Human Population Studies Research Articles

Mitochondrial Dysfunction in Obesity and Reproduction.

Mounting evidence suggests a role for mitochondrial dysfunction in the pathogenesis of many diseases, including type 2 diabetes, aging, and ovarian failure. Because of the central role of mitochondria in energy production, heme biosynthesis, calcium buffering, steroidogenesis, and apoptosis signaling within cells, understanding the molecular mechanisms behind mitochondrial dysregulation and its potential implications in disease is critical. This review will take a journey through the past and summarize what is known about mitochondrial dysfunction in various disorders, focusing on metabolic alterations and reproductive abnormalities. Evidence is presented from studies in different human populations, and rodents with genetic manipulations of pathways known to affect mitochondrial function.

Removing reference bias and improving indel calling in ancient DNA data analysis by mapping to a sequence variation graph

BackgroundDuring the last decade, the analysis of ancient DNA (aDNA) sequence has become a powerful tool for the study of past human populations. However, the degraded nature of aDNA means that aDNA molecules are short and frequently mutated by post-mortem chemical modifications. These features decrease read mapping accuracy and increase reference bias, in which reads containing non-reference alleles are less likely to be mapped than those containing reference alleles. Alternative approaches have been developed to replace the linear reference with a variation graph which includes known alternative variants at each genetic locus. Here, we evaluate the use of variation graph software vg to avoid reference bias for aDNA and compare with existing methods.ResultsWe use vg to align simulated and real aDNA samples to a variation graph containing 1000 Genome Project variants and compare with the same data aligned with bwa to the human linear reference genome. Using vg leads to a balanced allelic representation at polymorphic sites, effectively removing reference bias, and more sensitive variant detection in comparison with bwa, especially for insertions and deletions (indels). Alternative approaches that use relaxed bwa parameter settings or filter bwa alignments can also reduce bias but can have lower sensitivity than vg, particularly for indels.ConclusionsOur findings demonstrate that aligning aDNA sequences to variation graphs effectively mitigates the impact of reference bias when analyzing aDNA, while retaining mapping sensitivity and allowing detection of variation, in particular indel variation, that was previously missed.

Hepatitis E Virus Infection: Is It Really a Problem in Latin America?

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Optimizing the genetic prediction of the eye and hair color for North Eurasian populations

BackgroundPredicting the eye and hair color from genotype became an established and widely used tool in forensic genetics, as well as in studies of ancient human populations. However, the accuracy of this tool has been verified on the West and Central Europeans only, while populations from border regions between Europe and Asia (like Caucasus and Ural) also carry the light pigmentation phenotypes.ResultsWe phenotyped 286 samples collected across North Eurasia, genotyped them by the standard HIrisPlex-S markers and found that predictive power in Caucasus/Ural/West Siberian populations is reasonable but lower than that in West Europeans. As these populations have genetic ancestries different from that of West Europeans, we hypothesized they may carry a somewhat different allele spectrum. Thus, for all samples we performed the exome sequencing additionally enriched with the 53 genes and intergenic regions known to be associated with the eye/hair color. Our association analysis replicated the importance of the key previously known SNPs but also identified five new markers whose eye color prediction power for the studied populations is compatible with the two major previously well-known SNPs. Four out of these five SNPs lie within the HERС2 gene and the fifth in the intergenic region. These SNPs are found at high frequencies in most studied populations. The released dataset of exomes from Russian populations can be further used for population genetic and medical genetic studies.ConclusionsThis study demonstrated that precision of the established systems for eye/hair color prediction from a genotype is slightly lower for the populations from the border regions between Europe and Asia that for the West Europeans. However, this precision can be improved if some newly revealed predictive SNPs are added into the panel. We discuss that the replication of these pigmentation-associated SNPs on the independent North Eurasian sample is needed in the future studies.

The Genetic Architecture of Carbon Tetrachloride-Induced Liver Fibrosis in Mice.

