Use of aspirin, other nonsteroidal anti-inflammatory drugs and acetaminophen and risk of endometrial cancer: the Epidemiology of Endometrial Cancer Consortium.

BACKGROUND: Regular use of aspirin has been associated with a reduced risk of cancer at several sites but the data for endometrial cancer are conflicting. Evidence regarding use of other analgesics is limited. PATIENTS AND METHODS: We pooled individual-level data from seven cohort and five case-control studies participating in the Epidemiology of Endometrial Cancer Consortium including 7120 women with endometrial cancer and 16 069 controls. For overall analyses, study-specific odds ratios (ORs) and 95% confidence intervals (CI) were estimated using logistic regression and combined using random-effects meta-analysis; for stratified analyses, we used mixed-effects logistic regression with study as a random effect. RESULTS: At least weekly use of aspirin and non-aspirin nonsteroidal anti-inflammatory drugs (NSAIDs) was associated with an approximately 15% reduced risk of endometrial cancer among both overweight and obese women (OR = 0.86 [95% CI 0.76-0.98] and 0.86 [95% CI 0.76-0.97], respectively, for aspirin; 0.87 [95% CI 0.76-1.00] and 0.84 [0.74-0.96], respectively, for non-aspirin NSAIDs). There was no association among women of normal weight (body mass index < 25 kg/m2, Pheterogeneity = 0.04 for aspirin, Pheterogeneity = 0.003 for NSAIDs). Among overweight and obese women, the inverse association with aspirin was stronger for use 2-6 times/week (OR = 0.81, 95% CI 0.68-0.96) than for daily use (0.91, 0.80-1.03), possibly because a high proportion of daily users use low-dose formulations. There was no clear association with use of acetaminophen. CONCLUSION: Our pooled analysis provides further evidence that use of standard-dose aspirin or other NSAIDs may reduce risk of endometrial cancer among overweight and obese women.

Determinants of survival and attempted resection in patients with non-metastatic pancreatic cancer: An Australian population-based study.

BACKGROUND: There are indications that pancreatic cancer survival may differ according to sociodemographic factors, such as residential location. This may be due to differential access to curative resection. Understanding factors associated with the decision to offer a resection might enable strategies to increase the proportion of patients undergoing potentially curative surgery. METHODS: Data were extracted from medical records and cancer registries for patients diagnosed with pancreatic cancer between July 2009 and June 2011, living in one of two Australian states. Among patients clinically staged with non-metastatic disease we examined factors associated with survival using Cox proportional hazards models. To investigate survival differences we examined determinants of: 1) attempted surgical resection overall; 2) whether patients with locally advanced disease were classified as having resectable disease; and 3) attempted resection among those considered resectable. RESULTS: Data were collected for 786 eligible patients. Disease was considered locally advanced for 561 (71%) patients, 510 (65%) were classified as having potentially resectable disease and 365 (72%) of these had an attempted resection. Along with age, comorbidities and tumour stage, increasing remoteness of residence was associated with poorer survival. Remoteness of residence and review by a hepatobiliary surgeon were factors influencing the decision to offer surgery. CONCLUSIONS: This study indicated disparity in survival dependent on patients' residential location and access to a specialist hepatobiliary surgeon. Accurate clinical staging is a critical element in assessing surgical resectability and it is therefore crucial that all patients have access to specialised clinical services.

Increasing thyroid cancer incidence in Queensland, Australia 1982-2008 - true increase or overdiagnosis?

BACKGROUND: Thyroid cancer incidence has been increasing worldwide. Some suggest greater ascertainment of indolent tumours is the only driver, but others suggest there has been a true increase. Increases in Australia appear to have been among the largest in the world, so we investigated incidence trends in the Australian state of Queensland to help understand reasons for the rise. METHODS: Thyroid cancers diagnoses in Queensland 1982-2008 were ascertained from the Queensland Cancer Registry. We calculated age-standardized incidence rates (ASR) and used Poisson regression to estimate annual percentage change (APC) in thyroid cancer incidence by socio-demographic and tumour-related factors. RESULTS: Thyroid cancer ASR in Queensland increased from 2·2 to 10·6/100 000 between 1982 and 2008 equating to an APC of 5·5% [95% confidence interval (CI) 4·7-6·4] in men and 6·1% (95% CI 5·5-6·6) in women. The rise was evident, and did not significantly differ, across socio-economic and remoteness-of-residence categories. The largest increase seen was in the papillary subtype in women (APC 7·9%, 95% CI 7·3-8·5). Incidence of localized and more advanced-stage cancers rose over time although the increase was greater for early-stage cancers. CONCLUSION: There has been a marked increase in thyroid cancer incidence in Queensland. The increase is evident in men and women across all adult age groups, socio-economic strata and remoteness-of-residence categories as well as in localized and more advanced-stage cancers. Our results suggest 'overdiagnosis' may not entirely explain rising incidence. Contemporary aetiological data and individual-level information about diagnostic circumstances are required to further understand reasons for rising thyroid cancer incidence.

