Manganese- and Platinum-Driven Oxidative and Nitrosative Stress in Oxaliplatin-Associated CIPN with Special Reference to Ca4Mn(DPDP)5, MnDPDP and DPDP.

Platinum-containing chemotherapeutic drugs are efficacious in many forms of cancer but are dose-restricted by serious side effects, of which peripheral neuropathy induced by oxidative-nitrosative-stress-mediated chain reactions is most disturbing. Recently, hope has been raised regarding the catalytic antioxidants mangafodipir (MnDPDP) and calmangafodipir [Ca4Mn(DPDP)5; PledOx®], which by mimicking mitochondrial manganese superoxide dismutase (MnSOD) may be expected to overcome oxaliplatin-associated chemotherapy-induced peripheral neuropathy (CIPN). Unfortunately, two recent phase III studies (POLAR A and M trials) applying Ca4Mn(DPDP)5 in colorectal cancer (CRC) patients receiving multiple cycles of FOLFOX6 (5-FU + oxaliplatin) failed to demonstrate efficacy. Instead of an anticipated 50% reduction in the incidence of CIPN in patients co-treated with Ca4Mn(DPDP)5, a statistically significant increase of about 50% was seen. The current article deals with confusing differences between early and positive findings with MnDPDP in comparison to the recent findings with Ca4Mn(DPDP)5. The POLAR failure may also reveal important mechanisms behind oxaliplatin-associated CIPN itself. Thus, exacerbated neurotoxicity in patients receiving Ca4Mn(DPDP)5 may be explained by redox interactions between Pt2+ and Mn2+ and subtle oxidative-nitrosative chain reactions. In peripheral sensory nerves, Pt2+ presumably leads to oxidation of the Mn2+ from Ca4Mn(DPDP)5 as well as from Mn2+ in MnSOD and other endogenous sources. Thereafter, Mn3+ may be oxidized by peroxynitrite (ONOO-) into Mn4+, which drives site-specific nitration of tyrosine (Tyr) 34 in the MnSOD enzyme. Conformational changes of MnSOD then lead to the closure of the superoxide (O2•-) access channel. A similar metal-driven nitration of Tyr74 in cytochrome c will cause an irreversible disruption of electron transport. Altogether, these events may uncover important steps in the mechanism behind Pt2+-associated CIPN. There is little doubt that the efficacy of MnDPDP and its therapeutic improved counterpart Ca4Mn(DPDP)5 mainly depends on their MnSOD-mimetic activity when it comes to their potential use as rescue medicines during, e.g., acute myocardial infarction. However, pharmacokinetic considerations suggest that the efficacy of MnDPDP on Pt2+-associated neurotoxicity depends on another action of this drug. Electron paramagnetic resonance (EPR) studies have demonstrated that Pt2+ outcompetes Mn2+ and endogenous Zn2+ in binding to fodipir (DPDP), hence suggesting that the previously reported protective efficacy of MnDPDP against CIPN is a result of chelation and elimination of Pt2+ by DPDP, which in turn suggests that Mn2+ is unnecessary for efficacy when it comes to oxaliplatin-associated CIPN.

Linking loss of sodium-iodide symporter expression to DNA damage.

Radiotherapy of thyroid cancer with I-131 is abrogated by inherent loss of radioiodine uptake due to loss of sodium iodide symporter (NIS) expression in poorly differentiated tumor cells. It is also known that ionizing radiation per se down-regulates NIS (the stunning effect), but the mechanism is unknown. Here we investigated whether loss of NIS-mediated iodide transport may be elicited by DNA damage. Calicheamicin, a fungal toxin that specifically cleaves double-stranded DNA, induced a full scale DNA damage response mediated by the ataxia-telangiectasia mutated (ATM) kinase in quiescent normal thyrocytes. At sublethal concentrations (<1nM) calicheamicin blocked NIS mRNA expression and transepithelial iodide transport as stimulated by thyrotropin; loss of function occurred at a much faster rate than after I-131 irradiation. KU-55933, a selective ATM kinase inhibitor, partly rescued NIS expression and iodide transport in DNA-damaged cells. Prolonged ATM inhibition in healthy cells also repressed NIS-mediated iodide transport. ATM-dependent loss of iodide transport was counteracted by IGF-1. Together, these findings indicate that NIS, the major iodide transporter of the thyroid gland, is susceptible to DNA damage involving ATM-mediated mechanisms. This uncovers novel means of poor radioiodine uptake in thyroid cells subjected to extrinsic or intrinsic genotoxic stress.

