Hyperthermia induces injury to the intestinal mucosa in the mouse: evidence for an oxidative stress mechanism

Loss of the intestinal barrier is critical to the clinical course of heat illness, but the underlying mechanisms are still poorly understood. We tested the hypothesis that conditions characteristic of mild heatstroke in mice are associated with injury to the epithelial lining of the intestinal tract and comprise a critical component of barrier dysfunction. Anesthetized mice were gavaged with 4 kDa FITC-dextran (FD-4) and exposed to increasing core temperatures, briefly reaching 42.4°C, followed by 30 min recovery. Arterial samples were collected to measure FD-4 concentration in plasma (in vivo gastrointestinal permeability). The small intestines were then removed to measure histological evidence of injury. Hyperthermia resulted in a ≈2.5-fold elevation in plasma FD-4 and was always associated with significant histological evidence of injury to the epithelial lining compared with matched controls, particularly in the duodenum. When isolated intestinal segments from control animals were exposed to ≥41.5°C, marked increases in permeability were observed within 60 min. These changes were associated with release of lactate dehydrogenase, evidence of protein oxidation via carbonyl formation and histological damage. Coincubation with N-acetylcysteine protected in vitro permeability during hyperthermia and reduced histological damage and protein oxidation. Chelation of intracellular Ca(2+) to block tight junction opening during 41.5°C exposure failed to reduce the permeability of in vitro segments. The results demonstrate that hyperthermia exposure in mouse intestine, at temperatures at or below those necessary to induce mild heatstroke, cause rapid and substantial injury to the intestinal lining that may be attributed, in part, to oxidative stress.     

Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability

The effects of physiologically relevant increase in temperature (37-41 degrees C) on intestinal epithelial tight junction (TJ) barrier have not been previously studied. Additionally, the role of heat-shock proteins (HSPs) in the regulation of intestinal TJ barrier during heat stress remains unknown. Because heat-induced disturbance of intestinal TJ barrier could lead to endotoxemia and bacterial translocation during physiological thermal stress, the purpose of this study was to investigate the effects of modest, physiologically relevant increases in temperature (37-41 degrees C) on intestinal epithelial TJ barrier and to examine the protective role of HSPs on intestinal TJ barrier. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro intestinal epithelial model system to assess the effects of heat exposure on intestinal TJ barrier. Exposure of filter-grown Caco-2 monolayers to modest increases in temperatures (37-41 degrees C) resulted in a significant time- and temperature-dependent increases in Caco-2 TJ permeability. Exposure to modest heat (39 or 41 degrees C) resulted in rapid and sustained increases in HSP expression; and inhibition of HSP expression produced a marked increase in heat-induced increase in Caco-2 TJ permeability (P < 0.001). Heat exposure (41 degrees C) resulted in a compensatory increase in Caco-2 occludin protein expression and an increase in junctional localization. Inhibition of HSP expression prevented the compensatory upregulation of occludin protein expression and produced a marked disruption in junctional localization of occludin protein during heat stress. In conclusion, our findings demonstrate for the first time that a modest, physiologically relevant increase in temperature causes an increase in intestinal epithelial TJ permeability. Our data also show that HSPs play an important protective role in preventing the heat-induced disruption of intestinal TJ barrier and suggest that HSP mediated upregulation of occludin expression may be an important mechanism involved in the maintenance of intestinal epithelial TJ barrier function during heat stress.     

