LOW-MODERATE DRINKING WATER ARSENIC CONTAMINATION, CIGARETTE SMOKE, AND ADVERSE PREGNANCY OUTCOMES IN TIMIS COUNTY, ROMANIA-AMONRA

Project title:  LOW-MODERATE DRINKING WATER ARSENIC CONTAMINATION, CIGARETTE SMOKE, AND ADVERSE PREGNANCY OUTCOMES IN TIMIS COUNTY, ROMANIA

ACRONIM: AMOnRA (Arsenic Molecular Outcomes Risk Assessment)

Period: 03.2021-02.2024

Budget: 1200000

Project team members

  • Prof. Dr. Gurzău Eugen – Director proiect
  • Dr. Neamțiu Iulia
  • Prof. Bloom Michael
  • Dr. Gati Gabriel
  • Ing. Ch. Pop Cristian
  • Prof. Dr. Berindan Neagoe Ioana
  • Dr. Braicu Cornelia, CSII
  • 2 poziții vacante

Summary

Adverse pregnancy outcomes, including spontaneous loss, lower birth weight, and smaller birth size have been reported in association with use of groundwater contaminated by inorganic arsenic (iAs) above 10 µg/L, the current World Health Organization maximum contamination limit. The goal of our proposed project is to determine if such adverse pregnancy outcomes are increased in association with exposure to a mixture of cigarette smoke and low-moderate level iAs contamination in drinking water. The proposed project will fundamentally advance our knowledge of how low-moderate dose iAs exposure may increase risks for adverse pregnancy outcomes among women exposed to cigarette smoke and will identify potentially modifiable risk factors on which we can act to reduce associated health risks. This is an important public health concern, given the widespread distribution of drinking water iAs contamination and cigarette smoking in some populations. Our proposed project would be the only study to examine whether use of low-moderate iAs contaminated drinking water sources may increase risks for adverse pregnancy outcomes solely and in conjunction with cigarette smoke exposure. It is also the first to examine the relevance of genetic and nutritional factors as modifiers of these associations.

Project aim:

Our main goal is to understand the risks of low-moderate drinking water iAs exposure on pregnancy outcomes, and to identify factors that predispose women to iAs-associated reproductive toxicity in regions of low-moderate groundwater contamination.

Objectives:

  1. Use biomarkers of exposure to cigarette smoke and iAs to confirm and better define interaction effects on adverse pregnancy outcomes we detected in our previous pilot study.
  2. Perform an exploratory assessment of biomarkers of genetic susceptibility to identify key

 

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Expected results:

Expected results we anticipate, include: 1) biomarker confirmation of the interaction between cigarette smoke exposure and low-moderate drinking water inorganic arsenic (iAs) on adverse pregnancy outcomes; and 2) preliminary identification of genetic factors that predispose women to reproductive toxicity in association with low-moderate drinking water iAs exposure. When completed, our expectation is our study will provide data to develop interventions for reducing adverse pregnancy outcomes in populations with low-moderate drinking water iAs and cigarette smoke exposures.

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Work packages and timeline

Year Work packages and tasks Estimated results
2021 Work package 1 – Development of conceptual model and operational procedures, analyses of the biological specimens and design of the databases (part I)
Task 11 – Chemical analysis method development and elaboration of standard operation procedures for arsenic (As) biomonitoring

R 1.1 Standard operation procedures for arsenic biomonitoring

R 1.2 Acquisition procedures

Task 1.2 – Genetic analysis method development and elaboration of standard operation procedures for genetic assessment

R 1.3 Standard operation procedures for genetic assessment

R 1.4 Web page design

Task 13 – Development of the risk assessment model R 1.5 Risk assessment model
Task 1.4 – Determination of total and speciated As and cotinine in urine and analysis of blood Se, validation and quality control

R 1.6 Databases with As and Se results

R 1.7 Phase report

2022 Work package 2 – Analyses of the biological specimens and design of the databases (part II), development of the statistical models and data analyses (part I)
Task 2.1 – Genetic analysis of the biological specimens, validation and quality control R 2.1 Databases with genetic analysis results
Task 2.2 – Design of the databases and integration of the biomarkers in our previous study database

R 2.2 Integrated databases with all results 

Task 2.3 – Human As biomonitoring and exposure model

R 2.3 Arsenic biomonitoring and exposure model

R 2.4 Protocol for the statistical and toxicological analysis of the data

R 2.5 Preliminary results report

2023 Work package 3 – Development of the statistical models and data analyses (part II), dissemination of the results and development of future intervention strategies (part I)
Task 3.1 – Data management and analysis R 3.1 Databases and preliminary results report
Task 3.2 Interindividual genetic polymorphism in pregnant women exposed to arsenic via drinking water
Task 3.3 – Complex and integrated analysis of the final results by modelling, spatial distribution and GIS technics
Task 3.4 – Results dissemination (part I)

