ENVIRONMENTAL
RESEARCH
Cadmium
20. 183-191(1979)
Toxicity in the Free-Living Caenorhabditis elegans J.D. POPHAM AND J.M.
Department
of Biological
Nematode,
WEBSTER
Sciences, Simon Fraser Unir*ersity. British Columbia VSA 135, Canado
Burr7uh.v.
Vancouver.
Received August 22. 1978 The effect of cadmium on the fecundity, growth, and tine structure of the free-living nematode Caenorhabditis elegans was studied. High concentrations of cadmium significantly decreased the fecundity and growth of these organisms. Electron microscopy showed that cadmium modifies the structure of the mitochondria in the esophagus and intestine. causes the formation of inclusion bodies in the nucleus of esophageal cells. and alters the morphology of cytosomes in the intestinal cells. The results suggest that the decreased fecundity and growth of cadmium-exposed C. elegans may be due to cadmium interfering with nutrient uptake or assimilation or both.
INTRODUCTION
Except for a report on the effect of copper on nematodes during their extraction from soil with bronze screens (Hafkenscheid, 1971), and a report on lead toxicity of nematodes living in intertidal beach sand (Roberts and Maguire, 1976) there are few references to the toxic effects of heavy metals on these organisms. The investigation was carried out primarily to assess the potential of using Caenorhabditis elegans in heavy metal toxicity studies, and considers the effects of “short-term” (2-6 hr) and “long-term” (3.5 days) exposures to various levels of cadmium (lOeH to 3.26 x 10efi moles Cd/l0 ml of agar gel) upon fecundity and growth. Detailed consideration has also been given to the effect of cadmium on the ultrastructure of the alimentary system. The free-living, saprophagous nematode, such as C. elegans, offers several advantages over alternative test species for bioassay studies in that it is small (permitting electron microscopic examination of almost all tissues simultaneously), parthenogenic (permitting tests on an isogenic line of organisms), grows and matures quickly (maturing within 72 hr of hatching at 20°C). and is easily cultured. Furthermore, its tissues usually include only one or two cell types and so the effects of toxicants are readily studied at the ultrastructural level. MATERIALS
AND METHODS
An isogenic line of the Bristol, N2 strain of Caenorhabditis efegans was cultured on NG agar seeded with either the OP50 or the B strain of Escherichirr co/i following the procedure of Brenner (1974). Fecundity test. In order to determine the dose of cadmium (as CdCl, 2.5 H,O) which visibly affected the nematode, groups of 36 60-mm petri dishes were filled with 10 ml of NG agar and divided into six replicates of six treatments. The agar in each petri dish was covered with 0.4 ml of a 70% ethanolic solution such that each petri dish contained either zero, lo-*, 3.26 x 10mH. lo-‘, 3.26 x lo-‘. lo-“, or 3.26 X lO+ moles, respectively, of cadmium. After the solvent had evaporated. each 183 0013-93S1/79/050183-09$02,00/O Copyright All rights
& 1974 by Academic I’re~. Ini. of reproduction I” any form rc\rrved
184
POPHAM
AND
WEBSTER
plate was seeded with a drop of an aqueous suspension of E. coli, incubated for 48 hr at 30°C and inoculated with five mature nematodes. In the short-term exposure the exposed nematodes were removed after 2 or 6 hr, and 24 hr later the number of Fl generation larvae in 1.0 cm2 of the bacterial lawn on each plate were counted and the mean number of Fl progeny produced per adult per hour in each cadmium treatment was determined. In the long-term exposure the dishes were heated to 35°C 3.5 days after inoculation to kill the nematodes. The number of mature (Fl generation) nematodes in 1.0 cm2 of the bacterial lawn on each plate was counted and a 0.25 cm* area was sampled for the number of eggs and larvae (F2 generation) produced by these mature worms. Hence, the mean number of progeny produced per Fl adult exposed to the various cadmium treatments was determined. Dunnett’s test (1955) was used to determine the significance (P = 0.05) of the decreased fecundity resulting from short- and long-term exposure to the different concentrations of cadmium. Growth studies. In four separate trials ten mature 3-day-old nematodes were placed on petri dishes to which had been added either distilled