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In vivo supplementation with coenzyme Q₁₀ enhances the recovery of human lymphocytes from oxidative DNA damage¹

Rowett Research Institute, Buckburn, Aberdeen AB21 9SB, U.K. and
^* IRCCS, Rizzoli Orthopaedic Institute, Bologna, Italy

²Correspondence: Corso Mazzini, 27–63039 San Benedetto del Tr. (AP), Italy. E-mail: renalle{at}libero.it

SPECIFIC AIMS

This study was designed to investigate whether supplementation with CoQ₁₀ protects DNA against oxidative damage in lymphocytes oxidized in vitro with compounds inducing different oxidizing profiles; DNA recovery from oxidative damage was also studied. The hypothesis that CoQ₁₀ supplementation might modulate the repair enzyme activity will be tested on whole cell extract lymphocytes.

PRINCIPAL FINDINGS

1. Coenzyme Q₁₀ in vitro enrichment inhibits the formation of DNA strand breaks induced by atmospheric oxygen
The incubation of lymphocytes under atmospheric oxygen leads to a formation of DNA strand breaks (SBs) detectable within the first hour of exposure. Both ubiquinol-10 and ubiquinone-10 are able to prevent DNA SB formation, the enriched lymphocytes resulting in less damage and exhibiting a faster DNA repair rate than that of nonenriched control lymphocytes (Fig. 1a ). Conversely, the ubiquinol-10 or ubiquinone-10 neither prevents the endogenous formation of oxidized purine and pyrimidine bases nor affects their repair (Fig. 1b , c ). When cells are treated with Ro19–8022, oxidized purine bases are specifically formed, even though a small amount of DNA SBs and modified pyrimidine bases is also detectable. Enrichment of lymphocytes with ubiquinol-10 or ubiquinone-10 did not prevent formation of oxidized purine bases, the kinetic of DNA repair being similar in control and ubiquinol-10- or ubiquinone-10-enriched cells.

View larger version (20K): [in this window] [in a new window]   Figure 1. Kinetics of repair of DNA strand breaks (a), oxidized purine bases (b), and oxidized pyrimidine bases (c) in ubiquinone-10 or ubiquinol-10 in vitro enriched and control lymphocytes exposed to oxygen atmospheric. Lymphocytes suspended in Petri dishes with RPMI 1640 containing 10% FCS (1.0x106 cells/ml) were incubated at 37°C under normal atmosphere (5% of CO2) for 24 h. At regular interval times (0, 1, 3, 6, 24 h), aliquots of the samples were collected for the Comet assay. The kinetics of DNA repair were calculated as a percentage of the basal value. Results are expressed as mean ± SD of values obtained from four separate experiments. Control lymphocytes vs. CoQ10 and CoQ10H2-enriched lymphocytes, P < 0.05.

2. Coenzyme Q₁₀ in vivo supplementation increases ubiquinol-10 endogenous levels of lymphocytes, inhibits DNA SB formation, and enhances DNA SB recovery from oxidative damage induced by atmospheric oxygen
The orally intake of 100 or 300 mg/day of coenzyme Q₁₀ (CoQ₁₀) for two consecutive weeks increases the endogenous CoQ₁₀ cellular content by 45% and 144%, respectively. Reminiscent of the situation occurring in vitro, DNA of enriched lymphocytes is less damaged by the exposure to oxygen, as indicated by the lower amount of DNA SBs formed in CoQ₁₀-enriched cells compared with native lymphocytes and the ones isolated from washout plasma. The extent of DNA SB formation inversely relates to the concentration of CoQ₁₀ in plasma and cells (Fig. 2a , b ).

View larger version (28K): [in this window] [in a new window]   Figure 2. DNA strand break formation after oxygen exposure in relation to CoQ10 content in plasma (a) and lymphocytes (b). The antioxidant concentrations and DNA strand break formation were assayed for each subject before starting the supplementation (native lymphocytes), after 1 wk of supplementation with 100 mg/day of CoQ10 (100 CoQ10 lymphocytes), after 1 wk of supplementation with 300 mg/day (300 CoQ10 lymphocytes), and at 1 wk after the last supplementation (washout lymphocytes), respectively. Data were obtained by analyzing plasma or lymphocytes isolated from six donors and results are expressed as mean ± SD. Native lymphocytes vs. 100 and 300 CoQ10-enriched lymphocytes, P < 0.05

DNA SBs accumulated in native and washout lymphocytes were repaired at the similar rate of endogenous DNA SBs in CoQ₁₀-enriched lymphocytes. Conversely, in vivo supplementation of lymphocytes with CoQ₁₀ neither prevents the formation of endogenous oxidized purine and pyrimidine bases nor affects their repair.

