Identification of Intron Retention in the Slc16a3 Gene Transcript Encoding the Transporter MCT4 in the Brain of Aged and Alzheimer-Disease Model (APPswePS1dE9) Mice.

The monocarboxylate transporter 4 (MCT4; Slc16a3) is expressed in the central nervous system, notably by astrocytes. It is implicated in lactate release and the regulation of glycolytic flux. Whether its expression varies during normal and/or pathological aging is unclear. As the presence of its mature transcript in the brain of young and old mice was determined, an unexpectedly longer RT-PCR fragment was detected in the mouse frontal cortex and hippocampus at 12 vs. 3 months of age. Cultured astrocytes expressed the expected 516 base pair (bp) fragment but treatment with IL-1ß to mimic inflammation as can occur during aging led to the additional expression of a 928 bp fragment like that seen in aged mice. In contrast, cultured pericytes (a component of the blood-brain barrier) only exhibited the 516 bp fragment. Intriguingly, cultured endothelial cells constitutively expressed both fragments. When RT-PCR was performed on brain subregions of an Alzheimer mouse model (APPswePS1dE9), no fragment was detected at 3 months, while only the 928 bp fragment was present at 12 months. Sequencing of MCT4 RT-PCR products revealed the presence of a remaining intron between exon 2 and 3, giving rise to the longer fragment detected by RT-PCR. These results unravel the existence of intron retention for the MCT4 gene in the central nervous system. Such alternative splicing appears to increase with age in the brain and might be prominent in neurodegenerative diseases such as Alzheimer's disease. Hence, further studies in vitro and in vivo of intron 2 retention in the Slc16a3 gene transcript are required for adequate characterization concerning the biological roles of Slc16a3 isoforms in the context of aging and Alzheimer's disease pathology.

Evaluation of biochemical, hematological, and genotoxic parameters in mice exposed to individual and combined ethoprophos and cadmium.

Environmental contamination by complex mixtures of pesticides and metals is a major health problem in agriculture and industry. In real life scenarios, we are exposed to mixtures of chemicals rather than single chemicals, and therefore it is critical to assess their toxicity. The current work was conducted to assess the toxic effects of a low dose (2% median lethal dose) of ethoprophos (Etho, 0.16 mg kg-1 bw), and cadmium (Cd, 0.63 mg kg-1 bw); each alone or in combination on hematological, biochemical, and genotoxic parameters in male mice for one or four weeks. The tested toxicants resulted in a decline in body and organs weights, the most hematological indices, acetylcholine esterase activity, and the total protein content, while they significantly increased liver and kidney function parameters. Furthermore, they increased the mitotic index (MI), number of abnormal sperms, and chromosomes. In conclusion, Etho and Cd induce deleterious effects on all tested parameters in male mice which reflect more obvious impacts when both combined, particularly after 28 days of exposure. However, further research is needed to confirm toxicokinetic or toxicodynamic interactions between these two toxic compounds in the organisms.

Three Complex alleles associated with N1303K mutation and their molecular consequences.

Cystic Fibrosis (CF) in Arab Mediterranean countries has a different CFTR mutational profile if compared either to Caucasians or in the Arabian Peninsula. The c.3909C>G (N1303K, p.Asn1303Lys) mutation of the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR). This mutation represents a higher frequency in the Mediterranean countries in association with different polymorphisms or mutations in cis position constituting various complex alleles. N1303K mutation induces many phenotypes, especially pancreatic insufficiency from mild to severe and it is associated in cis with other polymorphisms. The aim of this investigation is therefore to screen complex alleles carrying N1303K mutation among Lebanese, Egyptian and French patients. All exons of the CFTR and their flanking regions were performed by PCR amplification, followed by automated direct DNA sequencing. Two complex alleles are more frequent corresponding to Wild Type and mutated haplotype. Besides that two other very rare complex alleles have been detected, one in Egyptian and French samples, and then another one in Lebanon samples. We have studied their impact on the CFTR mRNA splicing using a minigene strategy. Constructs containing wild-type and mutant CFTR cloned into the pTBNdeI hybride minigene have been expressed in HeLa, HT29 and HEK293 cells. RT-PCR analysis of mRNA using ß-globin-specific primers revealed that N1303K and the polymorphisms associated with cis induce weak abnormal splicing and a modification of the quality and the quantity of CFTR protein. These different associations of identified polymorphisms with N1303K in cis could have an impact on the severity of the disease.

Cytogenetic and testicular histological alterations induced by sulphur dioxide in dried apricot leather.

