Comp. Biochem. Physid. Vol. 8OC, No. 2, pp. 407410, 1985 Printed in Great Britain

0306~4492/85 $3.00 + 0.00 ‘0 1985 Pergamon Press Ltd

THE EFFECTS OF CADMIUM ON ALA-D ACTIVITY, GROWTH AND HAEMOGLOBIN CONTENT IN THE

WATER FLEA, DAPHNIA MAGNA

RUNE BERGLIND Department of Zoophysiology, University of Gateborg, Box 250 59, S-400 31 GBteborg, Sweden.

Telephone: 031-85 36 77

(Received 27 July 1984)

Abstract-l. The ALA-D activity, haemoglobin content and growth was studied in the water flea, Duphnia magna, exposed to 0, 0.1, 0.2, 0.4, 0.8 and 1.6 pg Cd/l.

2. The ALA-D activity in water fleas exposed to 0.2- 1.6 pg Cd/l fluctuated around the control value. The activity in animals exposed to 0.1 pg Cd/l decreased during the entire experiment.

3. After 16 days exposure to cadmium the haemoglobin content in water fleas ranged between 80 and 31% of control value. In animals exposed to 0.8 and 1.6 pg Cd/l the haemoglobin content decreased progressively during the experiment.

4. Growth was not affected by cadmium at these concentrations.

INTRODUCHON

Cadmium is a non-essential heavy metal which inter- acts with many cellular and biochemical processes (Pool, 1981). In these processes it may act as an antimetabolite towards calcium, iron and zinc, and the symptoms associated with intoxication are mani- fested as deficiencies in these metals. Metalloenzymes associated with zinc are inhibited by cadmium, prob- ably by displacing it from the enzyme (Venugopal and Luckey, 1978). Magnesium- and calcium- stimulated ATPases are also inhibited by cadmium (Webb, 1979). Other enzymes, however, are stimu- lated by cadmium and some show different responses depending upon species, organ or fraction under study. There are a few enzymes not affected by cadmium (Pool, 1981).

In the biosynthesis of haem (porphyrin) two early steps, delta-ALA (delta-amino-levulinic acid) and PBG (porphobilinogen) formation, are inhibited by cadmium (Pool, 1981). In cadmium-fed calves the erythrocyte ALA-D (delta-aminolevulinic acid dehy- dratase) activity was reduced (Lynch et al., 1976), but in fish this activity was stimulated by cadmium in uivo (Johansson-SjBbeck and Larsson, 1978).

The scope of the present study was to find out if ALA-D activity was affected by cadmium exposure in the water flea (Duphnia magna). Chronic intoxication of cadmium has been shown to lower the hae- moglobin value in workers and decrease growth in experimental animals (Venugopal and Luckey, 1978). In order to evaluate the sensitivity of ALA-D to cadmium relative to these parameters, the hae- moglobin content and growth were measured as well.

MATERIALS AND METHODS

Culture

Water fleas (Daphniu magna), maintained in cultures since 1977 at our laboratory, were used in this experiment. They

were grown in hard reconstituted water (250mg as CaCO,/l), supplemented with seawater (60 ml/25 1) at room temperature (20 f 1°C) and they were fed daily with 90 mg of a 2: 1 mixture of fish food (Dorswal granular fish food, Roswal Producte AG, Ziirich, W. Germany) and baby gruel (Findus, Sweden). The cultures were illuminated 12 hi aday bv daylight tubes (2 x 40 W) 60 cm above the surface and a&ate-d continuously. From ‘these cultures juveniles for the experiment with an age-range of O-18 hr were obtained from reproductive females of 3-4 weeks of age. Before the start of the experiment the juveniles were acclimated for 30 hr to the diluent and to the food.

Cadmium exposure

From the same culture of reproductive females three samples of 30-48 hr old juveniles were taken. They were exposed to cadmium (0, 0.1,0.2,0.4,0.8 and 1.6 pg Cd/l) for 17 hr up to 16 days in 8 1. glass beakers filled with 6 I. test solution. The diluent was hard reconstituted water (250 mg as CaCOJ, Table 1). The experiment was started by adding 9OC- 1200 juveniles into each beaker in order to give samples of 2OC-400mg water fleas (w.w.) for analysis of ALA-D activity and haemoglobin content. The length and hae- moglobin content per Duphnia were determined from sam- ples of 20 (17 and 44 hr exposure) or 10 animals (5, 8 and 16 days exposure).

Equal amounts of food were added daily to each beaker. The food was prepared from tropical fish food flakes (Tetra SM 80, Tetra Werke, FRG) and baby gruel (Findus, Sweden), 50 and 25 g respectively, which was homogenized in 1 1. deionized water. The homogenate was left for 1 hr in a tall beaker and the decanted supematant was fed to the water fleas in the experiment.

