1 Environmental Health Department, Occupational Health Program and 2 Biostatistics Department, Harvard School of Public Health, Boston, MA 02115, 3 Department of Nursing, School for Health Studies, Simmons College, Boston, MA 02115 4 National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341 5 Department of Biostatistical Science, Dana-Farber Cancer Institute, Boston, MA 02115 and 6 Vincent Memorial Obstetrics & Gynecology Service, Andrology Laboratory and In Vitro Fertilization Unit, Massachusetts General Hospital, Boston, MA 02114, USA
7 To whom correspondence should be addressed
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Abstract |
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Key words: environmental/epidemiology/hormones/human/phthalates
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Introduction |
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Gestational, lactational or pubertal exposures to di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBzP) and di-2-ethylhexyl phthalate (DEHP) in rodents demonstrate an anti-androgenic mechanism of toxicity, but not at the level of the androgen receptor (Gray et al., 1999, 2000
; Foster et al., 2001
). Exposure to DEHP from gestational day 14 to post-natal day 3 reduced fetal and neonatal testosterone production in rats, thus suggesting a possible mechanism for the reproductive toxicity of phthalates (Parks et al., 2000
).
Limited human data are available on the possible relationship between phthalates and testicular function. In previous studies on men who were partners in infertile relationships, we found an inverse doseresponse relationship between monobutyl phthalate (MBP), a metabolite of DBP, and sperm motility and concentration, and between monobenzyl phthalate (MBzP), a metabolite of BBzP, and sperm concentration (Duty et al., 2003a). We also found a relationship between monoethyl phthalate (MEP), a metabolite of diethyl phthalate (DEP), and increased sperm DNA damage measured using the neutral single cell gel electrophoresis (comet) assay (Duty et al., 2003b
). In the present study on men from the same study population, we explored the relationship between environmental exposure to phthalates and reproductive hormones.
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Materials and methods |
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Reproductive hormones
One non-fasting blood sample was drawn between 09:00 and 16:00. Blood samples were centrifuged and serum stored at 20°C until analysis. Serum was analysed for hormones as follows. Testosterone was measured directly using the Coat-A-Count RIA kit (Diagnostic Products, USA), which have inter-assay and intra-assay coefficients of variation (CV) of 12 and 10% respectively with a sensitivity of 4 ng/dl (0.139 nmol/l). The free androgen index (FAI) was calculated as the molar ratio of total testosterone to sex hormone-binding globulin (SHBG) (Wilke and Utley, 1987). SHBG was measured using a fully automated system (Immulite: DPC, Inc.) which uses a solid-phase two-site chemiluminescent enzyme immunometric assay. Inhibin B was measured using a commercially available, double antibody, enzyme-linked immunosorbent assay (Oxford Bioinnovation, UK) with inter-assay and intra-assay CV of 20 and 8% respectively, limit of detection (LOD) of 15.6 pg/ml and a functional sensitivity (20% CV) of 50 pg/ml (Groome et al., 1996
). Serum LH and FSH concentrations were determined by microparticle enzyme immunoassay using an automated Abbott AxSYM system (Abbott Laboratories, USA). The Second International Reference Preparation (WHO 71/223) was used as the reference standard. The assay sensitivity for LH and FSH were 1.2 and 1.1 IU/l respectively. The intra-assay CV for LH and FSH were <5 and <3% respectively, with inter-assay CV for both hormones of <9%.
Phthalate monoester metabolites in urine
The phthalate monoester metabolites were measured because of potential sample contamination from the parent diester and because some of the metabolites are believed to be the active toxicant as opposed to the parent diester compounds (Peck and Albro, 1982; Li et al., 1998
). Phthalate monoesters were measured in a single spot urine sample collected in a sterile specimen cup on the same day as the blood sample. The analytical approach has been described in detail and adapted recently to enable the detection of additional monoesters and improve efficiency of the analysis (Blount et al., 2000
; Silva et al., 2003
). Briefly, phthalate metabolite determination in urine involved enzymatic deconjugation of the metabolites from their glucuronidated form, solid-phase extraction followed by reversed-phase high-performance liquid chromatographyatmospheric pressure chemical ionizationtandem mass spectrometry using isotope dilution with 13C4 internal standards. Detection limits were in the low nanogram per millilitre range. One method blank, two quality control samples (human urine spiked with phthalate monoesters), and two sets of standards were analysed along with every 21 unknown urine samples. Analysts at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA, were blind to all information concerning subjects.
