Comparisons of Brain, Uterus, and Liver mRNA Expression for Cytochrome P450s, DNA Methyltransferase-1, and Catechol-O-Methyltransferase in P
http://www.100md.com
《毒物学科学杂志》
Health Canada, Healthy Environments and Consumer Safety Branch, Environmental & Occupational Toxicology Division, Ottawa, Ontario, Canada K1A 0L2
Reproductive Biology Unit and Division of Gynaecologic Oncology, Departments of Obstetrics & Gyneacology and Cellular & Molecular Medicine, University of Ottawa
Hormones, Growth and Development Program, Ottawa Health Research Institute, Ottawa, Ontario, Canada K1Y 4E9
ABSTRACT
Non-ortho polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) are ubiquitous environmental contaminants that exert their toxicity mostly through activation of the aryl-hydrocarbon receptor (AhR), and are referred to as AhR agonists. The objective was to study, by real time reverse-transcriptase–polymerase chain reaction (RT-PCR), the effects of postnatal exposure to a reconstituted mixture of AhR agonists present in breast milk (3 non-ortho PCBs, 6 PCDDs, and 7 PCDFs, referred to hereinafter as AhRM) on mRNA expression of estrogen receptor (ER), enzymes involved with the metabolism of estrogens [catechol-o-methyltransferase (Comt), cytochrome P450 (Cyp)1A1, 1B1 and 2B1], and DNA methyltransferase-1 (Dnmt1), in brain areas, liver and uterus of immature female rats. Neonates were exposed by gavage during postnatal day (PND) 1–20 with dosages equivalent to 1, 10, 100, and 1000 times the estimated average human exposure level, and were sacrificed at PND 21. None of the end points were affected in uterine cross-sections, or in samples of uterine tissue layers collected by laser capture microdissection. At 1000x, the AhRM reduced Dnmt1 mRNA abundance to 28% and 32% of control in the liver and hypothalamus, respectively. In the brain, Cyp1A1 was increased (409%) but ER was reduced (66%). Similarly, mRNA abundance for Comt isoforms was reduced in the liver (45%) and brain areas (55–70%). AhRM at 100x, the lowest effective dose, exerted a 220% increase in brain cortex Comt [membrane bound (Mb)], a 219% increase in hepatic Cyp1B1, and a 63% decrease in hepatic Comt (soluble (S)+Mb). These results support the possibility that early exposure to environmental contaminants could lead to effects mediated by changes in DNA methylation and/or estrogen metabolism and signaling.
Key Words: polychlorinated dibenzodioxins; polychlorinated dibenzofurans; rat; uterus; DNA methyltransferase; catechol-o-methyltransferase.
INTRODUCTION
Organochlorines, such as non-ortho polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) are ubiquitous environmental contaminants present in human tissues. Most of their toxic effects are exerted through the activation of the aryl-hydrocarbon receptor (AhR) (Safe, 1990), and they are referred to as AhR agonists. Usually humans receive their highest level of exposure during the in utero and postnatal periods, particularly breast-fed infants (Feeley and Brouwer, 2000; Solomon and Weiss, 2002). To improve risk assessment of exposure to these chemicals, additional information is required to understand the relative contributions of prenatal and postnatal exposure to the cumulative health risk derived from these two exposure periods.
Exposure to AhR agonists has been associated with long-term adverse health consequences, including an increased risk of cancer development, reproductive problems, learning difficulties, and immune, and endocrine system deficiencies. Among endocrine effects, AhR agonists act as antiestrogens, not by competing for the ligand domain of the estrogen receptor (ER), but by downregulating ER, increasing the metabolism of estradiol-17, inhibiting various growth factors and crosstalk between AhR and ER pathways (Safe, 1999), and through proteosomal degradation of the receptor (Wormke et al., 2003). In contrast, estrogen-like effects are induced by activated AhR interacting with the unliganded ER (Ohtake et al., 2003). Regulatory agencies suggest using the uterotrophic response in immature rats to detect chemicals with estrogenic or antiestrogenic properties (Kanno et al., 2001; 2003a; O'Connor et al., 2002; Owens et al., 2003; Owens and Koeter, 2003). However, there are concerns about the ability of a uterotrophic assay to detect antiestrogenic effects of 2,3,7,8-tetrachlorodibenzodioxin (TCDD) at PND 21 (Desaulniers et al., 2003; White et al., 1995), but not in slightly older rats (Romkes et al., 1987; Safe et al., 1991).
Inductions of hepatic CYP1A1 and "extrahepatic" CYP1B1, which metabolize estradiol-17 into toxic 2-OH- and 4-OH-catecholestrogens (CE), are classical effects of AhR activation (Badawi et al., 2000; Hayes et al., 1996; Jefcoate et al., 2000). Toxic CE, mostly 4-OH-CE, are implicated in estrogen-mediated carcinogenesis (Cavalieri et al., 2000), raising the possibility that inappropriate levels of CE may increase health risks. Catecholestrogens are rapidly methylated, thus detoxified, by the enzyme catechol-o-methyltransferase (COMT). In addition, 2-OH-estradiol is converted to 2-methoxyestradiol (Zacharia et al., 2004), a compound that has numerous beneficial effects on cardiovascular and renal functions (Dubey et al., 2004), on obesity (Tofovic et al., 2001), and in many cancer cells in vivo and in vitro (Seeger et al., 2004). Along with its predominant regulatory role in the reproductive organs, estradiol-17 is an important modulator of brain development (Ivanova and Beyer, 2003; Weiss, 2002), a neuroprotective factor, and a stimulator of ontogenesis in the central nervous system (Beyer et al., 2003). Despite the limited production of ovarian estradiol-17 during the postnatal period, the expression of aromatase, ER, and ER in neonatal brains peaks during the first 2 weeks after birth, and it reaches higher levels in males than in females (Ivanova and Beyer, 2000; Raab et al., 1999). These events also coincide with the highest levels of tyrosine hydroxylase (a rate-limiting enzyme of the catecholamine biosynthetic pathway) during the early postnatal period, suggesting estrogen control of neuronal dopamine synthesis and function (Hersey et al., 1982; Ivanova and Beyer, 2003). Catecholamine neurotransmitters are deactivated by methylation carried out by the same COMT involved in CE detoxification (Zhu, 2002). Exposure to environmental contaminants, particularly during the prenatal period, has been associated with long-term deleterious effects on brain function (Andersen et al., 2000; Schantz et al., 2003). The present study was devoted to exploration of possible effects of early exposure to environmental contaminants on the expression of enzymes involved with estrogen metabolism and signaling and the COMT system in the developing brain.
Changes in DNA methylation may lead to chromosome instability, X chromosome inactivation, modifications in centromere structure, as well as changes in genomic imprinting, gene expression, and tissue differentiation (Reik et al., 2001; Wolffe and Matzke, 1999). Only limited information is available on the effects of various toxicants (phenobarbital; trichloroethylene, di- and trichloroacetic acid, methoxyacetic acid, arsenite, cadmium, nickel, chromium, radiation, TCDD) on DNA methylation (Bombail et al., 2004; Carnell and Goodman, 2003; Moggs and Orphanides, 2004; Pogribny et al., 2004; Ray and Swanson, 2004; Wu et al., 2004). Abnormal DNA methylation has been associated with a number of cancers (Gaudet et al., 2003), with infertility (Cisneros, 2004), and with developmental (Egger et al., 2004), neurological (Costa et al., 2004), immunological (Yasunaga et al., 2004), and age-related (Oakes et al., 2003) disorders. It may originate during early development (Wu et al., 2004) or later during mitosis, when the methylation pattern of the original DNA strand is copied mostly by DNA methyltransferase-1 (DNMT1) onto the replicating DNA. DNA methylation reactions are catalyzed by families of DNA methyltransferases (DNMT-1, –2, –3). Three isoforms of DNMT1 have been described: one specific for somatic cells (DNMT1s), one affecting pachytene spermatocytes (DNMT1p), and a type that influences oocyte and preimplantation embryos (DNMT1o) (Ko et al., 2005). The role of DNMT2 remains obscure. The de novo methyltransferases, DNMT3a, 3b, and 3L, which catalyze the transfer of a methyl group to previously unmethylated DNA, are expressed during gametogenesis and embryogenesis (Bachman et al., 2003; Kaneda et al., 2004; Margot et al., 2003; Rhee et al., 2002). We are not aware of any reports describing the effects of exposure to AhR agonists on the expression of DNA methyltransferases.
