Janice Onuki

Inhibition of 5-aminolevulinic acid-induced DNA damage by melatonin, N1-acetyl-N2-formyl-5-methoxykynuramine, quercetin or resveratrol

Abstract:  Porphyrias are defined as either inborn or acquired diseases related to enzymatic deficiencies in the heme biosynthetic pathway. Lead poisoning, hereditary tyrosinemia, and acute intermittent porphyria (AIP) are characterized by the absence of photosensitivity and the accumulation of 5‐aminolevulinic acid (ALA) together with its increased urinary excretion. The main clinical manifestations of AIP are intermittent attacks of abdominal pain, neuromuscular weaknesses and neuropsychiatry alterations, and also an association with primary liver cancer, in which may be involved the oxidative potential of ALA which is able to cause DNA damage. The use of antioxidants in the treatment of ALA‐induced oxidative stress is not well established. In the current work, we show the antioxidant efficacy of several compounds including melatonin, quercetin, resveratrol and N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK), a melatonin oxidation product, in terms of their ability to limit DNA damage induced by ALA/Fe2+ in an in vitro system. Damage was measured by plasmid DNA strand breaks and detection of 8‐oxo, 7‐8‐dihydro,2′‐deoxyguanosine (8‐oxodGuo) by high‐performance liquid chromatography coupled with electrochemical detection. All compounds tested showed a dose‐dependent protective action against free radical damage. These results could be the first step toward studies of the possible use of these antioxidants in oxidative stress promoted by ALA or other pro‐oxidants.

Hydroxyl radicals are involved in the oxidation of isolated and cellular DNA bases by 5-aminolevulinic acid.

5‐Aminolevulinic acid (ALA) is a heme precursor, pathological accumulation of which is associated with liver cancer. We show that the reactive oxygen species produced upon ALA metal‐catalyzed oxidation promote the formation of several radical‐induced base degradation products in isolated DNA. The distribution of modified bases is similar to that obtained upon gamma irradiation. This observation strongly suggests the involvement of hydroxyl radicals in the ALA‐mediated DNA damage. Increased levels of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine and 5‐hydroxy‐2′‐deoxycytidine in organ DNA of rats chronically treated with ALA were observed. This is strongly suggestive of the implication of hydroxyl radicals in the ALA‐induced degradation of cellular DNA.

Supramolecular cationic tetraruthenated porphyrin induces single-strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in DNA in the presence of light.

Abstract— The aim of this investigation is the evaluation of DNA interaction of with tetraruthenated porphyrin (TRP) and of DNA damage in the presence of light. Direct‐fluorescence and electronic absorption measurements after incubation of DNA with TRP indicate strong binding between pBR322 DNA or calf thymus DNA with the modified porphyrin. Exposure of pBR322 DNA to TRP (up to 3 μM) and light leads to single‐strand break formation as determined by the conversion of the supercoiled form (form I) of the plasmid into the nicked circular form (form II). Oxidative DNA base damage was evaluated by the detection of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodGuo) after irradiation of calf thymus DNA in the presence of the TRP. The data demonstrated a dose and time dependence with each type of DNA damage. These data indicate (1) a specificity of the binding mode and (2) type I and II photoinduced mechanisms leading to strand scission activity and 8‐oxodGuo formation. Accordingly, singlet molecular oxygen formation, after TRP excitation, was confirmed by near‐infrared emission. From these investigations a potential application of TRP in photodynamic therapy is proposed.

5-Aminolevulinic acid induces single-strand breaks in plasmid pBR322 DNA in the presence of Fe2+ ions

5-Aminolevulinic acid (ALA), a heme precursor accumulated in chemical and inborn porphyrias, has been demonstrated to produce reactive oxygen species upon metal-catalyzed aerobic oxidation and to cause oxidative damage to proteins, liposomes and subcellular structures. Exposure of plasmid pBR322 DNA to ALA (0.01-3 mM) in the presence of 10 microM Fe2+ ions causes DNA single-strand breaks (ssb), revealed by agarose gel electrophoresis as an increase in the proportion of the open circular form (75 +/- 7.5% at 3 mM ALA) at the expense of the supercoiled form. Addition of either anti-oxidant enzymes such as superoxide dismutase (10 micrograms/ml) and catalase (20 micrograms/ml), or a metal chelator (DTPA, 2.5 mM), or a HO. scavenger (mannitol, 100 mM) inhibited the damage (by 30, 45, 55, and 81%, respectively), evidencing the involvement of O2-., H2O2 and HO. (by the Haber-Weiss reaction) in this process. Hydrogen peroxide (100 microM) or Fe2+ (10 microM) alone were of little effect on the extent of DNA ssb. The present data may shed light on the correlation reported between primary liver-cell carcinoma and intermittent acute porphyria.

