Genotoxic and cytotoxic effects induced by aluminum in the lymphocytes of the common carp (Cyprinus carpio)

Few studies have been made in regard to the effect of aluminum on the molecular and cellular structure and function of aquatic organisms; therefore, in the present report we determined the genotoxic and cytotoxic effects induced by the metal on the lymphocytes of carp (Cyprinus carpio). Three groups of fish were exposed to 0.05, 120, and 239 mg/L of aluminum (Al), respectively, by using Al₂ (SO₄)₃·7H₂O, and another group was included as control. The cells obtained were studied with the comet assay, flow cytometry, and the TUNEL method. With the first method we found a concentration and time dependent, significant increase in the amount of DNA damage induced by Al, and a higher damage when we evaluated the level of oxidized DNA. By applying flow cytometry we established that the metal induced a DNA content increase and ploidy modifications as well as apoptosis and disturbances of the cell cycle progression. With the last method we determined a significant increase in the amount of apoptotic cells, mainly in the 72–96 h period. Our results established that Al caused deleterious DNA and cellular effects in the tested organism, and they suggested the pertinence of evaluating toxicity induced by the metal in organisms living in contaminated water bodies.     

Trinucleotide repeat expansions catalyzed by human cell-free extracts

Trinucleotide repeat expansions cause 17 heritable human neurological disorders. In some diseases, somatic expansions occur in non-proliferating tissues such as brain where DNA replication is limited. This finding stimulated significant interest in replication-independent expansion mechanisms. Aberrant DNA repair is a likely source, based in part on mouse studies showing that somatic expansions are provoked by the DNA repair protein MutSβ (Msh2-Msh3 complex). Biochemical studies to date used cell-free extracts or purified DNA repair proteins to yield partial reactions at triplet repeats. The findings included expansions on one strand but not the other, or processing of DNA hairpin structures thought to be important intermediates in the expansion process. However, it has been difficult to recapitulate complete expansions in vitro, and the biochemical role of MutSβ remains controversial. Here, we use a novel in vitro assay to show that human cell-free extracts catalyze expansions and contractions of trinucleotide repeats without the requirement for DNA replication. The extract promotes a size range of expansions that is similar to certain diseases, and triplet repeat length and sequence govern expansions in vitro as in vivo. MutSβ stimulates expansions in the extract, consistent with aberrant repair of endogenous DNA damage as a source of expansions. Overall, this biochemical system retains the key characteristics of somatic expansions in humans and mice, suggesting that this important mutagenic process can be restored in the test tube.     

Physical mapping and cloning of RAD56

Here we report the physical mapping of the rad56-1 mutation to the NAT3 gene, which encodes the catalytic subunit of the NatB N-terminal acetyltransferase in Saccharomyces cerevisiae. Mutation of RAD56 causes sensitivity to X-rays, methyl methanesulfonate, zeocin, camptothecin and hydroxyurea, but not to UV light, suggesting that N-terminal acetylation of specific DNA repair proteins is important for efficient DNA repair.     

Repair of DNA damage induced by anthanthrene, a polycyclic aromatic hydrocarbon (PAH) without bay or fjord regions

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, formed during incomplete burning of coal, oil and gas. Several PAHs have carcinogenic and mutagenic potencies, but these compounds must be activated in order to exert their mutagenic effects. One of the principal pathways proposed for metabolic activation of PAHs involves the cytochrome P450 enzymes. The DNA damaging potential of cytochrome P450-activated PAHs is generally associated with their bay and fjord regions, and the DNA repair response of PAHs containing such regions has been thoroughly studied. However, little is known about the repair of DNA damage resulting from metabolites from PAHs without bay and fjord regions. We have investigated the six-ringed PAH anthanthrene (dibenzo[def,mno]chrysene), which does not posses bay or fjord motifs. We analyzed the repair profile of human cell extracts and of cell cultures in response to DNA damage induced by cytochrome P450-activated anthanthrene. In cell extracts, functional nucleotide excision repair (NER) and mismatch repair (MMR) activities were necessary to trigger a response to anthanthrene metabolite-induced DNA damage. In cell cultures, NER was responsible for the repair of anthanthrene metabolite-induced DNA damage. However, when the NER pathway was inactivated, a residual repair pathway performed the DNA repair.     

