Development of toxicogenomics applications for screening and risk assessment when exposed to contaminants (arsenic, lead and cadmium) in the environmental media

ACRONYM: PROMETHEuS (Predicted Risk of Metal Exposure Toxicogenomics Health and Environmental Screening)

Time period: 23.06.2022-22.06.2024

No. of institutions involved: two – Environmental Heath Center (CMS) (Coordinator) and Iuliu Hațieganu University of Medicine and Pharmacy (UMF) (Partner)

Total funding (lei): 1.450.251,00

Abstract

Toxic metals such as arsenic (As), cadmium (Cd) and lead (Pb) are widely found in the environment, households and at the workplaces, contaminating food and drinking water. This project is based on the concept of toxicogenomics, which aims to understand and predict toxicity data using transcriptomics data (microRNA). The main objective of this project is to develop a toxicological prediction model to be used in health risk assessment, a public priority at national and international level, to be commercially exploited on the domestic and international market. Thus, we aim to explore, develop and exploit the full potential of toxicogenomics in order to advance in in vitro tests based on mechanisms using human cells, to develop a toxicological model and also, to identify new biomarkers of susceptibility in exposure to toxic metals, to be used in population studies.

The element of originality and innovation of this project is given by the way in which emerging knowledge and associated tools may be used to improve research in the field of toxicogenomics, to be used in human health risks assessment. The uniform integration of the generated data (in vitro and patient biological samples) will allow a deep understanding of the interaction mechanisms at the cellular and molecular level using microRNA as biomarkers of susceptibility in exposure to toxic metals.

Objectiv

The main goal of the study is: development of a toxicological prediction model based on toxicogenomics data, by determining biomarkers of susceptibility (miRNAs and their target genes) to be used in the assessment of health risks associated with the exposure to specific environmental contaminants (As, Pb, Cd).

Expected results: 1) Identification of new biomarkers of susceptibility in exposure to specific contaminants (As, Pb, Cd), to be used in population studies; 2) Development of a unique panel of miRNAs and the most relevant target genes, validated for each contaminant; 3) Development of a common panel to be used for screening in exposure to investigated contaminants (As, Pb, Cd).

Work Package 1 (2022)

Results

Populational group and collection of the biological specimens – Study participants selection (women undergoing an in vitro fertilization procedure (IVF)), and the collection of biological specimens, was performed in a previously conducted pilot study. The biological specimens were collected and stored in a biobank, at -20 ºC and -80 ºC, to be processed and analyzed in this project, in order to determine the biomarkers of exposure to metals (As, Pb, Cd) using the High Performance Liquid Chromatography – Inductively Coupled Plasma Mass Spectrometry (HPLC-ICPMS)) technique. A metal exposure assessment questionnaire was developed and filled out.

Biotoxicological analysis – Biotoxicological analysis protocols of As, Pb and Cd were also developed, as well as the design of experimental toxicological study on cell lines, microRNA analysis protocol, and at the same time, the UMF partner team carried out the procedures for the acquisition of consumables and laboratory reagents in order to conduct the experimental study on cell lines and microRNA analysis (expected to be carried out in Work package 2/2023).

Work Package 2 (2023)

Results

Work package 2 includes the analysis of biological specimens, the creation of databases and the statistical processing of the biological samples analysis results (exposure and biomonitoring data), the bioinformatics processing (genomics data, molecular biomarkers) and the development of the metal biodistribution model within the complex analysis of Identification of specific molecular signatures to be integrated into the risk assessment when exposed to arsenic, lead and cadmium (part II).
A total of 47 urine samples (in which total As and Cd were determined), and also a number of 47 blood samples (in which Pb was determined) were analysed.

In approximately 19% of the analysed samples, the total As concentration levels determined in urine exceeded the reference value of 15 µg/l. Also, in approximately 15% of the analysed samples, Cd concentration levels determined in urine exceeded the reference value of 2 µg/l. The Pb concentrations determined in the blood were within the range of normal values (below 10 µg/dl).
The pharmacogenomics study aimed to identify specific molecular signatures, providing detailed insight into how these metals can influence gene expression (using microarray technology) and contribute to associated human health risks.
The bioinformatic analysis methods applied in this study allowed the exploration of subtle changes at the cellular level, but also of the significant ones at the transcriptional level. An altered molecular profile was identified as a result of exposure to selected metals.

