Reproductive toxicity following in utero and lactational exposure to a human-relevant phthalate mixture in rats.

This rodent (Wistar rats) study examined reproductive effects of in utero/lactational exposure to a mixture of 6 antiandrogenic phthalates (PMix): diisobutyl phthalate, di-n-butyl phthalate, diisopentyl phthalate, butylbenzyl phthalate, di-2-ethylhexyl phthalate, and diisononyl phthalate. The PMix was defined based on exposure data from pregnant women in Brazil. Experimental groups were established by extrapolating the estimated human dose to rats (0.1 mg/kg/day), followed by up to 3 additional doses corresponding to 5, 1000, and 5000 times the starting rat dose: 0 (control), 0.1, 0.5, 100, and 500 mg/kg/day. The fetal experiment assessed gestational exposure effects on fetal gonads, whereas the postnatal experiment evaluated reproductive parameters in males and females after in utero and lactational exposure. Prenatal exposure decreased fetal testicular testosterone production at 0.5 and 500 mg/kg/day. PMix 500 also reduced mRNA expression of steroidogenesis-related genes, upregulated transcript expression of the retinoic acid-degrading enzyme Cyp26b1, and increased multinucleated gonocytes incidence in fetal testes. Postnatal assessment revealed antiandrogenic effects at the highest dose, including reduced anogenital distance, nipple retention, and decreased weight of reproductive organs. Early puberty onset (preputial separation) was observed at the lowest dose in males. In contrast, females did not show significant changes in fetal and adult endpoints. Overall, the PMix recapitulated early and late male rat phthalate syndrome phenotypes at the highest dose, but also induced some subtle changes at lower doses, which warrant confirmation and mechanistic assessments. Our data support the use of epidemiologically defined mixtures for exposure risk assessments over traditional toxicological approaches.

Unexpected, ubiquitous exposure of pregnant Brazilian women to diisopentyl phthalate, one of the most potent antiandrogenic phthalates.

BACKGROUND: Human exposure to phthalates and other non-persistent chemicals in developing countries is largely unknown. A preliminary analysis of urinary samples from pregnant Brazilian women revealed the presence of metabolites of Diisopentyl phthalate (DiPeP). OBJECTIVES: Reliably quantify DiPeP metabolites in human urine and investigate the potential antiandrogenic activity of this phthalate in rats. METHODS: We initiated a pilot pregnancy cohort in Curitiba, Brazil, to examine phthalate exposure in urine samples collected in early pregnancy (n = 50) or pooled samples from early, mid and late pregnancy (n = 44). Our well established phthalate method was modified to include the primary DiPeP metabolite, monoisopentyl phthalate (MiPeP), and two additional secondary oxidized metabolites, 3OH-MiPeP and 4OH-MiPeP. In a parallel approach, we orally exposed pregnant rats to DiPeP or Di-n-butyl phthalate (DnBP; reference phthalate) at 0, 125, 250, and 500 mg/kg/day from gestation day 14 to 18 and measured ex vivo fetal testis testosterone production. RESULTS: We were able to detect and quantify specific DiPeP metabolites in nearly all (98%) of the early pregnancy urine samples and in all gestational pool samples with a median concentration for MiPeP of 3.65 and 3.15 µg/L, respectively, and for the two oxidized metabolites between 1.00 and 1.70 µg/L. All three urinary DiPeP metabolites were strongly correlated (r = 0.89 to 0.99). In the rat model, the effective dose (mg/kg/day) inhibiting fetal testosterone production by 50% (ED50 [95% confidence interval]) was 93.6 [62.9-139.3] for DiPeP which was significantly lower than for DnBP (220.3 [172.9-280.7]), highlighting the strong antiandrogenic potency of DiPeP within the spectrum of the phthalates. CONCLUSIONS: We unveiled and confirmed the exposure of pregnant Brazilian women to DiPeP via specific urinary metabolites. This unexpected and ubiquitous DiPeP exposure indicates to unique DiPeP exposure sources in Brazil. These exposures spark considerable concern because DiPeP is one of the most potent antiandrogenic phthalates.

Intrauterine Exposure to Paracetamol and Aniline Impairs Female Reproductive Development by Reducing Follicle Reserves and Fertility.

