annales d'économie et de statistique. – n° 85 – 2007 L'INTéGRATION BOURSIèRE INTERNATIONALE : TESTS ET EFFETS SUR LA DIVERSIFICATION AROURI Mohamed El Hedi* RéSUMé. – cet article étudie l'intégration boursière internationale des marchés développés et émergents et évalue ses effets sur la diversification.
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WorkingchildResearch Children's Health
Decrease in Anogenital Distance among Male Infants with Prenatal
Shanna H. Swan,1 Katharina M. Main,2 Fan Liu,3 Sara L. Stewart,3 Robin L. Kruse,3 Antonia M. Calafat,4
Catherine S. Mao,5 J. Bruce Redmon,6 Christine L. Ternand,7 Shannon Sullivan,8 J. Lynn Teague,9 and
the Study for Future Families Research Team*
Department of Obstetrics and Gynecology, University of Rochester, Rochester, New York, USA; Department of Growth and Reproduction, University of Copenhagen, Copenhagen, Denmark; 3Department of Family and Community Medicine, University ofMissouri-Columbia, Columbia, Missouri, USA; 4National Center for Environmental Health, Centers for Disease Control and Prevention,Division of Laboratory Sciences, Atlanta, Georgia, USA; 5Department of Pediatrics, Division of Endocrinology, Los Angeles BiomedicalResearch Institute at Harbor-UCLA Medical Center, Los Angeles, California, USA; Departments of 6Pediatrics and 7Medicine, Universityof Minnesota Medical School, Minneapolis, Minnesota, USA; 8Department of Pediatrics, University of Iowa, Iowa City, Iowa, USA;9Departments of Surgery (Urology) and Child Health, University of Missouri-Columbia, Columbia, Missouri, USA As with the Colón et al. study, contamination Prenatal phthalate exposure impairs testicular function and shortens anogenital distance (AGD) in
from diesters in laboratory equipment could male rodents. We present data from the first study to examine AGD and other genital measure-
not be excluded (Murature et al. 1987).
ments in relation to prenatal phthalate exposure in humans. A standardized measure of AGD was
More recent studies have examined phtha- obtained in 134 boys 2–36 months of age. AGD was significantly correlated with penile volume
late monoester metabolites in urine. Because (R = 0.27, p = 0.001) and the proportion of boys with incomplete testicular descent (R = 0.20,
urinary metabolites are not likely to be present p = 0.02). We defined the anogenital index (AGI) as AGD divided by weight at examination
as the result of contamination, these studies [AGI = AGD/weight (mm/kg)] and calculated the age-adjusted AGI by regression analysis. We
avoid this potential source of measurement examined nine phthalate monoester metabolites, measured in prenatal urine samples, as predictors
error. Duty et al. (2003a) reported dose– of age-adjusted AGI in regression and categorical analyses that included all participants with prena-
response relationships between tertiles of tal urine samples (n = 85). Urinary concentrations of four phthalate metabolites [monoethyl phtha-
monobutyl phthalate and sperm motility and late (MEP), mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), and monoisobutyl
sperm concentration, and between tertiles of phthalate (MiBP)] were inversely related to AGI. After adjusting for age at examination, p-values
monobenzyl phthalate (MBzP) and sperm for regression coefficients ranged from 0.007 to 0.097. Comparing boys with prenatal MBP con-
concentration. They also reported inverse dose– centration in the highest quartile with those in the lowest quartile, the odds ratio for a shorter than
response relationships between monoethyl expected AGI was 10.2 (95% confidence interval, 2.5 to 42.2). The corresponding odds ratios for
phthalate (MEP) and sperm DNA damage MEP, MBzP, and MiBP were 4.7, 3.8, and 9.1, respectively (all p-values < 0.05). We defined a
summary phthalate score to quantify joint exposure to these four phthalate metabolites. The age-
Address correspondence to S.H. Swan, University of adjusted AGI decreased significantly with increasing phthalate score (p-value for slope = 0.009).
Rochester, Department of Obstetrics and Gynecology, The associations between male genital development and phthalate exposure seen here are consistent
School of Medicine and Dentistry, 601 ElmwoodAve., Box 668, Rochester, NY 14642-8668 USA.
with the phthalate-related syndrome of incomplete virilization that has been reported in prenatally
Telephone: (585) 273-3521. Fax: (585) 275-7366.
exposed rodents. The median concentrations of phthalate metabolites that are associated with
short AGI and incomplete testicular descent are below those found in one-quarter of the female
*The Study for Future Families Research Team population of the United States, based on a nationwide sample. These data support the hypothesis
included, from the University of Missouri- that prenatal phthalate exposure at environmental levels can adversely affect male reproductive
Columbia: E.Z. Drobnis, B.S. Carter, D. Kelly, and development in humans. Key words: anogenital distance, benzylbutyl phthalate, dibutyl phthalate,
T.M. Simmons. Los Angeles Biomedical ResearchInstitute at Harbor-UCLA Medical Center: diethyl phthalate, monobenzyl phthalate, monoethyl phthalate, monoisobutyl phthalate, mono-n-
C. Wang, L. Lumbreras, S. Villanueva, M. Diaz- butyl phthalate, phthalates, prenatal exposure. Environ Health Perspect 113:1056–1061 (2005).
Romero, M.B. Lomeli, and E. Otero-Salazar. Cedars- doi:10.1289/ehp.8100 available via http://dx.doi.org/ [Online 27 May 2005]
Sinai Medical Center: C. Hobel and B. Brock.
University of Minnesota: C. Kwong and A. Muehlen.
