Temporal variability in urinary concentrations of phthalate metabolites, phytoestrogens and phenols among minority children in the United States.

BACKGROUND: Exposure to endocrine disruptors (EDs), including some phthalates, phytoestrogens and phenols can be quantified using biomarkers of exposure. However, reliability in the use of these biomarkers requires an understanding of the timeframe of exposure represented by one measurement. Data on the temporal variability of ED biomarkers are sparse, especially among children. OBJECTIVE: To evaluate intraindividual temporal variability in 19 individual urinary biomarkers (eight phthalate metabolites from six phthalate diesters, six phytoestrogens (two lignans and four isoflavones) and five phenols) among New York City children. METHODS: Healthy Hispanic and Black children (N=35; 6-10 years old) donated several urine samples over 6 months. To assess temporal variability we used three statistical methods: intraclass correlation coefficient (ICC), Spearman correlation coefficients (SCC) between concentrations measured at different timepoints, and surrogate category analysis to determine how well the tertile categories based on a single measurement represented a 6-month average concentration. RESULTS: Surrogate category analysis indicated that a single sample provides reliable ranking for all analytes; at least three of four surrogate samples predicted the 6-month mean concentration. Of the 19 analytes, the ICC was >0.2 for 18 analytes and >0.3 for 10 analytes. Correlations among sample concentrations throughout the 6-month period were observed for all analytes; 14 analyte concentrations were correlated at 16 weeks. CONCLUSIONS: The reasonable degree of temporal reliability and the wide range of concentrations of phthalate metabolites, phytoestrogens and phenols suggest that these biomarkers are appropriate for use in epidemiologic studies of environmental exposures in relation to health outcomes in children.

Assessment of human exposure to di-isodecyl phthalate using oxidative metabolites as biomarkers.

Di-isodecyl phthalate (DiDP), primarily used as a plasticiser, is a mixture of isomers with predominantly ten-carbon branched side chains. Assessment of DiDP exposure has not been conducted before because adequate biomarkers were lacking. In 129 adult volunteers with no known exposure to DiDP, the urinary concentrations of three oxidative metabolites of DiDP: monocarboxyisononyl phthalate (MCiNP), monooxoisodecyl phthalate (MOiDP) and monohydroxyisodecyl phthalate (MHiDP), previously identified in DiDP-dosed rats, were estimated by solid-phase extraction coupled to high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) using the respective oxidative metabolites of di(2-ethylhexyl)phthalate since authentic standards of the DiDP oxidative metabolites were unavailable. Interestingly, the hydrolytic monoester of DiDP, monoisodecyl phthalate (MiDP), was not detected in any of the samples, while MCiNP, MHiDP and MOiDP were detected in 98%, 96% and 85%, respectively, of the samples tested. MCiNP was excreted predominantly in its free form, whereas MOiDP was excreted as its glucuronide. MCiNP, MHiDP and MOiDP eluted as clusters of multiple peaks from the HPLC column probably due to the presence of numerous structurally similar isomers present in commercial DiDP formulations. The urinary concentrations of these oxidative metabolites correlated significantly (p < 0.0001) with each other, thus confirming a common precursor. The urinary concentrations of these DiDP oxidative metabolites also correlated significantly (p < 0.0001) with oxidative metabolites of di-isononyl phthalate (DiNP) suggesting the potential presence of DiNP isomers in commercial DiDP or simultaneous use of DiDP and DiNP in consumer products. The concentrations presented are semiquantitative estimates and should be interpreted cautiously. Nevertheless, the higher frequency of detection and higher urinary concentrations of MCiNP, MHiDP and MOiDP than of MiDP suggest that these oxidative metabolites are better biomarkers for DiDP exposure assessment than MiDP. These data also suggest that unless oxidative metabolites are measured, the prevalence of exposure to DiDP will probably be underestimated.

Measurement of eight urinary metabolites of di(2-ethylhexyl) phthalate as biomarkers for human exposure assessment.

