Accepted Manuscript Title: Maternal exposure to a mixture of di (2-ethylhexyl) phthalate (DEHP) and polychlorinated biphenyls (PCBs) causes reproductive dysfunction in adult male mouse offspring Author: Nadia Fiandanese Vitaliano Borromeo Anna Berrini Bernd Fischer Kristina Schaedlich Juliane-Susanne Schmidt Camillo Secchi Paola Pocar PII: DOI: Reference:
S0890-6238(16)30261-1 http://dx.doi.org/doi:10.1016/j.reprotox.2016.07.004 RTX 7351
To appear in:
Reproductive Toxicology
Received date: Revised date: Accepted date:
2-11-2015 28-6-2016 8-7-2016
Please cite this article as: Fiandanese Nadia, Borromeo Vitaliano, Berrini Anna, Fischer Bernd, Schaedlich Kristina, Schmidt Juliane-Susanne, Secchi Camillo, Pocar Paola.Maternal exposure to a mixture of di (2-ethylhexyl) phthalate (DEHP) and polychlorinated biphenyls (PCBs) causes reproductive dysfunction in adult male mouse offspring.Reproductive Toxicology http://dx.doi.org/10.1016/j.reprotox.2016.07.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Maternal exposure to a mixture of di (2-ethylhexyl) phthalate (DEHP) and polychlorinated biphenyls (PCBs) causes reproductive dysfunction in adult male mouse offspring
Nadia Fiandanesea, Vitaliano Borromeoa, Anna Berrinia, Bernd Fischerb, Kristina Schaedlichb, Juliane-Susanne Schmidtb, Camillo Secchia, and Paola Pocara,*
a
Dipartimento di Medicina Veterinaria. Università degli Studi di Milano, Italy
b
Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle,
Germany
*
Corresponding author and reprint requests: Dr. Paola Pocar, Dipartimento di Medicina Veterinaria.
Università degli Studi di Milano - Via Celoria 10, I-20133 Milano (Italy) - 20133 Milano (Italy). Email:
[email protected]
2
HIGHLIGHTS
DEHP/PCB mixture affects male reproductive health differently than single compounds
Mixture had a panel of synergistic, antagonistic or non-interactive effects
Results provide novel insights into the risks of developmental exposure to mixtures
3
ABSTRACT
We investigated the effects of maternal exposure to the plasticizer di(2-ethylhexyl) phthalate (DEHP) and the organic industrial compounds polychlorinated biphenyls (PCBs), singly and combined, on the reproductive function of male mouse offspring. Mice dams were exposed throughout pregnancy and lactation to 1 µg PCBs (101+118)/kg/day, 50 µg DEHP/kg/day, or the DEHP/PCB mixture in the diet. The mixture induced permanent alterations in adult F1 males’ reproductive health in a way, differently from the single compounds. Depending on the endpoint, we observed: (1) synergy in altering the gross and histological morphology of the testis; (2) antagonism on the expression levels of genes involved in pituitary-gonadal cross-talk; (3) non-interactions on sperm parameters and testosterone production. This study illustrates the complex action of a DEHP/PCB mixture, leading to a unique panel of effects on the male reproductive system, indicating the need for research on the reproductive hazards of combined endocrine disruptors.
Keywords: di(2-ethylhexyl) phthalate (DEHP), polychlorinated biphenyls (PCBs), testis, testosterone, mouse, EDs mixture
Abbreviations: AGD, ano-genital distance; COCs, cumulus-oocyte complexes; DEHP, di(2ethylhexyl)phthalate; DPC, day post-coitum; DSP, daily sperm production; ED, endocrine disruptor; ITT, intra-testicular testosterone; PCB, polychlorinated biphenyl; PND, post-natal day.
