Estrogen Target Gene Regulation and Coactivator Expression in Rat Uterus after Developmental Exposure to the Ultraviolet Filter 4-Methylbenz
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内分泌学杂志 2005年第5期
Institute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zurich, Switzerland
Address all correspondence and requests for reprints to: Walter Lichtensteiger, Group for Reproductive, Endocrine, and Environmental Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. E-mail: Walter.Lichtensteiger@access.unizh.ch.
Abstract
Because the estrogen receptor (ER) ligand type influences transactivation, it is important to obtain information on molecular actions of nonclassical ER agonists. UV filters from cosmetics represent new classes of endocrine active chemicals, including the preferential ER? ligands 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor. We studied estrogen target gene expression in uterus of Long Evans rats after developmental exposure to 4-MBC (0.7, 7, 24, and 47 mg/kg·d) administered in feed to the parent generation before mating, during pregnancy and lactation, and to the offspring until adulthood. 4-MBC altered steady-state levels of mRNAs encoding for ER, ER?, progesterone receptor (PR), IGF-I, androgen receptor, determined by real-time RT-PCR in uterus of 12-wk-old offspring. Western-blot analyses of the same tissue homogenates indicated changes in ER and PR but not ER? proteins. To assess sensitivity to estradiol (E2), offspring were ovariectomized on d 70, injected with E2 (10 or 50 μg/kg sc) on d 84, and killed 6 h later. Acute up-regulation of PR and IGF-I and down-regulation of ER and androgen receptor by E2 were dose-dependently reduced in 4-MBC-exposed rats. The reduced response to E2 was accompanied by reduced coactivator SRC-1 mRNA and protein levels. Our data indicate that developmental exposure to 4-MBC affects the regulation of estrogen target genes and the expression of nuclear receptor coregulators in uterus at mRNA and protein levels.
Introduction
ESTROGENIC AND ANTIANDROGENIC UV filters used in cosmetics represent a new group of endocrine-active chemicals (1, 2). UV filters are released into the environment and reach animals via the food chain and humans via a dual exposure, food chain, and direct application (3, 4). After the identification of estrogenic activity of several UV filters, including 4-methylbenzylidene camphor (4-MBC), in vitro and in immature rats (1), their activity has been confirmed in vitro (5, 6, 7) as well as in vivo in mammals (5) and fish (8, 9). 4-MBC binds competitively to estrogen receptors (ERs) (10) and stimulates transactivation (6, 7).
Endocrine-active UV screens are relevant from a toxicological point of view because of widespread exposure, and they are also of interest because their chemical structure differs from structures so far encountered with endocrine disrupters. There is good evidence that the chemical structure of ER ligand and the ER subtype influence transactivation at several different levels (11, 12, 13, 14), leading to different gene expression patterns (15), but there is little information on molecular effect patterns after chronic exposure to nonclassical ER ligands.
We are studying effects of various ER ligands on reproduction and development, parts of the life cycle that are very sensitive to endocrine disrupters, with focus on reproductive organs and central nervous system. 4-MBC was chosen as the chemical with the highest acute estrogenic in vivo activity in the first group of UV filters studied (1). In receptor binding experiments, it shows distinct preference for ER? (10). Developmental exposure to 4-MBC influences a number of endocrine parameters, including delayed puberty in males and changes in reproductive organ weights (16, 17).
In the present study, we investigated whether pre- and postnatal exposure of rats to 4-MBC affects the expression of estrogen-regulated genes at the mRNA and protein level in uterus of adult offspring. We observed significant, dose-dependent changes in the expression of mRNAs encoding for ER subtypes, progesterone receptor (PR), IGF-I, and androgen receptor (AR), and in protein levels and a reduced sensitivity to acute administration of estradiol (E2). The decrease in estrogen sensitivity was accompanied by reduced expression of the steroid receptor coactivator (SRC)-1 in the same dose range. SRC-1 was found to be one of the most sensitive parameters.
Materials and Methods
Chemicals
The UV filter 3-(4-methylbenzylidene) camphor (4-MBC; Eusolex 6300 CAS no. 36861-47-9, purity 99.7–99.9%) was purchased from Merck (Dietikon, Switzerland), and E2 was purchased from Calbiochem (Lucerne, Switzerland).
Experimental animals
The study was conducted on Long Evans rats (M?llegaard Breeding and Research Centre, Ejby, Denmark) bred in our laboratory under controlled light and dark cycle (lights on from 0200–1600 h) and temperature (22 C ± 1 C) with free access to food and water. The animal facility is run by the Institute of Laboratory Animal Science of the University of Zurich (Zurich, Switzerland). Microbiological checks are performed every 3 months. Animal maintenance and experiments were conducted according to the Swiss Law for the Protection of Animals and the Ethical Guidelines of the Swiss Academy of Medical Sciences.
Treatment schedule
Food pellets containing 4-MBC.
Food pellets with 4-MBC were prepared by Provimi Kliba AG (Kaiseraugst, Switzerland). Appropriate amounts of 4-MBC dissolved in cold-pressed soy oil (Morga, Ebnat-Kappel, Switzerland) were added to Provimi Kliba chow no. 3340 (formerly 3430) to achieve 4-MBC concentrations of 0.01, 0.1, 0.33, or 0.66 g/kg chow, yielding an average daily intake of 0.7, 7, 24, or 47 mg/kg body weight·d, respectively. Control pellets consisted of the same matrix (Provimi Kliba no. 3430) with 1% of soy oil added. The soy oil preparation (Morga) was devoid of detectable amounts of phytoestrogens (information of the manufacturer).
4-MBC exposure.
The study was designed to mimic exposure through the food chain. It corresponds to an extended one-generation study. Males and females of the parent generation (5–6 wk old) were fed for at least 10 wk before mating (including one spermatogenic cycle) with chow containing 4-MBC (0.01, 0.1, 0.33, and 0.66 g/kg chow) or with control chow. Treatment continued throughout pregnancy and in the offspring until adulthood. Experiments were performed on offspring of time-pregnant rats (1). The day of birth was defined as postnatal d 1. Pups were weaned at postnatal d 28, male and female littermates were raised in separate groups until adulthood. Different developmental endpoints and the onset of puberty were assessed (16, 17). The study was run in two series, control A together with 7, 24, and 47 mg/kg 4-MBC, and control low dose, together with 0.7 mg/kg 4-MBC (Table 1). The numbers of litters were eight and five in the two control groups and four to six in the 4-MBC-exposed groups.
TABLE 1. Uterine weight and body weight in 12-wk-old rat offspring after pre- and postnatal exposure to 4-MBC
Analysis of adult offspring under steady-state conditions.
Offspring were raised without further experimental manipulation except 4-MBC treatment. Females were killed by decapitation at 12 wk of age in diestrus. The uterus was quickly removed, weighed (wet weight), and frozen in liquid nitrogen until further analysis.
Estrogen challenge experiment in adult offspring.
At 10 wk of age, female offspring were taken out of several different litters of the control group and of 4-MBC-exposed groups (0.01, 0.1, and 0.33 g/kg chow) and ovariectomized under anesthesia (Hypnorm, Domitor, and Atropin). After 2 wk of recovery, at the age of 12 wk, they received a single sc injection of E2 (10 or 50 μg/kg in dimethylsulfoxide) or vehicle (dimethylsulfoxide) (injection volume 1 μl/g body weight) and were decapitated 6 h later for tissue dissection. Animals from the same litters were studied under steady-state conditions.
Real-time RT-PCR
RNA isolation and RT.
The uterus was homogenized in RNA lysis tissue (RLT) buffer from the RNeasy-mini kit (QIAGEN, Valencia, CA) by a polytron rotor-stator homogenizer. Total RNA was extracted using the RNeasy mini kit according to the manufacturer’s instructions; the RNA preparation was incubated with DNaseI (QIAGEN). Total RNA concentration was assessed by A260 and checked for DNA contamination on an ethidium bromide-stained 2.5% agarose gel. RNA was stored at –80 C until used. For RT, 10 μg RNA was used in a total volume of 100 μl containing 1x TaqMan RT buffer, 5.5 mM MgCL2, 500 μM of each dNTP, 2.5 μM random hexamer primers, 0.4 μM RNase inhibitor, and 1.25 μl MultiScribe TM reverse transcriptase (Applied Biosystems, Foster City, CA). The mixture was incubated for 10 min at 25 C followed by RT of 30 min at 48 C and RT inactivation of 5 min at 95 C.
Primers and TaqMan probes.
Primers and probes were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA) as described by the manufacturer and were synthesized by Microsynth (Balgach, Switzerland). To exclude amplification from possible DNA contamination, either the probes or the primers were designed to overlap exon junctions in cDNA regions derived from intron-bearing genes. All probes were labeled with the fluorescent dyes 6-carboxy-fluorescein as reporter and 6-carboxy-tetramethyl-rhodamine as quencher. Table 2 lists the sequences of primers and probes used.
TABLE 2. Forward primers (FP), reverse primers (RP), and TaqMan probes with corresponding GenBank accession no.
Real-time-PCR.
