Early Biological Effects Research Articles

Protocol for CARES–HAPIN: an ambidirectional cohort study on exposure to environmental tobacco smoke and risk of early childhood caries

IntroductionPrenatal and postnatal exposure to environmental tobacco smoke (ETS) has been linked with early childhood caries (ECC), but the specific molecular mechanisms and pathways remain largely unknown. The Caries Risk...

Abstract 2870: Analysis of cancer cells response to radiotherapy activated NBTXR3 nanoparticles

Abstract In 2011, NBTXR3 emerged as the first-in-class radioenhancer used in clinical practice for treating patients with locally advanced soft tissue sarcoma (LA-STS). NBTXR3 became the first radioenhancer to receive CE mark approval after successfully completing a phase II/III clinical study for LA-STS with a doubling of the pCR rate in the NBTXR3 arm compared with the control arm. NBTXR3 nanoparticles consist of a functionalized core of hafnium oxide, a high atomic number element, specifically designed to amplify the effects of radiotherapy (RT) within cells without additional side effects on healthy tissues. Leveraging this physical mechanism, RT-activated NBTXR3 (NBTXR3+RT) has demonstrated superior efficacy in destroying tumor cells and controlling tumor growth in numerous preclinical models and in human, surpassing the effects of RT alone, while maintaining a favorable safety profile. Preclinical studies have revealed that the benefits of NBTXR3 extend beyond mere radioenhancement and improved cancer cell destruction. Specifically, NBTXR3 has been reported to possess immunomodulatory properties through 1) improved induction of DNA damage, leading to better activation of the cGAS-STING pathway, 2) promotion of immunogenic cell death, 3) enhancement of immunopeptidome presentation, 4) generation of an antitumor immune response leading to the production of an abscopal effect mediated by CD8+ cytotoxic lymphocytes. Notably, a series of recent studies using a two-tumor mouse model of anti-PD1-resistant lung cancer that the addition of NBTXR3 significantly improved the efficacy of various treatment regimens (RT+anti-PD1 and combinations with other check-point inhibitors) in terms of tumor growth, abscopal effect, and survival. It has been demonstrated that same benefits can be achieved when NBTXR3 was combined with proton therapy. Moreover, all these studies (X-ray and proton) reported a robust activation of the antitumor immune response, restoration of the effectiveness of anti-PD1 therapy, and the induction of memory response in cured mice. However, our understanding of the biological events leading to these effects remains limited. To address this question, we conducted a series of in vitro studies that employed cytometry, microscopy, and RNA sequencing on various cancer models, both human and murine. We reported that NBTXR3 nanoparticles primarily accumulate within lysosomes following their uptake by tumor cells, leading to the activation of genes associated with the biogenesis, structure, and contents of these organelles. Upon activation by RT, NBTXR3 within lysosomes induces lysosomal membrane permeabilization (LMP), resulting in an increase in lipid peroxidation, inducing regulation of fatty acid oxidation genes, and impacting mitochondrial genes expression, such as respirasome. These findings provide a deeper insight into the early biological effects of NBTXR3+RT in comparison to RT alone. Citation Format: Jordan Da Silva, Celia Bienassis, Sebastien Paris. Analysis of cancer cells response to radiotherapy activated NBTXR3 nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2870.

Radiotherapy-activated NBTXR3 nanoparticles promote ferroptosis through induction of lysosomal membrane permeabilization

