Retinol supplementation induces DNA damage and modulates iron turnover in rat Sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular DNA damage probably involving cellular iron accumulation. Retinol (7μM) significantly induced DNA single strands breaks, DNA fragmentation and production of 8-oxo-7, 8-dihydro-2'-deoxyguanosine in cultured Sertoli cells. In contrast, lower doses seemed not to induce single-strands break in this experimental model. The breaks in DNA were inhibited by an iron scavenger; and 7μM retinol treatment modulated iron turnover leading to iron accumulation, suggesting that iron ions were required for the retinol cellular effects. These findings suggest that retinol-induced DNA damage was associated with the modulation of iron turnover, and these characteristics could be responsible for the increased incidence of lung cancer associated with retinoids supplementation.     

Mitogenic signaling mediated by oxidants in retinol treated sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol-induced oxidative stress is accompanied by cellular proliferation. Retinol (7 μM) significantly induced thiobarbituric acid reactive species (TBARS) formation, which was inhibited by trolox, superoxide dismutase, N-acetylcysteine and ethanol. This was accompanied by an increase in DNA synthesis and focus formation in cultured rat Sertoli cells. Antioxidants and ethanol inhibited retinol-induced DNA synthesis. Our findings suggest that retinol-induced oxidative stress was associated with cellular proliferation complementing our understanding of the significance of retinol supplementation in neoplastic transformation.     

Mitogenic signaling mediated by oxidants in retinol treated sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol-induced oxidative stress is accompanied by cellular proliferation. Retinol (7 μM) significantly induced thiobarbituric acid reactive species (TBARS) formation, which was inhibited by trolox, superoxide dismutase, N-acetylcysteine and ethanol. This was accompanied by an increase in DNA synthesis and focus formation in cultured rat Sertoli cells. Antioxidants and ethanol inhibited retinol-induced DNA synthesis. Our findings suggest that retinol-induced oxidative stress was associated with cellular proliferation complementing our understanding of the significance of retinol supplementation in neoplastic transformation.     

Retinol supplementation induces oxidative stress and modulates antioxidant enzyme activities in rat Sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular oxidative stress and modulated superoxide dismutase, catalase and glutathione peroxidase activities. Retinol (7 μM) significantly increased TBARS, conjugated dienes, and hydroperoxide-initiated chemiluminescence in cultured Sertoli cells. In response to retinol treatment superoxide dismutase, catalase and glutathione peroxidase activities increased. TBARS content and catalase activities were decreased by a free radical scavenger. These findings suggest that retinol may induce oxidative stress and modulate antioxidant enzyme activities in Sertoli cells.     

Retinol supplementation induces oxidative stress and modulates antioxidant enzyme activities in rat sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular oxidative stress and modulated superoxide dismutase, catalase and glutathione peroxidase activities. Retinol (7 μM) significantly increased TBARS, conjugated dienes, and hydroperoxide-initiated chemiluminescence in cultured Sertoli cells. In response to retinol treatment superoxide dismutase, catalase and glutathione peroxidase activities increased. TBARS content and catalase activities were decreased by a free radical scavenger. These findings suggest that retinol may induce oxidative stress and modulate antioxidant enzyme activities in Sertoli cells.     

Oxidative DNA damage by vitamin A and its derivative via superoxide generation

Recent intervention studies revealed that beta-carotene supplement to smokers resulted in a higher incidence of lung cancer. However, the causal mechanisms remain to be clarified. We reported here that vitamin A (retinol) and its derivative (retinal) caused cellular DNA cleavage detected by pulsed field gel electrophoresis. Retinol and retinal significantly induced 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in HL-60 cells but not in H(2)O(2)-resistant HP100 cells, suggesting the involvement of H(2)O(2) in cellular DNA damage. Experiments using (32)P-labeled isolated DNA demonstrated that retinol and retinal caused Cu(II)-mediated DNA damage, which was inhibited by catalase. UV-visible spectroscopic and electron spin resonance-trapping studies revealed the generation of superoxide and carbon-centered radicals, respectively. The superoxide generation during autoxidation of retinoids was significantly correlated with the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, although the yield of carbon-centered radicals was not necessarily related to the intensity of DNA damage. These findings suggest that superoxide generated by autoxidation of retinoids was dismutated to H(2)O(2), which was responsible for DNA damage in the presence of endogenous metals. Retinol and retinal have prooxidant abilities, which might lead to carcinogenesis of the supplements of beta-carotene.     

