Oxidative stress during diabetic pregnancy disrupts cardiac neural crest migration and causes outflow tract defects

BACKGROUND: Maternal diabetes increases risk for congenital malformations, particularly cardiac outflow tract defects. Maternal diabetes inhibits expression of Pax3 in neuroepithelium through hyperglycemia‐induced oxidative stress. The neuroepithelium gives rise to the neural crest, and Pax3 expression in cardiac neural crest (CNC) is required for CNC migration to the heart and for outflow tract septation. Here we tested whether maternal diabetes, through hyperglycemia‐induced oxidative stress, before the onset of CNC delamination, impairs CNC migration and cardiac outflow tract septation. METHODS: CNC migration was mapped in mouse embryos whose mothers were diabetic, or transiently hyperglycemic, or in which oxidative stress was transiently induced, using reporters linked to Pax3 expression. CNC apoptosis was examined by TUNEL assay. Outflow tract septation was examined histologically and by gross inspection. RESULTS: Few, if any, migrating CNC cells were observed in embryos of diabetic mice, and this was associated with increased apoptosis along the path of CNC migration. Outflow tract defects were significantly increased in fetuses of diabetic mice. Notably, induction of hyperglycemia or oxidative stress on the day prior to the onset of Pax3 expression and CNC migration also impaired CNC migration, increased apoptosis, and caused outflow tract defects. However, antioxidants administered on the day prior to the onset of Pax3 expression and CNC migration prevented these effects of hyperglycemia or oxidative stress. CONCLUSIONS: In diabetic pregnancy, oxidative stress, which inhibits expression of genes required for CNC viability, causes subsequent CNC depletion by apoptosis during migration, which leads to outflow tract defects. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Influence of acute heat stress on the development of GABAergic neurons in HPA-axies of mouse embryos

The damaging effects of acute heat stress in pregnant mice is well known, however, very little is known about the impact of heat on embryonic neurodevelopment and its dependence on the dam's physiological response to the stress. To study the changes in GABAergic neurons expression on the hypothalamo-pituitary-adrenocortical axis (HPA-axis), superoxide dismutase (SOD) activities and malondialdehyde (MDA) levels were measured in mouse embryos on E13-17 following acute, maternal heat stress. Blood samples and amniotic fluid from pregnant mice, and homogenates of whole embryos and embryo brains were collected for SOD and MDA analyses. SOD and MDA activities were measured in embryo and the sections by staining with anti-γ-aminobutyric acid-A- α1 receptor (GABAA receptor- α1), anti-γ-aminobutyric acid-B-1 receptor (GABAB receptor-R1) and anti-glutamate decarboxylase-65(GAD-65) antibodies. After the pregnant mice underwent acute heat stress on E13, the embryonic GABAergic neurons and GABA receptor expression were triggered immediately as a result of induction of the HPA-axis. This expression recovered to normal levels consistent with the control groups. However, the expression of the GABAA receptor in embryonic adrenal gland decreased continually. The SOD activity decreased in the embryonic brain and increased in the amniotic fluid after the heat stress, whereas the MDA levels increased in the maternal plasma only. Therefore, the GABAergic neurons in the developing HPA-axis of the embryos are susceptible to heat stress and the enhancement of SOD activities in the amniotic fluid might be a protective mechanism.     

Hyperglycemia induces oxidative stress and impairs axonal transport rates in mice

Background

While hyperglycemia-induced oxidative stress damages peripheral neurons, technical limitations have, in part, prevented in vivo studies to determine the effect of hyperglycemia on the neurons in the central nervous system (CNS). While olfactory dysfunction is indicated in diabetes, the effect of hyperglycemia on olfactory receptor neurons (ORNs) remains unknown. In this study, we utilized manganese enhanced MRI (MEMRI) to assess the impact of hyperglycemia on axonal transport rates in ORNs. We hypothesize that (i) hyperglycemia induces oxidative stress and is associated with reduced axonal transport rates in the ORNs and (ii) hyperglycemia-induced oxidative stress activates the p38 MAPK pathway in association with phosphorylation of tau protein leading to the axonal transport deficits.

Research Design and Methods

T₁-weighted MEMRI imaging was used to determine axonal transport rates post-streptozotocin injection in wildtype (WT) and superoxide dismutase 2 (SOD2) overexpressing C57Bl/6 mice. SOD2 overexpression reduces mitochondrial superoxide load. Dihydroethidium staining was used to quantify the reactive oxygen species (ROS), specifically, superoxide (SO). Protein and gene expression levels were determined using western blotting and Q-PCR analysis, respectively.