Breast-feeding and risk of epithelial ovarian cancer.

PURPOSE: Evidence suggests that breast-feeding may decrease the risk of epithelial ovarian cancer but it is not clear whether there is a relationship with duration of breast-feeding, patterns of breast-feeding, or particular histological subtypes of ovarian cancer. We sought to investigate these issues in detail. METHODS: Data from participants in a population-based study of ovarian cancer in western Washington State, USA (2002-2007) who had had at least one birth (881 cases and 1,345 controls) were used to assess relations between patterns of breast-feeding and ovarian cancer. Logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI). RESULTS: Women who ever breast-fed had a 22 % reduction in risk of ovarian cancer compared with those who never breast-fed (OR = 0.78, 95% CI 0.64-0.96) and risk reduction appeared greater with longer durations of feeding per child breast-fed (OR = 0.56, 95% CI 0.32-0.98 for 18 months average duration breast-feeding versus none). Introduction of supplementary feeds did not substantially alter these effects. The overall risk reduction appeared greatest for the endometrioid and clear cell subtypes (OR per month of average breast-feeding per child breast-fed = 0.944, 95% CI 0.903-0.987). CONCLUSIONS: Among women who have had the opportunity to breast-feed, ever breast-feeding and increasing durations of episodes of breast-feeding for each breast-fed child are associated with a decrease in the risk of ovarian cancer independent of numbers of births, which may be strongest for the endometrioid subtype.

Antibodies directed against merozoite surface protein-6 are induced by natural exposure to Plasmodium falciparum in a low transmission environment.

Malaria caused by Plasmodium falciparum is a major cause of global infant mortality, and there is currently no licensed vaccine that provides protection against infection or disease. Several P. falciparum vaccine targets have undergone early testing, but many more candidates remain with little data to support their development. Plasmodium falciparum Merozoite Surface Protein 6 (PfMSP6) is a candidate of particular interest because it is a member of the PfMSP3 multi-gene family, raising the possibility that vaccine-induced immune responses could cross-react across multiple family members. However, few immunoepidemiological studies of PfMSP6 have been carried out to measure domain-specific anti-PfMSP6 responses. This study investigated anti-PfMSP6 responses in P. falciparum-infected individuals from the Peruvian Amazon, using two different PfMSP6 N-terminal allele antigens and a single C-terminal domain antigen, and compared the responses with both PfMSP6 genotyping data and anti-PfMSP3 response data that had been previously generated for the same samples. Anti-PfMSP6 responses were detected despite the low transmission setting, but were less frequent and of considerably lower intensity than anti-PfMSP3 responses. There was a positive correlation between anti-PfMSP3 and PfMSP6 responses, suggesting that the possibility that PfMSP3 family antigens could induce cross-reactive responses requires further detailed investigation.

Marked intra-strain variation in response of Listeria monocytogenes dairy isolates to acid or salt stress and the effect of acid or salt adaptation on adherence to abiotic surfaces.

During food processing, and particularly in cheese manufacturing processes, Listeria monocytogenes may be exposed routinely to environments of low pH or high salt concentration. It has been suggested that these environmental conditions may contribute to bacterial adherence to abiotic surfaces and increased resistance to disinfection. In this study strains isolated from the environment of artisanal cheese-making dairies were used to investigate the behaviour of L. monocytogenes in response to acid and salt stress and clear differences between strains was observed. In planktonic culture, strains varied in resistance to low pH or high NaCl concentration and in the occurrence of an adaptive response to moderate acid or NaCl. There was dislocation in responses to salt and acid. Strains resistant, or adaptive, to acid were not resistant or adaptive to NaCl. The reverse also was observed. Exposure to moderate acid did not promote adherence to polystyrene but survival, at low pH or high NaCl concentration, of cells adherent to stainless steel was increased, even for strains that had no adaptive response planktonically, but the detail of these observations varied between strains. In contrast to acid adaptation, with some strains salt adaptation enhanced adherence of L. monocytogenes to polystyrene but this was not true for all strains. For some strains salt- or acid adaptation may enhance the survival of sessile cells exposed to hypochlorite disinfection.

