T. Chaveca

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.

Micronuclei and sister chromatid exchanges induced by capsaicin in human lymphocytes

Capsaicin is the main pungent and irritating component of hot peppers (species Capsicum annuum and C. frutescens). Genotoxicity and carcinogenicity studies evaluating capsaicin effects are sparse and contradictory. In this study, we investigated the genotoxicity of capsaicin (10-200 microM) in human peripheral blood lymphocytes using the cytokinesis-block micronucleus (CBMN) assay and the sister chromatid exchange (SCE) assay in the presence or absence of external metabolic activation. Capsaicin induced the formation of micronuclei (MN) in a dose-dependent manner in the cytokinesis-blocked lymphocytes. This increase was more evident in the absence of metabolic activation, with a maximum of 3.4-fold increase above the background. Some inter-individual variability was observed. The results for the SCE assay also show that capsaicin is genotoxic and in this case with a more homogeneous response among donors. This end-point, however, has proven to be less sensitive than the CBMN assay for capsaicin.

Genotoxicity assessment of aromatic amines and amides in genetically engineered V79 cells

A genetically engineered V79 cell line expressing rat CYP1A2 and another cell line expressing rat CYP1A2 as well as endogenous acetyltransferase activity, as well as CYP-deficient parental V79 cell lines, were used to assess the genotoxicity of the aromatic amines and amides 2-aminoanthracene, 2-aminofluorene, 2-acetylaminofluorene, 4-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoline, with chromosomal aberrations and sister chromatid exchanges as the end-points. None of the test compounds showed a clear effect on the frequency of chromosomal aberrations in any cell line used. Sister chromatid exchanges, however, were induced by 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene in the CYP1A2-proficient cells, but not in the CYP1A2-deficient cells. The presence of acetyltransferase activity enhanced the effect of 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene. 4-Acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoline did not induce sister chromatid exchanges in the investigated cell lines. The use of cell lines with defined metabolic capabilities seems to be a valuable tool to study specific metabolic pathways important in the activation of procarcinogens.

The role of poly(ADP-ribose)polymerase in the induction of sister chromatid exchanges and micronuclei by mitomycin C in Down's syndrome cells as compared to euploid cells

Inhibitors of poly(ADP-ribose)polymerase (PARP; EC 2.4.2.30), such as 3-aminobenzamide (3-AB), can be used to assess the role of the enzyme in the induction of DNA lesions in euploid cells as compared to cells of genetic conditions known to exhibit increased susceptibility to chemical or physical mutagens, such as Downs syndrome (DS) lymphocytes. We report in this work on the effect of PARP inhibition by 3-AB in the induction of sister chromatid exchanges (SCE) and micronuclei (MN) in DS lymphocytes as compared to lymphocytes from normal controls exposed in vitro to a gradient of mitomycin C (MMC). For both types of cells, DS and normal lymphocytes, MMC induces a significant increase in frequencies of SCE and MN in the absence and in the presence of 3-AB. In the presence of 3-AB the yield of SCE and MN induced by MMC was significantly higher in normal lymphocytes as compared to lymphocytes from DS patients. The molecular mechanisms by which 3-AB affects the yield of SCE and MN remains to be fully elucidated; however, it seems clear that DS patients display a different behavior in what concerns poly(ADP-ribosyl)ation as compared to normal individuals.

Preferential sensitivity of acrocentric chromosomes to the aneugenic effect of colchicine

In order to evaluate the predisposition to the aneuploidy-inducing agent colchicine (Col) on lymphocytes from trisomic 21 patients compared with their parents and with a control group of subjects without trisomic children, we performed the micronucleus (MN) assay associated with C-banding, CREST staining, and nucleolar organizing region (NOR)-banding. According to our results Col behaves as an aneugenic agent independently of the population studied for CREST and C-banding. The Col-induced MN exhibited a clear majority (> 80%) of positive NOR-MN, meaning that they contain a NOR region transcriptionally active or inactive. The same data were observed in trisomic 21 individuals, their parents, and the control group, without significant differences between them. These results seem to suggest a preferential effect of the aneugen Col on acrocentric chromosomes in all of the three groups studied.

DNA Damage and Oxygen Species

Various genes are involved in cell protection against oxidative stress (e.g., SOD, catalase, GSH peroxidase) and some genes recognize and repair oxidative damage to DNA (both base and sugar damage). Yet, the nature and relative rates of the reactions involving active oxygen species make it difficult to ascertain, in each particular situation, what reactants may act as the predominant genotoxicant and what DNA repair mechanisms may ensue.

Evaluation of some biomonitoring markers in occupationally exposed populations to acrylonitrile

In the present work we studied acrylonitrile (AN) occupationally exposed populations and respective control individuals working in a Portuguese plant producing acrylic textile fibers. Three subgroups of individuals were considered: controls (C), workers of the continuous polymerization (CP) area, and workers of equipment maintenance (MM). Besides aiming to contribute to a better understanding of the hazardous exposure of man to AN, the study aimed to help validate and optimize the use of a combination of methods applied to human populations exposed to genotoxic compounds. Three main compartments related to the dose or effect of the hazardous compound were evaluated using various assessment methods: 1) internal dose (genotoxicity in urine, indicators of oxidative stress, induction of cytochromes P450); 2) biological effective dose (hemoglobin adducts); and 3) early biological effects (chromosomal aberrations, sister chromatid exchanges). Although concern with exposure to AN has long been the subject of numerous studies, they have been carried out essentially in animals and using in vitro systems. The significant differences (P < 0.01) found in the chromosomal aberrations of MM are in agreement with the highly significant levels of hemoglobin adducts described in another study performed in the same population. Hemoglobin adducts were also sensitive in detecting a hazardous exposure in the case of CP. The results obtained for the lipid peroxidation indicator used seem to confirm the AN capability of inducing lipid peroxidation in vivo. From the results available it seems that chromosomal aberrations as well as hemoglobin adducts are accurate and sensitive biomonitoring markers for AN exposure.

Naturally contaminated shellfish samples: quantification of diarrhetic shellfish poisoning toxins in unhydrolysed and hydrolysed extracts and cytotoxicity assessment

Contamination of shellfish from the Portuguese coast with diarrhetic shellfish poisoning (DSP) toxins is a recurrent event, with most of the commercial bivalves contaminated with high percentages of esters of okadaic acid (OA) and dinophysistoxin‐2 (DTX2). This report describes the quantification of DSP toxins in unhydrolysed and hydrolysed extracts of several cockle and mussel samples naturally contaminated and the evaluation of their cytotoxicity profiles in V79 cells. The quantification of the acyl esters in the shellfish samples involved the cleavage of the ester bond through alkaline hydrolysis and the release of the parent toxins OA and DTX2. Unhydrolysed and hydrolysed extracts were then analyzed by liquid chromatography (LC) coupled with mass spectrometry (MS) for the detection and quantification of DSP toxins. The cytotoxicity of the analysed extracts was evaluated using the MTT reduction assay and compared with the cytotoxicity presented by different concentrations of OA standard (1–100 nm). OA exhibited marked cytotoxic effects and decreased cell viability in a dose dependent mode, with an IC50 of 27 nm. The cytotoxicity pattern of unhydrolysed extracts was clearly dependent on the concentration of free toxins. Moreover, the cytotoxicity of the esterified toxins present was revealed after their conversion into free toxins by alkaline hydrolysis. For the hydrolysed extracts of cockles and mussels, the cytotoxicity presented was mainly related to the concentration of OA and DTX2. Copyright