Reduction in free-radical-induced DNA strand breaks and base damage through fast chemical repair by flavonoids

This paper provides evidence that dietary flavonoids can repair a range of oxidative radical damages on DNA, and thus give protection against radical-induced strand breaks and base alterations. We have irradiated dilute aqueous solutions of plasmid DNA in the absence and presence of flavonoids (F) in a "constant *OH radical scavenging environment", k of 1.5 x 10(7) s(-1) by decreasing the concentration of TRIS buffer in relation to the concentration of added flavonoids. We have shown that the flavonoids can reduce the incidence of single-strand breaks in double-stranded DNA as well as residual base damage (assayed as additional single-strand breaks upon post-irradiation incubation with endonucleases) with dose modification factors of up to 2.0+/-0.2 at [F] < 100 microM by a mechanism other than through direct scavenging of *OH radicals. Pulse radiolysis measurements support the mechanism of electron transfer or H* atom transfer from the flavonoids to free radical sites on DNA which result in the fast chemical repair of some of the oxidative damage on DNA resulting from *OH radical attack. These in vitro assays point to a possible additional role for antioxidants in reducing DNA damage.     

Reduction in free-radical-induced DNA strand breaks and base damage through fast chemical repair by flavonoids

This paper provides evidence that dietary flavonoids can repair a range of oxidative radical damages on DNA, and thus give protection against radical-induced strand breaks and base alterations. We have irradiated dilute aqueous solutions of plasmid DNA in the absence and presence of flavonoids (F) in a “constant ^·OH radical scavenging environment”, k of 1.5 × 10⁷ s^-1 by decreasing the concentration of TRIS buffer in relation to the concentration of added flavonoids. We have shown that the flavonoids can reduce the incidence of single-strand breaks in double-stranded DNA as well as residual base damage (assayed as additional single-strand breaks upon post-irradiation incubation with endonucleases) with dose modification factors of up to 2.0 ± 0.2 at [F] < 100 μM by a mechanism other than through direct scavenging of ^·OH radicals. Pulse radiolysis measurements support the mechanism of electron transfer or H^· atom transfer from the flavonoids to free radical sites on DNA which result in the fast chemical repair of some of the oxidative damage on DNA resulting from ^·OH radical attack. These in vitro assays point to a possible additional role for antioxidants in reducing DNA damage.     

Intrauterine exposure to flavonoids modifies antioxidant status at adulthood and decreases oxidative stress-induced DNA damage

Maternal intake of flavonoids, known for their antioxidant properties, may affect the offspring's susceptibility to developing chronic diseases at adult age, especially those related to oxidative stress, via developmental programming. Therefore, we supplemented female mice with the flavonoids genistein and quercetin during gestation, to study their effect on the antioxidant capacity of lung and liver of adult offspring. Maternal intake of quercetin increased the expression of Nrf2 and Sod2 in fetal liver at gestational day 14.5. At adult age, in utero exposure to both flavonoids resulted in the increased expression of several enzymatic antioxidant genes, which was more pronounced in the liver than in the adult lung. Moreover, prenatal genistein exposure induced the nonenzymatic antioxidant capacity in the adult lung, partly by increasing glutathione levels. Prenatal exposure to both flavonoids resulted in significantly lower levels of oxidative stress-induced DNA damage in liver only. Our observations lead to the hypothesis that a preemptive trigger of the antioxidant defense system in utero had a persistent effect on antioxidant capacity and as a result decreased oxidative stress-induced DNA damage in the liver.     

