Formation Of Intracellular Reactive Species Research Articles

Extraction of phospholipid-rich fractions from egg yolk and development of liposomes entrapping a dietary polyphenol with neuroactive potential

A new protocol to obtain egg yolk phospholipids in ethanol is presented. Rutin-phospholipids nanoliposomes were prepared and characterized. The procedure takes advantage of the different solubility of egg yolk lipids in ethanol and acetone at low temperature, to efficiently obtain a phospholipid-rich fraction of high purity degree. The phospholipid content in the final fraction is 208.65 ± 26.46 μmol/g fresh egg yolk (16%), accounting for ca. 96% of the extract's dry weight. The phospholipid-rich fraction contains cholesterol (0.069–0.082 cholesterol/phospholipid molar ratio), and vestigial amounts of lutein and zeaxanthin (89.24 ± 9.76 and 14.9 ± 2.16 ng/g of fresh egg yolk, respectively). Saturated fatty acids dominate the extracted phospholipids (50% of egg's total yolk phospholipids), the levels of monounsaturated ranging from 20 to 25%, and polyunsaturated up to 35%. Rutin-liposomes, prepared with phospholipid-rich fraction, presented mean diameter <140 nm, negative surface charge (Zeta potential ~ −13 mV), and entrapment efficiency of rutin up to 87%. In human neuroblastoma cell line SH-SY5Y, rutin-liposomes (lipid 25 μM + rutin 16.7 μM) attenuated glutamate-induced cytotoxicity, in part by reducing the formation of intracellular reactive species, pointing to their potential application as new functional neuroprotective agents.

Medicinal species as MTDLs: Turnera diffusa Willd. Ex Schult inhibits CNS enzymes and delays glutamate excitotoxicity in SH-SY5Y cells via oxidative damage

One of the most promising approaches to confront the complexity of central nervous system disorders are new multi-target directed ligands (MTDLs). Five medicinal species (Cereus grandiflorus (L.) Mill., Hyssopus officinalis L., Acorus calamus L., Silybum marianum L. Gaertn. and Turnera diffusa Willd. Ex Schult), selected for their ethnopharmacological relevance, were object for in vitro screening. The aqueous extract of T. diffusa revealed the strongest neuroactive potential, inhibiting monoamine oxidase-A (IC50 = 129.80 ± 11.97 μg/mL), and acetyl- and butyrylcholinesterase (IC25 = 0.352 ± 0.011 and 0.370 ± 0.036 mg/mL, respectively). Its phenolic profile was established for the first time by HPLC-DAD-ESI/MSn. Twenty-six out of thirty-seven compounds were newly identified in this species. The pre-treatment with this flavonoid-rich extract promoted a rightward shift of the glutamate concentration neuronal cell (SH-SY5Y) death response curve. Furthermore, it significantly reduced the early phase formation of intracellular reactive species after glutamate and t-BHP exposure, suggesting that neuroprotection in SH-SY5Y cells was, in part, mediated by antioxidant mechanisms.

RNA as a new target for toxic and protective agents

In contrast to damage of genomic DNA and despite its potential to affect cell physiology, RNA damage is a poorly examined field in biomedical research. Potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. While less lethal than mutations in genome, such non-acutely lethal insults to cells have been recently associated with underlying mechanisms of several human chronic diseases. We investigated whether RNA damage could be related to the exposure of particular xenobiotics by testing the RNA-damaging activity of a series of chemicals with different mechanisms of action. Cultured human T-lymphoblastoid cells were treated with ethyl methanesulfonate (EMS), H2O2, doxorubicin, spermine, or S-nitroso-N-acetylpenicillamine (SNAP). Furthermore, we studied the potential protective activity of a pomegranate extract against RNA damage induced by different chemicals. Special attention has been paid to the protective mechanisms of the extract. The protective effect of pomegranate can be mediated by alterations of the rates of toxic agent absorption and uptake, by trapping of electrophiles as well as free radicals, and protection of nucleophilic sites in RNA. We used two different treatment protocols (pre- and co-treatment) for understanding the mechanism of the inhibitory activity of pomegranate. We demonstrated that total RNA is susceptible to chemical attack. A degradation of total RNA could be accomplished with doxorubicin, H2O2, spermine and SNAP. However, EMS, a well-known DNA-damaging agent, was devoid of RNA-damaging properties, while spermine and SNAP, although lacking of DNA-damaging properties, were able to damage RNA. Pomegranate reduced the RNA-damaging effect of doxorubicin, H2O2, and spermine. Its inhibitory activity could be related with its ability to forms complexes with doxorubicin and H2O2, or interacts with the intracellular formation of reactive species mediating their toxicity. For spermine, an alteration of the rates of spermine absorption and uptake can also be involved.

