Glaucia R. Martinez

Novel rhythms of N1-acetyl-N2-formyl-5-methoxykynuramine and its precursor melatonin in water hyacinth: importance for phytoremediation

N1‐acetyl‐N2‐formyl‐5‐methoxykynura‐mine (AMFK) is a major metabolite of melatonin in mammals. To investigate whether AFMK exists in plants, an aquatic plant, water hyacinth, was used. To achieve this, LC/MS/MS with a deuterated standard was employed. AFMK was identified in any plant for the first time. Both it and its precursor, melatonin, were rhythmic with peaks during the late light phase. These novel rhythms indicate that these molecules do not serve as the chemical signal of darkness as in animals but may relate to processes of photosynthesis or pho‐toprotection. These possibilities are supported by higher production of melatonin and AFMK in plants grown in sunlight (10, 000–15, 000 μW/cm2) compared to those grown under artificial light (400–450 μW/ cm2). Melatonin and AFMK, as potent free radical scavengers, may assist plants in coping with harsh environmental insults, including soil and water pollutants. High levels of melatonin and AFMK in water hyacinth may explain why this plant more easily tolerates environmental pollutants, including toxic chemicals and heavy metals and is successfully used in phytoremediation. These novel findings could lead to improvements in the phytoremediative capacity of plants by either stimulating endogenous melatonin synthesis or by adding melatonin to water/soil in which they are grown.—Dun‐Xian Tan, Lucien C. Manchester, Paolo Di Mascio, Glaucia R. Martinez, Fernanda M. Prado, and Russel J. Reiter. Novel rhythms of N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine and its precursor mel‐atonin in water hyacinth: importance for phytoremedia‐tion. FASEB J. 21, 1724–1729 (2007)

Singlet Oxygen Oxidation of Isolated and Cellular DNA: Product Formation and Mechanistic Insights

Abstract This survey focuses on recent aspects of the singlet oxygen oxidation of the guanine moiety of nucleosides, oligonucleotides, isolated and cellular DNA that has been shown to be the exclusive DNA target for this biologically relevant photogenerated oxidant. A large body of mechanistic data is now available from studies performed on nucleosides in both aprotic solvents and aqueous solutions. A common process to both reaction conditions is the formation of 8-oxo-7,8-dihydroguanine by reduction of 8-hydroperoxyguanine that arises from the rearrangement of initially formed endoperoxide across the 4,8-bond of the purine moiety. However, in organic solvent the hydroperoxide is converted as a major degradation pathway into a dioxirane that subsequently decomposes into a complex pattern of oxidation products. A different reaction that involved the formation of a highly reactive quinonoid intermediate consecutively to the loss of a water molecule from the 8-hydroperoxide has been shown to occur in aqueous solution. Subsequent addition of a water molecule at C5 leads to the generation of a spiroiminodihydantoin compound via a rearrangement that involves an acyl shift. However, in both isolated and cellular DNA the latter decomposition pathway is at the best a minor process, because only 8-oxo-7,8-dihydroguanine has been found to be generated. It is interesting to point out that singlet oxygen has been shown to contribute predominantly to the formation of 8-oxo-7,8-dihydroguanine in the DNA of bacterial and human cells upon exposure to UVA radiation. It may be added that the formation of secondary singlet-oxygen oxidation products of 8-oxo-7,8-dihydroguanine, including spiroiminodihydantoin and oxaluric acid that were characterized in nucleosides and oligonucleotide, respectively, have not yet been found in cellular DNA.

Damage to isolated DNA mediated by singlet oxygen

In the present work, we study the reaction of singlet oxygen (1O2) with isolated DNA. Emphasis is placed on the identification and quantitative measurement of the DNA modifications that are produced by the reaction of 1O2 with DNA. For this purpose, calf-thymus DNA was incubated with the endoperoxide of N,N′-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide, a chemical generator of 1O2. Thereafter, DNA was digested, and the resulting oxidized nucleosides were measured by means of a recently optimized high-performance-liquid-chromatography tandem-mass-spectrometry assay. It was found that, among the different DNA lesions observed, 7,8-dihydro-8-oxo-2′-deoxyguanosine is the major 1O2-mediated DNA-damage product. Interestingly, cyclobutane pyrimidine dimers, oxidized pyrimidine bases, 7,8-dihydro-8-oxo-2′-deoxyadenosine, and 2,6-diamino-5-formamido-4-hydroxypyrimidine are not formed, at least not in detectable amounts, following treatment of DNA with the 1O2 generator. The reported results strongly suggest that the decomposition of the endoperoxide provides a pure source of 1O2, and that reaction of 1O2 with isolated DNA induces the specific formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine.