Results

STZ-treated WT mice exhibited significantly reduced axonal transport rates and significantly higher levels of ROS, phosphorylated p38 MAPK and tau protein as compared to the WT vehicle treated controls and STZ-treated SOD2 mice. The gene expression levels of p38 MAPK and tau remained unchanged.

Conclusion

Increased oxidative stress in STZ-treated WT hyperglycemic mice activates the p38 MAPK pathway in association with phosphorylation of tau and attenuates axonal transport rates in the olfactory system. In STZ-treated SOD-overexpressing hyperglycemic mice in which superoxide levels are reduced, these deficits are reversed.     

Role of gp91phox-containing NADPH oxidase in left ventricular remodeling induced by intermittent hypoxic stress

Intermittent hypoxia due to sleep apnea syndrome is associated with cardiovascular diseases. However, the precise mechanisms by which intermittent hypoxic stress accelerates cardiovascular diseases are largely unclear. The aim of this study was to investigate the role of gp91(phox)-containing NADPH oxidase in the development of left ventricular (LV) remodeling induced by intermittent hypoxic stress in mice. Male gp91(phox)-deficient (gp91(-/-)) mice (n = 26) and wild-type (n = 39) mice at 7-12 wk of age were exposed to intermittent hypoxia (30 s of 4.5-5.5% O(2) followed by 30 s of 21% O(2) for 8 h/day during daytime) or normoxia for 10 days. Mean blood pressure and LV systolic and diastolic function were not changed by intermittent hypoxia in wild-type or gp91(-/-) mice, although right ventricular systolic pressure tended to be increased. In wild-type mice, intermittent hypoxic stress significantly increased the diameter of cardiomyocytes and interstitial fibrosis in LV myocardium. Furthermore, intermittent hypoxic stress increased superoxide production, 4-hydroxy-2-nonenal protein, TNF-alpha and transforming growth factor-beta mRNA, and NF-kappaB binding activity in wild-type, but not gp91(-/-), mice. These results suggest that gp91(phox)-containing NADPH oxidase plays a crucial role in the pathophysiology of intermittent hypoxia-induced LV remodeling through an increase of oxidative stress.     

Activation of the hexosamine pathway causes oxidative stress and abnormal embryo gene expression: involvement in diabetic teratogenesis

BACKGROUND: Oxidative stress is critical to the teratogenic effects of diabetic pregnancy, yet the specific biochemical pathways responsible for oxidative stress have not been fully elucidated. The hexosamine pathway is activated in many tissues during diabetes and could contribute to oxidative stress by inhibiting the pentose shunt pathway, thereby diminishing production of the cellular antioxidant, reduced glutathione (GSH). METHODS: To test the hypothesis that activation of the hexosamine pathway might contribute to the teratogenic effects of diabetic pregnancy, pregnant mice were injected with glucose, to induce hyperglycemia, or glucosamine, to directly activate the hexosamine pathway. Embryo tissue fragments were also cultured in physiological glucose, high glucose, or physiological glucose plus glucosamine, to test effects on oxidative stress and embryo gene expression. RESULTS: Glucosamine increased hexosamine synthesis and inhibited pentose shunt activity. There was a trend for transient hyperglycemia to have the same effects, but they did not reach statistical significance. However, both glucose and glucosamine significantly decreased GSH, and increased oxidative stress, as indicated by 2',7'-dichloro-dihydrofluorescein fluorescence. Glucose and glucosamine inhibited expression of Pax-3, a gene required for neural tube closure both in vivo and in vitro, and increased neural tube defects (NTDs) in vivo; these effects were prevented by GSH ethyl ester. High glucose and glucosamine inhibited Pax-3 expression by embryo culture, but culture in glutamine-free media to block the hexosamine pathway prevented the inhibition of Pax-3 expression by high glucose. CONCLUSIONS: Activation of the hexosamine pathway causes oxidative stress through depletion of GSH and consequent disruption of embryo gene expression. Activation of this pathway may contribute to diabetic teratogenesis.     

