Oxidative pathways in response to polyunsaturated aldehydes in the marine diatom Skeletonema marinoi (Bacillariophyceae)

Polyunsaturated aldehydes (PUA) have recently been shown to induce reactive oxygen species (ROS) and possibly reactive nitrogen species (RNS, e.g., peroxynitrite) in the diatom Skeletonema marinoi (S. marinoi), which produces high amounts of PUA. We now are attempting to acquire better understanding of which reactive molecular species are involved in the oxidative response of S. marinoi to PUA. We used flow cytometry, the dye dihydrorhodamine 123 (DHR) as the main indicator of ROS (but which is also known to partially detect RNS), and different scavengers and inhibitors of both nitric oxide (NO) synthesis and superoxide dismutase activity (SOD). Both the scavengers Tempol (for ROS) and uric acid (UA, for peroxynitrite) induced a lower DHR‐derived green fluorescence in S. marinoi cells exposed to the PUA, suggesting that both reactive species were produced. When PUA‐exposed S. marinoi cells were treated with the NO scavenger 2‐4‐carboxyphenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO), an opposite response was observed, with an increase in DHR‐derived green fluorescence. A higher DHR‐derived green fluorescence was also observed in the presence of sodium tungstate (ST), an inhibitor of NO production via nitrate reductase. In addition, two different SOD inhibitors, 2‐methoxyestradiol (2ME) and sodium diethyldithiocarbamate trihydrate (DETC), had an effect, with DETC inducing the strongest inhibition after 20 min. These results indicate the involvement of O₂^? generation and SOD activity in H₂O₂ formation (with downstream ROS generation dependent from H₂O₂) in response to PUA exposure. This is relevant as it refines the biological impact of PUA and identifies the specific molecules involved in the response. It is speculated that in PUA‐exposed S. marinoi cells, beyond a certain threshold of PUA, the intracellular antioxidant system is no longer able to cope with the excess of ROS, thus resulting in the observed accumulation of both O₂^?? and H₂O₂. This might be particularly relevant for population dynamics at sea, during blooms, when cell lysis increases and PUA are released. It can be envisioned that in the final stages of blooms, higher local PUA concentrations accumulate, which in turn induces intracellular ROS generation that ultimately leads to cell death and bloom decay.     

A Microtiter Plate Assay for Screening Antioxidant Activity in Extracts of Marine Organisms

A novel microtiter plate assay was developed to determine the total peroxyl radical-trapping activity of antioxidants extracted from marine organisms by measuring the inhibition rate of dye-substrate oxidation. We compared use of dihydrorhodamine-123, dihydrofluorescein, and dichlorodihydrofluorescein as reduced substrates for oxidation by peroxyl radicals generated from 2,2-azobis(2-amidinopropane) dihydrochloride. The oxidation products of these highly reactive substrates are intensely colored dyes that absorb maximally in the wavelength region, _max = 489 to 512 nm, and their concentrations were determined photometrically using a 96-well, microtiter plate reader. The microtiter plate method provides for concurrent multisample analysis with automated data storage, regression analyses, and calculation of oxidation inhibition rates. Dihydrorhodamine was selected as the preferred substrate for screening crude extracts, and typical assay results are presented. Novel lead antioxidants are selected from active extracts by chromatographic analysis with electrochemical detection.     

Induction of DNA damage by free radicals generated either by organic or inorganic arsenic (AsIII, MMAIII, and DMAIII) in cultures of B and T lymphocytes

The aim of this work is based in the premise that inorganic arsenic (As^III) and trivalentmethylated metabolites monomethylarsonous (MMA^III) and dimethylarsinous (DMA^III) participate in DNA damage through the generation of reactive oxygen species (ROS). We have utilized two lymphoblastic lines, Raji (B cells) and Jurkat (T cells), which were treated with the trivalent arsenic species (dose: 0–100 μM) and analyzed by two assays (comet assay and flow cytometry) in the determination of DNA damage and ROS effects in vivo. The results showed that the damage to the DNA and the generation of ROS are different in both cellular lines with respect to the dose of organic arsenic, and the order of damage is MMA^III>DMA^III>As^III. This fact suggests that the DMA^III is not always the more cytotoxic intermediary xenobiotic, as has already been reported in another study.     

