Effects of the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate on the wasp Bracon hebetor.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 3:529-538 (1986) Effects of the Tumor Promoter 12-0Tetradecanoy l-Phorbol-13-Acetate on the Wasp, Bracon hebetor R.G. Best and D.S. Grosch Department of Genetics, North Carolina State University, Raleigh, North Carolina A single oral dose of the tumor promoter, 12-O-tetradecanoyl-phorbol-13acetate (TPA), caused a rapid necrosis of the ovarioles, aberrations in the developmental sequence of oocytes, and a concomitant dose-dependent decline in egg production in the wasp, Bracon hebetor. TPA and its metabolities were found t o have a biological half life of 26.7 h, with a peak concentration in the ovarioles in 3 h. Damage to ovariole tissue was persistent despite the relatively short half life. Other tissues in the wasp were largely unaffected, although TPA induced lethargy that persisted until death. There was no shortening of life span. Inhibition of intercellular transport and metabolic cooperation may account for decreased fecundity and fertility, but interaction with a phorbol ester receptor i s more likely to account for developmental changes and central nervous system poisoning. Key words: anti-vitellogenic benzo(a)pyrene, braconid, habrobracon, TPA INTRODUCTION 12-0-Tetradecanoyl-phorbol-13-acetate has been widely studied in recent years because of its efficacy and potency as a tumor promotor in two-stage carcinogenesis studies with mice. In the mouse, the primary target for P A * is the phospholipid-sensitive, calcium-dependent protein kinase, kinase c [l]. *Abbreviations: BP = benzo(a)pyrene; ED, = the median effective dose that reduced egg production by 1/2; LSC = liquid scintillation counting; TPA = 12-O-Tetradecanoyl-phorbol-13acetate. Acknowledgments: The work presented here was supported by grant no. ES-07046 from the National institutes of Health. Paper No. 10019 of the Journal Series of the N.C. Agric. Res. Serv., Raleigh, N.C. Received October 15, 1985; accepted January28,1986. Address reprint requests to Dr. R.G. Best, Department of Genetics, Box 7614, North Carolina State University, Raleigh, North Carolina 27695-7614. 0 1986 Alan R. Liss, Inc. 530 Best and Crosch However, RNA, DNA, protein, phospholipid, polyamine synthesis, ornithine decarboxylase activity, and histone phosphorylation are increased markedly as well [2-51. Such a diverse range of pleiotropic effects confounded the identification of kinase c as the primary target for several years. In mammalian cell cultures, TPA causes an inhibition of intercellular communication and metabolic cooperation [6,7l, and has been shown to alter cellular differentiation both in vivo and in cell culture in chick , mouse , and human cells [lo].The similarity in response within a wide range of organisms suggests a common mode of action and possibly a common phorbol ester receptor molecule. Since protein kinase c is common to a variety of organism classes (including mammals, birds, amphibians, fish, molluscs, crustaceans, annelids, and insects [ll]),kinase c would be the most likely candidate. Reports of TPA effects in insects systems have been lacking, however. The purpose of this investigation was to study the effects of TPA in the ectoparasitic wasp, Brucon hebetor. This insect’s sequentially arranged oocytes and synchronized ovarioles make it well-suited for a study of the effects of TPA on the differentiation of the gametogenic cells of an adult insect. In addition, egg production and hatchability can be observed to provide information on genotoxicity. MATERIALS AND METHODS All experiments were conducted in B. hebetor (Say) (= Habrobracon juglandis (Ashmead) adult females. Stock #33L was selected for fecundity and egg viability. Eggs were collected daily and egg hatchability was determined by microscopic examination after 48 h incubation at 30°C in mineral oil. Benzo(a)pyrene and TPA were obtained from Sigma Chemical Company (St. Louis, MO). 