Background & AimsLiver fibrosis is a multifactorial trait that develops in response to chronic liver injury. Our aim was to characterize the genetic architecture of carbon tetrachloride (CCl4)-induced liver fibrosis using the Hybrid Mouse Diversity Panel, a panel of more than 100 genetically distinct mouse strains optimized for genome-wide association studies and systems genetics.MethodsChronic liver injury was induced by CCl4 injections twice weekly for 6 weeks. Four hundred thirty-seven mice received CCl4 and 256 received vehicle, after which animals were euthanized for liver histology and gene expression. Using automated digital image analysis, we quantified fibrosis as the collagen proportionate area of the whole section, excluding normal collagen.ResultsWe discovered broad variation in fibrosis among the Hybrid Mouse Diversity Panel strains, demonstrating a significant genetic influence. Genome-wide association analyses revealed significant and suggestive loci underlying susceptibility to fibrosis, some of which overlapped with loci identified in mouse crosses and human population studies. Liver global gene expression was assessed by RNA sequencing across the strains, and candidate genes were identified using differential expression and expression quantitative trait locus analyses. Gene set enrichment analyses identified the underlying pathways, of which stellate cell involvement was prominent, and coexpression network modeling identified modules associated with fibrosis.ConclusionsOur results provide a rich resource for the design of experiments to understand mechanisms underlying fibrosis and for rational strain selection when testing antifibrotic drugs.

Methodological Issues in Population-Based Studies of Multigenerational Associations.

Laboratory-based animal research has revealed a number of exposures with multigenerational effects-ones that affect the children and grandchildren of those directly exposed. An important task for epidemiology is to investigate these relationships in human populations. Without the relative control achieved in laboratory settings, however, population-based studies of multigenerational associations have had to use a broader range of study designs. Current strategies to obtain multigenerational data include exploiting birth registries and existing cohort studies, ascertaining exposures within them, and measuring outcomes across multiple generations. In this paper, we describe the methodological challenges inherent to multigenerational studies in human populations. After outlining standard taxonomy to facilitate discussion of study designs and target exposure associations, we highlight the methodological issues, focusing on the interplay between study design, analysis strategy, and the fact that outcomes may be related to family size. In a simulation study, we show that different multigenerational designs lead to estimates of different exposure associations with distinct scientific interpretations. Nevertheless, target associations can be recovered by incorporating (possibly) auxiliary information, and we provide insights into choosing an appropriate target association. Finally, we identify areas requiring further methodological development.

Abstract B48: Ovarian cancer risk in laying hens is reduced by dietary polyunsaturated fatty acids: Implications for soluble E-cadherin, de novo lipogenesis, and mitochondrial metabolism

