Transient Adaptive Response Research Articles

Outcome of critically ill patients with non-thyroidal illness

Background: During period of critical illness, there are diverse alterations in the hypothalamus-pituitarythyroid (HPT) axis. This diversity in critically ill patients and the etiological relationship between underlying disease and non-thyroidal illness (NTI) is poorly understood. The aims of this study were to examine the features of NTI and outcomes in critically ill patients admitted in Critical Care Medicine (CCM) Department, BIRDEM General Hospital.
 Methods: A total of 86 patients admitted to CCM department, BIRDEM General Hospital during the period of July to December 2015 , having nonthyroidal illness, detected by thyroid function tests during ICU stay were enrolled in this study. All patients discharged from hospital were followed up for a period of 6 months. Patients with known thyroid diseases or taking medications that affect thyroid function were excluded. Condition at hospital discharge and mortality in the ICU or later at home after discharge within next 6 months was assessed as outcomes.
 Results: Mean age of the study subjects was 63.87(±13.5)years and 45(52.3%)of the study subjects were female. Most of the study subjects had diabetes (84.88%) and hypertension (82.55%).Mean (±SD) of FT3 (pmol/l), FT4(pmol/l), TSH (uIU/ml) were 2.85(±1.35),12.74(±8.17) and 2.81(±8.57)respectively. Among the total study subjects 44.18% patients died in ICU and 2.32% patients after shifting to ward. Among the patients having pneumonia, Myocardial Infarction(MI) /Arrhythmia, Stroke, Sepsis and Gastrointestinal disease, 50.94% 51.02%, 56.0%,53.85%,37.50% died in hospital (ICU or after shifting toward).The 46 patients ,who were discharged from hospital were followed up for next 6 months.
 Conclusion: NTI is a transient adaptive response affecting individuals with acute and chronic illness and is more common among patients admitted in intensive care unit (ICU). The prognosis of patients having NTI depends on severity of thyroid dysfunction.
 Birdem Med J 2021; 11(1): 47-51

Emergent memory in cell signaling: Persistent adaptive dynamics in cascades can arise from the diversity of relaxation time-scales

The mitogen-activated protein kinase (MAPK) signaling cascade, an evolutionarily conserved motif present in all eukaryotic cells, is involved in coordinating crucial cellular functions. While the asymptotic dynamical behavior of the pathway stimulated by a time-invariant signal is relatively well-understood, we show using a computational model that it exhibits a rich repertoire of transient adaptive responses to changes in stimuli. When the signal is switched on, the response is characterized by long-lived modulations in frequency as well as amplitude. On withdrawing the stimulus, the activity decays over long timescales, exhibiting reverberations characterized by repeated spiking in the activated MAPK concentration. The long-term persistence of such post-stimulus activity suggests that the cascade retains memory of the signal for a significant duration following its removal. The molecular mechanism underlying the reverberatory activity is related to the existence of distinct relaxation rates for the different cascade components. This results in the imbalance of fluxes between different layers of the cascade, with the reuse of activated kinases as enzymes when they are released from sequestration in complexes. The persistent adaptive response, indicative of a cellular “short-term” memory, suggests that this ubiquitous signaling pathway plays an even more central role in information processing by eukaryotic cells.

Involvement of MAPK signalling in radioadaptive response in BALB/c mice exposed to low dose ionizing radiation

To investigate low dose ionizing radiation (LDIR)-induced adaptive response in lymphocytes of BALB/c mice and to elucidate related molecular mechanisms. Mice were exposed to a priming dose (PD) of 0.1 Gy and challenge dose (CD) of 2 Gy ionizing radiation. Proliferation response to mitogen concanavalin A was assessed using 3H thymidine incorporation and carboxyfluoresceinsuccinamidylester (CFSE) dye dilution. Early activation markers were assessed by flow cytometry, cytokines by ELISA, DNA damage by comet assay and mitogen activated protein kinase (MAPK) signaling by Western blotting. Radioadaptive response was observed in lymphocytes of mice exposed to PD prior to CD of ionizing radiation in terms of DNA damage, early activation markers CD69, CD71, cytokines IL-2, IFN-γ as well as proliferation. This effect was transient and observed 24 h after CD and not after 0 h or 72 h. Hyper activation of MAPK signaling pathways in lymphocytes from LDIR-exposed mice and abrogation by ERK and p38 inhibitors suggests the involvement of MAPK signaling in radioadaptive response. Our study demonstrates that LDIR-induced transient adaptive response was due to hyper activation of MAPK signaling. Our findings contribute towards the understanding of LDIR-induced adaptive response.

