Partial inhibition of estrogen-induced mammary carcinogenesis in rats by tamoxifen: balance between oxidant stress and estrogen responsiveness

Epidemiological and experimental evidences strongly support the role of estrogens in breast tumor development. Both estrogen receptor (ER)-dependent and ER-independent mechanisms are implicated in estrogen-induced breast carcinogenesis. Tamoxifen, a selective estrogen receptor modulator is widely used as chemoprotectant in human breast cancer. It binds to ERs and interferes with normal binding of estrogen to ERs. In the present study, we examined the effect of long-term tamoxifen treatment in the prevention of estrogen-induced breast cancer. Female ACI rats were treated with 17β-estradiol (E2), tamoxifen or with a combination of E2 and tamoxifen for eight months. Tissue levels of oxidative stress markers 8-iso-Prostane F(2α) (8-isoPGF(2α)), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, and oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG) were quantified in the mammary tissues of all the treatment groups and compared with age-matched controls. Levels of tamoxifen metabolizing enzymes cytochrome P450s as well as estrogen responsive genes were also quantified. At necropsy, breast tumors were detected in 44% of rats co-treated with tamoxifen+E2. No tumors were detected in the sham or tamoxifen only treatment groups whereas in the E2 only treatment group, the tumor incidence was 82%. Co-treatment with tamoxifen decreased GPx and catalase levels; did not completely inhibit E2-mediated oxidative DNA damage and estrogen-responsive genes monoamine oxygenase B1 (MaoB1) and cell death inducing DFF45 like effector C (Cidec) but differentially affected the levels of tamoxifen metabolizing enzymes. In summary, our studies suggest that although tamoxifen treatment inhibits estrogen-induced breast tumor development and increases the latency of tumor development, it does not completely abrogate breast tumor development in a rat model of estrogen-induced breast cancer. The inability of tamoxifen to completely inhibit E2-induced breast carcinogenesis may be because of increased estrogen-mediated oxidant burden.     

Suppression of calbindin D28K in estrogen-induced hamster renal tumors

It has been hypothesized that generation of reactive estrogen–quinone species and oxidative stress, both of which result from the metabolic activation of estrogens, plays an important role in estrogen-induced carcinogenesis. In the present investigation, we used an estrogen-induced hamster renal tumor model to identify gene(s) associated with oxidative stress that may be differentially expressed in estrogen-induced tumors compared with untreated controls. Hamsters were implanted with 17β-estradiol (E2) for 7 months. This treatment resulted in the development of target organ specific kidney tumors. Delta differential PCR technique on RNA isolated from estrogen-induced hamster renal tumors and untreated control kidneys identified a number of cDNA fragments that were differentially expressed in tumor RNA compared with untreated controls. We report the cloning of one of the differentially expressed cDNA fragments, the hamster calbindin-D28k (Cb28k) cDNA, and present a finding that both Cb28k mRNA and protein are suppressed in estrogen-induced hamster renal tumors compared with untreated controls. Cb28k is a Vitamin D3-dependent calcium binding protein that acts as a buffer to maintain intracellular calcium homeostasis, although its exact role is still not clear. Since Cb28k gene has been shown to be associated with providing cells resistance against oxidative stress, Cb28k may be an important biomarker in estrogen-mediated carcinogenesis and oxidative stress.     

Carcinogenicity of catechol estrogens in Syrian hamsters

Estradiol and other estrogens induce renal carcinoma in male Syrian hamsters. The mechanism of carcinogenesis still remains unclear. Activation of estrogens to catechol metabolites has in the past been postulated to play a role in estrogen-induced carcinogenesis. Therefore, the carcinogenic activity of catechol estrogens was investigated. After 175 days of treatment, 4-hydroxyestradiol was found to be as carcinogenic as estradiol in male Syrian hamsters (4/5 and 4/5 animals with kidney tumors, respectively). Animals treated with 2-hydroxyestradiol (0/5) or 2-methoxyestradiol (0/6) did not develop renal carcinoma. The catechol estrogens failed to be mutagenic in the Ames test (reversions of his- S. typhimurium to histidine prototrophy in the TA 100 strain). The lack of carcinogenic activity of 2-hydroxyestradiol was not due to a failure to stimulate estrogen-dependent tumor growth. Growth of H-301 cells, an estrogen-dependent hamster kidney tumor cell line, was supported in vivo by estrogens in the following order: estradiol greater than 4-hydroxyestradiol greater than 2-hydroxyestradiol. Stimulation of tumor growth by 2-methoxyestradiol was not detected. It was concluded that the carcinogenic activity of 4-hydroxyestradiol was consistent with a role of catechol metabolites in estrogen-induced carcinogenesis. However, the intrinsic carcinogenic or hormonal activity of 2-hydroxyestradiol probably can not be assessed accurately in vivo because of its rapid methylation and metabolic clearance.     

