Research
All EHP content is accessible to individuals with disabilities. A fully accessible (Section 508–compliant) HTML version of this article is available at http://dx.doi.org/10.1289/ehp.1306720.
Associations of Filaggrin Gene Loss-of-Function Variants with Urinary Phthalate Metabolites and Testicular Function in Young Danish Men Ulla Nordström Joensen,1 Niels Jørgensen,1 Michael Meldgaard,2 Hanne Frederiksen,1 Anna-Maria Andersson,1 Torkil Menné,3 Jeanne Duus Johansen,3 Berit Christina Carlsen,3 Steen Stender,2 Pal Bela Szecsi,2 Niels Erik Skakkebæk,1 Ewa Rajpert-De Meyts,1 and Jacob P. Thyssen 3 1Department
of Growth and Reproduction, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark; 2Department of Clinical Biochemistry, and 3National Allergy Research Centre, Department of Dermato-Allergology, Copenhagen University Hospital Gentofte, Copenhagen, Denmark
Background: Filaggrin is an epidermal protein that is crucial for skin barrier function. Up to 10% of Europeans and 5% of Asians carry at least one null allele in the filaggrin gene (FLG). Reduced expression of filaggrin in carriers of the null allele is associated with facilitated transfer of allergens across the epidermis. We hypothesized that these individuals may have increased transdermal uptake of endocrine disruptors, including phthalates. Objectives: We investigated urinary excretion of phthalate metabolites and testicular function in young men with and without FLG loss-of-function variants in a cross-sectional study of 861 young men from the general Danish population. Methods: All men were genotyped for FLG R501X, 2282del4, and R2447X loss-of-function variants. We measured urinary concentrations of 14 phthalate metabolites and serum levels of reproductive hormones. We also evaluated semen quality. Results: Sixty-five men (7.5%) carried at least one FLG‑null allele. FLG‑null carriers had signifi cantly higher urinary concentrations of several phthalate metabolites, including a 33% higher concentration of MnBP (mono-n-butyl phthalate; 95% CI: 16, 51%). FLG‑null variants were not significantly associated with reproductive hormones or semen quality parameters. Conclusion: This study provides evidence that carriers of FLG loss-of-function alleles may have higher internal exposure to phthalates, possibly due to increased transepidermal absorption. FLG loss-of-function variants may indicate susceptible populations for which special attention to trans epidermal absorption of chemicals and medication may be warranted. Citation: Joensen UN, Jørgensen N, Meldgaard M, Frederiksen H, Andersson AM, Menné T, Johansen JD, Carlsen BC, Stender S, Szecsi PB, Skakkebæk NE, Rajpert-De Meyts E, Thyssen JP. 2014. Associations of filaggrin gene loss-of-function variants with urinary phthalate metabolites and testicular function in young Danish men. Environ Health Perspect 122:345–350; http://dx.doi. org/10.1289/ehp.1306720
Introduction In human stratum corneum of the skin, protein-enriched corneocytes are embedded in a lipid-rich matrix that impedes egress of water and penetration of pathogenic micro organisms, allergens, and noxious chemicals. To maintain skin hydration, abundant intra cellular filaggrin proteins are hydrolyzed into amino acids and their deiminated products, collectively referred to as the “natural mois turizing factors” (Gruber et al. 2011). Filaggrin deficiency due to the presence of one or more loss-of-function variants in the filaggrin gene (FLG) is observed in approximately 10% of lightly pigmented Europeans and in a slightly lower proportion of Asians (Irvine et al. 2011). These variants cause ichthyosis vulgaris (Smith et al. 2006), which is characterized by xerosis, scaling, and keratosis pilaris as well as palmar and plantar hyperlinearity. Permeation of allergens seems to be increased in filaggrin-depleted skin (Fallon et al. 2009; Gruber et al. 2011; Scharschmidt et al. 2009), and an increased risk of atopic dermatitis, asthma, rhinitis, food allergies, and nickel sensitization have been observed (Brown et al. 2011; Palmer et al. 2006; Thyssen et al. 2010). Clinical trials are
currently being conducted to study whether primary prevention of atopic disorders is possible using topical therapy with moisturizers (Irvine et al. 2011). Diesters of 1,2-benzenedicarboxylic acid, commonly referred to as phthalates, are man-made chemicals used in a wide range of consumer products, including moisturizers and other cosmetics. Although phthalates are rapidly metabolized and excreted in the urine following absorption, humans are continuously exposed by skin contact with, for example, cosmetics, fragrances, solvents, and plastics (Wittassek et al. 2011), as well as through the diet and via inhalation. An inverse association between phthalate exposure and markers of testicular function has been reported in some human studies (Meeker et al. 2009; Mendiola et al. 2011). Animal studies have established that certain phthalates act as endocrine disruptors, resulting in developmental abnormalities of the male reproductive tract as well as inhibition of testicular testosterone production in prenatally or perinatally exposed animals (Boberg et al. 2011; Gray et al. 2000). Although the pathogenic effects of phthalate
Environmental Health Perspectives • volume 122 | number 4 | April 2014
exposure on testicular function in humans is not yet clear, certain phthalates inhibit testos terone synthesis in cultured adult human testes at concentrations estimated to be within the range observed in epidemiological studies (Desdoits-Lethimonier et al. 2012). In the present study, we addressed the question of whether male FLG‑null carriers would have increased internal exposure to phthalate metabolites, and if so, whether this would affect testicular function.
