Receptor Tyrosine Kinase Inhibitor Sunitinib as Novel Immunotherapy to Inhibit Myeloid-Derived Suppressor Cells for Treatment of Endometriosis.

Endometriosis is a common, benign, and hormone-dependent gynaecological disorder that displays altered immunoinflammatory profiles. Myeloid-derived suppressor cells (MDSCs) suppressed immunosurveillance in endometriosis in human and mouse model. Receptor tyrosine kinase inhibitor Sunitinib can induce MDSC apoptosis and suppress the progression of cancer. However, the effects of Sunitinib on MDSCs in endometriosis and the underlying mechanism are not clear. In this study, we employed an animal study of the endometriosis model in mice for treatment of Sunitinib. After syngeneic endometrium transplantation and treatment, endometriotic lesion volume, weight, and histology were compared. Peritoneal fluid, peripheral blood, and bone marrow MDSC subsets and their molecular signaling were monitored by flow cytometry. Peritoneal cytokines were assayed by ELISA. The gene expression profiles of isolated CD11b+Ly6G+Ly6Clo cells were studied by RNA sequencing. We found that Sunitinib significantly decreased the endometriotic lesion size and weight after 1 and 3 weeks, and decreased p-STAT3 activation in MDSCs after 1 week of treatment. In the first week, Sunitinib specifically increased the G-MDSC population in peritoneal fluid but the isolated CD11b+Ly6G+Ly6Clo MDSCs after Sunitinib treatment were presented as mature polynuclear MDSCs, while the control group had immature mononuclear MDSCs. Importantly, we found Sunitinib differentially suppressed gene expressions of immunosuppressive function and differentiation in peritoneal G-MDSCs. Apelin signaling pathway associated genes and inflammation related genes were upregulated, and amino acid metabolism regulator genes were downregulated in bone marrow G-MDSCs. For endometriotic lesions, the PPARG gene governing glucose metabolism and fatty acid storage, which is important for the development of endometriosis was upregulated. In conclusion, Sunitinib inhibited endometriotic lesions, by promoting peritoneal fluid MDSCs maturation and inhibiting the immunosuppressive function. These findings suggest that Sunitinib changed the immune microenvironment and inhibited the development of endometriosis, which has potential therapeutic effects as novel immunotherapy to promote MDSCs maturation, differentiation, and metabolism for the treatment of endometriosis.

Long noncoding RNA SAM promotes myoblast proliferation through stabilizing Sugt1 and facilitating kinetochore assembly.

The functional study of lncRNAs in skeletal muscle satellite cells (SCs) remains at the infancy stage. Here we identify SAM (Sugt1 asssociated muscle) lncRNA that is enriched in the proliferating myoblasts. Global deletion of SAM has no overt effect on mice but impairs adult muscle regeneration following acute damage; it also exacerbates the chronic injury-induced dystrophic phenotype in mdx mice. Consistently, inducible deletion of SAM in SCs leads to deficiency in muscle regeneration. Further examination reveals that SAM loss results in a cell-autonomous defect in the proliferative expansion of myoblasts. Mechanistically, we find SAM interacts and stabilizes Sugt1, a co-chaperon protein key to kinetochore assembly during cell division. Loss of SAM or Sugt1 both disrupts kinetochore assembly in mitotic cells due to the mislocalization of two components: Dsn1 and Hec1. Altogether, our findings identify SAM as a regulator of SC proliferation through facilitating Sugt1 mediated kinetochore assembly during cell division.

Effects of low-dose melamine exposure during pregnancy on maternal and fetal kidneys in rats.

Despite the previous reports on melamine contamination in high concentrations some years ago, there were not many studies on low-level exposure in daily life, particularly in pregnancy. We investigated the effect of low-dose melamine on the kidneys of the pregnant rats and their developing embryos/fetuses during various gestational stages namely implantation, gastrulation, organogenesis, maturation and whole pregnancy. Our results showed that the repeated low level of melamine (12.5, 25, and 50 mg/kg bw/d) during pregnancy did not cause obstruction of renal tubules although more precipitating crystals were found in the early gestational periods. Simple hyperplasia in the maternal tubules and pelvic epithelium were more prominent after exposed to melamine during the whole gestational period. Neonatal kidneys significantly suffered more from congestion in glomeruli and interstitium, dilated tubules and interstitial edema after melamine administration to the mother in the late and the whole gestational periods. A trend of advance of glomerular development in fetuses was also observed. We conclude that in utero exposure of low-level melamine could post a risk on the kidneys of the pregnant mother as well as the developing fetuses, which may further increase the possibility of other health problems later in life.

