The steady-state levels of oxidative DNA damage and of lipid peroxidation (F2-isoprostanes) are not correlated in healthy human subjects

Oxidative damage to DNA in human tissues can be determined by measuring multiple products of oxidative damage to the purine and pyrimidine bases using gas chromatography-mass spectrometry (GC-MS). Oxidative damage to lipids (lipid peroxidation) can be quantitated by the mass spectrometry-based determination of F₂-isoprostanes, specific end-products of the peroxidation of arachidonic acid residues in lipids. For both DNA base damage products and 8-epi prostaglandin F_2α (PGF_2α), there is a wide variation in levels between different healthy human subjects. We measured multiple products of oxidative damage to DNA bases in white cells, and 8-epi PGF_2α in plasma, from blood samples obtained from healthy human subjects in the UK and in Portugal. No correlation of 8-epi PGF_2α levels with levels of any modified DNA base (including 8-hydroxyguanine) was observed. We conclude that no single parameter can be measured as an index of “oxidative stress” or “oxidative damage” in vivo.     

An Assessment of Oxidative Damage to Proteins, Lipids, and DNA in Brain from Patients with Alzheimer's Disease

Abstract: Oxidative stress may contribute to neuronal loss in Alzheimer's disease (AD). The present study compares the levels of oxidative damage to proteins, lipids, and DNA bases from seven different brain areas of AD and matched control tissues by using a range of techniques. No differences in levels of lipid peroxidation were found in any of the brain regions by using two different assay systems. Overall, there was a trend for protein carbonyl levels to be increased in AD in frontal, occipital, parietal, and temporal lobe, middle temporal gyrus, and hippocampus, but a significant difference was found only in the parietal lobe. Gas chromatography-mass spectrometry was used to measure products of damage to all four DNA bases. Increased levels of some (8-hydroxyadenine, 8-hydroxyguanine, thymine glycol, Fapy-guanine, 5-hydroxyuracil, and Fapy-adenine), but not all, oxidized DNA bases were observed in parietal, temporal, occipital, and frontal lobe, superior temporal gyrus, and hippocampus. The baseline level of oxidative DNA damage in the temporal lobe was higher than in other brain regions in both control and AD brain. The finding of increased oxidative damage to protein and DNA strengthens the possibility that oxidative damage may play a role in the pathogenesis of AD in at least some key brain regions.     

Oxidative Damage to Proteins, Lipids, and DNA in Cortical Brain Regions from Patients with Dementia with Lewy Bodies

Abstract: Dementia with Lewy bodies (DLB) forms the second most common pathological subgroup of dementia after Alzheimer's disease. The present study compares the levels of oxidative damage to proteins, lipids, and DNA bases in cortical brain areas from patients with DLB with levels in matched control tissues. Overall, there was a trend for protein carbonyl levels to be increased in all areas, but a significant difference was found only in the parietal and temporal lobes. No differences were observed in the levels of lipid peroxidation. Measurement of products of damage to DNA bases showed increased levels of thymine glycol, 8-hydroxyguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, and xanthine. Xanthine levels were increased in the DLB group in the parietal, occipital, and temporal lobes, indicating that peroxynitrite or other deaminating species may be involved. The finding of increased protein carbonyls and increased DNA base products in cortical regions from DLB patients indicates that oxidative stress may play a role in DLB.     

Time course and attenuation of ischaemia-reperfusion induced oxidative injury by propofol in human renal transplantation

