DNA Damage In Primary Cultures Research Articles

Neuroprotective Effects of Rosmarinic Acid on Ciguatoxin in Primary Human Neurons

Ciguatoxin (CTX), is a toxic compound produced by microalgae (dinoflagellate) Gambierdiscus spp., and is bio-accumulated and bio-transformed through the marine food chain causing neurological deficits. To determine the mechanism of CTX-mediated cytotoxicity in human neurons, we measured extracellular lactate dehydrogenase (LDH) activity, intracellular levels of nicotinamide adenine dinucleotide (NAD(+)) and H2AX phosphorylation at serine 139 as a measure for DNA damage in primary cultures of human neurons treated with Pacific (P)-CTX-1B and P-CTX-3C. We found these marine toxins can induce a time and dose-dependent increase in extracellular LDH activity, with a concomitant decline in intracellular NAD(+) levels and increased DNA damage at the concentration range of 5-200nM. We also showed that pre- and post-treatment with rosmarinic acid (RA), the active constituent of the Heliotropium foertherianum (Boraginaceae) can attenuate CTX-mediated neurotoxicity. These results further highlight the potential of RA in the treatment of CTX-induced neurological deficits.

The specificity of UVA-induced DNA damage in human melanocytes

Exposure to solar UV radiation is the origin of most skin cancers, including deadly melanomas. Melanomas are quite different from keratinocyte-derived tumours and exhibit a different mutation spectrum in the activated oncogenes, possibly arising from a different class of DNA damage. In addition, some data suggest a role for UVA radiation in melanomagenesis. To get further insight into the molecular mechanisms underlying induction of melanoma, we quantified a series of UV-induced DNA damage in primary cultures of normal human melanocytes. The results were compared with those obtained in keratinocytes from the same donors. In the UVB range, the frequency and the distribution of pyrimidine dimers was the same in melanocytes and keratinocytes. UVA was also found to produce thymine cyclobutane dimer as the major DNA lesion with an equal efficiency in both cell types. In contrast, following UVA-irradiation a large difference was found for the yield of 8-oxo-7,8-dihydroguanine; the level of this product was 2.2-fold higher in melanocytes than in keratinocytes. The comet assay showed that the induction of strand breaks was equally efficient in both cell types but that the yield of Fpg-sensitive sites was larger in melanocytes. Our data show that, upon UVA irradiation, oxidative lesions contribute to a larger extent to DNA damage in melanocytes than in keratinocytes. We also observed that the basal level of oxidative lesions was higher in the melanocytes, in agreement with a higher oxidative stress that may be due to the production of melanin. The bulk of these results, combined with qPCR and cell survival data, may explain some of the differences in mutation spectrum and target genes between melanomas and carcinomas arising from keratinocytes.

TNF-α induced DNA damage in primary murine hepatocytes

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, usually arising from a background of chronic inflammatory disease. Tumor necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine produced in response to tissue injury, endotoxin exposure or infection and TNF-alpha signalling in hepatocytes is associated with an increase in oxidative stress. DNA is vulnerable to reactive oxygen species (ROS)-induced damage, which is highly mutagenic. Cells respond to DNA damage through the stabilisation of the tumor suppressor p53, which maintains genomic fidelity through induction of a cell cycle arrest in order to allow repair or elimination of the damaged cell through apoptosis. This study was carried out to determine if TNF-alpha caused oxidative DNA damage in primary cultures of murine hepatocytes and whether any damage would result in the induction of the tumor suppressor p53 and cell-cycle arrest. Using a modified Comet assay, to measure DNA damage we have demonstrated that TNF-alpha causes the formation of 8-oxo-deoxyguanosine (8-oxodG), an established marker of oxidative DNA damage, and a lesion associated with chronic hepatitis in human livers. In addition, the increase in DNA damage did not result in p53 stabilisation and TNF-alpha caused an increase in cell-cycle progression. We believe that this study indicates a possible putative role for TNF-alpha in the early stages of malignant transformation of hepatocytes.

Correlation between induction of DNA fragmentation in urinary bladder cells from rats and humans and tissue-specific carcinogenic activity

Seven chemicals, six of which are known to induce epithelial neoplasms of the urinary bladder in rats, were assayed for their ability to induce DNA damage in primary cultures of rat and human cells from urinary bladder mucosa, and in urinary bladder, liver and kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, were obtained in cells from both rats and humans with the following concentrations of five test compounds: 2-naphthylamine and N-nitrosodi- n-butylamine 0.5 and 1 mM, phenacetin 2 and 4 mM, cyclophosphamide from 2 to 8 mM, and o-toluidine 16 and 32 mM. Nitrilotriacetic acid (1–4 mM), a rat bladder carcinogen, and 4-aminobiphenyl (0.125–0.5 mM), a bladder carcinogen in humans but not in rats, gave a weak positive response in rats cells and a more marked response in humans cells. In terms of DNA-damaging potency, 4-aminobiphenyl, cyclophosphamide, phenacetin and 4 nitrilotriacetic acid were more active in human than in rat cells, whereas the converse occurred with 2-naphthylamine. Consistently with the results observed in vitro statistically significant dose-dependent increases in the average frequency of DNA breaks were detected in the urinary bladder mucosa of rats given p.o. single doses corresponding to 1 4 and 1 2 LD50 of six of the seven test compounds; the only one which gave a substantially negative response was 4-aminobiphenyl. With the exception of N-nitrosodi- n-butylamine which caused DNA damage in liver and of phenacetin and nitrilotriacetic acid which caused damage in kidney in agreement with their tumorigenic activity, any substantial evidence of DNA lesions in these two organs was absent in rats treated with 1 2 LD50 of the other 4 test compounds. These findings give evidence that urinary bladder genotoxic carcinogens may be identified by the DNA damage/Comet assay using as targets cells of urinary bladder mucosa, and show that the effect may be quantitatively different in cells from rats and from human donors.

