Kugaku a4, 141151. ACKNOWLEDGMENTS The authors are grateful to W. M. Hoskins and R. Miskus for reading the manuscript and for helpful criticism.
PARASITOLOGY
The Maria
12, 61-72 (1962)
Metabolic
Fate of C14-DDT in
Luisa
Moises
Dinamarca,
Department of Parasitology,
Agosin,
Triatoma
infestud2
and Amador
Biochemistry Section, Santiago, Chile
University
Neghme
of Chile,
(Submitted for publication, 10 August 1961) (1) Nymph and male T. infestans specimens differ in their rate of absorption, excretion and metabolism of Cr4-DDT. Nymphs absorb DDT to a lesser extent than males. Furthermore, DDT is excreted at a faster rate in nymphs than males. After 96 hours of intoxication, 27.5% of the absorbed DDT is converted to a mixture of five metabolites in nymphs, while only 4.7% of the absorbed DDT is accounted for by DDT-metabolites in males after the same time of intoxication. (2) DDT is metabolized into five metabolites in both nymph and male adult T. infestans. DDE is the major DDT-metabolite produced by nymphs, while in males, metabolite No. 5 is the most important one, DDE being found only in negligible amounts. Metabolite 3 has the same chromatographic identity as Kelthane, a DDT metabolite found in Drosophib and Blattella germanica. (3) It is postulated that the production of the more polar metabolites involves oxidative processes independent of the mechanism of formation of DDE. A tentative sequence is given for the formation of DDT metabolites in T. infestans and the availability of TPNH is suggested as a common lii for both the oxidation and the dehydrochlorination processes. (4) It is considered that the differences regarding DDT penetration, excretion, and metabolism shown by nymphs as compared to males, may account at least partly for the higher tolerance to DDT exhibited by nymph T. infestam.
Previous work in this laboratory has shown that nymph and male adult T. infestans specimens differ markedly in their susceptibility towards DDT, males being much more susceptible than nymphs (Agosin, Scaramelli, and Neghme, 1961). Apparently, this 1 Supported by grant E-2300 of the Division of Research Grants and Fellowships of the National Institutes of Health, U.S. Public Health Service, and by funds of the Commission for Scientific Research of the University of Chile. 2 Abbrevidons: DDT, 2,2-bis-(p-cblorophenyl)l,l,l-trichloroethane; DDE, 2,2-bis-(p-chlorophenyl)l,l-dicbloroethylene; DDA, 2,2-bis-(p-chlorophenyl) acetic acid ; DDD, 2,2-bis-(p-chlorophenyl)-l,ldichloroethane; Kelthane, 2,2-bis-(p-chlorophenyl)l,l,l-trichloroethanol; DBP, p,p’-dichlorobenzophenone; DBH, p,p’-dichlorobenzohydrol; GSSG, oxidized glutathione ; GSH, reduced glutathione ; TPN, oxidized triphosphopyridine nucleotide; TPNH, reduced triphosphopyridine nucleotide.
