Induction of apoptosis and NF-kappaB by quercetin in growing murine L1210 lymphocytic leukaemic cells potentiated by TNF-alpha

Polyphenol quercetin induced apoptosis in proliferating murine L1210 lymphocytic cells. DNA damage, as well as apoptosis and withdrawal from the cell cycle were transient. The above mentioned death promoting activity of quercetin was enhanced by physiological concentrations of TNF-alpha. At the same time, indices of cell viability dropped. However, the extent and tendency of the initially enhanced cell mortality steadily diminished throughout the experiment. After 12 h the G2/M phase reappeared. After 24 h all indices almost returned to control levels indicating either the selection of subpopulation of unaffected leukaemic cells or cells developing resistance to the treatment. A DNA ladder of oligonucleosomes was observed for apoptogenic treatments. We conclude that quercetin unmasked cell death, promoting the activity of TNF-alpha. However, after 12 and 24 h of exposure, surviving cells could complete the cell cycle and finally recover. At the same time, increased NF-kappaB activation was demonstrated by immunoblotting of the immunoreactive RelA/p65 subunit in nuclear extracts. Exposure to TNF-alpha or quercetin was crucial for increased activity of NF-kappaB, which may implicate an increasing resistance to their cytotoxicity.     

Reciprocal control of apoptosis and proliferation in cultured rat hepatoma arl-6 cells: Roles of nutrient supply, serum, and oxidative stress

Summary In order to understand how cancer cells accumulate, rat hepatoma ARL-6 cells were cultured for 8 d to identify factors involved in spontaneous cell proliferation and apoptosis. With increasing time in culture, the proportion of cells in the proliferative phases of the cell cycle and the rate of deoxyribonucleic acid (DNA) synthesis decreased. The waning of proliferation was associated with a gradual reduction of cell viability, and this was temporally related to the appearance of typical apoptotic morphology and DNA laddering. Medium replacement or supplementation with fetal calf serum (FCS) suppressed apoptosis, while medium change, but not fetal calf serum alone, enhanced cell proliferation. Apoptosis was also suppressed by dimethyl sulfoxide (DMSO), but supplementary glutathione was without effect. Expression of poly(adenosine diphosphate[ADP]-ribose)polymerase peaked on days 3–4 of culture, and was followed by a progressive decrease thereafter, consistent with proteolytic cleavage. This decrease was prevented to varying extents by complete medium replacement, FCS and DMSO, indicating a close temporal relationship between poly(ADP-ribose)polymerase activation and apoptosis. Expression of Fas and Bcl-2 did not change appreciably over the 8-d culture, but there was a gradual increase in Bax expression; medium change, FCS and DMSO all partly inhibited Bax expression. These data indicate that spontaneous apoptosis in cultured ARL-6 cells is inversely related to cell proliferation, and that nutrient supply, and to a lesser extent, serum-derived factors and oxidative stress modulate apoptosis in this system. Proteolytic cleavage of poly(ADP-ribose)polymerase and expression of Bax are likely to be mechanistically involved with the control of spontaneous apoptosis in ARL-6 cells, whereas changes in the levels of Fas and Bcl-2 do not play a role.     

Nicotine mediates oxidative stress and apoptosis through cross talk between NOX1 and Bcl-2 in lung epithelial cells

Nicotine contributes to the onset and progression of several pulmonary diseases. Among the various pathophysiological mechanisms triggered by nicotine, oxidative stress and cell death are reported in several cell types. We found that chronic exposure to nicotine (48 h) induced NOX1-dependent oxidative stress and apoptosis in primary pulmonary cells. In murine (MLE-12) and human (BEAS-2B) lung epithelial cell lines, nicotine acted as a sensitizer to cell death and synergistically enhanced apoptosis when cells were concomitantly exposed to hyperoxia. The precise signaling pathway was investigated in MLE-12 cells in which NOX1 was abrogated by a specific inhibitor or stably silenced by shRNA. In the early phase of exposure (1 h), nicotine mediated intracellular Ca²⁺ fluxes and activation of protein kinase C, which in its turn activated NOX1, leading to cellular and mitochondrial oxidative stress. The latter triggered the intrinsic apoptotic machinery by modulating the expression of Bcl-2 and Bax. Overexpression of Bcl-2 completely prevented nicotine’s detrimental effects, suggesting Bcl-2 as a downstream key regulator in nicotine/NOX1-induced cell damage. These results suggest that NOX1 is a major contributor to the generation of intracellular oxidative stress induced by nicotine and might be an important molecule to target in nicotine-related lung pathologies.     

