Hanne Frederiksen

Urinary excretion of phthalates and paraben after repeated whole-body topical application in humans

Diethyl phthalate (DEP), dibutyl phthalate (DBP) and butyl paraben (BP) are man-made chemicals used in personal care products, such as lotions and creams. Exposure to these chemicals causes a variety of adverse reproductive outcomes in animal studies. Humans can be exposed to these chemicals through dermal absorption, but there are no published data on absorption, metabolism, and excretion after dermal application. This study investigates urinary concentrations of BP and metabolites of DEP and DBP after topical application. In a 2-week single-blinded study, 26 healthy Caucasian male subjects were given a whole body topical application of basic cream 2 mg/cm(2) (control week) and then a cream containing 2% (w/w) of DEP, DBP and BP each (treatment week) daily. Twenty-four-hour urine samples were collected. Urinary total, and unconjugated BP, monoethyl phthalate (MEP) and monobutyl phthalate (MBP) metabolites were analysed by Liquid Chromatography-Tandem Mass Spectroscopy (LC-MS/MS). All 26 subjects showed increased excretion of MEP, MBP and BP following topical application. Total MEP, MBP and BP (mean +/- SEM) excreted in urine in the treatment week were, respectively, 41 +/- 1.9, 11.8 +/- 0.6 and 2.6 +/- 0.1 mg/24 h. On average 5.79, 1.82 and 0.32%, respectively, of the applied DEP, DBP and BP could be recovered in urine as MEP, MBP and BP. The concentration of the compounds peaked in urine 8-12 h after application. The fractions of unconjugated MEP, MBP, and BP were 78, 8.0 and 2.1%, respectively. Absorption of DEP, DBP and BP through skin could potentially contribute to adverse health effects. The three chemicals are systemically absorbed, metabolized and excreted in urine following application on the skin in a cream preparation. More DEP than DBP was absorbed, presumably because of a faster absorption rate for DEP.

Sunscreens: are they beneficial for health? An overview of endocrine disrupting properties of UV-filters

Today, topical application of sunscreens, containing ultraviolet-filters (UV-filters), is preferred protection against adverse effects of ultraviolet radiation. Evidently, use of sunscreens is effective in prevention of sunburns in various models. However, evidence for their protective effects against melanoma skin cancer is less conclusive. Three important observations prompted us to review the animal data and human studies on possible side effects of selected chemical UV-filters in cosmetics. (1) the utilization of sunscreens with UV-filters is increasing worldwide; (2) the incidence of the malignant disorder for which sunscreens should protect, malignant melanoma, is rapidly increasing and (3) an increasing number of experimental studies indicating that several UV-filters might have endocrine disruptive effects. The selected UV-filters we review in this article are benzophenone-3 (BP-3), 3-benzylidene camphor (3-BC), 3-(4-methyl-benzylidene) camphor (4-MBC), 2-ethylhexyl 4-methoxy cinnamate (OMC), Homosalate (HMS), 2-ethylhexyl 4-dimethylaminobenzoate (OD-PABA) and 4-aminobenzoic acid (PABA). The potential adverse effects induced by UV-filters in experimental animals include reproductive/developmental toxicity and disturbance of hypothalamic-pituitary-thyroid axis (HPT). Few human studies have investigated potential side effects of UV-filters, although human exposure is high as UV-filters in sunscreens are rapidly absorbed from the skin. One of the UV-filters, BP-3, has been found in 96% of urine samples in the US and several UV-filters in 85% of Swiss breast milk samples. It seems pertinent to evaluate whether exposure to UV-filters contribute to possible adverse effects on the developing organs of foetuses and children.

