Animal care, diets, and DEHP exposure. The experimental protocol was approved by the Animal Use and Care Committee (Regierungspräsidium Sachsen-Anhalt, Dessau, Germany). All experimental animals were treated humanely and with regard for alleviation of suffering. Mice were housed individually under controlled light and temperature conditions (12 hr light/dark cycle; 22 ± 1°C) with free access to food and water.
We randomly divided mature female C3H/N mice (purchased from Charles River, Sulzfeld, Germany) weighing 18–23 g into four groups and exposed them to different concentrations of dietary DEHP (0, 0.05, 5, and 500 mg/kg bw/day) for a period of 8 weeks. Food intake and weight gain were measured twice weekly.
For the production of DEHP-containing diets, DEHP (Sigma-Aldrich, Taufkirchen, Germany) was diluted in commercial sunflower oil (Maggi GmbH, Frankfurt/Main, Germany) and applied to rodent chow (Altromin, Lage, Germany). All DEHP containing diets were produced with the end volume of 60 g oil/1 kg diet preparation. The control diet contained the same amount of sunflower oil vehicle. Because of the same composition of the diets, experimental and control diets had the same caloric density. The DEHP concentration range (0.05, 5, and 500 mg/kg bw/day] included dose levels relevant to human exposure (Kavlock et al. 2002
), the NOAEL, and high doses known for adverse reproductive and developmental effects in animal studies (Moore et al. 2001
). The DEHP concentrations in animal diets were verified by an accredited laboratory (SGS GmbH Germany, Hamburg, Germany) before being used in the present study. For the DEHP-containing diets, actual DEHP ingestion had to be calculated to ensure that changes in food intake did not change the estimated DEHP intake. In a pilot study, we measured daily food intake in female C3H/N mice. Using these data we estimated the daily DEHP intake to be 0.00102 mg, 0.108 mg, and 10.60 mg DEHP for the 0.05-, 5-, and 500-mg/kg bw/day groups, respectively.
Effects of DEHP on F0 females (study I). In three independent experiments (two with 10 animals/group and one with 5 animals/group), females received the diets for 8 weeks, and body weights were recorded weekly. In the last week of exposure, female F0 mice were superovulated by intraperitoneal (ip) injection of 7.0 IU pregnant mare’s serum gonadotropin (PMSG), injected 48 hr later with 7.5 IU human chorionic gonadotropin (hCG; Calbiochem, Darmstadt, Germany), and mated with unexposed males. The vaginal plug was checked the next morning, and females were sacrificed by cervical dislocation 92 hr after hCG injection. We collected F1 embryos by flushing the excised uteri with 0.2 mL phosphate-buffered saline (PBS) and 4% polyvinyl alcohol. The morphology of embryos was assessed by light microscopy (KL 1500 LCD; Carl Zeiss, Oberkochen, Germany). Embryos with fragmented or disaggregated blastomeres were categorized as degenerated embryos. Preserved tissues (liver and visceral fat) were immediately frozen in liquid nitrogen and stored at –80°C until use. We collected blood specimens by heart puncture using Monovettes (Sarstedt, Nümbrecht, Germany), centrifuged blood samples (3,500 × g for 5 min), and stored the plasma at –20°C prior to analysis. We determined the plasma concentrations of leptin by enzyme-linked immunosorbent assay (ELISA; ChrystalChem, Downers Grove, IL, USA).
Effects of DEHP on F0 dams and in utero/lactationally exposed F1 offspring (study II). In two independent experiments (one with 10/animals/group and one with 5 animals/group), female mice were exposed to 0, 0.05, 5, or 500 mg DEHP/kg/day for 8 weeks. After the first week of feeding, the females were mated with unexposed males and the vaginal plug was checked. The times to pregnancy between dams ranged from 2 to 7 days. The time intervals between exposure and mating were the same for DEHP-exposed and control animals. The mice were weighed weekly and allowed to give birth. We detected spontaneous abortion by bloody residues and weight loss of dams. At weaning, dams were sacrificed by cervical dislocation.