PurposeRadiotherapy-activated NBTXR3 (NBTXR3 + RT) has demonstrated superior efficacy in cancer cell destruction and tumor growth control, compared to radiotherapy (RT), in preclinical and clinical settings. Previous studies highlighted the immunomodulatory properties of NBTXR3 + RT, such as modification of tumor cell immunogenicity/adjuvanticity, producing an effective local tumor control and abscopal effect, related to an enhanced antitumor immune response. Furthermore, NBTXR3 + RT has shown potential in restoring anti-PD1 efficacy in a refractory tumor model. However, the early events leading to these results, such as NBTXR3 endocytosis, intracellular trafficking and primary biological responses induced by NBTXR3 + RT remain poorly understood.MethodsWe analyzed by transmission electron microscopy endocytosis and intracellular localization of NBTXR3 nanoparticles after endocytosis in various cell lines, in vitro and in vivo. A kinetic of NBTXR3 endocytosis and its impact on lysosomes was conducted using LysoTracker staining, and a RNAseq analysis was performed. We investigated the ability of NBTXR3 + RT to induce lysosomal membrane permeabilization (LMP) and ferroptosis by analyzing lipid peroxidation. Additionally, we evaluated the recapture by cancer cells of NBTXR3 released from dead cells.ResultsNBTXR3 nanoparticles were rapidly internalized by cells mainly through macropinocytosis and in a less extend by clathrin-dependent endocytosis. NBTXR3-containing endosomes were then fused with lysosomes. The day following NBTXR3 addition, we measured a significant increase in LysoTracker lysosome labeling intensity, in vitro as in vivo. Following RT, a significant lysosomal membrane permeabilization (LMP) was measured exclusively in cells treated with NBTXR3 + RT, while RT had no effect. The day post-irradiation, a significant increase in lipid peroxidation, a biomarker of ferroptosis, was measured with NBTXR3 + RT compared to RT. Moreover, we demonstrated that NBTXR3 nanoparticles released from dead cells can be recaptured by cancer cells.ConclusionsOur findings provide novel insights into the early and specific biological effects induced by NBTXR3 + RT, especially LMP, not induced by RT in our models. The subsequent significant increase in lipid peroxidation partially explains the enhanced cancer cell killing capacity of NBTXR3 + RT compared to RT, potentially by promoting ferroptosis. This study improves our understanding of the cellular mechanisms underlying NBTXR3 + RT and highlights its potential as an agnostic therapeutic strategy for solid cancers treatment.Graphical

Air pollution and oxidative stress in adults suffering from airway diseases. Insights from the Gene Environment Interactions in Respiratory Diseases (GEIRD) multi-case control study

Air pollution is a leading risk factor for global mortality and morbidity. Oxidative stress is a key mechanism underlying air-pollution-mediated health effects, especially in the pathogenesis/exacerbation of airway impairments. However, evidence lacks on subgroups at higher risk of developing more severe outcomes in response to air pollution. This multi-centre study aims to evaluate the association between air pollution and oxidative stress in healthy adults and in patients affected by airway diseases from the Italian GEIRD (Gene Environment Interactions in Respiratory Diseases) multi-case control study. Overall, 1841 adults (49 % females, 20–83 years) were included from four Italian centres: Pavia, Sassari, Turin, and Verona. Following a 2-stage screening process, we identified 1273 cases of asthma, chronic bronchitis, rhinitis, or COPD and 568 controls. Systemic oxidative stress was quantified by urinary 8-isoprostane and 8-OH-dG. Individual residential exposures to NO2, PM10, PM2.5, and O3 were derived using an innovative five-stage machine-learning-based approach. Linear mixed regression models tested the association between oxidative stress biomarkers and air pollution tertiles, adjusting by age, sex, BMI, smoking, education and season, with recruiting centres as random intercept. Only cases exhibited higher levels of log-transformed 8-isoprostane and 8-OH-dG in association with NO2 (β: 0.30 95 % CI: 0.08–0.52 and 0.20 95 % CI: 0.03–0.37), PM10 (0.34 95 % CI: 0.12–0.55 and 0.21 95 % CI: 0.05–0.37) and PM2.5 (0.27 95 % CI: 0.09–0.49 and 0.18 95 % CI: 0.02–0.34) as compared to the first tertile of exposure. No significant associations were observed for summer O3. Our findings suggest that exposure to air pollution may increase systemic oxidative stress levels in people suffering from airway diseases. This introduces a potential novel approach available for future epidemiological studies and Public Health for effective prevention strategies oriented at the quantification of early biological effects in susceptible people, whose additional risk level might be currently underrated. Air-pollution-mediated exacerbations, driven by oxidative stress, still deserve our attention.

Safety and early biologic effects of phase 1 PERIO-01 trial of pressure-enabled drug delivery (PEDD) of TLR9 agonist SD-101 and immune checkpoint inhibition (ICI) in uveal melanoma metastatic to the liver (MUM).

