Determining oxidative and non-oxidative genotoxic effects driven by estuarine sediment contaminants on a human hepatoma cell line.

Estuarine sediments may be reservoirs of hydrophilic and hydrophobic pollutants, many of which are acknowledged genotoxicants, pro-mutagens and even potential carcinogens for humans. Still, studies aiming at narrowing the gap between ecological and human health risk of sediment-bound contaminant mixtures are scarce. Taking an impacted estuary as a case study (the Sado, SW Portugal), HepG2 (human hepatoma) cells were exposed in vitro for 48 h to extracts of sediments collected from two areas (urban/industrial and Triverine/agricultural), both contaminated by distinct mixtures of organic and inorganic toxicants, among which are found priority mutagens such as benzo[a]pyrene. Comparatively to a control test, extracts of sediments from both impacted areas produced deleterious effects in a dose-response manner. However, sediment extracts from the industrial area caused lower replication index plus higher cytotoxicity and genotoxicity (concerning total DNA strand breakage and clastogenesis), with emphasis on micronucleus induction. On the other hand, extracts from the rural area induced the highest oxidative damage to DNA, as revealed by the FPG (formamidopyrimidine-DNA glycosylase) enzyme in the Comet assay. Although the estuary, on its whole, has been classified as moderately contaminated, the results suggest that the sediments from the industrial area are significantly genotoxic and, furthermore, elicit permanent chromosome damage, thus potentially being more mutagenic than those from the rural area. The results are consistent with contamination by pro-mutagens like polycyclic aromatic hydrocarbons (PAHs), potentiated by metals. The sediments from the agriculture-influenced area likely owe their genotoxic effects to metals and other toxicants, probably pesticides and fertilizers, and able to induce reactive oxygen species without the formation of DNA strand breakage. The findings suggest that the mixtures of contaminants present in the assayed sediments are genotoxic to HepG2 cells, ultimately providing a useful approach to hazard identification and an effective line-of-evidence in the environmental monitoring of anthropogenically-impacted coastal ecosystems.

Human hepatoma cells exposed to estuarine sediment contaminant extracts permitted the differentiation between cytotoxic and pro-mutagenic fractions.

Complex toxicant mixtures present in estuarine sediments often render contaminant screening unfeasible and compromise determining causation. HepG2 cells were subjected to bioassays with sediment extracts obtained with a series of progressively polar solvents plus a crude extract. The sediments were collected from an impacted area of an estuary otherwise regarded as pristine, whose stressors result mostly from aquaculture effluents and hydrodynamic shifts that enhance particle deposition. Compared to a reference scenario, the most polar extracts yielded highest cytotoxicity while higher genotoxicity (including oxidative damage) was elicited by non-polar solvents. While the former caused effects similar to those expected from biocides, the latter triggered effects compatible with known pro-mutagens like PAHs, even though the overall levels of toxicants were considered of low risk. The results indicate that the approach may constitute an effective line-of-evidence to infer on the predominant set of hazardous contaminants present in complex environmental mixtures.

Nonsense mutations in the human beta-globin gene lead to unexpected levels of cytoplasmic mRNA accumulation.

Generally, nonsense codons 50 bp or more upstream of the 3'-most intron of the human beta-globin gene reduce mRNA abundance. In contrast, dominantly inherited beta-thalassemia is frequently associated with nonsense mutations in the last exon. In this work, murine erythroleukemia (MEL) cells were stably transfected with human beta-globin genes mutated within each of the 3 exons, namely at codons 15 (TGG-->TGA), 39 (C-->T), or 127 (C-->T). Primer extension analysis after erythroid differentiation induction showed codon 127 (C-->T) mRNA accumulated in the cytoplasm at approximately 20% of the normal mRNA level. Codon 39 (C-->T) mutation did not result in significant mRNA accumulation. Unexpectedly, codon 15 (TGG-->TGA) mRNA accumulated at approximately 90%. Concordant results were obtained when reticulocyte mRNA from 2 carriers for this mutation was studied. High mRNA accumulation of codon 15 nonsense-mutated gene was revealed to be independent of the type of nonsense mutation and the genomic background in which this mutation occurs. To investigate the effects of other nonsense mutations located in the first exon on the mRNA level, nonsense mutations at codons 5, 17, and 26 were also cloned and stably transfected into MEL cells. After erythroid differentiation induction, mRNAs with a mutation at codon 5 or 17 were detected at high levels, whereas the mutation at codon 26 led to low mRNA levels. These findings suggest that nonsense-mediated mRNA decay is not exclusively dependent on the localization of mutations relative to the 3'-most intron. Other factors may also contribute to determine the cytoplasmic nonsense-mutated mRNA level in erythroid cells. (Blood. 2000;96:2895-2901)

Cystic fibrosis F508del patients have apically localized CFTR in a reduced number of airway cells.