The effect of hyperthermia in vitro on vitality of rabbit preimplantation embryos

The aim of our study was to test the influence of short exposure (6 h) of preimplantation rabbit embryos to elevated temperatures (41.5 degrees C or 42.5 degrees C) in vitro on their developmental capacity. Fertilized eggs recovered from female oviducts at the pronuclear stage (19 hpc) were cultured at standard temperature (37.5 degrees C) until the morula stage (72 hpc). Afterwards, the embryos were divided into two groups, cultured for 6 h either at hyperthermic (41.5 degrees C or 42.5 degrees C) or standard temperature (control 37.5 degrees C), post-incubated overnight (16-20 h) at 37.5 degrees C and then evaluated for developmental stages, apoptosis (TUNEL), proliferation (cell number), actin cytoskeleton and presence of heat-shock proteins Hsp70. It was observed that hyperthermia at 41.5 degrees C did not alter progression of embryos to higher preimplantation stages (expanded and hatching/hatched blastocysts), rate of apoptosis, total cell number of blastocysts and structure of actin filament compared to 37.5 degrees C. Western-blotting revealed the presence of heat stress-induced 72 kDa fraction of Hsp70 proteins in granulosa cells (exposed to 41 degrees C) and embryos (exposed to 41.5 degrees C). Following the elevation of temperature to 42.5 degrees C embryo development was dramatically compromised. The embryos were arrested at the morula or early blastocyst stage, showed an increased rate of apoptosis and decreased total cell number compared to control. The structure of actin filaments in most of blastomeres was damaged and such blastomeres often contained apoptotic nuclei. In this group a presence of heat-stress-induced fraction of Hsp70 proteins had not been confirmed. This is the first report demonstrating a threshold of thermotolerance of rabbit preimplantation embryos to hyperthermic exposure in vitro. A detrimental effect of higher temperature on the embryo is probably associated with the loss of their ability to produce Hsp70 de novo, which leads to cytoskeleton alterations and enhanced apoptosis.     

Heat-induced thermal resistance and its relationship to lysosomal response.

Two separate effects of hyperthermia on mouse splenic lysosomes have been reported, dependent on the severity of the treatment. Heating to temperatures below 42.5 degrees C causes a transient increase in lysosomal acid phosphatase activity which can be correlated with the ability of moderate hyperthermia to potentiate X-ray damage. Heating to temperatures above 42.5 degrees C results in an immediate increase in lysosomal membrane permeability which may be involved in tissue necrosis. By giving a priming heat treatment at 41.8 degrees C, induced thermal resistance was demonstrated for the lysosomal membrane effect, but not for the enzyme activation. The degree of induced thermal resistance observed is similar to that reported for the cell-killing effect of heat on tissues in vivo and cells in vitro and occurs over a similar time course. The relevance of these results to the understanding of fractionated hyperthermia in cancer therapy is discussed.     

The differential response of the skin in young and old rats to a combination of X-rays and 'wet' or 'dry' hyperthermia

The left hind feet of groups of female rats aged 7, 14 and 52 weeks were irradiated at three dose levels of X-rays (20, 25 or 30 Gy). Hyperthermia (42.5 degrees C for 1 h) was carried out immediately following irradiation using either 'wet' or 'dry' heat, achieved by immersion in either water or fluorocarbon liquid. The results demonstrated that 'wet' heat produced a consistently greater enhancement of the irradiation damage than 'dry' heat. The thermal enhancement ratio for irradiation plus 'wet' heat was approximately 1.5 and for irradiation plus 'dry' heat it was in the range 1.17 to 1.39. Immersion of the feet in fluorocarbon liquid at 37 degrees C did not significantly modify the irradiation response of the skin. The lower thermal enhancement ratios obtained using immersion in fluorocarbon liquid at 42.5 degrees C are close to those obtained in large animal studies and also similar to the limited amount of data from clinical studies where microwave or ultrasound heating techniques were used. It has been demonstrated that there are large age-related differences in the response of the rat foot skin to irradiation alone. It has also been shown in the present study, using rats of the same age, that the response to irradiation plus hyperthermia was less age dependent. This finding may reflect the differing methods by which damage occurs in tissue after irradiation or hyperthermia.     

