Michael E. Rybak

Investigation of Relationships between Urinary Biomarkers of Phytoestrogens, Phthalates, and Phenols and Pubertal Stages in Girls

Background Hormonally active environmental agents may alter the course of pubertal development in girls, which is controlled by steroids and gonadotropins. Objectives We investigated associations of concurrent exposures from three chemical classes (phenols, phthalates, and phytoestrogens) with pubertal stages in a multiethnic longitudinal study of 1,151 girls from New York City, New York, greater Cincinnati, Ohio, and northern California who were 6–8 years of age at enrollment (2004–2007). Methods We measured urinary exposure biomarkers at visit 1 and examined associations with breast and pubic hair development (present or absent, assessed 1 year later) using multivariate adjusted prevalence ratios (PR) and 95% confidence intervals (CIs). Modification of biomarker associations by age-specific body mass index percentile (BMI%) was investigated, because adipose tissue is a source of peripubertal hormones. Results Breast development was present in 30% of girls, and 22% had pubic hair. High-molecular-weight phthalate (high MWP) metabolites were weakly associated with pubic hair development [adjusted PR, 0.94 (95% CI, 0.88–1.00), fifth vs. first quintile]. Small inverse associations were seen for daidzein with breast stage and for triclosan and high MWP with pubic hair stage; a positive trend was observed for low-molecular-weight phthalate biomarkers with breast and pubic hair development. Enterolactone attenuated BMI associations with breast development. In the first enterolactone quintile, for the association of high BMI with any development, the PR was 1.34 (95% CI, 1.23–1.45 vs. low BMI). There was no BMI association in the fifth, highest quintile of enterolactone. Conclusions Weak hormonally active xenobiotic agents investigated in this study had small associations with pubertal development, mainly among those agents detected at highest concentrations.

Development of a Standard Reference Material for Metabolomics Research

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.

Human aflatoxin exposure in Kenya, 2007: a cross-sectional study

Aflatoxins contaminate approximately 25% of agricultural products worldwide. They can cause liver failure and liver cancer. Kenya has experienced multiple aflatoxicosis outbreaks in recent years, often resulting in fatalities. However, the full extent of aflatoxin exposure in Kenya has been unknown. Our objective was to quantify aflatoxin exposure across Kenya. We analysed aflatoxin levels in serum specimens from the 2007 Kenya AIDS Indicator Survey – a nationally representative, cross-sectional serosurvey. KAIS collected 15,853 blood specimens. Of the 3180 human immunodeficiency virus-negative specimens with ≥1 mL sera, we randomly selected 600 specimens stratified by province and sex. We analysed serum specimens for aflatoxin albumin adducts by using isotope dilution MS/MS to quantify aflatoxin B1-lysine, and normalised with serum albumin. Aflatoxin concentrations were then compared by demographic, socioeconomic and geographic characteristics. We detected serum aflatoxin B1-lysine in 78% of serum specimens (range = <LOD–211 pg/mg albumin, median = 1.78 pg/mg albumin). Aflatoxin exposure did not vary by sex, age group, marital status, religion or socioeconomic characteristics. Aflatoxin exposure varied by province (p < 0.05); it was highest in Eastern (median = 7.87 pg/mg albumin) and Coast (median = 3.70 pg/mg albumin) provinces and lowest in Nyanza (median = <LOD) and Rift Valley (median = 0.70 pg/mg albumin) provinces. Our findings suggest that aflatoxin exposure is a public health problem throughout Kenya, and it could be substantially impacting human health. Wide-scale, evidence-based interventions are urgently needed to decrease exposure and subsequent health effects.

The CDC’s Second National Report on Biochemical Indicators of Diet and Nutrition in the U.S. Population Is a Valuable Tool for Researchers and Policy Makers

The CDCs National Report on Biochemical Indicators of Diet and Nutrition in the U.S. Population (Nutrition Report) is a serial publication that provides ongoing assessment of the populations nutritional status. The Nutrition Report presents data on blood and urine biomarker concentrations (selected water- and fat-soluble vitamins and nutrients, trace elements, dietary bioactive compounds) from a representative sample of the population participating in the NHANES. The Second Nutrition Report (released in 2012) contains reference information (means and percentiles) for 58 biomarkers measured during all or part of 2003-2006, stratified by age, sex, and race-ethnicity. Where available, we presented cutoff-based prevalence data during 2003-2006 and data on changes in biomarker concentrations or prevalence since 1999. Blood vitamin concentrations were generally higher in older (≥ 60 y) than in younger (20-39 y) adults and lower in Mexican Americans and non-Hispanic blacks than in non-Hispanic whites. Nearly 80% of Americans (aged ≥ 6 y) were not at risk of deficiencies in any of the 7 vitamins studied (vitamins A, B-6, B-12, C, D, and E and folate). Deficiency rates varied by age, sex, and race-ethnicity. Approximately 90% of women (aged 12-49 y) were not at risk of iron deficiency, but only 68% were not at risk of deficiencies in iron and all 7 vitamins. Young women (20-39 y) had median urine iodine concentrations bordering on insufficiency. First-time data are presented on plasma concentrations of 24 saturated and mono- and polyunsaturated fatty acids. Tabulation and graphical presentation of NHANES data in the Second Nutrition Report benefits those organizations involved in developing and evaluating nutrition policy.

