All-cause, cardiovascular disease and cancer mortality in the population of a large Italian area contaminated by perfluoroalkyl and polyfluoroalkyl substances (1980-2018).

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are associated with many adverse health conditions. Among the main effects is carcinogenicity in humans, which deserves to be further clarified. An evident association has been reported for kidney cancer and testicular cancer. In 2013, a large episode of surface, ground and drinking water contamination with PFAS was uncovered in three provinces of the Veneto Region (northern Italy) involving 30 municipalities and a population of about 150,000. We report on the temporal evolution of all-cause mortality and selected cause-specific mortality by calendar period and birth cohort in the local population between 1980 and 2018. METHODS: The Italian National Institute of Health pre-processed and made available anonymous data from the Italian National Institute of Statistics death certificate archives for residents of the provinces of Vicenza, Padua and Verona (males, n = 29,629; females, n = 29,518) who died between 1980 and 2018. Calendar period analysis was done by calculating standardised mortality ratios using the total population of the three provinces in the same calendar period as reference. The birth cohort analysis was performed using 20-84 years cumulative standardised mortality ratios. Exposure was defined as being resident in one of the 30 municipalities of the Red area, where the aqueduct supplying drinking water was fed by the contaminated groundwater. RESULTS: During the 34 years between 1985 (assumed as beginning date of water contamination) and 2018 (last year of availability of cause-specific mortality data), in the resident population of the Red area we observed 51,621 deaths vs. 47,731 expected (age- and sex-SMR: 108; 90% CI: 107-109). We found evidence of raised mortality from cardiovascular disease (in particular, heart diseases and ischemic heart disease) and malignant neoplastic diseases, including kidney cancer and testicular cancer. CONCLUSIONS: For the first time, an association of PFAS exposure with mortality from cardiovascular disease was formally demonstrated. The evidence regarding kidney cancer and testicular cancer is consistent with previously reported data.

Adipocyte signaling affects thyroid-specific gene expression via down-regulation of TTF-2/FOXE1.

Obesity affects thyroid gland function. Hypothyroidism, thyroid nodules, goiter, and thyroid cancer are more frequent in patients with higher BMI values. Although these data are supported by many clinical and epidemiological studies, our knowledge is very scarce at the molecular level. In this study, we present the first experimental evidence that adipocyte signaling downregulates the expression of thyroid-specific transcription factor 2 (TTF-2/FoxE1). It plays a crucial role in thyroid development and thyroid homeostasis and it is strictly connected to thyroid cancer as well. We provide in vivo and in vitro evidence that inhibition of TTF-2/FoxE1 gene expression is mediated by adipocyte signaling.

[Perfluoroalchilic food contamination in the PFAS high-risk area of the Veneto Region (Northern Italy)]. / Sostanze perfluoroalchiliche (PFAS) negli alimenti dell'area rossa del Veneto.

In a wide area among the provinces of Vicenza, Verona, and Padua (Veneto Region, Northern Italy), one of the larger known massive contamination of the environment by per- and polyfluoroalkyl substances (PFASs) occurred since the year 1965. The most important source of human exposure was through aquiferous and contaminated water supplies (the regional authorities distinguished red zone A - aquiferous - and red zone B - water supply) for a total of slightly less than two hundred thousand exposed people. Food contamination and food contribution to total human exposure were assessed by a food monitoring campaign on the years 2016-2017 by the National Health Institute sponsored by Veneto Region.Thanks to the availability of the individual records of the monitoring campaign, we evaluated the spatial distribution of PFAS contamination by food matrix. Generally speaking, PFAS contamination was widespread over the entire red zone (either A or B). Some zones appeared to be higher contaminated consistently with the water plume. Other higher contaminated zones were less consistent and such result raises questions about routes of environmental pollution.

TTF-1/Nkx2.1 functional connection with mutated EGFR relies on LRIG1 and ß-catenin pathways in lung cancer cells.

In non-small lung cancer, the expression of the transcription factor TTF-1/Nkx2.1 correlates with the presence of EGFR mutations, therefore TTF-1/Nkx2.1 expression is used to optimize an EGFR testing strategy and to guide clinical treatment. We investigate the molecular mechanisms underlying the functional connection between EGFR and TTF-1/Nkx2.1 gene expression in lung adenocarcinoma. Using the H1975 cell line as a non-small cell lung cancer model system and short hairpin RNA, we have selected clones with TTF-1/Nkx2.1 silenced expression. We have found that Leucine-rich immunoglobulin repeats-1 (LRIG1) gene is a direct target of TTF-1/Nkx2.1 and the transcription factor binding to the LRIG1 genomic sequence inhibits its gene expression. In TTF-1/Nkx2.1 depleted clones, we have found high levels of LRIG1 and decreased presence of EGFR protein. Furthermore, in TTF-1/Nkx2.1 depleted clones we detected a reduced ß-catenin level and we provide experimental evidence indicating that TTF-1/Nkx2.1 gene expression is regulated by ß-catenin. Published studies indicate that LRIG1 triggers EGFR degradation and that mutated EGFR induces ß-catenin activity. Hence, with the present study we show that mutated EGFR, enhancing ß-catenin, stimulates TTF-1/Nkx2.1 gene expression and, at the same time, TTF-1/Nkx2.1, down-regulating LRIG1, sustains EGFR pathway. Therefore, LRIG1 and ß-catenin mediate the functional connection between TTF-1/Nkx2.1 and mutated EGFR.