To amplify cDNA, RT samples were mixed with TaqMan PCR master mix (Applied Biosystems), optimized concentrations of primers and probes and distilled water were added to a final volume of 25 μl. Signals were monitored by an ABI PRISM 7700 Sequence Detector (Applied Biosystems). PCR cycle parameters were an initial denaturation step at 95 C for 10 min, followed by 40 cycles at 95 C for 15 sec and 60 C for 1min.
Sequence Detector Software SDS 2.0 (Applied Biosystems) was used for data analysis. After the reaction, the threshold line was set in the linear region of the plots above the baseline noise, and the threshold cycle (CT) was determined as the cycle number at which the threshold line crosses the amplification curve. The CT values obtained were exported to a Microsoft Excel spreadsheet for further analysis. Standard curve plots were constructed from a cDNA dilution series of a control animal, using Microsoft Excel as recommended in User Bulletin no. 2 for the ABI Prism 7700 Sequence Detection System (Applied Biosystems, 1997), with CT values plotted against the logarithm of the input amount of cDNA (equivalent to the amount of total RNA). The CT values obtained were used to calculate the initial input amount. Thereafter, the results were normalized to cyclophilin as reference gene and expressed as percentage of control. For comparison of levels of different control or reference groups (Table 3), additional measurements were performed with samples of these control groups on the same plate. Part of the data was calculated with the comparative CT method. CT was the difference of the CT values of the target gene being assayed and of the cyclophilin reference, whereas Ct represented the difference between the paired tissue samples, as calculated by the formula Ct = Ct of control tissue – Ct of treated tissue. The n-fold differential expression in a specific gene of a treated sample compared with the control was expressed as 2–Ct (User Bulletin 2, Applied Biosystems).
TABLE 3. Comparison of mRNA levels in untreated controls and 4-MBC-exposed groups after ovariectomy and vehicle injection
Choice of reference gene.
The use of housekeeping genes as internal control starts from the assumption that they vary comparatively little in response to experimental treatment, yet, variations can be considerable (18, 19). It is difficult to find an internal mRNA standard that is not affected by endocrine manipulation. Preliminary tests were performed on uteri of rats after E2 treatment, and mRNAs encoding for glyceraldehyde-3-phosphate dehydrogenase, ?-actin, hypoxanthine phosphoribosyl transferase, and cyclophilin were determined. An analysis of the data indicated that glyceraldehyde-3-phosphate dehydrogenase and ?-actin could be influenced by estrogen treatment and gonadectomy. Cyclophilin appeared to be more stable. Hypoxanthine phosphoribosyl transferase could also have been used but was not expressed in the appropriate CT range in some tissues. Therefore, cyclophilin was chosen as reference gene.
Western blot analysis.
Protein levels were analyzed in the same tissue homogenates that were used for mRNA analysis. Equal amounts of protein extracts were run on SDS-PAGE gels and transferred onto nitrocellulose membrane. After transfer, the membranes were blocked in 5% milk (blotting grade blocker nonfat dry milk, Bio-Rad Laboratories, Hercules, CA) in Tris-buffered saline-Tween 20 (TBST), then incubated overnight with the first antibody, anti-ER (ABR-Affinity BioReagents, Golden, CO), anti-ER?, anti-PR, anti-SRC-1, anti--Tubulin (Santa Cruz Biotech. Co., Santa Cruz, CA), diluted in 5% milk/TBST. After washes with TBST, the membranes were incubated for 1 h with the appropriate secondary antibodies conjugated to horseradish peroxidase. Proteins were detected using chemiluminescence (Pierce, Rockford, IL). Densitometrical analyses were performed using the AIDA-2D software (Raytest, Pittsburgh, PA). Results are expressed as the ratio of the protein of interest to -tubulin to correct for possible differences of protein loading between samples.
Statistical analysis
Experimental series A (untreated control, 7, 24, and 47 mg/kg 4-MBC) and B (untreated control, 0.7 mg/kg 4-MBC) were analyzed separately because they had been conducted during different periods. 4-MBC-exposed groups of series A and B were compared with their corresponding control groups. Data were expressed as percentage of control. For Western-blot data, values of treated samples were expressed as percentage of the mean of control samples of individual gels. To compare mRNA baseline levels, additional analyses were run (1) with intact untreated controls and ovariectomized, vehicle-injected untreated controls on the same plate (Table 4) (2) with vehicle-injected, ovariectomized animals of control and 4-MBC-exposed groups on the same plate (Table 3). Differences between treatment groups were determined for mRNA and proteins by one-way ANOVA with Bonferroni post hoc analysis of selected pair-wise comparisons and by Dunnett’s multiple comparison test for series A and by Student’s t test for series B (two groups) (GraphPad Prism 4, GraphPad Software Inc., San Diego, CA). Because the two control groups did not differ significantly from each other, controls were pooled in Figs. 1–6, but the levels of statistical significance refer to the separate analyses of the series A and B. P < 0.05 was taken as statistically significant. Linear trend analyses or regression analyses of log dose-response relationships were also performed.
TABLE 4. mRNA levels in uterus of intact and ovariectomized adult control offspring
FIG. 1. Levels of mRNAs encoding for PR, IGF-I, ER, ER?, and AR in uterus of adult (12 wk old) female rat offspring exposed during pre- and postnatal life to 4-MBC (0.7, 7, 24, and 47 mg/kg body weight·d in chow) and of rat offspring receiving control chow. Real-time RT-PCR values normalized to cyclophilin and expressed as percentage of the mean of the corresponding untreated controls [mean ± SD, n = 8 per group, pooled controls A (n = 8) + B (n = 8)]. *, P < 0.05; **, P < 0.01, compared with the corresponding untreated control A (for 7–47 mg/kg 4-MBC) or B (for 0.7 mg/kg).
FIG. 2. Levels of PR-A, PR-B, ER, and ER? protein determined by Western-blot analysis in uterine extracts of adult (12 wk old) female rat offspring exposed during pre- and postnatal life to 4-MBC (0.7, 7, 24, and 47 mg/kg body weight·d in chow) and of rat offspring receiving control chow. A, Representative Western blot. B, Protein levels determined by densitometry. Values are relative to -tubulin and expressed as percentage of the mean of the corresponding control (mean ± SD, n = 6–9 per group, pooled controls A + B). *, P < 0.05; **, P < 0.01, compared with corresponding untreated control A (for 7–47 mg/kg 4-MBC) or B (for 0.7 mg/kg).
FIG. 3. A, Induction of PR mRNA in uterus of ovariectomized adult female rat offspring 6 h after sc injection of E2 (10 or 50 μg/kg) or vehicle in untreated controls [pooled controls A (n = 8) + B (n = 8)] and in three 4-MBC treatment groups (0.7, 7, and 24 mg/kg·d). Values were normalized to cyclophilin and expressed as percentage of the corresponding vehicle-injected group (mean ± SD, n = 8). ***, Different from corresponding vehicle group, P < 0.001. B, Comparison of the up-regulation of PR mRNA by E2 in adult ovariectomized rat offspring of the control group and 4-MBC-exposed groups (0.7, 7, or 24 mg/kg·d). Values were normalized to cyclophilin and expressed as percentage of the corresponding vehicle-injected group (mean ± SD, n = 8). **, P < 0.01, compared with E2 effect in the corresponding 4-MBC-free control group (A for 7-24 mg/kg 4-MBC).
FIG. 4. A, Induction of IGF-I mRNA in uterus of ovariectomized adult female rat offspring 6 h after sc injection of E2 (10 or 50 μg/kg) or vehicle in untreated controls [pooled controls A (n = 8) + B (n = 8)] and in three 4-MBC treatment groups (0.7, 7, and 24 mg/kg·d). Values were normalized to cyclophilin and expressed as percentage of the corresponding vehicle-injected group (mean ± SD, n = 8). ***, Different from corresponding vehicle group, P < 0.001. B, Comparison of the up-regulation of IGF-I mRNA by E2 in adult ovariectomized rat offspring of the control group and 4-MBC-exposed groups (0.7, 7, or 24 mg/kg·d). Values were normalized to cyclophilin and expressed as percentage of the corresponding vehicle-injected group (mean ± SD, n = 8). **, P < 0.01, compared with E2 effect in the corresponding 4-MBC-free control group (A for 7-24 mg/kg 4-MBC).
FIG. 5. Comparison of the acute suppression of mRNAs encoding for ER (A), ER? (B), and AR (C) in uterus of adult ovariectomized female rat offspring 6 h after sc injection of E2 (10 or 50 μg/kg sc) in the untreated controls [pooled controls A (n = 8) + B (n = 8)] compared with corresponding 4-MBC-exposed groups (0.7, 7, or 24 mg/kg·d). Values were normalized to cyclophilin and expressed as percentage of the corresponding vehicle-injected group (mean ± SD, n = 8). **, P < 0.01, compared with E2 effect in the corresponding 4-MBC-free control group (A for 7-24 mg/kg 4-MBC).
