Paraquat (PQ) is a herbicide that preferentially accumulates in the lung and exerts its cytotoxicity via the generation of reactive oxygen species (ROS). There is no specific treatment for paraquat poisoning. Attempts have been made to increase the antioxidant status in the lung using antioxidants (e.g., superoxide dismutase, vitamin E, N-acetylcysteine) but the outcome from such treatments is limited. Encapsulation of antioxidants in liposomes improves their therapeutic potential against oxidant-induced lung damage because liposomes facilitate intracellular delivery and prolong the retention of entrapped agents inside the cell. In the present study, we compared the effectiveness of conventional N-acetylcysteine (NAC) and liposomal-NAC (L-NAC) against PQ-induced cytotoxicity and examined the mechanism(s) by which these antioxidant formulations conferred cytoprotection. The effects of NAC or L-NAC against PQ-induced cytotoxicity in A549 cells were assessed by measuring cellular PQ uptake, intracellular glutathione content, ROS levels, mitochondrial membrane potential, cellular gene expression, inflammatory cytokine release and cell viability. Pretreatment of cells with L-NAC was significantly more effective than pretreatment with the conventional drug in reducing PQ-induced cytotoxicity, as indicated by the biomarkers used in this study. Our results suggested that the delivery of NAC as a liposomal formulation improves its effectiveness in counteracting PQ-induced cytotoxicity.
Paraquat (PQ) is a herbicide that preferentially accumulates in the lung and exerts its cytotoxic effects via the generation of reactive oxygen species (ROS) [
Liposomes are phospholipid vesicles composed of lipid bilayers enclosing an aqueous compartment. Hydrophilic molecules can be encapsulated in the aqueous spaces, and lipophilic molecules can be incorporated into the lipid bilayers. Liposomes, in addition to their use as artificial membrane systems, are used for the selective delivery of antioxidants and other therapeutic drugs to different tissues in sufficient concentrations to be effective in ameliorating tissue injuries. The relative ease in incorporating hydrophilic and lipophilic therapeutic agents into liposomes, the possibility of directly delivering liposomes to an accessible body site, such as the lung, and the relative nonimmunogenicity and low toxicity of liposomes have rendered the liposomal system highly attractive for drug delivery [
In the present study, we compared the effectiveness of conventional NAC and liposomal-NAC (L-NAC) against PQ-induced cytotoxicity and examined the mechanism(s) by which these antioxidant formulations conferred their cytoprotection. N-Acetylcysteine is a low-molecular-weight thiol-containing antioxidant with free radical-scavenging properties [
Human alveolar type II-like epithelial A549 cells (ATCC no. CCL-185, American Type Culture Collection, Manassas, Va, USA) were maintained in Costar 0.2
To determine the cytotoxicity of NAC alone, cells were treated with control media or media containing different concentrations of NAC (0 to 50 mM final NAC concentrations). To determine the effect of NAC or L-NAC on the toxicity of PQ, cells were first pretreated with control, empty liposomes- (EL-) NAC-, or L-NAC-containing media (5.0 mM for 4 h), followed by treatment with control or PQ-containing media.
NAC (N-acetyl-L-cysteine, SigmaUltra > 99% TLC; Sigma-Aldrich) was dissolved in PBS and adjusted to pH 7.4 to produce a 0.1 M stock solution. Following filter sterilization (0.2
Liposomal-N-acetylcysteine (L-NAC) was prepared from a mixture of DPPC (dipalmitoylphosphatidylcholine) and NAC in a 7 : 3 molar ratio by using a dehydration-rehydration method as described in [
Cell viability was measured with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as previously described in [
The intracellular levels of NAC, PQ, and GSH were determined by an ultraperformance liquid chromatographic (UPLC) method using a Waters Acquity system equipped with a binary solvent manager, an automated sample manager, and a photodiode array detector (Waters, Milford, Mss, USA) as described previously by Mitsopoulos and Suntres [
The intracellular levels of reactive oxygen species were determined by staining the cells with CM-H2DCFDA (5-(and 6-) chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester) (Molecular Probes, Eugene, Ore, USA) in PBS as previously described in [
Mitochondrial membrane potential was assessed using the MitoProbe JC-1 Assay Kit for Flow Cytometry (Molecular Probes). Following challenge, cells were washed with PBS and stained for 30 minutes with JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide), a cationic dye that exhibits potential-dependent accumulation in mitochondria, under standard incubation conditions. Stained cells were detached from the plate surface and suspended in PBS for flow cytometric analysis using the FL1-H and FL2-H channels of a BD FACSCalibur Flow Cytometer (BD Biosciences) with BD CellQuest Pro software. A minimum of 10,000 gated events were acquired per sample. Mitochondrial depolarization was indicated by decreased red fluorescence intensity due to concentration-dependent formation of red fluorescent J-aggregates.
