Hydroxynonenal and uncoupling proteins: a model for protection against oxidative damage

In this mini review we summarize recent studies from our laboratory that show the involvement of superoxide and the lipid peroxidation product 4-hydroxynonenal in the regulation of mitochondrial uncoupling. Superoxide produced during mitochondrial respiration is a major cause of the cellular oxidative damage that may underlie degenerative diseases and ageing. Superoxide production is very sensitive to the magnitude of the mitochondrial protonmotive force, so can be strongly decreased by mild uncoupling. Superoxide is able to give rise to other reactive oxygen species, which elicit deleterious effects primarily by oxidizing intracellular components, including lipids, DNA and proteins. Superoxide-induced lipid peroxidation leads to the production of reactive aldehydes, including 4-hydroxynonenal. These aldehydic lipid peroxidation products are in turn able to modify proteins such as mitochondrial uncoupling proteins and the adenine nucleotide translocase, converting them into active proton transporters. This activation induces mild uncoupling and so diminishes mitochondrial superoxide production, hence protecting against disease and oxidative damage at the expense of energy production.     

Rescue of replication failure by Fanconi anaemia proteins

Chromosomal aberrations are often associated with incomplete genome duplication, for instance at common fragile sites, or as a consequence of chemical alterations in the DNA template that block replication forks. Studies of the cancer-prone disease Fanconi anaemia (FA) have provided important insights into the resolution of replication problems. The repair of interstrand DNA crosslinks induced by chemotherapy drugs is coupled with DNA replication and controlled by FA proteins. We discuss here the recent discovery of new FA-associated proteins and the development of new tractable repair systems that have dramatically improved our understanding of crosslink repair. We focus also on how FA proteins protect against replication failure in the context of fragile sites and on the identification of reactive metabolites that account for the development of Fanconi anaemia symptoms.     

Role of proteins in protection against radiation-induced damage in membranes

The effect of gamma irradiation on liposomes in the presence of a large number of commercially available proteins has been studied. Experiments were designed to demonstrate that the configuration of both acyl chain and cis C = C bonds created by lipid-protein associations are crucial in autocatalyzed radiation-induced lipid peroxidation. Raman spectroscopy was used to characterize these states. Raman spectra in the C-C stretching region show three prominent bands at 1064, 1090, and 1125 cm-1, assigned to trans, gauche, and trans C-C bonds, respectively. A single symmetrical C = C stretching band assigned to the cis isomer occurs at 1660 cm-1. The intensity ratios (I1064/I1090) and (I1660/I1440) are used as Raman probes to define the conformational states of acyl chains and C = C bonds, respectively. Our data show that the ratio (I1064/I1090) decreases in the presence of proteins, indicating that these proteins induce more gauche structures. Upon irradiation, the ratio (I1064/I1090) increases by about 30% in the absence of proteins and by about 15% in the presence of proteins. This shows that proteins retain the gauche structures in irradiated samples. The ratio (I1660/I1440) decreases in liposomes containing proteins, showing that proteins modify the configuration of cis C = C bonds. Upon irradiation, this ratio decreases by about 45-50% in samples without proteins and by about 10% in samples with proteins. These data show that proteins inhibit the radiation-induced configurational changes in the cis C = C bonds. The determination of radiation-induced peroxides (as malondialdehyde equivalents) in liposomes reveals that proteins inhibit the formation of peroxide products at low molar ratio and that the preventive capacity of different proteins is different. We conclude that proteins alter the conformation of both acyl chains and cis C = C bonds in liposomes and that these altered states are less sensitive to radiation-induced peroxidation.     

Ozone oxidative preconditioning: a protection against cellular damage by free radicals.

