Characterization and toxicity evaluation of air-borne particles released by grinding from two dental resin composites in vitro.

OBJECTIVE: The project aims to evaluate whether inhalation of particles released upon grinding of dental composites may pose a health hazard to dentists. The main objective of the study was to characterize the dust from polymer-based dental composites ground with different grain sized burs and investigate particle uptake and the potential cytotoxic effects in human bronchial cells. METHODS: Polymerized blocks of two dental composites, Filtek™ Z250 and Filtek™ Z500 from 3M™ ESPE, were ground with super coarse (black) and fine (red) burs inside a glass chamber. Ultrafine airborne dust concentration and particle size distribution was measured real-time during grinding with a scanning mobility particle sizer (SMPS). Filter-collected airborne particles were characterized with dynamic light scattering (DLS) and scanning electron microscopy (SEM). Human bronchial epithelial cells (HBEC-3KT) were exposed to the dusts in dose-effect experiments. Toxicity was measured with lactate dehydrogenase (LDH) assay and cell counting kit-8 (CCK8). Cellular uptake was observed with transmission electron microscopy (TEM). RESULTS: Airborne ultrafine particles showed that most particles were in the size range 15-35 nm (SMPS). SEM analysis proved that more than 80% of the particles have a minimum Feret diameter less than 1 µm. In solution (DLS), the particles have larger diameters and tend to agglomerate. Cell toxicity (LDH, CCK8) is shown after 48 h and 72 h exposure times and at the highest doses. TEM showed presence of the particles within the cell cytoplasm. SIGNIFICANCE: Prolonged and frequent exposure through inhalation may have negative health implications for dentists.

A rare missense mutation in CHRNA4 associates with smoking behavior and its consequences.

Using Icelandic whole-genome sequence data and an imputation approach we searched for rare sequence variants in CHRNA4 and tested them for association with nicotine dependence. We show that carriers of a rare missense variant (allele frequency=0.24%) within CHRNA4, encoding an R336C substitution, have greater risk of nicotine addiction than non-carriers as assessed by the Fagerstrom Test for Nicotine Dependence (P=1.2 × 10(-4)). The variant also confers risk of several serious smoking-related diseases previously shown to be associated with the D398N substitution in CHRNA5. We observed odds ratios (ORs) of 1.7-2.3 for lung cancer (LC; P=4.0 × 10(-4)), chronic obstructive pulmonary disease (COPD; P=9.3 × 10(-4)), peripheral artery disease (PAD; P=0.090) and abdominal aortic aneurysms (AAAs; P=0.12), and the variant associates strongly with the early-onset forms of LC (OR=4.49, P=2.2 × 10(-4)), COPD (OR=3.22, P=2.9 × 10(-4)), PAD (OR=3.47, P=9.2 × 10(-3)) and AAA (OR=6.44, P=6.3 × 10(-3)). Joint analysis of the four smoking-related diseases reveals significant association (P=6.8 × 10(-5)), particularly for early-onset cases (P=2.1 × 10(-7)). Our results are in agreement with functional studies showing that the human α4ß2 isoform of the channel containing R336C has less sensitivity for its agonists than the wild-type form following nicotine incubation.

Molecular characterization of a cancer-related single nucleotide polymorphism in the pro-inflammatory interleukin-1B gene.

Interleukin-1ß is a key pro-inflammatory cytokine that has been associated with chronic inflammation and inflammation-related cancer initiation and progression. There are inter-individual differences in IL1B expression which may be due to single nucleotide polymorphisms (SNPs) in the regulatory regions of the gene. We have previously shown that a SNP located in the promoter of the IL1B gene (the IL1B T-31C SNP) was associated with lung cancer risk. Interestingly, the presence of the C allele was also associated with reduced IL1B expression in normal lung tissue of lung cancer patients. In the present study, we found that differential binding patterns of nuclear proteins to oligonucleotide probes containing the IL1B -31C allele compared to those with the T allele were due to specific binding of the transcription factor Yin Yang 1 (YY1). We further found evidence that specific recruitment of YY1 to the -31C region of the IL1B promoter regulated IL1B gene expression using siRNA directed towards YY1. The results indicate that the presence of a C allele at the -31 position may lead to decreased expression of the IL1B gene due to a specific binding of YY1 in lung epithelial cells. Our study provides functional significance of allelic variation at a single locus in the IL1B promoter and contributes to understanding the regulation of IL1B in inflammation-related carcinogenesis.

