DNA damage and susceptibility to oxidative damage in lymphocytes: effects of carotenoids in vitro and in vivo.

Reports on the effects of carotenoids are conflicting. The present paper examines similarities and differences from contiguous studies in vitro and in vivo. Single-cell gel electrophoresis was used to measure the frequency of single-strand breaks (SSB) in the cell line MOLT-17 (as a model system) and human peripheral blood lymphocytes (PBL). MOLT-17 cells were supplemented with beta-carotene, lutein or lycopene at a range of concentrations (0.00-8.00 micromol/l) using a liposome delivery method. Uptake was dose-dependent. beta-Carotene concentration in the media had no effect on SSB in control cells, but incubation with lycopene or lutein (>2.00 micromol/l) increased the numbers of SSB in control cells. MOLT-17 DNA was less susceptible to oxidative damage (100 micromol H2O2/l, 5 min, 4 degrees C) following incubation with carotenoids between 0.50 and 1.00 micromol/l; at >1.00 micromol/l the effects were ambiguous. Apparently healthy male volunteers supplemented their habitual diets with lutein, beta-carotene or lycopene (natural isolate capsules, 15 mg/d, 4 weeks) in three independent studies, raising plasma concentrations to different extents. Lycopene and lutein had no effect on SSB in control PBL or following oxidative challenge. However, increased plasma beta-carotene was associated with more SSB in control cells whilst PBL DNA resistance to oxidative damage ex vivo was unaffected. These results suggest that the carotenoids are capable of exerting two overlapping but distinct effects: antioxidant protection by scavenging DNA-damaging free radicals and modulation of DNA repair mechanisms.

Evidence that dietary supplementation with carotenoids and carotenoid-rich foods modulates the DNA damage: repair balance in human lymphocytes.

Epidemiological evidence has shown that the habitual consumption of diets high in fruits and vegetables is associated with reduced risk of cancers. The challenge is to identify causal mechanisms of effect. The aim of the current study was to determine whether an increase in rate of removal of DNA single-strand breaks (SSB) following oxidative challenge could be provoked ex vivo in peripheral blood lymphocytes (PBL). The PBL were isolated from apparently healthy volunteers following dietary intervention with: (1) a mixed carotene capsule; (2) a daily portion of cooked minced carrots; (3) a matched placebo; (4) a portion of mandarin oranges; (5) vitamin C tablets. Single-cell gel electrophoresis was employed to measure baseline levels of SSB and DNA susceptibility to oxidative damage, and to monitor the number of SSB over 4 h, in both unchallenged and H2O2-treated PBL. The enzymatic capacity for repair of different types of DNA oxidative lesions was also measured using two related cell-free assays. There was no evidence that any of the dietary supplementation regimens altered baseline levels of SSB, provided any direct antioxidant protection or altered DNA repair capacity, with two exceptions: the number of SSB following exposure to H2O2 decreased more rapidly in PBL from volunteers given the mixed carotene capsules and repair patch synthesis activity in PBL increased from volunteers given the cooked carrots. These results suggest that carotenoids and carotenoid-rich foods can influence DNA damage:repair by modulation of discrete stages in the DNA repair mechanisms.

Increased cellular carotenoid levels reduce the persistence of DNA single-strand breaks after oxidative challenge.

Dietary antioxidants, such as the carotenoids, may protect DNA from oxidative damage. This has been proposed to explain the epidemiological association between higher consumption of fruits and vegetables, which are rich in antioxidants, and lower incidence of cancer. However, this remains to be demonstrated conclusively. The effects of carotenoid supplementation on 1) baseline DNA damage, 2) susceptibility of cellular DNA to oxidative attack, and 3) DNA repair were measured in the human lymphocyte cell line Molt-17. Baseline DNA damage, susceptibility to oxidant attack (100 mumol/l H2O2 for 5 min at 4 degrees C), and disappearance of DNA single-strand breaks (SSB) after oxidative challenge were monitored by single-cell gel electrophoresis. DNA repair patch synthesis activity in cell extracts was determined using assays that measure nucleotide incorporation during repair of oxidative lesions in template DNA. Unlike single-cell gel electrophoresis, the parameters measured with these assays are not dependent on strand break religation. There was no evidence that beta-carotene, lutein, or beta-cryptoxanthin supplementation protected cellular DNA from oxidation under basal conditions or after oxidative challenge. However, only carotenoid-supplemented cells exhibited a significant decrease in numbers of SSB over a 2-h period after treatment with H2O2. Carotenoid supplementation did not provoke any detectable change in repair patch synthesis activity. We conclude that supplementation with carotenoids at 8 mumol/l does not provide significant antioxidant protection for DNA in Molt-17 lymphocytes but may enhance recovery of cells from oxidative challenge, as measured by loss of SSB. We argue that these data are most consistent with carotenoids acting to enhance DNA strand break repair.