water, or 0.4 ml of either 10B7, lo+, 4 x 10e7 or 4 x lO-‘j moles cadmium. The nematodes were removed 2 hr later so that their resulting progeny were approximately synchronous in their development. At either 6- or 12-hr intervals 12 nematodes from each treatment were heat-killed in situ with a soldering iron and measured with a calibrated reticule. Equations for lines fitted by the method of least squares were then calculated for these data. At the end of the experiment the remaining nematodes were either removed to be heat-killed and photographed, or prepared for electron microscope examination. Ultrastructure studies. C. elegans specimens from the studies of cadmium on growth or fecundity were fixed overnight by flooding petri dishes of cultures with a solution of 5% glutaraldehyde buffered with 0.1 M sodium cacodylate adjusted to pH 7.4 with HCI. They were washed overnight in cacodylate-HCl buffer, fixed in cacodylate-buffered 2% osmium tetroxide for 4 hr, washed in distilled water, and stained en bloc with 2% uranyl acetate dissolved in 50% ethanol for 1 hr. The specimens were dehydrated in ethanol, transferred to epoxy propane, and infiltrated and embedded in Spurr’s resin (Polysciences). Silver/gold sections were cut and mounted on Formvar-coated loo-mesh copper grids and stained with uranyl acetate and Reynolds’ (1963) lead citrate solution. In order to minimize misinterpretation of electron micrographs of transverse sections of the intestine, only midsections of the nematodes were studied. RESULTS Fecundity Nematodes exposed briefly (2 and 6 hr) to cadmium required a ten times greater concentration (Table 1) to depress fecundity than did nematodes exposed to cadmium for 3.5 days (Table 2). For example, a 6-hr exposure to 3.26 x IO-’ moles cadmium per dish significantly decreased the fecundity of the adults, whereas a 3.5-day exposure to 1OV moles cadmium per dish was required to decrease the fecundity of their progeny. C. elegans growth rate shows the typical curvilinear relationship, and during the
CADMIUM
THY EFFECT OF DIFFERENT Cnrnorhabditis elrgans
-
Length of exposure (hr)
TOXlCITY
185
NEMATODES
TABLE 1 EXPOSURES FOR EACH CONCENTRATION OF CADMIUM ON MAWRt EXPRESSED AS THE MEAN NUMBER (*SE) OF Fl GENERATION LARVAE PRODUCED PER ADULT PER HOUR Concentration (moles/l0
0
IN
10 *
3.26
x 10 )i
of cadmium ml agar gel) lo- i
3.26 x 10 i ----
10 I1
3.26 x 10 I’
6 1.15 0.44
-
1.06 0.08 A = 0.293” 1.15 - (0.794 + 0.293)
0.79 0.09
1.30 0.10
= 0.63
0.48 0.14
0.42. 0.06
4.584 0.45
-
,> 0
2 6.60 0.48
6.25 0.60
6.75 0.56 6.60
5.80 0.76 A = 2.55 - (4.58 + 2.55)
6.07 0.60 = 0.53 > 0
” A. the allowable limit of Dunnett’s test: A = t s/Z/N (Dunnett, 1955). :’ Means which have significantly (P < 0.05) lower values than do the controls
first 72 hr growth approximates a straight line (Fig. 1, correlation coefficient being very close to 1) before plateauing on reaching maturity. Cadmium-treated nematodes grew similarly but the rate of growth decreased with increasing doses of cadmium as can be seen by comparing the slopes in the equations for the lines of regression in Fig. 1. Increasing concentrations of cadmium appeared to delay TABLE 2 THE EFFECT OF LONG-TERM EXPOSURE (3.5 DAYS) OF C‘aenorhabditis rlrgans TO DIFFERENT CONCENTRATIONS OF CADMIUM EXPRESSED AS THE MEAN NUMBER (t-SE) OF F2 GENERATION PROGENY PRODUCED PER ADULT Fl Concentration of cadmium (moles/ 10 ml agar gel)
Trial
0
10-x
3.26 x lo-”
10-7
3.26 x lo-’
10-t;
13.26 3.42
4.98 3.25
1.03” 0.06
-
-
-
-
-
-
x 10 ”
1
13.26 - (4.98 Trial
3.26
A = 3.292” + 7.825) = 0.455
i
0
2 6.55 0.65
5.72 1.42
4.36 0.60 6.55
3.03’ 0.82 0.67 0.27 A = 3.292 - (3.03 + 3.29) = 0.49 > 0
” A, the allowable limit of Dunnett’s test: A = t s/2/N (Dunnett. 1955) * Means which have significantly (P < 0.05) lower values than do the controls.
186
POPHAM
AND
WEBSTER
.
.
900 t 800
regression
equation
n
r
Jx = y + b(G) l -
0---
1
2
y,=744
20
,991
6
,996
4x10’
A-n
+302 (x-58.5)
IO-’ mo, Cd vx = 692 +256 (x-60.0)
Control
u-
mol Cdyx=635+253
(x-575)
12
,995 .999
(x-57.5)
6 12
10-~molCd~x=510+197(x-6a))
-----
4x10+
mot Cdy’,=470
+i35
I
I
I
I
I
I
1
3
4
5
6
7
8
9
966
DAYS FIG. 1. Growth rates of Caenorhabditis elegans exposed to the different concentrations of cadmium. Horizontal line marks length at maturity of untreated nematodes.