4
1. INTRODUCTION
Endocrine disruptors (EDs) are a large group of environmental pollutants that behave as agonists or antagonists of endogenous hormones and have long been suspected of being involved in reproductive defects in males and females [1]. The timing of ED exposure strongly influences the severity of the effect on the reproductive tract. The concept of a “fetal origin of adult disease”, firstly formulated by Barker & Osmond [2], suggests that the risk of developing certain diseases later during adult life is influenced not only by genetics and life-style factors, but also by environmental influences affecting early developmental stages. With its heightened sensitivity, the fetus is extremely vulnerable to environmental insults. Exposure to EDs during this critical window of early development can create interferences within biological systems, which may permanently influence disease outcomes throughout the lifespan of an organism [2, 3]. Thus investigation of the adverse effects of EDs on adult organ function and tissue metabolism after maternal exposure attracted increasing attention from scientists and policy makers [4]. Worryingly, many of the reproductive abnormalities resulting from developmental exposure only become apparent after puberty (long latency), and this is a significant obstacle to the identification of causal relationships. Phthalates (phthalic acid esters) and polychlorinated biphenyls (PCBs) are two typical classes of EDs that cause reproductive abnormalities in humans and animals [5, 6]. Their lipophilic nature means they can easily cross the placental barrier [7-9] and pass into breast milk [10-12], constituting a significant risk of damage for the fetus and newborns [13, 14]. Phthalates are plasticizers that are added to polymers, especially PVC, to impart softness and flexibility. Di(2-ethylhexyl) phthalate (DEHP), the most commonly used phthalate, is produced at the rate of around two million tons/year [15] and is widely employed in the manufacture of medical devices, clothing, packaging, food containers, personal care products and children’s toys [16].
5
Phthalates do not bind covalently to plastics so they easily migrate into the environment [17, 18]. As a result, the general population is unavoidably exposed to these compounds through ingestion, inhalation or skin absorption. Polychlorinated biphenyls (PCBs) are pollutants resulting from industrial processes and products; they have been used on a large scale for more than 40 years. Even though their production was banned at the end of the 1970s, they still persist in the environment and bio-accumulate in food chains on account of their chemical stability and lipophilicity. PCBs can be detected in plasma, tissue samples and breast milk of numerous species including humans [19-23]. The developing reproductive system of males is particularly susceptible to the endocrine disrupting activity of phthalates and PCBs and they have been linked to adverse health effects on male reproductive function in humans and animals [24-31]. We recently reported that exposure to these two xenobiotica during pregnancy and lactation leads to multiple long-lasting effects on the reproductive system in adult male mouse offspring, including reductions in gonadal weight, altered testicular morphology, and impaired semen quality and developmental competence [32, 33]. Their chemical nature and distribution in the environment means that DEHP and PCBs are highly likely to be taken up simultaneously by organisms. Both these ED classes are encountered in surface water, sediment of urban waters, and in sewage sludge [34-37] which is often recycled to land in increasing amounts in both Europe and the United States as landfill sites become less available [38, 39]. Soil acts as a long-term repository for these contaminants. Remobilization by volatilization from soil is an important mechanism of redistribution in the environment [40], leading to ubiquitous chronic exposure to these chemicals of organisms in any trophic level, including humans. Developing organisms are subject to the greatest PCB and DEHP burden from maternal exposure, an observation of particular concern. PCB 101 and 118, as well as phthalates are among the EDs that have been measured in human placenta and maternal milk [41, 42]. In sheep and mice,
6
both DEHP and the two selected PCB congeners were reported to accumulate preferentially in the offspring, more than their dams [43, 44]. To date, few studies have investigated the combined effects of their mixtures, and these interactions need to be clarified. Animals and humans are concurrently exposed to a large number of chemicals from multiple sources and through various routes [45]. Different chemicals, when acting simultaneously, may induce effects that can significantly differ in quality and/or magnitude from those after exposure to the individual toxicants [46]. This highlights the need for testing combinations of EDs to gain a further picture of the health risks of exposure to multiple sources of these substances. Studies with mixtures of EDs have identified a broad spectrum of combinatory effects in terms of interactions (synergism or antagonism) or non-interactions (dose addition or independent action) [47]. Research has mostly focused on exposure to chemicals acting on the same biological site, by the same mechanism (e.g. anti-androgens or xeno-estrogens) [48-51]. In contrast, there are only few reports about different classes of EDs sharing similar target organs but with different mechanisms of action. Therefore, the present study explored a range of biological endpoints to see whether concomitant exposure to DEHP and PCBs, which differ in chemical nature and do not necessarily share a narrowly defined mechanism of action, but all target the developing male reproductive system, cause adverse effects which can differ, quantitatively and qualitatively, from those after single exposure. We designed an experiment to compare the toxic effects of single and combined exposure to these two xenobiotica throughout pregnancy and lactation on the reproductive health of adult male offspring of CD-1 mice.