Rewarming affects EEG background in term newborns with hypoxic-ischemic encephalopathy undergoing therapeutic hypothermia.

To investigate how rewarming impacts the evolution of EEG background in neonates with hypoxic-ischemic encephalopathy (HIE) undergoing therapeutic hypothermia (TH). We recruited a retrospective cohort of 15 consecutive newborns with moderate (9) and severe (6) HIE monitored with a continuous EEG during TH and at least 12h after its end. EEG background was analyzed using conventional visual and quantitative EEG analysis methods including EEG discontinuity, absolute and relative spectral magnitudes. One patient with seizures on rewarming was excluded from analyses. Visual and quantitative analyses demonstrated significant changes in EEG background from pre- to post-rewarming, characterized by an increased EEG discontinuity, more pronounced in newborns with severe compared to moderate HIE. Neonates with moderate HIE also had an increase in the relative magnitude of slower delta and a decrease in higher frequency theta and alpha waves with rewarming. Rewarming affects EEG background in HIE newborns undergoing TH, which may represent a transient adaptive response or reflect an evolving brain injury. EEG background impairment induced by rewarming may represent a biomarker of evolving encephalopathy in HIE newborns undergoing TH and underscores the importance of continuously monitoring the brain health in critically ill neonates.

Shear stress regulates endothelial cell autophagy via redox regulation and Sirt1 expression.

Disturbed cell autophagy is found in various cardiovascular disease conditions. Biomechanical stimuli induced by laminar blood flow have important protective actions against the development of various vascular diseases. However, the impacts and underlying mechanisms of shear stress on the autophagic process in vascular endothelial cells (ECs) are not entirely understood. Here we investigated the impacts of shear stress on autophagy in human vascular ECs. We found that shear stress induced by laminar flow, but not that by oscillatory or low-magnitude flow, promoted autophagy. Time-course analysis and flow cessation experiments confirmed that this effect was not a transient adaptive stress response but appeared to be a sustained physiological action. Flow had no effect on the mammalian target of rapamycin-ULK pathway, whereas it significantly upregulated Sirt1 expression. Inhibition of Sirt1 blunted shear stress-induced autophagy. Overexpression of wild-type Sirt1, but not the deacetylase-dead mutant, was sufficient to induce autophagy in ECs. Using both of gain- and loss-of-function experiments, we showed that Sirt1-dependent activation of FoxO1 was critical in mediating shear stress-induced autophagy. Shear stress also induced deacetylation of Atg5 and Atg7. Moreover, shear stress-induced Sirt1 expression and autophagy were redox dependent, whereas Sirt1 might act as a redox-sensitive transducer mediating reactive oxygen species-elicited autophagy. Functionally, we demonstrated that flow-conditioned cells are more resistant to oxidant-induced cell injury, and this cytoprotective effect was abolished after inhibition of autophagy. In summary, these results suggest that Sirt1-mediated autophagy in ECs may be a novel mechanism by which laminar flow produces its vascular-protective actions.

Juvenile animal testing of hydroxypropyl-β-cyclodextrin in support of pediatric drug development.

Hydroxypropyl-β-cyclodextrin (HP-β-CD) is being explored as excipient for administration of poorly soluble NCE's in pediatrics. In support of pharmaceutical development, non-clinical studies were performed to investigate whether oral and intravenous administration of HP-β-CD showed a different response in juvenile rats versus adult rats. Juvenile rats received HP-β-CD via the intravenous route at dose levels of 50, 200 and 400mg/kg/day from postnatal day 16 to 44, or via oral gavage at 500, 1000 and 2000mg/kg/day from postnatal day 4 to 46. In addition to in vivo parameters, toxicokinetics and post-mortem evaluations were conducted. The main findings were related to the renal excretion of intact HP-β-CD and were regarded as non-adverse transient adaptive responses. The pathogenesis of the osmotic nephrosis-like changes are discussed. With increasing age a more effective renal clearance of HP-β-CD is present in line with the postnatal functional maturation of the kidney. In addition, following oral administration an increase in soft stools was seen which was related to osmotic water retention in the large intestine. The findings in the juvenile studies are very similar to those observed in previously performed adult rat studies at similar dose levels, same routes and similar or longer dose duration. No novel toxicity was seen in the juvenile studies.

