Viability of cultured human skin cells treated with 1,6-hexamethylene diisocyanate monomer and its oligomer isocyanurate in different culture media.

The isocyanate monomer 1,6-hexamethylene diisocyanate (HDI) and one of its trimers, HDI isocyanurate, are airway and skin sensitizers contained in polyurethane paint. The toxic response of cultured skin cells to these compounds was measured by evaluating the isocyanate concentrations at which 50% of the cells die (i.e., lethal concentration 50%, LC50) because the relative toxicity of each form of HDI should be considered when exposure limits of HDI-based paints are set. By using a luminescent ATP-viability assay, we compared the cytotoxic effects of HDI monomer and HDI isocyanurate on cultured human skin cells (keratinocytes, fibroblasts, and melanocytes) after 4-h isocyanate exposures using culture media with varying levels of nutrients in order to also determine the effects of media composition on isocyanate toxicity. Before analysis, experimental wells were normalized to controls containing cells that were cultured with the same vehicle and media. The measured mean LC50 values ranged from 5 to 200 µM across the experimental conditions, in which HDI isocyanurate in protein-devoid media was the most toxic to cells, producing the lowest LC50 values. For HDI monomer, keratinocytes were the most resistant to its toxicity and melanocytes were the most susceptible. However, when exposed to HDI isocyanurate, the opposite was observed, with melanocytes being the most resilient and the keratinocytes and fibroblasts were more susceptible. Depending on the type of skin cells, dose-response data indicated that HDI isocyanurate was 2-6 times more toxic than HDI monomer when using protein-devoid media whereas HDI isocyanurate was 4-13 times more toxic than HDI monomer when protein-rich media was used. Therefore, if the protein-devoid saline medium alone were used for these experiments, then a significant under-estimation of their relative toxicities in protein-rich environments would have resulted. This difference is because HDI monomer toxicity was more attenuated by the presence of protein in the culture media than HDI isocyanurate toxicity. Thus, conclusions based on comparative toxicity studies and consequent inference applied to potential human toxicity can be affected by in vitro culture media conditions. The physiochemical difference in reactivity of the two forms of HDI to biological molecules most likely explains the observed toxicity differences and may have implications for skin penetration, adverse effects like skin sensitization, and systemic responses like asthma. Future studies are warranted to investigate differences in the biological availability, cellular toxicity, and immunologic sensitization mechanisms for HDI monomer and HDI isocyanurate.

Influence of Genetic Variance on Biomarker Levels After Occupational Exposure to 1,6-Hexamethylene Diisocyanate Monomer and 1,6-Hexamethylene Diisocyanate Isocyanurate.

We evaluated the impact of genetic variance on biomarker levels in a population of workers in the automotive repair and refinishing industry who were exposed to respiratory sensitizers 1,6-hexamethylene diisocyanate (HDI) monomer and one of its trimers, HDI isocyanurate. The exposures and respective urine and plasma biomarkers 1,6-diaminohexane (HDA) and trisaminohexyl isocyanurate (TAHI) were measured in 33 workers; and genome-wide microarrays (Affymetrix 6.0) were used to genotype the workers' single-nucleotide polymorphisms (SNPs). Linear mixed model analyses have indicated that interindividual variations in both inhalation and skin exposures influenced these biomarker levels. Using exposure values as covariates and a false discovery rate < 0.10 to assess statistical significance, we observed that seven SNPs were associated with HDA in plasma, five were associated with HDA in urine, none reached significance for TAHI in plasma, and eight were associated with TAHI levels in urine. The different genotypes for the 20 significant SNPs accounted for 4- to 16-fold changes observed in biomarker levels. Associated gene functions include transcription regulation, calcium ion transport, vascular morphogenesis, and transforming growth factor beta signaling pathway, which may impact toxicokinetics indirectly by altering inflammation levels. Additionally, in an expanded analysis using a minor allele cutoff of 0.05 instead of 0.10, there were biomarker-associated SNPs within three genes that have been associated with isocyanate-induced asthma: ALK, DOCK2, and LHPP. We demonstrate that genetic variance impacts the biomarker levels in workers exposed to HDI monomer and HDI isocyanurate and that genetics can be used to refine exposure predictions in small cohorts when quantitative personal exposure and biomarker measurements are included in the models.

