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Leucocytes and leucopoietic capacity in goldfish, Carassius auratus, exposed to sublethal levels of cadmium
Aquatic Toxicology, 13 (1988) 141-154 Elsevier
141
AQT 00293
Leucocytes and leucopoietic capacity in goldfish, Carassius auratus, exposed to sublethal levels of cadmium A. Murad and A.H. Houston Department of Biological Sciences, Brock University, St. Catharines, Canada (Received 8 October 1987; revision received 1 April 1988; accepted after revision 4 April 1988) By comparison with animals in essentially cadmium-free water (< 2 ~tg Cd2÷/l) goldfish exposed for 3 wk to 90, 270 and 445 #g Cd2+/l (5, 15 and 25070 240-h LCso) exhibited significant reductions in total leucocyte counts. These were the result of decreases in lymphocyte and thrombocyte numbers. Mitogenic response to administered PHA as well as sharp decreases in blast cell numbers suggested that lympho- and thrombopenia reflect, in part at least, decreased proliferative capacity. By contrast, neutrophil, eosinophil and basophil numbers increased in cadmium-intoxicated fish. Cadmium apparently reduced PHA-related changes in granulocyte abundances. Key words: Goldfish; Carassius auratus; Cadmium; Leucocyte INTRODUCTION Significant c a d m i u m - i n d u c e d h e m a t o l o g i c a l effects can be detected in f r e s h w a t e r a n d m a r i n e fishes c h r o n i c a l l y e x p o s e d to low levels o f this e x t r e m e l y toxic h e a v y m e t a l . R e d u c t i o n s in b l o o d O2-carrying c a p a c i t y were, for e x a m p l e , o b s e r v e d b y J o h a n s s o n - S j o b e c k a n d L a r s s o n (1978) in f l o u n d e r , Pleuronectesflesus, e x p o s e d to as little as 5 # g / l . S t r o m b e r g et al. (1983) r e p o r t e d persistent lesioning o f h e m a t o p o i e t i c sites in f a t h e a d m i n n o w s , Pimephales promelas, e x p o s e d for 96 h to 12 p p m C D 2÷ . C o n s i s t e n t with this, H o u s t o n a n d Keen (1984) f o u n d significant, d o s e - d e p e n d e n t i m p a i r m e n t o f e r y t h r o p o i e t i c c a p a c i t y in g o l d f i s h , Carassius auratus, c h r o n i c a l l y e x p o s e d to c a d m i u m c o n c e n t r a t i o n s e q u i v a l e n t to 1-25°/0 o f the s o f t w a t e r 240-h LCso ( M c C a r t y et al., 1978). A l t h o u g h c o r r e s p o n d i n g studies o n leucocytes have been c a r r i e d out, their results are in conflict. G a r d n e r a n d Yevich (1970), for e x a m p l e , r e p o r t e d t h a t e x p o s u r e o f m u m m i c h o g , Fundulus heteroclitus, to 50% o f their 48-h LCso for up to 48 h p r o -
Correspondenceto: A.H. Houston, Department of Biological Sciences, Brock University, St. Catharines, Canada L2S 3AI. 0378-4274/88/$ 03.50 © Elsevier Science Publishers B.V. (Biomedical Division)
142 duced marked and progressive decreases in differential lymphocyte and thrombocyte counts with major increases in the overall granulocyte differential count. This was accompanied by increased incidence of morphological aberrances including nuclear deformation, extensive cytoplasmic vacuolization and reductions in the number and size of cytoplasmic granules and other inclusions. In sharp contrast to this, Srivastava and Mishra (1979) recorded increases of 50-85°7o in total leucocyte, thrombocyte and small and large lymphocyte numbers in Colisa fasciatus held for 90 h at 80°70 of their 96-h LCs0. In the present study, we examined the effects of 3-wk exposures to cadmium concentrations equivalent to 5, 15 and 25°7o of the 240-h LCso on absolute numbers and differential counts of thrombocytes and agranular and granular leucocytes in goldfish. In addition, consideration was given to the effects of cadmium intoxification on lymphopoietic capacity as measured by response to i.p. administration of the mitogenic agent, phytohemagglutinin (PHA). MATERIALS AND METHODS Goldfish ranging in weight from 7.8-36.2 g (mean ___ 1 SEM = 17.1 + 0.48 g) were obtained from a commercial supplier (Grassyforks Fisheries Co., Indiana). On arrival at the laboratory they were initially held for a 3-wk period in 600-1 fiberglass tanks equipped with high capacity ( - 5 1/min) styrofoam activated-charcoal filters. Duty-operated thermoregulators linked to refrigeration and heating units held water temperatures at 25.0 + 0.2°C, a widely accepted value for the thermal optimum of this species (Houston, 1982). Supplementary aeration provided oxygen levels > 6.8 mg/l or 80°70 saturation. Light-tight hoods incorporating timers accurate to +_ 7 min/day (corrected daily), and providing water surface light intensities of 90-120 Lx were used to establish a 12 h light:12 h darkness photoperiod regime. Animals were fed daily to satiation on a commercial ration (Purina Trout Chow), and excess food and fecal materials removed after cessation of feeding. As judged by general activity, feeding behaviour and the absence of obvious disease symptoms all were healthy during the study period. The cadmium content of dechlorinated St. Catharines city water, as determined by atomic absorption spectrophotometry, is relatively low (< 10/xg/l) as are zinc and copper levels (< 50 #g/l). However, pH (7.6-8.0) and total hardness (140-150 mg/1, as CaCo3) are such that added cadmium is rapidly lost a s C d C O 3 , Cd(OH)2 and other insoluble precipitates. Earlier studies involving cadmium loads of 1-60 mg/1, for example, revealed exponential decreases in dissolved cadmium concentrations to negligible levels within 24-48 h (McCarty et al., 1978). Hardness and conductivity increased in proportion to the amount of cadmium added, while total alkalinity and pH fell transiently, the latter by as much as 1.0-1.2 pH units. To avoid the complications associated with such changes this investigation was conducted with water of reduced hardness prepared by the dilution of aged and
143 dechlorinated city water with deionized, glass-distilled water. This had the following characteristics - total hardness: 20-22 mg/1 as CaCO3; total alkalinity: 14-18 mg/l as CaCO3; conductivity: 15-25/~mho/cm; pH: 7.4-7.8, cadmium: < 2 #g/l. Under such circumstances there is little precipitation and changes in water quality following cadmium addition are reduced (McCarty et al., 1978). Dissolved oxygen, total hardness and alkalinity, conductivity and pH were measured on alternate days throughout the study period. After the initial holding period, fish were transferred to 100-1 fiberglass test tanks containing softened water. Other conditions were as previously described. A period of 1 wk was provided for recovery from any transfer stresses. Aqueous analytical grade CdC12 was then added to bring test tank concentrations to 90, 270 and 445 /zg/1, i.e. 5%, 15% and 25% of the 240-h soft water LCso for this species (McCarthy et al., 1978). Following 3-wk exposures to these conditions all specimens were sampled. The study was replicated to provide samples of 10 to 12 animals at each test concentration. The effects of cadmium on leucopoiesis were investigated by reference to PHAinduced mitogenic response. Preliminary trials revealed that PHA administered intraperitoneally in 0.1 ml of modified Cortland saline (Houston et al., 1985) at dosages of 3-8/~g/g body weight elicited consistently detectable changes in total and differential leucocyte counts within 1 wk in goldfish ranging from 11.6-30.5 g in weight. Smaller amounts did not produce consistent results, while dosages exceeding approximately 10/zg/g body weight did not increase magnitude of response. The effective dose range could be achieved, with attention to specimen weight, by administration of 100 ttg PHA/specimen. Investigations on leucopoietic capacity were confined to the examination of animals exposed for 3 wk to 15% of the 240-h LCso (i.e., 270 #g Cd 2÷/1). A similar group was held during the same period in nominally cadmium-free water (i.e., < 2/zg Cd 2 ÷/1) under comparable conditions. One half of each group received PHA, the remainder an equivalent volume of saline. Sampling was carried out immediately prior to treatment (0 h) and 1, 2, 4, 8 and 12 days after injection. To avoid possible induction of artefacts by exposure to chemical anesthetics, specimens were stunned before sampling by a blow to the juncture of the cranium and vertebral column. The caudal peduncle was descaled, cleansed with alcohol and transected. Blood was quickly drawn by capillary action directly into anticoagulanttreated sampling tubes, the complete procedure requiring less than three minutes. Smears were immediately prepared, air-dried and fixed in acetone-free methanol for 5 min. These were subsequently stained with Leishman-Giemsa, Sudan Black-B or the periodic acid Schiff procedure (Christensen et al., 1978; Sigma Diagnostics Procedure Bulletins 380, 395, 1984). Leucocytes were identified by the morphological and staining characteristics described by Weinreb (1963), Weinreb and Weinreb (1969), Ellis (1977) and Christensen et al. (1978). Slides were examined at x 1000, and a minimum of two smears and 500 leucocytes considered for each specimen.
144 Total
leucocyte
hemocytometer.
counts
Whole
were
blood
made
in
was diluted
triplicate
using
1:200 with modified
the
Neubauer
Cortland
saline
( H o u s t o n et al., 1985) c o n t a i n i n g 20 m g / 1 W r i g h t ' s s t a i n . C o u n t s w e r e a c c e p t e d o n l y when subsequent examination of fixed slides revealed thrombocytes
of normal mor-
phology. All statistical treatments were based on procedures outlined by Sokal and Rohlf (1969). D e s c r i p t i v e s t a t i s t i c s w e r e b a s e d o n t h e u s e o f u n t r a n s f o r m e d Kolmogorov-Smirnov
test for normality
Barlett's test for homogeneity
of distribution
data. The
was routinely employed.
o f v a r i a n c e s w a s u s e d p r i o r t o all c o m p a r i s o n s ,
and
modified variance analyses used where necessary. RESULTS
Leucocyte types and abundances Thrombocytes, immature
lymphocytes, monocytes, neutrophils, eosinophils, basophils and
o r b l a s t cells w e r e i d e n t i f i a b l e w i t h o u t a m b i g u i t y o n m o s t o f t h e s m e a r s
examined. However,
s m a l l n u m b e r s o f cells c o u l d n o t b e p o s i t i v e l y i d e n t i f i e d , a n d
t h e s e h a v e b e e n r e c o r d e d as s u c h . In cadmium-free water lymphocytes were the most abundant
cell t y p e , c o m p r i s i n g
5 4 . 6 _+ 0 . 7 1 % o f t h e t o t a l c o u n t o f 4 4 . 9 2 + 0 . 7 1 7 cells × 1 0 3 / m m 3 ( T a b l e s I a n d II). T h e s e cells w e r e t y p i c a l l y c i r c u l a r t o o v o i d i n p r o f i l e a n d r a n g e d f r o m 4 t o 9 m/~ in largest dimension. The usually somewhat eccentric nucleus occupied most of the cell m a s s , a n d s t a i n e d d e e p l y p u r p l e . C y t o p l a s m orange. Pseudopodia
was normally light blue to slightly
w e r e e v i d e n t i n s o m e cells.
