Jorge Luis Fuentes

Analyses of genetic diversity in Cuban rice varieties using isozyme, RAPD and AFLP markers

A survey of the genetic diversity among the major cuban rice cultivars was conducted using isozyme, RAPD and AFLP markers. Polymorphisms were detected for esterases, peroxidases, alcohol dehydrogenases and polyphenoloxidases systems; 21 RAPD primers and four AFLP primer combinations. Heterozygosity arithmetic mean value (Hav(p)), the effective multiplex ratio (EMR) and the marker index (MI), were calculated for isozyme, RAPD and AFLP markers. The mean value of genetic similarity among the different varieties was 0.92 for isozyme, 0.73 for RAPD and 0.58 for AFLP analyses. Thus, AFLP were able to detect polymorphisms with higher efficiency than RAPD (+15%) and isozyme (+34%). Data from the isozyme, RAPD and AFLP analyses were used to compute matrices of genetic similarities. The efficiency of the UPGMA for the estimation of genetic relatedness among varieties was supported by cophenetic correlation coefficients. The resulting values indicated that the distortion level for the estimated similarities was minimal. The correlation coefficients obtained by the Mantel matrix correspondence test, which was used to compare the cophenetic matrices for the different markers, showed that estimated values of genetic relationship given for isozyme and RAPD markers (r = 0.89), as well as for AFLP and RAPD markers (r = 0.82) were properly related. However, AFLP and isozyme data showed only moderate correlation (r = 0.63). Although the genetic variability found among the different cultivars was low, both RAPD and AFLP markers proved to be efficient tools in assessing the genetic diversity of rice genotypes.

Antimutagenic mechanisms of Phyllanthus orbicularis when hydrogen peroxide is tested using Salmonella assay.

Phyllanthus orbicularis is a medicinal plant, endemic to Cuba, whose aqueous extract has proven antiviral properties and antimutagenic activities against aromatic amines and hydrogen peroxide. In addition, this plant extract presents antioxidant activity. In this paper, using the Salmonella assay with the experimental approaches of co-incubation, pre- and post-treatments, it is shown that the P. orbicularis extract protects bacterial cells from oxidative damage and mutation by some intracellular mechanism, irrespective of its antioxidant activity.

Assessment of the potential genotoxic risk of Phyllantus orbicularis HBK aqueous extract using in vitro and in vivo assays

Phyllanthus orbicularis HBK is an endemic Cuban plant whose aqueous extract has been proposed as an effective drug for the treatment of viral diseases. In addition, antimutagenic properties of this extract have also been reported. In the present study, the genotoxicity of this plant extract was assessed using different in vitro and in vivo assays. Results from SOS gene induction, gene reversion and conversion, and SMART assays clearly show that P. orbicularis aqueous extract does not induce either primary DNA damage or mutation. Additionally, no statistically significant difference was found in the percentage of chromosomal aberrations in Chinese hamster ovarian (CHO) cells treated with the plant extract. On the contrary, micronuclei and abnormal anaphase were induced by this extract in CHO cells. This genotoxic effect was related to a high cytotoxicity. Single spots were detected in the SMART assay. These results point to a possible aneugenic effect of the P. orbicularis aqueous extract at cytotoxic doses which are much higher than those seen by their antiviral and antimutagenic activities.

Studies on the antimutagenesis of Phyllanthus orbicularis: mechanisms involved against aromatic amines

Phyllanthus orbicularis is a medicinal plant, endemic to Cuba, whose aqueous extract has proven antiviral properties. This plant extract is being studied for treatment of viral diseases in animals and humans. Antimutagenic activities of this plant aqueous extract have been investigated as an additional and possible valuable property. Antimutagenesis was assayed against the mutagenic activity of m-phenylenediamine (m-PDA), 2-aminofluorene (2-AF), 1-aminopyrene (1-AP), 2-aminoanthracene (2-AA) and 9-aminophenantrene (9-AP) in Salmonella typhimurium (S. typhimurium) YG1024, in different co-treatment approaches. This plant extract produced a significant decrease of the mutagenesis mediated by these aromatic amines (AA) in the following order: m-PDA>2-AA>2-AF>9-AP>1-AP. Interactions with S9 enzymes and transformation of promutagenic amines and their mutagenic metabolites by chemical reactions to non-mutagenic compounds are proposed as possible mechanisms of antimutagenesis. Mutagenesis mediated by m-PDA was almost completely abolished when S9 mixture was co-incubated with the plant extract during 40 min, previous to the addition of the m-PDA and bacterial cells to the assay. Similar results were found with 2-AA and 1-AP, but the reduction of the mutation rate was not so dramatic. In contrast, the most significant antimutagenic effect against 2-AF and 9-AP was seen when these chemicals were co-incubated with the plant extract, before addition of the S9 mixture and bacterial cells to the assay. Therefore, inhibition or competition for S9 enzymes seems to be the main antimutagenic mechanism of this plant extract against m-PDA, 2-AA and 1-AP, whilst a chemical modification of 2-AF and 9-AP into non-promutagenic derivatives is likely to be the main mechanism of antimutagenesis against both compounds.

