Impact of Metal Contaminations

In order to have a comprehensive picture of the effects of metals on ROS production, this immune response was measured after in vitro exposure of A. rubens amoebocytes or after in vivo experimental contamination of starfishes. Furthermore, natural populations exposed to these contaminants were monitored in the field.

A first experiment was designed to screen the potential of several metals to affect ROS production in vitro. Amoebocytes were exposed to different concentrations of selected metals prior to measuring the unstimulated ROS production. Among all metals tested, only mercury and silver were shown to modulate the resting amoebocyte ROS production (linear regressions:

Metal concentration p<0.001, R2=0.277 and p=0.004, R2=0.165,for Hg and Ag respectively) (Fig. 2). The lack of ROS production inhibition by other metals (Al, Cr, Cd, Zn, Pb, Fe and Cu) is somewhat surprising since Larson et al. (1989) found that, among several types of xenobiotics, Cu was the most immunosuppressive in vitro. However, these authors used a contaminating copper concentration range of 200-2,500 ^g l-1. In our hands, the maximal copper concentration tested was

2 ^g l-1 which is closer to environmentally relevant concentrations. In order to compare the sensitivity of A. rubens amoebocytes with that of bivalve hemo-cytes,we calculated the IC50 values (the metal concentration inducing 50 % of response suppression) for mercury and silver. These values (14.8 and 23.0 ^g l-1 for Hg and Ag respectively) are 60-600 times lower than those obtained using the phagocytic activity of hemocytes of the clams Spisula polynyma and Mya arenaria (Brousseau et al. 2000; Fournier et al. 2000). It thus seems that starfish amoebocyte ROS production is highly sensitive to in vitro contamination by silver and mercury but not to other metals (at least at or close to environmentally relevant concentrations).

The impact of in vivo metal contaminations was studied along a metal contamination gradient occurring in a Norwegian fjord, the S0rfjord, where wastes of three smelters, built in the innermost part of the fjord, were discharged in this area for more than 60 years. Concentrations of mercury in fish and of cadmium and lead in mussels from this fjord resulted in public advisories to regulate human consumption (North Sea Task Force 1993).

ROS production was measured both in starfishes sampled along the gradient (long-term - life-long - contaminations) and in starfishes transferred up the gradient (short-term experimental contamination) (Coteur et al. 2003a). The production of ROS in starfishes from field populations increased along the pollution gradient in direct relation with the contamination of the starfishes by cadmium, lead and zinc (e.g. zinc, Fig. 3).

In contrast, when starfishes were transferred from the control site to the contaminated head of the fjord, the temporary accumulation of some metals such as zinc or cadmium in starfishes was accompanied by an inhibition of ROS production (Fig. 4).

Fig. 3. Bacteria-stimulated reactive oxygen species (ROS) production (mean ± SE, n=10) and zinc concentration in the pyloric caeca (mean ± SE, n=5) of starfishes collected along a metal pollution gradient in a Norwegian fjord. RLU Relative light units

Fig. 4. Effects of short-term transfer experiments up the gradient of metal contamination in a Norwegian fjord. Bacteria-stimulated reactive oxygen species (ROS) production (mean ± SE, n=10) and zinc concentration in the pyloric caeca (mean ± SE, n=5) of starfishes transferred from the uncontaminated site (opening of the fjord) to the most contaminated site (head of the fjord) over time. RLU Relative light units

Fig. 4. Effects of short-term transfer experiments up the gradient of metal contamination in a Norwegian fjord. Bacteria-stimulated reactive oxygen species (ROS) production (mean ± SE, n=10) and zinc concentration in the pyloric caeca (mean ± SE, n=5) of starfishes transferred from the uncontaminated site (opening of the fjord) to the most contaminated site (head of the fjord) over time. RLU Relative light units

Thus, it seems that, depending on the duration of exposure (several days or life-long), the effects of the same contaminants can range from a severe inhibition to a marked increase in ROS production. We hypothesised that the impact of metals in field conditions would occur in three phases: short-term inhibitory effects exerted by direct action of metals on the immune cells are followed by a recovery due to the induction of protective mechanisms and, eventually, when these mechanisms are overwhelmed, indirect stimulatory effects on the immune responses appear due to a global disruption of the animal physiology.

For studies on the impact of metal contamination on stress marker production, see Matranga et al., (this Vol.).

Impact of PCB Contaminations

Polychlorinated biphenyls are persistent organic contaminants of human origin which accumulate in the environment. These contaminants are highly toxic, particularly the non-ortho-substituted and mono-ortho-substituted congeners, the so-called coplanar PCB congeners (cPCBs) that can display a configuration very close to that of the highly toxic 2,3,7,8-tetrachlorodibenzo-dioxin (2,3,7,8-TCDD).

The effects of PCBs were tested by injecting coplanar or non-coplanar congeners in the coelomic cavity of the sea urchin Paracentrotus lividus (Coteur

Fig. 5. Bacteria-stimulated reactive oxygen species (ROS) production (mean ± SE, n=4) and PCB congener #126 (mean ± SD, n=4) concentrations in the body wall of starfishes maintained on sediments spiked with a mixture of PCB congeners, over exposure time. RLU Relative light units

et al. 2001) and of the starfish A. rubens (Danis et al., pers. comm.). In both studies, PCBs were found to increase the production of ROS; coplanar congeners being the most effective in this respect. At very high concentrations, however, a steep drop in ROS production was observed probably due to direct cellular toxicity (Danis et al., pers. comm.). Similarly, when starfishes were maintained on a sediment spiked with a mixture of PCB congeners, the amoe-bocyte ROS production followed closely the transient accumulation of coplanar congeners, such as congener #126, in the starfish tissues (Fig. 5).

Likewise, the contamination by coplanar congener #77 was found to increase the ROS production of starfishes exposed through water, sediments or food (Danis et al. 2003).

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