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Ultrastructural changes induced by low concentrations of DDT in the livers of the zebra fish and the guppy
Chem.-Biol. Interactions, 8 (1974) 25-30 0 Elsevier Scientific Publishing Company, Amsterdam-Printed
in The Netherlands
25
ULTRASTRUCTURAL CHANGES INDUCED BY LOW CONCENTRATIONS OF DDT IN THE LIVERS OF THE ZEBRAFISH AND THE GUPPY
PEDDRICK
WEB
Department of Anatomy, New Jersey Medical School, Newark, N,J. 07103 (U.S.A.) (Received March 16th, 1973) (Revision received April 30th, 1973) (Accepted August 6th. 1973)
SUMMARY
Following exposure to 0.3-3.0 ppb DDT, the livers of zebrafish and guppies were examined by electron microscopy. After 24 h in 1.0 ppb or 72 h in 0.3 ppb, zebrafish had a complete loss of glycogen, decrease in cell size and a cytoplasmic compartment filled with rough endoplasmic reticulum (ER). Guppies exposed for periods of up to 28 days showed only a gradual accumulation of smooth ER and no change in glycogen, lipid, rough ER or cell size. The relationship of these changes to the zebra&h’s sensitivity and the guppy’s tolerance to DDT is dicwssed.
INTRODUCTION
DDT induces a proliferation of the smooth ER in the liver of the rat’ and stimulates the drug-induced microsomal enzyme system which is associated with the smooth ERZ. In preparation for similar enzyme studies in teleost fishes, ultrastructural analysis was performed on the livers of the zebrafish (Bruc&&rk” rerio) and the guppy (Poecilia retidura) following exposure to DDT in water. The concentrations of DDT in water to which the fish were exposed were substantially less than the maximal amount found to occur in the field (up to 12 ppb in areas of DDT application3). MATERIALS AND METHODS
The fishes used in this experiment were maintained in Abbreviations: DDT. reticulum; ppm. ppb.
water. without mixing of species mlder conditions described previously”, at a maximum of I g of fish per I. Technical grade DDT (72f;, /),JI’-DDT, courtesy of Montrose Chemical Corp., Torrance, Cpiif,) was recrystallized from ethanol to yield > 987f, p,$-DDT as estimated by disappearance of minor spots on thin-layer chromatograms. Acetone solutions of DDT were added to the water to provide concentrations of 0.3, 1.0 and 3.0 ppb DDT. Acetone (0.05 ml/i) was added to control jars. The ja,*s were washed and fresh solutions prepared weekly. Additional fish were selected from the stock tanks for comparison. After I to 28 days of exposure, fish were sacrificed and liver fragments fixed in pi~ospl~ate-bll~ered gl~~taraldeilyde follo~~ed by post-fixation in 0~0,. Ethanoidehydrated, Epon-embedded tissues were sectioned, stained with uranyi acetate and lead citrate, and examined with Philips EM-100 and EM-300 electron microscopes. Cell size was measured on low-power n~icrograpi~s taken after ~lib~tioI1 of the microscope with a diffraction-grating replica. in parallel experiments, some fish of each species were exposed to [3H]DDT (0.145 mCi/mg, New England Nuclear Corp., Boston, Mass.) at a concentration of 1 ppb, using 0.4 g of frsit per 1. Livers from the zebrafish were esch divided into two parts, the smaiie’rof which were prepared for microscopy and the larger of which were pooled and homogenized in a glass tissue grinder and. the hexane-soluble portion an~~iyzedby liquid ~i~ltiilation (5eckn~~l~ LS-21). The guppies exposed to [“H]DDT were homogenized i/t toto and counted also, after their livers had been excised, pooled and separately prepared for scintillation counting. For r~~dioa~ltograpi~ic~~ll~~iysis, 1-1’ sections of liver from both species were placed on glass slides, coated with liford K-5 emulsion (ilford, Ltd., Iiford, England), exposed at 4” for 3--28 days, developed in Kodak Microdol-X and stained with toiuidine blue. A total of 40 zebrafish and 40 guppies were included in this study. Only male guppies were used since female guppies show cyclic changes in their livers associated with their reproductive cyciess.
