Luminol Research Articles

Ferrocene/ β-cyclodextrin based supramolecular nanogels as theranostic systems

A supramolecular redox responsive nanogel (NG) with the ability to sense cancer cells and loaded with a releasing therapeutic agent was synthesized using hostguest interactions between polyethylene glycol-grafted-β-cyclodextrin and ferrocene boronic acid. Cyclic voltammetry matched with other spectroscopy and microscopy methods provided strong indications regarding host-guest interactions and formation of the NG. Moreover, the biological properties of the NG were evaluated using fluorescence silencing, confocal laser scanning microscopy, and cell toxicity assays. Nanogel with spherical core-shell architecture and 100–200 nm sized nanoparticles showed high encapsulation efficiency for doxorubicin (DOX) and luminol (LU) as therapeutic and sensing agents. High therapeutic and sensing efficiencies were manifested by complete release of DOX and dramatic quenching of LU fluorescence triggered by 0.05 mM H2O2 (as an ROS component). The NGs showed high ROS sensitivity. Taking advantage of a high loading capacity, redox sensitivity, and biocompatibility, the NGs can be used as strong theranostic systems in inflammation-associated diseases.

AIE Nanozyme-Based Long Persistent Chemiluminescence and Fluorescence for POCT of Pathogenic Bacteria.

Pathogenic bacteria are widely distributed in diverse environments and significantly threaten human health. Point-of-care testing (POCT) is a valuable way for early warnings of bacteria threat. Herein, a chemiluminescence (CL)-based ratiometric sensing platform was constructed for sensitive POCT of bacteria according to a newly designed aggregation-induced emission (AIE) molecule. The new AIE molecule presents oxidase-like properties (named as AIEzyme) and can trigger long persistent CL of luminol (LUM) with strong intensity in the absence of H2O2. The CL emission can be monitored with the naked eye for over 2 h. The emission mechanism is explored and may be attributed to the persistent reactive oxygen species generation of the AIEzyme according to the cyclic energy transfer between the AIEzyme and luminol, which catalyzes CL of luminol. Based on the CL resonance energy transfer mechanism, an afterglow luminescence system is further developed, which is used to construct a ratiometric biosensor for detection of pathogenic bacteria. With a homemade holder as a detection room and a smartphone as an analyzer, the portable biosensing platform is used for quantitative POCT of bacteria in real samples with good recovery. The detection is free of H2O2 and an external excitation source, which not only simplifies the operation but reduces interference. Specifically, the long persistent luminescence and the ratiometric strategy can significantly improve accuracy, providing an instructive way for point-of-need analysis, for example, SARS-CoV-2 detection and bioimaging analysis.

Hydrogen Bond Organic Frameworks as Radical Reactors for Enhancement in ECL Efficiency and Their Ultrasensitive Biosensing.

Nowadays, electrochemiluminescence (ECL) efficiency of an organic emitter is closely related with its potential applications in food safety and environmental monitoring fields. In this work, 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (TATB) was self-assembled to form hydrogen bond organic frameworks (HOFs), which worked as ideal reactors to generate highly active oxygen-containing radicals, followed by linking with isoluminol (ILu) via amide bond (termed ILu-HOFs). After covalent assembly with aminated indium-tin oxide electrode (labeled NH2-ITO), the ECL efficiency of the ILu-HOFs NH2-ITO showed about a 23.4-time increase over that of ILu itself in the presence of H2O2. Meanwhile, the enhanced ECL mechanism was mainly studied by electron paramagnetic resonance, theoretical calculation, and electrochemistry. On the above foundation, an aptamer "sandwich" ECL biosensor was constructed for detecting isocarbophos (ICP) via in situ elimination of H2O2 with catalase-linked palladium nanocubes (CAT-Pd NCs). The as-built sensor showed a broad linear range (1 pM to 100 nM) and a low limit of detection (LOD) down to 0.4 pM, coupled with efficient assays of ICP in lake water and cucumber juice samples. This strategy provides an effective way for the synthesis of advanced ECL emitter, coupled by showing promising applications in environmental and food analysis.