DNA damage in breast epithelial cells: detection by the single-cell gel (comet) assay and induction by human mammary lipid extracts.

The presence of DNA damage in primary cultures of human mammary epithelial cells (HMECs), and the ability of extracts of human mammary lipid to cause such damage, has been investigated. Lipid extracts, prepared by a solid-phase procedure, and HMECs were obtained from breast tissue removed from healthy women (ages 18-50 years) who were resident in the UK and undergoing elective reduction mammoplasties. DNA single strand breaks (SSBs) were detected using the single-cell gel assay (comet assay) with alkaline electrophoresis (pH 12.3) and quantified by measuring comet tail length (CTL) (microm). Untreated HMECs and HMECs incubated (30 min, 37 degrees C) with a mammary lipid extract, with or without DNA-repair inhibitors hydroxyurea (HU) and cytosine arabinoside (ara-C), were examined. Ionizing radiation was used as a positive control. An active lipid extract gave a linear dose-response over the range 2.0-12.2 g equivalents. When MCL-5 cells, a line of metabolically-competent human lymphoblastoid cells, were used to compare the DNA-damaging properties of lipid extracts from six different donors, significant interindividual variations (median CTLs were 15.0, 53.5, 32.5, <4.0, <4.0 and 77.5 microm respectively) were observed. In eight subjects, the donors' HMECs were examined both before and after treatment with extracts of that donor's own lipid. Pre-existing DNA damage was detected in untreated HMECs from some donors (median CTLs 22.0-37.5 microm) that was not present in others (median CTLs 4.0-11.5 microm), and increases in CTL could be induced by incubation with the matching lipid extract (8 g equivalent) in more than half (five out of eight) the subjects examined (median CTL up to 111.0 microm). There was a tendency for the most active lipid extracts to be those obtained from donors whose HMECs also contained the most pre-existing DNA SSBs. The results of this pilot study may prove to be significant in relation to the initiation of breast cancer.

Report of a comparative study of DNA damage and repair assays in primary rat hepatocytes with five coded chemicals

We report the results of a collaborative study for the detection of chemical-induced DNA damage in primary cultures of rat hepatocytes. The methods include the detection of unscheduled DNA synthesis (UDS) with either autoradiography (5 laboratories) or liquid scintillation counting (2 laboratories) and the assessment of DNA single-strand breaks with the alkaline elution assay (1 laboratory). Interlaboratory standardization was omitted in order to prove the agreement of the assays under routine conditions. Five coded chemicals were tested. For 4 chemicals (2-acetylaminofluorene, thiourea, glycerine and potassium chloride) the UDS data were consistent in all laboratories, thus indicating a high consensus of the test systems applied in the different laboratories. Those 3 chemicals that were not expected to elicit genotoxic activity (thiourea, glycerine, and potassium chloride) yielded negative results in all laboratories. 2-Acetylaminofluorene, a known DNA-damaging agent in hepatocytes, gave strongly positive responses in all laboratories. In contrast, N-nitrosodiphenylamine led to equivocal responses.

DNA fragmentation by N-nitrosodimethylamine and methyl methanesulfonate in human hepatocyte primary cultures

The ability of N-nitrosodimethylamine (DMN) and methyl methanesulfonate (MMS) to induce DNA damage in primary cultures of human hepatocytes was examined by the alkaline elution technique. Both the agents induced a dose-dependent increase in DNA elution rate, but appreciable differences in the degree of response to the procarcinogen DMN were observed among cultures obtained from the livers of four patients. A comparative analysis of DNA fragmentation indicated a substantial similarity between human and concurrently studied rat hepatocytes in their response to both DMN and MMS.

Detection of DNA damage in primary cultures of rat hepatocytes following in vivo and in vitro exposure to genotoxic agents.

A simplified method for the quantitation of DNA damage in nonlabeled hepatocytes, using a fluorometric technique for the quantitation of DNA in conjunction with a modification of the alkaline elution technique of Kohn et al [1976], following chemical treatment in vitro and in vivo, is described. Freshly isolated hepatocytes were treated in vitro with 2-acetylaminofluorene, aflatoxin B1, and dimethylnitrosamine, then examined for DNA damage. Exposure to each of these compounds resulted in DNA damage. Hepatocytes isolated from rats treated with the hepatocarcinogens 2-acetylaminofluorene, benzidine, azoxymethane, dimethylhydrazine, dimethylnitrosamine, and diethylnitrosamine sustained DNA damage as evidenced by increased alkaline elution. DNA damage in hepatocytes was also observed as a result of treatment with methylmethanesulfonate and azaserine. The hepatotoxin carbon tetrachloride did not induce DNA damage in hepatocytes isolated from treated animals. A comparison of the induction of DNA damage and of unscheduled DNA synthesis in hepatocytes from the same animals revealed that in most cases the extent of elution of DNA from filters was proportional to the induction of DNA repair.