Follow up of intima-media thickness after severe early-onset preeclampsia.

Early-onset preeclampsia is associated with premature cardiovascular disease. We previously demonstrated that femoral intima-media thickness (IMT) and markers of cardiovascular disease were increased in women 1 year after early-onset preeclampsia. The current study measured (progression of) IMT, cardiovascular disease risk factors and markers of endothelial cell dysfunction 4-5 years postpartum in the same women. Case-control study. Formerly preeclamptic women. IMT of carotid and femoral arteries was measured by ultrasound, as a marker of subclinical atherosclerosis. Various conventional cardiovascular risk factors were determined, as well as serum markers of endothelial cell activation and inflammation. Values were compared with those 1 year after the first (preeclamptic) pregnancy. We included 17 formerly preeclamptic women (cases) and 16 controls. Mean interval between index delivery and day of investigation was 4.7 years for the cases and 4.3 years for the controls. Neither differences nor progression of IMT was observed between the cases and the controls. Increased blood pressure, body mass index, serum triglycerides and inflammatory markers were found in the cases compared with the controls. IMT was not increased in women with an almost 5-year history of severe preeclampsia as an indicator of increased cardiovascular risk. This study suggests a transient adaptive response of the arteries in formerly preeclamptic women. The persistence of cardiovascular risk factors in this group emphasizes the need for long-term follow-up.

Influence of A Continuous Very Low Dose of Gamma-Rays on Cell Proliferation, Apoptosis and Oxidative Stress.

We have previously shown a delay of death by lymphoma in SJL/J mice irradiated with continuous very low doses of ionizing radiation. In order to understand the mechanisms involved in this phenomenon, we have irradiated in vitro the Raw264.7 monocytic and the YAC-1 lymphoma cell lines at very low-dose rate of 4cGy.month-1. We have observed a transient increase in production of both free radicals and nitric oxide with a transient adaptive response during at least two weeks after the beginning of the irradiation. The slight decrease of Ki67 proliferation index observed during the second and third weeks of YAC-1 cells culture under irradiation was not significant but consistent with the shift of the proliferation assay curves of YAC-1cells at these same durations of culture. These in vitro results were in good agreement with the slightly decrease under irradiation of Ki67 proliferative index evaluated on lymphomatous lymph nodes of SJL/J mice. A significant decrease of YAC-1 cells apoptotic rate under radiation appeared after 4 weeks of culture.Therefore very small doses of gamma-irradiation are able to modify the cellular response. The main observations did not last with increasing time under irradiation, suggesting a transient adaptation of cells or organisms to this level of irradiation.

Epigenetics and Transcriptomics to Detect Adverse Drug Effects in Model Systems of Human Development

Prenatal exposure to environmental chemicals or drugs has been associated with functional or structural deficits and the development of diseases in later life. For example, developmental neurotoxicity (DNT) is triggered by lead, and this compound may predispose to neurodegenerative diseases in later life. The molecular memory for such late consequences of early exposure is not known, but epigenetic mechanisms (modification of the chromatin structure) could take this role. Examples and underlying mechanisms have been compiled here for the field of DNT. Moreover, we addressed the question as to what readout is suitable for addressing drug memory effects. We summarize how complex developmental processes can be modelled in vitro by using the differentiation of human stem cells. Although cellular models can never replicate the final human DNT phenotype, they can model the adverse effect that a chemical has on key biological processes essential for organ formation and function. Highly information-rich transcriptomics data may inform on these changes and form the bridge from in vitro models to human prediction. We compiled data showing that transcriptome analysis can indicate toxicity patterns of drugs. A crucial question to be answered in our systems is when and how transcriptome changes indicate adversity (as opposed to transient adaptive responses), and how drug-induced changes are perpetuated over time even after washout of the drug. We present evidence for the hypothesis that changes in the histone methylation pattern could represent the persistence detector of an early insult that is transformed to an adverse effect at later time-points in life.