Childhood Exposure to Phthalates: Associations with Thyroid Function, Insulin-like Growth Factor I, and Growth

Background Phthalates are widely used chemicals, and human exposure is extensive. Recent studies have indicated that phthalates may have thyroid-disrupting properties. Objective We aimed to assess concentrations of phthalate metabolites in urine samples from Danish children and to investigate the associations with thyroid function, insulin-like growth factor I (IGF-I), and growth. Methods In 845 children 4–9 years of age, we determined urinary concentrations of 12 phthalate metabolites and serum levels of thyroid-stimulating hormone, thyroid hormones, and IGF-I. Results Phthalate metabolites were detected in all urine samples, of which monobutyl phthalate was present in highest concentration. Phthalate metabolites were negatively associated with serum levels of free and total triiodothyronine, although statistically significant primarily in girls. Metabolites of di(2-ethylhexyl) phthalate and diisononyl phthalate were negatively associated with IGF-I in boys. Most phthalate metabolites were negatively associated with height, weight, body surface, and height gain in both sexes. Conclusions Our study showed negative associations between urinary phthalate concentrations and thyroid hormones, IGF-I, and growth in children. Although our study was not designed to reveal the mechanism of action, the overall coherent negative associations between urine phthalate and thyroid and growth parameters may suggest causative negative roles of phthalate exposures for child health.

Urinary excretion of phthalate metabolites, phenols and parabens in rural and urban Danish mother-child pairs.

Some phthalates, parabens and phenols have shown adverse endocrine disrupting effects in animal studies and are also suspected to be involved in human reproductive problems. However, knowledge about exposure sources and biomonitoring data in different subsets of populations are still scarce. Thus, in this study first morning urine samples were collected from 6 to 11 years Danish children and their mothers. The content of seven parabens, nine phenols and metabolites of eight different phthalates were analysed by LC-MS/MS. Two parabens, six phenols and metabolites from six phthalate diesters were measurable in more than 50%, 75% and 90% of the participants, respectively. Thus the children and their mothers were generally exposed simultaneously to a range of phthalates, phenols and parabens. In general, the levels were low but for several of the compounds extreme creatinine adjusted concentrations 100-500-fold higher than the median level were seen in some participants. Children were significantly higher exposed to bisphenol A (BPA) and some of the phthalates (DiBP, DnBP, BBzP, DEHP and DiNP) than their mothers, whereas mothers were higher exposed to compounds related to cosmetics and personal care products such as parabens (MeP, EtP and n-PrP), benzophenone-3, triclosan and diethyl phthalate. However, a very high correlation between mothers and their children was observed for all chemicals. A high individual exposure to one chemical was often associated with a high exposure to other of the chemicals and the possibility of combination effects of multiple simultaneous exposures cannot be excluded.

Parabens in urine, serum and seminal plasma from healthy Danish men determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Parabens are used as anti-microbial preservatives in a range of consumer products, especially in cosmetics. In vitro and animal studies have shown weak estrogenic and other endocrine disrupting effects of parabens, including reduced testosterone levels in exposed male rats. The knowledge of paraben exposure, distribution and excretion in humans is limited. In this study we determined the concentration of five parabens; methyl-, ethyl-, n-propyl-, n-butyl- and benzylparaben in urine, serum and seminal plasma samples from 60 healthy Danish men. To conduct the study a sensitive and specific method using LC–MS/MS for simultaneous determination of the five parabens was developed for all three different matrices. Highest concentrations of the parabens were found in urine, wherein methyl-, ethyl-, n-propyl- and n-butyl parabens were measurable in 98%, 80%, 98% and 83% of the men, respectively. Benzyl paraben was only measurable in urine from 7% of the men. Methyl- and n-propyl parabens were also measurable in the majority of serum and seminal plasma samples, whereas the other parabens could only be detected in some of the samples. In all the three matrices significant correlations between the parabens were seen. Furthermore, urinary paraben concentrations correlate to the paraben concentrations in both serum and seminal plasma.

