Oxidative stress and its effects during dehydration

Water is usually thought to be required for the living state, but several organisms are capable of surviving complete dehydration (anhydrobiotes). Elucidation of the mechanisms of tolerance against dehydration may lead to development of new methods for preserving biological materials that do not normally support drying, which is of enormous practical importance in industry, in clinical medicine as well as in agriculture. One of the molecular mechanisms of damage leading to death in desiccation-sensitive cells upon drying is free-radical attack to phospholipids, DNA and proteins. This review aims to summarize the strategies used by anhydrobiotes to cope with the danger of oxygen toxicity and to present our recent results about the importance of some antioxidant defense systems in the dehydration tolerance of Saccharomyces cerevisiae, a usual model in the study of stress response.     

Cutaneous interstitial nitric oxide concentration does not increase during heat stress in humans.

Inhibition of cutaneous nitric oxide (NO) synthase reduces the magnitude of cutaneous vasodilation during whole body heating in humans. However, this observation is insufficient to conclude that NO concentration increases in the skin during a heat stress. This study was designed to test the hypothesis that whole body heating increases cutaneous interstitial NO concentration. This was accomplished by placing 2 microdialysis membranes in the forearm dermal space of 12 subjects. Both membranes were perfused with lactated Ringer solutions at a rate of 2 microl/min. In both normothermia and during whole body heating via a water perfused suit, dialysate from these membranes were obtained and analyzed for NO using the chemiluminescence technique. In six of these subjects, after the heat stress, the membranes were perfused with a 1 M solution of acetylcholine to stimulate NO release. Dialysate from these trials was also assayed to quantify cutaneous interstitial NO concentration. Whole body heating increased skin temperature from 34.6 +/- 0.2 to 38.8 +/- 0.2 degrees C (P < 0.05), which increased sublingual temperature (36.4 +/- 0.1 to 37.6 +/- 0.1 degrees C; P < 0.05), heart rate (63 +/- 5 to 93 +/- 5 beats/min; P < 0.05), and skin blood flow over the membranes (21 +/- 4 to 88 +/- 10 perfusion units; P < 0.05). NO concentration in the dialysate did not increase significantly during of the heat stress (7.6 +/- 0.7 to 8.6 +/- 0.8 microM; P > 0.05). After the heat stress, administration of acetylcholine in the perfusate significantly increased skin blood flow (128 +/- 6 perfusion units) relative to both normothermic and heat stress values and significantly increased NO concentration in the dialysate (15.8 +/- 2.4 microM). These data suggest that whole body heating does not increase cutaneous interstitial NO concentration in forearm skin. Rather, NO may serve in a permissive role in facilitating the effects of an unknown neurotransmitter, leading to cutaneous vasodilation during a heat stress.     

Fluid replacement following dehydration reduces oxidative stress during recovery

To investigate the effects of hydration status on oxidative DNA damage and exercise performance, 10 subjects ran on a treadmill until exhaustion at 80% VO_2max during four different trials [control (C), 3% dehydration (D), 3% dehydration + water (W) or 3% dehydration + sports drink (S)]. Dehydration significantly decreased exercise time to exhaustion (D < C and S). Plasma MDA levels were significantly higher at pre-exercise in D than C. Plasma TAS was significantly lower at pre-exercise in C and S than in D, and was significantly lower in S than D at 60 min of recovery. Dehydration significantly increased oxidative DNA damage during exercise, but fluid replacement with water or sports drink alleviated it equally. These results suggest that (1) dehydration impairs exercise performance and increases DNA damage during exercise to exhaustion; and (2) fluid replacement prolongs exercise endurance and attenuates DNA damage.     

Regional hemodynamics during postexercise hypotension. II. Cutaneous circulation.

