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Background: Human exposure to bisphenol A (BPA) is widespread. After exposure, BPA is rapidly metabolized and eliminated in urine. Therefore, there is considerable within-person and between-person variability of BPA concentrations in spot urine samples. However, no information exists on the within-day variability of urinary BPA concentrations. Objectives: We examined the between-person and within-person and between-day and within-day variability in the urinary BPA concentrations of eight adults who collected all voids for 1 week to investigate the impact of sampling strategy in the exposure assessment of BPA using spot, first morning, or 24-hr urine collections. Methods: We determined the urinary concentrations of BPA using on-line solid-phase extraction coupled to isotope dilution high-performance liquid chromatography/tandem mass spectrometry. Results: The between-day and within-person variability was the primary contributor to the total variance both for first morning voids (77%) and 24-hr urine collections (88%). For the spot collections, we observed considerable within-day variance (70%), which outweighed the between-person (9%) and between-day and within-person (21%) variances. Conclusions: Regardless of the type of void (spot, first morning, 24-hr collection), urinary BPA concentrations for a given adult changed considerably—both within a day and for the 7 days of the study period. Single 24-hr urine collections accurately reflect daily exposure but can misrepresent variability in daily exposures over time. Of interest, when the population investigated is sufficiently large and samples are randomly collected relative to meal ingestion times and bladder emptying times, the single spot–sampling approach may adequately reflect the average exposure of the population to BPA.

Background Parabens are widely used as antimicrobial preservatives in cosmetics, pharmaceuticals, and food and beverage processing. Objectives We assessed exposure to methyl, ethyl, propyl, and butyl parabens in a representative sample of persons ≥ 6 years of age in the U.S. general population from the 2005–2006 National Health and Nutrition Examination Survey. Methods We analyzed 2,548 urine samples by using online solid-phase extraction coupled to isotope dilution–high-performance liquid chromatography/tandem mass spectrometry. Results We detected methyl paraben (MP) and propyl paraben (PP) in 99.1% and 92.7% of the samples, respectively. We detected ethyl (42.4%) and butyl (47%) parabens less frequently and at median concentrations at least one order of magnitude lower than MP (63.5 μg/L) and PP (8.7 μg/L). Least-square geometric mean (LSGM) concentrations of MP were significantly higher (p ≤ 0.01) among non-Hispanic blacks than among non-Hispanic whites except at older ages (≥ 60 years). Adolescent and adult females had significantly higher (p < 0.01) LSGM concentrations of MP and PP than did adolescent and adult males. Females were more likely than males [adjusted odds ratios (ORs) and 95% confidence intervals (CIs): MP, 3.2 (2.99–5.27); PP, 4.19 (2.34–7.49)] and non-Hispanic blacks were more likely than non-Hispanic whites [MP, 4.99 (2.62–9.50); PP, 3.6 (1.86–7.05)] to have concentrations above the 95th percentile. Conclusions The general U.S. population was exposed to several parabens during 2005–2006. Differences in the urinary concentrations of MP and PP by sex and race/ethnicity likely reflect the use of personal care products containing these compounds.

Nonylphenol (NP) has been widely used for more than 50 years in the synthesis of NP ethoxylates, which are important nonionic surfactants. NP is considered an endocrine disruptor based on in vitro and in vivo animal studies. However, the toxic effects of NP in humans are unknown. Information regarding the metabolic fate of 4-t-nonylphenol (4-tNP), a mixture of commercial NP branched isomers, in mammalian species is limited. This information is critical for the identification of adequate biomarkers of exposure to NP that could be used for exposure and risk assessment. We identified metabolites of one 4-tNP isomer, namely, 4-(3',6'-dimethyl-3'-heptyl) phenol (P363-NP), using rat and human liver microsomes. The P363-NP metabolites were extracted by on-line solid-phase extraction and then separated and detected using high-performance liquid chromatography/tandem mass spectrometry. Using the genuine standard, we unambiguously identified 4-(3',6'-dimethyl-3'-heptyl) catechol (P363-NC) as the main P363-NP metabolite when using human liver microsomes. Based on their chromatographic behavior and mass spectral fragmentation patterns, several other metabolites were tentatively identified, including a hydroxylated P363-NP with the alcohol functional group on the branched alkyl chain and its oxidative metabolite, a catechol with a hydroxylated alkyl side chain. Furthermore, the metabolite profile of P363-NP using rat and human enzymes was compared. Our findings suggest that P363-NC could be used as a biomarker to assess exposure to 4-tNP, although additional research to evaluate its suitability as a biomarker is warranted.

