What will happen if lysosomal enzyme leak out of the lysosome to cytoplasm?

14.2.4 Lysosomal responses, as unspecific effect biomarkers

Lysosomes are cell organelles specialised in digestion, of both endogenous and exogenous materials. Impairment of lysosomes and, hence, of food assimilation, can result in severe alterations of the cells and whole organisms. Lysosomal responses to pollutant exposure or, in general terms, to environmental stress, fall into essentially three categories: changes in lysosomal size; reduced membrane stability; and changes in lysosomal contents. Diverse sources of environmental stress (chemical pollution, salinity changes, elevated temperature, malnutrition, reproductive stress) induce, generally an increase in the size of digestive cell lysosomes (as reviewed in Cajaraville et al., 1995b); however decreases have also been reported (Cajaraville et al., 1995a; Marigómez et al., 1996a). The activity of hydrolases and the number of lysosomes are usually augmented (Moore et al., 1987; Cajaraville et al., 1989; Marigómez et al., 1996a; Etxeberria et al., 1994; and Krishnakumar et al., 1994). Likewise, it has also been reponsed extensively reported that environmental stressors reduce the stability of the lysosomal membrane; this is measured usually in terms of reduced labilisation period (Moore, 1976, 1985; Köhler, 1991; Regoli, 1992; Regoli et al., 1998; and Lowe and Fossato, 2000). In both piscine liver and the molluscan digestive gland, destabilisation of the lysosomal membrane and changes in lysosomal volume may be quantified, using histochemical methods (Cajaraville et al., 1995b). After cryopreservation, freezing and cryosectioning, tissue samples are processed for enzyme histochemistry of β-glucuronidase or hexosaminidase, respectively. Subsequently, in the lysosomal membrane stability (LMS) test (UNEP, 1997), the labilisation period is estimated generally on the basis of subjective grading. In the lysosomal structural changes (LSC) test, lysosomal size, numbers and other related parameters are measured, by image analysis (Cajaraville et al., 1991, 1995b). Finally, immunochemical approaches (based upon the use of specific antibodies against lysosomal enzymes) can be applied also to the measurement of lysosomal responses (Lekube et al., 2000).

3.3.7 Lysosomal membrane stability

Lysosomes are intracellular organelles easy to visualize at the microscope and are involved in diverse functions for cellular well-being [42], including cellular defense, recycling of macromolecules, and detoxification of xenobiotics [156,157]. Lysosomes are capable of sequestering contaminants to prevent oxidative damage that can lead to membrane destabilization [158]. In stable lysosomes, hydrolases are prevented from reacting with substrates by an intact membrane. Membrane stability decreases in response to stress and membrane permeability increases. Some chemicals are known to alter the stability of the lysosomal membrane [112], while the mechanism behind the alteration of the membrane stability remains incompletely understood [52]. Lysosomal membrane stability (LMS) is usually measured through the determination of the neutral red retention time (NRRT) assay, which is based on the fact that healthy lysosomes retain the dye longer than perturbed ones. Lysosomal damage can produce the leakage of the dye into the cytosol, indicating damage that may lead to cell death [42]. Therefore, the retention time of the neutral red dye in living hemocytes provides a reliable health index of the organism [157,159]. LMS is not only considered as a biomarker of effect (Fig. 1) but also is used as a screening biomarker to assess the health status of a variety of organisms [42], and is also used for the Tier-1 approach in wide-scale biomonitoring programs [42,63]. This biomarker has been included in the general guidelines for monitoring programs (Joint Assessment & Monitoring Programme-JAMP by OSPAR) and is proposed as a marine pollution index to evaluate stress responses in mollusks, as was established in the Mediterranean Pollution Programme (MEDPOL) and in the International Council for the Exploration of the Sea (ICES) [160,161]. The current evidence suggests that pharmaceutical compounds are able to disrupt the LMS in marine bivalves, indicating general stress (Table 1).