Oestradiol levels and superoxide dismutase activity in age-related cataract: a case-control study.

BACKGROUND: It has been suggested that the higher prevalence of cataract in women is caused by a withdrawal effect of oestrogen at menopause. In vitro studies have demonstrated protection of serum oestradiol (E2) against oxidative stress through upregulation of antioxidant enzymes, including superoxide dismutase (SOD). The purpose of the present study was to investigate E2 levels and SOD erythrocyte activity in patients with age-related cataract. METHODS: The studied subjects consisted of 103 patients with age-related cataract and 22 controls. Cataracts were classified as nuclear, cortical, or posterior subcapsular. Blood samples were collected and data on smoking, hormonal use, diabetes and age at menarche/menopause was obtained for all individuals. Serum oestradiol analyses were performed with radioimmunoassay (RIA) and SOD activity was measured in erythrocyte lysates. RESULTS: A negative correlation between age and E2 concentration was seen in a linear regression analysis. No correlation was seen between SOD activity and age or gender and no correlation between E2 levels and SOD activity was found using multiple linear regression. The mean level of E2 for all male subjects was 50.1 ± 16.3 pmol/L, significantly higher compared to 13.8 ± 11.8 pmol/L for postmenopausal women. CONCLUSION: The present study does not support a role for E2-induced effects on SOD in cataract formation. The findings of higher E2 levels in men than in postmenopausal women may suggest that decreased oestrogen at menopause is partially responsible for the gender-related difference in cataract prevalence. However, the latter can only be verified through prospective randomized trials using hormonal replacement therapy.

Exacerbated Neuropathy in POLAR A and M Trials Due to Redox Interaction of PledOx-Associated Mn2+ and Oxaliplatin-Associated Pt2.

Disappointing results from the POLAR A and M phase III trials involving colorectal cancer patients on chemotherapy with FOLFOX6 in curative (A) and palliative (M) settings have been reported by the principal investigators and the sponsor (PledPharma AB/Egetis Therapeutics AB). FOLFOX6, oxaliplatin in combination with 5-fluorouracil (5-FU), possesses superior tumoricidal activity in comparison to 5-FU alone, but suffers seriously from dose-limiting platinum-associated Chemotherapy-Induced Peripheral Neuropathy (CIPN). The aim of the POLAR trials was to demonstrate that PledOx [calmangafodipir; Ca4Mn(DPDP)5] reduced the incidence of persistent CIPN from 40% to 20%. However, this assumption was based on "explorative" data in the preceding PLIANT phase II trial, which did not mirror the expected incidence of unwanted toxicity in placebo patients. In POLAR A and M, the assessment of PledOx efficacy was conducted in patients that received at least six cycles of FOLFOX6, enabling analyses of efficacy in 239 A and 88 M patients. Instead of a hypothesized improvement from 40% to 20% incidence of persistent CIPN in the PledOx group, i.e., a 50% improvement, the real outcome was the opposite, i.e., an about 50% worsening in this bothersome toxicity. Mechanisms that may explain the disastrous outcome, with a statistically significant number of patients being seriously injured after having received PledOx, indicate interactions between two redox active metal cations, Pt2+ (oxaliplatin) and Mn2+ (PledOx). A far from surprising causal relationship that escaped prior detection by the study group and the sponsor. Most importantly, recently published data (ref 1) unequivocally indicate that the PLIANT study was not suited to base clinical phase III studies on. In conclusion, the POLAR and PLIANT trials show that PledOx and related manganese-containing compounds are unsuited for co-treatment with platinum-containing compounds. For use as a therapeutic adjunct in rescue treatment, like in ischemia-reperfusion of the heart or other organs, or in acetaminophen (paracetamol)-associated liver failure, there is little or nothing speaking against the use of PledOx or other PLED compounds. However, this must be thoroughly documented in more carefully designed clinical trials.

The Damaging Outcome of the POLAR Phase III Trials Was Due to Avoidable Time-Dependent Redox Interaction between Oxaliplatin and PledOx.

