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.

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 (P¼0·0333), a 1·2-fold increase of mt protein carbonyls (P¼0·0278) 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 (P¼ 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 mM-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-oxidanttreated FD hepatocytes with supplemental folic acid (10-1000 mM) reversed the mt oxidative defects described earlier and diminished superoxide overproduction. Increased supplemented levels of folic acid strongly correlated with decreased lipid peroxidation (g 20·824, P¼0·0001) and protein oxidative injuries (g 2 0·865, P¼ 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-oxidantelicited mt oxidative decay. The biochemical function of folate involved in one-carbon metabolism critical for cellular proliferation and nuclear DNA repair has been well documented (Shane, 1995). In the last decade, growing literature evidence suggests another potential role of folate on antioxidant action (Nakano et al. However, the basis for folate status and oxidative insults is unclear. Folic acid has been proposed to scavenge peroxyl radicals, azide radicals and hydroxyl radicals in an in vitro radical reaction model system (Joshi et al. 2001). Particularly, the intracellular superoxide-scavenging capability of folic acid was observed in various pro-oxidant-challenged cells such as homocysteine thiolactone-treated HL60 cells (Huang et al. 2002) and 7-ketocholesterol-treated U937 cells (Huang et al. 2004). Increased folate intake was found to improve endothelial function in patients with coronary artery disease, which is largely independent of homocysteine lowering action. Instead, reduction of intracellular endothelial superoxide may have ...

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.

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.