Loss of heterozygosity is related to p53 mutations and smoking in lung cancer.

Carcinogenesis results from an accumulation of several genetic alterations. Mutations in the p53 gene are frequent and occur at an early stage of lung carcinogenesis. Loss of multiple chromosomal regions is another genetic alteration frequently found in lung tumours. We have examined the association between p53 mutations, loss of heterozygosity (LOH) at frequently deleted loci in lung cancer, and tobacco exposure in 165 tumours from non-small cell lung cancer (NSCLC) patients. A highly significant association between p53 mutations and deletions on 3p, 5q, 9p, 11p and 17p was found. There was also a significant correlation between deletions at these loci. 86% of the tumours with concordant deletion in the 4 most involved loci (3p21, 5q11-13, 9p21 and 17p13) had p53 mutations as compared to only 8% of the tumours without deletions at the corresponding loci (P< 0.0001). Data were also examined in relation to smoking status of the patients and histology of the tumours. The frequency of deletions was significantly higher among smokers as compared to non-smokers. This difference was significant for the 3p21.3 (hMLH1 locus), 3p14.2 (FHIT locus), 5q11-13 (hMSH3 locus) and 9p21 (D9S157 locus). Tumours with deletions at the hMLH1 locus had higher levels of hydrophobic DNA adducts. Deletions were more common in squamous cell carcinomas than in adenocarcinomas. Covariate analysis revealed that histological type and p53 mutations were significant and independent parameters for predicting LOH status at several loci. In the pathogenesis of NSCLC exposure to tobacco carcinogens in addition to clonal selection may be the driving force in these alterations.

Nickel(II) induces microsatellite mutations in human lung cancer cell lines.

Nickel(II) is a human carcinogen causing respiratory cancers. The purpose of this study was to determine whether Ni(II) may induce microsatellite mutations in human cells. We transfected the three human lung tumor cell lines A427, HCC15 and NCI-H2009 with a mammalian expression vector containing a (CA)(13) repeat in the coding sequences of the reporter hygromycin gene (hyg). A total of 33 clones carrying the integrated vector derived from the three cell lines was investigated for spontaneous and Ni(II)-induced hygromycin-resistant (hyg(r)) reversion mutants. Significantly higher frequencies of hyg(r) reversion mutations were observed in Ni(II)-treated cells (NCI-H2009 and HCC-15) than control cells. In the majority of the colonies hyg(r) phenotype was due to mutations within the integrated (CA) repeat sequence. The type of mutations consisted of both contraction and expansion of the (CA) repeat unit. The finding that Ni(II) promotes microsatellite mutations raises the possibility that genetic instability may be a mechanism involved in nickel carcinogenesis.

Induction of microsatellite mutations by oxidative agents in human lung cancer cell lines.

Genomic instability has been associated with cancer development. Oxidative DNA damage seems to contribute to genetic instability observed in cancer. We have used human lung cancer cell lines carrying a plasmid vector containing a (CA)(13) microsatellite sequence to study frameshift mutations mediated by ROS-generating chemicals paraquat and hydrogen peroxide. Exposure of the cells to both paraquat and hydrogen peroxide resulted in significantly higher mutation frequencies compared with untreated control cells. Mutation frequencies up to 27-fold higher than the spontaneous mutation frequencies were obtained. The majority of the reversion mutants contained frameshift mutations within the target sequence. However, the pattern of deletions and additions was significantly different in the two cell lines. These results indicate that oxidative damage may play a role in instability of microsatellite sequences in vivo.