Human exposure to environmental phenols can be assessed by measuring the urinary concentrations of these compounds or their metabolites. Total concentrations, which include both free and conjugated (i.e., glucuronide and sulfated) species, are usually reported. Because conjugation may reduce the potential bioactivity of the compounds, measuring separately both the concentrations of free and conjugated species can be of interest. Data on the stability of these conjugated species in urine is critical if the concentrations of free and conjugated species are to be compared. Over a period of 6 months, we investigated the stability of the urinary conjugates of eight environmental phenols (bisphenol A, 2-hydroxy-4-metoxybenzophenone or benzophenone-3, triclosan, 2,5-dichlorophenol, methyl paraben, ethyl paraben, propyl paraben, and butyl paraben) at three storage conditions (room temperature, 4 degrees C, and -70 degrees C). After collection, conjugated species appeared to be stable for at least 7 days when the urine was stored at 4 degrees C, and for at least 180 days at -70 degrees C. By contrast, some of the environmental phenol conjugates commenced to degrade within 24 h after collection when the urine was stored at room temperature although the total concentrations remained relatively constant for at least 30 days. These results suggest that if the concentrations of free and conjugated species will be used for exposure assessment purposes, urine specimens collected for analysis of environmental phenols should be kept at room temperature for the shortest possible time after collection.

Parabens are esters of p-hydroxybenzoic acid, widely used as antimicrobial preservatives—especially against molds and yeast—in cosmetic products and pharmaceuticals and in food and beverage processing (Elder 1984; Orth 1980; Weber 1993). Cosmetics manufacturers and food processers use methyl and propyl parabens most extensively (Elder 1984; Jackson 1992). Parabens are popular because of their low toxicity and cost, broad inertness, and worldwide regulatory acceptance (Soni et al. 2005). Parabens are not mutagenic (Elder 1984), but their potential estrogenic activity—although many orders of magnitude lower than that of estrogen (Golden et al. 2005; Routledge et al. 1998)— has raised some concerns about their toxicity. In vitro data suggest that parabens demonstrate weakly estrogen activities in yeast-based assays (Miller et al. 2001; Nishihara et al. 2000; Routledge et al. 1998; Vinggaard et al. 2000), induce the growth of MCF-7 human breast cancer cells, and influence the expression of estrogen-dependent genes (Byford et al. 2002; Darbre et al. 2002, 2003; Okubo et al. 2001). On the other hand, increased uterine weights have been reported in immature mice after exposure to n-butyl, iso-butyl, and benzyl parabens (Darbre et al. 2002). Decreased excretion of testosterone and alterations in the male reproductive tract were observed in male rodents after exposure to butyl and propyl parabens (Oishi 2001, 2002a, 2002b) but not to methyl and ethyl parabens (Oishi 2004). Ethyl, propyl, and butyl parabens evoked estrogenic responses in sexually immature rainbow trout, although the estrogenic potency of ethyl paraben was weaker than that of propyl and butyl paraben (Pedersen et al. 2000). Data on human exposure to parabens are limited (Darbre et al. 2004; Makino 2003; National Toxicology Program 2004), and the toxic effects of parabens in humans are mostly unknown. The detection (but not concentration ranges) of methyl paraben in cord blood and breast milk has been reported (Makino 2003). A study on the presence of parabens in human breast tumors (Darbre et al. 2004) triggered debate over the use of parabens in cosmetics, particularly in underarm deodorants and antiperspirants, which may increase the incidence of breast cancer (Darbre 2001, 2004, 2006; Elder 1984; Golden et al. 2005; Harvey 2003, 2004; Harvey and Darbre 2004; Harvey and Everett 2004). Such findings, involving as they do the estrogenicity of parabens in animals and the presence of parabens in human breast tissue, have raised questions about the safety of widespread paraben use, and these questions prompted the Cosmetic Ingredient Review program (established in 1976 by the Cosmetics, Toiletry, and Fragrance Association), with the support of the Food and Drug Administration (FDA) and the Consumer Federation of America, to reevaluate paraben safety (Bergfeld et al. 2005). Furthermore, the National Institute of Environmental Health Sciences nominated butyl paraben for toxicological characterization, including reproductive toxicity studies (National Toxicology Program 2004). After intraduodenal or intravenous administration (2 mg/kg) in rats, parabens are mainly hydrolyzed to p-hydroxybenzoic acid, which can then be conjugated with glycine, glucuronide, and sulfate and excreted in urine (Cashman and Warshaw 2005; Kiwada et al. 1979, 1980; Soni et al. 2005). In rabbits, after oral administration of methyl-, ethyl-, propyl-, and butyl parabens at doses of 0.4 and 0.8 g/kg, a small percentage (0.2–0.9%) of the unchanged paraben was excreted in the urine (Tsukamoto and Terade 1960, 1962, 1964). Although human toxicokinetic data are limited, some reports state that after oral administration of propyl paraben (2 g for 5 days) to a human volunteer, only 17.4% of the administered dose was recovered as p-hydroxybenzoic acid and its conjugates in urine (Sabalitschka 1954). Another report concerning six preterm infants who were injected intramuscularly with a paraben-containing gentamicin formulation showed that the urinary excretion of methyl paraben could range from 13.2 to 88.1% (Hindmarsh et al. 1983). Most of the methyl paraben was excreted in urine in its conjugated form. Measuring p-hydroxybenzoic acid or its conjugates in urine may not represent the optimal approach for assessing human exposure to parabens. p-Hydroxybenzoic acid is a nonspecific metabolite of all parabens, and individual parabens are known to have quite different estrogenic bioactivities (Golden et al. 2005). In the present study, we evaluated the use of the free and conjugated species of the parent parabens as new biomarkers for human exposure to these compounds.

