Elsevier

Chemosphere

Volume 45, Issues 4–5, November 2001, Pages 399-407
Chemosphere

Ecocytological and toxicological responses to copper in Perna viridis (L.) (Bivalvia: Mytilidae) haemocyte lysosomal membranes

https://doi.org/10.1016/S0045-6535(01)00039-XGet rights and content

Abstract

Bivalve lysosomes are sites of intense intracellular digestion. Lysosomes accumulate many pollutants to high concentrations resulting in membrane destabilisation. Consequently, the elucidation of lysosomal membrane integrity utilising the neutral red assay has been used to good effect in pollution monitoring. Naturally occurring environmental stressors also have the potential to destabilise the membrane. Exposure to elevated copper concentrations and extremes of temperature, salinity, hypoxia, emersion and inadequate ration were investigated in haemocyte lysosomes from the tropical bivalve, Perna viridis. Elevated copper concentrations destabilised the membrane although responses were not entirely related to the exposure-concentration. Environmental stressors induced through higher thermal regimes (29°C and 35°C), hyposalinity (10–25‰) and prolonged emersion elicited significant lysosomal membrane destabilisation. Hypoxia and inadequate ration did not significantly effect membrane stability. The haemocyte lysosomal membranes were generally resistant to exogenous alterations within normal ranges and only showed significant labilisation at environmental extremes. P. viridis haemocyte lysosomal membrane biomarkers should, therefore, prove robust to natural stressors when deployed in marine monitoring programmes and thus prove a valuable, rapid, cost-effective cytological marker of pollution.

Introduction

Lysosomes are polymorphic, hydrolytic enzyme-containing, organelles with many intracellular and extracellular roles (de Duve and Wattiaux, 1966). Their major function in bivalves is food degradation in the well-developed lysosomal compartment of the digestive diverticula cells (Owen, 1972). Other functions, however, include protein and organelle turnover, thus contributing to cell economy (Owen, 1972), cellular defence (Adema et al., 1991), cell detoxication (Owen, 1972, George et al., 1977), gamete resorption (Bayne et al., 1978) and shell formation (Saleuddin and Petit, 1983).

Lysosomal hydrolases are capable of degrading any biological material (Goldman, 1976). Consequently, membrane labilisation and hydrolase release are potentially lethal to cells as these enzymes are capable of cellular component lysis (de Duve and Wattiaux, 1966, Goldman, 1976). Hydrolases are, therefore, predominantly sequestered in an inactive form within a thick membrane in order to prevent free-access to cellular constituents; this property is termed structure-linked latency in that hydrolase latency depends on the integrity of the bounding membrane (Dean, 1977). Nevertheless, selective cytosolic and extracellular hydrolase release does occur naturally in healthy mollusc cells and constitutes an essential cytological function (Adema et al., 1991). Only when severe membrane dysfunction and hence excess hydrolytic ctyosolic activity is evident will a pathological state ensue (Adema et al., 1991).

Many chemical, physical and environmental stressors are known to destabilise lysosomal membranes and injury is proportional to the magnitude of stress (Moore, 1985). Environmental stressors known to induce bivalve lysosomal membrane labilisation include elevated temperature (Moore, 1976), prolonged emersion (Moore et al., 1979), reproduction (Bayne et al., 1978), starvation and rapid salinity change (Bayne et al., 1976, Bayne et al., 1981). Lysosomes also play a terminal role in metal homeostasis through sequestration and storage of metal-binding metallothioneins (George et al., 1977). Prevention of cytotoxicity through metal sequestration also renders the lysosomal membrane particularly susceptible to excess ambient concentrations and membranes may become destabilised (Moore, 1985). The aforementioned stressors have usually been evaluated in bivalves either from tissue sections or isolated, cell-free lysosomes from the digestive diverticula. The digestive diverticula cells are a major interface between the bivalve and the environment and also the major site of nutrient storage and detoxication (Owen, 1972, Moore, 1976). Consequently, digestive diverticula cells are likely to be highly susceptible to deleterious environmental and endogenous modification. Recently, a technique devised to determine lysosomal membrane stability from living fish (Limanda limanda) hepatocytes has been adapted for mussel (Mytilus edulis) haemocytes (Lowe et al., 1992, Lowe et al., 1995a). Elucidation of lysosomal membrane stability from living cells is superior to tissue sections because the former will afford a better indication of animal condition (Lowe et al., 1992). Furthermore, mussel haemocytes can be obtained without sacrifice thus affording the opportunity of conducting non-destructive monitoring and the stability of lysosomal membranes has been used as a diagnostic tool to measure mussel (Perna viridis, Mytilus spp.) condition in field populations exposed to different degrees of pollution (Cheung et al., 1998, Nicholson, 1999a).

Lysosomal membrane responses determined in marine mussels have proven effective tools in the elucidation of cytological toxicity (Lowe et al., 1995b, Cheung et al., 1998, Nicholson, 1999b) and are being used increasingly as biomarkers in pollution monitoring. Natural environmental stressors, however, need to be also taken into account in such biomonitoring and environmental effects on mussel haemocyte lysosomes have not been reported upon. It is imperative, therefore, that lysosomal membrane variability associated with normal environmental change is not confused with pollution-induced cytotoxicity.

The present study investigated living P. viridis haemocytes in an attempt to elucidate whether environmental parameters that affect mussel digestive cell lysosomal stability can induce similar injury in cells capable of diapedesis and, potentially, capable of stress-avoidance. P. viridis were also exposed to elevated copper concentrations in order to further investigate the potential of using the haemocyte lysosomes in this species as a biomarker of metal pollution.

Section snippets

Methods and materials

P. viridis were obtained from a subtidal population in Kat O, New Territories, Hong Kong (Grid Reference [1980] 844050N; 849150E) and transported to the laboratory in humid cool boxes (transport time ∼ 150 min). They were cleaned of epibiota and habituated (for at least 2 h prior to experimentation) in natural, unfiltered, seawater in a flow-through tank (363 l, flow rate ∼ 6 l min−1). P. viridis were not fed in the experimental treatments as natural seawater was used which contained seston. P.

Results

P. viridis survived all experimental treatments, apart from emersion when mortality was high (40%). Haemocytes tended to cease spreading over the slides, i.e., pseudopodia were generally absent concomitantly with reduced lysosomal retention of the neutral red probe. Haemocyte viability was always high (normally > 80%) indicating that their extraction and storage did not compromise cell physiology.

Discussion

Haemocyte lysosomal membrane destabilisation assessed through the neutral red assay was indicative of enzyme-associated dysfunction (probably to the enzymes of the membrane-bound proton pump) (Lowe et al., 1992), because the copper and environmental stressors measured also affect enzyme function. P. viridis haemocyte lysosomal membranes were generally stable, apart from when individuals were exposed to environmental extremes and elevated ambient copper.

No significant hypoxia effect occurred in P

Acknowledgements

This work was supported by a studentship from The University of Hong Kong and represents some work conducted for the partial fulfillment of a Ph.D. degree. I would like to thank Prof. B. Morton for commenting on an earlier draft of this manuscript.

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