The light regime and temperature were as described for the culture. Test solutions were renewed Monday, Wednes- day and Friday. At each renewal, dissolved oxygen (DO, percentage of air saturation) and pH was measured in old and new solutions. Fresh solutions had DO and pH of 100% and 8.1-8.2, respectively. Old solutions ranged 66-87x and 7.2-7.5 during the entire experiment. However, on each renewal DO and pH values were equal in all beakers. All chemicals were of analytical grade and the solutions, includ- ing test solutions, were prepared in Milli Q Reagent Water (Milli Pore@). Cadmium chloride (CdCI, x 2: H,O) was

407

408 RUNE BERCLIND

Table 1. Chemical composition of hard reconstituted water used in the experiments with Daphnia magna

Ingredient C&I, x 2H20 M&30, x 7H,O NaHCO, KC1

Concentration 281 124 64.8 5.8

m/u Matrix: Milli Q Reagent Water (10 megohmjcm resistivity). Hardness: 250 mg/l as Ca/CO,.

used as toxicant. Test concentrations, expressed as pg Cd/l, are nominal values.

Biochemical analysis

For the analysis of ALA-D activity and haemoglobin content the samples of water fleas were homogenized by a glass-Teflon homogenizer in 2 ml ice-cold 0.2 M phosphate buffer (pH 6.8). Of the crude homogenate 1.5 ml was used for analysis of ALA-D activity, 0.3 and 0.2 ml for analysis of hemoglobin and protein content, respectively.

The ALA-D activity was determined according to the original procedure described by Bonsignore et al. (1965) as modified by Johansson-Sjobeck and Larsson (1978). The activity, pg PBG formed/mg protein/hr at 38”C, in cad- mium exposed animals was expressed as a percentage of control value.

For the analysis of haemoglobin the homogenate was diluted to 1 ml by phosphate buffer (0.2 M, pH 6.8) centri- fuged for 5 min at 5700 rpm (Wifug, Winkelcentrifug) and 0.75 ml of the supernatant was used for analysis. The haemoglobin content was also measured in homogenates of water fleas prepared by ultrasonication for 15 set of lo-20 animals in 1 ml ice-cold Milli Q Reagent Water. The homogenate was centrifuged for 25 min at 5700 rpm (Wifug) and 0.75 ml of the supernatant was used for analysis of haemoglobin.

The haemoglobin content was analysed according to Hildemann and Keighley (1955) by a spectrophotometer, measured as the absorbance (Abs) at 412 nm per mg protein and per animal, and expressed as a percentage of the control value. The total protein content in the crude homogenate was analysed according to the method of Lowry et al. (1951).

Growlh

To measure the growth, lo-20 animals from each concen- tration were sampled and photographed through a Zeiss preparation microscope. The length was measured from the top of the head to the base of the spine from photographic copies by means of a pair of Vernier callipers. These photographed animals were thereafter placed in vials with 1 ml water and disintegrated by ultrasonication and anal- ysed for haemoglobin content as described above.

Subcellular fiactioning

The influence of ultrasonication on the yield of hae- moglobin was investigated as follows.

Water fleas and liver from a starved female albino rat was homogenized by a glass-Teflon homogenizer in ice-cold 0.2 M phosphate buffer @H 6.8) in proportions of one part tissue and five parts buffer. One portion of homogenate was treated by ultrasonication (15 set per ml) and the other part was kept as control. Fractions were prepared by centrifu- ging at 1000 g for 10 min (cell and nuclear fraction), 15,000 g for 15 min (mitochondrial fraction) and l,OOO,OOOg for 60 min (microsomal fraction). Each pellet was resuspended up to the original volume in buffer by means of ultra- sonication. Samples of 300 and 50~1 were taken from the supematant and resuspended pellet fractions for analysis of haemoglobin and protein content, respectively. The volume of each supernatant was adjusted by buffer to the original volume before centrifugation. All operations were done at ONIC.

ALA -D % OF CONTROL

100

50

0 I 2 5 8 16

days

Fig. 1. Effects of cadium on ALA-D activity in Daphnia magna. 0 0.1, 0 0.2, 0 0.4, A 0.8, n 1.6pgCd/l.

RESULTS

Cadmium exposure

After 8 days’ exposure to cadmium (0.1, 0.2, 0.4, 0.8 and 1.6 pg Cd/l) the activity of ALA-D in Duph- niu magna was reduced to 70, 75, 68, 77 and 61% of control activity, respectively. After another 8 days the same enzyme activities were found to be 21, 101, 123, 141 and 128% of the control (Fig. 1).