Secondary metabolites in floral nectar reduce parasite infections in bumblebees

The synthesis of secondary metabolites is a hallmark of plant defence against herbivores. These compounds may be detrimental to consumers, but can also protect herbivores against parasites. Floral nectar commonly contains secondary metabolites, but little is known about the impacts of nectar chemistry on pollinators, including bees. We hypothesized that nectar secondary metabolites could reduce bee parasite infection. We inoculated individual bumblebees with Crithidia bombi, an intestinal parasite, and tested effects of eight naturally occurring nectar chemicals on parasite population growth. Secondary metabolites strongly reduced parasite load, with significant effects of alkaloids, terpenoids and iridoid glycosides ranging from 61 to 81%. Using microcolonies, we also investigated costs and benefits of consuming anabasine, the compound with the strongest effect on parasites, in infected and uninfected bees. Anabasine increased time to egg laying, and Crithidia reduced bee survival. However, anabasine consumption did not mitigate the negative effects of Crithidia, and Crithidia infection did not alter anabasine consumption. Our novel results highlight that although secondary metabolites may not rescue survival in infected bees, they may play a vital role in mediating Crithidia transmission within and between colonies by reducing Crithidia infection intensities.     

Impaired DNA repair and genomic stability in hereditary tyrosinemia type 1

The autosomal recessive disorder, hereditary tyrosinemia type 1 (HT1), is caused by a defective fumarylacetoacetate hydrolase enzyme. Consequently intermediate metabolites such as fumarylacetoacetate, succinylacetone and p-hydroxyphenylpyruvic acid accumulate. Characteristic to HT1 is the development of hepatocellular carcinoma, irrespective of dietary intervention or pharmacological treatment. Carcinogenesis may occur through a chromosomal instability mutator phenotype or a microsatellite instability phenotype, and deficient DNA repair may be a contributing factor thereof. The purpose of this study was to investigate the expression of DNA repair proteins, and the possible occurrence of microsatellite instability in HT1. Gene expression analyses show low expression of hOGG1 and ERCC1 in HT1 patient lymphocytes. Results from microsatellite instability analyses show allelic imbalance on chromosome 7 of the fah^−/− mouse genome, and instability of the D2S123, D5S346 and (possibly) D17S250 microsatellite markers, in HT1 patient lymphocytes.     

Host-mediated assay with yeast and rats using probenecid (Benemid®) to block the renal tubular excretion of cyclophosphamide metabolites

The genetic activity of cyclophosphamide (Cy) was tested in the host-mediated assay (injection of yeasts into the peritoneal cavity of rats) modified by the use of probenecid (Pro) (Benemid®) to block the renal tubular excretion of the genetically active metabolites. The genetic test system used was the induction of mitotic gene conversion in two unlinked loci of a diploid strain of Saccharomyces cerevisiae. By this method the genetic effect of Cy was doubled in comparison with the case of administering Cy alone. Compared with the animals which received only Pro, increases of conversion frequencies of 20 times in the ade2 locus and of 15 times in the trp5 locus were found.     

