## 4.3.11. Molecular epidemiology - I. Human biomonitoring

Author: Marja Lamoree

Reviewers: Michelle Plusquin and Adrian Covaci

Learning objectives:

You should be able to

- explain the purpose of human biomonitoring
- understand that the internal dose may come from different exposure routes
- describe the different steps in analytical methods and to clarify the specific requirements with regard to sampling, storage, sensitivity, throughput and accuracy
- clarify the role of metabolism in the distribution of samples in the human body and specify some sample matrices
- explain the role of ethics in human biomonitoring studies

Keywords: chemical analysis, human samples, exposure, ethics, cohort

Human biomonitoring

Human biomonitoring (HBM) involves the assessment of human exposure to natural and synthetic chemicals by the quantitative analysis of these compounds, their metabolites or reaction products in samples from human origin. Samples used in HBM can include blood, urine, faces, saliva, breast milk and sweat or other tissues, such as hair, nails and teeth.

The concentrations determined in human samples are a reflection of the exposure of an individual to the compounds analysed, also referred to as the internal dose. HBM data are collected to obtain insight into the population’s exposure to chemicals, often with the objective to integrate them with health data for health impact assessment in epidemiological studies. Often, specific age groups are addressed, such as neonates, toddlers, children, adolescents, adults and elderly. Human biomonitoring is an established method in occupational and environmental exposure assessment.

In several countries, HBM studies have been conducted for decades already, such as the German Environment Survey (GerES) and the National Health and Nutrition Examination Survey (NHANES) program in the United States. HBM programs may sometimes be conducted under the umbrella of the World Health Organization (WHO). Other examples are the Canadian Health Measures Survey, the Flemish Environment and Health Study and the Japan Environment and Children’s Study, the latter specifically focuses on young children. Children are considered to be more at risk for the adverse health effects of early exposure to chemical pollutants, because of their rapid growth and development and their limited metabolic capacity to detoxify harmful chemicals.

Table 1. Information sources for Human Biomonitoring (HBM) programmes

Programme | Internet link
German Environment Survey (GerES) | www.umweltbundesamt.de/en/topics/health/assessing-environmentally-related-health-risks/german-environmental-survey-geres
National Health and Nutrition Examination Survey (NHANES) | https://www.cdc.gov/nchs/nhanes/index.htm
WHO | www.euro.who.int/en/data-and-evidence/environment-and-health-information-system-enhis/activities/human-biomonitoring-survey
Canadian Health Measures Survey (CHMS) | http://www23.statcan.gc.ca/imdb/p2SV.pl?Function=getSurvey&Id=148760
Japan Environment and Children’s Study (JECS) | https://www.env.go.jp/en/chemi/hs/jecs/

Studies focusing on the impact of exposure to chemicals on health are conducted with the use of cohorts: groups of people that are enrolled in a certain study and volunteer to take part in the research program. Usually, apart from donating e.g. blood or urine samples, health measures, such as blood pressure, body weight, hormone levels, etc. but also data on diet, education, social background, economic status and lifestyle are collected, the latter through the use of questionnaires. A cross-sectional study aims at the acquisition of exposure and health data of the whole (volunteer) group at a defined moment, whereas in a longitudinal study follow-up studies are conducted with a certain frequency (i.e. every few years) in order to follow and evaluate the changes in exposure, describe time trends as well as study health and lifestyle on the longer term (see section on Environmental Epidemiology). To obtain sufficient statistical power to derive meaningful relationships between exposure and eventual (health) effects, the number of participants in HBM studies is often very large, i.e. ranging to 100,000 participants.

Because a lot of (sometimes sensitive) data is gathered from many individuals, ethics is an important aspect of any HBM study. Before starting a certain study involving HBM, a Medical Ethical Approval Committee needs to approve it. Applications to obtain approval require comprehensive documentation of i) the study protocol (what is exactly being investigated), ii) a statement regarding the safeguarding of the privacy and collected data of the individuals, the access of researchers to the data and the safe storage of all information and iii) an information letter for the volunteers explaining the aim of and procedures used in the study and their rights (e.g. to withdraw), so that they can give consent to be included in the study.

Chemical absorption, distribution, metabolism and excretion

Because chemicals often undergo metabolic transformation (see section on Xenobiotic metabolism and defence) after entering the body via ingestion, dermal absorption and inhalation, it is important to not only focus on the parent compound (= the compound to which the individual was exposed), but also include metabolites. Diet, socio-economic status, occupation, lifestyle and the environment all contribute to the exposure of humans, while age, gender, health status and weight of an individual define the effect of the exposure. HBM data provide an aggregation of all the different routes through which the individual was exposed. For an in-depth investigation of exposure sources, however, chemical analysis of e.g. diet (including drinking water), the indoor and outdoor environment are still necessary. Another important source of chemicals to which people are exposed in their day to day life are consumer products, such as electronics, furniture, textiles, etc., that may contain flame retardants, stain repellents, colorants and dyes, preservatives, among others.

