TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone in Xpa-deficient human TP53 knock-in mouse embryo fibroblasts

Abstract3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:C>T:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ. To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53-mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:C>T:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers’ lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity

Prediction of carcinogenic potential of chemicals using repeated-dose (13-week) toxicity data

AbstractSub-chronic toxicity studies of 163 non-genotoxic chemicals were evaluated in order to predict the tumour outcome of 24-month rat carcinogenicity studies obtained from the EFSA and ToxRef databases. Hundred eleven of the 148 chemicals that did not induce putative preneoplastic lesions in the sub-chronic study also did not induce tumours in the carcinogenicity study (True Negatives). Cellular hypertrophy appeared to be an unreliable predictor of carcinogenicity. The negative predictivity, the measure of the compounds evaluated that did not show any putative preneoplastic lesion in de sub-chronic studies and were negative in the carcinogenicity studies, was 75%, whereas the sensitivity, a measure of the sub-chronic study to predict a positive carcinogenicity outcome was only 5%. The specificity, the accuracy of the sub-chronic study to correctly identify non-carcinogens was 90%. When the chemicals which induced tumours generally considered not relevant for humans (33 out of 37 False Negatives) are classified as True Negatives, the negative predictivity amounts to 97%. Overall, the results of this retrospective study support the concept that chemicals showing no histopathological risk factors for neoplasia in a sub-chronic study in rats may be considered non-carcinogenic and do not require further testing in a carcinogenicity study

Utility of a next‐generation framework for assessment of genomic damage: A case study using the pharmaceutical drug candidate etoposide

We present a hypothetical case study to examine the use of a next-generation framework developed by the Genetic Toxicology Technical Committee of the Health and Environmental Sciences Institute for assessing the potential risk of genetic damage from a pharmaceutical perspective. We used etoposide, a genotoxic carcinogen, as a representative pharmaceutical for the purposes of this case study. Using the framework as guidance, we formulated a hypothetical scenario for the use of etoposide to illustrate the application of the framework to pharmaceuticals. We collected available data on etoposide considered relevant for assessment of genetic toxicity risk. From the data collected, we conducted a quantitative analysis to estimate margins of exposure (MOEs) to characterize the risk of genetic damage that could be used for decision-making regarding the predefined hypothetical use. We found the framework useful for guiding the selection of appropriate tests and selecting relevant endpoints that reflected the potential for genetic damage in patients. The risk characterization, presented as MOEs, allows decision makers to discern how much benefit is critical to balance any adverse effect(s) that may be induced by the pharmaceutical. Interestingly, pharmaceutical development already incorporates several aspects of the framework per regulations and health authority expectations. Moreover, we observed that quality dose response data can be obtained with carefully planned but routinely conducted genetic toxicity testing. This case study demonstrates the utility of the next-generation framework to quantitatively model human risk based on genetic damage, as applicable to pharmaceuticals

Hippocampal T2 hyperintensities on 7Tesla MRI

AbstractHippocampal focal T2 hyperintensities (HT2Hs), also referred to as hippocampal sulcal cavities, are a common finding on Magnetic Resonance (MR) images. There is uncertainty about their etiology and clinical significance. In this study we aimed to describe these HT2Hs in more detail using high resolution 7Tesla MR imaging, addressing 1) the MR signal characteristics of HT2Hs, 2) their occurrence frequency, 3) their location within the hippocampus, and 4) their relation with age. We also performed an explorative post-mortem study to examine the histology of HT2Hs.Fifty-eight persons without a history of invalidating neurological or psychiatric disease (mean age 64±8years; range 43–78years), recruited through their general practitioners, were included in this study. They all underwent 7Tesla MRI, including a T1, T2, and FLAIR image. MR signal characteristics of the HT2Hs were assessed on these images by two raters. Also, the location and number of the HT2Hs were assessed. In addition, four formalin-fixed brain slices from two subjects were scanned overnight. HT2Hs identified in these slices were subjected to histopathological analysis.HT2Hs were present in 97% of the subjects (median number per person 10; range 0–20). All HT2Hs detected on the T2 sequence were hypointense on T1 weighted images. Of all HT2Hs, 94% was hypointense and 6% hyperintense on FLAIR. FLAIR hypointense HT2Hs were all located in the vestigial sulcus of the hippocampus, FLAIR hyperintense HT2Hs in the hippocampal sulcus or the gray matter. Post-mortem MRI and histopathological analysis suggested that the hypointense HT2Hs on FLAIR were cavities filled with cerebrospinal fluid. A hyperintense HT2H on FLAIR proved to be a microinfarct upon microscopy.In conclusion, hippocampal T2Hs are extremely common and unrelated to age. They can be divided into two types (hypo- and hyperintense on FLAIR), probably with different etiology