20-[3H](N)-12-0-tetradecanoyl-phorbol-13-acetate with a specific activity of 250 pCilmmo1 was obtained from New England Nuclear (Boston, MA). Scintillation reagents were obtained from Fisher Scientific Co. (Raleigh, NC). Gamma radiation was produced by 6oC0. Biological half life was determined by feeding a 0.1% (wtlvol) [3H]-TPA meal to females deprived of food for 72 h and killing them at various times afterward. Retention of the labeled TPA was quantified by LSC following chemical digestion of the insect tissues. Wasps were killed and digested individually at 0, 3, 8, 12, 24, 48, 72, 96, and 124 h after feeding, using 16-17 wasps at each time point. Chemical treatments were administered by dissolving first in acetone, followed by suspension in 25% aqueous sucrose solution. Disposition of TPA in the wasp was determined by dissecting females fed 3H-labeled TPA and quantitating the label in various tissues by LSC. Groups of 5-6 wasps were dissected at 0, 1, 3, 8, 12, 24, 48, 72, and 96 h post treatment, and separated into four components: digestive tracts (intact), ovarioles, body contents, and excreta. The digestive tracts included the crop, foregut, Malpighian tubules, and hindgut. The body contents consisted of the head, exoskeleton, fat body, hemolymph, nerve chord, and poison gland. TPA Effects in Bracon 531 In addition, ovarioles were dissected from a series of TPA-treated females for microscopic examination of the oogenetic sequence of cells. Oocyte damage was quantitated by measuring daily egg production and hatchability over a 20-day span following treatment with TPA alone and in combination with BP or gamma radiation. To simplify the analysis, daily observations on each wasp were pooled into four 5-day periods. Conclusions on treatment interactions and comparisons between groups were based on F tests from an analysis of variance for each period for egg production and hatchability. RESULTS A plot of the log of the TPA (nglwasp) remaining in the animals against time after treatment with 3H-TPA yields a straight line of the form: ln(Y) = 6.197 - 0.0223 where Y is TPA (nglwasp) and t is the time in hours after consumption of a [3H]-TPAmeal (Fig. 1).The correlation coefficient is -0.968** with 5 degrees of freedom. From this line, the biological half life of TPA and its metabolites was determined to be 32.2 h in the adult wasp. The disposition of TPA in the wasp, determined by the movement of [3H]TPA after a single meal of labeled TPA, was studied for a period of 96 h. Within 1 h of treatment, ovarioles contained measurable amounts of TPA. Maximal levels were obtained in 3 h. TPA levels in the gut were maximal just after feeding and fell off with time. After 48 h, gut levels began to rise again and level off, possibly due to active removal of P A from the hemolymph by 6.0. 3.0 Ln b~i] (.o/l..p) 4.0 3.0 0 1; i4 I 41 72 96 124 HOURS AFTER TR€ATMENT Fig. 1. Retention of TPA in the wasp. The amount of TPA retained by the wasp following oral administration is plotted against time. Each point shown i s the mean of 16-17 observations. **Significant at the 99% level. 532 Best and Crosch the Malpighian tubules. TPA began to appear in the body (fat body, head, exoskeleton, nerve chord, hemolymph, and poison gland) 3 h after feeding. Absorption of TPA from the gut caused body levels to rise until 24 h, where they began to decline. TPA first appeared in the feces 24 h after ingestion (Fig. 2 ) . Dissections of treated females revealed a rapid necrosis of cells in the ovarioles after a single feeding of a 2.5 mglml TPA suspension (ca. 1pg TPAl wasp). This was also observable as an immediate decline in egg production that was persistent for the entire life of the animals. Even at lower doses, where a quantitative decline in egg production was not obvious, dissection and microscopic examination revealed damage and alterations in the developing oocytes. After 5 days, the normal arrangement of oocytes from least to most mature was disturbed. In treated animals, oocytes were observed that were less mature than expected from their position within the ovariole. Control animals did not show any deviation from the normal arrangement of oocytes. By day 12, most wasps had one or two of their four ovarioles completely degenerated while the remaining ones were fully functional (Fig. 3). The developmental aberrations, especially early on, were similar to those seen in avian and murine systems in that normal development did not proceed to terminal differentiation in all cells. Necrosis and other abnormalities were not observed in any of the tissues outside the ovarioles. The ingestion of a single dose of TPA ( 2 1.0 mg TPAlml sucrose solution) induced lethargy, which persisted until death, with no decrease in life-span. Egg production decreased in a dose-dependent manner (Fig. 4) following ingestion of a single meal containing 0.1, 0.5, 1.0, or 2.5 mg TPAlml. This response is described by the equation: o_p&p.