Growth modulation of human cells in vitro by mild oxidative stress and 1,4-dihydropyridine derivative antioxidants

Reactive oxygen species and lipid peroxidation products are not only cytotoxic but may also modulate signal transduction in cells. Accordingly, antioxidants may be considered as modifiers of cellular redox signaling. Therefore, the effects of two novel synthetic antioxidants, analogues of 1,4-dihydropyridine derivatives, cerebrocrast and Z41-74 were analysed in vitro on human osteosarcoma cell line HOS, the growth of which can be modulated by lipid peroxidation. The cells were pretreated with either cerebrocrast or Z41-74 and afterwards exposed to mild, copper induced lipid peroxidation or to 4-hydroxynonenal (HNE), the end product of lipid peroxidation. The results obtained have shown that both antioxidants exert growth modulating effects interfering with the lipid peroxidation. Namely, cells treated with antioxidants showed increased metabolic rate and cell growth, thereby attenuating the effects of lipid peroxidation. Such biomodulating effects of cerebrocrast and Z41-74 resembled growth modulating effects of HNE, suggesting that the antioxidants could eventually promote cellular adaptation to oxidative stress interacting with redox signaling and hydroxynonenal HNE-signal transduction pathways. This may be of particular relevance for better understanding the beneficial role of hydroxynonenal HNE in cell growth control. Therefore, cerebrocrast and Z41-74 could be convenient to study further oxidative homeostasis involving lipid peroxidation.     

Nicotinamide stimulates repair of DNA damage in human lymphocytes

Nicotinamide stimulates the amount of DNA repair synthesis that occurs when freshly isolated, normal human lymphocytes are treated with UV irradiation, N-methyl-N′-nitro-N-nitroso guanidine, or dimethyl sulfate. Stimulation of DNA repair synthesis is concentration dependent and reaches a maximum between 2 to 5 mM nicotinamide. In contrast, DNA synthesis in cells that have not been subjected to DNA damage is not affected by nicotinamide at concentrations below 2 mM and is inhibited by concentrations between 2 to 5 mM. In the same concentration range, nicotinic acid has no effect on the rate of DNA synthesis in the presence or absence of DNA damage.     

Alkylation damage, DNA repair and mutagenesis in human cells

17 human cell lines that differ significantly in level of O⁶-alkylguanine-DNA alkyltransferase (AGT) activity were identified by comparing their sensitivity to the cytotoxic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and determining the level of AGT activity in cell extracts from the various lines by measuring the decrease in radiolabeled O⁶-methylguanine from DNA, using high-performance liquid chromatography. 9 lines exhibited high levels of AGT activity, 2 showed an intermediate level (25–50% of the mean of those with the higher levels), and 6 exhibited very low or virtually undetectable levels of AGT. Included were several lines that are very deficient in capacity for nucleotide excision repair. When representatives from the 3 categories of cell lines defined by the level of AGT activity were compared for sensitivity to the cytotoxic and mutagenic effect of MNNG, they showed an inverse correlation between the degree of cell killing and frequency of mutants induced and the level of AGT activity. The cells' capacity for nucleotide excision repair did not affect these results. Exposure of cells with a high level of AGT activity to O⁶-methylguanine in the medium reduced the AGT activity 60–80%. These pre-treated cells exhibited a significantly higher frequency of MNNG-induced mutants than did cells that were not pre-treated, suggesting that the O⁶-methylguanine lesion in DNA is responsible for a significant proportion of the mutations induced. Cell strains containing substrates for assaying intrachromosomal homologous recombination were constructed using parental cell lines from each of the 3 categories of AGT activity. These strains showed an inverse correlation between the level of AGT activity and the frequency of MNNG-induced recombination. When various cell lines representing the 3 categories of AGT activity were compared for sensitivity to ethylnitrosourea, the results were consistent with AGT and nucleotide excision repair playing a role in preventing cell killing and mutation induction by this agent.     

