Enhanced protein synthesis in Triatoma infestans treated with DDT

EXPERIMENTAL

PX+.SITOLOGY

(1963)

16, 3 18-324

Enhanced

Protein

Synthesis

Treated Moises Chair

of

Chemistry

Agosin,

Luisa

in

with

Triatoma

DDT’,

Aravena,

and

2 Amador

and Department of Parasitology, Biochemistry Medicine, University of Chile, Santiago, Chile (Submitted

for

Publication,

4 February

infestans

Neghme Section,

School

of

1964)

AGOSIN, MOISES, hAVENA, LUISA, AND NEGK~CE, AMADOR. 1965. Enhanced protein synthesis in Triatonza injestans treated with DDT. Experimental Parasitology 16, 318-324. The effect of DDT and DDE, a DDT-non toxic analog, on protein biosynthesis in Triatoma injestans nymphs and adult specimens has been studied. DDT increases the in viz10 rate of incorporation of nn-leucine-l-C14 into total proteins, while a slight inhibitory effect is observed in adult males. DDE does not appreciably change the rate of protein biosynthesis in nymphs, although it appears to be inhibitory in males. lYympha1 microsomes and sarcosomes show the highest specific activity after nr-leucine-1-C’” injection. This activity is markedly increased by DDT, while DDE again is inhibitory. A cell-free system obtained from nymphal specimens incorporates m-leucine-l-Cl4 into pro.teins in the presence of ATP, an ATP-generating system, GTP, and magnesium ions, In vivo pretreatment with DDT at low concentrations increases the rate of DLleucine-l-C14 incorporation into protein by this system. In vitro added DDT also is stimulatory at very low concentrations, while higher ones are inhibitory. The results obtained indicate that protein biosynthesis is stimulated by DDT, but not DDE; an effect that is only, evident in nymphal T. infestam. This appears to be consistent with the inductive effect of DDT previously shown with N.4D-kinase in this organism (Ilevicky et al., 1964).

Work from this laboratory has shown that DDT and several analogs,but not any known DDT-metabolic products, increase the in viva levels of KAD-kinase in Triatoma infestans (Ilevicky et al., 1964). This effect was inter1 Abbreviations: DDT, Z,Z-bis-(p-chlorophenyl)l,l,l-trichloroethane; DDE, 2,2-bis-(p-chlorophenyl) -l,l-dichloroethylene; ATP, adenosine-S’-triphosphate ; WAD, nicotinamide adenine dinucleotide ; Tris buffer, tris (hydroxymethyl) aminomethane PEP, buffer; GTP, guanosine-S’-triphosphate; phosphoenol pyruvic acid; P.K, pyruvic kinase; CTP, cytidine-5’-triphosphate; ITP inosine-5’-triphosphate; UTP, uridine-5’-triphosphate; TCA, trichloroacetic acid; AMP, adenosine-S’-monophosphate. 2 Supported by grant AI-02300 Research Grants and Fellowships, Service.

of the Division of US. Public Health 318

preted as an enzyme induction rather than merely activation. However, to be consistent with this interpretation, those compoundsthat stimulate the activity of Xl-.%D-kinaseshould also stimulate protein synthesis ilz ZJ~ZV.The latter would also be reflected in the rate of incorporation of C”-amino acids into TCA insoluble proteins by microsomal fractions derived from T. infestans, under conditions similar to those described by Zamecnik and Keller (19.54). Furthermore, if the effect of DDT on protein synthesis were related to DDT-resistance, it should be expected that protein synthesis would be increased only in nymphal T. infestam, which are very tolerant to DDT, and not in adult specimens.which are very susceptible (Agosin et al., 1961). This paper reports the in viva effect of DDT

PROTEIN SYNTHESIS IN Triatoma infestans

-

3

!

A A h =NORMAL CH :DOT

NYMPHS

6

=DDE

16

MALES

3

6

FIG. 1. Effect of DDT and DDE in viva on nL-leucine-l-C14 mcorporation and males of Triatoma infestans. The values correspond to 3 determinations k 20 c.p.m.).

