José Rueff

Chemical features of flavonols affecting their genotoxicity. Potential implications in their use as therapeutical agents.

Flavonls are natural compounds present in edible plants and possess several biological activities that can be useful in drug design. Conversely some of these compounds have been shown to be genotoxic to prokaryotic and eukaryotic cells. In this study we tried to establish the chemical features responsible for the genotoxicity of flavonols and to study the conditions that can modulate their genotoxicity namely pH, the presence of antioxidants and metabolism. We assessed the induction of revertants in Salmonella typhimurium TA98 and the induction of Chromosomal aberrations in V79 cells by eight different flavonols and one catechin in the presence and in the absence of metabolizing systems. We have also studied the generation of hydroxyl radical by these flavonoids using the deoxyribose degradation assay. The results obtained in this study suggest that flavonols having a free hydroxyl group at position 3 of the C ring, a free hydroxyl group at position 7 of the A ring and a B ring with a catechol or pyrogallol structure, or a structure that after metabolic activation is transformed into a catechol or a pyrogallol, are flavonols whose genotoxicity in eukaryotic cells depends on their autooxidation. These flavonols can autooxidize when the pH value is slightly alkaline, such as in the intestine, and therefore can induce genotoxicity in humans. Given the above mentioned considerations it is necessary to clarify the mechanisms and the conditions that mediate the biological effects of flavonols before considering them as therapeutical agents.

Genotoxicity of quercetin in the micronucleus assay in mouse bone marrow erythrocytes, human lymphocytes, V79 cell line and identification of kinetochore-containing (CREST staining) micronuclei in human lymphocytes

Quercetin, a mutagenic flavonoid widely distributed in edible plants, was studied for the induction of micronuclei (MN). We have carried out the MN assay in bone marrow polychromatic erythrocytes in mice, in cytokinesis-blocked human lymphocytes and in cytokinesis-blocked V79 cells. MN assay in vitro was performed in the presence and in the absence of S9. To further extend the study, an antikinetochore antibody (CREST staining) was used to distinguish MN containing whole chromosomes (kinetochore positive) from those containing acentric fragments (kinetochore negative). When tested in vivo quercetin failed to induce micronuclei, a result which is in agreement with other published reports. When tested in vitro in V79 cells quercetin clearly induces micronuclei in the absence of S9 and also in the presence of S9 for the highest dose used. When tested in vitro in human lymphocytes quercetin shows a significant induction of micronuclei in the absence and in the presence of S9. The presence of S9 compared to its absence is not significant for any of the systems used. Both in the presence and absence of S9, quercetin appears to behave as a clastogenic agent in human lymphocytes inducing a significant majority of kinetochore-negative MN.

Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays

We present here the results obtained within the framework of an EU funded project aimed to develop and validate alternative metabolic activating systems to be used in short-term mutagenicity assays, in order to reduce the use of laboratory animals for toxicology testing. The activating systems studied were established cell lines (Hep G2, CHEL), genetically engineered V79 cell lines expressing specific rat cytochromes P450, erythrocyte-derived systems, CYP-mimetic chemical systems and plant homogenates. The metabolically competent cell lines were used as indicator cells for genotoxic effects as well as for the preparation of external activating systems using other indicator cells. The following endpoints were used: micronuclei, chromosomal aberrations and sister chromatid exchanges, mutations at the hprt locus, gene mutations in bacteria (Ames test), unscheduled DNA synthesis and DNA breaks detected in the comet assay. All metabolic systems employed activated some promutagens. With some of them, promutagens belonging to many different classes of chemicals were activated to genotoxicants, including carcinogens negative in liver S9-mediated assays. In other cases, the use of the new activating systems allowed the detection of mutagens at much lower substrate concentrations than in liver S9-mediated assays. Therefore, the alternative metabolizing systems, which do not require the use of laboratory animals, have a substantial potential in in vitro toxicology, in the basic genotoxicity testing as well as in the elucidation of activation mechanisms. However, since the data basis is much smaller for the new systems than for the activating systems produced from subcellular liver preparations, the overlapping use of both systems is recommended for the present and near future. For example, liver S9 preparations may be used with some indicator systems (e.g., bacterial mutagenicity), and metabolically competent mammalian cell lines may be used with other indicator systems (e.g., a cytogenetic endpoint) in a battery of basic tests.

Catechols from abietic acid synthesis and evaluation as bioactive compounds.

Catechols from abietic acid were prepared by a short and good yielding chemical process and further evaluated for several biological activities namely, antifungal, antitumoral, antimutagenic, antiviral, antiproliferative and inhibition of nitric oxide. Their properties were compared with those of carnosic acid (6), a naturally occurring catechol with an abietane skeleton and known to possess potent antioxidant activity, as well as anticancer and antiviral properties. From all the synthetic catechols tested compound 2 showed the best activities, stronger than carnosic acid.