Gene array analysis of cells challenged with 0.25 mM PQ for 4 h following pretreatment with control, NAC-containing or L-NAC-containing media (5.0 mM for 4 h) was performed as detailed previously in [
Relative expression, via microarray analysis, of genes involved with cellular stress and toxicity in cells challenged with 0.25 mM PQ for 4 h following pretreatment with control, NAC-containing, or L-NAC-containing media. Genes are listed in order of decreasing fold change in cells pretreated with control media and challenged with PQ. Fold change is expressed relative to untreated control cells using the housekeeping genes B2M, HPRT1, RPL13A, and GAPDH.
GeneBank accession no. | Gene name | Symbol | Fold change | |||||
Control media + PQ | NAC + PQ | LNAC + PQ | ||||||
NM_005953 | Metallothionein 2A | MT2A | 1.17 | 0.127 | −1.07 | 0.616 | 1.33 | 0.229 |
NM_002133 | Heme oxygenase (decycling) 1 | HMOX1 | 1.14 | 0.531 | 1.34 | 0.170 | −1.16 | 0.456 |
NM_000962 | Prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) | PTGS1 | 1.10 | 0.671 | 1.34 | 0.208 | 1.50 | 0.026** |
NM_001885 | Crystallin, alpha B | CRYAB | 1.07 | 0.847 | 1.36 | 0.190 | 1.18 | 0.510 |
NM_002574 | Peroxiredoxin 1 | PRDX1 | 1.07 | 0.505 | 1.05 | 0.367 | 1.04 | 0.662 |
NM_000454 | Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult)) | SOD1 | 1.04 | 0.343 | 1.05 | 0.332 | −1.07 | 0.200 |
NM_000581 | Glutathione peroxidase 1 | GPX1 | 1.01 | 0.812 | 1.05 | 0.395 | −1.08 | 0.286 |
NM_000849 | Glutathione S-transferase M3 (brain) | GSTM3 | −1.02 | 0.833 | −1.09 | 0.600 | −1.03 | 0.743 |
NM_001752 | Catalase | CAT | −1.03 | 0.740 | −1.31 | 0.136 | −1.16 | 0.168 |
NM_000637 | Glutathione reductase | GSR | −1.08 | 0.594 | −1.57 | 0.098 | −1.46 | 0.111 |
NM_005809 | Peroxiredoxin 2 | PRDX2 | −1.12 | 0.528 | −1.01 | 0.892 | −1.22 | 0.573 |
NM_001461 | Flavin-containing monooxygenase 5 | FMO5 | −1.28 | 0.154 | −1.53 | 0.072 | −1.60 | 0.065 |
NM_000499 | Cytochrome P450, family 1, subfamily A, polypeptide 1 | CYP1A1 | −1.40 | 0.057 | 1.09 | 0.583 | 4.44 | 0.003** |
NM_000941 | P450 (cytochrome) oxidoreductase | POR | −1.93 | 0.174 | 1.26 | 0.367 | −2.70 | 0.122 |
NM_001979 | Epoxide hydrolase 2, cytoplasmic | EPHX2 | −2.06 | 0.066 | −2.24 | 0.225 | −1.22 | 0.369 |
NM_000773 | Cytochrome P450, family 2, subfamily E, polypeptide 1 | CYP2E1 | — | — | — | — | — | — |
NM_000780 | Cytochrome P450, family 7, subfamily A, polypeptide 1 | CYP7A1 | — | — | — | — | — | — |