Destabilization of the lysosomal membrane has been identified in hemocytes of M. galloprovincialis after chronic exposure to NSAIDs namely, ibuprofen, diclofenac, ketoprofen, acetaminophen, and nimesulide [17,73]; effects have been also reported for Perna perna mussels [66]. The stimulant caffeine has been demonstrated to reduce the NRRT in hemocytes and digestive glands of M. galloprovincialis [36,54]. Similarly, the lipid regulator atorvastatin reduced the NRRT of hemocytes in M. edulis after 4 weeks of exposure [65]. In line with these results, a decrease of the LMS was observed in clams R. philippinarum exposed to carbamazepine, caffeine novobiocin tamoxifen [59,67], and ibuprofen [59,84]. The antidepressant fluoxetine induced a significant decrease of LMS in M. galloprovincialis [75] and in V. philippinarum [89].

LMS is therefore a biomarker of effect able to display the capacity of bivalves to respond to toxicity induced by pharmaceutical residues in seawater. In an environmental context, this biomarker has demonstrated to be a sensitive indicator of general stress, indicating that pharmaceuticals have entered the organism and are able to induce toxic effects. Due to this, its use is highly recommended.

2.28.2.4.3 Lysosomal stress and cathepsins

Lysosomes are a common target of various proapoptotic stimuli, including proinflammatory cytokines (TNF), lysosomotropic agents (hydroxychloroquine; O-methyl-serine dodecylamide hydrochloride; L-leucyl-L-leucine methyl ester), photosensitizers (N-aspartyl chlorin e6), quinolone antibiotics (ciprofloxacin; norfloxacin), amine aldehydes (3-aminopropanal), and redox-cycling quinones (5,8-dihydroxy-1,4-naphtho-quinone), just to name a few (Boya et al. 2003a,b; Deiss et al. 1996; Foghsgaard et al. 2001; Guicciardi et al. 2001; Kagedal et al. 2001; Li et al. 2000, 2003b; Reiners et al. 2002; Thiele and Lipsky 1990; Uchimoto et al. 1999). Lysosomes are particularly sensitive to oxidative stress as intralysosomal iron can catalyze fenton chemistry and the formation of highly reactive hydroxyl radicals that damage membranes and cause lysosomal rupture (Antunes et al. 2001). Lysosomal permeabilization invariably results in the release of various cathepsins, a number of which have been implicated in apoptosis, including cathepsin D (aspartic peptidase) and cathepsins B, C (DPPI), L, and S (cysteine proteases). Some reports suggest that cathepsins C and L may directly activate the effector caspase-3 (Bidere et al. 2002; Ishisaka et al. 1999), but the majority of studies indicate that cathepsins engage the intrinsic pathway via the cleavage of BID, which in turn promotes cytochrome c release and formation of the apoptosome (Stoka et al. 2001).

VII The LDL Receptor

The discovery of the LDL receptor pathway by Michael S. Brown and Joseph L. Goldstein represents the most significant triumph in the field of atherosclerosis research. In an extraordinary collaboration begun in 1972, they discovered that cells possess a high-affinity receptor that binds to the apo-B100 moiety of LDL. (They were awarded the Nobel Prize in 1985.)

Binding of LDL to its receptor results in rapid endocytosis and the formation of an endocytic vesicle (Fig. 8). The LDL and the receptor separate while in this vesicle and part ways; the receptor recycles and returns to the cell surface, while the LDL particle is delivered to the lysosome, where the protein and lipid moieties are degraded.

Hydrolysis of LDL cholesterol esters in the lysosome results in the release of free cholesterol, which exits the lysosome and exerts three important regulatory functions:

It suppresses cellular cholesterol synthesis by reducing the levels of the rate-limiting enzymes in the cholesterol biosynthetic pathway, principally 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase).

It enhances the re-esterification of cholesterol for storage in a cytoplasmic lipid droplet.

It inhibits production of new LDL receptor, thus diminishing the further supply of cholesterol to the cell.

The LDL receptor pathway assures a constant steady-state level of cellular cholesterol. This is accomplished both by adjusting cellular cholesterol synthesis according to ambient LDL levels and by altering LDL receptor number to limit the amount of LDL getting into cells. Like free fatty acids, unesterified cholesterol can be toxic to cells. The formation of cholesterol esters protects cells from cholesterol toxicity.

About two-thirds of LDL is catabolized by the liver. The rest is cleared by just about all other tissues. Steroid-producing tissues are especially active in LDL uptake. Adrenal cells (and presumably ovarian and testicular cells) do not synthesize cholesterol at rates sufficient to support high rates of steroidogenesis. They supplement their cholesterol supply by consuming cholesterol carried on LDL and HDL.

