Improvement of genetic stability in lymphocytes from Fanconi anemia patients through the combined effect of α-lipoic acid and N-acetylcysteine

Abstract

Fanconi Anemia (FA) is a rare genetic disorder, characterized by progressive bone marrow failure and increased predisposition to cancer. Despite being highly heterogeneous, all FA patients are hypersensitive to alkylating agents, in particular to 1,2:3,4-diepoxybutane (DEB), and to oxidative damage. Recent studies point to defective mitochondria in FA cells, which is closely related with increased production of reactive oxygen species (ROS) and concomitant depletion of antioxidant defenses, of which glutathione is a well-known biomarker.The objective of the present work is to evaluate the putative protective effect of α-lipoic acid (α-LA), a mitochondrial protective agent, and N-acetylcysteine (NAC), a direct antioxidant and a known precursor for glutathione synthesis, in spontaneous and DEB-induced chromosome instability (CI) in lymphocyte cultures from FA patients.For that purpose, lymphocyte cultures from 15 FA patients and 24 healthy controls were pre-treated with 20 μM α-LA, 500 μM NAC and α-LA plus NAC at the same concentrations, and some of them were exposed to DEB (0.05 μg/ml). A hundred metaphases per treatment were scored to estimate the relative frequency of spontaneous and DEB-induced chromosome breakage.The obtained results revealed that a cocktail of α-LA and NAC can drastically improve the genetic stability in FA lymphocytes in vitro, decreasing CI by 60% and 80% in cultures from FA patients and FA mosaic/chimera patients, respectively. These results suggest that the studied cocktail can be used as a prophylactic approach to delay progressive clinical symptoms in FA patients caused by CI, which can culminate in the delay of the progressive bone marrow failure and early cancer development.

Figure 1

CI pattern in metaphases from DEB-induced lymphocyte cultures from a healthy donor and FA patient. Chromosomes were stained in a 4% Giemsa solution. The images were observed with an optical microscope (Olympus CX31) and captured with a digital camera (Nikon Sightds-smc), with the software DP20-5E microscope digital camera. (A) Selected metaphase from a HD lymphocyte culture exposed to 0.2 μg/ml of DEB for 48 h. One chromatid break can be seen (asterisk). (B) Selected metaphase from a FA patient lymphocyte culture exposed to 0.05 μg/ml of DEB for 48 h. High level of chromosome instability can be visualized, especially being important the tri and tetraradial figures (arrow) that are the hallmark for the diagnosis of FA. It can also be seen a dicentric chromosome (head arrow) and 3 chromatid breaks (asterisks).

Figure 2

Effect of α-LA, NAC and α-LA + NAC on chromosome breaks in cultured lymphocytes from FA patients. Lymphocyte cultures were treated with 20 μM α-LA, 500 μM NAC, and both antioxidants simultaneously at the same concentrations. The results are presented as the relative mean of breaks per cell (percentage of control values). Comparison of the relative mean of breaks/cell was made between control group and each antioxidant treatment (*** P < 0.001 and **P < 0.01) and between treatments (P > 0.05). Results are representative of the mean of 14 experiments depicted from Table 2.

Figure 3

Effect of α-LA, NAC and α-LA + NAC on chromosome breaks in cultured lymphocytes from FA mosaics/chimeras. Lymphocyte cultures were treated with 20 μM α-LA, 500 μM NAC, and both antioxidants simultaneously at the same concentrations. The results are presented as the relative mean of breaks per cell (percentage of control values). Comparison of the relative mean of breaks/cell was made between control group and each antioxidant treatment (*** P < 0.001 and **P < 0.01) and between treatments (P > 0.05). Results are representative of the mean of 3 experiments depicted from Table 2.

Figure 4

Effect of α-LA + NAC on mitotic index of cultured lymphocytes from FA patients. Lymphocyte cultures were treated with 20 μM α-LA plus 500 μM NAC. Mitotic index was evaluated as described in “methods” and the results are presented as absolute frequencies depicted from Table 3 (*P < 0.05).

Figure 5

Effect of α-LA, NAC and α-LA + NAC on chromosome breaks in DEB-induced cultured lymphocytes from FA patients. Lymphocyte cultures were pre-treated with 20 μM α-LA, 500 μM NAC, and both antioxidants simultaneously at the same concentrations, 1.5 h before exposure to DEB 0.05 μg/ml for 48 h. The results are presented as the relative mean of breaks per cell (percentage of control values). Comparison of the relative mean of breaks/cell was made between control group and each antioxidant treatment (*** P < 0.001) and between treatments (P > 0.05). Results are representative of the mean of 14 experiments depicted from Table 4.

Figure 6

Comparison between the different studied groups according to the antioxidant treatments. Comparative analysis of the effect of α-LA (A), NAC (B) and α-LA plus NAC treatments was performed between lymphocyte cultures from the four studied groups: healthy donors, FA patients (spontaneous breaks), FA patients (DEB-induced breaks) and FA mosaic/chimera patients. All values were depicted from Figures 2, 3 and 5.