University of Iowa: A. Sparks, A. Wolf, J. Whitham, Diesters of phthalic acid, commonly referred et al. 2000), and DEHP (Gray et al. 2000; M. Hatterman-Zogg, and M. Maifeld.
to as phthalates, are widely used in industry Parks et al. 2000).
We thank the health care providers and study par- and commerce; they are used in personal care Despite the growing body of literature ticipants at University Physicians Clinic (Columbia, products (e.g., makeup, shampoo, and soaps), on phthalate reproductive toxicity and data MO), Fairview Riverside Women's Clinic (Minne-apolis, MN), Los Angeles Biomedical Research plastics, paints, and some pesticide formula- demonstrating extensive human exposure Institute at Harbor-UCLA Medical Center (Los tions. Consistent toxicologic evidence indi- (Silva et al. 2004a), few studies have examined Angeles, CA), Cedars-Sinai Medical Center (Los cates association between several of these the effects of these chemicals on human repro- Angeles, CA), and University of Iowa Hospitals and phthalate esters and reproductive effects.
ductive development. Colón et al. (2000) Clinics (Iowa City, IA). We also thank M. Silva, In particular, dibutyl phthalate (DBP), ben- reported elevated levels of several phthalates J. Reidy, E. Samandar, and J. Preau for phthalate zylbutyl phthalate (BzBP), di-2-ethylhexyl [including diethyl phthalate (DEP), DBP, and analyses and E. Gray, P. Foster, and D. Barr for theirguidance.
phthalate (DEHP), and di-isononyl phthalate DEHP] in serum samples from young girls This work was supported by grants from the U.S.
have been shown to disrupt reproductive tract with premature breast development. However, Environmental Protection Agency and the National development in male rodents in an antian- the timing of exposure was unknown and high Institutes of Health (R01-ES09916 to the University drogenic manner (Parks et al. 2000). Recent exposure levels may have reflected phthalate of Missouri, MO1-RR00400 to the University of studies have reported significant reductions contamination of serum samples (McKee and Minnesota, MO1-RR0425 to Harbor-UCLA in anogenital distance (AGD) in Sprague- Toxicology Research Task Group 2004). Until Medical Center) and by grant 18018278 from theState of Iowa to the University of Iowa.
Dawley rats after prenatal exposure at high recently, the only study of humans to evaluate The authors declare they have no competing doses to BzBP (Nagao et al. 2000; Tyl et al.
phthalate exposure and male reproductive 2004), DBP (Barlow and Foster 2003; Foster toxicity measured phthalate diesters in semen.
Received 7 March 2005; accepted 25 May 2005.
VOLUME 113 NUMBER 8 August 2005 • Environmental Health Perspectives Prenatal phthalate exposure and male anogenital distance measured using the neutral single-cell gel elec- institutions approved SFFI and SFFII, and all recoveries are near 100%), and precise with trophoresis (comet) assay (Duty et al. 2003b). In participants signed informed consents for each between-day relative standard deviations of this population of men attending an infertility < 10%. Quality control (QC) samples and clinic, increased urinary concentration of MBzP Physical examination. After standard
laboratory blanks were analyzed along with was also associated with decreased follicle stimu- anthropometric measurements (height, weight, unknown samples to monitor performance of lating hormone, whereas increases in monobutyl head circumference, and skin-fold thickness) the method. The metabolite concentrations phthalate were marginally associated with were obtained, a detailed examination of the reported here are from 85 prenatal maternal increased inhibin-B (Duty et al. 2005).
breast and genitals was conducted under the urine samples of a total of 214 that also Newborn male rodents have no scrotum, supervision of pediatric physicians who were included postnatal maternal and baby samples and the external genitalia are undeveloped; trained in its administration. Every attempt from the same mothers and their children.
only a genital tubercle is apparent for both was made to standardize the examination, The 214 samples were analyzed for phthalate sexes. The distance from the anus to the which was developed specifically for this study.
metabolites in six batches, none of which insertion of this tubercle, the AGD, is andro- These methods included training sessions had to be re-extracted for QC failures. Of the gen dependent and about twice as long in before and during the study and the use of stan- 214 samples, seven were re-extracted using males as in females. The AGD has been dardized equipment. Neither the pediatric < 1 mL of urine because concentrations of shown to be a sensitive measure of prenatal physicians nor the support staff had any knowl- MEP calculated using 1 mL were above the antiandrogen exposure (Rhees et al. 1997).
edge of the mother's phthalate concentrations.
linear range of the method.
Recently, Salazar-Martinez et al. (2004) stud- Boys' genital examinations included a Statistical analysis. After examining
ied AGD in 45 male and 42 female infants.
description of the testes and scrotum, location descriptive and summary statistics for all study They measured the distance from the anus to and size of each testicle, and measurement of variables, we explored models for AGD. We the base of the scrotum in males and from the penis. The placement of each testicle was fit several alternative measures of body size the anus to the base of the genitals (the four- initially coded in six categories; in the present (weight, height, and body mass index) and chette) in females. By these measures, AGD analysis, boys are dichotomized into those with both additive and multiplicative functions of was sexually dimorphic and about twice as normal testicular descent (placement of both these. We defined the anogenital index [AGI = long in males as in females. No other studies testes coded as normal or normal retractile) or AGD/weight (mm/kg)] as a weight-normalized have examined AGD among human males, with incomplete testicular descent (all other index of AGD.
although two other studies have evaluated cases). The scrotum was categorized as distinct AGD and AGI were modeled as both lin- AGD in female infants (Callegari et al. 1987; from surrounding tissue or not, and by size ear and quadratic functions of age. For babies Phillip et al. 1996).