FIG. 6. SRC-1 in uterus of adult (12 wk old) female rat offspring exposed during pre- and postnatal life to 4-MBC (0.7, 7, 24, and 47 mg/kg body weight·d in chow) and of rat offspring receiving control chow. A, SRC-1 mRNA levels determined by real-time RT PC. Values normalized to cyclophilin and expressed as percentage of the mean of the corresponding untreated controls. B, SRC-1 protein. Representative Western-blot and protein levels determined by densitometry. Values relative to -tubulin and expressed as percentage of the mean of the corresponding untreated control. Mean values ± SD (n = 8 for mRNA, n = 6 for protein; pooled controls). *, P < 0.05; **, P < 0.01, compared with corresponding untreated control A (for 7–47 mg/kg 4-MBC) or B (for 0.7 mg/kg 4-MBC).
Results
Effect of developmental exposure to 4-MBC on gene expression in uterus of intact adult offspring
mRNA analysis.
4-MBC was administered to Long Evans rats in the chow at concentrations yielding an average daily intake of 0.7, 7, 24, or 47 mg/kg body weight·d to the parent generation (F0) at least 10 wk before mating, during pregnancy and lactation, and to the F1 offspring until adulthood. The expression pattern of different estrogen-regulated genes, PR, IGF-I, ER, ER?, and AR, was examined by quantitative real-time PCR, with cyclophilin mRNA as reference, in the uterus of adult 4-MBC-exposed offspring in diestrus at 12 wk of age (steady-state condition, Fig. 1). Criteria for the selection of genes were their role in uterine function and the possibility of correlation with central nervous system and male reproductive organs.
4-MBC exposure significantly affected the steady-state levels of all mRNA species studied, except for ER? (PR, P = 0.0004; IGF-I, P < 0.0001; ER, P = 0.0069; AR, P = 0.0060). PR mRNA levels exhibited a marked dose-dependent down-regulation, statistically significant at 24 and 47 mg/kg·d 4-MBC (Fig. 1). IGF-I mRNA expression in uterus was significantly increased at 7 mg/kg·d 4-MBC but decreased at the highest dose (47 mg/kg·d). 4-MBC exposure further resulted in a dose-dependent decrease of ER mRNA (significant at 24 and 47 mg/kg·d) and AR mRNA (significant at 24 mg/kg·d) (Fig. 1). ER? mRNA also appeared to decrease in a dose-dependent manner, but the treatment effect was not significant (P = 0.0588). Treatment groups 7, 24, and 47 mg/kg 4-MBC were compared with control A, the 0.7 mg/kg group with low-dose control B. The two control groups did not differ significantly from each other.
Protein analysis.
To directly compare changes in mRNA and protein levels, ER, ER?, and PR protein were quantitated by Western-blot analysis in the same uterine tissue homogenates as used for real-time PCR analyses. Developmental exposure to 4-MBC resulted in a down-regulation of ER protein levels in uterus that was statistically significant at 47 mg/kg·d (Fig. 2). PR-A and PR-B protein showed a very similar dose-response relationship with a reduction at the 0.7 mg/kg·d dose that was significant for PR-A. At higher doses of 4-MBC, both PR proteins tended to increase rather than decrease (positive linear trend for PR-B, P = 0.032). Uterine ER? protein was not altered significantly.
Uterine weight.
Absolute and relative uterine weights and body weight of 4-MBC-exposed rat offspring studied at 12 wk of age did not differ significantly from untreated controls, except for the 24 mg/kg dosage group, which showed a slight increase in absolute and relative uterine weight (Table 1).
Acute response to E2 in adult ovariectomized rat offspring
To answer the question whether 4-MBC exposure could affect the sensitivity of estrogen-regulated genes to estrogen, offspring out of different litters of the untreated control groups A and B and of the 0.7, 7, and 24 mg/kg 4-MBC treatment groups were ovariectomized in adulthood, at 10 wk of age, injected with one of two doses of E2 (10 or 50 μg/kg sc) at 12 wk (the age studied under steady-state conditions) and decapitated 6 h after the injection (Table 5).
TABLE 5. Treatment schedule of estrogen challenge experiments
Ovariectomy did not affect uterine ER and ER? transcript levels as indicated by a comparison of mRNA levels of intact 12-wk-old offspring of the untreated control group with those of ovariectomized, vehicle-injected 12-wk-old untreated controls belonging to the same litters. In contrast, uterine IGF-I mRNA levels were moderately reduced, and a marked decrease was observed for mRNAs encoding for PR and AR (Table 4). mRNA levels of ovariectomized, vehicle-injected groups, representing the reference level of the challenge experiments, did not differ significantly between untreated controls and 4-MBC-exposed groups (Table 3).
PR mRNA.
In uterus of offspring that were not exposed to 4-MBC during ontogeny (untreated controls), a single sc injection of E2 (10 and 50 μg/kg) led to a significant induction of PR mRNA relative to the corresponding vehicle control within 6 h after the E2 injection (Fig. 3). Pre- and postnatally 4-MBC-exposed female offspring also showed a significant induction of PR mRNA by E2 (Fig. 3A), but the response to the lower dose of E2 (10 μg/kg) was reduced as a function of 4-MBC dose (Fig. 3B). The difference from offspring not exposed to 4-MBC was significant in the 7 and 24 mg/kg·d groups. The response to the higher E2 dose (50 μg/kg) was similar in magnitude across the various treatment groups.
IGF-I mRNA.
Subcutaneous administration of E2 to ovariectomized rats increased uterine IGF-I mRNA levels 6-fold compared with levels seen in vehicle controls (Fig. 4A). The IGF-I mRNA induction in animals exposed to 24 mg/kg·d 4-MBC was significantly smaller than the induction in ovariectomized controls after both doses of E2 (Fig. 4B).
ER mRNA.
ER mRNA was significantly down-regulated 6 h after both doses of E2 in controls as well as in 4-MBC-exposed groups (P < 0.001). However, down-regulation by 10 μg/kg E2 was dose-dependently reduced in offspring exposed to 4-MBC (Fig. 5A). The effect reached statistical significance at 24 mg/kg·d. In contrast to the 10 μg/kg E2 dose, no significant change of the response was seen after 50 μg/kg E2.
ER? mRNA.
ER? mRNA levels were strongly down-regulated in the uterus 6 h after E2 treatment in controls and 4-MBC-exposed animals (P < 0.001). 4-MBC exposure did not affect this response (Fig. 5B).
AR mRNA.
E2 administration greatly reduced the AR mRNA level in uterus in all experimental groups compared with the corresponding vehicle (P < 0.001). The inhibitory effect of E2 (10 and 50 μg/kg) on AR mRNA expression was dose-dependently diminished in 4-MBC-exposed offspring with a significant difference from controls at 24 mg/kg·d (Fig. 5C).
Effect of 4-MBC on SRC-1
To obtain more information about factors involved in changes of sensitivity to steroid hormones, we analyzed SRC-1 mRNA and protein levels in 4-MBC-exposed offspring. 4-MBC exposure affected SRC-1 mRNA levels (P = 0.0003), with a dose-dependent decrease that was statistically significant at the 24 mg/kg·d dose (Fig. 6A). Western-blot analysis demonstrated a significant decrease in SRC-1 protein levels, which was statistically significant at 0.7 and 47 mg/kg 4-MBC (Fig. 6B).
Discussion
The present study indicates that exposure to the UV filter 4-MBC throughout ontogeny until adulthood changes the steady-state expression of estrogen-regulated genes in the uterus of rat F1 offspring. 4-MBC exposure also affects the sensitivity of estrogen target genes to estrogen, and the expression of SRC-1. 4-MBC displays estrogenic activity in vitro on MCF-7 cells and in vivo in the uterotrophic assay on immature rats (1). The dose-response relationship suggests that 4-MBC is a partial ER agonist; in vivo, its maximum effect is 35% of that of the full ER agonist ethinylestradiol. Recent receptor binding data show a strong preference of 4-MBC for ER? (10). So far, it is not known whether there exist bioactive metabolites that might possibly exhibit a different effect spectrum; a carboxylated derivative has recently been identified as the major 4-MBC metabolite in female rats (20).
4-MBC was administered in the feed. To mimic a real-world situation, we used a natural ingredient open formula rodent diet (Kliba 3430) containing phytoestrogens (total genistein after hydrolysis approximately 135 mg/kg). The additional soy oil preparation that was used to incorporate 4-MBC into the chow and was also added in the same amount to the control chow did not contain detectable amounts of phytoestrogen according to informations by the manufacturer (Morga). Developmental studies on rats did not yield evidence for significant effects of phytoestrogen content in normal rodent diets on developmental parameters (21, 22), except for a small difference (12%) in anogenital distance noted in one study. Dietary phytoestrogens in the usual range also did not seem to exert significant influences on the sensitivity of the uterotrophic assay in immature rats (23, 24). This is in line with the low uterine control weights we regularly observed in immature rats on Kliba chow 3430 in uterotrophic assays (1). However, the effects of 4-MBC should be viewed as resulting from actions of a partial ER agonist on the background of endogenous steroidal estrogens and phytoestrogens taken up from food. There, the interaction with endogenous estrogens probably is more important than that with the low exogenous phytoestrogen background.
