Identification of chemicals of emerging concern in urine of Flemish adolescents using a new suspect screening workflow for LC-QTOF-MS.

An essential step in human biomonitoring or molecular epidemiology programs is to estimate human exposure to environmental chemicals. Despite significant progress in the capabilities of analytical methods, the number of pollutants and their metabolites keeps increasing continuously. Some of these relatively unknown chemicals of emerging concern (CECs) may pose significant health risks to humans and biota, but remain virtually undetected in traditional HBM-studies. Non-target and suspect screening techniques based on high-resolution mass spectrometry (HRMS) perform the detection and identification of compounds without any a priori compound selection or chemical information and provide a more holistic overview of human exposure. In this study, 50 urine samples (25 female and 25 male) from a larger cohort of the Flemish Environment and Health Study (FLEHS IV, 2016-2020) have been submitted to suspect screening analysis, with the aim of detecting and identifying new CECs. For this purpose, an analytical method has been developed, optimised and evaluated in terms of analytical performance. Satisfactory results were obtained in terms of reproducibility, sensitivity and quality control. Data-mining was performed through the combination of two different workflows. The use of two complementary workflows enhanced the number of identified compounds. As a result, 45 CECs have been identified with a level of confidence ranged between 3 and 1. Most of the identified compounds were metabolisation products, many of which were currently not included in the targeted measurements of FLEHS IV. The identified chemicals and metabolites could be used as candidate biomarkers of exposure in future studies. Overall, the newly developed suspect screening workflow of this pilot study provided complementary and promising results for future HBM-programs.

Neurobehavioural and cognitive effects of prenatal exposure to organochlorine compounds in three year old children.

BACKGROUND: We report data of a Belgian observational prospective cohort study regarding cognitive and behavioural development until the age of 36 months in relation to internal exposure to organochlorine pollutants [sum of polychlorinated biphenyls (sum PCB), dioxin-like activity, PCB118, PCB170, hexachlorobenzene (HCB) and p,p'-dichlorodiphenyldichloroethylene (DDE)] measured in cord blood. METHODS: Participants were recruited as part of an Flemish Environmental Health Survey (2002-2006). Two hundred and six mother-child pairs were recruited. Hundred twenty five toddlers [Reynell Taal Ontwikkelings Schalen (language development, RTOS), Snijders-Oomen Niet-verbale intelligentietest (non-verbal intelligence, SON), Bayley Scales, milestones, Infant Behaviour Questionnaire (IBQ), gender specific play behaviour, Neurobehavioral Evaluation System (NES)-attentional task] and their mothers [Home Observation Measurement of the Environment (HOME), Wechsler Abbreviated Scale of Intelligence (WASI), State-Trait Anxiety Inventory (STAI), general questionnaires] were tested. Statistical analysis was performed with the SPSS program. Much attention was paid to confounding factors. RESULTS: In the first years of development, higher organochlorine pollutants were associated with less active children (delayed crawling: sum PCB*HCB (p < 0.05), sumPCB*DDE (p < 0.1); delayed first steps alone: sum PCB (p < 0.5), PCB118 (p < 0.01), PCB170 (p < 0.01), HCB (p < 0.01); less switching between toys: sum PCB (p < 0.01); less switching between toys in boys: PCB118 (p < 0.01), sum PCB(p < 0.01)). At 12 months children with higher dioxin-like activity tended to show less fear responses(p < 0.1) (IBQ 12 months). At 36 months, a slower development of language comprehension (RTOS) was related to all organochlorine exposure parameters(p < 0.1 or p < 0.05) except DDE. Lower nonverbal IQ scores (SON) were related to PCB118 in boys only(p < 0.05 or p < 0.01). Less masculine and more non-gender specific play behaviour was associated with sum PCB in boys and girls at 36 months(p < 0.1). Moreover, PCB118 (p < 0.05), PCB170 (p < 0.1), HCB(p < 0.05) and DDE(p < 0.05) were associated with diminished masculine play behaviour in boys. CONCLUSION: Our data confirm the observations that neurobehavioral development of young children is adversely influenced by environmental concentrations of PCBs, especially in boys. In this context, observation of play behaviour seems to be a reliable, easy to perform and sensitive test to detect neurotoxic effects of chemicals like PCB's and dioxin-like compounds in very young children. On the basis of our results, we hypothesize that an underarrousal pattern may play a role in the spectrum of effects measured in toddlers prenatally exposed to PCBs and dioxin-like compounds.