(small or not). Penile width and (stretched) born at < 38 weeks, age at examination in the length were recorded, and penile volume [pro- first year was calculated from the estimated Materials and Methods
portional to (penile width/2)2 × penile length] date of conception instead of the birth date.
Study participants. Women included in our
was calculated. We recorded the AGD, meas- Once the best fitting model was identified, we study were originally recruited into the first ured from the center of the anus to the anterior plotted the expected AGI and its 25th and phase of the Study for Future Families (SFFI), a base of the penis. We also recorded the ano- 75th percentiles as a function of age. We cate- multicenter pregnancy cohort study, at prenatal scrotal distance (ASD), measured from the gorized boys in two ways: We dichotomized clinics in Los Angeles, California (Harbor- center of the anus to the posterior base of the boys into those with AGI smaller than or at UCLA and Cedars-Sinai), Minneapolis, scrotum. This latter measurement was used by least as large as expected, and we used the dif- Minnesota (University of Minnesota Health Salazar-Martinez et al. (2004), who refer to it ference between observed and expected AGI to Center), and Columbia, Missouri (University define three groups of boys, short (AGI < 25th Physicians), from September 1999 through Phthalate metabolite analysis. Urinary
percentile for age), intermediate (25th per- August 2002. Data collection is still ongoing phthalate metabolite analyses were carried centile ≤ AGI < 75th percentile), and long in Iowa, where a center was added late in out by the Division of Laboratory Sciences, (AGI ≥ 75th percentile for age) AGI. We also SFFI, so Iowa participants are not included in National Center for Environmental Health, calculated the proportion of boys in these three this analysis. Methods are described in detail Centers for Disease Control and Prevention groups with normal testicular descent (both elsewhere (Swan et al. 2003). Briefly, couples (CDC), which had no access to participant testes normal or normal retractile) and normal whose pregnancy was not medically assisted data. The analytical approach for the analysis scrotal (scrotum of normal size and distinct were eligible unless the woman or her partner of urinary phthalate metabolites (Silva et al.
from surrounding tissue). We calculated the was < 18 years of age, either partner did not 2004b) is a modification of previously pub- correlations between AGD and AGI and penile read and speak Spanish or English, or the lished methods (Silva et al. 2003). The analy- volume, testicular placement and scrotal para- father was unavailable or unknown. All partici- sis involves the enzymatic deconjugation of meters (size and distinctness from surrounding pants completed a questionnaire, most gave the phthalate metabolites from their glu- tissue). Our decision to use AGI as the measure blood samples, and after urine collection was curonidated form, automated on-line solid- of genital development was made, and cut added midway through the study, most also phase extraction, separation with high- points for categorical analyses of outcomes gave a urine sample.
performance liquid chromatography, and were selected, before obtaining phthalate Eighty-five percent of SFFI participants detection by isotope-dilution tandem mass metabolite values.
agreed to be recontacted, and we invited these spectrometry. This high-throughput method We used general linear models to explore mothers to take part in our follow-up study.
allows for the simultaneous quantification in the relationships between phthalate metabolite The family was eligible for the follow-up study human urine of the nine phthalate metabolites concentration (unadjusted for urine concentra- (SFFII) if the pregnancy ended in a live birth, reported in this work. Limits of detection tion) and genital parameters. Most metabolite the baby was 2–36 months of age, and the (LOD) are in the low nanogram per milliliter concentrations were above the LOD; those mother lived within 50 mi of the clinic and range. Isotopically labeled internal standards below the LOD were assigned the value LOD could attend at least one study visit. Here we were used along with conjugated internal stan- divided by the square root of 2, which has been report on results from the first study visit only.
dards to increase precision and accuracy of the recommended when the data are not highly Human subject committees at all participating measurements. The method is accurate (spiked skewed, as was the case here (Hornung and Environmental Health Perspectives • VOLUME 113 NUMBER 8 August 2005 Reed 1990). Metabolite concentrations were have genital measurements, no frank genital metabolites (other than MEP) were signifi- logarithmically transformed to normalize dis- malformations or disease were detected, and no cantly correlated (p < 0.005).
tributions. We examined several potentially parameters appeared grossly abnormal. The Regression analyses. We initially modeled
confounding factors including mother's ethnic- mean age at first examination was 15.9 months, AGD as a linear function of age and weight, ity and smoking status, time of day and season and mean weight was 10.5 kg (Table 2). Mean but this model fit poorly (adjusted R2 = 0.22).
in which the urine sample was collected, gesta- (± SD) AGD was 70.3 ± 11.0 mm, with a dis- We found that using AGI (AGD/weight) as a tional age at sample collection, and baby's tribution that was well approximated by a nor- function of age provided the best fit, as has weight at examination.
mal curve. Overall, 86.6% of boys had both been shown in rodent models (Vandenbergh We also categorized metabolite concentra- testes classified as normal or normal-retractile.
and Huggett 1995). The best-fitting model for tions into low (< 25th percentile), intermediate A prenatal urine sample was assayed for AGI includes linear and quadratic terms for (between the 25th and 75th percentiles), and phthalate metabolites for mothers of 85 of these age and is given by AGI = 10.8835 – 0.3798 high (≥ 75th percentile) categories and exam- boys. These mother–son pairs comprise the (age) + 0.0068 (age2) (adjusted R 2 = 0.61).
ined the odds ratio (OR) for smaller than data set for the analysis of AGD and phthalate Using this model, we calculated mean AGI expected AGI for babies with high compared metabolite concentration. Because urine collec- and its 5th, 25th, 75th, and 95th percentiles with low exposure, and medium compared tion began midway through SFFI, mothers with with low. On the basis of these regression and a stored urine sample were recruited later in We then examined models that included categorical analyses, we identified the phthalate the study, and their sons tended to be younger individual phthalate metabolites. Other than metabolites most strongly associated with AGI.