Uterine weight of adult offspring remained unaffected in most 4-MBC dosage groups, but a marginal increase was noted at 24 mg/kg. The 4-MBC present in the adult offspring would not be expected to affect this parameter because the doses were below that eliciting a uterotrophic response (1). However, the possibility of delayed developmental effects is suggested by observations on diethylstilbestrol (DES) and certain phytoestrogens. In comparatively high perinatal doses, DES and coumestrol induced an initial increase in uterine weight followed by subsequent growth impairment (25, 26, 27). Yet, developmental exposure to lower doses of DES (28) or E2 (29) did not affect uterine weight, indicating that even exposure to potent ER agonists does not necessarily result in changes in uterine weight in adult offspring. In consideration of the ER? preference of 4-MBC (10), it should also be noted that uterine weight appears to be little influenced by ER? agonists at doses that affect estrogen-regulated genes (30).
4-MBC exposure caused changes in the expression of estrogen-regulated genes. The presence of significant effects at the protein level, with maximal changes of 40% of control levels, supports the idea that the alterations in gene regulation may be of functional relevance. These alterations could be a consequence of developmental actions of 4-MBC, of direct effects of the chemical present in the adult offspring, or, possibly, of a combination of both of these. An analysis of the chronic condition is complicated by the cross talk between several of the genes studied (ER/IGF-I, ER/AR, and ER/PR).
ER exhibited a dose-dependent reduction of steady-state mRNA and protein levels that was more clear-cut for ER protein. The parallel change in ER mRNA and protein suggests an interaction of 4-MBC with transcriptional regulation, although additional regulatory effects at the protein level (31) cannot be excluded. In contrast to ER, ER? protein level remained unchanged, even though mRNA showed a slight decrease that was significant across the dose-response curve. Acute administration of ER agonists down-regulates ER and ER? in adult rodent uterus (32, 33), but for ER, this was shown to be followed by a normalization of ER levels upon prolonged estrogen exposure (34). Thus, a developmental action appears to offer a more plausible explanation for the reduced ER mRNA levels. Reduced uterine ER mRNA levels could result from perinatal exposure to an estrogenic chemical, as observed for DES (25), whereas the effect of neonatal exposure on uterine ER? is not quite clear (35).
PRs were also affected by 4-MBC exposure, but in contrast to ER, dose-response curves of PR mRNA and PR-A and PR-B proteins differed. Whereas PR mRNA was down-regulated with a monotonic dose-response curve, PR protein levels were significantly reduced at the lowest 4-MBC dose but returned to normal or slightly supranormal levels (significant positive linear trend) at higher doses. Because this pattern was identical for both PR-A and PR-B, it appears to be reliable. With a significant treatment effect at 0.7 mg/kg 4-MBC, PR-A protein appeared to be one of the most sensitive parameters.
The complex dose-response relationships of PR are not easy to interpret. The decrease in PR protein and mRNA levels at the lowest 4-MBC dose could be due to the reduced sensitivity to estrogenic stimulation observed in the E2 challenge experiments. At higher doses, direct ER agonistic actions of 4-MBC might overlay this effect. Why PR mRNA does not follow the same pattern is not known. Uterine PR can be up- or down-regulated by acute administration of classical ER agonists depending on cell type (36, 37, 38). It cannot be excluded that different uterine tissue compartments contributed differently to mRNA and protein analyses. Atypical acute responses of uterine PR mRNA have been reported for some xenoestrogens (15). Whether the ER? preference of 4-MBC (10) plays a role is difficult to judge because both ER and ER? appear to be involved in up- and down-regulation of PR (37, 38, 39). A possible AR-mediated effect on PR (40) can be excluded because 4-MBC is devoid of androgenic activity (2). To what extent actions of 4-MBC during early development might contribute to the present condition is not known. In the rat fetus, E2 and DES increased PR in mesenchymal cells of the middle part of the Mullerian duct that gives rise to the uterus, whereas DES decreased PR in epithelial cells (41). However, we did not observe a change in uterine PR mRNA levels in adult rat offspring after prenatal DES treatment (S. Durrer, unpublished data). Thus, the 4-MBC data do not appear to match either acute or delayed developmental effects of classical ER agonists.
Exposure to 4-MBC also resulted in reduced AR mRNA levels. Uterine AR are down-regulated by acute administration of estrogens (15, 42), ER, and ER? agonists (30). However, in immature rats, an acute inductive effect has been reported (39). Because data on uterine AR after prolonged developmental exposure to estrogenic compounds are missing, it cannot be decided whether the present AR data would be compatible with a developmental effect or whether an explanation should be sought in a direct action in the adult offspring.
The decrease in IGF-I mRNA at higher 4-MBC doses might be linked with the reduction of ER protein because activation of IGF-I signaling by estrogens appears to be mediated by ER (43). The ER? preference of 4-MBC may contribute to the decrease in IGF-I because ER? appears to exert a repressive role (36). The reason for the increase in mRNA levels at the lower 4-MBC dose is not known. Estrogenic substances, including steroidal estrogens, DES, and xenoestrogens such as genistein and bisphenol A, can elicit an acute induction of IGF-I mRNA in rodent uterus (43, 44), but this induction appears to be transient upon continued exposure to estrogen (45) and, thus, would not explain an increased level upon prolonged exposure to a partial agonist.
The sensitivity of target genes to hormones should provide more insight into possible alterations in gene regulation than steady-state mRNA levels. The E2 challenge experiments in adult offspring revealed significant changes in the acute responsiveness to E2 of four of five genes studied. Down-regulation of ER? (33) did not seem to be affected. Irrespective of whether acute administration of E2 was followed by mRNA up-regulation (PR and IGF-I) or down-regulation (ER and AR), the response to E2 was dose-dependently reduced in 4-MBC-exposed offspring. The levels of these four mRNA species were similar in vehicle-injected ovariectomized animals of untreated control and 4-MBC-exposed groups. This indicates that the mRNA response to E2 started from the same level in untreated and 4-MBC-exposed animals and that the differences in the acute response to E2 are related to 4-MBC exposure. For the two genes up-regulated by E2, PR, and IGF-I, this means that the response to E2 started from low mRNA levels because baseline mRNA levels were significantly reduced in ovariectomized vehicle-injected controls compared with untreated intact controls and, as mentioned above, were similar in vehicle-injected ovariectomized animals of the controls and 4-MBC-exposed groups. This should have allowed for a full-size response to E2.
One possible explanation of reduced sensitivity to E2 could be sought in an antagonistic activity of 4-MBC, which exhibits features of a partial agonist in vivo (1), provided tissue levels were high enough in the adult offspring. However, this idea would be difficult to reconcile with the direction of changes in steady-state levels of ER, AR, and IGF-I mRNA (low dose) and would be in contradiction to observations in the central nervous system of the same animals, where we observed enhanced sensitivity to E2 (46). Observations with DES indicate that developmental exposure to estrogenic chemicals may influence gene expression by alterations in methylation status (47, 48). Although such effects remain a possibility, it should be noted that perinatal DES treatment resulted in a different effect pattern with continuous overexpression of the target genes studied (lactoferrin and c-fos).
Changes in the sensitivity to steroid hormones might also be brought about by changes in the availability of steroid receptor coregulators. We studied the expression of SRC-1, a member of the SRC/p160 family (49), in 4-MBC-exposed offspring. Developmental exposure to 4-MBC reduced the expression of SRC-1 mRNA and protein. SRC-1 protein was significantly decreased in the dose range between 0.7 and 47 mg/kg 4-MBC, which includes the doses causing reduced inductory responses of IGF-I and PR genes to E2. The combination of reduced ER and SRC-1 levels may provide a sufficient explanation for the reduced responsiveness to E2 of the genes that are up-regulated by E2. Deletion of the SRC-1 gene was found to be accompanied by reduced responsiveness of mouse uterus to E2 (50), despite the continued presence of alternative coactivators. Uterine SRC-1 mRNA expression appears to be unresponsive to acute administration of E2 (51). This suggests that the down-regulation by prolonged exposure to 4-MBC may have resulted from a more complex mechanism. In this respect, the preferential binding of 4-MBC to ER? (10) may be of interest because SCR-1 has been reported to be the preferred partner of ER? (14).
In conclusion, our data demonstrate that exposure to the UV filter 4-MBC, an environmental xenoestrogen with preference for ER? during early development and postnatal life, can lead to significant changes in the expression of ERs, nuclear receptor coregulators, and estrogen target genes in uterus at mRNA and protein levels. The minimum effective dose of 4-MBC was 0.7 mg/kg (PR-A and SRC-1 protein). At the dose of 7 mg/kg, 4-MBC concentrations in adipose tissue of adult F1 offspring of the present study (17) were close to levels determined in fish. The 4-MBC-induced changes in steady-state expression levels are subtle, but it should be noted that in real world, the compound contributes to effects of a mixture of endocrine active chemicals. A most interesting observation of the present study is the change in coactivator SRC-1 expression, which was one of the most sensitive parameters. It illustrates that exposure to endocrine active chemicals can alter the regulation of additional proteins of the transactivation complex besides nuclear receptors. The reduction of estrogen sensitivity that may be due to the parallel decrease of SRC-1 and ER represents a potentially relevant regulatory dysfunction.
Acknowledgments
We thank Marianne Conscience and Vreni Haller for most valuable support in the conduction of the long-term animal experiments and Laurent Gelman (Unité CIG Sciences, University of Lausanne, Lausanne, Switzerland) for helpful support in Western-blot analyses.