WORK PACKAGE 3 (2024)                                                                                               

Results                                                                                                               

Work package 3 includes the validation of the molecular signature and the development of the conceptual model of consumer exposure.

The conceptual model for risk assessment includes: a) defining the geographical scale at which the assessment is conducted; b) identification of potentially exposed population groups and subgroups with increased susceptibility; c) characterization of methods and ways of exposure; d) description of how the exposure is evaluated; e) determining how hazard and dose response are assessed; f) description of how the risk is characterized.

Results (presented as cluster) of the analysis of follicular fluid samples for patients diagnosed with idiopathic infertility (IDIO) and, respectively, for those with Pelvic Inflammatory Disease (PID) and Polycystic Ovary Syndrome (Polycystic Ovary Syndrome PCOS) versus the control group

The evaluation of the miRNA profile using the microarray technique (Agilent technology) in women diagnosed with infertility of idiopathic cause (IDIO) and, respectively, in women diagnosed with pelvic inflammatory disease (PID) and polycystic ovary syndrome (PCOS) provides a deeper understanding of the molecular mechanisms involved in these disorders. Total RNA was isolated from the follicular fluid samples collected from the study participants. The bioinformatics analysis identified 21 differently expressed miRNAs, of which six overexpressed and 15 underexpressed, in the case of PID patients; 25 differently expressed miRNAs, of which 16 overexpressed and 9 underexpressed, in the case of PCOS patients; 34 differently expressed miRNAs, of which 10 overexpressed and 24 underexpressed, in the case of patients with idiopathic infertility (IDIO). Bioinformatics software (Ingenuity Pathway Analysis-IPA), specifically, prediction algorithms of the miRNA targets and network analyzes were used to validate the predicted interactions between miRNAs and target genes, and to confirm and prioritize relevant interactions. The miRNAs identified as being of interest were included in three molecular networks. The graphical representation of the most relevant molecular network facilitated the identification of the nodal genes, the type of interaction between them and their direct or indirect interaction products.

The identification of the miRNAs that are overexpressed or underexpressed compared to the control groups, allows the identification of key molecules involved in the pathogenesis of the investigated infertility disorders.

Project main results (2022-2024)

The assessment of the impact of toxic agents in the environment require a systematic approach to understand distribution, exposure, toxicity and potential risks to human health. In this regard, a model of exposure to cadmium (Cd) and lead (Pb) was developed using two normal colon cell lines, and the changes at the genomic and transcriptomic level were evaluated.

Based on the analysis of the molecular data, specific signatures of metal exposure were identified on each individual cell line. The heatmap representation in the figure below indicates important transcriptomic changes associated with lead exposure on an epithelial cell line (CCD-18Co).

The complex approach, from the point of view of human biotoxicology (biomarkers of exposure), genetics/genomics (from experimental models of exposure to Pb and Cd, to genomic analysis of biological samples) and last but not least, an advanced analytical component in the evaluation of the relationship/association between the exposure and the observed effects, have generated new and promising results for the development of surveillance and prevention programs and for obtaining information necessary for the development of new, advanced treatment methods/techniques.

The evaluation of the miRNA profile using the microarray technique (Agilent technology) in women diagnosed with infertility of idiopathic cause and, respectively, in women diagnosed with pelvic inflammatory disease and polycystic ovary syndrome provides a deeper understanding of the molecular mechanisms involved in these disorders. Analysis of the expression changes of these miRNAs and their target genes provides important information about how these molecules may affect certain mechanisms and relevant biological processes. These experimental data are extremely useful in providing opportunities for an improved diagnosis and prognosis of disorders associated with metal exposure (arsenic (As), Pb, Cd). Also, these results open the way for the development of personalized therapies that specifically target the molecular pathways which are affected in each type of disorder.