ToxSci Advance Access originally published online on March 7, 2003
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Toxicological Sciences 72, 242-252 (2003)
Copyright © 2003 by the Society of Toxicology
ENVIRONMENTAL TOXICOLOGY |
Gestational Exposure to Persistent Organic Pollutants: Maternal Liver Residues, Pregnancy Outcome, and Effects on Hepatic Gene Expression Profiles in the Dam and Fetus
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* Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montréal, Québec H3G 1Y6, Canada;
Toxicology Research Division, Health Products and Food Branch, Food Directorate, Health Canada, Tunney’s Pasture, Ottawa, Ontario K1A 0L2, Canada;
Departments of Cellular and Molecular Medicine, Obstetrics and Gynecology, University of Ottawa, Ottawa, Ontario, Canada; and
Departments of Pediatrics and Human Genetics, and
¶ Department of Obstetrics and Gynecology, McGill University, Montréal, Québec H3G 1Y6, Canada
Received November 29, 2002; accepted December 9, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dietary exposure of Inuit people to a mixture of pesticides and polychlorinated biphenyls, or persistent organic pollutants (POPs), during pregnancy is a public health concern. We examined the consequences of administering the mixture of 28 POPs found in the Inuit diet (at doses representing 10–1000 times dietary levels) by gavage to pregnant Sprague-Dawley rats either during gestation days 0–19 or 8–19. The levels of individual components of the POPs mixture in the maternal liver were measured by high-resolution mass spectrometry. On gestation day 20, dams were sacrificed and pregnancy outcome determined. RNA isolated from maternal and fetal livers was 32P-labeled for gene expression profiling. The concentrations of individual POPs were increased in maternal livers of dams gavaged with the 1000x POPs mixture by 10- to 500-fold. While exposure to POPs had no significant effects on pregnancy outcome, dramatic changes were observed in the gene expression profiles of both the maternal and fetal livers. The gene expression profiles of maternal and fetal female and male liver were distinct with respect to the numbers of transcripts detected, the genes expressed exclusively in control or POPs-exposed livers, and those for which expression was up- or downregulated. While different genes were affected in each group, the overall consequence of POPs exposure on hepatic gene expression profiles was to decrease both the numbers of genes expressed and the relative intensity of expression. Thus, in utero exposure to POPs alters hepatic gene expression in the dam and the fetus; these changes may have functional implications.
Key Words: persistent organic pollutants; microarray; liver; rat; fetus; pregnancy; gene expression; Inuit diet.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Persistent organic pollutants (POPs), comprised of pesticides such as mirex and polychlorinated biphenyls (PCBs), were some of the first industrial compounds identified in the environment. These organochlorine compounds were widely used in agriculture and manufacturing industries because of their unique physical and chemical properties. Many of them are lipid soluble, have a very long half-life, and are concentrated in adipose tissues, especially in species that are high in the food chain (Pocar et al., 2001
). Movement of air masses occurs from heavily industrialized parts of the world, where agricultural pesticides are used, to the Arctic. Inuits, who are indigenous to Canada and Greenland, have the highest body burden of PCBs (Bjerregaarde et al., 2001), presumably because they consume large amounts of sea mammal fat in their diet.
A number of studies have investigated the effects of exposure to single compounds or technical mixtures of organochlorines in rodents and other species. PCBs, especially the higher chlorinated congeners, may selectively induce the cytochrome P450s that catalyze the oxidation of many endogenous and exogenous substances (Parkinson et al., 1983a). Workers exposed to PCBs have an increased incidence of liver cancer (Brown, 1987). Exposure to persistent organic pollutants is of particular concern during pregnancy. PCBs cross the placenta (Foster et al., 2000; Waliszewski et al., 2000, 2001), exposing the developing fetus to these chemicals during organogenesis. Indeed, transplacental transfer of PCBs induced the expression of P450 isoenzymes and some protooncogenes (Borlakoglu et al., 1993). Toxaphene was toxic to embryos in the in vitro embryo culture system (equivalent to gestational days 10–12; Calciu and Chan, 1997). Maternal exposure to mirex resulted in cardiovascular-related perinatal deaths in the rat (Grabwoski and Payne, 1983). Female Swiss-Vancouver mice exposed to dieldrin had reduced fertility and increased preimplantation loss, correlating with dieldrin-induced hepatomegaly (Birgo and Bellward, 1975). Gestational exposure to polychlorinated biphenyls was reported to result in decreased length of gestation, supposedly resulting from increased uterine stimulation (Loch-Caruso, 2002), while in another report (White et al., 1983) a delay in the onset of parturition was documented.