RESULTS
The zebrafish was found to be quite sensitive to DDT. A concentration of 3 ppb was toxic to most zebrafish within 24 h. I ppb allowed survival for as long as a week and 0.3 ppb for as long as 16 days. The proliferation of smooth ER predicted by mammalian studies failed to materialize. Instead, there was rapid disappearance of giycogen from the hepatocytes. This giycogenoiysis was virtually compiete by 24 h at one ppb or by 72 h at 0.3 ppb. When the giycogen had disappeared, the cytoplasm of the hepatocytes contained closely packed rough ER as its principal component (Figs. I and 2). The occasional lipid droplets found in the hepatocytes had also disappeared. The size of the hepa-
Fig. I. An acetone-control zebrafish ~~~~~O~ytE. showing the glyco~~ granules fCly) which fill most of the cytoplasmic com~rtment. along with snrne rough eudoplasn~ic reticulum (RERI. An erythrocytc (Ety) is seen within a sinusoid. t 19 700 (bar 1 ~6). Fig. 2. Zebrafish hcpatocyt~ after 24 h in I.0 ppb DDT. The cells are reduced in sire and RER occupies most of the cytopiasmic c~lmpartrnct~t. N. nucleus. If) 700.
Fig. 3. Acetone-control guppy hepatocyles, showing the glycogen granules and lipid droplets (L) which fill most of the cyloplasmic compartment, along with some RER. I’ 19 700. Fig. 4. A guppy hcp~tocyte after ?I) days in I .Oppb DDT. Smooth endopl~smic reticulum (SER) is i~dic~tcd by arrows. .I 19 700. Inset: a dctuil of a cell in which the glyeogcn was poorly stained, allowing easy visualization of ribosomes in the endoplasmie reticulum. SER is seen interiningled with the RER. 38 600.
EFFECT OF
DDT
ON FISH LIVER
29
tocytes was reduced from a profile of 28.3 If 2.0” ,Mby 21 .O f 2.0* ,I” to one of 16.0 i 1.1* ,U by 10.4 5 0.8” if (d.f. = 38, t = 4.93 and 4.95 for width and length respectively, P -=c0.001 by r-test for independent observations). This reduction in cell size correlates with a decrease in gross size observed during excision. There were no changes observed in other organelles or in the bile canaliculi.
The guppy, which has been reported to have a 1Cday LD5, of I8 ppb6, had no grossly observable changes in the early days of exposure and was found to be suitable for long-term experiments. Observations after 7 to 28 days of exposure to all concentrations of DDT used in these experiments indicate that the guppy has no loss of either glycogen or lipid as was seen in the zebrafish, and that the only noticeable change was a gradual accumulation of a small amount of rough and smooth ER in the area of glycogen storage (Figs. 3 and 4). The amount of smooth ER which was present would appear to be both dose- and time-dependent. The size of the hepatocytes, 16.6 5 0.7 ,I( by 9.6 + 0.9 ,U in profile, was not altered by the treatments.
Following the 24-h exposure to I ppb [3H]DDT, the 8 pooled zebrafish livers contained 0.50 ppm DDT and the residual amount in the water was 0.098 ppb or 9.8 p,{of the original amount. 4 guppies exposed to I ppb [3H]DDT for I day COIIremaining in the water: this represented tained 0.59 ppm i/l tn~o with 0.19 ppb or I9 ‘,B,, 6.5 ppm in the pooled livers and 0.36 ppm in the remainder of the body. After I week, 8 guppies contained 0.76 ppm: this represented 6.8 ppm in the pooled livers and 0.64 ppm in the remainder of the bodies. The water content was reduced to 0.024 ppb (2.4”,,). Water standing without fish had 0.35 ppb or 35”,, remaining after 7 days, the difference presumably bein,0 L#idsorbed to the glass and detritus. This near-total uptake of available DDT from water compares favorably with other data presented for small fishes’.
