William E. Funk

Environmental and occupational exposure to chemicals and telomere length in human studies

Telomeres are complexes of tandem repeats of DNA (5′-TTAGGG-3′) and protein that cap eukaryotic chromosomes and play a critical role in chromosome stability. Telomeres shorten with aging and this process can be accelerated by increased oxidative stress and episodes of inflammation. Evidence is rapidly growing that telomere length (TL) may be affected by environmental chemicals that have frequently been associated with chronic diseases. In this article, we review the published data on TL in relation to environmental and occupational exposure to several chemicals based on our own and others’ studies. The environmental and occupational exposures associated with shorter TL include traffic-related air pollution (ie, particulate matter (PM), black carbon (BC), and benzene and toluene), polycyclic aromatic hydrocarbons (PAHs), N-nitrosamines, pesticides, lead, exposure in car mechanical workshops, and hazardous waste exposure. Arsenic, persistent organic pollutants (POPs) and short-term exposure to PM are associated with longer TL. We discuss the possible reasons for the differences in results, including time- and dose-related issues, study design, and possible mechanisms involved in telomere regulation. We also discuss the future directions and challenges for TL-related environmental and occupational health research, such as investigation of TL in subpopulations of blood leukocytes, and the study of genetic and epigenetic factors that may regulate telomere integrity using longitudinal designs.

Environmental and occupational exposure to chemicals and telomere length in human studies.

Telomeres are complexes of tandem repeats of DNA (5′-TTAGGG-3′) and protein that cap eukaryotic chromosomes and play a critical role in chromosome stability. Telomeres shorten with aging and this process can be accelerated by increased oxidative stress and episodes of inflammation. Evidence is rapidly growing that telomere length (TL) may be affected by environmental chemicals that have frequently been associated with chronic diseases. In this article, we review the published data on TL in relation to environmental and occupational exposure to several chemicals based on our own and others’ studies. The environmental and occupational exposures associated with shorter TL include traffic-related air pollution (ie, particulate matter (PM), black carbon (BC), and benzene and toluene), polycyclic aromatic hydrocarbons (PAHs), N-nitrosamines, pesticides, lead, exposure in car mechanical workshops, and hazardous waste exposure. Arsenic, persistent organic pollutants (POPs) and short-term exposure to PM are associated with longer TL. We discuss the possible reasons for the differences in results, including time- and dose-related issues, study design, and possible mechanisms involved in telomere regulation. We also discuss the future directions and challenges for TL-related environmental and occupational health research, such as investigation of TL in subpopulations of blood leukocytes, and the study of genetic and epigenetic factors that may regulate telomere integrity using longitudinal designs.

Indoor air pollutants and health in the United Arab Emirates

Background: Comprehensive global data on the health effects of indoor air pollutants are lacking. There are few large population-based multi–air pollutant health assessments. Further, little is known about indoor air health risks in the Middle East, especially in countries undergoing rapid economic development. Objectives: To provide multifactorial indoor air exposure and health data, we conducted a population-based study of indoor air pollution and health in the United Arab Emirates (UAE). Methods: We conducted a cross-sectional study in a population-based sample of 628 households in the UAE. Indoor air pollutants [sulfur dioxide (SO2), nitrogen dioxide (NO2), hydrogen sulfide (H2S), formaldehyde (HCHO), carbon monoxide (CO), and particulate matter] were measured using passive samplers over a 7-day period. Health information was collected from 1,590 household members via in-person interviews. Results: Participants in households with quantified SO2, NO2, and H2S (i.e., with measured concentrations above the limit of quantification) were twice as likely to report doctor-diagnosed asthma. Participants in homes with quantified SO2 were more likely to report wheezing symptoms {ever wheezing, prevalence odds ratio [POR] 1.79 [95% confidence interval (CI) 1.05, 3.05]; speech-limiting wheeze, POR 3.53 (95% CI: 1.06, 11.74)}. NO2 and H2S were similarly associated with wheezing symptoms. Quantified HCHO was associated with neurologic symptoms (difficulty concentrating POR 1.47; 95% CI: 1.02, 2.13). Burning incense daily was associated with increased headaches (POR 1.87; 95% CI: 1.09, 3.21), difficulty concentrating (POR 3.08; 95% CI: 1.70, 5.58), and forgetfulness (POR 2.68: 95% CI: 1.47, 4.89). Conclusions: This study provides new information regarding potential health risks from pollutants commonly found in indoor environments in the UAE and other countries. Multipollutant exposure and health assessments in cohort studies are needed to better characterize health effects of indoor air pollutants.

Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring

A method is described for profiling putative adducts (or other unknown covalent modifications) at the Cys34 locus of human serum albumin (HSA), which represents the preferred reaction site for small electrophilic species in human serum. By comparing profiles of putative HSA-Cys34 adducts across populations of interest it is theoretically possible to explore environmental causes of degenerative diseases and cancer caused by both exogenous and endogenous chemicals. We report a novel application of selected-reaction-monitoring (SRM) mass spectrometry, termed fixed-step SRM (FS-SRM), that allows detection of essentially all HSA-Cys34 modifications over a specified range of mass increases (added masses). After tryptic digestion, HSA-Cys34 adducts are contained in the third largest peptide (T3), which contains 21 amino acids and an average mass of 2433.87 Da. The FS-SRM method does not require that exact masses of T3 adducts be known in advance but rather uses a theoretical list of T3-adduct m/z values separated by a fixed increment of 1.5. In terms of added masses, each triply charged parent ion represents a bin of ±2.3 Da between 9.1 Da and 351.1 Da. Synthetic T3 adducts were used to optimize FS-SRM and to establish screening rules based upon selected b- and y-series fragment ions. An isotopically labeled T3 adduct is added to protein digests to facilitate quantification of putative adducts. We used FS-SRM to generate putative adduct profiles from six archived specimens of HSA that had been pooled by gender, race, and smoking status. An average of 66 putative adduct hits (out of a possible 77) were detected in these samples. Putative adducts covered a wide range of concentrations, were most abundant in the mass range below 100 Da, and were more abundant in smokers than in nonsmokers. With minor modifications, the FS-SRM methodology can be applied to other nucleophilic sites and proteins.

ENRICHMENT OF CYSTEINYL ADDUCTS OF HUMAN SERUM ALBUMIN

We report a method to enrich cysteinyl adducts of human serum albumin (HSA), representing biomarkers of exposure to systemic electrophiles. Because the major site of HSA adduction is the single free sulfhydryl group at Cys34, we used thiol-affinity resins to remove mercaptalbumin (i.e., unadducted HSA) from the cysteinyl adducts. Electrospray ionization mass spectrometry was used to detect mercaptalbumin and HSA-Cys34 modifications before and after enrichment of HSA. Differences in adduct content were detected across samples of freshly isolated, archived, and commercial HSA. Cysteinylated and glycosylated adducts were present in all samples, with abundances decreasing in the following order: commercial HSA>archived HSA>fresh HSA. After enrichment of HSA, mercaptalbumin was no longer observed in mass spectra. The ratios of HSA adducts post-/preenrichment, quantified via the Bradford assay and gel electrophoresis, were 0.029 mg adducts/mg HSA in fresh HSA and 0.323 mg adducts/mg HSA in archived HSA. The apparent elevation of adduct levels in archived samples could be due to differences in specimen preparation and storage rather than to differences in circulating HSA adducts. We conclude that thiol-affinity resins can efficiently remove mercaptalbumin from HSA samples prior to characterization and quantitation of protein adducts of reactive systemic electrophiles.

Release of the Pro-Inflammatory Markers by BEAS-2B Cells Following In Vitro Exposure to Biodiesel Extracts

Biodiesel, an alkyl ester of plant oils that can be used in an unmodified diesel engine, is the first renewable die- sel fuel alternative to become a commercially accepted part of our nations energy infrastructure. For traditional diesel fuel exhaust, it has been demonstrated that the particulate matter (PM) organic components play a role in acute inflamma- tory reactions. However, there have been only a few cytotoxicity and mutagenicity studies on biodiesel emissions. In this study, BEAS-2B cells, a transformed human airway epithelial cell line, were exposed in vitro to the PM organic extracts from Standard Reference Material (SRM) 1975, soy ethyl ester (SEE), soy methyl ester (SME), and petroleum diesel for 24 hours. This study demonstrated that the organic extracts of biodiesel PM in an aqueous solution can increase the re- lease of pro-inflammatory cytokines IL-8 and IL-6 by respiratory epithelial cells. On a microgram PM equivalent per ml (μg PM eq/ml) basis, exposure to biodiesel extracts was associated with a greater release of IL-8 and IL-6 relative to or- ganic extracts of two diesel PM samples. The dose range tested was not cytotoxic. It was also noted that the solvent ex- change method, which was used to prepare the aqueous exposure doses, may not be appropriate for the investigation of biodiesel extracts, though it has been used extensively in petroleum diesel research. A valuable new finding from these experiments is that the soluble organic fraction (SOF) of biodiesel PM begins to elicit a cytokine response in BEAS-2B cells at an exposure lower than petroleum diesel PM extract (approximately 40 μg PM eq/ml). However, more research is required to better characterize the potency of the organic fraction of biodiesel compared to petroleum diesel.