Tertiary butylhydroquinone alleviates gestational diabetes mellitus in C57BL/KsJ-Lep db/+ mice by suppression of oxidative stress

Gestational diabetes mellitus (GDM) is a common disorder characterized by abnormal glucose metabolism during pregnancy, affecting 2% to 5% of pregnant women. Currently, clinical treatment for GDM is very limited. The present study was designed to investigate the effect and underlying molecular mechanism of tertiary butylhydroquinone (TBHQ) in a pregnant C57BL/KsJ-Lep db/+ (referred to as db+) GDM mouse model. The results showed that nonpregnant db/+ mice did not show a diabetic phenotype, and TBHQ had no effect on glucose and insulin tolerance in these mice. Moreover, in db/+ pregnant mice exhibiting typical diabetes symptoms, such as hyperglycemia and hypoinsulinemia, TBHQ could remarkably decrease the blood glucose level, increase insulin level, and improve glucose and insulin intolerance. The results also revealed that TBHQ could inhibit oxidative stress in pregnant db/+ mice. Furthermore, TBHQ greatly improved offspring survival rate, glucose metabolism, and insulin tolerance. In addition, TBHQ inhibited oxidative stress by reducing malondialdehyde (MDA) and reactive oxygen species (ROS) levels and increased superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities. Moreover, we found that TBHQ activated the nuclear factor erythroid 2-related factor 2 (Nrf2), thereby increasing the levels of Nrf2, and ultimately upregulating the expression of heme oxygenase 1 (NO-1) and superoxide dismutase 2 (SOD2). In conclusion, our findings demonstrated that TBHQ alleviated GDM via Nrf2 activation.     

Diabetes in the Cohen Rat Intensifies the Fetal Pancreatic Damage Induced by the Diabetogenic High Sucrose Low Copper Diet

Intrauterine hyperglycemic environment could harm the fetus making it more susceptible to develop postnatal glucose intolerance. A possible mechanism is compromise of the fetal pancreatic development. We previously found that a high sucrose low copper diabetogenic diet induces type 2 diabetes in the Cohen diabetic sensitive rats, but not in the Sabra control rats. However, oxidative stress was observed in the placenta and term fetal liver of diabetic and nondiabetic controls. We now investigated whether the fetal pancreas is affected by this diet and whether the effects result from oxidative stress, maternal hyperglycemia, or both. Term fetal pancreases were evaluated for morphology, beta cells, oxidative stress, apoptosis, and DNA methylation. There were no microscopic changes in hematoxylin and eosin stained sections and beta cells immunostaining in the pancreas of fetuses of both strains. Fetuses of the sensitive strain fed diabetogenic diet had significantly higher activity of superoxide dismutase and catalase, elevated levels of low molecular weight antioxidants, and more intense immunostaining for nuclear factor kappa‐B and hypoxia inducing factor‐1α. Both strains fed diabetogenic diet had increased immunostaining for Bcl‐2‐like protein and caspase 3 and decreased immunostaining for 5‐methylcytosine in their islets and acini. Our data suggest that maternal diabetogenic diet alters apoptotic rate and epigenetic steady states in the term fetal pancreas, unrelated to maternal diabetes. Maternal hyperglycemia further increases pancreatic oxidative stress, aggravating the pancreatic damage. The diet‐induced insults to the fetal pancreas may be an important contributor to the high susceptibility to develop diabetes following metabolic intrauterine insults     

Hypoxic preconditioning increases survival and angiogenic potency of peripheral blood mononuclear cells via oxidative stress resistance

Cell-based angiogenesis is a promising treatment for ischemic diseases; however, the survival of implanted cells is impaired by oxidative stress in the ischemic microenvironment. We tested the hypothesis that hypoxic preconditioning of implanted cells enhances their resistance against oxidative stress, increasing cell survival and angiogenic potency after implantation into ischemic tissue. Mouse peripheral blood mononuclear cells (PBMNCs) were collected and subjected to hypoxic preconditioning by culture for 24 h in 2% O(2) at 33 degrees C. Hypoxic preconditioning of PBMNCs increased the expression of various genes related to antioxidant and survival signals remarkably. Compared with cells cultured under normoxia, the hypoxia-preconditioned PBMNCs showed significantly lower reactive oxygen species (ROS) accumulation and higher cell survival under oxidative stress induced by LY-83583 (a superoxide generator). Three days after intramuscular implantation into the ischemic hindlimbs of mice, survival of the hypoxia-preconditioned PBMNCs was high, whereas that of the normoxia-cultured PBMNCs was relatively low. Furthermore, 28 days after treatment microvessel density and blood flow in the ischemic hindlimbs were significantly better in the mice implanted with hypoxia-preconditioned PBMNCs than in those implanted with normoxia-cultured PBMNCs. Hypoxic preconditioning increased the survival and angiogenic potency of PBMNCs, through oxidative stress resistance mechanisms.     