Novel compound heterozygous mutations in SLC5A2 are responsible for autosomal recessive renal glucosuria.

Familial renal glucosuria is an inherited renal tubular disorder. A homozygous nonsense mutation in the SLC5A2 gene, encoding the sodium/glucose co-transporter SGLT2, has recently been identified in an affected child of consanguineous parents. We now report novel compound heterozygous mutations in the son of non-consanguineous parents. One allele has a p.Q167fsX186 mutation, which is expected to produce a truncated protein, and the other a p.N654S mutation involving a highly conserved residue. These findings confirm that mutations in the SLC5A2 gene are responsible for recessive renal glucosuria.

Association of Polymorphisms in Genes of the Homologous Recombination DNA Repair Pathway and Thyroid Cancer Risk

BACKGROUND Ionizing radiation exposure has been pointed out as a risk factor for thyroid cancer. The double-strand breaks induced by this carcinogen are usually repaired by homologous recombination repair pathway, a pathway that includes several polymorphic genes. Since there is a scarcity of data about the involvement of these gene polymorphisms in thyroid cancer susceptibility, we carried out a case-control study in a Caucasian Portuguese population. METHODS We genotyped 109 patients and 217 controls for the XRCC3 T241M, XRCC2 R188H, NBS1 E185Q, and RAD51 Ex1-59G>T polymorphisms to evaluate their potential main effects on risk for this pathology. RESULTS The results obtained showed that for the RAD51 Ex1-59G>T polymorphism, the homozigosity for the variant allele was associated with an almost significant increase of the odds ratio (OR) (adjusted OR = 1.9; confidence interval 95%: 1.0-3.5; p = 0.057). Additionaly, when the XRCC3 T241M data were analyzed concerning the presence of at least one wild-type allele, we observed that individuals homozygous for the variant allele had a higher risk for thyroid cancer (adjusted OR = 2.0; confidence interval 95%: 1.1-3.6; p = 0.026). When the data were analyzed according to the number of RAD51 Ex1-59G>T and XRCC3 T241M variant alleles, the coexistence of three or more variant alleles in either gene was associated to a significant higher risk (three variant alleles: adjusted OR = 2.9, p = 0.036; four variant alleles: adjusted OR = 8.0, p = 0.006). CONCLUSIONS Since XRCC3 is involved in the assembly and stabilization of RAD51 protein multimers at double-strand break sites, we cannot exclude that the interaction of both polymorphisms can lead to a decreased DNA repair capacity and consequently increased risk for thyroid cancer.

Association of common variants in mismatch repair genes and breast cancer susceptibility: a multigene study.

BackgroundMMR is responsible for the repair of base-base mismatches and insertion/deletion loops. Besides this, MMR is also associated with an anti-recombination function, suppressing homologous recombination. Losses of heterozygosity and/or microsatellite instability have been detected in a large number of skin samples from breast cancer patients, suggesting a potential role of MMR in breast cancer susceptibility.MethodsWe carried out a hospital-based case-control study in a Caucasian Portuguese population (287 cases and 547 controls) to estimate the susceptibility to non-familial breast cancer associated with some polymorphisms in mismatch repair genes (MSH3, MSH4, MSH6, MLH1, MLH3, PMS1 and MUTYH).ResultsUsing unconditional logistic regression we found that MLH3 (L844P, G>A) polymorphism GA (Leu/Pro) and AA (Pro/Pro) genotypes were associated with a decreased risk: OR = 0.65 (0.45-0.95) (p = 0.03) and OR = 0.62 (0.41-0.94) (p = 0.03), respectively.Analysis of two-way SNP interaction effects on breast cancer revealed two potential associations to breast cancer susceptibility: MSH3 Ala1045Thr/MSH6 Gly39Glu - AA/TC [OR = 0.43 (0.21-0.83), p = 0.01] associated with a decreased risk; and MSH4 Ala97Thr/MLH3 Leu844Pro - AG/AA [OR = 2.35 (1.23-4.49), p = 0.01], GG/AA [OR = 2.11 (1.12-3,98), p = 0.02], and GG/AG [adjusted OR = 1.88 (1.12-3.15), p = 0.02] all associated with an increased risk for breast cancer.ConclusionIt is possible that some of these common variants in MMR genes contribute significantly to breast cancer susceptibility. However, further studies with a large sample size will be needed to support our results.