Transcardiac 8-iso-prostaglandin F(2 alpha)generation from acute myocardial infarction heart: insight into abrupt reperfusion and oxidant stress

8-iso-prostaglandin F(2 alpha)(8-iso-PGF(2 alpha)), a representative isoprostane, has been reported to be a reliable marker for oxidant stress in vivo. To examine if 8-iso-PGF(2 alpha)is generated in patients with acute myocardial infarction (AMI), we measured the level of immunoreactive 8-iso PGF(2 alpha)in the great cardiac vein as well as classical eicosanoids, 6-keto-prostaglandin F(1 alpha)(6-keto-PGF(1 alpha)) and thromboxane B(2)(TXB(2)) in the process of urgent coronary balloon angioplasty. Fourteen patients with anterior AMI were divided into two groups: the totally occluded (n=7) and the already perfused groups (n=7). In the former, transient elevation of 8-iso-PGF(2 alpha)was observed immediately after the angioplasty, i.e. the ratio of post-angioplasty level to pre-level was approximately 2.4 for 8-iso-PGF(2 alpha), 14 for 6-keto-PGF(1 alpha), and 5 for TXB(2). In the already perfused group, the levels of these eicosanoids were unchanged. In the totally occluded group, peak creatine phosphokinase in a peripheral vein was correlated with the level of 8-iso-PGF(2 alpha)(r(2)=0.841, P<0.01), but not with those of the other two eicosanoids. In conclusion, transcardiac 8-iso-PGF(2 alpha)generation is a reliable marker for the size of myocardium exposed to oxidant stress.     

F2-isoprostane induced prostaglandin formation in the rabbit

F(2)-isoprostanes, non-enzymatic free radical mediated products of arachidonic acid, have shown to form during various oxidant stress status and have potent biological effects. This study investigates to what extent 8-iso-PGF(2alpha) (a major F(2)-isoprostane), a bioactive product of lipid peroxidation can modify endogenous prostaglandin F(2alpha) (PGF(2alpha)) formation since prostaglandins are inflammatory as well as potent vasoregulatory substances that modulate diverse important physiological functions, and also form during acute and chronic inflammation. An immediate appearance and disappearance of 8-iso-PGF(2alpha) was seen in both plasma and urine within a short interval after i.v. administration of 43 microg/kg of 8-iso-PGF(2alpha) to the rabbits. A successive but differential formation of PGF(2alpha) resulted in a rapid and pulsatile increase of plasma 15-keto-dihydro-PGF(2alpha), a major metabolite of primary PGF(2alpha). Later, this compound was excreted efficiently as intact compound into the urine during the 3 h of experiment. A 8-fold increase of PGF(2alpha) metabolite in plasma at 10 min and 12-fold increase in the urine at 30-60 after the i.v. administration of 8-iso-PGF(2alpha) was observed which continued throughout the 3 h of experiment. This observation suggests that pharmacologically administered or endogenously produced 8-iso-PGF(2alpha) during oxidant stress induces prostaglandin formation presumably through the classical cyclooxygenase-catalysed arachidonic acid oxidation which might be inflammatory itself to the cells and exerts further vasoconstrictive effects.     

