The Comet Assay as a Method of Assessment of Neurotoxicity

Frenzilli, Giada; Scarcelli, Vittoria; Fornai, Francesco; Paparelli, Antonio; Nigro, Marco

doi:10.1196/annals.1369.048

Cited by 13 publications

(6 citation statements)

References 23 publications

(25 reference statements)

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“…pX Propagates DNA Damage-In support of the interpretation that pX promotes DNA damage, we performed the comet assay, a method that detects single or double strand DNA breaks in single cells (55,56). pX expressed for 12-24 h increased to about 15% the number of cells displaying DNA tailing (Fig.…”

Section: Resultsmentioning

confidence: 99%

Hepatitis B Virus X Protein Increases the Cdt1-to-Geminin Ratio Inducing DNA Re-replication and Polyploidy

Rakotomalala¹,

Studach²,

Wang

et al. 2008

Journal of Biological Chemistry

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Hepatitis B virus X protein (pX) is implicated in hepatocellular carcinoma pathogenesis by an unknown mechanism. Employing the tetracycline-regulated pX-expressing 4pX-1 cell line, derived from the murine AML12 hepatocyte cell line, we demonstrate that pX induces partial polyploidy (>4N DNA). Depletion of p53 in 4pX-1 cells increases by 5-fold the polyploid cells in response to pX expression, indicating that p53 antagonizes pX-induced polyploidy. Dual-parameter flow cytometric analyses show pX-dependent bromodeoxyuridine (BrdUrd) incorporation in 4pX-1 cells containing 4N and >4N DNA, suggesting pX induces DNA re-replication. Interestingly, pX increases expression of endogenous replication initiation factors Cdc6 and Cdtl while suppressing geminin expression, a negative regulator of rereplication. In comparison to a geminin knockdown 4pX-1 cell line used as DNA re-replication control, the Cdt1/geminin ratio is greater in 4pX-1 cells expressing pX, indicating that pX promotes DNA re-replication. In support of this conclusion, pX-expressing 4pX-1 cells, similar to the geminin knockdown 4pX-1 cells, continue to incorporate BrdUrd in the G 2 phase and exhibit nuclear Cdc6 and MCM5 co-localization and the absence of geminin. In addition, pX expression activates the ATR kinase, the sensor of DNA re-replication, which in turn phosphorylates RAD17 and H2AX. Interestingly, phospho-H2AX-positive and BrdUrd -positive cells progress through mitosis, demonstrating a link between pX-induced DNA re-replication and polyploidy. Our studies highlight a novel function of pX that likely contributes to hepatocellular carcinoma pathogenesis. Chronic hepatitis B virus (HBV)4 infection results in the development of hepatocellular carcinoma (HCC) by the fourth or fifth decade (1) by an unknown mechanism. In HBV-mediated HCC the rate of chromosomal aberrations is significantly increased in comparison to HCC associated with other risk factors (2-4). However, the mechanism by which genomic changes initiate HCC development is not yet understood (5-7). Herein, employing the HBV X protein (pX) as the oncogenic signal, we investigate whether pX expression induces chromosomal abnormalities, resulting in HCC pathogenesis.The link between HBV-mediated HCC and pX is derived both from clinical evidence (8) as well as animal and cell culture transformation studies (9). Specifically, integration of HBV DNA into the host genome occurs at early steps of clonal tumor expansion, with most tumors displaying sustained expression of pX (8). Importantly, pX, which is essential for the viral life cycle (10), is a multifunctional protein inducing activation of the cellular mitogenic ras-raf-MAPK, c-Jun NH 2 -terminal kinase, and p38MAPK pathways (11) and transcription of select viral and cellular genes (9). These pX activities deregulate cellular gene expression, resulting either in unscheduled cell cycle progression (12) or apoptosis (13), depending on the growth conditions. Specifically, pX expression sensitizes the less-differentiated 4pX-1 hepatocyte cell ...

