Evaluating the impact of polycyclic aromatic hydrocarbon bioaccumulation in adipose tissue of obese women.

Polycyclic aromatic hydrocarbons (PAHs) are widespread across the environment and humans are unavoidably and constantly exposed to them. As lipophilic contaminants, these substances tend to accumulate in fatty tissues as adipose tissue and exposure to these endocrine disruptors has been associated with severe health hazards including prevalence and incidence of obesity. Previous studies have shown significantly higher concentrations of PAHs in adipose tissue compared to other human samples, such as urine and plasma, which are typically used for PAHs assessment. Therefore, conducting biomonitoring studies in adipose tissue is essential, although such studies are currently limited. In this study, the concentrations of 18 PAHs were measured in subcutaneous (scAT) and visceral adipose tissue (vAT) of 188 Portuguese obese females by high performance liquid chromatography (HPLC). The obtained results were then associated with the patient's data namely: 13 clinical, 4 social, and 42 biochemical parameters. Seventeen PAHs were present, at least, in one sample of both scAT and vAT, most of them with detection frequencies higher than 80%. Indeno [1,2,3-cd]pyrene (InP) was the only PAH never detected. Overall higher concentrations of PAHs were observed in scAT. Median concentrations of ∑PAHs were 32.2 ± 10.0 ng/g in scAT and 24.6 ± 10.0 ng/g in vAT. Thirty-six significant associations (7 with social, 18 with clinical, and 11 with biochemical parameters), including 21 Spearman's correlations were identified (12 positive and 9 negative correlations). Indicating the potential effects of PAHs on various parameters such as obesity evolution, body fat, number of adipocytes, total cholesterol, alkaline phosphatase, macrominerals, uric acid, sedimentation velocity, and luteinizing hormone. This study underscores the significance of biomonitoring PAH levels in adipose tissue and their potential effects on metabolic health. Further research is essential to fully comprehend the metabolic implications of PAHs in the human body and to develop strategies for obesity prevention and treatment.

Analysis of atorvastatin in environmental waters: Validation of an electrochemical molecularly imprinted polymer sensor with application of life cycle assessment.

The widespread presence of pharmaceuticals in wastewater effluents after treatment stands as a significant challenge faced in the field of wastewater management and public health. Governments and the scientific community have worked to meet this urgent need for effective solutions. Nevertheless, the development of detection strategies for pharmaceutical monitorization capable of delivering rapid, on-site, and sensitive responses remains an ongoing necessity. In this work, the performance of a previously developed molecularly imprinted polymer (MIP) based electrochemical sensor for detecting atorvastatin (ATV) in wastewater effluents and surface waters is presented. A simple preconcentration method followed by electrochemical measurements by differential pulse voltammetry (DPV) in 0.1 M phosphate buffer (pH = 7), was implemented. The analytical results were validated with those obtained on a set of 16 water samples by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Additionally, a life cycle assessment (LCA) was conducted to compare the environmental impact of both methodologies. The results obtained demonstrated that ATV detection using MIP-sensor was reliable when compared to the results found by UHPLC-MS/MS presenting a robust linear correlation coefficient of 0.843. The LCA results show that the novel MIP-sensor technique has lower associated environmental impacts than UHPLC-MS/MS, when the current analytical protocol for pharmaceuticals detection is applied. These findings highlight the potential of the developed MIP-sensor as an eco-friendly analytical tool for routine analysis and point-of-care monitoring of ATV in WWTP wastewater and surface water samples.

Optimization of a simple, effective, and greener methodology for polycyclic aromatic hydrocarbon extraction from human adipose tissue.