Role of Pi-Electron Density at the Interface of Small Molecule-Sensitized Solar Cells

Conventional dye-sensitized solar cells (DSSCs) involving charge-transfer interfaces face charge injection losses and offsets for TiO2-sensitizer band alignment. The direct charge-transfer mechanism in DSSCs with catechol (CT, 1,2-benzenediol)-based compounds minimizes the injection losses and eliminates band alignment issues, although the photovoltaic performance of the corresponding device is very poor due to the ultrafast (picosecond) recombination of photoexcited electrons. Just as in a natural photosystem, structural selectivity toward inhibition of this recombination needs to be defined. The interfacial electron density and back electron transfer kinetics at the molecular sensitizer–TiO2 interface play a significant role in the overall energy conversion efficiency. Herein, we identified, for the first time, that the π-electron cloud at the sensitizer–TiO2 interface facilitates the degree of recombination. Comparative density functional theory analyses confirmed that these electron clouds act as large recombination sites. Luminol (LM) and isoluminol (ILM) were employed as “small molecule” sensitizers without the cloud, having a secondary amine linker, which increased the photoenergy conversion efficiency of the single-step sensitization-based photovoltaic cell (type-II DSSC) by reducing the recombination. The device with LM exhibited a power-conversion efficiency (PCE) of ca. 1.11% (representing 363% improvement when compared to CT), the highest ever reported in this category. This understanding is insightful for the design of novel small molecular sensitizers for future DSSCs.

Synthesis, characterization and investigation of antibacterial activity for some new functionalized luminol derivatives

ABSTRACT. The present study describes synthesis of some new luminol derivatives, which might play an important role in biological active agents. These new synthesized compounds are functionalized based on luminol (LM) with different carboxylic drugs such as mefenamic acid, ibuprofen, diclofenac sodium, and ampicillin). Synthesis processes was conducted by converting carboxylic group in the investigated drugs into acid chloride group by reacting with SOCl2. Then the synthesized chloride drug derivatives were reacted with luminol in presences of DMSO and TEA to yield the final target molecules. These compounds were characterized using FTIR, NMR and CHNS techniques. Besides that, their physical properties and solubility were also investigated. Biological activity of the derivatives (TH1-TH4) was investigated using a pathogenic bacterium, Staphylococcus aureus (gram +ve), and Escherichia (E. coli) (gram -ve). The obtained results for antibacterial activity showed that TH1-TH4 derivatives have higher antibacterial activity against these types of bacteria in comparison with pure LM compound and the investigated drugs.
 
 KEY WORDS: Luminol, Mefenamic acid, Ibuprofen, Diclofenac sodium, Ampicillin
 
 Bull. Chem. Soc. Ethiop. 2023, 37(1), 159-169. 
 DOI: https://dx.doi.org/10.4314/bcse.v37i1.13

Lanthanide coordination polymer nanoparticles as a ratiometric fluorescence sensor for real-time and visual detection of tetracycline by a smartphone and test paper based on the analyte-triggered antenna effect and inner filter effect

Lanthanide coordination polymer nanoparticles (LML-Eu3+-GMP CPNPs) were fabricated as a ratiometric fluorescence sensor for real-time and visual assays of tetracycline (TC). The LML-Eu3+-GMP CPNPs were prepared by spontaneous self-assembly of Eu3+, luminol (LML) and guanosine 5′-monophosphate (GMP) at room temperature, which only emitted blue fluorescence dominated by LML, and no red characteristic fluorescence of Eu3+ was observed. In the presence of TC, TC can trigger the antenna effect (AE) and inner filter effect (IFE), the blue fluorescence of LML-Eu3+-GMP CPNPs is quenched by TC via IFE, and the red characteristic fluorescence of Eu3+ can be turned on through AE. When sodium citrate (Cit) is added to the sensing system, it can further enhance the red fluorescence of Eu3+. The dual-ligand CPNP sensor has a rapid fluorescence response, noticeable fluorescence color change, good selectivity, and high sensitivity with a detection limit of 3.4 nM. It was successfully applied to detect TC in honey, milk, lake water and tap water. Meanwhile, the portable smartphone and test paper were applied to develop POCT devices for real-time and visual detection of TC, which could offer a promising application for real-time and semiquantitative assays of TC.