We have previously examined the effects of postnatal exposure to reconstituted mixtures of the most abundant (19 PCBs, DDT, DDE) (Desaulniers et al., 2001; 2005), and "most toxic" (based on growth, and on liver and thyroid effects) breast milk contaminants [a mixture of AhR agonists: 6 PCDDs, 7 PCDFs, and 3 non-ortho PCBs (Desaulniers et al., 2003; 2004)] on organ weights, endocrine parameters, liver function, and mammary tumor development. To better understand tissue differences in dose–response effects of the mixture of AhR agonists, we here extend previous information by comparing mRNA abundance for Cyp1A1, 1B1, and 2B1, Comt, ER, and Dnmt1 in the liver, uterus, and brain in female rats at PND 21. Cyp1A1 and Cyp1B1 expression was assessed in all tissues as an indicator of "functional" AhR-mediated effects. Cyp1A1 and 1B1 mRNA abundance was not affected in complete uterine tissue samples, but because the uterus is composed of different tissue layers (perimetrium, myometrium, endometrium, and epithelium), the possibility was tested that effects could be occurring in specific layers. Consequently, a subjective assessment of ER and CYP1B1 protein abundance in the uterus was performed by immunohistochemistry (IHC), and these observations were corroborated by a quantitative assessment of the abundance of their mRNAs in uterine perimetrium, myometrium, endometrium, and epithelium, using samples collected by laser capture microdissection (LCM).
MATERIALS AND METHODS
Animal treatment.
A mixture of 3 PCBs, 6 PCDDs, and 7 PCDFs (referred as AhRM) in corn oil was prepared based on individual concentrations of each chemical found in one study of human breast milk (Table 1). Female Sprague-Dawley neonatal rats (10–13 per group) were gavaged at PND 1, 5, 10, 15, and 20, with either corn oil, or AhRM at a cumulative dose equivalent to 1, 10, 100, and 1000 times the estimated amount consumed by an infant over the first 24 days of life. Prepubertal rats were sacrificed at PND 21, a time at which rats are often euthanized when performing uterotrophic bioassays (Kanno et al., 2003b; Li and Hansen, 1997; White et al., 1995).
Tissue processing.
Brains were dissected out of the cranium and placed on dry ice until completely frozen to maintain structural integrity before storage. Sections of liver and uterus were snap frozen in liquid nitrogen immediately after dissection. All tissues were kept at –80°C until analyzed. The anterior half of a uterine horn was transferred into 10% neutral buffered formalin, held for 24 h, processed, and embedded in paraffin for immunohistochemical analysis.
Dissection of the different brain regions was performed on dry ice. The frontal portion of the brain anterior to the optic chiasm was discarded. Working backward, a 2-mm-thick cross section that contained the hippocampus (HC), the hypothalamic area (HT), and the cortex (C), was used to generate data identified as "Total brain tissue" (TBT) (Tables 2 and 3); subsequent sections were used to dissect tissues from individual brain areas.
Immunohistochemistry of the uterus.
Paraffin-embedded sections of uteri (5 μm) were mounted onto positively charged slides, deparaffinized in xylene and absolute ethanol, then rehydrated by successive immersions in 95% ethanol, 70% ethanol, and distilled water. After rehydration, the sections were microwaved at high power for 1 min, then at medium power for 14 min in citrate buffer (0.01 M, pH 6.0). All sections were washed (3 x 5 min in PBS), and immersed (RT; 10 min) in PBS with 1% (v/v) H2O2 to inhibit endogenous peroxidase activity. After a course of three additional PBS washes (5 min each), the sections were incubated (RT; 60 min) with 5% milk powder in PBS to minimize nonspecific binding and then kept at 4°C overnight with the primary antibodies [ER: rabbit polyclonal IgG (1:400; Santa Cruz Biotechnology, Santa Cruz, CA); CYP1B1: rabbit anti-human CPY1B1 (1:100, Alpha Diagnostic Intl. Inc, San Antonio, TX)]. The sections were incubated with biotinylated second antibody (RT, 30 min), washed with PBS, and then incubated with streptavidin-peroxidase (RT, 30 min). After washing, the specimens were immersed (RT, 10 min) in chromagen solution (Dako Diagnostics Canada Inc., Mississauga, ON), counterstained with hematoxylin, dehydrated in a series of alcohols and xylene, and covered with glass slips.
RNA isolation from whole uteri and brain areas.
Total RNA was isolated from liver and whole uteri, using the Mini RNeasy Kit (Qiagen, Valencia, CA). The RNeasy Lipid Tissue Mini Kit was used for RNA isolation from brain areas, according to the manufacturer's protocols (Qiagen). Contaminating genomic DNA was removed by DNase digestion using the RNase-Free DNase Kit (Qiagen). Quantification of total RNA from whole uteri was analyzed using RiboGreen RNA Quantitation Reagent Kit (Invitrogen Canada Inc., Burlington, ON), and the RNA from liver and brain area samples were measured with a spectrometer at 260 nm. RNA quality was assessed by RNA gel electrophoresis.
RNA isolation from LCM samples.
Uterine cryosections (10 μm) were prepared from frozen tissues embedded in Tissue-Tek O.C.T (Sakura Finetek Inc, Torrance, CA). Cryosections were mounted onto coated glass slides, fixed (1 min, 70% ethanol), stained for visualization of the different tissue layers, and dehydrated, using the protocol, slides, and reagents supplied with the HistoGene Frozen Section Staining Kit (Arcturus Bioscience Inc, Mountain View, CA). Cells from the uterine epithelium, endometrium, myometrium, and perimetrium were collected with a PixCell II Laser Capture Microdissection system (beam settings: 15 μm, 40–45 mW; Arcturus Bioscience Inc.). To verify the quality of the RNA after laser capture microdissection (LCM), samples of RNA were prepared from 200–400 uterine cells (one laser pulse equals approximately one cell), and the presence of a 2:1 ratio of 28S/18S RNA was verified from microcapillary electrophoresis analysis (RNA 6000 LabChips, Agilent 2100 Bioanalyzer; Agilent Technologies, Wilmington, DE). Other analyses were from LCM samples of 400–2000 pulses. The LCM samples were incubated (RT, 30 min) with 300 μl of lysis buffer. Total RNA was extracted with the RNeasy micro kit (Qiagen), and contaminating genomic DNA was removed by on-column DNase digestion during RNA purification, using the RNase-free DNase set (Qiagen).
Reverse transcription (RT).
Total RNA was reverse transcribed by random primer and MMLV reverse-transcriptase according to the manufacturer's protocol (Sensiscript RT kit; Qiagen). Non-RT control samples were prepared by omitting the reverse-transcriptase from the reaction mixture.
Primers for polymerase chain reaction (PCR).
The respective forward and reverse sequences of the primers, designed using Beacon Designer 2.0 (BioRad, Mississauga, ON) according to published rat gene sequences [ER (Spreafico et al., 1992); www.ncbi.nlm.nih.gov with the following loci NM_012540 [GenBank] (Cyp1A1), RNU09540(Cyp1B1), RATCYP450-J00719 (Cyp2B1), AF116344 [GenBank] (Dnmt1), NM_012531 [GenBank] (Comts), and NM_031144 [GenBank] (-actin)] are as follows: ER (5'-GCG-CCG-CCT-ACG-AGT-TTC-A-3' and 5'-GAC-CGT-AAG-TGA-TGC-TCG-ACT-G-3'), Cyp1A1 (5'-CTT-CAC-ACT-TAT-CGC-TAA-TGG-3' and 5'TTG-GGT-CTG-AGG-CTA-TGG-3'), Cyp1B1 (5'-GAG-TTG-GTG-GCA-GTG-TTG-3' and 5'-GCA-TCG-TCG-TGG-TTG-TAC-3'), Cyp2B1 (5'-TGA-TCT-TTG-CCA-ATG-GGG-AAC-3' and 5'-CCG-TTC-TTC-CAC-ACT-CCT-CT-3'), Dnmt1 (5'-AAC-GGA-ACA-CTC-TCT-CTC-ACT-CA-3' and 5'-TCA-CTG-TCC-GAC-TTG-CTC-CTC-3'), Comt [soluble form + membrane-bound form (S+Mb)] (5'-GCC-AGG-CTT-CTC-ACC-ATG-3' and 5'-CGT-CGT-ACT-TCT-TCT-TCA-GCT-3'), Comt (Mb) (5'-CTC-ATT-GGG-TCT-CCT-GTT-GTT-G-3' and 5'-AGG-TTG-TGG-ACT-GGC-TGC-3'), and -actin (5'-TCG-GCA-ATG-AGC-GGT-TCC-3' and 5'-CAG-CAC-TGT-GTT-GGC-ATA-GAG-3').