Exposure levels, determinants and risk assessment of organophosphate flame retardants and plasticizers in adolescents (14-15 years) from the Flemish Environment and Health Study.

The ubiquitous use of organophosphate flame retardants and plasticizers (PFRs) in a variety of consumer products has led to widespread human exposure. Since certain PFRs are developmental and carcinogenic toxicants, detailed exposure assessments are essential to investigate the risk associated with environmental exposure levels. However, such data are still lacking for European countries. In this study, concentrations of thirteen PFR metabolites were measured in urine samples from 600 adolescents from Flanders, Belgium. 1-Hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate (BCIPHIPP), diphenyl phosphate (DPHP), bis(1,3-dichloro-isopropyl) phosphate (BDCIPP), 2-hydroxyethyl bis(2-butoxyethyl) phosphate (BBOEHEP), 2-ethylhexyl phenyl phosphate (EHPHP) and 2-ethyl-5-hydroxyhexyl diphenyl phosphate (5-HO-EHDPHP) were frequently detected (>83%) in all participants. Comparisons with study populations from outside the EU showed that urinary levels of DPHP, BDCIPP and BCIPHIPP were generally within the same range. Only exposure to 2-ethylhexyl diphenyl phosphate (EHDPHP) was presumably higher in Flemish adolescents. However, determinants analysis through multivariate regression analyses did not reveal significant predictors that may explain this finding. Significantly higher levels of BDCIPP were observed in participants with new decorations at home, while adolescents with highly educated parents had higher levels of BBOEHEP and BDCIPP. Furthermore, multiple PFR metabolite concentrations followed a seasonal pattern. Estimated daily intakes (EDIs) were calculated from the internal dose by including fractions of urinary excretion (FUE) estimated in in vitro metabolism studies. EDIs ranged from 6.3 ng/kg bw/day for TBOEP to 567.7 ng/kg bw/day for EHDPHP, which were well below the available oral reference doses for all investigated PFRs. This suggests that the associated risk is low at present. This is the first report on internal exposure to seven commonly used PFRs in a European population.

Arsenic speciation in fish and shellfish from the North Sea (Southern bight) and Açu Port area (Brazil) and health risks related to seafood consumption.

In North Sea and Port Açu (Brazil) coastal areas, high arsenic (As) concentrations were observed in water, soil and sediments. Therefore, the impact of this contamination on fish and shellfish species bought from local fishermen was studied. Total As was assessed with ICP-OES (Brazil) and ICP-MS (North Sea) after microwave digestion. Toxic As was assessed with liquid chromatography-ICP-MS (Brazil) and hydride generation-atomic fluorescence spectrometry (North Sea). All analytical methods comply with Quality Assurance/Quality Control procedures. Several fish species have average Total As concentrations above 1 µg g-1 wet weight (ww), but the highest concentrations are found in less spotted dogfish, lemon sole and whelks from the North Sea, with respectively 50, 49 and 50 µg g-1 ww. High Total As concentrations correspond to high Toxic As concentrations, except for scallops having increased Toxic As concentrations. Toxic As fractions are highest in scallops (almost 10%) but rarely exceeds 2% in all other species. Liver samples were only analyzed in ray, dogfish and catfish and their Toxic As fractions are between 2 and 4 times higher than in muscle. For a consumption of 150 g of seafood, only 3 samples exceed the provisional total daily intake of 2 µg kg-1 bw, however, cancer risks are non-negligible. Using mean Toxic As concentrations for each of the different fish and shellfish species studied, Lifetime Cancer Risk values at the actual global seafood consumption rate of 54 g day-1 are above 10-4 for whelks, scallops, dogfish, ray and lemon sole.