Abstract Aim: The specific aim of our research is to understand the relationship between soluble E-cadherin (sE-cad) expression, de novo lipogenesis, and mitochondrial metabolism in ovarian tumors from White Leghorn laying hens, in response to enrichment with diets that contain high levels of polyunsaturated fatty acids (PUFAs). Background: The shedding of the 80 kd extracellular domain of E-cadherin (E-cad) causes the production of sE-cad, which is implicated as a putative ligand for growth, survival, and angiogenesis in solid tumors. Little is known with regard to the relationship between sE-cad production and de novo lipogenesis or mitochondrial metabolism in ovarian tumors. De novo lipogenesis has been argued to be indispensable for their growth and proliferation. Vast physiologic research indicates that de novo lipogenesis is inhibited by PUFAs, indicating that dietary PUFAs could provide negative feedback to an essential metabolic pathway in ovarian tumors. Epidemiologic studies of human populations indicate that ovarian cancer (OvCa) risk is reduced 30-40% by high dietary consumption of omega-3 or omega-6 PUFAs. Hypothesis: We hypothesize that high dietary PUFA consumption downregulates de novo lipogenesis in ovarian tumors of laying hens, correlating with reduced sE-cad production and increased mitochondrial oxidative phosphorylation (OXPHOS) capacity. Methods: We used qPCR to measure genes related to de novo lipogenesis and mitochondrial OXPHOS. Gas chromatography was used measure the fatty acid content in ovarian tumors. Western blot was used to measure E-cad protein fragments. Results: Our lab observed a 20-40% reduction of OvCa risk in laying hens that were supplemented with whole flaxseed, flaxseed oil, corn oil or fish oil. qPCR results indicate reduced expression of genes related to de novo lipogenesis (i.e., Fatty Acid Synthase). Gas chromatography results indicate that the content of C16:0, C18:0 and C18:1 fatty acids is similarly reduced by dietary PUFA enrichment. qPCR results also indicate increased expression of the catalytic subunits for complexes 3, 4, and 5 of the mitochondrial electron transport chain when diets are enriched with PUFAs. Lastly, although the expression of E-cad protein was unaffected by diet, we did detect a significant reduction of the 37 kd carboxy-terminal fragment of E-cad (CTF1-Ecad) in the fish oil diet. This is an important finding, because CTF1-E-cad is the complementary fragment of sE-cad. Conclusions: OvCa risk in hens is reduced by consumption of omega-3 and omega-6 PUFAs, and these risk reductions associate with reduced de novo lipogenesis, reduced CTF1-E-cad expression, and improved mitochondrial OXPHOS capacity. This study reveals an additional anti-ovarian cancer mechanism of a flaxseed-supplemented diet. (NIH-RO1AT005295) Citation Format: Chris Weston, Matthew Young, Karen Hales, Dale Buchanan Hales. Ovarian cancer risk in laying hens is reduced by dietary polyunsaturated fatty acids: Implications for soluble E-cadherin, de novo lipogenesis, and mitochondrial metabolism [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B48.

Serum dioxin levels and sperm DNA methylation age: Findings in Vietnam war veterans exposed to Agent Orange

Exposure to dioxin, a known endocrine disruptor and carcinogen, is associated with poor reproductive outcomes. Yet, few studies have explored the role of DNA methylation in these relationships. Utilizing a publicly available dataset from 37 male Air Force Health Study participants exposed to dioxin-contaminated Agent Orange during the Vietnam war, we cross-sectionally examined the relationship of serum dioxin levels with a novel DNA methylation-based measure of sperm age (DNAm-agesperm). DNAm-agesperm was calculated using CpG sites on the Illumina HumanMethylation450 BeadChip. We estimated associations of dioxin levels with DNAm-agesperm using linear regression models adjusted for chronological age, body mass index, and smoking status. Chronological age was highly correlated with DNAmagesperm (r = 0.80). In fully-adjusted linear models, a one percent increase in serum dioxin levels was significantly associated with a 0.0126-year (i.e. 4.6-day) increase in DNAm-agesperm (95%CI: 0.003, 0.022, p = 0.01). Further analyses demonstrated significant negative associations of dioxin levels (β = −0.0005, 95%CI: −0.0010, 0.00004, P = 0.03) and DNAm-agesperm (β = −0.02, 95%CI: −0.04, −0.001, P = 0.03) with methylation levels of FOXK2 – a gene previously reported to be hypomethylated in infertile men. In sum, we demonstrate associations of dioxin with increased methylation aging of sperm. DNAm-agesperm may provide utility for understanding how dioxin levels impact sperm health and potentially male reproductive capacity in human population studies. Moreover, our pilot study contributes further evidence that some environmental toxicants are associated with methylation aging. Additional studies are necessary to confirm these findings, and better characterize dioxin and sperm methylation relationships with male reproductive health.

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) and their effects on the ovary.