2. MATERIALS AND METHODS
2.1. Chemicals and food preparation
7
DEHP (CAS 117-81-7) was purchased from Sigma-Aldrich, Hamburg, Germany. PCB congeners 101 (CAS 37680-73-2) and 118 (CAS 31508-00-6) were purchased from LGC Standards GmbH (Wesel, Germany) and were certified 99.8% pure. PCB 101 is a di-ortho substituted PCB and is a typical non-coplanar PCB (e.g. non-dioxin-like effects); PCB 118, being a mono-ortho substituted PCB, can assume a partial coplanar conformation and may therefore share some effects with non-ortho PCBs (e.g. dioxin-like effects). The two congeners were diluted 1:1 in commercial sunflower oil for chow. DEHP and the PCBs, singly or combined, were diluted in commercial sunflower oil and used by a specialized company (Altromin, Lage, Germany) for preparing treated chow. Dams received a diet formulated to ensure mean daily intake as follows: control group, vehicle only; DEHP group, 50 µg DEHP/kg/day; PCB group, 1 µg PCB/kg/day; DEHP/PCB group, 50 µg DEHP + 1 µg PCB/kg/day. The amounts of DEHP (285.7 µg DEHP/kg/food) and PCBs (0.571 µg PCBs/kg/food) added to the chow were calculated in order to obtain the desired doses on the basis of the mean daily food intake in mice estimated in a preliminary study in the same physiological conditions as described (DEHP [33]; PCBs [32]). Each batch of diet was tested before use in an accredited laboratory (SGS Laboratory GmbH, Hamburg, Germany). The doses were selected so as to overlap the DEHP and PCB concentrations reported in the literature as environmental exposure in humans [16, 52]. The dose range for DEHP exposure was selected taking as reference value an amount close to the estimated daily intake of the general population (58 µg/kg/day) as reported by Kavlok et al. [16]. The dose range for PCB exposure was selected to overlap concentrations reported in the breast milk of women in industrialized countries (19 ng/g/fat of PCB 118, corresponding to a daily oral dose of 3.8 μg/kg bodyweight) [52].
2.2. Animals and treatments Virgin five-week-old female and six-week-old male CD-1 mice were purchased from Charles River (Calco, Italy) and allowed to acclimatize for two weeks. They were housed in the animal
8
facilities of the Department of Veterinary Science and Public Health, University of Milan, under controlled conditions (23 1°C, 12-h light/dark cycle). They received a standard rodent diet (4RF21, Charles River) and tap water ad libitum. Groups of two or three females were mated with one male and inspected daily for a mating plug. The day the vaginal plug was detected [0.5 day post-coitum (DPC)] each female was housed individually in type II cages with stainless steel covers and hardwood shavings as bedding and randomly assigned to one of four treatment groups. Treatment was continued from 0.5 dpc through lactation until weaning [post-natal day 21 (PND 21)]. Two or three pregnant mice were randomly assigned to each group, and the experiment was replicated at least three times (total seven to ten dams per treatment). Dams and lactating offspring were examined daily for clinical signs of toxicity. On PND 21, dams were euthanized by CO 2 inhalation and organs were collected. Litter size, sex ratio, weight of pups, and the number of viable pups were recorded. The liver, ovaries, and uterus were removed, weighed, and snap-frozen in liquid nitrogen for later analysis.