A chronic very low dose of gamma-rays alters cell adhesion

The biological effects of very low doses of ionising radiation are difficult to assess. We previously observed a delay of death by lymphoma in two different mouse strains continuously irradiated ( γ -rays) with a dose rate of 10 cGy year −1 . Cellular mechanisms likely to lead to slowing tumour growth were explored. Human lymphoid and epithelial cell lines (HL60 and MCF7) were irradiated in vitro at very low dose rate of 4 cGy month −1 . Proliferation was not modified in HL60 and MCF7 cells. However, irradiated MCF7 adherent cells showed a lower cell attachment to support partly related to a slight decrease in expression levels of α6 and β4 integrins. We also observed a transient adaptive response during at least two weeks after the beginning of the irradiation in both cell lines. These results demonstrate the ability of tiny amounts of gamma-irradiation to alter cell attachment to support and to induce an adaptive response.

P63 Morbidity and mortality conference using ACGME competencies: a case study in eclampsia

(1) Persistence of aberrant CVD risk factors and an accelerated progression of IMT. (2) Lower levels of circulating endothelial markers and impaired numbers of circulating endothelial progenitor cells (EPCs), indicating endothelial cell dysfunction and impaired vascular repair. Methods: 18 formerly preeclamptic women and 16 controls were included in our follow-up (study 2). Ultrasound measurements of carotid and femoral arteries and various classical cardiovascular risk factors were recorded. Blood samples were taken and whole blood was stained using anti-CD34, anti-CD133 and anti-vascular growth factor receptor antibodies. Flow cytometry was used to quantify EPCs (triple positive cells). Circulating endothelial markers (von Willebrand factor (vWF), ICAM-1) and inflammatory markers (hsCRP) were measured in plasma using ELISA. Results: See Table 1. Conclusion: This study suggests a transient adaptive response of the vasculature to a preeclamptic pregnancy which normalizes within years. Otherwise, the presence and persistence of cardiovascular risk factors in formerly preeclamptic women emphasizes the need to follow up these high-risk women in future.

The 24-hour respiratory quotient predicts energy intake and changes in body mass

To define the relationship between the respiratory quotient (RQ) and energy intake (EI) and to determine the impact of spontaneous locomotor activity (LMA) in the development of diet-induced obesity (DIO), we fed C57BL/6 mice a high-fat diet (HFD) for either 4 days or 17 wk and analyzed them using indirect calorimetry. Importantly, changes in body mass during calorimetry (DeltaM(b)) significantly covaried with RQ and EI; adjusting the data for DeltaM(b) permitted an analysis of the energy-balanced state. The 24-h RQ strongly predicted 24-h EI, and the slope of this relationship was diet dependent (HFD or chow) but independent of the HFD feeding period. Early-stage DIO was characterized by dark-period hyperphagia and fat storage, offset by greater light-period lipid oxidation; later stage DIO mice had a milder hyperphagia and lower substrate flexibility. Consequently, whereas 24-h RQ equaled the food quotient of the HFD in both early- and late-stage DIO, the range of RQ values was negatively correlated with, and mostly explained by, 24-h EI only in late-stage DIO. Lean and early-stage DIO mice had similar LMA values that were reduced in late-stage DIO. However, LMA significantly explained variance in total energy expenditure (EE) in only early-stage DIO mice. This indicated that the link between LMA and EE was a transient adaptive response to early DIO, whereas the later loss of LMA did not explain body weight gain in C57BL/6 DIO mice.

Free radical biology and medicine: it's a gas, man!

We review gases that can affect oxidative stress and that themselves may be radicals. We discuss O(2) toxicity, invoking superoxide, hydrogen peroxide, and the hydroxyl radical. We also discuss superoxide dismutase (SOD) and both ground-state, triplet oxygen ((3)O(2)), and the more energetic, reactive singlet oxygen ((1)O(2)). Nitric oxide ((*)NO) is a free radical with cell signaling functions. Besides its role as a vasorelaxant, (*)NO and related species have other functions. Other endogenously produced gases include carbon monoxide (CO), carbon dioxide (CO(2)), and hydrogen sulfide (H(2)S). Like (*)NO, these species impact free radical biochemistry. The coordinated regulation of these species suggests that they all are used in cell signaling. Nitric oxide, nitrogen dioxide, and the carbonate radical (CO(3)(*-)) react selectively at moderate rates with nonradicals, but react fast with a second radical. These reactions establish "cross talk" between reactive oxygen (ROS) and reactive nitrogen species (RNS). Some of these species can react to produce nitrated proteins and nitrolipids. It has been suggested that ozone is formed in vivo. However, the biomarkers that were used to probe for ozone reactions may be formed by non-ozone-dependent reactions. We discuss this fascinating problem in the section on ozone. Very low levels of ROS or RNS may be mitogenic, but very high levels cause an oxidative stress that can result in growth arrest (transient or permanent), apoptosis, or necrosis. Between these extremes, many of the gasses discussed in this review will induce transient adaptive responses in gene expression that enable cells and tissues to survive. Such adaptive mechanisms are thought to be of evolutionary importance.

