Inhibition of 5-aminolevulinic acid-induced DNA damage by melatonin, N 1 -acetyl-N 2 -formyl-5-methoxykynuramine, quercetin or resveratrol Introduction Porphyrias are defined as either inborn or acquired diseases related to enzymatic deficiencies in the heme biosynthetic pathway. Numerous genetic defects have been associated with each type of porphyria. These enzyme deficiencies result in excessive production and excretion of both porphyrins and their precursors [1–4]. Porphyrias are classified according to the main organ in which excess porphyrins are generated and to the occurrence or not of acute attacks and cutaneous lesions (photosensitivity) and/ or neurological alterations [5–7]. We have been studying porphyrias that are characterized by the absence of photosensitivity and the accumulation of 5-aminolevulinic acid (ALA) together with its increased urinary excretion. These include lead poisoning, hereditary tyrosinemia, and particularly, acute intermittent porphyria (AIP) [6]. Acute intermittent porphyria is an autosomal dominant disorder, characterized by a deficiency in porphobilinogen deaminase (PBGD) which leads to an increase in ALA and porphobilinogen levels in blood and urine. ALA especially accumulates in the liver and brain [8] and there is a higher incidence of this disease in women than men [9]. The prevalence of AIP is globally estimated as 1–3 cases per 100,000 inhabitants and Sweden is the country with the highest prevalence with 10 cases per 100,000 inhabitants [10]. The main clinical manifestations of AIP are intermittent attacks of abdominal pain, neuromuscular weaknesses and neuropsychiatry alterations [10]. It should be noted that some secondary diseases have been associated with AIP such as hypertension [11], renal failure [12] and higher incidence of hepatocellular carcinoma (HCC) [13]. The association between AIP and HCC was first noted by Lithner and Wetterberg [13]. Among 206 carriers of mutated PBGD, 11 cases were reported of HCC (5%). Hardell et al. [14] reported three cases of HCC among 11 individuals with AIP (27%). Since then, numerous studies have reported higher HCC incidence in AIP patients [15, 16]. Kauppinen and Mustajoki [16] estimated an increased risk of 61-fold for the development of HCC in AIP patients. There is no established explanation for the higher incidence of HCC in AIP patients. Several hypotheses have been proposed [13, 17]; but much evidence points to the oxidative potential of ALA as it causes DNA damage leading to mutations that could initiate a carcinogenic process [18–22]. 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 N 1 -acetyl-N 2 - formyl-5-methoxykynuramine (AFMK), a melatonin oxidation product, in terms of their ability to limit DNA damage induced by ALA/Fe 2+ 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. Janice Onuki, Eduardo A. Almeida, Marisa H. G. Medeiros and Paolo Di Mascio Departamento de Bioquı ´mica, Instituto de Quı ´mica, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, Brazil Key words: 5-aminolevulinic acid, acute intermittent porphyria, melatonin, N 1 -acetyl- N 2 -formyl-5-methoxykynuramine, quercetin, resveratrol Address reprint requests to Paolo Di Mascio, Departamento de Bioquı ´mica, Instituto de Quı ´mica, Universidade de Sa ˜o Paulo, CP 26077, CEP 05513-970, Sa ˜o Paulo, SP, Brasil. E-mail: pdmascio@iq.usp.br Received June 1, 2004; accepted August 25, 2004. J. Pineal Res. 2005; 38:107–115 Doi:10.1111/j.1600-079X.2004.00180.x Copyright Ó Blackwell Munksgaard, 2004 Journal of Pineal Research 107