Measurement of melatonin and its metabolites: importance for the evaluation of their biological roles.

Many physiologic changes related to lightdark cycles and antioxidant effects have been related to melatonin (N-acetyl-5methoxytryptamine) and its metabolites, N1-acetyl-N2-formyl-5methoxykynuramine AMK) and N1-acetyl-5methoxykynuramine AMK). In this review, we discuss some methodologies, in particular, those employing high-performance liquid chromatography tandem mass spectrometry (HP (MS/MS)assays to quantitatively determine melatonin, AMK, and AMK. These approaches offer a highly specific and an accurate quantification of melatonin and its metabolites. These characteristics are essential to point out correctly the biological effects of these compounds in physiological and pathological conditions.

DNA damage by 3,6-dihydropyrazine-2,5-dipropanoic acid, the cyclic dimerization product of 5-aminolevulinic acid.

Abstract 5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in lead poisoning and inborn porphyrias. It has been shown to produce reactive oxygen species upon metalcatalyzed aerobic oxidation and to cause oxidative damage to proteins, liposomes, DNA, and subcellular structures. Studies have also shown that ALA may condense to yield the cyclic product 3,6-dihydropyrazine 2,5-dipropanoic acid (DHPY). Here we propose that DHPY could be involved in DNA damage in the presence of high concentrations of ALA. Exposure of plasmid pUC19 DNA to low concentrations of DHPY (2 10 M) in the presence of 0.1 mM Cu[2+] ions causes DNA strand breaks, as demonstrated by agarose gel electrophoresis. It was also shown that in the presence of Cu[2+] ions DHPY is able to increase the oxidation of monomeric 2deoxyguanosine to form 8- oxo-7,8-dihydro-2deoxyguanosine as inferred from high performance liquid chromatography measurements using electrochemical detection. Addition of a metal chelator (bathocuproine, 0.5 mM), the DNA compacting polyamines spermidine (1 mM) and spermine (1 mM) or antioxidant enzymes such as superoxide dismutase (10 g/ml) and catalase (20 g/ml) protect the DNA against these damages. The data presented here are discussed with respect to the increased frequency of liver cancer in patients with acute intermittent porphyria.

Danos ao DNA promovidos por ácido 5-aminolevulínico: possível associação com o desenvolvimento de carcinoma hepatocelular em portadores de porfiria aguda intermitente

5-Aminolevulinic acid (ALA) is a heme precursor accumulated in acute intermittent porphyria (AIP), which might be associated with hepatocellular carcinoma (HCC) in symptomatic patients. Under metal catalyzed oxidation, ALA and its cyclic dimerization product, 3,6-dihydropyrazine-2,5-dipropanoic acid, produce reactive oxygen species that damage plasmid and calf thymus DNA bases, increase the steady state level of 8-oxo-7,8-dihydro-2´-deoxyguanosine in liver DNA and promote mitochondrial DNA damage. The final product of ALA, 4,5-dioxovaleric acid (DOVA), is able to alkylate guanine moieties, producing adducts. ALA and DOVA are mutagenic in bacteria. This review shows an up-to-date literature data that reinforce the hypothesis that the DNA damage induced by ALA may be associated with the development of HCC in AIP patients.

Measurement of 4,5-dioxovaleric acid by high-performance liquid chromatography and fluorescence detection

In this work we describe a sensitive method for the detection of 4,5-dioxovaleric acid (DOVA). 4,5-Dioxovaleric acid is derivatized with 2,3-diaminonaphthalene to form 3-(benzoquinoxalinyl-2)propionic acid (BZQ), a product with favorable UV absorbance and fluorescence properties. The high-performance liquid chromatographic method with UV absorbance and fluorescence detection is simple and its detection limit is approximately 100 fmol. This method was used to detect 4,5-dioxovaleric acid formation during metal-catalyzed 5-aminolevulinic acid (ALA) oxidation. Iron and ferritin were active in the formation of 4,5-dioxovaleric acid in the presence of 5-aminolevulinic acid. In addition, HPLC-MS-MS assay was used to characterize BZQ. The determination of 4,5-dioxovaleric acid is of great interest for the study of the mechanism of the metal-catalyzed damage of biomolecules by 5-aminolevulinic acid. This reaction may play a role in carcinogenesis after lead intoxication. The high frequency of liver cancer in acute intermittent porphyria patients may also be due to this reaction.