Interaction with Cytotoxicity Assays

The knowledge gathered so far consists of data based on a nonstandardized CNT material thus, all the studies reviewed in Table 7.1 have to be considered as isolated and not directly comparable experiments. In particular, from the divergent interpretation of the WST-1 assay output in two different studies, it is clear that a standardized CNT reference material used by all toxicologists is needed. It is also important to keep in mind that the employment of viability tests based on the 

Calcein AM (CAM) Live cells green stained Cell membrane Yesa [34] 

Coomassie Brilliant Blue G250 dye Binds specifically to tyrosine side chains of proteins Cell membrane No [33] 

CytoTox One Homogeneous LDH released by cells with damaged Cell membrane No [34] 

Membrane Integrity (CTO) membrane results in the conversion of resazurin into a fluorescent resorufin product  

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As recently reported, nanoparticulate carriers or other substances may interact with cytotoxicity measurement principles (see Note 3). Before performing cytotoxicity assays, it is therefore recommended to clarify whether there are any interactions between nanoparticles/delivery enhancers and test reagents. For MTT assay, it has been described that nanotubes interact with MTT-formazan crystals, therefore, the assay cannot be performed properly (21). In addition, other compounds may interfere by forming a precipitate or by their color and/or... 

Due to their high chemical reactivity, certain NMs can directly interact with the assay components and catalyze reactions, giving false-positive or false-negative results. For example, some NMs (e.g., single-wall carbon nanotubes, carbon black) have been shown to reduce the tetrazolium compounds in the MTT andXTT cytotoxicity assays into the colored formazan without any cells present [61, 62], Moreover, certain NPs, like gold or platinum, were found to oxidize directly NADH, the reaction substrate LDH cytotoxicity assay. 

Several factors are associated with the difficulty in assessing CNT toxicity. Among them, CNT interactions with components of the dispersing medium, the cytotoxicity assay employed, impurities, CNT surface chemistry, and dispersion state are the... 

Gay M, Montana AM, Moreno V, Prieto M-J, Perez JM, Alonso C (2006) Studies of interaction of trichloro r 2-cis-N, N-dimethyl-l-[6-(N , N -dimethyl-ammoniummethyl)-cyclohex-3-ene-l-yl]-methylammonium platinum(II) chloride with DNA effects on secondary and tertiary structures of DNA. Cytotoxic assays on human ovarian cancer cell lines, resistant and non-resistant to cisplatin. Bioorg Med Chem 14 1565-1572... 

Endpoint assays such as proliferation or cytotoxicity assays are routinely used for functional assessments. For these assessments, primary cells, transformed cells, or cells transfected with the target receptor are exposed to range of concentrations of the test article. Proliferation or cytoxicity is then measured using a variety of methods such as crystal violet vital dye staining, MTT/MTS incorporation, or a luminescence readout like ATP lite. In addition, assays that analyze phosphorylation of specific transcription factors, or release of specific cytokines and chemokines, are also common. Figure 9.5 illustrates the measure of functional consequences of receptor-test article interaction by quantifying cytokine release. Cells from the species under evaluation were cultured in the presence of serial dilutions of the test article or control reagents, and supernatants harvested for determination of cytokine levels by ELISA (i.e.,... 

Furthermore, the expression and function of the Pgp can be modulated by indirect mechanisms, such as interactions with membrane lipids [67] or inhibition of protein kinase C. The reversal of MDR is established using tumor cells lines that are made resistant by the exposure to an anticancer agent or by transfection of the mdrl or mrpl genes. The parameter most widely used to show the activity of MDR reversal agents is reversal factor (RF). This type of assay assumes that the reversal agent does not show inherent cytotoxicity at the concentrations tested. 

Liu et al. reported in 2003 on the synthesis and cytotoxicities of 3 -cyclopropane analogs with both Tot- and 7(3-OH and 2 R)- and 2 (5)-OH functionalities. Both 2 - R) isomers were 400 times less active than paclitaxel in A2780 cancer cell assays, and 2 -(5) isomers displayed even weaker cytotoxicities. Unsatisfactory results may have developed from small volumes of the 3 -cycloprapane, which is similar to 3 -methyl in 9(R)-dihydro paclitaxels as reported earlier. As mentioned, larger groups such as isobutyl or isobutenyl may interact with the receptor better. 

As part of a study to establish 3-azidoamsacrine (34) as a viable photoaffinity probe for amsacrine-DNA interactions, the cytotoxic and mutagenic properties of this compound were investigated utilizing mammalian cell-mutation assays with and without light activation [68]. Not surprisingly, 3-azidoamsacrine was cytotoxic to V79 Chinese hamster cells... 

The 9-anilino proflavine derivative was designed to optimize the interaction with the intramolecular G-quadruplex from human telomere and minimize that with duplex DNA. These compounds have 60 to 100 nM potency in a modified TRAP assay and corresponding low cytotoxicity (93). The triazines have been demonstrated to produce telomere shortening, which is associated with delayed growth arrest and cell senescence (80). The fluoroquinophenoxazines are redesigned topoisomerase II poisons that now interact more specifically with G-quadruplex structures, and this activity is correlated with production of anaphase bridges (78), a property also shared by the cationic porphyrin TMPyP4 (96) and... 

The bioactivity of costunolide (19) in both the DNA-damaging yeast bioassay and the cytotoxicity assay has led to the suggestion that it acted by interaction with DMA in some manner. The lack of DNA-damaging activity of the structurally related (-)-frullanolide (21) has led to the conclusion that the focus of activity in 19 is not simply the a, -unsaturated lactone moiety as suspected in many other cytotoxicity studies with a-methylene-Y-lactones [32]. 

The more potent cis isomers were also assayed for inhibition of tubulin polymerization, to investigate the correlation cytotoxic activity/ interaction with the microtubule system. 

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