Environ. Sci. Technol. 2009, 43, 4567–4573
Relation between Urinary Metabolites of Polycyclic Aromatic Hydrocarbons and Human Semen Quality YANKAI XIA,† YAN HAN,‡ PENGFEI ZHU,† SHOULIN WANG,† AIHUA GU,† LI WANG,† CHUNCHENG LU,† GUANGBO FU,§ L I N G S O N G , † A N D X I N R U W A N G * ,† Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing, China, National Center for STD Control, Chinese Academy of Medical Sciences and Peking Union Medical College Institute of Dermatology, Nanjing, China, and Huai’an First Affiliated Hospital of Nanjing Medical University, Huai’an, China
Received August 18, 2008. Revised manuscript received April 16, 2009. Accepted April 27, 2009.
Most of the general populations are exposed to polycyclic aromatic hydrocarbons (PAHs) at different levels. A limited number of studies have suggested that PAHs exposure may be associated with semen quality. To examine the association of four PAH metabolites, 1-hydroxynapthalene (1-N), 2-hydroxynapthalene (2-N), 1-hydroxypyrene (1-OHP) and 2-hydroxyfluorene (2-OHF) with altered semen quality, 542 subjects were recruited through the clinic following strict eligibility screening. Using LC-MS/MS, individual exposures were measured as spot urinary concentrations of PAH metabolites adjusted by creatinine (CR). Semen quality was assessed by semen volume, sperm concentration, sperm number per ejaculum, and sperm motility. First, we confirmed the variability of PAH metabolites in human urine. Our results showed that the median CR-adjusted concentrations of 1-N, 2-N, 1-OHP, 2-OHF were 2.35, 4.05, 1.14, and 2.89 µg/g of CR, respectively. Significant P-values for trend were found that men with higher 1-OHP (assessed as quintiles) were more likely to have below-reference sperm concentration and sperm number per ejaculum. These results indicate that PAHs exposure might be related to altered human semen quality.
Introduction Some researchers have hypothesized that compounds with endocrine disrupting effects may be associated with the suggested, although not confirmed, downward trend in male reproductive functions, particularly semen parameters (1-8). Furthermore, most of these studies suggested that semen quality varies by geographic location. These geographic variations may be caused by environmental exposures, lifestyle factors, or some unknown causes (9). Recently, several studies have reported the associations between * Corresponding author phone: +86-25-86862863; fax: +86-2586662863; e-mail:
[email protected]. † Nanjing Medical University. ‡ Chinese Academy of Medical Sciences and Peking Union Medical College Institute of Dermatology. § Huai’an First Affiliated Hospital of Nanjing Medical University. 10.1021/es9000642 CCC: $40.75
Published on Web 05/07/2009
2009 American Chemical Society
exposure to some common environmental chemicals and altered human semen quality (7, 10-13). Polycyclic aromatic hydrocarbons (PAHs) are a class of chemicals that are formed from the incomplete burning of coal, oil, gas, wood, garbage, or other organic substances, such as tobacco and charbroiled meat. Most PAHs are widespread in the atmosphere, soil and water in close proximity to humans, particularly in developing countries. Some reports showed high detection rates of PAH metabolites among different races and genders, reflecting ubiquitous exposure to the parent compounds among the general population (14-17). Exposure to PAHs usually occurs as mixtures and not to individual chemicals. PAH mixtures can be absorbed through the skin, respiratory tract, and gastrointestinal tract (18). It is difficult to assess the exact exposure levels from multiple routes. However, PAH metabolites measured in urine may reflect internal exposure and can be utilized as sensitive biomarkers of exposure, for example, hydroxypyrene (15, 19-22). In humans, the PAH biotransformation process begins with a cytochrome P450 (CYP)-mediated epoxidation of the molecule, then involves hydroxylation with the formation of diols, leading to different metabolites (18). PAHs are rapidly metabolized. For example, the half-life of 1-Hydroxypyrene is about 29 h (21). As a consequence of this rapid metabolism, the concentrations of PAHs in serum are considerably lower than the amount of metabolites excreted in urine. Therefore, the determination of urinary metabolites, which can reflect exposure to PAHs that has occurred within the previous few days, is useful for the estimation of PAHs exposure. The parent PAH can produce more than one kind of measurable urinary metabolites, but their hydro- metabolites are considered as appropriate exposure biomarkers, particularly hydroxypyrene (19-21) and hydroxynaphthalenes (22). These metabolites have shown estrogenic activities, particularly hydroxypyrene, with potencies much greater than those of the parent compound (23). However, it is generally believed that the AhR activity is the main cause of effects of PAHs and their metabolites (24, 25). Due to the limited detection rates of some PAH metabolites and previous studies of prevalent PAH exposures in China, we selected four PAH metabolites as our target analytes. They were 1-hydroxynapthalene (1-N, CAS No. 90-15-3, metabolite of naphthalene and carbaryl), 2-hydroxynapthalene (2-N, CAS No. 135-19-3, metabolite of naphthalene), 1-hydroxypyrene (1-OHP, CAS No. 5315-797, metabolite of pyrene), and 2-hydroxyfluorene (2-OHF, CAS No. 2443-58-5, metabolite of fluorene). Ubiquitously distributed PAHs are considered human mutagens and carcinogens and are classified as probable carcinogens by IARC, NTP, and EPA. In the reproductive system, PAHs have been implicated as causative agents in prostate (26) and cervical (27) cancers. The reproductive and developmental toxicities of PAHs have been investigated recently (24, 28-33). Animal and limited human studies also suggest possible associations between PAHs exposure and semen quality (24, 29, 30, 32-34). In occupational studies, the relationship between occupational exposure to PAHs and sperm dysfunction was also found (35). However, the potential impact of exposure to PAHs on human fertility remains controversial. It may be attributed to the differences between studies in regions, races, selected biomarkers, and exposure levels. Lack of adequate internal monitoring data of different PAHs from multiple routes has also limited understanding of their respective effects on male reproduction. Furthermore, the nonoccupational population is completely distinct from the occupational population for the VOL. 43, NO. 12, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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exposure level, time, and route. To determine whether PAH metabolite levels in the nonoccupational population is associated with altered semen parameters in adult men, and whether the relationships involving different PAH metabolites are consistent, we selected a study population without specific exposure to the compounds with reported reproductive toxicities.
