In silico prediction of the interaction of legacy and novel per- and poly-fluoroalkyl substances (PFAS) with selected human transporters and of their possible accumulation in the human body.

Per- and poly-fluorinated compounds constitute a wide group of fluorocarbon chemicals with widespread industrial applications, ranging from non-stick coating in cookware to water surfactants, from fire-fighting foams to water-repellent coatings on textiles. Presently, over 12,000 PFAS are known worldwide. In recent years, extensive research has focused on investigating the biological effects of these molecules on various organisms, including humans. Here, we conducted in silico simulations to examine the potential binding of a representative selection of PFAS to various human proteins known to be involved in chemical transportation and accumulation processes. Specifically, we targeted human serum albumin (HSA), transthyretin (TTR), thyroxine binding protein (TBG), fatty acid binding proteins (FABPs), organic anion transporters (OATs), aiming to assess the potential for bioaccumulation. Molecular docking simulations were employed for this purpose, supplemented by molecular dynamics (MD) simulations to account for protein flexibility, when necessary. Our findings indicate that so-called "legacy PFAS" such as PFOA or PFOS exhibit a higher propensity for interaction with the analysed human protein targets compared to newly formulated PFAS, characterised by higher branching and hydrophilicity, and possibly a higher accumulation in the human body.

Difficulties in establishing a causal link between chemical exposures and cancer cannot be overcome by court assessments.

Scientific data are often used in lawsuits to prove, or dismiss, causation by a claimed factor of a claimed disease. Recent media reports of million-dollar compensations awarded to some cancer patients who had been exposed to certain chemical substances motivated me to examine how solid the causal links really were. Here, I discuss the limitations of epidemiological research on cancer causation and highlight how new knowledge of cancer genetics makes it unrealistic to expect that cancer causation can be clearly demonstrated. I then present two exposure-cancer cases, namely talcum powder-ovarian cancer and glyphosate-non-Hodgkin lymphoma, that led to civil lawsuits decided, in the United States, in favor of the claimants. Both these cancers have several risk factors, among which the claimed exposure presents only a minor, if any, increased risk. Through these cases, I explain why the use of epidemiological data is inappropriate to define causal associations in complex diseases like cancer. I close by suggesting a fairer approach, called proportional liability, to resolving future cancer litigation cases.

Are strong opioids equally effective and safe in the treatment of chronic cancer pain? A multicenter randomized phase IV 'real life' trial on the variability of response to opioids.

BACKGROUND: Guidelines tend to consider morphine and morphine-like opioids comparable and interchangeable in the treatment of chronic cancer pain, but individual responses can vary. This study compared the analgesic efficacy, changes of therapy and safety profile over time of four strong opioids given for cancer pain. PATIENT AND METHODS: In this four-arm multicenter, randomized, comparative, of superiority, phase IV trial, oncological patients with moderate to severe pain requiring WHO step III opioids were randomly assigned to receive oral morphine or oxycodone or transdermal fentanyl or buprenorphine for 28 days. At each visit, pain intensity, modifications of therapy and adverse drug reactions (ADRs) were recorded. The primary efficacy end point was the proportion of nonresponders, meaning patients with worse or unchanged average pain intensity (API) between the first and last visit, measured on a 0-10 numerical rating scale. (NCT01809106). RESULTS: Forty-four centers participated in the trial and recruited 520 patients. Worst pain intensity and API decreased over 4 weeks with no significant differences between drugs. Nonresponders ranged from 11.5% (morphine) to 14.4% (buprenorphine). Appreciable changes were made in the treatment schedules over time. Each group required increases in the daily dose, from 32.7% (morphine) to 121.2% (transdermal fentanyl). Patients requiring adjuvant analgesics ranged from 68.9% (morphine) to 81.6% (oxycodone), switches varied from 22.1% (morphine) to 12% (oxycodone), discontinuation of treatment from 27% ( morphine) to 14.5% (fentanyl). ADRs were similar except for effects on the nervous system, which significantly prevailed with morphine. CONCLUSION: The main findings were the similarity in pain control, response rates and main adverse reactions among opioids. Changes in therapy schedules were notable over time. A considerable proportion of patients were nonresponders or poor responders. CLINICAL TRIAL REGISTRATION: NCT01809106 (https://clinicaltrials.gov/ct2/show/NCT01809106?term=cerp&rank=2).