The level of LDL receptor activity is affected by the steady-state level of cholesterol in a cell. Thus, any factors that increase or decrease the cholesterol level of a cell will affect the rate of LDL clearance from the circulation. This means that nutritional factors (proportion and type of dietary fat), hormonal status, pharmacological factors (drugs that inhibit cholesterol synthesis), and agents that affect bile acid metabolism all affect plasma cholesterol by influencing the level of expression of the LDL receptor.

7.13.7.4 Postendocytic Transport

After endocytosis, the endosomes containing the aminoglycosides fuse with lysosomes (Ford et al. 1994; Sandoval et al. 1998; Servais et al. 2005; Silverblatt and Kuehn 1979; Wedeen et al. 1983). Progressively, more and more aminoglycoside molecules are sequestered in the lysosomes resulting in a markedly high intralysosomal aminoglycoside concentration and a long renal cortical half-life (Fabre et al. 1976; Silverblatt and Kuehn 1979; Tulkens and Trouet 1977; Wedeen et al. 1983). Intralysosomal sequestration is not the sole metabolic fate for aminoglycosides, however. Elegant in vitro and in vivo studies by Sandoval et al. (1998) revealed that gentamicin not only localizes to the lysosome but also traffics to the Golgi complex (Sundin et al. 2001). In addition, a related study of renal ischemia showed increased trafficking of gentamicin to the Golgi complex, which may lend an explanation as to why hypotensive patients are more susceptible to aminoglycoside nephrotoxicity (Sandoval et al. 2002). Other cell culture research revealed that the localization of the gentamicin to the Golgi complex occurred within 15–30 min after exposure and accounted for 5–10% of the total cellular accumulation of gentamicin (Sandoval et al. 1998, 2000). A more compelling finding was that like Shiga toxin, gentamicin trafficked in a retrograde fashion through the Golgi complex to the ER (Lord and Roberts 1998; Sandoval and Molitoris 2004; Sandvig et al. 1994, 1996) (Figure 1). Furthermore, after retrograde transport to the ER, gentamicin was released to the cytosol and accumulated in the mitochondria and nucleus (Sandoval and Molitoris 2004) (Figures 2–4).

Prior to this body of research delineating the secondary intracellular pathway for gentamicin, researchers believed that PTC toxicity was secondary to aminoglycoside accumulation in the lysosomes followed by lysosomal rupture, release, and association with the protein synthetic machinery. Studies have demonstrated, however, that the reduction in protein synthesis occurs rapidly after aminoglycoside administration. As a result, the attenuation in protein synthesis cannot be attributed solely to lysosomal rupture, which occurs later in the time course of aminoglycoside nephrotoxicity (Bennett et al. 1988). The rapid trafficking of the aminoglycoside to the Golgi complex after administration with the subsequent disruption of protein sorting and synthesis is more consistent to what is observed experimentally and a more plausible explanation for the early damage that is observed in the PTC. Moreover, the observed colocalizations of the aminoglycoside to the mitochondria and nuclei after the retrograde transport to the ER and cytosolic release explain the perturbations in mitochondrial potential and protein synthesis within the PTC observed with aminoglycoside nephrotoxicity (Sandoval and Molitoris 2004).

Distribution

Microscopic observations show that inside renal epithelial cells, uranium localizes in lysosomes, where it precipitates, leading to the appearance of thin needles, either isolated or grouped in spherical clusters, sea-urchin-like electron-dense structures of approximately 1–2 μm in diameter in the cell cytoplasm. Electron-dense structures are never observed in the nuclei. Even though their number increases with time or concentration of contamination, their diameter never exceeds 2 μm. Such precipitates are certainly lethal if they appear in vivo, in the case of extremely acute exposure to uranium. Indeed, the uranium concentration required for them to occur is very high. The precipitates are progressively expelled from cells, leading to extracellular and intracellular precipitates.

In addition, research has recently revealed the presence of soluble uranium in the cytoplasm of cells. The presence of these two chemical forms of uranium inside the cells can come either from initial soluble uranium internalization and a subsequent partial precipitation or more probably from two parallel internalization pathways.

What happens if lysosome enzymes are leaked into a cell?

What can happen if the lysosome contents are released into the cytoplasm?

Is there a problem with leakage of lysosomal enzymes into the cytosol?

What would happen if lysosome membrane leaks its digestive enzyme in cytosol?