Evaluation of mouse preimplantation embryos exposed to oxidative stress cultured with insulin-like growth factor I and II, epidermal growth factor, insulin, transferrin and selenium

Blastocyst culture requires strictly defined culture media to sustain its viability and quality. Although blastocyst media are commercially available, they do not meet all the needs and research focused on blastocyst-promoting agents is on the way. The aims of the study were to evaluate the significance of insulin-like growth factors I (IGF-I) and II (IGF-II); epidermal growth factor (EGF) and a mixture of insulin, transferrin and selenium (ITS) on the development of embryos exposed to oxidative stress. C3B6F1 mice were stimulated with 5 IU of pregnant mare serum gonadotropin following by administration of 5 IU of equine chorionic gonadotropin and mating with DBA males. The mice were killed 40 h after eCG injection by cervical dislocation and then the 2 cell embryos were flushed out from the fallopian tubes. To evaluate whether the growth factors may compensate the unfavorable--oxidative milieu created by hydrogen peroxide (H2O2), the embryos were transferred to 1/ control medium, 2/ control medium+0.1 mM (H2O2) or 3/ control medium+H2O2 enriched with 10(-7) g/ml of IGF-I, IGF-II, EGF or a mixture of insulin (5x10(-6) g/ml), transferrin (5 x10(-6) g/ml) and selenium (5x10(-9) g/ml; ITS). Embryos were evaluated 96-144 hours following eCG injection. In the study the dynamics of embryo development and blastocyst cell numbers (including inner cell mass) were assessed. The morphological evaluation comprised viability and apoptosis (TUNEL). In oxidative stress setting, IGF-I, IGF-II, EGF and ITS minimized the negative influence of H2O2, and embryos developed faster than in control conditions. Blastocysts cultured with hydrogen peroxide and growth factors or ITS displayed normal morphology and had more cells--also within the inner cell mass--than those treated only with H2O2. The positive TUNEL reactions were sporadically observed in embryos cultured with hydrogen peroxide supplemented with growth factors. IGF-I, IGF-II, EGF and ITS have a positive effect on pre-implantation embryo development in detrimental culture conditions of oxidative stress.     

Developmental programming of cardiovascular dysfunction by prenatal hypoxia and oxidative stress

Fetal hypoxia is a common complication of pregnancy. It has been shown to programme cardiac and endothelial dysfunction in the offspring in adult life. However, the mechanisms via which this occurs remain elusive, precluding the identification of potential therapy. Using an integrative approach at the isolated organ, cellular and molecular levels, we tested the hypothesis that oxidative stress in the fetal heart and vasculature underlies the molecular basis via which prenatal hypoxia programmes cardiovascular dysfunction in later life. In a longitudinal study, the effects of maternal treatment of hypoxic (13% O(2)) pregnancy with an antioxidant on the cardiovascular system of the offspring at the end of gestation and at adulthood were studied. On day 6 of pregnancy, rats (n = 20 per group) were exposed to normoxia or hypoxia ± vitamin C. At gestational day 20, tissues were collected from 1 male fetus per litter per group (n = 10). The remaining 10 litters per group were allowed to deliver. At 4 months, tissues from 1 male adult offspring per litter per group were either perfusion fixed, frozen, or dissected for isolated organ preparations. In the fetus, hypoxic pregnancy promoted aortic thickening with enhanced nitrotyrosine staining and an increase in cardiac HSP70 expression. By adulthood, offspring of hypoxic pregnancy had markedly impaired NO-dependent relaxation in femoral resistance arteries, and increased myocardial contractility with sympathetic dominance. Maternal vitamin C prevented these effects in fetal and adult offspring of hypoxic pregnancy. The data offer insight to mechanism and thereby possible targets for intervention against developmental origins of cardiac and peripheral vascular dysfunction in offspring of risky pregnancy.     