METHODS

Third instar larvae (nymphs) and adult males were grown as previously described (Agosin et al., 1961). For studying the in vivo incorporation of Cr4-amino acids into total protein, groups of 5 nymphs (about 0.5 gm fresh weight) and 3 males (0.6 gm fresh weight) were treated with acetone solutions of DDT or DDE according to Agosin et al. (1963) at the dose of 300 pg per specimen. Groups treated only with acetone were used as controls. Simultaneously with the DDT or DDE application, 3.0 ul of an aqueous solution of

I6

HOURS

HOURS

and DDE, a DDT-non toxic analog, on the rate of incorporation of C14-leucineinto total protein, subcellular fractions, and on the activity of a cell-free system, as well as the in vitro effect of DDT on the latter system. In addition, some characteristic of protein synthesis in a cell-free system of T. infestans are briefly reported.

319

into total (standard

protein of nymphs error of the mean

nL-leucine-l-C14 were injected by meansof a microsyringe into the abdominal region of the insects, at the dose of 100 ng per gm fresh weight (600,000 c.p.m./gm fresh weight). At timed intervals, shown in Fig. 1, the insects were homogenizedin 10 volumes of 10% TCA in a Virtis homogenizer, and proteins obtained as described elsewhere (Agosin et al., 1963). For following the incorporation of radioactivity into subcellular fractions, groups of 10 insects were used. In this case, 5.0 ul of nn-leucine-l-Cl4 per specimen were injected at a dose of 165 ug per gram fresh weight (1 X 10m6 c.p.m.,/gm fresh weight). At timed intervals, shown in Fig. 2, the insects were homogenized in 20 ml 0.25 M sucrose per group in a Virtis homogenizer. The homogenates were then centrifuged at 600 X g X 10 minutes at 0-2°C. The pellet thus obtained, corresponding to the nuclei plus cellular debris, was washed twice with 2 ml 0.25 M sucrose and resuspended in 2.0 ml of the same medium. The supernatant fluid plus

320

AGOSIN,

DDT-TREATED

ARAVENA,

AND

NEGHME

NYMPHS 1

DDE

FIG. 2. Effect of DDT and DDE in V~JO on or,-leucine-l-Cl-’ fractions of nymphs of Triatoma infestans. The values correspond the mean -C 25 c.p.m.).

washings was centrifuged at 3,000 X g X 10 minutes. The precipitate, corresponding to the sarcosomes,was washed as the nuclear fraction and resuspended in 1.0 ml 0.25 M sucrose.The remaining supernatant fluid plus washings was centrifuged at 105,000 X g X 60 minutes in a Spinco centrifuge (Model L, rotor #30). The pellet obtained, which was taken to correspond to microsomes,was suspended in 1.0 ml 0.25 M sucrose.The remaining supernatant fluid corresponded to the

- Treated

nymphs

incorporation into protein of subcellular to 18 determinations (standard error of

soluble fraction. Proteins ‘were obtained from each fraction by adding an equal volume of 10% TCA following purification as previously described (Agosin et al., 1963). In vitro experiments were conducted with a cell-free system patterned after Zamecnik and Keller (1954). Homogenates were prepared from groups of 25 specimens each in a medium consisting of 0.05 M Tris buffer, pH 7.6, 0.154 M KCl, and 0.004 M MgC12. Microsomes were obtained by differential

PROTEIN

SYNTHESIS

centrifugation as indicated above. The microsomal pellet was homogenized with a glass rod and resuspended in the same homogenization medium in a volume such as to give a final protein concentration of 8 mg per milliliter. The supernatant fluids remaining after the 105,000 X g X 60 minutes centrifugation were used to prepare the t’pH 5 fraction” by adjusting the pH to 5.0 (glass electrode) with N acetic acid at 0-1°C. The precipitates thus obtained were centrifuged at 14,000 X g X 10 minutes at 0-1°C and resuspended in the same homogenizing medium to give a final protein concentration of 8 mg per milliliter. For purposes other than measuring radioactivity, protein was determined by a microKjeldahl procedure with 6.25 as calculation factor. In all other cases, protein was determined gravimetrically. The potassium salts of ATP, PEP and GTP (Sigma Chemical Co.) were used throughout In some cases, the tricyclohexylamine salt of PEP (Sigma Chemical Co.) was also used. P.K. was obtained from Sigma Chemical Co., and nL-leucine-l-C14 from the Radiochemical Centre, Ammersham, England. The rest of the reagents were of the highest purity commercially available. Measurement of the radioactivity was done as previously described (Agosin et al., 1963). All data were corrected for background and self-absorption by conventional procedures. All results given correspond to the average of 3 to 18 separate experiments. RESULTS