Cascade electrochemiluminescence-based integrated graphitic carbon nitride-encapsulated metal-organic framework nanozyme for prostate-specific antigen biosensing

Developing sensitive strategies to detect the biomarkers is responsible for early detection of a genetic disorder and cancer-related biomarkers. Herein, a rational design of high catalytic activity of artificial enzyme mimics with enhancing the robustness of functional nanozymes has been introduced to detect PSA as a model target. Using a separation/enrichment tool of capture antibody (cAb1)-modified magnetic nanoparticles (MNPs/cAb1), signal amplifier luminol (LUM)-loaded graphitic carbon nitride nanosheets (C3N4 NS)-encapsulated NH2-MIL(53)-Fe (NMF) (C3N4@LUM/NMF)) to label signal antibody (Ab2) as a specific targeting and a supporting platform of a nanoporous gold electrode (NPGE) with high-efficiency electron acceleration, a cascade signal amplification strategy has been achieved. In addition, the nanoscale proximity effect of confined LUM within the C3N4@NMF nanozyme and synergistically enhanced catalytic activity causes significantly to promote the electrochemiluminescence (ECL) of LUM owing to minimize the decomposition of highly active intermediates of •OH-generating reaction of C3N4@NMF nanozymes from H2O2. Taking the advantages of multiple-amplification, the present strategy shows PSA detection in dynamic range of 0.1 pg mL−1-60 ng mL−1 with 0.02 pg mL−1 limit of detection. This ECL-based nanozyme opens a new horizon for high sensitive targeted bioanalysis with functional nanomaterial design.

Establishment of specific IgE antibody detection method for Tyrophagus putrescentiae allergen based on nano-magnetic particle chemiluminescence analysis

Objective To establish a nanometer magnetic particle chemiluminescence methodfor the detection of specific IgE antibodies to Tyrophagus putrefaciens allergen. Methods According to the routine operation steps of chemiluminescence, chemiluminescence reaction system and suitable immune reaction conditions for detection of specific IgE of Tyrophagus putrescentiae allergen are established. Considering the performance of the chemiluminescence method established in this paper, the American Somerfly Phadia method wasused as the gold standard to evaluate the test performance of the established chemiluminescence method. Results In thechemiluminescence reaction system, Luminol concentration in luminescent substrate A solution is 0.4 mg/ml, Urea hydrogen peroxidein luminescent substrate B solutionis 0.2 mg/ml.The sensitivity of this luminescent reaction system to horseradish peroxidase (HRP) is very high, the minimum detectable HRP is 0.01 ng/ml.In the immune response, the room temperature light-shielding reaction was detected for 5 min after the addition of substrates A and B, and the detection values were effective within 5-30 min. 37 ℃ is optimum luminescence reaction temperature. Choose the range of 0.35-100 IU/ml as the standard curve. According to the results of 120 clinical trials, using Phadia (immunofluorescence method) as the gold standard, the chemiluminescence method established in this study has significant significance for the detection of Tyrophagus putrescentiae allergen. Conclusion A nanometer magnetic particle chemiluminescence method was successfully established for the detection of specific IgE antibodies to Tyrophagus putrescentiae allergen. Key words: Tyrophagus putrescentiae allergen; Nanometer magnetic particles; Chemiluminescence; Streptavidin; Biotin

The Antioxidant and Membrane Activities of the Benzotropalone Pigment Purpurogallin

