Université catholique de Louvain
Louvain Drug Research Institute > Cellular and Molecular Pharmacology
Cellular toxicity of antibiotics

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Understanding the cellular and molecular mechanisms of antibiotic toxicity is critical for improving our therapeutic arsenal and select drugs with the best benefit to risk ratio.

We focus our attention on the cellular lipidosis and apoptosis induced by antibiotics, and try to understand the corresponding underlying mechanisms, using a combination of biochemical, morphological and biophysical techniques.

These research programs are closely linked to those exploring drug-membrane interactions and intracellular chemotherapy (pharmacokinetics)



Main current research programs

Lipidosis induced by antibiotics accumulating in the lysosomes

The accumulation of antibiotics in lysosomes (by endocytosis or proton trapping) may cause metabolic alterations that can lead to cell toxicity. Three classes of antibiotics have been especially studied in this context.

1. Aminoglycosides

Aminoglycoside antibiotics were amongst the first antibiotics for which a lysosomal accumulation was demonstrated both in cultured cells and in renal proximal tubular cells in vivo. These antibiotics cause a lysosomal phospholipidosis (see Figure 1A) which we consider to be a hallmark of their renal toxicity. Phospholipidosis is the consequence of the binding of these cationic antibiotics to negatively-charged phospholipids in membrane, which is favored by the acidic pH of lysosomes. In vitro studies have shown that negative charges are essential for the activity of lysosomal phospholipases. The binding of aminglycosides to phospholipid bilayers in lysosomes may, therefore, results in a decreased activity of the phospholipases, which explains the accumulation of undegraded phospholipids (see model in Figure 1B).

In addition, aminoglycosides cause membrane permeabilization as well as membrane bilayer aggregation, and impair membrane fusion.

Figure 1A:

Ultrastructural alterations observed in renal proximal tubular cells of rats treated with low, therapeutic doses of gentamicin
The picture shows enlarged lysosomes filled by osmiophilc material.

At higher magnification, this material appears as apposed layers of lipids (myeloid bodies; see inset).

From Tulkens, 1986


Figure 1B:
Model describing the mechanism of inhibition of phospholipases by aminoglycosides. These polycationic antibiotiocs bind to acidic phospholipids, reducing the negatively charges required for the optimal activity of lysosomal phospholipases, and inhibiting thereby their activity.

From Mingeot-Leclercq, 1999

These studies are being extended to the evaluation of novel molecules with reduced binding to negatively-charged phospholipids and/or reduced accumulation by renal proximal tubular cells.

2. Macrolides and lipoglycopeptides

Macrolides also accumulate in lysosomes (by proton trapping), whereas the novel lipoglycopeptides (telavancin, oritavancin) enter lysosomes by adsorptive endocytosis.

By interacting with phospholipids, macrolides and lipoglycopeptides (especially those with a large cellular accumulation) induce a lysosomal phospholidosis (see Figure 2A). However, and in contrast with what has been described for aminoglycosides (see above), they also cause an accumulation of cholesterol and, probably, other lipids. This is particularly conspicuous in cells exposed to lipoglycopeptides (see Figure 2B). Similar alterations are seen in animals treated with large doses of these antibiotics.

Figure 2A:
Illustration of the ultrastuctural alterations induced by azithromycin in cultured fibroblats when maintained in the presence of 0.03-0.1 mg/L for 7 to 16 days.Upon high magnification, this material appears as lamellar, concentric structures which seem to originate from the lysosomal membrane.

From Van Bambeke et al, 1998

Figure 2B:
Accumulation of total cholesterol (left panel) and phospholipids (ight panel) in macrophages (open symbols) and fibroblasts (closed symbols) exposed to oritavancin (0-50 mg/L for up to 3 days) as a function of the cellular concentration of the drug (oritavancin accumulates in lysosomes by endocytosis, meaning that its concentration in these organelles could be several-fold higher than for the total cells.
From Van Bambeke et al, 2005

These studies are now extended to new molecules as a basis for their pre-clinical assessment for early toxicity related to perturbations of lipid metabolism.    