There is some anecdotal evidence that oxygen-ozone therapy may be beneficial in some human diseases. However so far only a few biochemical and pharmacodynamic mechanisms have been elucidated. On the basis of preliminary data we postulated that controlled ozone administration would promote an oxidative preconditioning preventing the hepatocellular damage mediated by free radicals. Six groups of rats were classified as follows: (1) negative control, using intraperitoneal sunflower oil; (2) positive control using carbon tetrachloride (CCl4) as an oxidative challenge; (3) oxygen-ozone, pretreatment via rectal insufflation (15 sessions) and after it, CCl4; (4) oxygen, as group 3 but using oxygen only; (5) control oxygen-ozone, as group 3, but without CCl4; group (6) control oxygen, as group 5, but using oxygen only. We have evaluated critical biochemical parameters such as levels of transaminase, cholinesterase, superoxide dismutase, catalase, phospholipase A, calcium dependent ATPase, reduced glutathione, glucose 6 phosphate dehydrogenase and lipid peroxidation. Interestingly, in spite of CCl4 administration, group 3 did not differ from group 1, while groups 2 and 4 showed significant differences from groups 1 and 3 and displayed hepatic damage. To our knowledge these are the first experimental results showing that repeated administration of ozone in atoxic doses is able to induce an adaptation to oxidative stress thus enabling the animals to maintain hepatocellular integrity after CCl4 poisoning.     

Tempol protects blood proteins and lipids against peroxynitrite-mediated oxidative damage

Oxidative stress is characterized by excessive production of various free radicals and reactive species among which, peroxynitrite is most frequently produced in several pathological conditions. Peroxynitrite is the product of the superoxide anion reaction with nitric oxide, which is reported to take place in the intravascular compartment. Several studies have reported that peroxynitrite targets red blood cells, platelets and plasma proteins, and induces various forms of oxidative damage. This in vitro study was designed to further characterize the types of oxidative damage induced in platelets and plasma proteins by peroxynitrite. This study also determined the ability of tempol to protect blood plasma and platelets against peroxynitrite-induced oxidative damage. The ability of various concentrations of tempol (25, 50, 75, and 100 µM) to antagonize peroxynitrite-induced oxidation was evaluated by measuring the levels of protein carbonyl groups and thiobarbituric-acid-reactive substances in experimental groups. Exposure of platelets and plasma to 100 µM peroxynitrite resulted in an increased levels of carbonyl groups and lipid peroxidation (P < 0.05). Tempol significantly inhibited carbonyl group formation in plasma and platelet proteins (P < 0.05). In addition, tempol significantly reduced the levels of lipid peroxidation in both plasma and platelet samples (P < 0.05). Thus, tempol has antioxidative properties against peroxynitrite-induced oxidative damage in blood plasma and platelets.     

The catecholic metal sequestering agent 1,2-dihydroxybenzene-3,5-disulfonate confers protection against oxidative cell damage.

Tiron (1,2-dihydroxybenzene-3,5-disulfonate), a nontoxic chelator of a variety of metals, is used to alleviate acute metal overload in animals. It is also oxidized to the EPR-detectable semiquinone radical by various biologically relevant oxidants, such as .OH, O2-., alkyl, and alkoxyl radicals. Since Tiron reacts with potentially toxic intracellular species and is also a metal chelator, we evaluated its protective effects in V79 cells subjected to various types of oxidative damage and attempted to distinguish the protection due to direct detoxification of intracellular radicals from that resulting from chelation of redox-active transition metals. We found that Tiron protects Chinese hamster V79 cells against both O2.(-)-induced (and H2O2 via dismutation of O2.-) and H2O2-induced cytotoxicity as measured by clonogenic assays. In experiments where Tiron was incubated with V79 cells and rinsed prior to exposure to HX/XO or H2O2, cytoprotection was observed, indicating that it protects against intracellular oxidative damage. On the other hand, Tiron did not protect V79 cells against the damage caused by ionizing radiation under aerobic conditions, which is predominantly mediated by H., .OH, and hydrated electrons in a metal-independent fashion. We demonstrate also that in in vitro studies, Tiron protects supercoiled DNA from metal-mediated superoxide-dependent strand breaks. We conclude that Tiron is a potentially useful protecting agent against the lethal effects of oxidative stress and suggest that it offers protection by chelating redox-active transition metal ions, in contrast to earlier reports where the protection by this compound in cellular systems subjected to oxidative damage has been interpreted as due to radical scavenging alone.     

