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Funding Information: This work was supported by the European Union's Horizon 2020 research and innovation program projects HBM4EU [grant number 733032 ]. Authors acknowledge the editorial assistance of Richard Davies. Publisher Copyright: © 2023 The Author(s) 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. publishersversion published

The proposed HBM4EU effort to assess human adverse health effects due to environmental chemicals exposure using biomarkers of effect, has continued with the implementation of these indicators, as described in D14.8. In the HBM4EU aligned studies, a broad panel of classical and novel biomarkers of effect (e.g. brain-derived neurotrophic function, kisspeptin, reproductive hormones, thyroid hormones, metabolic markers, oxidative stress markers, among others) has been implemented. However, some proposed biomarkers have not been validated and implemented, such as leptin and adiponectin. These important cytokines play a key role in the proper development of metabolic and reproductive function. Additionally, the relationships between exposure and effect biomarkers data in the HBM4EU aligned studies have also not been reported. Therefore, the aims of this deliverable were to show: i) the quality control and quality analysis of leptin and adiponectin, as well as their measured concentrations in the aligned studies; ii) the physiological validation of kisspeptin (serum protein concentrations, kiss54, and whole blood DNA methylation of the KISS1 gene), in a pilot study within the INMA-Granada cohort, to investigate its suitability as effect biomarker of the reproductive function prior to their application in HBM4EU aligned studies; and iii) the preliminary results of the exposure-effect analyses conducted in the Aligned studies. For this purpose, leptin and adiponectin protein levels were measured using different commercial enzymelinked immunosorbent assays (ELISA). The new quality control procedure for leptin measurements showed that the chosen ELISA kit was a reproducible and confident analytical technique. Adiponectin was then measured in adolescent’s serum samples from the aligned studies, including the FLEHS, PCB, BEA, and SLO CRP studies; as well as in children from the SLO CRP study. Leptin concentrations were also measured in adolescents from the FLEHS studies and in children from the SLO CRP studies. On the other hand, preliminary data with INMA-Granada blood and serum samples demonstrate the physiological feasibility of implementing new biomarkers of reproductive function, such as KISS, at different levels of biological organisation. The results showed that percentages of KISS1 DNA methylation were inversely correlated with the serum concentrations of kiss54 protein, as well as with the serum concentrations of the selected reproductive hormones (FSH, estradiol, testosterone, SHBG). Moreover, levels of kiss54 were positively correlated with all reproductive hormones. Thus, the pilot study with samples from INMA-Granada demonstrated the technical feasibility of implementing kisspeptin at different levels of biological organisation to assess the reproductive function in the HBM4EU aligned studies. Finally, when the kisspeptin effect biomarker (serum kiss54) data were analysed together with serum concentrations of reproductive hormones in the aligned studies, consistent results with the findings described in the pilot study were found, indicating that the novel effect biomarker KISS is a valuable marker of reproductive function useful for assessing possible alterations caused by environmental pollutants.