Mitochondrial DNA deletions of blood lymphocytes as genetic markers of low folate-related mitochondrial genotoxicity in peripheral tissues.

BACKGROUND: A low folate status and mitochondrial DNA (mtDNA) mutations are risk factors for various cancers and degenerative diseases. It is not known if lymphocytic mtDNA deletions can be used as genetic "markers" to reflect global mtDNA damage during folate deficiency. AIM OF THE STUDY: The aim of this study was to characterize folate-related mtDNA deletions in lymphocytes and their associations with mt genotoxicity in peripheral tissues. METHODS: Weaning Wistar rats were fed folate-deficient and folate-replete (control) diets for 2 and 4 weeks. Folate levels of blood lymphocytes and various tissues were assayed by the Lactobacillus casei method. mtDNA deletions were measured by a real-time polymerase chain reaction analysis of whole DNA extracts. RESULTS: Compared to the control counterparts, mtDNA deletions of lymphocytes increased by 3.5-fold (P < 0.05) after 4 weeks of folate deficiency. Lymphocytic mtDNA deletions were inversely associated with plasma (r = -0.619, P = 0.018), red blood cell (r = -0.668, P = 0.009), and lymphocytic folate levels (r = -0.536, P = 0.048). Frequencies of lymphatic mtDNA deletions were positively correlated with mtDNA deletions in tissues including the lungs (r = 0.803, P = 0.0005), muscles (r = 0.755, P = 0.001), heart (r = 0.633, P = 0.015), liver (r = 0.722, P = 0.003), kidneys (r = 0.737, P = 0.006), pancreas (r = 0.666, P = 0.009), and brain (r = 0.917, P < 0.0001). CONCLUSIONS: Our data demonstrate that accumulated mtDNA deletions of lymphocytes depended upon dietary folate deprivation. The accumulated mt deletions in lymphocytes closely reflected the mt genotoxicity in the peripheral tissues during folate deficiency.

Folate deprivation and copper exposure potentiate reactive oxygen species generation and chromosomal DNA loss but not mitochondrial DNA deletions in rat hepatocytes.

Both increased copper and reduced folate levels are commonly found in patients with liver diseases. To better understand the mechanisms by which folate deprivation interacts with copper to contribute to hepatocellular toxicity, rat primary hepatocytes were isolated, cultured in folate-deprived (FD) RPMI medium, and assayed for cytotoxicity after copper sulfate (CuSO4) exposure. MTT measurement and trypan blue assay showed that elevated CuSO4 levels aggravated cell death of folate-deprived but not folate-sufficient hepatocytes. CuSO4 treatment increased the levels of intracellular reactive oxygen species (ROS) by 3 times in FD hepatocytes and tripled the proportion of FD hepatocytes with hypodiploid DNA contents. Measurement of membrane phosphatidylserine exposure indicated that the CuSO4-mediated toxicity in FD hepatocytes was not mediated by the apoptotic pathway. Real-time polymerase chain reaction (PCR) analysis revealed that CuSO4 treatment did not increase the occurrence of a 4834-bp mtDNA (mtDNA4834) deletion in FD hepatocytes. Preincubation of FD hepatocytes with various concentrations of folate prior to CuSO4 treatment did not modulate the mtDNA4834 deletion. Taken together, the data suggest that elevated copper levels potentiate cell death of folate-deprived hepatocytes, which is primarily associated with increased ROS generation and chromosomal DNA loss. The cytotoxicity exerted by folate depletion and elevated copper levels, however, is not due to apoptosis or accumulated mtDNA4834 deletions in primary hepatocytes.

Changes in mitochondrial DNA deletion, content, and biogenesis in folate-deficient tissues of young rats depend on mitochondrial folate and oxidative DNA injuries.