Review: Endogenously Produced Volatiles for In Vitro Toxicity Testing Using Cell Lines.

Due to the ∼86,000 chemicals registered under the Toxic Substances Control Act and increasing ethical concerns regarding animal testing, it is not economically or technically feasible to screen every registered chemical for toxicity using animal-based toxicity assays. To address this challenge, regulatory agencies are investigating high-throughput screening in vitro methods to increase speed of toxicity testing, while reducing the overall cost. One approach for rapid toxicity testing currently being investigated is monitoring of volatile emissions produced by cell lines in culture. Such a metabolomics approach would measure gaseous emissions from a cell line and determine if such gaseous metabolites are altered upon exposure to a xenobiotic. Herein, we describe the history and rationale of monitoring endogenously produced volatiles for identification of pathologic conditions, as well as emerging applications in toxicity testing for such an approach.

The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts.

Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600­13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration: ClinicalTrials.gov NCT01087307.

DNA methylation modifies urine biomarker levels in 1,6-hexamethylene diisocyanate exposed workers: a pilot study.

DNA methylation may mediate inter-individual responses to chemical exposure and, thus, modify biomarker levels of exposure and effects. We analyzed inter-individual differences in inhalation and skin exposure to 1,6-hexamethylene diisocyanate (HDI) and urine biomarker 1,6-hexamethylene diamine (HDA) levels in 20 automotive spray-painters. Genome-wide 5-methyl cytosine (CpG) DNA methylation was assessed in each individual's peripheral blood mononuclear cells (PBMC) DNA using the Illumina 450K CpG array. Mediation analysis using linear regression models adjusted for age, ethnicity, and smoking was conducted to identify and assess the association between HDI exposure, CpG methylation, and urine HDA biomarker levels. We did not identify any CpGs common to HDI exposure and biomarker level suggesting that CpG methylation is a mediator that only partially explains the phenotype. Functional significance of genic- and intergenic-CpG methylation status was tested using protein-protein or protein-DNA interactions and gene-ontology enrichment to infer networks. Combined, the results suggest that methylation has the potential to affect HDI mass transport, permeation, and HDI metabolism. We demonstrate the potential use of PBMC methylation along with quantitative exposure and biomarker data to guide further investigation into the mediators of occupational exposure and biomarkers and its role in risk assessment.

Sequence-dependent effect of interruptions on microsatellite mutation rate in mismatch repair-deficient human cells.

Although microsatellite mutation rates generally increase with increasing length of the repeat tract, interruptions in a microsatellite may stabilize it. We have performed a direct analysis of the effect of microsatellite interruptions on mutation rate and spectrum in cultured mammalian cells. Two mononucleotide sequences (G(17) and A(17)) and a dinucleotide [(CA)(17)] were compared with interrupted repeats of the same size and with sequences of 8 repeat units. MMR-deficient (MMR(-)) cells were used for these studies to eliminate effects of this repair process. Mutation rates were determined by fluctuation analysis on cells containing a microsatellite sequence at the 5' end of an antibiotic-resistance gene; the vector carrying this sequence was integrated in the genome of the cells. In general, interrupted sequences had lower mutation rates than perfect ones of the same size, but the magnitude of the difference was dependent upon the sequence of the interrupting base(s). Some interrupted repeats had mutation rates that were lower than those of perfect sequences of the same length but similar to those of half the length. This suggests that interrupting bases effectively divide microsatellites into smaller repeat runs with mutational characteristics different from those of the corresponding full-length microsatellite. We conclude that interruptions decrease microsatellite mutation rate and influence the spectrum of frameshift mutations. The sequence of the interrupting base(s) determines the magnitude of the effect on mutation rate.

Variation in efficiency of DNA mismatch repair at different sites in the yeast genome.