Reorganization of ZO-1 by sodium-dependent glucose transporter activation after heat stress in LLC-PK1 cells

Heat stress (HS) induces activation of high-affinity sodium-dependent glucose transporter (SGLT1) in porcine renal LLC-PK(1) cells. In this study, we investigated the roles of SGLT1 activation in reorganization of zonula occludens-1 (ZO-1), a cytosolic tight junction (TJ) protein, after HS. HS (42 degrees C, 3 h) caused decrease in transepithelial electrical resistance (TER). Subsequent incubation at 37 degrees C for 12 h increased TER above pre-HS level. The treatment of phloridzin, a potent SGLT1 inhibitor, or the replacement of glucose with a nonmetabolizable glucose analog blocked the recovery of TER and increased the transepithelial flux of FITC-dextran (4,000 Da). Immunofluorescent staining of ZO-1 showed that HS diffused ZO-1 from cell contact to cytosolic sites. Furthermore, the fraction of ZO-1 was distributed from the Triton X-100 insoluble to the Triton X-100 soluble pool. After incubation at 37 degrees C for 12 h, cell contact and ZO-1 extractability with Triton X-100 returned to pre-HS conditions, but the recovery was completely prevented by phloridzin. Tyrosine kinases activity was increased by HS that was inhibited by phloridzin. Genistein and CGP77675, tyrosine kinases inhibitors, blocked the recovery of TER and increased the transepithelial flux of FITC-dextran. Furthermore, these inhibitors prevented the recovery of cell contact and ZO-1 extractability with Triton X-100 as same as phloridzin. These findings suggested that the activation of SGLT1 reorganized ZO-1 mediated by elevation of tyrosine kinases activity after heat injury.     

Expression of thermotolerance following microinjection of glutathione disulfide

Asynchronous Chinese hamster ovary cells were microinjected with glutathione disulfide (GSSG). Successfully injected cells were scored by coinjecting FITC-dextran with GSSG, followed by fluorescent microscopy. After microinjection, cells were incubated for 2.5 h at 37 degrees C to permit thermotolerance development and then heated at 45 degrees C for 40 min. Cellular heat sensitivity was quantitated by counting the number of grains per cell after labeling heated cells with tritiated amino acids and processing for autoradiography. The data show that microinjection of GSSG induced thermotolerance which increased the number of grains per cell up to 500% of controls. Cells that were exposed to similar concentrations of GSSG in culture medium without microinjection or microinjected without GSSG did not develop thermotolerance.     

Response of murine bone marrow granulocyte-macrophage colony-forming units to hyperthermia in situ

A detailed understanding of how bone marrow stem cell progenitors are affected by heat is prerequisite to predicting how whole-body or regional hyperthermia protocols may affect bone marrow function. This investigation reports the reproductive integrity of murine tibial bone marrow granulocyte-macrophage colony-forming units (CFU-GM) after in situ hyperthermia. Heat was applied by water bath immersion of the leg of male BALB/c mice anesthetized with 90 mg/kg pentobarbital given subcutaneously. Tibial and rectal temperatures were monitored in representative animals by microthermocouples (tip diameter approximately 100 microns). By approximately 3 min after immersion of the limb, marrow temperature was within 0.3 degree C of water bath temperature (O'Hara et al., Int. J. Hyperthermia 5, 589-601, 1989) and was within 0.1 degree C by 5 min after immersion. The CFU-GM were cultured in "lung-conditioned" McCoy's 5A medium supplemented with 15% fetal calf serum and 0.3% Bacto agar. In situ heating of tibial marrow to exposure temperatures of 42, 42.5, 43, 44, and 45 degrees C gave D0's (+/- 95% CI) of 91 +/- 44, 44 +/- 27, 27 +/- 2.2, 16 +/- 6, and 7 +/- 4 min, respectively. Heating to 41.5 degrees C for up to 180 min did not result in cytotoxicity. Development of thermotolerance after approximately 100 min of heating was apparent by the presence of a "resistant tail" of the 42 degrees C survival curve. A plot of D0 vs water bath temperature was bimodal with an inflection point at approximately 42.5 degrees C. The inactivation enthalpy for temperatures above 42.5 degrees C was 586 kJ/mol (140 kcal/mol) and for temperatures below 42.5 degrees C was estimated to be 1205 kJ/mol (288 kcal/mol). These results show that CFU-GM can be heated predictably in situ, can be inactivated with thermal exposures as low as 42 degrees C, and are capable of developing thermotolerance. These findings underscore the necessity to understand stem cell inactivation by hyperthermia in situ prior to widespread implementation of clinical hyperthermia protocols where bone marrow may be included in the treatment field.     