Hypoxia. 1. Intracellular sensors for oxygen and oxidative stress: novel therapeutic targets

A variety of human disorders, e.g., ischemic heart disease, stroke, kidney disease, eventually share the deleterious consequences of a common, hypoxic and oxidative stress pathway. In this review, we utilize recent information on the cellular defense mechanisms against hypoxia and oxidative stress with the hope to propose new therapeutic tools. The hypoxia-inducible factor (HIF) is a key player as it activates a broad range of genes protecting cells against hypoxia. Its level is determined by its degradation rate by intracellular oxygen sensors prolyl hydroxylases (PHDs). There are three different PHD isoforms (PHD1-3). Small molecule PHD inhibitors improve hypoxic injury in experimental animals but, unfortunately, may induce adverse effects associated with PHD2 inhibition, e.g., angiogenesis. As yet, no inhibitor specific for a distinct PHD isoform is currently available. Still, the specific disruption of the PHD1 gene is known to induce hypoxic tolerance, without angiogenesis and erythrocytosis, by reprogramming basal oxygen metabolism with an attendant decreased oxidative stress in hypoxic mitochondria. A specific PHD1 inhibitor might therefore offer a novel therapy against hypoxia. The nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulates the basal and inducible expression of numerous antioxidant stress genes. Disruption of its gene exacerbates oxidative tissue injury. Nrf2 activity is modulated by Kelch-like ECH-associated protein 1 (Keap1), an intracellular sensor for oxidative stress. Inhibitors of Keap 1 may prove therapeutic against oxidative tissue injury.     

Oxidative stress and diabetes in pregnant rats

A considerable amount of clinical and experimental evidence now exists suggesting the involvement of free radical-mediated oxidative processes in the pathogenesis of diabetic complications. If the diabetic state is associated with a generalized increase in oxidative stress, it might well be reflected in the alterations in embryonic and fetal development during pregnancy. In the present study, incidence of the malformed fetuses, biochemical parameters and antioxidant system activity of streptozotocin (STZ)-induced diabetic pregnant rats was investigated and the results obtained were compared with those of the control group (non-diabetic). Virgin female Wistar rats were injected with 40 mg/kg streptozotocin (STZ) before mating. All the females were killed on Day 21 of pregnancy and the fetuses were analyzed. A maternal blood sample was collected by venous puncture and the maternal liver was removed for biochemical measurement. The diabetic dams presented hyperglycemia, hyperlipemia, hypertriglyceridemia, hypercholesterolemia, hyperuricemia, decreased reduced glutathione (GSH), hepatic glycogen and superoxide dismutase (SOD) determinations. There was an increased incidence of skeletal and visceral malformation in fetuses from diabetic rats. Our findings suggest that oxidative stress occurs in the diabetic pregnant state, which might promote maternal homeostasis alterations. These diabetic complications might be a contributory factor to conceptus damage causing embryonic death (abortion/miscarriage) or the appearance of malformations in the fetuses of diabetic dams. Antioxidant treatment of women with diabetes may be important in future attempts to prevent congenital malformations.     

Decreased glucose transporter expression triggers BAX-dependent apoptosis in the murine blastocyst

We report that a decrease in facilitative glucose transporter (GLUT1) expression and reduced glucose transport trigger apoptosis in the murine blastocyst. Inhibition of GLUT1 expression either by high glucose conditions or with antisense oligodeoxynucleotides significantly lowers protein expression and function of GLUT1 and as a result induces a high rate of apoptosis at the blastocyst stage. Similar to wild-type mice, embryos from streptozotocin-induced diabetic Bax -/- mice experienced a significant decrease in glucose transport compared with embryos from non-diabetic Bax -/- mice. However, despite this decrease, these blastocysts demonstrate significantly fewer apoptotic nuclei as compared with blastocysts from hyperglycemic wild-type mice. This decrease in preimplantation apoptosis correlates with a decrease in resorptions and malformations among the infants of the hyperglycemic Bax -/- mice versus the Bax +/+ and +/- mice. These findings suggest that hyperglycemia by decreasing glucose transport acts as a cell death signal to trigger a BAX-dependent apoptotic cascade in the murine blastocyst. This work also supports the hypothesis that increased apoptosis at a blastocyst stage because of maternal hyperglycemia may result in loss of key progenitor cells and manifest as a resorption or malformation, two adverse pregnancy outcomes more common in diabetic women.     

