13 research outputs found
Long-Lasting Genomic Instability Following Arsenite Exposure inMammalian Cells: The Role of Reactive Oxygen Species
Previously, we reported that the progeny of mammalian cells, which has been exposed to sodium arsenite for two cell cycles, exhibited chromosomal instability and concurrent DNA hypomethylation, when they were subsequently investigated after two months of subculturing (about 120 cell generations) in arsenite-free medium. In this work, we continued our investigations of the long-lasting arsenite-induced genomic instability by analyzing additional endpoints at several time points during the cell expanded growth. In addition to the progressive increase of aneuploid cells, we also noted micronucleated and multinucleated cells that continued to accumulate up to the 50th cell generation, as well as dicentric chromosomes and/or telomeric associations and other complex chromosome rearrangements that began to appear much later, at the 90th cell generation following arsenite exposure. The increasing genomic instability was further characterized by an increased frequency of spontaneous mutations. Furthermore, the long-lasting genomic instability was related to elevated levels of reactive oxygen species (ROS), which at the 50th cell generation appeared higher than in stable parental cells. To gain additional insight into the continuing genomic instability, we examined several individual clones isolated at different time points from the growing cell population. Chromosomally and morphologically unstable cell clones, the number of which increased with the expanded growth,were also present at early phases of growth without arsenite. All genomically unstable clones exhibited higher ROS levels than untreated cells suggesting that oxidative stress is an important factor for the progression of genomic instability induced by arsenite
Genomic instability induced by a-pinene in Chinese hamster cell line
Here, we report the effects of exposure of mammalian cells to a-pinene, a bicyclic monoterpene used in insecticides, solvents and perfums. Morphological analysis, performed in V79-Cl3 cells exposed for 1 h to increasing concentrations (25 up to 50 mM) of a-pinene, indicated
a statistically significant increase in micronucleated and multinucleated cell frequencies; apoptotic cells were seen at 40 and 50 mM. This monoterpene caused genomic instability by interfering with mitotic process; in fact, 50% of cells (versus 19% of control cells) showed irregular mitosis with multipolar or incorrectly localised spindles.
Cytogenetic analysis demonstrated high-frequency hypodiploid metaphases as well as endoreduplicated cells and chromosome breaks. Clastogenic damage was prevalent over aneuploidogenic damage as demonstred by the higher proportion of kinetochore-negative micronuclei. Alkaline comet confirmed that monoterpene exposure caused DNA lesions in a concentration-dependent manner. This damage probably arose by increased reactive oxygen species (ROS) production. In order to assess the generation of ROS, the cells were incubated with CM-H2DCFDA and then
analysed by flow cytometry. Results demonstrated an increase in fluorescence intensity after a-pinene treatment indicating increased oxidative stress. On the whole, these findings strongly suggest that a-pinene is able to compromise genome stability preferentially through mitotic alterations and to damage DNA through ROS production
Cytochrome P450 2E1 variable number tandem repeat polymorphisms and health risks: A genotype-phenotype study in cancers associated with drinking and/or smoking
Cytochrome P450 2E1 (CYP2E1) is one of the main enzymes involved in the oxidation of ethanol and in the transformation of a number of potentially dangerous compounds. It has various polymorphic sites, one of which is a variable number tandem repeat (VNTR) polymorphism previously described in the 5'-flanking region. The aim of this study was to investigate the genotype-phenotype association between CYP2E1 VNTR polymorphisms and risky health habits in healthy subjects and to analyze the associations between these polymorphisms with drinking- and/or smoking-related cancers. We analyzed 166 healthy subjects by genotyping for the CYP2E1 VNTR polymorphism associated with drinking and/or smoking habits by the more sensitive restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) method, using the NlaIV restriction enzyme. Sixty cases of pancreatic adenocarcinoma (PA) and 66 with hepatocellular carcinoma (HCC), were also genotyped. Statistical analysis was carried out to investigate the genotype-phenotype associations and to compare certain genotypes and cancer. We found 7 genotypes both in the healthy subjects and patients. The A1/A1 genotype was observed to be mainly associated with non-drinkers and -smokers (87.5 and 75.0%, respectively); moreover it was never found in the PA or HCC patients. Conversely, a weak association between A2/A3 with smokers (45.8%) and A4/A4 with drinkers (53.9%) was detected. In addition, the A4/A4 genotype was found to be significantly associated to PA [odds ratio (OR)=3.25; 95% confidence interval (CI) 1.21-7.50]. Our data demonstrate that certain CYP2E1 VNTR genotypes are associated with drinking and/or smoking habits; consequently, they may contribute either to the decreased or increased risk of developing drinking- and/or smoking-related cancers. In particular, we hypothesize that the A1/A1 VNTR genotype may have a protective role against drinking- and/or smoking-related cancers, and that A4/A4 may be a high-risk genotype during the early stages of cancer
Genotoxicity of citrus wastewater in prokaryotic and eukaryotic cells and efficiency of heterogeneous photocatalysis by TiO2
The presence of (±)a-pinene, (+)b-pinene, (+)3-carene, and R-(+)limonene terpenes in wastewater of a citrus transformation factory was detected and analyzed, in a previous study, by using Solid Phase Microextraction (SPME) followed by GC analyses. Purpose of that research was to compare the genotoxic responses of mixtures of terpenes with the genotoxicity of the individual compounds, and the biological effects of actual wastewater. Genotoxicity was evaluated in the Salmonella reversion assay (Ames test) and in V79 cells by Comet assay. Ames tests indicated that the four single terpenes did not induce an increase of revertants frequency. On the contrary, the mixtures of terpenes caused, in the presence of metabolic activation, a highly significant increase of the revertants in TA100 strain in comparison to the control. The Comet assay showed a significant increase in DNA damage in V79 cells treated for 1 h with single or mixed terpenes. Moreover, the actual wastewater was found highly genotoxic in bacterial and mammalian cells. Photocatalytic tests completely photodegraded the pollutants present in aqueous wastewater and the initial high genotoxicity of samples of wastewater collected during the photocatalytic run, was completely lose in 3 h of irradiation
ALLELIC VARIANTS OF CYP2E1 GENE IN HEPATOCARCINOMA PATIENTS AND IN HEPATIC TUMOR CELL LINES
Background and Aims: Hepatic enzyme CYP2E1 is involved in the
metabolism of a number of exogenous and endogenous substances
(i.e. ethanol, drugs and chemical carcinogens). Being polymorphic,
CYP2E1 gene can give different xeno-metabolic capabilities in a
population and it is well known that inadequate or no enzymatic
deactivation of xenobiotics could induce an increased susceptibility
to disease and cancer. In particular, one of the 5 -flanking region
polymorphisms, able to differentiate CYP2E1 gene transcriptional
activity, is caused by the appearance/disappearance of RsaI and
PstI restriction sites, which generates two different alleles, namely
*C1(Rsa+/Pst−) and *C2(Rsa−/Pst+) respectively, reported to be in
complete linkage disequilibrium.