at examination (mean age, 12.6 months; age and age squared, no covariates altered We refer to these as AGI-associated phthalates.
interquartile range, 5–16 months). Summary regression coefficients for the phthalate metabo- Because phthalate metabolite concentra- statistics for all boys included in the analysis lites by > 15%, and none were included in final tions are highly correlated, and because our lim- of physical measurements, and the subset of models. All regression coefficients for individ- ited sample size prohibited us from examining boys for whom mothers' prenatal phthalate ual metabolites (logarithmically transformed to multiway interactions, we constructed a sum- concentrations were also available are shown normalize distributions) were negative (Table mary phthalate score to examine the effect of separately in Table 2.
4). MEP, mono-n-butyl phthalate (MBP), joint exposure to more than one AGI-associated All phthalate metabolites tested were above MBzP, and monoisobutyl phthalate (MiBP) phthalate. For this purpose, we used quartiles of the LOD in > 49% of women, and most tested were (inversely) related to AGI; p-values for metabolite concentration; values in the lowest were above the LOD in > 90% of the samples regression coefficients were between 0.007 quartile did not contribute to the sum, whereas (Table 3). Concentrations spanned four orders and 0.097. We also measured three metabo- higher values increased the sum one unit per of magnitude, from below the LOD (estimated lites of DEHP. Although the hydrolytic quartile. We divided this sum into three cate- value = 0.71 ng/mL) to 13,700 ng/mL for monoester metabolite mono-2-ethylhexyl gories: low (0–1, reflecting little or no exposure MEP. Means ranged from 2.68 for mono- phthalate (MEHP) was unrelated to AGI to AGI-associated phthalates), intermediate 3-carboxypropyl phthalate (MCPP) to 629.8 [regression coefficient = –0.05; 95% confi- (2–10), and high (11–12, reflecting high expo- for MEP. Three of the four AGI-associated dence interval (CI), –0.53 to 0.43], regression sure to all, or almost all, AGI-associated phtha- Table 1. Participants included in present analysis.
lates). We examined the magnitude of theresidual (observed – expected) AGI as a func- tion of this summary phthalate score.
potential participants All pregnancy outcomes (CA, MN, and MO) Potential participantsa Eligible for SFFII The population for the present analysis was SFFII participant identified from families recruited in California, Male babies only (CA, MN, and MO) Minnesota, or Missouri for whom data entry SFFII participant was complete by 17 December 2004, the cutoff With AGD, age, and weightb date for the present analysis. At that time, Prenatal urine samplec 654 participants from these three centers had aA potential participant is an SFFI participant from CA, MO, or MN who gave permission to be recontacted for future studies
completed SFFI and given permission to be and for whom all study data were entered by 17 December 2004. bBoys in twin births and boys with missing data or AGD
recontacted. Of these, 477 (72.9%) were eligi- measurements considered unreliable by pediatricians excluded. cUrine collection began midway through SFFI.
ble for SFFII and 346 (72.5%) participated Table 2. Characteristics of boys with complete physical examination.
(Table 1). SFFII participants were demographi- cally similar to nonparticipants except that non- participants were more likely to be Hispanicbecause of a lower eligibility rate (60%) in CA, All boys (n = 134) where most participants were Hispanic. Of the 172 boys born to these mothers, we excluded 5 boys in twin births, 10 boys with incomplete data, and 23 boys for whom AGD was not recorded [two whose mothers declined the geni- tal exam, with the remainder older boys (mean Boys whose mother's prenatal urine wasassayed for phthalate metabolites (n = 85) age, 19.6 months), for whom the study exam- iner felt the measurement was not reliable, usually because of the boys' activity level]. The remaining 134 boys comprise the sample used for the analysis of AGD and other genital meas- urements. Among the 134 boys for whom we VOLUME 113 NUMBER 8 August 2005 • Environmental Health Perspectives Prenatal phthalate exposure and male anogenital distance coefficients for the oxidative monoester MEP, MBzP, and MiBP were 4.7, 3.8, and –0.137). For the other phthalate metabolites, metabolites of DEHP, mono-2-ethyl-5-oxo- 9.1, respectively (all p-values < 0.05).
regression coefficients were less significant (all hexyl phthalate (MEOHP), and mono-2-ethyl- Other genital parameters. Degree of testic-
p-values between 0.11 and 0.97).
5-hydroxyhexyl phthalate (MEHHP) were of a ular descent was associated with AGD (R = Summary phthalate score. We used
magnitude comparable with those for MEP 0.20, p = 0.02). The proportions of boys with the summary phthalate score as defined in and MBzP (p-values = 0.114 and 0.145 for one or both testicles incompletely descended "Materials and Methods" to study the effect of MEOHP and MEHHP, respectively). AGI were 20.0, 9.5, and 5.9% for boys classified as joint exposure to more than one AGI-associated appeared to be independent of the concentra- having short, intermediate, and long AGI phthalate. The summary phthalate score was tions of monomethyl phthalate (MMP) and (p-value for short AGI compared with all other directly related to the proportion of boys with MCPP, metabolites of dimethyl phthalate and boys < 0.001). The proportion of boys with a short AGI (p = 0.001). Of the 10 boys whose di-n-octyl phthalate, respectively.
scrotum categorized as small and/or "not dis- phthalate scores were high (score = 11–12), all Categorical analyses. The 25 boys with
tinct from surrounding tissue" was also ele- but one had a short AGI. Conversely, of the AGI below the 25th percentile for age were vated for boys with short AGI (p < 0.001).