TABLE I Absolute leucocyte levels (cells × 103/mm 3) in goldfish exposed for 3 wk to < 2, 90, 270 and 445 /xg C2+/1 (i.e., 0.5, 15 and 25o70 of 240-h LCs0) a. Weight, g Cadmium Total count Thrombocytes Lymphocytes Monocytes Neutrophils Eosinophils Basophils Blast cells Unidentified cells
19.8 4- 1.51 <2
20.0 +_2.01 90
21.4 + 1.80 270
24.7 _+2.32 445
44.92 _+0.72 15.74 + 0.39 24.51 _+0.50 0.48 4- 0.09 2.12 4- 0.10 0.68 4- 0.04 0.15 4- 0.02 1.18 4- 0.08 0.05 4- 0.02
28.22 _+0.92 b 9.15 + 0.42 b 11.44 _+0.39 b 0.68 4- 0.09 d 5.30 4- 0.29 b 1.02 4- 0.08 b 0.41 +_0.038 0.17 4- 0.028 0.03 4- 0.01 d
22.85 + 0.71 b 7.30 _+0.30 b 9.21 + 0.48 b 0.55 4- 0.06 d 4.42 4- 0.25 b 0.97 4- 0.05 b 0.27 4- 0.02 c 0.11 4- 0.02 b 0.03 4- 0.01 d
22.52 + 0.72 b 6.99 _+0.25 b 9.31 4- 0.45 b 0.50 + 0.03 d 4.45 4- 0.29 b 0.85 4- 0.09 d 0.23 4- 0.0U 0.12 4- 0.01 b 0.01 4- 0.01 d
a Values reported as mean _+ 1 SE (N= 10). b Significantly different from control group at P<0.05. c Significantly different from control group at P < 0.01. a NS, not significantly different from control group at P<0.05.
145 T h e t h r o m b o c y t e s o f g o l d f i s h a r e e l o n g a t e , b u t v a r i a b l e in p r o f i l e , r a n g i n g f r o m o v a l t o o v a l - w i t h - t a i l - l i k e - p r o j e c t i o n s t o s p i n d l e s h a p e d . C o n s i d e r a b l e v a r i a t i o n in size was a l s o e v i d e n t , a n d m a j o r axis l e n g t h s o f u p t o 15 m # w e r e r e c o r d e d . N u c l e i w e r e d i s t i n c t l y e l o n g a t e d , r a n g i n g f r o m 4.5 t o 6.5 m # in l e n g t h a n d o c c u p y i n g m u c h o f t h e cell. A s v i s u a l i z e d b y R o m a n o w s k y s t a i n i n g p r o c e d u r e s n u c l e i w e r e i n t e n s e l y b l u i s h - p u r p l e , t h e c y t o p l a s m a l i g h t e r b l u i s h h u e . T h e 15.74 _+ 0.39 × 1 0 3 / m m 3 t h r o m b o c y t e s f o u n d in t h e c o n t r o l g r o u p s o f g o l d f i s h c o n s t i t u t e d 35.0 + 0 . 6 6 % o f the leucocyte population. Monocytes were uncommon,
a n d w e r e n o t i d e n t i f i e d in all p r e p a r a t i o n s .
On
a v e r a g e 0.48 _+ 0 . 8 6 ___ 103 c e l l s / m m 3 w e r e p r e s e n t (1.1 _+ 0.1807o). T h e s e w e r e l a r g e , r o u n d t o o v o i d cells r a n g i n g f r o m 12 t o 15 mtt in d i a m e t e r . N u c l e a r : c y t o p l a s m i c r a t i o s w e r e a p p r o x i m a t e l y 0.5. C y t o p l a s m was b l u e - g r a y , a n d o c c a s i o n a l l y e v i d e n c ed p s e u d o p o d - l i k e s t r u c t u r e s . V a c u o l e s w e r e u s u a l l y p r e s e n t , b u t v a r i e d in size a n d n u m b e r . I n s o m e i n s t a n c e s , t h e i r b r a i n - s h a p e d o r r e n i f o r m n u c l e i also a p p e a r e d to be vacuolated. N e u t r o p h i l s w e r e t h e m o s t c o m m o n o f t h e g r a n u l o c y t e t y p e s (2.12 + 0.103 × 10 ~ c e l l s / m m 3 ; 4.7 ___ 0.24o7o o f w h i t e cell p o p u l a t i o n ) . T h e s e cells w e r e n o r m a l l y 10-12 m ~ in d i a m e t e r . T h e i r f r e q u e n t l y e c c e n t r i c n u c l e i v a r i e d f r o m k i d n e y - s h a p e d to r i b b o n - l i k e in p r o f i l e w i t h f r o m 2 to 4 l o b e s e v i d e n t in m a n y cells. T h e f i n e l y granular cytoplasm was weakly acidophilic, strands and many neutrophilic granules.
with weakly basophilic regions or
E o s i n o p h i l s c o n s t i t u t e d 1.5 × 0 . 0 9 % o f t h e l e u c o c y t e p o p u l a t i o n (0.68 _+ 0.042 × 103 c e l l s / m m 3) a n d w e r e e v i d e n t in all s p e c i m e n s . T h e s e w e r e t h e s m a l l e s t o f t h e g r a n u l o c y t e s ; t h e i r r o u n d to s o m e w h a t o v o i d p r o f i l e s m e a s u r i n g f r o m 7 t o 9 m # . N u c l e i w e r e r e l a t i v e l y l a r g e , r o u n d to c r e s c e n t i c b u t n e v e r l o b u l a r a n d p u r p l e stain-
TABLE II Differential leucocyte counts (°7o of total white cell population) in goldfish exposed for 3 wk to <2, 90, 270 and 445/zg Cd/1 (i.e., 0, 5, 15 and 25% of 240-h LCs0)a. Cadmium Thrombocytes Lymphocytes Monocytes Neutrophils Eosinophils Basophils Blast cells Unidentified cells
<2 35.0 +_0.66 54.6 +_0.71 1.1 + 0.18 4.7 _+0.24 1.5 __.0.09 0.3 + 0.05 2.6 _+0.17 0.1 ___0.04
90 32.3 + 0.67 a 40.6+ 0.92 a 2.4 + 0.17 c 18.9 + 0.97 ~ 3.6 + 0.20¢ 1.5 + 0.10~ 0.6 + 0.07 ~ 0.1 + 0.04 d
a Values reported as mean + 1 sE ( N = 10). b Significantly different from control group at P<0.05. c Significantly different from control group at P<0.01. NS, not significantly different from control group at P<0.05.
270 31.9 + 0.55 c 40.1 + 0.23 c 2.4 + 0.25 c 19.5 _+1.24~ 4.3 + 0.24 ~ 1.2 + 0.09 c 0.5 + 0.09 ~ 0.1 __.0.04 a
445 31.0 + 0.04 c 41.3 + 1.17c 2.2 + 0.12 ~ 19.8 _+1.20c 4.0 + 0.31 c 1.0 +_0.08 ¢ 0.5 + 0.07 b 0.1 + 0.03 d
146
ing. Granules were large, abundant and coarse in texture. With Leishman-Giemsa the granules o f these cells stained a characteristic brick red color. The least c o m m o n identifiable leucocytes were basophils (0.3 + 0.05%; 0.15 +_ 0.024 × 103 cells/mm3). These were somewhat larger cells (11-14 m#) and typically round to ovoid in profile. Nuclei were usually eccentric, typically crescent-shaped and never lobed, and stained a moderately deep purple. The granular, mesh-like cytoplasm tended to be somewhat vacuolated and moderately basophilic. Granules were c o m m o n , relatively large and distinctly blue-staining. Blast cells were fairly c o m m o n (1.18 _+ 0.084 × 103 cells/mm3; 2.6 _ 0.17% of leucocytes). Over 90°7o of these were negative to Sudan Black B, and presumably lymphoid in commitment. A very small proportion (1 to 2%) displayed the weakly positive responses to PAS and Sudan Black B which are associated with the granulocyte lineage. Their paucity suggested that the granulocytes of goldfish are released to circulation at a comparatively mature stage of development. The remainder of these cells were not assignable to either general category.
Effects of cadmium on leucocyte abundances Exposure to sublethal cadmium concentrations p r o m p t e d sharp reductions in overall white cell numbers (Table I). After 21 days leucocyte counts at 5, 15 and 25% of LCs0 had fallen to 62.8, 50.8 and 50.1% of control values. L y m p h o c y t e numbers declined by - 5 3 . 3 , - 6 2 . 4 and - 6 2 . 0 % to 11.44 + 0.39, 9.21 + 0.48 and 9.31 + 0.45 × 103 cells/mm a. In each instance, analysis of variance revealed that these decreases were significant at the 0.01 level or better. Corresponding reductions for the next most abundant class of white blood cells, the thrombocytes, were - 41.9, - 5 3 . 6 and - 5 5 . 6 % , i.e. 9.15 +_ 0.42, 7.30 + 0.30 and 6.99 ___ 0.25 × 103 cells/mm 3. Again, all changes were significant at the 0.01 level or better. The greatest effect of cadmium was, however, seen in relation to blast cell numbers. At 5% of LCso (90 #g/l) blast cell counts dropped by 85.5% f r o m 1.18 ___ 0.08 to only 0.17 ___ 0.02 × 103 cells/mm 3. Somewhat larger reductions were observed at higher concentrations. Monocyte abundances ranged f r o m 0.48 _+ 0.09 to 0.68 _+ 0.06 × 103 cells/mm 3, but displayed no significant differences. Similarly, while the number of unidentified cells exhibited mean decreases from 0.05 +_ 0.02 to 0.01 + 0.01 x 103 cells/ram 3 these differences were not significant at the 0.05 level. By contrast, the granular leucocytes increased in both absolute and relative numbers. This was particularly evident at 5% of LCso. Some reduction was found at the higher cadmium concentrations but, except for eosinophils, the numbers of cells present remained significantly in excess of corresponding values in the control group. Thus, neutrophil numbers rose from 2.12 +__ 0.10 to 5.30 + 0.29 × 103 cells/mm 3. Eosinophils increased f r o m 0.68 + 0.04 to 1.02 + 0.08 × 103 cells/mm 3 and basophils from 0.15 + 0.02 to 0.41 +_ 0.03.