DNA damage by singlet oxygen and cellular protective mechanisms

Reactive oxygen species, as singlet oxygen ((1)O(2)) and hydrogen peroxide, are continuously generated by aerobic organisms, and react actively with biomolecules. At excessive amounts, (1)O(2) induces oxidative stress and shows carcinogenic and toxic effects due to oxidation of lipids, proteins and nucleic acids. Singlet oxygen is able to react with DNA molecule and may induce G to T transversions due to 8-oxodG generation. The nucleotide excision repair, base excision repair and mismatch repair have been implicated in the correction of DNA lesions induced by (1)O(2) both in prokaryotic and in eukaryotic cells. (1)O(2) is also able to induce the expression of genes involved with the cellular responses to oxidative stress, such as NF-κB, c-fos and c-jun, and genes involved with tissue damage and inflammation, as ICAM-1, interleukins 1 and 6. The studies outlined in this review reinforce the idea that (1)O(2) is one of the more dangerous reactive oxygen species to the cells, and deserves our attention.

Oxidation of melatonin by singlet molecular oxygen (O2(1deltag)) produces N1-acetyl-N2-formyl-5-methoxykynurenine.

Abstract: It has been shown that melatonin exhibits antioxidant properties. Chemical structures of some of the products formed by the interaction of melatonin with reactive oxygen and nitrogen species have been elucidated. Despite some evidence that the reaction of melatonin with singlet molecular oxygen (O2(1Δg)) produces N1‐acetyl‐N2‐formyl‐5‐methoxykynurenine (AFMK), it has not been fully documented. In this investigation, melatonin was oxidized by photosensitization with methylene blue or by a clean chemical source of O2(1Δg), the thermodecomposition of N,N′‐di(2,3‐dihydroxypropyl)‐1,4‐naphtalenedipropanamide (DHPNO2). The resulting product was characterized by high performance liquid chromatography, coupled to electrospray ionization mass spectrometry and also by 1H, 13C and dept135 nuclear magnetic resonance spectroscopy. An isotopically labeled DHPN18O2 was also prepared and used as a chemical source of labeled 18[O2(1Δg)] to unequivocally characterize the end product. The results uncovered by this work confirm the hypothesis that oxidation of melatonin by O2(1Δg) produces AFMK.

Singlet oxygen-mediated damage to cellular DNA determined by the comet assay associated with DNA repair enzymes.

Abstract The damage profile produced by the reaction of singlet molecular oxygen with cellular DNA was determined using the comet assay associated with DNA repair enzymes. Singlet oxygen was produced intracellularly by thermal decomposition of a watersoluble endoperoxide of a naphthalene derivative which is able to penetrate through the membrane into mammalian cells. We found that the DNA modifications produced by singlet oxygen were almost exclusively oxidised purines recognised by the formamidopyrimidine DNA N-glycosylase. In contrast, significant amounts of direct strand breaks and alkalilabile sites or oxidised pyrimidines, detectable by the bacterial endonuclease III, were not produced.

[18O]-labeled singlet oxygen as a tool for mechanistic studies of 8-oxo-7,8-dihydroguanine oxidative damage: detection of spiroiminodihydantoin, imidazolone and oxazolone derivatives.