<: ..... ................. ................. k +Z aiz 0 *). d8 0 3 0. . . . . O ds TlYE(hours) Fig. 2. Distribution of TPA in the wasp. Percentage of TPA in each tissue relative to the total TPA for all tissues after a single meal containing TPA. Gut contents included the crop, foregut, stomach, Malpighian tubules, and hindgut isolated intact. Body contents included fat body, poison gland, hemolymph, nerve chord, exoskeleton, and head. TPA Effects in Bracon 533 Fig. 3. Ovariole contents after a single meal containing TPA. Ovariole appearance at various times post-treatment at two doses of TPA. y = 4.12 - 2.34~ where y is the number of eggs laid per day per wasp during the first 5-day period of egg laying, and x is the In of the dose of TPA (mglml). This has a correlation coefficient of 0.54**with 473 degrees of freedom. The EDs0 with this endpoint is 0.55 mg TPAlml. There was some recovery from decreased egg production at lower doses (Table l),but higher doses caused an irreversible decrease in fecundity (Table 2). Pretreatment of wasps with 2,000 rads of gamma radiation or 1mglml of BP did not cause an enhanced fecundity 534 Best and Grosch 2.0- Ln Idore) (mew 1.0- 0- -1.0- -2.0- 0 I I I 2 4 b EGGS LAID (w-sp-l I I a 10 day-]) Fig. 4. TPA dose response curve. Points shown are mean values for each dose. The regression line is derived from the original data set with 473 degrees of freedom. TABLE 1. Egg Production and Hatchability Following BPTPA Treatment Combinations* Treatment group Period 1 Control BP P A Combination Period 2 Control BP TPA Combination Period 3 Control BP TPA Combination Period 4 Control BP TPA Combination Eggs f SE (wasp-' day-') Percent hatched f SE 10.93 f 0.42 9.42 f 0.40 6.31 f 0.40 5.77 f 0.37 93.4 94.0 79.9 78.8 f 0.8 f 0.8 f 1.7 f 1.8 12.56 f 0.58 9.73 f 0.48 9.67 f 0.48 10.38 f 0.45 92.8 94.9 91.4 94.2 f 0.8 f 0.7 f 1.0 f 0.7 15.72 f 0.56 14.93 f 0.60 13.63 f 0.54 15.13 f 0.58 87.9 f 0.9 90.0 f 0.8 89.4 f 0.9 89.4 f 0.8 19.25 f 0.69 19.92 f 0.61 17.30 f 0.60 18.72 f 0.60 88.8 88.2 87.9 86.0 f 0.7 f 0.7 f 0.8 k 0.9 *Treated groups are: control, TPA (0.5 mglml), BP (1.0 mglml benzo(a)pyrene), and combination (1.0 mglml BP followed by 0.5 mglml F A ) . Mean values for eggs laid per wasp per day f SE and percent hatched f SE for each treatment group in each group in each period. Daily observations were combined into 5-day periods to simplify analysis. TPA Effects in Bracon 535 TABLE 2. Egg Production and Hatchability Following TPAGamma Radiation Treatment Combinations* Treatment group Period 1 Control Gamma TPA Combination Period 2 Control Gamma P A Combination Period 3 Control Gamma TPA Combination Period 4 Control Gamma TPA Combination Eggs f SE (wasp-' day-') 8.19 9.10 3.18 4.04 f 0.42 Percent hatched f SE f 0.41 f 0.43 96.02 f 0.7 86.45 1.2 84.27 f 2.2 81.31 f 2.0 10.77 10.71 4.90 6.02 f 0.44 f 0.53 f 0.53 f 0.64 93.70 84.85 95.46 83.39 f 0.8 f 1.2 f 1.0 f 1.6 16.50 16.89 6.92 9.29 f 0.54 f 0.48 f 0.73 f 0.88 88.75 85.92 90.53 84.81 f 0.8 f 0.9 f 1.2 f 1.2 17.35 18.22 7.90 9.61 f 0.65 f 0.55 f 0.86 f 0.99 83.03 83.23 86.08 83.45 f 1.0 f 0.9 f 1.3 f 1.2 k 0.39 + *Treatment groups are: control, TPA (1.0 mglml), gamma (2,000 rads of gamma radiation), and combination (2,000 rads followed by 1.0 m g / d P A ) . Mean values for eggs laid per day per wasp f SE and percent hatched f SE for each treatment group in each period. Daily observations were combined into 5-day periods to simplify analysis. decline when followed with TPA treatment. Figure 5 compares the daily egg production