egg laying, and those exposed to the highest concentration of cadmium layed no eggs even though they were 5 days old. Nematodes exposed to the highest concentrations of cadmium (4 x 10W6moles per dish) never grew to the same length as the untreated nematodes (Fig. 1) and tended to resemble worms collected from starved, overcrowded cultures in which food availability was minimal (Fig. 2). Electron Microscope Studies The nematodes showed no visible ultrastructural effect of cadmium at 4 x lo-” moles per dish for 3 days, but the effects were very obvious after 5 days. Such cadmium treatment caused the occurrence of electron-dense pleomorphic structures in the esophageal muscle and salivary gland cells (Fig. 3) and in the intestinal cells (Figs. 7 and 8) of nuclear inclusions in the esophageal cells (Fig. 3), of structurally modified cytosomes in the intestinal cells (Figs. 7 and 8), and the shortening of the intestinal microvilli from about 1.8 pm in the untreated nematodes (Fig. 5) to about 0.4 pm in the treated ones (Figs. 7 and 8). No exam-
contra
I
FIG. 2. Gross morphology of Caenorhabdifis eleguns after the following treatments: Incubation in a nutrient medium for 5 days (top). incubation in a nutrient medium for 10 days with, therefore. less available food (center). or incubation in a nutrient medium plus 4 x lo-” moles per dish of cadmium for 5 days (bottom). Note that the cadmium-exposed nematode (bottom) resembles the one from the starved culture (center). FIG. 3. Section of esophagus of a Sday-old Caenorhabdiris elegans exposed since hatching to 4 x lo-” moles per dish of cadmium. Note the abundance of electron-dense pleomorphic structures (p) near the outer edge of the esophagus and the presence of homogeneous electron-dense inclusions (ni) in some nuclei. x6500.
FIG. 4. Section of esophagus of an untreated, S-day-old Caenorhabditis elegans. Note the absence of the pleomorphic structures. x6500. FIG. 5. Section of intestinal cells of an untreated S-day-old Caenorhabditis elegans. Note the length of the microvilli (mv) and the circular contours and homogeneous appearance of the electron-dense cytosomes (c). x5000. FIG. 6. Section of a portion of an esophageal cell from a cadmium-exposed (4 x 10mBmoles per dish elegans. Note the range in structure between the normal looking mitnrhnnfor 5 days) Caenorhabditis
FIG. 7. Section of a S-day-old Caenorhabdiris eiegans after exposure since hatching to IO-” moles per dish of cadmium. Note the presence of altered cytosomes (c) and abnormally short microvilli (mv) in the intestinal cells. ~20,000. FIG. 8. Muscle cells (ml. and intestinal cell of a 5-day-old Caenorhabditis elugans exposed since hatching to 4 x IO-” moles per dish cadmium. Note the presence of pleomorphic structures (pt. small microvilli (mv), and altered cytosomes (c). Mitocondrion (m). x20.000.
190
POPHAM
AND
WEBSTER
pies of cadmium-induced cellular injury could be found in the ovaries or the excretory tubules. The pleomorphic structures were electron-opaque and contained lamellae (Fig. 6). They occurred most frequently in the periphery of the esophageal muscle cells in close juxtaposition with mitochondria in cadmium-treated nematodes (Fig. 3). They did not occur in untreated nematodes (Fig. 4). Since mitochondria occurred intermediate in appearance between these structures and normal looking mitochondria (Fig. 6), it can be concluded that these bodies are coagulated or degenerated mitochondria. DISCUSSION
The fecundity and growth of C. elegans is significantly reduced by exposure to cadmium (Tables 1 and 2, Fig. 1). As the concentration of cadmium is increased the exposure necessary to cause a decrease in nematode fecundity becomes shorter. However, the results of the short-term exposure may be confounded by a behavioral response to an obnoxious stimulus as rhabditid nematodes are known to be attracted to some chemicals (Ward, 1973; Dusenbury et al., 1975) and repelled by others (Dusenbury, 197.5) including copper (Taniguchi, 1932) and zinc (Croll, 1976). The most obvious cellular lesions were the disrupted cytosomes and shortened microvilli in the intestinal cells and the altered mitochondria1 morphology in the esophageal muscle cells and intestinal cells. These lesions are similar to the ones observed in the kidney cells of the higher metazoa following cadmium exposure except that the mitochondria in these organisms usually appear swollen rather than electron dense (Castano, 1971; Nishizumi, 1972). Heavy metals in general are concentrated in lysosomes (Brun and Brunk, 1970; Fowler et al., 1975; Moore and Stebbing, 1976). Cadmium in particular has been shown to inhibit the rate of formation of primary lysosomes in cadmium-exposed mice cells (Mego and Cain, 1975) and to reduce the phagocytotic activity (a function of lysosome viability) in others (Graham et al., 1975). Shortening of the microvilli of Japanese quail intestinal cells (Richardson and Fox, 1974) and a decrease in the activity of alkaline phosphatase associated with microvilli of rat intestinal cells (Sugawara and Sugawara, 1975) following cadmium exposure also has been