Selected References on drug-induced lipidosis (by reverse chronological order; for a full reference list, see our publication list)




Molecular mechanisms of aminoglycoside-induced apoptosis

Beside phospholipidosis, aminoglycoside antibiotics also induce apoptosis. Animals treated with low, therapeutically relevant doses of aminoglycosides show both lysosomal phospholipidosis and apoptosis in proximal tubular cells (see Figure 3A). Apoptosis induced by aminoglycosides has been reproduced in vitro with LLC-PK1 and MDCK cells and found to be directly related to the amount of drug accumulated by the cells.

Our current data have shown that gentamicin destabilizes the lysosomal membrane, which could result in the release of the drug and lysosomal constituents such as cathepsins to the cytosol. In this context, the storage of gentamicin in lysosomes would actually appear as a protective mechanism rather than a toxic event, as long as the drug is prevented from moving from there to the cytosol.

The role of cytosolic gentamicin in triggering apoptosis was further documented by showing that its direct delivery to cells by electroporation induces apoptosis at much lower concentrations than what is required for cells incubated with the drug (see Figure 3B; electroporation can be used for fast screening of potentially less toxic aminoglycosides, as it requires only very low amounts of drugs). In parallel, ongoing reseach examines how gentamicin accumulated in lysosomes could cause membrane permeabilization, and, thereby trigger its own relase into the cytosol.

The next steps leading to apoptosis appear rather straightforward, and involve mitochondrial activation with the release of cytochrome c and activation of caspase-3, which can be prevented by overexpression of Bcl-2. Cytosolic gentamicin could act directly on mitochondria or indirectly through impairment of the proteosomal degradation of Bax (see Figure 3C).

Current studies examine the role of reactive oxygen species (ROS) formed in lysosomes by interaction of iron with gentamicin in triggering lysosomal membrane destablisation and in activating the apoptosis mitochondrial pathway.

Figure 3C:
Current view of the mechanism of apoptosis induced by gentamicin. The antibiotic is taken up into cells by receptor-mediated endoytosis (via megalin and, probably also, negtively-charged phospholids) and accumulates in lysosomes where it causes a phosphlipidosis. However, a small amount released released from lysosomes to cytosol (by membrane destabilisation), or reaching cytosol by retrograde traffic, will trigger apoptotic signals that will casue the activation of caspases. These signals could either be direct (activation of Bax and subsequent release of cytochrome c from michondria) or indirect (inhibition of the proteasome causing an increase of ubiquitinylated Bax protein) effects. Current studies also examine the role of reactive oxygen species (ROS) in this process.

From Servais et al, 2008b

Figure 3A. Light microscopy appearance of paraffin sections of kidney cortex of rats A: control; B and C: 10 mg/kg 10 days. B; section treated for TdT-mediated labeling of fragmented DNA (TUNEL) to evidence apoptosis (arrows). C: section stained with methyl green-pyronin (Brachet's staining); single arrows: apoptosis; circle: mitotic figures. Bar is 20 µm. From El Mouedden et al, 2000a

Figure 3B:
Nuclei of LLC-PK1 cells stained by DAPI. In the absence of gentamicin, both electroporated and incubated cells show a diffuse, finely reticulated, staining characteristic of euchromatin of diploid interphase animal cells. In contrast, cells electroporated or incubated in the presence of gentamicin show typical changes associated with apoptosis, consisting in the condensation and fragmentation of the nuclear material. From Servais et al, 2006

These studies are now extended to other antibiotics, including polycationic peptides and saponins, as means to screen for and assess their potential to cause early toxicities.

Selected references on drug-induced apoptosis (by reverse chronological order; for full reference list, see our publication list)


Additional information:  <tulkens@facm.ucl.ac.be>
Last significant update: December 27, 2010