DNA damage and DNA repair in cultured human cells exposed to chromate

DNA damage and DNA repair have been observed in cultured human skin fibroblasts exposed to potassium chromate but not to a chromic glycine complex. DNA repair synthesis (unscheduled incorporation of [3H]thymidine (TdR)) was measured in cells during or following exposure to chromate and was significant for chromate concentrations above 10(-6) M. Maximal DNA repair was observed at about 10(-4) M chromate. DNA repair capacity was found to be saturated at this concentration. Chromate was stable for at least 8 h in culture medium and produced approximately a linear increase in repair with duration of exposure. DNA damage as determined by alkaline sucrose gradient sedimentation was detected after treatment for 1.5 h with 5 . 10(-4) M chromate. Exposure to 10(-7) M chromate solution for 7 days inhibited colony formation while acute (1 h) treatment was toxic at 5 . 10(-6) M. The chromic glycine complex was toxic above 10(-3) M for a 1-week exposure but was not observably toxic after a 1-h treatment. These results indicate that chromate and not chromic compounds may be the carcinogenic form for man. The nature of the ultimate carcinogen is discussed. These findings illustrate the utility of the DNA repair technique to study the effects on human cells of inorganic carcinogens and mutagens.     

Pesticide induced DNA damage and its repair in cultured human cells.

The effects of pesticides on the induction of unscheduled DNA synthesis in SV-40 transformed human cells (VA-4) in culture with and without metabolic activation by liver microsomes was studied. Results showed that ten of the thirteen compounds examined either directly or upon metabolic activation induced unscheduled DNA synthesis in the human cell system used. The DNA repair kinetics and size of the repaired regions resulting from treatment with four of the chemicals (Carbaryl, Chlordane, Dieldrin and 2.4-D Fluid) were studied by 313 nm photolysis of repaired regions containing bromodeoxyuridine (BUdR). The size of the repaired regions differed between compounds but could generally be classified as either of the X-ray (short) or UV-type (long).     

Influence of iron-overload on DNA damage and its repair in human leukocytes in vitro

Iron is an important element that modulates the production of reactive oxygen species, which are thought to play a causative role in biological processes such as mutagenesis and carcinogenesis. The potential genotoxicity of dietary iron has been seldom studied in human leukocyte and only few reports have investigated in human colon tumor cells. Therefore, DNA damage and repair capacity of human leukocytes were examined using comet assay for screening the potential toxicity of various iron-overloads such as ferric-nitrilotriacetate (Fe-NTA), FeSO(4), hemoglobin and myoglobin, and compared with 200μM of H(2)O(2) and HNE. The iron-overloads tested were not cytotoxic in the range of 10-1000 microM by trypan blue exclusion assay. The exposure of leukocytes to Fe-NTA (500 and 1000 microM), FeSO(4) (250-1000 microM), hemoglobin (10 microM) and myoglobin (250 microM) for 30 min induced significantly higher DNA damage than NC. Treatment with 500 and 1000 microM of Fe-NTA showed a similar genotoxic effect to H(2)O(2), and a significant higher genotoxic effect than HNE. The genotoxicity of FeSO(4) (250-1000 microM), hemoglobin (10 microM) and myoglobin (250 microM) was not significantly different from that of H(2)O(2) and HNE. Iron-overloads generated DNA strand break were rejoined from the first 1h. Their genotoxic effect was not observed at 24h. These data from this study provide additional information on the genotoxicity of iron-overloads and self-repair capacity in human leukocytes.     

Active DNA damage eviction by HLTF stimulates nucleotide excision repair

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Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.     

Modulation of cellular defense processes in human lymphocytes in vitro by a 1,4-dihydropyridine derivative

The aim of this pilot study was to assess whether a compound of the β-carbonyl-1,4-dihydropyridine series (AV-153 or sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate), which has high efficiency in stimulating DNA repair, can simultaneously modulate apoptosis in human cells. Peripheral blood lymphocytes of healthy donors were used in this study. DNA strand-break rejoining was assessed with the alkaline comet assay after a 3-h incubation of lymphocytes in the presence of a wide range of concentrations of AV-153 (10⁻¹⁰–10⁻⁵ M). Apoptotic and micronucleated (MN) cells were scored in phytohaemagglutinin-stimulated lymphocytes after a 72-h incubation with AV-153, using the standard cytokinesis-blocked micronucleus test. The study revealed dual effects of AV-153 on cellular defense systems against endogenously generated DNA damage: the compound per se simultaneously reduces DNA strand breaks and stimulates apoptosis, with a maximal efficiency of 76% and 42%, respectively; in contrast, after genotoxic stress (2 Gy of gamma-radiation) AV-153 reduces DNA strand breaks, the number of MN cells and apoptotic cells in a similar dose-dependent manner. A maximal efficiency of 67% was found for reduction of DNA strand breaks, while for MN cells and apoptotic cells the efficiencies were, respectively, 47% and 44%. While limited in number, these preliminary studies show the direct correlation between the efficiency of AV-153 in reduction of radiation-induced DNA breaks and MN cells on one side, and in reduction of apoptosis on the other. It suggests that the major target of the compound's action on genotoxic stress is DNA repair, followed by reduction of the number of damaged cells entering apoptosis.     