Environmentally relevant human exposures are to mixtures of organochlorines, rather than to single compounds. There is relatively little information on the potential outcomes of such exposures. In particular, the full effects of dietary exposure during pregnancy to a cocktail of organochlorine compounds, as occurs in the Inuit, are yet to be investigated. The first objective of the present study was to quantify liver concentrations of POPs following dietary exposure to an organochlorine mixture based on the Inuit diet (Chan et al., 2000); the second was to evaluate the effects of exposure to this mixture on pregnancy outcome; the third objective was to evaluate the effects of POPs exposure on global gene expression in both the maternal and fetal livers.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
A mixture comprised of polychlorinated biphenyls and pesticides (referred to hereafter as POPs) found in the diet of the Inuit living on Broughton Island was prepared in our laboratory (Table 1
; Chan et al., 2000). The POPs mixture was prepared in corn oil for administration at 10, 100, and 1000 times the doses that reflect normal dietary exposure.
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Animal treatment.
Male (300–350 g) and female (200–250 g) Sprague-Dawley rats were obtained from Charles River Canada (St. Constant, Québec, Canada) and housed in the McIntyre Medical Building Animal Resource Centre at McGill University with a 14-h light:10-h dark cycle. The animals had free access to food (Purina chow 5012, Mondou Feeds, Montréal, Canada) and water. All animal handling and care followed the guidelines of the Canadian Council on Animal Care.
Virgin female rats in proestrus were mated overnight with males. Successful mating was indicated by the presence of spermatozoa in the vaginal smear on the following morning (day 0 of pregnancy). Pregnant rats were randomly divided into groups (n = 12) that were given a daily dose of vehicle or POPs (10x, 100x, or 1000x) by gavage, either from gestation days 0 to 19 or from gestation days 8 to 19. Control dams were given identical volumes of corn oil during the same period. Dams were weighed once every three days from day 0 and then on day 20 of pregnancy.
On gestation day 20, dams were euthanized with an overdose of diethyl ether by inhalation. Pieces of maternal liver were flash-frozen in liquid nitrogen and stored at -80°C for subsequent analysis of POPs concentrations and RNA extraction; fetal livers were similarly frozen for RNA extraction. The ovaries of dams were dissected, and the number of corpora lutea were counted. The two-horned uterus was removed and inspected for implantation and resorption sites; preimplantation loss was determined by subtracting the number of implantation sites from the number of corpora lutea, while postimplantation loss was determined by subtracting the number of fetuses from the number of implantation sites. Fetuses were individually weighed, examined for external malformations and the anogenital (A-G) distance was measured. Data were analyzed by two-way ANOVA.
Analysis of POPs concentrations in dams’ livers.
Approximately 1 g of liver was extracted at high speed with a PTA 10S generator by adding 20 ml of acetone:hexane (2:1, v/v) to each sample and homogenizing for approximately 1 min. The mixture was filtered through precleaned glass wool into a 250 ml round bottom flask (RBF). The extracts were concentrated to near dryness on a rotary evaporator and were dried by filtering them through a bed of precleaned anhydrous sodium sulfate (granular) into a second 250 ml RBF. Fat determination was done by concentrating to near dryness on a rotary evaporator, transferring the concentrated extract to a preweighed scintillation vial, allowing the solvent to dry overnight, and weighing the scintillation vial the next morning. The weight of the vial with oil minus the weight of the empty vial equals the amount of extractable lipid found in liver. The lipid was redissolved in approximately 1 ml of hexane and adsorption chromatography was conducted by using 12 x 20 cm columns dry packed with 6.0 g of Florisil deactivated with 1% water (w/w). The samples were applied to the columns and all POPs were eluted with 75 ml 30% dichloromethane/hexane. The extracts were concentrated to near dryness (rotary evaporator), transferred to a 15 ml centrifuge tube, and brought to a final volume of 1 ml. Samples were injected on a high resolution mass spectrometer (Fison GC 8060 with Fison Autospec-Ultima Mass Spectrometer).
