Dioxin Induces Genomic Instability in Mouse Embryonic Fibroblasts

Ionizing radiation and certain other exposures have been shown to induce genomic instability (GI), i.e., delayed genetic damage observed many cell generations later in the progeny of the exposed cells. The aim of this study was to investigate induction of GI by a nongenotoxic carcinogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mouse embryonic fibroblasts (C3H10T1/2) were exposed to 1, 10 or 100 nM TCDD for 2 days. Micronuclei (MN) and expression of selected cancer-related genes were assayed both immediately and at a delayed point in time (8 days). For comparison, similar experiments were done with cadmium, a known genotoxic agent. TCDD treatment induced an elevated frequency of MN at 8 days, but not directly after the exposure. TCDD-induced alterations in gene expression were also mostly delayed, with more changes observed at 8 days than at 2 days. Exposure to cadmium produced an opposite pattern of responses, with pronounced effects immediately after exposure but no increase in MN and few gene expression changes at 8 days. Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay), was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point. The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI. These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response

Genes and gene groups in Mouse Cancer PathwayFinder PCR array.

<p>Genes and gene groups in Mouse Cancer PathwayFinder PCR array.</p

Venn diagrams showing gene expression changes.

<p>Direct and delayed alterations in expression of genes in the mouse CancerFinder PCR array are shown after 2 days treatment with 10 nM TCDD or 1 µM cadmium and after further culture without exposure. Both ≥1.5-fold (left panel) and ≥2.0-fold (right panel) up- or downregulations are shown. Asterisk (*) indicates a low level of expression (Ct>30) and therefore less reproducible measurement.</p

Induction of micronuclei.

<p>The effect of 1, 10 or 100 nM TCDD (A) and 1 µM cadmium (B) on relative micronucleus (MN) frequency in mouse embryonal fibroblasts was determined immediately after exposure for 2 days and at the end of 6 days of recovery without exposure. Etoposide (0.025 µg/µl) was used as a positive control. Each column represents mean ± SE of two replicates in 3–4 independent experiments. Statistically significant differences are indicated by asterisks (*).</p

Effect of TCDD pretreatment on menadione-induced DNA damage and its repair.

<p>Comet tail moments were analyzed after TCDD exposure (1, 10 or 100 nM) for 2 days (A) and at the end of 6 days recovery time without exposure (B). After menadione treatment (40 µM) for one hour, cells were allowed to repair menadione-induced DNA damage for 0, 15, or 30 min. Each column represents mean ± SE of 400 Olive tail moments (OTM) in 4 independent experiments (A) or mean ± SE of 300 tail moments of 3 independent experiments (B). The effect of TCDD, tested over all TCDD doses and all three time points, was significant (p = 0.0009) when measured immediately after TCDD exposure, but not at 6 days after the end of exposure. The effect of menadione was significant (p<0.0001) in both cases.</p

The use of effect biomarkers in human biomonitoring studies: exposure to hexavalent chromium

Hexavalent chromium, Cr(VI) is a human carcinogen (Group 1, IARC), and its expo-sure has been associated with increased lung cancer risk, particularly in exposed workers. The general population may be exposed to Cr(VI) through food, drinking water and tobacco smoke. Under the Human Biomonitoring for Europe Initiative (HBM4EU), Cr(VI) has been considered a priority substance, indicating the need for generating and analyzing data on human exposure and effects, both as single sub-stance and in mixtures. Although many epidemiological studies have reported data on human exposure to Cr(VI), comparably fewer included effect biomarkers assess-ment. However, these biomarkers are central to identify early biological effects be-fore the onset of any adverse health effect. Additionally, biomarkers provide a link between human exposure and health outcomes, when considered in an adverse outcome pathway (AOP) perspective. In this work, we present the results of a critical review on the conventional and po-tentially new biomarkers for Cr(VI) early biological effects, which may be linked to adverse health outcomes in humans. The results show that the most frequently analyzed effect biomarkers concerning Cr(VI) exposure have been those associated with oxidative stress and genotoxicity (comet and micronucleus in blood cells). Urinary 8-isoprostane, a marker of lipid pe-roxidation, has also been used to relate Cr(VI) exposure to lung cancer. More recent-ly, single-gene alterations as well as omics-based biomarkers e.g., genomic or epigenomic changes and protein signatures, have been pointed as novel effect bi-omarkers, but they still need to be further developed and validated. In the literature revision, the most important knowledge gaps have also been identi-fied and discussed, such as the need of additional mechanistic data, in the perspec-tive of building an AOP for Cr(VI) occupational exposure and lung cancerWork co-funded by the HBM4EU project (GA 733032).N/

Biomonitoring for Occupational Exposure to Diisocyanates: A Systematic Review.

Diisocyanates are a group of chemicals that are widely used in occupational settings. They are known to induce various health effects, including skin- and respiratory tract sensitization resulting in allergic dermatitis and asthma. Exposure to diisocyanates has been studied in the past decades by using different types of biomonitoring markers and matrices. The aim of this review as part of the HBM4EU project was to assess: (i) which biomarkers and matrices have been used for biomonitoring diisocyanates and what are their strengths and limitations; (ii) what are (current) biomonitoring levels of the major diisocyanates (and metabolites) in workers; and (iii) to characterize potential research gaps. For this purpose we conducted a systematic literature search for the time period 2000-end 2018, thereby focussing on three types of diisocyanates which account for the vast majority of the total isocyanate market volume: hexamethylene diisocyanate (HDI), toluene diisocyanate (TDI), and 4,4'-methylenediphenyl diisocyanate (MDI). A total of 28 publications were identified which fulfilled the review inclusion criteria. The majority of these studies (93%) investigated the corresponding diamines in either urine or plasma, but adducts have also been investigated by several research groups. Studies on HDI were mostly in the motor vehicle repair industry [with urinary hexamethylene diamine result ranging from 0.03 to 146.5 µmol mol-1 creatinine]. For TDI, there is mostly data on foam production [results for urinary toluene diamine ranging from ~0.01 to 97 µmol mol-1 creatinine] whereas the available MDI data are mainly from the polyurethane industry (results for methylenediphenyl diamine range from 0.01 to 32.7 µmol mol-1 creatinine). About half of the studies published were prior to 2010 hence might not reflect current workplace exposure. There is large variability within and between studies and across sectors which could be potentially explained by several factors including worker or workplace variability, short half-lives of biomarkers, and differences in sampling strategies and analytical techniques. We identified several research gaps which could further be taken into account when studying diisocyanates biomonitoring levels: (i) the development of specific biomarkers is promising (e.g. to study oligomers of HDI which have been largely neglected to date) but needs more research before they can be widely applied, (ii) since analytical methods differ between studies a more uniform approach would make comparisons between studies easier, and (iii) dermal absorption seems a possible exposure route and needs to be further investigated. The use of MDI, TDI, and HDI has been recently proposed to be restricted in the European Union unless specific conditions for workers' training and risk management measures apply. This review has highlighted the need for a harmonized approach to establishing a baseline against which the success of the restriction can be evaluated.status: publishe