147
Mitogenic responses Responses of control and cadmium-exposed fish to saline and PHA administration are summarized in Table III. Comparison of pretreatment data with that of the first control group (Tables I and II) revealed no significant differences in either absolute or differential counts. Similarly, goldfish exposed to 15070 of LCs0 in this phase of the study exhibited changes in overall and specific leucocyte numbers which were comparable in kind and magnitude to those previously observed. PHA treatment led to significant changes in leucocyte status, with maximum variation typically found after four days. The effects of PHA were transient. Animals sampled 12 days after PHA treatment did not, in general, differ significantly from those injected with saline. Briefly summarized, normal goldfish appeared to respond to PHA administration with increases in total leucocyte count. This was largely the result of increases in blast cell and lymphocyte levels. Thrombocyte and monocyte numbers displayed relatively little change, while granular leucocyte abundances were variably affected. Assessment of cadmium influence on mitogenic response was, however, complicated by several factors. While differences between PHA-treated and salineinjected animals provide a means for isolating PHA from procedural effects, variable responses were observed. Thus, lymphocyte, thrombocyte and blast cells exhibited qualitatively different responses to PH A than did monocytes and the granular leucocytes. In addition, leucocyte status in cadmium-exposed fish differed significantly from that of the non-exposed animals in a number of respects. Accordingly, to examine cadmium effects, mean differences between PHA- and salinetreated animals were calculated. These are summarized in Fig. 1 for the 4-day period leading to maximum response. PHA appeared to specifically stimulate lymphopoiesis since increases in lymphocyte numbers followed PHA injection. Count differences between control and cadmium-exposed groups were best-fitted by the relationships: Lymphocyte difference = 7.35 (+ 0.54) + 5.410 (+ 0.608) log time (control) = 4.47 (+0.13)+ 3.102 (+0.142) log time (Cd2 +-exposed). Intensity of response to PH A is indicated by slope. That of exposed animals was over 40% less than was the case in the control group, and indicated cadmium suppression of lymphopoietic capacity. Much the same was true of blast cells as well. The slope difference was identical to that encountered with lymphocytes. blast cell difference = 0.83 ( ___0.01) + 1.118 ( ___0.013) log time (control) = 0.32 (+0.13)+0.642 (+0.146) log time (Cd2÷-exposed). PHA also affected thrombocyte formation:
Monocytes
Lymphocytes
Thrombocytes
Total leucocytes
Variable
270
<2
270
<2
270
<2
270
<2
/zg/1
[Cd 2 +]
Saline PHA Saline PHA
Saline PHA Saline PHA
Saline PHA Saline PHA
Saline PHA Saline PHA
Treatment
0.51 +0.06(15) a (1.2 _+0.1307o)b 0.51+_0.04(15) a (2.3 _+0.19°7o)b
24.76_+0.37(15) a (55.3 _+0.58°7o)b 9.30+0.33(15) a (41.1 + 0.91o7o)b
15.60_+0.27(15) a (34.8 -+ 0.49070)b 7.38+0.22(15) ~ (32.6 -+ 0.5307o)b
22.59_+0.50(15) a
44.81_+0.47(15) a
0
0.84+0.06 0.42 _+0.08 0.48-+0.07 0.47 _+0.05
20.44+_0.18 27.55 _+0.44 8.34_+0.08 12.75 + 0.26
14.17_+0.23 15.59 -+ 0.33 7.11+0.08 8.87 -+ 0.22
39.93-+0.23 48.11 --20.11 20.56--20.15 27.26 _+0.48
1
0.82+0.07 0.31 _+0.06 0.58_+0.05 0.70 -+0.09
18.83_+0.13 30.42 _+i).25 7.83_+0.12 14.56 + 0.41
13.86+0.28 15.86 -+ 0.13 6.59_+0.26 9.51 _+0.33
38.40--20.31 51.17--20.10 19.83_+0.44 30.12 _-20.27
2
4
0.85 _ + 0 . 0 8 0.11 _+0.06 0.54_+0.04 0.70 -+ 0.09
17.72_+0.22 32.33 -+ 0.17 8.22+0.12 16.93 + 0.36
13.16_+0.29 15.48 -+0.33 6.73_+0.16 10.92 _+0.28
36.98_+0.34 52.78_+0.20 20.17_+0.17 33.93 _+0.27
Post treatment, days
0.76_+0.03 0.31 _+0.06 0.45-+0.06 0.70 _+0.08
21.16-+0.22 30.88 _+0.16 9.01+0.12 14.94 -+0.18
11.52-+0.34 16.45 _+0.30 7.30_+0.21 20.54 -+ 0.29
41.02_+0.15 51.72+0.28 21.72-+0.15 31.36 _+0.26
8
0.76_+0.08 0.55 _+0.05 0.41_+0.01 0.50 _+0.02
23.38+0.49 25.79 + 0.17 9.20+0.07 10.84 _+0.20
15.31 +0.31 15.18 _+0.63 7.52-+0.27 7.95 _+0.23
43.76-+0.60 45.72-+0.15 21.94-+0.06 23.98 _+0.24
12
Total leucocyte numbers, thrombocytes, lymphocytes, monocytes, blast cells, neutrophils, eosinophils, basophils and unidentified cell types ( × 103/mm 3) in goldfish exposed to cadmium-free ( < 2 #g/l) water or 15°70 of 240-h LCso (270/~g/l) for 3 wk and treated with saline or phytohemagglutinin.
TABLE III
Saline PHA
Saline PHA
Saline PHA
Saline PHA
Saline PHA
Saline PHA
Saline PHA
Saline PHA
<2
270
<2
270
<2
270
< 2
270
270
Saline PHA Saline PHA
< 2
0.03 _+0.01(15) a (0.1 +_0 . 0 3 % ) b
0.05 + 0.02(15) a (0.1 + 0 . 0 4 % ) b
0.24 +- 0.02(15) a (1.0 _+0 . 0 9 % ) b
0.13+0.02(15) a (0.3 + 0 . 0 5 % ) b
0.89+-0.05(15) a (4.0 _+0.21%)b
0.65_+0.04(15) a (1.4_+ 0 . 0 8 % ) b
4.02 -+ 0.22(15) a (17.9-+ 1.03%) b
2.14+_0.07(15) a (4.08 +- 0 . 1 6 % ) b
0.95 _+0.10(15) a (2.1 _+0 . 2 2 % ) ~' 0.24 + 0.05(15) ~ (1.1 + 0 . 2 4 % ) b
a P r e t r e a t m e n t values (mean +- 1 SE ( s a m p l e size)). b Differential c o u n t (mean -+ I sE).
Unidentified
Basophils
Eosinophils
Neutrophils
Blast cell
0.06 -+ 0.03 0.06_+0.06 0.18+0,03 0.14+0.02 0.05 _-!-0.02
0.91 -+ 0.07 0.38-+0.12 0.77 _+0.06 0,79 -+ 0.05 0.09 -+ 0.02 0.13 _+0.03 0.19_+0.03 0.13 +- 0.03 0.06 + 0.02 0.07 _-4-0.03 0.09 + 0.02 0.04-+0.01
0.94 _+0.05 0.11+0.06 0.81 + 0.04 0.66+_0.04 0.16+0.02 0.07 + 0.04 0.17-+0.01 0.14+_0.02 0.03 -+ 0.02 0.04 -+ 0.04 0.04_+0.02 0.04+_0.01
0.98+0.11 0.20 +_0.06 0.85+0.04 0.76 + 0.07 0.16+_0.03 0.07 + 0.03 0.16_+0.02 0.13+0.02 0.04 _+0.02 0.10_+0.06 0.04+_0.02 0.04 _+0.02
0.83 ~ 0.07 0.48 + 0.06 0.80 _+0.08 0.76_+0.03 0.18+0.02 0.13_+0.09 0.15_+0.01 0.13+0.03 0.05 0.03 0.04 0.02
+ 0.02 +_0.03 +_0.02 __.0.01
0.61+0.21 0.80 _+0.08 0.78 +--0.07
2.58_+0.10 1.93--.0.13 3.30-+0.12 3.00_+0.16
3.44 -+ 0.09 1.58_+0.13 3.20+0.17 2.81 -+ 0.22
0.03 +- 0.03 0.03 + 0.02 0.02 + 0.02
0.84--- 0.06
2.16+0.21 3.17+_0.18 3.07_+0.14
2.59+0.09
0.78 -+ 0.02
1.34-+0.13 0.63+0.01
2.97+0.15 1.88+0.08 3.28 _+0.05 3.38_+0.21
0.78 + 0.07
2.67 _+0.09 2.31 + 0 . 7 7 3.13_+0.15 3.37+_0.15
0.62 + 0.03 1.17_+0.09
0.79_+0.07 2.07 + 0.05
0.68 -+ 0.07 3.06 -+ 0.29 0.46 + 0.04 1.73+-0.12
1.10_+0.08
0.73 + 0.05 2,32+_0.18 0.45+_0.10
0.74 _+0.07 1.57+0.14 0.51+-0.04 0.89_+0.07
7~
150
Lymphocytes
Total leucocytes 14 12
12
/~/+
10
o
,os
+
8 6
I/ I! II ii I/ ii i/ I~ H
4 2
# E
6
--
///
4
--
2
--
/i II II II
0
_
II #
Y
0
l
Thrombocytes
o
--
f
Blast
20
g
I.O 0.5 0
N T D-
L Monocytes 0 ~(-- + / j + ~ + -0.5
0
\°"'~-'-o---_~
- 1.0
/: f Eosinophil
"~+~+~+
-0.5 - 10
Neutrophils 0
c
-
-
Basophils
<\\
-0.5
~ +
0
"
\\ "+
- 0.02
-I.0
-0.04
-I.5
-0.06
-2.0
-008
\ ~+ \\
+
L
I
I
I
I
I
I
I
0
I
2
4
0
2
4
Days , post PHA administration Fig. 1. Mean differences (cells × 103/mm 3) in total leucocyte, lymphocyte, thrombocyte, blast cell, monocyte, neutrophil, eosinophil and basophil numbers in phytohemagglutinin- and saline-injected goldfish: control, O; cadmium, + .