RNA extraction.
Total RNA was extracted from the livers of control and 1000x POPs-exposed dams, male and female fetuses using Trizol reagent following protocols provided by the manufacturer (Invitrogen Canada Inc., Burlington, Canada). The quality of the DNAse-treated RNA was assessed by spectrophotometric reading and electrophoresis.
Preparation of radiolabeled cDNA and hybridization.
Total RNA (3.5 µg) was reverse transcribed with Moloney-murine-leukemia virus reverse transcriptase (MMLV-RT) in the presence of [
-32P]dATP (Amersham Pharmacia Biotec, Baie d’Urfé, Québec; 10 µCi/µl), as described previously (Aguilar-Mahecha et al., 2001). Probes were purified with nucleospin columns (Clontech, Palo Alto, CA) and hybridized to AtlasTM Rat 1.2 Array nylon membranes (Clontech) at 68°C overnight. Hybridization and washing conditions were as described previously (Aguilar-Mahecha et al., 2001). Five separate replicates were analyzed for control and POPS-treated livers from each group (n = 5/group).
Analysis of gene expression.
Hybridization intensity was captured using a phosphorimager (Storm, Molecular Dynamics, Sunnyvale, CA) after an overnight exposure of membranes to phosphorimaging intensifying screens. The intensity of each spot was quantified using the Atlas ImageTM version 2.0 software; these data were then imported to GeneSpringTM (version 4.0.7, Silicon Genetics, Redwood, CA) for further analysis. To control for experiment-to-experiment variations, the signal intensity for all genes on each membrane was averaged and the intensity of individual genes on that membrane normalized to this average value to give a relative intensity ratio for each gene. A gene was considered as being expressed if its intensity was at least two-fold the background intensity on each individual array, and it was expressed in at least three out of five replicates. An experiment-to-experiment normalization was done (GeneSpring) in order to minimize the variation from one experiment to the other; for each replicate array, the raw value of each gene was divided by the median intensity signal on its membrane to generate what is defined as the relative intensity for that gene. The expression of a gene was considered altered by treatment when the difference in expression was at least 1.5-fold (upregulated by 50% or downregulated by 33%) and was consistent in at least three out of five replicates.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Components of the POPs Mixture in the Diet and in the Dams’ Livers
The individual POPs and their relative contributions to the dietary mixture (Chan et al., 2000
) are identified in Table 1
. On a weight basis, the predominant components were toxaphene, pp'-DDE, oxychlordane, and PCB #153. With the exception of the chlordanes and toxaphene, all of the dietary POPs components were present in the livers of rats in the control group. Livers of the dams exposed to 1000x POPs during pregnancy (gestation days 0–19 or 8–19) had increased amounts of all residues. The concentrations of the POPs used in the dosing mixture found in the livers of control rats totaled less than 250 ppb, in comparison to the dosed dams in which the total concentrations were 104,000 and 71,000 ppb after exposure from gestation days 0–19 or 8–19, respectively.
With the notable exception of toxaphene, most of the organochlorines were present in the liver in the same proportion as in the dosing mixture. Toxaphene constituted 51% by weight of the dosing mixture, and yet it was evident at less than 3% of the total organochlorine residues in the livers. Other organochlorines that were at levels different from their dietary proportions are cis and trans-chlordane (
6% of their theoretical values), cis and trans-nonachlor ( 50% of their theoretical values), oxychlordane ( 140% of its theoretical value), ß- and 
-HCH ( 30% of their theoretical values), and PCB 32 and PCB 199 ( 12% of their theoretical values). Overall, the quantities of individual POPs in the livers from dams exposed from gestation days 8–19 were 70% of the level found in the gestation day 0–19 dams.