After an acute bout of exercise, there is an unexplained elevation in systemic vascular conductance that is not completely offset by an increase in cardiac output, resulting in a postexercise hypotension. The contributions of the splanchnic and renal circulations are examined in a companion paper (Pricher MP, Holowatz LA, Williams JT, Lockwood JM, and Halliwill JR. J Appl Physiol 97: 2065-2070, 2004). The purpose of this study was to determine the contribution of the cutaneous circulation in postexercise hypotension under thermoneutral conditions (approximately 23 degrees C). Arterial blood pressure was measured via an automated sphygmomanometer, internal temperature was measured via an ingestible pill, and skin temperature was measured with eight thermocouples. Red blood cell flux (laser-Doppler flowmetry) was monitored at four skin sites (chest, forearm, thigh, and leg), and cutaneous vascular conductance (CVC) was calculated (red blood cell flux/mean arterial pressure) and scaled as percent maximal CVC (local heating to 43 degrees C). Ten subjects [6 men and 4 women; age 23 +/- 1 yr; peak O(2) uptake (Vo(2 peak)) 45.8 +/- 2.0 ml.kg(-1).min(-1)] volunteered for this study. After supine rest (30 min), subjects exercised on a bicycle ergometer for 1 h at 60% of their Vo(2 peak) and were then positioned supine for 90 min. Exercise elicited a postexercise hypotension reaching a nadir at 46.0 +/- 4.5 min postexercise (77 +/- 1 vs. 82 +/- 2 mmHg preexercise; P < 0.05). Internal temperature increased (38.0 +/- 0.1 vs. 36.7 +/- 0.1 degrees C preexercise; P < 0.05), remaining elevated at 90 min postexercise (36.9 +/- 0.1 degrees C vs. preexercise; P < 0.05). CVC at all four skin sites was elevated by the exercise bout (P < 0.05), returning to preexercise values within 50 min postexercise (P > 0.05). Therefore, although transient changes in CVC occur postexercise, they do not appear to play an obligatory role in mediating postexercise hypotension under thermoneutral conditions.     

Liposomes destabilize tRNA during heat stress

Biomembranes play an important role in cellular response to heat stress. In this study, we focus on the interaction between liposomes and tRNA. Upon heat treatment we determined circular dichroism spectra of tRNA in presence of liposomes prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and cholesterol (Ch). To compare thermal stability, midpoint temperature (T_m) of tRNA was calculated from normalized θ₂₀₈. Addition of POPC/Ch liposomes decreased the T_m value of tRNA from 48°C to 38°C. We conclude that POPC/Ch liposomes interact with tRNA and destabilize its conformation under heat stress.     

Metabolism during starvation/dehydration stress in two species of galagos

Two species of galagos (G. senegalensis moholi andG. garnettii) were subjected to dehydration and starvation stress in order to determine whether, as is common in other animals, these hypometabolic prosimians would lower their metabolic rate even further. Dehydration was confirmed by losses in body mass, a decrease in fecal water content and a rise in urine osmolality. At the height of dehydration, 20 to 25% reduction in body mass, 30 to 40% reduction in fecal water content and urine osmolality ranging from 1.8 to 3.5 Osmol kg⁻¹ H₂O, were recorded in some of the animals. Basal metabolic rate of 0.536 ml O₂ (g·h)⁻¹ inG. s. moholi and 0.302 ml O₂ (g·h)⁻¹ inG. garnettii were recorded, representing 50 to 42% reduction in metabolic rate, respectively, compared with mass specific values. In none of the tested animals did we observe significant reduction in basal metabolism during dehydration/starvation stress compared with the rates observed during the control period. Basal metabolism in the bushbabies seems to have reached the lowest level and no further adjustment is apparently possible as a strategy for energy saving during starvation and/or dehydration stress.     