A subset of genetic susceptibility variants for colorectal cancer also has prognostic value.

We investigated the possible influence of 86 single-nucleotide polymorphisms (SNPs), known to associate with the risk of colorectal cancer (CRC), on overall survival and time to recurrence (TTR) in 733 Italian CRC patients followed up for up to 84 months after surgery. In the Cox multivariate analysis, adjusted for gender, age, pathological stage and adjuvant chemotherapy (yes/no), the risk of death significantly increased by rare allele count (P<0.05) for rs1801133 (MTHFR), rs4939827 (SMAD7), rs2306283 (SLCO1B1) and rs12898159 (BMP4), whereas for rs736775 (GPX3) the opposite was observed. Two additional SNPs associated with TTR, namely rs16892766 (downstream of EIF3H) and rs10749971 (COLCA2). Our findings show that some genetic variants previously found to associate with CRC risk are also associated with survival after treatment. The identification of alleles defining subgroups of patients with worse clinical outcome may have application in developing pharmacogenetic strategies aimed at personalizing CRC treatment.

Genetic linkage analysis identifies Pas1 as the common locus modulating lung tumorigenesis and acute inflammatory response in mice.

Selective breeding for the acute inflammatory response (AIR) generated two mouse lines characterized by maximum (AIRmax) and minimum (AIRmin) responses, explained by the additive effect of alleles differentially fixed in quantitative trait loci (QTLs). These mice also differ in their susceptibility to lung tumorigenesis, raising the possibility that the same loci are involved in the control of both phenotypes. To map the QTLs responsible for the different phenotypes, we carried out a genome-wide linkage analysis using single-nucleotide polymorphism arrays in a pedigree consisting of 802 mice, including 693 (AIRmax × AIRmin)F2 intercross mice treated with urethane and phenotyped for AIR and lung tumor multiplicity. We mapped five loci on chromosomes 4, 6, 7, 11 and 13 linked to AIR (logarithm of odds (LOD)=3.56, 3.52, 15.74, 7.74 and 3.34, respectively) and two loci linked to lung tumor multiplicity, on chromosomes 6 and 18 (LOD=12.18 and 4.69, respectively). The known pulmonary adenoma susceptibility 1 (Pas1) locus on chromosome 6 was the only locus linked to both phenotypes, suggesting that alleles of this locus were differentially fixed during breeding and selection of AIR mice. These results represent a step toward understanding the link between inflammation and cancer.

Genetic clustering of European cancer patients indicates that opioid-mediated pain relief is independent of ancestry.

The European Pharmacogenetic Opioid Study (EPOS) of a large series of European cancer patients treated with opioids was carried out to assess the influence of genetics on cancer pain relief. As response to opioid therapy was associated with the patients' country of origin, we tested whether population stratification might represent a confounding factor in the analysis of genetic control of response to opioid therapy. From the whole EPOS series representing 2294 patients' genotypes for 379 single-nucleotide polymorphisms (SNPs), we extracted 117 autosomal SNPs with minor allele frequency>0.28 to obtain highly informative genetic markers, and analyzed the SNPs in 1724 individuals showing <20% missing genotypes. Use of the AWclust program to detect clusters of genetically related individuals in the EPOS series showed that the 117-SNP panel distinguished four main European subgroups statistically associated with ethnicity, but not with country of origin or with the pain relief phenotype. Subethnic European groups of genetically related individuals exist that can be correctly identified using an ∼100-SNP panel. Such genetic clustering may control for admixture in association studies and may allow discrimination between genetic and environmental effects on phenotypes showing association with country of origin, as in the case of pain relief.

Association study by genetic clustering detects multiple inflammatory response loci in non-inbred mice