Pregnancy Outcome
Pregnancy outcome was evaluated after treatment of Sprague-Dawley rats with the POPs dietary mixtures equivalent to 10, 100, and 1000x the Inuit dietary levels of these compounds (Table 2). The pregnant females that received POPs from gestation days 8–19 (at all doses) gained significantly less body weight than those treated from gestation days 0–19 (p < 0.05; Table 1); however, maternal serum thyroxine and triiodothyronine were not significantly different across the treatment groups (data not shown). The litter sizes and incidences of preimplantation and postimplantation loss/litter were not different in any group. Neither fetal weights nor sex ratios were changed by in utero exposure to POPs. Exposure to POPs did not induce external malformations in the fetuses.
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Hepatic Gene Expression Profile
Of the 1176 genes spotted on the microarrays, 29% were detected in the livers of dams, while only 23 and 19% were detected in female and male fetal livers, respectively (Fig. 1
). Generally, the genes that were highly expressed in the male fetuses were also highly expressed in the female fetuses; a notable exception to this is gastric inhibitory polypeptide precursor, which was highly expressed only in the male fetal liver.
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After treatment with 1000x POPs, nearly 90 genes were not detected in the liver of dams, while only a few genes (9) became detectable. While 326 genes were expressed in the livers of control dams, there were only 246 expressed in the livers of the treated dams (Fig. 1
, top graph). In contrast, the number of genes in the liver of female pups that became undetectable was nearly identical to the number that was brought into the range of detectability by treatment of the dams with POPs (Fig. 1, middle graph). In male pups, just over 10% of the genes, many of which are involved in metabolism, were expressed exclusively in the livers of the control male fetuses, while 24 genes, including proteasome component C13 precursor, G1/S-specific cyclins D2 and E, growth hormone receptor and early growth response protein 1, were expressed exclusively in the livers of male fetuses exposed to POPs.
Regulation of Gene Expression Resulting from Exposure to POPs
Of the genes that were expressed in common in the livers of control and treated dams, more than 85% were not affected (less than 1.5-fold change) as a result of treatment, while nearly 12% were decreased, and less than 2% were increased 1.5-fold (Fig. 2). The repressed genes included transcripts for protease inhibitors, growth regulators, metabolism-regulators, stress-response, hemostasis, ion-channel/transport (Table 3). In contrast, the four genes that showed an increased expression did not belong to any single grouping (Table 3).
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A strikingly similar response to treatment with POPs was seen in the liver of female fetuses as that of the dams, with percentages of genes that were unaffected, decreased, and increased being 86, 11, and 3, respectively. In contrast, fewer genes (less than 6%) in the liver of male pups were affected by treatment, and all but one (liver fatty acid-binding protein) showed a decreased response (Fig. 2
, Table 3).
Gene Families
Cytochromes P-450.
The cytochrome P450 proteins are largely responsible for metabolism of xenobiotics and endogenously produced substances. P450s 1A1 and 1B1 were expressed at background intensity in both dam and fetal livers, irrespective of exposure to 1000x POPs (Fig. 3). P450 2A1, which in the rat is inducible by 3-methylcholanthrene, was downregulated by 1.5-fold in the dams as a result of POPs treatment; however, it was not affected by POPs exposure in female or male fetal livers. P450 2C7 was highly expressed in the livers of both control and treated dams; transcripts for this gene were absent in the liver of female fetuses, whether treated or not. In contrast, this transcript was present in the livers of control male fetuses but became undetectable after in utero treatment with POPs. In the dams, 2C22 and 2C23 (arachidonic acid epoxygenase) were downregulated by at least 1.5-fold. In the fetuses, 2C22 was expressed only in the livers of females exposed in utero to POPs, while 2C23 was highly expressed in fetal livers, but not altered by POPS exposure. Interestingly, the intensity of expression of 2C23 was about twice as high in male as compared to female fetuses. Cytochrome P450 3A1 expression was near or below the level of detection in dam and fetal livers. The expression of cytochrome 4A3 was not altered in dams by POPs treatment; while this gene was expressed in the livers of control (female and male) fetuses, it was not detected in the livers of male fetuses that were exposed to POPs. In the maternal liver, NADPH-cytochrome P450 reductase expression was reduced by 1.5-fold; this transcript was not detected in fetal livers.
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Hepatic function associated genes.