Gene-environment interactions in human lung cancer.

Gene-environment interactions are thought to be critical for several diseases such as cancer, diabetes, heart disease and asthma. Cancer is a result of multiple gene-environment interactions occurring over several decades. During tumor development the cell accumulates multiple genetic changes, which generate the transformed phenotype, i.e. a cell with increased genetic instability. Lung cancer is a useful model for the study of the interplay between genetic factors and environmental exposure since the primary etiology is well established. Several polymorphic enzymes that may be important determinants of susceptibility have been demonstrated. Data also provide evidence for sex differences in lung cancer susceptibility. Furthermore, certain chemical carcinogens may contribute to the carcinogenic process in the lung epithelial cells by inducing genomic instability either directly or indirectly through inflammatory processes.

DNA mismatch binding in human lung tumor cell lines.

Defects in mismatch repair (MMR) genes have been involved in several types of sporadic and hereditary cancers. In order to elucidate the role of MMR in human lung carcinogenesis we examined DNA mismatch binding in cell-free extracts of seven lung tumor cell lines and five corresponding lymphoblastoid cell lines from lung cancer patients. Using the technique of bandshift assay we have demonstrated that 2/7 of the tumor cell lines are aberrant in binding to specific DNA mismatches while all lymphoblastoid cell lines were proficient in binding to all tested mismatches. Both extracts were aberrant in binding to G/T mismatch whereas one of the cell lines showed deficiency in binding to the C:A mismatches as well. Immunoblotting analysis showed that all known DNA mismatch repair (MMR) proteins were present in these extracts. The cell line deficient in binding to both G:T and C:A mismatches showed microsatellite instability (MSI) in tumor DNA and higher resistance to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This report indicates that DNA mismatch binding deficiencies may be implicated in at least a subgroup of human lung cancer.

Hras1 VNTR alleles as susceptibility markers for lung cancer: relationship to microsatellite instability in tumors.

PURPOSE: To further evaluate lung cancer risk associated with rare Hras1 VNTR alleles and possible biological mechanisms. MATERIALS AND METHODS: The Hras1 VNTR was genotyped in 295 lung cancer patients and 500 healthy controls by PCR and high resolution electrophoresis. Microsatellite alterations were examined in 168 tumors by PCR and capillary electrophoresis. RESULTS: 35 Hras1 VNTR alleles were found, of which 24 were defined as rare. A relative risk of 3.3 (95% CI; 1.9-6.0) associated with rare alleles was obtained using the total groups. Increased risk was significant both for males and females. When a matched control group was used, a relative risk of 12.7 (95% CI; 1.7-93.9) was calculated for individuals with rare alleles at the Hras1 VNTR locus. A low frequency of microsatellite alterations was observed (4.7%) in lung tumors. The frequency of altered microsatellite loci was higher among patients with rare Hras1 VNTR alleles than among patients with common alleles. CONCLUSION: Rare Hras1 VNTR alleles are associated with lung cancer risk, and a genetic mechanism which increases allelic diversity may be involved.

A hereditary genetic marker closely associated with microsatellite instability in lung cancer.

Alterations in 5 microsatellite loci were analyzed in tumors from 137 patients with primary non-small cell lung carcinomas that were also genotyped for the Hras1 variable number of tandem repeats (VNTR) locus. Twenty-nine patients (21%) had changes in at least one microsatellite locus. A majority of these cases (24 of 29, 83%) had VNTR alleles classified as rare in the population. The frequency of these rare alleles were significantly higher among lung cancer patients than in healthy controls (P = 0.016 or 1.80; 95% confidence interval = 1.13-2.85). Microsatellite alterations were significantly more frequent among patients with at least one rare Hras1 VNTR allele (24 of 40, 60%) compared to patients with two common alleles (5 of 97, 5%; P < 0.001 or 27.6; 95% confidence interval = 8.18-82.9). Microsatellite alterations were also more frequent among patients below 50 years of age (8 of 21, 38%) than for older patients (21 of 112, 19%).