3. CoQ₁₀ supplementation enhances DNA repair enzyme activity
The activity of DNA repair enzymes has been assessed by incubating lymphocyte extracts containing DNA enzyme repair with a substrate consisting of oxidized purine bases derived from lymphocytes previously exposed to Ro 19–8022. DNA repair enzymes introduce breaks at sites of oxidized purines and enzyme activity is reflected by the number of DNA breaks. CoQ₁₀ supplementation enhances DNA repair activity, which is markedly higher in cellular extracts from CoQ₁₀-enriched lymphocytes than in that of native and washout lymphocytes, 148 ± 25 au. vs. 55 ± 15 au. and 94 ± 21 au., P < 0.05, respectively.

CONCLUSIONS

There are at least two ways to explain the effect of antioxidants on the recovery from oxidative DNA damage. Thus, the ability of an antioxidant to enhance the recovery may be due to a stimulation of the activity of repair enzymes or a protection against oxidation. Our results show that both ubiquinol-10 and ubiquinone-10 are capable of reacting with oxygen, as indicated by no additional formation of DNA strand breaks in enriched lymphocytes when exposed to atmosphere. The ability of CoQ₁₀ to increase the DNA repair rate is probably due to an inhibition of additional damage by protecting the cells against further oxidation. Such an effect is likely ascribed to the known antioxidant activity of ubiquinol-10. On the contrary, the capability of ubiquinone-10 to protect DNA from oxidative damage remains unclear. We hypothesize that the enrichment of cells with ubiquinone-10 yielded an ordering and condensing effect on cell membranes, and thus may have restricted the number of radicals capable of reaching the cells’ DNA. The assessment of repair capacity in a sample extract indicates that lymphocytes in vivo enriched with CoQ₁₀ are endowed with a high capacity of DNA repair compared to native cells. Changes in the redox state of transcriptional factors have been proposed as a mechanism regulating the extent of DNA binding activity, which in turn modulates various events occurring in cells, including proliferation and apoptosis. The redox mechanism implicated in enzyme trans-activation could explain the property of ubiquinol-10 in enhancing the DNA repair enzyme activity and protecting DNA from oxidative damage. After CoQ₁₀ supplementation, most of the antioxidant is present in its reduced form in plasma and lymphocytes (85–98% and 65–70%, respectively), and can be involved into redox reactions. In conclusion, we demonstrated the ability of in vitro and in vivo CoQ₁₀ supplementation in inhibiting oxidative DNA damage and enhancing DNA repair enzyme activity in cultured lymphocyte. On the basis of our results and consistent with data previously shown by others, we propose a hypothetical scheme illustrating the preventive role of CoQ₁₀ against DNA damage (Fig. 3 ). However, further investigations will be required to clarify the precise mechanism(s) by which CoQ₁₀ may modulate gene expression of DNA repair enzymes.

View larger version (33K): [in this window] [in a new window]   Figure 3. Hypothetical scheme illustrating the role of CoQ10 in the prevention of DNA oxidative damage. 1) CoQ10 may prevent DNA damage by either directly scavenging ROS or restricting the number of ROS able to reach DNA due to its ordering and condensing effect within the lipid bilayer of cell membrane; 2) CoQ10 inhibits lipid peroxidation by a chain-breaking mechanism, preventing the propagation of peroxidation processes that can generate harmful radicals able to reach DNA, and by recycling -tocopherol; 3) CoQ10 may play a role in the trans-activation of DNA repair enzymes by affecting the cellular redox status involved in the regulation of transcriptional factors, such as NF-B, and of regulatory proteins governing the transcriptional regulation of formamidopyrimidine (Fpg) DNA glycosylase, a base excision repair protein. ROS, reactive oxygen species; Q, ubiquinone-10; QH2, ubiquinol-10: QH., ubisemiquinone-10

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0694fje ; to cite this article, use FASEB J. (April 6, 2001) 10.1096/fj.00-0694fje

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