11 boys whose scores were low (score = 0 or 1), classified as having a short AGI. This group had AGD was significantly associated with penile only one had a short AGI. The ORs for having an AGI that was, on average, 18.3% (range, volume (R = 0.27, p = 0.001), and penile vol- a short AGI for high summary phthalate score 10–32%) shorter than expected based on the ume divided by weight was correlated with compared with low (OR = 90.0; 95% CI, final regression model. Boys with AGI ≥ 75th AGI (R = 0.43, p = 0.001). Testicular volume, 4.88 to 1,659), and high compared with percentile of expected were classified as having a which was measured by orchidometer, is not medium (29.4; 95% CI, 3.4 to 251) were large long AGI, and boys with AGI between the shown here because participating physicians and significant, although the confidence inter- 25th and 75th percentile of expected were con- considered the measurement to be unreliable— vals were very wide. These data are shown sidered intermediate. Boys' weight and age did a decision made before analyses of phthalate graphically in Figure 1.
not differ appreciably among these groups.
Table 5 shows mean and median values ASD was, on average, 47% as long as for the AGI-associated metabolites for boys in AGD, and these two measurements were cor- In the recent National Health and Nutrition the short, intermediate, and long categories of related (R = 0.47, p < 0.0001). However, the Examination Survey (NHANES 1999–2000), AGI. We calculated the ORs for short AGI for model predicting ASD as a function of baby's most of the general population in the United each monoester metabolite (Table 6). For high age and weight fit poorly (adjusted R2 = 0.10).
States had measurable exposure to multiple compared with low concentration of MBP, The fit for the model using ASD/weight as a phthalates (CDC 2003; Silva et al. 2004a).
the OR for a short AGI was 10.2 (95% CI, function of age and age squared was better The samples in the present study and in 2.5 to 42.2), whereas for medium concentra- (adjusted R2 = 0.47) but did not fit as well as NHANES were both analyzed using compara- tion compared with low the OR was 3.8 (95% the model using AGI (R2 = 0.61). ASD/weight ble methods and standards by the same labora- CI, 1.2 to 12.3). The corresponding ORs for was associated with MEP concentration (regres- tory, although the specific metabolites that high compared with low concentration of sion coefficient = –0.429; 95% CI, –0.722 to were measured in the two studies differedsomewhat. We compared the medians and Table 3. Percentiles of phthalate monoester metabolites.
75th percentiles of the AGI-associated phtha-late metabolite concentrations among two Percentile (ng/mL) groups of mothers in our study (those whose Monoester metabolite Percent > LODa boys fell in the short AGI group and all others) Phthalate monoester metabolite with those of females in the NHANES sample (Table 7). In the analysis of the NHANES samples, monobutyl phthalate includes both MBP and MiBP, which were measured sepa- rately in our study. Metabolite concentrations for mothers of boys with short AGI were con- Metabolites of DEHP sistently higher than those of other mothers.
Compared with women in the NHANES sam- ple, metabolite concentrations for our popula-tion were somewhat lower. However, our aLOD for all metabolites was between 0.95 and 1.07 ng/mL.
population cannot be directly compared with Phthalate score 0–1 Table 4. Regression analyses of AGI on log10 monoester metabolite concentration, controlling for age and
Phthalate score 2–10 age squared.
Phthalate score 11–12 Log10 monoester metabolite concentration Monoester metabolite 0.031 (–1.126 to –0.057) 0.097 (–0.851 to 0.072) AGI (mm/kg)
0.461 (–0.973 to 0.445) 0.145 (–0.935 to 0.140) 0.833 (–0.530 to 0.428) 0.114 (–0.925 to 0.101) 0.017 (–0.726 to –0.074) 0.007 (–1.309 to –0.220) Boy's age (months)
0.383 (–0.924 to 0.359) –0.0951 (0.035) 0.009 (–0.165 to –0.025) Figure 1. Mean AGI (mm/kg) in relation to boys' age
at examination (months).
aPhthalate score measures joint exposure to MBP, MBzP, MEP, and MiBP; see "Statistical analysis."
Environmental Health Perspectives • VOLUME 113 NUMBER 8 August 2005 NHANES: the proportion of pregnant women more sensitive to trenbolone by an order of human studies suggesting reproductive toxic- in the NHANES sample is unknown, and age magnitude (Neumann 1976). This greater ity (Colón et al. 2000; Duty et al. 2003b; distributions differ. Nonetheless, these data sensitivity is thought to be a result of rodents' Main KM, unpublished data). It is therefore demonstrate that the four AGI-associated higher metabolic rate and more rapid inactiva- uncertain whether the absence of data in phthalate metabolites are prevalent in the U.S.
tion of toxicants, both of which have been rodents showing reproductive toxicity is the female population, and levels were not unusu- shown to be inversely related to body size result of failure to detect it, unmeasured con- ally high among mothers whose sons had a (White and Seymour 2005).
founding in human studies, or interspecies In light of the toxicologic literature differences in response to these compounds.