Increased urinary excretion of 8-iso-prostaglandin F2alpha in patients with HFE-related hemochromatosis: a case-control study

The hypothesis according to which iron overload could be harmful has been extensively and controversially discussed in the literature. One underlying pathological mechanism may be elevated oxidative stress. Thus, we studied the correlation between hemochromatosis and an established marker of oxidative stress, 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha, iPF2alpha-III, 15-F2t-IsoP). We enrolled 21 patients with hemochromatosis, positive for the homozygous C282Y mutation in the HFE gene, and 21 healthy controls frequency-matched by age and gender in a case-control study design. The objective was to show that iron overload in HFE-related hemochromatosis is associated with increased oxidative stress assessed through 8-iso-PGF(2alpha) urinary excretion, and that oxidative stress is impacted by iron-removal treatment (phlebotomy). Study parameters were transferrin saturation, 8-iso-PGF(2alpha) urine excretion, transferrin, ferritin, serum iron, and vitamins A and E for all participants. Iron concentration in the liver and non-transferrin-bound iron were measured in patients only. We found a significant difference in 8-iso-PGF2alpha in patients (245 [interquartile range 157-348] pg/mg creatinine) compared with controls (128 [106-191] pg/mg creatinine, P = 0.002). Vitamin A was significantly reduced in cases (0.34 [0.25-1.83] microg/ml compared to 3.00 [2.11-3.39] microg/ml, P < 0.001), while vitamin E did not show a significant difference in cases (14.7 [11.5-18.1] microg/ml) compared with controls (14.9 [13.1-19.2] microg/ml, P = 0.52). After phlebotomy treatment and normalization of the iron parameters in the hemochromatosis group, serum vitamin A levels were significantly increased (1.36 [1.08-1.97] microg/ml, P = 0.035 vs. baseline, P < 0.001 vs. controls) and 8-iso-PGF2alpha urinary excretion was lowered to control levels (146 [117-198] pg/mg creatinine, P = 0.38 vs. controls). In our study, HFE-related hemochromatosis was associated with increased oxidative stress and hypovitaminemia A in C282Y homozygotes. The increased oxidative stress was reversible by normalization of the iron load by phlebotomy. Thus, phlebotomy is an effective and adequate means for reducing oxidative stress in these patients.     

Resveratrol inhibits isoprostane production in young and aged rat brain

Resveratrol (3,5,4-trihydroxystilbene), a viniferin polyphenolic compound, has been shown to have neuroprotective effects and we tested its possible antioxidant activity in young and aged rat brain, evaluating, in vitro, synaptosomal 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha) production as a marker of oxidative stress. We found that in young rat brain synaptosomes resveratrol perfusion had no effect on basal 8-iso-PGF2alpha production, but quenched to basal levels the increased 8-iso-PGF2alpha production induced by hydrogen peroxide. On the other hand, in aged rats, resveratrol was able to decrease 8-iso-PGF2alpha production both basally and after hydrogen peroxide-induced oxidative stimulus. In conclusion, our findings show that the antioxidant effects of resveratrol in rat brain could play a neuroprotective role in aging, when the increased burden of oxidative stress is faced by defective antioxidant mechanisms.     

Oxidative stress and 8-iso-prostaglandin F(2alpha) induce ectodomain shedding of CD163 and release of tumor necrosis factor-alpha from human monocytes

CD163 is a membrane glycoprotein of the cysteine-rich scavenger receptor superfamily. Upon an inflammatory stimulus CD163 undergoes ectodomain shedding and the soluble protein has been shown to play a role in downregulation of inflammation. The purpose of the present study was to identify a physiological activator of CD163 shedding that is consistently present under inflammatory conditions. Therefore, we elucidated whether oxidative stress or 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) is involved in shedding of CD163. Oxidative stress induced by H(2)O(2) or a NO donor as well as 8-iso-PGF(2alpha) induced significant shedding of CD163. In contrast, release of CD163 was not stimulated by PGF(2alpha). We identified both calcium and reactive oxygen species as common cellular mediators of CD163 release. Since shedding of both CD163 and tumor necrosis factor-alpha (TNFalpha) is known to be mediated by a TIMP-3-sensitive metalloproteinase we examined whether release of TNFalpha was induced by the same mediators that trigger shedding of CD163. Only oxidative stress generated by H(2)O(2) as well as 8-iso-PGF(2alpha) and PGF(2alpha) enhanced TNFalpha secretion. Thus, we identified novel common and divergent activators of shedding of CD163 and TNFalpha. These inducers of shedding are present in inflammation and might play an important role in membrane protein cleavage.     