patterns of treated (2.5 mg TPAlml) and untreated wasps over the first 10 days of egg laying. Whereas egg production climbed sharply in the first 3 days of egg laying from 6.1 to 13.9 eggs per wasp in control animals, TPA-treated animals started much lower (0.8 eggs per wasp) and climbed gradually to a high of only 4.2 eggs per wasp on day 6. Recovery of treated animals to control values was not achieved during the life of the animals. Embryo viability was decreased in wasps treated with TPA at doses of 0.5 (Table 1)and 1.0 (Table 2) mg TPAlml. Unlike egg production, however, egg hatchability returned to normal after the first period of egg laying (days 1-5). Neither BP (Table 1)nor gamma radiation (Table 2) interacted synergistically with TPA when wasps were pretreated. DISCUSSION TPA alone caused a decrease in egg hatchability during the first period of egg laying at 0.5 mglml (Table l), and during the first and second periods at 1.0 mglml (Table 2), but this was not synergistic to the decrease in hatchabil- Best and Grosch 536 7 16- . . .-.. ........ ....* 14- .................... -.=--.-. ..... --.._ . ..... . ...:. 1% ....... CONTROL 10- # EWS 8- .... /.-............... ... 6- *.............. .................... 4- ..... ......... .............. ... .*. ........ ...Y 1- .-*A.. *..- Y.. II.. 07 1 1 4 3 mn 4 4 UTER i 6 6 7 7 a 9 9 I RIATMENT Fig. 5. Daily egg production in treated and untreated females. Mean numbers of eggs laid per wasp in control animals (0-0) and animals treated with 2.5 mg/ml TPA (A-A) during the first 10 days of egg laying following a single feeding. ity induced by pretreatment with gamma radiation (Table 2). Likewise, pretreatment with 1mglml BP had no effect on PA-induced egg hatchability. The complete lack of synergy between TPA and two tumor initiators (BP and gamma radiation) confirms in the wasp what has been known in the mouse: that TPA does not act primarily through alterations in DNA repair or maturation of premutational lesions, as was once suspected. If this were the case, TPA would be expected to synergistically interact with both BP and gamma radiation to decrease fecundity and egg viability. TFA ' does appear to be weakly genotoxic by itself, however. The presence and function of kinase c has not yet been demonstrated in the wasp; however, work in the cockroach has shown that this enzyme is found in insects [ll]. The localization of kinase c in the nervous system of the cockroach, and the observation of TPA preferential binding to the brain tissue in mice [l], may be related to the observed nervous system depression in the wasp. Since no role has been established for kinase c in the wasp as yet, it is not known what if any of the observed changes in oocyte development are attributable to the enzyme. In the adult female wasp, cell proliferation and differentiation is restricted to the ovarioles. All other cells in the adult wasp are fully differentiated at the time of eclosure. TPA caused aberrations in the differentiation of oocytes, and necrosis of oocytes and nurse cells within the ovarioles, while fully differentiated cells remained healthy, and life span was not reduced. Egg production and hatchability were both markedly reduced immediately following treatment with TPA. A rapid decline in egg production and concomitant necrosis of the ovarioles is a typical response to exposure of the female TPA Effects in Bracon 537 wasp to anti-vitellogenic agents that may interfere with yolk production and/ or deposition during vitellogenesis by interfering with the production of RNA , amino acids , or proteins , as postulated in previous studies. Typically, however, anti-vitellogenic agents caused a nadir in egg production on day 3. TPA more closely resembles fluoroacetate in that it exerts its strongest effect on day 1. Fluoroacetate blocks egg production by inhibiting the conversion of citrate to isocitrate . TPA differs from all of these compounds in that egg production never fully recovers after treatment. Egg production is an involved process in which a complex of interrelated metabolic pathways and cellular events converge, and different modes of action may produce the same end