In vivo effect of DDT and DDE on amino acid incorporation into total proteins. DDT significantly increased the incorporation of DL-leucine- 1-Cl4 in z&o into total protein in nymphal T. infestam, while in males it proved to be slightly inhibitory. On the other hand, DDE did not appreciably change the specific activity of protein in nymphs, as compared to the control insects. However, although in males some increase in the amount of radioactivity incorporated was evident after 3

IN

Triatoma injestans

321

hours of DDE treatment; it was markedly inhibitory after 18 hours (Fig. 1). In vivo efiect of DDT and DDE on DLleucine-l-P4 incorporation into protein of subcellular fractions. Since no stimulatory effect of DDT on the incorporation of radioactivity into total protein in males was observed, this and the rest of the experiments were conducted only with nymphal specimens, where a stimulatory effect was clearly evident (Fig. 1) . It is well known that after in viva injection of a labeled amino acid, the microsomal fraction of the liver has a higher specific activity than any other fraction in mammalians (Borsook et al., 1950). Apparently, the same happens in insect tissues. Thus, after 6 hours of nL-leucine-l-Cl4 injection, and more markedly after 18 hours, the microsomal fraction isolated from whole nymphs was the one with the highest specific activity (Fig. 2). Sarcosomes had also a fairly high specific activity, but contrary to what happened with the microsomal fraction, its specific activity tended to decrease 6 hours after the amino acid injection. The nuclear and soluble fractions had the lowest specific activity. DDT treatment markedly increased the incorporation of nL-leucine-l-C14 into microsomes and sarcosomes, the effect being evident after 3 hours of intoxication. On the other hand, DDE somewhat inhibited the rate of incorporation of the labeled amino acid into subcellular fractions. This was very appreciable in the microsomal fraction, which had a specific activity lower than the sarcosomes. Effect of DDT and DDE in vivo pretreatment on in vitro protein synthesis. Before attempting to study protein synthesis in a cellfree system derived from normal and DDT or DDE intoxicated insects, preliminary experiments were conducted to determine optimal conditions for the system. The best homogenizing medium was the one described under “Methods,” composed of 0.004 M MgCln, 0.154 A4 KCl, and 0.05 M Tris buffer, pH 7.6. Addition of sucrose to the medium in concentrations varying from 0.25 to 0.44 M did

322

AGOSIN,

ARAVENA.

not modify the Cl4 incorporation into proteins. Phenylthiourea, at the concentration of 1 X 10T3 M, which has been shown necessary for demonstrating amino acid incorporation in a cell-free system of Drosophila (Jenny et al., 1962), was not stimulatory, but rather inhibitory. Maximal incorporation was obtained when the “pH 5 fraction” was precipitated at pH 5.0 with iv acetic acid. When this was done at pH 4.7, only about 50% of the incorporation was obtained. Optimal pH for the incubation system was found to be 7.6 with Tris buffer. Table I shows some factors affectFactors

TABLE I dfecting Incorporation of m-Leucine-I-Cl4 into the Protein of LVymph T. infestans Cell-Free System

Conditions Complete -ATP -GTP -PEP -Mg++ -“pH 5 -microsomes

system

P.K (a) fraction”

Average specific activity c.p.m./mg protein -C S.E. 34 2 5 k 17 5 13 k 13 k Sk 28 -c