Abstract—The phenolic compound purpurogallin (PPG) is found in oak nutgalls and is a red pigment with a benzotropolone ring structure. PPG shows pronounced cytoprotective and anti-inflammatory activities. PPG might be effective in preventing and treating diverse systemic inflammatory diseases, affecting functional activities of various cell metabolic and signaling pathways that are associated with inflammation. A study was made to compare the antioxidant (free radical) properties for PPG, dihydroquercetin (DHQ), ascorbic acid, and trolox using two test systems, hemoglobin (Hb)–H2O2–luminol (LM) and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH)–LM. PPG was additionally tested for its effect on the permeability of flat lipid bilayer membranes (LBMs). Moderate antioxidant activity was observed for PPG in the Hb–H2O2–LM system; its neutralizing potential towards hydroxyl and superoxide anions was comparable with that of ascorbic acid. The compounds were ranked according to the efficiency of their antioxidant activity in the system: DHQ > trolox > ascorbic acid > PPG. Interestingly, when the AAPH–LM system was used to test antioxidant activity, PPG did not show neutralizing activity towards peroxyl radicals formed in the system. PPG did not significantly change the permeability of flat LBMs in a dose range of 1.5–30 μM, suggesting lack of a primary membrane effect. Putative mechanisms and characteristics of cytoprotective, antioxidant, and anti-inflammatory activities of PPG are discussed.

The effects of oral nutritional formula enriched with arginine, omega 3 fatty acids and nucleotides on methotrexate-induced experimental intestinal mucositis

The aim of this study to investigate the effect of an immunonutritional (IN) oral formula enriched with arginine, omega-3 fatty acids and nucleotides in methotrexate (MTX)-induced experimental intestinal mucositis model. In the study, 32 rats were divided into four groups consisting eight animals in each. Control group fed for 5 days with only saline with gavage, IN group fed for 5 days with an oral IN formula three times a day, MTX group with an intraperitoneal single dose MTX (20 mg/kg), followed by saline with gavage, MTX-IN group with a single dose of MTX, followed by an oral IN formula three times a day. The blood and jejunal tissue sample were collected and then the rats were sacrificed on the sixth day of the study. The level of TNF-α, IL-1β in serum, luminol, lusigenin, glutathione, myeloperoxidase, malondialdehyde and Na-K+ATPase in the jejunal tissue samples were analyzed. Histopathological examination was performed in the jejunal tissue samples. In the MTX group, TNF-α, IL-1β levels in serum, and luminol, lusigenin, malondialdehyde levels, and myeloperoxidase activity in tissue samples were found significantly higher than the control group. Glutathione and Na-K+ATPase levels were lower in the jejunal tissue of the MTX group compared to control group. However, the supplementation of IN with the MTX resulted in a significant increase in glutathione and Na-K+ATPase levels. Severe epithelium loss and inflammatory cell increase were observed in the MTX group on histological examination, whereas these parameters were regressed in the MTX-IN group. Increasing in the mitosis rate of enterocytes and inflammatory cell density decreased with the IN. In conclusion, this study shows that chemotherapy has adverse effects on intestinal mucosa and the IN formula has a protective effect of on MTX-associated intestinal damage.

Cold Environment Exacerbates Brain Pathology and Oxidative Stress Following Traumatic Brain Injuries: Potential Therapeutic Effects of Nanowired Antioxidant Compound H-290/51.

The possibility that traumatic brain injury (TBI) occurring in a cold environment exacerbates brain pathology and oxidative stress was examined in our rat model. TBI was inflicted by making a longitudinal incision into the right parietal cerebral cortex (2mm deep and 4mm long) in cold-acclimatized rats (5°C for 3h daily for 5weeks) or animals at room temperature under Equithesin anesthesia. TBI in cold-exposed rats exhibited pronounced increase in brain lucigenin (LCG), luminol (LUM), and malondialdehyde (MDA) and marked pronounced decrease in glutathione (GTH) as compared to identical TBI at room temperature. The magnitude and intensity of BBB breakdown to radioiodine and Evans blue albumin, edema formation, and neuronal injuries were also exacerbated in cold-exposed rats after injury as compared to room temperature. Nanowired delivery of H-290/51 (50mg/kg) 6 and 8h after injury in cold-exposed group significantly thwarted brain pathology and oxidative stress whereas normal delivery of H-290/51 was neuroprotective after TBI at room temperature only. These observations are the first to demonstrate that (i) cold aggravates the pathophysiology of TBI possibly due to an enhanced production of oxidative stress, (ii) and in such conditions, nanodelivery of antioxidant compound has superior neuroprotective effects, not reported earlier.