Human exposure to endocrine disrupting chemicals and fertility: A case-control study in male subfertility patients.

BACKGROUND: Dioxins, PCBs, chlorinated pesticides, brominated flame retardants, bisphenol A, triclosan, perfluorinated compounds and phthalates are known as endocrine disrupting chemicals (EDCs). OBJECTIVES: The aim of our study was to investigate whether higher exposure to EDCs is associated with increased subfertility in men. METHODS: We measured biomarkers of exposure in 163 men, recruited through four fertility clinics. According to WHO guidelines, we used a total motility count (TMC) of 20 million as cut-off value. We assigned patients to the case group when two semen samples - collected at least one week apart - had a TMC<20 and to the control group when both samples had a TMC≥20. To estimate the risk of subfertility and alteration in sex hormone concentrations we used multivariable-adjusted analysis, using logistic and linear regressions, respectively. RESULTS: For an IQR increase in serum oxychlordane, the odds ratio for subfertility was 1.98 (95% CI: 1.07; 3.69). Furthermore, men with serum levels of BDE209 above the quantification limit had an odds of 7.22 (1.03; 50.6) for subfertility compared with those having values below the LOQ. Urinary levels of phthalates and triclosan were negatively associated with inhibin B and positively with LH. Urinary bisphenol A correlated negatively with testosterone levels. CONCLUSIONS: Our study in men showed that internal body concentrations of endocrine disrupting chemicals are associated with an increased risk of subfertility together with alterations in hormone levels. The results emphasize the importance to reduce chemicals in the environment in order to safeguard male fertility.

Causes of genome instability: the effect of low dose chemical exposures in modern society.

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome's integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.

Internal exposure to organochlorine pollutants and cadmium and self-reported health status: a prospective study.

In this paper, based on the Flemish biomonitoring programs, we describe the associations between internal exposure to organochlorine pollutants and to cadmium (measured in 2004-2005 for adults aged 50-65 years) and self-reported health status obtained through a questionnaire in November 2011. Dioxin-like activity in serum showed a significant positive association with risk of cancer for women. After adjustment for confounders and covariates, the odds ratio for an exposure equal to the 90th percentile was 2.4 times higher than for an exposure equal to the 10th percentile. For both men and women dioxin-like activity and serum hexachlorobenzene (HCB) showed a significant positive association with risk of diabetes and of hypertension. Detailed analysis suggested that an increase in BMI might be part of the mechanism through which HCB contributes to diabetes and hypertension. Serum dichlorodiphenyldichloroethylene (p,p'-DDE) concentration showed a significant positive association with diabetes and hypertension in men, but not in women. Serum polychlorinated biphenyl (PCB) 118 showed a significant positive association with diabetes in both men and women, and after adjustment for correlated exposures, also with hypertension in men. Urinary cadmium concentrations showed a significant positive association with hypertension. Urinary cadmium concentrations were (in 2004-2005) significantly higher in persons who felt in less than good health (in 2011) than in persons who felt in very good health. After adjustment for correlated exposures (to HCB, p,p'-DDE and PCB118) marker PCBs showed a significant negative association with diabetes and hypertension. Serum p,p'-DDE showed in men a significant negative association with risk of diseases based on atheromata. Our findings suggest that exposure to pollutants can lead to an important increase in the risk of diseases such as cancer, diabetes and hypertension. Some pollutants may possibly also decrease the risk of some health problems, although this requires confirmation by other approaches.

Trace metal concentrations measured in blood and urine of adolescents in Flanders, Belgium: reference population and case studies Genk-Zuid and Menen.