Ischaemia-reperfusion injury resulting from interruption and restoration of blood flow might be related to free radical mediated oxidative stress and inflammation, and subsequently to post-surgery related complications. We studied the impact of renal transplantation on oxidative stress and inflammation by measuring F(2)-isoprostanes and prostaglandin F(2alpha), respectively, during transplantation and post-surgery. Additionally, due to earlier observations, two dissimilar anaesthetic agents (thiopentone and propofol) were compared to determine their antioxidative capacity rather than their anaesthetic properties. Blood samples were collected before, post-intubation, immediately, 30, 60,120, 240 min, and 12 and 24 h after reperfusion. Oxidative stress and inflammatory response were detected by measuring 8-iso-PGF(2alpha) (a major F(2)-isoprostane and a biomarker of oxidative stress) and 15-keto-dihydro-PGF(2alpha) (a major metabolite of PGF(2alpha) and a biomarker of COX-mediated inflammatory response), respectively. Reperfusion of the transplanted graft significantly increased plasma levels of 8-iso-PGF(2alpha). PGF(2alpha) metabolite levels, although elevated, did not reach statistical significance. In addition, significantly lower levels of 8-iso-PGF(2a) were observed in the propofol group compared to the thiopentone group. Together, these findings underline an augmented oxidative stress activity following an inflammatory response after human renal transplantation. Furthermore, propofol a well-known anaesthetic, counteracted oxidative stress by lowering the formation of a major F(2)-isoprostane.     

Lipid peroxidation cannot be used as a universal criterion of oxidative stress

Oxidative stress is a term used to denote the imbalance between the concentrations of reactive oxygen and nitrogen species and the defense mechanisms of the body. Although it is generally accepted that such an imbalance plays a pivotal role in many pathologies, the term "oxidative stress" remains ill defined. In an attempt to evaluate the relationship between various assays of oxidative stress, we have analyzed the correlations between the results reported in those publications in which "oxidative stress" has been assayed by at least two methods. We found good correlations between the concentrations of several peroxidation products, including malondialdehyde, F2-Isoprostanes, lipid hydroperoxides, conjugated dienes, glutathione and protein carbonyls, but not with other criteria of "individual oxidative status" such as the concentration of antioxidants and products of DNA fragmentation (the "comet" assay). In light of these findings, we divide the assays used for evaluation of "oxidative stress" into the following three categories: (i) assays based on measuring the concentrations of oxidation products of lipids, proteins and DNA, as well as the concentrations of antioxidants, (ii) assays used to evaluate the oxidative and reductive capacity of biological fluids and (iii) assays used to evaluate the ex vivo susceptibility of lipids to oxidation upon their exposure to a source of free radicals. Our analyses demonstrate that oxidative stress cannot be defined in universal terms. Two results are of special interest:1.the commonly used criteria based on lipid peroxidation can not be regarded as a general estimate of the individual "oxidative status".2.the levels of antioxidants exhibit a non-monotonic relation with other criteria for oxidative stress. Further research is required to evaluate the significance of the latter finding.     

Loss of oxidized and chlorinated bases in DNA treated with reactive oxygen species: implications for assessment of oxidative damage in vivo

Oxidative damage to DNA has been reported to occur in a wide variety of disease states. The most widely used "marker" for oxidative DNA damage is 8-hydroxyguanine. However, the use of only one marker has limitations. Exposure of calf thymus DNA to an .OH-generating system (CuCl(2), ascorbate, H(2)O(2)) or to hypochlorous acid (HOCl), led to the extensive production of multiple oxidized or chlorinated DNA base products, as measured by gas chromatography-mass spectrometry. The addition of peroxynitrite (ONOO(-)) (<200 microM) or SIN-1 (1mM) to oxidized DNA led to the extensive loss of 8-hydroxyguanine, 5-hydroxycytosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2-hydroxyadenine, 8-hydroxyadenine, and 4,6-diamino-5-formamidopyrimidine were lost at higher ONOO(-) concentrations (>200 microM). Exposure of DNA to HOCl led to the generation of 5-Cl uracil and 8-Cl adenine and addition of ONOO(-) (<200 microM) or SIN-1 (1mM) led to an extensive loss of 8-Cl adenine and a small loss of 5-Cl uracil at higher concentrations (>500 microM). An .OH-generating system (CuCl(2)/ascorbate/H(2)O(2)) could also destroy these chlorinated species. Treatment of oxidized or chlorinated DNA with acidified nitrite (NO(2)(-), pH 3) led to substantial loss of various base lesions, in particular 8-OH guanine, 5-OH cytosine, thymine glycol, and 8-Cl adenine. Our data indicate the possibility that when ONOO(-), nitrite in regions of low pH or .OH are produced at sites of inflammation, levels of certain damaged DNA bases could represent an underestimate of ongoing DNA damage. This study emphasizes the need to examine more than one modified DNA base when assessing the role of reactive species in human disease.     