difference in response to DDT is not related to enzyme content of male and nymph specimens. The latter have, in general, a lower enzyme content than males, at least in respect to glycolytic and pentose phosphate pathway enzymes (Agosin et d., 1961). The possibility that key enzymes which are inhibited by DDT in males are not affected in nymphs is supported by the fact that in males some glycolytic and pentose phosphate pathway enzymes are inhibited in vivo as well as in vitro by DDT. The corresponding nymphal enzymes are not affected (Agosin et al., 1961). These observations suggest that the hypothetical DDT-sensitive key enzyme(s) may be structurally different in male and nymph T. infestans as seems to be the case for triose phosphate dehydrogenase (Agosin, Neghme, and Scaramelli, 1960). However, other factors in addition to differ61
DINAMARCA,AGOSIN,ANDNEGHME
62
ential enzymatic inhibition may be involved in the lesser susceptibility of nymphs to DDT. These factors, such as differences in the rate of absorption and excretion of DDT, and/or different or more efficient detoxifying mechanisms in nymphs have been explored in the present studies. The results obtained indicate that DDT is absorbed less and excreted faster in nymphs than in males. Furthermore, although both nymph and males metabolize DDT to at least five metabolites, the rate of production of these metabolites is much higher in the nymph. It is hoped that these studies will contribute to our understanding of the mode of action of DDT, and the mechanism involved in the differences of susceptibility towards DDT exhibited by the various developmental stages of T. infestans. METHODS
Insect Material Laboratory reared T. infestans specimens were used throughout. They were maintained as indicated elsewhere (Agosin et al., 1961). The experimental work was done with adult males and third instar larvae (nymphs) that were starved at least one week before being used. Groups of 3.5 specimens were treated with C14-DDT. Twenty microliters of an acetone C14-DDT solution, containing 16.8 ug of DDT (2,380 c.p.m. per pg) were topically applied to the ventral abdominal region of the insects by means of a micropipette. At 6, 24, 48, 72, and 96 hours, five surviving specimens were selected for analysis. The dead nymph or male specimens were discarded. After application of C14-DDT, the insects were kept as previously described (Agosin et aZ., 1961). As a control, the amount of applied C14-DDT was determined in several experiments by extracting and measuring the radioactivity immediately after the topical application of the insecticide. Extraction
Procedures
At the above indicated time intervals, the insects selected for analysis were rinsed three times individually in about 5 ml of acetone to eliminate any external insecticide. The abdomen of the insects was gently pressed
with a forceps and the fecal matter obtained pooled with the feces present in the insects container. The rinsed insects were then homogenized in about 10 ml of chloroform in a semimicro Virtis homogenizer. About 2 ml of water was added to the homogenate which was then extracted three times each with about 20ml of chloroform in a separatory funnel. The combined chloroform layers, filtered through anhydrous sodium sulfate, were concentrated to dryness under a stream of hot air. The insect container and excreta were extracted for 48 hours with 10 ml of a solution of 80 ml acetone, 20 ml water and 1 ml 1 N sulfuric acid, according to Lindquist and Dahm (1956). The mixture was then extracted three times with chloroform, the chloroform layer filtered through anhydrous sodium sulfate, and the filtrate concentrated as above. The efficiency of the extraction procedure for both insect and fecal material was 98 to 99%. Chromatographic Procedures The dry extracts, dissolved in a minimum volume of acetone, were spotted on Whatman #l one-inch-wide filter paper strips, and chromatographically purified by the procedure of Menn, Eldefrawi, and Gordon (1960) with 100% acetonitrile. The purified extracts from the strips were made up to a volume of 1 ml with acetone and an aliquot was taken for measuring the radioactivity. The remaining extract was again concentrated to a minimum volume and chromatographed with betamethoxypropionitrile as stationary phase and iso-octane saturated with beta-methoxypropionitrile as mobile phase (Gordon, 1960; I/M system). In some special cases, 75% N,N-dimethyl-formamide in water as mobile phase, and 10% mineral oil in chloroform as stationary phase (D/M system) or isopropyl ether saturated with glutaronitrile as mobile phase and 15% glutaronitrile in acetone as stationary phase (E/G system) were also used. The dye reference system of Gordon (1960) formed by a mixture of Oil Blue N (A + B), Oil Orange 2311, and Sudan Yellow RRA was used with the I/M system. Confirming Gordon’s work (1960) the position of insecticide spots relative to that of the different dyes was constant. After develop-
METABOLIC
FATE
OF DDT IN TRIATOMA