Antioxidant activity of Melilotus officinalis extract investigated by means of the radical scavenging activity, the chemiluminescence of human neutrophil bursts and lipoperoxidation assay

Plants have been useful sources of new biologically active compounds. However, traditional ethno-medical approaches may draw attention to one or other clinical claim, but only specific scientific investigation can validate their effects. The aim of this study was to examine whether an extract of Melilotus officinalis leaves interferes with reactive oxygen/nitrogen species (ROS/RNS) during the course of human neutrophil respiratory bursts, and to establish the lowest concentration at which it still has antioxidant activity by means of luminol amplified chemiluminescence (LACL). We also studied its ability to counteract lipid peroxidation (LPO) in human cells. Before investigating its antioxidant effects on human cells, we analysed its scavenging activity against ABTS●+, hydroxyl radical, superoxide anion, and Fremy’s salt (the last three by means of electron paramagnetic resonance [EPR] spectrometry). The extract of M. officinalis exerted its anti-ROS/RNS activity in a concentration-dependent manner, with significant effects being observed for even very low concentration: 20 μg/ml without L-arginine (L-Arg) and 10 μg/ml when L-Arg was added to the formyl-methionyl-leucyl-phenylalanine (fMLP) test. LPO assay confirmed these results, which were paralleled by the EPR study. These findings are interesting for improving the antioxidant network and restoring redox balance in human cells, and extend the possibility of using plant-derived molecules to antagonise the oxidative stress generated in living organisms. Key words: Melilotus officinalis extract, antioxidant activity, human neutrophils, respiratory bursts, chemiluminescence, lipoperoxidation, electron paramagnetic resonance.

Chemiluminescence of phthalhydrazide derivatives in organized media: Interactions with surfactants and cyclodextrins

The chemiluminescent oxidation of some phthalhydrazide derivatives, luminol (LUM), isoluminol (ISOL), N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and N-(6-aminohexyl)-N-ethylisoluminol (AHEI), has been carried out in micellar media and in the presence of natural cyclodextrins (CDs), using Co(II) as catalyst and H2O2 as oxidant. The cationic cetyltrimethylammonium bromide (CTAB) and, in a lesser extent, the anionic sodium dodecyl sulfate (SDS) produce quenching in the chemiluminescence (CL) of all the luminophores at concentrations above the cmc, whereas slight enhancements are attained with the non-ionic pentaethylene glycol monododecyl ether (C12E5). On the contrary, the incorporation of the CDs to the reaction produces a remarkable intensification of the CL. Binding of the luminophores to the macrocycles and the micelles has been studied by Pulsed-Gradient-Spin-Echo-NMR (PGSE-NMR) and fluorescence anisotropy. The cationic CTAB decreases the emission mainly due to charge compensation as a result of the association of the luminophores and the luminescent intermediates to the micelles. The presence of the alkyl substituents of ABEI and AHEI provides an additional hydrophobic contribution to the binding process. SDS quenches this reaction as the micelles retain Co(II) on their anionic layer. The protection and stabilization of these luminophores or their luminescent intermediates provided by the cavities of the CDs make this family of cyclic oligosaccharides much more suitable agents than the surfactants for enhancing the CL in aqueous media for this specific reaction.

Abstract 5483: Targeted intracellular photodynamic therapy (TIP) reduces tumor mass and prolongs survival in a mouse model of cancer

Abstract Targeted Intracellular Photodynamic Therapy (TIP) is a novel advance on traditional photodynamic therapy (PDT) in which photosensitizer activation is afforded by chemiluminescence rather than by laser beam. This advance allows PDT access to target cells essentially anywhere in the body. TIP also has advantages over bioluminescence as there is no need for transfection of cells with enzymes such as luciferase. It can therefore be more easily applied both in vitro and in vivo and has potential as a new targeted cancer therapeutic modality that could be applied to a wide variety of cancers. We have previously described in vitro the efficacy and specificity of TIP toward murine leukemic cells (Brit. J. Cancer, 95; 189-196, 2006). We now report that TIP is effective in vivo using a murine model of cancer. To target the photosensitizer, Protoporphyrin IX was conjugated to Transferrin (Trnsf-PrP). Balb/c mice were injected subcutaneously with the mouse hybridomas U13.6 or U7.6, both of which overexpress the Transferrin receptor CD71. The use of Trnsf-PrP resulted in rapid uptake of photosensitizer into cells and conjugate was retained for over 48hrs. Animals were treated with TIP by injecting Trnsf-PrP and the chemiluminescent agent Luminol (LM) under reduced light conditions. Experiments were performed whereby different parameters of the system were varied, including: time of treatment initiation after tumor cell inoculation; dose and number of Trnsf-PrP and luminol treatments; and time intervals between treatments. Initial experiments in which TIP was performed at the time of tumor inoculation showed that this procedure completely abolished tumor growth. In subsequent experiments where tumors were palpable before treatment began, various treatment protocols were tested. TIP reduced tumor mass by over 40% and increased survival by 50%. These results demonstrate that TIP is active against cancer cells in vivo and encourages the further development of the modality as a targeted cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5483.

