New Insights and Perspectives in Fanconi Anemia Research.

Fanconi anemia is a rare, cancer-prone disease with mutations in 22 genes. The primary defect results in altered DNA repair mechanisms that fuel a severe proinflammatory condition in the bone marrow, leading to cellular depletion of the hematopoietic system and eventually to bone marrow failure. During the past three decades, a plethora of dysfunctions have been highlighted in the Fanconi anemia phenotype, but recent research allows us to glimpse an even more complex scenario where defective lipid metabolism could have important consequences in hematopoietic stem cell differentiation.

The role of CSA in the response to oxidative DNA damage in human cells.

Cockayne syndrome (CS) is a rare genetic disease characterized by severe growth, mental retardation and pronounced cachexia. CS is most frequently due to mutations in either of two genes, CSB and CSA. Evidence for a role of CSB protein in the repair of oxidative DNA damage has been provided recently. Here, we show that CSA is also involved in the response to oxidative stress. CS-A human primary fibroblasts and keratinocytes showed hypersensitivity to potassium bromate, a specific inducer of oxidative damage. This was associated with inefficient repair of oxidatively induced DNA lesions, namely 8-hydroxyguanine (8-OH-Gua) and (5'S)-8,5'-cyclo 2'-deoxyadenosine. Expression of the wild-type CSA in the CS-A cell line CS3BE significantly decreased the steady-state level of 8-OH-Gua and increased its repair rate following oxidant treatment. CS-A cell extracts showed normal 8-OH-Gua cleavage activity in an in vitro assay, whereas CS-B cell extracts were confirmed to be defective. Our data provide the first in vivo evidence that CSA protein contributes to prevent accumulation of various oxidized DNA bases and underline specific functions of CSB not shared with CSA. These findings support the hypothesis that defective repair of oxidative DNA damage is involved in the clinical features of CS patients.

Chronic sub-lethal oxidative stress by spermine oxidase overactivity induces continuous DNA repair and hypersensitivity to radiation exposure.

In the aging process and in most degenerative diseases, the oxidant by-products of cellular metabolism lead to oxidative stress. Oxidative stress plays an important role in switching from cell proliferation to its opposite outcome, cell death. The metabolic pathways in charge of the interconversion and degradation of the polyamines are responsible for oxidant by-products. In the past few years, spermine metabolism has been found closely related to DNA oxidation and apoptosis. Moreover, that the ectopical expression of murine spermine oxidase induced DNA damage in the neuroblastoma cell line, and this was uncoupled with any increase in cell mortality, thus suggests an activation of DNA repair. In this work, we provide new evidence showing that only spermine oxidase overactivity can deliver sub-lethal chronic DNA damage and repair without affecting transcriptional and enzymatic levels of the PA key regulatory enzymes ODC and SSAT. Chronic sub-lethal DNA damage is below the cell cycle arrest induction threshold, but is able to activate apurinic/apyrimidinic endonuclease protein (APE1) and gamma H2AX. Of therapeutic interest, the chronic sub-lethal DNA damage and activation of the repair processes are in turn responsible for inducing hypersensitivity after exposure to radiation with no induction of adaptive response to damage.

Direct oxidative DNA damage, apoptosis and radio sensitivity by spermine oxidase activities in mouse neuroblastoma cells.

In mammals, the polyamines affect cell growth, differentiation, and apoptosis; their levels are increased in malignant and proliferating cells, thus justifying an interest in a chemotherapeutic approach to cancer. The flavoprotein SMO is the most recently characterized catabolic enzyme, preferentially oxidizing SPM to SPD, 3-aminopropanal and H(2)O(2). In this report, we describe a novel functional characterization of the recently cloned splice variant isoforms from mouse brain, encoding, among others, the nuclear co-localized spermine oxidase mSMOmu. The over-expression of the active isoforms mSMOalpha and mSMOmu, and the inactive mSMOdelta and mSMOgamma in mouse neuroblastoma cells, demonstrated the first evidence of the direct oxidative DNA damage by the SMO activities, either alone or, in a higher extent, when associated with radiation exposure, thus working as radio sensitizer. These effects were reverted by treatment with 50 muM and 100 muM doses of the inhibitor of SMO activity MDL 72,527. The over-expression of all SMO isoforms failed to influence the expression of the regulating enzymes of polyamines metabolism ODC and SSAT. Dealing with the unbalanced tissue specific SMO activities, these results could indicate a new direction to tailor chemotherapy-associated radiotherapy, improving dose-rate protocol and allowing the modulation of deleterious side effects on healthy tissues.