On 2 July 2021, highly negative results were reported from the POLAR A and M phase III trials in patients with colorectal cancer, treated with an oxaliplatin-based regimen and co-treated with calmangafodipir (CaM; PledOx®; PledPharma AB/Egetis Therapeutics AB) or placebo. The results revealed persistent chemotherapy-induced peripheral neuropathy (CIPN) in 54.8% of the patients treated with PledOx, compared with 40.0% of the patients treated with the placebo (p < 0.05), i.e., a 37% increase in incidence of the side effect that the trial was aimed to prevent. The damaging outcome of the trials differed diametrically from an in-parallel conducted mice study and from a clinical trial with mangafodipir, the active ingredient of CaM. According to the authors of the POLAR report, the etiology of the profound increase in CIPN in the PledOx arm is unclear. However, these devastating effects are presumably explained by intravenous administrations of PledOx and oxaliplatin being too close in time and, thereby, causing unfavorable redox interactions between Mn2+ and Pt2-. In the mice study as well as in the preceding phase II clinical trial (PLIANT), PledOx was administered 10 min before the start of the oxaliplatin infusion; this was clearly an administration procedure, where the devastating interactions between PledOx and oxaliplatin could be avoided. However, when it comes to the POLAR trials, PledOx was administered, for incomprehensible reasons, "on Top of Modified FOLFOX6" at day one, i.e., after the two-hour oxaliplatin infusion instead of before oxaliplatin. This is a time point when the plasma concentration of oxaliplatin and Pt2+-metabolites is at its highest, and where the risk of devastating redox interactions between PledOx and oxaliplatin, in turn, is at its highest.

Nuclear translocation and calpain-dependent reduction of Bcl-2 after neonatal cerebral hypoxia-ischemia.

Apoptosis-related mechanisms are important in the pathophysiology of hypoxic-ischemic injury in the neonatal brain. Caspases are the major executioners of apoptosis, but there are a number of upstream players that influence the cell death pathways. The Bcl-2 family proteins are important modulators of mitochondrial permeability, working either to promote or prevent apoptosis. In this study we focused on the anti-apoptotic Bcl-2 protein after neonatal cerebral hypoxia-ischemia (HI) in 8-day-old rats. Bcl-2 translocated to nuclei and accumulated there over the first 24h of reperfusion after HI, as judged by immunohistochemistry and immuno-electron microscopy. We also found that the total level of Bcl-2 decreased after HI in vivo and after ionophore challenge in cultured human neuroblastoma (IMR-32) cells in vitro. Furthermore, the Bcl-2 reduction was calpain-dependent, because it could be prevented by the calpain inhibitor CX295 both in vivo and in vitro, suggesting cross-talk between excitotoxic and apoptotic mechanisms.

May Mangafodipir or Other SOD Mimetics Contribute to Better Care in COVID-19 Patients?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by massive inflammation of the arterial endothelium accompanied by vasoconstriction and widespread pulmonary micro thrombi. As a result, due to the destruction of nitric oxide (•NO) by inflammatory superoxide (O2•-), pulmonary •NO concentration ceases, resulting in uncontrolled platelet aggregation and massive thrombosis, which kills the patients. Introducing •NO by inhalation (INO) may replace the loss of endothelium-derived •NO. The first results from clinical trials with INO in SARS-CoV-2 patients show a rapid and sustained improvement in cardiopulmonary function and decreased inflammation. An ongoing phase III study is expected to confirm the method's efficacy. INO may hence become a first line treatment in SARS-CoV-2 patients. However, due to the rapid inactivation of •NO by deoxyhemoglobin to nitrate, pulmonary administration of •NO will not protect remote organs. Another INO-related pharmacological approach to protect SARS-CoV-2 patients from developing life-threatening disease is to inhibit the O2•--driven destruction of •NO by neutralizing inflammatory O2•-. By making use of low molecular weight compounds that mimic the action of the enzyme manganese superoxide dismutase (MnSOD). The MnSOD mimetics of the so-called porphyrin type (e.g., AEOL 10150), salen type (e.g., EUK-8) and cyclic polyamine type (e.g., M40419, today known as GC4419 and avasopasem manganese) have all been shown to positively affect the inflammatory response in lung epithelial cells in preclinical models of chronic obstructive pulmonary disease. The Manganese diPyridoxyL EthylDiamine (MnPLED)-type mangafodipir (manganese dipyridoxyl diphosphate-MnDPDP), a magnetic resonance imaging (MRI) contrast agent that possesses MnSOD mimetic activity, has shown promising results in various forms of inflammation, in preclinical as well as clinical settings. Intravenously administration of mangafodipir will, in contrast to INO, reach remote organs and may hence become an important supplement to INO. From the authors' viewpoint, it appears logical to test mangafodipr in COVID-19 patients at risk of developing life-threatening SARS-CoV-2. Five days after submission of the current manuscript, Galera Pharmaceuticals Inc. announced the dosing of the first patient in a randomized, double-blind pilot phase II clinical trial with GC4419 for COVID-19. The study was first posted on ClinicalTrials.gov (Identifier: NCT04555096) 18 September 2020.

