Aeromonas hydrophila inhibits autophagy triggering cytosolic translocation of mtDNA which activates the pro-apoptotic caspase-1/IL-1ß-nitric oxide axis in headkidney macrophages.

The molecular mechanisms underlying Aeromonas hydrophila-pathogenesis are not well understood. Using head kidney macrophages (HKM) of Clarias gariepinus, we previously reported the role of ER-stress in A. hydrophila-induced pathogenesis. Here, we report that PI3K/PLC-induced cytosolic-Ca2+ imbalance induces the expression of pro-apoptotic ER-stress marker, CHOP in A. hydrophila-infected HKM. CHOP promotes HKM apoptosis by inhibiting AKT activation and enhancing JNK signaling. Elevated mitochondrial ROS (mtROS) was recorded which declined significantly by ameliorating ER-stress and in the presence of ER-Ca2+ release modulators (2-APB and dantrolene) and mitochondrial-Ca2+ uptake inhibitor, Ru360, together suggesting the role of ER-mitochondrial Ca2+ dynamics in mtROS generation. Inhibiting mtROS production reduced HKM death implicating the pro-apoptotic role of mtROS in A. hydrophila-pathogenesis. The expression of autophagic proteins (LC3B, beclin-1, and atg 5) was suppressed in the infected HKM. Our results with autophagy-inducer rapamycin demonstrated that impaired autophagy favored the cytosolic accumulation of mitochondrial DNA (mtDNA) and the process depended on mtROS levels. Enhanced caspase-1 activity and IL-1ß production was detected and transfection studies coupled with pharmacological inhibitors implicated mtROS/mtDNA axis to be crucial for activating the caspase-1/IL-1ß cascade in infected HKM. RNAi studies further suggested the involvement of IL-1ß in generating pro-apoptotic NO in A. hydrophila-infected HKM. Our study suggests a novel role of ER-mitochondria cross-talk in regulating A. hydrophila pathogenesis. Based on our observations, we conclude that A. hydrophila induces ER-stress and inhibits mitophagy resulting in mitochondrial dysfunction which leads to mtROS production and translocation of mtDNA into cytosol triggering the activation of caspase-1/IL-1ß-mediated NO production, culminating in HKM apoptosis.

Arsenic induced alteration in Mrp-1 like activity leads to zebrafish hepatocyte apoptosis: The cellular GSH connection.

Multidrug-resistance protein-1 facilitates the efflux of arsenic conjugated with reduced glutathione nonetheless; the relation between Mrp-1 ATPase activity and cellular GSH levels is contentious. To study this, Mrp-1-ATPase activity was measured in 5 µM arsenic trioxide exposed zebrafish hepatocytes (ZFH) and correlated with intracellular GSH levels. Alongside, mrp-1 gene expression as well as Mrp-1 protein level was also monitored. Diverse mode of Mrp-1 inhibition was reflected from differential level of Km and Vmax of Mrp-1 at different time points. 3 h post-arsenic treatment demonstrated non-competitive inhibition. At 6 h, there was significant increase in Km and ZFH death, suggesting reduced binding affinity of Mrp-1 for ATP. Increased caspase-9-cytochromeC-ATP levels (putative apoptosome), reinforced ZFH apoptosis. The increase in Vmax coupled with reduced substrate affinity of Mrp-1 suggests malfunctioning in arsenic- tolerance mechanisms. We posit the triggering glutathione level regulate arsenic tolerance in ZFH. Irreversible impairment of ATP binding to Mrp-1 culminates in arsenic-induced ZFH apoptosis.

Aeromonas hydrophila-induced alterations in cytosolic calcium activate pro-apoptotic cPKC-MEK1/2-TNFα axis in infected headkidney macrophages of Clarias gariepinus.