Dietary antioxidants fail in protection against oxidative genetic damage in in vitro evaluation

Carcinogenesis is believed to be induced through the oxidative damage of DNA, and antioxidants are expected to suppress it. So, the polyphenolic antioxidants in daily foods were investigated to see whether they protect against genetic damage by active oxygen. In the evaluation, we used a bioassay and a chemical determination, a Salmonella mutagenicity test for mutation by a N-hydroxyl radical from one of the dietary carcinogens 3-amino-1-methyl-5H-pyrido[4,3-b]indole and the formation of 8-hydroxyl (8-OHdG) from 2'-deoxyguanosine (2'-dG) in a Fenton OH-radical generating system. Thirty-one antioxidants including flavonoids were compared in terms of radical-trapping activity with bacterial DNA and 2'-dG. Antioxidants inhibited the mutation but the IC50 values were in the mM order. Against 8-OHdG formation, only alpha-tocopherol had a suppressive effect with an IC50 of 1.5 microM. Thus, except alpha-tocopherol, the dietary antioxidants did not scavenge the biological radicals faster than bacterial DNA and intact 2'-dG, indicating that they failed to prevent oxidative gene damage and probably carcinogenesis.     

Exercise preconditioning against hydrogen peroxide-induced oxidative damage in proteins of rat myocardium

Both regular physical exercise and low levels of H(2)O(2) administration result in increased resistance to oxidative stress. We measured the accumulation of reactive carbonyl derivatives and the activities of proteasome complex and DT-diaphorase in cardiac muscle of trained and untrained rats after chronic i.p. administration of 1 ml t-butyl H(2)O(2) (1 mmol/kg for 3 weeks every second day). Twenty-four rats were randomly assigned to a control group administered with saline, control administered with H(2)O(2), and exercised administered either saline or H(2)O(2). The activity of DT-diaphorase significantly increased in H(2)O(2) administered and exercised groups, indicating that an increase in H(2)O(2) levels stimulate the activity of this enzyme. The cardiac muscle of H(2)O(2) administered nonexercised animals accumulated significantly more carbonyl than control group (P < 0.05). The exercise and H(2)O(2) administration resulted in less oxidatively modified protein than found in nonexercised groups (P < 0.05). The peptide-like activity of proteasome complex was induced by the treatment of H(2)O(2) and exercise and exercise potentiate the effect of H(2)O(2). On the other hand, the chymotrypsin-like and trypsin-like activities were stimulated only by physical training and H(2)O(2) administration. The data suggest that chronic administration of H(2)O(2) after exercise training decreases the accumulation of carbonyl groups below the steady-state level and induces the activity of proteasome and DT-diaphorase. Hence, the stimulating effect of physical exercise on free radical generation is an important phenomenon of the exercise-induced adaptation process since it increases resistance to oxidative stress. Regular exercise training is a valuable physiological means of preconditioning the myocardium to prolonged oxidative stress.     

Thioredoxin-1 contributes to protection against DON-induced oxidative damage in HepG2 cells

Leucocytes are susceptible to the toxic effects of deoxynivalenol (DON), which is a trichothecene mycotoxin produced by a number of fungi including Fusarium species. One mechanism of action is mediated by reactive oxygen species (ROS). The liver is an important target for toxicity caused by foreign compounds including mycotoxins. On the other hand, little is known about the influence of the redox state on hepatocytes treated with DON. The present study investigated the effect of DON on the cytosolic redox state and antioxidative system in the human hepatoma cell line HepG2. The cell viability of human monocyte cell line THP-1 or leukemia cell line KU812 treated with 2.5 and 5???mol/l DON were significantly reduced. However, HepG2 cells showed no toxic effects under the same conditions and did not exhibit an increased oxidative state. Further experiments showed that thioredoxin-1 (Trx-1) protein levels but not glutathione increased in the cells treated with 10???mol/l DON. In addition, the enhancement of Trx-1 content was repressed by antioxidants. These results suggest that DON-induced accumulation of Trx-1 in HepG2 cells plays one of the key roles in protection against cytotoxicity caused by DON and that the mechanism may be mediated by the antioxidant properties of Trx-1.     