Hypothesis: exposure to endocrine-disrupting chemicals may interfere with timing of puberty

A recent decline in onset of puberty - especially among girls - has been observed, first in the US in the mid-1990s and now also in Europe. The development of breast tissue in girls occurs at a much younger age and the incidence of precocious puberty (PP) is increasing. Genetic factors and increasing prevalence of adiposity may contribute, but environmental factors are also likely to be involved. In particular, the widespread presence of endocrine-disrupting chemicals (EDCs) is suspected to contribute to the trend of earlier pubertal onset. The factors regulating the physiological onset of normal puberty are poorly understood. This hampers investigation of the possible role of environmental influences. There are many types of EDCs. One chemical may have more than one mode of action and the effects may depend on dose and duration of the exposure, as well as the developmental stage of the exposed individual. There may also be a wide range of genetic susceptibility to EDCs. Human exposure scenarios are complex and our knowledge about effects of mixtures of EDCs is limited. Importantly, the consequences of an exposure may not be apparent at the actual time of exposure, but may manifest later in life. Most known EDCs have oestrogenic and/or anti-androgenic actions and only few have androgenic or anti-oestrogenic effects. Thus, it appears plausible that they interfere with normal onset of puberty. The age at menarche has only declined by a few months whereas the age at breast development has declined by 1 year; thus, the time span from initiation of breast development to menarche has increased. This may indicate an oestrogen-like effect without concomitant central activation of the hypothalamic-pituitary axis. The effects may differ between boys and girls, as there are sex differences in age at onset of puberty, hormonal profiles and prevalence of precocius puberty.

Urinary excretion of phthalate metabolites in 129 healthy Danish children and adolescents: estimation of daily phthalate intake.

BACKGROUND Phthalates are a group of chemicals with widespread use in the industrial production of numerous consumer products. They are suspected to be involved in male reproductive health problems and have also been associated with several other health problems in children including obesity and asthma. OBJECTIVES To study the urinary excretion of phthalate metabolites in Danish children recruited from the general population, and to estimate the daily intake of phthalates in this segment of the population. METHOD One 24 h urine sample and to consecutive first morning urine samples were collected from 129 healthy Danish children and adolescents (range 6-21 yrs). The concentrations of 11 phthalate metabolites of 5 different phthalate diesters were analyzed by liquid chromatography-tandem mass spectrometry. RESULTS The analyzed metabolites were detectable in almost all 24h urine samples. The median concentrations of monoethyl phthalate (MEP), monobenzyl phthalate (MBzP) and the sums of the two monobutyl phthalate isoforms (∑MBP(i+n)), metabolites of di-(2-ethylhexyl) phthalate (∑DEHPm) and of di-iso-nonyl phthalate (∑DiNPm) were 29, 17, 111, 107 and 31 ng/mL, respectively. The youngest children were generally more exposed to phthalates than older children and adolescents (except diethyl phthalate (DEP)). Boys were more exposed than girls. The median estimated daily intake of phthalate diesters was: 4.29 (dibutyl phthalate isoforms (DBP(i+n))), 4.04 (DEHP), 1.70 (DiNP), 1.09 (DEP) and 0.62 (butylbenzyl phthalate (BBzP)), all calculated as μg/kg body weight/24h. Between 40% and 48% of the absolute amount of phthalate metabolites excreted over 24h were excreted in first morning urine voids. CONCLUSION Danish children are exposed simultaneously to multiple phthalates. The highest exposure levels were found for DBP(i+n) and DEHP, which in animal models are the known most potent anti-androgenic phthalates. The combined exposure to the two isoforms of DBP, which have similar endocrine-disrupting potencies in animal models, exceeded the TDI for di-n-butyl phthalate (DnBP) in several of the younger children.

Human urinary excretion of non-persistent environmental chemicals: an overview of Danish data collected between 2006 and 2012