reported. It seems likely, therefore, that once cadmium has entered the nematodes it poisons them by interfering with the uptake and metabolism of nutrients, and so the nematodes appear to be “physiologically starved” (Fig. 2). Such “starvation” is manifest in the smaller size of the worms, in the development of smaller ovaries, and in the delay in egg laying as compared to untreated nematodes; the cadmium-treated worms resembled the worms on the crowded dishes which contained almost no food. Castano and Vigliani (1972) showed that cadmium intoxication inhibited the uptake of protein by cells, which in nematodes could be one of the causes of small ovaries and, hence, of reduced fecundity. Although the precise amount of cadmium available to the nematodes in the petri dishes of these experiments is not known, the results suggest that relatively high concentrations of cadmium are needed to induce a toxic response in the nematodes. High concentrations of mercury, copper, and zinc salts are required also in order to decrease fecundity in C. elegans (Popham and Webster, 1976). Since results of ecological studies suggest that the free-living nematodes are more
CADMIUM
TOXICITY
IN
NEMATODES
191
resistant than other meiofauna, such as copepods, to both organic (Wormald. 1975) and inorganic pollutants (Roberts and Maguire, 1976), they may not be sensitive organisms for bioassay studies. On the other hand such hardiness appears useful when comparing different types of lesions resulting from exposure to different heavy metals (Popham and Webster, 1977). REFERENCES Brenner. S. ( 1974). The genetics of Caenorhabditis elegans. Genc,rics 77, 7 I-94. Brun. A.. and Brunk. U. (1970). Histochemical indications for lysosomal localization of heavy metals in normal rat brain and liver. J. Hisrochem. Cytochem. 18, 820-827. Castano, P. (1971). Chronic intoxication by cadmium experimentally induced in rabbits. A study of kidney ultrastructure. Pathof. Microbial. 37, 280-301. Castano, P.. and Vigliani. E. C. (1972). Cadmium nephropathy: Ultrastructural observations (Horseradish Peroxidase). J. Occup. Med. 14, 125- 128. Croll. N. A. (1976). Behavioral Coordination of Nematodes. In “The Organisation of Nematodes” (N. A. Croll. Ed.). pp. 343-361. Academic Press. London. Dunnett. C. W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Srat. Ass. 50, 1096-1121. Dusenbery. D. B. (1975). The avoidance of D-tryptophan by the nematode Cnenwhubditi.s e/egan.c. .I. Exp. Zoo/. 193, 413-418. Dusenbery. D. B., Sheridan, R. E.. and Russell. R. L. (1975). Chemotaxis-defective mutants of the nematode Cuenorhabditis elegans. Genetics 80, 297-309. Epstein, J.. Castillo. .J.. Himmelhoch. S., and Zuckerman. B. M. (19711. Ultrastructural studies on Cuenorhabditis
briggsae.
J. Nematol.
3, 69-78.
Fowler. B. A.. Wolfe. D. A., and Hetter. W. F. (1975). Mercury and iron uptake by cytosomes in mantle epithelial cells of Quahog clams (Mercenaria mercencrria) exposed to mercury. .I. F&/I. Res. Bd. Canad.
32, 1767-1775.
Graham. J. A.. Garner. D. E.. Waters, M. D., and Coffin. D. 2. (1975). Effect of trace metals on phagocytosis by alveolar macrophages. Inject. Immrtnol. 11, 1278- 1283. Hafkenscheid. H. H. M. (1971). Influence of Cu++ Ions on Trickodorirs pachydermrts and an extraction method to obtain active specimens. Nematologicu 17, 535-541. Mego, J. L.. and Cain. J. A. (1975). An effect of cadmium on heterolysosome formation and function in mice. Biochem. Pharmacol. 24, 1227-1232. Moore. M. N.. and Stebbing, A. R. D. (1976). The quantitative cytochemical effects of three metal ions on a lysosomal hydrolase of a hydroid. J. Mar. Biol. Ass. U.K. 56, 99551005. Nishizumi, M. (1972). Electron microscopic study of cadmium nephrotoxicity in the rat. Arch. Enl?ron. Health 24, 215-225. Popham. J. D.. and Webster, J. M. (1976). Comparative toxicity of heavy metals. with special reference to cadmium. on Caenorhabdifis rlegnns, 372-373. 9th Annual Meeting of the Society of Invertebrate Pathologists. Popham. J. D.. and Webster. J. M. (1977). Ultrastructural changes associated with heavy metal intoxication of the nematode. Caenorhabditis elegans. Proc. Microsc. Sot. Canad. 4, 44-45. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. Richardson. M. E.. and Fox, M. R. S. (1974). Dietary cadmium and enteropathy in the Japanese Quail. Lab. Invest. 31, 722-731. Roberts. D., and Maguire. C. (1976). Interactions of lead with sediments and meiofauna. Mar. Poll/r/. Buli. 11, 21 l-214. Sugawara, N.. and Sugawara. C. (197.5). Effect of cadmium in vi\,cr and in vitro on intestinal brush border ALPase and ATPase. Bull. Environ. Contam. To.rico/. 14, 653-656. Taniguchi ( 1932). Notes in the chemotactic response of Rhabdifis ,fi/iformis Butschlei. Prr)c. fmp. Acad.
f Tokyo)
9, 432-435.
Ward, S. W. (1973). Chemotaxis by the nematode Caenorhabditis e/egaru. Identification of attractants and analysis of the response by use of mutants. Proc. Nat. Acad. Sci. USA 70, 817-821. Wormald, A. P. (197%. Effect of a spill of marine diesel oil on the meiofauna of a sandy beach at Picnic Bay. Hong Kong. Enriron. Pollut. 11, 117- 129.
