A Comparison of the Genotoxic Effects of Gold Nanoparticles Functionalized with Seven Different Ligands in Cultured Human Hepatocellular Carcinoma Cells.

Gold nanoparticles (GNPs) have shown great potential in diagnostic and therapeutic applications in diseases, such as cancer. Despite GNP versatility, there is conflicting data regarding the toxicity of their overall functionalization chemistry for improved biocompatibility. This study aimed to determine the possible genotoxic effects of functionalized GNPs in Human hepatocellular carcinoma (HepG2) cells. GNPs were synthesized and biofunctionalized with seven common molecules used for biomedical applications. These ligands were bovine serum albumin (BSA), poly(sodium 4-styrene sulfonate) (PSSNA), trisodium citrate (citrate), mercaptoundecanoic acid (MUA), glutathione (GSH), polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG). Before in vitro genotoxicity assessment, inductively coupled plasma mass spectrometry was used to determine GNP cellular internalization quantitatively, followed by cell-based assays; WST-1 to find IC 30 and ApoPercentage for apoptotic induction time-points. The effect of the GNPs on cell growth in real-time was determined by using xCELLigence, followed by a comet assay for genotoxicity determination. The HepG2 cells experienced genotoxicity for all GNP ligands; however, they were able to initiate repair mechanisms and recover DNA damage, except for two functionalization chemistries.

Using a medium-throughput comet assay to evaluate the global DNA methylation status of single cells.

The comet assay is a simple and cost effective technique, commonly used to analyze and quantify DNA damage in individual cells. The versatility of the comet assay allows introduction of various modifications to the basic technique. The difference in the methylation sensitivity of the isoschizomeric restriction enzymes HpaII and MspI are used to demonstrate the ability of the comet assay to measure the global DNA methylation level of individual cells when using cell cultures. In the experiments described here, a medium-throughput comet assay and methylation sensitive comet assay are combined to produce a methylation sensitive medium-throughput comet assay to measure changes in the global DNA methylation pattern in individual cells under various growth conditions.

Point mutation instability (PIN) mutator phenotype as model for true back mutations seen in hereditary tyrosinemia type 1 - a hypothesis.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder affecting fumarylacetoacetate hydrolase (FAH), the last enzyme in the tyrosine catabolism pathway. The liver mosaicism observed in HT1 patients is due to the reversion to the wild type of one allele of the original point mutation in fah. It is generally accepted that these reversions are true back mutations; however, the mechanism is still unresolved. Previous reports excluded intragenic recombination, mitotic recombination, or homologous recombination with a pseudogene as possible mechanisms of mutation reversion in HT1. Sequence analysis did not reveal DNA motifs, tandem repeats or other sequence peculiarities that may be involved in mutation reversion. We propose the hypothesis that a point mutation instability mutator (PIN) phenotype brought about by the sustained stress environment created by the accumulating metabolites in the cell is the driver of the true back mutations in HT1. The metabolites accumulating in HT1 create a sustained stress environment by activating the extracellular signal-regulated kinase (ERK) and AKT survival pathways, inducing aberrant mitosis and development of death resistant cells, depleting glutathione, and impairing DNA ligase IV and possibly DNA polymerases δ and ε. This continual production of proliferative and stress-related survival signals in the cellular environment coupled with the mutagenicity of FAA, may instigate a mutator phenotype and could end in tumorigenesis and/or mutation reversion. The establishment of a PIN-mutator phenotype therefore not only seems to be a possible mechanism underlying the true back mutations, but also contributes to explaining the clinical heterogeneity seen in hereditary tyrosinemia type 1.

Increased excretion of c4-carnitine species after a therapeutic acetylsalicylic Acid dose: evidence for an inhibitory effect on short-chain Fatty Acid metabolism.

Acetylsalicylic acid and/or its metabolites are implicated to have various effects on metabolism and, especially, on mitochondrial function. These effects include both inhibitory and stimulatory effects. We investigated the effect of both combined and separate oral acetylsalicylic acid and acetaminophen administration at therapeutic doses on the urinary metabolite profile of human subjects. In this paper, we provided in vivo evidence, in human subjects, of a statistically significant increase in isobutyrylcarnitine after the administration of a therapeutic dose of acetylsalicylic acid. We, therefore, propose an inhibitory effect of acetylsalicylic acid on the short-chain fatty acid metabolism, possibly at the level of isobutyryl-CoA dehydrogenase.

O6-methylguanine-DNA methyltransferase (MGMT): can function explain a suicidal mechanism?

Why does O(6)-methylguanine-DNA methyltransferase (MGMT), an indispensable DNA repair enzyme, have a mechanism which seems to run counter to its importance? This enzyme is key to the removal of detrimental alkyl adducts from guanine bases. Although the mechanism is well known, an unusual feature surrounds its mode of action, which is its so-called suicidal endpoint. In addition, induction of MGMT is highly variable and its kinetics is atypical. These features raise some questions on the seemingly paradoxical mechanism. In this manuscript we point out that, although there is ample literature regarding the "how" of the MGMT enzyme, we found a lack of information on "why" this specific mechanism is in place. We then ask whether we know all there is to know about MGMT, or if perhaps there is a further as yet unknown function for MGMT, or if the suicidal mechanism may play some kind of protective role in the cell.

Mitochondrial DNA replication and OXPHOS gene transcription show varied responsiveness to Rieske protein knockdown in 143B cells.