The human Werner syndrome protein stimulates repair of oxidative DNA base damage by the DNA glycosylase NEIL1

The mammalian DNA glycosylase, NEIL1, specific for repair of oxidatively damaged bases in the genome via the base excision repair pathway, is activated by reactive oxygen species and prevents toxicity due to radiation. We show here that the Werner syndrome protein (WRN), a member of the RecQ family of DNA helicases, associates with NEIL1 in the early damage-sensing step of base excision repair. WRN stimulates NEIL1 in excision of oxidative lesions from bubble DNA substrates. The binary interaction between NEIL1 and WRN (K(D) = 60 nM) involves C-terminal residues 288-349 of NEIL1 and the RecQ C-terminal (RQC) region of WRN, and is independent of the helicase activity WRN. Exposure to oxidative stress enhances the NEIL-WRN association concomitant with their strong nuclear co-localization. WRN-depleted cells accumulate some prototypical oxidized bases (e.g. 8-oxoguanine, FapyG, and FapyA) indicating a physiological function of WRN in oxidative damage repair in mammalian genomes. Interestingly, WRN deficiency does not have an additive effect on in vivo damage accumulation in NEIL1 knockdown cells suggesting that WRN participates in the same repair pathway as NEIL1.     

The relationship between DNA repair after alkylation damage and in vitro aging in human T-lymphocytes

In vitro cultures of human peripheral blood lymphocytes, both unstimulated G0 cells as well as phytohemagglutinin (PHA) stimulated cells, have been used in the investigation of DNA repair following different types of damage, including that induced by ultraviolet light, ionizing radiation, and chemical agents. We report here repair of DNA damage in cultured normal human T-lymphocytes after treatment with the alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or methanesulfonate (MMS), as measured by the alkaline elution technique. T-lymphocytes cultured with different sources of T-cell growth factors (TCGFs) were shown to have similar repair levels, as measured by loss of single-strand breaks. However, diminished repair was observed with in vitro culture age when T-cells were cultured with PHA and TCGF for a 3-week period. Cell-cycle analysis performed on asynchronously growing cultures indicated that differential repair with in vitro aging was not cell-cycle-related. Fluorescence Activated Cell Sorting (FACS) was used to determine the percentages of CD4+ and CD8+ T-cell subsets present during the in vitro culture periods. Cultures consisted primarily of CD4+ cells until day 20 when the percentage of CD8+ cells increased to approximately 50% of the T-cell population. Neither the absolute percentages of CD4+ and CD8+ cells not the CD4+/CD8+ ratios correlated with repair rates of cultured T-cells. Therefore, the observed decreases in the repair of alkylating agent-induced damage are not due solely to the change in the CD4+/CD8+ ratio.     

Metal-induced DNA damage and repair in human diploid fibroblasts and Chinese hamster ovary cells

Cloning efficiency and DNA strand breaks induction were compared in human diploid fibroblasts (HSBP) and chinese hamster ovary (CHO) cells treated with various metal salts. Cadmium (Cd2+), nickel (Ni2+) and chromate (Cr2O7) reduced the cloning efficiency of HSBP cells more than that of CHO cells whereas the reverse was true after treatment with mercury (Hg2+), manganese (Mn2+) and cobalt (Co2+). The effects on cloning efficiency did not consistently correlate with DNA strand breaking activity as all metals except Cr(VI) were more effective at producing DNA strand breaks in CHO cells than in human cells. The differential responses of the two cell types was shown to be only partially due to differences in cellular uptake of metals. DNA breaks induced in human cells by Hg2+ and Cr2O7 were shown most likely to be alkaline labile sites rather than true strand breaks since no damage was detected in a nick translation assay which measures the amount of free 3'-OH terminals. Damage induced by Mn2+ and Co2+, however, appeared to be comprised at least in part by true DNA strand breaks. DNA damage was also induced in HSBP cells following treatment with selenium but only in the presence of reduced glutathione. These studies indicate that DNA damage is not as major a consequence following some metal treatments in human cells as it appears to be in rodent cells. This suggests that rodent models for risk estimation of metal-induced tumorigenesis may not always be appropriate for extrapolation to humans.     