Antibodies against 7-methyldeoxyguanosine: its detection in rat peripheral blood lymphocyte DNA and potential applications to molecular epidemiology.

Polyclonal antibodies have been raised against the imidazole ring-open form of 7-methyldeoxyguanosine (7-mdGua). A combined high performance liquid chromatography/immunoassay method has been developed using these antibodies which provides a specific and sensitive way to quantitate 7-mdGua in DNA. Following enzyme hydrolysis and chromatographic purification of 7-mdGua, the adduct is quantitatively converted to the ring-open form and can be measured at levels as low as 0.05 pmol by immunoassay. With 1 mg of DNA a level below 1 adduct per 10(7) normal deoxynucleosides can be measured. Using DNA modified by radiolabeled carcinogens, a good correlation between 7-mdGua levels, as measured by immunoassay or radioactivity, was obtained. In rats treated with dimethylnitrosamine (0.4 and 1.0 mg/kg), both 7-mdGua and O6-methyldeoxyguanosine were detected in peripheral blood lymphocyte DNA. In addition the levels of both adducts at time points up to 48 h posttreatment were very similar to those seen in liver DNA from the same animals. The measurement of 7-mdGua, quantitatively the major methylation adduct, in small cell samples such as lymphocytes has great potential in determining the exposure of humans to environmental methylating agents such as nitrosamines.

Oxidative stress in myelin sheath: The other face of the extramitochondrial oxidative phosphorylation ability.

Oxidative phosphorylation (OXPHOS) is not only the main source of ATP for the cell, but also a major source of reactive oxygen species (ROS), which lead to oxidative stress. At present, mitochondria are considered the organelles responsible for the OXPHOS, but in the last years we have demonstrated that it can also occur outside the mitochondrion. Myelin sheath is able to conduct an aerobic metabolism, producing ATP that we have hypothesized is transferred to the axon, to support its energetic demand. In this work, spectrophotometric, cytofluorimetric, and luminometric analyses were employed to investigate the oxidative stress production in isolated myelin, as far as its respiratory activity is concerned. We have evaluated the levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), markers of lipid peroxidation, as well as of hydrogen peroxide (H2O2), marker of ROS production. To assess the presence of endogenous antioxidant systems, superoxide dismutase, catalase, and glutathione peroxidase activities were assayed. The effect of certain uncoupling or antioxidant molecules on oxidative stress in myelin was also investigated. We report that isolated myelin produces high levels of MDA, 4-HNE, and H2O2, likely through the pathway composed by Complex I-III-IV, but it also contains active superoxide dismutase, catalase, and glutathione peroxidase, as antioxidant defense. Uncoupling compounds or Complex I inhibitors increase oxidative stress, while antioxidant compounds limit ROS generation. Data may shed new light on the role of myelin sheath in physiology and pathology. In particular, it can be presumed that the axonal degeneration associated with myelin loss in demyelinating diseases is related to oxidative stress caused by impaired OXPHOS.

Oxidative DNA damage in circulating leukocytes occurs as an early event in chronic HCV infection.