We tested the possibility to map loci affecting the acute inflammatory response (AIR) in an (AIRmax AIRmin) F2 intercrossmouse population derived from non-inbred parents, by association analysis in the absence of pedigree information. Using 1064 autosomal single nucleotide polymorphisms (SNPs), we clustered the intercross population into 12 groups of genetically related individuals. Association analysis adjusted for genetic clusters allowed to identify two loci, inflammatory response modulator 1 (Irm1) on chromosome 7 previously detected by genetic linkage analysis in the F2 mice, and a new locus onchromosome 5 (Irm2), linked to the number of infiltrating cells in subcutaneous inflammatory exudates (Irm1: P»6.3 10 7; Irm2: P»8.2 10 5) and interleukin 1 beta (IL-1b) production (Irm1: P»1.9 10 16; Irm2: P»1.1 10 6). Use of a polygenic model based on additive effects of the rare alleles of 15 or 18 SNPs associated at suggestive genome-wide statistical threshold(Po3.4 10 3) with the number of infiltrating cells or IL-1b production, respectively, allowed prediction of the inflammatory response of progenitor AIR mice. Our findings suggest the usefulness of association analysis in combination with genetic clustering to map loci affecting complex phenotypes in non-inbred animal species.

Association study by genetic clustering detects multiple inflammatory response loci in non-inbred mice.

We tested the possibility to map loci affecting the acute inflammatory response (AIR) in an (AIRmax × AIRmin) F2 intercross mouse population derived from non-inbred parents, by association analysis in the absence of pedigree information. Using 1064 autosomal single nucleotide polymorphisms (SNPs), we clustered the intercross population into 12 groups of genetically related individuals. Association analysis adjusted for genetic clusters allowed to identify two loci, inflammatory response modulator 1 (Irm1) on chromosome 7 previously detected by genetic linkage analysis in the F2 mice, and a new locus on chromosome 5 (Irm2), linked to the number of infiltrating cells in subcutaneous inflammatory exudates (Irm1: P=6.3 × 10(-7); Irm2: P=8.2 × 10(-5)) and interleukin 1 beta (IL-1ß) production (Irm1: P=1.9 × 10(-16); Irm2: P=1.1 × 10(-6)). Use of a polygenic model based on additive effects of the rare alleles of 15 or 18 SNPs associated at suggestive genome-wide statistical threshold (P<3.4 × 10(-3)) with the number of infiltrating cells or IL-1ß production, respectively, allowed prediction of the inflammatory response of progenitor AIR mice. Our findings suggest the usefulness of association analysis in combination with genetic clustering to map loci affecting complex phenotypes in non-inbred animal species.

Cis-acting genomic elements of the Pas1 locus control Kras mutability in lung tumors.

The Pas1 locus is the major tumor modifier of lung tumorigenesis in mouse inbred strains. Of six genes contained in a conserved haplotype, three (Casc1, Kras and Ifltd1) have been proposed as Pas1 candidates, but mechanistic evidence is sparse. Herein, we examined urethane-induced lung tumorigenesis in a new mouse model developed by replacing the Kras gene with an Hras gene in the susceptible A/J-type Pas1 locus and crossing these mice with either C57BL/6J or A/J mice. Heterozygous mice carrying the Hras-replacement gene were more susceptible than wild-type mice to lung carcinogenesis, indicating that Hras replacement not only compensates for Kras functions, but is more active. Indeed, most lung tumors carried a Gln61Leu mutation in the Hras-replacement gene, whereas no mutations were observed in the endogenous Hras gene. Thus, the context of the Kras locus determined mutability of ras genes. In mice carrying the Hras-replacement gene, the mutation frequency affecting the wild-type Kras gene was much higher when this gene was located in the A/J type than in the C57BL/6J-type Pas1 locus (12 versus 0%, -log P=5.0). These findings identify cis-acting elements in the Pas1 locus as the functional components controlling genetic susceptibility to lung tumorigenesis by modulating mutability of the Kras gene.

BHLHB3: a candidate tumor suppressor in lung cancer.

BHLHB3 is a basic helix-loop-helix (bHLH) domain-containing protein that acts as a transcriptional repressor. We found that BHLHB3 transcript levels were low in three human lung cancer cell lines and downregulated in human lung adenocarcinomas as compared to normal lung tissue. BHLHB3 gene overexpression inhibited colony formation of A549, NCI-H520 and NCI-H596 lung cancer cells. The reduced colony growth was likely due to inhibition of cell proliferation as suggested by the downregulation of cyclin D1 (CCND1) expression in NCI-H520 cells transfected to overexpress the BHLHB3 gene; no evidence of apoptosis was observed. These results point to the potential role of the BHLHB3 protein as a tumor suppressor for lung cancer.

AZGP1 mRNA levels in normal human lung tissue correlate with lung cancer disease status.