In the Flemish human biomonitoring programme FLEHS II (2007-2011) trace metals (Cd, Pb, Cr, Ni, Cu, Mn, Tl, Sb, As and toxic relevant arsenic) were analysed in the blood and urine of adolescents (14-15 years old) in the reference population in Flanders and in areas of important industrial activities: Genk-Zuid and Menen. After adjustment of the results for confounding factors, the adolescents living in Genk-Zuid had higher levels of Cr, Cu and Tl in blood, higher levels of Cd and toxic relevant arsenic (TRA) in urine, but lower levels of Ni in blood and Sb in urine compared to the reference population. In Menen higher levels of Cd and Cu in urine, higher concentrations of Tl in blood but lower concentrations of Pb in blood and lower Ni, Sb and As in urine were found compared to the reference population. For both the reference population and the hot spots the concentrations are within the ranges found in other countries. Compared to the previous biomonitoring programme FLEHS I (2002-2006) a decrease in the concentrations of Cd and especially of Pb in blood was observed. However, it cannot be excluded that differences between the two campaigns are partially due to different sampling strategies.

Prenatal exposure to environmental contaminants and behavioural problems at age 7-8years.

Animal studies showed that the developing brain is particularly sensitive to chemical exposure. Human studies carried out in areas with high exposures have proven neurodevelopmental disorders in relation to e.g. lead and PCBs. Whether these chemicals are associated with behavioural problems in childhood at current environmental levels is not well known. Therefore, we assessed the association between prenatal exposure to lead, cadmium, PCBs, dioxin-like compounds, HCB and p,p'-DDE and behavioural problems in 7-8year old children. Prenatal exposure data were obtained from the Flemish mother-new-born cohort. Lead, cadmium, PCBs, dioxin-like compounds, HCB and p,p'-DDE were analysed in cord blood. When the child reached 7-8years, 270 mothers completed the Strengths and Difficulties Questionnaire assessing their children's behavioural health. We found that doubling the prenatal lead exposure (cord blood lead levels) was associated with a 3.43 times higher risk for hyperactivity in both boys and girls. In addition, total difficulties were 5.08 times more likely in the highest tertile for prenatal lead exposure compared to the lowest tertile. In girls, total difficulties were 4.92 more likely when doubling cord blood p,p'-DDE, whereas no significant association was found in boys. Further, we noted in boys a 1.53 times higher risk for emotional problems when doubling cord blood cadmium, whereas no significant association was found in girls. These results indicate that the presence of environmental contaminants influences the mental health of the next generation.

Opening the research agenda for selection of hot spots for human biomonitoring research in Belgium: a participatory research project.

BACKGROUND: In order to select priority hotspots for environment and health research in Flanders (Belgium), an open procedure was organized. Environment and health hotspots are strong polluting point sources with possible health effects for residents living in the vicinity of the hot spot. The selection procedure was part of the work of the Flemish Centre of Expertise for Environment and Health, which investigates the relation between environmental pollution and human health. The project is funded and steered by the Flemish government. METHODS: The involvement of other actors than merely experts is inspired by the 'analytical-deliberative' approach of the National Research Council in the United States and the extended peer community approach. These approaches stress the importance of involving different expert- and social perspectives in order to increase the knowledge base on complex issues. In the procedure used in the project a combination of expert and stakeholder input was essential. The final decision was supported by a multi-criteria analysis of expert assessment and stakeholder advice. RESULTS: The endeavour was challenging from the start because of the complicated ambition of including a diversity of actors, potential hotspots, concerns and assessment criteria, but nevertheless the procedure proved its value in both structuring and informing the decision-making process. Moreover the process gained the support of most actors participating in the process, even though the final selection could not satisfy all preferences. CONCLUSIONS: Opening the research agenda exemplifies the value of inter- and transdisciplinary cooperation as well as the need for a well-structured and negotiated procedure that combines relevant factors and actors with pragmatism. The value of such a process also needs to prove itself in practice after the procedure has been completed: the tension between an ambition of openness on the one hand and a more closed attitude amongst experts on the other will continue to play a role even after closure.

The relation between the estimated dietary intake of PCDD/Fs and levels in blood in a Flemish population (50-65 years).