Radiosensitivity and thermosensitization of thermotolerant Chinese hamster cells and RIF-1 tumors

CHO cells subline HA-1 were made thermotolerant by a priming heat treatment (43 degrees C, 30 min). Later, 4, 16, or 24 hr, they were either irradiated or heated (43 degrees C, 30 min) and irradiated. Thermotolerance had no effect on the radiation sensitivity of the cells as measured by the D0 value of the clonogenic survival curve. However, the N value of the curve (width of shoulder) showed a significant increase at 24 hr, indicating an increased capacity to accumulate sublethal damage. This indicates that the fractionation schedule 43 degrees C, 30 min + 37 degrees C, 24 hr + 43 degrees C, 30 min + X ray required approximately 100 rad more radiation than 43 degrees C, 30 min + X ray to reduce survival to the same level. The same priming treatment was given to RIF-1 tumors growing in C3H mice. Later, 24 hr, when the tumors were either irradiated or heated (43 degrees C, 30 min) and irradiated, it was found that thermotolerance had no effect on the radiosensitivity of the cells as measured by in vitro assay. However, thermal radiosensitization was not apparent 24 hr after the priming treatment.     

Effects of gamma radiation and hyperthermia on DNA repair synthesis and the level of NAD+ in cultured human mononuclear leukocytes

DNA repair has been investigated, estimated by unscheduled DNA synthesis (UDS) and the cellular NAD+ pool, after exposing human mononuclear leukocytes to hyperthermia and gamma radiation separately and in combination. It was found that gamma radiation induced a decline in UDS with increasing temperature through the temperature region studied (37-45 degrees C). At 42.5 degrees C the gamma-ray-induced UDS was reduced to about 70% of that at 37 degrees C. Following gamma-ray damage the NAD+ pool dropped to about 20% of control values. Without hyperthermic treatment the cells completely recovered to the original level within 5 hr. Moderate hyperthermia (42.5 degrees C for 45 min) followed by gamma-ray damage altered the kinetics so that even after 8 hr the NAD+ pool had recovered to only 70% of the original level. After heat treatment at 44 degrees C for 45 min prior to gamma radiation the cells did not recover at all, presumably because of the cytotoxic effects from the combined treatment.     

Differential role of Rho GTPases in intestinal epithelial barrier regulation in vitro

Maintenance of intestinal epithelial barrier functions is crucial to prevent systemic contamination by microbes that penetrate from the gut lumen. GTPases of the Rho-family such as RhoA, Rac1, and Cdc42 are known to be critically involved in the regulation of intestinal epithelial barrier functions. However, it is still unclear whether inactivation or activation of these GTPases exerts barrier protection or not. We tested the effects of Rho GTPase activities on intestinal epithelial barrier functions by using the bacterial toxins cytotoxic necrotizing factor 1 (CNF-1), toxin B, C3 transferase (C3 TF), and lethal toxin (LT) in an in vitro model of the intestinal epithelial barrier. Incubation of cell monolayers with CNF-1 for 3 h induced exclusive activation of RhoA whereas Rac1 and Cdc42 activities were unchanged. As revealed by FITC-dextran flux and measurements of transepithelial electrical resistance (TER) intestinal epithelial permeability was significantly increased under these conditions. Inhibition of Rho kinase via Y27632 blocked barrier destabilization of CNF-1 after 3 h. In contrast, after 24 h of incubation with CNF-1 only Rac1 and Cdc42 but not RhoA were activated which resulted in intestinal epithelial barrier stabilization. Toxin B to inactivate RhoA, Rac1, and Cdc42 as well as Rac1 inhibitor LT increased intestinal epithelial permeability. Similar effects were observed after inhibition of RhoA/Rho kinase signaling by C3 TF or Y27632. Taken together, these data demonstrate that both activation and inactivation of RhoA signaling increased paracellular permeability whereas activation of Rac1 and Cdc42 correlated with stabilized barrier functions.     