F1 offspring were exposed to DEHP only via placenta and breast milk until weaning. At weaning on postnatal day (PND) 21, we sexed all pups and recorded their body weights. The mean body weight was calculated for at least 12 litters/treatment group (control: 25 females, 22 males; 0.05 mg DEHP: 28 females, 27 males; 5 mg DEHP: 22 females, 23 males). For intergenerational analysis, we randomly seleced 12 F1 males and 12 F1 females per treatment group on PND21; these mice received standard chow without DEHP (Altromin). On PND84 we recorded their body weights. Sexually mature F1 females (PND84) were stimulated by ip injection of 7.0 IU PMSG, injected 48 hr later with 7.5 IU hCG, and mated with unexposed males. Ninety-two hours after hCG administration, F1 females were sacrificed by cervical dislocation and tissues were collected as described for study I.
Reagents for molecular biology. We purchased the Superscript II Reverse Transcriptase (RT) kit, deoxyribonucleotide triphosphate (dNTPs), and Taq polymerase from Invitrogen (Karlsruhe, Germany); restriction enzymes from New England Biolabs (Frankfurt, Germany); random primers from Roche (Mannheim, Germany); and RNAse inhibitor from Promega (Mannheim, Germany).
RNA extraction and RT reaction.
We extracted total RNA from liver and visceral fat tissue as described previously by Chomczynski and Sacchi (1987)
. RNA was treated with DNAse for 1 hr. The amount of total RNA was determined spectrophotometrically at 260 nm and RNA purity was determined by the 230/280 nm ratio. RNA was used only if the 260/280 nm ratio was > 1.8. Three micrograms of total RNA were reverse transcribed in a volume of 20 μL containing 10 mM dNTPs, 0.1 M dithiothereitol, 200 units superscript II, 20 units RNAse inhibitor, 1 μL random primer, and 2 μL RT buffer at 42°C for 1 hr, followed by incubation at 70°C for 10 min. As a control for DNA contamination, 2 μg RNA was amplified by polymerase chain reaction (PCR) without the RT reaction. This control reaction was performed for each primer combination and in all PCR amplifications. Resulting PCR products were separated by electrophoresis. To confirm the amplification product, the resulting RT-PCR products were visualized in a 2.0% agarose gel and sequenced.
Quantitative real-time PCR (qRT-PCR). We analyzed samples by qRT-PCR using an iQ5 Optical System (Bio-Rad Laboratories, Herts, UK) and SYBR green master mix (Eurogentec, Köln, Germany), a double-stranded DNA–specific fluorescent dye. Primer sequences are listed in . Each assay included duplicates of each cDNA sample and a “no-template” control. The parameter cycle threshold (CT) is defined as the cycle number at which fluorescence intensity exceeds a fixed threshold. Absolute mRNA expression was calculated by means of specific plasmid standards, using serial dilutions (108, 107, 106, 105). The expression of the housekeeping gene 18S rRNA was used to normalize samples for the amount of cDNA used per reaction. To confirm the amplification, the resulting qRT-PCR products were analyzed by dissociation curves and visualized in an agarose gel.
Primers used for real time RT-PCR.
Histological examination of visceral fat tissue. Visceral fat tissue from control and DEHP-exposed mice was fixed in PBS containing 4% paraformaldehyde (pH 7.5) for 24 hr, embedded in paraffin, sectioned (6 µm), and stained with hematoxylin/eosin. The slides were examined by light microscopy (BZ-8100; Keyence Germany, Frankfurt, Germany); we analyzed three fields in three histological sections from each animal.
Statistical analysis. Statistical differences between groups in study I (except for body weight gain) and study II were tested using one-way analysis of variance (ANOVA) followed by Duncan’s post hoc test using SigmaPlot, version 11.0 (Systat Software GmbH, Erkrath, Germany). Differences between groups were considered statistically significant if p < 0.05. Data are presented as mean ± SE. Data for body weight gain (study I) were analyzed by a generalized linear model (GLM; SPSS Statistics 17; IBM Deutschland GmbH, Ehningen, Germany) using the DEHP feeding period, experimental replicate, and DEHP treatment group as factors. Differences in body weight gain of DEHP-exposed groups compared with the control group during the DEHP feeding period were adjusted to the experimental replicate. We used the Greenhouse–Geisser correction to correct for violations of sphericity, which is the extension of the variance homogeneity assumption needed for ANOVA in the case of repeated measurement ANOVA. Controls were used as the reference group.