Promoting effect of beta-mercaptoethanol on in vitro development under oxidative stress and cystine uptake of bovine embryos

The effects of beta-mercaptoethanol (beta-ME) on in vitro development under oxidative stress and cystine uptake of bovine embryos were investigated. Bovine 1-cell embryos obtained by in vitro fertilization were cultured in TCM-199 or synthetic oviductal fluid (SOF) in 20% O(2) supplemented with beta-ME. Addition of beta-ME significantly (P < 0.01) promoted embryo development when cultured in both TCM-199 and SOF under high levels of O(2), to almost the same rates when they were cultured in 5% O(2). To investigate whether the growth-promoting effect of beta-ME was related to cystine uptake, which is an important amino acid for intracellular glutathione (GSH) synthesis, 1-cell, 8-cell, morula, and blastocyst stage embryos were incubated in cystine, cysteine-free TCM-199 containing radioisotope-labeled cystine supplemented with or without beta-ME. It was found that cystine uptake was consistently low in each embryo stage incubated without beta-ME. In contrast, addition of beta-ME significantly (P < 0.05 to 0.0001) promoted cystine uptake in each stage of embryo development. This increase of cystine uptake by beta-ME was significantly inhibited by supplementation of buthionine sulfoximine, a specific inhibitor of GSH biosynthesis (P < 0.0001). High-performance liquid chromatography (HPLC) analysis clearly revealed a decrease of cystine in culture medium after supplementation by beta-ME, thereby forming another peak. HPLC analysis also showed the incorporated cystine by supplementation of beta-ME was possibly metabolized for GSH synthesis in the embryos. These results indicate that beta-ME has a protective effect in embryo development against oxidative stress and that the effect of beta-ME is associated with the promotion of cystine uptake of low availability in embryos.     

Maternal voluntary exercise mitigates oxidative stress and incidence of congenital heart defects in pre‐gestational diabetes

Women with pre‐gestational diabetes have a higher risk of producing children with congenital heart defects (CHDs), caused predominantly by hyperglycemia‐induced oxidative stress. In this study, we evaluated if exercise during pregnancy could mitigate oxidative stress and reduce the incidence of CHDs in the offspring of diabetic mice. Female mice were treated with streptozotocin to induce pre‐gestational diabetes, then mated with healthy males to produce offspring. They were also given access to running wheels 1 week before mating and allowed to exercise voluntarily until E18.5. Heart morphology, gene expression, and oxidative stress were assessed in foetal hearts. Maternal voluntary exercise results in a significantly lower incidence of CHDs from 59.5% to 25%. Additionally, diabetes‐induced defects in coronary artery and capillary morphogenesis were also lower with exercise. Myocardial cell proliferation and epithelial‐mesenchymal transition at E12.5 was significantly lower with pre‐gestational diabetes which was mitigated with maternal exercise. Cardiac gene expression of Notch1, Snail1, Gata4 and Cyclin D1 was significantly higher in the embryos of diabetic mice that exercised compared to the non‐exercised group. Furthermore, maternal exercise produced lower reactive oxygen species (ROS) and oxidative stress in the foetal heart. In conclusion, maternal exercise mitigates ROS and oxidative damage in the foetal heart, and results in a lower incidence of CHDs in the offspring of pre‐gestational diabetes. Exercise may be an effective intervention to compliment clinical management and further minimize CHD risk in mothers with diabetes.     

Increased expression of stress proteins in the surf clam Donax variabilis following hydrogen sulfide exposure

Endogenous free radical production and resulting oxidative damage may result from exposure to hypoxia, hyperoxia, or hydrogen sulfide. Previous investigations of sulfide-induced oxidative damage have produced conflicting results, perhaps because these studies utilized species presumably adapted to sulfide. We examined the effects of sulfide, hypoxia and hyperoxia on the surf clam Donax variabilis to test whether these stressors induce a cellular response to oxidative stress. These clams inhabit high-energy sandy beaches and are unlikely to have specific adaptations to these stressors. In duplicate flow-through experiments performed in fall and spring, clams were exposed to normoxia (22 kPa P(O(2))), hypoxia (10 kPa), hyperoxia (37 kPa), or sulfide with normoxia ( approximately 100 mumol L(-1), 22 kPa respectively) for 24 h. We quantified whole-animal expression of three antioxidants (Cu/Zn and Mn superoxide dismutases, glutathione peroxidase), a lipid peroxidation marker (4-hydroxy-2E-nonenol-adducted protein), a DNA repair enzyme (OGG1-m), four heat shock proteins (small Hsp, Hsp60, Hsp70, and mitochondrial Hsp70), ubiquitin, and actin. Clams exposed to sulfide showed upregulation of the greatest number of stress proteins and the pattern was consistent with a cellular response to oxidative stress. Furthermore, there was a marked seasonality, with greater stress protein expression in clams from the spring.