NM_002021 | Flavin-containing monooxygenase | FMO1 | — | — | — | — | — | — |
NM_005347 | Heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa) | HSPA5 | 1.35 | 0.004** | 1.24 | 0.026** | −1.00 | 0.983 |
NM_007034 | DnaJ (Hsp40) homolog, subfamily B, member 4 | DNAJB4 | 1.32 | 0.052 | 1.10 | 0.568 | −1.02 | 0.813 |
NM_001539 | DnaJ (Hsp40) homolog, subfamily A, member 1 | DNAJA1 | 1.29 | 0.048** | 1.12 | 0.439 | −1.08 | 0.443 |
NM_005526 | Heat shock transcription factor 1 | HSF1 | 1.14 | 0.074 | 1.08 | 0.253 | 1.13 | 0.354 |
NM_006644 | Heat shock 105 kDa/110 kDa protein 1 | HSPH1 | 1.14 | 0.253 | 1.03 | 0.551 | 1.03 | 0.612 |
NM_001 | Heat shock protein 90 kDa alpha (cytosolic), class A member 2 | HSP90AA2 | 1.08 | 0.200 | 1.05 | 0.557 | 1.09 | 0.279 |
NM_002157 | Heat shock 10 kDa protein 1 (chaperonin 10) | HSPE1 | 1.07 | 0.414 | −1.06 | 0.367 | −1.07 | 0.503 |
NM_002156 | Heat shock 60 kDa protein 1 (chaperonin) | HSPD1 | 1.06 | 0.671 | −1.15 | 0.312 | −1.08 | 0.406 |
NM_006597 | Heat shock 70 kDa protein 8 | HSPA8 | 1.02 | 0.922 | −1.09 | 0.599 | −1.33 | 0.060 |
NM_021979 | Heat shock 70 kDa protein 2 | HSPA2 | 1.01 | 0.908 | −1.40 | 0.004** | −1.46 | 0.003** |
NM_002154 | Heat shock 70 kDa protein 4 | HSPA4 | −1.06 | 0.523 | −1.47 | 0.036** | −1.71 | 0.004** |
NM_007355 | Heat shock protein 90 kDa alpha (cytosolic), class B member 1 | HSP90AB1 | −1.07 | 0.666 | 1.00 | 0.995 | −1.61 | 0.058 |
NM_005345 | Heat shock 70 kDa protein 1A | HSPA1A | −1.09 | 0.478 | −1.29 | 0.102 | 1.00 | 0.957 |
NM_001540 | Heat shock 27 kDa protein 1 | HSPB1 | −1.24 | 0.227 | −1.00 | 0.924 | −1.28 | 0.218 |
NM_005527 | Heat shock 70 kDa protein 1-like | HSPA1L | −1.29 | 0.084 | −1.65 | 0.032** | −1.42 | 0.077 |
NM_002155 | Heat shock 70 kDa protein 6 (HSP70B′) | HSPA6 | — | — | — | — | — | — |
NM_001964 | Early growth response 1 | EGR1 | 1.97 | 0.039** | 1.70 | 0.117 | −1.24 | 0.395 |
NM_005190 | Cyclin C | CCNC | 1.30 | 0.226 | 1.02 | 0.995 | 1.18 | 0.561 |
NM_182649 | Proliferating cell nuclear antigen | PCNA | 1.14 | 0.283 | −1.08 | 0.461 | −1.04 | 0.678 |
NM_053056 | Cyclin D1 | CCND1 | −1.03 | 0.885 | −1.28 | 0.225 | −1.02 | 0.803 |
NM_004060 | Cyclin G1 | CCNG1 | −1.05 | 0.410 | −1.02 | 0.316 | 1.07 | 0.435 |
NM_005225 | E2F transcription factor 1 | E2F1 | −1.28 | 0.473 | −1.16 | 0.450 | −1.94 | 0.107 |
NM_004864 | Growth differentiation factor 15 | GDF15 | 1.91 | 0.000** | 1.96 | 0.003** | 1.37 | 0.045** |
NM_004083 | DNA-damage-inducible transcript 3 | DDIT3 | 1.87 | 0.000** | 2.17 | 0.000** | 1.46 | 0.020** |