UNLABELLED: Chronic hepatitis C virus (HCV) infection is associated with an increased production of reactive oxygen species within the liver that are responsible for the oxidation of intracellular macromolecules. To ascertain whether the increased risk of hepatocellular carcinoma in individuals with chronic HCV infection is related to an accumulation of oxidative DNA damage, the 8-hydroxydeoxyguanosine (8-OHdG) content in the DNA of liver tissue and leukocytes of 87 individuals with HCV- or HBV-related liver disease and of 10 healthy controls was measured. Serum levels of thiobarbituric acid reactive substances (TBARS) were also assessed as an index of lipid peroxidation. RESULTS: The 8-OHdG content in the circulating leukocytes correlated with that of liver tissue (r = 0.618, p < .0004). HCV patients had the highest median 8-OHdG levels (p < .0004). 8-OHdG leukocyte levels in HCV patients were higher than in HBV patients (p < .04) and they significantly correlated with the clinical diagnosis (p < .025), the serum ferritin levels (p < .05), and the amount of liver steatosis (p < .001). No correlation was found with age, gender, history of drinking or smoking, ALT or GGT levels, ESR, alpha-1, or gamma-globulin level and Ishak score. TBARS levels were significantly higher in cirrhotics than in noncirrhotics (p < .01). CONCLUSIONS: The 8-OHdG level in circulating leukocytes is a reliable marker of oxidative stress occurring in the liver of individuals with chronic HCV infection. DNA oxidative damage appears to be an early and unique event in the natural history of HCV-related hepatitis. This injury increases the risk of genomic damage and may be one of the important factors involved in the carcinogenic process in cases of HCV-related chronic liver disease.

Time resolved fluoroimmunoassay of 7-methyl-2'-deoxyguanosine imidazole (ring open).

A solid-phase competitive time-resolved fluoroimmunoassay for 7-methyl-2'-deoxyguanosine imidazole (ring open) is described, based on highly specific hemocyanin carrier rabbit antibodies, modified with europium chelates. Eu3+ photoluminescence was detected in a novel micellar solution. The assay was validated both by comparing it with an ELISA and by analysing DNA samples, alkylated either 'in vitro' or 'in vivo' by dimethyl sulfate. The proposed assay proved to be sensitive, simple and reliable. It should be of value, together with other immunoassays for methylated DNA bases, in assessing human environmental exposure to alkylating agents such as nitrosamines, thereby providing a powerful tool in epidemiological investigations.

Time-resolved fluoroimmunoassay.

A method is reported to set up a standard competitive TR-FIA. A simple and inexpensive way to prepare reagents and carry out operations is presented as well, with the aim to make it possible to perform a very sensitive analytical procedure in a personalized way within a nondedicated biochemistry laboratory. This protocol is general and can be easily modified with consideration to the analytical target. Once the antibody is available, both its labeling with diethylenetriaminepentaacetic acid dianhydride and Eu3+, and the setting-up of the assay with measurement of europium ion time-resolved fluorescence in a home-made enhancement solution become feasible.

Genotoxic effects of the carbamate insecticide methomyl. I. In vitro studies with pure compound and the technical formulation "Lannate 25".

The carbamate insecticide methomyl and the methomyl-containing technical formulation "Lannate 25" were tested on whole blood human lymphocyte cultures. Both products induced dose-dependent increases in chromosome aberrations and micronuclei. Lannate 25 induced DNA damage as measured by the alkaline elution assay and hydroxylation of guanine at the C8 position. Sister chromatid exchanges were not increased significantly with either product. Overall, the technical formulation was more active than the pure compound, when compared at similar concentrations of active principle. Moreover, a different ratio of CREST-positive/CREST-negative micronuclei was observed with the two products, pure methomyl being relatively more active than Lannate 25 in the induction of CREST-positive micronuclei. On the basis of these results, previous evaluations of methomyl as a nongenotoxic compound should be reconsidered.

Genotoxic effects of the carbamate insecticide, methyomyl. II. In vivo studies with pure compound and the technical formulation, "Lannate 25".

The carbamate insecticide, methomyl, and the methomyl-containing technical formulation, "Lannate 25", were tested for the induction of DNA damage in vivo. Swiss CD1 mice were treated intraperitoneally with test substances and the following tests were performed: alkaline elution of liver and kidney DNA, 8-hydroxyguanosine detection in liver DNA, and 32P-postlabelling analysis of DNA adducts in liver DNA. The clastogenic activity of the two pesticide preparations was also evaluated as micronucleus frequency in bone marrow. No DNA adducts were detected in liver DNA of mice treated with pure methomyl, while a dose-related increase in DNA adducts was found in Lannate 25-treated animals. All other tests were positive with both methomyl and Lannate 25. A summary of genotoxic activity of methomyl is also presented. The hypothesis that the observed genotoxic effects of methomyl are induced indirectly, through formation of active oxygen species, is discussed.