Methods Study population. All Danish men are called to a compulsory examination the year of their 18th birthday to determine fitness for military service. On the day they went for this examination, men residing in the Copenhagen, Denmark, area were asked to participate in a study on semen quality. A total of 881 men volunteered during 2007–2009 for a study on urinary phthalate excretion and testicular function (Joensen et al. 2012). The partici pation rate was approximately 30%, which is higher than in other population-based semen-quality studies (Jørgensen et al. 2002). Address correspondence to U.N. Joensen, Department of Growth and Reproduction, Copenhagen University Hospital (Rigshospitalet), Section 5064, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Telephone: 45 2613 7760. E-mail: ulla.nordstroem.joensen@ rh.regionh.dk Supplemental Material is available online (http:// dx.doi.org/10.1289/ehp.1306720). We thank all the participants. We also thank O. Nielsen, I.D. Garn, and A. Main. The authors received financial support from the European Commission (DEER; FP7-2007-212844), the Danish Agency for Science, Technology and Innovation (27107068 and 09-067180), Rigshospitalet (961506336), the University of Copenhagen, the Danish Ministry of Health, the Danish Environmental Protection Agency (MST‑621‑00013), and the Kirsten and Freddy Johansen Foundation (95-103-72087). Funding for equipment was provided by the Velux Foundation. The funding organizations played no role in the design and conduct of the study; in collection, management, analysis, and interpretation of the data; or in the presentation, review, or approval of the manuscript. The authors declare they have no actual or potential competing financial interests. Received: 27 February 2013; Accepted: 23 December 2013; Advance Publication: 3 January 2014; Final Publication: 1 April 2014.
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Basic study details have been described previously (Jørgensen et al. 2012). Each man underwent a physical examination focusing on reproductive development, completed a questionnaire, and gave samples of spot urine, semen, and blood, in most cases all within 1 hr. Ejaculation abstinence period and time of blood sampling were recorded. All urine, semen, and blood samples were collected between 0840 hours and 1230 hours (median time, 1000 hours). In 2011, FLG genotyping was performed according to a protocol developed for the present study. DNA samples from 13 of the 881 men were missing or of poor quality; for another 7 men, the urine sample was too small to analyze osmolality. This left 861 men with complete data on FLG variant status and other main outcomes. All laboratory analyses were carried out on coded samples. The research protocol was approved by the Danish National Committee on Biomedical Research Ethics (no. H-KF-289428). Participants gave written informed consent before participation. Questionnaire. Questions included information on lifestyle factors (e.g., smoking and alcohol consumption) and medical history. The responses were reviewed with the partici pants to clarify missing or equivocal information. Participants provided information on use of prescription and nonprescription medication within the past 3 months, but were not asked to further specify the time of use. Thus, men reporting medicine intake within the past 3 months may not have taken any within ≥ 24 hr. Ethnicity was deduced from the selfreported country of birth of the participant and his parents; 83 men were categorized as other than European [i.e., they themselves or one or both of their parents were born outside of European countries (20 from African countries, 16 from Asian countries, 4 from Greenland, 11 from Latin American countries, and 32 from Middle Eastern countries)]. Participants were asked to report any previous diseases, with the focus on reproductive disorders. The questionnaire included a specific question about previous diagnosis of asthma and hay fever but no specific questions about eczema, food allergies, or use of moisturizers or other personal care products. FLG genotyping. DNA was purified from blood samples and stored at –80°C until analys is. FLG genotyping was performed according to the method of Meldgaard et al. (2012), with slight modifications. Briefly, three regions covering the mutations R501X, 2282del4, and R2447X of the FLG were asymmetrically amplified from genomic DNA by polymerase chain reaction (PCR) using DNA-tagged allele-specific primers. The obtained single-stranded PCR products were hybridized with MagPlex-C micro