Materials and Methods Subject Recruitment. Study subjects were diagnosed with unexplained male factor infertility from affiliated hospitals of Nanjing Medical University between March 2004 and October 2007 (NJMU Infertile Study). The protocol and consent form were approved by the Institutional Review Board of Nanjing Medical University prior to the study. All activities involving human subjects were done under full compliance with government policies and the Helsinki Declaration. Consecutive eligible men (with wives not diagnosed as infertile) were recruited to participate. Of those approached, 92.8% consented (640 participants). There were no significant differences in sampling numbers among years and seasons. After the study procedures were explained and all questions were answered, subjects signed informed consent forms. A complete physical examination including height and weight was performed, and a questionnaire was used to collect information including personal background, lifestyle factors, occupational and environmental exposures, genetic risk factors, sexual and procreate state, medical history, and physical activity. Men with abnormal sexual and ejaculatory functions, immune infertility, semen nonliquefaction, medical history of risk factors for infertility (e.g., varicocele, postvasectomy, or orchidopexy), and receiving treatment for infertility (e.g., hormonal treatments) were excluded from the study (57 of 640 subjects). Men with other known causes related to male infertility, such as genetic disease, infection, occupational exposure to PAHs, or other agents suspected to be associated with male reproduction, were also excluded (31 of 583 subjects). Furthermore, to avoid azoospermia or severe oligozoospermia caused by Y chromosome microdeletions, we excluded subjects with Y chromosome microdeletions of azoospermia factor (AZF) region (10 of 552 subjects, microdeletion rate was 1.81%). All of the participants claimed that their life styles and environments had not been changed in several months leading up to sample collection. A single spot urine sample was collected from each subject on the same day as the semen sample. Urine samples were frozen at -20 °C until analyses for PAH metabolites. Chemicals and Instrumentation. Urinary concentrations of PAH metabolites were analyzed by a sensitive and selective LC-MS/MS (Waters 2695 and Waters Quattro Premier, USA). Due to the limited detection rate, we only selected four metabolites (1-N, 2-N, 1-OHP (g99.0%, Acros, Gorcanics, NJ) and 2-OHF(g98.0%, Sigma-Aldrich, U.S.)) for further analysis. Urinary PAH Metabolites Measurement. The analyses were basically performed as described by Xu et al. (36). The analytes underwent hydrolysis by using β-glucuronidase/ arylsulfatase (98%, Sigma-Aldrich, England) and separated from the matrix by solid-phase extraction, and the metabolites were detected by LC-MS/MS (see SI Figure S1). The calibration was carried out using pooled urine to which known amounts of PAH metabolites were added and was processed and analyzed in the same manner as the samples. The correlation appeared to be linear between 1 and 100 µg/L for each of the PAH metabolites (r > 0.99). The limit of detection for 1-OHP was 0.15 µg/L, and was 0.3 µg/L for the 1-N, 2-N, and 2-OHF. The relative standard deviation (RSD) of the within-series imprecision was between 3.3 and 12.3% at a spiked concentration of 3, 8, and 80 µg/L and the relative 4568
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recovery was between 80.8 and 122.7% (n ) 5) depending on the different spiked concentrations (details of measurement are shown in SI). Urinary Creatinine (CR) Concentrations Measurement. CR concentrations were used to adjust PAH concentrations for variable urine dilution in spot samples. CR concentrations of urine were measured photometrically using kinetic colorimetric assay technology with an automated chemistry analyzer (7020 Hitachi, Japan). Samples with CR concentrations above 300 or below 30 mg/dL were considered too concentrated or too dilute to provide valid results and were excluded from the primary analysis (37). Variability of Urinary PAH Metabolites. According to the half-life of PAHs, we collected consecutive urina sanguinis from six healthy young men on the first, second, third, fourth, fifth, sixth, seventh, ninth, 11th, and 13th days in two weeks to evaluate the variabilities of these four PAH metabolites. During these days, all of the donors did not change their life styles and environments. Collected urinary samples were divided into two groups (including seven sampling days, respectively), GroupA for every other day variability analysis, GroupB for every day variability analysis. CR concentrations were used to adjust for variable urine dilution in all samples when measuring PAH metabolites. The individual intraclass correlation coefficient (ICC) of metabolite levels of seven sampling days was used to indicate the variabilities of these metabolites in human urine. Semen Analysis. Semen samples were obtained in a private room by masturbation into a sterile wide-mouth and metal-free glass container after a recommended 2-day sexual abstinence. After liquefaction at 37 °C for 30 min, conventional semen analysis was conducted in accordance with guidelines in the WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction (38) including semen volume, sperm concentration, sperm number per ejaculum, and sperm motility by using Microcell slide and the computer-aided semen analysis (CASA, WLJY 9000, Weili New Century Science and Tech Dev.). Percent motile sperm was defined as WHO grade “A” sperm (rapidly progressive with a velocity g25 µm/sec at 37 °C) plus grade “B” sperm (slow/sluggish progressive with a velocity g5 µm/ sec but