BACKGROUNDPerfluoroalkyl and polyfluoroalkyl substances (PFAS) are found widespread in drinking water, foods, food packaging materials and other consumer products. Several PFAS have been identified as endocrine-disrupting chemicals based on their ability to interfere with normal reproductive function and hormonal signalling. Experimental models and epidemiologic studies suggest that PFAS exposures target the ovary and represent major risks for women’s health.OBJECTIVE AND RATIONALEThis review summarises human population and toxicological studies on the association between PFAS exposure and ovarian function.SEARCH METHODSA comprehensive review was performed by searching PubMed. Search terms included an extensive list of PFAS and health terms ranging from general keywords (e.g. ovarian, reproductive, follicle, oocyte) to specific keywords (including menarche, menstrual cycle, menopause, primary ovarian insufficiency/premature ovarian failure, steroid hormones), based on the authors’ knowledge of the topic and key terms.OUTCOMESClinical evidence demonstrates the presence of PFAS in follicular fluid and their ability to pass through the blood–follicle barrier. Although some studies found no evidence associating PFAS exposure with disruption in ovarian function, numerous epidemiologic studies, mostly with cross-sectional study designs, have identified associations of higher PFAS exposure with later menarche, irregular menstrual cycles, longer cycle length, earlier age of menopause and reduced levels of oestrogens and androgens. Adverse effects of PFAS on ovarian folliculogenesis and steroidogenesis have been confirmed in experimental models. Based on laboratory research findings, PFAS could diminish ovarian reserve and reduce endogenous hormone synthesis through activating peroxisome proliferator-activated receptors, disrupting gap junction intercellular communication between oocyte and granulosa cells, inducing thyroid hormone deficiency, antagonising ovarian enzyme activities involved in ovarian steroidogenesis or inhibiting kisspeptin signalling in the hypothalamus.WIDER IMPLICATIONSThe published literature supports associations between PFAS exposure and adverse reproductive outcomes; however, the evidence remains insufficient to infer a causal relationship between PFAS exposure and ovarian disorders. Thus, more research is warranted. PFAS are of significant concern because these chemicals are ubiquitous and persistent in the environment and in humans. Moreover, susceptible groups, such as foetuses and pregnant women, may be exposed to harmful combinations of chemicals that include PFAS. However, the role environmental exposures play in reproductive disorders has received little attention by the medical community. To better understand the potential risk of PFAS on human ovarian function, additional experimental studies using PFAS doses equivalent to the exposure levels found in the general human population and mixtures of compounds are required. Prospective investigations in human populations are also warranted to ensure the temporality of PFAS exposure and health endpoints and to minimise the possibility of reverse causality.

Metabolomics and Proteomics in Type 2 Diabetes.

The persistent increase in the worldwide burden of type 2 diabetes mellitus (T2D) and the accompanying rise of its complications, including cardiovascular disease, necessitates our understanding of the metabolic disturbances that cause diabetes mellitus. Metabolomics and proteomics, facilitated by recent advances in high-throughput technologies, have given us unprecedented insight into circulating biomarkers of T2D even over a decade before overt disease. These markers may be effective tools for diabetes mellitus screening, diagnosis, and prognosis. As participants of metabolic pathways, metabolite and protein markers may also highlight pathways involved in T2D development. The integration of metabolomics and proteomics with genomics in multiomics strategies provides an analytical method that can begin to decipher causal associations. These methods are not without their limitations; however, with careful study design and sample handling, these methods represent powerful scientific tools that can be leveraged for the study of T2D. In this article, we aim to give a timely overview of circulating metabolomics and proteomics findings with T2D observed in large human population studies to provide the reader with a snapshot into these emerging fields of research.

Chen A, ed. Stress Resilience: Molecular and Behavioral Aspects. London, England: Academic Press; 2020.

Chen A, ed. Stress Resilience: Molecular and Behavioral Aspects. London, England: Academic Press; 2020.

PIG-A gene mutation as a genotoxicity biomarker in human population studies: An investigation in lead-exposed workers.