2521 Background: Delivery barriers due to intratumoral pressure and immunosuppression related to myeloid-derived suppressor cells (MDSC) in liver tumors have created challenges for immunotherapy. TLR9 agonists seem to improve response to ICI. PERIO-01 is a first-in-man trial of PEDD of SD-101 using hepatic arterial infusion (HAI) with ICI in MUM. Methods: PERIO-01 is an open-label phase 1 trial of SD-101 given by HAI in MUM (NCT04935229). The study consists of dose-escalation cohorts of SD-101 alone (Cohort A), with nivolumab (Cohort B), or nivolumab + ipilimumab (Cohort C). SD-101 is delivered over 2 cycles, with 3 weekly doses per cycle. Blood, liver metastasis (LM) and normal liver biopsies are collected for correlative studies. Results: At data cutoff, 33 patients were enrolled, 30 of whom received at least one dose of SD-101, 13 in Cohort A (2, 4, and 8 mg), 15 patients in Cohort B (2 mg and 4 mg) and 2 patients in Cohort C (2 mg). The median age was 63 years of equal gender. Only 3 patients were treatment-naïve and 2 were HLA-A*-02:01+ who received prior tebentafusp. Nine subjects had 4-7 LM and 4 had > 10. The median index LM size was 4.7 cm. Only one patient experienced a grade ≥3 adverse event (AE) related to SD-101, which was increased liver enzymes. All AEs related to cytokine release syndrome have been low-grade with the most common being fever (9), chills (5), and dizziness (4). Treatment resulted in high liver drug levels (median at 2 mg = 1540 ng/g and 8 mg = 2325 ng/g, p = 0.035), with only transient exposure in the periphery ( < 4 hours) and a maximum peak serum level of 554 mg/ml 30 minutes post-infusion. Increases in serum IL-18 and IFNγ were noted, with highest levels at 8 mg. Expansion of natural killer cells were detected in 9 of 10 patients from flow cytometry data peripherally. Monocytic MDSC levels were decreased in 5 of 5 patients on immunofluorescence, and NanoString analysis revealed decreases in ARG-1 and IDO-1 gene levels up to 100 days from initial treatment. Broad immunostimulatory gene expression changes were noted in tumor and normal liver following SD-101, including increases in IFNB1 and IL-9. Using the available samples from 13 patients, 7 of 10 had decrease in ctDNA, with complete clearance in 3, along with circulating tumor cell decreases in 6 of 13. In cohort B at 2 mg, 5 of 6 with available response data have stable disease with a median duration of disease control of 12 weeks (range = 7.5-24) at data cutoff. Conclusions: HAI of SD-101 has been well tolerated and associated with encouraging immunologic activity. Evidence of biologic effects at the lower doses of SD-101 with nivolumab is encouraging and enrollment with escalation continues in Cohorts B and C. Clinical trial information: NCT04935229 .

Per- and polyfluoroalkyl substances (PFAS) and immune system-related diseases: results from the Flemish Environment and Health Study (FLEHS) 2008–2014

BackgroundThe successive FLEHS campaigns assess internal exposure to pollutants and associated early biological and health effects in participants of different age groups.Materials and methodsMother–newborn pairs (N = 220 in 2008–2009, age 18–42 years; N = 269 in 2013–2014, age 18–44 years), 197 adolescents 14–15 years (2010–2011), 201 adults 20–40 years (2008–2009) and 205 adults 50–65 years (2014) were recruited. For the various groups of subjects different sets of PFAS were assessed. Perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorobutane sulfonate (PFBS) were determined in cord plasma and peripheral serum as these were the PFAS compounds for which we had access to high quality measurements and which were expected to be present in the highest concentrations. Participants filled out a questionnaire based on the European Community Respiratory Health Survey questionnaire on asthma and allergy. In these cross-sectional studies associations were assessed using stepwise multiple logistic regression, with confounders (including smoking and familial occurrence of the disease) and potential covariates selected on the basis of experience in our previous studies and a literature search. Forest plots of odds ratios summarize the associations between the various PFAS on the one hand and the different immune outcomes on the other hand.ResultsFor several self-reported immune system-related diseases inverse associations with PFAS serum concentrations were observed. These inverse associations were more pronounced in mothers and adults than in adolescents. A significant inverse association was observed in adults and mothers (for mothers based on measurements on cord plasma) between PFNA, PFOS, and PFHxS and asthma (for mothers also for PFOA), in mothers between PFHxS, PFNA and PFOS and allergic rhinitis, in mothers and adults between PFHxS and PFOS and some forms of allergy (for mothers also for PFOA), in adults between PFOA and eczema, and in adolescents between PFOS and systemic allergy.ConclusionInternal exposure to PFAS was associated with changes in immunological processes consistent with what has been reported in the literature. Whereas these changes were observed in many publications to be associated with adverse health effects, our findings suggest that they can also lead to inverse associations with certain immune system-related diseases.