Studies report that fetal exposure to paracetamol/acetaminophen by maternal consumption can interfere with male reproductive development. Moreover, recent biomonitoring data report widespread presence of paracetamol in German and Danish populations, suggesting exposure via secondary (nonpharmaceutical) sources, such as metabolic conversion from the ubiquitous industrial compound aniline. In this study, we investigated the extent to which paracetamol and aniline can interfere with female reproductive development. Intrauterine exposure to paracetamol by gavage of pregnant dams resulted in shortening of the anogenital distance in adult offspring, suggesting that fetal hormone signaling had been disturbed. Female offspring of paracetamol-exposed mothers had ovaries with diminished follicle reserve and reduced fertility. Fetal gonads of exposed animals had also reduced gonocyte numbers, suggesting that the reduced follicle count in adults could be due to early disruption of germ cell development. However, ex vivo cultures of ovaries from 12.5 days post coitum fetuses showed no decrease in proliferation or expression following exposure to paracetamol. This suggests that the effect of paracetamol occurs prior to this developmental stage. Accordingly, using embryonic stem cells as a proxy for primordial germ cells we show that paracetamol is an inhibitor of cellular proliferation, but without cytotoxic effects. Collectively, our data show that intrauterine exposure to paracetamol at levels commonly observed in pregnant women, as well as its precursor aniline, may block primordial germ cell proliferation, ultimately leading to reduced follicle reserves and compromised reproductive capacity later in life.

Human metabolism and excretion kinetics of aniline after a single oral dose.

Aniline is an important source material in the chemical industry (e.g., rubber, pesticides, and pharmaceuticals). The general population is known to be ubiquitously exposed to aniline. Thus, assessment of aniline exposure is of both occupational and environmental relevance. Knowledge on human metabolism of aniline is scarce. We orally dosed four healthy male volunteers (two fast and two slow acetylators) with 5 mg isotope-labeled aniline, consecutively collected all urine samples over a period of 2 days, and investigated the renal excretion of aniline and its metabolites by LS-MS/MS and GC-MS. After enzymatic hydrolysis of glucuronide and sulfate conjugates, N-acetyl-4-aminophenol was the predominant urinary aniline metabolite representing 55.7-68.9 % of the oral dose, followed by the mercapturic acid conjugate of N-acetyl-4-aminophenol accounting for 2.5-6.1 %. Acetanilide and free aniline were found only in minor amounts accounting for 0.14-0.36 % of the dose. Overall, these four biomarkers excreted in urine over 48 h post-dose represented 62.4-72.1 % of the oral aniline dose. Elimination half-times were 3.4-4.3 h for N-acetyl-4-aminophenol, 4.1-5.5 h for the mercapturic acid conjugate, and 1.3-1.6 and 0.6-1.2 h for acetanilide and free aniline, respectively. Urinary maximum concentrations of N-acetyl-4-aminophenol were reached after about 4 h and maximum concentrations of the mercapturic acid conjugate after about 6 h, whereas concentrations of acetanilide and free aniline peaked after about 1 h. The present study is one of the first to provide reliable urinary excretion factors for aniline and its metabolites in humans after oral dosage, including data on the predominant urinary metabolite N-acetyl-4-aminophenol, also known as an analgesic under the name paracetamol/acetaminophen.

Mercury analysis in hair: Comparability and quality assessment within the transnational COPHES/DEMOCOPHES project.

Human biomonitoring (HBM) is an effective tool for assessing actual exposure to chemicals that takes into account all routes of intake. Although hair analysis is considered to be an optimal biomarker for assessing mercury exposure, the lack of harmonization as regards sampling and analytical procedures has often limited the comparison of data at national and international level. The European-funded projects COPHES and DEMOCOPHES developed and tested a harmonized European approach to Human Biomonitoring in response to the European Environment and Health Action Plan. Herein we describe the quality assurance program (QAP) for assessing mercury levels in hair samples from more than 1800 mother-child pairs recruited in 17 European countries. To ensure the comparability of the results, standard operating procedures (SOPs) for sampling and for mercury analysis were drafted and distributed to participating laboratories. Training sessions were organized for field workers and four external quality-assessment exercises (ICI/EQUAS), followed by the corresponding web conferences, were organized between March 2011 and February 2012. ICI/EQUAS used native hair samples at two mercury concentration ranges (0.20-0.71 and 0.80-1.63) per exercise. The results revealed relative standard deviations of 7.87-13.55% and 4.04-11.31% for the low and high mercury concentration ranges, respectively. A total of 16 out of 18 participating laboratories the QAP requirements and were allowed to analyze samples from the DEMOCOPHES pilot study. Web conferences after each ICI/EQUAS revealed this to be a new and effective tool for improving analytical performance and increasing capacity building. The procedure developed and tested in COPHES/DEMOCOPHES would be optimal for application on a global scale as regards implementation of the Minamata Convention on Mercury.