The role of selenium peroxidases in the protection against oxidative damage of membranes

The present review deals with the chemical properties of selenium in relation to its antioxidant properties and its reactivity in biological systems. The interaction of selenite with thiols and glutathione and the reactivity of selenocompounds with hydroperoxides are described. After a short survey on distribution, metabolism and organification of selenium, the role of this element as a component of the two seleno-dependent glutathione peroxidases is described. The main features of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are also reviewed. Both enzymes reduce different hydroperoxides to the corresponding alcohols and the major difference is the reduction of lipid hydroperoxides in membrane matrix catalyzed only by the phospholipid hydroperoxide glutathione peroxidase. However, in spite of the different specificity for the peroxidic substrates, the kinetic mechanism of both glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase seems identical and proceeds through a tert-uni ping pong mechanism. In the reaction cycle, indeed, as supported by the kinetic data, the oxidation of the ionized selenol by the hydroperoxide yields a selenenic acid that in turn is reduced back by two reactions with reduced glutathione. Special emphasis has been given to the role of selenium-dependent glutathione peroxidases in the prevention of membrane lipid peroxidation. While glutathione peroxidase is able to reduce hydrogen peroxide and other hydroperoxides possibly present in the soluble compartment of the cell, this enzyme fails to inhibit microsomal lipid peroxidation induced by NADPH or ascorbate and iron complexes. On the other hand, phospholipid hydroperoxide glutathione peroxidase, by reducing the phospholipid hydroperoxides in the membranes, actively prevents lipid peroxidation, provided a normal content of vitamin E is present in the membranes. In fact, by preventing the free radical generation from lipid hydroperoxides, phospholipid hydroperoxide glutathione peroxidase decreases the vitamin E requirement necessary to inhibit lipid peroxidation. Finally, the possible regulatory role of the selenoperoxidases on the arachidonic acid cascade enzymes (cyclooxygenase and lipoxygenase) is discussed.     

Spermine and spermidine mediate protection against oxidative damage caused by hydrogen peroxide

Summary. The polyamines spermidine and spermine have been hypothesized to possess different functions in the protection of DNA from reactive oxygen species. The growth and survival of mouse fibroblasts unable to synthesize spermine were compared to their normal counterparts in their native and polyamine-depleted states in response to oxidative stress. The results of these studies suggest that when present at normal or supraphysiological concentrations, either spermidine or spermine can protect cells from reactive oxygen species. However, when polyamine pools are pharmacologically manipulated to produce cells with low levels of predominately spermine or spermidine, spermine appears to be more effective. Importantly, when cells are depleted of both glutathione and endogenous polyamines, they exhibit increased sensitivity to hydrogen peroxide as compared to glutathione depletion alone, suggesting that polyamines not only play a role in protecting cells from oxidative stress but this role is distinct from that played by glutathione.     

Garlic provides protection to mice heart against isoproterenol-induced oxidative damage: role of nitric oxide

Garlic has been widely recognized as a cardioprotective agent. However, the molecular mechanism of its cardioprotective effects is not well established. Here we hypothesized that aqueous garlic homogenate may mediate cardioprotection via nitric oxide (NO). Mice were fed with saline and aqueous garlic homogenate (250 and 500 mgkg(-1)day(-1) orally) for 30 days. In another set of experiment, mice were pre-treated with saline, aqueous garlic homogenate (AGH) (250 mgkg(-1)day(-1) for 30 days), and AGH (30 days) along with L-NAME (20 mgkg(-1)day(-1) i.p. for last 7 days) before inducing acute myocardial infarction by isoproterenol (s.c. injection of isoproterenol 150 mgkg(-1)day(-1) for 2 days) and sacrificed after 48 h. Dose dependent increase in serum NO level was observed after garlic 250 and 500 mgkg(-1) dose feeding. While no change in serum SGPT and SGOT level, a significant decrease in serum LDH level was observed after garlic feeding. Garlic-induced NO formation was further confirmed in human aortic endothelial cells (HAEC). Administration of isoproterenol caused a significant decrease in endogenous antioxidants i.e., myocardial catalase, GSH and GPx activity, and mitochondrial enzyme activities like citrate synthase and β hydroxyacyl CoA dehydrogenase. All those deleterious cardiac changes induced by isoproterenol were significantly attenuated by garlic homogenate. However this beneficial effect of garlic was blunted when garlic was administered with L-NAME, a nonspecific inhibitor of nitric oxide synthase (NOS). Further, a significant increase in myocardial TBARS and decrease in total antioxidant activity was observed in L-NAME treated group compared to isoproterenol treated group. Administration of L-NAME in mice from control group lowered serum and cardiac NO levels without any change of oxidative stress parameters. In conclusion, our study provides novel evidence that garlic homogenate is protective in myocardial infarction via NO-signaling pathway in mice.     