Implementation of effect biomarkers in human biomonitoring studies: A systematic approach synergizing toxicological and epidemiological knowledge.

Human biomonitoring (HBM) studies have highlighted widespread daily exposure to environmental chemicals. Some of these are suspected to contribute to adverse health outcomes such as reproductive, neurological, and metabolic disorders, among other developmental and chronic impairments. One of the objectives of the H2020 European Human Biomonitoring Initiative (HBM4EU) was the development of informative effect biomarkers for application in a more systematic and harmonized way in large-scale European HBM studies. The inclusion of effect biomarkers would complement exposure data with mechanistically-based information on early and late adverse effects. For this purpose, a stepwise strategy was developed to identify and implement a panel of validated effect biomarkers in European HBM studies. This work offers an overview of the complete procedure followed, from comprehensive literature search strategies, selection of criteria for effect biomarkers and their classification and prioritization, based on toxicological data and adverse outcomes, to pilot studies for their analytical, physiological, and epidemiological validation. We present the example of one study that demonstrated the mediating role of the effect biomarker status of brain-derived neurotrophic factor BDNF in the longitudinal association between infant bisphenol A (BPA) exposure and behavioral function in adolescence. A panel of effect biomarkers has been implemented in the HBM4EU Aligned Studies as main outcomes, including traditional oxidative stress, reproductive, and thyroid hormone biomarkers. Novel biomarkers of effect, such as DNA methylation status of BDNF and kisspeptin (KISS) genes were also evaluated as molecular markers of neurological and reproductive health, respectively. A panel of effect biomarkers has also been applied in HBM4EU occupational studies, such as micronucleus analysis in lymphocytes and reticulocytes, whole blood comet assay, and malondialdehyde, 8-oxo-2'-deoxyguanosine and untargeted metabolomic profile in urine, to investigate, for example, biological changes in response to hexavalent chromium Cr(VI) exposure. The use of effect biomarkers in HBM4EU has demonstrated their ability to detect early biological effects of chemical exposure and to identify subgroups that are at higher risk. The roadmap developed in HBM4EU confirms the utility of effect biomarkers, and support one of the main objectives of HBM research, which is to link exposure biomarkers to mechanistically validated effect and susceptibility biomarkers in order to better understand the public health implications of human exposure to environmental chemicals.

Characterization of the external exposome and its contribution to the clinical respiratory and early biological effects in children: The PROMESA cohort study protocol.

Air pollution contains a mixture of different pollutants from multiple sources. However, the interaction of these pollutants with other environmental exposures, as well as their harmful effects on children under five in tropical countries, is not well known. This study aims to characterize the external exposome (ambient and indoor exposures) and its contribution to clinical respiratory and early biological effects in children. A cohort study will be conducted on children under five (n = 500) with a one-year follow-up. Enrolled children will be followed monthly (phone call) and at months 6 and 12 (in person) post-enrolment with upper and lower Acute Respiratory Infections (ARI) examinations, asthma development, asthma control, and genotoxic damage. The asthma diagnosis will be pediatric pulmonologist-based and a standardized protocol will be used. Exposure, effect, and susceptibility biomarkers will be measured on buccal cells samples. For environmental exposures PM2.5 will be sampled, and questionnaires, geographic information, dispersion models and Land Use Regression models for PM2.5 and NO2 will be used. Different statistical methods that include Bayesian and machine learning techniques will be used for the ambient and indoor exposures-and outcomes. This study was approved by the ethics committee at Universidad Pontificia Bolivariana. To estimate i) The toxic effect of particulate matter transcending the approach based on pollutant concentration levels; ii) The risk of developing an upper and lower ARI, based on different exposure windows; iii) A baseline of early biological damage in children under five, and describe its progression after a one-year follow-up; and iv) How physical and chemical PM2.5 characteristics influence toxicity and children's health.