Polycyclic aromatic hydrocarbons (PAHs) are environmentally persistent organic pollutants formed during incomplete combustion and pyrolysis processes. Humans are continuously exposed to PAHs which are linked to severe health effects such as diabetes, cancer, infertility, and poor foetal development, amongst others. PAHs are lipophilic compounds prone to accumulating in adipose tissue. Even though adipose tissue is the ideal matrix to assess over time accumulation of lipophilic pollutants, only a few analytical methods have been developed for this matrix. Aiming to reduce the existent gap, a method for the extraction of PAHs from adipose tissue samples using ultrasound-assisted extraction (UAE) was developed. The behaviour of PAHs (retention, adsorption, and volatilization) over several steps of the analytical procedure was studied. Validation tests were performed on the optimized method. PAHs were quantified using a high performance liquid chromatography (HPLC) system equipped with a photodiode array (PDA) and fluorescence (FLD) detector inline. The method achieved a low matrix effect and presents low method detection (MDL) and quantification (MQL) limits, showing suitability for a selective and sensitive determination of PAHs in adipose tissue. The extraction is performed with 0.4 g of adipose tissue and 6 mL of n-hexane and it does not require clean-up afterwards. Additionally, an Eco-Scale score of 74 and an Analytical GREEnness score of 0.66 were obtained. The method achieved is effective, simpler, greener, and easy to perform, being an alternative to conventional extraction methods. Furthermore, this method can be used as a multi-analyte methodology since it has been previously validated by the authors for the analysis of other lipophilic compounds. Naphthalene (Naph), acenaphthene (Ace), fluorene (Flu), phenanthrene (Phe), anthracene (Ant), fluoranthene (Fln), pyrene (Pyr) and benzo[k]fluoranthene (B[k]Ft) were found in all the tested adipose tissue samples.

Seasonal and Spatial Comparison of Polycyclic Aromatic Hydrocarbons Among Decapod Shrimp from Coastal Portugal.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous global pollutants that are lipophilic, bioaccumulative and can be both endocrine disruptors and carcinogens. In this study, we measured 18 PAHs among decapod shrimp samples (Palaemon spp.) collected during the spring and autumn from multiple locations along the Portuguese coastal, including local markets and aquaculture farms. ΣPAH concentrations in samples ranged from 3.14 to 320.48 ng/g (ww) with the highest value in this range detected in a sample provided from an aquaculture facility. Fifteen of the 18 scanned PAHs were present in shrimp samples. However, detectable concentrations (0.07 ng/g ww) of the carcinogenic benzo-a-pyrene occurred for a single sample only and approached method detection limits. No significant differences in shrimp ΣPAH concentrations were evident between the spring and autumn seasons. Target hazard quotient estimation suggested low probability of adverse health effects to consumers through consumption of shrimp from the study collection locations.

Evaluation of the Biological Potential of Himanthalia elongata (L.) S.F.Gray and Eisenia bicyclis (Kjellman) Setchell Subcritical Water Extracts.

Neuroprotection is a need that remains unmet in treating chronic neurodegenerative disorders, despite decades of extensive research. To find new neuroprotective compounds, extracts of Himanthalia elongata (L.) S.F.Gray and of Eisenia bicyclis (Kjellman) Setchell were obtained through subcritical water extraction applying a four-step temperature gradient. The fractions obtained were screened against brain enzymes involved in neurodegenerative etiology, namely in Alzheimer's and Parkinson's diseases, and against reactive oxygen and nitrogen species, all contributing factors to the progression of neurodegeneration. Results showed no significant enzyme inhibition but strong radical scavenging activities, particularly in the fourth fraction, extracted at the highest temperature (250 °C), highlighting their ability to retard oxidative and nitrosative stresses. At higher temperatures, fractions were composed of phenolic compounds and Maillard reaction products, a combination that contributed to their antioxidant activity and, consequently, their neuroprotective properties. All fractions were evaluated for the presence of iodine, 14 organochlorine and 7 organophosphorus pesticides, and pharmaceuticals used in Alzheimer's and Parkinson's diseases (14), psychiatric drugs (8), and metabolites (8). The fractions studied did not present any of the screened contaminants, and only fraction 1 of E. bicyclis should be used with caution due to iodine content.

Multi-residue analysis of fifty pesticides in river waters and in wastewaters.

Three sampling campaigns were performed in the Lis River (Leiria, Portugal) in February of 2018, November of 2018, and May of 2019. River water and wastewater (influent and effluent) samples of two wastewater treatment plants were target of the study. A total of 25 samples were collected and 50 pesticides were monitored, including organochlorines, triazines, pyrethroids, and organophosphorus, among others. Most of the detected pesticides were insecticides and mainly organochlorines. Concentrations between 1.29 and 2134 ng/L were found. Aldrin, γ-HCH, and cypermethrin were detected in some samples in µg/L, being γ-HCH the pesticide most frequently detected with concentration in µg/L level. The pesticides with the highest detection frequency were (i) cypermethrin, HCB, methoxychlor, and ζ-HCH in river waters; (ii) isoproturon, cypermethrin, methoxychlor, pyrimethanil, γ-HCH, dieldrin, diuron, α-HCH, and α-endosulfan in effluents; and (iii) diuron and isoproturon in influents. The detection of the organochlorides and their degradation products is a consequence of their persistence in the environment, as their usage has long been prohibited in the European Union. Pesticides were grouped by their types in herbicides, insecticides, or fungicides and the detection and concentration for each type were discussed with the climatic conditions. Pesticide toxicity index was determined in the samples collected in the river.