RESEARCH
Cadmium
20. 183-191(1979)
Toxicity in the Free-Living Caenorhabditis elegans J.D. POPHAM AND J.M.
Department
of Biological
Nematode,
WEBSTER
Sciences, Simon Fraser Unir*ersity. British Columbia VSA 135, Canado
Burr7uh.v.
Vancouver.
Received August 22. 1978 The effect of cadmium on the fecundity, growth, and tine structure of the free-living nematode Caenorhabditis elegans was studied. High concentrations of cadmium significantly decreased the fecundity and growth of these organisms. Electron microscopy showed that cadmium modifies the structure of the mitochondria in the esophagus and intestine. causes the formation of inclusion bodies in the nucleus of esophageal cells. and alters the morphology of cytosomes in the intestinal cells. The results suggest that the decreased fecundity and growth of cadmium-exposed C. elegans may be due to cadmium interfering with nutrient uptake or assimilation or both.
INTRODUCTION
Except for a report on the effect of copper on nematodes during their extraction from soil with bronze screens (Hafkenscheid, 1971), and a report on lead toxicity of nematodes living in intertidal beach sand (Roberts and Maguire, 1976) there are few references to the toxic effects of heavy metals on these organisms. The investigation was carried out primarily to assess the potential of using Caenorhabditis elegans in heavy metal toxicity studies, and considers the effects of “short-term” (2-6 hr) and “long-term” (3.5 days) exposures to various levels of cadmium (lOeH to 3.26 x 10efi moles Cd/l0 ml of agar gel) upon fecundity and growth. Detailed consideration has also been given to the effect of cadmium on the ultrastructure of the alimentary system. The free-living, saprophagous nematode, such as C. elegans, offers several advantages over alternative test species for bioassay studies in that it is small (permitting electron microscopic examination of almost all tissues simultaneously), parthenogenic (permitting tests on an isogenic line of organisms), grows and matures quickly (maturing within 72 hr of hatching at 20°C). and is easily cultured. Furthermore, its tissues usually include only one or two cell types and so the effects of toxicants are readily studied at the ultrastructural level. MATERIALS
AND METHODS
An isogenic line of the Bristol, N2 strain of Caenorhabditis efegans was cultured on NG agar seeded with either the OP50 or the B strain of Escherichirr co/i following the procedure of Brenner (1974). Fecundity test. In order to determine the dose of cadmium (as CdCl, 2.5 H,O) which visibly affected the nematode, groups of 36 60-mm petri dishes were filled with 10 ml of NG agar and divided into six replicates of six treatments. The agar in each petri dish was covered with 0.4 ml of a 70% ethanolic solution such that each petri dish contained either zero, lo-*, 3.26 x 10mH. lo-‘, 3.26 x lo-‘. lo-“, or 3.26 X lO+ moles, respectively, of cadmium. After the solvent had evaporated. each 183 0013-93S1/79/050183-09$02,00/O Copyright All rights
& 1974 by Academic I’re~. Ini. of reproduction I” any form rc\rrved
184
POPHAM
AND
WEBSTER
plate was seeded with a drop of an aqueous suspension of E. coli, incubated for 48 hr at 30°C and inoculated with five mature nematodes. In the short-term exposure the exposed nematodes were removed after 2 or 6 hr, and 24 hr later the number of Fl generation larvae in 1.0 cm2 of the bacterial lawn on each plate were counted and the mean number of Fl progeny produced per adult per hour in each cadmium treatment was determined. In the long-term exposure the dishes were heated to 35°C 3.5 days after inoculation to kill the nematodes. The number of mature (Fl generation) nematodes in 1.0 cm2 of the bacterial lawn on each plate was counted and a 0.25 cm* area was sampled for the number of eggs and larvae (F2 generation) produced by these mature worms. Hence, the mean number of progeny produced per Fl adult exposed to the various cadmium treatments was determined. Dunnett’s test (1955) was used to determine the significance (P = 0.05) of the decreased fecundity resulting from short- and long-term exposure to the different concentrations of cadmium. Growth studies. In four separate trials ten mature 3-day-old nematodes were placed on petri dishes to which had been added either distilled water, or 