NM_000389 | Cyclin-dependent kinase inhibitor 1A (p21, Cip1) | CDKN1A | 1.50 | 0.002** | 1.72 | 0.001** | 1.88 | 0.000** |
NM_001924 | Growth arrest and DNA-damage-inducible, alpha | GADD45A | 1.29 | 0.125 | 1.50 | 0.044** | −1.01 | 0.942 |
NM_002392 | Mdm2, transformed 3T3 cell double minute 2, p53-binding protein (mouse) | MDM2 | 1.26 | 0.119 | −1.01 | 0.999 | 1.25 | 0.112 |
NM_000546 | Tumor protein p53 | TP53 | 1.14 | 0.294 | −1.10 | 0.546 | −1.11 | 0.353 |
NM_002178 | Insulin-like growth factor-binding protein 6 | IGFBP6 | −1.19 | 0.186 | 1.04 | 0.761 | −1.58 | 0.032** |
NM_001562 | Interleukin 18 (interferon-gamma-inducing factor) | IL18 | 1.49 | 0.010** | 1.26 | 0.106 | 1.28 | 0.016** |
NM_000575 | Interleukin 1, alpha | IL1A | 1.47 | 0.085 | 1.18 | 0.481 | 1.88 | 0.020** |
NM_000602 | Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 | SERPINE1 | 1.38 | 0.035** | 1.34 | 0.096 | 1.01 | 0.976 |
NM_000595 | Lymphotoxin alpha (TNF superfamily, member 1) | LTA | 1.22 | 0.458 | 1.38 | 0.141 | −2.19 | 0.060 |
NM_003998 | Nuclear factor of kappa light polypeptide gene enhancer in B cells 1 (p105) | NFKB1 | 1.22 | 0.004** | 1.12 | 0.092 | 1.36 | 0.000** |
NM_002415 | Macrophage migration inhibitory factor (glycosylation-inhibiting factor) | MIF | 1.02 | 0.505 | 1.13 | 0.043** | −1.06 | 0.331 |
NM_000576 | Interleukin 1, beta | IL1B | −1.02 | 0.948 | −1.69 | 0.357 | −1.02 | 0.936 |
NM_002989 | Chemokine (C-C motif) ligand 21 | CCL21 | — | — | — | — | — | — |
NM_002983 | Chemokine (C-C motif) ligand 3 | CCL3 | — | — | — | — | — | — |
NM_002984 | Chemokine (C-C motif) ligand 4 | CCL4 | — | — | — | — | — | — |
NM_001565 | Chemokine (C-X-C motif) ligand 10 | CXCL10 | — | — | — | — | — | — |
NM_000051 | Ataxia telangiectasia mutated | ATM | 1.21 | 0.318 | −1.38 | 0.197 | −1.07 | 0.727 |
NM_005431 | X-ray repair complementing defective repair in Chinese hamster cells 2 | XRCC2 | 1.19 | 0.125 | −1.26 | 0.261 | 1.10 | 0.303 |
NM_003362 | Uracil-DNA glycosylase | UNG | 1.11 | 0.315 | −1.01 | 0.882 | −1.01 | 0.890 |
NM_000122 | Excision repair cross-complementing rodent repair deficiency, complementation group 3 (xeroderma pigmentosum group B complementing) | ERCC3 | 1.07 | 0.612 | −1.26 | 0.271 | −1.20 | 0.229 |
NM_005053 | RAD23 homolog A ( | RAD23A | 1.04 | 0.764 | −1.00 | 0.956 | −1.23 | 0.265 |
NM_007194 | CHK2 checkpoint homolog ( | CHEK2 | 1.03 | 0.844 | −1.32 | 0.111 | −1.23 | 0.100 |