Biomonitoring of N-ethyl-2-pyrrolidone in automobile varnishers.

N-alkyl-2-pyrrolidones are important organic solvents for varnishes in industry. This study investigates exposure to N-ethyl-2-pyrrolidone (NEP) in varnishing of hard plastic components in an automobile plant. Two specific biomarkers of exposure, 5-hydroxy-N-ethyl-2-pyrrolidone (5-HNEP) and 2-hydroxy-N-ethylsuccinimide (2-HESI), were analyzed in urine samples of 14 workers. For this purpose, pre-shift, post-shift and next day pre-shift urine samples were collected midweek. Twelve workers performed regular work tasks (loading, wiping and packing), whereas two workers performed special work tasks including cleaning the sprayer system with organic solvents containing N-alkyl-2-pyrrolidones. Spot urine samples of nine non-exposed persons of the same plant served as controls. Median post-shift urinary levels of workers with regular work tasks (5-HNEP: 0.15 mg/L; 2-HESI: 0.19 mg/L) were ∼5-fold higher compared to the controls (0.03 mg/L each). Continuously increasing metabolite levels, from pre-shift via post-shift to pre-shift samples of the following day, were observed in particular for the two workers with the special working tasks. Maximum levels were 31.01 mg/L (5-HNEP) and 8.45 mg/L (2-HESI). No clear trend was evident for workers with regular working tasks. In summary, we were able to show that workers can be exposed to NEP during varnishing tasks in the automobile industry.

The European COPHES/DEMOCOPHES project: towards transnational comparability and reliability of human biomonitoring results.

COPHES/DEMOCOPHES has its origins in the European Environment and Health Action Plan of 2004 to "develop a coherent approach on human biomonitoring (HBM) in Europe". Within this twin-project it was targeted to collect specimens from 120 mother-child-pairs in each of the 17 participating European countries. These specimens were investigated for six biomarkers (mercury in hair; creatinine, cotinine, cadmium, phthalate metabolites and bisphenol A in urine). The results for mercury in hair are described in a separate paper. Each participating member state was requested to contract laboratories, for capacity building reasons ideally within its borders, carrying out the chemical analyses. To ensure comparability of analytical data a Quality Assurance Unit (QAU) was established which provided the participating laboratories with standard operating procedures (SOP) and with control material. This material was specially prepared from native, non-spiked, pooled urine samples and was tested for homogeneity and stability. Four external quality assessment exercises were carried out. Highly esteemed laboratories from all over the world served as reference laboratories. Web conferences after each external quality assessment exercise functioned as a new and effective tool to improve analytical performance, to build capacity and to educate less experienced laboratories. Of the 38 laboratories participating in the quality assurance exercises 14 laboratories qualified for cadmium, 14 for creatinine, 9 for cotinine, 7 for phthalate metabolites and 5 for bisphenol A in urine. In the last of the four external quality assessment exercises the laboratories that qualified for DEMOCOPHES performed the determinations in urine with relative standard deviations (low/high concentration) of 18.0/2.1% for cotinine, 14.8/5.1% for cadmium, 4.7/3.4% for creatinine. Relative standard deviations for the newly emerging biomarkers were higher, with values between 13.5 and 20.5% for bisphenol A and between 18.9 and 45.3% for the phthalate metabolites. Plausibility control of the HBM results of all participating countries disclosed analytical shortcomings in the determination of Cd when using certain ICP/MS methods. Results were corrected by reanalyzes. The COPHES/DEMOCOPHES project for the first time succeeded in performing a harmonized pan-European HBM project. All data raised have to be regarded as utmost reliable according to the highest international state of the art, since highly renowned laboratories functioned as reference laboratories. The procedure described here, that has shown its success, can be used as a blueprint for future transnational, multicentre HBM projects.

Phthalate exposure in pregnant women and newborns - the urinary metabolite excretion pattern differs distinctly.