R 3.2 Abstracts presented at professional meetings

R 3.3 Articles published in national and international peer-reviewed journals

Task 3.5 – Translating science into policy – elaboration and development of intervention strategies

R 3.4 Intervention strategies

R 3.5 Phase report

2024 Etapa IV - Dissemination of the results and development of future intervention strategies (part II)
Task 4.1 – Results dissemination (part II)

R 4.1 Abstracts presented at professional meetings

R 4.2 Articles published in national and international peer-reviewed journals

R 4.3 Final report

 

Work package 2021

Results

Study participants and biological specimen collection

Study participants selection (pregnant women who lost the pregnancy (cases) and pregnant women who carried the pregnancy and delivered their babies (controls)), and the collection of biological specimens (urine and blood) from the participants, was performed in a previously conducted pilot study funded by the National Institute of Environmental Health Sciences (NIEHS) in the USA. The biological specimens were collected and stored in a biobank, at -20 ºC, to be processed and analyzed in this project.

Processing and analysis of the biological specimens

Total arsenic (As) and As species were determined in 20 of the urine samples collected by High Performance Liquid Chromatography - Inductively Coupled Plasma Mass Spectrometry (HPLC-ICPMS).

Screenshot 2021 11 15 at 15.33.47

În 20 din probele de urină colectate s-a determinat arsenul (As) total și speciile de As prin tehnica cu plasmă cuplată inductiv cu spectrometrie de masă combinată cu cromatografie lichidă de înaltă performanță (High Performance Liquid Chromatography - Inductively Coupled Plasma Mass Spectrometry (HPLC-ICPMS)).

Screenshot 2022 02 02 at 08.53.55

Summary of work package 1

Adverse pregnancy outcomes, including spontaneous loss, lower birth weight, and smaller birth size have been reported in association with use of groundwater contaminated by inorganic arsenic (iAs) above 10 µg/L, the current World Health Organization maximum contamination limit. The goal of our proposed project is to determine if such adverse pregnancy outcomes are increased in association with exposure to a mixture of cigarette smoke and low-moderate level iAs contamination in drinking water.

Work package 1 conducted during the year 2021, included the conceptual model of risk assessment when exposed to iAs in drinking water, standard operating procedures and partial results of the biological specimen analysis, and the design of databases. The study has a case-control epidemiological design.

The risk assessment model for metal exposure includes: a) defining the geographical scale at which the assessment will be performed; b) identification of potentially exposed population groups and subgroups with increased susceptibility; c) characterization of the pathways and routes of exposure; d) description of how the exposure will be assessed; e) determining how the hazard and dose response of the receptor will be assessed; f) description of way by which the risk will be characterized.

Human internal exposure to an environmental contaminant may be assessed by analyzing a sample from a biological fluid or tissue. This analysis aims to determine the substance itself, its metabolites, enzymes or other biological substances or responses influenced by that specific contaminant, representing what we refer to as biomarkers or indicators of internal dose. The role of biomarkers is to summarize complex information in a simplified and useful form, and to facilitate the identification of a status, trends, and correlations.

In the particular case of iAs exposure, the biomarkers that we are measuring in this project are the total urinary As and As species, using the High Performance Liquid Chromatography - Inductively Coupled Plasma Mass Spectrometry (HPLC-ICPMS) technique. We also determine the cotinine in urine, a metabolite of nicotine commonly used as a biomarker for exposure to cigarette smoke, by Liquid Cromatography - Mass Spectrometry (LC-MS) și and the blood selenium (Se) as an indicator of the nutritional status, using the ICP-MS technique. In order to provide a high degree of certainty as regards the analytical results, a series of validation parameters, imposed by national and international forums in the field, were determined (by experimental and statistical analysis) (e.g. selectivity, specificity; limit of detection, limit of quantification; work domain and linearity; sensitivity; accuracy; accuracy expressed in repeatability and reproducibility conditions; recovery; robustness; measurement uncertainty).

In 20% of the analyzed samples, the total As determined in urine exceeded the reference value of 15 µg/l.

The main area of interest in many biological and pharmacological studies is the detection of mutations for the diagnosis of genetic diseases and cancer. Many diseases have, in addition to germline mutations, somatic mutations that are not inherited and are not passed on to offspring. DPCR LNA Mutation assays with the QIAcuity dPCR platform provide accurate detection of low-frequency mutations found in a sample with a large number of healthy cells. Partitions for the wild-type (WT) and mutant (MUT) sequences in the dPCR reaction allow the MUT to be separated from the WT sequence, leading to positive partitions and amplification of the PCR signal in the dPCR, but also to the correct estimation of the concentration based on the presence or absence of the signal.

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