Natural Cyclodextrins as Efficient Boosters of the Chemiluminescence of Luminol and Isoluminol: Exploration of Potential Applications

The chemiluminescent oxidation of luminol (LUM) and isoluminol (ISOL) is notably enhanced, both in intensity and duration, in the presence of natural cyclodextrins (alpha-, beta-, gamma-CD). The experiments have considered some of the most widespread applications of these compounds: the determination of metal cations and the revealing of bloodstains by oxidation with hydrogen peroxide in alkaline solution in the presence of Co(II), Fe(III), human hemoglobin, and blood, in order to explore potential applications. The largest enhancement in the emitted intensity occurs for the reaction of LUM with Co(II) in the presence of beta-CD. The use of the more soluble gamma-CD permits to expand the range of concentration and obtain more intense emission, although soluble derivatives of the beta-CD (methyl, hydroxypropyl-beta-CD, and a soluble cross-linked epichlorhydrin polymer) do not improve the chemiluminescence (CL) yield. In the case of hemoglobin and diluted human blood, the CDs aid in producing more light but only at high concentration of CDs, with a more lasting luminescence, up to three times longer. The changes in CL when glucose is used instead, much lower than with any of the CDs, imply that the cyclic structure of these oligosaccharides plays a key factor in the boosting of the emission. The results are explained in terms of the binding between the luminescent intermediate of the reaction, 3-aminophthalate (3-AP) and the CD, rather than to the luminescent reactant itself. The association constants obtained by steady-state fluorescence by assuming 1:1 stoichiometries reveal that the most stable association occurs between beta-CD and the intermediate, in accordance with the trend in the chemiluminescence. The topology of the complex deduced via ROESY experiments confirms a shallow inclusion of the double-charged intermediate by the primary rim of the CD, which accounts for the low stability of the complexes.

In vitro investigations on the differential pro-oxidant and/or antioxidant properties of cyclosporin A and tacrolimus in human and rat liver microsomes

The use of cyclosporin A (CSA) and tacrolimus (TAC) in organ transplantation and in the therapy of immune disorders is often hampered by adverse effects, mainly nephro-, hepato- and neurotoxicity. For the development of these side effects, among others, an increased formation of reactive oxygen species, probably generated by the cytochrome P450 (CYP) system, has been accused. Since in this respect literature data are inconsistent, in the present study possible pro- and/or antioxidant effects of CSA and TAC and the involvement of the CYP system were re-evaluated in vitro. Effects of CSA and TAC were examined on CYP mediated oxidase functions by stimulated lipid peroxidation (LPO), H2O2 production, and lucigenin (LC) or luminol (LM) amplified chemiluminescence (CL) in liver microsomes of either untreated rats or of rats treated with beta-naphthoflavone (BNF), phenobarbital (PB) or dexamethasone (DEX) and in human liver microsomes. In rat liver microsomes, CSA displayed pro-oxidant properties (though only very slightly), whereas in human liver microsomes small antioxidant effects were seen. With TAC in both species the antioxidant capacity prevailed. Treatment of rats with BNF or DEX caused an increase in the pro-oxidant effects of CSA with respect to LPO or LM-CL, whereas in liver microsomes of DEX-treated rats H2O2 production and LC-CL were diminished. CSA seems to have both pro-oxidant and antioxidant properties, whereas with TAC mainly an antioxidant capacity was seen. The CYP system seems to be involved in the pro-oxidant influence of CSA. Whether pro-oxidant or antioxidant effects predominate may depend on the antioxidant capacity of a tissue and on the CYP isoforms mainly present.