BRCA1 role in the mitigation of radiotoxicity and chromosomal instability through repair of clustered DNA lesions

Oxidatively-induced clustered DNA lesions are considered the signature of any ionizing radiation like the ones human beings are exposed daily from various environmental sources (medical X-rays, radon, etc.). To evaluate the role of BRCA1 deficiencies in the mitigation of radiation-induced toxicity and chromosomal instability we have used two human breast cancer cell lines, the BRCA1 deficient HCC1937 cells and as a control the BRCA1 wild-type MCF-7 cells. As an additional control for the DNA damage repair measurements, the HCC1937 cells with partially reconstituted BRCA1 expression were used. Since clustered DNA damage is considered the signature of ionizing radiation, we have measured the repair of double strand breaks (DSBs), non-DSB bistranded oxidative clustered DNA lesions (OCDLs) as well as single strand breaks (SSBs) in cells exposed to radiotherapy-relevant γ-ray doses. Parallel measurements were performed in the accumulation of chromatid and isochromatid breaks. For the measurement of OCDL repair, we have used a novel adaptation of the denaturing single cell gel electrophoresis (Comet assay) and pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. Independent monitoring of the γ-H2AX foci was also performed while metaphase chromatid lesions were measured as an indicator of chromosomal instability. HCC1937 cells showed a significant accumulation of all types of DNA damage and chromatid breaks compared to MCF-7 while BRCA1 partial expression contributed significantly in the overall repair of OCDLs. These results further support the biological significance of repair resistant clustered DNA damage leading to chromosomal instability. The current results combined with previous findings on the minimized ability of base clusters to induce cell death (mainly induced by DSBs), enhance the potential association of OCDLs with breast cancer development especially in the case of a BRCA1 deficiency leading to the survival of breast cells carrying a high load of unrepaired DNA damage clusters.     

The spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast

The formation of RNA–DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.     

Effects of ageing on nuclear DNA integrity and metabolism in mussel cells (Mytilus edulis L.)

As the age of a cell increases, so does the potential for DNA damage. Recent theories on ageing suggest accumulative DNA damage is the primary cause of cellular senescence, possibly due to the decreased ability of DNA to act as a template for gene expression.In this paper we investigate the effects of ageing on the level of nuclear DNA damage in tissues of wild mussels of three different age groups; 2–4 years (group I), 6–8 years (group II) and 10 years (group III). In the digestive gland and haemolymph cells, a significant age-dependent increase of DNA damage was observed, as evaluated by the fluorimetric alkaline DNA unwinding technique, which is able to detect both direct single strand DNA breaks as well as alkali-labile apurinic sites.In addition, the rate of DNA polymerase activity was studied in order to determine whether DNA damage was dependent on DNA alteration, or because of a reduced rate of DNA repair. Unscheduled DNA repair synthesis in isolated nuclei of digestive gland cells in older mussels, was significantly decreased in comparison to younger mussels (−42% in group II and −37% in group III, p<0.01). In the digestive gland, salt extraction gives a slight, but significant, decrease of aphidicolin-sensitive DNA polymerase activity in age group III of −25%, p<0.05.Finally, we looked at the age variation in relation to oxidative stress. This was evaluated by measuring malondialdehyde accumulation in mussel cells. Digestive gland cells of group III, showed a significant age-related increase in malondialdehyde content of 170%, p<0.01, indicative of enhanced peroxidative processes.Taken together, these data suggest that the accumulation of DNA damage in group II is mainly dependent on the impairment of DNA repair systems. This is contrary to group III DNA damage, where a possible relationship between oxidative stress and alteration of nuclear DNA metabolism is found, probably deriving from an antioxidant defence decline.     

A Strain of Rosa damascena Cultured Cells Resistant to Ultraviolet Light

A strain of cultured cells of Rosa damascena Mill. which showed unusual resistance to damage by short wave (254 nanometers) ultraviolet radiation was isolated. The resistant cells were 2.2 to 2.8 times larger and had about twice the amount of DNA and more chromosomes than the parental, sensitive cells. The resistant cells also produced larger quantities of polyphenolic compounds, principally flavonoids, during the later phases of culture growth. At 10 days, resistant cells had 4 times more nonflavonoid polyphenolics and 14 times more flavonoids than parental cells. The resistance, which was also observed only in the later phases of culture growth, was best correlated with the production of polyphenolics, which apparently shielded ultraviolet-sensitive target molecules from damage.