Lipid peroxidation is increased in paraoxonase L55 homozygotes compared with M-allele carriers

Human serum paraoxonase (PON) is an antioxidative enzyme, which circulates on high-density lipoproteins and appears to use oxidized phospholipids as physiological substrates. PON M/L55 substitution changes the ability of PON to prevent lipid oxidation. Urinary 8-iso-PGF(2alpha) (one of F2 -isoprostanes) may represent a non-invasive in vivo index of free radical generation and we propose that PON might influence the biosynthesis of 8-iso-PGF(2alpha) in the vasculature. We studied the urinary excretion of 8-iso-PGF(2alpha) and related it to PON M/L55 genotypes in patients with type 2 diabetes mellitus (n = 55) and non-diabetic control subjects (n = 55). Urinary 8-iso-PGF(2alpha) was determined by competitive ELISA and the PON genotype by a PCR based restriction enzyme digestion method. LL homozygotes were compared to M-allele carriers (ML heterozygotes and MM homozygotes). The urinary excretion of 8-iso-PGF(2alpha) among non-diabetic non-smoking LL homozygotes was 3995.5 +/- 3352.8 ng/24-hour and among M-allele carriers 1689.8 +/- 1051.3 ng/24-hour (p = 0.017, ANCOVA; gender, hypertension, total cholesterol, triglycerides and LDL cholesterol as covariates). The excretion of 8-iso-PGF(2alpha), was increased in type 2 diabetes mellitus compared to non-diabetic control subjects. PON may thus protect against oxidative stress by destroying some biologically active lipids. Excretion of 8-iso-PGF(2alpha) is increased in type 2 diabetes, which may reflect oxidant injury.     

Effect of garlic supplementation on oxidized low density lipoproteins and lipid peroxidation in patients of essential hypertension

Reactive oxygen species (ROS) has been implicated in the pathogenesis of many diseases including hypertension. Therefore, certain compounds with antioxidative capacity are believed to be protective against such diseases. Some components of garlic are known to possess antioxidative properties. Therefore, in the present study we investigated the effect of short-term garlic supplementation in essential hypertensive patients (EH) on indices of oxidative stress. Twenty patients of EH as diagnosed by JNC VI criteria (Group I) and 20 age and sex-matched normotensive controls were enrolled for the study. Both groups were given garlic pearls (GP) in a dose of 250 mg per day for 2 months. Baseline samples were obtained at start of the study, i.e. 0 day, and thereafter, 2 months (follow-up). Lipids and lipoprotein subfractions, plasma-oxidized low-density lipoproteins (ox-LDL), plasma and urinary concentration of 8-iso-Prostaglandin F2alpha (8-iso-PGF2alpha) as a biomarker of oxidative stress in vivo, and the total antioxidant status (TOS) of these individuals were determined. We observed a moderate hypercholesterolemia and a significantly raised blood pressure in hypertensive patients as compared to the controls. The indices of oxidative stress, i.e. plasma ox-LDL and plasma and urinary concentration of 8-iso-PGF2alpha were significantly increased in EH group. Further, hypertensive patients had a significantly low TOS as compared to the control group. With in 2 months of GP supplementation, there was a significant decline in both systolic (SBP) and diastolic blood pressures (DBP) and a significant reduction in ox-LDL and 8-iso-PGF2alpha levels in Group I patients. Further, a moderate increase in the TOS was also observed in this group as compared to their control counterparts. These findings suggest that dietary supplementation of garlic may be beneficial in reducing blood pressure and oxidative stress in hypertensive individuals.     