0.4 ml of either 10B7, lo+, 4 x 10e7 or 4 x lO-‘j moles cadmium. The nematodes were removed 2 hr later so that their resulting progeny were approximately synchronous in their development. At either 6- or 12-hr intervals 12 nematodes from each treatment were heat-killed in situ with a soldering iron and measured with a calibrated reticule. Equations for lines fitted by the method of least squares were then calculated for these data. At the end of the experiment the remaining nematodes were either removed to be heat-killed and photographed, or prepared for electron microscope examination. Ultrastructure studies. C. elegans specimens from the studies of cadmium on growth or fecundity were fixed overnight by flooding petri dishes of cultures with a solution of 5% glutaraldehyde buffered with 0.1 M sodium cacodylate adjusted to pH 7.4 with HCI. They were washed overnight in cacodylate-HCl buffer, fixed in cacodylate-buffered 2% osmium tetroxide for 4 hr, washed in distilled water, and stained en bloc with 2% uranyl acetate dissolved in 50% ethanol for 1 hr. The specimens were dehydrated in ethanol, transferred to epoxy propane, and infiltrated and embedded in Spurr’s resin (Polysciences). Silver/gold sections were cut and mounted on Formvar-coated loo-mesh copper grids and stained with uranyl acetate and Reynolds’ (1963) lead citrate solution. In order to minimize misinterpretation of electron micrographs of transverse sections of the intestine, only midsections of the nematodes were studied. RESULTS Fecundity Nematodes exposed briefly (2 and 6 hr) to cadmium required a ten times greater concentration (Table 1) to depress fecundity than did nematodes exposed to cadmium for 3.5 days (Table 2). For example, a 6-hr exposure to 3.26 x IO-’ moles cadmium per dish significantly decreased the fecundity of the adults, whereas a 3.5-day exposure to 1OV moles cadmium per dish was required to decrease the fecundity of their progeny. C. elegans growth rate shows the typical curvilinear relationship, and during the
CADMIUM
THY EFFECT OF DIFFERENT Cnrnorhabditis elrgans
-
Length of exposure (hr)
TOXlCITY
185
NEMATODES
TABLE 1 EXPOSURES FOR EACH CONCENTRATION OF CADMIUM ON MAWRt EXPRESSED AS THE MEAN NUMBER (*SE) OF Fl GENERATION LARVAE PRODUCED PER ADULT PER HOUR Concentration (moles/l0
0
IN
10 *
3.26
x 10 )i
of cadmium ml agar gel) lo- i
3.26 x 10 i ----
10 I1
3.26 x 10 I’
6 1.15 0.44
-
1.06 0.08 A = 0.293” 1.15 - (0.794 + 0.293)
0.79 0.09
1.30 0.10
= 0.63
0.48 0.14
0.42. 0.06
4.584 0.45
-
,> 0
2 6.60 0.48
6.25 0.60
6.75 0.56 6.60
5.80 0.76 A = 2.55 - (4.58 + 2.55)
6.07 0.60 = 0.53 > 0
” A. the allowable limit of Dunnett’s test: A = t s/Z/N (Dunnett, 1955). :’ Means which have significantly (P < 0.05) lower values than do the controls
first 72 hr growth approximates a straight line (Fig. 1, correlation coefficient being very close to 1) before plateauing on reaching maturity. Cadmium-treated nematodes grew similarly but the rate of growth decreased with increasing doses of cadmium as can be seen by comparing the slopes in the equations for the lines of regression in Fig. 1. Increasing concentrations of cadmium appeared to delay TABLE 2 THE EFFECT OF LONG-TERM EXPOSURE (3.5 DAYS) OF C‘aenorhabditis rlrgans TO DIFFERENT CONCENTRATIONS OF CADMIUM EXPRESSED AS THE MEAN NUMBER (t-SE) OF F2 GENERATION PROGENY PRODUCED PER ADULT Fl Concentration of cadmium (moles/ 10 ml agar gel)
Trial
0
10-x
3.26 x lo-”
10-7
3.26 x lo-’
10-t;
13.26 3.42
4.98 3.25
1.03” 0.06
-
-
-
-
-
-
x 10 ”
1
13.26 - (4.98 Trial
3.26
A = 3.292” + 7.825) = 0.455
i
0
2 6.55 0.65
5.72 1.42
4.36 0.60 6.55
3.03’ 0.82 0.67 0.27 A = 3.292 - (3.03 + 3.29) = 0.49 > 0
” A, the allowable limit of Dunnett’s test: A = t s/2/N (Dunnett. 1955) * Means which have significantly (P < 0.05) lower values than do the controls.
186
POPHAM
AND
WEBSTER
.
.
900 t 800
regression
equation
n
r
Jx = y + b(G) l -
0---
1
2
y,=744
20
,991
6
,996
4x10’
A-n
+302 (x-58.5)
IO-’ mo, Cd vx = 692 +256 (x-60.0)
Control
u-
mol Cdyx=635+253
(x-575)
12
,995 .999
(x-57.5)
6 12
10-~molCd~x=510+197(x-6a))
-----
4x10+
mot Cdy’,=470
+i35
I
I
I
I
I
I
1
3
4
5
6
7
8
9
966
DAYS FIG. 1. Growth rates of Caenorhabditis elegans exposed to the different concentrations of cadmium. Horizontal line marks length at maturity of untreated nematodes.
