Error-corrected next generation sequencing - Promises and challenges for genotoxicity and cancer risk assessment.

Error-corrected Next Generation Sequencing (ecNGS) is rapidly emerging as a valuable, highly sensitive and accurate method for detecting and characterizing mutations in any cell type, tissue or organism from which DNA can be isolated. Recent mutagenicity and carcinogenicity studies have used ecNGS to quantify drug-/chemical-induced mutations and mutational spectra associated with cancer risk. ecNGS has potential applications in genotoxicity assessment as a new readout for traditional models, for mutagenesis studies in 3D organotypic cultures, and for detecting off-target effects of gene editing tools. Additionally, early data suggest that ecNGS can measure clonal expansion of mutations as a mechanism-agnostic early marker of carcinogenic potential and can evaluate mutational load directly in human biomonitoring studies. In this review, we discuss promising applications, challenges, limitations, and key data initiatives needed to enable regulatory testing and adoption of ecNGS - including for advancing safety assessment, augmenting weight-of-evidence for mutagenicity and carcinogenicity mechanisms, identifying early biomarkers of cancer risk, and managing human health risk from chemical exposures.

Assessment of the DNA damaging potential of environmental chemicals using a quantitative high-throughput screening approach to measure p53 activation.

Genotoxicity potential is a critical component of any comprehensive toxicological profile. Compounds that induce DNA or chromosomal damage often activate p53, a transcription factor essential to cell cycle regulation. Thus, within the US Tox21 Program, we screened a library of ∼10,000 (∼8,300 unique) environmental compounds and drugs for activation of the p53-signaling pathway using a quantitative high-throughput screening assay employing HCT-116 cells (p53+/+ ) containing a stably integrated ß-lactamase reporter gene under control of the p53 response element (p53RE). Cells were exposed (-S9) for 16 hr at 15 concentrations (generally 1.2 nM to 92 µM) three times, independently. Excluding compounds that failed analytical chemistry analysis or were suspected of inducing assay interference, 365 (4.7%) of 7,849 unique compounds were concluded to activate p53. As part of an in-depth characterization of our results, we first compared them with results from traditional in vitro genotoxicity assays (bacterial mutation, chromosomal aberration); ∼15% of known, direct-acting genotoxicants in our library activated the p53RE. Mining the Comparative Toxicogenomics Database revealed that these p53 actives were significantly associated with increased expression of p53 downstream genes involved in DNA damage responses. Furthermore, 53 chemical substructures associated with genotoxicity were enriched in certain classes of p53 actives, for example, anthracyclines (antineoplastics) and vinca alkaloids (tubulin disruptors). Interestingly, the tubulin disruptors manifested unusual nonmonotonic concentration response curves suggesting activity through a unique p53 regulatory mechanism. Through the analysis of our results, we aim to define a role for this assay as one component of a comprehensive toxicological characterization of large compound libraries. Environ. Mol. Mutagen. 58:494-507, 2017. © 2017 Wiley Periodicals, Inc.

Estrogenic and anti-estrogenic activity of off-the-shelf hair and skin care products.

Use of personal care products is widespread in the United States but tends to be greater among African Americans than whites. Of special concern is the possible hazard of absorption of chemicals with estrogenic activity (EA) or anti-EA (AEA) in these products. Such exposure may have adverse health effects, especially when it occurs during developmental windows (e.g., prepubertally) when estrogen levels are low. We assessed the ethanol extracts of eight commonly used hair and skin products popular among African Americans for EA and AEA using a cell proliferation assay with the estrogen sensitive MCF-7:WS8 cell line derived from a human breast cancer. Four of the eight personal care products tested (Oil Hair Lotion, Extra-dry Skin Lotion, Intensive Skin Lotion, Petroleum Jelly) demonstrated detectable EA, whereas three (Placenta Hair Conditioner, Tea-Tree Hair Conditioner, Cocoa Butter Skin Cream) exhibited AEA. Our data indicate that hair and skin care products can have EA or AEA, and suggest that laboratory studies are warranted to investigate the in vivo activity of such products under chronic exposure conditions as well as epidemiologic studies to investigate potential adverse health effects that might be associated with use of such products.

Dose-response assessment of four genotoxic chemicals in a combined mouse and rat micronucleus (MN) and Comet assay protocol.

The in vivo micronucleus (MN) assay has proven to be an effective measure of genotoxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic damage using the MN assay provides a limited genotoxicity profile. The in vivo alkaline (pH >13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues following administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophosphamide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24-hr intervals (VS was administered to rats for 3 days); animals were euthanized 4 hr after the last administration. All compounds induced significant increases in micronucleated reticulocytes (MN-RET) in the peripheral blood of mice, and all but ACM induced MN-RET in rats. EMS and ACM induced significant increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced DNA damage was detected in leukocytes and duodenum cells. VS, a spindle fiber disrupting agent, was negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program (NTP) is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern.

Assessing human germ-cell mutagenesis in the Postgenome Era: a celebration of the legacy of William Lawson (Bill) Russell.

Birth defects, de novo genetic diseases, and chromosomal abnormality syndromes occur in approximately 5% of all live births, and affected children suffer from a broad range of lifelong health consequences. Despite the social and medical impact of these defects, and the 8 decades of research in animal systems that have identified numerous germ-cell mutagens, no human germ-cell mutagen has been confirmed to date. There is now a growing consensus that the inability to detect human germ-cell mutagens is due to technological limitations in the detection of random mutations rather than biological differences between animal and human susceptibility. A multidisciplinary workshop responding to this challenge convened at The Jackson Laboratory in Bar Harbor, Maine. The purpose of the workshop was to assess the applicability of an emerging repertoire of genomic technologies to studies of human germ-cell mutagenesis. Workshop participants recommended large-scale human germ-cell mutation studies be conducted using samples from donors with high-dose exposures, such as cancer survivors. Within this high-risk cohort, parents and children could be evaluated for heritable changes in (a) DNA sequence and chromosomal structure, (b) repeat sequences and minisatellites, and (c) global gene expression profiles and pathways. Participants also advocated the establishment of a bio-bank of human tissue samples from donors with well-characterized exposure, including medical and reproductive histories. This mutational resource could support large-scale, multiple-endpoint studies. Additional studies could involve the examination of transgenerational effects associated with changes in imprinting and methylation patterns, nucleotide repeats, and mitochondrial DNA mutations. The further development of animal models and the integration of these with human studies are necessary to provide molecular insights into the mechanisms of germ-cell mutations and to identify prevention strategies. Furthermore, scientific specialty groups should be convened to review and prioritize the evidence for germ-cell mutagenicity from common environmental, occupational, medical, and lifestyle exposures. Workshop attendees agreed on the need for a full-scale assault to address key fundamental questions in human germ-cell environmental mutagenesis. These include, but are not limited to, the following: Do human germ-cell mutagens exist? What are the risks to future generations? Are some parents at higher risk than others for acquiring and transmitting germ-cell mutations? Obtaining answers to these, and other critical questions, will require strong support from relevant funding agencies, in addition to the engagement of scientists outside the fields of genomics and germ-cell mutagenesis.