Massimiliano Delpero

Evolution of the beta defensin 2 gene in primates

With the aim of further investigating the molecular evolution of beta defensin genes, after having analysed beta defensin 1 (DEFB1) in humans and several nonhuman primate species, we have studied the evolution of the beta defensin 2 gene (DEFB2), which codifies for a peptide with antimicrobial and chemoattractant activity, in humans and 16 primate species. We have found evidence of positive selection during the evolution of orthologous DEFB2 genes at two points on a phylogenetic tree relating these primates: during the divergence of the platyrrhines from the catarrhines and during the divergence of the Cercopithecidae from the Hylobatidae, Great Apes and humans. Furthermore, amino acid variations in Old World Monkeys seem to centre either on residues that are involved in oligomerisation in the human molecule, or that are conserved (40–80%) in beta-defensins in general. It is thus likely that these variations affect the biological function of the molecules and suggest that their synthesis and functional analysis might reveal interesting new information as to their role in innate immunity.

Combined Analysis of Chromosomal Aberrations and Glutathione S-transferase M1 and T1 Polymorphisms in Pathologists Occupationally Exposed to Formaldehyde

The formaldehyde (FA) genotoxic potential in occupationally exposed individuals is conflicting. A relevant indoor-air FA pollution was found in hospitals and scientific institutions where FA is used as a bactericide and tissue preservative. In the present study, we evaluated the frequency of chromosomal aberrations (CAs) in peripheral blood lymphocytes from workers in pathology wards who have been exposed to FA, compared with a group of unexposed subjects. The subjects were also analyzed for the GSTM1 and GSTT1 metabolic gene polymorphisms. The exposed subjects showed a significant increase in the frequency of CA per cell and in the percentage of cells with aberrations compared to control subjects. The different GST genotypes did not affect the level of cytogenetic damage since CA frequencies were not statistically different between the GST “null” genotypes and the GST “positives”. The generalized linear models showed that the number of CAs and cells with CAs increased with age, but, independent of age, it was significantly higher in the experimental rather than in the control group. Cubic-spline regression confirmed the linear relationship between CAs and age, but it provided evidence for a non-linear relationship between CAs and the number of years of FA exposure. Similar results were observed when the model included the number of cells with CAs as dependent variables. Our results demonstrate that air FA induces CAs even consequently to low levels of daily exposure, indicating an increased risk of genetic damage for workers exposed to this air pollutant.

Mitochondrial Sequences as Indicators of Generic Classification in Bush Babies

Systematic relationships among the African bush babies are not well understood. Various generic designations are currently in use. Some authors refer all species to a single genus (Galago), while others recognize ≤4 genera. Phylogenetic reconstructions based on morphology, karyology, allozymes and vocal repertoires have generated inconsistent hypotheses of relationship. We analyzed partial sequences of three mitochondrial genes (270 bp from cytochrome b, 387 bp from 12S rRNA, and 241 bp from 16S rRNA, total 898 bp) to resolve some uncertainties. We sampled taxa from each of three genera: Galagoides alleni, G. demidoff and G. zanzibaricus; Galago senegalensis, G. gallarum and G. moholi; and Otolemur crassicaudatus and O. garnettii. Outgroup taxa were Asian lorises: Nycticebus coucang and Loris tardigradus. We analyzed sequences separately and in combination, and modeled phylogenies using maximum parsimony, weighted parsimony, neighbor-joining and maximum-likelihood. We obtained some variation in phylogenetic inference depending on sequence and analytical method, but the results also gave strong phylogenetic signals. The lesser bush babies invariably formed a clade, showing evidence of very recent radiation. The greater bush babies also formed a clade, marked by somewhat greater interspecific genetic distances, which was allied with Galagoides alleni in most instances. Galagoides demidoff and G. zanzibaricus are not closely related, though both diverged early in the history of the group. A genus comprising Galagoides alleni, G. demidoff and G. zanzibaricus is not supported by our data. The most likely alliance for Galagoides alleni is within the genus Otolemur. Of the three partial sequences employed in the study, 16S rRNA gave the most consistent results, while cytochrome b was least informative.