NM_001923 | Damage-specific DNA-binding protein 1, 127 kDa | DDB1 | −1.11 | 0.403 | −1.26 | 0.241 | −1.54 | 0.041** |
NM_001983 | Excision repair cross-complementing rodent repair deficiency, complementation group 1 (includes overlapping antisense sequence) | ERCC1 | −1.16 | 0.463 | 1.08 | 0.666 | −1.48 | 0.278 |
NM_006297 | X-ray repair complementing defective repair in Chinese hamster cells 1 | XRCC1 | −1.25 | 0.235 | −1.29 | 0.226 | −1.64 | 0.071 |
NM_007120 | UDP glucuronosyltransferase 1 family, polypeptide A4 | UGT1A4 | −1.30 | 0.319 | −2.04 | 0.071 | −1.50 | 0.283 |
NM_001230 | Caspase 10, apoptosis-related cysteine peptidase | CASP10 | 1.71 | 0.006** | 1.25 | 0.194 | 1.35 | 0.110 |
NM_001154 | Annexin A5 | ANXA5 | 1.42 | 0.025** | 1.17 | 0.073 | 1.42 | 0.004** |
NM_020529 | Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha | NFKBIA | 1.20 | 0.097 | 1.35 | 0.014** | 1.47 | 0.017** |
NM_001228 | Caspase 8, apoptosis-related cysteine peptidase | CASP8 | 1.15 | 0.184 | −1.08 | 0.571 | 1.36 | 0.017** |
NM_004324 | BCL2-associated X protein | BAX | −1.04 | 0.656 | −1.12 | 0.503 | −1.30 | 0.069 |
NM_003810 | Tumor necrosis factor (ligand) superfamily, member 10 | TNFSF10 | −1.09 | 0.788 | −1.15 | 0.444 | −1.53 | 0.110 |
NM_033292 | Caspase 1, apoptosis-related cysteine peptidase (interleukin 1, beta, convertase) | CASP1 | −1.11 | 0.818 | −1.24 | 0.150 | −1.19 | 0.627 |
NM_001065 | Tumor necrosis factor receptor superfamily, member 1A | TNFRSF1A | −1.29 | 0.235 | −1.15 | 0.610 | −1.64 | 0.081 |
NM_138578 | BCL2-like 1 | BCL2L1 | −1.46 | 0.028** | −1.37 | 0.257 | −1.42 | 0.340 |
NM_001101 | Actin, beta | ACTB | 1.35 | 0.018** | −1.17 | 0.251 | 1.23 | 0.152 |
NM_004048 | Beta-2-microglobulin | B2M | — | — | — | — | — | — |
NM_000194 | Hypoxanthine phosphoribosyltransferase 1 (Lesch-Nyhan syndrome) | HPRT1 | — | — | — | — | — | — |
NM_012423 | Ribosomal protein L13a | RPL13A | — | — | — | — | — | — |
NM_002046 | Glyceraldehyde-3-phosphate dehydrogenase | GAPDH | — | — | — | — | — | — |
Conventional RT-PCR analysis of cells challenged as indicated previously was performed using the RT2 qPCR Primer Assays (Table
Relative expression, via conventional RT-PCR analysis, of genes involved with cellular stress and toxicity in cells challenged with 0.25 mM PQ for 4 h following pretreatment with control on NAC-, L-NAC- or EL-containing media. Fold change is expressed relative to the respective untreated time control using the housekeeping gene RPL13A.
