Development of low-density oligonucleotide microarrays for detecting mutations causing Wilson's disease.

BACKGROUND & OBJECTIVES: Wilson's disease (WD) is an autosomal recessive disorder caused by mutations in ATP7B, a copper transporter gene, leading to hepatic and neuropsychiatric manifestations due to copper accumulation. If diagnosed early, WD patients can be managed by medicines reducing morbidity and mortality. Diagnosis of this disease requires a combination of tests and at times is inconclusive due to overlap of the symptoms with other disorders. Genetic testing is the preferred alternative in such cases particularly for individuals with a family history. Use of DNA microarray for detecting mutations in ATP7B gene is gaining popularity because of the advantages it offers in terms of throughput and sensitivity. This study attempts to establish the quality analysis procedures for microarray based diagnosis of Wilson's disease. METHODS: A home-made microarrayer was used to print oligonucleotide based low-density microarrays for addressing 62 mutations causing Wilson's disease reported from Indian population. Inter- and intra- array comparisons were used to study quality of the arrays. The arrays were validated by using mutant samples generated by site directed mutagenesis. RESULTS: The hybridization reaction were found to be consistent across the surface of a given microarray. Our results have shown that 52 °C post-hybridization wash yields better reproducibility across experiments compared to 42 °C. Our arrays have shown > 80 per cent sensitivity in detecting these 62 mutations. INTERPRETATION & CONCLUSIONS: The present results demonstrate the design and evaluation of a low-density microarray for the detection of 62 mutations in ATP7B gene, and show that a microarray based approach can be cost-effective for detecting a large number of mutations simultaneously. This study also provides information on some of the important parameters required for microarray based diagnosis of genetic disorders.

Differential immunotoxic effects of ethanol on murine EL-4 lymphoma and normal lymphocytes is mediated through increased ROS production and activation of p38MAPK.

Ethanol has been used to achieve thymic depletion in myasthenia gravis patients. Ethanol (95%) has also been used widely in the therapy of many tumors including hepatocellular carcinoma. In light of these findings, we delineated the differential immunotoxic behavior and mechanism of lower concentration of ethanol towards murine EL-4 lymphoma and its normal counterpart lymphocytes. EL-4 lymphoma and normal lymphocytes were cultured with ethanol (0%-5%) for 6 h and cytotoxicity was measured by various methods. EL-4 cells treated with ethanol showed concentration-dependent loss of viability at 2%-5% ethanol concentration and exhibit proliferative arrest at preG1 stage. Acridine-orange and ethidium-bromide staining indicated that ethanol induced death in EL-4 cells, by induction of both apoptosis and necrosis which was further supported by findings of DNA-fragmentation and trypan blue dye exclusion test. However, treatment of lymphocytes with similar concentration of ethanol did not show any death-associated parameters. Furthermore, ethanol induced significantly higher ROS generation in EL-4 cells as compared to lymphocytes and caused PARP cleavage and activation of apoptotic proteins like p53 and Bax, in EL-4 cells and not in normal lymphocytes. In addition, ethanol exposure to EL-4 cells led to phosphorylation of p38MAPK, and upregulation of death receptor Fas (CD95). Taken together, these results suggest that ethanol upto a concentration of 5% caused no significant immunotoxicity towards normal lymphocytes and induced cell death in EL-4 cells via phosphorylation of p38MAPK and regulation of p53 leading to further activation of both extrinsic (Fas) and intrinsic (Bax) apoptotic markers.

Plumbagin inhibits proliferative and inflammatory responses of T cells independent of ROS generation but by modulating intracellular thiols.

Plumbagin inhibited activation, proliferation, cytokine production, and graft-versus-host disease in lymphocytes and inhibited growth of tumor cells by suppressing nuclear factor-kappaB (NF-kappaB). Plumbagin was also shown to induce reactive oxygen species (ROS) generation in tumor cells via an unknown mechanism. Present report describes a novel role of cellular redox in modulation of immune responses in normal lymphocytes by plumbagin. Plumbagin depleted glutathione (GSH) levels that led to increase in ROS generation. The decrease in GSH levels was due to direct reaction of plumbagin with GSH as evinced by mass spectrometric and HPLC analysis. Further, addition of plumbagin to cells resulted in decrease in free thiol groups on proteins and increase in glutathionylation of proteins. The suppression of mitogen-induced T-cell proliferation and cytokine (IL-2/IL-4/IL-6/IFN-gamma) production by plumbagin was abrogated by thiol antioxidants but not by non-thiol antioxidants confirming that thiols but not ROS play an important role in biological activity of plumbagin. Plumbagin also abrogated mitogen-induced phosphorylation of ERK, IKK, and degradation of IkappaB-alpha. However, it did not affect phosphorylation of P38, JNK, and AKT. Our results for the first time show that antiproliferative effects of plumbagin are mediated by modulation of cellular redox. These results provide a rationale for application of thiol-depleting agents as anti-inflammatory drugs.