A Composite Molecular Phylogeny of Living Lemuroid Primates

Lemuroid phylogeny is a source of lively debate among primatologists. Reconstructions based on morphological, physiological, behavioural and molecular data have yielded a diverse array of tree topologies with few nodes in common. In the last decade, molecular phylogenetic studies have grown in popularity, and a wide range of sequences has been brought to bear on the problem, but consensus has remained elusive. We present an analysis based on a composite molecular data set of approx. 6,400 bp assembled from the National Center for Biotechnology Information (NCBI) database, including both mitochondrial and nuclear genes, and diverse analytical methods. Our analysis consolidates some of the nodes that were insecure in previous reconstructions, but is still equivocal on the placement of some taxa. We conducted a similar analysis of a composite data set of approx. 3,600 bp to investigate the controversial relationships within the family Lemuridae. Here our analysis was more successful; only the position of Eulemur coronatus remained uncertain.

Increased frequency of chromosomal aberrations and sister chromatid exchanges in peripheral lymphocytes of radiology technicians chronically exposed to low levels of ionizing radiations

Chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) frequencies were estimated in peripheral lymphocytes from 21 radiology technicians, and from 21 non-exposed control subjects. We exclusively considered individuals who neither smoke nor consume drugs or alcohol for a period of at least two years prior to the analysis. Significant differences were found between exposed and controls in terms of SCEs and CAs frequencies. Technicians showed a significant higher number of high-frequency individuals (HFIs) with respect to the control group. Nevertheless, the mean frequency of SCEs observed among technician HFIs did not significantly differ with respect to that observed among control HFIs. Vice versa, the non-HFIs belonging to technicians group showed a statistically higher difference in the SCEs/NSM value with respect to the non-HFIs belonging to control group. Since the differences in the SCEs frequencies between the two groups are due to non-HFIs, our results seem to indicate a general genotoxic effect of the IR, not affected by HFIs. Among technicians, the level of chromosome damage correlated neither with years of radiation exposure nor with the age of the subjects. Vice versa, in the control group, a positive correlation was found between the number of SCEs and age. In both samples the gender status did not influence the frequencies of CAs and SCEs. Our results suggest that chronic long-term exposure to low doses of ionizing radiation could increase the CAs and SCEs frequencies. This study reinforces the relevance of the biomonitoring of hospital workers chronically exposed to ionizing radiation.

Polymorphisms of cytochrome P450 1A1, glutathione s-transferases M1 and T1 genes in ouangolodougou (Northern Ivory Coast)

In this study, the frequencies of CYP1A1, GSTM1, and GSTT1 gene polymorphisms were determined in 133 healthy individuals from Ouangolodougou, a small rural town situated in the north of the Ivory Coast. As appeared in several published studies, ethnic differences in these frequencies have been found to play an important role in the metabolism of a relevant number of human carcinogens. In the studied sample, the frequencies of Ile/Ile (wild type), Ile/Val (heterozygous variant), and Val/Val (homozygous variant) CYP1A1 genotypes were 0.271, 0.692, and 0.037, respectively. Frequencies of GSTM1 and GSTT1 null genotypes were 0.361 and 0.331, respectively. No significant differences were noted between men and women. In contrast to published data for Africans, CYP1A1 *Val Allele frequency (0.383) was significantly high (p < 0.001) in this specific population. For the GSTT1 null genotype, no differences were found between the studied and other African populations, the contrary to what occurred for the GSTM1 null genotype in relation to Gambia and Egypt.

DAT1 VNTR polymorphisms in a European and an African population: identification of a new allele.

ABSTRACT Polymorphism frequencies of the dopamine transporter gene (DAT1) hypervariable region have been analyzed in a sample of Italian and Ivory Coast individuals. The 3′untranslated region (UTR) of DAT1 includes a variable number of tandem repeats (VNTR) of a 40-bp monomer, ranging from 3 to 13 repeats in Caucasian and African populations. In our sample we found alleles with 3 to 16 repeats, and the most common alleles were the 10-repeat (DAT1*10) and the 9-repeat (DAT1*9) alleles. We also found two rare alleles in the Italian population and four in the Ivory Coast population. For the first time the new allele DAT1*16 is described in the Ivorians. The Ivory Coast population was not in Hardy-Weinberg equilibrium for the DAT1 locus because of a deficit of heterozygote genotypes. The observed heterozygosity of the Ivorian population was half that of the Italians. The lower observed heterozygosity and deviation from Hardy-Weinberg equilibrium could be the result of microevolutionary trends, such as genetic drift and/or inbreeding, acting on the relatively small and isolated population sampled for this study, although some sort of selective pressures acting against the shorter alleles cannot be excluded. This evidence, in association with the reduced polymorphism shown by the DAT1 VNTR compared to other VNTRs, seems to indicate that the DAT1 locus may be under some selective pressure.