The radioautographs did not demonstrate any exposed grains above background level. It was assumed that the [3H]DDT was not bound to the tissue in such a way as to resist solution by the solvents used for dehydration and embedding (ethanol and propylene oxide). DISCUSSION
Two teleost fishes, the DDT-sensitive zebrafish and the DDT-tolerant guppy, responded differently to low levels of DDT in respect to the ultrastructure of their hepatic parenchyma. The zebrafish showed, in addition to a lossof lipid, a glycogenolysis which was as extensive after 24 h at I ppb or 72 h at 0.3 ppb as was complete fasting for I4 days4. Since the rate of glycogenolysis was, therefore, several times greater *
S.E.M.
PEDDRICK WEIS
30 than metabolic requirements,
even with the increase in general activity co~oniy acknowledged to be caused by DDT, it would appear that equilibria were upset in rate-limiting step(s) in giycogenoiysis. A qualitatively similar response was found in rabbits, in which 5 days of treatment with phenobarbital caused a 40% decrease in the amount of giycoger8. A proliferation of smooth ER was also noted in that experiment. The greater amount of lipid found in the guppy, plus the ability to develop smooth ER, may be related to that species’ tolerance to DDT. Indeed, FABACHER AND CHAMBERS~ reported that the degree of tolerance to DDT in Gambusia afjnis was proportional to the amount of body fat. This report of the guppies’ response to DDT supports their contention that lipid droplets sequester the fat-soluble insecticide and give the fish a modicum of protection from the toxic effects. The guppy liver responded with a smooth ER proliferation as has been reported for rats’, although the time required is much longer than the 3-day period observed in the rat and the results are much less obvious. Since the smooth ER is the site of the drug-induced microsomal enzymes which detoxify insecticides, perhaps these fish are able to handle the toxic burden in the same manner, if not to the same extent, as mammals. The zebrafish does not have a significant amount of hepatic lipid. Perhaps if it did, sufficient initial protection would be given by sequestration of DD f to allow time for proliferation of smooth ER to detoxifjr the drug, as in the guppy. ACKNOWLEDGMENTS
thank Messrs. N. URAY and M. SILVERSTEI~~for technical assistance. This research was supported by institutional awards from the American Cancer Society and the National Science Foundation. I
REFERENCES P. ORTEGA, Light and electron microscopy of dichlomdiphenyltrichloroethane (DDT) poisoning in the rat liver, tnb. In~*rst.. I5 f1966) 657-679. J. C. STREET, Organochlorine insecticides and the stimulation of liver microsomal enzymes, Ann. N. Y. Acad. Sci., 160 (1969) 274-290. M. T. FINLEY, D. E. FERGUSON AND J. L. LUDKE, Possible selective mechanisms in the development of insecticide-resistant fish, Pestir. Manif. J., 3 (1970) 2 12-2 18. P. WEIS, Hepatic ultrastructure in two species of normal, fasted and gravid teleost fishes, Am J. Anar.. 133 (1972) 317-332. J. P. ZAHND, Modifications hbpatiques liees au cycle ovarien chez deux poissons ovovivipares: Xiphophoroas helferi et Lebistes reticulates, Arch. Anat. Microsc. Morph&. Esptl., 48 (1959)
23 I-259. S. F. KING, Some effects of DDT on the guppy and the brown Service, Speciul Sci. Rept.-Fisheries NV. 399, 1962, 21 pp. A. V. HOLDEN, A study of the absorption
of 14C-labeiled
DDT
trout,
U.S. Fish and Wildlife
from water by fish, Am.
Appl.
Biol., 50 {1962) 467-477. H. REMMER AND H. J. MERKER, Morphological cells with enzyme induction after pretreatment
Meetly:,
changes in the endopiasmic reticulum of the liver with several drugs, Proc. 2nd Intern. Pharmrcol.
Prague. 4 (1965) 299-307.