Gender differences in steady-state levels of oxidative damage to DNA in healthy individuals

Oxidative damage to DNA has often been used as a biomarker for oxidative stress and more specifically for cancer risk. Indeed, the measurement of oxidative damage to DNA, particularly of 8-hydroxyguanine (8OHG) and 8-hydroxy-2'-deoxyguanosine (8OHdG), has been adopted as a method for establishing the effects of antioxidant supplementation towards protection from certain cancers, cardiovascular and neuro-degenerative diseases, both in patients and healthy individuals. However, reported levels of 8OHdG or 8OHG vary considerably, possibly due to the different methodologies used, and only few data are available for the non-smoking and the female population. In this paper, steady-state levels of oxidative damage to DNA measured in a group of 20 males and 19 females are reported. Significant gender differences in levels of modified DNA bases such as 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine), 8-hydroxyadenine (8OHA) and 5-hydroxycytosine (5OHC), measured by gas chromatography-mass spectrometry (GC/MS), were observed. The results are discussed in relation to the Vitamin C and iron status of the subjects and to the existing, yet limited, literature data. The role of gender in predisposition to oxidative damage to DNA needs to be addressed in future studies.     

Oxidative DNA Damage in the Parkinsonian Brain: An Apparent Selective Increase in 8-Hydroxyguanine Levels in Substantia Nigra

Abstract: Oxidative damage has been implicated in the pathology of Parkinson's disease (PD), e.g., rises in the level of the DNA damage product, 8-hydroxy-2'-deoxyguanosine, have been reported. However, many other products result from oxidative DNA damage, and the pattern of products can be diagnostic of the oxidizing species. Gas chromatography/mass spectrometry was used to examine products of oxidation and deamination of all four DNA bases in control and PD brains. Products were detected in all brain regions examined, both normal and PD. Analysis showed that levels of 8-hydroxyguanine (8-OHG) tended to be elevated and levels of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine) tended to be decreased in PD. The most striking difference was a rise in 8-OHG in PD substantia nigra ( p = 0.0002); rises in other base oxidation/deamination products were not evident, showing that elevation in 8-OHG is unlikely to be due to peroxynitrite (ONOO⁻) or hydroxyl radicals (OH^•), or to be a prooxidant effect of treatment with l -Dopa. However, some or all of the rise in 8-OHG could be due to a change in 8-OHG/FAPy guanine ratios rather than to an increase in total oxidative guanine damage.     

Repair of products of oxidative DNA base damage in human cells.

Oxidative DNA damage is the most frequent type of damage encountered by aerobic cells and may play an important role in biological processes such as mutagenesis, carcinogenesis and aging in humans. Oxidative damage generates a myriad of modifications in DNA. We investigated the cellular repair of DNA base damage products in DNA of cultured human lymphoblast cells, which were exposed to oxidative stress by H2O2. This DNA-damaging agent is known to cause base modifications in genomic DNA of mammalian cells [Dizdaroglu, M., Nackerdien, Z., Chao, B.-C., Gajewski, E. and Rao, G. (1991) Arch. Biochem. Biophys. 285, 388-390]. Following treatment with H2O2, the culture medium was freed from H2O2 and cells were incubated for time periods ranging from 10 min to 6 h. DNA was isolated from control cells, hydrogen peroxide-treated cells and cells incubated after H2O2 exposure. DNA samples were analyzed by gas chromatography/isotope-dilution mass spectrometry. Eleven modified bases were identified and quantified. The results showed a significant formation of these DNA base products upon H2O2-treatment of cells. Subsequent incubation of cells caused a time-dependent excision of these products from cellular DNA. The cell viability did not change significantly by various treatments. There were distinct differences between the kinetics of excision of individual products. The observed excisions were attributed to DNA repair in cells. The rate of repair of purine lesions was slower than that of pyrimidine lesions. Most of the identified products are known to possess various premutagenic properties. The results of this work may contribute to the understanding of the cellular repair of oxidative DNA damage in human and other mammalian cells.     

