DNA damage induced by alloisoleucine and other metabolites in maple syrup urine disease and protective effect of l-carnitine.

Maple syrup urine disease (MSUD) is an inherited deficiency of the branched-chain α-keto dehydrogenase complex, characterized by accumulation of the branched-chain amino acids (BCAAs) and their respective branched chain α-keto-acids (BCKAs), as well as by the presence of alloisoleucine (Allo). Studies have shown that oxidative stress is involved in the pathophysiology of MSUD. In this work, we investigated using the comet assay whether Allo, BCAAs and BCKAs could induce in vitro DNA damage, as well as the influence of l-Carnitine (L-Car) upon DNA damage. We also evaluated urinary 8-hydroxydeoguanosine (8-OHdG) levels, an oxidative DNA damage biomarker, in MSUD patients submitted to a restricted diet supplemented or not with L-Car. All tested concentrations of metabolites (separated or incubated together) induced in vitro DNA damage, and the co-treatment with L-Car reduced these effects. We found that Allo induced the higher DNA damage class and verified a potentiation of DNA damage induced by synergistic action between metabolites. In vivo, it was observed a significant increase in 8-OHdG levels, which was reversed by L-Car. We demonstrated for the first time that oxidative DNA damage is induced not only by BCAAs and BCKAs but also by Allo and we reinforce the protective effect of L-Car.

Inflammatory profile in X-linked adrenoleukodystrophy patients: Understanding disease progression.

X-linked adrenoleukodystrophy (X-ALD) is an inherited disease characterized by progressive inflammatory demyelization in the brain, adrenal insufficiency, and an abnormal accumulation of very long chain fatty acids (VLCFA) in tissue and body fluids. Considering that inflammation might be involved in pathophysiology of X-ALD, we aimed to investigate pro- and anti-inflammatory cytokines in plasma from three different male phenotypes (CCER, AMN, and asymptomatic individuals). Our results showed that asymptomatic patients presented increased levels of pro-inflammatory cytokines IL-1ß, IL-2, IL-8, and TNF-α and the last one was also higher in AMN phenotype. Besides, asymptomatic patients presented higher levels of anti-inflammatory cytokines IL-4 and IL-10. AMN patients presented higher levels of IL-2, IL-5, and IL-4. We might hypothesize that inflammation in X-ALD is related to plasmatic VLCFA concentration, since there were positive correlations between C26:0 plasmatic levels and pro-inflammatory cytokines in asymptomatic and AMN patients and negative correlation between anti-inflammatory cytokine and C24:0/C22:0 ratio in AMN patients. The present work yields experimental evidence that there is an inflammatory imbalance associated Th1, (IL-2, IL-6, and IFN-γ), Th2 (IL-4 and IL-10), and macrophages response (TNF-α and IL-1ß) in the periphery of asymptomatic and AMN patients, and there is correlation between VLCFA plasmatic levels and inflammatory mediators in X-ALD. Furthermore, we might also speculate that the increase of plasmatic cytokines in asymptomatic patients could be considered an early biomarker of brain damage and maybe also a predictor of disease progression.

DNA damage in homocystinuria: 8-oxo-, 8-dihydro-2'-deoxyguanosine levels in cystathionine-ß-synthase deficient patients and the in vitro protective effect of N-acetyl­L­cysteine

Introduction: Homocysteine (Hcy) tissue accumulation occurs in a metabolic disease characterized biochemically by cystathionine ß-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 CBS­deficient 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.

Experimental evidence of oxidative stress in patients with l-2-hydroxyglutaric aciduria and that l-carnitine attenuates in vitro DNA damage caused by d-2-hydroxyglutaric and l-2-hydroxyglutaric acids.

d-2-hydroxyglutaric (D-2-HGA) and l-2-hydroxyglutaric (L-2-HGA) acidurias are rare neurometabolic disorders biochemically characterized by increased levels of d-2-hydroxyglutaric acid (D-2-HG) and l-2-hydroxyglutaric acid (L-2-HG) respectively, in biological fluids and tissues. These diseases are caused by mutations in the specific enzymes involved in the metabolic pathways of these organic acids. In the present work, we first investigated whether D-2-HG and L-2-HGA could provoke DNA oxidative damage in blood leukocytes and whether l-carnitine (LC) could prevent the in vitro DNA damage induced by these organic acids. It was verified that 50µM of D-2-HG and 30µM of L-2-HG significantly induced DNA damage that was prevented by 30 and 150µM of LC. We also evaluated oxidative stress parameters in urine of L-2-HGA patients and observed a significant increase of oxidized guanine species and di-tyrosine, biomarkers of oxidative DNA and protein damage, respectively. In contrast, no significant changes of urinary isoprostanes and reactive nitrogen species levels were observed in these patients. Taken together, our data indicate the involvement of oxidative damage, especially on DNA, in patients affected by these diseases and the protective effect of LC.