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Address all correspondence and requests for reprints to: Walter Lichtensteiger, Group for Reproductive, Endocrine, and Environmental Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. E-mail: Walter.Lichtensteiger@access.unizh.ch.
Abstract
Because the estrogen receptor (ER) ligand type influences transactivation, it is important to obtain information on molecular actions of nonclassical ER agonists. UV filters from cosmetics represent new classes of endocrine active chemicals, including the preferential ER? ligands 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor. We studied estrogen target gene expression in uterus of Long Evans rats after developmental exposure to 4-MBC (0.7, 7, 24, and 47 mg/kg·d) administered in feed to the parent generation before mating, during pregnancy and lactation, and to the offspring until adulthood. 4-MBC altered steady-state levels of mRNAs encoding for ER, ER?, progesterone receptor (PR), IGF-I, androgen receptor, determined by real-time RT-PCR in uterus of 12-wk-old offspring. Western-blot analyses of the same tissue homogenates indicated changes in ER and PR but not ER? proteins. To assess sensitivity to estradiol (E2), offspring were ovariectomized on d 70, injected with E2 (10 or 50 μg/kg sc) on d 84, and killed 6 h later. Acute up-regulation of PR and IGF-I and down-regulation of ER and androgen receptor by E2 were dose-dependently reduced in 4-MBC-exposed rats. The reduced response to E2 was accompanied by reduced coactivator SRC-1 mRNA and protein levels. Our data indicate that developmental exposure to 4-MBC affects the regulation of estrogen target genes and the expression of nuclear receptor coregulators in uterus at mRNA and protein levels.
Introduction
ESTROGENIC AND ANTIANDROGENIC UV filters used in cosmetics represent a new group of endocrine-active chemicals (1, 2). UV filters are released into the environment and reach animals via the food chain and humans via a dual exposure, food chain, and direct application (3, 4). After the identification of estrogenic activity of several UV filters, including 4-methylbenzylidene camphor (4-MBC), in vitro and in immature rats (1), their activity has been confirmed in vitro (5, 6, 7) as well as in vivo in mammals (5) and fish (8, 9). 4-MBC binds competitively to estrogen receptors (ERs) (10) and stimulates transactivation (6, 7).
Endocrine-active UV screens are relevant from a toxicological point of view because of widespread exposure, and they are also of interest because their chemical structure differs from structures so far encountered with endocrine disrupters. There is good evidence that the chemical structure of ER ligand and the ER subtype influence transactivation at several different levels (11, 12, 13, 14), leading to different gene expression patterns (15), but there is little information on molecular effect patterns after chronic exposure to nonclassical ER ligands.
We are studying effects of various ER ligands on reproduction and development, parts of the life cycle that are very sensitive to endocrine disrupters, with focus on reproductive organs and central nervous system. 4-MBC was chosen as the chemical with the highest acute estrogenic in vivo activity in the first group of UV filters studied (1). In receptor binding experiments, it shows distinct preference for ER? (10). Developmental exposure to 4-MBC influences a number of endocrine parameters, including delayed puberty in males and changes in reproductive organ weights (16, 17).
In the present study, we investigated whether pre- and postnatal exposure of rats to 4-MBC affects the expression of estrogen-regulated genes at the mRNA and protein level in uterus of adult offspring. We observed significant, dose-dependent changes in the expression of mRNAs encoding for ER subtypes, progesterone receptor (PR), IGF-I, and androgen receptor (AR), and in protein levels and a reduced sensitivity to acute administration of estradiol (E2). The decrease in estrogen sensitivity was accompanied by reduced expression of the steroid receptor coactivator (SRC)-1 in the same dose range. SRC-1 was found to be one of the most sensitive parameters.
Materials and Methods
Chemicals
The UV filter 3-(4-methylbenzylidene) camphor (4-MBC; Eusolex 6300 CAS no. 36861-47-9, purity 99.7–99.9%) was purchased from Merck (Dietikon, Switzerland), and E2 was purchased from Calbiochem (Lucerne, Switzerland).
Experimental animals
The study was conducted on Long Evans rats (M?llegaard Breeding and Research Centre, Ejby, Denmark) bred in our laboratory under controlled light and dark cycle (lights on from 0200–1600 h) and temperature (22 C ± 1 C) with free access to food and water. The animal facility is run by the Institute of Laboratory Animal Science of the University of Zurich (Zurich, Switzerland). Microbiological checks are performed every 3 months. Animal maintenance and experiments were conducted according to the Swiss Law for the Protection of Animals and the Ethical Guidelines of the Swiss Academy of Medical Sciences.
Treatment schedule
Food pellets containing 4-MBC.
Food pellets with 4-MBC were prepared by Provimi Kliba AG (Kaiseraugst, Switzerland). Appropriate amounts of 4-MBC dissolved in cold-pressed soy oil (Morga, Ebnat-Kappel, Switzerland) were added to Provimi Kliba chow no. 3340 (formerly 3430) to achieve 4-MBC concentrations of 0.01, 0.1, 0.33, or 0.66 g/kg chow, yielding an average daily intake of 0.7, 7, 24, or 47 mg/kg body weight·d, respectively. Control pellets consisted of the same matrix (Provimi Kliba no. 3430) with 1% of soy oil added. The soy oil preparation (Morga) was devoid of detectable amounts of phytoestrogens (information of the manufacturer).
4-MBC exposure.
The study was designed to mimic exposure through the food chain. It corresponds to an extended one-generation study. Males and females of the parent generation (5–6 wk old) were fed for at least 10 wk before mating (including one spermatogenic cycle) with chow containing 4-MBC (0.01, 0.1, 0.33, and 0.66 g/kg chow) or with control chow. Treatment continued throughout pregnancy and in the offspring until adulthood. Experiments were performed on offspring of time-pregnant rats (1). The day of birth was defined as postnatal d 1. Pups were weaned at postnatal d 28, male and female littermates were raised in separate groups until adulthood. Different developmental endpoints and the onset of puberty were assessed (16, 17). The study was run in two series, control A together with 7, 24, and 47 mg/kg 4-MBC, and control low dose, together with 0.7 mg/kg 4-MBC (Table 1). The numbers of litters were eight and five in the two control groups and four to six in the 4-MBC-exposed groups.
TABLE 1. Uterine weight and body weight in 12-wk-old rat offspring after pre- and postnatal exposure to 4-MBC
Analysis of adult offspring under steady-state conditions.
Offspring were raised without further experimental manipulation except 4-MBC treatment. Females were killed by decapitation at 12 wk of age in diestrus. The uterus was quickly removed, weighed (wet weight), and frozen in liquid nitrogen until further analysis.
Estrogen challenge experiment in adult offspring.
At 10 wk of age, female offspring were taken out of several different litters of the control group and of 4-MBC-exposed groups (0.01, 0.1, and 0.33 g/kg chow) and ovariectomized under anesthesia (Hypnorm, Domitor, and Atropin). After 2 wk of recovery, at the age of 12 wk, they received a single sc injection of E2 (10 or 50 μg/kg in dimethylsulfoxide) or vehicle (dimethylsulfoxide) (injection volume 1 μl/g body weight) and were decapitated 6 h later for tissue dissection. Animals from the same litters were studied under steady-state conditions.
Real-time RT-PCR
RNA isolation and RT.
The uterus was homogenized in RNA lysis tissue (RLT) buffer from the RNeasy-mini kit (QIAGEN, Valencia, CA) by a polytron rotor-stator homogenizer. Total RNA was extracted using the RNeasy mini kit according to the manufacturer’s instructions; the RNA preparation was incubated with DNaseI (QIAGEN). Total RNA concentration was assessed by A260 and checked for DNA contamination on an ethidium bromide-stained 2.5% agarose gel. RNA was stored at –80 C until used. For RT, 10 μg RNA was used in a total volume of 100 μl containing 1x TaqMan RT buffer, 5.5 mM MgCL2, 500 μM of each dNTP, 2.5 μM random hexamer primers, 0.4 μM RNase inhibitor, and 1.25 μl MultiScribe TM reverse transcriptase (Applied Biosystems, Foster City, CA). The mixture was incubated for 10 min at 25 C followed by RT of 30 min at 48 C and RT inactivation of 5 min at 95 C.
Primers and TaqMan probes.
Primers and probes were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA) as described by the manufacturer and were synthesized by Microsynth (Balgach, Switzerland). To exclude amplification from possible DNA contamination, either the probes or the primers were designed to overlap exon junctions in cDNA regions derived from intron-bearing genes. All probes were labeled with the fluorescent dyes 6-carboxy-fluorescein as reporter and 6-carboxy-tetramethyl-rhodamine as quencher. Table 2 lists the sequences of primers and probes used.
TABLE 2. Forward primers (FP), reverse primers (RP), and TaqMan probes with corresponding GenBank accession no.
Real-time-PCR.
To amplify cDNA, RT samples were mixed with TaqMan PCR master mix (Applied Biosystems), optimized concentrations of primers and probes and distilled water were added to a final volume of 25 μl. Signals were monitored by an ABI PRISM 7700 Sequence Detector (Applied Biosystems). PCR cycle parameters were an initial denaturation step at 95 C for 10 min, followed by 40 cycles at 95 C for 15 sec and 60 C for 1min.