Transient increase of de novo amino acid synthesis and its physiological significance in water-stressed white clover.

In white clover (Trifolium repens L. cv. Regal) the kinetics of de novo synthesis of amino acid and protein were compared by tracing 15N under well-watered (control) or water-deficit conditions. The physiological relationship between ammonia concentration, in response to the change in leaf water parameters, and de novo synthesis of amino acid and protein was also assessed. Leaf and root dry mass were not significantly affected for the first 3 d, whereas metabolic parameters such as total N and ammonia were significantly affected within the first day of water-deficit treatment. Inhibitory effect of water deficit on N acquisition from the soil was significant throughout the experimental period. Water deficit induced a significant increase in ammonia concentration in leaves during the first 3 d, and in roots for only the first day. In both leaves and roots, an increase in de novo amino acid synthesis, which peaked in leaves within the first 3 d of water-deficit treatment (Ψw ≥ -1.18 MPa), was observed. The rate of decrease in de novo protein synthesis gradually accelerated as the duration of the water-deficit treatment increased. There was a significant positive relationship between ammonia production and the increase in de novo amino acid synthesis during the first 3-d period, but not during the later period (day 3-day 7). This experiment clearly indicates that the increase in de novo amino acid synthesis caused by water deficit is a transient adaptive response occurring during the first few days and that it is associated with the increased ammonia concentrations, which in turn arise in response to a decrease in de novo protein synthesis.

Possible transient adaptive response to mitomycin C in peripheral lymphocytes from thyroid cancer patients after iodine-131 therapy.

Our study attempted to assess the possible induction and persistence of an adaptive response in lymphocytes of thyroidectomized thyroid cancer patients treated with 131I (2,590 MBq, corresponding to whole body doses in the range of 200-300 mGy), to a testing dose of mitomycin C (MMC) in vitro. The cytogenetic endpoint studied was the induction of micronuclei in cytokinesis-blocked peripheral blood lymphocytes, immediately before treatment and 1, 6 and 24 months after therapy. One month after therapy, induction of micronucleated cytokinesis-blocked lymphocytes ( per thousand ) by MMC was lower (34.6 +/- 7.7) than before therapy (52.1 +/- 5.0). In 7 of 11 patients this reduction was significant. However, at 6 months, induction of micronuclei was markedly higher (133.1 +/- 13.6). This significant increase was observed regardless of the decrease at 1 month. At 24 months, the frequency of micronucleated cells decreased (84.8 +/- 5.5), but remained higher than before treatment. The results obtained 1 month after therapy could reflect adaptation due to radiation, or a higher rate of early apoptosis or cell death, with bone marrow suppression, visible as a lower response in vitro towards MMC. At 6 months, recovery of the lymphocyte population may occur, and higher responses to MMC in vitro could reflect higher chromosomal instability in the previously irradiated stem cells with a concomitant disappearance of adaptation, whereas at 24 months the results show a tendency to return to pretherapy values.

The essential role of calcium in induction of the DSCR1 (ADAPT78) gene.