Although not identical, AGD in pups is for MBP, MBzP, and MiBP (Ema et al. 2003; DEHP has been shown to shorten AGD most similar to AGD as we defined it in this Foster et al. 1980, 1981; Gray et al. 2000; (Gray et al. 2000) and reduce testosterone study. In rodents, AGD has been shown to be Nakahara et al. 2003), our data suggest that (Parks et al. 2000). Although MEHP was not one of the most sensitive end points for phtha- the end points affected by these phthalates are associated with AGD in our data, the associa- lates such as DBP (Mylchreest et al. 2000) and quite consistent across species. A boy with tions for the oxidative metabolites of DEHP other antiandrogens such as flutamide (Barlow short AGI has, on average, an AGI that is 18% (MEOHP and MEHHP) were of comparable and Foster 2003; McIntyre et al. 2001) and shorter than expected based on his age and magnitude with those for metabolites of DBP finasteride (Bowman et al. 2003). It is difficult weight as well as an increased likelihood of tes- and BzBP, although not statistically signifi- to compare the dose to humans from low- ticular maldescent, small and indistinct scro- cant. Thus, it is unclear whether MEOHP level, ongoing, environmental exposure with tum, and smaller penile size. These changes in and MEHHP are (inversely) associated with that delivered to rodents experimentally in AGD and testicular descent are consistent with AGI, although associations are of borderline a narrow window of gestation. Nonetheless, those reported in rodent studies after high-dose statistical significance because of our sample it is likely that the doses to which our par- phthalate exposure (Ema et al. 2003; Gray size, or whether human and rodent responses ticipants were exposed are lower than those et al. 2000; Mylchreest et al. 2000). The lack to this phthalate and its metabolites differ.
used in toxicologic settings, suggesting that of association for MCPP and MMP, which Masculinization of external male genitalia, humans may be more sensitive to prenatal have not been widely studied, is not inconsis- represented by longer AGD, is controlled by phthalate exposure than rodents. This greater tent with the toxicologic literature.
dihydrotestosterone (Clark et al. 1990). Ema sensitivity in humans has been observed for With respect to DEP and its metabolite and Miyawaki (2001) demonstrated that other toxicants. For example, humans are MEP, we note that there are three other this metabolite of testosterone is markedlydecreased by prenatal administration of MBP, Table 5. Mean (median) phthalate monoester metabolite levels by AGI category.
suggesting that MBP acts as an antiandrogen.
AGI category [mean (median; ng/mL)] AGD in male rodents is associated with other Monoester metabolite Intermediateb (n = 43) adverse developmental effects (Foster and McIntyre 2002) and some phthalate-induced changes have been shown to be permanent.
For example, Barlow et al. (2004) report that prenatal exposure to 500 mg/kg/day DBP aLong, AGI ≥ 75th percentile of expected AGI. bIntermediate, 25th percentile ≤ AGI < 75th percentile of expected AGI.
resulted in permanently decreased AGD and cShort, AGI < 25th percentile of expected AGI.
testicular dysgenesis. They also report that Table 6. OR (95% CI) for AGI less than expected from regression model, by monoester metabolite level.
in utero DBP exposure induced proliferative Monoester metabolite Level (percentile) AGI < expected Leydig cell lesions. Follow-up of exposed chil-dren until adulthood will be required to determine whether long-term effects, includ- 3.8 (1.2 to 12.3) 10.2 (2.5 to 42.2) ing testicular dysgenesis, are seen in humans after prenatal phthalate exposure.
3.1 (1.002 to 9.8) Several recent studies of the variability of 3.8 (1.03 to 13.9) phthalate monoester concentration in human samples suggest that phthalate concentration in humans is fairly stable, perhaps reflecting 4.7 (1.2 to 17.4) habitual use of phthalate-containing household 3.4 (1.1 to 10.5) and consumer products (Colón et al. 2000; 9.1 (2.3 to 35.7) Hauser et al. 2004; Hoppin et al. 2002). These Low, < 25th percentile; medium, ≥ 25th and < 75th percentile; high, ≥ 75th percentile.
studies lend support to the use of a singlesample for exposure assessment. We obtained Table 7. Concentrations of four phthalate metabolites in three groups of women (ng/mL).
only a single prenatal urine sample from each woman, and most samples were obtained Monoester metabolite quite late in pregnancy (mean = 28.3 weeks).
Therefore, the measured phthalate metabolite levels may not reflect exposure during the most sensitive developmental window, resulting in some degree of exposure misclassification.
However, unless this misclassification varied systematically with outcome, such errors would bias the effect estimate toward the null. In aFemales only (CDC 2003). bMBP in the NHANES analysis includes both MBP and MiBP; in this study these metabolites
fact, the categorical analysis, which should be were measured separately.
less sensitive to such misclassification, showed VOLUME 113 NUMBER 8 August 2005 • Environmental Health Perspectives Prenatal phthalate exposure and male anogenital distance stronger associations than did the continuous genital morphology is altered by antiandro- Parks L. 2000. Perinatal exposure to the phthalates DEHP, gens, including some phthalates. We report BBP, and DINP, but not DEP, DMP, or DOTP, alters sexualdifferentiation of the male rat. Toxicol Sci 58:350–365.
Our analysis is based on a single measure of that AGD, the most sensitive marker of anti- Hauser R, Meeker JD, Park S, Silva MJ, Calafat AM. 2004.
AGD, and the reliability of this measurement androgen action in toxicologic studies, is Temporal variability of urinary phthalate metabolite lev- in humans has not been established. During shortened and testicular descent impaired in els in men of reproductive age. Environ Health Perspect112:1734–1740.
two training sessions, three study physicians boys whose mothers had elevated prenatal Hoppin JA, Brock JW, Davis BJ, Baird DD. 2002. Reproducibility each measured AGD in four male infants phthalate exposure. These changes in male of urinary phthalate metabolites in first morning urinesamples. Environ Health Perspect 110:515–518.