Negative cell cycle control of human T cells by β-galactoside binding protein (βGBP): Induction of programmed cell death in leukaemic cells

The cell cycle is negatively regulated by diverse molecular events which originate in part from the interaction of secreted proteins with specific cell surface receptors. By exerting negative control on cell proliferation, these factors can help maintain cell number balance both through growth restraints and the induction of apoptosis and may thus contribute to prevent or control tumourigenesis. Here we report that βGBP, a negative growth factor which controls transition from S phase into G₂, causes an S/G₂ growth arrest in both normal and leukaemic T cells. However, in leukaemic T cells but not in normal T lymphocytes, growth arrest is followed by apoptosis. Analysis of possible mechanisms of induction of apoptosis does not support Fas and Fas L as having a main role but points instead to Bcl-2 and Bax. The induction of apoptosis in leukaemic T cells is characterised by the decrease of Bcl-2 and consequent predominance of Bax. By contrast, in the normal T cells, which do not enter apoptosis, the quantitative relationship of Bcl-2 to Bax remains unchanged. The ability of βGBP to selectively induce apoptosis in leukaemic cells suggests that βGBP may play a role in cancer surveillance and that its use has potential therapeutic implications. J Cell Physiol 178:102–108, 1999. © 1999 Wiley-Liss, Inc.     

Bcl-2 protects against hyperoxia-induced apoptosis through inhibition of the mitochondria-dependent pathway

Bcl-2 is an antiapoptotic molecule that prevents oxidative stress damage and cell death. We investigated the possible protective mechanisms mediated by Bcl-2 during hyperoxia-induced cell death in L929 cells. In these cells, hyperoxia promoted apoptosis without DNA fragmentation. Overexpression of Bcl-2 significantly protected cells from oxygen-induced apoptosis, as shown by measurement of lactate dehydrogenase release, quantification of apoptotic nuclei, and detection of Annexin-V-positive cells. Bcl-2 partially prevented mitochondrial damage and interfered with the mitochondrial proapoptotic signaling pathway: it reduced Bax translocation to mitochondria, decreased the release of cytochrome c, and inhibited caspase 3 activation. However, treatment with the caspase inhibitor Z-VAD.fmk failed to rescue the cells from death, indicating that protection provided by Bcl-2 was due not only to caspase inhibition. Bcl-2 also prevented the release of mitochondrial apoptotic inducing factor, a mediator of caspase-independent apoptosis, correlating with the absence of oligonucleosomal DNA fragmentation. In addition, Bcl-2-overexpressing cells showed significantly higher intracellular amounts of glutathione after 72 h of oxygen exposure. In conclusion, our results demonstrate that the overexpression of Bcl-2 is able to prevent hyperoxia-induced cell death, by affecting mitochondria-dependent apoptotic pathways and increasing intracellular antioxidant compounds.     

Neuroprotective effect of carnosine on primary culture of rat cerebellar cells under oxidative stress

Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatographymass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.     

Apoptotic and autophagic responses to Bcl-2 inhibition and photodamage.

Among the cellular responses to photodamage initiated by photodynamic therapy (PDT) are autophagy and apoptosis. While autophagy is a reversible process that can be both a survival and a death pathway, apoptosis is irreversible, leading only to cell death. In this study, we followed the fate of mouse leukemia L1210 cells after photodamage to the endoplasmic reticulum (ER) using a porphycene photosensitizer, where Bcl-2 was among the PDT targets. In wild-type cells, we observed a rapid wave of autophagy, presumed to represent the recycling of some damaged organelles, followed by apoptosis. Using shRNA technology, we created a Bax knockdown line (L1210/Bax(-)). In the latter cell line, we found a marked decrease in apoptosis after photodamage or pharmacologic inactivation of Bcl-2 function, but this did not affect PDT efficacy. Loss of viability was associated with a highly-vacuolated morphology consistent with autophagic cell death. Previous studies indicated pro-survival attributes of autophagy after low-dose PDT, suggesting that autophagy may be responsible for the 'shoulder' on the dose-response curve. It appears that attempts at extensive recycling of damaged organelles are associated with cell death, and that this phenomenon is amplified when apoptosis is suppressed.     