Evolutionary studies have suggested that mutation rates vary significantly at different positions in the eukaryotic genome. The mechanism that is responsible for this context-dependence of mutation rates is not understood. We demonstrate experimentally that frameshift mutation rates in yeast microsatellites depend on the genomic context and that this variation primarily reflects the context-dependence of the efficiency of DNA mismatch repair. We measured the stability of a 16.5-repeat polyGT tract by using a reporter gene (URA3-GT) in which the microsatellite was inserted in-frame into the yeast URA3 gene. We constructed 10 isogenic yeast strains with the reporter gene at different locations in the genome. Rates of frameshift mutations that abolished the correct reading frame of this gene were determined by fluctuation analysis. A 16-fold difference was found among these strains. We made mismatch-repair-deficient (msh2) derivatives of six of the strains. Mutation rates were elevated for all of these strains, but the differences in rates among the strains were substantially reduced. The simplest interpretation of this result is that the efficiency of DNA mismatch repair varies in different regions of the genome, perhaps reflecting some aspect of chromosome structure.

Sequence dependent instability of mononucleotide microsatellites in cultured mismatch repair proficient and deficient mammalian cells.

We have measured the mutation rates of G(17) and A(17) repeat sequences in cultured mammalian cells with and without mismatch repair and have compared these rates to those of a (CA)(17) repeat sequence. Plasmids containing microsatellites that disrupt the reading frame of a downstream neomycin-resistance gene were introduced into the cells by transfection and revertants were selected using the neomycin analog G418. Comparison of mutation rates within cell lines showed that the mutation rates of A(17) and (CA)(17) sequences were similar in the mismatch repair proficient cells, but the mutation rate of G(17) was significantly higher than that of either A(17) or (CA)(17). In the mismatch repair deficient cells, the G(17) and (CA)(17) mutation rates were similar and were significantly higher than the A(17) rate. PCR analysis of the mutants showed that 1 bp insertions predominated in both mononucleotide repeats in the mismatch repair proficient cells; in mismatch repair deficient cells, 2 bp deletions were the most common mutation in the A(17) sequence, but 1 bp insertions and 2 bp deletions were equally represented in the G(17) sequence. These results indicate that a G(17) repeat is less stable than an A(17) repeat in both mismatch repair proficient and mismatch repair deficient mammalian cells. This observation implies that the replication fidelity is lower in G(17) repeats.

Relative rates of insertion and deletion mutations in dinucleotide repeats of various lengths in mismatch repair proficient mouse and mismatch repair deficient human cells.

Microsatellites are DNA elements composed of short tandem repeats of 1-5bp. These sequences are particularly prone to frameshift mutation by insertion-deletion loop formation during replication. The mismatch repair system is responsible for correcting these replication errors, and microsatellite mutation rates are significantly elevated in the absence of mismatch repair. We have investigated the effect of varying the number of repeats in a (CA)n microsatellite on mutation rates in cultured mammalian cells proficient or deficient in mismatch repair. We have also compared the relative rates of single-repeat insertions and deletions in these cells. Two plasmid vectors were constructed for each repeat unit number (n=8, 17, and 30), such that the microsatellites, placed upstream of a bacterial neomycin resistance gene (neo), disrupted the reading frame of the gene in the (-1) or (+1) direction. Plasmids were introduced separately into the cells, where they integrated into the cellular genome. Mutation rates were determined by selection of clones with frameshift mutations in the microsatellite that restored the reading frame of the neo gene. We found that mutation rates were significantly higher for (CA)17 and (CA)30 tracts than for (CA)8 tracts in both mismatch repair proficient (mouse) and deficient (human) cells. A mutational bias favoring insertions was generally observed. In both (CA)17 and (CA)30 tracts, single-repeat insertion rates were higher than single-repeat deletion rates with or without mismatch repair; deletions of multiple repeat units (> or =8bp) were observed in these tracts, where as deletions this large were not found in the (CA)8 tract. Single-repeat mutations of both types were made at similar rates in (CA)8 tracts in human mismatch repair deficient (MMR-) cells, but single-repeat insertion rates were higher than single-repeat deletion rates in mouse mismatch repair proficient (MMR+) cells. Results of these direct studies on microsatellite mutations in cultured cells should be useful for refinement of mathematical models for microsatellite evolution.