PmMGST3, a novel microsomal glutathione S-transferase gene in the dinoflagellate Prorocentrum minimum, is a potential biomarker of oxidative stress

In this study, we evaluated a novel microsomal glutathione S-transferase3 (MGST3) gene from the dinoflagellate Prorocentrum minimum, and examined its expression pattern in response to copper-and nickel-induced stresses. The full length of PmMGST3 was 732 bp, ranging from the dinoflagellate splice leader (DinoSL) sequence to the poly (A) tail, covering a 441-bp ORF, 97-bp 5′UTR, and 194-bp 3′UTR. The PmMGST3 was up-regulated by metals, including copper and nickel. The highest up-regulation levels of the PmMGST3 were found under 0.1 mg/L copper and 0.5 mg/L nickel treatment, respectively. In addition, the PmMGST3 was gradually up-regulated by 0.1 mg/L copper with increasing exposure time. Furthermore, ROS production and reduced GSH was measured in the copper treated cells. A significant increased ROS production and reduced GSH were found in the copper treated cells. These results suggest that PmMGST3 may be related to defense mechanisms associated with oxidative stress in dinoflagellates.     

Antifungal effect with apoptotic mechanism(s) of Styraxjaponoside C

The antifungal effects and mechanisms of Styraxjaponoside C were investigated. Styraxjaponoside C was active against several human pathogens, including Candida albicans. Styraxjaponoside C induced a series of cellular changes characteristic of apoptosis in C. albicans, including increased reactive oxygen species (ROS) production, measured by DHR-123 staining; phosphatidylserine externalization, visualized by Annexin V staining; DNA fragmentation, as seen by TUNEL; and plasma membrane depolarization, observed by DiBAC₄(3) staining. The plasma membrane depolarization is likely to be associated with production of ROS. The current study suggests that Styraxjaponoside C exerts an antifungal effect by promoting apoptosis.     

KatG from Salmonella Typhimurium is a peroxynitritase

Pathogenic bacteria elicit protective responses to oxidative and nitrosative stresses. Although such responses are generally distinct, it was recently reported in Mycobacterium tuberculosis that catalase-peroxidase (KatG), a classical defence against peroxides, also exhibits peroxynitritase activity. Here, the katG gene from Salmonella Typhimurium was cloned and protein purified and characterised. An increase in the rate of decomposition of peroxynitrite was observed for KatG from the enterobacterium with a second-order rate constant of 4.2 × 10⁴ M⁻¹ s⁻¹ at pH 7.4, 25 °C. This enzyme was able to reduce dihydrorhodamine oxidation by peroxynitrite to ∼83%. Given the peroxynitritase activity demonstrated here it is likely that KatG may play a wider role in the detoxification of oxidative stresses than previously thought.     

Oxidised LDL modulate adipogenesis in 3T3-L1 preadipocytes by affecting the balance between cell proliferation and differentiation

The effects of oxidised LDL (oxLDL) on cell proliferation, apoptosis and hormone-induced differentiation have been evaluated for the first time in 3T3-L1 preadipocytes. Unlike control cells, oxLDL-treated preadipocytes showed a high proliferation rate, a low apoptosis level, and an impaired differentiation process with an increased preadipocyte factor-1 (Pref-1) mRNA expression at late times. By silencing Pref-1 mRNA or inhibiting its expression with an increased dexamethasone concentration, differentiation occurred as usual, which demonstrates the key role of Pref-1 overexpression. The results suggest a specific action of oxLDL on the adipogenesis inhibitor Pref-1, as indicated also by its reappearance in mature adipocytes treated with oxLDL. The inhibitory effects of oxLDL on differentiation required oxLDL uptake by CD36, and were associated with lipoprotein lipids. These results point to oxLDL as a modulator of adipose tissue mass and as possible link between obesity and its clinical complications.     

A biochemical fluorometric method for assessing the oxidative properties of HDL

Most current assays of HDL functional properties are cell-based. We have developed a fluorometric biochemical assay based on the oxidation of dihydrorhodamine 123 (DHR) by HDL. This cell-free assay assesses the intrinsic ability of HDL to be oxidized by measuring increasing fluorescence due to DHR oxidation over time. The assay distinguishes the oxidative potential of HDL taken from different persons, and the results are reproducible. Direct comparison of this measurement correlated well with results obtained using a validated cell-based assay (r(2) = 0.62, P < 0.001). The assay can be scaled from a 96-well format to a 384-well format and, therefore, is suitable for high-throughput implementation. This new fluorometric method offers an inexpensive, accurate, and rapid means for determining the oxidative properties of HDL that is applicable to large-scale clinical studies.