Quantification of fluoroquinolones in wastewaters by liquid chromatography-tandem mass spectrometry.

Antibiotics are the most consumed therapeutic classes worldwide and are released to the environment in their original form as well as potentially active metabolites and/or degradation products. Consequences of the occurrence of these compounds in the environment are primarily related to bacterial resistance development. This work presents a validated analytical method based on solid phase extraction (SPE) using HLB cartridges, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantification of seven different fluoroquinolone antibiotics, namely ciprofloxacin (CPF), enrofloxacin (ENR), lomefloxacin (LOM), norfloxacin (NOR), ofloxacin (OFL), prulifloxacin (PLF) and moxifloxacin (MOX) and its application to detect the target compounds in influents and effluents of wastewater treatment plants (WWTP). Linearity was established through calibration curves in solvent and matrix match using internal calibration method in the range of 50-1300 ng L-1 and all the fluoroquinolones showed good linear fit (r2 ≥ 0.991). Accuracy ranged between 80.3 and 92.9%, precision was comprised between 7.2 and 14.6%, and 10.7 and 18.1% for intra- and inter-batch determinations, respectively. Method detection and quantification limits ranged from 6.7 to 59.0 ng L-1 and 22.3-196.6 ng L-1, respectively. Influents and effluents of fifteen WWTPs of North of Portugal were analyzed. OFL was the fluoroquinolone found at the highest concentration, up to 4587.0 ng L-1 and 987.9 ng L-1, in influent and effluent, respectively. NOR and PLF were not detected.

Antibiotics and antidepressants occurrence in surface waters and sediments collected in the north of Portugal.

To our knowledge, this is the first study in Portugal for the analysis of both antibiotics and psychiatric drugs in river waters from Douro and Leça rivers and its sediments. Samples were extracted using solid phase extraction and QuEChERS procedures and the analysis was performed using liquid chromatography with tandem mass spectrometry. Higher number of pharmaceuticals and higher concentrations were found in the Leça river for the two matrices under study. The highest detection frequency for the twenty-seven pharmaceuticals was observed for fluoxetine with 83.3% in both matrices. None of the studied antibiotics were detected in water collected in the Douro river however six antibiotics (azithromycin, ciprofloxacin, clarithromycin, moxifloxacin, ofloxacin, and trimethoprim) were found in the Leça river. Further, the analysis of sediments exhibits the occurrence of sulfamethoxypyridazine in the Douro river and azithromycin in the Leça river. The highest concentration was observed in Leça river for azithromycin in the river water (2,819 ng/L) and sediments (43.2 ng/g). Carbamazepine, citalopram, fluoxetine, sertraline, trazodone, and venlafaxine were the psychiatric drugs detected in river waters and sediments with more diazepam as water river contamination. Concentrations ranged from

Monitoring survey of caffeine in surface waters (Lis River) and wastewaters located at Leiria Town in Portugal.

Investigation during 11-month period was performed to study the presence of caffeine in the Lis River in Leiria Town in Portugal, and a monitoring during 9-month period was realized to check the contribution of the human pollution of two wastewater treatment plants (WWTPs) that discharge their effluents to the studied river. The samples were collected in five sampling points along the river and in two influents and two effluents of the studied WWTPs. Caffeine was detected in all ninety-one collected samples. The caffeine concentration ranged from 25.3 to 321 ng/L in the river samples, from 112 to 1927 ng/L in the WWTP effluents, and from 9478 to 83,901 ng/L in the WWTP influents. The highest concentration in the river was detected in the two sampling points located after the effluent discharge points and reached 315 and 321 ng/L. Risk assessment was performed for three trophic levels using the risk quotient calculation and revealed that caffeine do not cause toxic effect on Daphnia magna and on fish but could be possibly toxic to algae. The results proved that caffeine can be an effective indicator of human-born pollution.

Evaluation of the adsorption potential of biochars prepared from forest and agri-food wastes for the removal of fluoxetine.