Evidence in animal models has suggested an association between susceptibility to lung tumorigenesis and gene-expression profiles in normal lung. Here, we compared RNA pools from normal lung tissue of lung adenocarcinoma patients (cases) or non-lung cancer patients (controls) by hybridization of whole-human genome expression arrays. Principal component analysis identified a gene-expression signature of 85 genes that distinguishes cases from controls as well as smokers from nonsmokers. Elevated mRNA levels of one of these genes, AZGP1, were significantly associated with disease status. These results support the hypothesis that differences in the gene-expression levels of the normal tissue may be predictive of genetic predisposition to lung cancer in humans.

Specific gene expression profiles distinguish among functional allelic variants of the mouse Pthlh gene in transfected human cancer cells.

The mouse parathyroid hormone-like hormone (Pthlh) gene encodes three allelic variants characterized by amino acid substitutions that are associated with susceptibility (Pthlh(Pro)) or resistance (Pthlh(Thr) and Pthlh(SerAspTyr)) to two-stage skin carcinogenesis and to modulation of cell migration in vitro in transfected human cancer cells. cDNA microarray hybridization analysis of 8473 transcript clones revealed a similar gene expression profile for the Pthlh(Thr) and Pthlh(SerAspTyr) alleles but a distinct pattern for the Pthlh(Pro) allele, suggesting an association between a specific gene expression profile and biological function of the Pthlh alleles. Some of the genes modulated by the Pthlh alleles, e.g., ANXA1, CCL2, FN1 and TFF3, play a role in cell migration and may represent candidate targets for this Pthlh function. Our study demonstrates the potential usefulness of gene expression profiling of genetic variants for the functional characterization of candidate cancer modifier genes.

Identification of RASSF8 as a candidate lung tumor suppressor gene.

The RASSF8 gene, which maps close to the KRAS2 gene, contains a RAS-associated domain and encodes a protein that is evolutionarily conserved from fish to humans. Analysis of the RASSF8 transcript revealed a complex expression pattern of 5'-UTR mRNA isoforms in normal lung and in lung adenocarcinomas (ADCAs), with no apparent differences. However, RASSF8 gene transcript levels were approximately seven-fold-lower in lung ADCAs as compared to normal lung tissue. Expression of RASSF8 protein by transfected lung cancer cells led to inhibition of anchorage-independent growth in soft agar in A549 cells and reduction of clonogenic activity in NCI-H520 cells. These results raise the possibility protein encoded by RASSF8 is a novel tumor suppressor for lung cancer.

A new polymorphism (Ser362Thr) of the L-myc gene is not associated with lung adenocarcinoma risk and prognosis.

The non-coding variation in the second intron of the L-myc gene, generating an EcoRI polymorphism, is associated with lung cancer risk and prognosis. We carried out sequence analysis of the L-myc gene in lung adenocarcinoma (ADCA) patients to identify functional polymorphisms and identified a new single nucleotide polymorphism (SNP) in the third exon of the gene causing a Ser362Thr conservative amino acid change in the C-terminus of the encoded protein. This polymorphism showed significant linkage disequilibrium with the L-myc EcoRI polymorphism located at 1751 bp distance. Genotyping of the Ser362Thr SNP in 220 Italian ADCA patients and in 230 general population controls revealed a similar low frequency (0.10-0.11) of the Thr allele in both groups. The multivariate odds ratio was 0.68 (95% confidence interval (CI) 0.38-1.22). In the ADCA patients, no significant association between the Ser/Thr polymorphism and survival was observed. Thus, the present results do not support candidacy of the L-myc Ser362Thr polymorphism for the functional polymorphism of the L-myc genomic region.

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

Linkage disequilibrium pattern in the L-myc gene in Italian and Japanese non-small-cell lung-cancer patients.

Italian and Japanese non-small-cell lung-cancer patients were genotyped for an intragenic L-myc EcoRI restriction site polymorphism previously reported to be associated with lung-tumor prognosis in Asian populations but not in Caucasians. Screening of the L-myc sequence in Italian samples allowed identification of 2 additional 3'-UTR SNPs, located 2.3-3.0 kb from the EcoRI polymorphism, but no coding polymorphism was found. No significant association was found between any of the 3 SNPs and lung-tumor prognosis in Italian patients, consistent with the reported difference between Caucasian and Asian populations. Moreover, the newly discovered polymorphisms in the Italian group were not present in Japanese patients. Significant LD between EcoRI and the 2 other SNPs was detected in the Italian population, whereas no significant LD between the 2 3'-UTR markers was detected despite their close proximity (0.7 kb). Thus, the disparate conclusions about the role of L-myc polymorphism in tumor prognosis among different populations may rest in population-specific LD between the functional gene and the L-myc polymorphism.