Adverse reproductive effects of maternal low-dose melamine exposure during pregnancy in rats.

Melamine is a heterocyclic, aromatic amine and nitrogen-enriched environmental toxicant, found in not only adulterated foodstuffs but also industrial household tableware and paints. Previous studies demonstrated adverse effects of high-dose melamine on human infants and pregnant animals, but effects of low-dose melamine on pregnancy have not been reported. In this study, reproductive effects of low-dose melamine were investigated in pregnant rats. Melamine in the range of 12.5-50 mg/kg was administered to pregnant rats at different gestational stages. Maternal weight gain was not significantly affected, and other maternal morbidity was not observed. Low-dose melamine exposure during pregnancy increased fetal size but reduced somite number in gastrulation (GD8.5-GD10.5) and organogenesis (GD10.5-GD16.5) periods, and increased incidence of stillbirth in whole gestational period (GD0.5 to delivery). Embryotoxicity of melamine was further confirmed by whole embryo culture in vitro that melamine retarded embryonic growth, impaired development of brain and heart, and induced open neural tube and atrioventricular defects with increased apoptosis. In conclusion, adverse reproductive effects of low-dose melamine during pregnancy were identified in the developing rat embryos and the perinatal effects of melamine were gestational and developmental stage dependent. Detailed hazard and risk assessment of melamine in reproduction system are warrant. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 131-138, 2017.

Determination of exogenous epigallocatechin gallate peracetate in mouse plasma using liquid chromatography with quadrupole time-of-flight mass spectrometry.

A robust method for the quantitation of epigallocatechin gallate peracetate in plasma for pharmacokinetic studies is lacking. We have developed a validated method to quantify this compound using liquid chromatography with quadrupole time-of-flight mass spectrometry with isopropanol and tert-butyl methyl ether (3:10) extraction and thin-layer chromatography purification. The epigallocatechin gallate peracetate-1-(13) C8 isotope was used as an internal standard. The linear range (r(2) > 0.9950) was from 0.05 to 100.00 µg/mL. The lower limit of quantification of the method was 0.05 µg/mL. Reproducibility, coefficient of variation, was between 0.7 and 12.6% (n = 6), accuracy between 83.7 and 104.6% (n = 5), and recovery ranged from 82.4 to 109.0% (n = 4). Ion suppression was approximately 40%. No mass spectral peaks were found to interfere between the standard and internal standard or the blank plasma extracts. Epigallocatechin gallate peracetate in plasma was stably stored at -80°C over three months even after three freeze-thaw cycles. Extracts were stable in the sampler at 4°C for over 48 h. Plasma levels were maintained at 1.36 µg/mL for 360 min after intraorbital intravenous injection at 50 mg/kg in mice. This method can be used to reliably measure epigallocatechin gallate peracetate in plasma for pharmacokinetic studies.

Serotonin receptor 6 mediates defective brain development in monoamine oxidase A-deficient mouse embryos.

Monoamine oxidases A and B (MAO-A and MAO-B) are enzymes of the outer mitochondrial membrane that metabolize biogenic amines. In the adult central nervous system, MAOs have important functions for neurotransmitter homeostasis. Expression of MAO isoforms has been detected in the developing embryo. However, suppression of MAO-B does not induce developmental alterations. In contrast, targeted inhibition and knockdown of MAO-A expression (E7.5-E10.5) caused structural abnormalities in the brain. Here we explored the molecular mechanisms underlying defective brain development induced by MAO-A knockdown during in vitro embryogenesis. The developmental alterations were paralleled by diminished apoptotic activity in the affected neuronal structures. Moreover, dysfunctional MAO-A expression led to elevated levels of embryonic serotonin (5-hydroxytryptamine (5-HT)), and we found that knockdown of serotonin receptor-6 (5-Htr6) expression or pharmacologic inhibition of 5-Htr6 activity rescued the MAO-A knockdown phenotype and restored apoptotic activity in the developing brain. Our data suggest that excessive 5-Htr6 activation reduces activation of caspase-3 and -9 of the intrinsic apoptotic pathway and enhances expression of antiapoptotic proteins Bcl-2 and Bcl-XL. Moreover, we found that elevated 5-HT levels in MAO-A knockdown embryos coincided with an enhanced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and a reduction of proliferating cell numbers. In summary, our findings suggest that excessive 5-HT in MAO-A-deficient mouse embryos triggers cellular signaling cascades via 5-Htr6, which suppresses developmental apoptosis in the brain and thus induces developmental retardations.