As indicated above, two sets of primers were designed to determine the abundance of the enzyme COMT, which exists as a soluble and a membrane-bound isoform, the latter possessing an extended NH2 terminal end for anchoring the enzyme to the membrane. Both forms originate from the same gene, which expresses two mRNA species with different tissue distributions. The expression of the transcripts is regulated by at least two promoters (Tenhunen et al., 1993). Here, a first set of primers was designed from the carboxy terminal region to amplify both the soluble and membrane-bound forms, Comt (S+Mb). The second set of primers is specific to the NH2 terminal portion to provide a measurement of only the membrane-bound form, Comt (Mb).
Quantitative real-time PCR analysis.
The PCR reaction volume (25 μl) contained 4 μl of RT products, 0.5 unit HotStarTaq DNA polymerase (Qiagen) in 1x QuantiTect SYBR Green Master Mixture, and 0.5 μM each of the forward and reverse primers. The PCR amplification cycles included denaturation (95°C, 15 min; to activate the HotStarTaq DNA polymerase and to minimize primer-dimers contribution), and amplifications [over 40–50 cycles including denaturation (94°C; 30 s), annealing (55°C; 30 s), and extension (72°C; 1 min)]. All PCR reactions were performed in duplicate. Non-RT control and negative control samples (without template) were processed in the same manner. The specificity of the amplifications was verified by melting curve analysis for all samples, and occasionally by agarose gel electrophoresis. Amplifications of target gene sequences were compared against serial dilutions of known quantities of their purified cDNA fragments, and normalized to the abundance of the house-keeping gene -actin. The amount of total RNA from the LCM samples was too low to be quantified spectrometrically, and specific gene mRNA abundance was thus reported as a ratio to -actin mRNA within the same sample.
Statistical analysis.
All analyses were performed using JMP software (SAS Institute Inc, 1998). The normality of the data was verified by goodness-of-fit Shapiro-Wilk W tests. Data that failed this test were log transformed and the normality was retested. If normality of the transformed data failed, it was analyzed using the nonparametric Mann and Whitney U-test (comparison of two groups) or the Kruskal-Wallis rank sum test (comparison of three or more groups). Brain data were analyzed by two-way analysis of variance (ANOVA) for treatment effect, tissue area effect, and their interactions. Two-way ANOVA was also used to test differences in the expression of ER, or Cyp1B1 mRNA between age groups and uterine tissue layers, or treatment and uterine tissue layers. Respective interactions were also assessed. Differences between group means were further tested by Tukey honestly significant difference (HSD) tests. Values were considered to be significantly different when p 0.05.
RESULTS
The most abundant brain mRNA was Comt (S+Mb), and the least abundant was Cyp1A1 (Table 2). Compared to respective controls, the 1000x dose reduced Comt (S+Mb) mRNA abundance in TBT (70%), HC (55%), and C (63%). Cyp1A1 mRNA abundance was significantly increased in all brain tissues: TBT (345%), HT (409%), HC (340%), C (250%). ER mRNA was more abundant in HT than in C or HC, and 1000x reduced ER mRNA abundance in all brain areas [TBT (51%), HT (37%), HC (34%)], except in the cortex (66%) where the difference did not reach statistical significance. Dnmt1 mRNA abundance was similar in all brain regions, and although 1000x reduced it in all regions, statistical significance was reached only in HT (32%). The 1000x dosage had no effects on mRNA abundance for Comt (Mb), Cyp1B1, and Cyp2B1.
The comparison of brain mRNA abundance between the control and the 100x groups was achieved in a second analysis, presented in Table 3. Interestingly, increases in Comt (Mb) were detected in all brain areas, although statistical significance was observed only in C (220%). The 100x dose induced slight but nonsignificant increases in Cyp1A1 and had no effect on ER mRNA. The higher, but not statistically different, abundance of ER mRNA in HT compared with other brain areas (Table 3), confirms the similar observation in Table 2. AhRM at 100x had no effect on Comt (S+Mb) and Dnmt1 mRNA abundance. Cyp1B1 and 2B1, unaffected by the 1000x dose, were not tested at 100x. Given the absence of important effects induced by the 100x dose, samples from lower dose groups were not analyzed.
In the liver, AhRM treatments reduced the abundance of mRNA for Comt (S+Mb) [100x (63%), 1000x (45%)], whereas that of Comt (Mb) was not affected (Table 4). Although mRNAs for Comt isoforms are more abundant in liver than in brain, the proportion of Comt (Mb) relative to that of Comt (S+Mb) is usually similar in both tissues [liver (1.3%), TBT (1.6%), HT (1%), HC (0.7%), C (1.7%)]. The higher abundance of Comt (S+Mb) compared to Comt (Mb) mRNA suggests that most of the Comt are of the soluble (S) form in both tissues. While the AhRM treatments induced a dose–response increase in Cyp1B1 mRNA abundance [100x (219%), 1000x (475%)], the mRNA of Dnmt1 was reduced [1000x (28%)]. Given the absence of effects induced by the 10x dose, samples from the 1x dose groups were not analyzed.
In contrast to its effect on brain and liver, the 1000x dose had no effects on the end points measured in the uterine tissues. Briefly, the abundance of mRNA attoM/femtoM -actin in control (n = 6) and 1000x (n = 7) uterine samples were 0.15 ± 0.05 and 0.19 ± 0.03 of Cyp1A1, and 0.5 ± 0.1 and 0.7 ± 0.1 of Cyp1B1. Visualization of the CYP1B1 and ER proteins in individual uterine layers by immunohistochemistry (IHC) revealed that they were coexpressed in the perimetrium and the endometrium, with low signals in the myometrium and luminal epithelium (Fig. 1A and 1C). For this analysis, Cyp1A1 was not considered given that it was the least abundant mRNA. In contrast to young female rats at PND 21, adult rat ER was found in the luminal and glandular epithelium (Fig. 1B). Immunohistochemistry is subjective and does not easily permit the assessment of treatment effects. Thus, a laser capture microdissection technique was initially developed to compare ER mRNA abundance between PND 21 uterine tissue and adult uterine tissue (Fig. 2A). ER mRNA was significantly more abundant in the luminal epithelium of adult rats than in PND 21 females [two-way ANOVA (tissue layers, p < 0.0001; tissue layer x age interaction, p = 0.001); Tukey HSD test (p < 0.05)]. Uterine glands were rarely visible at PND 21, and thus ER mRNA abundance was analyzed only in glandular epithelium of adult rats. In accordance with IHC observations of ER (Fig. 1A), mRNA abundance was significantly higher in the perimetrium than in the endometrium (p < 0.05, Fig. 2A). The endometrium had significantly more ER mRNA than the luminal epithelium or the myometrium (p < 0.05). Cyp1B1 mRNA was significantly more abundant in the perimetrium than in the endometrium (p < 0.05, Fig. 2B). No difference in Cyp1B1 mRNA abundance was observed between the myometrium, endometrium, and luminal epithelium. The 1000x dose had no effects on the abundance of ER and Cyp1B1 mRNAs in the different uterine tissue layers.
DISCUSSION
The lowest effective dose of AhRM was equivalent to 100x the estimated average human exposure level during the first 24 days of life. This dose affected both the liver and the brain, but based on the magnitude of changes, the liver might be considered one of the most sensitive organs. The 100x dose increased mRNA expression of hepatic Cyp1B1 (Table 4) and Cyp1A1 [as well as activity (Desaulniers et al., 2003)], and it decreased that of Comt (S+Mb), suggesting that the COMT enzyme system might be as sensitive as that of CYPs. However, the magnitude of the changes was much greater for CYP1A1 [657% induction by 100x (Desaulniers et al., 2003)]. The rat uterus at PND 21 is not a sensitive organ, as indicated by the apparent lack of effects of AhRM on mRNA abundance (ER, Cyp1A1, Cyp1B1), on protein expressions (ER, CYP1B1) (current results), and on uterine growth (Desaulniers et al., 2003). Uneven distribution of the AhR agonists among tissues could contribute to tissue differences in the magnitude of the responses. Other investigators compared the tissue distribution of radioactive coplanar and non-coplanar ortho-PCBs in weanling female rats at PND 21, and revealed that coplanar PCBs accumulate in the liver but not in the brain, whereas non-coplanar PCBs do accumulate in the brain (Saghir et al., 1999; 2000). The congeners of PCBs, PCDDs, and PCDFs used in the current studies have coplanar structures and might have accumulated preferentially in the liver.