ment, the chromatograms were scanned in a model ClOO-A Nuclear Chicago Actigraph with a thin-window D-47 Flow-Counter, with a speed of 0.75 inches/mm and a slit of l/4 inch. The metabolites were located, eluted with 100% acetonitrile and rechromatographed with the above chromatographic systems. Finally, when the metabolites were clearly separated, they were eluted with 100% acetonitrile, and the radioactivity measured on planchets at infinite thinness in a Model 6000 Nuclear Chicago Dynacon Electrometer. Standard corrections were applied in measuring the radioactivity. Non-radioactive chromatographic reference compounds were detected with Mitchell’s silver nitrate reagent (1957). Reagents 2-C14-ethane-labelled DDT obtained through the courtesy of the Communicable Diseases Center, Savannah, Georgia, was used at the beginning of this work. However, the specific activity was low for our purposes. Therefore, most of the results given here were obtained with ring-labelled-C14-DDT, prepared in the laboratory (Miskus, 1959). The dyes were a gift of Dr. H. T. Gordon. Chromatographically pure DDE, DBP, DBH, DDD, DDA, were kindly provided by R. Miskus. The rest of the reagents were of the highest purity commercially available. RESULTS
Rate of Absorption and Excretion and Metabolites
of DDT
The rate of absorption of DDT as well as the rate of excretion of DDT and metabolites was found to be different in nymph as compared to male T. infestans (Fig. 1). Six hours after DDT application, only about 11% of the applied C14-DDT was absorbed in nymphs, while 27% was found in males. At 72 hours the percentage of absorbed DDT was fairly similar in male and nymphal specimens, but while the amount of C14-DDT absorbed at 96 hours was the same as at 72 hours in nymphs, it continued to increase almost linearly in males (Fig. 1). In experiments not shown in Fig. 1, the level of the absorbed radioactivity remained constant up to at least 480 hours in nymphs. On the other hand, the
63
absorbed radioactivity continued increasing up to 120 hours in males, at which time 100% of the insects were dead. The excretion of DDT and metabolites in nymphs increased rapidly to a fairly constant level, a steadystate between internal and excreted radioactivity being reached at about 72 hours after application of the insecticide (Fig. 1). This steady-state was maintained up to 480 hours. In males, the excretion of radioactivity was always below 5% of the applied dose, a steady-state between internal and excreted radioactivity not being reached at any moment. The rate of absorption and excretion of DDT and metabolites in nymphs appears to be fairly similar to what has been reported for the Madeira roach (Lindquist and Dahm, 1956) while in males the overall rate of absorption and excretion can be compared with the rate observed for the American cockroach (Robbins and Dahm, 1955). Figure 2 shows the plotting of absorbed radioactivity, that is internal plus excreted radioactivity, and the radioactivity of internal and excreted metabolites plus the excreted unchanged Cl4-DDT. It is evident from the data that over 37% of the absorbed DDT is either metabolized or excreted in nymphs, while only slightly over 9% is metabolized or excreted in male specimens. This marked difference may partly account for the higher susceptibility of males to DDT. A similar situation has been reported for the Madeiraroach as compared with the American-roach (Robbins and Dahm, 1955). Chromatographic Separation of Metabolites The paper chromatography of internal extracts of C14-DDT treated T. infestans nymphs revealed five metabolites. The characteristic movement of these metabolites in the I/M chromatographic system is shown in Fig. 3. Figure 3 shows an R, scale for the four dyes used, and a reference for the known pure compounds together with the relative movement of the five metabolites. As can be seen, metabolites 1, 2, and 3 are more polar in this system than DDT, while metabolites 4 and 5 are less polar. Figure 4
64
DINAMARCA,
AGOSIN, AND NEGHME
::
80
2 2 :: ? 2 4
60
2
07
2 t - 40
40
ABSORBED
% INTERNAL
RADIOACTIVITY 2 85 E 20
20
INTEdNAL
RAOIOAC7IVI7Y
2 z! 0 0
24
48
hours
72
96
0
24
FIG. 1. Rate of absorption and excretion treated with ring-labelled-C14-DDT.
40
hours
of intoxication of radioactivity
in nymph
72
of intoxication
and male
T. infestans topically
NYMPH SPECIMENS
Unabsorbed
"
L.
DD 1
.(I
hours of intoxication FIG. 2. The relationship of internal time intervals in nymph and male T.
‘nabsorbed
72
96
24
DDT
4a
7.2
96
hours of intoxico tion unchanged DDT versus metabolized or excreted infestans specimens treated with C14-DDT.
DDT
at various
METABOLIC FATE OF DDT IN TRIATOMA
l.O--
q
0.9 -o.a--
T 10
O Oil Blue 8
Q9 0.8
0.7-Q6--
0.7
0
m
m
Oil Blue A
0.5--
!3
t
0
!xl
OilOrangt
El:
pJ
0
El
m
0.2-O.l--
0.5
1 0.4
0.4-0.3--
0.6
Sudah Yellow
q
0.3
Q2
0.1
1
FIG. 3. Chromatographic solubilities of dyes, reference compounds and T. infestans metabolites in the I/M system. Standards: l,DDA; 2,DBH; 3,Kelthane ; 4,DDD ; 5,DBP ; 6,DDT; t,DDE.