Melamine in prenatal and postnatal organs in rats.

Melamine can be transferred to fetus in utero through placenta and to infant ex utero by breast feeding. In this study, we characterized the pharmacokinetics of melamine in prenatal and postnatal organs in rats. Single bolus of melamine was administered to pregnant rats at different gestational stages and to infants at different postnatal stages. Distribution of melamine in maternal serum was about 30% higher in late pregnancy than that in early pregnancy; and it was 2 folds higher in postnatal serum in early infants in young adulthood. Distribution of melamine in all postnatal organs was higher than that in prenatal organs. Postnatal kidneys in early infants had the highest maximum concentration and the lowest clearance of melamine than the other postnatal organs. It may relate to the high vulnerability to the toxicity of melamine in this population.

Prodrug of green tea epigallocatechin-3-gallate (Pro-EGCG) as a potent anti-angiogenesis agent for endometriosis in mice.

Green tea epigallocatechin-3-gallate (EGCG) can inhibit angiogenesis and development of an experimental endometriosis model in mice, but it suffers from poor bioavailability. A prodrug of EGCG (pro-EGCG, EGCG octaacetate) is utilized to enhance the stability and bioavailability of EGCG in vivo. In this study, the potential of pro-EGCG as a potent anti-angiogenesis agent for endometriosis in mice was investigated. Homologous endometrium was subcutaneously transplanted into mice to receive either saline, vitamin E, EGCG or pro-EGCG treatment for 4 weeks. The growth of the endometrial implants were monitored by IVIS(®) non-invasive in vivo imaging during the interventions. Angiogenesis of the endometriotic lesions was determined by Cellvizio(®) in vivo imaging and SCANCO(®) Microfil microtomography. The bioavailability, anti-oxidation and anti-angiogenesis capacities of the treatments were measured in plasma and lesions. The implants with adjacent outer subcutaneous and inner abdominal muscle layers were collected for histological, microvessel and apoptosis examinations. The result showed that EGCG and pro-EGCG significantly decreased the growth of endometrial implants from the 2nd week to the 4th week of intervention. EGCG and pro-EGCG significantly reduced the lesion size and weight, inhibited functional and structural microvessels in the lesions, and enhanced lesion apoptosis at the end of interventions. The inhibition by pro-EGCG in all the angiogenesis parameters was significantly greater than that by EGCG, and pro-EGCG also had better bioavailability and greater anti-oxidation and anti-angiogenesis capacities than EGCG. Ovarian follicles and uterine endometrial glands were not affected by either EGCG or pro-EGCG. Vitamin E had no effect on endometriosis. In conclusion, pro-EGCG significantly inhibited the development, growth and angiogenesis of experimental endometriosis in mice with high efficacy, bioavailability, anti-oxidation and anti-angiogenesis capacities. Pro-EGCG could be a potent anti-angiogenesis agent for endometriosis.

Green tea epigallocatechin-3-gallate inhibits angiogenesis and suppresses vascular endothelial growth factor C/vascular endothelial growth factor receptor 2 expression and signaling in experimental endometriosis in vivo.

OBJECTIVE: To investigate the antiangiogenesis mechanism of epigallocatechin-3-gallate (EGCG) in an endometriosis model in vivo. DESIGN: Animal studies. SETTING: University laboratory. ANIMAL(S): Human endometrium from women with endometriosis (n = 10) was transplanted into immunocompromised mice. INTERVENTION(S): Mice (n = 30) were randomly treated with EGCG, vitamin E (antioxidant control), or vehicle (negative control) for microvessel imaging. MAIN OUTCOME MEASURE(S): Endometriotic implants were collected for angiogenesis microarray and pathway analysis. Differentially expressed angiogenesis molecules were confirmed by quantitative polymerase chain reaction, Western blot, and immunohistochemistry. Effects of EGCG on angiogenesis signal transduction were further characterized in a human endothelial cell line. Microvessel parameters and the angiogenesis signaling pathway in endometriotic implants and endothelial cells were studied. RESULT(S): EGCG, but not vitamin E, inhibited microvessels in endometriotic implants. EGCG selectively suppressed vascular endothelial growth factor C (VEGFC) and tyrosine kinase receptor VEGF receptor 2 (VEGFR2) expression. EGCG down-regulated VEGFC/VEGFR2 signaling through c-JUN, interferon-γ, matrix metalloproteinase 9, and chemokine (C-X-C motif) ligand 3 pathways for endothelial proliferation, inflammatory response, and mobility. EGCG also suppressed VEGFC expression and reduced VEGFR2 and ERK activation in endothelial cells. VEGFC supplementation attenuated the inhibitory effects by EGCG. CONCLUSION(S): EGCG inhibited angiogenesis and suppressed VEGFC/VEGFR2 expression and signaling pathway in experimental endometriosis in vivo and endothelial cells in vitro.