L-Arginine reverses radiation-induced immune dysfunction: the need for optimum treatment window.

The aim of the present study was to investigate the protective efficacy of l-arginine in mitigating the injury induced by 2 Gy of total-body gamma radiation (TBI). Mice exposed to radiation (TBI group) had significantly decreased spleen weight, splenocyte numbers and bone marrow cellularity. Administration of l-arginine 2 h after TBI (TBI + l-arginine group) was effective in reducing the radiation-induced depletion of spleen and bone marrow cellularity but was not effective when administered before TBI (l-arginine + TBI group). The radiation-induced decrease in Con A-induced spleen cell proliferation, specific antibody response of spleen B cells to sheep red blood cells, and spleen RNA content was reversed in mice in the TBI + l-arginine group. The radiation-induced increase in serum TNF-alpha levels, serum nitrate/nitrite (NOx) levels, spleen DNA fragmentation, spleen nitric oxide synthase (NOS) activity, spleen inducible NOS (iNOS) activity, and hepatic iNOS activity was reversed in mice in the TBI + l-arginine group. l-Arginine administered before TBI could not reverse these changes. Mice in the TBI + l-arginine group had significantly increased spleen arginase activity compared to mice from either the TBI or l-arginine + TBI group. The results suggest the importance of the time of administration of l-arginine and the l-arginine pathway in mitigating the radiation-induced host immune dysfunction.

Immunomodulatory and radioprotective effects of lignans derived from fresh nutmeg mace (Myristica fragrans) in mammalian splenocytes.

Recently, the lignans present in the aqueous extract of fresh nutmeg mace (aril of the fruit of Myristica fragrans) were shown to possess antioxidant properties in cell free systems and protected PUC18 plasmid against radiation-induced DNA damage. The present report describes the immunomodulatory and radiomodifying properties of lignans present in the aqueous extract of fresh nutmeg mace in mammalian splenocytes. These macelignans (ML) inhibited the proliferation of splenocytes in response to polyclonal T cell mitogen concanavalin A (Con A). This inhibition of proliferation was due to cell cycle arrest in G1 phase and augmentation of apoptosis as shown by increase in pre G1 cells. The increase in activation induced cell death by ML was dose dependent. It was found to inhibit the transcription of IL-2 and IL-4 genes in response to Con A. The production of IL-2, IL-4 and IFN-gamma cytokines was significantly inhibited by ML in Con A-stimulated lymphocytes in a dose dependent manner. ML protected splenocytes against radiation-induced intracellular ROS production in a dose dependent manner. ML was not cytotoxic towards lymphocytes. On the contrary, it significantly inhibited the radiation-induced DNA damage in splenocytes as indicated by decrease in DNA fragmentation. To our knowledge, this is the first report showing the antioxidant, radioprotective and immunomodulatory effects of lignans in mammalian cells.

Antitumor immunity, V.BCG-induced growth inhibition of murine tumors. Effect of hydrocortisone, antiserum against theta antigen, and gamma-irradiated BCG.

Dimethylbenzdithionsaphthene (DBDN)-induced fibrosarcoma transplantable in syngeneic Swiss mice was a weakly immunogenic tumor and did not regress spontaneously. BCG from India, when incubated with the lethal dose of DBDN-induced fibrosarcoma and subsequently inoculated sc into the susceptible animal markedly inhibited growth of the tumor. Animals that did not show any tumors developed tumor-specific immunity. BCG, if administered separately and not inoculated with the tumor cells, did not show similar response. The close contact between the BCG organism and the tumor cell seemed to be an essential requirement for BCG-induced tumor-growth inhibition. Hydrocortisone-treated animals showed tumor growth even when a BCG-tumor admixture was incorporated, indicating the immunologic nature of the phenomenon. Animals preinoculated with anti-O-serum also showed tumor growth when the BCG-tumor admixture was given. This, however, pointed out a major role of O-bearing lymmphocytes in BCG-induced-tumor-growth inhibition.