346
beads (Luminex, Austin, TX, USA) carrying the DNA tags as probes, and subsequently analyzed on a Bio-Plex 200 system (Bio‑Rad, Hercules, CA, USA). Deviation from the Hardy-Weinberg equilibrium was tested for all three FLG‑null variants using a free online calculator (Online Encyclopedia for Genetic Epidemiology 2008). Participants were classified as FLG-null carriers if they carried at least one of the null FLG alleles that were evaluated (R501X, 2282del4, or R2447X). Osmolality analyses. To assess urinary dilution, we measured urinary osmolality using the freezing point depression method and an autom atic cryoscopic osmometer (Osmomat® 030; Gonotec, Berlin, Germany). For every 12 samples measured, a standard urine pool was also measured. Mean urinary osmolality for this standard pool (n = 73) was 0.83 Osm/kg with a relative standard deviation (RSD) of 1.63%. Concentrations of phthalate metabolites were adjusted for osmolality by dividing the measured metabolite concentration with the urinary osmolality of that sample and multiplying the result by the median osmolality of the population (0.86 Osm/kg). Because urine density is close to 1 kg/L, 1 Osm/kg was assumed to equal 1 Osm/L. Only samples with phthalate metabolite concentrations > LOD (limit of detection) were adjusted in this way; samples
All EHP content is accessible to individuals with disabilities. A fully accessible (Section 508–compliant) HTML version of this article is available at http://dx.doi.org/10.1289/ehp.1306720.
Associations of Filaggrin Gene Loss-of-Function Variants with Urinary Phthalate Metabolites and Testicular Function in Young Danish Men Ulla Nordström Joensen,1 Niels Jørgensen,1 Michael Meldgaard,2 Hanne Frederiksen,1 Anna-Maria Andersson,1 Torkil Menné,3 Jeanne Duus Johansen,3 Berit Christina Carlsen,3 Steen Stender,2 Pal Bela Szecsi,2 Niels Erik Skakkebæk,1 Ewa Rajpert-De Meyts,1 and Jacob P. Thyssen 3 1Department
of Growth and Reproduction, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark; 2Department of Clinical Biochemistry, and 3National Allergy Research Centre, Department of Dermato-Allergology, Copenhagen University Hospital Gentofte, Copenhagen, Denmark
Background: Filaggrin is an epidermal protein that is crucial for skin barrier function. Up to 10% of Europeans and 5% of Asians carry at least one null allele in the filaggrin gene (FLG). Reduced expression of filaggrin in carriers of the null allele is associated with facilitated transfer of allergens across the epidermis. We hypothesized that these individuals may have increased transdermal uptake of endocrine disruptors, including phthalates. Objectives: We investigated urinary excretion of phthalate metabolites and testicular function in young men with and without FLG loss-of-function variants in a cross-sectional study of 861 young men from the general Danish population. Methods: All men were genotyped for FLG R501X, 2282del4, and R2447X loss-of-function variants. We measured urinary concentrations of 14 phthalate metabolites and serum levels of reproductive hormones. We also evaluated semen quality. Results: Sixty-five men (7.5%) carried at least one FLG‑null allele. FLG‑null carriers had signifi cantly higher urinary concentrations of several phthalate metabolites, including a 33% higher concentration of MnBP (mono-n-butyl phthalate; 95% CI: 16, 51%). FLG‑null variants were not significantly associated with reproductive hormones or semen quality parameters. Conclusion: This study provides evidence that carriers of FLG loss-of-function alleles may have higher internal exposure to phthalates, possibly due to increased transepidermal absorption. FLG loss-of-function variants may indicate susceptible populations for which special attention to trans epidermal absorption of chemicals and medication may be warranted. Citation: Joensen UN, Jørgensen N, Meldgaard M, Frederiksen H, Andersson AM, Menné T, Johansen JD, Carlsen BC, Stender S, Szecsi PB, Skakkebæk NE, Rajpert-De Meyts E, Thyssen JP. 2014. Associations of filaggrin gene loss-of-function variants with urinary phthalate metabolites and testicular function in young Danish men. Environ Health Perspect 122:345–350; http://dx.doi. org/10.1289/ehp.1306720