The relative intensity of expression of hepatic function related genes is depicted in Figure 4
. Long chain acyl-CoA synthase was highly expressed in dams’ livers, expressed in the female fetal livers, but undetected in the liver of the male fetuses. Thrombin was also highly expressed in the livers of dams and fetuses; expression of this gene was downregulated by 1.5-fold in dams but not altered in the fetuses. The patterns of expression of liver fructose-metabolizing enzymes (fructose bisphosphate aldolase B and fructose-16-bisphosphatase) were distinct. The former was highly expressed in both dams and fetuses while the latter was detected only in dams; neither was affected by POPs exposure. Treatment with POPs also did not affect the intensity of expression of hydroxysteroid sulfotransferase A, catalyzing the sulfation of alcohol, hydroxysteroids, and other substrates; the relative abundance of this transcript was high in the livers of dams but below detection in the fetal livers. Fibrinogen ß subunit was highly expressed in the livers of dams and fetuses, but its expression was not changed by exposure to POPs. Liver fatty acid-binding protein was downregulated by 1.5-fold in dams, unchanged in the female fetal livers but up regulated by at least 1.5-fold in the livers of male fetuses.
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Stress response and apoptosis-related genes.
Plasma glutathione peroxidase (selenoprotein) expression was downregulated by exposure to POPs in the dams’ livers; this transcript was not detected in the fetal livers (Fig. 5
). Glutathione reductase and synthase were expressed at low levels in dams and female fetuses and expression was not altered by POPs; glutathione synthase was not detected in the livers of the male fetuses, nor was the reductase detected in the livers of male fetuses that were exposed to POPs in utero. Heat shock 90-kDa protein ß (HSP 90-ß) and clusterin were expressed in both maternal and fetal livers and unaltered by POPs exposure. In dams and female but not male fetuses, the hepatic expression of TNFR 1 was reduced 1.5-fold by POPs exposure. Stress response-related Cu-Zn-containing superoxide dismutase 1 (SOD1) was highly expressed in both maternal and fetal livers; expression of this gene was downregulated by maternal exposure to POPs exclusively in the female fetal livers. Ceruloplasmin precursor was highly expressed in the livers of dams and fetuses; expression was downregulated 1.5-fold in dams and male fetuses but not altered in female fetuses.
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Growth and cell cycle-related genes.
The expression of growth hormone receptor was highest in the maternal liver and downregulated by at least 1.5-fold by POPs treatment; this transcript was low or undetected in the fetal livers (Fig. 6
). Nuclear tyrosine phosphatase was expressed in dams and fetuses and reduced by 1.5-fold in the dams in response to POPs exposure. Early growth response protein 1 was also highly expressed in the dams’ livers and low or not detected in the livers of control or treated fetuses. Hepatocyte growth factor receptor was expressed in both dams and fetuses but expression was downregulated only in the female fetal livers. A number of the cell cycle genes were expressed much more highly in the fetal livers than in the dams. The expression of one, G1/S-specific cyclin D3, was reduced by POPs exposure in the female but not male fetal livers; M-phase inducer phosphatase 2 and proliferating cell nuclear antigen were highly expressed in the fetal livers but unaffected by treatment (data not shown). A differential pattern of expression of the insulin-like growth factors was observed in the dams compared with the fetuses. IGF I was expressed in dams, where it was upregulated by POP exposure, but it was not detected in the livers of the fetuses. IGF II was not detected in the maternal liver, but highly expressed in the fetal livers; POPs exposure did not affect the expression of this gene.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The POPs components administered in this study were based on the organochlorines present in the diet of the Inuit living on Broughton Island in Canada (Chan et al., 2000
). The treatment regimen mimics the consequences of exposure to 10 to 1000 times the dietary exposure among the Inuit. To the best of our knowledge, this is the first comprehensive report of the effects of Inuit diet-based organochlorine levels on pregnancy outcome, fetal development, and global hepatic gene expression. The disposition of POPs in the livers of the dams during the dosing windows indicated that the total organochlorine load in the liver was related to the length of dosing, since the levels after exposure from gestation days 0–19 were generally higher compared with the levels after treatment from gestation days 8–19. The POPs with significant differences in concentration between gestation days 0–19 and 8–19 were always present in lower concentrations in the livers of the latter group of dams.