Parabens (alkyl esters of p-hydroxybenzoic acid) are widely used as antimicrobial preservatives in cosmetic products, pharmaceuticals, and food processing. However, weak estrogenicity of some parabens has been revealed from several studies. Human exposure to parabens may be assessed by measuring the conjugated or free species of these compounds or their metabolites in urine. We have developed a method using on-line solid phase extraction-high performance liquid chromatography-isotope dilution tandem mass spectrometry with peak focusing to measure the urinary concentrations of methyl, ethyl, propyl, n- and iso- butyl, and benzyl parabens. This method has good reproducibility and accuracy with detection limits for all analytes below 0.2ng/mL in 100microL of urine, and permits quick and accurate analysis of a large number of samples in epidemiologic studies for assessing the prevalence of human exposure to parabens. Using this method, we detected methyl, ethyl, and propyl parabens, mostly as conjugated species, in 22 urine samples collected from anonymous adults.

The free and conjugated urinary species of non-persistent environmental chemicals or their breakdown products are valid human exposure biomarkers. For convenience, disposable diapers and other absorbent materials are widely used to collect urine specimens from infants and young toddlers. However, the extent to which the different urinary species of the target analytes and other components are recovered after the urine is extracted from these absorbent materials is unknown. In this proof-of-concept study, we investigated the extraction recovery from disposable diapers, cotton pads, and gauzes of the free versus glucuronidated urinary species of three example chemicals: bisphenol A, triclosan, and 4-methylumbelliferone. Although the glucuronides were almost fully recovered, the free species were not. Our results suggest that, in addition to other sampling considerations, the binding affinity and extraction recovery of the target biomarkers to the material used to collect the urine should be considered. Alternative collection approaches that do not require such an extraction (e.g., urine bags routinely used in hospitals) may be worth exploring. Despite its shortcomings, having urinary concentrations for biomonitoring considerably strengthens the exposure assessment, particularly for infants and young toddlers, and the benefits of including biomonitoring data outweigh their potential limitations.

Parabens (esters of p-hydroxybenzoic acid) and triclosan are widely used as preservatives and antimicrobial agents, respectively, in personal care products, pharmaceuticals, and food processing. Because of their widespread use and potential risk to human health, assessing human exposure to these compounds in breastfed infants is of interest. We developed a sensitive method, using a unique on-line solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry system with peak focusing feature, to measure in human milk the concentrations of five parabens (methyl-, ethyl-, propyl-, butyl-, and benzyl parabens), triclosan, and six other environmental phenols: bisphenol A (BPA); ortho-phenylphenol (OPP); 2,4-dichlorophenol; 2,5-dichlorophenol; 2,4,5-trichlorophenol; and 2-hydroxy-4-methoxybenzophenone (BP-3). The method, validated by use of breast milk pooled samples, shows good reproducibility (inter-day coefficient of variations ranging from 3.5% to 16.3%) and accuracy (spiked recoveries ranging from 84% to 119% at four spiking levels). The detection limits for most of the analytes are below 1 ng mL(-1) in 100 microL of milk. We tested the usefulness of the method by measuring the concentrations of these twelve compounds in four human milk samples. We detected methyl paraben, propyl paraben, triclosan, BPA, OPP, and BP-3 in some of the samples tested. The free species of these compounds appear to be the most prevalent in milk. Nevertheless, to demonstrate the utility of these measures for exposure and risk assessment purposes, additional data about sampling and storage of the milk, and on the stability of the analytes in milk, are needed.