Use of single-strand conformation polymorphism analysis to examine the variability of the rpoS sequence in environmental isolates of Salmonellae.

The natural environment places its resident microflora under stress, which may often result in adaptation by the microflora in order to increase the probability of survival. One such mechanism that has been postulated involves rpoS, which encodes a sigma factor that is known to enhance survival upon exposure to stress. The present work aimed to examine the genetic variability of rpoS in a selection of Salmonella enterica subspecies environmental isolates with an automated single-strand conformation polymorphism analysis technique. The results indicated that sequence variation does occur and that these changes are mainly located in two areas: at the center and near the end of the coding region. The variability was generally at the single-base level, although one strain (S. arizonae) did demonstrate significant differences in nucleotide sequence.

In vivo production of nitric oxide in rats after administration of hydroxyurea.

The metabolism of nitrovasodilators such as glyceryl trinitrate and nitroprusside provides the active moiety of these drugs (that is, nitric oxide). This process is not limited to the known nitrovasodilators, but also occurs with nitroaromatic antimicrobials. Here we report that the administration of hydroxyurea, an antitumor drug, to rats at pharmacological doses formed detectable nitrosyl hemoglobin, which increased with dose. At higher doses, nitrosyl hemoprotein complexes could also be detected in liver tissue. [15N]hydroxyurea was synthesized and compared with [14N]hydroxyurea. These observations verified that nitric oxide detected as nitrosyl hemoglobin or nitrosyl hemoprotein complexes in rats was the result of the metabolism of hydroxyurea. The time course and dose-dependence of nitric oxide generation were also investigated. Hydroxyurea's antineoplastic activity is caused by its direct action on ribonucleotide reductase, the rate-limiting enzyme in DNA synthesis. Because nitric oxide also inhibits ribonucleotide reductase, this metabolite may supplement this action of hydroxyurea. In addition, the known ability of hydroxyurea to ease the pain of sickle cell anemia patients may be the result of vasodilation by the drug-derived nitric oxide.

Detection of free radical metabolite formation using in vivo EPR spectroscopy: evidence of rat hemoglobin thiyl radical formation following administration of phenylhydrazine.

The spin-trapping technique in conjunction with a low-frequency electron paramagnetic (or spin) resonance (EPR or ESR) spectrometer was used to detect the hemoglobin thiyl free radical in living rats using a whole body resonator. The hemoglobin thiyl free radical was formed following the intragastric administration of phenylhydrazine at the LD50 dose of 188 mg/kg. The hemoglobin thiyl free radical was then trapped by preinjected 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which formed the DMPO/hemoglobin thiyl-free radical adduct in the blood. The time course of the in vivo formation and disappearance of the spin adduct was followed. The DMPO/hemoglobin thiyl free radical was detected in blood samples using 9.5 GHz (X-band) and 1.1 GHz (L-band) EPR at room temperature and 77 K. Pretreatment of rats with ascorbate and diethylmaleate (DEM) decreased the signal intensity of the DMPO/hemoglobin thiyl free radical spin adduct. The incubation of ascorbate or DEM at 37 degrees C with rat blood containing preformed DMPO/hemoglobin thiyl radical adduct showed that there was no effect of DEM on the free radical concentration, while ascorbate reduced the radical adduct. This study provided direct evidence of the formation of the DMPO/hemoglobin thiyl free radical in vivo and enabled us to study this formation in living animals free of any artifacts that can occur when using ex vivo methods.

Tumor necrosis factor-alpha and nitric oxide production in endotoxin-primed rats administered carbon tetrachloride.