Monoamine oxidase a expression is vital for embryonic brain development by modulating developmental apoptosis.

Monoamine oxidases (MAO-A, MAO-B) metabolize biogenic amines and have been implicated in neuronal apoptosis. Although apoptosis is an important process in embryo development, the role of MAO isoenzymes has not been investigated in detail. We found that expression of MAO-A and MAO-B can be detected early on during embryo development. Expression levels remained constant until around midgestation but then dropped to almost undetectable levels toward birth. Similar expression kinetics were observed in the brain. Isoform-specific expression silencing of MAO-A mediated by siRNA during in vitro embryogenesis induced developmental defects, as indicated by a reduction of the crown rump length and impaired cerebral development. These alterations were paralleled by elevated serotonin levels. Similar abnormalities were observed when embryos were cultured in the presence of the MAO-A inhibitor clorgyline or when the transcriptional inhibitor of MAO-A expression R1 was overexpressed. In contrast, no such alterations were detected when expression of MAO-B was knocked down. To explore the underlying mechanisms for the developmental abnormalities in MAO-A knockdown embryos, we quantified the degree of developmental apoptosis in the developing brain. MAO-A knockdown reduced the number of apoptotic cells in the neuroepithelium, which coincided with impaired activation of caspases 3 and 9. Moreover, we observed reduced cyclin D1 levels as an indicator of impaired cell proliferation in MAO-A knockdown embryos. This data highlights MAO-A as a vital regulator of embryonic brain development.

Association of hemopexin in tear film and conjunctival macrophages with vernal keratoconjunctivitis.

OBJECTIVE: Vernal keratoconjunctivitis (VKC) is a chronic allergic inflammatory disease with unclear etiology and pathogenesis. We investigated the tear film proteome of patients with VKC to understand the pathologic characteristics of VKC. METHODS: Tear samples were collected from healthy volunteers and patients with VKC. Electrophoresis was performed to display the tear proteomic profiles according to VKC severity. The identities of differentially expressed proteins were analyzed by mass spectrometry and quantified by enzyme-linked immunosorbent assay. Impression cytology was performed on VKC conjunctival samples to demonstrate the cellular protein expression. Allergic sensitization was performed in mice to study the pathologic role of these proteins in VKC. RESULTS: Hemopexin, an inflammatory protein, was elevated in the tear film of patients with VKC. The increased hemopexin concentration in VKC tears was significantly associated with disease severity. Impression cytology showed specific high hemopexin expression in dekeratinized conjunctival epithelium and necrotic macrophages in patients with VKC. Immunohistochemical examination of normal lacrimal tissues from mice showed that hemopexin was not expressed in any lacrimal apparatus. Under systemic and topical sensitization and challenge using hemopexin in mice, the affected eye had mild to moderate bead discharge, chemosis, and edema with excessive macrophage infiltration and conjunctival necrosis. CONCLUSION: An association exists between tear hemopexin and the development and pathologic effects of VKC. CLINICAL RELEVANCE: Increased hemopexin may have a role in the development of VKC.

Green tea catechins and their oxidative protection in the rat eye.

Catechins, active constituents of green tea, are well-known antioxidative natural products. It was proposed that green tea extract (GTE) consumption could benefit the eye, and the pharmacokinetics of catechins and oxidation status in rat eye were investigated after oral administration. Sprague-Dawley rats were fed GTE and sacrificed at different time intervals. Their eyes were dissected into cornea, lens, retina, choroid-sclera, vitreous humor, and aqueous humor for analysis of catechins and 8-epi-isoprostane by HPLC-ECD and GC-NCI-MS, respectively. Catechins were differentially distributed in eye tissues. Gallocatechin was present at the highest concentration in the retina, 22729.4 +/- 4229.4 pmol/g, and epigallocatechin in aqueous humor at 602.9 +/- 116.7 nM. The corresponding area-under-curves were 207,000 pmol x h/g and 2035.0 +/- 531.7 nM x h, respectively. The time of maximum concentration of the catechins varied from 0.5 to 12.2 h. Significant reductions in 8-epi-isoprostane levels were found in the compartments except the choroid-sclera or plasma, indicating antioxidative activities of catechins in these tissues.