2.3. F1 offspring On PND 21, all pups were sexed and body weight was recorded. Male pups from each litter were housed in groups for another three weeks. Standard pellet food (Charles River 4RF21) and tap water were available ad libitum. On PND 42, at least three males per litter were randomly selected for autopsy. In all, 238 F1 males were examined (45-70 per treatment group). Males were euthanized by CO2 inhalation followed by cervical dislocation, and the external genitalia were examined for malformations; testicular position was recorded after opening the abdominal cavity. Pituitaries and testes were removed and weighed, and the mean weight was used in subsequent analyses. Then organs were either snap-frozen in liquid nitrogen or fixed in Bouin’s fixative for later analysis.
9
Care and experimental procedures with mice were in accordance with accepted standards of humane animal care following Italian national regulations, and were approved by the University of Milan ethics committee.
2.4. Intra-testicular testosterone (ITT) Intra-testicular testosterone (ITT) levels were measured in at least eight adult mice from each group as previously described [53]. Briefly, half the testis was weighed and homogenized for 2 min in 500 μL ice-cold phosphate buffer (50 mM, pH 7.4). To precipitate nuclei and cell debris, samples were then centrifuged at 18000 g for 10 min at 4°C and supernatant was collected and stored at 80°C until analysis. ITT was assayed on 25 μL of the supernatant by a competitive ELISA (Testosterone ELISA, Diametra, Italy) following the manufacturer’s instructions. All samples were run in duplicate and all groups were assayed in the same run in order to avoid inter-assay variation.
2.5. Daily sperm production (DSP) Daily sperm production was determined by hemocytometric counts of spermatid heads according to Thayer et al. [54], with minor modifications. Briefly, half the frozen left or right testis from control and treated animals was thawed, weighed and homogenized for 3 min in 15 mL of 0.05% Triton X-100 in 0.9% saline. Step 14-16 spermatids (stage II-VIII) survive this treatment and can be counted with a hemocytometer. To count them, 40 µL of homogenate for each mouse were diluted with 60 µL of saline and 100 µL of 0.4% Trypan blue, which stains spermatids and facilitates counting; 7-µL samples were then placed in a Neubauer chamber and counted twice at 400X magnification under a light microscope. The average number of homogenization-resistant spermatid heads per sample was used to calculate the total spermatids per testis, which was then divided by the sample weight to give spermatids per gram of testis. Since developing spermatids spend 4.84 days in step 14-16 during spermatogenesis in the mouse, the spermatids per gram were
10
divided by 4.84 to obtain the DSP per gram of testis. The mean DSP for each group of 12 animals was calculated and compared for statistical significance.
2.6. Epididymal sperm count and dead: live ratio. Sperm was obtained from the cauda epididymis of adult offspring. Both cauda were dissected out from the body and transferred into 500 µL of previously equilibrated Whittingham medium (37°C at 5% CO2 in air). Sperm was passively released into the culture medium by puncturing the cauda three to four times with a 27G needle. Some samples were diluted (1:100) with water, and sperm was counted in a Neubauer chamber. Other samples were diluted (1:20) with 0.9% NaCl and stained by a modified Kovacs-Foote method [55]. Briefly, one drop of dilute sample was mixed on a microscope slide with one drop of iso-osmotic 0.2% Trypan blue (Sigma T-8154) and smeared with the edge of another slide. The slides were vertically air-dried, then fixed for 2 min with fixative solution (86 mL 1N HCl plus 14 mL 37% formaldehyde solution and 0.2 g neutral red [Fluka, 72210]), and rinsed with tap and distilled water. Finally, the slides were air-dried and covered with Eukitt (Fluka, 03989) and a coverslip. Stained smears were examined by light microscopy at 400X magnification. The status of the heads and tails of at least 100 spermatozoa was classified in each smear. Sperm with white or pale pink heads (intact plasma membrane) were classified as alive, and those with black to dark-purple heads (damaged membrane) as dead.