CHIP interacts with heat shock factor 1 during heat stress

Heat shock factor 1 (HSF1) is a major transactivator of heat shock genes in response to stress and mediates cell protection against various harmful conditions. In this study, we identified the interaction of CHIP (carboxyl terminus of the heat shock cognate protein 70-interacting protein) with the N-terminus of HSF1. Using GST full-down assay, we found that CHIP directly interacts with C-terminal deleted HSF1 (a.a. 1-290) but not with full-length HSF1 under non-stressed conditions. Interestingly, interaction of CHIP with full-length HSF1 was induced by heat shock treatment. The structural change of HSF1 was observed under heat stressed conditions by CD spectra. These observations demonstrate the direct interaction between HSF1 and CHIP and this interaction requires conformational change of HSF1 by heat stress.     

Relationship of dehydration rate to drought avoidance,dehydration tolerance and dehydration avoidance of cabbage leaves,and to their acclimation during drought-induced water stress*

Abstract. Drought avoidance due to cuticular control increases with leaf number to a maximum in the intermediate leaves, decreasing to a minimum in the upper leaves. Dehydrated intermediate leaves do not rehydrate detectably when floated on water for several days. Excision of their petioles when submerged, permits full rehydration, presumably via the xylem. Stressing the plant by withholding water for 1–3 weeks fails to increase this already high resistance to water movement through the leaf surface. It does, however, markedly decrease the loss of water from the fully rehydrated (100% RWC) leaves during the first hour of dehydration, presumably due to a more rapid stomatal closure than in the non-stressed leaves. Dehydration tolerance increases with leaf number, without an intermediate maximum. The intermediate and upper leaves were markedly more tolerant of dehydration after drought-induced stress than when non-stressed. Dehydration tolerance in some cases, was inversely proportional to dehydration rate. It was possible, however, to equalize the rates of dehydration of drought-stressed and non-drought-stressed leaves without affecting the greater tolerance of the drought-stressed leaves. Dehydration avoidance by osmotic adjustment was markedly developed in the slowly dehydrated attached leaves of drought-stressed plants, but not in the rapidly dehydrated excised leaves. This is evidence of drought acclimation. It must, therefore, be concluded that the slow dehydration of the drought-stressed plants also leads to the increase in dehydration tolerance by permitting drought-induced acclimation. The overall drought resistance of cabbage leaves depends on the three components: drought avoidance, dehydration avoidance and dehydration tolerance. The latter two increase during acclimation but the cuticular control of drought avoidance does not.     

Effects of dehydration and heat stress on food intake and dry matter digestibility in East African ruminants

Comparative investigations were made between wild and domestic ruminants from arid and semi-arid regions and those species from non-arid areas in an attempt to evaluate the adaptations of these ruminants in terms of the effects of heat stress and dehydration on food intake and digestibility. The effect of (a) an intermittent heat load (a daily light cycle of 12 h at 22 degrees C and 12 h at 40 degrees C) compared to 22 degrees C throughout the day and (b) dehydration level of 15% weight loss, with and without the heat load, on the intake and digestibility of a poor quality hay was investigated in the Grant's gazelle, Oryx, the domestic Turkana goats, fat-tailed sheep, zebu cattle, Thomson's gazelle and wildebeest. The intermittent heat load with water available ad libitum depressed the food intake of zebu cattle and Turkana goats by more than 40%. It had no significant effect on the food intake of the other species. The Thomson's and Grants gazelle, oryx, wildebeest and fat-tailed sheep appear well adapted to withstanding a periodic heat load. Dehydration at 22 degrees C caused a marked depression on food intake of all the species investigated. Dehydration together with a heat load caused no further reduction in the food intake by the Grants's gazelle, oryx, and goats but it did cause a further reduction in the intake in the other species. The small non-domestic ruminants (i.e. Grant's and Thomson's gazelle) appear much more digestive efficient than any of their domestic counterpart.     