Innate immune response by ficolin binding in apoptotic placenta is associated with the clinical syndrome of preeclampsia.

BACKGROUND: Unidentified circulating factors derived from placenta are thought to be responsible for the exaggerated systemic inflammation leading to preeclampsia. Our aim was to identify the circulating factors present in preeclampsia and to investigate their relationship to the underlying systemic immune response responsible for the associated clinical manifestations. METHODS: We obtained blood samples from pregnant women with and without preeclampsia and performed comparative proteomic analyses to identify the abnormal circulating factors by 2-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight for protein separation and identification. In placentas from preeclamptic pregnancies, we evaluated the potential role of the candidate proteins identified by Western and immunohistochemical analysis. We also used proinflammatory cytokine antibody arrays to investigate local and systemic immune responses. RESULTS: We found that ficolins, the pattern-recognition proteins involved in the lectin-complement pathway, were differentially expressed in plasma from preeclamptic pregnancies. Ficolins were present in low concentrations in plasma but at high concentrations in the placenta, particularly in syncytiotrophoblasts undergoing apoptosis. The binding of ficolins in apoptotic trophoblasts induced innate immunity through local and systemic cytokine activation and correlated with the clinical manifestation of preeclampsia. CONCLUSIONS: We identified specific in vivo circulating factors derived from the placenta that are responsible for the local immune recognition and systemic inflammatory response in the development of clinical manifestations of preeclampsia. These findings may have predictive value and also therapeutic applications to lessen adverse clinical outcomes of preeclampsia.

Pharmacokinetic studies of green tea catechins in maternal plasma and fetuses in rats.

We carried out a pharmacokinetic study to determine the levels and profiles of catechins in pregnant rats and their fetuses after ingestion of green tea extract (GTE). We measured total catechin levels after enzyme digestions. Dams, at 15.5 days of gestation, were fed with GTE and catechins were measured in the maternal plasma, placenta, and fetus 0, 0.5, 1, 2, 3, 5, 8, 10, 12, 16, and 20 h after maternal GTE intake. The pharmacokinetic changes were analyzed by non compartmental models. We found that maternal plasma concentrations of catechins were about 10 times higher than in placenta and 50-100 times higher than in the fetus. AUC and Cmax levels of (-)-epicatechin (EC) were the highest in plasma while the levels of (-)-epigallocatechin gallate (EGCG) were the highest in the placenta and the fetus. The exposure level of catechin derivatives was higher than the gallate derivatives in maternal plasma after normalization but reversed in the placenta and fetus. The absorption of epi-isomers in plasma was found to be more favorable than their non epi-isomer counterparts. EGCG had the highest level of exposure (AUC) and the highest Cmax in the fetus, implying it may have potential for in utero antioxidant protection.

Metabolomic and bioinformatic analyses in asphyxiated neonates.

OBJECTIVES: We tested the application of bioinformatic algorithms in studying the metabolomic profiles of neonatal urine samples with clinical evidence of severe asphyxia at birth and subsequent neurodevelopmental handicap. DESIGN AND METHODS: The clinical outcomes of 256 newborns that required direct admission to neonatal intensive care unit for respiratory support or did not require direct admission were studied. Urinary metabolite profiles were measured by high throughput mass spectrometry and analyzed by bioinformatic methods. RESULTS: We found a positive relationship between suppressed biochemical networks involved in macromolecular synthesis and birth asphyxia associated with significant neonatal oxidative stress and morbidity. The metabolomic discriminators between good neonatal outcome and poor neonatal outcome were established using hierarchical clustering analysis. Concentrations of eight urinary organic acids in distinct biochemical pathways were elevated and significantly associated with the prognosis of neurodevelopmental handicap with high sensitivity and specificity: ethylmalonate, 3-hydroxy-3-methylglutarate, 2-hydroxy-glutarate and 2-oxo-glutarate were associated with good neonatal outcome, whereas glutarate, methylmalonate, 3-hydroxy-butyrate and orotate were associated with poor outcome. CONCLUSIONS: The data demonstrated the potential application of bioinformatics methods in this metabolomic study and proved its clinical relevance.

Meconium-stained liquor during labor is associated with raised neonatal cord blood 8-iso-prostaglandin F2alpha concentration.