Involvement of DNA polymerase beta in protection against the cytotoxicity of oxidative DNA damage

We had shown previously that DNA polymerase beta (beta-pol) null mouse fibroblasts, deficient in base excision repair (BER), are hypersensitive to monofunctional methylating agents but not to hydrogen peroxide (H2O2). This is surprising because beta-pol is thought to be involved in BER of oxidative as well as methylated DNA damage. We confirm these findings here in early-passage cells. However, with time in culture, beta-pol null cells become hypersensitive to H2O2 and other reactive oxygen species-generating agents. Analysis of in vitro BER reveals a strong deficiency in single-nucleotide BER of 8-oxoguanine (8-oxoG) by both early- and late-passage beta-pol null cell extracts. Therefore, in early-passage wild-type and beta-pol null cells, the capacity for single-nucleotide BER of 8-oxoG does not correlate with cellular sensitivity to H2O2. Expression of beta-pol protein in the late-passage null cells almost completely reverses the H2O2-hypersensitivity phenotype. Methoxyamine (MX) treatment sensitizes late-passage wild-type cells to H2O2 as expected for beta-pol-mediated single-nucleotide BER; however in beta-pol null cells, MX has no effect. The data indicate a role(s) of beta-pol-dependent repair in protection against the cytotoxicity of oxidative DNA damage in wild-type cells.     

Bax expression and apoptotic cell death in Fanconi anaemia peripheral blood lymphocytes

OBJECTIVE: Deregulated apoptosis might be involved in some of the features of Fanconi anaemia (FA). The possibility that the pro-apoptotic Bax protein could be involved in an increased susceptibility to apoptosis in FA patients was investigated. MATERIALS AND METHODS: Intracellular Bax expression, Bcl-2 expression (an anti-apoptotic protein) and cell death were analysed in 26 FA peripheral blood lymphocyte samples. RESULTS: Most FA samples (69%) displayed increased levels of Bax and were more susceptible to both spontaneous apoptosis and mitogen activation-induced cell death. Two subgroups were identified: one presented elevated levels of Bax (n = 18), whereas the other (n = 8), had Bax levels lower than controls. Two subgroups based on Bcl-2 expression were also identified: one with normal and another with high Bcl-2 expression. No inverse correlation was found between Bcl-2 levels and Bax expression. A clear difference in susceptibility to induced cell death could be observed between control and FA samples. The best correlation was observed between high levels of Bax and mitogen-induced apoptosis of cells; these displayed characteristics of necrosis secondary to apoptosis, suggesting that the intrinsic apoptotic pathway was being activated. CONCLUSION: Despite increased susceptibility to cell death induction, there was no correlation between Bax levels, chromosome breakage, haematological parameters or androgen therapy. The importance of apoptosis and Bax expression in the clinical development of FA awaits clarification.     

The roles of thioredoxin in protection against oxidative stress-induced apoptosis in SH-SY5Y cells.

Using models of serum deprivation and 1-methyl-4-phenylpyridinium (MPP(+)), we investigated the mechanism by which thioredoxin (Trx) exerts its antiapoptotic protection in human neuroblastoma cells (SH-SY5Y) and preconditioning-induced neuroprotection. We showed that SH-SY5Y cells are highly sensitive to oxidative stress and responsive to both extracellularly administered and preconditioning-induced Trx. Serum deprivation and MPP(+) produced an elevation in the hydroxyl radicals, malondialdehyde and 4-hydroxy-2,3-nonenal (HNE), causing the cells to undergo mitochondria-mediated apoptosis. Trx in the submicromolar range blocked the observed apoptosis via a multiphasic protection mechanism that includes the suppression of cytochrome c release (most likely via the induction of Bcl-2), the inhibition of procaspase-9 and procaspase-3 activation, and the elevated level of Mn-SOD. The reduced form of Trx suppresses the serum-free-induced hydroxyl radicals, lipid peroxidation, and apoptosis, indicating that H(2)O(2) is removed by Trx peroxidase. The participation of Trx in preconditioning-induced neuroprotection is supported by the observation that inhibition of Trx synthesis with antisense oligonucleotides or of Trx reductase drastically reduced the hormesis effect. This effect of Trx-mediated hormesis against oxidative stress-induced apoptosis is striking. It induced a 30-fold shift in LD(50) in the MPP(+)-induced neurotoxicity.     