Sequence Detector Software SDS 2.0 (Applied Biosystems) was used for data analysis. After the reaction, the threshold line was set in the linear region of the plots above the baseline noise, and the threshold cycle (CT) was determined as the cycle number at which the threshold line crosses the amplification curve. The CT values obtained were exported to a Microsoft Excel spreadsheet for further analysis. Standard curve plots were constructed from a cDNA dilution series of a control animal, using Microsoft Excel as recommended in User Bulletin no. 2 for the ABI Prism 7700 Sequence Detection System (Applied Biosystems, 1997), with CT values plotted against the logarithm of the input amount of cDNA (equivalent to the amount of total RNA). The CT values obtained were used to calculate the initial input amount. Thereafter, the results were normalized to cyclophilin as reference gene and expressed as percentage of control. For comparison of levels of different control or reference groups (Table 3), additional measurements were performed with samples of these control groups on the same plate. Part of the data was calculated with the comparative CT method. CT was the difference of the CT values of the target gene being assayed and of the cyclophilin reference, whereas Ct represented the difference between the paired tissue samples, as calculated by the formula Ct = Ct of control tissue – Ct of treated tissue. The n-fold differential expression in a specific gene of a treated sample compared with the control was expressed as 2–Ct (User Bulletin 2, Applied Biosystems).
TABLE 3. Comparison of mRNA levels in untreated controls and 4-MBC-exposed groups after ovariectomy and vehicle injection
Choice of reference gene.
The use of housekeeping genes as internal control starts from the assumption that they vary comparatively little in response to experimental treatment, yet, variations can be considerable (18, 19). It is difficult to find an internal mRNA standard that is not affected by endocrine manipulation. Preliminary tests were performed on uteri of rats after E2 treatment, and mRNAs encoding for glyceraldehyde-3-phosphate dehydrogenase, ?-actin, hypoxanthine phosphoribosyl transferase, and cyclophilin were determined. An analysis of the data indicated that glyceraldehyde-3-phosphate dehydrogenase and ?-actin could be influenced by estrogen treatment and gonadectomy. Cyclophilin appeared to be more stable. Hypoxanthine phosphoribosyl transferase could also have been used but was not expressed in the appropriate CT range in some tissues. Therefore, cyclophilin was chosen as reference gene.
Western blot analysis.
Protein levels were analyzed in the same tissue homogenates that were used for mRNA analysis. Equal amounts of protein extracts were run on SDS-PAGE gels and transferred onto nitrocellulose membrane. After transfer, the membranes were blocked in 5% milk (blotting grade blocker nonfat dry milk, Bio-Rad Laboratories, Hercules, CA) in Tris-buffered saline-Tween 20 (TBST), then incubated overnight with the first antibody, anti-ER (ABR-Affinity BioReagents, Golden, CO), anti-ER?, anti-PR, anti-SRC-1, anti--Tubulin (Santa Cruz Biotech. Co., Santa Cruz, CA), diluted in 5% milk/TBST. After washes with TBST, the membranes were incubated for 1 h with the appropriate secondary antibodies conjugated to horseradish peroxidase. Proteins were detected using chemiluminescence (Pierce, Rockford, IL). Densitometrical analyses were performed using the AIDA-2D software (Raytest, Pittsburgh, PA). Results are expressed as the ratio of the protein of interest to -tubulin to correct for possible differences of protein loading between samples.
Statistical analysis
Experimental series A (untreated control, 7, 24, and 47 mg/kg 4-MBC) and B (untreated control, 0.7 mg/kg 4-MBC) were analyzed separately because they had been conducted during different periods. 4-MBC-exposed groups of series A and B were compared with their corresponding control groups. Data were expressed as percentage of control. For Western-blot data, values of treated samples were expressed as percentage of the mean of control samples of individual gels. To compare mRNA baseline levels, additional analyses were run (1) with intact untreated controls and ovariectomized, vehicle-injected untreated controls on the same plate (Table 4) (2) with vehicle-injected, ovariectomized animals of control and 4-MBC-exposed groups on the same plate (Table 3). Differences between treatment groups were determined for mRNA and proteins by one-way ANOVA with Bonferroni post hoc analysis of selected pair-wise comparisons and by Dunnett’s multiple comparison test for series A and by Student’s t test for series B (two groups) (GraphPad Prism 4, GraphPad Software Inc., San Diego, CA). Because the two control groups did not differ significantly from each other, controls were pooled in Figs. 1–6, but the levels of statistical significance refer to the separate analyses of the series A and B. P < 0.05 was taken as statistically significant. Linear trend analyses or regression analyses of log dose-response relationships were also performed.
TABLE 4. mRNA levels in uterus of intact and ovariectomized adult control offspring
FIG. 1. Levels of mRNAs encoding for PR, IGF-I, ER, ER?, and AR in uterus of adult (12 wk old) female rat offspring exposed during pre- and postnatal life to 4-MBC (0.7, 7, 24, and 47 mg/kg body weight·d in chow) and of rat offspring receiving control chow. Real-time RT-PCR values normalized to cyclophilin and expressed as percentage of the mean of the corresponding untreated controls [mean ± SD, n = 8 per group, pooled controls A (n = 8) + B (n = 8)]. *, P < 0.05; **, P < 0.01, compared with the corresponding untreated control A (for 7–47 mg/kg 4-MBC) or B (for 0.7 mg/kg).
FIG. 2. Levels of PR-A, PR-B, ER, and ER? protein determined by Western-blot analysis in uterine extracts of adult (12 wk old) female rat offspring exposed during pre- and postnatal life to 4-MBC (0.7, 7, 24, and 47 mg/kg body weight·d in chow) and of rat offspring receiving control chow. A, Representative Western blot. B, Protein levels determined by densitometry. Values are relative to -tubulin and expressed as percentage of the mean of the corresponding control (mean ± SD, n = 6–9 per group, pooled controls A + B). *, P < 0.05; **, P < 0.01, compared with corresponding untreated control A (for 7–47 mg/kg 4-MBC) or B (for 0.7 mg/kg).
FIG. 3. A, Induction of PR mRNA in uterus of ovariectomized adult female rat offspring 6 h after sc injection of E2 (10 or 50 μg/kg) or vehicle in untreated controls [pooled controls A (n = 8) + B (n = 8)] and in three 4-MBC treatment groups (0.7, 7, and 24 mg/kg·d). Values were normalized to cyclophilin and expressed as percentage of the corresponding vehicle-injected group (mean ± SD, n = 8). ***, Different from corresponding vehicle group, P < 0.001. B, Comparison of the up-regulation of PR mRNA by E2 in adult ovariectomized rat offspring of the control group and 4-MBC-exposed groups (0.7, 7, or 24 mg/kg·d). Values were normalized to cyclophilin and expressed as percentage of the corresponding vehicle-injected group (mean ± SD, n = 8). **, P < 0.01, compared with E2 effect in the corresponding 4-MBC-free control group (A for 7-24 mg/kg 4-MBC).
FIG. 4. A, Induction of IGF-I mRNA in uterus of ovariectomized adult female rat offspring 6 h after sc injection of E2 (10 or 50 μg/kg) or vehicle in untreated controls [pooled controls A (n = 8) + B (n = 8)] and in three 4-MBC treatment groups (0.7, 7, and 24 mg/kg·d). Values were normalized to cyclophilin and expressed as percentage of the corresponding vehicle-injected group (mean ± SD, n = 8). ***, Different from corresponding vehicle group, P < 0.001. B, Comparison of the up-regulation of IGF-I mRNA by E2 in adult ovariectomized rat offspring of the control group and 4-MBC-exposed groups (0.7, 7, or 24 mg/kg·d). Values were normalized to cyclophilin and expressed as percentage of the corresponding vehicle-injected group (mean ± SD, n = 8). **, P < 0.01, compared with E2 effect in the corresponding 4-MBC-free control group (A for 7-24 mg/kg 4-MBC).
FIG. 5. Comparison of the acute suppression of mRNAs encoding for ER (A), ER? (B), and AR (C) in uterus of adult ovariectomized female rat offspring 6 h after sc injection of E2 (10 or 50 μg/kg sc) in the untreated controls [pooled controls A (n = 8) + B (n = 8)] compared with corresponding 4-MBC-exposed groups (0.7, 7, or 24 mg/kg·d). Values were normalized to cyclophilin and expressed as percentage of the corresponding vehicle-injected group (mean ± SD, n = 8). **, P < 0.01, compared with E2 effect in the corresponding 4-MBC-free control group (A for 7-24 mg/kg 4-MBC).
FIG. 6. SRC-1 in uterus of adult (12 wk old) female rat offspring exposed during pre- and postnatal life to 4-MBC (0.7, 7, 24, and 47 mg/kg body weight·d in chow) and of rat offspring receiving control chow. A, SRC-1 mRNA levels determined by real-time RT PC. Values normalized to cyclophilin and expressed as percentage of the mean of the corresponding untreated controls. B, SRC-1 protein. Representative Western-blot and protein levels determined by densitometry. Values relative to -tubulin and expressed as percentage of the mean of the corresponding untreated control. Mean values ± SD (n = 8 for mRNA, n = 6 for protein; pooled controls). *, P < 0.05; **, P < 0.01, compared with corresponding untreated control A (for 7–47 mg/kg 4-MBC) or B (for 0.7 mg/kg 4-MBC).