GeneBank | Gene name | Symbol | Fold change | |||||||
Control media + PQ | NAC + PQ | LNAC + PQ | EL + PQ | |||||||
NM_00584 | Interleukin 8 | IL8 | 2.23 | 0.007** | 2.01 | 0.001** | 1.74 | 0.010** | 2.15 | 0.009** |
NM_002746 | Mitogen-activated protein kinase 3 | MAPK3 | 1.47 | 0.404 | 1.45 | 0.472 | 1.86 | 0.151 | 1.55 | 0.378 |
NM_002228 | Jun oncogene | JUN | 1.40 | 0.240 | 1.63 | 0.049** | 1.36 | 0.174 | 1.09 | 0.606 |
NM_002750 | Mitogen-activated protein kinase 8 | MAPK8 | 1.18 | 0.847 | 1.45 | 0.544 | 1.03 | 0.907 | −1.08 | 0.820 |
NM_000660 | Transforming growth factor, beta 1 | TGFB1 | 1.13 | 0.593 | 1.34 | 0.245 | 1.26 | 0.316 | 1.07 | 0.664 |
NM_005252 | V-fos FBJ murine osteosarcoma viral oncogene homolog | FOS | 1.02 | 0.866 | 1.63 | 0.417 | 1.09 | 0.958 | 1.04 | 0.737 |
NM_001315 | Mitogen-activated protein kinase 14 | MAPK14 | −1.03 | 0.781 | 1.24 | 0.590 | −1.01 | 0.930 | −1.33 | 0.911 |
NM_000572 | Interleukin 10 | IL10 | −2.15 | 0.079 | −1.82 | 0.126 | 1.09 | 0.889 | −1.60 | 0.177 |
NM_000576 | Interleukin 1, beta | IL1B | 1.35 | 0.677 | 1.73 | 0.476 | 1.77 | 0.452 | 1.40 | 0.656 |
NM_020529 | Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha | NFKBIA | 1.31 | 0.271 | 1.46 | 0.179 | 1.69 | 0.087 | 1.55 | 0.205 |
NM_000499 | Cytochrome P450, family 1, subfamily A, polypeptide 1 | CYP1A1 | 1.12 | 0.950 | 1.10 | 0.925 | 2.26 | 0.155 | 1.77 | 0.400 |
NM_000575 | Interleukin 1, alpha | IL1A | 1.08 | 0.802 | 1.48 | 0.308 | 1.65 | 0.317 | 1.36 | 0.446 |
NM_000594 | Tumor necrosis factor (TNF superfamily, member 2) | TNF | −1.00 | 0.942 | 1.34 | 0.255 | 1.32 | 0.134 | 1.36 | 0.098 |
NM_000454 | Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult)) | SOD1 | −1.04 | 0.962 | 1.02 | 0.784 | −1.43 | 0.263 | −1.24 | 0.297 |
NM_001752 | Catalase | CAT | −1.12 | 0.852 | −1.21 | 0.402 | −1.30 | 0.299 | −1.25 | 0.466 |
Cells seeded into sterile 25 cm2 culture flasks (Corning) at 1.35 × 106 cells/flask were incubated to 80% confluence overnight, then washed with PBS and pretreated with control, NAC-containing or L-NAC-containing media (5.0 mM for 4 h) followed by challenge with control or PQ-containing media (0.25 or 1.0 mM for 4 h). Following incubation, media of treated cells were analyzed for cytokine levels using a Human Grp I Cytokine 7-Plex Panel kit (Bio-Rad) specific for interleukin (IL)-1
Data are presented as mean ± S.E.M (
Challenge of A549 cells with NAC at concentrations ranging from 0 to 10 mM did not have any effect on cell viability 24 h after NAC exposure. However, a 30% decrease in viability relative to control cells was observed following treatment with 50.0 mM NAC (Figure
Effect of NAC on the cell viability (a) and uptake of NAC (b) in A549 cells. The viability of cells treated for 24 h with increasing concentrations of NAC was assessed using the MTT assay. Bars represent mean ± S.E.M. of 3 independent experiments performed in octuplet. *denotes significant difference relative to control
The uptake of NAC by A549 cells was assessed using UPLC following treatment with 5.0 mM NAC- or L-NAC-containing media for 0, 1, 2, 4, 8, and 24 h (Figure
Challenge of A549 cells with PQ resulted in concentration-dependent decreases in cell viability (Figure
Effect of EL, NAC, or L-NAC pretreatment on viability of PQ-challenged cells. The viability of cells pretreated with control media (no pretreatment), or 5.0 mM NAC- (NAC Pretreatment), L-NAC- (L-NAC pretreatment) or empty liposome-containing media (EL pretreatment) for 4 h prior to 24 h PQ challenge (0, 0.1, 0.5, or 1.0 mM) was assessed using the MTT assay. Bars represent mean ± S.E.M. of 3 independent experiments performed in octuplet. *denotes significant difference relative to cells with no pretreatment
Exposure of cells to increasing concentrations of PQ for 24 h significantly decreased intracellular GSH content, which correlated with increases in cellular PQ uptake, as measured by UPLC (Figure