Figure 2 illustrates the haemoglobin content (Abs,,, per mg protein, percentage of control) after 1, 5, 8 and 16 days exposure to cadmium (0.1, 0.2, 0.4, 0.8 and 1.6 pg Cd/l). After 16 days’ exposure the haemoglobin content was 80, 66, 73, 43 and 31% of control, respectively. In water fleas exposed to 0.8 and 1.6 pg Cd/l the haemoglobin content decreased progressively during the experiment, but in the lower concentrations (0.1, 0.2 and 0.4 pg Cd/l) no dose- dependent decrease was found after 1, 5 and 8 days.

The homogenization of water fleas by ultra- sonication gave a pattern of cadmium response on haemogloblin, different from the pattern that glass- Teflon homogenization gave. On the eighth day of exposure, the haemoglobin analysis of the water fleas gave values 1.5-2 times above the control value for the exposed animals. After 16 days the haemoglobin content had declined and varied between 12 and 92% of control (Fig. 3).

The growth of the water fleas showed no response to cadmium in any of the concentrations used in the experiment (Table 2).

SubcelIular fractions

The haemoglobin content was below the detection limit in the non-sonicated portion of the Duphnia

Effects of cadmium on Daphnia 409

HAEMOGLOBIN HAEMOGLOBIN

% OF CONTROL 36 OF CONTROL

200

100

C

150

100

50

0

l

0

&\A 8 .___ __

4 _____ _____ __ --------

a :+

\ I

I 2 5 8 16 days

Fig. 2. Effects of cadium on haemoglobin content in Daph- nia magna homogenized by a glass-Teflon homogenizer. c]

0.1, . 0.2, 0 0.4, A 0.8, II 1.6pgCd/l.

homogenate and supernatants. The raw homogenate and 1000 and 100,OOOg supernatants of the ultra- sonicated rat liver homogenate showed higher hae- moglobin values than the non-sonicated. Ultra- sonication increased the yield of haemoglobin in the 1000 and 100,000 g pellets and the 100,OOOg pellet of the Daphnia and rat liver homogenates, respectively.

DISCUSSION

The present study has shown that the water flea, Daphnia magna, responds with a fluctuation around the control value of ALA-D activity and a decrease in haemoglobin content after exposure to low concen- trations of cadmium {range: 0.1, 0.2, 0.4, 0.8 and 1.6 pg Cd/l, Figs I,2 and 3). No effect on growth was found (Table 2). After 16 days’ exposure the ALA-D activity and the haemoglobin content showed a dose- dependent response to cadmium (Figs 1 and 2). The haemoglobin analysis of the homogenates prepared by ultrasonication showed a different pattern of response to cadmium (Fig. 3) than corresponding glass-Teflon homogenates (Fig. 2). The ultra- sonicated preparation showed an increase in hae- moglobin content with a maximum on day 8 and on day 16 the content had decreased to values below the

a

I 2 5 8 16 days

Fig. 3. Effects of cadimn on haemoglobin content in Daph- nia magna homogenized by ultrasonication. q 0.1, l 0.2,

0 0.4, A 0.8, n 1.6pgCd/l.

control value (Fig. 3). Preparing homogenates by ultrasonication probably brings haem-proteins like cytochromes and myog~obins into solution together with haemoglobin, making them available for anal- ysis and expressed as haemoglobin. Ultrasonicated glass-Teflon homogenates of water fleas and rat liver showed higher haemoglobin values in the raw ho- mogenate and supernatants than the respective non- sonicated ones did. Therefore the haemoglobin anal- ysed from glass-Teflon homogenates of water fleas probably does reflect the “true” haemoglobin content while ultrasonicated homogenates reflect the total amount of haem-proteins. The increase of “hae- moglobin” content in the ultrasonicated preparation after 8 days (Fig. 3) may therefore be an indication of a compensatory increase of cytochromes and myog~obins and other haem-proteins due to a cadmium-me~ated uncoupling of oxidative phos- phorylation (Brown et al., 1982) rather than of an increased amount of haemoglobin.

The interaction of Cd with Ca, Fe and Zn can

Table 2. Mean bodv leneth (mm> + SD of Dadmia mama exuosed to cadmium for 1-16 davs

Days of exDosare 0 a.1

Nominal concentration (pg Cd/l ) 0.2 0.4 0.8 1.6

1’ 0.99*0.11 1.1oio.09 1.02*0.12 1.06&00.13 0.98 kO.12 0.95+0.10

(19) (20) (20) (19) (19) (21)

2t 1.08 k 0.08 1.11 *a.10 1.11 +a.10 1.08 i_ 0.08 l.loio.ll 0.14+0.10

(24) (20) (21) (18) (26) (20)

St 2.04 k 0.12 2.14 f 0.23 1.98 + 0.18 2.11 f0.17 2.17 +0.08 2.04 5 0.16

(10) (10) (W GO) (10) (IO)

gf 2.2iO.18 2.16+0.35 2.35 f: 0.14 2.44f0.19 2.37 + 0.14 2.20 i 0.27

(10) (10) (10) (10) (10) (IO)

16t 2.71 kO.13 2.74 + 0.11 2.70 + 0.11 2.68 + 0.46 2.76 + 0.20 2.77 ?;O.lS

(10) (10) (10) (IO) (10) (10)

Mean body length at start of the test: *l.O i: 0.10 (22), to.93 k 0.09 (19), $0.99 + 0.08 (19).