Results
Effect of developmental exposure to 4-MBC on gene expression in uterus of intact adult offspring
mRNA analysis.
4-MBC was administered to Long Evans rats in the chow at concentrations yielding an average daily intake of 0.7, 7, 24, or 47 mg/kg body weight·d to the parent generation (F0) at least 10 wk before mating, during pregnancy and lactation, and to the F1 offspring until adulthood. The expression pattern of different estrogen-regulated genes, PR, IGF-I, ER, ER?, and AR, was examined by quantitative real-time PCR, with cyclophilin mRNA as reference, in the uterus of adult 4-MBC-exposed offspring in diestrus at 12 wk of age (steady-state condition, Fig. 1). Criteria for the selection of genes were their role in uterine function and the possibility of correlation with central nervous system and male reproductive organs.
4-MBC exposure significantly affected the steady-state levels of all mRNA species studied, except for ER? (PR, P = 0.0004; IGF-I, P < 0.0001; ER, P = 0.0069; AR, P = 0.0060). PR mRNA levels exhibited a marked dose-dependent down-regulation, statistically significant at 24 and 47 mg/kg·d 4-MBC (Fig. 1). IGF-I mRNA expression in uterus was significantly increased at 7 mg/kg·d 4-MBC but decreased at the highest dose (47 mg/kg·d). 4-MBC exposure further resulted in a dose-dependent decrease of ER mRNA (significant at 24 and 47 mg/kg·d) and AR mRNA (significant at 24 mg/kg·d) (Fig. 1). ER? mRNA also appeared to decrease in a dose-dependent manner, but the treatment effect was not significant (P = 0.0588). Treatment groups 7, 24, and 47 mg/kg 4-MBC were compared with control A, the 0.7 mg/kg group with low-dose control B. The two control groups did not differ significantly from each other.
Protein analysis.
To directly compare changes in mRNA and protein levels, ER, ER?, and PR protein were quantitated by Western-blot analysis in the same uterine tissue homogenates as used for real-time PCR analyses. Developmental exposure to 4-MBC resulted in a down-regulation of ER protein levels in uterus that was statistically significant at 47 mg/kg·d (Fig. 2). PR-A and PR-B protein showed a very similar dose-response relationship with a reduction at the 0.7 mg/kg·d dose that was significant for PR-A. At higher doses of 4-MBC, both PR proteins tended to increase rather than decrease (positive linear trend for PR-B, P = 0.032). Uterine ER? protein was not altered significantly.
Uterine weight.
Absolute and relative uterine weights and body weight of 4-MBC-exposed rat offspring studied at 12 wk of age did not differ significantly from untreated controls, except for the 24 mg/kg dosage group, which showed a slight increase in absolute and relative uterine weight (Table 1).
Acute response to E2 in adult ovariectomized rat offspring
To answer the question whether 4-MBC exposure could affect the sensitivity of estrogen-regulated genes to estrogen, offspring out of different litters of the untreated control groups A and B and of the 0.7, 7, and 24 mg/kg 4-MBC treatment groups were ovariectomized in adulthood, at 10 wk of age, injected with one of two doses of E2 (10 or 50 μg/kg sc) at 12 wk (the age studied under steady-state conditions) and decapitated 6 h after the injection (Table 5).
TABLE 5. Treatment schedule of estrogen challenge experiments
Ovariectomy did not affect uterine ER and ER? transcript levels as indicated by a comparison of mRNA levels of intact 12-wk-old offspring of the untreated control group with those of ovariectomized, vehicle-injected 12-wk-old untreated controls belonging to the same litters. In contrast, uterine IGF-I mRNA levels were moderately reduced, and a marked decrease was observed for mRNAs encoding for PR and AR (Table 4). mRNA levels of ovariectomized, vehicle-injected groups, representing the reference level of the challenge experiments, did not differ significantly between untreated controls and 4-MBC-exposed groups (Table 3).
PR mRNA.
In uterus of offspring that were not exposed to 4-MBC during ontogeny (untreated controls), a single sc injection of E2 (10 and 50 μg/kg) led to a significant induction of PR mRNA relative to the corresponding vehicle control within 6 h after the E2 injection (Fig. 3). Pre- and postnatally 4-MBC-exposed female offspring also showed a significant induction of PR mRNA by E2 (Fig. 3A), but the response to the lower dose of E2 (10 μg/kg) was reduced as a function of 4-MBC dose (Fig. 3B). The difference from offspring not exposed to 4-MBC was significant in the 7 and 24 mg/kg·d groups. The response to the higher E2 dose (50 μg/kg) was similar in magnitude across the various treatment groups.
IGF-I mRNA.
Subcutaneous administration of E2 to ovariectomized rats increased uterine IGF-I mRNA levels 6-fold compared with levels seen in vehicle controls (Fig. 4A). The IGF-I mRNA induction in animals exposed to 24 mg/kg·d 4-MBC was significantly smaller than the induction in ovariectomized controls after both doses of E2 (Fig. 4B).
ER mRNA.
ER mRNA was significantly down-regulated 6 h after both doses of E2 in controls as well as in 4-MBC-exposed groups (P < 0.001). However, down-regulation by 10 μg/kg E2 was dose-dependently reduced in offspring exposed to 4-MBC (Fig. 5A). The effect reached statistical significance at 24 mg/kg·d. In contrast to the 10 μg/kg E2 dose, no significant change of the response was seen after 50 μg/kg E2.
ER? mRNA.
ER? mRNA levels were strongly down-regulated in the uterus 6 h after E2 treatment in controls and 4-MBC-exposed animals (P < 0.001). 4-MBC exposure did not affect this response (Fig. 5B).
AR mRNA.
E2 administration greatly reduced the AR mRNA level in uterus in all experimental groups compared with the corresponding vehicle (P < 0.001). The inhibitory effect of E2 (10 and 50 μg/kg) on AR mRNA expression was dose-dependently diminished in 4-MBC-exposed offspring with a significant difference from controls at 24 mg/kg·d (Fig. 5C).
Effect of 4-MBC on SRC-1
To obtain more information about factors involved in changes of sensitivity to steroid hormones, we analyzed SRC-1 mRNA and protein levels in 4-MBC-exposed offspring. 4-MBC exposure affected SRC-1 mRNA levels (P = 0.0003), with a dose-dependent decrease that was statistically significant at the 24 mg/kg·d dose (Fig. 6A). Western-blot analysis demonstrated a significant decrease in SRC-1 protein levels, which was statistically significant at 0.7 and 47 mg/kg 4-MBC (Fig. 6B).
Discussion
The present study indicates that exposure to the UV filter 4-MBC throughout ontogeny until adulthood changes the steady-state expression of estrogen-regulated genes in the uterus of rat F1 offspring. 4-MBC exposure also affects the sensitivity of estrogen target genes to estrogen, and the expression of SRC-1. 4-MBC displays estrogenic activity in vitro on MCF-7 cells and in vivo in the uterotrophic assay on immature rats (1). The dose-response relationship suggests that 4-MBC is a partial ER agonist; in vivo, its maximum effect is 35% of that of the full ER agonist ethinylestradiol. Recent receptor binding data show a strong preference of 4-MBC for ER? (10). So far, it is not known whether there exist bioactive metabolites that might possibly exhibit a different effect spectrum; a carboxylated derivative has recently been identified as the major 4-MBC metabolite in female rats (20).
4-MBC was administered in the feed. To mimic a real-world situation, we used a natural ingredient open formula rodent diet (Kliba 3430) containing phytoestrogens (total genistein after hydrolysis approximately 135 mg/kg). The additional soy oil preparation that was used to incorporate 4-MBC into the chow and was also added in the same amount to the control chow did not contain detectable amounts of phytoestrogen according to informations by the manufacturer (Morga). Developmental studies on rats did not yield evidence for significant effects of phytoestrogen content in normal rodent diets on developmental parameters (21, 22), except for a small difference (12%) in anogenital distance noted in one study. Dietary phytoestrogens in the usual range also did not seem to exert significant influences on the sensitivity of the uterotrophic assay in immature rats (23, 24). This is in line with the low uterine control weights we regularly observed in immature rats on Kliba chow 3430 in uterotrophic assays (1). However, the effects of 4-MBC should be viewed as resulting from actions of a partial ER agonist on the background of endogenous steroidal estrogens and phytoestrogens taken up from food. There, the interaction with endogenous estrogens probably is more important than that with the low exogenous phytoestrogen background.