In addition to appearing less sensitive, the brain response was different from that of the liver. Whereas in the liver a dose–response decrease in Comt (S+Mb) with no effects on Comt (Mb) was observed, in the brain the Comt (Mb) mRNA level was increased by AhRM 100x, particularly in the cortex, but not at the 1000x dose (Table 3). Reminiscent of hormesis effects (Calabrese and Baldwin, 2003), the biological significance of this discrepancy should be investigated further. The COMT system transforms toxic CE into non-toxic methoxyestrogens; it converts L-3,4-dihydroxyphenylalanine (L-DOPA; the precursor of the catecholamine neurotransmitters), into 3-methoxytyrosine, and it deactivates dopamine, epinephrine, and norepinephrine into homovanillic acid, metanephrine, and normetanephrine, respectively (Goldstein et al., 2003). The COMT system constitutes an important part of the enzymatic "blood–brain barrier" against peripherally produced catecholamines (Goldstein et al., 2003). An increase in brain Comt (Mb) mRNA at a 100x dose is suggestive of an adaptive response. Decreased activity of COMT in the periphery (as suggested by the reduced hepatic Comt (S+Mb) abundance), might be associated with an increased level of catecholamines reaching the brain, so that the latter compensates by increasing COMT production. A decrease in hepatic COMT activity is further supported by the observation that Aroclor 1254, a commercial mixture of PCBs, decreased methylation of CE, presumably as a result of inhibition of COMT activities by catechol metabolites of PCBs (Garner et al., 2000). The mRNA results (Tables 2–4) provide an additional mechanism supporting an in vivo reduction in COMT activity.
Based on the current results showing mRNA abundance from the brain and liver, and the previously increased hepatic mRNA, protein, and activity of CYP1A1 (Desaulniers et al., 2003), it is possible that AhRM increased the activity of both CYP1A1 and 1B1 and that it raised the production of toxic CE. The latter, usually labile, might have persisted longer in brain and liver given the reduction in COMT. These changes are believed to result in oxidative stress and estrogen-induced mutagenic events (Cavalieri et al., 2000; Li et al., 2004). The AhR agonists present in the mixture are known hepatocarcinogens that affect female rats to a greater extent than their male counterparts (Mayes et al., 1998). The current findings are consistent with the previous suggestion that the higher hepatocarcinogenic sensitivity in the female is associated with chronic estrogen metabolism–dependent oxidative imbalance (Wyde et al., 2001). DNA methylation affects chromosome stability and is associated with oncogenesis (Eden et al., 2003). Thus, along with changes in COMT and CYPs, the AhRM-induced reduction in Dnmt1 mRNA abundance in the liver (and perhaps changes in DNA methylation) might promote hepatic carcinogenesis and may possibly serve as an early indicator of this process.
The observed changes in mRNA abundance for the current set of genes might suggest adverse effects on brain development. AhRM reduced Dnmt1 mRNA levels, and might be associated with abnormal DNA methylation. Changes in DNA methylation can affect tissue differentiation, nervous system development, and intellectual disorders (Costa et al., 2004; Egger et al., 2004). The mechanism by which AhR agonists could alter Dnmt1 expression is unknown. However, an involvement of TCDD and the transcription factor Sp1 was suggested (Wu et al., 2004) given that Sp1 is a regulator of Dnmt1 expression (Kishikawa et al., 2003), and transplacental exposure to TCDD affected the temporal sequence in Sp1 expression in the brain (Nayyar et al., 2002). Changes in the expression of estrogen-metabolizing enzymes in the brain may influence neural development and function because CE have been reported to be neurotoxic (Brawer et al., 1993). COMT is a therapeutic target in the pathogenesis of various diseases, including neurodegenerative (Zhu, 2002). In agreement with the results of other studies (Pinzone et al., 2004), ER mRNA was particularly abundant in the hypothalamic area. AhRM decreased ER mRNA abundance in most brain areas, and that reduction might decrease estrogen signaling. Locally produced estradiol-17 acts as a stimulator of ontogenic processes in the central nervous system (Beyer et al., 2003), and ER, ER, and aromatase expression peaks during the first 2 weeks after birth (Ivanova and Beyer, 2000; Raab et al., 1999). Later, ER also acts as a neuroprotective factor (Kajta and Beyer, 2003). Whether AhRM alters gene responses to developmental cues in the young brains and induces long-term adverse effects remains to be investigated.
The current observations support the concept that CYP1B1 is the major extrahepatic CYP1 family member (Murray et al., 2001), with a higher abundance of Cyp1B1 mRNA than Cyp1A1 in the brain and the uterus, but lower levels in the liver. However, while AhRM induced the expression of both enzymes in the liver, only Cyp1A1 was increased in the brain. This finding is in agreement with the notion that a highly inducible, functional CYP1A1 is present in the brain (Granberg et al., 2003). In contrast, CYP1B1, but not CYP1A1, can be induced in the mammary gland by AhR agonists (Christou et al., 1995). Although these CYPs are important inducible detoxification enzymes, they may have other biological functions (e.g., embryo activation [Paria et al., 1998]), which may be tissue-specific. Finally, even though CYP2B1 is one of the most abundant CYP in the brain, and although it is induced by phenobarbital in the cortex (Upadhya et al., 2002), CYP2B1 brain expression was not significantly affected by AhRM, suggesting that it is not a sensitive target in this organ.
AhRM did not change uterine mRNA levels, and it had no effects on basal or 17-ethynylestradiol-stimulated uterine growth, despite its clear effects on other tissues ([Desaulniers et al., 2003], and the present study). This suggests that the rat uterus at PND 21 is relatively insensitive to AhR agonists. The possibility of tissue layer specific effects undetected when analyzing the entire uterus was tested, but this did not reveal any changes. When comparing IHC observations with mRNA expression in the different uterine tissue layers (using LCM and real-time RT-PCR), we observed that ER and Cyp1B1 mRNA and proteins were coexpressed in the immature rat uterus, and their abundance differed in the various uterine tissue layers; for example, mRNA expression was highest in the vascularized perimetrium and lowest in the luminal epithelium (Figs. 1 and 2). The constitutive expression of CYP1B1 observed by IHC and mRNA analysis supports a role of CYP1B1 in uterine physiology (Paria et al., 1998). The expression of ER mRNA shows a developmental difference between 21-day-old and adult female rats, with the presence of glandular development containing ER-positive cells in the adult only, and higher levels of luminal epithelial ER expression in the adults. These age differences do not prevent the ER system from being inducible at PND 21, as demonstrated by the uterotrophic effect of 17-ethynylestradiol (Desaulniers et al., 2003). In contrast, the absence of effects of AhRM on uterine Cyp1A1 and 1B1 suggests that the AhR system may not be functional at PND 21. It can be argued that the dosage of AhRM was not sufficient to induce effects in the uterus, but White et al. (1995) also failed to demonstrate antiestrogenic effects of TCDD at PND 21 at a dose of 80 μg/kg, suggesting that the dose of AhRM was not likely a factor. AhR agonists have been shown in the uteri of slightly older rats to have antiestrogenic activities attributable to crosstalk between ER and AhR signaling (Safe, 1999). Pharmacodynamic differences might exist depending on age variations. The current findings, together with our previous results (Desaulniers et al., 2003), suggest that antiestrogenic effects of AhRM is tissue- and age- specific. It is of interest to note that at this prepubertal age the uterus has been exposed to limited levels of endogenous estrogen (Li and Hansen, 1997) and, as in the case of breast cancer cells (Spink et al., 2003), might require pre-exposure to estrogens for the induction of CYP1A1 and 1B1.
CONCLUSIONS
To our knowledge, the findings presented here may be the first demonstration of Comt and Dnmt1 mRNAs as targets for adverse effects of exposure to AhR agonists. Perinatal changes in COMT (alone or in combination with a decrease in ER-signaling) and DNMT systems could have important carcinogenic and developmental effects, and thus might be important complementary indicators of toxicity. These results support our previous findings (Desaulniers et al., 2003) that dosages of AhRM (non-ortho PCBs, PCDDs, PCDFs) equivalent to 100 times the average human exposure level over the first 24 days of life are required to induce statistically significant changes in female rats. This dose of AhRM altered the mRNA expression for Comt, Cyp1A1, and Cyp1B1 in liver and brain cortex, but not in the uterus. The liver is one of the most sensitive organs in which to measure indicators of these effects. Tissue-specific changes in the abundance of these mRNAs substantiate the notion that abnormal metabolism of estrogens and DNA methylation could be important consequences of exposure to AhR agonists.
ACKNOWLEDGMENTS
The authors are grateful to L. Casavant, Dr. M. Charbonneau, J. Cole, Dr. G. Douglas, A. Lee, Y. Li, T. Odorozzi, K. Pazterko, L. Shao, Dr. K. Soumano, Dr. R. Vincent, and G. Zu for their technical advice and assistance, and to Drs. Guillaume Pelletier and Raymond Poon for suggested improvements to the manuscript. This study was funded by Health Canada, Toxic Substances Research Initiative (TSRI) grant no. 045 and Northern Contaminants Program (NCP) grant no. H16-2004.