Twelve biochars from forest and agri-food wastes (pruning of Quercus ilex, Eucalyptus grandis, Pinus pinaster, Quercus suber, Malus pumila, Prunus spinosa, Cydonia oblonga, Eriobotrya japonica, Juglans regia, Actinidia deliciosa, Citrus sinensis and Vitis vinifera) were investigated as potential low-cost and renewable adsorbents for removal of a commonly used pharmaceutical, fluoxetine. Preliminary adsorption experiments allowed to select the most promising adsorbents, Quercus ilex, Cydonia oblonga, Eucalyptus, Juglans regia and Vitis vinifera pruning material. They were characterized by proximate, elemental and mineral analysis, thermogravimetric analysis, Fourier transform infrared spectroscopy, determination of specific surface area and pH at the point of zero charge. Batch and equilibrium studies were performed, and the influence of pH was evaluated. The equilibrium was reached in less than 15 min in all systems. The maximum adsorption capacity obtained was 6.41 mg/g for the Eucalyptus biochar, which also demonstrated a good behavior in continuous mode (packed column).

Mineral Content of Various Portuguese Breads: Characterization, Dietary Intake, and Discriminant Analysis.

The chemical composition and daily mineral intake (DMI) of six macro (calcium, magnesium, sodium, potassium, phosphorous, and chloride) and four microminerals (copper, iron, manganese, and zinc) were determined in four types of Portuguese breads (white wheat, maize, wheat/maize, and maize/rye breads). Samples were processed with microwave assisted digestion and mineral composition was determined with a high-resolution continuum-source atomic absorption spectrometer with flame and graphite furnace. Bread contributes to an equilibrated diet since it is rich in several minerals (0.21 mg/100 g of copper in wheat bread to 537 mg/100 g of sodium in maize/rye bread). Maize/rye bread presented the highest content of all minerals (except phosphorous and chloride), while the lowest levels were mainly found in wheat bread. Median sodium concentrations (422-537 mg/100 g) represented more than 28% of the recommended daily allowance, being in close range of the maximum Portuguese limit (550 mg/100 g). Maize/rye bread exhibited the highest DMI of manganese (181%), sodium (36%), magnesium (32%), copper (32%), zinc (24%), iron (22%), potassium (20%), and calcium (3.0%). A Principal Component Analysis (PCA) model based on the mineral content allowed the differentiation among white wheat, maize, and maize/rye bread. Zinc, magnesium, manganese, iron, phosphorus, potassium, copper, and calcium proved to be good chemical markers to differentiate bread compositions.

Assessment of 83 pharmaceuticals in WWTP influent and effluent samples by UHPLC-MS/MS: Hourly variation.

The removal efficiency of pharmaceuticals in wastewater treatment plants (WWTPs) is variable and some of these compounds pass these plants almost intact and others presenting a removal efficiency close to 100%. Their incomplete removal results in a continuous discharge of pharmaceuticals into the environment. To assess the profile of contamination of influents and effluents over a day, a set of 83 pharmaceuticals were evaluated hourly in a WWTP in Leiria, Portugal. The composite samples of the influent and effluent were also collected. Concentrations varied from 1 in WWTP influents, and carbamazepine, fluoxetine, sertraline the pharmaceuticals with an RQ > 1 in WWTP effluents.

Anthropogenic contamination of Portuguese coastal waters during the bathing season: Assessment using caffeine as a chemical marker.

Bathing water quality standards are based on international standards and legislations. However, in Europe, only the microbiological parameters are to be accomplished. Recent research has focused on chemical indicators that can determine human fecal contaminants in water. Therefore, the suitability of caffeine as a chemical marker of seawater pollution in the north Portuguese coastal area in the Atlantic Ocean during the bathing season was assessed in this study. The quality of water from the coasts of five cities was monitored. 101 seawater samples were collected from 14 beaches, and their water quality was classified as sufficient, good, and excellent. Caffeine was detected in all samples in concentration range of 18 to 525ng/L. The highest average concentration of caffeine was found in seawater samples collected in July from beaches classified as having sufficient water quality, which were located in cities with high population density and high tourist affluence.

Analysis of pharmaceutical adulterants in plant food supplements by UHPLC-MS/MS.