The distribution of a chemical in the body is highly dependent on its physico-chemical properties, such as lipophilicity/hydrophilicity and persistence, while also phase I and Phase II transformation (see section on Xenobiotic metabolism and defence) play a determining role, see Figure. 1. For lipophilic compounds (e.g. section on POPs) storage occurs in fat tissue, while moderately lipophilic to hydrophilic compounds are excreted after metabolic transformation, or in unchanged form. Based on these considerations, a proper choice for sampling of the appropriate matrix can be made, i.e. some chemicals are best measured in urine, while for others blood may be better suitable.

Figure 1. Distribution biotransformation of a compound (xenobiotic) in the body leading to storage or excretion.

For the design of the sampling campaign, the properties of the compounds to be analyzed should be taken into account. In case of volatility, airtight sampling containers should be used, while for light-sensitive compounds amber coloured glassware is the optimal choice.

Ideally, after collection, the samples need to be stored under the correct conditions as quickly as possible, in order the avoid degradation caused by thermal instability or by biodegradation caused by remaining enzyme activity in the sample (e.g. in blood or breast milk samples). Labeling and storage of the large quantities of samples generally included in HBM studies are important parts of the sampling campaign (see for video: https://www.youtube.com/watch?v=FQjKKvAhhjM).

Chemical analysis of human samples for exposure assessment

Typically, for the determination of the concentrations of compounds to which people are exposed and the corresponding metabolites formed in the human body, analytical techniques such as liquid and gas chromatography (LC and GC, respectively) coupled to mass spectrometry (MS) are applied. Chromatography is used for the separation of the compounds, while MS is used to detect the compounds. Prior to the analysis using LC- or GC-MS, the sample is pretreated (e.g. particles are removed) and extracted, i.e. the compounds to be analysed are concentrated in a small volume while sample matrix constituents that may interfere with the analysis (e.g. lipids, proteins) are removed, resulting in an extract that is ready to be injected onto chromatographic system.

In Figure 2 a schematic representation is given of all steps in the analytical procedure.

Figure 2. Schematic representation of the analytical procedure typically used for the quantitative determination of chemicals and their metabolites in human samples.

The analytical methods to quantify concentrations of chemicals in order to assess human exposure need to be of a high quality due to the specific nature of HBM studies. The compounds to be analysed are usually present in very low concentrations (i.e. in the order of pg/L for cord blood), and the sample volumes are small. For some matrices, the small sample volumes is dictated by the fact that sample availability is not unlimited, e.g. for blood. Another factor that governs the limited sample volume available is the costs that are related to the requirement of dedicated, long term storage space at conditions of -20 ⁰C or even -80 ⁰C to ensure sample integrity and stability.

The compounds on which HBM studies often focus are those to which we are exposed in daily life. This implies that the analytical procedure should be able to deal with contamination of the sample with the compounds to be analysed, due to the presence of the compounds in our surroundings. Higher background contamination leads to a decreased capacity to detect low concentrations, thus negatively impacting the quality of the studies. Examples of compounds that have been monitored frequently in human urine are phthalates, such as diethyl hexyl phthalate or shortly DEHP. DEHP is a chemical used in many consumer products and therefore contamination of the samples with DEHP from the surroundings severely influences the analytical measurements. One way around this is to focus on the metabolites of DEHP after Phase I or II metabolism: this guarantees that the chemical has passed the human body and has undergone a metabolic transformation, and its detection is not due to contamination from the background, which results in a more reliable exposure metric. When the analytical method is designed for the quantitative analysis of metabolites, an enzymatic step for the deconjugation of the Phase II metabolites should be included (see section on Xenobiotic metabolism and defence).

Because the generated data, i.e. the concentrations of the compounds in the human samples, are used to determine parameters like average/median exposure levels, the detection frequency of specific compounds and highest/lowest exposure levels, the accuracy of the measurements should be high. In addition, analytical methods used for HBM should be capable of high throughput, i.e. the time needed per analysis should be low, because of the large numbers of samples that are typically analysed, in the order of a hundred to a few thousand samples, depending on the study.

Summarizing, HBM data support the assessment of temporal trends and spatial patterns in human exposure, sheds light on subpopulations that are at risk and provides insight into the effectiveness of measures to reduce or even prevent adverse health effects due to chemical exposure.

Background information:

HBM4EU project info: www.hbm4eu.eu; video: https://www.youtube.com/watch?v=DmC1v6EAeAM&feature=youtu.be.