We aimed to characterize folate-related changes in mitochondrial (mt) DNA of various tissues of young rats. Weaning Wistar rats were fed folate-deficient (FD) or folate-replete (control) diet for 2 or 4 wk. The mtDNA 4834-bp large deletion (mtDNA(4834) deletion) and mtDNA content were analyzed by quantitative real-time PCR. Compared with pooled 2-wk and 4-wk control groups, 4-wk folate deprivation significantly increased the frequency of the mtDNA(4834) deletion in pancreas, heart, brain, liver, and kidney and reduced mtDNA contents in brain, heart, and liver (P < 0.05). Decreased mt folate levels were correlated with increased mtDNA(4834) deletion frequency in tissues from FD rats after 2 wk (r = -0.380, P = 0.001) and 4 wk FD (r = -0.275, P = 0.033) and with reduced mtDNA content after 4 wk (r = 0.513, P = 0.005). In liver of 4-wk FD rats, the accumulated mtDNA large deletions and decline in mtDNA accompanied increased expressions of messenger RNAs (mRNA) of factors that regulate mtDNA proliferation and transcription, including nuclear respiratory factor 1, mt transcriptional factor A, mt single-strand DNA-binding protein, and mt polymerase r. In parallel, expression of mRNA for nuclear-encoded cytochrome c oxidase subunits (CcOX) IV, V, cytochrome c, and mtDNA-encoded CcOX III increased significantly. This enhanced mt biogenesis in 4-wk FD liver coincided with an elevated ratio of 8 hydroxydeoxyguanosine (8-OHdG):deoxyguanosine (dG) (2.67 +/- 1.41) relative to the controls (0.99 +/- 0.36; P = 0.0002). The 8-OHdG:dG levels in FD liver were correlated with liver mt folate (r = -0.819, P < 0.001), mtDNA deletions (r = 0.580, P = 0.001), and mtDNA contents (r = -0.395, P = 0.045). Thus, folate deprivation induced aberrant changes of mtDNA(4834) deletion and mtDNA content in a manner that was dependent on mt folate and oxidative DNA injuries. The folate-related mt biogenesis provides a molecular mechanism to compensate mtDNA impairment in FD tissues.

Folate deprivation promotes mitochondrial oxidative decay: DNA large deletions, cytochrome c oxidase dysfunction, membrane depolarization and superoxide overproduction in rat liver.

Little is known about the biological effect of folate in the protection against mitochondrial (mt) oxidative decay. The objective of the present study was to examine the consequence of folate deprivation on mt oxidative degeneration, and the mechanistic link underlying the relationship. Male Wistar rats were fed with an amino acid-defined diet containing either 8 (control) or 0 (folate-deficient, FD) mg folic acid/kg diet. After a 4-week FD feeding period, significant elevation in oxidative stress was observed inside the liver mitochondria with a 77% decrease in mt folate level (P<0.001), a 28 % reduction in glutathione peroxidase activity (P= 0.0333), a 1.2-fold increase of mt protein carbonyls (P=0.0278) and an accumulated 4834 bp large-scale deletion in mtDNA. The elicited oxidative injuries in FD liver mitochondria were associated with 30 % reduction of cytochrome c oxidase (CcOX) activity (P=0.0264). The defective CcOX activity in FD hepatocytes coincided with mt membrane potential dissipation and intracellular superoxide elevation. Exposure of FD hepatocytes to pro-oxidant challenge (32 microM-copper sulphate for 48 h) led to a further loss in CcOX activity and mt membrane potential with a simultaneous increase in superoxide production. Preincubation of pro-oxidant-treated FD hepatocytes with supplemental folic acid (10-1000 microM) reversed the mt oxidative defects described earlier and diminished superoxide overproduction. Increased supplemented levels of folic acid strongly correlated with decreased lipid peroxidation (gamma - 0.824, P=0.0001) and protein oxidative injuries (gamma -0.865, P=0.0001) in pro-oxidant-challenged FD liver mitochondria. Taken together, the results demonstrated that folate deprivation induces oxidative stress in liver mitochondria, which is associated with CcOX dysfunction, membrane depolarization and superoxide overproduction. The antioxidant activity of supplemental folic acid may partially, if not fully, contribute to the amelioration of pro-oxidant-elicited mt oxidative decay.