Elsevier

Toxicology in Vitro

Volume 25, Issue 3, April 2011, Pages 715-723
Toxicology in Vitro

A high content screening assay for identifying lysosomotropic compounds

https://doi.org/10.1016/j.tiv.2010.12.010Get rights and content

Abstract

Lysosomes are acidic organelles that are essential for the degradation of old organelles and engulfed microbes. Furthermore, lysosomes play a key role in cell death. Lipophilic or amphiphilic compounds with a basic moiety can become protonated and trapped within lysosomes, causing lysosomal dysfunction. Therefore, high-throughput screens to detect lysosomotropism, the accumulation of compounds in lysosomes, are desirable.

Hence, we developed a 96-well format, high content screening assay that measures lysosomotropism and cytotoxicity by quantitative image analysis. Forty drugs, including antidepressants, antipsychotics, antiarrhythmics and anticancer agents, were tested for their effects on lysosomotropism and cytotoxicity in H9c2 cells. The assay correctly identified drugs known to cause lysosomotropism and revealed novel information showing that the anticancer drugs, gefitinib, lapatinib, and dasatinib, caused lysosomotropism. Although structurally and pharmacologically diverse, drugs that were lysosomotropic shared certain physicochemical properties, possessing a ClogP > 2 and a basic pKa between 6.5 and 11. In contrast, drugs which did not lie in this physicochemical property space were not lysosomotropic. The assay is a robust, rapid screen that can be used to identify lysosomotropic, as well as, cytotoxic compounds, and can be positioned within a screening paradigm to understand the role of lysosomotropism as a contributor to drug-induced toxicity.

Introduction

Lysosomes are conventionally known as the cell’s recycling center as they are essential for the digestion of old organelles and engulfed microbes. Three processes, endocytosis, phagocytosis and autophagy, are involved in delivering macromolecules to lysosomes, in which lie enzymes that function at pH 4.5 (de Duve, 1983, Luzio et al., 2007, Turk and Turk, 2009). More than 50 lysosomal enzymes are responsible for the breakdown of lipids, phospholipids, glycolipids, proteins, nucleic acids and sugars. Genetic disorders involving defects in lysosomal enzymes can affect many organs and tissues and can sometimes be fatal (Walkley, 2009). Remarkably, lysosomes are also important players in triggering programmed cell death since lysosomal enzymes known as cathepsins are involved in both mitochondria-dependent apoptosis and mitochondria-independent programmed cell death (Kroemer and Jaattela, 2005, Kirkegaard and Jaattela, 2009). Furthermore, rupture of lysosomes leading to release of cathepsins can cause necrosis (Kirkegaard and Jaattela, 2009, Groth-Pedersen and Jaattela, 2010).

Compounds that accumulate in lysosomes are called lysosomotropic agents, and their accumulation within lysosomes is known as lysosomotropism (de Duve et al., 1974). Compounds with both a lipophilic moiety and a basic moiety have a propensity to cause lysosomotropism (de Duve et al., 1974). Within the neutral pH of the cytoplasm, these compounds are free to penetrate membranes including those of lysosomes, but once inside the acidic environment of lysosomes, they become protonated and are unable to cross the lysosomal membrane to re-enter the cytoplasm. Hence, these compounds become trapped, leading to intra-lysosomal compound concentrations which are much higher than those found in the cytoplasm (Kaufmann and Krise, 2007). Many drugs given in the treatment of central nervous system disorders are cationic amphiphilic compounds and it is, thus, not surprising that they have been reported to be lysosomotropic (Daniel, 2003).

How lysosomotropism might cause drug-induced toxicity is not well understood. Cationic amphiphilic compounds have been known to cause phospholipidosis, a phenomenon in which excessive accumulation of phospholipids occurs within lysosomes (Reasor et al., 2006). Phospholipidosis occurs predominantly in the brain and in organs which are lysosome-rich such as the lungs, liver, and kidney (Kodavanti and Mehendale, 1990). However, it is not clear if phospholipidosis is a manifestation of toxicity or merely an adaptive response to drugs (Reasor et al., 2006).