A method based on the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) extraction and ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was successfully developed and validated for the analysis of 26 pharmaceutical compounds belonging to different therapeutic classes (anorexics, stimulants, anxiolytics, antidepressants and laxatives), which are all prone to be illegally added into weight-loss plant food supplements (PFS) for their pharmacological activity. Internal standard calibration with six isotopically labelled compounds rendered good linearity in the range of 5 to 1000µg/l, depending on the compound, and good sensitivity with limits of quantification in the range of 0.02-9.80µg/l. Recoveries were assessed for all the 16 samples analysed and were found between 70% and 120% for over 90% of the analytes. The average recovery value was 90.8%, for the different studied matrices (liquids, liquid ampoules, tablets and capsules), with RSD values lower than 10% for all forms. The changes introduced to the QuEChERS procedure maintained the good performance characteristics of the extraction method while preserving the chromatographic system for the introduction of unwanted matrix compounds. Synephrine was the only compound detected and quantified in one sample, but at a very low concentration (768µg/l) and its presence may be due to the plant extracts used in the formulation, as synephrine is known to be a natural constituent of Citrus aurantium amara. Despite none of the 16 evaluated samples were found to be adulterated by the illegal addition of the drugs included in this work, the developed methodology can be very useful for monitoring the adulteration of weight-loss PFS.

Presence of pharmaceuticals in the Lis river (Portugal): Sources, fate and seasonal variation.

The occurrence of 33 pharmaceuticals and metabolites was evaluated along the Lis river and in the influents and effluents of two wastewater treatment plants (WWTPs) located along the river. Results indicate that pharmaceuticals, such as ibuprofen, ketoprofen, carbamazepine and fluoxetine, and the metabolite salicylic acid are widespread along the Lis river, showing 100% of detection frequency, at levels up to 1.3µgL-1. The number of molecules detected increased along the river, with 11 molecules in the source, 15 upstream WWTP 1, 16 downstream WWTP 1 and upstream WWTP 2 and 19 downstream WWTP 2. The highest concentrations were often found downstream near the river mouth. Different possible sources of contamination of the Lis river were identified, namely WWTP effluents, untreated wastewaters and livestock production. Nevertheless, the discharge of WWTP effluents appeared to be the most pronounced, given that, in general, it was noticed an increase in the concentration of pharmaceuticals downstream of the WWTPs. WWTP effluents contributed with a total mass load of pharmaceuticals into the Lis river between 470 and 2317mg/d/1000 inhabitants. Non-steroidal anti-inflammatory drugs/analgesics were the therapeutic group with a high contribution to the total mass load of pharmaceuticals entering the Lis river, followed by psychiatric drugs and antibiotics. No seasonal variation was observed for the detected concentrations of pharmaceuticals. At the levels detected in the Lis river, sulfamethoxazole, clarithromycin, azithromycin and ibuprofen showed to have potential risk for aquatic organisms. These findings show that further studies embracing different environmental compartments (water, sediment and biota) are needed, in order to evaluate the partition/distribution of pharmaceuticals, their metabolites and transformation products in the environment as well as to predict their possible impact to non-target organisms and, in a last instance, to human health.

Optimization of the Ion Source-Mass Spectrometry Parameters in Non-Steroidal Anti-Inflammatory and Analgesic Pharmaceuticals Analysis by a Design of Experiments Approach.

The flow rates of drying and nebulizing gas, heat block and desolvation line temperatures and interface voltage are potential electrospray ionization parameters as they may enhance sensitivity of the mass spectrometer. The conditions that give higher sensitivity of 13 pharmaceuticals were explored. First, Plackett-Burman design was implemented to screen significant factors, and it was concluded that interface voltage and nebulizing gas flow were the only factors that influence the intensity signal for all pharmaceuticals. This fractionated factorial design was projected to set a full 2(2) factorial design with center points. The lack-of-fit test proved to be significant. Then, a central composite face-centered design was conducted. Finally, a stepwise multiple linear regression and subsequently an optimization problem solving were carried out. Two main drug clusters were found concerning the signal intensities of all runs of the augmented factorial design. p-Aminophenol, salicylic acid, and nimesulide constitute one cluster as a result of showing much higher sensitivity than the remaining drugs. The other cluster is more homogeneous with some sub-clusters comprising one pharmaceutical and its respective metabolite. It was observed that instrumental signal increased when both significant factors increased with maximum signal occurring when both codified factors are set at level +1. It was also found that, for most of the pharmaceuticals, interface voltage influences the intensity of the instrument more than the nebulizing gas flowrate. The only exceptions refer to nimesulide where the relative importance of the factors is reversed and still salicylic acid where both factors equally influence the instrumental signal. Graphical Abstract ᅟ.