1.0 0.5 4.0 4.0 2.0 2.0 1.0

Complete system, in kmoles: ATP, 10; Tris buffer, pH 7.6, 50; MgCI,, 9 (calculated) ; GTP, 2.5; PEP, 10; P.K., 5 fig; m-leucine-I-Cl’, 1.0 (specific activ1.2 X 10” c.p.m./pmole) ; Microsomes, 4 mg ity, protein; “pH fraction,” 4 mg protein. Final volume, 1.55 ml. Incubation, 60 minutes at 37°C. (a) Microsomes and “pH 5 fraction” prepared in the absence of Mg+ f The figures are averages of 10 determinations each.

ing the incorporation of nL-leucine-l-C1l into protein by the T. in~cstans cell-free system under optimal conditions. The incorporation was dependent on ATP and on a ATP-generating system, as well as magnesium ions. Contrary to what has been reported for Drosophila (Jenny et al., 1962), GTP was also required. It should be added that, according to data not shown in Table I, addition of reduced glutathione, at the final concentration of 1 ,y 1O-a &f to the optimal system, proved to be inhibitory; while P-mercaptoethanol, at the

AND

NEGHME

same final concentration, was without effect. The addition of a mixture of 19 natural unlabeled L-amino acids (1 pmole each) was also without noticeable effect. ATP could not be replaced by CTP, ITP, or UTP. Best results were obtained when microsomes and the “pH 5 fraction” were added in a 1: 1 protein ratio. The omission of microsomes in the system of Table I resulted in a decrease in the rate of isotope incorporation as compared to the complete system. Although this decrease was rather small, it was consistently obtained. Once the requirements for m-leucine-1-C” incorporation into protein by the cell-free system were defined, nymphs were intoxicated with 300 pg DDT or DDE per specimen: and after 3 hours, the extent of incorporation of DL-leucine- 1-C 1-1by the cell-free system was determined with the complete system of Table 1. Surprisingly enough, no effect of DDT was observed, while DDE was slightly inhibitory. Since a problem of insecticide concentrations might be involved in these results, insects were intoxicated with DDT or DDE at various doses. Figure 3 shows that the cell-free system derived from nymphs previously treated with varying doses of DDT is more active than the system derived from control insects at the range of 25 to 50 pg DDT. On the other hand, DDE was again inhibitory, including at lower concentrations where its effect was even more evident. DDT effect in vitro. The experiments described in Fig. 3 indicated that DDT increases the nL-leucine-l-C14 incorporation into protein by the cell-free system, but only at lower doses than those previously shown to stimulate in viz)0 protein synthesis (Figs. 1 and 2). The possibility that DDT in vitro at low concentrations could also stimulate protein synthesis was tested by adding various DDT concentrations in ethylenglycol to the cell-free system. The results shown in Table I1 indicate that a small increase in specific activity is obtained with 2.2 1-18DDT, but that higher concentrations are frankly inhibitory.

PROTEIN

SYNTHESIS

IN

Triatoma infestans

323

insecticide in T. infestans. Increased amino acid incorporation into protein is not itself proof of a stimulation of net or protein synthesis de nova, but it agrees quite well with -.DDT the increased activity of NAD-kinase in -= DDE nymphs of T. infestans, where protein synthesis very possibly is involved (Ilevicky et al., 1964). The in vitro effect of DDT suggests a close relationship to the direct action of the insecticide rather to simply a result of the modification of some normal regulatory mechanism. It is interesting to note that DDE, a non-toxic DDT-analog and a product of DDT-metabolism by T. infestans (Dinamarca et aZ., 1962), does not share the DDT effects. On the contrary, it would appear as fairly inhibitory (Figs. 2 and 3). This suggestsa rather delicate enzymic control mechanism. The stimulatory effect of DDT on protein synthesis would be regu)Lg INSECTICIDE lated by the enzymic product of DDT-deFIG. 3. Effect of DDT and DDE in viva prehydrochlorinase (Lipke and Kearns, 1959). treatment on amino acid incorporation by the cellIt should be added that attempts to demonfree system of nymphs of Triatomu infestans. The strate induction of the latter enzyme have values correspond to 3 experiments (standard error been so far unsuccessful (Ilevicky et al., of the mean & 25%). 1964). A puzzling observation is the increased TABLE II amino acid incorporation into protein in a The in Vitro Effect of DDT on the Zncorpovation cell-free system by pretreatment with DDT of m-Leucine-I-C14 in Nymphal in rather low concentrations, while higher T. infestans Cell-Free System concentrations are without apparent effect Specific activity, (Fig. 3). Obviously, it is difficult to extrapoc.p.m./mg protein late the results obtained in vitro to the whole DDT (percentage of insect. It is possible that in order to show (UK) the controls) DDT stimulation of protein synthesis in the 2.2 120 cell-free system, different in vitro conditions 4.4 99 are required, especially with higher doses. 8.8 88 This possibility seemsto be supported by the 17.7 54 fact that DDT activates the cell-free in vitro Conditions as for the complete system of Table I. DDT added in ethylenglycol. The figures for specific system only at lower concentrations, where activity represent averages of 3 determinations each. optimal conditions of the system would not The standard error of the mean was & 5. be too different, while higher DDT concentrations are frankly inhibitory (Table II). DISCUSSION Nymphs of T. infestans are very tolerant The finding that DDT stimulates amino to DDT, while males are extremely suscepacid incorporation into protein in vivo as well tible (Agosin et al., 1961). In this connection, as irt vitro is further evidence of a special it is interesting to note that the stimulation pattern of enzyme activity induced by the of protein synthesis by DDT occurs only in