65
shows a radiogram of nymph internal extracts 6 hours after application of C14-DDT. When the radioactivity between 0.0 and 0.45 RF was eluted with acetonitrile and rechromatographed with the I/M system, metabolites 2 and 3 were detected, while the radioactivity between 0.45 and 0.72 RF was found to be only DDT (Fig. 5). Metabolite 5 of Fig. 4 is apparently a single one in the I/M system. A radiogram of nymphal internal extracts 48 hours after application of DDT is shown in Fig. 6. Rechromatography of the region between 0.0 and 0.38 R,s shows the presence of three metabolites: number 1, which did not move from the origin; number 2, which almost coincides with the Sudan yellow spot, and number 3 between the Sudan yellow and Oil orange spots. (Fig. 7). Rechromatography of the region between 0.38 and 0.94 Rts showed the presence of another DDT metabolite, metabolite number 4 (Fig. 8). All these metabolites were detected at 24, 48, 72, and 96 hours. However metabolite 5 was not found after 24 hours of intoxication (Table I). Metabolite 5 was from the beginning very distinctive in males, as shown in Fig. 9. Metabolites 2 and 3 were also detected but in
Solvent front
I
I
1.0
.9
8 I
.8
I
I
.7
.6
.s
I
I
I
.4
.3
I
-2
I
.I
I
W
Rf
FIG. 4. Radiogram of nymph internal extracts after 6 hours of topical application of ring-labelled-WDDT. Chromatographic system, I/M. The dotted line corresponds to the background radioactivity.
66
DINAMARCA,
AGOSIN,
AND
NEGHM’R
Solvent front
Origin I oil
BlueB
0
1.0
.9
.8
Oil Blue A
Oil
Sudan Yellow
(
.7
.6
.C
.S
.3
.2
.I
0.0
5 FIG. 5. Radiogram of the eluate between 0.0 and 0.45 R,s obtained from the chromatogram Chromatographic system, I/M. The dotted line corresponds to the background radioactivity.
Solvent front
Origin
\ Oil Orange
1.0
II .9
I
I
.8
.7
Sudan Yellow
I
I .6
.S
of Fig. 4.
.4
.3
.2
.l
I .O
Rf FIG. 6.
DDT.
Radiogram of nymph internal extracts after 48 hours of topical application Chromatographic system, I/M. The dotted line corresponds to the background
of ring-labelled-C14radioactivity.
METABOLIC
FATE
OF DDT IN
67
TRIATOMA
Solvent front
1.0
.9
.8
.7
.6
.5
4
.3
.2
.I
QO
FIG. 7. Radiogram of the eluate between 0.0 and 0.38 R,s obtained from the chromatogram Chromatographic system, I/M. The dotted line corresponds to the background radioactivity.
of Fig. 6.
Solvent Sudan Yellow
1.0
.9
.8
.7
33
.!i
I .4
I .3
.2
.i
Origia
OD
% FIG. 8. Same as Fig. 7, but the eluate was obtained from 0.38 to 0.94 R,s from chromatogram The dotted line corresponds to the background radioactivity.
of F.ig. 6.
6 24 48 72 96
Hours of intoxication
0.0 0.0 0.0 0.7 0.9
1
0.6 1.6 1.7 0.7 0.9
2
3
0.0 0.0 0.4 0.5 0.3
4
5
3.4 3.3 2.3 3.7 1.2
metaholites
0.5 1.0 0.5 0.7 0.2
Internal
95.5 94.1 95.1 94.4 97.4
DDT
Males
Rate of Production
0.0 0.0 0.0 0.4 0.5
1 0.0 0.0 0.0 0.6 0.4
2
TABLE I C14-DDT-Metabolites
Internal
0.0 0.0 0.0 0.2 0.3 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0
100.0 100.0 100.0 98.8 98.8
0.0 4.1 1.7 0.8 0.5
0.1 1.9 4.0 5.9 1.8
0.1 5.4 8.2 14.5 8.1
0.0 8.1 4.0 4.6 13.1
4
metabolites
in Male and Nymph
3 4 5 DDT 1 2 3 Percentage of the absorbed radioactivity
metaholites
and Excreted
Excreted
of Internal
0.3 0.3 0.0 0.0 0.0
5
99.5 80.2 82.1 74.2 76.5
DDT
Nymphs
0.0 0.0 0.0 0.0 0.0
1
0.0 0.0 0.0 0.0 0.0
3
Excreted
0.0 1.6 2.8 4.7 2.4
2
T. infestans Specimens
0.0 14.3 14.0 6.3 1.6
4
0.0 0.0 0.0 0.0 0.0
5
metabolites
100.0 84.1 83.2 89.0 96.0
DDT
Ori Solvent 1 front
I l.0
I .9
I .a
I
1
.7
.6
I
.s
I
1
.4
.3
L
gi I n
I
.2
.l
0.0
FIG. 9. Radiogram of male internal extracts after 6 hours of topical application of ring-labelled W-
DDT.