Analysis of random PCR-originated mutants of the yeast Ste2 and Ste3 receptors.

The G protein-coupled receptors Ste2 and Ste3 bind α- and a-factor, respectively, in Saccharomyces cerevisiae. These receptors share a similar conformation, with seven transmembrane segments, three intracellular loops, a C-terminus tail, and three extracellular loops. However, the amino acid sequences of these two receptors bear no resemblance to each other. Coincidently the two ligands, α- and a-factor, have different sequences. Both receptors activate the same G protein. To identify amino acid residues that are important for signal transduction, the STE2 and STE3 genes were mutagenized by a random PCR-based method. Mutant receptors were analyzed in MATα cells mutated in the ITC1 gene, whose product represses transcription of a-specific genes in MATα. Expression of STE2 or STE3 in these cells results in autocrine activation of the mating pathway, since this strain produces the Ste2 receptor in addition to its specific ligand, α-factor. It also produces a-factor in addition to its specific receptor, Ste3. Therefore, this strain provides a convenient model to analyze mutants of both receptors in the same background. Many hyperactive mutations were found in STE3, whereas none was detected in STE2. This result is consistent with the different strategies that the two genes have adopted to be expressed.

Functional characterization of two novel mutations in TTF-1/NKX2.1 homeodomain in patients with benign hereditary chorea.

The thyroid transcription factor 1 (TTF-1) is encoded, on chromosome 14q13, by the gene termed TITF-1/NKX2.1. Mutations in this gene have been associated with chorea, hypothyroidism, and lung disease, all included in the "brain-thyroid-lung syndrome." We here describe two cases of novel missense mutations [NM_003317.3:c.516G>T and c.623G>C resulting in p.(Gln172His) and p.(Trp208Ser), respectively] in TITF-1/NKX2-1 in non-consanguineous patients. We provide a functional study of the role of the two mutations on the TTF-1 ability to bind DNA and to trans-activate both thyroid and lung specific gene promoters. Our results confirm the difficulty to correlate the TTF-1 activity with the clinical phenotype of affected patients and highlight the need to increase the limited knowledge we have on the activity of TTF-1 in neuronal cells.

Yeast pheromone receptor genes STE2 and STE3 are differently regulated at the transcription and polyadenylation level.

The orderly expression of specific genes is the basis for cell differentiation. Saccharomyces cerevisiae has two haploid mating types, a and α cells, in which the mating-specific genes are differentially expressed. When a and α cells are committed to mate, their growth is arrested. Here we show that a cryptic polyadenylation site is present inside the coding region of the a-specific STE2 gene, encoding the receptor for the α-factor. The two cell types produce an incomplete STE2 transcript, but only a cells generate full-length STE2 mRNA. We eliminated the cryptic poly(A) signal, thereby allowing the production of a complete STE2 mRNA in α cells. We mutagenized α cells and isolated a mutant producing full-length STE2 mRNA. The mutation occurred in the ITC1 gene, whose product, together with the product of ISW2, is known to repress STE2 transcriptional initiation. We propose that the regulation of the yeast mating genes is achieved through a concerted mechanism involving transcriptional and posttranscriptional events. In particular, the early poly(A) site in STE2 could contribute to a complete shutoff of its expression in α cells, avoiding autocrine activation and growth arrest. Remarkably, no cryptic poly(A) sites are present in the a-factor receptor STE3 gene, indicating that S. cerevisiae has devised different strategies to regulate the two receptor genes. It is predictable that a correlation between the repression of a gene and the presence of a cryptic poly(A) site could also be found in other organisms, especially when expression of that gene may be harmful.

Splicing of mRNA mediated by tRNA sequences in mouse cells.

tRNA splicing is essential for the formation of tRNAs and therefore for gene expression. A circularly permuted sequence of an amber-suppressor pre-tRNA gene was inserted into the sequence encoding the mouse NEMO protein. We demonstrated that, in mouse cells, the hybrid pre-tRNA/pre-mRNAs can be spliced precisely at the sites of the pre-tRNA intron. This splicing reaction produces functional tRNAs that suppress amber codons as well as translatable mRNAs that sustain the NF-kappaB activation pathway. The RNA molecules extracted from mouse cells were amplified by RT-PCR, and their sequences were determined, confirming the identity of the splice junctions. We then applied the Archaea-express technology, in which an archaeal RNA endonuclease is expressed in mouse cells. We show that both the endogenous eukaryal endonuclease and the archaeal one cleave the hybrid pre-tRNA/pre-mRNAs in the same manner with an additive effect.

Exonucleolytic degradation of double-stranded RNA by an activity in Xenopus laevis germinal vesicles.

We have identified, in extracts from Xenopus laevis germinal vesicles, a 5' exonuclease activity that cleaves double-stranded RNA (dsRNA). Features of the 5' ends of dsRNAs determine whether the strands are symmetrically or asymmetrically degraded. The activity hydrolyzes in the 5' to 3' direction, releasing 5'-mononucleotides processively, favoring strands with 5'-monophosphate termini; molecules with capped ends are resistant to digestion. Because of its ability to processively digest dsRNA to mononucleotides, we have named the exonuclease Chipper, which could cooperate or compete with Dicer (an endonuclease that produces molecules with a 5'-phosphate) in the processing of dsRNA.