Expression level and function analysis of serum miRNAs in workers with occupational exposure to benzene series

Benzene series are ubiquitous in industrial production and daily life, and can have an impact on health even at low concentrations. miRNAs have been proved to be a biomarker of a variety of diseases and carcinogens. The purpose of this study was to explore the distribution characteristics and biological function of miRNAs in subjects exposed to benzene series. In this study, serum miRNAs were measured in 247 occupationally exposed subjects and 256 controls. The relationship between cumulative exposure dose of benzene series and miRNAs was analyzed by Generalized linear model, Spearman's rank correlation, and chi-square test for trend. The function of MiRNAs target gene was analyzed by means of bioinformatics method. The results showed that the expressions of miR-181a-5p, 221-3p, 223-3p, and 342-3p were down-regulated, whilst the expression of miR-638 was up-regulated in the occupational exposure group. miR-181a-5p, 221-3p, 223-3p, 342-3p, and 638 showed dose-response relationship with benzene series, and were closely related to multiple tumor pathways. miR-181a-5p, 221-3p, 223-3p, 342-3p, and 638 may be involved in the carcinogenic process of benzene series, and can be used to evaluate the early biological effects and monitor the exposure level of benzene series. miRNAs are potential biomarkers of benzene series exposure.

Characterization of the External Exposome and its Contribution to the Clinical Respiratory and Early Biological Effects in Children: PROMESA cohort in Colombia

Characterization of the External Exposome and its Contribution to the Clinical Respiratory and Early Biological Effects in Children: PROMESA cohort in Colombia

Exposure to engineered nanomaterials and early biological effects measured in the exhaled breath condensate: First results from the NanoExplore cohort

BACKGROUND AND AIM Activities involving engineered nanomaterials (ENM) may raise concerns of occupational exposure and subsequent health effects. We aimed to build a multicenter prospective cohort to assess the potential effects of ENM exposure using biomarkers of early effects measured in exhaled breath condensate (EBC). METHODS We recruited seven centers in three countries (Switzerland, Spain, and Italy), where we conducted a 4-day exposure monitoring campaign and two EBC samplings, at the beginning (T1) and end (T2) of working week. The recruited workers were split in three groups depending on their exposure and compared in terms of their baseline individual and professional characteristics, and health status. EBC biomarkers of oxidative and nitrosative stress (malondialdehyde, 8-isoprostane, nitrotyrosine), systemic inflammation (High-Sensitivity C-Reactive Protein (hsCRP)), activation of pro-fibrotic cascade and interstitial lung disease (Krebs von den Lungen glycoprotein 6 (KL6)) and inflammatory cytokines (Interleukins (IL-10, IL-1β), Tumor necrosis factor (TNF-α)) were analyzed using multilevel mixed interval regression models. RESULTS The cohort included 140 participants: 43 non-exposed, 55 with negligible to low exposure, and 43 with medium to high exposure. The latter were gradually more often men, older, and in overweight than lowly exposed and non-exposed workers. A statistically significant change between T1 and T2 average concentrations was observed for malondialdehyde and KL6. Between-group differences were observed for all biomarkers but KL6 and nitrotyrosine. In multivariate models adjusted for age, sex and body mass index, a linear exposure-response relationship was observed for malondialdehyde, 8-isoprostane, IL-10, IL-1β, and TNF-α. ENM exposure was also associated with higher hs-CRP and KL6 levels, but not monotonically. CONCLUSION These first results suggest that ENM exposure might lead to early biological effects in workers’ airways. These findings need confirmation and a caution interpretation of their clinical significance, as most effects are non-specific and likely reversible.

HBM4EU Chromates Study-Genotoxicity and Oxidative Stress Biomarkers in Workers Exposed to Hexavalent Chromium.