Serum electrolyte and enzyme responses to heat stress and dehydration in the fowl (Gallus domesticus)

1. Serum electrolytes, enzymes and various metabolites were determined in the hyperthermic and dehydrated fowl. 2. In normally-hydrated fowls, heat stress did not significantly affect blood constituents. 3. Water deprivation for 48 hr (dehydration) significantly (p less than 0.05) increased Na+, osmolality, SGPT and T3-retention. 4. During hyperthermic dehydration, Na+, Cl-, osmolality (p less than 0.01), BUN, glucose, T3-retention (p less than 0.02) and uric acid (p less than 0.001) significantly increased. 5. The present findings are consistent with the suggestion that changes in Na+/Ca2+ ratio might raise the hypothalamic thermoregulatory set-point and support our previous findings that acclimated fowls could efficiently regulate body temperature and acid-base status while avoiding extreme metabolic and enzymatic changes during heat exposure and dehydration.     

Plasma volume during heat stress and exercise in women

Five women were studied during exercise and passive heating to determine whether PV dynamics were affected by the menstrual cycle. The exercise bout (80% VO2 peak) on a modified cycle ergometer and the passive heat stress were done in a hot environment (Ta = 50 degrees C, Pw = 1.61 kPa) during the follicular and luteal phase. Esophageal temperature (Tes) was measured continuously. Blood samples were drawn after each 0.2 degree C increase in Tes and VO2 was measured at that time. Initial PV was estimated at rest during the follicular phase. PV changes from rest were calculated at each Tes from Hb and Hct. During passive heating, PV decreased by a mean volume of 156 (+/- 80) ml to 2.83 (+/- 0.09) l in the follicular phase. During the luteal phase, there was a larger volume reduction (300 +/- 100 ml) during passive heating, and the final PV was lower than in the follicular phase and averaged 2.47 +/- 0.18 l. During exercise, PV decreased 463 (+/- 90) ml to 2.50 (+/- 0.11) l in the follicular and 381 (+/- 70) ml to 2.50 (+/- 0.23) l in the luteal phase. These data indicate that there is a menstrual cycle effect on PV dynamics during passive heating such that more fluid is shifted out of the vasculature during the luteal phase. During severe exercise there is a greater fluid loss during the follicular phase, yet the final PV is not different between phases.     

Transient sequestration of TORC1 into stress granules during heat stress

The target of rapamycin complex 1 (TORC1) is a central kinase that coordinates nutrient availability with eukaryotic cell growth. Although TORC1 signaling is repressed by various stresses in yeast, the underlying mechanisms remain elusive. Here we report that TORC1 signaling upon heat stress is regulated by stress granules (SGs), which are cytoplasmic foci formed under certain stresses. Ectopic formation of SGs achieved by Pbp1 overexpression in unstressed cells sequesters TORC1 in this compartment, thereby blunting TORC1 signaling. Upon heat stress, a physiological SG-inducing condition, TORC1 is also recruited to SGs, which delays reactivation of TORC1 signaling during recovery from heat stress. Moreover, TORC1 reactivation is directed through SG disassembly, suggesting that SGs act as a key determinant for TORC1 reactivation during recovery from heat stress. Furthermore, this mechanism contributes to reduction of heat-induced mutations. Thus, TORC1 signaling is coupled to heat-induced SGs to protect cells from DNA damage.     

C-fos expression in rat brain during heat stress

Rats, under urethane anesthesia, 0, 20, 40 or 80 min after the start of heat stress (42°C) were sacrificed for determination of c-fos expression in different brain regions. In situ hybridization and immunocytochemistry methods were used, respectively, for determination of c-fos mRNA and protein, respectively. In general, either colon temperature (T_CO), mean arterial pressure (MAP), local cerebral blood flow (CBF) or c-fos expression in different brain regions (including the preoptic area, supraoptic nuclei, paraventricular nuclei, thalamus, amygdala, nucleus tract solitarii, area postrema and ventrolateral medulla) increased at 20–40 min after the start of heat exposure. However, the heatstroke, which appears as profound decreases in both MAP and local CBF and increases in T_CO, was produced 80 min after heat stress. The c-fos expression was heavily induced in all these brain regions after the onset of heatstroke. The data suggest that c-fos expression in rat brain during heatstroke is associated with hyperthermia, arterial hypotension or cerebral ischemia.     