The rodent Pig-a gene mutation assay has demonstrated remarkable sensitivity in identifying in vivo mutagens, while much less is known about the value of the human PIG-A assay for risk assessment. To obtain more evidence of its potential as a predictive biomarker for carcinogen exposure, we investigated PIG-A mutant frequencies (MFs), along with performing the Comet assay and micronucleus (MN) test, in 267 workers occupationally exposed to lead. Multivariate Poisson regression showed that total red blood cell PIG-A MFs were significantly higher in lead-exposed workers (10.90 ± 10.7 × 10-6 ) than in a general population that we studied previously (5.25 ± 3.6 × 10-6 ) (p < .0001). In contrast, there was no increase in lymphocyte MN frequency or in DNA damage as measured by percentage comet tail intensity in whole blood cells. Current year worker blood lead levels (BLL), an exposure biomarker, were elevated (232.6 ± 104.6 μg/L, median: 225.4 μg/L); a cumulative blood lead index (CBLI) also was calculated based on a combination of current and historical worker BLL data. Chi-square testing indicated that PIG-A MFs were significantly related to CBLI (p = .0249), but independent of current year BLL (p = .4276). However, % comet tail intensity and MN frequencies were better associated with current year BLL than CBLI. This study indicates that the PIG-A assay could serve as biomarker to detect the genotoxic effects of lead exposure and demonstrates that a battery of genotoxicity biomarkers having mechanistic complementarity may be useful for comprehensively monitoring human carcinogenic risk.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster.

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

Pharmacology and physiological function of the orphan GPRC6A receptor

The G protein-coupled receptor GPRC6A (GPCR, Class C, group 6, subtype A) is a Gq/11 -coupled receptor widely expressed in human and rodent tissues. The proposed endogenous ligands are L-amino acids, divalent cations, osteocalcin and testosterone. This MiniReview provides an updated overview of the literature including the latest in vitro and in vivo studies. GPRC6A forms homodimers, it undergoes constitutive internalization, and very interestingly, the reason for the intracellular retention of the human receptor has been revealed. Multiple physiological functions of GPRC6A have been suggested based on studies using three different global GPRC6A knockout (KO) mouse models where exon II, exon VI or the full locus has been deleted. The newest studies on the full locus GPRC6A KO model show intact glucose and bone homoeostasis with a minor reduction in serum osteocalcin levels. Unfortunately, the physiological function of the receptor remains elusive due to a general lack of consensus/validation of reported phenotypes of the different KO models, and more research is thus warranted to uncover the physiological function. Recent discoveries of human genetic variants that cause either a premature stop codon or an intracellular retention of the receptor point towards human population studies as the preferred approach to continue studies on the function of GPRC6A.

Allograft inflammatory factor-1-like is not essential for age dependent weight gain or HFD-induced obesity and glucose insensitivity

The allograft inflammatory factor (AIF) gene family consists of two identified paralogs – AIF1 and AIF1-like (AIF1L). The encoded proteins, AIF1 and AIF1L, are 80% similar in sequence and show conserved tertiary structure. While studies in human populations suggest links between AIF1 and metabolic diseases such as obesity and diabetes, such associations with AIF1L have not been reported. Drawing parallels based on structural similarity, we postulated that AIF1L might contribute to metabolic disorders, and studied it using mouse models. Here we report that AIF1L is expressed in major adipose depots and kidney but was not detectable in liver or skeletal muscle; in notable contrast to AIF1, AIF1L was also not found in spleen. Studies of AIF1L deficient mice showed no obvious postnatal developmental phenotype. In response to high fat diet (HFD) feeding for 6 or 18 weeks, WT and AIF1L deficient mice gained weight similarly, showed no differences in fat or lean mass accumulation, and displayed no changes in energy expenditure or systemic glucose handling. These findings indicate that AIF1L is not essential for the development of obesity or impaired glucose handling due to HFD, and advance understanding of this little-studied gene and its place in the AIF gene family.

