Estrogen sensitivity of target genes and expression of nuclear receptor co-regulators in rat prostate after pre- and postnatal exposure to the ultraviolet filter 4-methylbenzylidene camphor

Abstract

Background and objectives: In previous studies, we found that the ultraviolet filter 4-methyl-benzylidene camphor (4-MBC) exhibits estrogenic activity, is a preferential estrogen receptor (ER)-beta ligand, and interferes with development of female reproductive organs and brain of both sexes in rats. Here, we report effects on male development.

Methods: 4-MBC (0.7, 7, 24, 47 mg/kg/day) was administered in chow to the parent generation before mating, during gestation and lactation, and to offspring until adulthood. mRNA was determined in prostate lobes by real-time reverse transcription-polymerase chain reaction and protein was determined by Western blot analysis.

Results: 4-MBC delayed male puberty, decreased adult prostate weight, and slightly increased testis weight. Androgen receptor (AR), insulin-like growth factor-1 (IGF-1), ER-alpha, and ER-beta expression in prostate were altered at mRNA and protein levels, with stronger effects in dorsolateral than ventral prostate. To assess sensitivity of target genes to estrogens, offspring were castrated on postnatal day 70, injected with 17beta-estradiol (E(2); 10 or 50 microg/kg, sc) or vehicle on postnatal day 84, and sacrificed 6 hr later. Acute repression of AR and IGF-1 mRNAs by E(2), studied in ventral prostate, was reduced by 4-MBC exposure. This was accompanied by reduced co-repressor N-CoR (nuclear receptor co-repressor) protein in ventral and dorsolateral prostate, whereas steroid receptor coactivator-1 (SRC-1) protein levels were unaffected.

Conclusions: Our data indicate that 4-MBC affects development of male reproductive functions and organs, with a lowest observed adverse effect level of 0.7 mg/kg. Nuclear receptor coregulators were revealed as targets for endocrine disruptors, as shown for N-CoR in prostate and SRC-1 in uterus. This may have widespread effects on gene regulation.

Figure 1

Representative Western blots from dorsolateral prostate of untreated and 4-MBC (0.7, 7, 24 mg/kg/day)-exposed 12-week-old rat offspring. Actin = reference protein. Molecular masses of proteins verified by prestained SDS-PAGE standards (Broad Range, Bio-Rad Laboratories).

Figure 2

Levels of mRNAs encoding for insulin-like growth factor-1 (IGF-1), androgen receptor (AR, ER-α , and ER-β in dorsolateral (A) and ventral (B) prostate of untreated adult (12-week-old) rat offspring and offspring exposed to 4-MBC; 0.7, 7, 24, 47 mg/kg/day in chow. Real-time RT-PCR values were normalized to cyclophilin and are expressed as percentage of the mean of the corresponding untreated control (C) [mean ± SE, n = 8, pooled controls A (n = 8) + B (n = 8)]. Different from corresponding control A (for 7, 24, 47 mg/kg) or B (for 0.7 mg/kg), *p < 0.05, **p < 0.01.

Figure 3

AR, ER-β and ER-α protein levels in dorsolateral prostate (DP) and ventral prostate (VP) of untreated and 4-MBC (0.7, 7, 24 mg/kg/day)-exposed 12-week-old rat offspring. Proteins analyzed by Western blot in the same homogenates as used for mRNA determination, quantitated by densitometry relative to actin, expressed as percentage of the mean of the corresponding control (mean ± SE, n = 7–9). ER-α protein was not detectable in ventral lobe homogenate. Different from control: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

(A ) Insulin-like growth factor-1 (IGF-1) mRNA 6 hr after sc injection of E₂ [10 μg/kg (10 E₂) or 50 μg/kg (50 E₂)] or vehicle in ventral prostate of castrated 12-week-old rat offspring from control and 4-MBC (0.7, 7, 24 mg/kg/day)-exposed groups. Values normalized to cyclophilin, as percentage of the corresponding vehicle-injected group (mean ± SE, n = 8). (B) Magnitude of down-regulation of IGF-1 mRNA by E₂ in ventral prostate of castrated adult rat offspring of control and 4-MBC (0.7, 7, 24 mg/kg/day)-exposed groups. Values normalized to cyclophilin, as percentage of the corresponding vehicle-injected group [mean ± SE, n = 8, control = pooled control groups A (n = 8) + B (n = 8)]. Separate statistics for 0.7-mg/kg group vs. control B, and 7- and 24-mg/kg groups vs. control A. Different from corresponding vehicle-injected group: ^ap < 0.05, ^aaap < 0.001. Magnitude of E₂ effect different from control: ^b< 0.05, ^bbp < 0.01.

Figure 5

Magnitude of acute suppression of mRNAs encoding for AR (A), ER-α (B) and ER-β (C) in ventral prostate of castrated adult rat offspring from control (C) and 4-MBC (0.7, 7, 24 mg/kg/day)-exposed groups 6 hr after sc injection of E₂ [10 μg/kg (10 E₂) or 50 μg/kg (50 E₂)]. Values normalized to cyclophilin, as percentage of the corresponding vehicle-injected group [mean ± SE, n = 8; control = pooled control groups A (n = 8) + B (n = 8)]. Separate statistical analysis for 0.7-mg/kg group vs. control B, and 7- and 24-mg/kg groups vs. control A: ^aSignificant suppression of mRNA by E₂ compared with vehicle, p < 0.05. ^bMagnitude of suppression by E₂ different from untreated control, p < 0.05.

Figure 6

Steroid receptor coactivator-1 (SRC-1) and nuclear receptor corepressor (N-CoR) protein levels (Western-blot) in dorsolateral (dorsal + lateral) prostate (DP) and ventral prostate (VP) of untreated and 4-MBC (0.7, 7, 24 mg/kg/day)-exposed 12-week-old rat offspring. Proteins analyzed in the same homogenates as used for mRNA determination, quantitated by densitometry relative to actin, expressed as percentage of the mean of the corresponding control (mean ± SE, n = 7–9). Different from control: *p < 0.05, ***p < 0.001.