Effect of NAC or L-NAC pretreatment on intracellular levels of GSH (a), PQ (b), and ROS (c) in PQ-challenged cells. Cells pretreated for 4 h with control media (No Pretreatment) or 5.0 mM NAC- (NAC Pretreatment) or L-NAC-containing media (L-NAC Pretreatment) were challenged with increasing PQ concentrations (0, 0.1, 0.5, 1.0, and 5.0 mM) for 24 h. Cells were harvested and lysed for concomitant measurement of intracellular GSH content (a) and PQ uptake (b) via UPLC analysis and normalized to total protein. For the measurement of ROS, cells were stained for 30 min posttreatment with the cell permeable CM-H2DCFDA fluorescent dye specific for oxidative species. Adherent cells were scraped and analyzed flow cytometrically using the FL1-H channel. Bars represent mean ± S.E.M. of 3 independent experiments. *denotes significant difference relative to cells with no pretreatment
The mitochondrial membrane potential of cells challenged with 0.25 mM PQ for 4 h was significantly decreased relative to untreated control cells and was further decreased following 1.0 mM PQ challenge. Pretreatment with L-NAC was effective in preventing the decreases of mitochondrial membrane potential in both 0.25 and 1.0 mM PQ-challenged cells, returning it to basal levels in the former, as well as increasing it nearly 2-fold when compared to untreated control cells (Figure
Effect of NAC or L-NAC pretreatment on mitochondrial membrane potential following PQ challenge. Cells pretreated for 4 h with control media or 5.0 mM NAC- (N) or L-NAC-containing media (L) were challenged with 0, 0.25, or 1.0 mM PQ for 4 h. Cells were stained for 30 min posttreatment with the cell permeable JC-1 fluorescent dye. Bars represent mean ± S.E.M. of 3 independent experiments. *denotes significant difference relative to cells with no pretreatment
Levels of IL-8 secreted by cells exposed to 0.25 mM and 1.0 mM PQ were significantly increased relative to untreated control cells (Figure
Effect of NAC or L-NAC pretreatment on IL-8 levels after PQ exposure. Cells pretreated for 4 h with control media or 5.0 mM NAC- (N) or L-NAC-containing media (L) were challenged with 0, 0.25, or 1.0 mM PQ for 4 h. Cell culture supernatants were collected immediately following challenge and concomitantly analyzed for IL-8 using the Bio-Plex suspension array system. Bars represent mean ± S.E.M. of 3 independent experiments. *denotes significant difference relative to cells with no pretreatment
Changes in gene expression were assessed using a gene array designed to study genes involved with cellular stress and toxicity. The magnitude of gene expression in cells pretreated with NAC or L-NAC prior to 0.25 mM PQ challenge for 4 h was generally decreased relative to challenged cells with no pretreatment (Figure
Effect of NAC or L-NAC pretreatment on the magnitude of gene expression in PQ-challenged cells. RNA was extracted from cells challenged with 0 or 0.25 mM PQ for 4 h following pretreatment with 5.0 mM NAC- or L-NAC-containing media and analyzed via quantitative reverse-transcription PCR using a gene array. The magnitude of expression of each gene is expressed on a scale ranging from minimal (intense green) to maximal (intense red) expression (
The expression of many oxidative or metabolic stress-related genes was not significantly altered under any of the studied conditions, with the exception of CYP1A1 and PTGS1 being significantly upregulated in PQ-challenged cells pretreated with L-NAC. The expression of all studied heat shock genes remained more or less unchanged in PQ-treated cells but HSPA2, HSPA4, and HSPA1L were each significantly downregulated with NAC or L-NAC pretreatment. The expression of EGR1 was increased 2.0-fold in PQ challenged cells in the absence of antioxidant pretreatment but its expression was maintained at control levels with L-NAC pretreatment.