egg laying, and those exposed to the highest concentration of cadmium layed no eggs even though they were 5 days old. Nematodes exposed to the highest concentrations of cadmium (4 x 10W6moles per dish) never grew to the same length as the untreated nematodes (Fig. 1) and tended to resemble worms collected from starved, overcrowded cultures in which food availability was minimal (Fig. 2). Electron Microscope Studies The nematodes showed no visible ultrastructural effect of cadmium at 4 x lo-” moles per dish for 3 days, but the effects were very obvious after 5 days. Such cadmium treatment caused the occurrence of electron-dense pleomorphic structures in the esophageal muscle and salivary gland cells (Fig. 3) and in the intestinal cells (Figs. 7 and 8) of nuclear inclusions in the esophageal cells (Fig. 3), of structurally modified cytosomes in the intestinal cells (Figs. 7 and 8), and the shortening of the intestinal microvilli from about 1.8 pm in the untreated nematodes (Fig. 5) to about 0.4 pm in the treated ones (Figs. 7 and 8). No exam-
contra
I
FIG. 2. Gross morphology of Caenorhabdifis eleguns after the following treatments: Incubation in a nutrient medium for 5 days (top). incubation in a nutrient medium for 10 days with, therefore. less available food (center). or incubation in a nutrient medium plus 4 x lo-” moles per dish of cadmium for 5 days (bottom). Note that the cadmium-exposed nematode (bottom) resembles the one from the starved culture (center). FIG. 3. Section of esophagus of a Sday-old Caenorhabdiris elegans exposed since hatching to 4 x lo-” moles per dish of cadmium. Note the abundance of electron-dense pleomorphic structures (p) near the outer edge of the esophagus and the presence of homogeneous electron-dense inclusions (ni) in some nuclei. x6500.
FIG. 4. Section of esophagus of an untreated, S-day-old Caenorhabditis elegans. Note the absence of the pleomorphic structures. x6500. FIG. 5. Section of intestinal cells of an untreated S-day-old Caenorhabditis elegans. Note the length of the microvilli (mv) and the circular contours and homogeneous appearance of the electron-dense cytosomes (c). x5000. FIG. 6. Section of a portion of an esophageal cell from a cadmium-exposed (4 x 10mBmoles per dish elegans. Note the range in structure between the normal looking mitnrhnnfor 5 days) Caenorhabditis
FIG. 7. Section of a S-day-old Caenorhabdiris eiegans after exposure since hatching to IO-” moles per dish of cadmium. Note the presence of altered cytosomes (c) and abnormally short microvilli (mv) in the intestinal cells. ~20,000. FIG. 8. Muscle cells (ml. and intestinal cell of a 5-day-old Caenorhabditis elugans exposed since hatching to 4 x IO-” moles per dish cadmium. Note the presence of pleomorphic structures (pt. small microvilli (mv), and altered cytosomes (c). Mitocondrion (m). x20.000.
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pies of cadmium-induced cellular injury could be found in the ovaries or the excretory tubules. The pleomorphic structures were electron-opaque and contained lamellae (Fig. 6). They occurred most frequently in the periphery of the esophageal muscle cells in close juxtaposition with mitochondria in cadmium-treated nematodes (Fig. 3). They did not occur in untreated nematodes (Fig. 4). Since mitochondria occurred intermediate in appearance between these structures and normal looking mitochondria (Fig. 6), it can be concluded that these bodies are coagulated or degenerated mitochondria. DISCUSSION
The fecundity and growth of C. elegans is significantly reduced by exposure to cadmium (Tables 1 and 2, Fig. 1). As the concentration of cadmium is increased the exposure necessary to cause a decrease in nematode fecundity becomes shorter. However, the results of the short-term exposure may be confounded by a behavioral response to an obnoxious stimulus as rhabditid nematodes are known to be attracted to some chemicals (Ward, 1973; Dusenbury et al., 1975) and repelled by others (Dusenbury, 197.5) including copper (Taniguchi, 1932) and zinc (Croll, 1976). The most obvious cellular lesions were the disrupted cytosomes and shortened microvilli in the intestinal cells and the altered mitochondria1 morphology in the esophageal muscle cells and intestinal cells. These lesions are similar to the ones observed in the kidney cells of the higher metazoa following cadmium exposure except that the mitochondria in these organisms usually appear swollen rather than electron dense (Castano, 1971; Nishizumi, 1972). Heavy metals in general are concentrated in lysosomes (Brun and Brunk, 1970; Fowler et al., 1975; Moore and Stebbing, 1976). Cadmium in particular has been shown to inhibit the rate of formation of primary lysosomes in cadmium-exposed mice cells (Mego and Cain, 1975) and to reduce the phagocytotic activity (a function of lysosome viability) in others (Graham et al., 1975). Shortening of the microvilli of Japanese quail intestinal cells (Richardson and Fox, 1974) and a decrease in the activity of alkaline phosphatase associated with microvilli of rat intestinal cells (Sugawara and Sugawara, 1975) following cadmium exposure also has been reported. It seems likely, therefore, that