Increase in rat intestinal permeability to endotoxin during hyperthermia

Victims of heat stroke exhibit several clinical features which are also encountered in endotoxaemia. In order to investigate these similarities hyperthermic rats were used to explore the possibility that high body temperature results in increased permeability of intestinal wall to endotoxin. 125I endotoxin was introduced into intestinal segments taken from non-heat exposed rats. The segments were then incubated at 37 degrees C or 45 degrees C. Intestinal segments from heat stressed rats were similarly prepared and incubated at 37 degrees C. Leakage of endotoxin from segments taken from heat stressed rats was three times greater than from those from non-heat stressed rats, as were the segments from non-heat stressed rats which were incubated at 45 degrees C. These results indicate that the intestinal membrane is damaged by heat and that an increase in outward leakage of microbial endotoxins from the gut then occurs. This might contribute to the pathophysiological picture of heatstroke.     

Breakdown of mucin as barrier to digestive enzymes in the ischemic rat small intestine

Loss of integrity of the epithelial/mucosal barrier in the small intestine has been associated with different pathologies that originate and/or develop in the gastrointestinal tract. We showed recently that mucin, the main protein in the mucus layer, is disrupted during early periods of intestinal ischemia. This event is accompanied by entry of pancreatic digestive enzymes into the intestinal wall. We hypothesize that the mucin-containing mucus layer is the main barrier preventing digestive enzymes from contacting the epithelium. Mucin breakdown may render the epithelium accessible to pancreatic enzymes, causing its disruption and increased permeability. The objective of this study was to investigate the role of mucin as a protection for epithelial integrity and function. A rat model of 30 min splanchnic arterial occlusion (SAO) was used to study the degradation of two mucin isoforms (mucin 2 and 13) and two epithelial membrane proteins (E-cadherin and toll-like receptor 4, TLR4). In addition, the role of digestive enzymes in mucin breakdown was assessed in this model by luminal inhibition with acarbose, tranexamic acid, or nafamostat mesilate. Furthermore, the protective effect of the mucin layer against trypsin-mediated disruption of the intestinal epithelium was studied in vitro. Rats after SAO showed degradation of mucin 2 and fragmentation of mucin 13, which was not prevented by protease inhibition. Mucin breakdown was accompanied by increased intestinal permeability to FITC-dextran as well as degradation of E-cadherin and TLR4. Addition of mucin to intestinal epithelial cells in vitro protected against trypsin-mediated degradation of E-cadherin and TLR4 and reduced permeability of FITC-dextran across the monolayer. These results indicate that mucin plays an important role in the preservation of the mucosal barrier and that ischemia but not digestive enzymes disturbs mucin integrity, while digestive enzymes actively mediate epithelial cell disruption.     

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.     

Hyperthermic killing and hyperthermic radiosensitization in Chinese hamster ovary cells: effects of pH and thermal tolerance

