InVitro Toxicity Endpoints

In order to assess the potential toxicity of chemical substances, several in-vitro tests have been developed by measuring different biological endpoints, which are summarized in Table 4.2.

Over recent years, research investigations into apoptosis have provided extensive knowledge of the mechanisms involved in cell death, and this in turn has led to the development of mechanistically based endpoints. Many morphological and biological changes that may occur at the cellular membrane, nucleus, specific

Table 4.2 Common biological endpoints assessed by in-vitro toxicity tests (from [28, 42, 50, 52, 56-59]).

Biological endpoint

Detection method

Cell morphology

Cell size and shape

Cell-cell contacts

Nuclear number, size, shape and inclusions

Nuclear or cytoplasmic vacuolation

Cell viability

Trypan blue dye exclusion

Diacetyl fluorescein uptake

Cell counting

Replating efficiency

Cell metabolism

Mitochondrial integrity (MTT, MTS and XTT tetrazolium salt assays)

Lysosome and Golgi body activity (Neutral red uptake)

Cofactor depletion (e.g., ATP content)

Membrane leakage

Loss of enzymes (e.g., LDH), ions or cofactors (e.g., Ca2+, K+, NADPH)

Leakage of pre-labeled markers (e.g., 51Cr or fluorescein)

Cell proliferation

Cell counting

Total protein content (e.g., methylene blue, Coomassie blue,

kenacid blue)

DNA content (e.g., Hoechst 33342)

Colony formation

Cell adhesion

Attachment to culture surface

Detachment from culture surface

Cell-cell adhesion

Radioisotope

Thymidine incorporation into DNA

incorporation

Uridine incorporation into RNA

Amino acids incorporation into proteins

proteases and DNA level can be used as biological endpoints for measuring apoptosis [52, 59]. Moreover, the rapid progress in genomic, transcriptomic (gene expression) and proteomic technologies has created a unique powerful tool in toxicological investigations [44].

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