Tomato consumption modulates oxidative DNA damage in humans.

Consumption of a single serving of tomatoes by healthy human volunteers was sufficient to alter levels of oxidative DNA base damage in white cell DNA within 24 h. Levels of the mutagenic oxidized purine base 8-hydroxyguanine decreased, especially in those subjects whose initial levels of this base were higher than the mean. However, total DNA base damage remained unchanged since levels of 8-hydroxyadenine rose. The ability of tomato consumption to modulate oxidative DNA damage in the short term may indicate why daily consumption of fruits and vegetables is beneficial in decreasing cancer incidence.     

Peroxyl radical mediated oxidative DNA base damage: implications for lipid peroxidation induced mutagenesis

Endogenous DNA damage induced by lipid peroxidation is believed to play a critical role in carcinogenesis. Lipid peroxidation generates free radical intermediates (primarily peroxyl radicals, ROO(*)) and electrophilic aldehydes as the principal genotoxicants. Although detailed information is available on the role of aldehyde base adducts in mutagenesis and carcinogenesis, the contribution of peroxyl radical mediated DNA base damage is less well understood. In the present study we have mapped oxidative base damage induced by peroxyl radicals in the supF tRNA gene and correlated this information with peroxidation-induced mutations in several human fibroblast cell lines. Nearly identical patterns of oxidative base damage were obtained from reaction of DNA with either peroxidizing arachidonic acid (20:4omega6) or peroxyl radicals generated by thermolysis of ABIP in the presence of oxygen. Oxidative base damage primarily occurred at G and C. Transversions at GC base pairs in the supF gene were the major base substitution detected in all cell lines. Peroxyl radical induced tandem mutations were also observed. Many mutation hot spots coincided with sites of mapped oxidative lesions, although in some cases hot spots occurred adjacent to the damaged base. Evidence is presented for the involvement of 8-oxodG in the oxidation of DNA by ROO(*). These results are used to interpret some key features of previously published mutation spectra induced by lipid peroxidation in human cells.     

Identification of metabolites from type III F2-isoprostane diastereoisomers by mass spectrometry

F(2)-isoprostanes (F(2)-iPs) are prostaglandin (PG)-like products of non-enzymatic free radical-catalyzed peroxidation of arachidonic acid that are now widely used as indices of lipid peroxidation in vivo. Knowledge of the metabolic fate of F(2)-iPs in vivo is still scant, despite its importance for defining their overall formation and biological effects in vivo. Type III F(2)-iPs, which are diastereoisomers of cyclooxygenase-derived PGF(2alpha), may be metabolized through the pathways of PG metabolism. We therefore studied the in vitro metabolism of eight synthetic Type III F(2)-iP diastereoisomers in comparison with PGF(2alpha). We used gas chromatography-mass spectrometry and high performance liquid chromatography-electrospray-tandem mass spectrometry for structural identification of metabolites formed after incubation of the various compounds with isolated rat hepatocytes. PGF(2alpha) was metabolized to several known products, resulting from a combination of beta-oxidation, reduction of Delta(5) and/or Delta(13) double bonds, and 15-OH oxidation, plus other novel products deriving from conjugation with taurine of PGF(2alpha) and its metabolites. Of the eight F(2)-iP diastereoisomers, some were processed similarly to PGF(2alpha), whereas others showed peculiar metabolic profiles according to specific stereochemical configurations.These data represent the first evidence of biodegradation of selected Type III F(2)-iP isomers other than 8-epi-PGF(2alpha), through known and novel pathways of PGF(2alpha) metabolism. The analytical characterization of these products may serve as a basis for identifying the most significant products formed in vivo.     