A throughput method using the quick easy cheap effective rugged safe method for the quantification of ibuprofen and its main metabolites in soils.

Quick, easy, cheap, effective, rugged, and safe extraction is a modern sample preparation method that involves a number of steps with a low susceptibility to error. The efficiency of a laboratory can be improved not only through labor reduction and consumable savings but also through the use of high-throughput methods and the reduction of wastes. In commercially available kits, different salts and buffers are mixed in anhydrous packages. The composition of these kits are optimized and fixed for particular applications by the suppliers. In this work three model compounds (ibuprofen and two of its main metabolites: hydroxyibuprofen and carboxyibuprofen) were chosen and the amount of each salt (magnesium sulfate, sodium chloride, sodium citrate, and disodium citrate sesquihydrate) in the quick, easy, cheap, effective, rugged, and safe content was optimized for three different soil samples (soils A, B, and C) with different organic carbon contents, using a 2(4) factorial design. The optimized extraction procedure was applied to 12 soil samples; ranging from river sediments to agricultural soils. Based on the analysis 100 samples, a price reduction of 5.1- (soil B), 5.7- (Soil C), and 6.1-fold (soil A) was achieved without compromising the performance of the method when compared to commercial kits.

Assessment of non-steroidal anti-inflammatory and analgesic pharmaceuticals in seawaters of North of Portugal: occurrence and environmental risk.

The occurrence of seven pharmaceuticals and two metabolites belonging to non-steroidal anti-inflammatory drugs and analgesics therapeutic classes was studied in seawaters. A total of 101 samples covering fourteen beaches and five cities were evaluated in order to assess the spatial distribution of pharmaceuticals among north Portuguese coast. Seawaters were selected in order to embrace different bathing water quality (excellent, good and sufficient). Acetaminophen, ketoprofen and the metabolite hydroxyibuprofen were detected in all the seawater samples at maximum concentrations of 584, 89.7 and 287 ng L(-1), respectively. Carboxyibuprofen had the highest seawater concentration (1227 ng L(-1)). The temporal distribution of the selected pharmaceuticals during the bathing season showed that, in general, higher concentrations were detected in August and September. The environmental risk posed by the pharmaceuticals detected in seawaters towards different trophic levels (fish, daphnids and algae) was also assessed. Only diclofenac showed hazard quotients above one for fish, representing a potential risk for aquatic organisms. These results were observed in seawaters classified as excellent bathing water. Additional data is needed in order to support the identification and prioritization of risks posed by pharmaceuticals in marine environment.

Role of oxidative stress-induced systemic and cavernosal molecular alterations in the progression of diabetic erectile dysfunction.

BACKGROUND: Erectile dysfunction (ED) is a prevalent complication of diabetes, and oxidative stress is an important feature of diabetic ED. Oxidative stress-induced damage plays a pivotal role in the development of tissue alterations. However, the deleterious effects of oxidative stress in the corpus cavernosum with the progression of diabetes remain unclear. The aim of this study was to evaluate systemic and penile oxidative stress status in the early and late stages of diabetes. METHODS: Male Wistar streptozotocin-diabetic rats (and age-matched controls) were examined 2 (early) and 8 weeks (late) after the induction of diabetes. Systemic oxidative stress was evaluated by urinary H2 O2 and the ratio of circulating reduced/oxidized glutathione (GSH/GSSG). Penile oxidative status was assessed by H2 O2 production and 3-nitrotyrosine (3-NT) formation. Cavernosal endothelial nitric oxide synthase (eNOS) was analyzed by quantitative immunohistochemistry. Dual immunofluorescence was also performed for 3-NT and α-smooth muscle actin (α-SMA) and eNOS-α-SMA. RESULTS: There was a significant increase in urinary H2 O2 levels in both diabetic groups. The plasma GSH/GSSG ratio was significantly augmented in late diabetes. In cavernosal tissue, H2 O2 production was significantly increased in late diabetes. Reactivity for 3-NT was located predominantly in cavernosal smooth muscle (SM) and was significantly reduced in late diabetes. Quantitative immunohistochemistry revealed a significant decrease in eNOS levels in cavernosal SM and endothelium in late diabetes. CONCLUSIONS: The findings indicate that the noxious effects of oxidative stress are more prominent in late diabetes. Increased penile protein oxidative modifications and decreased eNOS expression may be responsible for structural and/or functional deregulation, contributing to the progression of diabetes-associated ED.