Hemoglobin Adducts of Benzene Oxide in Neonatal and Adult Dried Blood Spots

Adducts of reactive chemicals with hemoglobin (Hb) or human serum albumin can be used as biomarkers of internal doses of carcinogens. Because dried blood spots are easier to collect and store than conventional venous blood samples, they encourage applications of biomarkers of exposure in large epidemiologic studies. In addition, neonatal dried blood spot can be used to investigate chemical exposures in utero. Here, we report a simple method to isolate Hb from dried blood spot with high recovery and purity using the addition of ethanol to aqueous dried blood spot extracts. To prove the concept that dried blood spot–derived proteins can be used to assay for adducts, we measured Hb adducts of benzene oxide, a reactive metabolite of the ubiquitous air pollutant benzene in nine neonatal and nine adult dried blood spots (from volunteer subjects), using a gas chromatography–mass spectrometry method that we had previously developed. For comparison, benzene oxide–Hb adducts were measured in the same nine adult subjects using Hb that had been isolated and purified using our conventional method for venous blood. The geometric mean of benzene oxide–Hb levels in all dried blood spot samples ranged from 27.7 to 33.1 pmol/g globin. Neither of the comparisons of mean (logged) benzene oxide–Hb levels between sources (adult conventional versus adult dried blood spot and adult dried blood spot versus newborn dried blood spot) showed a significant difference. Based upon the estimated variance of the benzene oxide–Hb levels, we had 80% power to detect a 1.7-fold difference in geometric mean levels of benzene oxide–Hb in our sample of nine subjects. (Cancer Epidemiol Biomarkers Prev 2008;17(8):1896–901)

Quantification of arsenic, lead, mercury and cadmium in newborn dried blood spots

Abstract Exposures to heavy metals during fetal and perinatal development are of particular concern. Yet, the health impacts of exposures to toxic metals during these early stages of human development are not well understood due to the paucity of in vivo human data. Dried blood spots (DBS), collected by public health departments to screen for inherited metabolic errors and other disorders, are routinely archived and can be used for exposure assessment. Here we report an improved method for quantifying arsenic, lead, mercury and cadmium in newborn DBS to facilitate epidemiologic research on the health effects of early exposures to toxic metals.

Estimating common parameters of lognormally distributed environmental and biomonitoring data: Harmonizing disparate statistics from publications

The progression of science is driven by the accumulation of knowledge and builds upon published work of others. Another important feature is to place current results into the context of previous observations. The published literature, however, often does not provide sufficient direct information for the reader to interpret the results beyond the scope of that particular article. Authors tend to provide only summary statistics in various forms, such as means and standard deviations, median and range, quartiles, 95% confidence intervals, and so on, rather than providing measurement data. Second, essentially all environmental and biomonitoring measurements have an underlying lognormal distribution, so certain published statistical characterizations may be inappropriate for comparisons. The aim of this study was to review and develop direct conversions of different descriptions of data into a standard format comprised of the geometric mean (GM) and the geometric standard deviation (GSD) and then demonstrate how, under the assumption of lognormal distribution, these parameters are used to answer questions of confidence intervals, exceedance levels, and statistical differences among distributions. A wide variety of real-world measurement data sets was reviewed, and it was demonstrated that these data sets are indeed of lognormal character, thus making them amenable to these methods. Potential errors incurred from making retrospective estimates from disparate summary statistics are described. In addition to providing tools to interpret “other people’s data,” this review should also be seen as a cautionary tale for publishing one’s own data to make it as useful as possible for other researchers.

Quantification of anti-Müllerian hormone (AMH) in dried blood spots: validation of a minimally invasive method for assessing ovarian reserve

BACKGROUND Biological markers of ovarian reserve have the potential to advance research on fecundability, infertility and reproductive aging. Anti-Müllerian hormone (AMH) has emerged as a clinically useful measure of ovarian reserve, but the requirement for venous blood is an obstacle to application in non-clinical settings. This paper validates a new method for quantifying AMH in dried blood spot (DBS) samples--drops of whole blood collected on filter paper following a simple finger stick. METHODS Matched serum and DBS samples were obtained from n=101 women of reproductive age, and AMH values were compared using regression analyses and scatter plots. The precision, reliability, linearity, recovery and lower detection limit of the DBS assay were evaluated, as well as the stability of AMH in DBS across a range of storage conditions. RESULTS There was a strong agreement between AMH concentrations measured in DBS and serum samples across the entire assay range. Analysis of within-assay (percent coefficient of variation, 4.7-6.5%) and between-assay (3.5-7.2%) variability indicated a high level of assay precision and reliability, respectively. The minimum detectable dose of AMH was 0.065 ng/ml. Concentrations of AMH remained stable in DBS samples stored for 2 weeks at room temperature, and for 4 weeks when refrigerated. CONCLUSIONS The DBS assay performs at a level that is comparable to serum-based methods, with the advantage of lower burdens and costs associated with blood collection that may be advantageous for research in clinical as well as non-clinical settings on the causes and consequences of variation in ovarian reserve.