To quantitatively relate heat killing and heat radiosensitization, asynchronous or G1 Chinese hamster ovary (CHO) cells at pH 7.1 or 6.75 were heated and/or X-irradiated 10 min later. Since no progression of G1 cells into S phase occurred during the heat and radiation treatments, cell cycle artifacts were minimized. However, results obtained for asynchronous and G1 cells were similar. Hyperthermic radiosensitization was expressed as the thermal enhancement factor (TEF), defined as the ratio of the D0 of the radiation survival curve to that of the D0 of the radiation survival curve for heat plus radiation. The TEF increased continuously with increased heat killing at 45.5 degrees C, and for a given amount of heat killing, the amount of heat radiosensitization was the same for both pH's. When cells were heated chronically at 42.4 degrees C at pH 7.4, the TEF increased initially to 2.0-2.5 and then returned to near 1.0 during continued heating as thermal tolerance developed for both heat killing and heat radiosensitization. However, the shoulder (Dq) of the radiation survival curve for heat plus radiation did not manifest thermal tolerance; i.e., it decreased continuously with increased heat killing, independent of temperature, pH, or the development of thermotolerance. These results suggest that heat killing and heat radiosensitization have a target(s) in common (TEF results), along with either a different target(s) or a difference in the manifestation of heat damage (Dq results). For clinical considerations, the interaction between heat and radiation was expressed as (1) the thermal enhancement ratio (TER), which is the dose of X rays alone divided by the dose of X rays combined with heat to obtain an isosurvival, e.g., 10(-4), and (2) the thermal gain factor (TGF), the ratio of the TER at pH 6.75 to the TER at pH 7.4. Since low pH reduced the rate of development of thermal tolerance during heating at low temperatures, low pH enhanced heat killing more at 42-42.5 degrees C than at 45.5 degrees C where thermal tolerance did not develop. Therefore, the increase in the TGF after chronic heating at 42-42.5 degrees C was greater than after acute heating at 45.5 degrees C, due primarily to the increase in heat killing causing an even greater increase in heat radiosensitization. These findings agree with animal experiments suggesting that in the clinic, a therapeutic gain for tumor cells at low pH may be greater for temperatures of 42-42.5 degrees C than of 45.5 degrees C.     

Investigation of thermotolerance in mouse testis

The effect of a two-fraction heat treatment on mouse testis has been assessed by measuring testis weight loss at 1 week after treatment. The rate of repair of 'sublethal' heat damage following the first treatment was dependent on the severity of the treatment. Using a primary treatment of 41.5 degrees C for 30 min, the weight loss following a test treatment of 41.5 degrees C for 30 min returned to that of the test treatment alone within an interval of 16-24 h. Using a milder primary treatment of 40.0 degrees C for 30 min, repair of sublethal heat damage appeared to be complete by 1-2 h. When a single test treatment was used, there was no evidence of heat-induced thermal resistance (thermotolerance) following primary treatments of 40.0 or 41.5 degrees C for 30 min, for periods up to 24 h between treatments. A small degree of thermotolerance could, however, be demonstrated following the most severe primary treatment used if full dose: effect curves were obtained. Thermotolerance, manifest as a decrease in slope, was maximal at approximately 4 h after the primary treatment. The results are discussed with reference to other normal tissue data.     

Effect of hypothermia on cell kinetics and response to hyperthermia and X rays

Hyperthermia is a potent radio enhancer. Studies using hypothermia in combination with irradiation have given confusing results due to lack of uniformity in experimental design. This report shows that hypothermia might have potential significance in the treatment of malignant cells with both thermo- and radiotherapy. Reuber H35 hepatoma cells, clone KRC-7 were used to study the effect of hypothermia on cell kinetics and subsequent response to hyperthermia and/or X rays. Cells were incubated at 8.5 degrees C or between 25 and 37 degrees C for 24 hr prior to hyperthermia or irradiation. Hypothermia caused sensitization to both hyperthermia and X rays. Maximum sensitization was observed between 25 and 30 degrees C and no sensitization was found at 8.5 degrees C. At 25 degrees C maximum sensitization was achieved in approximately 24 hr, cell proliferation was almost completely blocked, and cells gradually accumulated in the G2 phase of the cell cycle. In contrast to the effect of hypothermia on either hyperthermia or X rays alone, thermal radiosensitization was decreased in hypothermically pretreated cells (24 hr at 25 degrees C) compared to control cells (37 degrees C). The expression of thermotolerance and the rate of development at 37 degrees C after an initial heating at 42.5 degrees C were not influenced after preincubation at 25 degrees C for 24 hr. The expression of thermotolerance for heat or heat plus X rays during incubation at 41 degrees C occurred in a significantly smaller number of cells after 24 hr preincubation at 25 degrees C. The enhanced thermo- and radiosensitivity in hypothermically treated cells disappeared in approximately 6 hr after return to 37 degrees C.     

