The current study was undertaken in order to gain further insight into the mechanisms involved in paraoxon cytotoxicity through alterations in protein regulation. For our proteomic analysis, the cells were treated with 20 µM paraoxon for 48 h since the MTT assays indicated there was no significant decrease in cell viability at 72 h. Cell viability was adversely affected at paraoxon concentrations greater than 500 nM at the 96 h time point, however, SH-SY5Y cells showed normal viability with paraoxon concentrations as high as 100 µM at 24 h, which is consistent with earlier reports with undifferentiated and differentiated cells (21
). Although cell viability was unaffected, treatment with 20 µM paraoxon inhibits acetylcholinesterase in SH-SY5Y cell lines within one hour (25
). Interestingly, 24 h exposure to 5, 10, and 50 µM paraoxon appeared to have a modest proliferative effect on cell growth as indicated by an increase in absorbance at these time points. This observation is contrary to that found for human hepatoma HepG2 cells that show a reduction in cell proliferation after exposure to 100 µM methyl paraoxon (29
A sub-proteome fractionation approach was undertaken to facilitate the identification of low abundance proteins and aid separation of individual proteins by their locus of compartment. These advantages can be offset, in part, by carryover of certain proteins that are distributed into more than one cellular compartment thereby complicating measures in the expression changes. Sixteen proteins changed in expression level > 1.3-fold following exposure of SH-SY5Y cells to 20 µM paraoxon at 48 h () and these proteins were found to be located in the four different cellular compartments (). This is a significant finding indicating that paraoxon exposure results in cell-wide changes in protein expression. The total number of proteins that changed in expression roughly correlates with the fifteen genes that were identified by gene array in SH-SY5Y cells treated with paraoxon (3 µM and 30 µM) for 24 h (17
Based on our sub-proteome analysis, our results indicate that paraoxon exposure leads to protein expression changes throughout cell compartments and not just AChE and serine hydrolase-type proteins. Good to excellent (18–53%) sequence coverage was determined by MALDI MS analyses and all protein spots were in good agreement with the theoretical molecular weight and pI. As such, the proteomic analysis identified a number of proteins that may be sensitive to paraoxon exposure, only one of which had been previously uncovered during a gene expression study (17
). A few proteins changed in expression three-fold or greater including ATPase (membrane fraction), beta actin (membrane fraction) and histone H2B.1 (nuclear fraction). During the tryptic peptide analysis by mass spectrometry, no evidence was obtained that proteins were directly phosphorylated by paraoxon although the possibility that covalent modification may have occurred cannot be excluded.
To address the possibility that overall protein expression changes due to paraoxon exposure may differ from those determined using a subproteome fractionation approach, western blot analysis was conducted for three of the identified proteins using a total protein extraction. SH-SY5Y cells were exposed to 20 µM paraoxon and the cells collected at several different time points to investigate dynamic expression patterns for HSP 90, hnRNP c1/c2 and the ATP synthase β-subunit, which displayed large changes in expression as determined by the 2-D gel analysis.
HSP 90 showed a decrease in expression of 1.3 to 1.7-fold in the cytosolic fraction following paraoxon exposure but when analyzed by Western blot analysis the expression change showed an increase 2.0 to 2.6-fold. This discrepancy has not been resolved although it is possible that isolated cytosolic HSP 90 expression differs from protein located throughout the cell. HSP 90 has been reported to play a role in various cellular processes including signal transduction, protein folding, and protein degradation (30
). The results observed by Western blot analysis agrees with previous studies using cultured PC-12 cells, which found that HSP90 was induced by a variety of different pesticides, including the organophosphate chlorpyrifos, and believed to be a cellular protection response to oxidative stress (32
Following paraoxon exposure, the heterogeneous nuclear ribonucleoprotein C (C1/C2) (hnRNP c1/c2) was up-regulated from 1.6 to 2.8-fold when analyzed from the membrane fraction and validated by Western blot by a 1.2 to 1.3-fold increase from total protein. HnRNP c1/c2 belongs to the RNA-binding protein family (33
) and is primarily located in the nucleus but may also be found in the cytoplasm. It was therefore surprising that hnRNP c1/c2 was found in the membrane fraction although the detergents used to sub-fractionate the proteome may have played a role in distribution to this compartment. HnRNP c1/c2 may be associated with pre-mRNA processing, metabolism and transport (34
), cellular homeostasis (34
), plays a role in cell cycle regulation (35
), and hypothesized to be associated with disease pathways such as cancer (36
) and schizophrenia (38
). Down-regulation of hnRNP c1/c2 by siRNA in HeLa cells resulted in cells hypersensitivity to a variety of cellular stresses including H2