Urinary excretion of 8-iso-PGF(2 alpha) in three patients during sepsis,recovery and state of health

Sepsis is known to be associated with oxidative stress. Novel markers of oxidative stress are now believed to be F2-isoprostanes which are produced in situ in phospholipids and subsequently released into circulation and excreted in the urine. This study, therefore, sought to investigate whether the excretion of the isoprostane, 8-iso-PGF(2 alpha), is elevated during sepsis. The excretion of 8-iso-PGF(2 alpha), in the 24 h urine of three patients was studied in the septic stage, during mobilisation and in the state of health by a radioimmunological method. Extrapolating the urinary excretion of 8-iso-PGF(2 alpha) over time showed an insignificant variation in the excretion values during 24 h. The amount of mean 24 h urinary 8-iso-PGF(2 alpha) was about similar in the septic stage and in the state of health but increased remarkably during mobilisation in two of the patients. We suggest that mobilisation of septic patients can be associated with an increase of oxidative stress which may stem from an increase in oxygen consumption and/or from a depletion of antioxidants leading to the enhanced formation of free radicals.     

Impact of long-term hormone replacement therapy on in vivo and in vitro markers of lipid oxidation

Postmenopausal hormone replacement therapy (HRT) with estrogen has been suggested to inhibit oxidation of low-density lipoprotein (LDL) in vitro, but progestins may oppose this effect. We studied whether estrogen HRT and combined HRT with estrogen and progestin differ in their ability to resist in vivo and in vitro oxidation of lipids. Study group included 15 women on oestradiol valerate (mean age 56 years, treatment duration 10.5 years) and 15 women on combined HRT with oestradiol valerate and levonorgestrel (mean age 58 years, treatment duration 11.3 years). In addition to lipid and apolipoprotein concentrations, the lagtime of LDL to oxidation, the rate of the propagation phase and the maximum concentration of conjugated dienes were recorded as indices of LDL susceptibility to copper-induced oxidation in vitro. As an in vivo marker of oxidative stress we measured 24-h excretion of urinary 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha). All measurements were done after long-term HRT (baseline), after 4 weeks pause and again 3 weeks after reintroduction of HRT. High-density lipoprotein (HDL) cholesterol and apolipoprotein AI concentrations were significantly higher and LDL to HDL ratio significantly lower after long-term oestradiol valerate therapy than after combined therapy. Simultaneously, the triglyceride and lipoprotein (a) levels were higher in the estrogen group. Susceptibility of LDL to oxidation and the level of 8-iso-PGF2alpha were similar in both groups at all measurement points, and treatment group was not a statistically significant determinant of these markers at baseline. According to these results, estrogen and combined HRT do not differ in their abilities to oppose LDL oxidation in vitro or systemic oxidative stress in vivo, but have differential effects on blood lipids.     

Oxidative stress and myocardial damage during elective percutaneous coronary interventions and coronary angiography. A comparison of blood-borne isoprostane and troponin release

The role of oxidative stress in clinical cardiology is still controversial. The aims of the present study were to examine if minor ischaemic episodes as may occur during elective percutaneous coronary intervention (PCI) induce oxidative stress and, eventually, if oxygen stress correlates with myocardial injury. Thirty eight and nine patients underwent PCI and diagnostic coronary angiography, respectively. Peripheral blood was sampled at different time points for plasma analyses of: 8-iso-PGF2alpha (free radical-mediated oxidative stress); 15-keto-dihydro-PGF2alpha (cyclooxygenase-mediated inflammation); troponin-T (myocardial injury); hsCRP, vitamin A and vitamin E; and, total antioxidants status (TAS). In both groups 8-iso-PGF2alpha increased transiently by approximately 80% (p < 0.001) during the procedure. There was a minor troponin-T release (p < 0.001) after PCI, but no correlation with 8-iso-PGF2alpha. Troponin-T did not increase after angiography. 15-keto-dihydro-PGF2alpha decreased by 50% after ended procedure, but increased by 100% after 24 h compared to baseline. hsCRP increased significantly (p < 0.001) from baseline to the next day in the PCI-group, but not in the angiography group. Vitamins and TAS decreased slightly after the procedures. It is concluded that a moderate oxidative stress was induced by both elective PCI and coronary angiography but that no correlation was found between oxidative stress and myocardial injury in this setting. This indicates that other mechanisms than ischaemia-reperfusion episodes caused an elevation in plasma isoprostane such like the injury at a vascular site mutual for both procedures. A secondary finding from the study was elevated markers of early inflammatory response, not only after PCI, but also after angiography.     