Lectin potentiation of BCG-contact-mediated antitumor action.

Concanavalin A (Con A) potentiated the BCG-contact-mediated antitumor action when both Con A (10 microgram) and BCG (50 micrograms) were injected with fibrosarcoma cells (5 x 10(4)) into inbred Swiss mice. BCG (50 micrograms) alone had no antitumor effect. The Con A-potentiated BCG-contact-mediated phenomenon was shown to be immunologically mediated, inasmuch as tumors developed in immunosuppressed mice inoculated with tumor cells mixed with Con A and BCG. The same dose of 50 micrograms BCG was effective in controlling the growth of 10(6) fibrosarcoma cells in the presence of Con A. The specific inhibitor of Con A, alpha-methyl D-mannoside, abrogated the potentiation of the BCG effect seen with Con A. Con A also increased the antitumor effect of BCG in sarcoma 180 ascites tumor. Another lectin, peanut agglutinin, also induced an antitumor effect in the presence of BCG only when the tumor cells were treated with Vibrio cholerae neuraminidase.

Studies on metastases. I. Role of sensitization and immunosuppression.

A normally nonmetastatic tumor metastasized to the regional axillary lymph nodes following severe immunosuppression of inbred Swiss mice. The extent of metastasis was dependent on the severity of lymphocyte depletion and the initial number of tumor cells injected. Sensitization of the host to the tumor was important in prevention of metastasis: Metastasis was induced only if the immunosuppressive treatment occurred within 3 days of tumor inoculation. Antitumor immunity was adoptively transferred by the splenocytes from animals given immunosuppressive treatment with X-rays or hydrocortisone acetate on day 14. Though spleen cells from animals given both immunosuppressants did not have this property, that no metastasis was observed even in doubly immunosuppressed mice indicated sensitization to tumor cells. Lymphocyte number was important also: Injection of splenocytes from normal to tumor-bearing animals significantly reduced the occurrence of metastases.

Enhanced host resistance to transplantable murine lymphosarcoma in Swiss mice by combined immunostimulation with BCG and polyinosinic-polycytidylic acid.

Combined immunostimulation with BCG and double-stranded polyinosinic-polycytidylic acid (poly I . poly C) was more effective than single-modality immunostimulation in suppressing tumor growth in inbred Swiss mice. BCG sensitization followed by administration of poly I . poly C on the day of tumor cell injection significantly prolonged the survival period against parental lymphosarcoma (LS) and its ascites variant (LS-A). BCG and poly I . poly C given together on the day of tumor cell injection suppressed only LS-A and not LS. BCG or poly I . poly C given alone did not result in tumor cures. Silica injection given 2 days before poly I . poly C injection completely abrogated the antitumor effect of sequential treatment with BCG and poly I . poly C. Silica treatment given on and beyond the day of poly I . poly C injection did not abrogate the antitumor effect. This observation indicated that intact macrophage effector function was essential at the time of tumor cell inoculation to obtain an effective antitumor action.

Therapeutic treatment with L-arginine rescues mice from heat stroke-induced death: physiological and molecular mechanisms.

Heat stroke-induced death is a major killer worldwide. Mice were subjected to acute heat stress by exposing them to whole-body hyperthermia (WBH) treatment and were used as a model to study heat stroke. Administration of L-arginine (L-arg, 120 mg/kg, i.p) 2 h after the cessation of WBH rescued the mice from heat-induced death and reduced the hypothermia. Heat shock protein 70 levels in the liver were increased significantly in heat-stressed mice administered L-arg compared with the heat-stressed group. WBH induced apoptosis, as indicated by DNA fragmentation, and increased levels of p53 and caspase-3 activity, which were significantly reduced by the administration of L-arg. The levels of inducible nitric oxide synthase in the liver, nitrite, and inflammatory cytokines like interleukin 1beta and tumor necrosis factor-alpha in the serum increased in WBH-treated mice. The levels of the above markers of heat stress significantly decreased in L-arg-treated mice. Kinin-B1 receptor (kinin-B1R) in cardiac tissue that is upregulated in heat stressed mice was significantly lower in L-arg-administered mice. These data suggest the potential use of L-arg, a nonessential amino acid that is used as an enteral diet supplement, to treat heat stroke-related injury when administered at the appropriate dose and time.