Survival and in vitro fertility of boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase.

In the present study the potential benefit of reactive oxygen species (ROS)-scavenging enzymes superoxide dismutase (SOD) and catalase (CAT) when cryopreserving boar spermatozoa was evaluated. Pooled ejaculate sperm-rich fractions collected from 3 fertile boars were frozen in a split design, after being extended in a conventional freezing extender (control) or the same extender supplemented with SOD (150 or 300 IU/mL, experiment 1), CAT (200 or 400 IU/mL, experiment 2), or SOD + CAT in combination (150 + 200 or 300 + 400 IU/mL, experiment 3). Irrespective of the concentration used, SOD and CAT, alone or in combination, significantly improved postthaw sperm survival, in terms of total sperm motility (assessed with CASA) and viability (assessed with a triple stain; propidium iodide/R123/fluorescein isothiocyanate-labeled peanut agglutinin). Moreover, CAT alone, at a concentration of 400 IU/mL, or in combination with SOD, at concentrations of 200 and 400 UI/mL, improved the ability of frozen-thawed spermatozoa to produce embryos in vitro (zygote cleavage and blastocyst formation as end points). Additional data of ROS generation (luminol- and lucigenin-dependent chemiluminescence) and membrane lipid peroxidation (malondialdehyde [MDA] production) indicated that SOD and CAT reduced postthaw ROS generation by boar spermatozoa, without any influence on MDA production.

Estradiol, testosterone, dehydroepiandrosterone and androstenedione: novel derivatives and enantiomers. Interactions with rat liver microsomal cytochrome P450 and antioxidant/radical scavenger activities in vitro

Interactions of 27 steroids, among them 17 derivatives such as ethers, sulfates and amidosulfonates derived from 17β- and 17α-estradiol, from testosterone and α- and β-dihydrotesosterone and from dehydroepiandrosterone with rat liver microsomal cytochromes P450 (P450) were investigated in vitro by assessing binding to P450 and effects on P450 mediated monooxygenase functions as measured by different model reactions: ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD) and ethylmorphine N-demethylation (EMND). With the exception of 17α-estradiol-3-dimethylamidosulfonate, estrone, its -3-methylether and -3-amidosulfonate and testosterone, all other steroids displayed type I or reverse type I binding to P450. All steroids inhibited EROD activity in micromolar concentrations. An additional strong inhibition of ECOD and EMND activities was only demonstrated for the androgens and progestins. Estriol, estrone and mestranol displayed less inhibitory actions on the model reactions than estradiol. No major differences in comparison to the parent compounds were noted with the other derivatives. The only exceptions were 17β-(8,9-dehydro-14α,15α-methylene)estradiol, which displayed stronger effects than estradiol, and dehydroepiandrosterone-3-sulfate, which was less effective than dehydroepiandrosterone. Possible antioxidant properties of the steroids were examined by the stimulated lipid peroxidation (LPO), H 2O 2 production, and lucigenin (LC) and luminol (LM) amplified chemiluminescence (CL) using rat liver microsomes. Additionally, the influence on rat whole blood chemiluminescence (WB-CL) was assessed. All the estrogens, but not their methylethers and amidosulfonates inhibited LPO in micromolar concentrations. The effects on the other oxidase model reactions or on WB-CL were less distinct. Only ethinylestradiol and 17β-(8,9-dehydro-14α,15α-methylene)estradiol displayed a strong inhibitory action on all model reactions. With the exception of dehydroepiandrosterone-3-sulfate, which in general had only weak effects, the androgen and progestin derivatives, in contrast, strongly decreased H 2O 2 formation and LM- and LC-CL, but were mostly ineffective on LPO and WB-CL.