Chromatographic system, I/M.
The dotted line corresponds to the background radioactivity.
l-
Solvent front
Origin
Oil
Blue B
1sNoQ
Oil Blue A
udan Yellow
00
I
e3ti .----,---
----B---m-
1
1.0
e--w
I 1
.9
4
.7
-------
I
I
.6
.s
I
.4
--
I
.3
--
I I
.2
--_
*
.l
01)
Rf FIG. 10. Radiogram of the eluate between 0.0 and 0.4 RF obtained from a chromatogram of male internal extracts after 72 hours of topical application of ring-labelled Cl4-DDT. Chromatographic system, I/M. The dotted line corresponds to the background radioactivity.
70
DINAMARCA,
AGOSIN,
small amounts at 6 hours. A typical radiochromatogram is shown in Fig. 10 which corresponds to rechromatography of the radioactivity found between 0.0 and 0.4 Rfs in chromatograms of internal male extracts after 72 hours of Cl*-DDT application. Metabolite 4 was present in very small amounts and was detected only after 48 hours of intoxication, while metabolite 1 was found only at 72 hours of intoxication (Table I). The chromatographic resolution of fecal extracts showed the presence of metabolites 2 and 4 during all intoxication periods in nymphs, while in males only metabolites 1, 2, and 3 were detected in feces after 72 hours of DDT application. As shown in Fig. 3 metabolite 4 has the same chromatographic mobility as DDE. When metabolite 4 obtained from internal and external nymph extracts as well as internal male extracts was eluted from paper chromatograms and rechromatographed in the D/M and the E/G system, its position corresponded clearly to DDE. Furthermore, co-chromatography of metabolite 4 with non-radioactive DDE in the I/M system showed that the radioactivity coincides with the spot produced by DDE when treated with Mitchell’s reagent (1957). These observations indicate that metabolite 4 found in T. infestans can be identified as DDE. The remaining four metabolites have as yet not been identified. However metabolites 1 and 2 correspond closely from a chromatographic point of view to metabolites 1 and 2 described by Hoskins et al. (1958) in Blattella germanica. Metabolite 1 and 2 of T. infestans apparently do not correspond to DDA (Fig. 3). However metabolite 2 has the same solubility characteristics as DBH in the I/M system. Further chromatographic studies of metabolite 2 will be made in order to check chromatographic identity with DBH. Metabolite 3 possesses a chromatographic identity with metabolite 4, as indicated by Hoskins (1958), and has the same mobility as Kelthane, as seen in Fig. 3. Kelthane has been reported as a major DDT-metabolite in Drosophila (Tsukamoto, 1959). We do not have any clue as to the identity of metabolite 5 as yet.