Present state of knowledge, mostly based on heterologous expression studies, indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) protein bearing the F508del mutation is misprocessed and mislocalized in the cytoplasm, unable to reach the cell surface. Recently, however, it was described that protein levels and localization are similar between F508del and wild-type CFTR in airway and intestinal tissues, but not in the sweat glands. In this study, we used immunocytochemistry with three different anti-CFTR antibodies to investigate endogenous CFTR expression and localization in nasal epithelial cells from F508del homozygous patients, F508del carriers, and non-CF individuals. On average, 300 cells were observed per individual. No significant differences were observed for cell type distributions among CF, carrier, and non-CF samples; epithelial cells made up approximately 80% to 95% of all cells present. CFTR was detected mostly in the apical region (AR) of the tall columnar epithelial (TCE) cells, ciliated or nonciliated. By confocal microscopy analysis, we show that the CFTR apical region-staining does not overlap with either anti-calnexin (endoplasmic reticulum), anti-p58 (Golgi), or anti-tubulin (cilia) stainings. The median from results with three antibodies indicate that the apical localization of CFTR happens in 22% of TCE cells from F508del homozygous patients with CF (n = 12), in 42% of cells from F508del carriers (n = 20), and in 56% of cells from healthy individuals (n = 12). Statistical analysis indicates that differences are significant among all groups studied and for the three antibodies (p < 0.05). These results confirm the presence of CFTR in the apical region of airway cells from F508del homozygous patients; however, they also reveal that the number of cells in which this occurs is significantly lower than in F508del carriers and much lower than in healthy individuals. These findings may have an impact on the design of novel pharmacological strategies aimed at circumventing the CF defect caused by the F508del mutation.

Cystic fibrosis patients with the 3272-26A-->G mutation have mild disease, leaky alternative mRNA splicing, and CFTR protein at the cell membrane.

We characterized the 3272-26A-->G mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, creating an alternative acceptor splice site in intron 17a, that competes with the normal one, although we predict from consensus values, with lower efficiency. We analyzed five Cystic Fibrosis (CF) Portuguese patients with the 3272-26A-->G/F508del genotype. Besides clinical and haplotype characterization of those patients, we report here results from CFTR transcript analysis in nasal brushings from all five patients. RT-PCR analysis supports alternative splicing in all patients and carriers, but not in controls. By sequencing, we determined that the alternative transcript includes 25 nucleotides from intron 17a, which predictively cause frameshift and a premature stop codon. The use of this alternative splice site causes a reduction in the levels of normal transcripts from the allele with this mutation and, most probably, of normal protein as well. By immunocytochemistry of both epithelial primary cell cultures and slices from CF polyps, CFTR protein is detected at the cell membrane, with three different antibodies. Ussing chamber analysis of one nasal polyp shows a high sodium absorption, characteristic of CF. Altogether, the results suggest that the main defect caused by the 3272-26A-->G mutation is a reduction in normal CFTR transcripts and protein and therefore this mutation should be included in class V, according to Zielenski and Tsui.

Long-term follow-up of a family with autosomal dominant polycystic kidney disease type 3.

BACKGROUND: Autosomal dominant polycystic kidney disease is one of the most common hereditary diseases in man with an estimated prevalence of 1:1000. At least three genetic loci are responsible for the development of the disease. PKD1 localized to 16p13 is the most common gene, contributing to almost 85% of all cases, is associated with the most severe form. PKD2, localized to 4q21-23, responsible for almost all the remaining cases, is associated with a milder form. Up to now, only five families have been reported unlinked to the two most common genetic defects, and therefore little is known about the clinical findings of the non-PKD1/PKD2 families. METHODS: In this report we describe the clinical findings of 18 patients of a non-PKD1/PKD2 family, with a mean follow-up of 52 months (range 3-133 months) in our outpatient clinic. RESULTS: Of the 10 patients older than 40 years, nine were hypertensive; in this age group eight patients exhibited renal failure (two of them were on dialysis) and six had hepatic cysts. In eight patients younger than 40 years, the only clinical finding was hypertension in two. Considerable variation in the rate of progression to renal failure among members of this family was found; on the other hand, some patients did not exhibit any signs of progression. CONCLUSION: This family exhibits a more aggressive phenotype, in contrast with the majority of the described non-PKD1/non-PKD2 families.

Heterogeneous ethnic distribution of the 844ins68 in the cystathionine beta-synthase gene.

A novel mutation in the cystathionine beta-synthase (CBS) gene (a 68-bp insertion in the coding region of exon 8: 844ins68) was recently described, but its prevalence in different human populations is unknown. We analyzed the prevalence of the 68-bp insertion in the CBS gene in 405 unrelated individuals (810 chromosomes) of European, African, Asian and Amerindian origins. PCR amplification of a DNA fragment containing exon 8 of the CBS gene was employed. In addition, screening for the T833C CBS mutation by BsrI digestion was carried out in all samples bearing the 844ins68 mutation, since both mutations were previously reported to be associated in cis. The insertion was found in heterozygosity in 14 out of 104 whites (13.5%), was absent among Asians, and was found solely in 1 out of 220 Amerindian chromosomes analyzed, whereas a much higher prevalence was observed among blacks (37.7% of heterozygotes and 4% of mutant homozygotes). Furthermore, the T833C CBS mutation was found to cosegregate in cis with 844ins68 in all carriers of the insertion. The finding of the double mutant among blacks and Caucasians suggests that it antedated the racial divergence between Africans and non-Africans, and provides evidence for a partly or completely neutralizing effect conferred by the 68-bp insertion, since it allows the skipping of the T833C mutation. Our study represents the first analysis of the 844ins68 insertion in the CBS gene in different human populations, and reveals an extensive ethnic and geographic variability associated with this mutation.