Nitrobenzylthioinosine-insensitive uridine transport in human lymphoblastoid and murine leukemia cells

Both human lymphoblastoid (RPMI 6410) and murine leukemia (L1210) cells were found to have a component of uridine transport which is insensitive to the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). In both cell lines NBMPR-insensitive uridine transport is inhibited by other nucleosides and by the sulfhydryl reagent p-chloromercuribenzenesulfonate. In RPMI 6410 cells NBMPR-insensitive transport accounts for only 2% of the initial rate of uridine transport. In contrast, 20% of the initial rate of transport of L1210 cells is insensitive to NBMPR, and uridine uptake over longer periods (10 min) is completely insensitive to NBMPR.     

TRAF2 phosphorylation promotes NF-κB-dependent gene expression and inhibits oxidative stress-induced cell death

Tumor necrosis factor α (TNF-α) receptor-associated factor 2 (TRAF2) regulates activation of the c-Jun N-terminal kinase (JNK)/c-Jun and the inhibitor of κB kinase (IKK)/nuclear factor κB (NF-κB) signaling cascades in response to TNF-α stimulation. Gene knockout studies have revealed that TRAF2 inhibits TNF-α-induced cell death but promotes oxidative stress-induced apoptosis. Here we report that TNF-α and oxidative stress both induce TRAF2 phosphorylation at serines 11 and 55 and that this dual phosphorylation promotes the prolonged phase of IKK activation while inhibiting the prolonged phase of JNK activation. Prolonged IKK activation trigged by TNF-α plays an essential role in efficient expression of a subset of NF-κB target genes but has no substantial role in TNF-α-induced cell death. On the other hand, TRAF2 phosphorylation in response to oxidative stress significantly promotes cell survival by inducing prolonged IKK activation and by inhibiting the prolonged phase of JNK activation. Notably, stable expression of phospho-null mutant TRAF2 in cancer cells leads to an increase in the basal and inducible JNK activation and B-cell lymphoma 2 (Bcl-2) phosphorylation. In addition, exposure of cells expressing phospho-null mutant TRAF2 to sublethal oxidative stress results in a rapid degradation of Bcl-2 and cellular inhibitor of apoptosis 1 as well as significantly increased cell death. These results suggest that TRAF2 phosphorylation is essential for cell survival under conditions of oxidative stress.     

Augmentation of monocyte intracellular ascorbate in vitro protects cells from oxidative damage and inflammatory responses

Ascorbic acid is present as a primary antioxidant in plasma and within cells, protecting both cytosolic and membrane components of cells from oxidative damage. The effects of intracellular ascorbic acid on F(2)-isoprostanes (biomarkers of oxidative stress) and monocyte chemoattractant protein-1 (marker of inflammatory responses) production in monocytic THP-1 cells were investigated under conditions of 2,2'-Azobis(2-methylpropionamidine)dihydrochloride (AAPH) induced oxidative stress. Cells cultured under normal conditions have extremely low ascorbate levels and the intracellular ascorbate can be augmented significantly by adding ascorbate to the culture medium. While AAPH treatment reduced cell viability, increased F(2)-isoprostanes and MCP-1 production, the presence of intracellular ascorbic acid maintained high cell viability and attenuated both F(2)-isoprostanes and MCP-1 production. Measurement of intracellular ascorbic acid and its oxidised products showed that intracellular ASC was oxidised to a significantly greater extent during AAPH treatment and may be utilised to protect the cells under conditions of oxidative stress. This study demonstrates the importance of intracellular ascorbate, which may be lacking under normal cell culture conditions, under conditions of increased oxidative stress.     

Oxalomalate, a competitive inhibitor of NADP+ -dependent isocitrate dehydrogenase, regulates lipid peroxidation-mediated apoptosis in U937 cells

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Recently, we demonstrated that the control of cytosolic redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) through to supply NADPH for antioxidant systems. The protective role of IDPc against lipid peroxidation-mediated apoptosis in U937 cells was investigated in control and cells pre-treated with oxlalomalate, a competitive inhibitor of IDPc. Upon exposure to 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the susceptibility to apoptosis was higher in oxalomalate-treated cells as compared to control cells. The results suggest that IDPc plays an important protective role in apoptosis of U937 cells induced by lipid peroxidation-mediated oxidative stress.     

Cell surface properties of L1210 leukaemia cells.