result. In the wasp, maturation of oocytes is dependent on transport of yolk proteins, mitochondria, lipids, endoplasmic reticulum, and ribosomes from the follicular epithelial and nurse cells to the oocytes [lq.A block in transport from these cells to the oocytes would result in decreaseed egg production and viability. The extent to which this actually occurs in the wasp following treatment with TPA has not been determined. Because of the wide range of biochemical and cellular changes that follow TPA treatment in this and other organisms, it is likely that the observed changes in gametogenic cells in the wasp following TPA treatment are not the result of a single metabolic change, but a combination of several changes. Inhibition of metabolic cooperation between cells may account for decreased egg production and viability, but changes in cellular differentiation and the central nervous system poisoning are more likely the result of TPA binding to protein kinase c. LITERATURE CITED 1. Ashendel CH, Staller JM, Boutwell RK: Protein kinase activity associated with a phorbol ester receptor purified from mouse brain. Cancer Res 43, 33 (1983). 2. Baud WM, Sedgwick JA, Boutwell RK: Effect of phorbol and four diesters of phorbol on the incorporation of tritiated precursors into DNA, RNA, and protein in mouse epidermis. Cancer Res 32, 1434 (1971). 3. Rohrschneider LR, O’Brien DH, Boutwell RK: The stimulation of phospholipid metabolism in mouse skin following phorbol ester treatment. Biochim Biophys Acta 280, 57 (1972). 4. O’Brien TG, Simsiman RC, Boutwell RK: Introduction of the polyamine biosynthetic enzymes in mouse epidermis by tumor-promoting agents. Cancer Res 35, 1662 (1975). 5. Raineri R, Simsiman RC, Boutwell RK: Stimulation of the phosphorylation of mouse epidermal histone by tumor-promoting agents. Cancer Res 33, 134 (1973). 6. Yotti LP, Chang CC, Trosko JE: Elimination of metabolic cooperation in Chinese hamster cells by a tumor promoter. Science 206, 1089 (1979). 7. Murray AW, Fitzgerald DJ: Tumor promoters inhibit metabolic cooperation in cocultures of epidermal and 3T3 cells. Biochem Biophys Res Commun 91, 395 (1979). 8. Holtzer H, Pacifici M, Payette R, Croop J, Dlugosa A, Toyama Y: TPA reversibly blocks the differentiation of chick myogenic, chondragenic, and melanogenic cells. In: Carcinogenesis, A Comprehensive Survey. Hecker E, Fusenig NE, Kunz W, Marks F, Theilman HW, eds. Raven Press, New York, Vol. 7, pp 347 (1982). 9. Giovanni R, O’Brien TG, Diamond L: Tumor promoters inhibit spontaneous differentiation of Friend erythroleukemia cells in culture. Proc Natl Acad Sci USA 74, 2894 (1977). 10. Lotem J, Sachs L: Regulation of normal differentiation in mouse and human myeloid leukemia cells by phorbol esters and the mechanism of tumor promotion. Proc Natl Acad Sci USA 76, 5158 (1979). 538 Best and Grosch 11. Kuo JF, Anderson RGG, Wise BC, Mackerlova L, Salomonsson I, Brackett N, Katoh N, Shoji M, Wrenn RW: Calcium-dependent protein kinase: Widespread occurrence in various tissues and phyla of the animal kingdom and comparison of effects of phospholipid, calmodulin, and trifluoroperazine. Proc Natl Acad Sci USA 77, 7039 (1980). 12. Wissinger WL, Grosch DS: Influence of juvenile hormone analogues on reproductive peformance in the wasp, Habrobrucon juglandis. J Insect Physiol22, 1559 (1975). 13. Mirsalis JC, Grosch DS: The effects of three folic acid antagonists on reproduction of Habrobrucon juglandis. Ann Entomol SOC Am 72, 559 (1978). 14. Kratsas RG, Grosch DS: Contrasts in cell type sensitivity to alanosine demonstrated by altered patterns of Bracon oviposition, hatchability and egg morphology. J Econ Entomol 67, 577 (1974). 15. Smith GJ, Grosch DS: Fluoroacetate-induced changes in the fecundity and fertility of Bracon hebetor females. J Econ Entomol69, 521 (1976). 16. Peters RA:Mechanism of the toxicity of the active constituent of Dichapetalum cymosum and related compounds. Adv Enzymol 28, 113(1957). 17. Cassidy JD, King RC: Ovarian development in the wasp, Habrobrucon juglandis (Ashmead) (Hymenoptera: Braconidae) I. The origin and differentiation of the oocyte-nurse cell complex. Biol Bull 243, 483 (1972).