Comment on "Calmangafodipir Reduces Sensory Alterations and Prevents Intraepidermal Nerve Fibers Loss in a Mouse Model of Oxaliplatin Induced Peripheral Neurotoxicity" Antioxidants 2020, 9, 594.

We have with enthusiasm read the article "Calmangafodipir Reduces Sensory Alterations and Prevents Intraepidermal Nerve Fibers Loss in a Mouse Model of Oxaliplatin Induced Peripheral Neurotoxicity"[...].

Evidence that fodipir (DPDP) binds neurotoxic Pt2+ with a high affinity: An electron paramagnetic resonance study.

Oxaliplatin typically causes acute neuropathic problems, which may, in a dose-dependent manner, develop into a chronic form of chemotherapy-induced peripheral neuropathy (CIPN), which is associated with retention of Pt2+ in the dorsal root ganglion. A clinical study by Coriat and co-workers suggests that co-treatment with mangafodipir [Manganese(II) DiPyridoxyl DiPhosphate; MnDPDP] cures ongoing CIPN. These authors anticipated that it is the manganese superoxide dismutase mimetic activity of MnDPDP that explains its curative activity. However, this is questionable from a pharmacokinetic perspective. Another, but until recently undisclosed possibility is that Pt2+ outcompetes Mn2+/Ca2+/Zn2+ for binding to DPDP or its dephosphorylated metabolite PLED (diPyridoxyL EthylDiamine) and transforms toxic Pt2+ into a non-toxic complex, which can be readily excreted from the body. We have used electron paramagnetic resonance guided competition experiments between MnDPDP (10logKML ≈ 15) and K2PtCl4, and between MnDPDP and ZnCl2 (10logKML ≈ 19), respectively, in order to obtain an estimate the 10logKML of PtDPDP. Optical absorption spectroscopy revealed a unique absorption line at 255 nm for PtDPDP. The experimental data suggest that PtDPDP has a higher formation constant than that of ZnDPDP, i.e., higher than 19. The present results suggest that DPDP/PLED has a high enough affinity for Pt2+ acting as an efficacious drug in chronic Pt2+-associated CIPN.

Effects of 17ß-Estradiol on Activity, Gene and Protein Expression of Superoxide Dismutases in Primary Cultured Human Lens Epithelial Cells.

PURPOSE: Protective effects of estradiol against H2O2-induced oxidative stress have been demonstrated in lens epithelial cells. The purpose of this study was to investigate the effects of 17ß-estradiol (E2) on the different superoxide dismutase (SOD) isoenzymes, SOD-1, SOD-2, and SOD-3, as well as estrogen receptors (ERs), ERα and ERß, in primary cultured human lens epithelial cells (HLECs). MATERIALS AND METHODS: HLECs were exposed to 0.1 µM or 1 µM E2 for 1.5 h and 24 h after which the effects were studied. Protein expression and immunolocalization of SOD-1, SOD-2, ERα, and ERß were studied with Western blot and immunocytochemistry. Total SOD activity was measured, and gene expression analyses were performed for SOD1, SOD2, and SOD3. RESULTS: Increased SOD activity was seen after 1.5 h exposure to both 0.1 µM and 1 µM E2. There were no significant changes in protein or gene expression of the different SODs. Immunolabeling of SOD-1 was evident in the cytosol and nucleus; whereas, SOD-2 was localized in the mitochondria. Both ERα and ERß were immunolocalized to the nucleus, and mitochondrial localization of ERß was evident by colocalization with MitoTracker. Both ERα and ERß showed altered protein expression levels after exposure to E2. CONCLUSIONS: The observed increase in SOD activity after exposure to E2 without accompanying increase in gene or protein expression supports a role for E2 in protection against oxidative stress mediated through non-genomic mechanisms.