Tumor necrosis factor-alpha (TNF alpha) is elevated in the sera of rats administered non-lethal doses of carbon tetrachloride (CCl4) followed by endotoxin. Elevated TNF alpha levels are correlated with the increased release of hepatic enzymes indicating hepatic damage. Under these conditions, nitric oxide (NO) was also produced in the liver as evidenced by the formation of nitrosyl complexes which were measured by electron paramagnetic resonance (EPR) spectroscopy. Decreased nitrosyl complex formation occurred in livers following treatment with either an inhibitor or macrophage activation (gadolinium trichloride; GdCl3), an inhibitor of cytokine responses (dexamethasone) or a NO synthase inhibitor (NG-monomethyl-L-arginine; 1-NMA), GdCl3 or dexamethasone treatment decreased, while 1-NMA treatment increased, TNF alpha serum level. Taken together, these data suggest that TNF alpha and NO are induced following CCl4 and LPS exposure and may be important regulators in the hepatotoxicity of this liver injury model.

Targets of nitric oxide in a mouse model of liver inflammation by Corynebacterium parvum.

Treatment of mice with Corynebacterium parvum induces chronic inflammation. This treatment followed by an injection of lipopolysaccharide (LPS) produces hepatic necrosis and death. We examined liver tissue by using electron paramagnetic resonance (EPR) spectroscopy and found that, in addition to the previously reported nonheme nitrosyl complexes, heme nitrosyl complexes were also formed. Hemoglobin nitrosyl complexes measured in the whole blood of mice treated with C. parvum were not increased after additional LPS treatment. However, this treatment significantly increased the heme nitrosyl complexes in the liver, whereas the nonheme nitrosyl complex concentration was unaffected. EPR signals from whole blood and liver tissues from mice treated with C. parvum and C. parvum + LPS were inhibited by prolonged treatment with NG-monomethyl-L-arginine (L-NMA). Nitric oxide (.NO) is known to bind to cytochrome P450 heme, and we consistently found a suppression of EPR signals attributable to ferric low-spin cytochrome P450/P420 peaks in the livers of mice treated with C. parvum and C. parvum + LPS. By performing analyses of EPR spectra obtained from hepatocytes exposed to .NO, we were able to unambiguously identify EPR signals attributable to cytochrome P420 and nonheme nitrosyl complexes in the livers of both treatments. Deconvolution of the composite in vivo EPR spectra indicated that hemoglobin nitrosyl complexes contributed weakly in the C. parvum livers, but threefold more in the C. parvum + LPS livers, suggesting that hemorrhage may have occurred. Experiments with L-NMA treatment revealed that this additional .NO production did not correlate with hepatic necrosis and onset of death. Immunoprecipitation of liver cytosols from C. parvum- and (C. parvum + LPS)-treated mice using an antibody against mouse inducible nitric oxide synthase showed that this enzyme was indeed present in the cytosolic fractions and was absent in those from control livers. Our novel detection of cytochrome P420 nitrosyl complex in vivo may be linked to any role of hepatic P450's functions during liver inflammation.

The metabolism of 17 beta-estradiol by lactoperoxidase: a possible source of oxidative stress in breast cancer.

Electron spin resonance (ESR) spectroscopy and oxygen consumption measurements using a Clark-type oxygen electrode have been used to study the metabolism of the estrogen 17 beta-estradiol by lactoperoxidase. Evidence for a one-electron oxidation of estradiol to its reactive phenoxyl radical intermediate is presented. The phenoxyl radical metabolite abstracts hydrogen from reduced glutathione generating the glutathione thiyl radical, which is spin trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and subsequently detected by ESR spectroscopy. In the absence of DMPO, molecular oxygen is consumed by a sequence of reactions initiated by the glutathione thiyl radical. Similarly, the estradiol phenoxyl radical abstracts hydrogen from reduced beta-nicotinamide-adenine dinucleotide (NADH) to generate the NAD. radical. The NAD. radical is not spin trapped by DMPO, but instead reduces molecular oxygen to the superoxide radical, which is then spin-trapped by DMPO. The superoxide generated may either spontaneously dismutate to form hydrogen peroxide or react with another NADH to form NAD., thus propagating a chain reaction leading to oxygen consumption and hydrogen peroxide accumulation. Ascorbate inhibits oxygen consumption when estradiol is metabolized in the presence of either glutathione or NADH by reducing radical intermediates back to their parent molecules and forming the relatively stable ascorbate radical. These results demonstrate that the futile metabolism of micromolar quantities of estradiol catalyzes the oxidation of much greater concentrations of biochemical reducing cofactors, such as glutathione and NADH, with hydrogen peroxide produced as a consequence. The accumulation of intracellular hydrogen peroxide could explain the hydroxyl radical-induced DNA base lesions recently reported for female breast cancer tissue.