Evolutionary life history theory as an organising framework for cohort studies: insights from the Cebu Longitudinal Health and Nutrition Survey

ABSTRACTBy tracking a group of individuals through time, cohort studies provide fundamental insights into the developmental time course and causes of health and disease. Evolutionary life history theory seeks to explain patterns of growth, development, reproduction and senescence, and inspires a range of hypotheses that are testable using the longitudinal data from cohort studies. Here we review two decades of life history theory-motivated work conducted in collaboration with the Cebu Longitudinal Health and Nutrition Survey (CLHNS), a birth cohort study that enrolled more than 3000 pregnant women in the Philippines in 1983 and has since followed these women, their offspring and grandoffspring. This work has provided evidence that reproduction carries “costs” to cellular maintenance functions, potentially speeding senescence, and revealed an unusual form of genetic plasticity in which the length of telomeres inherited across generations is influenced by reproductive timing in paternal ancestors. Men in Cebu experience hormonal and behavioural changes in conjunction with changes in relationship and fatherhood status that are consistent with predictions based upon other species that practice bi-parental care. The theoretical expectation that early life cues of mortality or environmental unpredictability will motivate a “fast” life history strategy are confirmed for behavioural components of reproductive decision making, but not for maturational tempo, while our work points to a broader capacity for early life developmental calibration of systems like immunity, reproductive biology and metabolism. Our CLHNS findings illustrate the power of life history theory as an integrative, lifecourse framework to guide longitudinal studies of human populations.

DNA Methylation-Based Biomarkers of Environmental Exposures for Human Population Studies.

This manuscript orients the reader to the underlying motivations of environmental biomarker development for human population studies and provides the foundation for applying these novel biomarkers in future research. In this review, we focus our attention on the DNA methylation-based biomarkers of (i) smoking, among adults and pregnant women, (ii) lifetime cannabis use, (iii) alcohol consumption, and (iv) cumulative exposure to lead. Prior environmental exposures and lifestyle modulate DNA methylation levels. Exposure-related DNA methylation changes can either be persistent or reversible once the exposure is no longer present, and this combination of both persistent and reversible changes has essential value for biomarker development. Here, we present available biomarkers representing past and cumulative exposures using individual DNA methylation profiles. In the present work, we describe how the field of environmental epigenetics can leverage machine learning algorithms to develop exposure biomarkers and reduce problems of misreporting exposures or limited access technology. We emphasize the crucial role of the individual DNA methylation profiles in those predictions, providing a summary of each biomarker, and highlighting their advantages, and limitations. Future research can cautiously leverage these DNA methylation-based biomarkers to understand the onset and progression of diseases.

Mercury Speciation in Whole Blood and Dried Blood Spots from Capillary and Venous Sources.

There is interest in measuring total mercury (THg) and methylmercury (MeHg) in dried blood spots (DBS) though more research is required to evaluate mercury (Hg) speciation in DBS and to validate the agreement between blood sources (venous vs capillary) and matrices (whole blood vs DBS). Therefore, the present study aimed to develop, evaluate, and validate a DBS-based method to assess MeHg and inorganic mercury (InHg) exposure in human population studies. First, we used volume-controlled (40 μL) paired DBS-whole blood samples to develop an analytical method that involved the extraction and quantification of MeHg and InHg with gas chromatography-cold vapor atomic fluorescence spectrometry (GC-CVAFS). Next, we carried out a validation study using paired DBS-whole blood samples from venous and capillary sources from 49 volunteers with Hg exposures similar to background populations (i.e., MeHg in venous whole blood ranged from 0.2 to 3 μg/L with a median value of 0.8). The limits of detection were higher for InHg (1.9 and 1.1 μg/L in DBS and whole blood, respectively) than MeHg (0.3 and 0.2 μg/L in DBS and whole blood, respectively). The MeHg concentrations among blood sources and matrices were highly correlated (r ranged from 0.85 to 0.95), with no constant bias (intercept ranged from -0.05 to 0.13 μg/L) and small proportional bias (slopes ranged from 0.92 to 1.08). Bland-Altman plots indicated little bias between MeHg measurements with 82-98% of the cases meeting the analytical acceptance criterion of 35% maximum allowed difference. Our results indicate that measures of MeHg in capillary DBS reflect concentrations in the gold standard (i.e., venous whole blood) and that DBS is a suitable tool for assessing MeHg exposure in human population studies, but that more work is required to assess InHg exposures.