Human metabolism of di(2-ethylhexyl) phthalate (DEHP) is complex and yields mono(2-ethylhexyl) phthalate (MEHP) and numerous oxidative metabolites. The oxidative metabolites, mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP) and mono(2-carboxymethylhexyl) phthalate (MCMHP), have been considered to be better biomarkers for DEHP exposure assessment than MEHP because urinary levels of these metabolites are generally higher than MEHP, and their measurements are not subject to contamination. The urinary levels of the above metabolites, and of three other recently identified DEHP oxidative metabolites, mono(2-ethyl-3-carboxypropyl) phthalate (MECPrP), mono-2-(1-oxoethylhexyl) phthalate (MOEHP), and mono(2-ethyl-4-carboxybutyl) phthalate (MECBP), were measured in 129 adults. MECPP, MCMHP and MEHHP were present in all the samples analysed. MEHP and the other oxidative metabolites were detected less frequently: MEOHP (99%), MECBP (88%), MECPrP (84%), MEHP (83%) and MOEHP (77%). The levels of all DEHP metabolites were highly correlated (p<0.0001) with each other, confirming a common parent. The ? and ?-1 oxidative metabolites (MECPP, MCMHP, MEHHP and MEOHP) comprised 87.1% of all metabolites measured, and thus are most likely the best biomarkers for DEHP exposure assessment. The percentage of the unglucuronidated free form excreted in urine was higher for the ester linkage carboxylated DEHP metabolites compared with alcoholic and ketonic DEHP metabolites. The percentage of the unglucuronidated free form excreted in urine was higher for the DEHP metabolites with a carboxylated ester side-chain compared with alcoholic and ketonic metabolites. Further, differences were found between the DEHP metabolite profile between this adult population and that of six neonates exposed to high doses of DEHP through extensive medical treatment. In the neonates, MEHP represented 0.6% and MECPP 65.5% of the eight DEHP metabolites measured compared to 6.6% (MEHP) and 31.8% (MECPP) in the adults. Whether the observed differences reflect differences in route/duration of the exposure, age and/or health status of the individuals is presently unknown.

Quantitative detection of eight phthalate metabolites in human urine using HPLC-APCI-MS/MS.

Because of the ubiquity of phthalates and their potential role in increasing risk for cancer and reproductive dysfunction, the need for human exposure assessment studies is urgent. In response to this need, we developed a high-throughput, robust, sensitive, accurate, and precise assay for simultaneous measurement of trace levels of eight phthalate metabolites in human urine by HPLC-MS/MS. Human urine samples were processed using enzymatic deconjugation of the glucuronides followed by solid-phase extraction. The eluate was concentrated, and the phthalate metabolites were chromatographically resolved by reversed-phase HPLC, detected by APCI-tandem mass spectrometry, and quantified by isotope dilution. This selective analytical method permits rapid detection (7.7 min total run time) of eight urinary metabolites of the most commonly used phthalates with detection limits in the low nanagram per milliliter range. Assay precision was improved by incorporating 13C4-labeled internal standards for each of the eight analytes, as well as a conjugated internal standard to monitor deconjugation efficiency. This selective, sensitive, and rapid method will help elucidate potential associations (if any) between human exposure to phthalates and adverse health effects.

Detection of mosaicism in amniotic fluid cultures: a CYTO2000 collaborative study.

PURPOSE: To evaluate the assumptions on which the American College of Medical Genetics (ACMG) Standards and Guidelines for detecting mosaicism in amniotic fluid cultures are based. METHODS: Data from 653 cases of amniotic fluid mosaicism were collected from 26 laboratories. A chi-square goodness-of-fit test was used to compare the observed number of mosaic cases with the expected number based on binomial distribution theory. RESULTS: Comparison of observed data from the in situ colony cases with the expected distribution of cases detected based on the binomial distribution did not reveal a significant difference (P = 0.525). CONCLUSIONS: The empirical data fit the binomial distribution. Therefore, binomial theory can be used as an initial discussion point for determining whether ACMG Standards and Guidelines are adequate for detecting mosaicism.

International Commission for Protection Against Environmental Mutagens and Carcinogens. Deoxyribonucleoside triphosphate levels: a critical factor in the maintenance of genetic stability.

DNA precursor pool imbalances can elicit a variety of genetic effects and modulate the genotoxicity of certain DNA-damaging agents. These and other observations indicate that the control of DNA precursor concentrations is essential for the maintenance of genetic stability, and suggest that factors which offset this control may contribute to environmental mutagenesis and carcinogenesis. In this article, we review the biochemical and genetic mechanisms responsible for regulating the production and relative amounts of intracellular DNA precursors, describe the many outcomes of perturbations in DNA precursor levels, and discuss implications of such imbalances for sensitivity to DNA-damaging agents, population monitoring, and human diseases.

Vitamin C intake influences the bleomycin-induced chromosome damage assay: implications for detection of cancer susceptibility and chromosome breakage syndromes.

Supplementation with 1 g of vitamin C (ascorbic acid) per day decreased the amount of chromosome damage induced in lymphocytes by an exposure to bleomycin during the last 5 h of cell culture. We did not see such changes in lymphocytes from control individuals samples at the same time but not taking vitamin C supplements. This bleomycin assay has been proposed as a test for cancer susceptibility. A similar assay for genetic instability may be useful in detecting heterozygotes for chromosome-breakage syndromes (for example, Fanconi anemia or ataxia telangiectasia). Even though our sample size is small and our results should be interpreted cautiously, statistically significant effects were found with vitamin C supplementation. It would, therefore, be prudent to consider dietary and perhaps other lifestyle factors when interpreting of results from this bleomycin assay and related assays for genetic instability.

Complete culture medium is better than low folate medium for detecting increased chromosome aberrations in smokers in 48-h lymphocyte cultures.