Vitamin A (retinol) downregulates the receptor for advanced glycation endproducts (RAGE) by oxidant-dependent activation of p38 MAPK and NF-kB in human lung cancer A549 cells

As an essential component of the diet, retinol supplementation is often considered harmless and its application is poorly controlled. However, recent works demonstrated that retinol may induce a wide array of deleterious effects, especially when doses used are elevated. Controlled clinical trials have demonstrated that retinol supplementation increased the incidence of lung cancer and mortality in smokers. Experimental works in cell cultures and animal models showed that retinol may induce free radical production, oxidative stress and extensive biomolecular damage. Here, we evaluated the effect of retinol on the regulation of the receptor for advanced glycation end-products (RAGE) in the human lung cancer cell line A549. RAGE is constitutively expressed in lungs and was observed to be down-regulated in lung cancer patients. A549 cells were treated with retinol doses reported as physiologic (2 μM) or therapeutic (5, 10 or 20 μM). Retinol at 10 and 20 μM increased free radical production, oxidative damage and antioxidant enzyme activity in A549 cells. These doses also downregulated RAGE expression. Antioxidant co-treatment with Trolox®, a hydrophilic analog of α-tocopherol, reversed the effects of retinol on oxidative parameters and RAGE downregulation. The effect of retinol on RAGE was mediated by p38 MAPK activation, as blockade of p38 with PD169316 (10 μM), SB203580 (10 μM) or siRNA to either p38α (MAPK14) or p38β (MAPK11) reversed the effect of retinol on RAGE. Trolox also inhibited p38 phosphorylation, indicating that retinol induced a redox-dependent activation of this MAPK. Besides, we observed that NF-kB acted as a downstream effector of p38 in RAGE downregulation by retinol, as NF-kB inhibition by SN50 (100 μg/mL) and siRNA to p65 blocked the effect of retinol on RAGE, and p38 inhibitors reversed NF-kB activation. Taken together, our results indicate a pro-oxidant effect of retinol on A549 cells, and suggest that modulation of RAGE expression by retinol is mediated by the redox-dependent activation of p38/NF-kB signaling pathway.     

Life-long supplementation with a low dosage of coenzyme Q10 in the rat: effects on antioxidant status and DNA damage

Life-long low-dosage supplementation of coenzyme Q(10) (CoQ(10)) is studied in relation to the antioxidant status and DNA damage. Thirty-two male rats were assigned into two experimental groups differing in the supplementation or not with 0.7 mg/kg/day of CoQ(10). Eight rats per group were killed at 6 and 24 months. Plasma retinol, alpha-tocopherol, coenzyme Q, total antioxidant capacity and fatty acids were analysed. DNA strand breaks were studied in peripheral blood lymphocytes. Aging and supplementation led to significantly higher values for CoQ homologues, retinol and alpha-tocopherol. No difference in total antioxidant capacity was detected at 6 months but significantly lower values were found in aged control animals. Similar DNA strand breaks levels were found at 6 months. Aging led to significantly higher DNA strand breaks levels in both groups but animals supplemented with CoQ(10) led to a significantly lower increase in that marker. Aged rats showed significantly higher polyunsaturated fatty acids. This study demonstrates that lifelong intake of a low dosage of CoQ(10) enhances plasma levels of CoQ(9), CoQ(10), alpha-tocopherol and retinol. In addition, CoQ(10) supplementation attenuates the age-related fall in total antioxidant capacity of plasma and the increase in DNA damage in peripheral blood lymphocytes.     