Stress responses have been of great interest to biologists for many years, both as interesting phenomena in their own right, and as tools to study gene expression. Stresses such as heat shock, cold shock, oxidative stress, glucose deprivation, heavy metals, and anoxia have all been studied. We have been studying transient adaptive responses to oxidative stress, in both prokaryotes and dividing eukaryotic cells, for over 10 years [1–5]. Oxidative stress involves responses to oxidants, or to agents that cause oxidation via intracellular redox reactions. Severe oxidative stress can result in compromise of vital cell or organismal functions, or even loss of viability. The transient adaptive response model to stress involves first finding a “challenge” level of stress that will either kill a large proportion of cells, or significantly inhibit cell growth or division. Having established the degree of stress required for this cytotoxicity, new cell cultures are “pre-treated” with various low stress exposures, for various time periods, in an attempt to induce an adaptive response. The pre-treated cells are then exposed to the challenge stress level and their resistance is measured. Through much trial and error (and not a little luck) an effective pre-treatment stress level can usually be found that will induce significant short-term protection against a subsequent stress challenge: this is a transient adaptive response. Importantly, such transient adaptation must be further tested to ensure that the cells become sensitive to stress again over time (de-adaptation), to insure that mutation has not caused a permanent change in stress phenotype. In the last few years we have discovered several new oxidant stress-inducible genes, all of which provide partial protection against acute oxidative stress. These include: Adapt15, Adapt33, Adapt66, Adapt73, Adapt78, and Adapt116. All of these genes contain a calcium response element, all can be induced by calcium ionophores such as A23187 in the presence of calcium; and by other agents, such as thapsigargin and cyclopiazonic acid, that increase intracellular calcium concentrations. In contrast, cell-permeant calcium chelators, such as BAPTA-AM, can completely block the induction of all the ‘adapt’ genes. Of all the adapt genes, Adapt78 is the most responsive to calcium signaling. Adapt78 was simultaneously discovered by Fuentes et al. and called Down Syndrome Critical Region 1 gene, or DSCR1 (subsequently called Down Syndrome Candidate Region 1) [6,7]. Recently we have discovered

Staging of Cytoskeletal and β-Amyloid Changes in Human Isocortex Reveals Biphasic Synaptic Protein Response during Progression of Alzheimer’s Disease

We have examined the relationships between dementia, loss of synaptic proteins, changes in the cytoskeleton, and deposition of beta-amyloid plaques in the neocortex in a clinicopathologically staged epidemiological cohort using a combination of biochemical and morphometric techniques. We report that loss of synaptic proteins is a late-stage phenomenon, occurring only at Braak stages 5 and 6, or at moderate to severe clinical grades of dementia. Loss of synaptic proteins was seen only after the emergence of the full spectrum of tau and beta-amyloid pathology in the neocortex at stage 4, but not in the presence of beta-amyloid plaques alone. Contrary to previous studies, we report increases in the levels of synaptophysin, syntaxin, and SNAP-25 at stage 3 and of alpha-synuclein and MAP2 at stage 4. Minimal and mild clinical grades of dementia were associated with either unchanged or elevated levels of synaptic proteins in the neocortex. Progressive aggregation of paired helical filament (PHF)-tau protein could be detected biochemically from stage 2 onwards, and this was earliest change relative to the normal aging background defined by Braak stage 1 that we were able to detect in the neocortex. These results are consistent with the possibility that failure of axonal transport associated with early aggregation of tau protein elicits a transient adaptive synaptic response to partial de-afferentation that may be mediated by trophic factors. This early abnormality in cytoskeletal function may contribute directly to the earliest clinically detectable stages of dementia.

The broad spectrum of responses to oxidants in proliferating cells: a new paradigm for oxidative stress.

Proliferating mammalian cells exhibit a broad spectrum of responses to oxidative stress, depending on the stress level encountered. Very low levels of hydrogen peroxide, e.g., 3 to 15 microM, or 0.1 to 0.5 micromol/10(7) cells, cause a significant mitogenic response, 25% to 45 % growth stimulation. Greater concentrations of H2O2, 120 to 150 microM, or 2 to 5 micromol/10(7) cells, cause a temporary growth arrest that appears to protect cells from excess energy use and DNA damage. After 4-6 h of temporary growth arrest, many cells will exhibit up to a 40-fold transient adaptive response in which genes for oxidant protection and damage repair are preferentially expressed. After 18 h of H2O2 adaptation (including the 4-6 h of temporary growth arrest) cells exhibit maximal protection against oxidative stress. The H2O2 originally added is metabolized within 30-40 min, and if no more is added the cells will gradually de-adapt, so that by 36 h after the initial H2O2 stimulus they have returned to their original level of H2O2 sensitivity. At H2O2 concentrations of 250 to 400 microM, or 9 to 14 micromol/10(7) cells, mammalian fibroblasts are not able to adapt but instead enter a permanently growth-arrested state in which they appear to perform most normal cell functions but never divide again. This state of permanent growth arrest has often been confused with cell death in toxicity studies relying solely on cell proliferation assays as measures of viability. If the oxidative stress level is further increased to 0.5 to 1.0 mM H2O2, or 15 to 30 micromol/10(7) cells, apoptosis results. This oxidative stress-induced apoptosis involves nuclear condensation, loss of mitochondrial transmembrane potential, degradation/down-regulation of mitochondrial mRNAs and rRNAs, and degradation/laddering of both nuclear and mitochondrial DNA. At very high H2O2 concentrations of 5.0 to 10.0 mM, or 150 to 300 micromol/10(7) cells and above, cell membranes disintegrate, proteins and nucleic acids denature, and necrosis swiftly follows. Cultured cells grown in 20% oxygen are essentially preadapted or preselected to survive under conditions of oxidative stress. If cells are instead grown in 3% oxygen, much closer to physiological cellular levels, they are more sensitive to an oxidative challenge but exhibit far less accumulated oxidant damage. This broad spectrum of cellular responses to oxidant stress, depending on the amount of oxidant applied and the concentration of oxygen in the cell culture system, provides for a new paradigm of cellular oxidative stress responses.