2.7. In vitro fertilization and embryo culture Females were super-ovulated by intraperitoneal (i.p.) injection of 3.5 IU Folligon (PMSG, Intervet International), followed 48 h later by an i.p. injection of 5 IU Chorulon (hCG, Intervet). Spermatozoa were collected as described above and capacitated for 60 min in Whittingham medium (37°C at 5% CO2 in air). Cumulus-oocyte complexes (COCs) were recovered from oviducts 14 h after hCG injection, in M2 medium (Sigma-Aldrich). After rinsing in Whittingham medium, COCs
11
were co-incubated with 2*106 capacitated spermatozoa. Putatively fertilized eggs (6 h after insemination) were then transferred to 250-L drops of M16 medium (Sigma-Aldrich), covered with paraffin oil and incubated at 37°C with 5% CO2 in air for another 96 h. Cleavage and blastocyst rates were assessed respectively 24 h and 96 h after insemination.
2.8. Histological analysis and Leydig cell count Testes from at least six mice per group were fixed in Bouin’s fixative, dehydrated, and embedded in paraffin. Then 5-µm serial microscopic sections were prepared and at least six slides from each testis were stained with hematoxylin-eosin for histological analysis; another six were immunostained for Leydig cell count. Each measurement was based on at least 20 fields per section. Histological analysis: In cross-sections of randomly selected tubular profiles that were round, staging analysis of the seminiferous tubules was done according to Meistrich and Hess (2013). The tubules were assigned to one of two groups based on spermiogenesis phases (group 1: stages I-VIII – maturation and spermiation; group 2: stages IX-XII – Golgi, Cap and acrosome phase). Percentages of each of the two groups of tubules were compared with those of the control group examined at the same age. The diameters of the tubules and epithelium thickness were measured using light microscopy, according to Koruji et al. [56]. Leydig cell count: After dewaxing, the sections were incubated in 3% hydrogen peroxide (H2O2) in methanol at room temperature for 30 min in the dark, then incubated overnight at 4°C with anti-3 β-hydroxysteroid dehydrogenase (3 β-HSD) rabbit IgG (Santa Cruz, 1:100 dilution in 3% BSA PBS/0.1% Tween 20). Sections were then treated with 10% normal donkey serum PBS/0.1% Tween 20 for 25 min, followed by an HRP-conjugated goat anti-rabbit antibody (Jackson Laboratories, Newmarket, UK) for 1 h at room temperature. The resulting immunocomplex was visualized by incubation with diaminobenzidine (Sigma). The sections were counterstained with Mayer’s hematoxylin. The total number of Leydig cells in testes was calculated based on the numbers of 3 β-HSD-positive cells in the sections. Sections were divided into 25 fields and 14
12
randomly chosen fields were analyzed. Leydig cells were counted in the interstitial space between tubules that were round, and results were expressed as the mean number of Leydig cells per seminiferous tubule.