Vitamin A treatment induces apoptosis through an oxidant-dependent activation of the mitochondrial pathway

Even though retinoids are widely used as adjuvant in chemotherapeutic interventions to improve cancer cell death, their mechanism(s) of action involves multiple overlapping pathways that remain unclear. We have previously shown that vitamin A, the natural precursor of the retinoids, induces oxidative-dependent cytochrome c release from isolated mitochondria, suggesting a putative mechanism for apoptosis activation. Using Sertoli cells in culture, we show that retinol causes mitochondrial-dependent apoptosis, involving oxidative stress. Apoptosis was evaluated by nuclear morphology, DNA fragmentation, and caspase-3/7 activity. Retinol induced oxidant- and time-dependent imbalance of several mitochondrial parameters, cytochrome c release and caspase-3/7 activation, leading cells to commit apoptosis. All parameters tested were attenuated or blocked by trolox co-administration, suggesting that retinol induces apoptosis through oxidative damage, which mitochondria plays a pivotal role.     

Hepatic iron accumulation is not directly associated with induction of DNA strand breaks in the liver cells of Long-Evans Cinnamon (LEC) rats.

Effects of accumulation of copper and iron on induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats that spontaneously develop fulminant hepatitis. Copper and iron accumulated in the liver of LEC rats in an age-dependent manner from 4 to 15 weeks. Low-iron diet prevented the accumulation of iron in the liver, but did not prevent accumulation of copper. The amounts of DNA strand breaks that were estimated by comet assay in the liver cells of rats fed standard diet increased with age from 4 to 15 weeks. No significant differences were observed in the proportions of LEC rat liver cells without tail and the average lengths of tail momentum in the comet images between LEC rats that had been fed standard MF diet and low-iron diet. These results support the idea that accumulation of iron is not directly associated with the induction of DNA damage in the liver cells of LEC rats.     

Genotoxicity,recombinogenicity and cellular preneoplasic transformation induced by vitamin A supplementation

In spite of being one of the first vitamins to be discovered, the full range of biological activities of Vitamin A remains incomplete. A growing body of evidence has demonstrated an apparent enhancement of carcinogenesis, induced by dietary retinol. Since DNA damage is a well-recognized inducer of carcinogenesis, the aim of this study was to test the possible genotoxic effect of dietary retinol, using different types of bioassays. Retinol caused an increased recombinogenic activity in Drosophila melanogaster larvae as measured by the SMART test. In mammalian cell cultures, retinol supplementation-induced DNA double-strands breaks (DSB) and single-strands breaks (SSB), cell cycle progression and proliferative focus formation in terminal-differentiated rat Sertoli cells and increased DNA fragmentation in Chinese hamster lung fibroblasts (V79 cells), as measured by the comet assay. Altogether, our results suggest that retinol causes DNA damage and chromosomal rearrangements, which may disturbs key physiological processes and lead to cell cycle progression and preneoplasic transformation of terminal-differentiated mammalian cells.     

Deposition of iron and beta-amyloid plaques is associated with cortical cellular damage in rabbits fed with long-term cholesterol-enriched diets

Hypercholesterolemia is a potential trigger of Alzheimer's disease, and is thought to increase brain levels of beta-amyloid (Abeta) and iron. However, animal models to address the mechanisms by which Abeta and iron accumulation may cause neuronal damage are poorly defined. To address this question, we fed adult rabbits a 1% cholesterol-enriched diet for 7 months. This diet was associated with increased regional deposition of both iron and Abeta peptide in the brain. Iron preferentially accumulated around Abeta plaques in the adjacent cortex, but was not found in the hippocampus. Co-localization of iron and Abeta was accompanied by apoptosis, DNA damage, blood-brain barrier (BBB) disruption, as well as dysregulation in the level of the iron-regulatory proteins, ferritin and heme-oxygenase-1. We further demonstrate that the cholesterol diet-induced apoptosis is mediated by the activation of the endoplasmic reticulum stress pathway, involving the down-regulation of the endoplasmic reticulum chaperones, calreticulin, grp78 and grp94, and the activation of the growth and arrest DNA damage protein, gadd153. Our results suggest that BBB damage and disturbances in iron metabolism may render the cortex more vulnerable than the hippocampus to the cholesterol-induced cellular stress.     