Introduction In human stratum corneum of the skin, protein-enriched corneocytes are embedded in a lipid-rich matrix that impedes egress of water and penetration of pathogenic micro organisms, allergens, and noxious chemicals. To maintain skin hydration, abundant intra cellular filaggrin proteins are hydrolyzed into amino acids and their deiminated products, collectively referred to as the “natural mois turizing factors” (Gruber et al. 2011). Filaggrin deficiency due to the presence of one or more loss-of-function variants in the filaggrin gene (FLG) is observed in approximately 10% of lightly pigmented Europeans and in a slightly lower proportion of Asians (Irvine et al. 2011). These variants cause ichthyosis vulgaris (Smith et al. 2006), which is characterized by xerosis, scaling, and keratosis pilaris as well as palmar and plantar hyperlinearity. Permeation of allergens seems to be increased in filaggrin-depleted skin (Fallon et al. 2009; Gruber et al. 2011; Scharschmidt et al. 2009), and an increased risk of atopic dermatitis, asthma, rhinitis, food allergies, and nickel sensitization have been observed (Brown et al. 2011; Palmer et al. 2006; Thyssen et al. 2010). Clinical trials are
currently being conducted to study whether primary prevention of atopic disorders is possible using topical therapy with moisturizers (Irvine et al. 2011). Diesters of 1,2-benzenedicarboxylic acid, commonly referred to as phthalates, are man-made chemicals used in a wide range of consumer products, including moisturizers and other cosmetics. Although phthalates are rapidly metabolized and excreted in the urine following absorption, humans are continuously exposed by skin contact with, for example, cosmetics, fragrances, solvents, and plastics (Wittassek et al. 2011), as well as through the diet and via inhalation. An inverse association between phthalate exposure and markers of testicular function has been reported in some human studies (Meeker et al. 2009; Mendiola et al. 2011). Animal studies have established that certain phthalates act as endocrine disruptors, resulting in developmental abnormalities of the male reproductive tract as well as inhibition of testicular testosterone production in prenatally or perinatally exposed animals (Boberg et al. 2011; Gray et al. 2000). Although the pathogenic effects of phthalate
Environmental Health Perspectives • volume 122 | number 4 | April 2014
exposure on testicular function in humans is not yet clear, certain phthalates inhibit testos terone synthesis in cultured adult human testes at concentrations estimated to be within the range observed in epidemiological studies (Desdoits-Lethimonier et al. 2012). In the present study, we addressed the question of whether male FLG‑null carriers would have increased internal exposure to phthalate metabolites, and if so, whether this would affect testicular function.
Methods Study population. All Danish men are called to a compulsory examination the year of their 18th birthday to determine fitness for military service. On the day they went for this examination, men residing in the Copenhagen, Denmark, area were asked to participate in a study on semen quality. A total of 881 men volunteered during 2007–2009 for a study on urinary phthalate excretion and testicular function (Joensen et al. 2012). The partici pation rate was approximately 30%, which is higher than in other population-based semen-quality studies (Jørgensen et al. 2002). Address correspondence to U.N. Joensen, Department of Growth and Reproduction, Copenhagen University Hospital (Rigshospitalet), Section 5064, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Telephone: 45 2613 7760. E-mail: ulla.nordstroem.joensen@ rh.regionh.dk Supplemental Material is available online (http:// dx.doi.org/10.1289/ehp.1306720). We thank all the participants. We also thank O. Nielsen, I.D. Garn, and A. Main. The authors received financial support from the European Commission (DEER; FP7-2007-212844), the Danish Agency for Science, Technology and Innovation (27107068 and 09-067180), Rigshospitalet (961506336), the University of Copenhagen, the Danish Ministry of Health, the Danish Environmental Protection Agency (MST‑621‑00013), and the Kirsten and Freddy Johansen Foundation (95-103-72087). Funding for equipment was provided by the Velux Foundation. The funding organizations played no role in the design and conduct of the study; in collection, management, analysis, and interpretation of the data; or in the presentation, review, or approval of the manuscript. The authors declare they have no actual or potential competing financial interests. Received: 27 February 2013; Accepted: 23 December 2013; Advance Publication: 3 January 2014; Final Publication: 1 April 2014.