Alterations in intracellular-calcium (Ca2+)i homeostasis is critical to Aeromonas hydrophila-induced headkidney macrophages (HKM) apoptosis of Clarias gariepinus, though the implications are poorly understood. Here, we describe the role of intermediate molecules of Ca2+-signaling pathway that are involved in HKM apoptosis. We observed phosphoinositide-3-kinase/phospholipase C is critical for (Ca2+)i release in infected HKM. Heightened protein kinase-C (PKC) activity and phosphorylation of MEK1/2-ERK1/2 was noted which declined in presence of 2-APB, Go6976 and PD98059, inhibitors to IP3-receptor, conventional PKC isoforms (cPKC) and MEK1/2 respectively implicating Ca2+/cPKC/MEK-ERK1/2 axis imperative in A. hydrophila-induced HKM apoptosis. Significant tumor necrosis factor-α (TNFα) production and its subsequent reduction in presence of MEK-ERK1/2 inhibitor U0126 suggested TNFα production downstream to cPKC-mediated signaling via MEK1/2-ERK1/2 pathway. RNAi and inhibitor studies established the role of TNFα in inducing caspase-8-mediated apoptosis of infected HKM. We conclude, alterations in A. hydrophila-induced (Ca2+)i alterations activate cPKC-MEK1/2-ERK1/2-TNFα signaling cascade triggering HKM apoptosis.

Arsenic-induced instrumental genes of apoptotic signal amplification in death-survival interplay.

Arsenic is a global health concern at present and it is well reported for causing systemic toxicity. It is also well known for generation of free radical and inducing apoptosis in different cell types. Paradoxically arsenic is reported to be a susceptible carcinogen as well. There are several reports demonstrating diverse mechanism of apoptosis in different cell types. However, the universal scenario of instrumental genes and their interaction leading to amplification of apoptotic signal are yet to be completely uncovered, which is predicted here. Conventional studies on signaling pathway aided by time and concentration kinetics data are inadequate for prediction of anchored genes for apoptotic signal amplification. Therefore, expression profile-based approach is adopted. Core apoptosis related and glutathione metabolism genes in 1 and 10 µM of arsenic-treated HepG2 cells were analyzed after 12 h of incubation. An arsenic treatment of 1 µM exhibits no cell death at 12 h, whereas 10 µM arsenic treatment reveals around 50% cell death at 12 h. Results depict 28 and 44 affected genes in 1 and 10 µM arsenic-treated cells, respectively. Early initiation of apoptotic signaling is detected in no cell death regimens (at 1 µM), whereas amplified apoptotic signal is demonstrated at 50% cell death regimens (at 10 µM). Instrumental genes involved in progression of apoptosis in the concourse of cell death and survival is designated from the responsive genes common to both the condition. We predict the initiation process is fairly aided by the activation of intrinsic pathway, which is amplified via TNF signaling and extrinsic pathway. Furthermore, regulatory genes involved in interplay between apoptosis/anti-apoptosis and their interactions are demonstrated here.

Arsenic trioxide and lead acetate induce apoptosis in adult rat hepatic stem cells.

In the present study, the toxicity of arsenic trioxide and lead acetate was assessed in adult hepatic stem cells induced in the 2-acetyl-aminofluorene/partial hepatectomy rat model. Isolated oval cells were incubated separately for 6 h with 40 muM each of arsenic trioxide and lead acetate. 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay denoted significant time-dependent cell death in arsenic and lead treated oval cells. The degree of stress imposed by these metals was evidenced by induction of heat shock protein (HSP) 70 and HSP 90. Arsenic and lead were found to trigger apoptosis as revealed by DNA ladder formation, Western blots of apoptotic factors, and reverse transcriptase polymerase chain reaction analyses of bax and bcl-2. Results clearly indicate that both arsenic and lead induced apoptosis is caspase-mediated and accompanied by extracellular signal-regulated kinase (ERK) dephosphorylation. Full-length BH3-interacting-domain death agonist expression in presence of caspase 3 inhibitor unravels a direct involvement of caspase in As and Pb induced apoptosis. Expression patterns of apoptosis inducing factor, B cell lymphoma-2 (Bcl-2) antagonist of cell death, Bcl-2-associated X protein, and Bcl2 also signify mitochondrial regulation of apoptosis effected by lead and arsenic. It is concluded that stimulation of caspase cascade and simultaneous ERK dephosphorylation are the most significant operative pathways directly associated with apoptotic signals triggered by arsenic and lead in the oval cells.