Some phthalates are endocrine disruptors and reproductive and developmental toxicants. Data on newborn phthalate exposure and elimination characteristics are scarce. We determined 21 urinary phthalate metabolites (indicating exposure to 11 parent phthalates) in two study approaches: in the first approach we collected the urine of 20 healthy newborns at days 2-5 post partum together with 47 urine samples of 7 women during pregnancy. In the second fine tuned approach we collected first urine samples of 9 healthy newborns together with their mother's urine shortly before birth. To ensure full and contamination free collection of the newborns first urines we used special adhesive urine bags for children. All urine samples revealed ubiquitous exposures to phthalates comparable to other populations. Metabolite levels in the newborns first day urine samples were generally lower than in all other samples. However, the newborns urines (both first and day 2-5 urines) showed a metabolite pattern distinctly different from the maternal and general population samples: in the newborns urines the carboxy-metabolites of the long chain phthalates (DEHP, DiNP, DiDP) were the by far dominant metabolites with a relative share in the metabolite spectrum up to 6 times higher than in maternal urine. Oppositely, for the short chain phthalates (DBP, DiBP) oxidized metabolites seemed to be less favored than the simple monoesters in the newborns urines. The skewed metabolite distribution in the newborns urine warrants further investigation in terms of early phthalate metabolism, the quantity of internal phthalate exposure of the fetus/newborn and its possible health effects.

Phthalate exposure during cold plastisol application--a human biomonitoring study.

The phthalates DEHP (Diethylhexyl phthalate), DiNP (Diisononyl phthalate) and DiDP (Diisodecyl phthalate) are constituents of plastisols. We sought to obtain first data on occupational exposures to the above phthalates by analyzing their metabolites in pre- and post-shift urine samples from 5 workers in a car manufacturing plant engaged in seam sealing with a DINP based plastisol. Pre-shift samples were collected after a work-free period of at least 2 days. As a comparison group we investigated 10 employees from the same plant. The comparison group had phthalate exposures in the range of the general German population. All plastisol workers had post shift values of DiNP and DiDP metabolites that were approx. 20-times higher, and pre-shift values that were approx. 5-10 times higher than those of the general background exposure. Post-shift values of DiNP metabolites were (median [maximum]: OH-MiNP: 117 [442] µg/L; oxo-MiNP: 44.3 [175] µg/L; carboxy-MiNP: 57.8 [286]µg/L), pre shift values were (OH-MiNP: 26 [164] µg/L; oxo-MiNP 12.9 [68.6] µg/L; carboxy-MiNP: 32.3 [103] µg/L), compared to the comparison group (OH-MiNP: 6.2 [33]µg/L; oxo-MiNP: 2.8 [16] µg/L; carboxy-MiNP: 6.5 [31] µg/L). DiDP values were generally lower. Regarding DEHP we found no significant work related exposure. The dermal exposure route might play an important role for phthalates in plastisols, with possible influences on distribution and elimination kinetics and therefore data interpretation.

Assessing exposure to phthalates - the human biomonitoring approach.

Some phthalates are developmental and reproductive toxicants in animals. Exposure to phthalates is considered to be potentially harmful to human health as well. Based on a comprehensive literature research, we present an overview of the sources of human phthalate exposure and results of exposure assessments with special focus on human biomonitoring data. Among the general population, there is widespread exposure to a number of phthalates. Foodstuff is the major source of phthalate exposure, particularly for the long-chain phthalates such as di(2-ethylhexyl) phthalate. For short-chain phthalates such as di-n-butyl-phthalate, additional pathways are of relevance. In general, children are exposed to higher phthalate doses than adults. Especially, high exposures can occur through some medications or medical devices. By comparing exposure data with existing limit values, one can also assess the risks associated with exposure to phthalates. Within the general population, some individuals exceed tolerable daily intake values for one or more phthalates. In high exposure groups, (intensive medical care, medications) tolerable daily intake transgressions can be substantial. Recent findings from animal studies suggest that a cumulative risk assessment for phthalates is warranted, and a cumulative exposure assessment to phthalates via human biomonitoring is a major step into this direction.

Internal phthalate exposure over the last two decades--a retrospective human biomonitoring study.