D. L. FABACHEK AND
H. CHAMBERS, A possible mechanism fish, B&I. Envirorr. Coniam Tasieui. 6, 11971) 372-376.
of insecticide
resistance in mosquito-
in The Netherlands
25
ULTRASTRUCTURAL CHANGES INDUCED BY LOW CONCENTRATIONS OF DDT IN THE LIVERS OF THE ZEBRAFISH AND THE GUPPY
PEDDRICK
WEB
Department of Anatomy, New Jersey Medical School, Newark, N,J. 07103 (U.S.A.) (Received March 16th, 1973) (Revision received April 30th, 1973) (Accepted August 6th. 1973)
SUMMARY
Following exposure to 0.3-3.0 ppb DDT, the livers of zebrafish and guppies were examined by electron microscopy. After 24 h in 1.0 ppb or 72 h in 0.3 ppb, zebrafish had a complete loss of glycogen, decrease in cell size and a cytoplasmic compartment filled with rough endoplasmic reticulum (ER). Guppies exposed for periods of up to 28 days showed only a gradual accumulation of smooth ER and no change in glycogen, lipid, rough ER or cell size. The relationship of these changes to the zebra&h’s sensitivity and the guppy’s tolerance to DDT is dicwssed.
INTRODUCTION
DDT induces a proliferation of the smooth ER in the liver of the rat’ and stimulates the drug-induced microsomal enzyme system which is associated with the smooth ERZ. In preparation for similar enzyme studies in teleost fishes, ultrastructural analysis was performed on the livers of the zebrafish (Bruc&&rk” rerio) and the guppy (Poecilia retidura) following exposure to DDT in water. The concentrations of DDT in water to which the fish were exposed were substantially less than the maximal amount found to occur in the field (up to 12 ppb in areas of DDT application3). MATERIALS AND METHODS
The fishes used in this experiment were maintained in Abbreviations: DDT. reticulum; ppm. ppb.
water. without mixing of species mlder conditions described previously”, at a maximum of I g of fish per I. Technical grade DDT (72f;, /),JI’-DDT, courtesy of Montrose Chemical Corp., Torrance, Cpiif,) was recrystallized from ethanol to yield > 987f, p,$-DDT as estimated by disappearance of minor spots on thin-layer chromatograms. Acetone solutions of DDT were added to the water to provide concentrations of 0.3, 1.0 and 3.0 ppb DDT. Acetone (0.05 ml/i) was added to control jars. The ja,*s were washed and fresh solutions prepared weekly. Additional fish were selected from the stock tanks for comparison. After I to 28 days of exposure, fish were sacrificed and liver fragments fixed in pi~ospl~ate-bll~ered gl~~taraldeilyde follo~~ed by post-fixation in 0~0,. Ethanoidehydrated, Epon-embedded tissues were sectioned, stained with uranyi acetate and lead citrate, and examined with Philips EM-100 and EM-300 electron microscopes. Cell size was measured on low-power n~icrograpi~s taken after ~lib~tioI1 of the microscope with a diffraction-grating replica. in parallel experiments, some fish of each species were exposed to [3H]DDT (0.145 mCi/mg, New England Nuclear Corp., Boston, Mass.) at a concentration of 1 ppb, using 0.4 g of frsit per 1. Livers from the zebrafish were esch divided into two parts, the smaiie’rof which were prepared for microscopy and the larger of which were pooled and homogenized in a glass tissue grinder and. the hexane-soluble portion an~~iyzedby liquid ~i~ltiilation (5eckn~~l~ LS-21). The guppies exposed to [“H]DDT were homogenized i/t toto and counted also, after their livers had been excised, pooled and separately prepared for scintillation counting. For r~~dioa~ltograpi~ic~~ll~~iysis, 1-1’ sections of liver from both species were placed on glass slides, coated with liford K-5 emulsion (ilford, Ltd., Iiford, England), exposed at 4” for 3--28 days, developed in Kodak Microdol-X and stained with toiuidine blue. A total of 40 zebrafish and 40 guppies were included in this study. Only male guppies were used since female guppies show cyclic changes in their livers associated with their reproductive cyciess.