DNA strand breaks and chromosomal aberrations induced by H2O2 and 60Co γ-radiation

DNA strand breaks and chromosomal aberrations (CAs) were studied in human cells treated with hydrogen peroxide or with ionizing radiation. DNA strand breaks could be produced at dose levels of H2O2 much lower than those which induced CAs. Doses as low as 0.5 mM of H2O2 produced about as many DNA strand breaks as 2 Gy of 60Co gamma-radiation. On the other hand, as much as 20 mM H2O2 produced only half as many CAs as 1 Gy of 60Co gamma-radiation. The different mechanisms involved in the production of human genetic damage by H2O2 and gamma-radiation are discussed.

Development of imatinib and dasatinib resistance: dynamics of expression of drug transporters ABCB1, ABCC1, ABCG2, MVP, and SLC22A1

About 20% of patients with chronic myeloid leukemia (CML) do not respond to treatment with imatinib either initially or because of acquired resistance. To study the development of CML drug resistance, an in vitro experimental system comprising cell lines with different resistance levels was established by exposing K562 cells to increasing concentrations of imatinib and dasatinib anticancer agents. The mRNA levels of BCR– ABL1 and of genes involved in drug transport or redistribution (ABCB1, ABCC1, ABCC3, ABCG2, MVP, and SLC22A1) were measured and the ABL1 kinase domain sequenced. Results excluded BCR– ABL1 overexpression and mutations as relevant resistance mechanisms. Most studied transporters were overexpressed in the majority of resistant cell lines. Their expression pattern was dynamic: varying with resistance level and chronic drug exposure. Studied efflux transporters may have an important role at the initial stages of resistance, but after prolonged exposure and for higher doses of drugs other mechanisms might take place.

Genetic effects and biotoxicity monitoring of occupational styrene exposure

Styrene is a commercially important chemical widely used in the manufacture of synthetic rubber, resins, polyesters, and plastics. The highest levels of human exposure to styrene occurs in occupational settings, especially during the production of reinforced plastic products, which involve manual lay-up or spray-up operations. In these settings, absorption of styrene occurs mainly through inhalation and, to a minor extent, via skin contact. A variety of biological markers (biomarkers) have been developed for genotoxic agents. Three types of biomarkers are identified: biomarkers of exposure, of effect and of susceptibility. Biomarkers of exposure measure the chemical itself or its metabolites in body fluids. Biomarkers of effect measure indicators of damage by the exposure. Biomarkers of effect are generally pre-clinical indicators of abnormalities and the most frequently used in genotoxicity assessment are sister chromatid exchanges (SCEs), chromosomal aberrations (CAs) and micronuclei (MN). More recently, the use of the single cell gel electrophoresis assay (SCGE), or comet assay has been proposed as a useful biomarker for early effects. The third type is a biomarker of susceptibility, which indicates that the individual is vulnerable to the effect of a xenobiotic or to the effects of a group of such compounds. This type of biomarkers are related with individual genetic polymorphisms that could lead to different capacities to activate, detoxify or repair DNA lesions arising from exposure to chemical carcinogens. Styrene metabolism involves cytochrome P450 enzymes (CYP)-mediated that by oxidation convert styrene to its reactive metabolite styrene-7,8-oxide (SO) which is capable of binding covalently with macromolecules and is considered to be directly responsible for the genotoxic effects of styrene. SO, is mainly hydrolysed to styrene glycol by the microsomal epoxide hydrolase (mEH), and subsequently oxidized by alcohol and aldehyde dehydrogenases to the main urinary metabolites, mandelic acid (MA) and phenylglyoxylic acid (PGA) (major pathway). MA and PGA represent more than 95% of the urinary metabolites of styrene. Further transformation of MA and PGA, via transamination of alpha-keto- and alpha-hydroxyacids into the corresponding amino acid, leads to phenylglycine. Evidence of the carcinogenicity of styrene has been reported in studies with mice. Epidemiologic evidence does not suggest a causal association between styrene and any forms of cancer in humans. However, the possibility of a small elevation of risk for one or more cancers cannot be ruled out. The International Agency for Research on Cancer (IARC) has designated styrene as possibly carcinogenic to humans (group 2B). Concern about the potential carcinogenicity of styrene stems largely from the ability of its metabolite, SO to bind covalently to DNA and to its activity in a variety of genotoxicity test systems. SO has been classified by IARC in group 2A, probably carcinogenic to humans. Styrene exposure has been reported to cause an increase in DNA and haemoglobin adducts and in the frequency of CAs; there is less evidence for an association between styrene exposure and the frequency of SCEs. This article thoroughly reviews all available published data on the genetic effects of styrene and the biotoxicity markers of exposure monitoring.