The role of oxidative stress in developmental and reproductive toxicity of tamoxifen.

The antiestrogen tamoxifen (TAM) is widely used as a drug against breast cancer and is currently being tested as a chemopreventive agent. However, a number of studies showed genotoxic and carcinogenic effects of TAM. These effects are thought to be related to oxygen radical overproduction which occurs during TAM metabolic activation. There is no evidence, thus far, on TAM toxicity to embryos and gametes. The present study was designed to elucidate the mechanisms of TAM-induced developmental, reproductive and cytogenetic toxicity towards sea urchin (SU) embryos with regard to the possibility of TAM-initiated oxidative stress. Embryo cultures from SU were subjected to long-term (throughout embryogenesis) or short-term (two hours) incubation with TAM at concentrations from 10(-8) to 10(-5) M. The experiments on TAM-induced toxicity to gametes were carried out with SU sperm, or unfertilized eggs, suspended in TAM (10(-8) to 10(-6) M). To assess the effects of TAM to embryos or to gametes, developmental defects, embryonic mortality, fertilization success, and cytogenetic abnormalities were scored. Oxidative damage to DNA and lipids was detected by measurements of 8OHdG levels and lipid peroxidation, respectively. Reactive oxygen species (ROS) production by eggs and embryos was recorded by luminol-dependent chemiluminescence (LDCL) and cytochrome c reduction methods. The changes in activities of SU superoxide dismutase (SOD) and catalase were also evaluated. TAM exerted: a) early embryonic mortality to exposed embryos and to the offspring of exposed eggs; b) developmental defects to the offspring of exposed sperm; c) decrease in sperm fertilization success, and d) cytogenetic effects in the offspring of exposed sperm or eggs. These morphological effects corresponded to the state of oxidative stress in SU embryos (increased oxidative damage to DNA and lipids and induction of antioxidant enzymes). Since TAM did increase significantly ROS production by embryos, it is suggested that TAM may be metabolically activated by SU embryonic oxidases and peroxidases, which in turn could be induced by TAM. The present study provides further support to the utilization of the SU system as a useful model to help elucidate mechanisms of chemical teratogenesis and carcinogenesis.

Defective splicing induced by 4NQO in the hamster hprt gene.

The molecular analysis of mutations affecting mRNA processing may contribute to a better understanding of the splicing mechanism through the identification of genomic sequences necessary for the recognition of splice sites. In this paper we report the sequence analysis of 14 splice mutants induced by 4-nitroquinoline 1-oxide (4NQO) at the hamster hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus. We show that mutations at the 3' acceptor splice site or at the first or fifth base of the 5' donor splice site are responsible for exon skipping. In addition, mutations in exon sequences also determine the skipping of one or more exons. Our data indicate that point mutations in intron regions at either side of an internal exon may induce the skipping of the same exon, supporting a model where the exon is the unit of early spliceosome assembly. Furthermore, they suggest that the splicing of hprt mRNA precursors may proceed through a clustering of exons 2, 3 and 4 which are then spliced in a concerted way.

Tissue dose, DNA adducts, oxidative DNA damage and CYP1A-immunopositive proteins in mussels exposed to waterborne benzo[a]pyrene.

A collaborative study was performed on Mediterranean mussels (Mytilus galloprovincialis) exposed to a wide dose-range (0.5-1000 ppb) of benzo[a]pyrene (B[a]P). We selected this model polycyclic aromatic hydrocarbon in order to confirm the formation of a specific DNA adduct, previously detected in gill DNA, and to clarify the in vivo effects of this mutagenic chemical requiring host-metabolism in mussels. B[a]P concentration reached consistently higher values in the digestive gland than in other analyzed tissues of mussels exposed to B[a]P for 2 or 3 days. With the exception of some values at 1000 ppb of B[a]P. DNA adduct levels increased significantly with the dose in gills and digestive gland and ranged from 0.054 to 0.789 adducts per 10(8) nucleotides (mean values per dose-point). Conversely, more complex dose-response relationships were found by detecting in parallel the levels of an oxidative DNA lesion (8-OHdG) and of CYP1A-immunopositive proteins (the latter measured in the digestive gland only). Overall, the formation of DNA adducts, the evidence of oxidative DNA damage, and changes in CYP1A-immunopositive protein levels support the hypothesis that B[a]P can induce DNA damage in mussels through a number of different molecular mechanisms.