Rice NTRC is a high-efficiency redox system for chloroplast protection against oxidative damage

One of the mechanisms plants have developed for chloroplast protection against oxidative damage involves a 2-Cys peroxiredoxin, which has been proposed to be reduced by ferredoxin and plastid thioredoxins, Trx x and CDSP32, the FTR/Trx pathway. We show that rice (Oryza sativa) chloroplast NADPH THIOREDOXIN REDUCTASE (NTRC), with a thioredoxin domain, uses NADPH to reduce the chloroplast 2-Cys peroxiredoxin BAS1, which then reduces hydrogen peroxide. The presence of both NTR and Trx-like domains in a single polypeptide is absolutely required for the high catalytic efficiency of NTRC. An Arabidopsis thaliana knockout mutant for NTRC shows irregular mesophyll cell shape, abnormal chloroplast structure, and unbalanced BAS1 redox state, resulting in impaired photosynthesis rate under low light. Constitutive expression of wild-type NTRC in mutant transgenic lines rescued this phenotype. Moreover, prolonged darkness followed by light/dark incubation produced an increase in hydrogen peroxide and lipid peroxidation in leaves and accelerated senescence of NTRC-deficient plants. We propose that NTRC constitutes an alternative system for chloroplast protection against oxidative damage, using NADPH as the source of reducing power. Since no light-driven reduced ferredoxin is produced at night, the NTRC-BAS1 pathway may be a key detoxification system during darkness, with NADPH produced by the oxidative pentose phosphate pathway as the source of reducing power.     

Human polynucleotide phosphorylase reduces oxidative RNA damage and protects HeLa cell against oxidative stress

We examined HeLa cell viability and RNA oxidative damage in response to hydrogen peroxide (H₂O₂) treatment. The level of damaged RNA, measured by the content of 8-hydroxyguanosine (7,8-dihydro-8-oxoguanosine, 8-oxoG), increases depending on H₂O₂ dosage and is inversely correlated with cell viability. The elevated level of 8-oxoG in RNA decreases after removal of oxidative challenge, suggesting the existence of surveillance mechanism(s) for cleaning up oxidized RNA. Human polynucleotide phosphorylase (hPNPase), an exoribonuclease primarily located in mitochondria, has been previously shown to bind 8-oxoG-RNA with high affinity. The role of hPNPase in HeLa cell under oxidative stress conditions is examined here. Overexpression of hPNPase reduces RNA oxidation and increases cell viability against H₂O₂ insult. Conversely, hPNPase knockdown decreases viability and increases 8-oxoG level in HeLa cell exposed to H₂O₂. Our results suggest that hPNPase plays an important role in protecting cells and limiting damaged RNA under oxidative stress.     

The interplay of Fanconi anemia proteins in the DNA damage response

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by chromosome instability and cancer predisposition. At least 11 complementation groups for FA have been identified, and eight FA genes have been cloned. Interestingly, the eight known FA proteins cooperate in a common pathway leading to the interaction of monoubiquitinated FANCD2 and BRCA2 in damaged chromatin. Disruption of this pathway results in the clinical and cellular abnormalities common to all FA subtypes. This review will examine the interaction of the cloned FA proteins with each other and with other DNA damage response proteins (i.e., ATM, ATR, and NBS1). Also, somatic (acquired) disruption of the FA pathway in human tumors appears to account for their chromosome instability and crosslinker hypersensitivity.     

Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins

Fanconi anaemia (FA) has recently become an attractive model to study breast cancer susceptibility (BRCA) genes, as three FA genes, FANCD1, FANCN and FANCJ, are identical to the BRCA genes BRCA2, PALB2 and BRIP1. Increasing evidence shows that FA proteins function as signal transducers and DNA-processing molecules in a DNA-damage response network. This network consists of many proteins that maintain genome integrity, including ataxia telangiectasia and Rad3 related protein (ATR), Bloom syndrome protein (BLM), and BRCA1. Now that the gene that is defective in the thirteenth and last assigned FA complementation group (FANCI) has been identified, I discuss what is known about FA proteins and their interactive network, and what remains to be discovered.