(oxidative stress) (34
Results from our proteomic analysis indicated that the ATPase beta chain demonstrated a 3.1-fold decrease in spot density, which was contrary to the 1.66-fold increase found using Western blot analysis based on total protein at the same time point. Although ATPase expression is clearly altered by paraoxon exposure, it is difficult to account for the reversed responses. The ATP synthase β-subunit is part of a multi-subunit complex found in mitochondria that catalyzes ATP synthesis utilizing an electrochemical gradient of protons across the inner membrane during oxidative phosphorylation (39
) and some early reports suggested that mitochondria may be a target for reactive OPs resulting in a decrease in ATP synthesis (21
The proteomics study was designed to obtain information about changes in protein expression levels in SH-SY5Y cells following exposure to paraoxon. To more accurately identify those changes, a sub-proteome fractionation approach was employed to separate paraoxon-affected proteins based on their cellular compartment. In addition to improved identification accuracy, the fractionation was also envisioned to assist with the analysis of low abundance proteins and/or better understand how paraoxon might alter protein expression changes based on the cellular localization. However, certain inherent problems in the sub-proteome extraction led to difficulties in correlating and validating protein regulation changes as noted for the three proteins selected for Western blot analysis. For example, many proteins likely appear in more than one of the cell fractions, extraction efficiency can be poor, and some proteins may exist in different post-translationally modified forms in different cellular compartments. Therefore, measures of sub-proteome expression changes may not correlate well with whole cell expression changes, and further interpretation should take the limitations of each experimental approach into account.
Although reactive OPs like paraoxon are known to inhibit AChE to initiate organism level toxicity, it is well known that other individual proteins and cellular pathways are affected by interaction with OPs and may or may not be related to AChE inhibition (13
). Further, the concentration of OP required to inhibit AChE is typically much less than the lethal dose of OP indicating that other cellular pathways are affected by OP exposure (25
). It is likely that, as AChE inhibition occurs, cells respond by initiating responses such as oxidative stress. Many environmental toxicants induce stress responses in affected cells and these stress responses can be broken down into several broad categories, such as oxidative stress, DNA damage, heat shock (chaperone), and inflammation, among others (40
). Certain OPs have been shown to cause oxidative damage in cell culture and animal model systems, as well as oxidative stress-like symptoms in human populations (41
). Protein expression changes uncovered in this study suggest that exposure to OPs may induce a cellular stress response. Whether the oxidative stress response elicited by OPs is due to downstream effects of AChE inhibition or to other direct targets of OP phosphorylation is not presently known.
Tropomyosin and actin both changed in regulation following exposure to paraoxon, and have been previously shown to have altered regulation in response to several toxicants, including the herbicide atrazine (42
). Likewise, exposure of rat aortic smooth muscle cells to homocysteine, which causes oxidative stress, affected protein expression of tropomyosin, actin, and prohibitin (43
), although prohibitin has also been shown to be up-regulated following oxidative stress (44
). In the proteomic analysis, FSCN1, actin, and tropomyosin all changed in expression in response to paraoxon exposure and are all implicated in cytoskeletal rearrangement pathways (45
) and although this pathway was not observed in the course of this exposure. A decrease in the expression of FSCN1 has been shown to be associated with the suppression of cellular proliferation, possibly indicative of slowing down of cell division (46
). Alpha enolase, which was down-regulated 2.3-fold in SH-SY5Y at 48 h following exposure, was briefly examined to see if this enzyme interacts directly with paraoxon. However, the activity of recombinant alpha enolase was largely unaffected by paraoxon at concentrations up to 10 mM (data not shown).
In order to fully elucidate the molecular mechanisms involved in OP toxicity, a better understanding is needed of the molecular events following OP exposure. The results from this study indicate that exposure to paraoxon resulted in the altered expression of a number of proteins involved in a diverse array of cellular processes. Further, it was found that protein expression changes may vary between sub-proteome analysis and gene expression analysis (17
) and conclusions using either method should be independently validated. The significance of these protein expression changes to the overall paraoxon cytotoxicity remains to be determined and future studies need to be aimed at determining whether these expression changes are a specific result of paraoxon exposure or due to a general cellular stress response.