Evidence of increased reactive species formation by retinol, but not retinoic acid, in PC12 cells

The biological effects of vitamin A (retinol) are generally ascribed to the activation of nuclear retinoid receptors by retinoic acid (RA), considered the most biologically active retinoid. However, it is not established whether the cytotoxic effects of vitamin A are due to retinoid receptors activation by RA. Vitamin A-related toxicity is associated with cellular redox modifications, often leading to severe oxidative damage, but the role of RA in this effect is also uncertain. We therefore studied the formation of intracellular reactive species induced by retinol and retinoic acid in PC12 cells, using an in vitro dichlorofluorescein (DCFH) fluorescence real-time assay. We observed that retinol, but not retinoic acid, induced a steady increase in DCF-based fluorescence over 60min of incubation, and this increase was reversed by antioxidant (N-acetyl-cysteine and α-tocopherol) pre-treatment. This effect was also inhibited by the iron chelator 1,10-phenantroline and the impermeable calcium chelator EGTA. These results suggest that vitamin A-associated cytotoxicity is probably related to an oxidant mechanism dependent on iron and calcium, and the formation of intracellular reactive species is related to retinol, but not to RA.

Dopamine-Dependent Iron Toxicity in Cells Derived from Rat Hypothalamus

We report a new and specific mechanism for iron-mediated neurotoxicity using RCHT cells, which were derived from rat hypothalamus. RCHT cells exhibit immunofluorescent-positive markers for dopamine beta-hydroxylase and the norepinephrine transporter, NET. In the present study, we observed that iron-induced neurotoxicity in RCHT cells was dependent on (i) formation of an Fe-dopamine complex (100 microM FeCl3:100 microM dopamine); (ii) specific uptake of the Fe-dopamine complex into RCHT cells via NET (79+/-2 pmol 59Fe/mg/min; P<0.05), since the uptake of the 59Fe-dopamine complex by the cells was inhibited by 30 microM reboxetine, a specific NET inhibitor (78% inhibition, P<0.001); and (iii) intracellular oxidation of dopamine present in the Fe-dopamine complex to aminochrome; (iv) inhibition of DT-diaphorase, since incubation of RCHT cells with 100 microM Fe-dopamine complex in the presence of 100 microM dicoumarol, an inhibitor of DT-diaphorase, induced significant cell death (51+/-5%; P<0.001). However, this cell death was reduced by 75% when the cells were incubated in the presence of 30 microM reboxetine (P<0.01). No significant cell death was observed when the cells were incubated with 100 microM dopamine, 100 microM Fe-Dopamine complex, 100 microM dicoumarol, or 100 microM FeCl3 (8.3+/-2, 9+/-4, 8.5+/-3, or 9.7+/-2% of control, respectively). ESR studies using the spin trapping agent DMPO showed no formation of hydroxyl radicals when the cells were incubated with 100 microM FeCl3 alone. However, using the same ESR technique, the formation of hydroxyl radicals and a carbon-centered radical was detected when the cells were incubated with 100 microM Fe-dopamine complex in the presence of 100 microM dicoumarol. These studies suggest that iron can induce cell toxicity by a mechanism that requires the formation and NET-mediated uptake of an Fe-dopamine complex, ultimately resulting in the intracellular formation of reactive species.