Use of intercross outbred mice and single nucleotide polymorphisms to map skin cancer modifier loci.

Car-R and Car-S outbred mouse lines, phenotypically selected for resistance and susceptibility to skin carcinogenesis respectively, show significant linkage disequilibrium (LD) at genetic markers mapping on chromosomal regions where skin cancer modifier loci (Skts3, Skts1, and Psl1 on Chrs 5, 7, and 9 respectively) have been mapped in standard crosses. Analysis of these regions for genetic linkage with skin cancer phenotypes in 245 (Car-R x Car-S)F2 intercross mice, by using single nucleotide polymorphisms (SNPs), revealed significant linkage at a possible allelic form of the Skts1 locus, whose mapping region was shortened to a <5.5-cM interval near the Tyr locus. The Car-derived Skts1 locus was linked with papilloma multiplicity and latency by a recessive inheritance of the susceptibility allele. Putative loci on Chr 5 (Skts3) and 9 (Psl1) showed no significant linkage. These results point to the important role of the Stks1 locus in mouse skin tumorigenesis in independent crosses. The shortened Skts1 mapping region should facilitate the identification of candidate genes.

A cancer modifier role for parathyroid hormone-related protein.

The parathyroid hormone-related protein (PTHrP) gene (Pthlh) maps in the distal region of mouse chromosome 6 that contains a quantitative trait locus associated with genetic predisposition to skin tumorigenesis. Here, we report a genetic polymorphism located in the osteostatin encoding region of the Pthlh gene and that produces Thr/ Pro PTHrP variants. PthlhThr and PthlhPro alleles were significantly linked with resistance and susceptibility to skin carcinogenesis in phenotypically selected Car-R and Car-S outbred mice. Transfection of human NCI-H520 squamous cell carcinoma cells with the PthlhPro allele resulted in cells growing in clusters, tending to pile up, and growing at a significantly faster rate in nude mice than non-transfected and PthlhThr-transfected cells. These results point to the role of the Pthlh gene as a cancer modifier gene in skin tumorigenesis.

Linkage disequilibrium and haplotype mapping of a skin cancer susceptibility locus in outbred mice.

Car-R (carcinogenesis-resistant) and Car-S (carcinogenesis-susceptible) outbred mice, obtained by phenotypic selection from an initial intercross of eight inbred strains, show a >100-fold difference in their susceptibility to two-stage skin tumorigenesis. We found that the lines carry a high degree of genetic polymorphism. with an average heterozygosity of 0.39. This polymorphism allowed the use of linkage disequilibrium (LD) and haplotype analysis for the mapping of a skin cancer modifier locus on Chr 7, in a short region of 6 cM, around the Tyr gene. Car-S mice inherited the susceptibility allele at this locus from the A/J, BALB/c, SJL/J, and SWR/J strains. Our results demonstrate the feasibility and usefulness of mapping disease genes by LD in phenotypically selected, genetically heterogeneous animals.

Mapping of melanoma modifier loci in RET transgenic mice.

Transgenic mice carrying the RET oncogene under the control of the metallothionein promoter exhibit severe pigmentation of the whole skin and melanocytic tumors. The genetic background influences melanoma development in RET mice; founder mice crossed with BALB/c mice show decreased incidence and increased latency of melanocytic tumors, whereas progeny of C57BL/6 mice show the opposite effect. Using partially congenic RET mice on a C57BL/6 genetic background (N3/RET mice), we studied genetic linkage in (N3/RETxBALB/c)xN3/RET backcross mice. We mapped three melanoma modifier loci, on chromosome 1 (Melm1 and Melm2) and chromosome 11 (Melm3), that are linked with early melanoma incidence and latency. Mapping of Melm loci and of five additional regions on chromosomes 6, 8, 9, 12, and 13 indicated allelic imbalance in N3/RET mice, with a significant excess of BALB/c alleles, suggesting the presence of additional putative melanoma modifier loci on these chromosomes.