Cardioprotective properties of a 1,4-dihydropyridine derivative, glutapyrone, in deep hypothermia

Effect of the derivative of 1,4-dihydropyridine-glutapyron on the activity of Ca2(+)-ATPase, lipid peroxidation and formation of the high-energy phosphate in the myocardium under deep hypothermia was investigated. Analysis of chemiluminescence parameters and changes of malondialdehyde production as a measure of peroxidation has shown high antioxidant activity of glutapyron under deep hypothermia. The inhibition of peroxidation by glutapyron takes place in the lipids of erythrocyte and heart mitochondrial membranes. Due to antioxidant activity glutapyron is able to inhibit initiation of free radical lipid oxidation, to stabilize membrane structure and to preserve function of membrane integrated proteins. In the aggregate these actins promote activity maintenance of high-energy phosphate production and transport reactions in heart under deep hypothermia.     

In vitro repair synthesis of BCNU-induced DNA damage

The nitrosoureas including BCNU are potent chemotherapeutic drugs and have been used extensively for treatment of brain tumors and other neoplasias but the mechanisms of action for the DNA lesions created and their repair are still unclear. We have recently determined the in vitro repair of BCNU-treated DNA with cellular extracts and with DNA modifying enzymes. BCNU not only caused an increase in breaks in plasmid DNA, but an increase in cross-linked DNA was also observed after restriction enzyme digestion followed by gel electrophoresis. When HeLa cell-extracts were incubated with BCNU-treated DNA, 5-10 fold increases in DNA repair synthesis were observed as compared with untreated control. Substantial increases in 5'OH and 3'OH sites of the breaks were also found in BCNU-treated DNA as determined by the 10-20 fold increases in labeling with T4-DNA kinase and by endogenous polymerases, while the amount of ligatable sites were at a minimal. When the repair capacity of two glioma cell lines (UWR1 and UWR3) with differential BCNU sensitivity, and cells from a chromosomal breakage disease, Bloom's syndrome (BS), were assessed, the activities of the two glioma cells were about 20-30% of the normal lymphoblastoid cells and HeLa cells, whereas no difference was observed in BS cells. However, differential patterns of DNA bands were observed in the glioma samples suggesting cell-type specific capacities of repair synthesis. These data are in accordance with the concept that BCNU creates multiple DNA lesions and suggests different cell types may develop a variety of repair capabilities.     

Novel 1,4-dihydropyridine induces apoptosis in human cancer cells through overexpression of Sirtuin1

1,4-Dihydropyridines (1,4-DHPs) are important as a class of heterocyclic compounds that exhibit wide range of biological actions. Many of its derivatives are already characterized as medicinally important drugs and used worldwide. In this study, we have screened some novel Hantzsch 1,4-DHP compounds using both in silico (QSAR and Pharmacophore) and in vitro (cytotoxic screening). 1,4-DHP showed selective cytotoxicity against five human cancerous cell lines; A375, A549, HeLa, HepG2 and SH-SY5Y but limited effect towards normal skin keratinocyte (HaCaT), lung fibroblast (WL-38) and healthy peripheral blood mononuclear cells. In A375 and HepG2 cells, one of the 1,4-DHP derivative (DHP-8) was found to inhibit cell proliferation, and simultaneously increased the apoptotic population as well as mitochondrial membrane depolarization. Furthermore, the mitochondrial signal was triggered with the activation of cleaved Caspase9, Caspase3 and PARP. The treatment with DHP-8 also increased the expression level of SIRT1, subsequently decreasing the level of pAKT^ser473 and survivin. Reduced pAKT^ser473 expression led to decrease the phosphorylated inactive form of GSK3β^ser9 and as a result, proteasomal degradation of Mcl-1 occurred in both the cell lines. Here, we suggest that the apoptotic effect of DHP-8 in A375 and HepG2 cells was mediated by AKT and survivin pathways through SIRT1 activation. The involvement of DHP-8 in SIRT1 activation was further verified by co-treatment of nicotinamide with DHP-8 in both A375 and HepG2 cells. Overall, this study emphasizes the possible potential and therapeutic role of DHP-8 in skin and liver cancer.     