Usefulness of the SOS Chromotest in the study of medicinal plants as radioprotectors

Purpose: The aim of this work is to investigate the usefulness of a modified protocol of the SOS Chromotest to detect antigenotoxicity activities against γ-rays of plant extracts with proven antioxidant activity, and to elucidate the antigenotoxic mechanisms involved in radioprotection using this system. Materials and methods: The methodology developed was assayed with amifostine, the most studied radioprotector, and with Phyllanthus orbicularis HBK, Cymbopogon citratus (DC) Stapf and Pinus caribaea Morelet extracts, using pre- and post-treatment procedures. Results: The P. caribaea and C. citratus extracts were antigenotoxic against γ-rays when the cells were pre-treated with both extracts, suggesting a possible antigenotoxic action through a free radical scavenging mechanisms. Amifostine and the P. orbicularis extract were also antigenotoxic under pre- and post-treatment conditions, indicating that several antimutagenic components of this plant extract may also operate by some intracellular mechanism, unlike its antioxidant activity. Conclusions: The results have demonstrated the usefulness of the modified SOS Chromotest assay in the screening of phytochemical radioprotectors as well as in the study of their antimutagenic mechanisms.

Genetic diversity analysis of rice varieties (Oryza sativa L.) based on morphological, pedigree and DNA polymorphism data

The diversity within 20 rice varieties used as progenitors in Cuban rice breeding programmes was analysed with respect to agro-morphological traits, pedigree and DNA markers. Eleven agro-morphological traits were scored, and phenotypic (Euclidian) distances between the rice varieties were calculated. Sixty random amplified polymorphism DNA (RAPD) and 115 amplified fragment length polymorphism (AFLP) bands served to determine Dice’s distance estimates. Cluster analyses were performed based on genetic distance matrices using the unweighted pair-group method of arithmetical means (UPGMA) as the clustering method. This analysis showed five phenotypic, six genealogical, five RAPD and six AFLP diversity groups. Genetic diversity estimates based on RAPD data, but not on AFLP, efficiently represented the genetic parentage and phenotypic diversity between rice varieties. Combined diversity estimates allowed the identification of 11 different genetic pools and permitted a more effective separation of the progenitor set than those obtained solely by phenotypic and genealogical information. The results of this study stress the necessity to diversify rice parental stocks for further breeding purposes.

Antigenotoxic Effect Against Ultraviolet Radiation Induced DNA Damage of the Essential Oils from Lippia Species

The antigenotoxicity against ultraviolet radiation (UV)‐induced DNA damage of essential oils (EO) from Lippia species was studied using SOS Chromotest. Based on the minimum concentration that significantly inhibits genotoxicity, the genoprotective potential of EO from highest to lowest was Lippia graveolens, thymol‐RC ≈ Lippia origanoides, carvacrol‐RC ≈ L. origanoides, thymol‐RC > Lippia alba, citral‐RC ≈ Lippia citriodora, citral‐RC ≈ Lippia micromera, thymol‐RC > L. alba, myrcenone‐RC. EO from L. alba, carvone/limonene‐RC, L. origanoides, α‐phellandrene‐RC and L. dulcis, trans‐β‐caryophyllene‐RC did not reduce the UV genotoxicity at any of the doses tested. A gas chromatography with flame ionization detection analysis (GC‐FID) was conducted to evaluate the solubility of the major EO constituents under our experimental conditions. GC‐FID analysis showed that, at least partially, major EO constituents were water‐soluble and therefore, they were related with the antigenotoxicity detected for EO. Constituents such as p‐cymene, geraniol, carvacrol, thymol, citral and 1,8‐cineole showed antigenotoxicity. The antioxidant activity of EO constituents was also determined using the oxygen radical antioxidant capacity (ORAC) assay. The results showed that the antigenotoxicity of the EO constituents was unconnected with their antioxidant activity. The antigenotoxicity to different constituent binary mixtures suggests that synergistic effects can occur in some of the studied EO.