Chromosome aberrations associated with CAD gene amplification in Chinese hamster cultured cells.

Eleven sublines with increasing resistance to N-phosphonacetyl-L-aspartate (PALA) were isolated from the V79,B7 Chinese hamster cell line. Aspartate transcarbamylase activity and CAD gene copy number increased with increasing resistance of sublines. In situ hybridization with a DNA probe for the CAD gene showed that the amplified sequences resided in the terminal region of a marker chromosome with elongated q arms. This region stained homogeneously after G-banding. A high incidence of both numerical and structural chromosome aberrations was found in PALA-resistant cells. In hyperdiploid and polyploid cells, containing 2 copies of the marker chromosome, dicentrics were found at a very high frequency. As indicated by in situ hybridization and G-banding, they originated from a rearrangement involving 2 homologous marker chromosomes.

DNA adducts and chronic degenerative disease. Pathogenetic relevance and implications in preventive medicine.

Chronic degenerative diseases are the leading causes of death in developed countries. Their control is exceedingly difficult due to their multiplicity and diversity, the interconnection with a network of multiple risk factors and protective factors, the long latency and multistep pathogenesis, and the multifocal localization. Adducts to nuclear DNA are biomarkers evaluating the biologically effective dose, reflecting an enhanced risk of developing a mutation-related disease more realistically than the external exposure dose. The localization and accumulation of these promutagenic lesions in different organs are the composite result of several factors, including (a) toxicokinetics (first-pass effect); (b) local and distant metabolism; (c) efficiency and fidelity of DNA repair; and (d) cell proliferation rate. The last factor will affect not only the dilution of DNA adducts but also the possible evolution towards either destructive processes, such as emphysema or cardiomyopathies, or proliferative processes, such as benign or malignant tumors at various sites. They also include heart tumors affecting fetal myocytes after transplacental exposure to DNA-binding agents, blood vessel tumors, and atherosclerotic plaques. In this article, particular emphasis is given to molecular alterations in the heart, which is the preferential target for the formation of DNA adducts in smokers, and in human aorta, where an extensive molecular epidemiology project is documenting the systematic presence of adducts to the nuclear DNA of smooth muscle cells from atherosclerotic lesions, and their significant correlation with known atherogenic risk factors. Exocyclic DNA adducts resulting from lipid peroxidation, and age-related indigenous adducts (I-compounds) may also originate from endogenous sources, chronic infections and infestations, and inflammatory processes. Type II I-compounds are bulky DNA lesions resulting from oxidative stress, whereas type II-compounds are presumably normal DNA modifications, which display positive correlations with median life span and are decreased in cancer and other pathological conditions. Profiles of type II-compounds strongly depend on diet and are related to the antidegenerative effects of caloric/ dietary restriction. Even broader is the possible meaning of adducts to mitochondrial DNA, which have been detected in rodents exposed to genotoxic agents and complex mixtures, as well as in untreated rodents, in larger amounts when compared to the nuclear DNA of the same cells. Mutations in mitochondrial DNA increase the number of oxidative phosphorylation-defective cells, especially in energy-requiring postmitotic tissues such as brain, heart and skeletal muscle, thereby playing an important role in aging and a variety of chronic degenerative diseases. A decreased formation of DNA adducts is an indicator of reduced risk of developing the associated disease. Therefore, these molecular dosimeters can be used as biomarkers in the prevention of chronic degenerative diseases, pursued either by avoiding exposure to adduct-forming agents or by using chemopreventive agents. Interventions addressed to the human organism by means of dietary measures or pharmacological agents have encountered a broad consensus in the area of cardiovascular diseases, and are deserving a growing interest also in cancer prevention. The efficacy of chemopreventive agents can be assessed by evaluating inhibition of nuclear DNA or mitochondrial DNA adduct formation in vitro, in animal models, and in phase II clinical trials in high-risk individuals.