Dose-response relationship between body temperature and birth defects in radiofrequency-irradiated rats

Five groups of pregnant Sprague-Dawley rats were irradiated for 10-40 min on gestation day 9 in a 27.12-MHz radiofrequency field at a magnetic field strength of 55 A/m and an electric field strength of 300 V/m. The specific absorption rate was 10.8 +/- 0.3 W/kg. Exposures were terminated after the rat's colonic temperature reached 41.0 degrees C, 41.5 degrees C, 42.0 degrees C, 42.5 degrees C, or 43.0 degrees C. A control group was sham irradiated at 0 A/m and 0 V/m on gestation day 9, whereas a second control group was untreated. The incidence of both birth defects and prenatal death was directly related to maternal body temperature once a temperature threshold was exceeded. The temperature threshold for both types of effects was approximately 41.5 degrees C. A few pregnant rats died after exposure to 43.0 degrees C, and higher temperatures were nearly always lethal.     

Wnt/β-catenin-mediated heat exposure inhibits intestinal epithelial cell proliferation and stem cell expansion through endoplasmic reticulum stress

Heat stress induced by continuous high ambient temperatures or strenuous exercise in humans and animals leads to intestinal epithelial damage through the induction of intracellular stress response. However, the precise mechanisms involved in the regulation of intestinal epithelial cell injury, especially intestinal stem cells (ISCs), remain unclear. Thereby, in vitro a confluent monolayer of IPEC-J2 cells was exposed to the high temperatures (39, 40, and 41°C), the IPEC-J2 cell proliferation, apoptosis, differentiation, and barrier were determined, as well as the expression of GRP78, which is a marker protein of endoplasmic reticulum stress (ERS). The Wnt/β-catenin pathway-mediated regenerative response was validated using R-spondin 1 (Rspo1). And ex-vivo, three-dimensional cultured enteroids were developed from piglet jejunal crypt and employed to assess the ISC activity under heat exposure. The results showed that exposure to 41°C for 72 hr, rather than 39°C and 40°C, decreased IPEC-J2 cell viability, inhibited cell proliferation and differentiation, induced ERS and cell apoptosis, damaged barrier function and restricted the Wnt/β-catenin pathway. Nevertheless, Wnt/β-catenin reactivation via Rspo1 protects the intestinal epithelium from heat exposure-induced injury. Furthermore, exposure to 41°C for 24 hr reduced ISC activity, stimulated crypt-cell apoptosis, upregulated the expression of GRP78 and caspase-3, and downregulated the expression of β-catenin, Lgr5, Bmi1, Ki67, KRT20, ZO-1, occludin, and claudin-1. Taken together, we conclude that heat exposure induces ERS and downregulates the Wnt/β-catenin signaling pathway to disrupt epithelial integrity by inhibiting the intestinal epithelial cell proliferation and stem cell expansion.     

Morphological response and survival of hepatoma cells during fractionated hyperthermia: effect of glycerol

Reuber H35 rat hepatoma cells rounded and became spherical during hyperthermia at 42.5 degrees C. When returned to 37 degrees C, the cells recovered and spread out again. As soon as the cells had recovered from the morphologically expressed stress, they expressed tolerance to a second hyperthermia treatment as measured by the same end point. Fractionated hyperthermia made the cells thermotolerant as judged by both the morphological and the cell survival response. Glycerol protected the cells against heat damage as measured by less morphological alteration and decreased cell lethality. Protection depended on the glycerol concentration and maximal protection was observed at 6-8%. After heating in the presence of 7% glycerol, cells expressed thermotolerance at an earlier time than in the absence of glycerol, although the rates of development were approximately similar. Cell survival data and morphological responses showed good correlation.