151 thrombocyte difference = 1.21 (_+ 0.21) + 0.866 (___0.233) log time (control) = 1.74 ( + 0.04) + 1.753 ( + 0.046) log time (Cd 2 ÷ -exposed). In this instance, however, response was less in the control group than in goldfish exposed to cadmium. Finally, overall leucocyte numbers were affected. Total leucocyte difference = 8.94( _+0.36) + 5.064( _+0.400) log time (control) = 6.74( + 0.05) + 5.093( ___0.050) log time (Cd 2 ÷ -exposed). This largely reflects the difference between effects on the apparent rates of proliferation of the two most prevalent cell types. The granular leucocytes and monocytes responded in the opposite fashion. Difference counts in control fish tended to be negative; i.e. fewer neutrophils, eosinophils and basophils were present in PHA-treated animals than in those injected with saline. With exposure to cadmium, however, differences between these groups were reduced. DISCUSSION As initially noted, reported changes in the leucocyte status of fish exposed to cadmium differ sharply (e.g. Gardner and Yevich, 1970; Srivastava and Misra, 1979). The findings o f the present study support those of Gardner and Yevich (1970). Furthermore, they are consistent with the fact that, in fishes as in higher vertebrates, a variety of challenges invoke similar variations in leucocyte abundances (Ellis 1981). Lymphocytopenia, for example, commonly follows stress imposition, and the occurrence of this condition following exposure to cadmium was not unanticipated. Less expected was the absence of a clear-cut dose-response relationship. Specimens exposed to cadmium concentrations of 90, 270 and 445 #g/l exhibited decreases of 53.3, 62.4 and 62.0% respectively after 3 wk. This argues for a very low threshold for cadmium action, but also suggests that some mechanism(s) may curtail the effect(s) of cadmium. It is tempting to speculate that the latter may stem from lymphocyte heterogeneity, and differences in the cadmium sensitivity of lymphocyte subpopulations (Jurd, 1985; Ruglys, 1985). Reduction in lymphocyte numbers can be brought about in two ways; by decreasing mean cellular life span and through impairment of proliferative capacity. Although these cells are short-lived (Ellis 1977), there is little information on factors affecting their longevity. The observations of the present study do not provide a basis for comment on this possibility. Responses to P H A administration were, however, compatible with cadmium suppression of lymphopoietic capacity. These cells differentiate from the lymphoid lineage. Most of the blast cells observed displayed the histochemical characteristics of lymphoid progenitor cells, and were
152 reduced to a greater extent than any other leucocyte type. Given validity of current views on lymphocyte formation such observations are mutually consistent. Moreover, they point to the probability of even greater reductions in lymphocyte numbers with prolonged exposure. The lymphocytes of fishes are thought to develop from an initial thymic stem cell population which subsequently colonizes other sites, and most notably the spleen and pronephros (Ellis, 1977). Stromberg et al. (1983) observed lesioning of these hematopoietic areas following cadmium exposure, and this also is consistent with observed reductions in lymphocyte abundance and proliferative capacity. The functional consequences of these effects are not entirely clear. Ellis (1977) concluded that existing evidence supported the view that fish lymphocytes were the executive cells of specific immune responses. However, he did not accept the view that lymphocytes were also phagocytic as suggested by Weinreb and Weinreb (1969) and Klontz (1972). Ellis (1977) contended that phagocytosis and immunoactivity are incompatible functions, and suggested that earlier reports of phagocytosis may have involved cell misidentifications. In any event, however, some diminution in resistance to pathogen challenge would be anticipated in fish exposed to even relatively dilute levels of this highly toxic pollutant. The thrombocytes of fishes are functional counterparts of the mammalian platelet, and consequently central to the clotting process. Fange (1968) postulated a phagocytic function as well. Support for this was provided by Ferguson (1976), who found that intravenously administered carbon microparticles subsequently appeared within these cells. However, Ellis (1977) felt that particle uptake was probably attributable to passive entrapment by the extensive peripheral vesicular system. Furthermore, Ellis (1977) also emphasized the difficulties of reconciling functions as disparate as phagocytosis and clot formation in a single cell. The latter argument also has pertinence in the question of thrombocyte origin. Current hypotheses tend, for the most part, to reflect earlier suggestions that thrombocytes differentiate from small lymphocytes (e.g. Gardner and Yevich, 1969) or that both derive from a common stem cell (e.g. Klontz, 1972). That PH A treatment led to proliferation of both lymphocytes and thrombocytes suggests similarities of origin. On the other hand, the opposed effects of cadmium on response to PHA points to differences in differentiation. In any event, thrombocyte numbers declined sharply following exposure to even relatively low levels of cadmium. Some impairment of clot-forming ability, and therefore increased susceptibility to injury would be expected. The relationship between lymphocytes and thrombocytes, the most abundant leucocyte types in fishes is controversial. Watson et al. (1963) reported that lymphocytes constituted 30%o of the white cell population in goldfish. Weinreb and Weinreb (1969) identified 71-82% of the leucocytes of this species as lymphocytes, 3-13%0 as thrombocytes. In sharp contrast to this, Gardner and Yevich (1969) reported that lymphocytes made up only 2-13% of the white cells of three cyprinodont species, thrombocytes from 82-95 %. Reported lymphocyte:thrombocyte ratios
153 vary by an order of magnitude from 25:1 to 2:1. Using immunofluorescent techniques rather than the usual Romanowsky stains Ellis (1976) observed lower lymphocyte:thrombocyte ratios. The lymphocyte and thrombocyte counts obtained in this study for undisturbed control goldfish suggest a ratio of about 1.5:1. The basis o f these discrepancies may lie in the relative fragility of thrombocytes. Gardner and Yevich (1968), who examined living preparations as well as stained smears, noted the readiness with which thrombocytes initiated a cytoplasmic spreading process which quickly led to the formation of long thread-like projections. Smear preparation frequently stripped these away, leaving nuclei largely denuded of cytoplasm and easily mistaken for small lymphocytes. It was for this reason that leucocyte data in this study was accepted only for samples in which thrombocytes of normal morphology were subsequently identified on stained slides. Stress-induced lymphopenia in fishes is commonly accompanied by neutrophilia (Ellis, 1981), and this proved to be the case in the present study. Teleostean neutrophils are mobile and phagocytic and are formed in granulopoietic areas of the kidney (Ellis, 1977). In view of the observations of Stromberg et al. (1983) response to cadmium challenge may be attributable to increased longevity in circulation, release from storage sites or to differential sensitivity of lympho- and granulopoietic cells. The last of these is compatible with observed responses to P H A treatment. Similar considerations also apply to the eosinophilia and basophilia encountered following exposure to cadmium. However, the functional consequences of this are unclear, for the roles played by such cells are not well understood. In summary, leucocyte status in goldfish is sensitive to even low concentrations of cadmium. Total white numbers are reduced, principally as a result of reductions in lymphocyte and thrombocyte numbers. Lymphopoietic capacity is apparently more affected than is the case with thrombocytes. Major increases in neutro-, eosino- and basophit numbers take place, and various ratios relating agranulocyte and granulocyte abundances are sharply altered. For example, E granulocytes:~ agranulocytes rises almost 5-fold, and may be of diagnostic value. From the functional viewpoint, these changes would be compatible with reductions in resistance to injury and pathogen challenge. We are, however, unaware of studies which unequivocally demonstrate that this actually occurs. ACKNOWLEDGEMENTS The financial support provided by the Natural Sciences and Engineering Research Council through Individual Operating Grant A6972 to A.H. Houston is gratefully acknowledged. REFERENCES Christensen, G.M., J.T. Fiandt and B.A. Poeschl, 1978. Cells, proteins and certain physical-chemical properties of brook trout (Salvelinusfontinalis) blood. J. Fish Biol. 12, 51-60.