The surface properties of vincristine-colchicine sensitive and resistant L1210 leukaemic cells have been studied using concanavalin A mediated agglutination assay as well as electron microscopic visualization of concanavalin A receptors. 3H-colchicine uptake by the sensitive and resistant lines has also been compared. The resistant L1210 leukaemic cells proved less agglutinable than the sensitive ones at the same concanavalin A concentration. Previous treatments with either colchicine, vincristine or chlorpromazine caused a marked decrease in the agglutinability of the sensitive L1210 leukaemic cells, while agglutination of the resistant ones was lowered slightly by the same treatments. The 3H-colchicine uptake of the sensitive cells was three times higher than that of the resistant ones.     

Alteration in p53 pathway and defect in apoptosis contribute independently to cisplatin-resistance

The accumulation of molecular genetic defects selected during the adaptation process in the development of cisplatin-resistance was studied using progressive cisplatin-resistant variants (L1210/DDP2, L1210/DDP5, L1210/DDP10) derived from a murine leukemia cell line (L1210/0). Of these cell lines, only the most resistant L1210/DDP10 was cross-resistant to etoposide and deficient in apoptosis induced by these two drugs, indicating that resistance to DNA-damaging agents correlates with a defect in apoptosis. This defect was tightly associated with the loss of a Ca2+/Mg2+-dependent nuclear endonuclease activity present in the less cisplatin-resistant cells. Evidence is presented that p53-dependent function (a) is lost not only in the apoptosis defective L1210/DDP10 cells, but also in the apoptosis susceptible L1210/DDP5 cells; (b) is unrelated to drug-induced cell cycle perturbations. These results suggest that deficiency in the p53 pathway and resistance to DNA-damaging agents due to a defect in apoptosis are independent events.     

The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis

Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H(2)O(2). The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H(2)O(2)-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H(2)O(2) treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H(2)O(2)-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H(2)O(2)-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H(2)O(2) activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.     

Hydrogen peroxide-induced apoptosis in human gastric carcinoma MGC803 cells

Hydrogen peroxide (H(2)O(2)), a representative ROS, has been used to study the apoptosis of cancer cells to oxidative stress. In this study, we exploited the cellular and molecular mechanisms involved in H(2)O(2)-induced apoptosis in human gastric carcinoma MGC803 cells. Exposure of cells to H(2)O(2) might cause significant viability loss and the increase in apoptotic rate. Treatment with 0.4 mmol/L H(2)O(2) up-regulated Bax but down-regulated Bcl-2 in a time-dependent manner, while Bcl-xL expression remained unchanged. Our results also showed that the levels of Fas and Fas-L were increased, the pro-caspase-3 and pro-caspase-9 were down-regulated in H(2)O(2)-treated MGC803 cells. Under H(2)O(2) stress, we found that the protein p53 also participated in MGC803 cells apoptosis. Taken together, the present study indicated that Fas-mediated cell surface death receptor pathway and mitochondria-mediated pathway may participate in regulating the MGC803 cells apoptosis under oxidative stress.     

Apoptosis and adaptive responses to oxidative stress in human endothelial cells exposed to cyclosporin A correlate with BCL-2 expression levels.

Treatment of transplanted patients with cyclosporin A (CSA) may cause adverse effects such as nephrotoxicity and hypertension. As CSA is known to induce oxidative stress in several tissues, it may cause vascular problems by triggering oxidative stress in endothelial cells (EC). However, oxidative stress has been reported for acute exposure to CSA concentrations exceeding its clinical range, whereas immunosuppression requires life-long treatment with therapeutic concentrations. We therefore compared the effects of 21 h pharmacological (200 microM) vs. 8 days clinical (0.5-2.5 microM) doses of CSA on cultured human EC. Pharmacological doses of CSA cause a decrease in cell density via apoptosis and a down-regulation of the antiapoptotic protein Bcl-2. However, these effects are independent of CSA-induced oxidative stress. In contrast, therapeutic concentrations of CSA cause Bcl-2 up-regulation and modification of EC morphology, both effects blocked by antioxidants. Therefore, a low level of oxidants may act in EC as second messengers that up-regulate Bcl-2, thus promoting survival of impaired EC. Our data suggest that the oxidative stress induced by clinical concentrations of CSA may be involved in the adverse effects of the drug on the vascular system of transplanted patients via an adaptive response involving Bcl-2 up-regulation rather than an apoptotic process     

A Free Radical Initiator, 2,2′-azobis (2-aminopropane) Dihydrochloride Enhances Hyperthermia-induced Apoptosis in Human Uterine Cervical Cancer Cell Lines