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Reproductive Biology Unit and Division of Gynaecologic Oncology, Departments of Obstetrics & Gyneacology and Cellular & Molecular Medicine, University of Ottawa
Hormones, Growth and Development Program, Ottawa Health Research Institute, Ottawa, Ontario, Canada K1Y 4E9
ABSTRACT
Non-ortho polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) are ubiquitous environmental contaminants that exert their toxicity mostly through activation of the aryl-hydrocarbon receptor (AhR), and are referred to as AhR agonists. The objective was to study, by real time reverse-transcriptase–polymerase chain reaction (RT-PCR), the effects of postnatal exposure to a reconstituted mixture of AhR agonists present in breast milk (3 non-ortho PCBs, 6 PCDDs, and 7 PCDFs, referred to hereinafter as AhRM) on mRNA expression of estrogen receptor (ER), enzymes involved with the metabolism of estrogens [catechol-o-methyltransferase (Comt), cytochrome P450 (Cyp)1A1, 1B1 and 2B1], and DNA methyltransferase-1 (Dnmt1), in brain areas, liver and uterus of immature female rats. Neonates were exposed by gavage during postnatal day (PND) 1–20 with dosages equivalent to 1, 10, 100, and 1000 times the estimated average human exposure level, and were sacrificed at PND 21. None of the end points were affected in uterine cross-sections, or in samples of uterine tissue layers collected by laser capture microdissection. At 1000x, the AhRM reduced Dnmt1 mRNA abundance to 28% and 32% of control in the liver and hypothalamus, respectively. In the brain, Cyp1A1 was increased (409%) but ER was reduced (66%). Similarly, mRNA abundance for Comt isoforms was reduced in the liver (45%) and brain areas (55–70%). AhRM at 100x, the lowest effective dose, exerted a 220% increase in brain cortex Comt [membrane bound (Mb)], a 219% increase in hepatic Cyp1B1, and a 63% decrease in hepatic Comt (soluble (S)+Mb). These results support the possibility that early exposure to environmental contaminants could lead to effects mediated by changes in DNA methylation and/or estrogen metabolism and signaling.
Key Words: polychlorinated dibenzodioxins; polychlorinated dibenzofurans; rat; uterus; DNA methyltransferase; catechol-o-methyltransferase.
INTRODUCTION
Organochlorines, such as non-ortho polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) are ubiquitous environmental contaminants present in human tissues. Most of their toxic effects are exerted through the activation of the aryl-hydrocarbon receptor (AhR) (Safe, 1990), and they are referred to as AhR agonists. Usually humans receive their highest level of exposure during the in utero and postnatal periods, particularly breast-fed infants (Feeley and Brouwer, 2000; Solomon and Weiss, 2002). To improve risk assessment of exposure to these chemicals, additional information is required to understand the relative contributions of prenatal and postnatal exposure to the cumulative health risk derived from these two exposure periods.
Exposure to AhR agonists has been associated with long-term adverse health consequences, including an increased risk of cancer development, reproductive problems, learning difficulties, and immune, and endocrine system deficiencies. Among endocrine effects, AhR agonists act as antiestrogens, not by competing for the ligand domain of the estrogen receptor (ER), but by downregulating ER, increasing the metabolism of estradiol-17, inhibiting various growth factors and crosstalk between AhR and ER pathways (Safe, 1999), and through proteosomal degradation of the receptor (Wormke et al., 2003). In contrast, estrogen-like effects are induced by activated AhR interacting with the unliganded ER (Ohtake et al., 2003). Regulatory agencies suggest using the uterotrophic response in immature rats to detect chemicals with estrogenic or antiestrogenic properties (Kanno et al., 2001; 2003a; O'Connor et al., 2002; Owens et al., 2003; Owens and Koeter, 2003). However, there are concerns about the ability of a uterotrophic assay to detect antiestrogenic effects of 2,3,7,8-tetrachlorodibenzodioxin (TCDD) at PND 21 (Desaulniers et al., 2003; White et al., 1995), but not in slightly older rats (Romkes et al., 1987; Safe et al., 1991).
Inductions of hepatic CYP1A1 and "extrahepatic" CYP1B1, which metabolize estradiol-17 into toxic 2-OH- and 4-OH-catecholestrogens (CE), are classical effects of AhR activation (Badawi et al., 2000; Hayes et al., 1996; Jefcoate et al., 2000). Toxic CE, mostly 4-OH-CE, are implicated in estrogen-mediated carcinogenesis (Cavalieri et al., 2000), raising the possibility that inappropriate levels of CE may increase health risks. Catecholestrogens are rapidly methylated, thus detoxified, by the enzyme catechol-o-methyltransferase (COMT). In addition, 2-OH-estradiol is converted to 2-methoxyestradiol (Zacharia et al., 2004), a compound that has numerous beneficial effects on cardiovascular and renal functions (Dubey et al., 2004), on obesity (Tofovic et al., 2001), and in many cancer cells in vivo and in vitro (Seeger et al., 2004). Along with its predominant regulatory role in the reproductive organs, estradiol-17 is an important modulator of brain development (Ivanova and Beyer, 2003; Weiss, 2002), a neuroprotective factor, and a stimulator of ontogenesis in the central nervous system (Beyer et al., 2003). Despite the limited production of ovarian estradiol-17 during the postnatal period, the expression of aromatase, ER, and ER in neonatal brains peaks during the first 2 weeks after birth, and it reaches higher levels in males than in females (Ivanova and Beyer, 2000; Raab et al., 1999). These events also coincide with the highest levels of tyrosine hydroxylase (a rate-limiting enzyme of the catecholamine biosynthetic pathway) during the early postnatal period, suggesting estrogen control of neuronal dopamine synthesis and function (Hersey et al., 1982; Ivanova and Beyer, 2003). Catecholamine neurotransmitters are deactivated by methylation carried out by the same COMT involved in CE detoxification (Zhu, 2002). Exposure to environmental contaminants, particularly during the prenatal period, has been associated with long-term deleterious effects on brain function (Andersen et al., 2000; Schantz et al., 2003). The present study was devoted to exploration of possible effects of early exposure to environmental contaminants on the expression of enzymes involved with estrogen metabolism and signaling and the COMT system in the developing brain.
Changes in DNA methylation may lead to chromosome instability, X chromosome inactivation, modifications in centromere structure, as well as changes in genomic imprinting, gene expression, and tissue differentiation (Reik et al., 2001; Wolffe and Matzke, 1999). Only limited information is available on the effects of various toxicants (phenobarbital; trichloroethylene, di- and trichloroacetic acid, methoxyacetic acid, arsenite, cadmium, nickel, chromium, radiation, TCDD) on DNA methylation (Bombail et al., 2004; Carnell and Goodman, 2003; Moggs and Orphanides, 2004; Pogribny et al., 2004; Ray and Swanson, 2004; Wu et al., 2004). Abnormal DNA methylation has been associated with a number of cancers (Gaudet et al., 2003), with infertility (Cisneros, 2004), and with developmental (Egger et al., 2004), neurological (Costa et al., 2004), immunological (Yasunaga et al., 2004), and age-related (Oakes et al., 2003) disorders. It may originate during early development (Wu et al., 2004) or later during mitosis, when the methylation pattern of the original DNA strand is copied mostly by DNA methyltransferase-1 (DNMT1) onto the replicating DNA. DNA methylation reactions are catalyzed by families of DNA methyltransferases (DNMT-1, –2, –3). Three isoforms of DNMT1 have been described: one specific for somatic cells (DNMT1s), one affecting pachytene spermatocytes (DNMT1p), and a type that influences oocyte and preimplantation embryos (DNMT1o) (Ko et al., 2005). The role of DNMT2 remains obscure. The de novo methyltransferases, DNMT3a, 3b, and 3L, which catalyze the transfer of a methyl group to previously unmethylated DNA, are expressed during gametogenesis and embryogenesis (Bachman et al., 2003; Kaneda et al., 2004; Margot et al., 2003; Rhee et al., 2002). We are not aware of any reports describing the effects of exposure to AhR agonists on the expression of DNA methyltransferases.