Oxidative stress in neonates: evaluation using specific biomarkers

Increased oxidative stress has been implicated in pathogenesis of serious diseases in neonates. We measured urinary levels of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative DNA damage), acrolein-lysine adduct (a marker of lipid peroxidation and oxidative protein damage), and nitrite/nitrate (a marker of endogenous nitric oxide formation) in one-month-old neonates to examine the status of oxidative stress and its relationship to the degree of prematurity and clinical condition in neonates. Study subjects comprised three groups: healthy term neonates, clinically stable preterm neonates requiring no supplemental oxygen, and clinically sick preterm neonates requiring supplemental oxygen and ventilator support. Urinary levels of 8-hydroxy-2'-deoxyguanosine and acrolein-lysine adduct were significantly higher in sick preterm neonates than those of stable preterm and healthy term neonates. In the sick preterm group, neonates developing active retinopathy showed significantly higher levels of acrolein-lysine adduct than the other neonates without retinopathy. There were no significant differences in both urinary markers of oxidative stress between stable preterm and healthy term neonates. The urinary nitrite/nitrate levels were not significantly different among the three groups, suggesting no difference in endogenous nitric oxide formation. Collectively, these results provide evidence of augmentation of oxidative damage to DNA, lipids and proteins, especially in clinically sick preterm neonates.     

Oxidative DNA damage in mild cognitive impairment and late-stage Alzheimer's disease

Increasing evidence supports a role for oxidative DNA damage in aging and several neurodegenerative diseases including Alzheimer's disease (AD). Attack of DNA by reactive oxygen species (ROS), particularly hydroxyl radicals, can lead to strand breaks, DNA–DNA and DNA–protein cross-linking, and formation of at least 20 modified bases adducts. In addition, α,β-unsaturated aldehydic by-products of lipid peroxidation including 4-hydroxynonenal and acrolein can interact with DNA bases leading to the formation of bulky exocyclic adducts. Modification of DNA bases by direct interaction with ROS or aldehydes can lead to mutations and altered protein synthesis. Several studies of DNA base adducts in late-stage AD (LAD) brain show elevations of 8-hydroxyguanine (8-OHG), 8-hydroxyadenine (8-OHA), 5-hydroxycytosine (5-OHC), and 5-hydroxyuracil, a chemical degradation product of cytosine, in both nuclear and mitochondrial DNA (mtDNA) isolated from vulnerable regions of LAD brain compared to age-matched normal control subjects. Previous studies also show elevations of acrolein/guanine adducts in the hippocampus of LAD subjects compared to age-matched controls. In addition, studies of base excision repair show a decline in repair of 8-OHG in vulnerable regions of LAD brain. Our recent studies show elevated 8-OHG, 8-OHA, and 5,6-diamino-5-formamidopyrimidine in both nuclear and mtDNA isolated from vulnerable brain regions in amnestic mild cognitive impairment, the earliest clinical manifestation of AD, suggesting that oxidative DNA damage is an early event in AD and is not merely a secondary phenomenon.     

Thyroid hormone-induced oxidative damage on lipids, glutathione and DNA in the mouse heart

Oxygen radicals of mitochondrial origin are involved in oxidative damage. In order to analyze the possible relationship between metabolic rate, oxidative stress and oxidative damage, OF1 female mice were rendered hyper- and hypothyroid by chronic administration of 0.0012% L-thyroxine (T₄) and 0.05% 6-n-propyl-2-thiouracil (PTU), respectively, in their drinking water for 5 weeks.