Antioxidant pretreatment of cells subsequently challenged with PQ had an effect on genes related to growth arrest and senescence as well. Briefly, GDF15 and DDIT3 were both significantly upregulated 1.9-fold following PQ challenge and were modulated with L-NAC (1.4- and 1.5-fold, resp.), but not NAC (2.0- and 2.2-fold, resp.), pretreatment. CDKN1A was upregulated 1.5-fold following PQ challenge and was progressively upregulated further with both NAC (1.7-fold) and L-NAC (1.9-fold) pretreatment. Also significantly altered were the expression patterns of several inflammatory genes. IL18 was upregulated 1.5-fold in PQ-challenged cells, but its expression was modulated with NAC or L-NAC pretreatment. Using individual primer assays with conventional RT-PCR, we found IL8 to be significantly upregulated (2.2-fold) in PQ-challenged cells with no pretreatment, but it was progressively modulated with NAC (2.0-fold) and L-NAC (1.7-fold) pretreatment. It is worth noting that the IL10 gene, which codes for the anti-inflammatory cytokine IL-10, was down-regulated in PQ-challenged cells, an effect reversed by L-NAC, but not NAC, pretreatment. Finally, the expression of many apoptosis signalling genes was not altered under the studied conditions with the exception of CASP10, which was upregulated 1.7-fold in PQ-challenged cells with no pretreatment and was modulated by both NAC and L-NAC pretreatment. The effect of L-NAC was confirmed to not be a direct result of the lipids composing the liposomes as pretreatment with empty liposomes did not alter the expression compared to PQ-challenged cells with no pretreatment (Table
Conventional RT-PCR assays were performed to validate findings obtained from the gene arrays. Similar gene expression patterns were observed for the majority of the genes (e.g., CAT, CYP1A1, IL1A, NFKBIA, and SOD1) analyzed by both methods.
Figure
Validation of RNA integrity (a) and assessment of PCR gene product quality (b). Aliquots of extracted RNA from control or PQ-challenged A549 cells with or without pretreatment were assessed for RNA concentration and integrity using the Experion automated electrophoresis station. A representative electropherogram displays 18 and 28 S rRNA peaks. Representative first-derivative dissociation curves of amplified PCR product of 4 h control and PQ-challenged cells pretreated with control media or NAC- or L-NAC-containing media are depicted.
Challenge of A549 cells with empty liposomes was not toxic to cells. Also, pretreatment of cells with empty liposomes did not confer any protection against PQ-induced cytotoxicity (Figure
The results of the present study showed that exposure of A549 cells to PQ
In order to assess the cytoprotective effects of both the conventional and liposomal NAC formulations, we first investigated their optimal treatment conditions in A549 cells. NAC has been reported to exhibit cytotoxicity at variable concentrations depending on cell type: 10 mM NAC was nontoxic in human bronchial epithelial cells [
Both NAC formulations conferred protection against PQ-induced cytotoxicity but generally L-NAC was more effective than the conventional NAC formulation in limiting the PQ-induced decreases of cellular GSH content (Figure
The mitochondria are thought to be essential targets of PQ and important in its toxicity. In fact, there is evidence that PQ disrupts the mitochondrial electron transfer chain resulting in a reduction of metabolic function, and it is suggested that lesions due to PQ first occur in the mitochondria [
The maintenance of cellular redox status is crucial for cellular homeostasis, and its dysregulation towards a more oxidized intracellular environment is associated with aberrant transcriptional activation and gene expression affecting several processes such as cell growth, differentiation, and inflammation [
Paraquat administration has been shown to be associated with an infiltration of neutrophils in lung [
Oxidative stress, which occurs when the redox homeostasis within the cell is altered, is a key pleiotropic modulator which may be involved in the upregulation and/or downregulation of several genes [
In conclusion, the results of the present study suggest that pretreatment of A549 cells with NAC, both in its conventional and liposomal form, conferred cytoprotection against PQ-induced toxicity. This was mainly attributed to its ability to ameliorate cellular redox status (i.e., intracellular GSH content and ROS levels) and was independent of PQ uptake. These protective effects were more evident in cells pretreated with L-NAC, which is attributed, at least in part, to the increased NAC levels achieved via liposomal delivery.
The authors declare that there is no conflict of interest.
This work was supported by a Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC no. 312533-2008).