RESULTS
The zebrafish was found to be quite sensitive to DDT. A concentration of 3 ppb was toxic to most zebrafish within 24 h. I ppb allowed survival for as long as a week and 0.3 ppb for as long as 16 days. The proliferation of smooth ER predicted by mammalian studies failed to materialize. Instead, there was rapid disappearance of giycogen from the hepatocytes. This giycogenoiysis was virtually compiete by 24 h at one ppb or by 72 h at 0.3 ppb. When the giycogen had disappeared, the cytoplasm of the hepatocytes contained closely packed rough ER as its principal component (Figs. I and 2). The occasional lipid droplets found in the hepatocytes had also disappeared. The size of the hepa-
Fig. I. An acetone-control zebrafish ~~~~~O~ytE. showing the glyco~~ granules fCly) which fill most of the cytoplasmic com~rtment. along with snrne rough eudoplasn~ic reticulum (RERI. An erythrocytc (Ety) is seen within a sinusoid. t 19 700 (bar 1 ~6). Fig. 2. Zebrafish hcpatocyt~ after 24 h in I.0 ppb DDT. The cells are reduced in sire and RER occupies most of the cytopiasmic c~lmpartrnct~t. N. nucleus. If) 700.
Fig. 3. Acetone-control guppy hepatocyles, showing the glycogen granules and lipid droplets (L) which fill most of the cyloplasmic compartment, along with some RER. I’ 19 700. Fig. 4. A guppy hcp~tocyte after ?I) days in I .Oppb DDT. Smooth endopl~smic reticulum (SER) is i~dic~tcd by arrows. .I 19 700. Inset: a dctuil of a cell in which the glyeogcn was poorly stained, allowing easy visualization of ribosomes in the endoplasmie reticulum. SER is seen interiningled with the RER. 38 600.
EFFECT OF
DDT
ON FISH LIVER
29
tocytes was reduced from a profile of 28.3 If 2.0” ,Mby 21 .O f 2.0* ,I” to one of 16.0 i 1.1* ,U by 10.4 5 0.8” if (d.f. = 38, t = 4.93 and 4.95 for width and length respectively, P -=c0.001 by r-test for independent observations). This reduction in cell size correlates with a decrease in gross size observed during excision. There were no changes observed in other organelles or in the bile canaliculi.
The guppy, which has been reported to have a 1Cday LD5, of I8 ppb6, had no grossly observable changes in the early days of exposure and was found to be suitable for long-term experiments. Observations after 7 to 28 days of exposure to all concentrations of DDT used in these experiments indicate that the guppy has no loss of either glycogen or lipid as was seen in the zebrafish, and that the only noticeable change was a gradual accumulation of a small amount of rough and smooth ER in the area of glycogen storage (Figs. 3 and 4). The amount of smooth ER which was present would appear to be both dose- and time-dependent. The size of the hepatocytes, 16.6 5 0.7 ,I( by 9.6 + 0.9 ,U in profile, was not altered by the treatments.
Following the 24-h exposure to I ppb [3H]DDT, the 8 pooled zebrafish livers contained 0.50 ppm DDT and the residual amount in the water was 0.098 ppb or 9.8 p,{of the original amount. 4 guppies exposed to I ppb [3H]DDT for I day COIIremaining in the water: this represented tained 0.59 ppm i/l tn~o with 0.19 ppb or I9 ‘,B,, 6.5 ppm in the pooled livers and 0.36 ppm in the remainder of the body. After I week, 8 guppies contained 0.76 ppm: this represented 6.8 ppm in the pooled livers and 0.64 ppm in the remainder of the bodies. The water content was reduced to 0.024 ppb (2.4”,,). Water standing without fish had 0.35 ppb or 35”,, remaining after 7 days, the difference presumably bein,0 L#idsorbed to the glass and detritus. This near-total uptake of available DDT from water compares favorably with other data presented for small fishes’.
