Toxicity and Drug Testing

ion, oxidation sulfhydryl and carbon chain destruction. Cd can also decompose the unsaturated fatty acid into malondialdehyde (MAD) by peroxiding and cause biological macromolecules to crosslink into abnormal macromolecules which degrade membrane structure and alter the membrane permeability (Shukla et al., 1989). After the crabs E. sinensis were exposed to Cd, many changes appeared in the R-cell in hepatopancreas, such as organells decrease, mitochondria damage, endoplasmic reticulum expansion, and thinning of the cytoplasm matrix (Wang L. et al., 2001). Cd can partly disintegrate the mitochondrial cristae of neurosecretory cells in E. sinensis (Li et al., 2008). Whenever injected into the crab S. yangtsekiense, Cd resulted in damage to the organells with membrane structure, and the mitochondria was damaged first, which suggested that mitochondria was a sensitive organelle to Cd that could be used to show the amount of damage caused by Cd (Wang L. et al., 2002a,b). Cd could cause the morpha of female ovaries to change markedly in S. henanese, such as the increase of fragmentations and adherences. The oval prosenchyma of egg cells became significantly larger. Egg membrane were much thicker. At the same time, the particulate protuterances on the surface of eggs cells decreased. The boundary between egg cells became more and more unclear. These morphological changes may be a form of self-preservation in eggs which can reduce the damage through self-adjustment, whereas with the increase of Cd dosage, the irreconcilable morpha damage would become much larger (Meng, 2006). Toxic Effects of Cadmium on Crabs and Shrimps 225 1.4 The influence of Cd on ovarian development in crabs and shrimps 1.4.1 The influence of Cd on ovarian development Studies regarding the effects of Cd on ovarian development in crabs and shrimps have been conducted since the 1990s. The majority of experiments showed that Cd inhibited ovarian growth, reduced hatch rates of the fertilized eggs and led to embryonic deformity. Reddy et al. (1997) found Cd could inhibit 5-HT-induced ovarian maturation in the red swamp crayfish, Procambarus clarkia. Lee et al. (1996) documented that Cd deformed eyespots, reduced hatching success, and inhibited growth of oocytes of Callinectes sapidus. Naqvi et al. (1993) reported that P. clarkia treated with Cd hatched 48 eggs with a hatching rate of only 17%. In comparison, untreated individuals hatched 203 eggs with a hatching rate of 95%. Some results were not consistent with the above observations. For exemple, red swamp crayfish fed with duckweeds containing Cd for 14 d had significantly bigger ovary index and total fat content than the respective groups fed with unpolluted duckweeds (Devi et al., 1996). 1.4.2 The mechanism for the iInfluence of Cd on ovarian development There are different views regarding the mechanism of how Cd affects ovary development. Reddy et al. (1997) suggested that the inhibition of Cd on ovarian maturation in P. clarkii was due to the metal inhibiting 5Hydroxytryptamine (5-HT)-stimulated gonad-stimulating hormone (GSH) release, and preventing the ovaries from responding to this hormone. Rodriguez et al. (2000) studied the effect of Cd on oocyte growth of the fiddler crab U. pugilator during the slow vitellogenesis phase of ovarian maturation of this crab. Only when eyestalks were present (intact crabs in vivo experiments or in the incubation media in vitro experiments) , the oocyte growth was inhibited by Cd. So the authors suggested that Cd could act to increase the secretion of the gonad-inhibiting hormone (GIH) from the sinus gland in the eyestalks, and then GIH inhibited the oocytes directly or indirectly. On the contrary, no significant (P >0.05) change of the gonadosomatic index was observed with intact female crab Chasmagnathus granulata exposed to 0.5 mg/L Cd, whereas eyestalkablated exposed females showed significantly (P<0.05) lower gonadosomatic index values than their respective controls. This indicated that Cd interfered with extra-eyestalk hormones. The experimental results shows a possible interference of Cd with the transduction pathway of methyl farnesoate or 17-hydroxyprogesterone.On the other hand, Cd has an inhibitory effect on GIH secretion from the eyestalk. 2. The reproductive toxicity of the Cd to the Chinese crab E. sinensis The ovarian growth in the Chinese crab is a process with oogonium multiplication, oocyte enlargement and yolk protein synthesis. It is the basis for the development of follow-up individual and is regulated by their own complex endocrine system. In the condition of internal hormone imbalance or external hormonal stimulation, the process of yolk synthesis will be affected. The gonad-inhibiting hormone (GIH), gonad-stimulating hormone (GSH), methyl ester (MF), progesterone and estradiol in the body can adjust ovarian development together. The existence of heavy metals in water as environment endocrine disruptors will cause certain damage for the shrimps and crabs. In this section, ovarian index (OI), oocyte diameter and yolk protein accumulation, GIH, progesterone and estradiol levels in hemolymph were meassured and ovarian ultrastructural changes were observed after E. sinensis was treated with Cd. The influence of Cd on ovarian development and its Toxicity and Drug Testing 226 mechanism are discussed. The discussion provides information regarding the effects of environmental endocrinal disrupter such as heavy metal on the health of animals and human. Juvenile female crabs for this experiment were obtained from Baiyangdian Lake, Hebei province, China. In the laboratory, the crabs were maintained for at least 2 weeks prior to the start of an experiment in fresh water, prepared to have a temperature of 25 oC and were fed uncooked potatoes daily. During the experiment, crabs were distributed into 3 groups of 15 crabs per group. The first group served as the control. Other animals were exposed to Cd concentrations of 0.25 and 0.50 mg/L (Cd added as CdCl2•2.5H2O). The duration of exposure was 12 d. After exposure, the OI, oocyte diameter, yolk protein, GIH, progesterone and estradiol levels in hemolymph were meassured and ovarian ultrastructural changes were observed. The results showed crabs exposed to 0.50 mg Cd/L had significiantly smaller ovarian index than controls. The difference between crabs exposed to 0.25 mg/L and controls were not significiant. The influence of Cd on OI presented the dose-effect relations. The influence of Cd on oocyte diameter had the similar regularity. OI (%) oocyte diameter (μm) controls 0.503±0.162 50.729±2.254 0.25mg/L Cd 0.293±0.149 45.792±1.599 0.50mg/L Cd 0.241±0.026* 40.771±2.097* * Significant difference to control group (P < 0.05) Table 1. The effect of Cd on ovarian index and oocyte diameter Fig. 1. Native PAGE maps of vitellin 1. map of native PAGE with CBB staining of ovary crude extracts in controls; 2. map of native PAGE with CBB staining of ovary crude extracts exposed to 0.25 mg/L Cd; and 3. map of native PAGE with CBB staining of ovary crude extracts exposed to 0.50 mg/L Cd 1 2 3