Genetic, biochemical and phenotypic diversity is a hallmark of OXPHOS deficiencies. Among the responses frequently reported for these deficiencies is differential expression of several genes involved in mitochondrial biogenesis. These responses are often associated with elevated oxygen radical production. The genetic diversity of tissue and cell lines used in these investigations, however, complicates the interpretation of observations. We investigated mtDNA copy number and selected transcriptional responses after inducing stable Rieske protein knockdown in 143B cells. Despite the significant loss of complex III activity, hydrogen peroxide levels remained comparable to controls. Furthermore, no significant change in mtDNA copy number was observed. Mitochondrial L-strand- and D-loop transcript levels remained unchanged, while the H-strand transcript for COXII was reduced. With the exception of mitochondrial single-stranded binding protein (mtSSB), which was reduced, no transcriptional changes of the mtDNA replication and transcription machinery were observed. Notably however, a selection of nuclear-encoded OXPHOS gene transcripts was generally reduced (statistically insignificant), except for NDUFS3 and COX4I1 transcripts, which were significantly reduced. From these results we conclude that the induction of a low superoxide producing complex III deficiency in 143B cells has an insignificant effect on mtDNA replication and function, but that expression of OXPHOS genes is generally down regulated. This may indicate a lowering of mitochondrial biogenesis and a shift towards anaerobic energy metabolism to improve cellular survival.

Assessing the DNA methylation status of single cells with the comet assay.

The comet assay (single cell gel electrophoresis) is a cost-effective, sensitive, and simple technique that is traditionally used for analyzing and quantifying DNA damage in individual cells. The aim of this study was to determine whether the comet assay could be modified to detect changes in the levels of DNA methylation in single cells. We used the difference in methylation sensitivity of the isoschizomeric restriction endonucleases HpaII and MspI to demonstrate the feasibility of the comet assay to measure the global DNA methylation level of individual cells. The results were verified with the well-established cytosine extension assay. We were able to show variations in DNA methylation after treatment of cultured cells with 5-azacytidine and succinylacetone, an accumulating metabolite in human tyrosinemia type I.

Is the role of circulating DNA as a biomarker of cancer being prematurely overrated?

BACKGROUND: Circulating DNA is utilized widely as a genetic biomarker in a variety of pathological conditions, mainly in cancerous conditions. Quantification of circulating DNA and identifying the frequencies of a variety of mutations, microsatellite alterations and gene promoter methylation are the main foci of research on circulating DNA. CONTENT: A compilation of research reports available to us were reviewed to highlight the rather great variety of methods presently used to isolate circulating DNA, the lack of uniformity in presenting and interpreting quantitative research data as well as the virtual absence of information regarding the structure and function of circulating DNA. CONCLUSIONS: The information compelled us to conclude that the application of circulating DNA as an unambiguous biomarker is currently overrated. We therefore emphasize the need for elucidating the prevailing questions regarding the origin, function and significance of these nucleic acid molecules before utilizing circulating DNA as a biomarker.

In vitro antioxidant, antimutagenic and genoprotective activity of Rosa roxburghii fruit extract.

The antioxidant properties of the fruit of the Rosa roxburghii (RR) plant have been associated with several putative health promoting effects. The possible cytotoxic, mutagenic/antimutagenic and genotoxic effects of RR fruit extract were investigated. The effect on antioxidant status and protection against induced oxidative stress were also investigated using primary rat hepatocytes. A RR fruit extract containing 45 g/L total ascorbic acid and 65 g/L total polyphenols was used in this study. Dilutions up to 0.08% (v/v) increased significantly the antioxidant status in primary rat hepatocytes. The glutathione redox state was decreased with RR treatment but was increased in Chang liver cells and MT-2 lymphoblast. No cyto- or genotoxicity were observed at levels of up to 5% (v/v) of the fruit extract. In addition, a significant protection against t-BHP induced oxidative stress was observed in primary rat hepatocytes. The Ames test revealed no mutagenic activity using the Salmonella typhimurium strains TA98, TA100 and TA102. A significant antimutagenic effect of the extract was observed against the metabolic activated mutagens 2-acetylaminofluorene and aflatoxin B1 and to a lesser extent against methyl methanesulfonate. It is concluded that these results support the associated health promoting potential of Rosa Roxburghii fruit and in particular against oxidative stress.

The molecular relationship between deficient UDP-galactose uridyl transferase (GALT) and ceramide galactosyltransferase (CGT) enzyme function: a possible cause for poor long-term prognosis in classic galactosemia.

Classic galactosemia is an autosomal recessive disorder that is caused by activity deficiency of the UDP-galactose uridyl transferase (GALT). The clinical spectrum of classic galactosemia differs according to the type and number of mutations in the GALT gene. Short-term clinical symptoms such as jaundice, hepatomegaly, splenomegaly and E. coli sepsis are typically associated with classic galactosemia. These symptoms are often severe but quickly ameliorate with dietary restriction of galactose. However, long-term symptoms such as mental retardation and primary ovarian failure do not resolve irrespective of dietary intervention or the period of initial dietary intervention. There seem to be an association between deficient galactosylation of cerebrosides and classic galactosemia. Galactocerebrosides and glucocerebrosides are the primary products of the enzyme UDP-galactose:cerebroside galactosyl transferase (CGT). There has been an observation of deficient galactosylation coupled with over glucosylation in the brain tissue specimens sampled from deceased classic galactosemia patients. The plausible mechanism with which the association between GALT and CGT had not been explained before. Yet, UDP-galactose serves as the product of GALT as well as a substrate for CGT. In classic galactosemia, there is a consistent deficiency in cerebroside galactosylation. We postulate that the molecular link between defective GALT enzyme, which result in classic galactosemia; and the cerebroside galactosyl transferase, which is responsible for galactosylation of cerebrosides is dependent on the cellular concentrations of UDP-galactose. We further hypothesize that a threshold concentration of UDP-galactose exist below which the integrity of cerebroside galactosylation suffers.