Relationship between the adaptive response to oxidants and stable menadione-resistance in Chinese hamster ovary cell lines.

To study the genetic changes that generate resistance to oxidants in mammalian cells, we isolated cell lines that are resistant to the naphthoquinone, menadione, from a Chinese hamster ovary cell line (CHO-K1). Corss-resistance to other oxidants (H2O2 and sodium arsenite) was observed. The IC50 of menadione (measured using a clonogenic assay) was 7.8-fold greater for one menadione-resistant cell line (MRc40) than for CHO-K1. Acquisition of resistance was associated with elevations of 2- and 3.2-fold in the low molecular weight thiols, glutathione and cysteine, respectively. Further, characterization demonstrated significant changes in the expression of enzymes associated with the oxidative stress response and with protection against oxidizing agents. The expressions of glutathione S-transferase pi (GST pi), glutathione peroxidase (GPX) and heme oxygenase mRNAs were all increased. Accompanying these changes the enzyme activity of GST pi, GPX and gamma-glutamyl transpeptidase (gamma-GT) were also elevated. Interestingly, in a revertant cell line heme oxygenase overexpression approached wild-type levels. Intriguingly, similar changes in gene expression seen in the menadione-resistant cells were also observed in wild-type cells following transient oxidative stress, indicating that the observed changes in the resistant line may be due to the immortalization of a normally transient adaptive stress response.

Transient Adaptation to Oxidative Stress in Yeast

Adaptive responses to the oxidative stress of hydrogen peroxide (H 2O 2) were studied in the yeast Saccharomyces cerevisciae strain RZ53. Our results show that the growth of naive cells is readily arrested by H 2O 2 challenge. In contrast, cells that have been preexposed to relatively low H 2O 2 priming treatments ( i.e., cells that have first been pretreated with low H 2O 2 concentrations) are able to survive a subsequent challenge dose and continue to divide at normal rates. The most effective adaptation was observed with the following conditions: 5 × 10 6 cells/ml at pretreatment, pretreatment or priming peroxide addition of 0.4 mM H 2O 2, interval between pretreatment and challenge of 45 min, challenge peroxide concentration of 0.8 mM H 2O 2 for 2 h. Under these conditions cells that were challenged without pretreatment exhibited a 90% loss of plating efficiency. In contrast, peroxide-pretreated cells grew and divided at rates that were actually 15-30% faster than those of nonpretreated cells, and some 90-100% of such pretreated cells continued to divide at normal rates even following exposure to the H 2O 2 challenge concentration. The increased H 2O 2 resistance of pretreated cells was transient, being readily reversed during 60-90 min of growth in the absence of H 2O 2. Furthermore, cells that were allowed to deadapt over a 4-h period again exhibited a transient adaptive response when reexposed to H 2O 2 pretreatment. These results, plus the high survival rates (90-100%) of H 2O 2 pretreated and challenged cells, demonstrates that our results represent a true transient adaptation, rather than a selection for any preexisting peroxide resistant subpopulation. H 2O 2 adaptation required protein synthesis as evidenced by studies with the translational inhibitor, cycloheximide. At least 21 proteins exhibited increased expression following H 2O 2 adaptation, while the expression of some 8 other proteins was decreased. Adaptation is now widely reported in bacterial strains and has also been observed in some mammalian cell lines. We propose that the basis for such adaptive responses rests in increased expression of genes that encode protective enzymes and repair enzymes.