Environmental phenols and pubertal development in girls.

Environmental exposures to many phenols are documented worldwide and exposures can be quite high (>1 μM of urine metabolites). Phenols have a range of hormonal activity, but knowledge of effects on child reproductive development is limited, coming mostly from cross-sectional studies. We undertook a prospective study of pubertal development among 1239 girls recruited at three U.S. sites when they were 6-8 years old and were followed annually for 7 years to determine age at first breast or pubic hair development. Ten phenols were measured in urine collected at enrollment (benzophenone-3, enterolactone, bisphenol A, three parabens (methyl-, ethyl-, propyl-), 2,5-dichlorophenol, triclosan, genistein, daidzein). We used multivariable adjusted Cox proportional hazards ratios (HR (95% confidence intervals)) and Kaplan-Meier survival analyses to estimate relative risk of earlier or later age at puberty associated with phenol exposures. For enterolactone and benzophenone-3, girls experienced breast development 5-6 months later, adjusted HR 0.79 (0.64-0.98) and HR 0.80 (0.65-0.98) respectively for the 5th vs 1st quintiles of urinary biomarkers (μg/g-creatinine). Earlier breast development was seen for triclosan and 2,5-dichlorophenol: 4-9 months sooner for 5th vs 1st quintiles of urinary concentrations (HR 1.17 (0.96-1.43) and HR 1.37 (1.09-1.72), respectively). Association of breast development with enterolactone, but not the other three phenols, was mediated by body size. These phenols may be antiadipogens (benzophenone-3 and enterolactone) or thyroid agonists (triclosan and 2,5-dichlorophenol), and their ubiquity and relatively high levels in children would benefit from further investigation to confirm these findings and to establish whether there are certain windows of susceptibility during which exposure can affect pubertal development.

Direct Determination of Metals in Soils and Sediments by Induction Heating-Electrothermal Vaporization (IH-ETV) Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES)

The application of an induction heating (IH) electrothermal vaporization (ETV) sample introduction arrangement for the determination of As, Cd, Cu, Mn, Pb, and Zn in soils and sediments by inductively coupled plasma-optical emission spectrometry (ICP-OES) is presented. Samples were deposited either directly as a solid or by means of slurry sampling into graphite cups that were then positioned in a radio-frequency (RF)-field and vaporized in a carrier flow of 15% (v/v) SF6-Ar. Four certified reference materials (CRMs) were examined: two soil samples—SRM 2710 and SRM 2711 (NIST); and two marine sediments—MESS-2 and PACS-2 (NRC Canada). In general, sample delivery was simpler and observed signal precision was better with slurry sampling when compared to the analysis of the solid directly, with peak area RSDs ranging from 4–16% (n = 6). Plots of intensity vs. certified concentration for the four CRMs were linear with log-log slopes of 0.98–1.02 and r2 values ≥ 0.995 for As, Cu, Pb, and Zn. Recoveries of 80–105% were achieved for the above elements in SRM 2711 by using an external standards curve constructed from the 3 remaining CRMs. Aqueous standard solutions were used for the analysis of all 4 CRMs by standard additions, resulting in recoveries ranging from 54–139% (average recovery of (101 ± 15)%) across all six determined elements in all four samples.

Dietary flavonol intake is associated with age of puberty in a longitudinal cohort of girls

Lignans and flavonols are dietary phytoestrogens found at high concentrations in the Western Diet. They have potential to influence the timing of puberty. We hypothesized that greater consumption of these 2 phytoestrogens would be related to later age at pubertal onset among girls. Pubertal assessment and 24-hour diet recall data were available for 1178 girls, ages 6 to 8 years (mean 7.3 years) in the Breast Cancer and Environment Research Project Puberty Study. Lignan and flavonol intakes were mainly derived from fruit and vegetable consumption. Average consumption was 6.5 mg/d for flavonols and 0.6 mg/d for lignans. Highest flavonol consumption (>5 mg/d) was associated with later breast development (adjusted hazards ratio [HR]: 0.74, 95% CI: [0.61-0.91]) compared to 2 to 5 mg/d (adjusted HR: 0.84, 95% CI: [0.70-1.0]) and <2 mg/d (referent group; P-trend = .006). Flavonol intake was not associated with pubic hair development. Lignan intake was not associated with either breast or pubic hair development. Dietary intake was only weakly correlated with urinary enterolactone, a biomarker for lignans (RS = 0.13). Consistent with biologic properties of phytoestrogens that indicate hormonal activity, their consumption may be associated with reproductive end points, even in childhood.