DNA methylation markers associated with type 2 diabetes, fasting glucose and HbA(1c) levels:a systematic review and replication in a case-control sample of the Lifelines study

AIMS/HYPOTHESIS: Epigenetic mechanisms may play an important role in the aetiology of type 2 diabetes. Recent epigenome-wide association studies (EWASs) identified several DNA methylation markers associated with type 2 diabetes, fasting glucose and HbA1c levels. Here we present a systematic review of these studies and attempt to replicate the CpG sites (CpGs) with the most significant associations from these EWASs in a case-control sample of the Lifelines study.METHODS: We performed a systematic literature search in PubMed and EMBASE for EWASs to test the association between DNA methylation and type 2 diabetes and/or glycaemic traits and reviewed the search results. For replication purposes we selected 100 unique CpGs identified in peripheral blood, pancreas, adipose tissue and liver from 15 EWASs, using study-specific Bonferroni-corrected significance thresholds. Methylation data (Illumina 450K array) in whole blood from 100 type 2 diabetic individuals and 100 control individuals from the Lifelines study were available. Multivariate linear models were used to examine the associations of the specific CpGs with type 2 diabetes and glycaemic traits.RESULTS: From the 52 CpGs identified in blood and selected for replication, 15 CpGs showed nominally significant associations with type 2 diabetes in the Lifelines sample (p &lt; 0.05). The results for five CpGs (in ABCG1, LOXL2, TXNIP, SLC1A5 and SREBF1) remained significant after a stringent multiple-testing correction (changes in methylation from -3% up to 3.6%, p &lt; 0.0009). All associations were directionally consistent with the original EWAS results. None of the selected CpGs from the tissue-specific EWASs were replicated in our methylation data from whole blood. We were also unable to replicate any of the CpGs associated with HbA1c levels in the healthy control individuals of our sample, while two CpGs (in ABCG1 and CCDC57) for fasting glucose were replicated at a nominal significance level (p &lt; 0.05).CONCLUSIONS/INTERPRETATION: A number of differentially methylated CpGs reported to be associated with type 2 diabetes in the EWAS literature were replicated in blood and show promise for clinical use as disease biomarkers. However, more prospective studies are needed to support the robustness of these findings.</p

Biomarkers of effect as determined in human biomonitoring studies on hexavalent chromium and cadmium in the period 2008–2020

This research was supported by funding from the European Union's Horizon 2020 research and innovation Programme under grant agreement No 733032 HBM4EU.A number of human biomonitoring (HBM) studies have presented data on exposure to hexavalent chromium [Cr (VI)] and cadmium (Cd), but comparatively few include results on effect biomarkers. The latter are needed to identify associations between exposure and adverse outcomes (AOs) in order to assess public health implications. To support improved derivation of EU regulation and policy making, it is of great importance to identify the most reliable effect biomarkers for these heavy metals that can be used in HBM studies. In the framework of the Human Biomonitoring for Europe (HBM4EU) initiative, our study aim was to identify effect biomarkers linking Cr(VI) and Cd exposure to selected AOs including cancer, immunotoxicity, oxidative stress, and omics/epigenetics. A comprehensive PubMed search identified recent HBM studies, in which effect biomarkers were examined. Validity and applicability of the markers in HBM studies are discussed. The most frequently analysed effect biomarkers regarding Cr(VI) exposure and its association with cancer were those indicating oxidative stress (e.g., 8-hydroxy-2 & rsquo;-deoxyguanosine (8-OHdG), malondialdehyde (MDA), glutathione (GSH)) and DNA or chromosomal damage (comet and micronucleus assays). With respect to Cd and to some extent Cr, 0-2-microglobulin (B2-MG) and N-acetyl-0-D-glucosaminidase (NAG) are well-established, sensitive, and the most common effect biomarkers to relate Cd or Cr exposure to renal tubular dysfunction. Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule (KIM)-1 could serve as sensitive biomarkers of acute kidney injury in response to both metals, but need further investigation in HBM studies. Omics-based biomarkers, i.e., changes in the (epi-)genome, transcriptome, proteome, and metabolome associated with Cr and/or Cd exposure, are promising effect biomarkers, but more HBM data are needed to confirm their significance. The combination of established effect markers and omics biomarkers may represent the strongest approach, especially if based on knowledge of mechanistic principles. To this aim, also mechanistic data were collected to provide guidance on the use of more sensitive and specific effect biomarkers. This also led to the identification of knowledge gaps relevant to the direction of future research.European Commission 733032 HBM4E