The radioautographs did not demonstrate any exposed grains above background level. It was assumed that the [3H]DDT was not bound to the tissue in such a way as to resist solution by the solvents used for dehydration and embedding (ethanol and propylene oxide). DISCUSSION
Two teleost fishes, the DDT-sensitive zebrafish and the DDT-tolerant guppy, responded differently to low levels of DDT in respect to the ultrastructure of their hepatic parenchyma. The zebrafish showed, in addition to a lossof lipid, a glycogenolysis which was as extensive after 24 h at I ppb or 72 h at 0.3 ppb as was complete fasting for I4 days4. Since the rate of glycogenolysis was, therefore, several times greater *
S.E.M.
PEDDRICK WEIS
30 than metabolic requirements,
even with the increase in general activity co~oniy acknowledged to be caused by DDT, it would appear that equilibria were upset in rate-limiting step(s) in giycogenoiysis. A qualitatively similar response was found in rabbits, in which 5 days of treatment with phenobarbital caused a 40% decrease in the amount of giycoger8. A proliferation of smooth ER was also noted in that experiment. The greater amount of lipid found in the guppy, plus the ability to develop smooth ER, may be related to that species’ tolerance to DDT. Indeed, FABACHER AND CHAMBERS~ reported that the degree of tolerance to DDT in Gambusia afjnis was proportional to the amount of body fat. This report of the guppies’ response to DDT supports their contention that lipid droplets sequester the fat-soluble insecticide and give the fish a modicum of protection from the toxic effects. The guppy liver responded with a smooth ER proliferation as has been reported for rats’, although the time required is much longer than the 3-day period observed in the rat and the results are much less obvious. Since the smooth ER is the site of the drug-induced microsomal enzymes which detoxify insecticides, perhaps these fish are able to handle the toxic burden in the same manner, if not to the same extent, as mammals. The zebrafish does not have a significant amount of hepatic lipid. Perhaps if it did, sufficient initial protection would be given by sequestration of DD f to allow time for proliferation of smooth ER to detoxifjr the drug, as in the guppy. ACKNOWLEDGMENTS
thank Messrs. N. URAY and M. SILVERSTEI~~for technical assistance. This research was supported by institutional awards from the American Cancer Society and the National Science Foundation. I
REFERENCES P. ORTEGA, Light and electron microscopy of dichlomdiphenyltrichloroethane (DDT) poisoning in the rat liver, tnb. In~*rst.. I5 f1966) 657-679. J. C. STREET, Organochlorine insecticides and the stimulation of liver microsomal enzymes, Ann. N. Y. Acad. Sci., 160 (1969) 274-290. M. T. FINLEY, D. E. FERGUSON AND J. L. LUDKE, Possible selective mechanisms in the development of insecticide-resistant fish, Pestir. Manif. J., 3 (1970) 2 12-2 18. P. WEIS, Hepatic ultrastructure in two species of normal, fasted and gravid teleost fishes, Am J. Anar.. 133 (1972) 317-332. J. P. ZAHND, Modifications hbpatiques liees au cycle ovarien chez deux poissons ovovivipares: Xiphophoroas helferi et Lebistes reticulates, Arch. Anat. Microsc. Morph&. Esptl., 48 (1959)
23 I-259. S. F. KING, Some effects of DDT on the guppy and the brown Service, Speciul Sci. Rept.-Fisheries NV. 399, 1962, 21 pp. A. V. HOLDEN, A study of the absorption
of 14C-labeiled
DDT
trout,
U.S. Fish and Wildlife
from water by fish, Am.
Appl.
Biol., 50 {1962) 467-477. H. REMMER AND H. J. MERKER, Morphological cells with enzyme induction after pretreatment
Meetly:,
changes in the endopiasmic reticulum of the liver with several drugs, Proc. 2nd Intern. Pharmrcol.
Prague. 4 (1965) 299-307.
D. L. FABACHEK AND
H. CHAMBERS, A possible mechanism fish, B&I. Envirorr. Coniam Tasieui. 6, 11971) 372-376.
of insecticide
resistance in mosquito-