The use of ELISA to monitor amplified hemolysis by the combined action of osmotic stress and radiation: potential applications.

A new assay has been developed to study the osmotic fragility of red blood cells (RBCs) and the involvement of oxygen-derived free radicals and other oxidant species in causing human red blood cell hemolysis. The amount of hemoglobin released into the supernatant, which is a measure of human red blood cell hemolysis, is monitored using an ELISA reader. This ELISA-based osmotic fragility test compared well with the established osmotic fragility test, with the added advantage of significantly reduced time and the requirement of only 60 mul of blood. This small amount of blood was collected fresh by finger puncture and was immediately diluted 50 times with PBS, thus eliminating the use of anticoagulants and the subsequent washings. Since exposure of RBCs to 400 Gy gamma radiation caused less than 5% hemolysis 24 h after irradiation, the RBC hemolysis induced by gamma radiation was amplified by irradiating the cell in hypotonic saline. The method was validated by examining the protective effect of Trolox, an analog of vitamin E and reduced glutathione (GSH), a well-known radioprotector, against human RBC hemolysis caused by the combined action of radiation and osmotic stress. Trolox, a known membrane stabilizer and an antioxidant, and GSH offered significant protection. This new method, which is simple and requires significantly less time and fewer RBCs, may offer the ability to study the effects of antioxidants and membrane stabilizers on human red blood cell hemolysis induced by radiation and oxidative stress and assess the osmotic fragility of erythrocytes.

Migration inhibition of normal rat thymocytes as an in vitro method for detecting cell-mediated immunity in rat and mouse.

Normal rat thymocytes were used as a migrating population to study the effect of migration inhibition factor (MIF) released by sheep red blood cell (SRBC) immune lymphocytes. Normal thymocytes (migrating cells), SRBC (target antigen) and SRBC immune lymphocytes (effector cells) were packed in capillary tubes and their migration studied. This migration inhibition assay proved to be a very good in vitro correlate of specific delayed type hypersensitivity against SRBC, a particulate antigen, in rats. Normal rat thymocytes could also be used to study the specific immune reactivity of murine lymphocytes sensitised to SRBC. SRBC immune rat thymocytes functioned as both the migrating cell population and MIF producing cells. This assay permitted study of the interaction between thymocytes and lymphocytes from rats in producing enhanced inhibition of cellular migration. A major advantage of the technique is the possibility of assaying a large number of replicates from abundant normal thymocytes.

Adoptive immunotherapy of murine lymphosarcoma: comparative study using allo-immune or tumor-immune T cells.

The antitumor effect of allosensitization with lymphocytes and skin graft of DBA/2 mice was evaluated using immunogeneic, transplantable Lymphosarcoma (LS-A) syngeneic to Swiss mice. A dose dependent tumor inhibitory effect in terms of tumor free mice was observed in mice sensitized i.p. with lymphocyte doses between 10-100 million per animal. Sensitization with allogeneic primary skin graft was more effective than lymphocyte immunization. The antitumor immunity could be adoptively transferred in syngeneic Swiss mice using either allo-immune or tumor-immune T cells. Analysis of T cell phenotypes using monoclonal antibodies against cell surface markers CD4 and CD8, indicated absolute dependence on the CD4+ T cells subset in tumor cure in case of allo-immune as well as tumor-immune T cells. CD8+ T cell subset was found essential only in case of allo-immune T cell therapy. Immunosuppression of mice with whole body gamma irradiation (4 Gy), 6 hr before transfer of allo-immune or tumor-immune T cells did not abrogate the therapeutic ability of allo-immune or tumor-immune T cells. Our results suggest that allosensitization could be an effective method of generating effector lymphocyte populations that might be used to treat tumors that exhibit detectable immunogenecity.

Effect of multigeneration alcohol feeding on murine immune system.