Influence of subchronic administration of catechol estrogens on the formation of reactive oxygen species in rat liver microsomes

Metabolic pathways of estrogens are the formation of catechol estrogens (CE; 2- and 4-hydroxy-estrogens), redox cycling of CE and free radical generation, mediated through cytochrome P450 (P450) oxidase/reductase activity. In previous investigations subchronic administration of estrogens showed prooxidative and antioxidative activities in rat liver microsomes (BARTH et al. 1999). To find out whether or not catechol metabolites are responsible for prooxidative activity, we checked 2- and 4-hydroxy-estradiol (2OH-E2 and 4OH-E2) and the non-catechol metabolite 6alpha-hydroxy-estradiol (6alpha-OH-E2) for formation of reactive oxygen species in liver microsomes of 30-day-old male Wistar rats after 5 days treatment (1, 10 mg/kg b. wt. orally, once a day). The results were compared with those after treatment of the rats with estradiol (E2), estradiol valerate (E2V) and ethinylestradiol (EE2). In liver homogenates glutathione and lipid peroxides were determined, in microsomes NADPH-Fe++-stimulated lipid peroxidation (LPO), H2O2 generation and lucigenin (LUC) and luminol (LUM) amplified chemiluminescence (CL) were investigated. In liver 9000 x g supernatants monooxygenase activities were measured. The two catechol estrogens did not show any antioxidative activity, whereas 6alpha-OH-E2 significantly diminished lipid peroxides in the liver as well as LPO and LUM-CL in liver microsomes. Among estrogens, only EE2 showed antioxidative activity. Both CE inhibited ethoxycoumarin O-deethylation. Peroxidative activity as enhanced LUC-CL was found after 2OH-E2 (1 mg/kg b.wt.) and E2, but 10 times higher doses of both CE did not change LUC-CL. Microsomal H2O2 generation was enhanced by E2, E2V and both CE, not by 6alpha-OH-E2. The lower level of H2O2 enhancement caused by CE in comparison to E2 and E2V together with unchanged LUC-CL after high CE doses did not unequivocally prove the CE to be mainly responsible for the prooxidative activities of E2 and E2V in liver microsomes, at least in 30-day-old male rats. Unchanged GSH in the liver after CE administration supports this hypothesis.

Influence of oestrogens on formation of reactive oxygen species in liver microsomes of differently aged male Wistar rats

Metabolic pathways of oestrogens are the formation of catechol oestrogens (CE; 2- and 4-hydroxy-oestrogens), redox cycling of CE and free radical generation, mediated through cytochrome P450 (P450) oxidase/reductase activity. We checked the oestrogens oestradiol (E2), oestradiol valerate (E2V) and ethinyloestradiol (EE2) for formation of reactive oxygen species in vitro and ex vivo in male Wistar rats in dependence on age. In liver microsomes of 10-, 30-, 60- and 270-day-old rats the influence of E2, E2V and EE2 (10(-7)-10(-3) M) on NADPH-Fe(++)-stimulated lipid peroxidation (LPO), H2O2 generation and lucigenin (LUC) and luminol (LUM) amplified chemiluminescence (CL) was investigated. The same parameters, additionally P450 content and monooxygenase activities were measured in liver 9000 x g supernatants after subchronic administration of the oestrogens (1, 10 mg/kg b. wt. orally). The most important results are the strong inhibitory capacities of the oestrogens in vitro on LPO in the order of E2V < E2 < EE2, most pronounced in 10-, 60- and 270-day-old animals. In microsomes of 30-day-old rats with the highest control LPO the antioxidative effect of the oestrogens was lower. Whereas the H2O2 generation was not changed by E2, enhanced by E2V, but diminished by EE2 in all age groups, CL(LUC) and CL(LUM) were inhibited in the order of E2 < E2V < EE2. Also after subchronical treatment of the rats the antioxidative action of the oestrogens was evident, microsomal LPO was inhibited in the order of E2 < E2V < EE2. All oestrogens inhibited ethylmorphine N-demethylation. But enhanced H2O2 generation and increased CL(LUC) also indicate a formation of reactive oxygen species by these oestrogens. Obviously in vitro the antioxidative phenolic structure of the oestrogens dominates, whereas after in vivo administration the dose- and age-dependent biotransformation produces prooxidative in addition to antioxidative structures.