Risk assessment of the mixture of hexavalent chromium, nickel and polycyclic aromatic hydrocarbons using occupational exposure data extracted from the literature

Na maioria dos contextos laborais ocorre, em geral, exposição a misturas de substâncias químicas. Este estudo, inserido na iniciativa HBM4EU, visou avaliar o risco de exposição a misturas de crómio hexavalente [Cr ( VI ) ], níquel (Ni ) e/ou hicrocarbonetos aromáticos policíclicos (HAPs ) a partir de dados de exposição reportados em estudos realizados em várias indústrias na Europa. Para a avaliação do risco de misturas binárias (Cr e Ni ) e ternárias (Cr, Ni e HAPs ) calcularam-se os Quocientes de Risco (QR) e a Soma dos Quocientes de Risco (SQR) a partir dos dados de biomonitorização humana (BMH) e de medições dessas substâncias no ar. É geralmente aceite que um valor de QR ou SQR superior a 1 indica um risco acrescido para a saúde. Para a maioria das medições de exposição a partir de biomarcadores urinários obtiveram-se valores médios de SQR>1, sendo que os respetivos QR calculados para cada uma das substâncias individuais por vezes não excederam os valores limite (QR<1). Estes resultados mostram não só que existe um risco acrescido para a saúde decorrente da coexposição a estas substâncias em contexto ocupacional, como também que esse risco é frequentemente subestimado no processo clássico de avaliação de risco substância a substância. Com este estudo demonstrou-se a relevância de avaliar o risco decorrente da exposição a misturas, particularmente com base em dados de BMH, sendo esta uma abordagem mais realista do contexto ocupacional e que permite uma melhor caracterização do risco com vista a uma proteção mais eficaz da saúde dos trabalhadores.In most occupational settings exposure to chemical mixtures generally occurs. This study, developed under the HBM4EU Initiative, aimed to assess the risk from occupational exposure to mixtures of hexavalent chromium [Cr ( VI )], nickel ( Ni ) and/or polycyclic aromatic hydrocarbons (PAHs ), based on exposure data collected from studies carried out in various European industries. To assess the risk of binary (Cr and Ni ) and ternary (Cr, Ni and PAHs ) mixtures, Risk Quotients ( RQ) and Sum of Risk Quotients (SQR ) were calculated from human biomonitoring data (HBM) and measurements of these substances in the air. A QR or SQR value greater than 1 has been considered to indicate an increased health risk. For most exposure measurements from urinary biomarkers, mean values of SQR>1 were obtained, with the respective QR calculated for each of the individual substances sometimes not exceeding the threshold values (QR<1). These results show not only that there is an increased health risk arising from co-exposure to these substances in an occupational context, but also that this risk is often underestimated in the classic substance-by-substance risk assessment process. This study demonstrated the relevance of applying a mixture risk assessment, particularly based on HBM data, which is a more realistic approach to the occupational context and allows for a better risk characterization with a view to more effective protection of workers' health.Este estudo recebeu financiamento do programa de investigação e inovação Horizonte 2020 da União Europeia sob o acordo de subvenção n.º 733032 e recebeu cofinanciamento das organizações dos autores.info:eu-repo/semantics/publishedVersio

Risk assessment of the mixture of hexavalent chromium, nickel and polycyclic aromatic hydrocarbons using occupational exposure data extracted from the literature

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Utility of a next generation framework for assessment of genomic damage: A case study using the industrial chemical benzene

We recently published a next generation framework for assessing the risk of genomic damage via exposure to chemical substances. The framework entails a systematic approach with the aim to quantify risk levels for substances that induce genomic damage contributing to human adverse health outcomes. Here, we evaluated the utility of the framework for assessing the risk for industrial chemicals, using the case of benzene. Benzene is a well‐studied substance that is generally considered a genotoxic carcinogen and is known to cause leukemia. The case study limits its focus on occupational and general population health as it relates to benzene exposure. Using the framework as guidance, available data on benzene considered relevant for assessment of genetic damage were collected. Based on these data, we were able to conduct quantitative analyses for relevant data sets to estimate acceptable exposure levels and to characterize the risk of genetic damage. Key observations include the need for robust exposure assessments, the importance of information on toxicokinetic properties, and the benefits of cheminformatics. The framework points to the need for further improvement on understanding of the mechanism(s) of action involved, which would also provide support for the use of targeted tests rather than a prescribed set of assays. Overall, this case study demonstrates the utility of the next generation framework to quantitatively model human risk on the basis of genetic damage, thereby enabling a new, innovative risk assessment concept. Environ. Mol. Mutagen. 61:94–113, 2020. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.JRC.F.3-Chemicals Safety and Alternative Method