A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA

We have isolated a Saccharomyces cerevisiae mutant that shows an increased tendency to form cytoplasmic petites (respiration-deficient ρ⁻ or ρ⁰ mutants) in response to treatment of cells growing on a solid medium with the DNA-damaging agent methyl methanesulfonate or ultraviolet light. The mutation in this strain, atm1-1, was found to cause a single amino acid substitution in ATM1, a nuclear gene that encodes the mitochondrial ATP-binding cassette (ABC) transporter. When the mutant cells were grown in liquid glucose medium, they accumulated free iron within the mitochondria and at the same time gave rise to spontaneous cytoplasmic petite mutants, as seen previously in cells carrying a mutation in a gene homologous to the human gene responsible for Friedreich's ataxia. Analysis of the effects of free iron and malonic acid (an inhibitor of oxidative respiration in mitochondria) on the incidence of petites among the mutant cells indicated that spontaneous induction of petites was a consequence of oxidative stress rather than a direct effect of either a defect in the ATM1 gene or the accumulation of free iron. We observed an increase in the incidence of strand breaks in the mitochondrial DNA of the atm1-1 mutant cells. Furthermore, we found that rates of induction of petites and accumulation of strand breaks in mitochondrial DNA were enhanced in the atm1-1 mutant by the introduction of another mutation, mhr1-1, which results in a deficiency in mitochondrial DNA repair. These observations indicate that spontaneous induction of petites in the atm1-1 mutant is a consequence of oxidative damage to mitochondrial DNA mediated by enhanced accumulation of mitochondrial iron. Received: 26 March 1999 / Accepted: 29 June 1999     

BMI1 is recruited to DNA breaks and contributes to DNA damage-induced H2A ubiquitination and repair

DNA damage activates signaling pathways that lead to modification of local chromatin and recruitment of DNA repair proteins. Multiple DNA repair proteins having ubiquitin ligase activity are recruited to sites of DNA damage, where they ubiquitinate histones and other substrates. This DNA damage-induced histone ubiquitination is thought to play a critical role in mediating the DNA damage response. We now report that the polycomb protein BMI1 is rapidly recruited to sites of DNA damage, where it persists for more than 8 h. The sustained localization of BMI1 to damage sites is dependent on intact ATM and ATR and requires H2AX phosphorylation and recruitment of RNF8. BMI1 is required for DNA damage-induced ubiquitination of histone H2A at lysine 119. Loss of BMI1 leads to impaired repair of DNA double-strand breaks by homologous recombination and the accumulation of cells in G(2)/M. These data support a crucial role for BMI1 in the cellular response to DNA damage.     

Regulation of Rad17 Protein Turnover Unveils an Impact of Rad17-APC Cascade in Breast Carcinogenesis and Treatment

Aberrant regulation of DNA damage checkpoint function leads to genome instability that in turn can predispose cellular tissues to become cancerous. Previous works from us and others demonstrated the role of Rad17 in either activation or termination of DNA damage checkpoint function. In the current study, we have revealed the unexpected accumulation of Rad17 in various types of breast cancer cell lines as well as human breast cancer tissues. We observed that Rad17 protein turnover rate in breast epithelial cells is much faster than in breast cancer cells, where the turnover of Rad17 is regulated by the Cdh1/APC pathway. We further observed that Rad17-mediated checkpoint function is modulated by proteolysis. Stabilization of Rad17 disrupts cellular response to chemotherapeutic drug-induced DNA damage and enhances cellular transformation. In addition, manipulation of Rad17 by RNA interference or stabilization of Rad17 significantly sensitize breast cancer cell to various chemotherapeutic drugs. Our present results indicate the manipulation of Rad17 proteolysis could be a valuable approach to sensitize breast cancer cell to the chemotherapeutic treatment despite of the critical role in governing DNA damage response and cellular recovery from genotoxic stress.     

