Exposure of humans and other mammals to hyperthermic conditions elicits many physiological responses to stress in various tissues leading to profound injuries, which eventually result in death. It has been suggested that hyperthermia may increase oxidative stress in tissues to form reactive oxygen species harmful to cellular functions. By using transgenic mice with human antioxidant genes, we demonstrate that the overproduction of glutathione peroxidase (GP, both extracellular and intracellular) leads to a thermosensitive phenotype, whereas the overproduction of Cu,Znsuperoxide dismutase has no effect on the thermosensitivity of transgenic mice. Induction of HSP70 in brain, lung, and muscle in GP transgenic mice at elevated temperature was significantly inhibited in comparison to normal animals. Measurement of peroxide production in regions normally displaying induction of HSP70 under hyperthermia revealed high levels of peroxides in normal mice and low levels in GP transgenic mice. There was also a significant difference between normal and intracellular GP transgenic mice in level of prostaglandin E2 in hypothalamus and cerebellum. These data suggest direct participation of peroxides in induction of cytoprotective proteins (HSP70) and cellular mechanisms regulating body temperature. GP transgenic mice provide a model for studying thermoregulation and processes involving actions of hydroxy and lipid peroxides in mammals.

Abstract-In response to endotoxemia induced by administration of lipopolysaccharide, a complex series of reactions occurs in mammalian tissues. During this inflammation response, cells produce different mediators, such as reactive oxygen species, a number of arachidonic acid metabolites, and cytokines. The reactive oxygen species thus generated have been suggested to produce tissue injury as a result of macromolecular damage or by interfering with regulatory processes. They may also act as important signaling molecules to induce redox-sensitive genes. We report here that transgenic mice overexpressing 2 major forms of human glutathione peroxidases (GPs), intra-and extracellular GP, are able to modulate host response during endotoxemic conditions. We show that these animals have a decreased hypotension and increased survival rate after administration of a high dosage of lipopolysaccharide. Overexpression of GPs alters vascular permeability and production of cytokines (interleukin-1␤ and tumor necrosis factor-␣) and NO, affects arachidonic acid metabolism, and inhibits leukocyte migration. These results suggest an important role for peroxides in pathogenesis during endotoxemia, and GPs, by regulating their level, may prove to be good candidates for antioxidant therapy to protect against such injury. 2 There is experimental evidence that suggests oxidative damage in pathogenesis of endotoxemia. 3 To evaluate the role of glutathione peroxidase (GP) in protection against endotoxemia in vivo, we have used transgenic mice overexpressing human extracellular (GPxP) and intracellular (GPx1) GP. In the present study, we report that these animals show an increased rate of survival after administration of a large dosage of LPS. Mice with human GPs are able to modulate levels of lipid peroxidation and inflammatory mediators. These transgenic animals represent a new model system for investigation of the role of GPs and reactive oxygen species (ROS) in endotoxemia and other inflammatory disorders. Materials and Methods Animals and TreatmentsHeterozygous transgenic mice 5 to 6 months old in a C57BL/ 6ϫCBA/J background overexpressing human GPxP (strain 17) and human GPx1 (strain 23) and their normal littermates were bred in our facility. We described the generation of these mice previously. 4 For survival studies, the mice were injected with LPS at a dose of 25 mg/kg (Escherichia coli serotype 0111:B4; Sigma). Arterial blood pressure and heart rate were measured using the Kent tail blood pressure system. For characterization of cytokine production and leukocyte distribution, blood was collected from the retro-orbital cavity of a group of animals dosed with LPS (16 mg/kg of body weight). Plasma was analyzed for cytokine (tumor necrosis factor-␣ [TNF-␣] and interleukin-1␤ [IL-1␤]) production with ELISA kits from R&D Systems. Vascular permeability was determined by assessing tissue accrual of Evan's Blue, as described previously. 5 Sixteen hours after PBS or LPS injection, the animals were administered 25 mg/kg Evan's Blue by tail ...

Biochemical SocietyTransactions ( 1 996) 24 535s C49 ENDOTOXEMIA IN TRANSGENIC MICE WITH ANTIOXIDANT ENZYMES 0. Mlrochnitchenko. U. Palnltkar a n d M. lnouye R o b e r t Wood J o h n s o n M e d l c a l School, U n l v e r s l t y of M e d l c l n e a n d D e n t i s t r yof N e w J e r s e y , Plscataway, New Jersey, USA Reactive oxygen species (ROS) are known to be important mediators of cellular injury during inflammation, either by causing macromolecular damage or by interfering with extraand intracellular regulatory processes. We are using transgenic mice overexpressing human antioxidant enzymes:Cu,Zn superoxide dismutase (SOD) and two types of glutathione peroxidases (GP), intracellular (GPE) and extracellular (GPP), to investigate whether antioxidant enzymes can modulate host inflammatory response to lipopolysacharide (LPS). Groups of normal and transgenic mice were administered a large dose of LPS and mortality rates compared.Data obtained indicate that GP mice were more protected against LPS mediated toxicity. This effect was more profound in GPP mice, indicating the critical effect of GP production in the bloodstream. SOD transgenics exhibited a similar response as normal mice. Levels of TNFa and IL-1p in blood of normal and GP mice as well as level of IL-lp mRNA in different tissues were measured. Our data show that GP transgenics produced an increased level of blood TNFa but a lower level of IL-Ip in comparison to the normal mice. Levels of lipid peroxide were significantly modulated in blood and tissues of GP transgenic mice. Since adherence of leukocytes to endothelium is a critical event in their migration to the inflammation foci, we determined the expression of integrins in GPP transgenic mice after LPS treatment. ICAM-1 mRNA and protein expression in different tissues of transgenic mice was reduced which in turn led to the lower migration of neutrophils into the tissues of transgenic mice as compared to normal animals. The results prove that ox-redox balance at the endothelium cell surface is a critical factor modulating adhesive interactions of these cells. These data provide new insights into the role of the antioxidant enzymes regulating levels of ROS during endotoxin-induced multiple organ failure. LS2 9JTPhotodynamic Therapy (PDT) is a cancer therapy which uses reactive oxygen species produced by illuminating photosensitisers. localised in tumours. with light (Dolphin, 1994) Macrophages have been suggested t o play an important role in this therapy due to the high concentration o f photosensitising drug accumulated in these cells, in particular the tumour-associated population These cells may also play a part in inflammatory and immune responses which influence the outcome o f the treatment (Hamblin and Newman, 1994) This work demonstrates PDT o f a macrophage cell line, PuS18. using Polyhaematoporphyrin (PHP), one form o f the clinically approved drug, and a zinc pyridinium phthalocyanine (PPC). after illumination with a white light source, results in a rapid reduction o f cellular glutath...