Uterine weight of adult offspring remained unaffected in most 4-MBC dosage groups, but a marginal increase was noted at 24 mg/kg. The 4-MBC present in the adult offspring would not be expected to affect this parameter because the doses were below that eliciting a uterotrophic response (1). However, the possibility of delayed developmental effects is suggested by observations on diethylstilbestrol (DES) and certain phytoestrogens. In comparatively high perinatal doses, DES and coumestrol induced an initial increase in uterine weight followed by subsequent growth impairment (25, 26, 27). Yet, developmental exposure to lower doses of DES (28) or E2 (29) did not affect uterine weight, indicating that even exposure to potent ER agonists does not necessarily result in changes in uterine weight in adult offspring. In consideration of the ER? preference of 4-MBC (10), it should also be noted that uterine weight appears to be little influenced by ER? agonists at doses that affect estrogen-regulated genes (30).
4-MBC exposure caused changes in the expression of estrogen-regulated genes. The presence of significant effects at the protein level, with maximal changes of 40% of control levels, supports the idea that the alterations in gene regulation may be of functional relevance. These alterations could be a consequence of developmental actions of 4-MBC, of direct effects of the chemical present in the adult offspring, or, possibly, of a combination of both of these. An analysis of the chronic condition is complicated by the cross talk between several of the genes studied (ER/IGF-I, ER/AR, and ER/PR).
ER exhibited a dose-dependent reduction of steady-state mRNA and protein levels that was more clear-cut for ER protein. The parallel change in ER mRNA and protein suggests an interaction of 4-MBC with transcriptional regulation, although additional regulatory effects at the protein level (31) cannot be excluded. In contrast to ER, ER? protein level remained unchanged, even though mRNA showed a slight decrease that was significant across the dose-response curve. Acute administration of ER agonists down-regulates ER and ER? in adult rodent uterus (32, 33), but for ER, this was shown to be followed by a normalization of ER levels upon prolonged estrogen exposure (34). Thus, a developmental action appears to offer a more plausible explanation for the reduced ER mRNA levels. Reduced uterine ER mRNA levels could result from perinatal exposure to an estrogenic chemical, as observed for DES (25), whereas the effect of neonatal exposure on uterine ER? is not quite clear (35).
PRs were also affected by 4-MBC exposure, but in contrast to ER, dose-response curves of PR mRNA and PR-A and PR-B proteins differed. Whereas PR mRNA was down-regulated with a monotonic dose-response curve, PR protein levels were significantly reduced at the lowest 4-MBC dose but returned to normal or slightly supranormal levels (significant positive linear trend) at higher doses. Because this pattern was identical for both PR-A and PR-B, it appears to be reliable. With a significant treatment effect at 0.7 mg/kg 4-MBC, PR-A protein appeared to be one of the most sensitive parameters.
The complex dose-response relationships of PR are not easy to interpret. The decrease in PR protein and mRNA levels at the lowest 4-MBC dose could be due to the reduced sensitivity to estrogenic stimulation observed in the E2 challenge experiments. At higher doses, direct ER agonistic actions of 4-MBC might overlay this effect. Why PR mRNA does not follow the same pattern is not known. Uterine PR can be up- or down-regulated by acute administration of classical ER agonists depending on cell type (36, 37, 38). It cannot be excluded that different uterine tissue compartments contributed differently to mRNA and protein analyses. Atypical acute responses of uterine PR mRNA have been reported for some xenoestrogens (15). Whether the ER? preference of 4-MBC (10) plays a role is difficult to judge because both ER and ER? appear to be involved in up- and down-regulation of PR (37, 38, 39). A possible AR-mediated effect on PR (40) can be excluded because 4-MBC is devoid of androgenic activity (2). To what extent actions of 4-MBC during early development might contribute to the present condition is not known. In the rat fetus, E2 and DES increased PR in mesenchymal cells of the middle part of the Mullerian duct that gives rise to the uterus, whereas DES decreased PR in epithelial cells (41). However, we did not observe a change in uterine PR mRNA levels in adult rat offspring after prenatal DES treatment (S. Durrer, unpublished data). Thus, the 4-MBC data do not appear to match either acute or delayed developmental effects of classical ER agonists.
Exposure to 4-MBC also resulted in reduced AR mRNA levels. Uterine AR are down-regulated by acute administration of estrogens (15, 42), ER, and ER? agonists (30). However, in immature rats, an acute inductive effect has been reported (39). Because data on uterine AR after prolonged developmental exposure to estrogenic compounds are missing, it cannot be decided whether the present AR data would be compatible with a developmental effect or whether an explanation should be sought in a direct action in the adult offspring.
The decrease in IGF-I mRNA at higher 4-MBC doses might be linked with the reduction of ER protein because activation of IGF-I signaling by estrogens appears to be mediated by ER (43). The ER? preference of 4-MBC may contribute to the decrease in IGF-I because ER? appears to exert a repressive role (36). The reason for the increase in mRNA levels at the lower 4-MBC dose is not known. Estrogenic substances, including steroidal estrogens, DES, and xenoestrogens such as genistein and bisphenol A, can elicit an acute induction of IGF-I mRNA in rodent uterus (43, 44), but this induction appears to be transient upon continued exposure to estrogen (45) and, thus, would not explain an increased level upon prolonged exposure to a partial agonist.
The sensitivity of target genes to hormones should provide more insight into possible alterations in gene regulation than steady-state mRNA levels. The E2 challenge experiments in adult offspring revealed significant changes in the acute responsiveness to E2 of four of five genes studied. Down-regulation of ER? (33) did not seem to be affected. Irrespective of whether acute administration of E2 was followed by mRNA up-regulation (PR and IGF-I) or down-regulation (ER and AR), the response to E2 was dose-dependently reduced in 4-MBC-exposed offspring. The levels of these four mRNA species were similar in vehicle-injected ovariectomized animals of untreated control and 4-MBC-exposed groups. This indicates that the mRNA response to E2 started from the same level in untreated and 4-MBC-exposed animals and that the differences in the acute response to E2 are related to 4-MBC exposure. For the two genes up-regulated by E2, PR, and IGF-I, this means that the response to E2 started from low mRNA levels because baseline mRNA levels were significantly reduced in ovariectomized vehicle-injected controls compared with untreated intact controls and, as mentioned above, were similar in vehicle-injected ovariectomized animals of the controls and 4-MBC-exposed groups. This should have allowed for a full-size response to E2.
One possible explanation of reduced sensitivity to E2 could be sought in an antagonistic activity of 4-MBC, which exhibits features of a partial agonist in vivo (1), provided tissue levels were high enough in the adult offspring. However, this idea would be difficult to reconcile with the direction of changes in steady-state levels of ER, AR, and IGF-I mRNA (low dose) and would be in contradiction to observations in the central nervous system of the same animals, where we observed enhanced sensitivity to E2 (46). Observations with DES indicate that developmental exposure to estrogenic chemicals may influence gene expression by alterations in methylation status (47, 48). Although such effects remain a possibility, it should be noted that perinatal DES treatment resulted in a different effect pattern with continuous overexpression of the target genes studied (lactoferrin and c-fos).
Changes in the sensitivity to steroid hormones might also be brought about by changes in the availability of steroid receptor coregulators. We studied the expression of SRC-1, a member of the SRC/p160 family (49), in 4-MBC-exposed offspring. Developmental exposure to 4-MBC reduced the expression of SRC-1 mRNA and protein. SRC-1 protein was significantly decreased in the dose range between 0.7 and 47 mg/kg 4-MBC, which includes the doses causing reduced inductory responses of IGF-I and PR genes to E2. The combination of reduced ER and SRC-1 levels may provide a sufficient explanation for the reduced responsiveness to E2 of the genes that are up-regulated by E2. Deletion of the SRC-1 gene was found to be accompanied by reduced responsiveness of mouse uterus to E2 (50), despite the continued presence of alternative coactivators. Uterine SRC-1 mRNA expression appears to be unresponsive to acute administration of E2 (51). This suggests that the down-regulation by prolonged exposure to 4-MBC may have resulted from a more complex mechanism. In this respect, the preferential binding of 4-MBC to ER? (10) may be of interest because SCR-1 has been reported to be the preferred partner of ER? (14).
In conclusion, our data demonstrate that exposure to the UV filter 4-MBC, an environmental xenoestrogen with preference for ER? during early development and postnatal life, can lead to significant changes in the expression of ERs, nuclear receptor coregulators, and estrogen target genes in uterus at mRNA and protein levels. The minimum effective dose of 4-MBC was 0.7 mg/kg (PR-A and SRC-1 protein). At the dose of 7 mg/kg, 4-MBC concentrations in adipose tissue of adult F1 offspring of the present study (17) were close to levels determined in fish. The 4-MBC-induced changes in steady-state expression levels are subtle, but it should be noted that in real world, the compound contributes to effects of a mixture of endocrine active chemicals. A most interesting observation of the present study is the change in coactivator SRC-1 expression, which was one of the most sensitive parameters. It illustrates that exposure to endocrine active chemicals can alter the regulation of additional proteins of the transactivation complex besides nuclear receptors. The reduction of estrogen sensitivity that may be due to the parallel decrease of SRC-1 and ER represents a potentially relevant regulatory dysfunction.
Acknowledgments
We thank Marianne Conscience and Vreni Haller for most valuable support in the conduction of the long-term animal experiments and Laurent Gelman (Unité CIG Sciences, University of Lausanne, Lausanne, Switzerland) for helpful support in Western-blot analyses.
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