Individual components exhibiting deviations from the theoretical distribution based on their proportions in the dosing mixture were toxaphene, the chlordane family of chemicals, ß- and -HCHs, and PCBs 32 and 199. All other components exhibited levels proportionate to those in the dosing mixture. In the case of toxaphene, this is most probably due to the rapid metabolism and excretion of toxaphene in mammals (Roney and Navarro, 1996); the persistence of toxaphene is measured in hours, compared with years for most of the organochlorines in the dosing mixture. Concentrations of the chlordanes were mostly decreased, whereas oxychlordane increased, reflecting the metabolic conversion of cis and trans-chlordane and cis and trans-nonachlor to oxychlordane, known to accumulate in the environment and in mammals and believed to be the metabolic end product for these chemicals (Abadin et al., 1994). HCHs are metabolized extensively by mammalian systems and, therefore, it was anticipated that ß- and -HCHs would not be found as the parent compound in the dams’ livers (Choudhary et al., 1999). With the exception of PCBs 32 and 199, all the other PCBs were present in liver at levels very close to their proportions in the dosing mixture; this is in agreement with their persistent, unchanged status in the body for extended periods of time (Faroon and Olson, 2000).
The body weight gains of dams gavaged with 1000x POPs for 20 days were not different from controls; this finding is consistent with previous reports (Arnold et al., 2000). The significantly lower body weight gain in dams that received POPs from days 8–19 compared to days 0–19 may be animal handling-related (Marti et al., 1994). Dams gavaged from day 0 had presumably adjusted to handling by the end of the experiment while those gavaged from day 8 may not have adjusted yet.
The incidences of pre- and postimplantation loss were not different between the control and dams treated with POPs, supporting previous reports (Backlin et al., 1998) that exposure to POPs altered neither sex ratio nor fetal viability, although conflicting reports exist in literature (Rice, 1999; Simmons et al., 1984). These discrepancies may be dose-related, but may also reflect differences in the effects of the single compounds administered in the previous studies and the effects of the mixture of organochlorines used in the present study.
In addition to the accumulation of POPs in the maternal liver, exposure to this organochlorine mixture resulted in a significant decrease in the numbers of genes expressed at the level of detection; of the transcripts that were detected in both control and POPs-exposed livers, more were downregulated than upregulated. These data suggest that POPs may inhibit transcription and/or destabilize RNA transcripts. It is conceivable that some components of the POPs mixture may interfere with DNA directly to inhibit the transcription of the affected genes (Morrell et al., 2000); in vivo administration of a polycyclic aromatic hydrocarbon suppressed transcription of the cytochrome P450 2C11 gene in the rat (Lee and Riddick, 2000). However, the possibility of reduced posttranscriptional RNA stability cannot be ruled out since the oxidation/reduction (redox) status of a cell modulates the stability of RNA (Miller et al., 1993) and organochlorines are known to affect cellular redox status (Bachowski et al., 1998).
In utero exposure to POPS had a less dramatic impact on liver gene expression in the fetus compared to the dams; a gender specific difference was observed with the female fetal liver being more susceptible than the male liver to the effects of POPs exposure. Although POPs accumulation in the fetal liver was not measured in the present study, previous reports found that fetal levels were considerably lower than maternal concentrations (Muckle et al., 2001). This may be responsible, in part, for the lower susceptibility of the fetal liver to POPs exposure-related effects on gene expression.