154 Ellis, A.E., 1976. Leucocytes and related cells in the plaice, Pleuronectes platessa. J. Fish Biol. 8, 143-156. Ellis, A.E., 1977. The leucocytes of fish: a review. J. Fish Biol. 11,453-491. Ellis, A.E., 1981. Stress and the modulation of defence mechanisms in fish. In: Stress and fish, edited by A.P. Pickering, Academic Press, New York, pp. 147-169. Fange, R., 1968. The formation of eosinophilic granulocytes in the eosophygeal lymphomyeloid tissue in the elasmobranchs. Acta Zool. Stockholm 49, 155-161. Ferguson, H.W., 1976. The ultrastructure of plaice leucocytes. J. Fish Biol. 8, 139-142. Gardner, G.A. and P.P. Yevich, 1968. Studies on the blood morphology of three estuarine cyprinodontiform fishes. J. Fish. Res. Board Can. 26, 433-447. Gardner, R.G. and P.P. Yevich, 1970. Histological and hematological responses of an estuarine teleost to cadmium. J. Fish. Res. Board Can. 27, 2185-2196. Houston, A.H., 1982. Thermal effects upon fishes, Nat. Res. Counc. Can. Pub. No. NRCC 18566 of the Environ. Secret., pp. 1-200. Houston, A.H. and J.E. Keen, 1984. Cadmium inhibition of erythropoiesis in goldfish, Carassius auratus. Can. J. Fish. Aquat Sci., 41, 1829-1834. Houston, A.H., C.A.M. McCullough, J. Keen, C. Maddalena and J. Edwards, 1985. Rainbow trout red cells in vitro. Comp. Biochem. Physiol. 81A, 555-565. Johansson-Sjobeck, M.L. and A. Larsson, 1978. The effect of cadmium on the hematology and on the activity of 6-aminolevulinic acid dehydrate (ALA-D) in blood and hematopoietic tissues of flounder, Pleuronectesflesus L. Environ. Res. 17, 191-204. Jurd, R.D. 1985. Specialization in the teleost and anuran immune response: a comparative critique. In: Fish immunology, edited by M.J. Manning and M.F. Tatner, Academic Press, New York, pp. 9-28. Klontz, G.W., 1972. Haematological techniques and the immune response in rainbow trout. In: Diseases of fish, edited by L.E. Mawdesley-Thomas, Academic Press, New York, pp. 89-99. McCarthy, L.S., A.H. Houston and J.A.C. Henry, 1978. Toxicity of cadmium to goldfish, Carassius auratus, in hard and soft water. J. Fish. Res. Board Can. 45, 35-42. Ruglys, M.P., 1985. lmmunosuppression and immunological tolerance in carp. In: Fish immunology, edited by M.J. Manning and M.F. Tatner, Academic Press, New York, pp. 357-368. Sokal, R.R. and F.J. Rohlf, 1969. Biometry. W.H. Freeman and Co., San Francisco. Srivastava, A.K. and S. Mishra, 1979. Blood dyscrasia in a teleost fish, Colisafasciatus, associated with cadmium poisoning. J. Comp. Pathol. 89, 1-5. Stromberg, P.C., J.G. Ferrante and S. Carter, 1983. Pathology of lethal and sublethal exposure of fathead minnows, Pimephalespromelas, to cadmium: a model for aquatic toxicity assessment. J. Toxicol. Environ. Health 11,247-259. Watson, L.J., I.L. Schechmeister and L.L. Jackson, 1963. The haematology of goldfish (Carassius auratus). Cytologia 28, 118-130. Weinreb, E.L., 1963. Studies on the fine structure of teleost blood cells. I. Perhipheral blood. Anat. Rec. 147, 219-238. Weinreb, E.L. and S. Weinreb, 1969. A study of experimentally induced endocytosis in a teleost. I. Light microscopy of peripheral blood cell response. Zoologica N.Y. 54, 25-34.
141
AQT 00293
Leucocytes and leucopoietic capacity in goldfish, Carassius auratus, exposed to sublethal levels of cadmium A. Murad and A.H. Houston Department of Biological Sciences, Brock University, St. Catharines, Canada (Received 8 October 1987; revision received 1 April 1988; accepted after revision 4 April 1988) By comparison with animals in essentially cadmium-free water (< 2 ~tg Cd2÷/l) goldfish exposed for 3 wk to 90, 270 and 445 #g Cd2+/l (5, 15 and 25070 240-h LCso) exhibited significant reductions in total leucocyte counts. These were the result of decreases in lymphocyte and thrombocyte numbers. Mitogenic response to administered PHA as well as sharp decreases in blast cell numbers suggested that lympho- and thrombopenia reflect, in part at least, decreased proliferative capacity. By contrast, neutrophil, eosinophil and basophil numbers increased in cadmium-intoxicated fish. Cadmium apparently reduced PHA-related changes in granulocyte abundances. Key words: Goldfish; Carassius auratus; Cadmium; Leucocyte INTRODUCTION Significant c a d m i u m - i n d u c e d h e m a t o l o g i c a l effects can be detected in f r e s h w a t e r a n d m a r i n e fishes c h r o n i c a l l y e x p o s e d to low levels o f this e x t r e m e l y toxic h e a v y m e t a l . R e d u c t i o n s in b l o o d O2-carrying c a p a c i t y were, for e x a m p l e , o b s e r v e d b y J o h a n s s o n - S j o b e c k a n d L a r s s o n (1978) in f l o u n d e r , Pleuronectesflesus, e x p o s e d to as little as 5 # g / l . S t r o m b e r g et al. (1983) r e p o r t e d persistent lesioning o f h e m a t o p o i e t i c sites in f a t h e a d m i n n o w s , Pimephales promelas, e x p o s e d for 96 h to 12 p p m C D 2÷ . C o n s i s t e n t with this, H o u s t o n a n d Keen (1984) f o u n d significant, d o s e - d e p e n d e n t i m p a i r m e n t o f e r y t h r o p o i e t i c c a p a c i t y in g o l d f i s h , Carassius auratus, c h r o n i c a l l y e x p o s e d to c a d m i u m c o n c e n t r a t i o n s e q u i v a l e n t to 1-25°/0 o f the s o f t w a t e r 240-h LCso ( M c C a r t y et al., 1978). A l t h o u g h c o r r e s p o n d i n g studies o n leucocytes have been c a r r i e d out, their results are in conflict. G a r d n e r a n d Yevich (1970), for e x a m p l e , r e p o r t e d t h a t e x p o s u r e o f m u m m i c h o g , Fundulus heteroclitus, to 50% o f their 48-h LCso for up to 48 h p r o -
Correspondenceto: A.H. Houston, Department of Biological Sciences, Brock University, St. Catharines, Canada L2S 3AI. 0378-4274/88/$ 03.50 © Elsevier Science Publishers B.V. (Biomedical Division)
142 duced marked and progressive decreases in differential lymphocyte and thrombocyte counts with major increases in the overall granulocyte differential count. This was accompanied by increased incidence of morphological aberrances including nuclear deformation, extensive cytoplasmic vacuolization and reductions in the number and size of cytoplasmic granules and other inclusions. In sharp contrast to this, Srivastava and Mishra (1979) recorded increases of 50-85°7o in total leucocyte, thrombocyte and small and large lymphocyte numbers in Colisa fasciatus held for 90 h at 80°70 of their 96-h LCs0. In the present study, we examined the effects of 3-wk exposures to cadmium concentrations equivalent to 5, 15 and 25°7o of the 240-h LCso on absolute numbers and differential counts of thrombocytes and agranular and granular leucocytes in goldfish. In addition, consideration was given to the effects of cadmium intoxification on lymphopoietic capacity as measured by response to i.p. administration of the mitogenic agent, phytohemagglutinin (PHA). MATERIALS AND METHODS Goldfish ranging in weight from 7.8-36.2 g (mean ___ 1 SEM = 17.1 + 0.48 g) were obtained from a commercial supplier (Grassyforks Fisheries Co., Indiana). On arrival at the laboratory they were initially held for a 3-wk period in 600-1 fiberglass tanks equipped with high capacity ( - 5 1/min) styrofoam activated-charcoal filters. Duty-operated thermoregulators linked to refrigeration and heating units held water temperatures at 25.0 + 0.2°C, a widely accepted value for the thermal optimum of this species (Houston, 1982). Supplementary aeration provided oxygen levels > 6.8 mg/l or 80°70 saturation. Light-tight hoods incorporating timers accurate to +_ 7 min/day (corrected daily), and providing water surface light intensities of 90-120 Lx were used to establish a 12 h light:12 h darkness photoperiod regime. Animals were fed daily to satiation on a commercial ration (Purina Trout Chow), and excess food and fecal materials removed after cessation of feeding. As judged by general activity, feeding behaviour and the absence of obvious disease symptoms all were healthy during the study period. The cadmium content of dechlorinated St. Catharines city water, as determined by atomic absorption spectrophotometry, is relatively low (< 10/xg/l) as are zinc and copper levels (< 50 #g/l). However, pH (7.6-8.0) and total hardness (140-150 mg/1, as CaCo3) are such that added cadmium is rapidly lost a s C d C O 3 , Cd(OH)2 and other insoluble precipitates. Earlier studies involving cadmium loads of 1-60 mg/1, for example, revealed exponential decreases in dissolved cadmium concentrations to negligible levels within 24-48 h (McCarty et al., 1978). Hardness and conductivity increased in proportion to the amount of cadmium added, while total alkalinity and pH fell transiently, the latter by as much as 1.0-1.2 pH units. To avoid the complications associated with such changes this investigation was conducted with water of reduced hardness prepared by the dilution of aged and
143 dechlorinated city water with deionized, glass-distilled water. This had the following characteristics - total hardness: 20-22 mg/1 as CaCO3; total alkalinity: 14-18 mg/l as CaCO3; conductivity: 15-25/~mho/cm; pH: 7.4-7.8, cadmium: < 2 #g/l. Under such circumstances there is little precipitation and changes in water quality following cadmium addition are reduced (McCarty et al., 1978). Dissolved oxygen, total hardness and alkalinity, conductivity and pH were measured on alternate days throughout the study period. After the initial holding period, fish were transferred to 100-1 fiberglass test tanks containing softened water. Other conditions were as previously described. A period of 1 wk was provided for recovery from any transfer stresses. Aqueous analytical grade CdC12 was then added to bring test tank concentrations to 90, 270 and 445 /zg/1, i.e. 5%, 15% and 25% of the 240-h soft water LCso for this species (McCarthy et al., 1978). Following 3-wk exposures to these conditions all specimens were sampled. The study was replicated to provide samples of 10 to 12 animals at each test concentration. The effects of cadmium on leucopoiesis were investigated by reference to PHAinduced mitogenic response. Preliminary trials revealed that PHA administered intraperitoneally in 0.1 ml of modified Cortland saline (Houston et al., 1985) at dosages of 3-8/~g/g body weight elicited consistently detectable changes in total and differential leucocyte counts within 1 wk in goldfish ranging from 11.6-30.5 g in weight. Smaller amounts did not produce consistent results, while dosages exceeding approximately 10/zg/g body weight did not increase magnitude of response. The effective dose range could be achieved, with attention to specimen weight, by administration of 100 ttg PHA/specimen. Investigations on leucopoietic capacity were confined to the examination of animals exposed for 3 wk to 15% of the 240-h LCso (i.e., 270 #g Cd 2÷/1). A similar group was held during the same period in nominally cadmium-free water (i.e., < 2/zg Cd 2 ÷/1) under comparable conditions. One half of each group received PHA, the remainder an equivalent volume of saline. Sampling was carried out immediately prior to treatment (0 h) and 1, 2, 4, 8 and 12 days after injection. To avoid possible induction of artefacts by exposure to chemical anesthetics, specimens were stunned before sampling by a blow to the juncture of the cranium and vertebral column. The caudal peduncle was descaled, cleansed with alcohol and transected. Blood was quickly drawn by capillary action directly into anticoagulanttreated sampling tubes, the complete procedure requiring less than three minutes. Smears were immediately prepared, air-dried and fixed in acetone-free methanol for 5 min. These were subsequently stained with Leishman-Giemsa, Sudan Black-B or the periodic acid Schiff procedure (Christensen et al., 1978; Sigma Diagnostics Procedure Bulletins 380, 395, 1984). Leucocytes were identified by the morphological and staining characteristics described by Weinreb (1963), Weinreb and Weinreb (1969), Ellis (1977) and Christensen et al. (1978). Slides were examined at x 1000, and a minimum of two smears and 500 leucocytes considered for each specimen.