Nitric oxide production during endotoxic shock in carbon tetrachloride-treated rats.

Earlier studies showed that hepatotoxicant-treated experimental animals were more susceptible than controls to the lethal effects of bacterial endotoxin. The exact mechanisms of this effect were not understood. In this paper we showed that nitric oxide (.NO) was produced in whole blood and in liver tissues of rats that had been treated with a nonlethal dose of CCl4 (1.3 g/kg) followed by a low dose of lipopolysaccharide (LPS) (100 micrograms/kg). EPR spectroscopy was used in this study to detect nitrosyl-protein complexes. Hemoglobin-nitrosyl complexes were detected in both whole blood and liver. By performing analyses of EPR spectra obtained from hepatocytes exposed to .NO, we were able to identify EPR signals attributable to nitrosyl-cytochrome P420 in rat liver. We found that nitrosyl complex formation in red blood cells and liver was inhibited by treatment with NG-mono-methyl-L-arginine, suggesting enzymatic biosynthesis of .NO. A small but significant inhibition of nitrosyl complex formation by gadolinium trichloride pretreatment was found in the liver, suggesting that Kupffer cells were also involved in .NO biosynthesis, because this treatment decreased Kupffer cells. There was a synergistic effect of CCl4 and LPS on the serum levels of the hepatic enzymes aspartate aminotransferase, alanine amino-transferase, lactate dehydrogenase, and sorbitol dehydrogenase, which are indices of parenchymal cell damage. NG-Mono-methyl-L-arginine treatment increased these hepatic enzyme activities, suggesting a protective role for .NO. EPR resonances at g approximately 2.48, 2.29, and 1.91, due to low-spin cytochromes P450/P420 (FE3+), were decreased in the livers of LPS-induced rats that had been previously treated with CCl4, indicating cytochrome P450/P420 destruction or at least a change in the valence state of the cytochrome P450/P420 heme groups to Fe2+ in the presence of .NO. Because nitrosyl-cytochrome P450 is not stable, the concomitant detection of nitrosyl-cytochrome P420 (Fe2+) could account, at least in part, for the decrease of the ferric low-spin heme groups. Our novel observations of hepatic nitrosyl species suggest that .NO plays an important role during hepatic injury caused by CCl4 in hosts exposed to endotoxin.

Role of metallothionein in zinc(II) and chromium(III) mediated tolerance to carbon tetrachloride hepatotoxicity: evidence against a trichloromethyl radical-scavenging mechanism.

The .CCl3 radical generated during the metabolism of CCl4 is readily spin trapped in vivo and in vitro by phenyl N-tert-butylnitrone (PBN) to form the stable PBN/.CCl3 radical adduct, which can then be extracted into organic solvents and detected by ESR spectroscopy. We have used this technique to examine the proposed protective roles of Zn(II), Cr(III), and metallothionein (MT) against carbon tetrachloride toxicity in vivo. Hepatic MT, which is induced by Zn(II), has been proposed to protect against CCl4-induced cellular damage by scavenging the free radical metabolites formed. CCl4-induced hepatotoxicity was significantly suppressed in male Sprague-Dawley rats pretreated with a single dose of 5 mg/kg Zn(II) or Cr(III) according to standard serum assays for liver-specific enzymes, and hepatic MT was elevated after pretreatment with either Zn(II) or Cr(III). In vitro, no difference was detected in either the amount of CCl4-derived free radical metabolites formed or the rate at which they were formed by microsomes from rats pretreated 24 h in advance with 5 mg/kg Zn(II) or Cr(III). Extraction of rat liver with 2:1 chloroform/methanol 1 h after the administration of a 0.8 mL/kg intraperitoneal or intragastric dose of CCl4 also revealed no difference in the amount of trichloromethyl radical spin trapped in vivo following pretreatment with either Zn(II) or Cr(III). These results suggest that pretreatment with either Zn(II) or Cr(III) does not affect CCl4 metabolism nor does the MT significantly scavenge the trichloromethyl free radical metabolite.

Electron spin resonance evidence for free radical generation in copper-treated vitamin E- and selenium-deficient rats: in vivo spin-trapping investigation.

