Electron‐microscopic localization of lipophilic chemicals by an antibody‐based detection system using the blue mussel Mytilus edulis as a model system

Abstract

Identification of lipophilic chemicals requires the development of new techniques to detect these compounds at the cellular and subcellular levels. To address this issue, we have developed a combinational protocol using antibodies directed against chemicals together with a multiple signal amplification system (catalyzed signal amplification/gold-substituted silver-intensified peroxidase) to detect the lipophilic compounds at their subcellular sites of accumulation by transmission electron microscopy. As a model organism, we exposed the blue mussel Mytilus edulis to the polycyclic aromatic hydrocarbon phenanthrene and the polychlorinated biphenyl mixture Aroclor 1254 for up to 10 d. The aim of these exposure experiments was to analyze the cellular targets and sites of accumulation in the digestive system by transmission electron microscopy. The endo-lysosomal system of digestive cells and mitochondria of epithelial cells appeared to be the preferred accumulation sites for the lipophilic compounds studied. The antibody-based approach to detect lipophilic chemicals provides an important insight into the pathways of uptake and accumulation as well as mechanisms of toxicity.

Application of a battery of biomarkers in mussel digestive gland to assess long-term effects of the Prestige oil spill in Galicia and the Bay of Biscay: Lysosomal responses

“…22,23 Additionally, lysosomes are known to accumulate a diverse range of toxic metals and organic chemicals. [24][25][26] Lysosomal responses to environmental stress include reduced membrane stability and increased lysosomal size (lysosomal enlargement). 27 Lysosomal integrity has been employed as a general marker of pollutant-induced stress in several laboratory [28][29][30] and field 15,27,[31][32][33] studies.…”

Cellular localization of lead using an antibody‐based detection system and enzyme activity changes in the gills and digestive gland of the blue mussel Mytilus edulis

“…Moreover, metal localization facilitates interpretation of responses of detoxifying enzymes and antiox‐idative processes at specific tissue sites. Antibody‐based detection techniques have been previously applied for the localization of organic contaminants at the light and electron microscopic levels [12,19,20]. In the present study, antibodies against Pb were successfully used for the visualization of this metal at the subcellular level.…”

“…After washing, unspecific antibody‐binding sites were blocked with 1% bovine serum albumin, 0.2% fish gelatine, and 0.05% saponin in phosphate‐buffered saline for 2 h. Tissue sections were incubated with the monoclonal antibodies to Pb (anti‐Pb 1:1000 = lot specific) from Biodesign (Saco, ME, USA) at 4°C overnight. The secondary detection step and embedding procedure for electron microscopy was performed as described in detail in the study of Einsporn and Koehler [12]. For electron microscopy, ultrathin sections were cut with an ultracut microtome (Reichert‐Jung, Wetzlar, Germany) and collected on formvar‐coated Cu grids.…”

“…Localization of Pb at the ultrastructural level was investigated with an antibody-based detection system for contaminants. A similar detection technique was developed in a previous study for the visualization of organic contaminants [12]. This technique is very sensitive, with a high amplification potential, and allows the localization of the chemicals under study by electron microscopy.…”

Immunodiagnostics and immunosensor design (IUPAC Technical Report)

“…After tissue fixation and embedding, ultrathin tissue slides are made and incubated with gold-colloid labelled antibody. Under the electron microscope, the gold particles are visualized as round, electron-dense spots [122,123].…”