Sulphur dioxide (SO2) is used as a preservative in food to prevent its discolouration, and to inhibit the growth of bacteria. Little data is available concerning its in vivo hazardous impact.The present study is therefore designed to examine the cyto-genotoxic potential and the testicular histological alterations in adult mice, induced by SO2 present in the dried apricot leather used to prepare the oriental drink Qamar Al-Deen. Two different forms of drinks were tested; cold and boiled drinks. Animals were placed into 4 groups. The first group received distilled water as a negative control.The second and third groups received orally the drink for 28 days in the form of a cold and a boiled drink, respectively. Animals of the fourth group received cyclophosphamide, they were used as a positive control for cyto-genotoxic tests. The chromosomal aberrations, as well as sperm abnormalities, were significantly elevated in animals that received the two different drink preparations. The mitotic index significantly decreased in comparison with negative and positive controls. Furthermore, histological examination showed different degrees of alterations in the testis. Our results suggest that the presence of SO2 inside the apricot leather might be responsible for these changes. Thus, these remarkable hazardous effects of SO2 on male albino mice could be used as a potential guide for the prediction of its human health impact. Furthermore, consumers could be advised to prevent excessive consumption of the drink (Qamar Al-Deen) prepared from dried apricot leather.

Effect of N-acetylcysteine on attenuation of chlropyrifos and its methyl analogue toxicity in male rats.

The attenuating effect of 150 mg/kg of N-acetylcysteine (NAC) against the oral administration of 7.88 and 202.07 mg/kg/day for 14 days of either chlropyrifos-ethyl (CPE-E) or chlropyrifos-methyl (CPF-M), respectively, in male rat was investigated using biochemical and genetic markers. Biomarkers such as acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), paraoxonase (PON), adenosine 5'-triphosphatase (ATP-ase), glutathione-S-transferase (GST), catalase (CAT), glutathione reduced (GSH) in serum showed a significant decline in their levels, while calcium (Ca+2), cytochrome C reduction (CYC-R), lipid peroxidation (LPO), nitric oxide (NO) levels showed a significant increase in serum of treated rats. Regarding the genotoxic parameters, when rats are treated either with CPE-E or CPF-M, liver DNA, chromosomal aberration (CA), and micronucleated polychromatic erythrocytes (MnPCE) significantly increased, while the mitotic index (MI) and polychromatic erythrocytes (PCE)/ normochromatic erythrocytes (NCE) ratio were significantly decreased. However, the administration of NAC following the intoxication of CPF-E or CPF-M attenuated the tested biochemical and genotoxic markers. It can be concluded that NAC can be used to ameliorate the toxicity of certain organophosphorus compounds such as CPF-E and CPF-M.

Role of biomarkers in the evaluation of cadmium and ethoprophos combination in male mice.

This work aimed to study the risk assessment procedures of a combination of single and repeated dose of the widely used pesticide, ethoprophos (Etho) and heavy metal cadmium (Cd), on the hematological, biochemical, reproduction and cytogenetic parameters in male mice. The results revealed that the sub-lethal dose (1/50 LD50) of the tested toxic substances (Etho and/or Cd) reduced the body and organ weights, the most hematological profile and the activity of acetylcholine esterase (AChE). The tested pollutants significantly increased the parameters of liver function, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as renal function tests, including creatinine and urea. In addition, they have deleterious effects on reproductive function tests by stimulating the number of sperm abnormalities (SA) and cytogenetic assays by increasing the frequency of chromosomal abnormalities (CA) and the mitotic index (MI). The overall results of this exploratory study suggest that the co-existence of the two tested compounds (Etho and Cd) had the propensity to cause a more pronounced effect than that of each compound alone on all the battery measured biomarkers, especially in the repeated treatment (14 doses) than that in the single one. Also, the combination of a range of simple and sensitive assays as endpoints gives a comprehensive picture and provides better insights to evaluate the potential effects of other commonly encountered environmental pollutants.

Impact of citrate- and chitosan-capped gold nanoparticles on the liver of Swiss albino mice: Histological and cyto-genotoxic study.