OBJECTIVE: The purpose of this study was to compare the umbilical arterial 8-iso-prostaglandin F2alpha, concentrations between pregnancies that were complicated by moderate or thick meconium-stained liquor and those with clear liquor. STUDY DESIGN: Umbilical cord arterial blood samples were collected from 247 singleton pregnancies with either moderate or thick meconium-stained liquor at any stage of labor or clear liquor at all stages of labor for the determination of the total 8-iso-prostaglandins F2alpha concentration. RESULTS: The median total 8-iso-prostaglandins F2alpha concentration of the meconium-stained liquor group was significantly higher than that of the control group (719.2 vs 115.8 pg/mL). Among the meconium-stained liquor group, those who had a change from "clear liquor" at early labor to "moderate/ thick meconium-stained liquor" at late first stage or at delivery (late meconium-stained liquor group) had higher 8-iso-prostaglandins F2alpha concentration, compared with those who had moderate/ thick meconium-stained liquor since early labor (early meconium-stained liquor group; 959.8 vs 499.9 pg/mL). With the use of multiple regression analysis, meconium-stained liquor, duration of second stage of labor, and abnormal fetal heart tracings were independent determinants of cord blood 8-iso-prostaglandins F2alpha concentration. CONCLUSION: Moderate or thick meconium-stained liquor is an independent factor for increased oxidative stress in pregnancy.

Determination of catechins and catechin gallates in tissues by liquid chromatography with coulometric array detection and selective solid phase extraction.

Catechins levels in organ tissues, particularly liver, determined by published methods are unexpectedly low, probably due to the release of oxidative enzymes, metal ions and reactive metabolites from tissue cells during homogenization and to the pro-oxidant effects of ascorbic acid during sample processing in the presence of metal ions. We describe a new method for simultaneous analysis of eight catechins in tissue: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (CG), (-)-epicatechin gallate (ECG), (-)-gallocatechin gallate (GCG) and (-)-epigallocatechin gallate (EGCG) (Fig. 1). The new extraction procedure utilized a methanol/ethylacetate/dithionite (2:1:3) mixture during homogenization for simultaneous enzyme precipitation and antioxidant protection. Selective solid phase extraction was used to remove most interfering bio-matrices. Reversed phase HPLC with CoulArray detection was used to determine the eight catechins simultaneously within 25 min. Good linearity (>0.9922) was obtained in the range 20-4000 ng/g. The coefficients of variance (CV) were less than 5%. Absolute recovery ranged from 62 to 96%, accuracy 92.5 +/- 4.5 to 104.9 +/- 6%. The detection limit was 5 ng/g. This method is capable for determining catechins in rat tissues of liver, brain, spleen, and kidney. The method is robust, reproducible, with high recovery, and has been validated for both in vitro and in vivo sample analysis.

In vitro exposure to carbon dioxide induces oxidative stress in human peritoneal mesothelial cells.

BACKGROUND: The objective of this study was to verify whether in vitro exposure of human peritoneal mesothelial cells to carbon dioxide (CO(2)) influences the levels of 8-isoprostaglandin F(2alpha) (8-iso-PGF(2alpha)), a marker of oxidative stress. METHODS: Mesothelial cells were exposed to either: (i). 100% CO(2) for 4 h; (ii). 100% helium (an alternative gas with which to create hypoxic conditions) for 4 h; (iii). 100% CO(2) for 24 h; or (iv). standard conditions (control). After gas exposure, mesothelial cells were returned to standard conditions and harvested immediately (T(0)), and at 1-(T(1)) and 3 (T(3)) h afterwards. Cell viability and culture medium pH were monitored throughout the experiments. 8-iso-PGF(2alpha) was assayed by enzyme-linked immunosorbent assay (ELISA). RESULTS: Exposure to CO(2) decreased the culture medium pH whereas helium increased the pH. 8-iso-PGF(2alpha) levels in all treated groups were significantly higher than in the control group: in the 4 h CO(2) group at T(1); in the 24 h CO(2) group at T(0) and T(1); and in the 4 h helium group at T(0), T(1) and T(3). 8-iso-PGF(2alpha) levels following 4 h CO(2) exposure were significantly lower than after 24 h CO(2) exposure at T(1), and lower than following 4 h helium exposure at all time points. CONCLUSIONS: Exposure to both CO(2) and helium induces oxidative stress in mesothelial cells. Hypoxia-reoxygenation may play a role in this process.