Chromosomal damage in peripheral blood lymphocytes from nurses occupationally exposed to chemicals

In the present study, we evaluated the induced genome damage in peripheral blood lymphocytes from a sample of nurses occupationally exposed to low doses of different chemicals. A comprehensive multi-biomarker approach using cytogenetic endpoints was employed for analyzing chromosomal aberrations (CAs) and sister chromatid exchange (SCE) assay. The study included 20 nurses and 20 control subjects matched in age, gender and smoking habits. Nurses were exposed to different chemicals, such as cytostatic drugs, anaesthetics, formaldehyde and other sterilizing gases. Significant differences were found between exposure group and control group in terms of SCEs frequency (p < 0.001) but not in terms of replication index value (p = 0.845) and CAs (p = 0.236). Regression analyses indicated that the age and the exposure years did not influence the amount of the chromosomal damage among nurses. Vice versa, among controls, a positive correlation was found between the number of SCEs and age. In conclusion, our results suggest that a continuous long-term exposure to low doses of chemicals could result in increased levels of SCEs among nurses. This data emphasize the importance of biomonitoring nurses and other hospital workers handling drugs.

Micronucleus frequency in human lymphocytes after exposure to diphenylamine in vitro.

Diphenylamine (DPA) is an antioxidant compound that occurs naturally in several vegetables. It is widely applied in agriculture for preservation of the quality of apples and pears, and used for controlling superficial scald, a disorder that renders fruits of a number of apple cultivars unfit for the market. Because of its anti-oxidative properties, DPA also has several industrial applications. The potential genotoxic effect of DPA on human lymphocytes has previously been investigated in only two studies, which focused on detection of chromosome aberrations and sister chromatid exchange, respectively. In the present analysis, we evaluated micronucleus (MN) formation in freshly isolated human peripheral lymphocytes exposed to different concentrations (0.625, 1.25, 2.50, 5.0 and 10.0μg/ml) of DPA. Peripheral venous blood was collected from ten healthy subjects, and a total of 10,000 bi-nucleated cells were analyzed. Results indicated that DPA significantly increased the micronucleus frequency at concentrations of 1.25μg/ml and higher. Significant differences in the MN frequency were also found between the lower dose (0.625μg/ml) and all other doses tested, with the exception of 1.25μg/ml. Our results indicate a potential cytogenetic effect of DPA on human cells in vitro and require further in vivo studies to clarify the actual genotoxicity of this compound and the consequent risks for human health.

Genotyping for cytokine polymorphisms in a Northern Ivory Coast population reveals a high frequency of the heterozygote genotypes for the TNF-α-308G/A SNP

Cytokine polymorphisms influence the outcomes of parasitic diseases and vary among populations because of their different evolutionary histories and selective pressures imposed by host–pathogen interactions. In this frame, we investigated the frequencies of TNF‐α (‐308G/A), TGF‐β1 (codon 10C/T, codon 25C/G) and IL‐10 (‐1082A/G) SNPs in 133 individuals from Ouangolodougou, a rural village in Northern Ivory Coast, where malaria and other parasitic diseases are endemic. The SNPs alleles were determined by ARMS‐PCR methodology. Allele frequencies of the SNPs investigated were as follows: IL 10 ‐1082G = 0.741 and ‐1082A = 0.259; TGF‐β1 Codon 10 C = 0.835 and T = 0.165; TGF‐β1 Codon 25 G = 0.782 and C = 0.218. For the TNF‐α gene, we found high frequencies of the ‐308A allele (0.305) and heterozygote genotypes (0.594), with a consequent deviation from the Hardy–Weinberg equilibrium. The high heterozygosity at the TNF‐α locus suggests a possible selective advantage of the heterozygote genomes, associated with intermediate levels of TNF‐α expression, against the infectious agents endemic in Western Africa.