Phthalate monoester levels were normalized for urine dilution by specific gravity (SG) adjustment using the following formula: Pc=P[(1.0241)/SG1)] where Pc is the SG-corrected phthalate concentration (ng/ml), P is the observed phthalate concentration (ng/ml) and SG is the specific gravity of the urine sample (Boeniger et al., 1993; Teass et al., 1998
). Specific gravity was measured using a hand-held refractometer (National Instrument Company, Inc., USA), which was calibrated with deionized water before each measurement.
Statistical analyses
Statistical Analysis Software (SAS) version 8.1 (SAS Institute Inc., Cary, NC, USA) was used for data analysis. Descriptive and summary statistics were generated, outcomes were assessed for outliers and general distributional shape and the associations between covariates and hormone levels were explored for evidence of non-linearity. In preliminary analyses, scatter-plots and Spearman correlation coefficients were used to explore the association between each hormone concentration and each phthalate metabolite concentration. Multiple linear regression analysis was then performed adjusting for appropriate covariates. Residuals were then checked for normality, homogeneity and lack of pattern with respect to covariates of interest. As possible covariates, we considered smoking status (i.e. current and former versus never), race, age, body mass index (BMI), previous infertility evaluation (i.e. yes or no), prior ability to impregnate a partner (i.e. yes or no), season (spring, summer and fall versus winter) and time of day the blood was drawn [morning (09:00 to 12:59) and afternoon (13:00 to 16:00)]; the inclusion of specific covariates in the multivariate models was based on statistical and biological considerations (Hosmer and Lemeshow, 1989). Age and BMI were modelled as continuous independent variables after evaluating appropriateness using a quadratic term, all others as dummy variables. For the secondary analyses, we excluded urine samples with specific gravity values <1.010 (too diluted) or >1.030 (too concentrated) (Teass et al., 1998
).
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Results |
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Secondary analysis
In secondary analyses, subjects with urine samples that were too concentrated or too dilute (specific gravity <1.010 or >1.030 respectively) were excluded. The associations between the concentrations of MBzP and FSH and between MBP and inhibin B were essentially unchanged. The association between MEHP and testosterone became weaker [0.42 (95% CI: 1.05, 0.21); P=0.19]. There were suggestive associations between MEP and testosterone and between MMP and FSH. For an IQR change in MEP and MMP, testosterone increased 0.73 nmol/ml (95% CI: 0.05, 1.52; P=0.07) and FSH increased by a multiplicative factor of 1.09 (95% CI: 0.99, 1.20; P=0.07) respectively. For the median of testosterone (14.2 nmol/ml) and FSH (7.3 IU/l), this represents a 5% (95% CI: 0, 11) and a 9% (95% CI: 1, 20) increase respectively. We also excluded one subject with an extreme MBP value and results were unchanged.
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Discussion |
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There are limited toxicological data on exposure of adult animals to phthalates, including BBzP, DBP and DEHP, and effects on reproductive hormones. In a study on adult male Fisher 344 rats, Agarwal et al. (1985) evaluated the potential effects BBzP had on male rats' reproductive health following dietary exposure to this phthalate. Plasma testosterone levels were significantly reduced following BBzP exposure, whereas plasma FSH levels increased in a dose-dependent fashion. LH levels were also increased. Marked degenerative changes in the testicular Leydig cells were not observed (Agarwal et al., 1985
). The increased FSH and LH concentrations in rats with reduced serum testosterone indicated responsiveness of the negative feedback system and suggested that pituitaryhypothalamic function was not impaired. BBzP metabolizes primarily to MBzP; MBP is also a minor metabolite of BBzP.
Although we found associations between some phthalate metabolites and serum hormone levels in the present study, there were several potential limitations in our design and methodology. We used a single blood and one-spot urine sample to measure hormone and phthalate metabolite concentrations respectively. Despite the diurnal and pulsatile fluctuations in serum hormone levels, in population studies a single blood sample can be used to provide a reliable measure of testosterone and FSH over both short and long time-periods (Bain et al., 1987; Vermeulen and Verdonck, 1992
; Schrader et al., 1993
). Additionally, requiring multiple blood samples may limit participation rates in epidemiological studies (Schrader et al., 1993
).