Hyperthyroidism significantly increased the sensitivity to lipid peroxidation in the heart, although the endogenous levels of lipid peroxidation were not altered. Thyroid hormone-induced oxidative stress also resulted in higher levels of GSSG and GSSG/GSH ratio. Oxidative damage to mitochondrial DNA was greater than that to genomic DNA. Hyperthyroidism decreased oxidative damage to genomic DNA. Hypothyroidism did not modify oxidative damage in the lipid fraction but significantly decreased GSSG and GSSG/GSH ratio and oxidative damage to mitochondrial DNA.

These results indicate that thyroid hormones modulate oxidative damage to lipids and DNA, and cellular redox potential in the mouse heart. A higher oxidative stress in the hyperthyroid group is presumably neutralized in the case of nuclear DNA by an increase in repair activity, thus protecting this key molecule. Treatment with PTU, a thyroid hormone inhibitor, reduced oxidative damage in the different cell compartments.     

Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer's disease

Increasing evidence suggests that oxidative stress is associated with normal aging and several neurodegenerative diseases, including Alzheimer's disease (AD). Here we quantified multiple oxidized bases in nuclear and mitochondrial DNA of frontal, parietal, and temporal lobes and cerebellum from short postmortem interval AD brain and age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring (GC/MS-SIM) and stable labeled internal standards. Nuclear and mitochondrial DNA were extracted from eight AD and eight age-matched control subjects. We found that levels of multiple oxidized bases in AD brain specimens were significantly (p < 0.05) higher in frontal, parietal, and temporal lobes compared to control subjects and that mitochondrial DNA had approximately 10-fold higher levels of oxidized bases than nuclear DNA. These data are consistent with higher levels of oxidative stress in mitochondria. Eight-hydroxyguanine, a widely studied biomarker of DNA damage, was approximately 10-fold higher than other oxidized base adducts in both AD and control subjects. DNA from temporal lobe showed the most oxidative damage, whereas cerebellum was only slightly affected in AD brains. These results suggest that oxidative damage to mitochondrial DNA may contribute to the neurodegeneration of AD.     

Garlic supplementation prevents oxidative DNA damage in essential hypertension

Oxygen-free radicals and other oxygen/nitrogen species are constantly generated in the human body. Most are intercepted by antioxidant defences and perform useful metabolic roles, whereas others escape to damage biomolecules like DNA, lipids and proteins. Garlic has been shown to contain antioxidant phytochemicals that prevent oxidative damage. These include unique water-soluble organosulphur compounds, lipid-soluble organosulphur compounds and flavonoids. Therefore, in the present study, we have tried to explore the antioxidant effect of garlic supplementation on oxidative stress-induced DNA damage, nitric oxide (NO) and superoxide generation and on the total antioxidant status (TAS) in patients of essential hypertension (EH). Twenty patients of EH as diagnosed by JNC VI criteria (Group I) and 20 age and sex-matched normotensive controls (Group II) were enrolled in the study. Both groups were given garlic pearls (GP) in a dose of 250 mg per day for 2 months. Baseline samples were taken at the start of the study, i.e. 0 day, and thereafter 2 months follow-up. 8-Hydroxy-2′-deoxyguanosine (8-OHdG), lipids, lipid peroxidation (MDA), NO and antioxidant vitamins A, E and C were determined. A moderate decline in blood pressure (BP) and a significant reduction in 8-OHdG, NO levels and lipid peroxidation were observed in Group I subjects with GP supplementation. Further, a significant increase in vitamin levels and TAS was also observed in this group as compared to the control subjects. These findings point out the beneficial effects of garlic supplementation in reducing blood pressure and counteracting oxidative stress, and thereby, offering cardioprotection in essential hypertensives.     