Differential expression pattern of heat shock protein 70 gene in tissues and heat stress phenotypes in goats during peak heat stress period

It has been established that the synthesis of heat shock protein 70 (Hsp70) is temperature-dependent. The Hsp70 response is considered as a cellular thermometer in response to heat stress and other stimuli. The variation in Hsp70 gene expression has been positively correlated with thermotolerance in Drosophila melanogaster, Caenorhabditis elegans, rodents and human. Goats have a wide range of ecological adaptability due to their anatomical and physiological characteristics; however, the productivity of the individual declines during thermal stress. The present study was carried out to analyze the expression of heat shock proteins in different tissues and to contrast heat stress phenotypes in response to chronic heat stress. The investigation has been carried out in Jamunapari, Barbari, Jakhrana and Sirohi goats. These breeds differ in size, coat colour and production performance. The heat stress assessment in goats was carried out at a temperature humidity index (THI) ranging from 85.36–89.80 over the period. Phenotyping for heat stress susceptibility was carried out by combining respiration rate (RR) and heart rate (HR). Based on the distribution of RR and HR over the breeds in the population, individual animals were recognized as heat stress-susceptible (HSS) and heat stress-tolerant (HST). Based on their physiological responses, the selected animals were slaughtered for tissue collection during peak heat stress periods. The tissue samples from different organs such as liver, spleen, heart, testis, brain and lungs were collected and stored at ?70 °C for future use. Hsp70 concentrations were analyzed from tissue extract with ELISA. mRNA expression levels were evaluated using the SYBR green method. Kidney, liver and heart had 1.5–2.0-fold higher Hsp70 concentrations as compared to other organs in the tissue extracts. Similarly, the gene expression pattern of Hsp70 in different organs indicated that the liver, spleen, brain and kidney exhibited 5.94, 4.96, 5.29 and 2.63-fold higher expression than control. Liver and brain tissues showed the highest gene expression at mRNA levels as compared to kidney, spleen and heart. HST individuals had higher levels of mRNA level expression than HSS individuals in all breeds. The Sirohi breed showed the highest (6.3-fold) mRNA expression levels as compared to the other three breeds, indicating the better heat stress regulation activity in the breed.     

Protein expression during heat stress in thermo-intolerant and thermo-tolerant diatoms

Diatoms (Chrysophyta) are photosynthetic microorganisms that are abundant in the natural environment and often associated with specific habitat and water quality conditions. Their significance as bioindicators and as exploitable sources of fine chemicals makes them desirable candidates for the study of stress responses. The protein expression of a thermo-intolerant (Phaeodactylum tricornutum) and thermo-tolerant (Chaetoceros muelleri) diatom following exposure to elevated temperature was investigated using one- and two-dimensional gel electrophoresis and Western blot analysis. It was determined using SDS PAGE with ³⁵S-methionine labeled proteins and Western blot analysis using pea HSP70 antisera that higher temperatures and longer duration treatment were required to cause a noticeable stress response in C. muelleri compared to P. tricornutum. This may be explained by C. muelleri possessing higher amounts of constitutively expressed heat shock proteins, which allows these cells to rapidly adjust to temperature increases. Two-dimensional gel electrophoresis revealed that putative small heat shock proteins (smHSPs) may appear to play a role during heat stress in both diatoms, which is similar to the response in plants. SDS PAGE data are also presented characterizing the recovery of P. tricornutum after heat shock. These results suggest that there is a lag period between heat shock and stress protein synthesis in these thermo-intolerant cells. This supports the hypothesis that cells without higher amounts of constitutively expressed stress proteins have a greater sensitivity to increased temperature. Work is underway to identify particular stress proteins responsible for conveying thermo-tolerance and to determine if overexpression of these genes in thermo-intolerant diatoms affects their temperature sensitivity.