A study was conducted within the European Human Biomonitoring Initiative (HBM4EU) to characterize occupational exposure to Cr(VI). Herein we present the results of biomarkers of genotoxicity and oxidative stress, including micronucleus analysis in lymphocytes and reticulocytes, the comet assay in whole blood, and malondialdehyde and 8-oxo-2′-deoxyguanosine in urine. Workers from several Cr(VI)-related industrial activities and controls from industrial (within company) and non-industrial (outwith company) environments were included. The significantly increased genotoxicity (p = 0.03 for MN in lymphocytes and reticulocytes; p < 0.001 for comet assay data) and oxidative stress levels (p = 0.007 and p < 0.001 for MDA and 8-OHdG levels in pre-shift urine samples, respectively) that were detected in the exposed workers over the outwith company controls suggest that Cr(VI) exposure might still represent a health risk, particularly, for chrome painters and electrolytic bath platers, despite the low Cr exposure. The within-company controls displayed DNA and chromosomal damage levels that were comparable to those of the exposed group, highlighting the relevance of considering all industry workers as potentially exposed. The use of effect biomarkers proved their capacity to detect the early biological effects from low Cr(VI) exposure, and to contribute to identifying subgroups that are at higher risk. Overall, this study reinforces the need for further re-evaluation of the occupational exposure limit and better application of protection measures. However, it also raised some additional questions and unexplained inconsistencies that need follow-up studies to be clarified.

Novel biomonitoring of early biological effects upon benzene low-level exposure

Novel biomonitoring of early biological effects upon benzene low-level exposure

Occupational exposure and markers of genetic damage, systemic inflammation and lung function: a Danish cross-sectional study among air force personnel

Air force ground crew personnel are potentially exposed to fuels and lubricants, as raw materials, vapours and combustion exhaust emissions, during operation and maintenance of aircrafts. This study investigated exposure levels and biomarkers of effects for employees at a Danish air force military base. We enrolled self-reported healthy and non-smoking employees (n = 79) and grouped them by exposure based on job function, considered to be potentially exposed (aircraft engineers, crew chiefs, fuel operators and munition specialists) or as reference group with minimal occupational exposure (avionics and office workers). We measured exposure levels to polycyclic aromatic hydrocarbons (PAHs) and organophosphate esters (OPEs) by silicone bands and skin wipes (PAHs only) as well as urinary excretion of PAH metabolites (OH-PAHs). Additionally, we assessed exposure levels of ultrafine particles (UFPs) in the breathing zone for specific job functions. As biomarkers of effect, we assessed lung function, plasma levels of acute phase inflammatory markers, and genetic damage levels in peripheral blood cells. Exposure levels of total PAHs, OPEs and OH-PAHs did not differ between exposure groups or job functions, with low correlations between PAHs in different matrices. Among the measured job functions, the UFP levels were higher for the crew chiefs. The exposure level of the PAH fluorene was significantly higher for the exposed group than the reference group (15.9 ± 23.7 ng/g per 24 h vs 5.28 ± 7.87 ng/g per 24 h, p = 0.007), as was the OPE triphenyl phosphate (305 ± 606 vs 19.7 ± 33.8 ng/g per 24 h, p = 0.011). The OPE tris(1,3-dichlor-2-propyl)phosphate had a higher mean in the exposed group (60.7 ± 135 ng/g per 24 h) compared to the reference group (8.89 ± 15.7 ng/g per 24 h) but did not reach significance. No evidence of effects for biomarkers of systemic inflammation, genetic damage or lung function was found. Overall, our biomonitoring study show limited evidence of occupational exposure of air force ground crew personnel to UFPs, PAHs and OPEs. Furthermore, the OH-PAHs and the assessed biomarkers of early biological effects did not differ between exposed and reference groups.