Determination of di-n-butyl adipate (DnBA) metabolites as possible biomarkers of exposure in human urine by online-SPE-LC-MS/MS

Di-n-butyl adipate (DnBA) is an alternative to the anti-androgenic and strictly regulated di-n-butyl phthalate (DnBP) used as a cosmetic ingredient, plasticizer, and in various articles of everyday life. Hence, exposures of the general population have to be expected. Currently, biomarkers of DnBA exposure and methods for their determination are not available. Here, we describe a sensitive, rugged and precise analytical method for the determination of the DnBA monoester metabolite mono-n-butyl adipate (MnBA), as well as its potential downstream metabolites 3-hydroxy-mono-n-butyl adipate (3OH-MnBA) and 3-carboxy-mono-n-propyl adipate (3cx-MnPrA) in human urine. Glucuronic acid conjugates present in urine were deconjugated using a pure β-glucuronidase. The metabolites were then analyzed by liquid chromatography on a C18 column with superficially porous particles coupled to electrospray ionization-triple quadrupole-tandem mass spectrometry, applying online turbulent flow chromatography for analyte enrichment and matrix depletion (online-SPE-LC-MS/MS). The metabolites were quantified using stable isotope dilution analysis with limits of quantification of 0.05 µg/L (MnBA), 0.1 µg/L (3OH-MnBA), and 0.5 µg/L (3cx-MnPrA). Method imprecision in urinary matrix was below 7% (coefficient of variation) for all analytes. Mean relative recoveries were between 93% and 107%. The suitability of the DnBA metabolites as biomarkers of exposure was demonstrated after dermal application of a commercially available sunscreen containing DnBA. Maximum concentrations were reached 6.5 h after dose (219 µg/L 3cx-MnPrA, 91 µg/L MnBA, and 3.9 µg/L 3OH-MnBA). Elimination kinetics were similar for all three metabolites. We were able to quantify 3cx-MnPrA and MnBA until 4 d after sunscreen application. In a sample set of 35 urine samples from the general German population, 3cx-MnPrA was quantified in 94% (median 2.54 µg/L, maximum 78.3 µg/L) and MnBA in 3% (median < LOQ, maximum 0.18 µg/L) of the samples. The method will be applied in future human metabolism and human biomonitoring population studies.

Functional analysis of candidate genes from genome-wide association studies of hearing

The underlying causes of age-related hearing loss (ARHL) are not well understood, but it is clear from heritability estimates that genetics plays a role in addition to environmental factors. Genome-wide association studies (GWAS) in human populations can point to candidate genes that may be involved in ARHL, but follow-up analysis is needed to assess the role of these genes in the disease process. Some genetic variants may contribute a small amount to a disease, while other variants may have a large effect size, but the genetic architecture of ARHL is not yet well-defined. In this study, we asked if a set of 17 candidate genes highlighted by early GWAS reports of ARHL have detectable effects on hearing by knocking down expression levels of each gene in the mouse and analysing auditory function. We found two of the genes have an impact on hearing. Mutation of Dclk1 led to late-onset progressive increase in ABR thresholds and the A430005L14Rik (C1orf174) mutants showed worse recovery from noise-induced damage than controls. We did not detect any abnormal responses in the remaining 15 mutant lines either in thresholds or from our battery of suprathreshold ABR tests, and we discuss the possible reasons for this.