We have previously examined the effects of postnatal exposure to reconstituted mixtures of the most abundant (19 PCBs, DDT, DDE) (Desaulniers et al., 2001; 2005), and "most toxic" (based on growth, and on liver and thyroid effects) breast milk contaminants [a mixture of AhR agonists: 6 PCDDs, 7 PCDFs, and 3 non-ortho PCBs (Desaulniers et al., 2003; 2004)] on organ weights, endocrine parameters, liver function, and mammary tumor development. To better understand tissue differences in dose–response effects of the mixture of AhR agonists, we here extend previous information by comparing mRNA abundance for Cyp1A1, 1B1, and 2B1, Comt, ER, and Dnmt1 in the liver, uterus, and brain in female rats at PND 21. Cyp1A1 and Cyp1B1 expression was assessed in all tissues as an indicator of "functional" AhR-mediated effects. Cyp1A1 and 1B1 mRNA abundance was not affected in complete uterine tissue samples, but because the uterus is composed of different tissue layers (perimetrium, myometrium, endometrium, and epithelium), the possibility was tested that effects could be occurring in specific layers. Consequently, a subjective assessment of ER and CYP1B1 protein abundance in the uterus was performed by immunohistochemistry (IHC), and these observations were corroborated by a quantitative assessment of the abundance of their mRNAs in uterine perimetrium, myometrium, endometrium, and epithelium, using samples collected by laser capture microdissection (LCM).
MATERIALS AND METHODS
Animal treatment.
A mixture of 3 PCBs, 6 PCDDs, and 7 PCDFs (referred as AhRM) in corn oil was prepared based on individual concentrations of each chemical found in one study of human breast milk (Table 1). Female Sprague-Dawley neonatal rats (10–13 per group) were gavaged at PND 1, 5, 10, 15, and 20, with either corn oil, or AhRM at a cumulative dose equivalent to 1, 10, 100, and 1000 times the estimated amount consumed by an infant over the first 24 days of life. Prepubertal rats were sacrificed at PND 21, a time at which rats are often euthanized when performing uterotrophic bioassays (Kanno et al., 2003b; Li and Hansen, 1997; White et al., 1995).
Tissue processing.
Brains were dissected out of the cranium and placed on dry ice until completely frozen to maintain structural integrity before storage. Sections of liver and uterus were snap frozen in liquid nitrogen immediately after dissection. All tissues were kept at –80°C until analyzed. The anterior half of a uterine horn was transferred into 10% neutral buffered formalin, held for 24 h, processed, and embedded in paraffin for immunohistochemical analysis.
Dissection of the different brain regions was performed on dry ice. The frontal portion of the brain anterior to the optic chiasm was discarded. Working backward, a 2-mm-thick cross section that contained the hippocampus (HC), the hypothalamic area (HT), and the cortex (C), was used to generate data identified as "Total brain tissue" (TBT) (Tables 2 and 3); subsequent sections were used to dissect tissues from individual brain areas.
Immunohistochemistry of the uterus.
Paraffin-embedded sections of uteri (5 μm) were mounted onto positively charged slides, deparaffinized in xylene and absolute ethanol, then rehydrated by successive immersions in 95% ethanol, 70% ethanol, and distilled water. After rehydration, the sections were microwaved at high power for 1 min, then at medium power for 14 min in citrate buffer (0.01 M, pH 6.0). All sections were washed (3 x 5 min in PBS), and immersed (RT; 10 min) in PBS with 1% (v/v) H2O2 to inhibit endogenous peroxidase activity. After a course of three additional PBS washes (5 min each), the sections were incubated (RT; 60 min) with 5% milk powder in PBS to minimize nonspecific binding and then kept at 4°C overnight with the primary antibodies [ER: rabbit polyclonal IgG (1:400; Santa Cruz Biotechnology, Santa Cruz, CA); CYP1B1: rabbit anti-human CPY1B1 (1:100, Alpha Diagnostic Intl. Inc, San Antonio, TX)]. The sections were incubated with biotinylated second antibody (RT, 30 min), washed with PBS, and then incubated with streptavidin-peroxidase (RT, 30 min). After washing, the specimens were immersed (RT, 10 min) in chromagen solution (Dako Diagnostics Canada Inc., Mississauga, ON), counterstained with hematoxylin, dehydrated in a series of alcohols and xylene, and covered with glass slips.
RNA isolation from whole uteri and brain areas.
Total RNA was isolated from liver and whole uteri, using the Mini RNeasy Kit (Qiagen, Valencia, CA). The RNeasy Lipid Tissue Mini Kit was used for RNA isolation from brain areas, according to the manufacturer's protocols (Qiagen). Contaminating genomic DNA was removed by DNase digestion using the RNase-Free DNase Kit (Qiagen). Quantification of total RNA from whole uteri was analyzed using RiboGreen RNA Quantitation Reagent Kit (Invitrogen Canada Inc., Burlington, ON), and the RNA from liver and brain area samples were measured with a spectrometer at 260 nm. RNA quality was assessed by RNA gel electrophoresis.
RNA isolation from LCM samples.
Uterine cryosections (10 μm) were prepared from frozen tissues embedded in Tissue-Tek O.C.T (Sakura Finetek Inc, Torrance, CA). Cryosections were mounted onto coated glass slides, fixed (1 min, 70% ethanol), stained for visualization of the different tissue layers, and dehydrated, using the protocol, slides, and reagents supplied with the HistoGene Frozen Section Staining Kit (Arcturus Bioscience Inc, Mountain View, CA). Cells from the uterine epithelium, endometrium, myometrium, and perimetrium were collected with a PixCell II Laser Capture Microdissection system (beam settings: 15 μm, 40–45 mW; Arcturus Bioscience Inc.). To verify the quality of the RNA after laser capture microdissection (LCM), samples of RNA were prepared from 200–400 uterine cells (one laser pulse equals approximately one cell), and the presence of a 2:1 ratio of 28S/18S RNA was verified from microcapillary electrophoresis analysis (RNA 6000 LabChips, Agilent 2100 Bioanalyzer; Agilent Technologies, Wilmington, DE). Other analyses were from LCM samples of 400–2000 pulses. The LCM samples were incubated (RT, 30 min) with 300 μl of lysis buffer. Total RNA was extracted with the RNeasy micro kit (Qiagen), and contaminating genomic DNA was removed by on-column DNase digestion during RNA purification, using the RNase-free DNase set (Qiagen).
Reverse transcription (RT).
Total RNA was reverse transcribed by random primer and MMLV reverse-transcriptase according to the manufacturer's protocol (Sensiscript RT kit; Qiagen). Non-RT control samples were prepared by omitting the reverse-transcriptase from the reaction mixture.
Primers for polymerase chain reaction (PCR).
The respective forward and reverse sequences of the primers, designed using Beacon Designer 2.0 (BioRad, Mississauga, ON) according to published rat gene sequences [ER (Spreafico et al., 1992); www.ncbi.nlm.nih.gov with the following loci NM_012540 [GenBank] (Cyp1A1), RNU09540(Cyp1B1), RATCYP450-J00719 (Cyp2B1), AF116344 [GenBank] (Dnmt1), NM_012531 [GenBank] (Comts), and NM_031144 [GenBank] (-actin)] are as follows: ER (5'-GCG-CCG-CCT-ACG-AGT-TTC-A-3' and 5'-GAC-CGT-AAG-TGA-TGC-TCG-ACT-G-3'), Cyp1A1 (5'-CTT-CAC-ACT-TAT-CGC-TAA-TGG-3' and 5'TTG-GGT-CTG-AGG-CTA-TGG-3'), Cyp1B1 (5'-GAG-TTG-GTG-GCA-GTG-TTG-3' and 5'-GCA-TCG-TCG-TGG-TTG-TAC-3'), Cyp2B1 (5'-TGA-TCT-TTG-CCA-ATG-GGG-AAC-3' and 5'-CCG-TTC-TTC-CAC-ACT-CCT-CT-3'), Dnmt1 (5'-AAC-GGA-ACA-CTC-TCT-CTC-ACT-CA-3' and 5'-TCA-CTG-TCC-GAC-TTG-CTC-CTC-3'), Comt [soluble form + membrane-bound form (S+Mb)] (5'-GCC-AGG-CTT-CTC-ACC-ATG-3' and 5'-CGT-CGT-ACT-TCT-TCT-TCA-GCT-3'), Comt (Mb) (5'-CTC-ATT-GGG-TCT-CCT-GTT-GTT-G-3' and 5'-AGG-TTG-TGG-ACT-GGC-TGC-3'), and -actin (5'-TCG-GCA-ATG-AGC-GGT-TCC-3' and 5'-CAG-CAC-TGT-GTT-GGC-ATA-GAG-3').