Abstract A watersoluble [18O]labeled endoperoxide derived from N,Ndi(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide (DHPN18O2) has been shown to act as a clean chemical source of [18O]labeled molecular singlet oxygen. This allows the assessment of the singlet oxygen (1O2) reactivity toward biological targets such as DNA. The present work focuses on the qualitative identification of the main 1O2-oxidation products of 8-oxo-7,8-dihydro-2deoxyguanosine, which was achieved using high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLCESIMS/MS). Thus, the [18O]labeled and unlabeled imidazolone and oxazolone, together with the diastereoisomeric spiroiminodihydantoin nucleosides, were detected as the main degradation products. In addition, a modified nucleoside that exhibits similar features as those of the oxidized guanidinohydantoin molecule was detected. Our data strongly suggest that the imidazolone and oxazolone nucleosides are generated via the rearrangement of an unstable 5-hydroperoxide intermediate. Interestingly, the combined use of appropriate tools, including isotopically labeled singlet oxygen and the high resolution HPLCESIMS/MS technique, has allowed to shed new light on the 1O2- mediated oxidation reactions of guanine DNA components.

Melanin Photosensitization and the Effect of Visible Light on Epithelial Cells

Protecting human skin from sun exposure is a complex issue that involves unclear aspects of the interaction between light and tissue. A persistent misconception is that visible light is safe for the skin, although several lines of evidence suggest otherwise. Here, we show that visible light can damage melanocytes through melanin photosensitization and singlet oxygen (1O2) generation, thus decreasing cell viability, increasing membrane permeability, and causing both DNA photo-oxidation and necro-apoptotic cell death. UVA (355 nm) and visible (532 nm) light photosensitize 1O2 with similar yields, and pheomelanin is more efficient than eumelanin at generating 1O2 and resisting photobleaching. Although melanin can protect against the cellular damage induced by UVB, exposure to visible light leads to pre-mutagenic DNA lesions (i.e., Fpg- and Endo III-sensitive modifications); these DNA lesions may be mutagenic and may cause photoaging, as well as other health problems, such as skin cancer.

Hemiparkinsonian rats rotate toward the side with the weaker dopaminergic neurotransmission

Rats with unilateral lesion of the substantia nigra pars compacta (SNpc) have been used as a model of Parkinsons disease. Depending on the lesion protocol and on the drug challenge, these rats rotate in opposite directions. The aim of the present study was to propose a model to explain how critical factors determine the direction of these turns. Unilateral lesion of the SNpc was induced with 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Separate analysis showed that neither the type of neurotoxin nor the site of lesion along the nigrostriatal pathway was able to predict the direction of the turns these rats made after they were challenged with apomorphine. However, the combination of these two factors determined the magnitude of the lesion estimated by tyrosine-hydroxylase immunohistochemistry and HPLC-ED measurement of striatal dopamine. Very small lesions did not cause turns, medium-size lesions caused ipsiversive turns, and large lesions caused contraversive turns. Large-size SNpc lesions resulted in an increased binding of [(3)H]raclopride to D2 receptors, while medium-size lesions reduced the binding of [(3)H]SCH-23390 D1 receptors in the ipsilateral striatum. These results are coherent with the model proposing that after challenged with a dopamine receptor agonist, unilaterally SNpc-lesioned rats rotate toward the side with the weaker activation of dopamine receptors. This activation is weaker on the lesioned side in animals with small SNpc lesions due to the loss of dopamine, but stronger in animals with large lesions due to dopamine receptor supersensitivity.

Involvement of catalase in the apoptotic mechanism induced by apigenin in HepG2 human hepatoma cells

Apigenin has been reported to inhibit proliferation of cancer cells; however, the mechanism underlying its action is not completely understood. Here, we evaluated the effects of apigenin on the levels of expression and activity of antioxidant enzymes, and the involvement of ROS in the mechanism of cell death induced by apigenin in HepG2 human hepatoma cells. Upon treatment with apigenin, HepG2 cells displayed a reduction in cell viability in a dose- and time-dependent manner, and some morphological changes. In addition, apigenin treatment induced ROS generation and significantly decreased the mRNA levels and activity of catalase and levels of intracellular GSH. On the other hand, apigenin treatment did not alter the expression or activity levels of other antioxidant enzymes. Addition of exogenous catalase significantly reduced the effects of apigenin on HepG2 cell death. We also demonstrated that HepG2 cells are more sensitive to apigenin-mediated cell death than are primary cultures of mouse hepatocytes, suggesting a differential toxic effect of this agent in tumor cells. Our results suggest that apigenin-induced apoptosis in HepG2 cells may be mediated by a H(2)O(2)-dependent pathway via reduction of the antioxidant defenses.