Antifungal efficacy of Thevetia peruviana leaf extract against Alternaria solani and characterization of novel inhibitory compounds by Gas Chromatography-Mass Spectrometry analysis

Alternaria solani, a plant pathogenic fungus causes significant economical losses of potato crop. The disease is controlled primarily through some traditional methods and most commonly via the application of chemical fungicides. Fungicides treatment is not protected as chemicals pollute environment, effect health vulnerability in humans and when these harmful chemicals enter into the food chain become hazardous to all living entities. Recent efforts have focused on developing environmentally safe, long-lasting, and effective biocontrol methods for the management of plant diseases. Present research focus on screening of crude and partially purified leaf extract of Thevetia peruviana for the presence of antifungal efficacy against Alternarai solani. It was observed that 100% alcoholic crude and alcoholic fraction of partially purified extract showed maximum inhibitory activity which is due to the presence of different secondary metabolites, revealed by phytochemical screening. Active column fraction (possess best antifungal activity against Alternaria solani) was subjected to Gas Chromatography-Mass Spectrometry (GS-MS) analysis. On the basis of peaks matching of GC-MS chromatogram with available data base showed the presence of benzoic acid and oxo-benzoate in active fraction of Thevetia peruviana leaf extract which is already known chemical among the phytochemicals described for antimicrobial activity. Further research on development of herbal formulation from the same would be very helpful environment friendly approach to manage concern crop disease.     

Influence of single-stranded DNA-binding protein on recA induction in Escherichia coli

Two ssb mutants of Escherichia coli, whic carry a lesion in the single-strand DNA-binding protein (SSB), are sensitive to UV-irradiation. We have investigated the influence of SSB on the “SOS” repair pathway by examining the levels of recA protein synthesis. These strains fail to induced normal levels of recA protein after treatment with nalidixic acid or ultraviolet light. The level of recA protein synthesis in wild-type cells is about three times greater than ssb cells. This deficiency in ssb mutants occurs in all strains and at all temperatures tested (30–41.5°). In contrast, the ssb-1 mutant has no effect on temperature-induced recA induction in a recA441 (tif-1) strain. Cells carrying ssb⁺ plasmids and overproducing normal DNA-binding protein surprisingly are moderated UV-sensitive and have reduced levels of recA protein synthesis. Together these results establish that single-strand DNA-binding protein is involved in the induction of recA, and accounts, at least in part, for the UV sensivitiy of ssb mutant. Three possible mechanisms to explain the role of SSB are discussed.     

Clinical Significance of a Point Mutation in DNA Polymerase Beta (POLB) Gene in Gastric Cancer

Gastric cancer (GC) is a major cause of global cancer mortality. Genetic variations in DNA repair genes can modulate DNA repair capability and, consequently, have been associated with risk of developing cancer. We have previously identified a T to C point mutation at nucleotide 889 (T889C) in DNA polymerase beta (POLB) gene, a key enzyme involved in base excision repair in primary GCs. The purpose of this study was to evaluate the mutation and expression of POLB in a larger cohort and to identify possible prognostic roles of the POLB alterations in GC. Primary GC specimens and their matched normal adjacent tissues were collected at the time of surgery. DNA, RNA and protein samples were isolated from GC specimens and cell lines. Mutations were detected by PCR-RFLP/DHPLC and sequencing analysis. POLB gene expression was examined by RT-PCR, tissue microarray, Western blotting and immunofluorescence assays. The function of the mutation was evaluated by chemosensitivity, MTT, Transwell matrigel invasion and host cell reactivation assays. The T889C mutation was detected in 18 (10.17%) of 177 GC patients. And the T889C mutation was associated with POLB overexpression, lymph nodes metastases and poor tumor differentiation. In addition, patients with- the mutation had significantly shorter survival time than those without-, following postoperative chemotherapy. Furthermore, cell lines with T889C mutation in POLB gene were more resistant to the treatment of 5-fluorouracil, cisplatin and epirubicin than those with wild type POLB. Forced expression of POLB gene with T889C mutation resulted in enhanced cell proliferation, invasion and resistance to anticancer drugs, along with increased DNA repair capability. These results suggest that POLB gene with T889C mutation in surgically resected primary gastric tissues may be clinically useful for predicting responsiveness to chemotherapy in patients with GC. The POLB gene alteration may serve as a prognostic biomarker for GC.     