Mangafodipir a Selective Cytoprotectant - with Special Reference to Oxaliplatin and Its Association to Chemotherapy-Induced Peripheral Neuropathy (CIPN).

Oxaliplatin, in combination with 5-fluorouracil plus folinate (or capecitabine), has increased survival substantially in stage III colorectal cancer and prolonged life in stage IV patients, but its use is compromised because of severe toxicity. Chemotherapy-induced peripheral neuropathy (CIPN) is the most problematic dose-limiting toxicity of oxaliplatin. Oncologists included for years calcium and magnesium infusion as part of clinical practice for preventing CIPN. Results from a phase III prospective study published in 2014, however, overturned this practice. No other treatments have been clinically proven to prevent this toxicity. There is a body of evidence that CIPN is caused by cellular oxidative stress. Clinical and preclinical data suggest that the manganese chelate and superoxide dismutase mimetic mangafodipir (MnDPDP) is an efficacious inhibitor of CIPN and other conditions caused by cellular oxidative stress, without interfering negatively with the tumoricidal activity of chemotherapy. MnPLED, the metabolite of MnDPDP, attacks cellular oxidative stress at several critical levels. Firstly, MnPLED catalyzes dismutation of superoxide (O2•-), and secondly, having a tremendous high affinity for iron (and copper), PLED binds and disarms redox active iron/copper, which is involved in several detrimental oxidative steps. A case report from 2009 and a recent feasibility study suggest that MnDPDP may prevent or even cure oxaliplatin-induced CIPN. Preliminary results from a phase II study (PLIANT) suggest efficacy also of calmangafodipir, but these results are according to available data obscured by a surprisingly low number of adverse events and a seemingly lower than expected efficacy of FOLFOX.

Methylenetetrahydrofolate reductase genetic polymorphisms in patients with cataract.

PURPOSE: Hyperhomocysteinemia is commonly associated with polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene. The level of homocysteine can be lowered by dietary intake of folate. A protective effect of folate supplementation has been reported against cataract. Here we investigate MTHFR polymorphisms in human cataract. DESIGN: Retrospective case-control association study. METHODS: Patients with nuclear (n = 77), cortical (n = 155), posterior subcapsular (n = 119), and mixed (n = 151) cataract, and 187 controls were analyzed for the MTHFR 677C-->T and 1298A-->C polymorphisms using minisequencing technique. RESULTS: The wild-type MTHFR 677CC/1298AA genotype was strongly overrepresented among cataract cases (P = .003). This effect was most pronounced in the mixed cataract group (P < .001). Hyperhomocysteinemia-associated genotypes had similar frequencies in cataract and control groups. CONCLUSIONS: The previously reported protective effect of folate against cataract is not due to overrepresentation of hyperhomocysteinemia-associated MTHFR genotypes. Instead, the strong predominance of wild-type MTHFR in cataract may suggest impaired DNA synthesis as a cataractogenic factor.

Calmangafodipir [Ca4Mn(DPDP)5], mangafodipir (MnDPDP) and MnPLED with special reference to their SOD mimetic and therapeutic properties.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) participate in pathological tissue damage. Mitochondrial manganese superoxide dismutase (MnSOD) normally keeps ROS and RNS in check. During development of mangafodipir (MnDPDP) as a magnetic resonance imaging (MRI) contrast agent, it was discovered that MnDPDP and its metabolite manganese pyridoxyl ethyldiamine (MnPLED) possessed SOD mimetic activity. MnDPDP has been tested as a chemotherapy adjunct in cancer patients and as an adjunct to percutaneous coronary intervention in patients with myocardial infarctions, with promising results. Whereas MRI contrast depends on release of Mn(2+), the SOD mimetic activity depends on Mn(2+) that remains bound to DPDP or PLED. Calmangafodipir [Ca4Mn(DPDP)5] is stabilized with respect to Mn(2+) and has superior therapeutic activity. Ca4Mn(DPDP)5 is presently being explored as a chemotherapy adjunct in a clinical multicenter Phase II study in patients with metastatic colorectal cancer.