(mean age, 8.1 months). The mean AGD for infants, associated with prenatal exposure to Hornung RW, Reed LD. 1990. Estimation of average concentra- these measurements was 58.6 mm, SD was some of the same phthalate metabolites that tion in the presence of nondectable values. Appl Occup (within infant) 4.2 mm, and coefficient of vari- cause similar alterations in male rodents, sug- Environ Hyg 5:46–51.
McIntyre BS, Barlow NJ, Foster PM. 2001. Androgen-mediated ation of 7.2%, suggesting that AGD can be gest that commonly used phthalates may development in male rat offspring exposed to flutamide measured reliably. Use of this measurement in undervirilize humans as well as rodents.
in utero: permanence and correlation of early postnatal larger studies in a range of diverse populations, changes in anogenital distance and nipple retention withmalformations in androgen-dependent tissues. Toxicol Sci with many more such training sessions, will be needed to obtain normative data.
McKee RH, Toxicology Research Task Group. 2004. Phthalate exposure and early thelarche. Environ Health Perspect Although it might have been ideal to Barlow NJ, Foster PM. 2003. Pathogenesis of male reproductive tract lesions from gestation through adulthood following examine babies shortly after birth, the timing in utero exposure to di(n-butyl) phthalate. Toxicol Pathol Murature DA, Tang SY, Steinhardt G, Dougherty RC. 1987.
of grant funding did not allow this. Babies Phthalate esters and semen quality parameters. BiomedEnviron Mass Spectrom 14:473–477.
were born to SFFI mothers as early as January Barlow NJ, McIntyre BS, Foster PM. 2004. Male reproductive tract lesions at 6, 12, and 18 months of age following Mylchreest E, Wallace DG, Cattley RC, Foster PMD. 2000. Dose- 2000, and the first baby visits did not occur in utero exposure to di(n-butyl) phthalate. Toxicol Pathol dependent alterations in androgen-regulated male repro- until April 2002. To maximize the number ductive development in rats exposed to di(n-butyl) phthalate Bowman CJ, Barlow NJ, Turner KJ, Wallace DG, Foster PM.
during late gestation. Toxicol Sci 55:143–151.
of children participating, we allowed recruit- 2003. Effects of in utero exposure to finasteride on andro- Nagao T, Ohta R, Marumo H, Shindo T, Yoshimura S, Ono H.
ment over a range of ages. On the other hand, gen-dependent reproductive development in the male rat.
2000. Effect of butyl benzyl phthalate in Sprague-Dawleyrats after gavage administration: a two-generation repro- because the use of AGD in humans is new, Toxicol Sci 74:393–406.
Callegari C, Everett S, Ross M, Brasel JA. 1987. Anogenital ductive study. Reprod Toxicol 14:513–532.
the optimal timing for this measurement is ratio: measure of fetal virilization in premature and full- Nakahara H, Shono T, Suita S. 2003. Reproductive toxicity evalu- not known. Our data suggest that measure- term newborn infants. J Pediatr 111:240–243.
ation of dietary butyl benzyl phthalate (BBP) in rats. FukuokaIgaku Zasshi 94:331–337.
ments are reliable and informative in young CDC. 2003. Second National Report on Human Exposure to Environmental Chemicals. Atlanta, GA:Centers for Disease Neumann F. 1976. Pharmacological and endocrinological stud- children at least until 18 months, when AGD Control and Prevention, National Center for Environmental ies on anabolic agents. Environ Qual Saf 5(suppl)253–264.
Parks LG, Ostby JS, Lambright CR, Abbott BD, Klinefelter GR, becomes more difficult to obtain reliably. Its Health, Division of Laboratory Sciences.
Clark RL, Antonello JM, Grossman SJ, Wise LD, Anderson C, Barlow NJ, et al. 2000. The plasticizer diethylhexyl phtha- value in adolescents and adults has yet to be Bagdon WJ, et al. 1990. External genitalia abnormalities in late induces malformations by decreasing fetal testos- male rats exposed in utero to finasteride, a 5 alpha-reduc- terone synthesis during sexual differentiation in the malerat. Toxicol Sci 58:339–349.
We note that phthalate metabolite levels tase inhibitor. Teratology 42:91–100.
Colón I, Caro D, Bourdony CJ, Rosario O. 2000. Identification Phillip M, De Boer C, Pilpel D, Karplus M, Sofer S. 1996. Clitoral were highly correlated, and most women were of phthalate esters in the serum of young Puerto Rican and penile sizes of full term newborns in two different exposed to all metabolites at detectable levels.
girls with premature breast development. Environ Health ethnic groups. J Pediatr Endocrinol Metab 9:175–179.
Rhees RW, Kirk BA, Sephton S, Lephart ED. 1997. Effects of pre- Gray et al. (2000) suggested that risk assess- Duty SM, Calafat AM, Silva MJ, Ryan L, Hauser R. 2005. Phthalate natal testosterone on sexual behavior, reproductive mor- ments for phthalate-induced reproductive toxi- exposure and reproductive hormones in adult men. Hum phology and LH secretion in the female rat. Dev Neurosci19:430–437.
city should consider phthalates as a group and Duty SM, Silva MJ, Barr DB, Brock JW, Ryan L, Chen Z, et al.
Salazar-Martinez E, Romano-Riquer P, Yanez-Marquez E, include exposures from multiple sources. The 2003a. Phthalate exposure and human semen parameters.
Longnecker MP, Hernandez-Avila M. 2004. Anogenital dis- score we use reflects joint exposure to the four tance in human male and female newborns: a descriptive,cross-sectional study. Environ Health 3:8; doi:10.1186/1476- AGI-associated phthalates, and our results sug- Duty SM, Singh NP, Silva MJ, Barr DB, Brock JW, Ryan L, et al.