144 Total
leucocyte
hemocytometer.
counts
Whole
were
blood
made
in
was diluted
triplicate
using
1:200 with modified
the
Neubauer
Cortland
saline
( H o u s t o n et al., 1985) c o n t a i n i n g 20 m g / 1 W r i g h t ' s s t a i n . C o u n t s w e r e a c c e p t e d o n l y when subsequent examination of fixed slides revealed thrombocytes
of normal mor-
phology. All statistical treatments were based on procedures outlined by Sokal and Rohlf (1969). D e s c r i p t i v e s t a t i s t i c s w e r e b a s e d o n t h e u s e o f u n t r a n s f o r m e d Kolmogorov-Smirnov
test for normality
Barlett's test for homogeneity
of distribution
data. The
was routinely employed.
o f v a r i a n c e s w a s u s e d p r i o r t o all c o m p a r i s o n s ,
and
modified variance analyses used where necessary. RESULTS
Leucocyte types and abundances Thrombocytes, immature
lymphocytes, monocytes, neutrophils, eosinophils, basophils and
o r b l a s t cells w e r e i d e n t i f i a b l e w i t h o u t a m b i g u i t y o n m o s t o f t h e s m e a r s
examined. However,
s m a l l n u m b e r s o f cells c o u l d n o t b e p o s i t i v e l y i d e n t i f i e d , a n d
t h e s e h a v e b e e n r e c o r d e d as s u c h . In cadmium-free water lymphocytes were the most abundant
cell t y p e , c o m p r i s i n g
5 4 . 6 _+ 0 . 7 1 % o f t h e t o t a l c o u n t o f 4 4 . 9 2 + 0 . 7 1 7 cells × 1 0 3 / m m 3 ( T a b l e s I a n d II). T h e s e cells w e r e t y p i c a l l y c i r c u l a r t o o v o i d i n p r o f i l e a n d r a n g e d f r o m 4 t o 9 m/~ in largest dimension. The usually somewhat eccentric nucleus occupied most of the cell m a s s , a n d s t a i n e d d e e p l y p u r p l e . C y t o p l a s m orange. Pseudopodia
was normally light blue to slightly
w e r e e v i d e n t i n s o m e cells.
TABLE I Absolute leucocyte levels (cells × 103/mm 3) in goldfish exposed for 3 wk to < 2, 90, 270 and 445 /xg C2+/1 (i.e., 0.5, 15 and 25o70 of 240-h LCs0) a. Weight, g Cadmium Total count Thrombocytes Lymphocytes Monocytes Neutrophils Eosinophils Basophils Blast cells Unidentified cells
19.8 4- 1.51 <2
20.0 +_2.01 90
21.4 + 1.80 270
24.7 _+2.32 445
44.92 _+0.72 15.74 + 0.39 24.51 _+0.50 0.48 4- 0.09 2.12 4- 0.10 0.68 4- 0.04 0.15 4- 0.02 1.18 4- 0.08 0.05 4- 0.02
28.22 _+0.92 b 9.15 + 0.42 b 11.44 _+0.39 b 0.68 4- 0.09 d 5.30 4- 0.29 b 1.02 4- 0.08 b 0.41 +_0.038 0.17 4- 0.028 0.03 4- 0.01 d
22.85 + 0.71 b 7.30 _+0.30 b 9.21 + 0.48 b 0.55 4- 0.06 d 4.42 4- 0.25 b 0.97 4- 0.05 b 0.27 4- 0.02 c 0.11 4- 0.02 b 0.03 4- 0.01 d
22.52 + 0.72 b 6.99 _+0.25 b 9.31 4- 0.45 b 0.50 + 0.03 d 4.45 4- 0.29 b 0.85 4- 0.09 d 0.23 4- 0.0U 0.12 4- 0.01 b 0.01 4- 0.01 d
a Values reported as mean _+ 1 SE (N= 10). b Significantly different from control group at P<0.05. c Significantly different from control group at P < 0.01. a NS, not significantly different from control group at P<0.05.
145 T h e t h r o m b o c y t e s o f g o l d f i s h a r e e l o n g a t e , b u t v a r i a b l e in p r o f i l e , r a n g i n g f r o m o v a l t o o v a l - w i t h - t a i l - l i k e - p r o j e c t i o n s t o s p i n d l e s h a p e d . C o n s i d e r a b l e v a r i a t i o n in size was a l s o e v i d e n t , a n d m a j o r axis l e n g t h s o f u p t o 15 m # w e r e r e c o r d e d . N u c l e i w e r e d i s t i n c t l y e l o n g a t e d , r a n g i n g f r o m 4.5 t o 6.5 m # in l e n g t h a n d o c c u p y i n g m u c h o f t h e cell. A s v i s u a l i z e d b y R o m a n o w s k y s t a i n i n g p r o c e d u r e s n u c l e i w e r e i n t e n s e l y b l u i s h - p u r p l e , t h e c y t o p l a s m a l i g h t e r b l u i s h h u e . T h e 15.74 _+ 0.39 × 1 0 3 / m m 3 t h r o m b o c y t e s f o u n d in t h e c o n t r o l g r o u p s o f g o l d f i s h c o n s t i t u t e d 35.0 + 0 . 6 6 % o f the leucocyte population. Monocytes were uncommon,
a n d w e r e n o t i d e n t i f i e d in all p r e p a r a t i o n s .
On
a v e r a g e 0.48 _+ 0 . 8 6 ___ 103 c e l l s / m m 3 w e r e p r e s e n t (1.1 _+ 0.1807o). T h e s e w e r e l a r g e , r o u n d t o o v o i d cells r a n g i n g f r o m 12 t o 15 mtt in d i a m e t e r . N u c l e a r : c y t o p l a s m i c r a t i o s w e r e a p p r o x i m a t e l y 0.5. C y t o p l a s m was b l u e - g r a y , a n d o c c a s i o n a l l y e v i d e n c ed p s e u d o p o d - l i k e s t r u c t u r e s . V a c u o l e s w e r e u s u a l l y p r e s e n t , b u t v a r i e d in size a n d n u m b e r . I n s o m e i n s t a n c e s , t h e i r b r a i n - s h a p e d o r r e n i f o r m n u c l e i also a p p e a r e d to be vacuolated. N e u t r o p h i l s w e r e t h e m o s t c o m m o n o f t h e g r a n u l o c y t e t y p e s (2.12 + 0.103 × 10 ~ c e l l s / m m 3 ; 4.7 ___ 0.24o7o o f w h i t e cell p o p u l a t i o n ) . T h e s e cells w e r e n o r m a l l y 10-12 m ~ in d i a m e t e r . T h e i r f r e q u e n t l y e c c e n t r i c n u c l e i v a r i e d f r o m k i d n e y - s h a p e d to r i b b o n - l i k e in p r o f i l e w i t h f r o m 2 to 4 l o b e s e v i d e n t in m a n y cells. T h e f i n e l y granular cytoplasm was weakly acidophilic, strands and many neutrophilic granules.
with weakly basophilic regions or
E o s i n o p h i l s c o n s t i t u t e d 1.5 × 0 . 0 9 % o f t h e l e u c o c y t e p o p u l a t i o n (0.68 _+ 0.042 × 103 c e l l s / m m 3) a n d w e r e e v i d e n t in all s p e c i m e n s . T h e s e w e r e t h e s m a l l e s t o f t h e g r a n u l o c y t e s ; t h e i r r o u n d to s o m e w h a t o v o i d p r o f i l e s m e a s u r i n g f r o m 7 t o 9 m # . N u c l e i w e r e r e l a t i v e l y l a r g e , r o u n d to c r e s c e n t i c b u t n e v e r l o b u l a r a n d p u r p l e stain-
TABLE II Differential leucocyte counts (°7o of total white cell population) in goldfish exposed for 3 wk to <2, 90, 270 and 445/zg Cd/1 (i.e., 0, 5, 15 and 25% of 240-h LCs0)a. Cadmium Thrombocytes Lymphocytes Monocytes Neutrophils Eosinophils Basophils Blast cells Unidentified cells
<2 35.0 +_0.66 54.6 +_0.71 1.1 + 0.18 4.7 _+0.24 1.5 __.0.09 0.3 + 0.05 2.6 _+0.17 0.1 ___0.04
90 32.3 + 0.67 a 40.6+ 0.92 a 2.4 + 0.17 c 18.9 + 0.97 ~ 3.6 + 0.20¢ 1.5 + 0.10~ 0.6 + 0.07 ~ 0.1 + 0.04 d
a Values reported as mean + 1 sE ( N = 10). b Significantly different from control group at P<0.05. c Significantly different from control group at P<0.01. NS, not significantly different from control group at P<0.05.
270 31.9 + 0.55 c 40.1 + 0.23 c 2.4 + 0.25 c 19.5 _+1.24~ 4.3 + 0.24 ~ 1.2 + 0.09 c 0.5 + 0.09 ~ 0.1 __.0.04 a
445 31.0 + 0.04 c 41.3 + 1.17c 2.2 + 0.12 ~ 19.8 _+1.20c 4.0 + 0.31 c 1.0 +_0.08 ¢ 0.5 + 0.07 b 0.1 + 0.03 d
146
ing. Granules were large, abundant and coarse in texture. With Leishman-Giemsa the granules o f these cells stained a characteristic brick red color. The least c o m m o n identifiable leucocytes were basophils (0.3 + 0.05%; 0.15 +_ 0.024 × 103 cells/mm3). These were somewhat larger cells (11-14 m#) and typically round to ovoid in profile. Nuclei were usually eccentric, typically crescent-shaped and never lobed, and stained a moderately deep purple. The granular, mesh-like cytoplasm tended to be somewhat vacuolated and moderately basophilic. Granules were c o m m o n , relatively large and distinctly blue-staining. Blast cells were fairly c o m m o n (1.18 _+ 0.084 × 103 cells/mm3; 2.6 _ 0.17% of leucocytes). Over 90°7o of these were negative to Sudan Black B, and presumably lymphoid in commitment. A very small proportion (1 to 2%) displayed the weakly positive responses to PAS and Sudan Black B which are associated with the granulocyte lineage. Their paucity suggested that the granulocytes of goldfish are released to circulation at a comparatively mature stage of development. The remainder of these cells were not assignable to either general category.