Oxidative stress and inflammatory response during and following coronary interventions for acute myocardial infarction

BACKGROUND: In acute myocardial infarction (AMI) treated with percutaneous coronary intervention (PCI), myocardial injury results from complex processes during both ischemia and reperfusion. Release of reactive oxygen species (ROS) may contribute to the accumulated myocardial damage. AIMS: To examine by frequent sampling of peripheral blood oxidative stress and early inflammation in patients undergoing primary PCI for AMI. Secondly, to assess whether a correlation exists between these parameters and the extent of myocardial damage. METHODS: Sixteen patients undergoing primary PCI within 6 h of AMI onset were included. Peripheral blood was sampled at start of procedure (t0) and repeatedly over 24 h following reperfusion. Main plasma analyses were: 8-iso-PGF2alpha (oxidative stress), 15-keto-dihydro-PGF2alpha (cyclooxygenase-mediated inflammation); and troponin-T (myocardial injury). Additional analyses included: total antioxidant status (TAS); vitamins; hsCRP and lipids. RESULTS: 8-Iso-PGF2alpha increased following restoration of blood flow, returned to t0 values after 3 h and was reduced below t0 the following day. TAS decreased significantly from t0 to the next day. There was no significant correlation between 8-iso-PGF2alpha and troponin T values. 15-Keto-dihydro-PGF2alpha was elevated during the first hour. There was a major rise in hsCRP after 24 h. CONCLUSION: Following reperfusion by primary PCI in AMI, oxidative stress and an inflammatory response are induced immediately. A rise in 8-iso-PGF2a during ischemia indicate that ROS generation may also take place during severely reduced coronary blood flow and hypoxia. No direct relationship between 8-iso-PGF2alpha or 15-keto-dihydro-PGF2alpha and troponin T was evident. The present study adds to the increasingly complex pathophysiological roles of ROS acting both as signal molecules and as mediators of tissue injury.     

Radioimmunological measurement of F(2)-isoprostanes after hydrolysis of lipids in tissues

8-Iso-prostaglandin F(2 alpha)(8-iso-PGF(2 alpha)) is a major isoprostane formed in vivo mainly by non-enzymatic peroxidation of arachidonic acid and a potential biomarker of oxidative injury. We have recently reported development of a specific radioimmunoassay for the measurement of free 8-iso-PGF(2 alpha)in plasma and urine. The aim of this study was to employ this radioimmunoassay to analyze the total amount of 8-iso-PGF(2 alpha)(sum of free and esterified) in liver tissues by using alkaline hydrolysis, and to apply it in an experimental model of carbon tetrachloride-induced lipid peroxidation in rats. Basal levels of total 8-iso-PGF(2 alpha)in hydrolyzed liver tissues of control rats were 6.5 times higher than the levels of free 8-iso-PGF(2 alpha). At maximum formation of total 8-iso-PGF(2 alpha)in the livers of carbon tetrachloride-treated rats, total levels of 8-iso-PGF(2 alpha)were almost 13 times higher than the levels of free 8-iso-PGF(2 alpha). In conclusion, high levels of 8-iso-PGF(2 alpha)in tissues can be quantified after hydrolysis both at basal conditions and in a model of increased lipid peroxidation. The methodology for measurement of total levels of 8-iso-PGF(2 alpha)in tissues may be suitable for future investigations of the location of oxidative injury in the body.     

Identification of 8-iso-prostaglandin F(2alpha) in rat brain neuronal endings: a possible marker of membrane phospholipid peroxidation