Structure and function of the DNA ligases encoded by the mammalian LIG3 gene

Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target.     

A Comparison of DNA Extraction Methods using Petunia hybrida Tissues

Extraction of DNA from plant tissue is often problematic, as many plants contain high levels of secondary metabolites that can interfere with downstream applications, such as the PCR. Removal of these secondary metabolites usually requires further purification of the DNA using organic solvents or other toxic substances. In this study, we have compared two methods of DNA purification: the cetyltrimethylammonium bromide (CTAB) method that uses the ionic detergent hexadecyltrimethylammonium bromide and chloroform-isoamyl alcohol and the Edwards method that uses the anionic detergent SDS and isopropyl alcohol. Our results show that the Edwards method works better than the CTAB method for extracting DNA from tissues of Petunia hybrida. For six of the eight tissues, the Edwards method yielded more DNA than the CTAB method. In four of the tissues, this difference was statistically significant, and the Edwards method yielded 27–80% more DNA than the CTAB method. Among the different tissues tested, we found that buds, 4 days before anthesis, had the highest DNA concentrations and that buds and reproductive tissue, in general, yielded higher DNA concentrations than other tissues. In addition, DNA extracted using the Edwards method was more consistently PCR-amplified than that of CTAB-extracted DNA. Based on these results, we recommend using the Edwards method to extract DNA from plant tissues and to use buds and reproductive structures for highest DNA yields.     

Analysis of intracellular doxorubicin and its metabolites by ultra-high-performance liquid chromatography

Doxorubicin, a highly effective anticancer drug, produces severe side effect such as cardiotoxicity, which is mainly caused by its metabolite, doxorubicinol. While in vitro studies by measuring cellular concentration of doxorubicin have been reported, there have been no reports on measuring cellular concentration of the metabolites. In this report, we developed a sensitive and high-throughput method for measuring cellular concentrations of doxorubicin and its metabolites by ultra-high-performance liquid chromatography. The method achieved more than 96% recovery of doxorubicin and its metabolites from cell homogenates. Using simple separation conditions, doxorubicin and its three main metabolites, and the internal standard, were separated within 3 min. The method has a limit of quantification of 17.4 pg (32.0 fmol) injected doxorubicin. This high sensitivity enables the detection and intracellular quantification of doxorubicin and its metabolite, doxorubicinol, in cell homogenates, and its use will facilitate studies of the relationship between doxorubicin pharmacokinetics and therapeutic outcome.     

In vitro eradication of abasic site-mediated DNA–peptide/protein cross-links by Escherichia coli long-patch base excision repair

Apurinic/apyrimidinic (AP or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA–protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication. Therefore, these adducts must be promptly eradicated to maintain genome integrity. Herein, we used in vitro reconstitution experiments with chemically synthesized, stable, and site-specific Schiff base AP-peptide/protein cross-link analogs to demonstrate for the first time that this type of DPC can be repaired by Escherichia coli (E. coli) long-patch base excision repair. We demonstrated that the repair process requires a minimum of three enzymes and five consecutive steps, including: (1) 5′-DNA strand incision of the DPC by endonuclease IV; (2 to 4) strand-displacement DNA synthesis, removal of the 5′-deoxyribose phosphate-peptide/protein adduct-containing flap, and gap-filling DNA synthesis by DNA polymerase I; and (5) strand ligation by a ligase. We further demonstrated that endonuclease IV plays a major role in incising an AP-peptide cross-link within E. coli cell extracts. We also report that eradicating model AP-protein (11.2–36.1 kDa) DPCs is less efficient than that of an AP-peptide_10mer cross-link, supporting the emerging model that proteolysis is likely required for efficient DPC repair.     