Effects of cadmium on leucocyte abundances Exposure to sublethal cadmium concentrations p r o m p t e d sharp reductions in overall white cell numbers (Table I). After 21 days leucocyte counts at 5, 15 and 25% of LCs0 had fallen to 62.8, 50.8 and 50.1% of control values. L y m p h o c y t e numbers declined by - 5 3 . 3 , - 6 2 . 4 and - 6 2 . 0 % to 11.44 + 0.39, 9.21 + 0.48 and 9.31 + 0.45 × 103 cells/mm a. In each instance, analysis of variance revealed that these decreases were significant at the 0.01 level or better. Corresponding reductions for the next most abundant class of white blood cells, the thrombocytes, were - 41.9, - 5 3 . 6 and - 5 5 . 6 % , i.e. 9.15 +_ 0.42, 7.30 + 0.30 and 6.99 ___ 0.25 × 103 cells/mm 3. Again, all changes were significant at the 0.01 level or better. The greatest effect of cadmium was, however, seen in relation to blast cell numbers. At 5% of LCso (90 #g/l) blast cell counts dropped by 85.5% f r o m 1.18 ___ 0.08 to only 0.17 ___ 0.02 × 103 cells/mm 3. Somewhat larger reductions were observed at higher concentrations. Monocyte abundances ranged f r o m 0.48 _+ 0.09 to 0.68 _+ 0.06 × 103 cells/mm 3, but displayed no significant differences. Similarly, while the number of unidentified cells exhibited mean decreases from 0.05 +_ 0.02 to 0.01 + 0.01 x 103 cells/ram 3 these differences were not significant at the 0.05 level. By contrast, the granular leucocytes increased in both absolute and relative numbers. This was particularly evident at 5% of LCso. Some reduction was found at the higher cadmium concentrations but, except for eosinophils, the numbers of cells present remained significantly in excess of corresponding values in the control group. Thus, neutrophil numbers rose from 2.12 +__ 0.10 to 5.30 + 0.29 × 103 cells/mm 3. Eosinophils increased f r o m 0.68 + 0.04 to 1.02 + 0.08 × 103 cells/mm 3 and basophils from 0.15 + 0.02 to 0.41 +_ 0.03.
147
Mitogenic responses Responses of control and cadmium-exposed fish to saline and PHA administration are summarized in Table III. Comparison of pretreatment data with that of the first control group (Tables I and II) revealed no significant differences in either absolute or differential counts. Similarly, goldfish exposed to 15070 of LCs0 in this phase of the study exhibited changes in overall and specific leucocyte numbers which were comparable in kind and magnitude to those previously observed. PHA treatment led to significant changes in leucocyte status, with maximum variation typically found after four days. The effects of PHA were transient. Animals sampled 12 days after PHA treatment did not, in general, differ significantly from those injected with saline. Briefly summarized, normal goldfish appeared to respond to PHA administration with increases in total leucocyte count. This was largely the result of increases in blast cell and lymphocyte levels. Thrombocyte and monocyte numbers displayed relatively little change, while granular leucocyte abundances were variably affected. Assessment of cadmium influence on mitogenic response was, however, complicated by several factors. While differences between PHA-treated and salineinjected animals provide a means for isolating PHA from procedural effects, variable responses were observed. Thus, lymphocyte, thrombocyte and blast cells exhibited qualitatively different responses to PH A than did monocytes and the granular leucocytes. In addition, leucocyte status in cadmium-exposed fish differed significantly from that of the non-exposed animals in a number of respects. Accordingly, to examine cadmium effects, mean differences between PHA- and salinetreated animals were calculated. These are summarized in Fig. 1 for the 4-day period leading to maximum response. PHA appeared to specifically stimulate lymphopoiesis since increases in lymphocyte numbers followed PHA injection. Count differences between control and cadmium-exposed groups were best-fitted by the relationships: Lymphocyte difference = 7.35 (+ 0.54) + 5.410 (+ 0.608) log time (control) = 4.47 (+0.13)+ 3.102 (+0.142) log time (Cd2 +-exposed). Intensity of response to PH A is indicated by slope. That of exposed animals was over 40% less than was the case in the control group, and indicated cadmium suppression of lymphopoietic capacity. Much the same was true of blast cells as well. The slope difference was identical to that encountered with lymphocytes. blast cell difference = 0.83 ( ___0.01) + 1.118 ( ___0.013) log time (control) = 0.32 (+0.13)+0.642 (+0.146) log time (Cd2÷-exposed). PHA also affected thrombocyte formation:
Monocytes
Lymphocytes
Thrombocytes
Total leucocytes
Variable
270
<2
270
<2
270
<2
270
<2
/zg/1
[Cd 2 +]
Saline PHA Saline PHA
Saline PHA Saline PHA
Saline PHA Saline PHA
Saline PHA Saline PHA
Treatment
0.51 +0.06(15) a (1.2 _+0.1307o)b 0.51+_0.04(15) a (2.3 _+0.19°7o)b
24.76_+0.37(15) a (55.3 _+0.58°7o)b 9.30+0.33(15) a (41.1 + 0.91o7o)b
15.60_+0.27(15) a (34.8 -+ 0.49070)b 7.38+0.22(15) ~ (32.6 -+ 0.5307o)b
22.59_+0.50(15) a
44.81_+0.47(15) a
0
0.84+0.06 0.42 _+0.08 0.48-+0.07 0.47 _+0.05
20.44+_0.18 27.55 _+0.44 8.34_+0.08 12.75 + 0.26
14.17_+0.23 15.59 -+ 0.33 7.11+0.08 8.87 -+ 0.22
39.93-+0.23 48.11 --20.11 20.56--20.15 27.26 _+0.48
1
0.82+0.07 0.31 _+0.06 0.58_+0.05 0.70 -+0.09
18.83_+0.13 30.42 _+i).25 7.83_+0.12 14.56 + 0.41
13.86+0.28 15.86 -+ 0.13 6.59_+0.26 9.51 _+0.33
38.40--20.31 51.17--20.10 19.83_+0.44 30.12 _-20.27
2
4
0.85 _ + 0 . 0 8 0.11 _+0.06 0.54_+0.04 0.70 -+ 0.09
17.72_+0.22 32.33 -+ 0.17 8.22+0.12 16.93 + 0.36
13.16_+0.29 15.48 -+0.33 6.73_+0.16 10.92 _+0.28
36.98_+0.34 52.78_+0.20 20.17_+0.17 33.93 _+0.27
Post treatment, days
0.76_+0.03 0.31 _+0.06 0.45-+0.06 0.70 _+0.08
21.16-+0.22 30.88 _+0.16 9.01+0.12 14.94 -+0.18
11.52-+0.34 16.45 _+0.30 7.30_+0.21 20.54 -+ 0.29
41.02_+0.15 51.72+0.28 21.72-+0.15 31.36 _+0.26
8
0.76_+0.08 0.55 _+0.05 0.41_+0.01 0.50 _+0.02
23.38+0.49 25.79 + 0.17 9.20+0.07 10.84 _+0.20
15.31 +0.31 15.18 _+0.63 7.52-+0.27 7.95 _+0.23
43.76-+0.60 45.72-+0.15 21.94-+0.06 23.98 _+0.24
12
Total leucocyte numbers, thrombocytes, lymphocytes, monocytes, blast cells, neutrophils, eosinophils, basophils and unidentified cell types ( × 103/mm 3) in goldfish exposed to cadmium-free ( < 2 #g/l) water or 15°70 of 240-h LCso (270/~g/l) for 3 wk and treated with saline or phytohemagglutinin.
TABLE III
Saline PHA
Saline PHA
Saline PHA
Saline PHA
Saline PHA
Saline PHA
Saline PHA
Saline PHA
<2
270
<2
270
<2
270
< 2
270
270
Saline PHA Saline PHA
< 2
0.03 _+0.01(15) a (0.1 +_0 . 0 3 % ) b
0.05 + 0.02(15) a (0.1 + 0 . 0 4 % ) b
0.24 +- 0.02(15) a (1.0 _+0 . 0 9 % ) b
0.13+0.02(15) a (0.3 + 0 . 0 5 % ) b
0.89+-0.05(15) a (4.0 _+0.21%)b
0.65_+0.04(15) a (1.4_+ 0 . 0 8 % ) b
4.02 -+ 0.22(15) a (17.9-+ 1.03%) b
2.14+_0.07(15) a (4.08 +- 0 . 1 6 % ) b
0.95 _+0.10(15) a (2.1 _+0 . 2 2 % ) ~' 0.24 + 0.05(15) ~ (1.1 + 0 . 2 4 % ) b
a P r e t r e a t m e n t values (mean +- 1 SE ( s a m p l e size)). b Differential c o u n t (mean -+ I sE).