Mapping histological levels of 8-hydroxy-2′-deoxyguanosine in female reproductive organs

Oxidative stress is associated with many disease states including gynecologic disease. This process can damage lipids, proteins and DNA. The present study highlights the role of oxidative stress induced DNA damage as measured by 8-hydroxy-2-deoxyguanosine in development of benign gynecological conditions (BGC). Our aim was to map the oxidative DNA damage on female reproductive organs and highlight the high amount found in a variety of benign gynecologic disorders. Seventeen biopsy specimens from female pelvic organs were divided in two groups: healthy organs tissue and BGC tissue. Healthy organs biopsy tissue included the cervix, tubes, uterus, peritoneum, and topic endometrium in secretory phase. Benign gynecological biopsy tissue included hydrosalpinges, leiomyoma, adenomyosis and tubal cysts. Immunohistochemical staining showed significantly higher levels of DNA damage between BGC and healthy organs [19.36 % (6.20; 32.51) vs. 4.61 % (0.63; 8.53); P < 0.0344]. Our results highlight the involvement of oxidative stress DNA damage in female benign pelvic disease. Hydrosalpinges, leiomyoma, and adenomyosis exhibit the highest amounts of oxidative DNA damage in the pelvic cavity.     

Oxidative Stress Markers Induced by Hyperosmolarity in Primary Human Corneal Epithelial Cells

Oxidative stress has been known to be involved in pathogenesis of dry eye disease. However, few studies have comprehensively investigated the relationship between hyperosmolarity and oxidative damage in human ocular surface. This study was to explore whether and how hyperosmolarity induces oxidative stress markers in primary human corneal epithelial cells (HCECs). Primary HCECs were established from donor limbal explants. The hyperosmolarity model was made in HCECs cultured in isosmolar (312 mOsM) or hyperosmotic (350, 400, 450 mOsM) media. Production of reactive oxygen species (ROS), oxidative damage markers, oxygenases and anti-oxidative enzymes were analyzed by DCFDA kit, RT-qPCR, immunofluorescent and immunohistochemical staining and Western blotting. Compared to isosmolar medium, ROS production significantly increased at time- and osmolarity-dependent manner in HCECs exposed to media with increasing osmolarities (350–450 mOsM). Hyperosmolarity significantly induced oxidative damage markers in cell membrane with increased toxic products of lipid peroxidation, 4–hydroxynonenal (4-HNE) and malondialdehyde (MDA), and in nuclear and mitochondria DNA with increased aconitase-2 and 8-OHdG. Hyperosmotic stress also increased the mRNA expression and protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2), but reduced the levels of antioxidant enzymes, superoxide dismutase-1 (SOD1), and glutathione peroxidase-1 (GPX1). In conclusion, our comprehensive findings demonstrate that hyperosmolarity induces oxidative stress in HCECs by stimulating ROS production and disrupting the balance of oxygenases and antioxidant enzymes, which in turn cause cell damage with increased oxidative markers in membrane lipid peroxidation and mitochondrial DNA damage.     

Intra-day variation of in vivo prostaglandin F2alpha formation in healthy subjects

Prostaglandin F(2alpha) (PGF(2alpha)) is a major stable prostaglandin formed in vivo in physiological and pathophysiological situations and has mainly potent vasoconstrictive and pro-inflammatory properties. PGF(2alpha) is now used as an indicator of acute and chronic inflammation in human clinical settings but the extent of daily variation of PGF(2alpha)in vivo in healthy humans is unknown. We quantified levels of the PGF(2alpha) metabolite 15-keto-dihydro-PGF(2alpha) in 10 healthy males and females in spot urine samples during the day (including morning urine sample) and in 24-h urine during the same day. The intra-day coefficient of variation was 20.9%. However, the total mean value of 15-keto-dihydro-PGF(2alpha) in urine collected in the morning did not significantly differ from the mean level of 15-keto-dihydro-PGF(2alpha) in the 24-h urine samples in the 10 subjects. 15-Keto-dihydro-PGF(2alpha) levels in morning urine showed a positive linear correlation with levels of 15-keto-dihydro-PGF(2alpha) in 24-h urine (R=0.72, P<0.05). In conclusion, formation of PGF(2alpha) shows a biological variation within the day in healthy humans which should not be overlooked when planning a clinical study. Single morning urine samples can be used as an alternative to 24-h urine collections for quantification of PGF(2alpha) formation to simplify the sampling regime in larger clinical studies.