2.9. RNA isolation and RT-PCR Total RNA was isolated from the half-testes from at least ten mice using Trizol (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. Total RNA was checked for integrity and DNA contamination using an UV spectrophotometer and 1.3% agarose gel electrophoresis. Total RNA (1 g) extracted from each sample was used to synthesize the cDNA using a SuperScript kit (Invitrogen). The reverse transcription reaction was run at 42°C for 1 h, and terminated by heating at 94°C for 2 min. Polyadenylated [poly(A)]+RNA from at least ten pituitaries per treatment group was extracted using a Dynabeads mRNA DIRECT kit (Deutsche Dynal, Hamburg, Germany). Briefly, single pituitaries were lysed for 10 min at room temperature in 200 L lysis buffer [100 mmol Tris-HCl (pH 8), 500 mmol LiCl, 10 mmol EDTA, 1% (wt/vol) sodium dodecyl sulfate, and 5 mmol dithiothreitol]. After lysis, 10 L prewashed Dynabeads-oligo(deoxythymidine) were pipetted into the tube and poly(A)+RNA binding to oligo(deoxythymidine) was hybridized for 5 min at room temperature. The beads were then separated with a Dynal MPC-E magnetic separator and washed twice with 50 L washing buffer A [10 mmol Tris-HCl (pH 8), 0.15 mmol LiCl, 1 mmol EDTA, and 0.1% (wt/vol) sodium dodecyl sulfate] and three times with 50 L washing buffer B [10 mmol Tris-HCl (pH 8), 0.15 mm LiCl, and 1 mmol EDTA]. Poly(A)+RNA were eluted from the beads by incubation in 11 L diethylpyrocarbonate-treated sterile water at 65°C for 2 min. Aliquots were immediately used for reverse transcription with the PCR Core Kit (PerkinElmer Corp., Wellesley, MA), using 2.5 µmol random hexamers to obtain the widest array of cDNA. The reverse
13
transcription reaction was run in a final volume of 20 L at 25°C for 10 min and 4°C for 1 h, followed by a denaturation step at 99°C for 5 min and immediate cooling on ice. Table 1 lists the primers and PCR conditions for target genes. Transcripts were selected based on the observation that both phthalates and PCBs affect steroidogenesis, directly or indirectly, altering the patterns of gene expression that regulate cholesterol transport and homeostasis (Ldlr, Scarb1, Star), testosterone biosynthesis (Cyp11a1, Cyp17a1, Cyp19a1) and the pituitary-gonadal cross-talk (Lhb, Fshb, Lh-r, Fsh-r) [57-59]. For each set of primers, the optimal cycle number at which the transcript was exponentially amplified was established by running a linear cycle series and the number of PCR cycles was kept within this range. Approximately 1 L cDNA per sample was used for amplification. The cDNA fragments were generated by initial denaturation at 94°C for 3 min. The PCR products were separated by electrophoresis on 1.3% agarose gel and detected under UV light. To normalize signals from different RNA samples, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts were co-amplified as an internal standard. Quantitative expression was analyzed with Quantity One software using the software’s Volume Analysis Report (Bio-Rad Laboratories, Inc., Hercules, CA).
2.10.
Statistical analysis
Data were analyzed using GraphPad Prism software 5.03 (GraphPad Software, San Diego, CA). The mean number of pups per litter was calculated for at least five mating pairs per treatment group, and mean body and organ weights were calculated for at least five litters per group. Because all the pups within a litter had received the same treatments, for statistical analysis, the experimental unit was the whole litter, and the unit of statistical analysis was the litter mean. Differences between the means for litter size, AGD, organ weight, semen parameters, and gene expression were tested by the D’Agostino and Pearson normality test to confirm Gaussian distribution, then analyzed by oneway ANOVA, with statistical significance P ≤0.05. When ANOVA gave a significant P value, treatments were compared using the Newman-Keuls test in the post- hoc analysis. Data for in vitro
14
embryo culture were analyzed by binary logistic regression, taking controls as the reference group. Experiments were replicated at least three times, and each replicate was fitted as a factor. The loglikelihood ratio statistic was used to detect between-treatment differences with dosage as an explanatory variable. Significance was P ≤0.05.
3. RESULTS
3.1. Effects of ED exposure on dams and pregnancy outcome There were no treatment-related deaths or other signs of general toxicity in dams treated with PCBs, DEHP or the DEHP/PCB mixture, and no effect on their body weights during pregnancy and lactation. Treatments had no significant effects on gestational length, litter size, litter sex ratio (male/female), and post-birth survival index throughout lactation (data not shown).
3.2. Morphological endpoints in male offspring Table 2 shows the effects of pre- and peri-natal treatment with PCB, DEHP and the DEHP/PCB mixture on male pup testis weight. Absolute and relative testis weights were significantly lower in the PCB and DEHP groups (P