Unidentified
Basophils
Eosinophils
Neutrophils
Blast cell
0.06 -+ 0.03 0.06_+0.06 0.18+0,03 0.14+0.02 0.05 _-!-0.02
0.91 -+ 0.07 0.38-+0.12 0.77 _+0.06 0,79 -+ 0.05 0.09 -+ 0.02 0.13 _+0.03 0.19_+0.03 0.13 +- 0.03 0.06 + 0.02 0.07 _-4-0.03 0.09 + 0.02 0.04-+0.01
0.94 _+0.05 0.11+0.06 0.81 + 0.04 0.66+_0.04 0.16+0.02 0.07 + 0.04 0.17-+0.01 0.14+_0.02 0.03 -+ 0.02 0.04 -+ 0.04 0.04_+0.02 0.04+_0.01
0.98+0.11 0.20 +_0.06 0.85+0.04 0.76 + 0.07 0.16+_0.03 0.07 + 0.03 0.16_+0.02 0.13+0.02 0.04 _+0.02 0.10_+0.06 0.04+_0.02 0.04 _+0.02
0.83 ~ 0.07 0.48 + 0.06 0.80 _+0.08 0.76_+0.03 0.18+0.02 0.13_+0.09 0.15_+0.01 0.13+0.03 0.05 0.03 0.04 0.02
+ 0.02 +_0.03 +_0.02 __.0.01
0.61+0.21 0.80 _+0.08 0.78 +--0.07
2.58_+0.10 1.93--.0.13 3.30-+0.12 3.00_+0.16
3.44 -+ 0.09 1.58_+0.13 3.20+0.17 2.81 -+ 0.22
0.03 +- 0.03 0.03 + 0.02 0.02 + 0.02
0.84--- 0.06
2.16+0.21 3.17+_0.18 3.07_+0.14
2.59+0.09
0.78 -+ 0.02
1.34-+0.13 0.63+0.01
2.97+0.15 1.88+0.08 3.28 _+0.05 3.38_+0.21
0.78 + 0.07
2.67 _+0.09 2.31 + 0 . 7 7 3.13_+0.15 3.37+_0.15
0.62 + 0.03 1.17_+0.09
0.79_+0.07 2.07 + 0.05
0.68 -+ 0.07 3.06 -+ 0.29 0.46 + 0.04 1.73+-0.12
1.10_+0.08
0.73 + 0.05 2,32+_0.18 0.45+_0.10
0.74 _+0.07 1.57+0.14 0.51+-0.04 0.89_+0.07
7~
150
Lymphocytes
Total leucocytes 14 12
12
/~/+
10
o
,os
+
8 6
I/ I! II ii I/ ii i/ I~ H
4 2
# E
6
--
///
4
--
2
--
/i II II II
0
_
II #
Y
0
l
Thrombocytes
o
--
f
Blast
20
g
I.O 0.5 0
N T D-
L Monocytes 0 ~(-- + / j + ~ + -0.5
0
\°"'~-'-o---_~
- 1.0
/: f Eosinophil
"~+~+~+
-0.5 - 10
Neutrophils 0
c
-
-
Basophils
<\\
-0.5
~ +
0
"
\\ "+
- 0.02
-I.0
-0.04
-I.5
-0.06
-2.0
-008
\ ~+ \\
+
L
I
I
I
I
I
I
I
0
I
2
4
0
2
4
Days , post PHA administration Fig. 1. Mean differences (cells × 103/mm 3) in total leucocyte, lymphocyte, thrombocyte, blast cell, monocyte, neutrophil, eosinophil and basophil numbers in phytohemagglutinin- and saline-injected goldfish: control, O; cadmium, + .
151 thrombocyte difference = 1.21 (_+ 0.21) + 0.866 (___0.233) log time (control) = 1.74 ( + 0.04) + 1.753 ( + 0.046) log time (Cd 2 ÷ -exposed). In this instance, however, response was less in the control group than in goldfish exposed to cadmium. Finally, overall leucocyte numbers were affected. Total leucocyte difference = 8.94( _+0.36) + 5.064( _+0.400) log time (control) = 6.74( + 0.05) + 5.093( ___0.050) log time (Cd 2 ÷ -exposed). This largely reflects the difference between effects on the apparent rates of proliferation of the two most prevalent cell types. The granular leucocytes and monocytes responded in the opposite fashion. Difference counts in control fish tended to be negative; i.e. fewer neutrophils, eosinophils and basophils were present in PHA-treated animals than in those injected with saline. With exposure to cadmium, however, differences between these groups were reduced. DISCUSSION As initially noted, reported changes in the leucocyte status of fish exposed to cadmium differ sharply (e.g. Gardner and Yevich, 1970; Srivastava and Misra, 1979). The findings o f the present study support those of Gardner and Yevich (1970). Furthermore, they are consistent with the fact that, in fishes as in higher vertebrates, a variety of challenges invoke similar variations in leucocyte abundances (Ellis 1981). Lymphocytopenia, for example, commonly follows stress imposition, and the occurrence of this condition following exposure to cadmium was not unanticipated. Less expected was the absence of a clear-cut dose-response relationship. Specimens exposed to cadmium concentrations of 90, 270 and 445 #g/l exhibited decreases of 53.3, 62.4 and 62.0% respectively after 3 wk. This argues for a very low threshold for cadmium action, but also suggests that some mechanism(s) may curtail the effect(s) of cadmium. It is tempting to speculate that the latter may stem from lymphocyte heterogeneity, and differences in the cadmium sensitivity of lymphocyte subpopulations (Jurd, 1985; Ruglys, 1985). Reduction in lymphocyte numbers can be brought about in two ways; by decreasing mean cellular life span and through impairment of proliferative capacity. Although these cells are short-lived (Ellis 1977), there is little information on factors affecting their longevity. The observations of the present study do not provide a basis for comment on this possibility. Responses to P H A administration were, however, compatible with cadmium suppression of lymphopoietic capacity. These cells differentiate from the lymphoid lineage. Most of the blast cells observed displayed the histochemical characteristics of lymphoid progenitor cells, and were
152 reduced to a greater extent than any other leucocyte type. Given validity of current views on lymphocyte formation such observations are mutually consistent. Moreover, they point to the probability of even greater reductions in lymphocyte numbers with prolonged exposure. The lymphocytes of fishes are thought to develop from an initial thymic stem cell population which subsequently colonizes other sites, and most notably the spleen and pronephros (Ellis, 1977). Stromberg et al. (1983) observed lesioning of these hematopoietic areas following cadmium exposure, and this also is consistent with observed reductions in lymphocyte abundance and proliferative capacity. The functional consequences of these effects are not entirely clear. Ellis (1977) concluded that existing evidence supported the view that fish lymphocytes were the executive cells of specific immune responses. However, he did not accept the view that lymphocytes were also phagocytic as suggested by Weinreb and Weinreb (1969) and Klontz (1972). Ellis (1977) contended that phagocytosis and immunoactivity are incompatible functions, and suggested that earlier reports of phagocytosis may have involved cell misidentifications. In any event, however, some diminution in resistance to pathogen challenge would be anticipated in fish exposed to even relatively dilute levels of this highly toxic pollutant. The thrombocytes of fishes are functional counterparts of the mammalian platelet, and consequently central to the clotting process. Fange (1968) postulated a phagocytic function as well. Support for this was provided by Ferguson (1976), who found that intravenously administered carbon microparticles subsequently appeared within these cells. However, Ellis (1977) felt that particle uptake was probably attributable to passive entrapment by the extensive peripheral vesicular system. Furthermore, Ellis (1977) also emphasized the difficulties of reconciling functions as disparate as phagocytosis and clot formation in a single cell. The latter argument also has pertinence in the question of thrombocyte origin. Current hypotheses tend, for the most part, to reflect earlier suggestions that thrombocytes differentiate from small lymphocytes (e.g. Gardner and Yevich, 1969) or that both derive from a common stem cell (e.g. Klontz, 1972). That PH A treatment led to proliferation of both lymphocytes and thrombocytes suggests similarities of origin. On the other hand, the opposed effects of cadmium on response to PHA points to differences in differentiation. In any event, thrombocyte numbers declined sharply following exposure to even relatively low levels of cadmium. Some impairment of clot-forming ability, and therefore increased susceptibility to injury would be expected. The relationship between lymphocytes and thrombocytes, the most abundant leucocyte types in fishes is controversial. Watson et al. (1963) reported that lymphocytes constituted 30%o of the white cell population in goldfish. Weinreb and Weinreb (1969) identified 71-82% of the leucocytes of this species as lymphocytes, 3-13%0 as thrombocytes. In sharp contrast to this, Gardner and Yevich (1969) reported that lymphocytes made up only 2-13% of the white cells of three cyprinodont species, thrombocytes from 82-95 %. Reported lymphocyte:thrombocyte ratios
153 vary by an order of magnitude from 25:1 to 2:1. Using immunofluorescent techniques rather than the usual Romanowsky stains Ellis (1976) observed lower lymphocyte:thrombocyte ratios. The lymphocyte and thrombocyte counts obtained in this study for undisturbed control goldfish suggest a ratio of about 1.5:1. The basis o f these discrepancies may lie in the relative fragility of thrombocytes. Gardner and Yevich (1968), who examined living preparations as well as stained smears, noted the readiness with which thrombocytes initiated a cytoplasmic spreading process which quickly led to the formation of long thread-like projections. Smear preparation frequently stripped these away, leaving nuclei largely denuded of cytoplasm and easily mistaken for small lymphocytes. It was for this reason that leucocyte data in this study was accepted only for samples in which thrombocytes of normal morphology were subsequently identified on stained slides. Stress-induced lymphopenia in fishes is commonly accompanied by neutrophilia (Ellis, 1981), and this proved to be the case in the present study. Teleostean neutrophils are mobile and phagocytic and are formed in granulopoietic areas of the kidney (Ellis, 1977). In view of the observations of Stromberg et al. (1983) response to cadmium challenge may be attributable to increased longevity in circulation, release from storage sites or to differential sensitivity of lympho- and granulopoietic cells. The last of these is compatible with observed responses to P H A treatment. Similar considerations also apply to the eosinophilia and basophilia encountered following exposure to cadmium. However, the functional consequences of this are unclear, for the roles played by such cells are not well understood. In summary, leucocyte status in goldfish is sensitive to even low concentrations of cadmium. Total white numbers are reduced, principally as a result of reductions in lymphocyte and thrombocyte numbers. Lymphopoietic capacity is apparently more affected than is the case with thrombocytes. Major increases in neutro-, eosino- and basophit numbers take place, and various ratios relating agranulocyte and granulocyte abundances are sharply altered. For example, E granulocytes:~ agranulocytes rises almost 5-fold, and may be of diagnostic value. From the functional viewpoint, these changes would be compatible with reductions in resistance to injury and pathogen challenge. We are, however, unaware of studies which unequivocally demonstrate that this actually occurs. ACKNOWLEDGEMENTS The financial support provided by the Natural Sciences and Engineering Research Council through Individual Operating Grant A6972 to A.H. Houston is gratefully acknowledged. REFERENCES Christensen, G.M., J.T. Fiandt and B.A. Poeschl, 1978. Cells, proteins and certain physical-chemical properties of brook trout (Salvelinusfontinalis) blood. J. Fish Biol. 12, 51-60.
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