AGOSIN,

324

ARAVENA,

nymphs. This suggests a close relationship between this phenomenon and resistance. The results in Fig. 2 suggest that the increased protein synthesis observed in the sarcosomal fraction has some relationship to the effect of DDT on the microsomal system, as it seems to be the case for the thyroxine stimulation of protein synthesis (Sokoloff et al., 1963). Experiments are now being conducted to determine at what step of the pathway leading to the incorporation of amino acid into microsomal protein is the DDT effect localized. REFERENCES AGOSIN, M., SCARAMELLI, N., AND NEGHME, A. 1961. Intermediary carbohydrate metabolism of Triatoma infestans (Insecta; Hemiptera). I. Glycolytic and pentose phosphate pathway enzymes and the effect of DDT. Comparative Biochemistry and Physiology 2, 143-159. AGOSIN, M., SCARAMELLI, N., DINAMARCA, M. L., AND ARAVENA, L. 1963. Intermediary carbohydrate metabolism in Triatoma infestans (Insecta ; Hemiptera). II. The metabolism of C14-glucose in Triatoma infestans nymphs and the effect of DDT. Comparative Biochemistry and Physiology 8, 311-320. BORSOOK, H., DEASY, C. L., HAAGEN-SMIT, A. J.,

AND

NEGHME

KEIGHLEY, G., AND LOWY, P. H. 1950. Metabolism of C14-labeled lycine, L-histidine, L-leucine and t-lysine. The Journal of Biological Chemistry 187, 839-848. DINAMARCA, M. L., AGOSIN, M., AND NEGHME, A. 1962. The metabolic fate of C14-DDT in Triutoma infestans. Experimental Parasitology 12, 61-72. ILEVICKY, J., DINAMARCA, M. L., AND AGOSIN, M. 1964. Activity of NAD-kinase of nymph Triatoma infestans upon treatment with DDT and other compounds. Comparative Biochemistry and Physiology 11, 291-301. JENNY, E., HICKLIN, A., AND LEUTHARD, F. 1962. In vitro-Einbau radioaktiver amino iuren in die proteine von Drosophila-puppen. Helvetica Chimica Acta. 45, 2014-2020. LIPKE, H., AND KEARNS, C. W. 1959. DDT dehydrochlorinase. I. Isolation, chemical properties, and spectrophotometric assay. The Journal of Biological Chemistry 234, 2123-2128. SOKOLOFF, L., KAUFMAN, S., CAMPBELL, P. L., FRANCIS, C. M., AND GELBOIM, H. V. 1963. Thyroxine stimulation of amino acid incorporation into protein. The Journal of Biological Chemistry 288, 1432-1437. ZAMECNIK, P. C., AND KELLER, E. B. 1954. Relation between phosphate energy donors and incorporation of labeled amino acids into proteins. The Journal of Biological Chemistry 209, 337-354.