Sociodemographic and Lifestyle Variables Are Compound- and Class-Specific Correlates of Urine Phytoestrogen Concentrations in the U.S. Population

Isoflavones and lignans are plant-derived dietary compounds generally believed to be beneficial to human health. We investigated the extent to which sociodemographic (age, sex, race-ethnicity, education, and income) and lifestyle variables (smoking, alcohol consumption, BMI, physical activity, and dietary supplement use) were correlates of spot urine concentration for daidzein, genistein, O-desmethylangolensin (DMA), equol, enterodiol, and enterolactone in the U.S. population aged ≥ 20 y (NHANES 2003-2006). We performed correlation analyses with continuous variables and calculated stratified unadjusted geometric means for each sociodemographic and lifestyle variable. We used bivariate significance testing and covariate adjustment by use of multiple regression models to identify influential variables and used β coefficients to estimate relative effects. Urine creatinine was also included in our analyses because of its use in correcting for variable dilution in spot urine samples. We observed many significant (P < 0.05) associations with the sociodemographic and lifestyle variables that withstood covariate adjustment. Smoking was a significant correlate of urine DMA and enterolactone, with concentrations at least 25% lower in smokers vs. nonsmokers. Consumers of 1 daily alcoholic drink vs. none were estimated to have 18-21% lower urine equol and DMA concentrations. A 25% increase in BMI was associated with a 21% lower urine enterolactone concentration, and increasing physical activity was associated with a >6% higher urine enterolactone concentration. Dietary supplement use was not significantly associated with any of the urine phytoestrogens. Overall, we found that relationships between sociodemographic and lifestyle variables and urine phytoestrogen concentration were highly compound and class specific.

Development and characterization of induction heating–electrothermal vaporization (IH-ETV) sample introduction for inductively coupled plasma spectrometry

Abstract A general study of performance attributes was conducted for a prototypical electrothermal vaporization (ETV) sample introduction system, in which induction heating (IH) was used to facilitate the drying, pyrolysis, and vaporization of samples from long, undercut graphite cup probes in a radio-frequency (RF) induction field. In the first part of this study, experiments were carried out to determine the heating characteristics and temperature control aspects of an IH-ETV arrangement. Using a remote-sensing infrared thermocouple, it was determined that a 3/8-inch (9.53-mm) outer diameter graphite cup sample probe could be heated to a maximum temperature of 1860°C in the induction field of the IH-ETV under full forward power (1.5 kW). The IH-ETV device was found to have a rapid heating response (1/e time-constant of 2.0±0.2 s) that was independent of the initial/final temperatures chosen. Linear temperature control was possible by regulating either the DC voltage applied to the plate or the current flowing to the grid of the RF generator oscillator tube. The second part of this work consisted of studies to establish benchmarks, such as limits of detection (LOD) with inductively coupled plasma optical emission spectrometry (ICP-OES) and transport efficiency for analyte vaporization under several x–Ar mixed gas atmospheres [where x=15% N2, 10% O2, HCl (sparged), or 15% SF6 (v/v)]. In general, reproducible transient signals with evolution times of 5–15 s were seen for the vaporization of most elements studied, with peak area intensity and reproducibility generally being the best with SF6–Ar. A 10-fold increase in transport efficiency was seen for refractory carbide-forming analytes (Cr, V) when vaporization was conducted in a halogenous (x=HCl, SF6) versus non-halogenous (x=N2, O2) environment, with a two-fold improvement being observed for most other non-refractory elements (Cd, Cu, Fe, Mn, Ni, Pb, Zn). The transport of arsenic proved to be a special case unto itself, with its transport efficiency increasing to 90% when vaporized in the presence of SF6 (a three-fold improvement over all other carrier mixtures) due to the formation of volatile AsF5. Using the concept of transport-independent analyte sensitivity, the introduction of the aforementioned x–Ar mixtures into the center channel of the plasma appeared to not have any appreciable effect on plasma excitation conditions, with N2 being the only exception (a 70% reduction in transport-independent sensitivity was observed). Detection limits ranged from 0.08 to 70 ng absolute and were highly dependent on the analyte studied and the vaporization atmosphere used. Based on the above information, the feasibility of an induction heating arrangement for thermal sample introduction was evaluated, with recommendations being made for the future design of IH-ETV instrumentation.

Associations of urinary phthalate and phenol biomarkers with menarche in a multiethnic cohort of young girls

To study potential environmental influences on puberty in girls, we investigated urinary biomarkers in relation to age at menarche. Phenols and phthalates were measured at baseline (6-8 years of age). Menarche was ascertained over 11 years for 1051 girls with menarche and biomarkers. Hazards ratios were estimated from Cox models adjusted for race/ethnicity and caregiver education (aHR, 95% confidence intervals [CI] for 5th vs 1st quintile urinary biomarker concentrations). 2,5-Dichlorophenol was associated with earlier menarche (aHR 1.34 [1.06-1.71]); enterolactone was associated with later menarche (aHR 0.82 [0.66-1.03]), as was mono-3-carboxypropyl phthalate (MCPP) (aHR 0.73 [0.59-0.91]); the three p-trends were <0.05. Menarche differed by 4-7 months across this range. Enterolactone and MCPP associations were stronger in girls with below-median body mass index. These analytes were also associated with age at breast development in this cohort. Findings from this prospective study suggest that some childhood exposures are associated with pubertal timing.