The present study aimed to disclose the histological alterations and cyto-genotoxic potential induced by citrate- and chitosan-capped AuNPs on liver of adult Swiss albino mice. Animals were randomly divided into 8 groups. The first two groups were intraperitoneally (i.p) injected with physiological saline once and left for 10 days and every other day for 21 days, respectively, and kept as negative control groups. While the third and fourth groups were injected i.p with a single dose of 2 mg/kg of citrate- and chitosan-capped AuNPs, respectively, and left for 10 days. The fifth and sixth groups were injected i.p every other day for 21 days with 200 µg/kg of citrate- and chitosan-capped AuNPs, respectively. Animals of the seventh and eighth groups were injected i.p with 50 mg/kg cyclophosphamide once and left for 10 days and with 20 mg/kg cyclophosphamide every other day for 21 days, respectively. The livers of mice were dissected and processed for microscopic examination and for analyzing the expression of inflammation-related genes using RT-PCR. In addition, bone marrow samples were taken to investigate the mitotic index and the chromosomal aberrations. The present study showed various degrees of structural changes in the liver of animals received AuNPs. Such changes were more prominent in animals treated with a single dose of AuNPs, particularly with citrate-capped AuNPs as compared to chitosan-capped AuNPs. Furthermore, genotoxic analysis did not reveal any genotoxicity for AuNPs with both coats. Therefore, chitosan-capped AuNPs were less hepatotoxic than citrate-capped ones. However, it has not been proven that AuNPs are genotoxic by both coats.

Complexity of phenotypes induced by p.Asn1303Lys-CFTR correlates with difficulty to rescue and activate this protein.

Cystic Fibrosis is the most common recessive autosomal rare disease found in Caucasian. It is caused by mutations on the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) that encodes for a protein located on the apical membrane of epithelial cells. c.3909C>G (p.Asn1303Lys) is one of the most common worldwide mutations located in nucleotide binding domain 2. The effect of the p.Asn1303Lys mutation on misprocessing was studied by immunofluorescence and western blotting analysis in presence and absence of treatment. To evaluate the functionality of potentially rescued p.Asn1303Lys-CFTR, we assessed the channel activity by radioactive iodide efflux. No recovery of the activity was observed in transfected cultured cells treated with VX-809. Thus, our results suggest that multiple drugs may be needed for the treatment of c.3909C>G patients in order to correct and activate p.Asn1303Lys-CFTR as it shows folding and functional defects.

In cellulo analyses of the p.Val322Ala mutation on the CFTR protein conformation and activity.

Cystic fibrosis is caused by mutations on the Cystic Fibrosis Transmembrane conductance Regulator gene (CFTR). Exonic mutations may have variable effect on the CFTR protein and may alter the normal localization of CFTR on the apical membrane of epithelial cells or/and its function as a chloride channel. Identifying the effect of a missense mutation can be a first step in helping the medical counseling and the therapeutic strategies. In this study, the effect of the c.965T>C exon 8 mutation that induces a valine-to-alanine substitution (p.Val322Ala) into the fifth helix of the first membrane spanning domain was determined by in silico and in cellulo analyses. The confocal microscopy analyses and functionality test showed, in the tested cell line, that this mutation should have no impact on the function of the p.Val322Ala-CFTR protein. However, regarding the importance of this Val322 amino acid in the CFTR protein, precautions and individual follow-up are still required when c.965T>C if associated with other mutation(s).

N1303K (c.3909C>G) mutation and splicing: implication of its c.[744-33GATT(6); 869+11C>T] complex allele in CFTR exon 7 aberrant splicing.

Cystic Fibrosis is the most common recessive autosomal rare disease found in Caucasians. It is caused by mutations on the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) that encodes a protein located on the apical membrane of epithelial cells. c.3909C>G (p.Asn1303Lys, old nomenclature: N1303K) is one of the most common worldwide mutations. This mutation has been found at high frequencies in the Mediterranean countries with the highest frequency in the Lebanese population. Therefore, on the genetic level, we conducted a complete CFTR gene screening on c.3909C>G Lebanese patients. The complex allele c.[744-33GATT(6); 869+11C>T] was always associated with the c.3909C>G mutation in cis in the Lebanese population. In cellulo splicing studies, realized by hybrid minigene constructs, revealed no impact of the c.3909C>G mutation on the splicing process, whereas the associated complex allele induces minor exon skipping.

Multi-physiopathological consequences of the c.1392G>T CFTR mutation revealed by clinical and cellular investigations.