As indicated above, two sets of primers were designed to determine the abundance of the enzyme COMT, which exists as a soluble and a membrane-bound isoform, the latter possessing an extended NH2 terminal end for anchoring the enzyme to the membrane. Both forms originate from the same gene, which expresses two mRNA species with different tissue distributions. The expression of the transcripts is regulated by at least two promoters (Tenhunen et al., 1993). Here, a first set of primers was designed from the carboxy terminal region to amplify both the soluble and membrane-bound forms, Comt (S+Mb). The second set of primers is specific to the NH2 terminal portion to provide a measurement of only the membrane-bound form, Comt (Mb).
Quantitative real-time PCR analysis.
The PCR reaction volume (25 μl) contained 4 μl of RT products, 0.5 unit HotStarTaq DNA polymerase (Qiagen) in 1x QuantiTect SYBR Green Master Mixture, and 0.5 μM each of the forward and reverse primers. The PCR amplification cycles included denaturation (95°C, 15 min; to activate the HotStarTaq DNA polymerase and to minimize primer-dimers contribution), and amplifications [over 40–50 cycles including denaturation (94°C; 30 s), annealing (55°C; 30 s), and extension (72°C; 1 min)]. All PCR reactions were performed in duplicate. Non-RT control and negative control samples (without template) were processed in the same manner. The specificity of the amplifications was verified by melting curve analysis for all samples, and occasionally by agarose gel electrophoresis. Amplifications of target gene sequences were compared against serial dilutions of known quantities of their purified cDNA fragments, and normalized to the abundance of the house-keeping gene -actin. The amount of total RNA from the LCM samples was too low to be quantified spectrometrically, and specific gene mRNA abundance was thus reported as a ratio to -actin mRNA within the same sample.
Statistical analysis.
All analyses were performed using JMP software (SAS Institute Inc, 1998). The normality of the data was verified by goodness-of-fit Shapiro-Wilk W tests. Data that failed this test were log transformed and the normality was retested. If normality of the transformed data failed, it was analyzed using the nonparametric Mann and Whitney U-test (comparison of two groups) or the Kruskal-Wallis rank sum test (comparison of three or more groups). Brain data were analyzed by two-way analysis of variance (ANOVA) for treatment effect, tissue area effect, and their interactions. Two-way ANOVA was also used to test differences in the expression of ER, or Cyp1B1 mRNA between age groups and uterine tissue layers, or treatment and uterine tissue layers. Respective interactions were also assessed. Differences between group means were further tested by Tukey honestly significant difference (HSD) tests. Values were considered to be significantly different when p 0.05.
RESULTS
The most abundant brain mRNA was Comt (S+Mb), and the least abundant was Cyp1A1 (Table 2). Compared to respective controls, the 1000x dose reduced Comt (S+Mb) mRNA abundance in TBT (70%), HC (55%), and C (63%). Cyp1A1 mRNA abundance was significantly increased in all brain tissues: TBT (345%), HT (409%), HC (340%), C (250%). ER mRNA was more abundant in HT than in C or HC, and 1000x reduced ER mRNA abundance in all brain areas [TBT (51%), HT (37%), HC (34%)], except in the cortex (66%) where the difference did not reach statistical significance. Dnmt1 mRNA abundance was similar in all brain regions, and although 1000x reduced it in all regions, statistical significance was reached only in HT (32%). The 1000x dosage had no effects on mRNA abundance for Comt (Mb), Cyp1B1, and Cyp2B1.
The comparison of brain mRNA abundance between the control and the 100x groups was achieved in a second analysis, presented in Table 3. Interestingly, increases in Comt (Mb) were detected in all brain areas, although statistical significance was observed only in C (220%). The 100x dose induced slight but nonsignificant increases in Cyp1A1 and had no effect on ER mRNA. The higher, but not statistically different, abundance of ER mRNA in HT compared with other brain areas (Table 3), confirms the similar observation in Table 2. AhRM at 100x had no effect on Comt (S+Mb) and Dnmt1 mRNA abundance. Cyp1B1 and 2B1, unaffected by the 1000x dose, were not tested at 100x. Given the absence of important effects induced by the 100x dose, samples from lower dose groups were not analyzed.
In the liver, AhRM treatments reduced the abundance of mRNA for Comt (S+Mb) [100x (63%), 1000x (45%)], whereas that of Comt (Mb) was not affected (Table 4). Although mRNAs for Comt isoforms are more abundant in liver than in brain, the proportion of Comt (Mb) relative to that of Comt (S+Mb) is usually similar in both tissues [liver (1.3%), TBT (1.6%), HT (1%), HC (0.7%), C (1.7%)]. The higher abundance of Comt (S+Mb) compared to Comt (Mb) mRNA suggests that most of the Comt are of the soluble (S) form in both tissues. While the AhRM treatments induced a dose–response increase in Cyp1B1 mRNA abundance [100x (219%), 1000x (475%)], the mRNA of Dnmt1 was reduced [1000x (28%)]. Given the absence of effects induced by the 10x dose, samples from the 1x dose groups were not analyzed.
In contrast to its effect on brain and liver, the 1000x dose had no effects on the end points measured in the uterine tissues. Briefly, the abundance of mRNA attoM/femtoM -actin in control (n = 6) and 1000x (n = 7) uterine samples were 0.15 ± 0.05 and 0.19 ± 0.03 of Cyp1A1, and 0.5 ± 0.1 and 0.7 ± 0.1 of Cyp1B1. Visualization of the CYP1B1 and ER proteins in individual uterine layers by immunohistochemistry (IHC) revealed that they were coexpressed in the perimetrium and the endometrium, with low signals in the myometrium and luminal epithelium (Fig. 1A and 1C). For this analysis, Cyp1A1 was not considered given that it was the least abundant mRNA. In contrast to young female rats at PND 21, adult rat ER was found in the luminal and glandular epithelium (Fig. 1B). Immunohistochemistry is subjective and does not easily permit the assessment of treatment effects. Thus, a laser capture microdissection technique was initially developed to compare ER mRNA abundance between PND 21 uterine tissue and adult uterine tissue (Fig. 2A). ER mRNA was significantly more abundant in the luminal epithelium of adult rats than in PND 21 females [two-way ANOVA (tissue layers, p < 0.0001; tissue layer x age interaction, p = 0.001); Tukey HSD test (p < 0.05)]. Uterine glands were rarely visible at PND 21, and thus ER mRNA abundance was analyzed only in glandular epithelium of adult rats. In accordance with IHC observations of ER (Fig. 1A), mRNA abundance was significantly higher in the perimetrium than in the endometrium (p < 0.05, Fig. 2A). The endometrium had significantly more ER mRNA than the luminal epithelium or the myometrium (p < 0.05). Cyp1B1 mRNA was significantly more abundant in the perimetrium than in the endometrium (p < 0.05, Fig. 2B). No difference in Cyp1B1 mRNA abundance was observed between the myometrium, endometrium, and luminal epithelium. The 1000x dose had no effects on the abundance of ER and Cyp1B1 mRNAs in the different uterine tissue layers.
DISCUSSION
The lowest effective dose of AhRM was equivalent to 100x the estimated average human exposure level during the first 24 days of life. This dose affected both the liver and the brain, but based on the magnitude of changes, the liver might be considered one of the most sensitive organs. The 100x dose increased mRNA expression of hepatic Cyp1B1 (Table 4) and Cyp1A1 [as well as activity (Desaulniers et al., 2003)], and it decreased that of Comt (S+Mb), suggesting that the COMT enzyme system might be as sensitive as that of CYPs. However, the magnitude of the changes was much greater for CYP1A1 [657% induction by 100x (Desaulniers et al., 2003)]. The rat uterus at PND 21 is not a sensitive organ, as indicated by the apparent lack of effects of AhRM on mRNA abundance (ER, Cyp1A1, Cyp1B1), on protein expressions (ER, CYP1B1) (current results), and on uterine growth (Desaulniers et al., 2003). Uneven distribution of the AhR agonists among tissues could contribute to tissue differences in the magnitude of the responses. Other investigators compared the tissue distribution of radioactive coplanar and non-coplanar ortho-PCBs in weanling female rats at PND 21, and revealed that coplanar PCBs accumulate in the liver but not in the brain, whereas non-coplanar PCBs do accumulate in the brain (Saghir et al., 1999; 2000). The congeners of PCBs, PCDDs, and PCDFs used in the current studies have coplanar structures and might have accumulated preferentially in the liver.
