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  • Immunofluorescence detection and localization of B[a]P and TCDD in earthworm tissues.

Immunofluorescence detection and localization of B[a]P and TCDD in earthworm tissues.

Chemosphere (2014-01-15)
Susanna Sforzini, Michael N Moore, Marta Boeri, Emilio Benfenati, Andrea Colombo, Aldo Viarengo
ABSTRACT

An immunohistochemical method using antibodies against polycyclic aromatic hydrocarbons (PAHs) and dioxins was developed on frozen tissue sections of the earthworm Eisenia andrei exposed to environmentally relevant concentrations of benzo[a]pyrene (B[a]P) (0.1, 10, 50 ppm) and 2,3,7,8-tetrachloro-dibenzo-para-dioxin (TCDD) (0.01, 0.1, 2 ppb) in spiked standard soils. The concentrations of B[a]P and TCDD in E. andrei exposed to the same conditions were also measured using analytical chemical procedures. The results demonstrated that tissues of worms exposed to even minimal amount of B[a]P and TCDD reacted positively and specifically to anti-PAHs and -dioxins antibody. Immunofluorescence revealed a much more intense staining for the gut compared to the body wall; moreover, positively immunoreactive amoeboid coelomocytes were also observed, i.e. cells in which we have previously demonstrated the occurrence of genotoxic damage. The double immunolabelling with antibodies against B[a]P/TCDD and the lysosomal enzyme cathepsin D demonstrated the lysosomal accumulation of the organic xenobiotic compounds, in particular in the cells of the chloragogenous tissue as well as in coelomocytes, involved into detoxification and protection of animals against toxic chemicals. The method described is timesaving, not expensive and easily applicable.

MATERIALS
Product Number
Brand
Product Description

Supelco
Benzo[a]pyrene, certified reference material, TraceCERT®, Manufactured by: Sigma-Aldrich Production GmbH, Switzerland
Supelco
Benzo[a]pyrene, analytical standard, for environmental analysis
Sigma-Aldrich
Benzo[a]pyrene, ≥96% (HPLC)
Supelco
Benzo[a]pyrene solution, 100 μg/mL in cyclohexane, analytical standard
Supelco
Benzo[a]pyrene solution, certified reference material, TraceCERT®, 200 μg/mL in methylene chloride
Supelco
Benzo[a]pyrene solution, certified reference material, TraceCERT®, 1000 μg/mL in acetone