Acetylcholine-induced vasodilation may depend entirely upon NO in the femoral artery of young piglets.

1 To characterize agonist-induced relaxation in femoral artery rings from young piglets, we compared the effect of a NOS-inhibitor N(omega)-nitro-L-arginine (L-NOARG), an NO-inactivator oxyhaemoglobin (HbO) and a soluble guanyl cyclase(sGC)-inhibitor 1H-[1,2,4]Oxadiazolo-[4,3,-alpha]quinoxalin-1-one (ODQ) on acetylcholine(ACh)-induced relaxation. The involvement of K(+) channel activation was studied on relaxations induced by ACh, the two NO donors sodium nitroprusside (SNP) and diethylamine (DEA) NONOate, and the cell membrane permeable guanosine 3'5' cyclic monophosphate (cGMP) analogue 8-Br-cGMP. 2 Full reversal of phenylephrine-mediated precontraction was induced by ACh (1 nM-1 microM) (pD(2) 8.2+/-0.01 and R(max) 98.7+/-0.3%). L-NOARG (100 microM) partly inhibited relaxation (pD(2) 7.4+/-0.02 and R(max) 49.6+/-0.8%). The L-NOARG/indomethacin(IM)-resistant response displayed characteristics typical for endothelium-derived hyperpolarizing factor (EDHF), being sensitive to a combination of the K(+) channel blockers charybdotoxin (CTX) (0.1 microM) and apamin (0.3 microM). 3 ODQ (10 microM) abolished relaxations induced by ACh and SNP. L-NOARG/IM-resistant relaxations to ACh were abolished by HbO (20 microM). 4 Ouabain (1 microM) significantly inhibited ACh-induced L-NOARG/IM-resistant relaxations and relaxations induced by SNP (10 microM) and 8-Br-cGMP (0.1 mM). A combination of ouabain and Ba(2+) (30 microM) almost abolished L-NOARG/IM-resistant ACh-induced relaxation (R(max) 7.7+/-2.5% vs 23.4+/-6.4%, with and without Ba(2+), respectively, P<0.05). 5 The present study demonstrates that in femoral artery rings from young piglets, despite an L-NOARG/IM-resistant component sensitive to K(+) channel blockade with CTX and apamin, ACh-induced relaxation is abolished by sGC-inhibition or a combination of L-NOARG and HbO. These findings suggest that relaxation can be fully explained by the NO/cGMP pathway.

Selenium has a protective role in caspase-3-dependent apoptosis induced by H2O2 in primary cultured pig thyrocytes.

OBJECTIVE: Hydrogen peroxide (H2O2), necessary for thyroid hormonogenesis, is produced at the apical surface of the thyroid follicular epithelium. Excess H2O2 is potentially cytotoxic and may contribute to the development of hypothyroidism, e.g. in severe selenium deficiency. Yet it is unclear how H2O2 contributes to thyroid cell death. DESIGN AND METHODS: H2O2-induced apoptosis and necrosis were studied in primary cultured pig thyroid cells. Glutathione peroxidase (GPx) activity was altered by culture in low serum with or without selenite substitution. Apoptosis was evaluated by spectrofluorometric measurement of caspase-3-specific substrate cleavage, and by analysis of DNA fragmentation by agarose gel electrophoresis. Necrosis was detected by 51Cr release from prelabeled cells. RESULTS: Exogenous H2O2 dose-dependently (100-400 micromol/l) activated caspase-3 within 3-12 h, and DNA degradation was observed after 24 h. The potency of H2O2 to induce apoptosis was low compared with that of staurosporine, a strong proapoptotic agent. H2O2-treated cells with reduced GPx activity showed increased caspase-3 activation. Incubation of serum-starved cells with selenite (10-100 nmol/l) normalized the GPx activity and reduced the activation of caspase-3 by H2O2. High H2O2 concentrations (400-800 micromol/l) were required to obtain necrosis. The H2O2-induced necrosis was exaggerated by both low GPx activity and catalase inhibition. CONCLUSIONS: Cytotoxic effects of H2O2 on thyroid cells include caspase-3-dependent apoptosis that occurs at H2O2 concentrations insufficient to induce necrosis. Selenium deficiency aggravates the apoptotic response, probably due to impaired capacity of GPx to degrade H2O2.