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

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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

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.

Work packages and timeline

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).

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)).

Scientific Report of work package 1 - 2021

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.

Scientific Report of work package 2 - 2022

Work package 2 comprises the analysis of biological specimens and database design (part II), and the development of statistical models and data analysis (part I).

The analyzed population sample included 300 participants (150 cases and 150 controls) with ages between 18 and 44 years, but the biochemical analyzes could only be performed in the case of participants from whom biological samples could be collected. A total of 227 urine samples were analyzed in which arsenic (As), As species and cotinine were determined, and 204 blood samples were analyzed for Se concentrations.

The databases were created in Microsoft Excel, and then, the information was imported into STATA 17 statistical package, for the statistical processing to be conducted.

In approximately 19% of the analyzed samples, the total As concentration determined in urine exceeded the reference value of 15 µg/l.

Graphical representation of median, 25th and 75th percentile, minimum and maximum concentrations of urinary As species and blood Se (µg/l)

Selenium (Se) concentrations determined in blood fell within the normal values range (except for 8 values). Values considered normal for the Se in the blood are in the interval of 50-120 µg/l, values below 40 µg/l usually indicating a Se deficiency associated with the loss of glutathione-peroxidase activity. Within the investigated sample, no values below 40 μg/l were recorded and only eight of the participants had values above 120 μg/l.

Most of the non-smoking patients had urinary cotinine values below 5 μg/l (limit of detection for the analytical method). Urinary cotinine levels were statistically significantly correlated with active smoking since pregnant, and passive smoking in the house, respectively.
Total As concentrations (µg/l) measured in urine (as a biomarker of As exposure) were positively and statistically significantly associated with the level of inorganic As concentrations (µg/l) measured in both primary and secondary drinking water sources, both being used for water consumption by the study participants.

DNA was obtained for all tested samples and dPCR analysis was performed for five "Single Nucleotide Polymorphism" (SNP), for all tested samples. Of the five SNPs analyzed, rs3740400 was the most frequently identified in the analyzed samples, followed by rs1046778 and rs3740393. The least frequently identified SNP of the five tested, is rs3740390.