Isoprostanes are a family of prostaglandin (PG) F and E isomers generated by free-radical attack from membrane bound arachidonic acid. We measured detectable levels of 8-iso-PGF(2alpha) in the perfusates of synaptosomes obtained from different areas of the rat brain cortex. A small but significant release of this isoprostane was found under basal conditions from all the areas explored; being lower in the dorsal cortex in respect to the frontal, parietal and occipital areas. Exposure of synaptosomes to a phospholipase A(2) activator, i.e. calcium-ionophore A23187, an oxidant agent, such as hydrogen peroxide or amyloid beta-peptide did not modify 8-iso-PGF(2alpha) release when these stimuli were applied separately. However, either hydrogen peroxide or amyloid beta-peptide increased 8-iso-PGF(2alpha) release in a dose-dependent manner, when given in the presence of the calcium-ionophore A23187. Synaptosome treatment with a non-selective cyclooxygenase inhibitor (fenoprofen) did not modify 8-iso-PGF(2alpha) release in any way, but treatment with a water soluble antioxidant (Trolox C) completely suppressed isoprostane release under basal conditions, as well as after the oxidant injury induced either by hydrogen peroxide or amyloid beta-peptide. We conclude that, in neuronal endings, 8-iso-PGF(2alpha) is generated under basal conditions and its formation may be increased in a dose-dependent fashion by oxidant stimuli through a cyclooxygenase-independent mechanism involving free radical-catalyzed oxidation of arachidonic acid on membrane phospholipids.     

Antioxidant butylated hydroxyanisole inhibits estrogen‐induced breast carcinogenesis in female ACI rats

Exposure to estrogens is suggested to be a risk factor in human breast cancer development. The mechanisms underlying estrogen‐induced cancer have not been fully elucidated. Both estrogen receptor (ER)‐mediated proliferative processes and ER‐independent generation of oxidative stress are suggested to play important roles in estrogen‐induced breast carcinogenesis. In the current study, we investigated the role of oxidative stress in breast carcinogenesis using the ACI rat model of mammary tumorigenesis. Female ACI rats were treated with 17β‐estradiol (E₂), butylated hydroxyanisole (BHA), or a combination of E₂ + BHA for up to 240 days. Cotreatment of rats with E₂ + BHA reduced estrogen‐induced breast tumor development with tumor incidence of 24%, a significant decrease relative to E₂ where tumor incidence was 82%. Proliferative changes in the breast tissue of E₂ + BHA‐treated animals were similar to those observed in E₂‐treated animals. Tissue levels of 8‐isoprostane, a marker of oxidant stress, as well as the activities of antioxidant enzymes including glutathione peroxidase, superoxide dismutase, and catalase were quantified in the breast tissues of rats treated with E₂ + BHA and compared to activity levels found in E₂‐treated animals and respective age‐matched controls. Cotreatment with BHA inhibited E₂‐mediated increases in 8‐isoprostane levels as well as activities of antioxidant enzymes. In summary, these data suggest that estrogen‐mediated oxidant stress plays a critical role in the development of estrogen‐dependent breast cancers and BHA inhibits E₂‐dependent breast carcinogenesis by decreasing oxidant stress. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:202–211, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20281     

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.     

Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells

There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention.     

Isoprostanes, prostaglandins and tocopherols in pre-eclampsia, normal pregnancy and non-pregnancy

This study is designed to evaluate whether oxidative stress and inflammation are involved in severe pre-eclampsia compared to normal pregnancy and non-pregnancy. We have measured plasma and urinary levels of 8-iso-PGF2alpha, a major isoprostane as an indicator of oxidative stress; plasma and urinary 15-keto-dihydro-PGF2alpha, a major metabolite of cyclooxygenase-catalysed PGF2alpha as an indicator of inflammatory response, and plasma -alpha-and -gamma-tocopherol in 18 pre-eclamptic, 19 normal pregnancy and 20 non-pregnant women. Pregnant women had significantly higher levels of 8-iso-PGF2alpha and PGF2alpha metabolite as compared to the non-pregnancy. Levels of 8-iso-PGF2alpha in the pre-eclamptic women did not differ from the normal pregnancy but PGF2alpha metabolite levels were significantly higher in normal pregnancy. On the other hand, gamma-tocopherol levels were significantly lower in pre-eclampsia than normal pregnancy. In contrast, the concentration of alpha-tocopherol was very similar between the groups. alpha-and gamma-tocopherol levels were significantly lower in pregnancy compared to non-pregnancy. Although no direct evidence of oxidative stress and inflammatory response was observed in severe pre-eclampsia, a reduction of gamma-tocopherol suggests the possible precedence of oxidative stress in this condition. Higher levels of isoprostanes and prostaglandin metabolite in late pregnancy suggest the importance of both free radicals and cyclooxygenase-catalysed oxidation products in normal biological processes of pregnancy.