The defense mechanisms in mammalian cells against oxidative damage in nucleic acids and their involvement in the suppression of mutagenesis and cell death

To counteract oxidative damage in nucleic acids, mammalian cells are equipped with several defense mechanisms. We herein review that MTH1, MUTYH and OGG1 play important roles in mammalian cells avoiding an accumulation of oxidative DNA damage, both in the nuclear and mitochondrial genomes, thereby suppressing carcinogenesis and cell death. MTH1 efficiently hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP, 8-oxo-dATP and 2-hydroxy (OH)-dATP, to the monophosphates, thus avoiding the incorporation of such oxidized nucleotides into the nuclear and mitochondrial genomes. OGG1 excises 8-oxoG in DNA as a DNA glycosylase and thus minimizes the accumulation of 8-oxoG in the cellular genomes. MUTYH excises adenine opposite 8-oxoG, and thus suppresses 8-oxoG-induced mutagenesis. MUTYH also possesses a 2-OH-A DNA glycosylase activity for excising 2-OH-A incorporated into the cellular genomes. Increased susceptibilities to spontaneous carcinogenesis of the liver, lung or intestine were observed in MTH1-, OGG1- and MUTYH-null mice, respectively. The increased occurrence of lung tumors in OGG1-null mice was abolished by the concomitant disruption of the Mth1 gene, indicating that an increased accumulation of 8-oxoG and/or 2-OH-A might cause cell death. Furthermore, these defense mechanisms also likely play an important role in neuroprotection.     

Critical roles of ring finger protein RNF8 in replication stress responses

Histone ubiquitylation is emerging as an important protective component in cellular responses to DNA damage. The ubiquitin ligases RNF8 and RNF168 assemble ubiquitin chains onto histone molecules surrounding DNA breaks and facilitate retention of DNA repair proteins. Although RNF8 and RNF168 play important roles in repair of DNA double strand breaks, their requirement for cell protection from replication stress is largely unknown. In this study, we uncovered RNF168-independent roles of RNF8 in repair of replication inhibition-induced DNA damage. We showed that RNF8 depletion, but not RNF168 depletion, hyper-sensitized cells to hydroxyurea and aphidicolin treatment. Consistently, hydroxyurea induced persistent single strand DNA lesions and sustained CHK1 activation in RNF8-depleted cells. In line with strict requirement for RAD51-dependent repair of hydroxyurea-stalled replication forks, RNF8 depletion compromised RAD51 accumulation onto single strand DNA lesions, suggesting that impaired replication fork repair may underlie the enhanced cellular sensitivity to replication arrest observed in RNF8-depleted cells. In total, our study highlights the differential requirement for the ubiquitin ligase RNF8 in facilitating repair of replication stress-associated DNA damage.     

Role of mitochondrial calcium uniporter‐mediated Ca2+ and iron accumulation in traumatic brain injury

Previous studies have suggested that the cellular Ca²⁺ and iron homeostasis, which can be regulated by mitochondrial calcium uniporter (MCU), is associated with oxidative stress, apoptosis and many neurological diseases. However, little is known about the role of MCU‐mediated Ca²⁺ and iron accumulation in traumatic brain injury (TBI). Under physiological conditions, MCU can be inhibited by ruthenium red (RR) and activated by spermine (Sper). In the present study, we used RR and Sper to reveal the role of MCU in mouse and neuron TBI models. Our results suggested that the Ca²⁺ and iron concentrations were obviously increased after TBI. In addition, TBI models showed a significant generation of reactive oxygen species (ROS), decrease in adenosine triphosphate (ATP), deformation of mitochondria, up‐regulation of deoxyribonucleic acid (DNA) damage and increase in apoptosis. Blockage of MCU by RR prevented Ca²⁺ and iron accumulation, abated the level of oxidative stress, improved the energy supply, stabilized mitochondria, reduced DNA damage and decreased apoptosis both in vivo and in vitro. Interestingly, Sper did not increase cellular Ca²⁺ and iron concentrations, but suppressed the Ca²⁺ and iron accumulation to benefit the mice in vivo. However, Sper had no significant impact on TBI in vitro. Taken together, our data demonstrated for the first time that blockage of MCU‐mediated Ca²⁺ and iron accumulation was essential for TBI. These findings indicated that MCU could be a novel therapeutic target for treating TBI.     