[Mucoviscidosis with respiratory symptomatology in the neonatal period]. / Mucoviscidose com sintomatologia respiratória no período neonatal.

A case of cystic fibrosis presented in the neonatal period with respiratory symptomatology associated with early pancreatic insufficiency is reported. The CFTR gene molecular analysis was found to be a compound heterozygotes for delta F508 and G542X. The rarity of this mode of presentation and the inclusion of this entity in the differential diagnosis for neonatal respiratory distress syndrome is emphasised. The pathogenesis and some therapeutic aspects carried out in our patient, which might have improved the life expectancy of patients with this disease, are discussed.

Missense mutation R1066C in the second transmembrane domain of CFTR causes a severe cystic fibrosis phenotype: study of 19 heterozygous and 2 homozygous patients.

We report the clinical features of 21 unrelated cystic fibrosis (CF) patients from Portugal and Spain, who carry the mutation R1066C in the CFTR gene. The current age of the patients was higher in the R1066C/any mutation group (P < 0.01), as compared to the deltaF508/deltaF508 group. Poor values for lung radiological involvement (Chrispin-Norman) and general status (Shwachman-Kulcycki) were observed in the R1066C/any mutation group (P < 0.005 and P < 0.0004). A slightly, but not significantly worse lung function was found in the R1066C/any mutation group when compared with the deltaF508/deltaF508 patients. No significant differences were detected regarding the age at diagnosis, sweat Cl-values, or percentiles of height and weight between the two groups. Neither were significant differences observed regarding sex, meconium ileus (4.7% vs. 11.1%), dehydration (10.5% vs. 14.7%), or pancreatic insufficiency (PI) (100% vs. 97.8%). The proportion of patients with lung colonization by bacterial pathogens was slightly, but not significantly higher in the R1066C/any mutation group (70.0%), as compared with the deltaF508/deltaF508 group (57.5%). Other clinical complications were significantly more frequent in the R1066C/any mutation patients(P < 0.02) than in the deltaF508/deltaF508 group. The two homozygous R1066C/R1066C patients died at the ages of 3 months and 7 years. The data presented in this study clearly demonstrate that the R1066C mutation is responsible for a severe phenotype similar to that observed in homozygous deltaF508 patients. The poor clinical scores and complications of patients with the R1066C mutation are probably related to their slightly longer survival.

Complex cystic fibrosis allele R334W-R1158X results in reduced levels of correctly processed mRNA in a pancreatic sufficient patient.

CFTR alleles containing two mutations have been very rarely found in cystic fibrosis (CF) patients. They provide an opportunity to study the effect of two in cis-interacting gene defects on gene expression. Here, we describe a three-generation CF family with a complex CFTR allele that has not been previously described, containing the missense mutation R334W in exon 7 and the nonsense mutation R1158X in exon 19. Lymphocyte RNA analysis showed that (1) the mRNA corresponding to the complex allele is present although at markedly reduced levels; and (2) the nonsense mutation does not lead to detectable skipping of exon 19. The clinical picture of the patients with the genotype R334W-R1158X/delta F508 is characterized by pancreatic sufficiency and an atypical course of the disease.

A fertile male with cystic fibrosis: molecular genetic analysis.

A family study is presented in which the father of a girl with severe cystic fibrosis (CF) was also found to have CF but was mildly affected. He was diagnosed with three positive sweat tests including one after suppression with fludrocortisone. Genetic analysis showed that he is a compound heterozygote with the delta F508 CF mutation associated with haplotype B and a second CF mutation associated with haplotype C. In this unusual, fertile CF male, the late age of diagnosis (30 years) and the mild clinical picture suggest that the compound genotype (delta F508/other CF mutation) determines a much less severe form of the disease which might have gone unnoticed in the absence of a severely affected child. The implications of these findings for genetic counselling of families with CF are discussed.

Cystic fibrosis in the Portuguese population: haplotype distribution and molecular pathology.

The aim of this study was to obtain an estimate of the frequency of the delta F508 mutation in the Portuguese population, and of the tightness of its association with specific haplotypes. Furthermore, the genotype/clinical phenotype relationship and the feasibility of prenatal diagnosis were also investigated. The analysis of 42 cystic fibrosis (CF) families revealed that (1) 52% of CF chromosomes carry the deletion of codon 508; (2) there seems to be a positive correlation between the occurrence of the delta F508 mutation and the severity of the disease; and (3) fully informative prenatal diagnosis can be offered in 76% of at-risk pregnancies by using both genomic and allele specific oligonucleotide probes.