Monitoring repair of DNA damage in cell lines and human peripheral blood mononuclear cells

We introduce a method to follow DNA repair that is suitable for both clinical and laboratory samples. An episomal construct with a unique 8-oxoguanine (8-oxoG) base at a defined position was prepared in vitro using single-stranded phage harboring a 678-bp tract from exons 5 to 9 of the human P53 gene. Mixing curve experiments showed that the real-time PCR method has a linear response to damage, suggesting that it is useful for DNA repair studies. The episomal construct with a unique 8-oxoG base was introduced into AD293 cells or human peripheral blood mononuclear cells, and plasmids were recovered as a function of time. The quantitative real-time PCR assay demonstrated that repair of the 8-oxoG was 80% complete in less than 48 h in AD293 cells. Transfection of small interfering RNAs down-regulated OGG1 expression in AD293 cells and reduced the repair of 8-oxoG to 30%. Transfection of the episome into unstimulated white blood cells showed that 8-oxoG repair had a half-life of 2 to 5 h. This method is a rapid, reproducible, and robust way to monitor repair of specific adducts in virtually any cell type.     

DNA damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes

In cultured human lymphocytes we determined the ability of nitrilotriacetic acid (NTA) to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. In phytohemagglutinin-stimulated lymphocytes a strong inhibition of DNA replication was induced by NTA concentrations above 10(-3) M, which was accompanied by a marked cell lethality, whereas at lower doses the incorporation of tritiated thymidine (3H-TdR) into DNA or treated cells was slightly increased in comparison to untreated cells. When, after NTA pretreatment, UDS was determined by scintillation spectrometry or autoradiography in unstimulated G0 lymphocytes, UV-irradiated or unirradiated, an increased incorporation of 3H-TdR was observed, positively correlated with the NTA doses. This effect was only partially due to the expansion of the intracellular TdR pool as a consequence of the stimulation of 3H-TdR uptake by NTA. Even after normalization of the scintillometric data by the radioactivities of the soluble nucleotide fraction, significant increase of DNA repair synthesis was detected after treatment with 7.5 x 10(-3)-10(-2) M NTA.     

A quinoxaline 1,4-di-N-oxide derivative induces DNA oxidative damage not attenuated by vitamin C and E treatment

Some anticancer compounds are pro-drugs which give rise to toxic species through enzymatic reduction. The quinoxaline-di-N-oxide derivative Q-85 HCl (7-chloro-3-[[(N,N-dimethylamino)propyl]amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxide hydrochloride) is a bioreductive compound selectively toxic in hypoxia. Due to the possibility of secondary tumors the study of the genotoxic capability of antitumoral drugs is very important. The aim of this study was to assess the ability of Q-85 HCl to produce reactive oxygen species (ROS) and oxidative DNA damage in Caco-2 cells, both in hypoxia and in well-oxygenated conditions. Secondly, we attempted to evaluate the effect of vitamins C and E under hypoxic and normoxic conditions, in order to determine if these antioxidant substances modify Q-85 HCl effect in hypoxic cells or possibly exert a protective action in normal cells. Caco-2 cells were treated with Q-85 HCl for 2h, at high concentrations in normoxia (0.1-5 microM) and at low concentrations in hypoxia (0.002-0.1 microM). In normoxia, a dose-related significant increase in intracellular ROS level was evident; in hypoxia all the concentrations produced very high level of ROS. Just after the treatment and 24h later, oxidative DNA damage was evaluated by the modified comet assay after post-digestion of the cells with formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (Endo III). Q-85 HCl treatment evoked a significant dose-dependent increase in the total comet score of the cells both in hypoxia and normoxia, indicating that this compound or some metabolite is able to oxidize purine and pyrimidine bases. After 24h DNA damage caused by the compound was completely repaired with only one exception: cells treated with the highest concentration of Q-85 HCl in hypoxia and post-digested with FPG. Vitamin C (5-100 microM) and vitamin E (500-400 microM) did not have a pro-oxidant effect in Caco-2 cells. Treatment of cells with vitamin C (10 microM) or vitamin E (100 microM) did not significantly reduce oxidative DNA damage in hypoxia and normoxia. In conclusion, the use of these vitamins would not hinder toxicity against hypoxic cells, but a protective effect in normoxic cells was not evident.