Congenital disorders sharing oxidative stress and cancer proneness as phenotypic hallmarks: prospects for joint research in pharmacology.

In spite of very distinct genotypic assets, a number of congenital conditions include oxidative stress as a phenotypic hallmark. These disorders include Fanconi's anaemia, ataxia telangiectasia, xeroderma pigmentosum and Bloom's syndrome, as well as two frequent congenital conditions: Down's syndrome and cystic fibrosis. Cancer proneness is a clinical feature shared by these disorders, while other manifestations include early ageing, neurological symptoms or congenital malformations. The onset of oxidative stress has been related to excess formation, or defective detoxification, of reactive oxygen species (ROS). This can arise from either the abnormal expression or inducibility of ROS-detoxifying enzymes, or by defective absorption of nutrient antioxidants. Resulting oxidative injury has been characterized through: (i) DNA, protein or lipid oxidative damage; (ii) excess ROS formation (in vitro and ex vivo); (iii) sensitivity to oxygen-related toxicity; (iv) improvement of cellular defects by either hypoxia or antioxidants; and (v) circumstantial evidence for in vivo oxidative stress (as e.g. clastogenic factors). Investigations conducted so far have been confined to individual disorders. Comparative studies of selected indicators for oxidative stress could provide further insights into the pathogenesis of each individual condition. Such a unified approach may have wide-ranging consequences for studies of ageing and cancer.

Diepoxybutane and mitomycin C toxicity is associated with the induction of oxidative DNA damage in sea urchin embryos.

Diepoxybutane (DEB)- and mitomycin C (MMC)-associated toxicity was investigated in embryos from the sea urchin (SU) species Sphaerechinus granularis. DEB- and MMC-induced toxicity resulted in S. granularis embryos and larvae at concentrations ranging 10(-5) to 10(-4) M DEB, and 3 x 10(-6) to 3 x 10(-5) M MMC, in terms of larval malformations, developmental arrest and mortality. The formation of DNA oxidative damage, 8-hydroxy-2'-deoxyguanosine (8-OHdG) was measured in DEB- and in MMC-exposed embryos (at gastrula stage). A dose-dependent increase in 8-OHdG levels was observed that was significantly correlated with DEB- and MMC-induced developmental defects. The results lend further support to the body of evidence associating both DEB and MMC toxicity with oxidative stress, including DNA oxidative damage.

Loss of MUTYH function in human cells leads to accumulation of oxidative damage and genetic instability.

The DNA glycosylase MUTYH (mutY homolog (Escherichia coli)) counteracts the mutagenic effects of 8-oxo-7,8-dihydroguanine (8-oxodG) by removing adenine (A) misincorporated opposite the oxidized purine. Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated adenomatous polyposis (MAP). Here we designed new tools to investigate the biochemical defects and biological consequences associated with different MUTYH mutations in human cells. To identify phenotype(s) associated with MUTYH mutations, lymphoblastoid cell lines (LCLs) were derived from seven MAP patients harboring missense as well as truncating mutations in MUTYH. These included homozygous p.Arg245His, p.Gly264TrpfsX7 or compound heterozygous variants (p.Gly396Asp/Arg245Cys, p.Gly396Asp/Tyr179Cys, p.Gly396Asp/Glu410GlyfsX43, p.Gly264TrpfsX7/Ala385ProfsX23 and p.Gly264TrpfsX7/Glu480del). DNA glycosylase assays of MAP LCL extracts confirmed that all these variants were defective in removing A from an 8-oxoG:A DNA substrate, but retained wild-type OGG1 activity. As a consequence of this defect, MAP LCLs accumulated DNA 8-oxodG in their genome and exhibited a fourfold increase in spontaneous mutagenesis at the PIG-A gene compared with LCLs from healthy donors. They were also hypermutable by KBrO3--a source of DNA 8-oxodG--indicating that the relatively modest spontaneous mutator phenotype associated with MUTYH loss can be significantly enhanced by conditions of oxidative stress. These observations identify accumulation of DNA 8-oxodG and a mutator phenotype as likely contributors to the pathogenesis of MUTYH variants.