410 RUNE BERGLIND

cause symptoms associated with deficiencies in these metals (Venugopal and Luckey, 1978). A defective intestinal absorption of iron inhibited haemoglobin formation and led to anaemia in cadmium-fed rats (Venugopal and Luckey, 1978). In Daphniu the ab- sorption and excretion of iron, and the synthesis of haem as well, are localized to the gut. The fat cells, ovary and gut diverticula are other sites involved in the synthesis, storage and catabolism of haem and haemoglobin in D~~~~~~ (Smaridge, 1956). Further- more the diverticula of the gut are thought to be an important site of secretion of enzymes, digestion and absorption in Dqhniu (Schultz and Kennedy, 1976). Furthermore when water fleas (D. magna) were exposed to sublethal cadmium concentrations (12-50 pg Cd/l) their diverticula were shrunk and peristalsis was paralysed (Griffiths, 1980). The effect of cadmium on specific parts of the gut in D. tnugnu (Grifhths, 1980) and the inhibition of intestinal ab- sorption of iron in the rat (Venugopal and Luckey, 1978) suggest that the lowered haemoglobin content was caused by a cadmium-induced defective absorp- tion of iron and not because of decreased ALA-D activity leading to a deficiency in the porphyrin precursor to haem. Water fleas exposed to 0.1 ~_lg Cd/l showed the lowest ALA-D activity values but only a moderate decrease of haemoglobin content (Figs 1 and 2).

The effect of cadmium on ALA-D activity in water fleas exposed to 0.2-I .6 ,ug Cd/l were characterized by fluctuations around the control value (Fig. l), while ALA-D activity in animals exposed to 0.1 pg Cd/l decreased during the entire experiment. A similar regression over time of ALA-D activity has been shown in erythrocytes from cadmium-fed calves (Lynch et al., 1976). Water fleas accumulate cadmium at a constant rate, directly related to the concen- tration in the water (Bertram, 1980). Like vertebrates, another cladoceran. China macrocopa, synthesizes a Cd-binding cysteine rich, small protein (metal- lothionein, mol. wt: 9000 daltons) when exposed to cadmium (Yamamura et al., 1983). The binding to metallothionein is believed to be a detoxication of cadmium. The diversity in response (i.e. ALA-D activity) to cadmium between water fleas exposed to 0.2- 1.6 pg Cd/l and those exposed to 0.1~8 Cd/l may be because of a difference in the degree of induction and the rate of synthesis of metallo- thionein. The inhibition of the ALA-D activity in the water fleas exposed to 0.1 pg Cd/l was very similar to the accumulation curve for cadmium shown by Bertram (1980). It is possible that at this concen- tration the accumulated cadmium was not detoxified by, or inducing synthesis of, metallothionein. An explanation may be that cadmium was bound to and thus affected larger metallo-proteins like enzymes associated with zinc (e.g. alkaline phosphatases and ALA-D) (Venugopal and Luckey, 1978, Fine& ef al., 1975). Further studies need to be done to determine if there exists any threshold for induction of Cd- metallothionein in Daphnia magna.

The present study has shown that cadmium at very low concentrations affects the respiratory physiology of Daphnia magna. Since cadmium acts as an anti-

metabolite for iron the most probable explanation for the lowered haemoglobin values was a decreased net uptake of iron, The progressive decrease of ALA-D activity in water fleas exposed to 0.1 p g Cdjl suggests that this concentration of cadmium is too low to induce synthesis of Cd-metallothionein for protection against intoxication. The synthesis of haemoglobin and the activity of ALA-D showed a similar sensi- tivity to cadmium. The absence of a dose-response of ALA-D to cadmium needs to be clarified and prob- ably the induction, and synthesis of metallothionein may be of importance in this respect.

Acknowledgemen&--This work has been supported by Anna Ahrenbergs fond for vetenskapliga m.i?. iindamdl and Valdemar och Emmy Gustafssons Naturvirdsfond. I thank Eva Nilsson for performing the experiments, Birgitta Vallander for preparing the figures and Dr Gciran Dave for scientific discussion and reading the manuscript.

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