Mice belonging to F8, F12, F14 and F20 generation of a multigeneration study reared on 20% (v/v) ethanol in water as the sole drinking source were investigated for their immune competence using various parameters. The results indicated lack of any significant effect on delayed type hypersensitivity to dinitro fluorobenzene (DNFB) or sheep red blood cells (SRBC) in mice consuming ethanol. Further, alloskin graft and tumor graft response was similar in both ethanol and water fed mice. Humoral response to SRBC was also intact. However, NK cell activity was reduced significantly in ethanol fed mice. Phagocytic index as assessed by the carbon clearance test was also reduced considerably in mice consuming ethanol. The results clearly indicate that ethanol per se has a significant effect on the nonspecific limb of the immune system, in chronically fed mice.

Radioprotective effect of whole-body hyperthermia on mice exposed to lethal doses of total-body gamma irradiation.

The whole-body hyperthermia (40 degrees C, 1 hr) 20-48 hr prior to total-body irradiation (TBI) with 9 Gy gamma rays gave 80% protection as assessed by survival of the animals. However this was reduced to 50% when mice were irradiated 7 or 15 days after hyperthermia. The local hyperthermia (42 degrees C, 1 hr) given prior to irradiation, on the other hand, did not show any protective effect. The whole-body or local hyperthermia given after TBI had no protective effect on survival of animals.

Allosensitization induced suppression of various murine tumors: role of non-H-2 antigens in antitumor immunity.

Presence of alloantigens on various murine tumors was tested by tumor rejection in allosensitized Swiss mice. The results indicated the presence of alloantigen on immunogenic tumors like chemically induced fibrosarcoma (FS), ascitic sarcoma 180 (S 180) and immunogenic variant of lymphosarcoma (LS-A) in Swiss mice, while these antigens could not be detected by this procedure on spontaneous lymphosarcoma (LS). Allosensitization with skin graft was found to offer quantitatively higher antitumor resistance than the allosensitization achieved by allogeneic lymphocytes. Antitumor effect was not seen when tumor cells were inoculated earlier than day 3 of grafting. Further, host immunosuppression with whole body irradiation up to day of 3 of skin grafting abrogated the antitumor effect. H-2 compatible and non-H-2 incompatible skin graft sensitization of host could offer resistance against both S 180 and LS-A. Further, tumor immune mice rejected H-2 compatible, non-H-2 incompatible skin graft significantly earlier.

Plumbagin, a vitamin K3 analogue, abrogates lipopolysaccharide-induced oxidative stress, inflammation and endotoxic shock via NF-κB suppression.

Plumbagin has been reported to modulate cellular redox status and suppress NF-κB. In the present study, we investigated the effect of plumbagin on lipopolysaccharide (LPS)-induced endotoxic shock, oxidative stress and inflammatory parameters in vitro and in vivo. Plumbagin inhibited LPS-induced nitric oxide, TNF-α, IL-6 and prostaglandin-E2 production in a concentration-dependent manner in RAW 264.7 cells without inducing any cell death. Plumbagin modulated cellular redox status in RAW cells. Plumbagin treatment significantly reduced MAPkinase and NF-κB activation in macrophages. Plumbagin prevented mice from endotoxic shock-associated mortality and decreased serum levels of pro-inflammatory markers. Plumbagin administration ameliorated LPS-induced oxidative stress in peritoneal macrophages and splenocytes. Plumbagin also attenuated endotoxic shock-associated changes in liver and lung histopathology and decreased the activation of ERK and NF-κB in liver. These findings demonstrate the efficacy of plumbagin in preventing LPS-induced endotoxemia and also provide mechanistic insights into the anti-inflammatory effects of plumbagin.

Bilirubin augments radiation injury and leads to increased infection and mortality in mice: molecular mechanisms.