In a retrospective human biomonitoring study we analyzed 24h urine samples taken from the German Environmental Specimen Bank for Human Tissues (ESBHum), which were collected from 634 subjects (predominantly students, age range 20-29 years, 326 females, 308 males) in 9 years between 1988 and 2003 (each n >or= 60), for the concentrations of primary and/or secondary metabolites of di-n-butyl phthalate (DnBP), di-iso-butyl phthalate (DiBP), butylbenzyl phthalate (BBzP), di(2-ethylhexyl) phthalate (DEHP) and di-iso-nonyl phthalate (DiNP). Based on the urinary metabolite excretion we estimated daily intakes of the parent phthalates and investigated the chronological course of the phthalate exposure. In over 98% of the urine samples metabolites of all five phthalates were detectable indicating a ubiquitous exposure of the German population to all five phthalates throughout the last 20 years. The median daily intakes in the subsets between 1988 and 1993 were quite constant for DnBP (approx. 7 microg/kg bw/d) and DEHP (approx. 4 microg/kg bw/d). However, from 1996 the median levels of both phthalates decreased continuously until 2003 (DnBP 1.9 microg/kg bw/d; DEHP 2.4 microg/kg bw/d). By contrast, the daily intake values for DiBP were slightly increasing over the whole time frame investigated (median 1988: 1.1 microg/kg bw/d; median 2003: 1.4 microg/kg bw/d), approximating the levels for DnBP and DEHP. For BBzP we observed slightly decreasing values, even though the medians as of 1998 levelled off at around 0.2 microg/kg bw/d. Regarding daily DiNP exposure we found continuously increasing values, with the lowest median being 0.20 microg/kg bw/d for the subset of 1988 and the highest median for 2003 being twice as high. The trends observed in phthalate exposure may be associated with a change in production and usage pattern. Female subjects exhibited significantly higher daily intakes for the dibutyl phthalates (DnBP p=0.013; DiBP p=0.004). Compared to data from US National Health and Nutrition Examination Surveys (NHANES) exposure levels of the dibutyl phthalates were generally higher in our German study population, while levels of BBzP were somewhat lower. Overall, for a considerable 14% of the subjects we observed daily DnBP intakes above the tolerable daily intake (TDI) value deduced by the European Food Safety Authority (EFSA) (10 microg/kg bw/d). However, the frequency of exceedance decreased during the years and was beneath 2% in the 2003 subset. Even though transgressions of the exposure limit values of the EFSA and the US Environmental Protection Agency (US EPA) occurred only in a relatively small share of the subjects, one has to take into account the cumulative exposure to all phthalates investigated and possible dose-additive endocrine effects of these phthalates.

Pilot study on the naphthalene exposure of German adults and children by means of urinary 1- and 2-naphthol levels.

Concentrations of 1- and 2-naphthol were measured in urine of 72 adults and 35 young children from Germany to assess the internal exposure to naphthalene of the general population. Naphthols could be detected in more than 90% of the urine samples. Levels of naphthols (sum of 1- and 2-naphthol) were 4-fold higher in smokers (median: 37.6 microg/g creatinine) compared to non-smoking adults (8.2 microg/g creatinine). On a creatinine basis young children had slightly lower naphthol levels in urine compared with adults (7.5 microg/g creatinine). Preliminary reference values for the sum of 1- and 2- naphthol in urine as means of the 95th percentile are proposed: 41.2 microg/g creatinine (non-smoking adults) and 23.5 microg/g creatinine (young children). It is concluded that 1- and 2-naphthol levels in urine are suitable for human biomonitoring of the naphthalene exposure in environmental medicine.

Analysis of ethenoguanine adducts in human urine using high performance liquid chromatography-tandem mass spectrometry.

Several chemical carcinogens, such as vinyl chloride and ethyl carbamate, can react with DNA to form etheno-adducts in vitro and in vivo, which can be repaired through the base excision repair pathway, and then excreted with the urine. A specific and sensitive method, based on high performance liquid chromatography electrospray ionization tandem mass spectrometry, was developed for the detection of ethenoguanines (1,N2-ethenoguanine and its isomer N2,3 ethenoguanine) in urine. Urine samples were obtained from 13 healthy subjects not occupationally exposed to industrial chemicals. A confirmatory GC/MS method was also applied. Ethenoguanine isomers excreted with the urine were in the low nmol/l range (<0.3-8 nmol/l). Since occupational exposure to chemicals that may form etheno-adducts can be ruled out, endogenously produced intermediates, such as 2,3-epoxy-4-hydroxynonanal, may be responsible for the formation of etheno-adducts in human DNA. The background level of the general population has to be taken into account, especially in the investigation of persons occupationally exposed to etheno-adduct forming chemicals.