Effects of air pollution on chlorophyll content and morphology of lichens transplanted around a paper industry (Morelia, Mexico)

We evaluated and compared the physiological and morphological response to pollution of two epiphytic lichen species, the foliose lichen Flavopunctelia praesignis and the fruticose lichen Usnea ceratina. Lichen samples were collected at remote areas and transplanted at different distances and directions from a paper industry in Morelia (Michoacán, Mexico). Lichen transplants were exposed for 4 months (1) around the industrial area and (2) in their native habitats (control sites). Changes of total chlorophyll content between samples before and after exposure, and morphological damage in the lichen thalli were investigated. Lichens showed species-specific responses. Flavopunctelia praesignis increased in total chlorophyll content after exposure around the industrial area and in control site. This suggests that total chlorophyll changes by a seasonal effect than pollution effect. On the other hand, chlorophyll content of U. ceratina did not change significantly after exposure. Bleaching, changes in color, deformations, and necrosis of lichen thalli were better visible in U. ceratina near the paper industry. We conclude that U. ceratina is a more pollution-sensitive species than F. praesignis. Morphological damage in the lichen thalli would be a suitable indicator for monitoring early biological effects of air pollution caused by the paper industry.

HBM4EU chromates study - Reflection and lessons learnt from designing and undertaking a collaborative European biomonitoring study on occupational exposure to hexavalent chromium.

The EU human biomonitoring initiative, HBM4EU, aims to co-ordinate and advance human biomonitoring (HBM) across Europe. As part of HBM4EU, we presented a protocol for a multicentre study to characterize occupational exposure to hexavalent chromium (Cr(VI)) in nine European countries (HBM4EU chromates study). This study intended to collect data on current occupational exposure and to test new indicators for chromium (Cr) biomonitoring (Cr(VI) in exhaled breath condensate and Cr in red blood cells), in addition to traditional urinary total Cr analyses. Also, data from occupational hygiene samples and biomarkers of early biological effects, including genetic and epigenetic effects, was obtained, complementing the biomonitoring information. Data collection and analysis was completed, with the project findings being made separately available. As HBM4EU prepares to embark on further European wide biomonitoring studies, we considered it important to reflect on the experiences gained through our harmonised approach. Several practical aspects are highlighted for improvement in future studies, e.g., more thorough/earlier training on the implementation of standard operating procedures for field researchers, training on the use of the data entry template, as well as improved company communications. The HBM4EU chromates study team considered that the study had successfully demonstrated the feasibility of conducting a harmonised multicentre investigation able to achieve the research aims and objectives. This was largely attributable to the engaged multidisciplinary network, committed to deliver clearly understood goals. Such networks take time and investment to develop, but are priceless in terms of their ability to deliver and facilitate knowledge sharing and collaboration.

Environmental Genotoxicity Assessment Using Micronucleus (and Nuclear Abnormalities) Test on Intertidal Mussel Perumytilus purpuratus: A Tool for Biomonitoring the Chilean Coast.

The release of pollutants is increasing along the coast of Chile, and the use of biomarkers in biomonitoring programs is essential to assess the early biological effects of marine contamination. The Micronucleus (MN) test was carried out using hemocytes of the mussel Perumytilus purpuratus from two sites in northern-central Chile (La Pampilla and Totoralillo). Nuclear abnormalities were assessed, and high frequencies of micronucleus (10.7-14.4‰) and other abnormalities were found (51.9-76.6‰). These values tended to be higher in La Pampilla, possibly due to the large presence of pollution sources in that site. However, considerably high values were observed in both sites. P. purpuratus is a suitable bioindicator and further monitoring along the Chilean coast using this species should be developed using the MN test and/or other biomarkers to comprehend the effects of human activities on the coastal environment of Chile.

The Gut Microbial Community Structure of the North American River Otter (Lontra canadensis) in the Alberta Oil Sands Region in Canada: Relationship with Local Environmental Variables and Metal Body Burden