Methods: To confirm the existence of a correlation between some
particular CYP2E1 genotypes/haplotypes and hepatocarcinoma, we
determined CYP2E1 PstI/RsaI genotypes/haplotypes by RFLP-PCR in
a cohort of central western Sicily hepatocarcinoma patients and in
a population of healthy students from the same geographic area.
Results: In hepatocarcinoma patients, modal genotype association
was Rsa++/Pst−−, corresponding to CYP2E1 *C1/*C1 haplotype,
whereas the Rsa+−/Pst−+ association, equivalent to CYP2E1 *C1/*C2
haplotype, resulted to have the lowest frequency both in patients
and in controls. Moreover, both in patients and in controls, noncanonical
genotype associations were frequent and arose from
a no-linkage disequilibrium between the two polymorphic sites.
Other authors reported this finding as a rare occurrence. Thus,
from analysis of only one restriction site, Rsa++ genotype was
approximately 1.5-fold more frequent in patients than in controls,
and the non-canonical Rsa+− genotype was found relatively
frequent in patients. Moreover, HuH7 and HA22T transformed
hepatocarcinoma cell lines also showed the Rsa+− genotype.
Conclusions: These results suggest that the presence in CYP2E1
genotype of at least one allele with an Rsa I restriction site is
correlated with hepatocarcinoma. As this site is known a consensus
sequence for some specific CYP gene transcription factors, like
HNF-1, it may be supposed that a single nucleotide polymorphism
can alter the possibility of HNF-1 to bind CYP2E1 promoter. This
could determine a marked change in the transcriptional activity
of the gene, incompetence in xenobiotic metabolism or in toxic
substance deactivation and an increased susceptibility to neoplastic
diseases, such as hepatocarcinoma
Biological effects of alpha-pinene in cultured mammalian cells
In this work we report the effects of exposure of mammalian cells to α-pinene, a bicyclic monoterpene founded in essential oils and used in insecticides, solvents, perfumes, etc.. Morphological analysis,
performed in V79 cells exposed to increasing doses(25μM up to 50μM) of α-pinene, indicated a increase of dose-related nuclear abnormalities; apoptotic cells were
seen at higher doses. Immunofluorescence with anti β-
tubulin antibody showed that monoterpene induced
genomic instability by interfering with mitotic process;
in fact, 50% (vs 19% in the control cells) of irregular
mitosis with multipolar or not correctly localized
spindles were detected, suggesting that α-pinene affects
cell stability by disturbing chromosome segregation.
Cytogenetic analysis demonstrated that frequency of
hypodiploid metaphases increased in a dose-dependent
manner and, moreover, α-pinene induced
endoreduplicated cells and double strand breaks.
Alkaline comet confirmed that monoterpene exposure
induced DNA lesions; in fact, OTM increased
significantly in a dose-dependent manner. In order to
assess whether the severe DNA damage evidenced by
comet assay was originated through the ROS
production, cells were incubated with CM-H2DCFDA
and then analysed by flow cytometry. Results
demonstrated an increase in fluorescence intensity after
α-pinene treatment indicating an increased oxidative
stress. On the whole, these findings strongly suggest
that α-pinene is able to compromise genome stability
preferentially through mitotic alterations and to damage
DNA through reactive oxigen species production
Biological effects of photodegratation by TiO2 of terpenes present in industrial wastewater
The aim of this work was to study the biological effects of four monoterpenes, i.e. α-pinene, β-pinene, 3-carene and d-limonene present in the wastewater of a citrus transformation factory. The study was carried out by exposing V79 Chinese hamster cells to single terpene or to the mixture of four terpenes at concentrations corresponding to those in the wastewater evaluated by head space solid phase micro extraction and gas chromatography (HS-SPME-GC) analyses. Treatments with single or combined terpenes similarly affected cell vitality, but only the combined treatments induced the 6-thioguanine resistant mutants. Moreover the photocatalytic degradation of the four terpenes was successfully achieved with the photocatalyst TiO(2) Degussa P25 in both the actual effluent and in synthetic solutions