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Joensen et al.
Basic study details have been described previously (Jørgensen et al. 2012). Each man underwent a physical examination focusing on reproductive development, completed a questionnaire, and gave samples of spot urine, semen, and blood, in most cases all within 1 hr. Ejaculation abstinence period and time of blood sampling were recorded. All urine, semen, and blood samples were collected between 0840 hours and 1230 hours (median time, 1000 hours). In 2011, FLG genotyping was performed according to a protocol developed for the present study. DNA samples from 13 of the 881 men were missing or of poor quality; for another 7 men, the urine sample was too small to analyze osmolality. This left 861 men with complete data on FLG variant status and other main outcomes. All laboratory analyses were carried out on coded samples. The research protocol was approved by the Danish National Committee on Biomedical Research Ethics (no. H-KF-289428). Participants gave written informed consent before participation. Questionnaire. Questions included information on lifestyle factors (e.g., smoking and alcohol consumption) and medical history. The responses were reviewed with the partici pants to clarify missing or equivocal information. Participants provided information on use of prescription and nonprescription medication within the past 3 months, but were not asked to further specify the time of use. Thus, men reporting medicine intake within the past 3 months may not have taken any within ≥ 24 hr. Ethnicity was deduced from the selfreported country of birth of the participant and his parents; 83 men were categorized as other than European [i.e., they themselves or one or both of their parents were born outside of European countries (20 from African countries, 16 from Asian countries, 4 from Greenland, 11 from Latin American countries, and 32 from Middle Eastern countries)]. Participants were asked to report any previous diseases, with the focus on reproductive disorders. The questionnaire included a specific question about previous diagnosis of asthma and hay fever but no specific questions about eczema, food allergies, or use of moisturizers or other personal care products. FLG genotyping. DNA was purified from blood samples and stored at –80°C until analys is. FLG genotyping was performed according to the method of Meldgaard et al. (2012), with slight modifications. Briefly, three regions covering the mutations R501X, 2282del4, and R2447X of the FLG were asymmetrically amplified from genomic DNA by polymerase chain reaction (PCR) using DNA-tagged allele-specific primers. The obtained single-stranded PCR products were hybridized with MagPlex-C micro
346
beads (Luminex, Austin, TX, USA) carrying the DNA tags as probes, and subsequently analyzed on a Bio-Plex 200 system (Bio‑Rad, Hercules, CA, USA). Deviation from the Hardy-Weinberg equilibrium was tested for all three FLG‑null variants using a free online calculator (Online Encyclopedia for Genetic Epidemiology 2008). Participants were classified as FLG-null carriers if they carried at least one of the null FLG alleles that were evaluated (R501X, 2282del4, or R2447X). Osmolality analyses. To assess urinary dilution, we measured urinary osmolality using the freezing point depression method and an autom atic cryoscopic osmometer (Osmomat® 030; Gonotec, Berlin, Germany). For every 12 samples measured, a standard urine pool was also measured. Mean urinary osmolality for this standard pool (n = 73) was 0.83 Osm/kg with a relative standard deviation (RSD) of 1.63%. Concentrations of phthalate metabolites were adjusted for osmolality by dividing the measured metabolite concentration with the urinary osmolality of that sample and multiplying the result by the median osmolality of the population (0.86 Osm/kg). Because urine density is close to 1 kg/L, 1 Osm/kg was assumed to equal 1 Osm/L. Only samples with phthalate metabolite concentrations > LOD (limit of detection) were adjusted in this way; samples