The Alberta Oil Sands Region in Canada is home to one of the largest oil bitumen deposits in the world. The North American river otter (Lontra canadensis) is a top predator with a small home range and is sensitive to disturbances; it has been designated as a sentinel species for the potential impacts of the natural resource exploitation on freshwater ecosystems in the Alberta Oil Sands Region. With an increasing interest in noninvasive biomarkers, recent studies suggest that gut microbiota can be used as a potential biomarker of early biological effects on aquatic wildlife. The goal of the present study was to determine the river otter gut microbial structure related to environmental variables characterizing mining activities and metal body burden. We obtained 18 trapped animals from and surrounding the surface mineable area of the Alberta Oil Sands Region. The gut microbial community structure was characterized using high-throughput sequencing of 16S rRNA gene amplicon analyses. Trace metal concentrations in the liver were measured by inductively coupled plasma-mass spectrometry. Our study revealed that the gut bacteria of river otters in the Alberta Oil Sands Region clustered in 4 groups dominated by Peptostreptococcaceae, Carnobacteriaceae, Enterobacteriaceae, Clostridiaceae, and Nostocaceae. We show that arsenic, barium, rubidium, liver-body weight ratio, and δ15 N were associated with each cluster. When comparing affected versus less affected sites, we show that river otter gut bacterial community and structure are significantly related to trophic level of the river otter but not to Alberta Oil Sands Region mining activities. Our study reveals that the gut bacterial dynamics can provide insights into the diet and habitat use of river otters but that more work is needed to use it as a pollution biomarker. Environ Toxicol Chem 2020;39:2516-2526. © 2020 SETAC.

A comparison of the early therapeutic effects of allogeneic bone marrow-derived mesenchymal stem cells and calcitonin on the healing of surgically induced mandibular bone defects in osteoporotic rats

ObjectivesThis study aimed to evaluate and compare the early biological effects of allogeneic bone marrow-derived mesenchymal stem cells (BMSCs) versus salmon calcitonin (SC) on healing of surgically induced mandibular bone defects in osteoporotic rats. MethodsSixty-one female albino rats were included in this study, four of them were used for BMSCs isolation. The remaining 57 rats were divided into 4 groups. Group I (negative control), 12 rats received a vehicle injection after which a unilateral mandibular defect was created in each rat. Osteoporosis was induced in the remaining 45 rats then rats were randomly allocated into 3 equal groups (15 each). Surgical defects were created as in group I. The defects were left to heal spontaneously in group II; positive control. While in group III each defect was filled with an absorbable hemostatic gelatin sponge loaded by 10 IU of injectable SC and in group IV the sponge was seeded by 0.5 × 106 BMSCs. Rats were euthanized at 1st, 2nd, and 4th week postsurgically. Hematoxylin and eosin, Masson’s trichrome, picrosirius, and alizarin red s stains were used, followed by statistical analysis. ResultsBMSCs-treatment showed marked enhanced bone healing. Moreover, collagen fibers and calcium deposits area percentages were statistically significantly higher when compared to the other groups particularly at 2 and 4 weeks. ConclusionsLocal application of bone marrow-derived mesenchymal stem cells and salmon calcitonin may be an effective therapy for treatment of osteoporotic bone defects, with privilege to the stem cells in terms of quantity and quality of regenerated bone.

Early genotoxic effects from exposure to environmental pollutants young people from South Italy

Abstract Children and young people are particularly sensitive to the environmental pollution which is closely related to degenerative diseases. Several studies show that a genotoxic damage during young age can increase the risk of chronic diseases in adulthood. The young people are more vulnerable than adults to the environmental pollutants because they spend more time outdoors, they have immaturity of some organs and of the mechanisms involved in the cellular repair. In the present study, the early biological effects of exposure to a particularly polluted area of Southern Italy were evaluated in 200 children (6-10 year-old) and 100 young people (18-25 year-old). This area, worldwide known as Sarno basin, is characterized by strong anthropization, many agro-food processing industries, massive use of fertilizers and pesticides in agricultural practices and a strong river pollution. The comet assay was chosen because it reflects cumulative exposure to a variety of environmental factors and it was performed on salivary leukocytes in the children selected for the survey, while in the young people the DNA damage was evaluated in human peripheral blood lymphocytes. As in previous studies were not find significant differences between salivary leukocytes and blood lymphocytes we preferred the sampling of saliva for the children to avoid bloody practices. Furthermore, before cell sampling the children's parents were interviewed using an ad hoc questionnaire designed to gather additional information about exposure sources. A questionnaire was administered also to the young people to have more information on their lifestyle and some characteristic of the area of exposure (vehicular traffic and so on). The results showed a clear damage from exposure in the children differently from young people. Key messages Comet assay was performed in vitro on lymphocytes of 200 children (6-10 year-old) and 100 young people (18-25 year-old) exposed to a particularly polluted area of Southern Italy. An evident DNA damage was observed in lymphocytes coming from children; no genetic material alterations were observed in young people.