Hyperthermia-induced apoptosis and its enhancement in the presence of a temperature-dependent free radical initiator, 2,2′-azobis (2-aminopropane) dihydrochloride (AAPH) were examined in human uterine cervical cancer cell lines, CaSki and HeLa. When both cell lines were treated with hyperthermia at 44°C for 60?min, minimal apoptosis was observed. When combined with nontoxic AAPH (50?mM), significant enhancement of apoptosis was observed, where the initial rate of free radical formation was about twice as high than that at 37°C. Augmentation of the growth delay, lipid peroxidation (LPO), activation of caspase-3 and increase in [Ca²⁺]i were also observed after the combined treatment. A water-soluble vitamin E, Trolox, blocked the increase in [Ca²⁺]i and an intracellular Ca²⁺ chelator, BAPTA-AM, prevented the DNA fragmentation induced by the combination. Cytochrome c release was also revealed by fluorescence microscopy. However, no significant change in mitochondrial membrane potential and expression of Bax and Bcl-2 was observed. A slight increase in Fas expression was observed only in CaSki cells after the combined treatment. These results indicate that hyperthermia and AAPH induce enhanced apoptosis and subsequent cell killing via two pathways; a pathway dependent on increase in LPO and [Ca²⁺]i, and a pathway associated with cytochrome c release and subsequent caspase activation without changes of mitochondrial membrane potential and Bax/Bcl-2 expression in these cell lines. Since it is known that cancer cells are generally resistant to physical and chemical stress-induced apoptosis, free radical generators like AAPH appear to be a useful thermosensitizer for hyperthermic cancer therapy.     

A free radical initiator, 2,2'-azobis (2-aminopropane) dihydrochloride enhances hyperthermia-induced apoptosis in human uterine cervical cancer cell lines

Hyperthermia-induced apoptosis and its enhancement in the presence of a temperature-dependent free radical initiator, 2,2'-azobis (2-aminopropane) dihydrochloride (AAPH) were examined in human uterine cervical cancer cell lines, CaSki and HeLa. When both cell lines were treated with hyperthermia at 44°C for 60 min, minimal apoptosis was observed. When combined with nontoxic AAPH (50 mM), significant enhancement of apoptosis was observed, where the initial rate of free radical formation was about twice as high than that at 37°C. Augmentation of the growth delay, lipid peroxidation (LPO), activation of caspase-3 and increase in [Ca₂₊]i were also observed after the combined treatment. A water-soluble vitamin E, Trolox, blocked the increase in [Ca₂₊]i and an intracellular Ca₂₊ chelator, BAPTA-AM, prevented the DNA fragmentation induced by the combination. Cytochrome c release was also revealed by fluorescence microscopy. However, no significant change in mitochondrial membrane potential and expression of Bax and Bcl-2 was observed. A slight increase in Fas expression was observed only in CaSki cells after the combined treatment. These results indicate that hyperthermia and AAPH induce enhanced apoptosis and subsequent cell killing via two pathways; a pathway dependent on increase in LPO and [Ca₂₊]i, and a pathway associated with cytochrome c release and subsequent caspase activation without changes of mitochondrial membrane potential and Bax/Bcl-2 expression in these cell lines. Since it is known that cancer cells are generally resistant to physical and chemical stress-induced apoptosis, free radical generators like AAPH appear to be a useful thermosensitizer for hyperthermic cancer therapy.     

Fasudil Mesylate Protects PC12 Cells from Oxidative Stress Injury via the Bax-Mediated Pathway

We previously reported that fasudil mesylate (FM) improves neurological deficit and neuronal damage in rats with ischemia following middle cerebral artery occlusion and reperfusion in vivo. In this study, the properties of FM on hydrogen peroxide (H₂O₂)-induced oxidative stress insult in cultured PC12 cells as well as the underlying mechanisms were investigated in vitro. Pretreatment with FM (5, 10 μM) prior to H₂O₂ exposure significantly elevated cell viability, reduced cell apoptosis by MTT assay, LDH assay, Hoechst 33258 dye staining, and FM also decreased the accumulation of reactive oxygen species (ROS) by DCFH-DA staining and NBT test. Furthermore, FM also reversed the upregulation of Bax/Bcl-2 ratio, the downstream cascade following ROS. FM protected PC12 cells from oxidative stress insult via down-regulating the Bax/Bcl-2 ratio. These findings indicate that a direct effect of fasudil mesylate on PC12 cells may be partly responsible for its protective effect against oxidative stress injury.