We compared chromosome breakage in parallel, 48-h lymphocyte cultures established from smokers and nonsmokers using minimal essential medium (MEM) and MEM without folate (MEM-FA). There was a statistically significant, higher frequency of aberrations for smokers than for nonsmokers in cells cultured in MEM, but not in those cultured in MEM-FA. Thus, these data support the recommendation of the World Health Organization (1985) that population monitoring studies for exposure assessment should not use a low folate medium.

Increased chromosome fragility as a consequence of blood folate levels, smoking status, and coffee consumption.

Chromosome fragility in 96 h, low-folate cultures was found to be associated with smoking status, coffee consumption, and blood folate level. The higher proportion of cells with chromosome aberrations in cigarette smokers was attributable to lower red cell folate levels in smokers compared with nonsmokers. There was a positive linear relationship between the average cups of coffee consumed per day and the proportion of cells with aberrations. This association was independent of the effects of smoking and red cell folate level. These data suggest that smoking history, coffee consumption, and red cell folate level are important considerations for the design and interpretation of fragile site studies in cancer cytogenetics.

Effects of folate in culture medium on common fragile sites in lymphocyte chromosomes from normal and leukemic children.

The expression of common fragile sites at 69 bands was evaluated in 20 normal children and in 15 children with newly diagnosed acute leukemia using medium with folate (FA+) and without folate (FA-). As expected, the FA- medium significantly increased expression of aberrations in all study groups but the differences were larger for normal children than leukemic children. The major effect of the FA- medium was a generalized increase in aberration frequency over all sites rather than site-specific increases. A tendency toward clumping of aberrations within cells was exhibited in both media. Aberrations were seen at 81% (FA+) and 83% (FA-) of the 69 bands, with 4 sites - 3p14, 6p21, 9q13, and 17q23 - recorded in most of the study individuals. In addition, 12 sites not previously recorded as common or rare sites had significant levels of expression in at least one study group.

Role of deoxyuridine incorporation and DNA repair in the expression of human chromosomal fragile sites.

This paper is a discussion of the possible roles of deoxyuridine incorporation into DNA and DNA-repair processes in the expression of the folate-sensitive, common chromosomal fragile sites. Expression of aberrations at these sites increases under conditions expected to increase deoxyuridine incorporation into the chromosome. It is likely that this abnormal base is removed by an excision-repair process that results in transient chromosome breaks; these breaks are seen as chromosome aberrations if repair is not completed before metaphase. Analogous events may account for other types of chromosome aberrations including the so-called "spontaneous" aberrations, the rare folate-sensitive fragile sites, and fragile sites induced by other means.

Increased sister chromatid exchange associated with smoking and coffee consumption.

Sister chromatid exchange (SCE) is a very sensitive cytogenetic assay for detecting exposure to chemical mutagens and carcinogens. One application of SCE is the monitoring of populations believed to be exposed to such agents. We have, however, relatively little knowledge about common lifestyle factors that may influence SCE and therefore complicate any study designed to examine the effects of exposure to genotoxins. In this study, we assessed the effect of cigarette smoking and coffee consumption on SCE. Smoking was associated with an increase of approximately 2 SCEs per cell and a decrease in cell proliferation. A positive linear relationship between SCE and coffee consumption was also observed. This effect was similar for smokers and nonsmokers. Additionally, the folic acid content of cell culture medium seemed to affect neither SCE nor cell proliferation.

Folate- and deoxyuridine-sensitive chromatid breakage may result from DNA repair during G2.

A substantial increase in chromosome breakage was seen in proliferating human lymphocytes treated with 1-beta-D-arabinofuranosyl cytosine (cytosine arabinoside, cytarabine, araC) during the last 2 h of culture. The increase was larger in low folate media than in high folate media and larger still in low folate media supplemented by deoxyuridine. Similar results were found when cells were exposed to aphidicolin for the last 2 h of culture. The results provide additional evidence that folate-sensitive chromosome breakage is a consequence of abnormal DNA synthesis (in particular incorporation of deoxyuridine) and subsequent attempts during G2 to repair the abnormal DNA.

Deoxyuridine increases folate-sensitive fragile site expression in human lymphocytes.

Deoxyuridine (dU) increases chromosome breakage at folic acid (FA)-sensitive common fragile sites (mostly 3p14 and 16q23) in human lymphocytes. This dU-related increase can be suppressed by thymidine or FA. These results suggest that the mechanism of fragile site expression in low FA medium involves misincorporation of dU into DNA.

Folic acid and chromosome breakage. IV. Variance estimates from longitudinal studies of normal individuals and their implications for sample size and power to detect differences between populations.

The frequency of chromosome aberrations was studied in minimal essential medium (MEM) with and without folic acid (FA) in lymphocytes of 4 normal individuals, each sampled 12 times over a 1-year period. The cells cultured without FA had significantly more breaks and gaps. In both media about 75% of aberrations were classified as gaps. Calculations based on variance estimates suggest that the use of medium without FA could enhance the statistical power to distinguish differences in proportions of chromosome breakage between groups in the same study.