Delayed kinetics of DNA double-strand break processing in normal and pathological aging

Accumulation of DNA damage may play an essential role in both cellular senescence and organismal aging. The ability of cells to sense and repair DNA damage declines with age. However, the underlying molecular mechanism for this age-dependent decline is still elusive. To understand quantitative and qualitative changes in the DNA damage response during human aging, DNA damage-induced foci of phosphorylated histone H2AX (γ-H2AX), which occurs specifically at sites of DNA double-strand breaks (DSBs) and eroded telomeres, were examined in human young and senescing fibroblasts, and in lymphocytes of peripheral blood. Here, we show that the incidence of endogenous γ-H2AX foci increases with age. Fibroblasts taken from patients with Werner syndrome, a disorder associated with premature aging, genomic instability and increased incidence of cancer, exhibited considerably higher incidence of γ-H2AX foci than those taken from normal donors of comparable age. Further increases in γ-H2AX focal incidence occurred in culture as both normal and Werner syndrome fibroblasts progressed toward senescence. The rates of recruitment of DSB repair proteins to γ-H2AX foci correlated inversely with age for both normal and Werner syndrome donors, perhaps due in part to the slower growth of γ-H2AX foci in older donors. Because genomic stability may depend on the efficient processing of DSBs, and hence the rapid formation of γ-H2AX foci and the rapid accumulation of DSB repair proteins on these foci at sites of nascent DSBs, our findings suggest that decreasing efficiency in these processes may contribute to genome instability associated with normal and pathological aging.     

Genotoxicity of hydroquinone in A549 cells

Hydroquinone (HQ) is found in natural and anthropogenic sources including food, cosmetics, cigarette smoke, and industrial products. In addition to ingestion and dermal absorption, human exposure to HQ may also occur by inhaling cigarette smoke or polluted air. The adverse effects of HQ on respiratory systems have been studied, but genotoxicity HQ on human lung cells is unclear. The aim of this study was to investigate the cytotoxicity and genotoxicity of HQ in human lung alveolar epithelial cells (A549). We found that HQ induced a dose response in cell growth inhibition and DNA damage which was associated with an increase in oxidative stress. Cytotoxicity results demonstrated that HQ was most toxic after 24 h (LC₅₀?=?33 μM) and less toxic after 1 h exposure (LC₅₀?=?59 μM). Genotoxicity of HQ was measured using the Comet assay, H2AX phosphorylation, and chromosome aberration formation. Results from the comet assay revealed that DNA damage was highest during the earlier hours of exposure (1 and 6 h) and thereafter was reduced. A similar pattern was observed for H2AX phosphorylation suggesting that damage DNA may be repaired in later exposure hours. An increase in chromosomal aberration corresponded with maximal DNA damage which further confirmed the genotoxic effects of HQ. To investigate whether oxidative stress was involved in the cytotoxic and genotoxic effects of HQ, cellular glutathione and 8-Oxo-deoguanisone (8-Oxo-dG) formation were measured. A decrease in the reduced glutathione (GSH) and an increase oxidized glutathione (GSSG) was observed during the early hours of exposure which corresponded with elevated 8-Oxo-dG adducts. Together these results demonstrate that HQ exerts its cytotoxic and genotoxic effects in A549 lung cells, probably through DNA damage via oxidative stress.