From genetic diversity to metabolic unity: studies on the biosynthesis of aurafurones and aurafuron-like structures in myxobacteria and streptomycetes

The myxobacterial polyketide secondary metabolites aurafuron A and B were identified by genome mining in the myxobacterial strain Stigmatella aurantiaca DW4/3-1. The compounds contain an unusual furanone moiety and resemble metabolites isolated from soil-dwelling and marine actinobacteria, a fungus and mollusks. We describe here the cloning and functional analysis of the aurafuron biosynthetic gene cluster, including site-directed mutagenesis and feeding studies using labeled precursors. The polyketide core of the aurafurones is assembled by a modular polyketide synthase (PKS). As with many such systems described from myxobacteria, the aurafuron PKS exhibits a number of unusual features, including the apparent iterative use of a module, redundant modules and domains, a trans acting dehydratase and the absence of a terminal thioesterase domain. Four oxidoreductases are encoded within the gene locus, some of which likely participate in formation of the furanone moiety via a Baeyer-Villiger type oxidation. Indeed, inactivation of a gene encoding a cytochrome P₄₅₀ monooxygenase completely abolished production of both compounds. We also compare the complete gene locus to biosynthetic gene clusters from two Streptomyces sp., which produce close structural analogues of the aurafurones. A portion of the post-PKS biosynthetic machinery is strikingly similar in all three cases, in contrast to the PKS genes, which are highly divergent. Phylogenetic analysis of the ketosynthase domains further indicates that the PKSs have developed independently (polyphyletically) during evolution. These findings point to a currently unknown but important biological function of aurafuron-like compounds for the producing organisms.     

Control of DNA end resection by yeast Hmo1p affects efficiency of DNA end-joining

The primary pathways for DNA double strand break (DSB) repair are homologous recombination (HR) and non-homologous end–joining (NHEJ). The choice between HR and NHEJ is influenced by the extent of DNA end resection, as extensive resection is required for HR but repressive to NHEJ. Conversely, association of the DNA end-binding protein Ku, which is integral to classical NHEJ, inhibits resection. In absence of key NHEJ components, a third repair pathway is exposed; this alternative-end joining (A-EJ) is a highly error-prone process that uses micro-homologies at the breakpoints and is initiated by DNA end resection. In Saccharomyces cerevisiae, the high mobility group protein Hmo1p has been implicated in controlling DNA end resection, suggesting its potential role in repair pathway choice. Using a plasmid end-joining assay, we show here that absence of Hmo1p results in reduced repair efficiency and accuracy, indicating that Hmo1p promotes end-joining; this effect is only observed on DNA with protruding ends. Notably, inhibition of DNA end resection in an hmo1Δ strain restores repair efficiency to the levels observed in wild-type cells. In absence of Ku, HMO1 deletion also reduces repair efficiency further, while inhibition of resection restores repair efficiency to the levels observed in kuΔ. We suggest that Hmo1p functions to control DNA end resection, thereby preventing error-prone A-EJ repair and directing repairs towards classical NHEJ. The very low efficiency of DSB repair in kuΔhmo1Δ cells further suggests that excessive DNA resection is inhibitory for A-EJ.     

Repair of bulky DNA lesions deriving from polycyclic aromatic hydrocarbons

Genomic DNA is damaged by a variety of factors exerting an adverse effect on human health, such as environmental pollution, UV light, ionizing radiation, and toxic compounds. Air pollution with products of incomplete combustion of hydrocarbon fuels and wastes of various industries are main sources of polycyclic aromatic hydrocarbons, whose metabolites can damage DNA by forming bulky DNA adducts, which potentially lead to mutations and cancer. Nucleotide excision repair is the main pathway that eliminates these lesions in eukaryotic cells. The excision efficiency of bulky adducts depends on many factors, including the structure of a bulky substituent and the degree of DNA double helix distortion induced by a lesion. Clustered DNA lesions are the most dangerous for the cell. Several DNA repair systems cooperate to recognize and remove such lesions. The review focuses on the mechanisms that repair DNA with single and clustered bulky lesions, taking the natural carcinogen benzo[a]pyrene as an example.