Dioxin-like activity was measured in the serum of 1425 Flemish men and women via the CALUX assay. The adults, aged between 50 and 65 years, participated in a large biomonitoring program, executed by the Flemish Center of Expertise for Environment and Health between 2002 and 2006. Within the context of this biomonitoring program also dietary intake of dioxin-like contaminants was assessed through a food frequency questionnaire. The relation between the estimated dietary intake and the dioxin-like activity in serum was evaluated using multivariate analyses: a logistic model was performed on the total population, while a linear regression analysis was done on the subsample with quantifiable dioxin activity levels in serum. Region, gender, age, BMI, smoking status, as well as dietary habits were entered in the model, with dioxin level as an outcome estimate. Both the logistic and linear model confirmed the contribution of dietary intake to the dioxin activity measured in serum. Also BMI and region were found to be associated with dioxin activity levels.

Pollutant effects on genotoxic parameters and tumor-associated protein levels in adults: a cross sectional study.

BACKGROUND: This study intended to investigate whether residence in areas polluted by heavy industry, waste incineration, a high density of traffic and housing or intensive use of pesticides, could contribute to the high incidence of cancer observed in Flanders. METHODS: Subjects were 1583 residents aged 50-65 from 9 areas with different types of pollution. Cadmium, lead, p,p'-DDE, hexachlorobenzene, PCBs and dioxin-like activity (Calux test) were measured in blood, and cadmium, t,t'-muconic acid and 1-hydroxypyrene in urine. Effect biomarkers were prostate specific antigen, carcinoembryonic antigen and p53 protein serum levels, number of micronuclei per 1000 binucleated peripheral blood cells, DNA damage (comet assay) in peripheral blood cells and 8-hydroxy-deoxyguanosine in urine. Confounding factors were taken into account. RESULTS: Overall significant differences between areas were found for carcinoembryonic antigen, micronuclei, 8-hydroxy-deoxyguanosine and DNA damage. Compared to a rural area with mainly fruit production, effect biomarkers were often significantly elevated around waste incinerators, in the cities of Antwerp and Ghent, in industrial areas and also in other rural areas. Within an industrial area DNA strand break levels were almost three times higher close to industrial installations than 5 kilometres upwind of the main industrial installations (p < 0.0001). Positive exposure-effect relationships were found for carcinoembryonic antigen (urinary cadmium, t,t'-muconic acid, 1-hydroxypyrene and blood lead), micronuclei (PCB118), DNA damage (PCB118) and 8-hydroxy-deoxyguanosine (t,t'-muconic acid, 1-hydroxypyrene). Also, we found significant associations between values of PSA above the p90 and higher values of urinary cadmium, between values of p53 above the p90 and higher serum levels of p,p'-DDE, hexachlorobenzene and marker PCBs (PCB 138, 153 and 180) and between serum levels of p,p'-DDE above the p90 and higher serum values of carcinoembryonic antigen. Significant associations were also found between effect biomarkers and occupational or lifestyle parameters. CONCLUSION: Levels of internal exposure, and residence near waste incinerators, in cities, or close to important industries, but not in areas with intensive use of pesticides, showed positive correlations with biomarkers associated with carcinogenesis and thus probably contribute to risk of cancer. In some rural areas, the levels of these biomarkers were not lower than in the rest of Flanders.

Endocrine disruptors and abnormalities of pubertal development.

Onset and development of puberty is regulated by the neuroendocrine system. Population-based studies worldwide have observed secular trends towards earlier puberty development. These changes are apparently caused by environmental factors such as improved socio-economic status, improved health care and nutrition. However, they may also partly result from endocrine-disrupting chemicals in the environment. Epidemiological studies have investigated the relationship between pubertal development and exposure to endocrine-disrupting chemicals (polychlorinated biphenyls, polybrominated biphenyls, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, phthalate esters, furans and the pesticide endosulfan). Associations with both perinatal and postnatal exposure have been reported. Studies in experimental animals support some of these findings and point to differential endocrine regulatory mechanisms linked to pubertal development acting in the perinatal and the pre-pubertal period. Pubertal development is naturally associated with growth and body composition. There is increasing evidence for a link between prenatal development and pubertal onset. In girls born small for gestational age (SGA), pubertal onset and age at menarche often are advanced, especially if there has been an extensive catch-up growth during the first months of life. In utero growth retardation may have multiple causes including exposure to xenobiotic substances as was suggested for some endocrine-disrupting chemicals. An abnormal perinatal environment of children born SGA may alter the endocrine status and the sensitivity of the receptors for endocrine and metabolic signalling that may have effects on maturation of brain and gonads. However, the causal pathways and the molecular mechanisms that may link the pubertal growth pattern of children born SGA, pubertal development and endocrine-disrupting chemicals need further study.