AND
NEGHME
Quantitative
Aspects of DDT-Metabolites Production
It is evident from Table I that metabolite 4, i.e., DDE, represents, from a quantitative standpoint, the most important DDT-metabolite in nymphs. Almost 21% of the absorbed radioactivity is found as DDE in the first 24 hours of intoxication. Over 14% of the produced DDE is found in the excreta. At 48 and 72 hours the rate of DDE production decreases in nymphs, only about 11% of the absorbed radioactivity being found as DDE. At 96 hours there is apparently an accumulation of DDE in nymphs, since of the 18% of radioactivity found as DDE, 13% is not excreted. Concomitant to the decrease in DDE production, a fairly rapid increase in the formation of metabolites 2 and 3 is observed; of these, only metabolite 2 is partially excreted after 24 hours. Over 25% of the absorbed radioactivity after 72 hours of intoxication is found as metabolites 2 and 3. Metabolites 1 and 5 are not excreted in nymphs. Metabolite 5 is found only during the first 24 hours of intoxication and metabolite 1 after increasing to 4.1% at 24 hours, decreases at 96 hours to a low 0.5%. Among the metabolites produced by male T. infestam, only number 5 accumulates to a significant extent. Small amounts of DDE are found after 48 hours of intoxication only in internal extracts. Very small amounts of metabolites 1, 2, and 3 are also produced and they appear in the excreta only after 72 hours of intoxication (Table I). Metabolite 5 is produced at a fairly constant rate, but none is excreted (Table I). DISCUSSION
The present studies clearly indicate that at least two mechanisms are involved in the higher resistance towards DDT exhibited by nymph as compared to male T. infestans. The first is a permeability factor, and the second, a detoxication process. It is evident that DDT penetrates at a lower rate in nymphs than in males (Fig. 1). Therefore it is safe to assume that the higher resistance shown by nymphs can be partially attributed to failure of some of the applied DDT to penetrate the nymphal cuticle. Furthermore, DDT is excreted at a faster rate in nymph than in male T. infestans.
METABOLIC
FATE
OF DDT IN
This explanation is supported by several observations by other workers; thus, the larvae of Tragoderma granarium is resistant to DDT because of a lack of penetration of the insecticide through the cuticle (Pradhan et al., 1952). Sternburg and Kearns (1958) found that Argyrotaenia velutinana absorbs DDT at an extremely low rate, which together with an efficient detoxifying mechanism, makes them resistant to DDT. Detoxication of DDT is also an important factor in the higher resistance of nymphs to DDT. Apparently the ability of nymphs to convert DDT to DDE to a much larger extent than males renders them less susceptible to DDT. Conversion of DDT to DDE has been implied as the mechanism of resistance to this insecticide by the house-fly (Perry and Hoskins, 1950). The enzyme involved in this conversion has been purified to a great extent (Lipke and Kearns, 1959) and although it is also found in susceptible house-fly strains, the amounts there are very low (Lipke and Kearns, 1959). Whether the same situation occurs in male T. infestans as compared to nymphs, is at present uncertain. There is the possibility that the smaller in vivo conversion of DDT to DDE exhibited by males might not be due to a smaller enzyme content, but to an inhibition by DDT of ancillary enzymatic reactions. It is known that reduced glutathione is required by DDT-dehydrochlorinase for activity (Lipke and Kearns, 1959). Inhibition of reactions supplying reduced glutathione would result in a lower activity of DDT-dehydrochlorinase. However, Moorefield (1958) reported that in a susceptible house fly strain, the larvae contained considerable DDT-dehydrochlorinase, but the adults lacked it. Whether conversion of DDT to the more polar metabolites (Fig. 3 and Table I) represents a protective mechanism, is difficult to establish with the available data. Similar metabolites have been found in a fairly susceptible strain of Blattella germanica (Hoskins et al., 1958). In this case, no DDE was detected and it would appear consequently that these polar metabolites would not be related to resistance. Metabolite 3 of T. infestans seems to correspond closely to metabolite 4 of Hoskins et al. (1958) and to the
71
TRIATOMA
Kelthane produced by Drosophila (Tsukamoto, 1959). Its in vitro production by a microsomal preparation of Blattella germanica has been shown by Agosin et al. (1961a). The system required TPNH, molecular oxygen, nicotinamide, and magnesium ions. The in V&O availability of TPNH could be a limiting factor for the production of Kelthane. Supporting this possibility, unpublished observations in this laboratory have shown that the pentose phosphate pathway is stimulated by DDT to a greater extent in nymph than in male T. infestans. Since this pathway is an important source of TPNH, this could explain why metabolite 3 and the other polar metabolites are produced in greater amounts in nymphs (Table I). Available evidence suggests that the mechanism of production of DDE is independent of the pathways involved in the formation of metabolites more polar than DDT. DDE is produced by a dehydrochlorinating enzyme (Lipke and Kearns, 1959), while the polar metabolites apparently are produced by oxidative processes involving hydroxylations (Agosin et al., 1961a). Assuming that the oxidative processes leading to the formation of polar metabolites of male and nymph T. infestans specimens are qualitatively similar, one is tempted to postulate, on the basis of the results shown in Table I, the metabolic sequence of Fig. 11. A common link for both
r
.