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Section: Resultsmentioning

confidence: 99%

Hepatitis B Virus X Protein Increases the Cdt1-to-Geminin Ratio Inducing DNA Re-replication and Polyploidy

Rakotomalala¹,

Studach²,

Wang

et al. 2008

Journal of Biological Chemistry

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“…It may represent the direct effect of some damaging agent on these molecules. In this manner, alkaline comet assay and carbonyl formation allow the detection of DNA and protein damages, respectively [39][40][41]. In the present study we also investigated the effect of intrastriatal GAA infusion (in vivo studies) and the addition of this compound at assay medium (in vitro studies) on comet assay (DNA damage) and carbonyl content (protein oxidation) in striatum of rats.…”

Section: Discussionmentioning

confidence: 99%

Guanidinoacetate Decreases Antioxidant Defenses and Total Protein Sulfhydryl Content in Striatum of Rats

et al. 2008

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Guanidinoacetate methyltransferase (GAMT) deficiency is an inherited neurometabolic disorder biochemically characterized by tissue accumulation of guanidinoacetate (GAA) and depletion of creatine. Affected patients present epilepsy and mental retardation whose pathogeny is unclear. In the present study we investigated the in vitro and in vivo (intrastriatal administration) effects of GAA on some oxidative stress parameters in rat striatum. Sixty-day-old rats were used for intrastriatal infusion of GAA. For the in vitro studies, 60-day-old Wistar rats were killed by decapitation and the striatum was pre-incubated for 1 h at 37 degrees C in the presence of GAA at final concentrations ranging from 10 to 100 microM. Parameters of oxidative stress such as total radical-trapping antioxidant potential (TRAP), antioxidant enzymes (SOD, GPx, and CAT), protein carbonyl and thiol contents were measured. DNA damage was also evaluated. Results showed that GAA administration (in vivo studies) or the addition of 100 microM GAA to assays (in vitro studies) significantly decreased TRAP, SOD activity, and total thiol levels in rat striatum. In contrast, this guanidino compound did not alter protein carbonyl content and the activities of CAT and GPx. DNA damage was not found after intrastriatal administration of GAA. The data indicate that the metabolite accumulating in GAMT deficiency decreases antioxidant capacity and total thiol content in the striatum. It is therefore presumed that this pathomechanism may contribute at least in part to the pathophysiology of the brain injury observed in patients affected by GAMT deficiency.

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“…Some of these methods are based on the detection of chromosome breakage (reviewed in [70]) including the Comet assay (see [71][72][73] for review). Other approaches measure the activity of nDNA repair enzymes [74,75] or the amount of damage localized to a plasmid (i.e., to exogenous DNA) that a cell can "reactivate" or repair (socalled host cell reactivation; reviewed in [76]).…”

Section: Nuclear Dna Repairmentioning

confidence: 99%

Accumulation of nuclear DNA damage or neuron loss: Molecular basis for a new approach to understanding selective neuronal vulnerability in neurodegenerative diseases

et al. 2008

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According to a long-standing hypothesis, aging is mainly caused by accumulation of nuclear (n) DNA damage in differentiated cells such as neurons due to insufficient nDNA repair during lifetime. In line with this hypothesis it was until recently widely accepted that neuron loss is a general consequence of normal aging, explaining some degree of decline in brain function during aging. However, with the advent of more accurate procedures for counting neurons, it is currently widely accepted that there is widespread preservation of neuron numbers in the aging brain, and the changes that do occur are relatively specific to certain brain regions and types of neurons. Whether accumulation of nDNA damage and decline in nDNA repair is a general phenomenon in the aging brain or also shows cell-type specificity is, however, not known. It has not been possible to address this issue with the biochemical and molecular-biological methods available to study nDNA damage and nDNA repair. Rather, it was the introduction of autoradiographic methods to study quantitatively the relative amounts of nDNA damage (measured as nDNA single-strand breaks) and nDNA repair (measured as unscheduled DNA synthesis) on tissue sections that made it possible to address this question in a cell-type-specific manner under physiological conditions. The results of these studies revealed a formerly unknown inverse relationship between age-related accumulation of nDNA damage and age-related impairment in nDNA repair on the one hand, and the age-related, selective, loss of neurons on the other hand. This inverse relation may not only reflect a fundamental process of aging in the central nervous system but also provide the molecular basis for a new approach to understand the selective neuronal vulnerability in neurodegenerative diseases, particularly Alzheimer's disease.

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The Comet Assay as a Method of Assessment of Neurotoxicity

Cited by 13 publications

References 23 publications

Hepatitis B Virus X Protein Increases the Cdt1-to-Geminin Ratio Inducing DNA Re-replication and Polyploidy

Hepatitis B Virus X Protein Increases the Cdt1-to-Geminin Ratio Inducing DNA Re-replication and Polyploidy

Guanidinoacetate Decreases Antioxidant Defenses and Total Protein Sulfhydryl Content in Striatum of Rats

Accumulation of nuclear DNA damage or neuron loss: Molecular basis for a new approach to understanding selective neuronal vulnerability in neurodegenerative diseases

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