The ESR spin-trapping technique has been used to investigate free radical generation in copper-challenged rats deficient in vitamin E and/or selenium. Radical adduct excreted in the bile was detected only from copper-challenged rats deficient in both vitamin E and selenium. The phenyl-N-t-butylnitrone radical adduct has hyperfine coupling constants of aN = 15.36 G and a beta H = 2.50 G and arises from the trapping of a radical formed from an endogenous molecular species. The induction of this radical species in vivo may be important in the increased toxicity of copper in rats deficient in both vitamin E and selenium. These findings support the proposal that dietary selenium and vitamin E can protect against lipid peroxidation and copper toxicity. The results obtained suggest that the presence of only one of these nutrients in the diet is enough to prevent the formation of this radical adduct at ESR-detectable levels, and they provide the most direct ESR evidence yet obtained for the involvement of in vivo lipid peroxidation in the toxicity of copper.

Nitric oxide formation during light-induced decomposition of phenyl N-tert-butylnitrone.

Phenyl N-tert-butylnitrone (PBN) is a spin trap commonly employed in free radical research. PBN has been shown to have adverse and beneficial effects on various biological systems. We report here evidence that photolysis (or even ambient light) decomposes PBN to nitric oxide in aqueous solutions. Non-heme and heme proteins have been employed to form nitrosyl complexes, which were detected using EPR spectroscopy. Concomitantly, nitrite formation was detected after light-induced decomposition of PBN. In addition, we found that tert-nitrosobutane and decomposed PBN caused an activation of guanylate cyclase. We propose a mechanism where PBN is decomposed by light to tert-nitrosobutane. The latter compound is, in turn, decomposed to nitric oxide. This study suggests the possibility that PBN or PBN radical adducts may be sources of nitric oxide in biological environments. When using PBN as a spin trap in biological samples, not only is the trapping of reactive free radicals operative, but nitric oxide produced from PBN decomposition may play an important role in altering biological functions.

Electron spin resonance spin-trapping investigation into the effects of paraquat and desferrioxamine on hydroxyl radical generation during acute iron poisoning.

We have previously described a secondary radical-trapping technique for the detection of in vivo hydroxyl radical generation during acute iron overload. With this technique, the hydroxyl radical (.OH) reacts with dimethylsulfoxide to form the methyl radical (.CH3), which is then detected by ESR spectroscopy as its adduct with the spin trap phenyl-N-tert-butylnitrone in the bile of treated animals. In this study, we report both the individual and combined effects of the futile-cycling agent paraquat (PQ2+) and the iron-chelating agent desferrioxamine (DFO) on iron-dependent .OH generation. Interactions between iron and the partially reduced oxygen species superoxide and hydrogen peroxide, which are generated during the metabolism of PQ2+, might be expected to stimulate .OH generation to a level above that seen in the presence of the metal ion alone. Although PQ2+ was often found to promote further .OH generation when administered to animals also given iron, the large variation observed between individual animals in response to the reagent meant that the effect was not statistically significant (p < 0.05). DFO was found to abolish iron-dependent .OH generation, both in the presence and in the absence of PQ2+. This is believed to result from the chelation of iron by DFO, to form an essentially redox-inert iron(III) complex that is unable to catalyze .OH radical formation. In addition, it was found that the iron(II) complex of DFO can reduce PQ2+ to its radical cation in vitro, indicating, therefore, that the chelation of iron by DFO may not necessarily prevent its participation in free radical reactions.

Fatty acid radical formation in rats administered oxidized fatty acids: in vivo spin trapping investigation.

We report in vivo evidence for fatty acid-derived free radical metabolite formation in bile of rats dosed with spin traps and oxidized polyunsaturated fatty acids (PUFA). When rats were dosed with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and oxidized PUFA, the DMPO thiyl radical adduct was formed due to a reaction between oxidized PUFA and/or its metabolites with biliary glutathione. In vitro experiments were performed to determine the conditions necessary for the elimination of radical adduct formation by ex vivo reactions. Fatty acid-derived radical adducts of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were detected in vivo in bile samples collected into a mixture of iodoacetamide, desferrioxamine, and glutathione peroxidase. Upon the administration of oxidized 13C-algal fatty acids and 4-POBN, the EPR spectrum of the radical adducts present in the bile exhibited hyperfine couplings due to 13C. Our data demonstrate that the carbon-centered radical adducts observed in in vivo experiments are unequivocally derived from oxidized PUFA. This in vivo evidence for PUFA-derived free radical formation supports the proposal that processes involving free radicals may be the molecular basis for the previously described cytotoxicity of dietary oxidized PUFA.