once cadmium has entered the nematodes it poisons them by interfering with the uptake and metabolism of nutrients, and so the nematodes appear to be “physiologically starved” (Fig. 2). Such “starvation” is manifest in the smaller size of the worms, in the development of smaller ovaries, and in the delay in egg laying as compared to untreated nematodes; the cadmium-treated worms resembled the worms on the crowded dishes which contained almost no food. Castano and Vigliani (1972) showed that cadmium intoxication inhibited the uptake of protein by cells, which in nematodes could be one of the causes of small ovaries and, hence, of reduced fecundity. Although the precise amount of cadmium available to the nematodes in the petri dishes of these experiments is not known, the results suggest that relatively high concentrations of cadmium are needed to induce a toxic response in the nematodes. High concentrations of mercury, copper, and zinc salts are required also in order to decrease fecundity in C. elegans (Popham and Webster, 1976). Since results of ecological studies suggest that the free-living nematodes are more
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resistant than other meiofauna, such as copepods, to both organic (Wormald. 1975) and inorganic pollutants (Roberts and Maguire, 1976), they may not be sensitive organisms for bioassay studies. On the other hand such hardiness appears useful when comparing different types of lesions resulting from exposure to different heavy metals (Popham and Webster, 1977). REFERENCES Brenner. S. ( 1974). The genetics of Caenorhabditis elegans. Genc,rics 77, 7 I-94. Brun. A.. and Brunk. U. (1970). Histochemical indications for lysosomal localization of heavy metals in normal rat brain and liver. J. Hisrochem. Cytochem. 18, 820-827. Castano, P. (1971). Chronic intoxication by cadmium experimentally induced in rabbits. A study of kidney ultrastructure. Pathof. Microbial. 37, 280-301. Castano, P.. and Vigliani. E. C. (1972). Cadmium nephropathy: Ultrastructural observations (Horseradish Peroxidase). J. Occup. Med. 14, 125- 128. Croll. N. A. (1976). Behavioral Coordination of Nematodes. In “The Organisation of Nematodes” (N. A. Croll. Ed.). pp. 343-361. Academic Press. London. Dunnett. C. W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Srat. Ass. 50, 1096-1121. Dusenbery. D. B. (1975). The avoidance of D-tryptophan by the nematode Cnenwhubditi.s e/egan.c. .I. Exp. Zoo/. 193, 413-418. Dusenbery. D. B., Sheridan, R. E.. and Russell. R. L. (1975). Chemotaxis-defective mutants of the nematode Cuenorhabditis elegans. Genetics 80, 297-309. Epstein, J.. Castillo. .J.. Himmelhoch. S., and Zuckerman. B. M. (19711. Ultrastructural studies on Cuenorhabditis
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Fowler. B. A.. Wolfe. D. A., and Hetter. W. F. (1975). Mercury and iron uptake by cytosomes in mantle epithelial cells of Quahog clams (Mercenaria mercencrria) exposed to mercury. .I. F&/I. Res. Bd. Canad.
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Graham. J. A.. Garner. D. E.. Waters, M. D., and Coffin. D. 2. (1975). Effect of trace metals on phagocytosis by alveolar macrophages. Inject. Immrtnol. 11, 1278- 1283. Hafkenscheid. H. H. M. (1971). Influence of Cu++ Ions on Trickodorirs pachydermrts and an extraction method to obtain active specimens. Nematologicu 17, 535-541. Mego, J. L.. and Cain. J. A. (1975). An effect of cadmium on heterolysosome formation and function in mice. Biochem. Pharmacol. 24, 1227-1232. Moore. M. N.. and Stebbing, A. R. D. (1976). The quantitative cytochemical effects of three metal ions on a lysosomal hydrolase of a hydroid. J. Mar. Biol. Ass. U.K. 56, 99551005. Nishizumi, M. (1972). Electron microscopic study of cadmium nephrotoxicity in the rat. Arch. Enl?ron. Health 24, 215-225. Popham. J. D.. and Webster, J. M. (1976). Comparative toxicity of heavy metals. with special reference to cadmium. on Caenorhabdifis rlegnns, 372-373. 9th Annual Meeting of the Society of Invertebrate Pathologists. Popham. J. D.. and Webster. J. M. (1977). Ultrastructural changes associated with heavy metal intoxication of the nematode. Caenorhabditis elegans. Proc. Microsc. Sot. Canad. 4, 44-45. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. Richardson. M. E.. and Fox, M. R. S. (1974). Dietary cadmium and enteropathy in the Japanese Quail. Lab. Invest. 31, 722-731. Roberts. D., and Maguire. C. (1976). Interactions of lead with sediments and meiofauna. Mar. Poll/r/. Buli. 11, 21 l-214. Sugawara, N.. and Sugawara. C. (197.5). Effect of cadmium in vi\,cr and in vitro on intestinal brush border ALPase and ATPase. Bull. Environ. Contam. To.rico/. 14, 653-656. Taniguchi ( 1932). Notes in the chemotactic response of Rhabdifis ,fi/iformis Butschlei. Prr)c. fmp. Acad.
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Ward, S. W. (1973). Chemotaxis by the nematode Caenorhabditis e/egaru. Identification of attractants and analysis of the response by use of mutants. Proc. Nat. Acad. Sci. USA 70, 817-821. Wormald, A. P. (197%. Effect of a spill of marine diesel oil on the meiofauna of a sandy beach at Picnic Bay. Hong Kong. Enriron. Pollut. 11, 117- 129.