Several non-persistent industrial chemicals have shown endocrine disrupting effects in animal studies and are suspected to be involved in human reproductive disorders. Among the non-persistent chemicals that have been discussed intensively during the past years are phthalates, bisphenol A (BPA), triclosan (TCS), and parabens because of their anti-androgenic and/or estrogenic effects. Phthalates are plasticizers used in numerous industrial products. Bisphenol A is the main component of polycarbonate plastics and epoxy resins. Parabens and TCS are antimicrobial preservatives and other phenols such as benzophenone-3 (BP-3) act as a UV-screener, while chlorophenols and phenyl phenols are used as pesticides and fungicides in agriculture. In spite of the widespread use of industrial chemicals, knowledge of exposure sources and human biomonitoring studies among different segments of the population is very limited. In Denmark, we have no survey programs for non-persistent environmental chemicals, unlike some countries such as the USA (NHANES) and Germany (GerES). However, we have analyzed the excretion of seven parabens, nine phenols, and the metabolites of eight different phthalates in urine samples collected over the past 6 years from four Danish cohorts. Here, we present biomonitoring data on more than 3600 Danish children, adolescents, young men, and pregnant women from the general population. Our study shows that nearly all Danes were exposed to the six most common phthalates, to BPA, TCS, and BP-3, and to at least two of the parabens. The exposure to other non-persistent chemicals was also widespread. Our data indicate decreasing excretion of two common phthalates (di-n-butyl phthalate and di-(2-ethylhexyl) phthalate) over time.

Phthalate excretion pattern and testicular function: a study of 881 healthy Danish men.

Background: In animals, some phthalates impair male reproductive development and function. Epidemiological studies have reported inconsistent evidence of associations between phthalates and markers of human testicular function. Objectives: We aimed to provide estimates of the effects of phthalate exposure on reproductive hormone levels and semen quality in healthy men. Methods: A total of 881 men gave urine, serum, and semen samples. Serum levels of testosterone, estradiol (E2), sex hormone-binding globulin (SHBG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and inhibin-B; semen quality; and urinary concentrations of 14 phthalate metabolites, including metabolites of di(2-ethylhexyl) phthalate (DEHP) and diisononyl phthalate (DiNP), were assessed. The proportions of DEHP and DiNP excreted as their respective primary metabolites [mono(2-ethylhexyl) phthalate (MEHP) and mono-isononyl phthalate (MiNP)] were calculated and expressed as percentages (%MEHP and %MiNP, respectively). Results: The free androgen index was 15% lower [95% confidence interval (CI): –23, –8%] for men in the highest %MiNP quartile compared to the lowest quartile (p < 0.001) after adjusting for confounders, and 9% lower (95% CI: –16, –1%) in the highest %MEHP quartile (p = 0.02). %MEHP and %MiNP were negatively associated with the ratio of testosterone/LH and testosterone/FSH. %MEHP was negatively associated with total testosterone, free testosterone, and ratio of testosterone/E2. %MiNP was positively associated with SHBG. There was little evidence of associations between urinary phthalate metabolites or sums of phthalates with reproductive hormones or semen quality Conclusion: Our data suggest that both testosterone production and pituitary–hypothalamic feedback may be compromised in individuals excreting a high proportion of primary metabolites of long-chained phthalates relative to the proportion of secondary metabolites.

Do Parabens Have the Ability to Interfere with Steroidogenesis

The effects of ethyl and butyl paraben on steroidogenesis were evaluated in rats exposed in utero. Pregnant Wistar rats were dosed from gestational day (GD) 7 to GD 21, followed by examination of the dams, and the fetuses. Additionally, both parabens were tested in vitro in the H295R steroidogenesis assay and in the T-screen assay, the later to test for their ability to act as thyroid hormone receptor agonist or antagonist. In the in utero exposure toxicity study, neither ethyl nor butyl paraben showed any treatment-related effects on testosterone production, anogenital distance, or testicular histopathology. However, butyl paraben caused a significant decrease in the mRNA expression level of estradiol receptor-beta in fetal ovaries, and also significantly decreased the mRNA expression of steroidogenic acute regulatory protein and peripheral benzodiazepine receptor in the adrenal glands. In vitro butyl paraben increased the proliferation of the GH3 cells in the T-Screen assay, thereby acting as a weak thyroid hormone receptor agonist. In the adrenal H295R steroidogenesis assay both ethyl and butyl paraben caused a significant increase in the progesterone formation. Overall, the results indicate that butyl paraben might have the ability to act as endocrine disruptor by interfering with the transport of cholesterol to the mitochondrion, thereby interfering with steroidogenesis, but also that the two tested parabens do not show clear endocrine disrupting capabilities in our short-term in vivo experiment.