In addition to appearing less sensitive, the brain response was different from that of the liver. Whereas in the liver a dose–response decrease in Comt (S+Mb) with no effects on Comt (Mb) was observed, in the brain the Comt (Mb) mRNA level was increased by AhRM 100x, particularly in the cortex, but not at the 1000x dose (Table 3). Reminiscent of hormesis effects (Calabrese and Baldwin, 2003), the biological significance of this discrepancy should be investigated further. The COMT system transforms toxic CE into non-toxic methoxyestrogens; it converts L-3,4-dihydroxyphenylalanine (L-DOPA; the precursor of the catecholamine neurotransmitters), into 3-methoxytyrosine, and it deactivates dopamine, epinephrine, and norepinephrine into homovanillic acid, metanephrine, and normetanephrine, respectively (Goldstein et al., 2003). The COMT system constitutes an important part of the enzymatic "blood–brain barrier" against peripherally produced catecholamines (Goldstein et al., 2003). An increase in brain Comt (Mb) mRNA at a 100x dose is suggestive of an adaptive response. Decreased activity of COMT in the periphery (as suggested by the reduced hepatic Comt (S+Mb) abundance), might be associated with an increased level of catecholamines reaching the brain, so that the latter compensates by increasing COMT production. A decrease in hepatic COMT activity is further supported by the observation that Aroclor 1254, a commercial mixture of PCBs, decreased methylation of CE, presumably as a result of inhibition of COMT activities by catechol metabolites of PCBs (Garner et al., 2000). The mRNA results (Tables 2–4) provide an additional mechanism supporting an in vivo reduction in COMT activity.
Based on the current results showing mRNA abundance from the brain and liver, and the previously increased hepatic mRNA, protein, and activity of CYP1A1 (Desaulniers et al., 2003), it is possible that AhRM increased the activity of both CYP1A1 and 1B1 and that it raised the production of toxic CE. The latter, usually labile, might have persisted longer in brain and liver given the reduction in COMT. These changes are believed to result in oxidative stress and estrogen-induced mutagenic events (Cavalieri et al., 2000; Li et al., 2004). The AhR agonists present in the mixture are known hepatocarcinogens that affect female rats to a greater extent than their male counterparts (Mayes et al., 1998). The current findings are consistent with the previous suggestion that the higher hepatocarcinogenic sensitivity in the female is associated with chronic estrogen metabolism–dependent oxidative imbalance (Wyde et al., 2001). DNA methylation affects chromosome stability and is associated with oncogenesis (Eden et al., 2003). Thus, along with changes in COMT and CYPs, the AhRM-induced reduction in Dnmt1 mRNA abundance in the liver (and perhaps changes in DNA methylation) might promote hepatic carcinogenesis and may possibly serve as an early indicator of this process.
The observed changes in mRNA abundance for the current set of genes might suggest adverse effects on brain development. AhRM reduced Dnmt1 mRNA levels, and might be associated with abnormal DNA methylation. Changes in DNA methylation can affect tissue differentiation, nervous system development, and intellectual disorders (Costa et al., 2004; Egger et al., 2004). The mechanism by which AhR agonists could alter Dnmt1 expression is unknown. However, an involvement of TCDD and the transcription factor Sp1 was suggested (Wu et al., 2004) given that Sp1 is a regulator of Dnmt1 expression (Kishikawa et al., 2003), and transplacental exposure to TCDD affected the temporal sequence in Sp1 expression in the brain (Nayyar et al., 2002). Changes in the expression of estrogen-metabolizing enzymes in the brain may influence neural development and function because CE have been reported to be neurotoxic (Brawer et al., 1993). COMT is a therapeutic target in the pathogenesis of various diseases, including neurodegenerative (Zhu, 2002). In agreement with the results of other studies (Pinzone et al., 2004), ER mRNA was particularly abundant in the hypothalamic area. AhRM decreased ER mRNA abundance in most brain areas, and that reduction might decrease estrogen signaling. Locally produced estradiol-17 acts as a stimulator of ontogenic processes in the central nervous system (Beyer et al., 2003), and ER, ER, and aromatase expression peaks during the first 2 weeks after birth (Ivanova and Beyer, 2000; Raab et al., 1999). Later, ER also acts as a neuroprotective factor (Kajta and Beyer, 2003). Whether AhRM alters gene responses to developmental cues in the young brains and induces long-term adverse effects remains to be investigated.
The current observations support the concept that CYP1B1 is the major extrahepatic CYP1 family member (Murray et al., 2001), with a higher abundance of Cyp1B1 mRNA than Cyp1A1 in the brain and the uterus, but lower levels in the liver. However, while AhRM induced the expression of both enzymes in the liver, only Cyp1A1 was increased in the brain. This finding is in agreement with the notion that a highly inducible, functional CYP1A1 is present in the brain (Granberg et al., 2003). In contrast, CYP1B1, but not CYP1A1, can be induced in the mammary gland by AhR agonists (Christou et al., 1995). Although these CYPs are important inducible detoxification enzymes, they may have other biological functions (e.g., embryo activation [Paria et al., 1998]), which may be tissue-specific. Finally, even though CYP2B1 is one of the most abundant CYP in the brain, and although it is induced by phenobarbital in the cortex (Upadhya et al., 2002), CYP2B1 brain expression was not significantly affected by AhRM, suggesting that it is not a sensitive target in this organ.
AhRM did not change uterine mRNA levels, and it had no effects on basal or 17-ethynylestradiol-stimulated uterine growth, despite its clear effects on other tissues ([Desaulniers et al., 2003], and the present study). This suggests that the rat uterus at PND 21 is relatively insensitive to AhR agonists. The possibility of tissue layer specific effects undetected when analyzing the entire uterus was tested, but this did not reveal any changes. When comparing IHC observations with mRNA expression in the different uterine tissue layers (using LCM and real-time RT-PCR), we observed that ER and Cyp1B1 mRNA and proteins were coexpressed in the immature rat uterus, and their abundance differed in the various uterine tissue layers; for example, mRNA expression was highest in the vascularized perimetrium and lowest in the luminal epithelium (Figs. 1 and 2). The constitutive expression of CYP1B1 observed by IHC and mRNA analysis supports a role of CYP1B1 in uterine physiology (Paria et al., 1998). The expression of ER mRNA shows a developmental difference between 21-day-old and adult female rats, with the presence of glandular development containing ER-positive cells in the adult only, and higher levels of luminal epithelial ER expression in the adults. These age differences do not prevent the ER system from being inducible at PND 21, as demonstrated by the uterotrophic effect of 17-ethynylestradiol (Desaulniers et al., 2003). In contrast, the absence of effects of AhRM on uterine Cyp1A1 and 1B1 suggests that the AhR system may not be functional at PND 21. It can be argued that the dosage of AhRM was not sufficient to induce effects in the uterus, but White et al. (1995) also failed to demonstrate antiestrogenic effects of TCDD at PND 21 at a dose of 80 μg/kg, suggesting that the dose of AhRM was not likely a factor. AhR agonists have been shown in the uteri of slightly older rats to have antiestrogenic activities attributable to crosstalk between ER and AhR signaling (Safe, 1999). Pharmacodynamic differences might exist depending on age variations. The current findings, together with our previous results (Desaulniers et al., 2003), suggest that antiestrogenic effects of AhRM is tissue- and age- specific. It is of interest to note that at this prepubertal age the uterus has been exposed to limited levels of endogenous estrogen (Li and Hansen, 1997) and, as in the case of breast cancer cells (Spink et al., 2003), might require pre-exposure to estrogens for the induction of CYP1A1 and 1B1.
CONCLUSIONS
To our knowledge, the findings presented here may be the first demonstration of Comt and Dnmt1 mRNAs as targets for adverse effects of exposure to AhR agonists. Perinatal changes in COMT (alone or in combination with a decrease in ER-signaling) and DNMT systems could have important carcinogenic and developmental effects, and thus might be important complementary indicators of toxicity. These results support our previous findings (Desaulniers et al., 2003) that dosages of AhRM (non-ortho PCBs, PCDDs, PCDFs) equivalent to 100 times the average human exposure level over the first 24 days of life are required to induce statistically significant changes in female rats. This dose of AhRM altered the mRNA expression for Comt, Cyp1A1, and Cyp1B1 in liver and brain cortex, but not in the uterus. The liver is one of the most sensitive organs in which to measure indicators of these effects. Tissue-specific changes in the abundance of these mRNAs substantiate the notion that abnormal metabolism of estrogens and DNA methylation could be important consequences of exposure to AhR agonists.
ACKNOWLEDGMENTS
The authors are grateful to L. Casavant, Dr. M. Charbonneau, J. Cole, Dr. G. Douglas, A. Lee, Y. Li, T. Odorozzi, K. Pazterko, L. Shao, Dr. K. Soumano, Dr. R. Vincent, and G. Zu for their technical advice and assistance, and to Drs. Guillaume Pelletier and Raymond Poon for suggested improvements to the manuscript. This study was funded by Health Canada, Toxic Substances Research Initiative (TSRI) grant no. 045 and Northern Contaminants Program (NCP) grant no. H16-2004.
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