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A fenilcetonúria (PKU) é um erro inato do metabolismo dos aminoácidos, causada pela atividade deficiente ou nula da fenilalanina hidroxilase, enzima responsável pela hidroxilação da fenilalanina em tirosina. Como consequência, ocorre o acúmulo da fenilalanina (Phe) e seus metabólitos tóxicos, ácido fenilacético (PAA), ácido fenillático (PLA) e ácido fenilpirúvico (PPA) no sangue e tecidos dos pacientes fenilcetonúricos. O tratamento para a PKU consiste em uma dieta restrita em Phe e proteínas, suplementada com uma fórmula especial, contendo aminoácidos (exceto Phe), vitaminas e minerais. Apesar dos mecanismos relacionados ao dano neurológico ainda não estarem bem elucidados, estudos vêm mostrando que o acúmulo de metabólitos tóxicos e o estresse oxidativo podem estar relacionados com a fisiopatologia da doença. No presente trabalho, investigamos in vitro os efeitos da Phe e seus metabólitos, bem como o efeito da L-carnitina (LC) sobre o dano ao DNA em células gliais C6 e leucócitos de indivíduos saudáveis, a citotoxicidade (através do ensaio vermelho neutro) e a geração de espécies reativas de oxigênio (ERO) (através do ensaio da oxidação da diclorofluoresceína - DCF) em células gliais. Demonstramos, pela primeira vez, que a Phe, PAA, PLA e PPA induzem, in vitro, dano ao DNA em células gliais e, verificamos que o pré-tratamento in vitro com a LC reduz significativamente esse dano. Verificamos também que a Phe e seus metabólitos, in vitro, aumentaram o dano ao DNA em leucócitos de indivíduos saudáveis quando comparado ao grupo controle e averiguamos que o co-tratamento com a LC reduz significativamente o dano gerado. Verificamos no ensaio de citotoxicidade que a Phe, PAA, PLA, PPA e LC não prejudicaram a viabilidade celular das células gliais. Observamos aumento da geração de ERO em células gliais induzidas pela Phe, PAA, PLA e PPA quando comparada ao grupo controle e verificamos que o pré-tratamento com LC não reduz as ERO geradas pela PPA e pela maior concentração de PLA nas células gliais. O presente trabalho fornece evidências experimentais de que o dano ao DNA e a geração de ERO podem ser induzidas pela Phe e seus metabólitos e que a LC protege em nível periférico e central contra esse dano, podendo ser considerada como uma terapia adjuvante adequada para evitar danos neurológicos na PKU. Phenylketonuria (PKU) is an inborn error of amino acid metabolism, caused by deficient or absent of phenylalanine hydroxylase activity, enzyme responsible for hydroxylation of phenylalanine (Phe) to tyrosine. As consequence, the accumulation of phenylalanine (Phe) and its toxic metabolites, phenylacetic (PAA), phenyllactic (PLA) and phenylpyruvic (PPA) acids, in blood and tissues of phenylketonuric patients occurs. The treatment for PKU consists in a Phe and protein-restricted diet, supplemented with a special formula containing amino acids (except Phe), vitamins and minerals. Although the mechanisms related to neurological damage are still poor understood, studies have been shown that the accumulation of toxic metabolites and oxidative stress may be related to the pathophysiology of disease. In the present work, we investigated the in vitro effect of Phe and its metabolites, as well as the effect of L-carnitine (LC) on DNA damage in C6 glial cells and white blood cells from healthy individuals, cytotoxicity (by neutral red assay) and reactive oxygen species (ERO) generation (by dichlorofluorescein – DCF - oxidation assay) in glial cells. We demonstrated, for the first time, that in vitro effect of Phe, PAA, PLA and PPA in glial cells induced DNA damage and we found that pre-treatment with LC added to glial cells significantly reduces DNA damage. We also verified that in vitro effect of Phe and its metabolites increased DNA damage in healthy leukocytes when compared to the control group and we found that co-treatment with LC added to white blood cells from healthy individuals significantly reduces this damage. We verified in cytotoxicity assay that Phe, PAA, PLA, PPA and LC did not impair the cell viability of glial cells. Next, we demonstrated increase in ROS generation in glial cells induced by Phe, PAA, PLA and PPA when compared to the control group and we verified that pre-treatment with LC doesn't reduce the ROS generated by PPA and the higher tested concentration of PLA in glial cells. The present work yields experimental evidence that DNA damage and ROS generation occur and can be induced by Phe and its metabolites and that LC protects at peripheral and central level against this damage and might be considered as an adjuvant therapy to prevent neurological damage in PKU.

Introduction: Homocysteine (Hcy) tissue accumulation occurs in a metabolic disease characterized biochemically by cystathionine β-synthase (CBS) deficiency and clinically by mental retardation, vascular problems, and skeletal abnormalities. Previous studies indicate the occurrence of DNA damage secondary to hyperhomocysteinemia and it was observed that DNA damage occurs in leukocytes from CBS-deficient patients. This study aimed to investigate whether an oxidative mechanism could be involved in DNA damage previously found and investigated the in vitro effect of N-acety-L-cysteine (NAC) on DNA damage caused by high Hcy levels. Methods: We evaluated a biomarker of oxidative DNA damage in the urine of CBS‑deficient patients, as well as the in vitro effect of NAC on DNA damage caused by high levels of Hcy. Moreover, a biomarker of lipid oxidative damage was also measured in urine of CBS deficient patients. Results: There was an increase in parameters of DNA (8-oxo-7,8-dihydro-2’- deoxyguanosine) and lipid (15-F2t-isoprostanes levels) oxidative damage in CBS-deficient patients when compared to controls. In addition, a significant positive correlation was found between 15-F2t-isoprostanes levels and total Hcy concentrations. Besides, an in vitro protective effect of NAC at concentrations of 1 and 5 mM was observed on DNA damage caused by Hcy 50 μM and 200 μM. Additionally, we showed a decrease in sulfhydryl content in plasma from CBS-deficient patients when compared to controls. Discussion: These results demonstrated that DNA damage occurs by an oxidative mechanism in CBS deficiency together with lipid oxidative damage, highlighting the NAC beneficial action upon DNA oxidative process, contributing with a new treatment perspective of the patients affected by classic homocystinuria.