This study combines a clinical approach and multiple level cellular analyses to determine the physiopathological consequences of the c.1392G>T (p.Lys464Asn) CFTR exon 10 mutation, detected in a CF patient with a frameshift deletion in trans and a TG(11)T(5) in cis. Minigene experiment, with different TG(m)T(n) alleles, and nasal cell mRNA extracts were used to study the impact of c.1392G>T on splicing in both in cellulo and in vivo studies. The processing and localization of p.Lys464Asn protein were evaluated, in cellulo, by western blotting analyses and confocal microscopy. Clinical and channel exploration tests were performed on the patient to determine the exact CF phenotype profile and the CFTR chloride transport activity. c.1392G>T affects exon 10 splicing by inducing its complete deletion and encoding a frameshift transcript. The polymorphism TG(11)T(5) aggravates the effects of this mutation on aberrant splicing. Analysis of mRNA obtained from parental airway epithelial cells confirmed these in cellulo results. At the protein level the p.Lys464Asn protein showed neither maturated form nor membrane localization. Furthermore, the in vivo channel tests confirmed the absence of CFTR activity. Thus, the c.1392G>T mutation alone or in association with the TG repeats and the poly T tract revealed obvious impacts on splicing and CFTR protein processing and functionality. The c.[T(5); 1392G>T] complex allele contributes to the CF phenotype by affecting splicing and inducing a severe misprocessing defect. These results demonstrate that the classical CFTR mutations classification is not sufficient: in vivo and in cellulo studies of a possible complex allele in a patient are required to provide correct CFTR mutation classification, adequate medical counseling, and adapted therapeutic strategies.

Consequences of partial duplications of the human CFTR gene on cf diagnosis: mutations or ectopic variations.

CFTR exon 10 and its flanking regions are duplicated in the human genome. These duplications present mutations compared to the normal exon 10 sequence. Due to the polymorphic sequence of the 3' intron 9 sequence, it may appear difficult to sequence exon 10 and some mutations described in this exon could, in fact, be variations observed in an ectopic duplicated sequence. In our previous work we described a methodology to carry out PCR only of exon 10 and not of ectopic regions. In this work, we analyzed mutations described in the CF data base as being CFTR mutations but also found in ectopic regions: c.1392G>T, c.1338_1339delAT, c.1235delC, and c.1247A>G. We have shown that these mutations appear to be authentic mutations in CFTR exon 10 and not ectopic variations in analyzed patients. These mutations validate the usefulness of our new strategy in the mutation analysis of this region of CFTR.

CFTR mutation combinations producing frequent complex alleles with different clinical and functional outcomes.

Genotype-phenotype correlations in cystic fibrosis (CF) may be difficult to establish because of phenotype variability, which is associated with certain CF transmembrane conductance regulator (CFTR) gene mutations and the existence of complex alleles. To elucidate the clinical significance of complex alleles involving p.Gly149Arg, p.Asp443Tyr, p.Gly576Ala, and p.Arg668Cys, we performed a collaborative genotype-phenotype correlation study, collected epidemiological data, and investigated structure-function relationships for single and natural complex mutants, p.[Gly576Ala;Arg668Cys], p.[Gly149Arg;Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala;Arg668Cys]. Among 153 patients carrying at least one of these mutations, only three had classical CF and all carried p.Gly149Arg in the triple mutant. Sixty-four had isolated infertility and seven were healthy individuals with a severe mutation in trans, but none had p.Gly149Arg. Functional studies performed on all single and natural complex mutants showed that (1) p.Gly149Arg results in a severe misprocessing defect; (2) p.Asp443Tyr moderately alters CFTR maturation; and (3) p.Gly576Ala, a known splicing mutant, and p.Arg668Cys mildly alter CFTR chloride conductance. Overall, the results consistently show the contribution of p.Gly149Arg to the CF phenotype, and suggest that p.[Arg668Cys], p.[Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala;Arg668Cys] are associated with CFTR-related disorders. The present study emphasizes the importance of comprehensive genotype-phenotype and functional studies in elucidating the impact of mutations on clinical phenotype.

Influence of the duplication of CFTR exon 9 and its flanking sequences on diagnosis of cystic fibrosis mutations.

The DNA sequences of seven regions in the human genome were examined for sequence identity with exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is mutated in cystic fibrosis, and its intronic boundaries. These sequences were 95% to 96% homologous. Based on this nucleotide sequence similarity, PCR primers for CFTR exon 9 can potentially anneal with other homologous sequences in the human genome. Sequence alignment analysis of the CFTR exon 9 homologous sequences revealed that five registered mutations in the Cystic Fibrosis Mutation Database may be due to the undesired annealing of primers to a homologous sequence, resulting in inappropriate PCR amplification. For this reason, we propose that certain pseudomutations may result from the similarity between CFTR exon 9 (and its flanking introns) and related sequences in the human genome. Here we show that two mutations previously described in the CFTR database (c.1392 + 6insC; c.1392 + 12G>A) were inappropriately attributed to two individuals who sought carrier testing. A more detailed study by either direct sequencing or subcloning and sequencing of PCR products using specially designed primers revealed that these apparent mutations were not, in fact, present in CFTR. In addition, we present new PCR conditions that permit specific amplification of CFTR exon 9 and its flanking regions.