A potential mechanism by which lysosomotropic compounds might cause toxicity is via inhibition of autophagy. Autophagy is a homeostatic process by which old or damaged intracellular components are enveloped in double-membraned vesicles called autophagosomes which, then, fuse with lysosomes to form autophagolysosomes (Rubinsztein et al., 2007). The contents of autophagolysosomes are subsequently degraded by lysosomal acid hydrolases (Rubinsztein et al., 2007). Lysosomotropic compounds could conceivably inhibit the degradation of autophagolysosomes by increasing the pH of lysosomes and thereby impairing lysosomal acid hydrolases. Indeed, the lysosomotropic drug, chloroquine, has been reported to inhibit the degradation of autophagolysosomes (Walls et al., 2010).

Early safety assessments of new chemical entities are becoming prevalent in the pharmaceutical industry as a means to reduce compound attrition caused by toxicity. Although there is a limited understanding of how lysosomotropism contributes to toxicity, an association between the two has been observed (Walls et al., 2010), and therefore assays that identify lysosomal impairment are desired. Hence, the aim of this study was to develop a high content screening (HCS) assay that identifies compounds which cause lysosomotropism. The assay was multiplexed so that cytotoxicity could also be measured. Two commercially available fluorescent dyes, LysoTracker® Red DND-99 and Hoechst 33342, were used to measure lysosomotropism and cytotoxicity, respectively, in a rat cardiomyocyte-derived cell line, H9c2. Under physiological conditions, LysoTracker® Red, a fluorophore linked to a weak base, readily enters acidic organelles such as lysosomes. However, in the presence of either a lipophilic basic compound or cationic amphiphilic compound, competition between the compound and the LysoTracker dye occurs, resulting in a pH-independent decrease in the fluorescence signal of the dye (Lemieux et al., 2004). We tested the effect of forty drugs including antidepressants, antipsychotics, anti-arrhythmics, and anticancer agents on H9c2 cells in the multiplexed HCS assay. The assay correctly identified several drugs that are known to be lysosomotropic agents and also revealed novel information showing that some, but not all, of the tyrosine kinase inhibitors that we tested caused lysosomotropism. The HCS assay was robust, rapid, and sensitive, and can be implemented in large-scale compound screening to identify lysosomotropic, as well as, cytotoxic compounds.

Section snippets

Materials and methods

Chemicals were obtained from Axxora LLC (San Diego, CA), Toronto Research Chemicals (Toronto, Canada), Sigma (St. Louis, MO) and Pfizer’s chemical sample bank (Groton, CT). Cell culture media and supplements were from Invitrogen (Carlsbad, CA).

The fluorescent dyes, LysoTracker® Red DND-99 and Hoechst 33342, were from Invitrogen.

High content screening assay performance

The aim of this study was to develop a rapid cell-based high content screening (HCS) assay that measures lysosomotropism. The assay was multiplexed so that cytotoxicity could also be measured. A fluorescent dye, LysoTracker® Red DND-99, which has been reported to accumulate in acidic organelles including lysosomes (Lemieux et al., 2004) was used for detecting lysosomotropism. The fluorescent dye, Hoechst 33342, was used to counterstain nuclei and, thus, evaluate cytotoxicity.

In order to choose

Discussion

The aim of the study described here was to develop a rapid high content screening assay that measures lysosomotropism and cytotoxicity. Methods for detecting lysosomotropism have been described before. One of these involved isolation of lysosomes from tissues followed by measurement of the uptake of radioactive compounds (Ishizaki et al., 1996). This technique, together with competition experiments, showed that cationic amphiphilic compounds such as imipramine, chlorpromazine and propranolol

Conflict of interest

There is no conflict of interest.

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