FIG. 11. Postulated sequence for the production of DDT-metabolites in 2’. infestens and the relationship with normal metabolic reactions of the insect.
metabolic pathways would be TPNH, which is required for DDT-hydroxylation (Agosin et al., 1961a) and for the production of reduced glutathione probably through glutathione-reductase. TPNH would be produced mainly by the reactions of the pentose phos-
72
DINAMARCA,
AGOSIN,
phate pathway which have been previously demonstrated in T. infestans (Agosin et al., 1961). Once the various metabolites are properly identified, attempts to isolate the enzyme(s) responsible for their production would be undertaken, and the validity of the metabolic sequence of Fig. 11 could be tested. REFERENCES ACOSIN, M., NECHME, A., AND SCARAMELLI, N. 1960. Non-identity of triosephosphate dehydrogenase of adult male and nymph development stages of Triutomu infestans. Neture 186, 1052-1053. AGOSIN, M., SCARAIMELLI,N., AND NEGHME, A. 1961. Intermediary carbohydrate metabolism of Tubatoma infestans. I. Glycolytic and pentose phosphate pathway enzymes and the effect of DDT. Comparative Biochemistry and Physiology 2, 143-159. AGOSIN, M., MICHAELI, D., MISXUS, R., NAGASAWA,S., AND HOSKINS, W. M. 1961a. A new DDT-metabolizing enzyme in the German Cockroach. Journal of Economic Entomology 64, 340-342. GORDON, H. T. 1960. Personal communication. HOSKINS, W. M., MISKUS, R., AND ELDEFRAWI, M. E. 1958. The biochemistry of DDT resistance in insects. Seminar on the susceptibility of insects to insecticides. Panama, Pan-American Health Orga&ation 239-253. LINDQUIST, M., AND DAECM, P. A. 1956. Metabolism of radioactive DDT by the American Roach and European Corn Borer. Joud of Ecolzomic Entomology 49, 579-584. LIPKE, H., AND KEARNS, C. W. 1959. DDT dehydrochlorinase. 1. Isolation, chemical properties, and spectrophotometric assay. Journal of Biological Chemistry 234, 2123-2128.
AND
NEGHME
MENN, J., ELDEFRAWI, M. E., AND GORDON, H. T. 1960. Prechromatographic purification of insecticides from insect tissue extracts. Journal of Agricultural and Food Chemistry 8, 41-42. MISKUS, R. 1959. Personal communication. MITCHELL, L. C. 1957. Note on the detection of chlorinated organic pesticides in the paper chromatogram. JournaZ of the Association of Oficiol Agricultural Ch,emists 40, 891-2. MOOREFIELD, H. 1958. The origin of DDT resistance in the house fly. Contributions of the Boyce Thompson Institute 19, 403-409. PERRY, A. S., AND HOSKINS, W. M. 1950. The detoxification of DDT by resistant house flies and inhibition of this process by piperonyl cyclonene. Science 111, 600-601. PRADHAN, S., NAIR, M., AND KRISCHMASWANMI, S. 1952. Lipoid solubility as a factor in the toxicity of contact insecticides. Nature 170, 619-620. ROBBINS, W. E., AND DAHM, P. A. 1955. Absorption and excretion, distribution and metabolism of C14-labelled DDT by the American Cockroach. Journal of Agricultural and Food Chemists 3, 500-508. STERNBURG,J., AND KEARNS, C. W. 1958. Metabolic fate of DDT when applied to certain naturally tolerant insects. Journal of Economic Entomology 46, 497-508. TSUKILMOTO, M. 1959. Metabolic fate of DDT in Drosophila melanoguster. I. Identification of a non DDE metabolite. Botyu-Kugaku a4, 141151. ACKNOWLEDGMENTS The authors are grateful to W. M. Hoskins and R. Miskus for reading the manuscript and for helpful criticism.