Our earlier results demonstrated that clinically relevant concentrations of unconjugated bilirubin (UCB) possessed immunotoxic effects. Whole-body irradiation (WBI) with 1 to 6 Gy leads to acute radiation syndrome, immunosuppression, and makes the host susceptible to infection. Since hyperbilirubinemia has been shown to be associated with several types of cancer, the present studies were undertaken to evaluate the radiomodifying effects of UCB in radiation-exposed mice having elevated levels of UCB. Pretreatment of splenic lymphocytes with UCB (1-50 µM at UCB/BSA ratio <1) augmented radiation-induced DNA strand breaks, MMP loss, calcium release, and apoptosis. Combination treatment of mice with UCB (50mg/kg bw) followed by WBI (2 Gy) 0.5h later, resulted in significantly increased splenic atrophy, bone marrow aplasia, decreased counts of peritoneal exudate cells, and different splenocyte subsets such as CD3+ T, CD4+ T, CD8+ T, CD19+ B, and CD14+ macrophages as compared to either UCB or WBI treatment. Hematological studies showed that WBI-induced lymphopenia, thrombocytopenia, and neutropenia were further aggravated in the combination treatment group. UCB pretreatment of mice potentiated WBI-induced apoptosis and decreased WBI-induced loss of functional response of various immune cells leading to augmentation of immunosuppression and infection susceptibility caused by WBI. In an acute bacterial peritonitis model, UCB pretreatment of mice significantly increased WBI-induced proinflammatory cytokines, nitric oxide, and peritoneal bacterial load resulting in increased infection and death. Studies using the pharmacological inhibitor of p38MAPK demonstrated the involvement of p38MAPK activation in the inflammatory cascade of peritonitis. These findings should prove useful in understanding the potential risk to hyperbilirubinemic patients during radiotherapy and victims of acute radiation exposure in the course of radiation accidents.

Immunomodulatory and immunotoxic effects of bilirubin: molecular mechanisms.

The immunomodulatory and immunotoxic effects of purified UCB have not been evaluated previously at clinically relevant UCB concentrations and UCB:BSA ratios. To delineate the molecular mechanism of UCB-induced immunomodulation, immune cells were exposed to clinically relevant concentrations of UCB. It inhibited LPS-induced B cell proliferation and cytokine production from splenic macrophages. UCB (≥25 µM) was toxic to unfractionated splenocytes, splenic T cells, B cells, macrophages, LPS-stimulated CD19(+) B cells, human PBMCs, and RBCs. Purified UCB also was found to be toxic to splenocytes and human PBMCs. UCB induced necrosis and apoptosis in splenocytes. UCB activated the extrinsic and intrinsic pathways of apoptosis, as reflected by the markers, such as CD95, caspase-8, Bax, MMP, cytoplasmic Ca(+2), caspase-3, and DNA fragmentation. UCB depleted GSH and activated p38MAPK. NAC, caspase inhibitors, and p38MAPK inhibitor attenuated the UCB-induced apoptosis. In vivo administration of ≥25 mg/kbw UCB induced atrophy of spleen, depletion of bone marrow cells, and leukopenia and decreased lymphocyte count and the T and B cell response to mitogens. UCB administration to mice led to induction of oxidative stress, activation of p38MAPK, and cell death in splenocytes. These parameters were attenuated by the injection of NAC and the p38MAPK inhibitor. Our results demonstrate for the first time that clinically relevant concentrations of UCB induce apoptosis and necrosis in immune cells by depleting cellular GSH. These findings should prove useful in understanding the immunosuppression associated with hyperbilirubinemia.

Pro-oxidants ameliorate radiation-induced apoptosis through activation of the calcium-ERK1/2-Nrf2 pathway.

There are no reports describing the ability of pro-oxidants to protect against radiation-induced apoptosis. Activation of the redox-sensitive transcription factor Nrf2 by low levels of ROS is known to protect against oxidative stress-induced cell death. In this study, hydrogen peroxide, diethylmaleate, and 1,4-naphthoquinone (NQ) exhibited complete protection against radiation-induced cell death in lymphocytes as estimated by propidium iodide staining. Radioprotection by NQ was demonstrated by inhibition of caspase activation, decrease in cell size, DNA fragmentation, nuclear blebbing, and clonogenic assay. Interestingly, NQ offered protection to lymphocytes even when added to cells postirradiation. NQ increased intracellular ROS levels and decreased GSH levels. NQ activated Nrf2 and increased the expression of the cytoprotective gene heme oxygenase-1 in lymphocytes. NQ increased ERK phosphorylation, which is upstream of Nrf2, and this ERK activation was through increased intracellular calcium levels. Administration of NQ to mice offered protection against whole-body irradiation (WBI)-induced apoptosis in splenic lymphocytes and loss of viability of spleen and bone marrow cells. It restored WBI-mediated changes in hematological parameters and functional responses of lymphocytes. Importantly, NQ rescued mice against WBI-induced mortality. These results demonstrated that a pro-oxidant such as NQ can protect against radiation-induced apoptosis by activation of multiple prosurvival mechanisms including activation of the calcium-ERK1/2-Nrf2 pathway.