A single-spot urine sample was used to measure urinary levels of phthalate monoester metabolites. The development of internal dose biomarkers of phthalate exposure allows for accurate assessments of human exposure since urinary concentrations of these metabolites represent an integrative measure of exposure to phthalates from multiple sources and pathways. Since humans rapidly metabolize phthalate diesters to their respective monoesters, phthalates do not bioaccumulate (Peck and Albro, 1982; ATSDR, 2002
; Koo et al., 2002
). Biological half-lives of phthalates are on the order of 1 day or less and hence represent exposure for no more than the few days preceding the collection of the urine specimen. Since most health endpoints of interest are likely affected by exposures over time-periods longer than a few days, information on the temporal variability of urinary levels of phthalate monoesters is needed to optimize exposure assessment in epidemiological studies. There are limited published data on the temporal variability of urinary phthalate monoester concentrations. A recent study documented acceptable reproducibility of urinary phthalate monoester levels in two first-morning urine specimens collected for 2 consecutive days; day-to-day intra-class correlation coefficients ranged from 0.5 to 0.8 (Hoppin et al., 2002
). Time intervals beyond a couple of days were not explored. If there is substantial temporal variability in urinary phthalate monoester levels, the associations between phthalate metabolite levels in urine and reproductive hormones may be attenuated.
Although the men in the present study may not be representative of men from the general population in Massachusetts, generalizability of the results is not necessarily limited. It is a misconception that generalization from a study group depends on the study group's being a representative subgroup of the target population (Rothman and Greenland, 1998). For generalizability to be limited, the associations between reproductive hormones and phthalates in this clinic population would have to differ from the associations within the larger general population. Therefore, we would need to speculate that, compared to others, men visiting this andrology clinic display an altered hormonal response to phthalates. Currently, there is no reason to suspect that the susceptibility to phthalates in the men who visit this andrology clinic is different to that from men who visit other clinics or men from the general population. However, until the results of the present study are replicated in larger and more diverse populations, the generalizability of our results will remain unclear.
We attempted to synthesize the present findings with the results from our previous work investigating the association between phthalates and several other reproductive endpoints. Although we found evidence that MEP exposure at environmental levels was associated with DNA damage in sperm (Duty et al., 2003b), there was little evidence that MEP was associated with any change in semen parameters (count, motility or morphology) (Duty et al., 2003a
), or with changes in hormone levels. The implications of these findings are unclear, but suggest that sperm DNA damage may result from mechanisms unrelated to alterations in semen quality or hormone profiles.
In our earlier studies, we also found inverse relationships between both MBP and MBzP and sperm concentration and between MBP and sperm motility (Duty et al., 2003a). Because these two phthalate metabolites are known Sertoli cell toxicants, we hypothesize that inhibin B, produced by Sertoli cells, would be inversely related to these phthalate monoesters. However, in the present study, inhibin B did not decrease but rather increased with higher MBP levels and there was no concurrent increase in FSH levels. Additionally, higher MBzP exposure was associated with a decrease in FSH but no change in inhibin B level. In short, although higher MBP and MBzP exposures were associated with lower sperm counts and motility in our previous work (Duty et al., 2003a
), FSH and inhibin B levels did not change in the expected direction. The pattern of change of inhibin B and FSH are inconsistent with other studies which showed that serum inhibin B, in combination with serum FSH levels, were a sensitive marker of impaired spermatogenesis (Uhler et al., 2003
). In one of these studies, an inhibin B level <80 pg/ml, in combination with an FSH level >10 mIU/ml, was 100% predictive for sperm concentrations <20 x 106/ml (Jensen et al., 1997
). At present, it is unclear why our results were inconsistent with earlier studies supporting the utility of inhibin B and FSH as markers of impaired spermatogenesis.
In conclusion, although we found associations between urinary concentrations of MBP and MBzP and altered serum levels of inhibin B and FSH, the hormone concentrations did not change in the expected patterns. Therefore, it is unclear whether these associations represent physiologically relevant alterations in these hormone levels, or whether they represent associations found as a result of conducting multiple comparisons. Our current understanding of how phthalate exposure affects the interrelationships between hormones, semen parameters and sperm DNA damage is limited and requires further investigation. Enrollment of additional men is ongoing in our study and we plan to perform further analyses on a larger dataset to revisit the preliminary associations found in this report, as well as the associations reported in our other previous studies on phthalate exposure and semen quality and sperm DNA damage.
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Acknowledgements |
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References |
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Submitted on May 6, 2004; resubmitted on August 2, 2004; accepted on November 3, 2004.