Radioprotective effect of sodium diethyldithiocarbamate (DDC) and S-2-aminoethyl-isothioronicadenosin-5-triphosphate (adeturon) in γ-irradiated Escherichia coli cells

The effect of sodium diethyldithiocarbamate (DDC) and S-2-aminoethyl-isothiouronicadenosin-5-triphosphate (adeturon) in the induction of Escherichia coli SOS response promoted by gamma-irradiation was studied by measuring the induction of sulA gene and the induction of lambda prophage. Furthermore, as a way of measure the exonuclease activity in gamma-irradiated cells in the presence or absence of both compounds, the DNA degradation was determined. Adeturon did not affected DNA degradation, but inhibited the induction of the SOS functions studied. On the contrary, DDC inhibited DNA degradation as well as the induction of the sulA gene, but enhanced lambda induction in E. coli lysogenic strains. These results indicate that both compounds diminish the DNA damage produced by gamma-irradiation and also suggest that the mechanisms of radioprotection must be different. Thus, radioprotection mediated by DDC should involve free hydroxyl radical scavenging and a minor activity of exonuclease. The enhancement of phage induction in E. coli cells that DDC produces could be attributed to its quelant effect and this would not be not probably directly related to radioprotection. Adeturon, as thiols, may serve also as scavenging agent of free hydroxyl radicals, diminishing indirectly the DNA damage level. In addition, adeturon must interact with DNA in the same form that other aminothiol compounds do it. This interaction, mediated by amino groups of adeturon, may serve to concentrate these compounds near of the DNA damage site, increasing the potential for the thiol portion of the molecule to donate hydrogen, decreasing the damage level on DNA molecule. However, adeturon do not modify the exonuclease activity. Some topic about the possible clinical application of both compounds are discussed.

Survival and SOS response induction in ultraviolet B irradiated Escherichia coli cells with defective repair mechanisms

Abstract Purpose In this paper, the contribution of different genes involved in DNA repair for both survival and SOS induction in Escherichia coli mutants exposed to ultraviolet B radiation (UVB, [wavelength range 280–315 nm]) was evaluated. Materials and methods E. coli strains defective in uvrA, oxyR, recO, recN, recJ, exoX, recB, recD or xonA genes were used to determine cell survival. All strains also had the genetic sulA::lacZ fusion, which allowed for the quantification of SOS induction through the SOS Chromotest. Results Five gene products were particularly important for survival, as follows: UvrA > RecB > RecO > RecJ > XonA. Strains defective in uvrA and recJ genes showed elevated SOS induction compared with the wild type, which remained stable for up to 240 min after UVB-irradiation. In addition, E. coli strains carrying the recO or recN mutation showed no SOS induction. Conclusions The nucleotide excision and DNA recombination pathways were equally used to repair UVB-induced DNA damage in E. coli cells. The sulA gene was not turned off in strains defective in UvrA and RecJ. RecO protein was essential for processing DNA damage prior to SOS induction. In this study, the roles of DNA repair proteins and their contributions to the mechanisms that induce SOS genes in E. coli are proposed.

Amifostine protection against induced DNA damage in γ‐irradiated Escherichia coli cells depend on recN DNA repair gene product activity

Amifostine is the most effective radioprotector known and the only one accepted for clinical use in cancer radiotherapy. In this work, the antigenotoxic effect of amifostine against γ‐rays was studied in Escherichia coli cells deficient in DNA damage repair activities. Assays of irradiated cells treated with amifostine showed that the drug reduced the genotoxicity induced by radiation in E. coli wild‐type genotypes and in uvr, recF, recB, recB‐recC‐recF mutant strains, but not in recN defective cells. Thus, the mechanism of DNA protection by amifostine against γ‐radiation‐induced genotoxicity appears to involve participation of the RecN protein that facilitates repair of DNA double‐strand breaks. The results are discussed in relation to amifostines chemopreventive potential.