Organochlorines, especially the highly chlorinated compounds, are cytochrome P-450 monooxygenase inducers (Parkinson et al., 1983b). Certain components of the POPs mixture used in this study exhibit phenobarbital-type induction; these include hexachlorocyclohexane, HCH; cis-chlordane, CCHL; dieldrin, DIELD; p, p'-1, 1-dichloro-2, 2'-bis(p-chlorophenyl) ethylene, DDE; p, p'-1, 1, 1-trichloro-2, 2'-bis (p-chlorophenyl) ethylene, DDT; MIREX and PCBs 101, 99, 153, 183, and 180. Others, such as hexachlorobenzene and PCBs 118, 105, 138, and 156, exhibit mixed-type induction (Ah receptor activation in addition to phenobarbital-type induction). Therefore, exposure to these chemicals would be anticipated to induce the expression of CYP 1A1/2, 2A, 2B, and 3A in the liver. The absence of induction (lack of a measurable increase in mRNA abundance) of these cytochrome P450s by POPs could be the result of the "mixture" administered. In a mixture of PCBs, noninducing/weak-inducing PCBs competitively inhibit the effects of the strong inducing PCBs in the liver; the strong inducers then become very susceptible to elimination by the liver (Chu et al., 2001; Lee et al., 2002), consequently reducing the induction effects of organochlorine exposure. The daily cumulative organochlorine dosage in the 1000x POPs group of 2.17 ppm is within the dietary no-effect levels of 1–5 ppm for CYP induction by organochlorine pesticides and PCBs (den Tonkelaar and Van Esch, 1974). The failure of POPs exposure to induce xenobiotic-metabolizing enzymes in dams and fetuses was therefore not surprising. Moreover, late pregnancy is associated with a reduced ability of the liver to metabolize foreign compounds (Dean and Stock, 1975; Symons et al., 1982). POPs accumulated in the livers of exposed dams, suggesting that metabolism was slow.
In the present study, expression of the TNFR 1 gene, which codes for a death receptor protein involved in liver remodeling, was downregulated in POPs-exposed maternal and female fetal livers. Mice deficient in TNFR 1 have reduced apoptosis (Sheikh and Fornace, 2000). Downregulation of apoptosis-enhancing genes such as TNFR 1 may contribute to an increased incidence of liver tumors (Hemming et al., 1993) and cancers (Brown, 1987) associated with exposure to PCBs. Ornithine decarboxylase, the enzyme responsible for the synthesis of putrescine, a polyamine essential for liver regeneration (Russell et al., 1976), was not expressed in the livers of dams and female fetuses following exposure to POPs.
Exposure to POPs may alter the hepatic transcriptional profile by triggering free-radical damage. The expression of ceruloplasmin precursor, an extracellular antioxidant (Gutteridge and Stock, 1981) that binds most of the copper in blood, was downregulated in the maternal and male fetal livers by POPs. This downregulation by POPs exposure might be expected to contribute to free radical-related hepatic pathology. Copper-zinc-containing superoxide dismutase (Cu-Zn-SOD), highly expressed in dams and fetuses, was downregulated in the female fetal liver by POPs, as was thioredoxin peroxidase 1. In dams, plasma glutathione peroxidase precursor was downregulated.
There were remarkable differences in the expression of hepatic function-related and cell cycle genes in the maternal liver compared to those of the fetal liver. The liver is mainly a hematopoietic organ during fetal life (Zaret, 2002) and a homeostatic organ in postnatal life. This function-related pattern of gene expression is typified by the expression of liver-type aldolase (B) and liver long chain fatty acid-CoA ligase, two genes that are highly expressed in the maternal liver, but undetected in the livers of the fetuses. Downregulation by POPs of the expression of some function-related genes in the maternal liver, such as thrombin, may prolong blood coagulation since thrombin mediates fibrin clot formation. Other metabolic functions of the liver may be altered by downregulation of the expression of liver fatty acid-binding protein that binds a variety of other molecules (Hertzel and Bernlohr, 2000) in addition to regulating long chain fatty acid transport and metabolism.
Thus, dietary POPs accumulated in the liver in a dose- and component-dependent manner; higher hepatic levels were observed with a longer exposure. While exposure to dietary persistent organic pollutants during pregnancy did not exert overt maternal toxicity or adversely affect pregnancy outcome, there were major effects on both maternal and fetal hepatic gene expression. The alteration of gene expression profiles in the liver as a consequence of POPs exposure could significantly impact on hepatic function in mothers and their offspring. If the progeny do demonstrate persistent changes in liver function as they mature, exposure in utero to POPS may be one example of the fetal basis for an adult disease.
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ACKNOWLEDGMENTS |
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These studies were funded by grant no. 152 from the Toxic Substances Research Initiative (TSRI) of Canada. We thank Daniel Cyr and Johanne Barthelemy for the serum thyroid hormone level measurements. J.M.T. is an Investigator of the Canadian Institutes of Health Research and Scholar of the Fonds de la recherche en santé du Québec.
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NOTES |
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1 To whom correspondence should be addressed. Fax: (514) 398-7120. E-mail: bhales{at}pharma.mcgill.ca.
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