2003b. The relationship between environmental exposures to 069X-3-8 [Online 13 September 2004].
gest that joint exposure may convey greater phthalates and DNA damage in human sperm using the neu- Silva MJ, Barr DB, Reidy JA, Malek NA, Hodge CC, Caudill SP, than additive risk, but larger sample sizes are tral comet assay. Environ Health Perspect 111:1164–1169.
et al. 2004a. Urinary levels of seven phthalate metabolites in Ema M, Miyawaki E. 2001. Adverse effects on development of the U.S. population from the National Health and Nutrition needed to confirm this.
the reproductive system in male offspring of rats given Examination Survey (NHANES) 1999–2000. Environ Health Gray and Foster (2003) refer to a "phtha- monobutyl phthalate, a metabolite of dibutyl phthalate, Silva MJ, Malek NA, Hodge CC, Reidy JA, Kato K, Barr DB, et al.
late syndrome" characterized by testicular, epi- during late pregnancy. Reprod Toxicol 15:189–194.
Ema M, Miyawaki E, Hirose A, Kamata E. 2003. Decreased 2003. Improved quantitative detection of 11 urinary phtha- didymal, and gubernacular cord agenesis as well anogenital distance and increased incidence of unde- late metabolites in humans using liquid chromatography- as decreased AGD, and stress the importance of scended testes in fetuses of rats given monobenzyl phtha- atmospheric pressure chemical ionization tandem massspectrometry. J Chromatogr B Analyt Technol Biomed Life evaluating all components of a syndrome so late, a major metabolite of butyl benzyl phthalate. ReprodToxicol 17:407–412.
that affected animals are not misidentified. It Fisher JS. 2004. Environmental anti-androgens and male repro- Silva MJ, Slakman AR, Reidy JA, Preau JL Jr, Herbert AR, Samandar E, et al. 2004b. Analysis of human urine for fif- has recently been suggested (Fisher 2004) that ductive health: focus on phthalates and testicular dysgene-sis syndrome. Reproduction 127:305–315.
teen phthalate metabolites using automated solid-phase this "phthalate syndrome" shares many features Foster PM, Cattley RC, Mylchreest E. 2000. Effects of di-n-butyl extraction. J Chromatogr B Analyt Technol Biomed Life Sci with the human testicular dysgenesis syndrome phthalate (DBP) on male reproductive development in the Skakkebaek NE, Rajpert-De Meyts E, Main KM. 2001. Testicular proposed by Skakkebaek et al. (2001) to follow rat: implications for human risk assessment. Food ChemToxicol 38:S97–S99.
dysgenesis syndrome: an increasingly common develop- chemically induced disruption of embryonic Foster PM, Lake BG, Thomas LV, Cook MW, Gangolli SD. 1981.
mental disorder with environmental aspects. Hum Reprod programming and gonadal development during Studies on the testicular effects and zinc excretion pro- duced by various isomers of monobutyl-o-phthalate in the Swan SH, Brazil C, Drobnis EZ, Liu F, Kruse RL, Hatch M, et al.
fetal life. The present findings, though based on rat. Chem Biol Interact 34:233–238.
2003. Geographic differences in semen quality of fertile U.S.
small numbers, provide the first data in humans Foster PM, McIntyre BS. 2002. Endocrine active agents: implica- males. Environ Health Perspect 111:414–420.
Tyl RW, Myers CB, Marr MC, Fail PA, Seely JC, Brine DR, et al.
linking measured levels of prenatal phthalates to tions of adverse and non-adverse changes. Toxicol Pathol30:59–65.
2004. Reproductive toxicity evaluation of dietary butyl ben- outcomes that are consistent with this proposed Foster PM, Thomas LV, Cook MW, Gangolli SD. 1980. Study of zyl phthalate (BBP) in rats. Reprod Toxicol 18:241–264.
the testicular effects and changes in zinc excretion pro- Vandenbergh JG, Huggett CL. 1995. The anogenital distance index, a predictor of the intrauterine position effects This is the first study to look at subtle duced by some n-alkyl phthalates in the rat. Toxicol ApplPharmacol 54:392–398.
on reproduction in female house mice. Lab Anim Sci patterns of genital morphology in humans in Gray LE Jr, Foster PMD. 2003. Significance of experimental White CR, Seymour RS. 2005. Allometric scaling of mammalian relation to any prenatal exposure. It was moti- studies for assessing adverse effects of endocrine-disrupting chemicals. Pure Appl Chem 75:2125–2141.
metabolism. J Exp Biol 208:1611–1619.
vated by toxicologic studies showing that Gray LE Jr, Ostby J, Furr J, Price M, Veeramachaneni DNR, Environmental Health Perspectives • VOLUME 113 NUMBER 8 August 2005
R ESEARCH ARTICLE The effect of mirtazapine on methotrexate-inducedtoxicity in rat liver Bunyami Ozogula, Abdullah Kisaoglua, Mehmet Ibrahim Turanb,∗, Durdu Altunerc, Ebru Senerd,Nihal Cetine, Cengiz Ozturke,f a Department of Surgery, Faculty of Medicine, Ataturk University, 25240, Erzurum, Turkeyb Department of Paediatrics, Faculty of Medicine, Ataturk University, 25240, Erzurum, Turkeyc Department of Pharmacology, Faculty of Medicine, Recep Tayyip Erdogan University, 53100, Rize, Turkeyd Department of Pathology, Erzurum Region Education and Research Hospital, Erzurum, Turkeye Department of Pharmacology-Toxicology, Faculty of Veterinary Medicine, Ataturk University, 25240,