The need to develop centers for environmental oncology.

The scale and scope of the cancer problem in the United States today is much greater than four decades ago when the formal war against the disease was first announced. Patterns of the disease are not fully explained by known risk factors. Much progress has been made in understanding the molecular basis of carcinogenesis, particularly the near consensus (realization) that virtually all cancers arise from an accumulation of genetic mutations and the more recent recognition of the role of inflammation and the tissue microenvironment, in particular for hormone-dependent cancers. However, most genetic mutations that contribute to cancer are not inherited, and thus must be attributable to accumulation of somatic mutations and epigenetic changes, from as yet poorly understood environmental factors, that certainly cannot be explained entirely by tobacco, use and arise over the course of a lifetime. Much of the national effort to control cancer has focused on detecting and treating the disease--not on seeking approaches to prevent cases from arising. Given this reality, we present a cross-disciplinary framework for establishing comprehensive research and policy centers focused on environmental oncology to be based at selected academic cancer centers across the country. The principal goal of such centers is to improve the ability to prevent cancer, by developing effective interventions based on insights obtained from epidemiology, including molecular epidemiology and basic scientific research on genomic, metabolomic, and other biomarkers of exposure, susceptibility, and disease. As the needed scientific evidence for environmental factors contributing to cancer is revealed, these academic centers will develop specific interventions and/or policy recommendations regarding ways to lower the burden of cancer, based on existing information about cancer hazards in the personal, occupational, and general environment. Ultimately the centers will improve the ability to identify and control the underlying causes of the occurrence of cancer and its progression.

Endogenous DNA damage in humans: a review of quantitative data.

DNA damage plays a major role in mutagenesis, carcinogenesis and ageing. The vast majority of mutations in human tissues are certainly of endogenous origin. A thorough knowledge of the types and prevalence of endogenous DNA damage is thus essential for an understanding of the interactions of endogenous processes with exogenous agents and the influence of damage of endogenous origin on the induction of cancer and other diseases. In particular, this seems important in risk evaluation concerning exogenous agents that also occur endogenously or that, although chemically different from endogenous ones, generate the same DNA adducts. This knowledge may also be crucial to the development of rational chemopreventive strategies. A list of endogenous DNA-damaging agents, processes and DNA adduct levels is presented. For the sake of comparison, DNA adduct levels are expressed in a standardized way, including the number of adducts per 10(6) nt. This list comprises numerous reactive oxygen species and products generated as a consequence (e.g. lipid peroxides), endogenous reactive chemicals (e.g. aldehydes and S-adenosylmethionine), and chemical DNA instability (e.g. depurination). The respective roles of endogenous versus exogenous DNA damage in carcinogenesis are discussed.

Immunologic and other biological parameters as a function of smoking status and of residence in areas differing in terms of air pollution.

In the industrial region of La Louvière (Belgium), healthy persons, presenting at a consultation of preventive medicine, showed differences as a function of smoking and of residence in one of four areas differing in sources of emissions, population density and green zones. Smokers had significantly (P/=2.25, significantly so for the Power Station-Landfill area. A non-significant but consistent trend towards higher lymphocyte and CD8 lys counts and higher complement C3c serum levels further suggested an association between residence in a more polluted area and immunologic features. The number of CD3+CD56+ lys microl(-1), reported to be increased in cancer patients, showed a negative correlation with the CD4/CD8 ratio (r(2)=0.132, P<0.0001).