An environmentally relevant mixture of organochlorines, their metabolites and effects on preimplantation development of porcine embryos

Reproductive Toxicology 25 (2008) 361–366

Contents lists available at ScienceDirect

Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox

An environmentally relevant mixture of organochlorines, their metabolites and effects on preimplantation development of porcine embryos ´ Celine Campagna a , Pierre Ayotte b , Marc-Andre´ Sirard a , Janice L. Bailey a,∗ a b

Centre de recherche en biologie de la reproduction, D´epartement des sciences animales, Pavillon Paul-Comtois, Universit´e Laval, Ste-Foy, Qu´ebec, QC, Canada G1V 0A6 Unit´e de recherche en sant´e publique (CHUL-CHUQ), D´epartement de m´edecine sociale et pr´eventive, Universit´e Laval, Qu´ebec, QC, Canada

a r t i c l e

i n f o

Article history: Received 21 August 2007 Received in revised form 1 March 2008 Accepted 18 March 2008 Available online 27 March 2008 Keywords: Organochlorines Metabolites PCB DDE Mixture Embryo Blastocyst Porcine

a b s t r a c t Environmental exposure of human populations to organochlorines is still widespread despite several international regulations banning or restricting their use. This study tested the hypothesis that an environmentally relevant complex mixture of organochlorines comprising polychlorinated biphenyls (PCBs), technical chlordane, dichlorodiphenyldichloroethylene and 12 other components is toxic for porcine embryos (at relative concentrations of 1–10 000-fold the environmental organochlorine levels of contamination or 4.2 ␮g/l total PCBs). We also tested the embryotoxicity of a metabolised organochlorine mixture (relative concentrations of 0.9, 1.8, 2.7, 3.6 and 4.5 ␮g/l hydroxy-PCBs (OH-PCBs)) obtained by extracting plasma samples from sows treated with the native mixture. Embryos produced in vitro were exposed to either the organochlorine mixture or the metabolised extract for 9 days. The organochlorine mixture reduced embryonic development at the 10 000× concentration (relative concentration of 42 mg/l PCBs; p = 0.05). The organochlorine mixture also reduced the mean number of blastomeres per expanded blastocyst in a dose-dependent manner (p = 0.038) but did not induce blastomere apoptosis (p > 0.05). In contrast, the metabolised extract did not affect development or blastomere number at the concentrations tested, although the highest level of this mixture (4.5 ␮g/l OH-PCBs) was still very low (i.e. similar to the 1× concentration of the organochlorine mixture, which also did not alter embryo parameters). These data lead to the conclusion that while high concentrations of the native organochlorine mixture are toxic for porcine embryos, concentrations of either the native or the metabolised mixture that bear some relevance to exposure of human populations in the Arctic were without observable effect. © 2008 Elsevier Inc. All rights reserved.

1. Introduction Organochlorine compounds, such as dichlorodiphenyltrichloroethane (DDT), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), are well known for their detrimental effects on the health of humans and wildlife. Since the use and production of those compounds have been restricted or banned, their concentration in the Arctic biota has started to slowly decrease [1]. Their presence in the environment and food, however, is still significant and will remain so for the future decades since their half-lives are generally very long [1]. In the Arctic circumpolar circle, including Canada, Alaska, Russia, Norway, Greenland, Iceland, Sweden and Finland, the concentration of organochlorines in the environment and biota is still high [1]. These chemicals are biomagnified in the Arctic food web up to the higher predators: Arctic cod, marine mammals, polar bears and humans.

∗ Corresponding author. Tel.: +1 418 656 2131x3354; fax: +1 418 656 3766. E-mail address: [email protected] (J.L. Bailey). 0890-6238/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2008.03.003

For the populations that rely on Arctic sea products as the main food source, contamination is inevitable. High blood levels of organochlorines have been reported in Northern Canada [2], Greenland [3] and Russia [4]. Organochlorine concentrations in breast milk from Inuit women in Nunavik are also very high [2], supporting the fact that women of procreating age are contaminated, thus creating an exposure risk for their progeny [5]. The in utero exposure of an embryo starts as early as the moment of fertilization. Previous reports describe in utero exposure to organochlorines as single compounds or as mixtures [6–8]. In these studies, it is impossible to determine pre- versus post-implantation effects of the organochlorine exposure. The impact of preimplantation exposure should not be underestimated, since contamination of ovarian, oviductal or uterine tissues or fluids has been reported [9–11]. The preimplantation embryo is therefore directly exposed to the mother’s contaminants before the placenta is formed. Preimplantation is a critical window for the development of the future individual, therefore, exposure to contaminants during this stage might impair future physiology and health [12–16]. Exposing porcine embryos to a mixture of organochlorines metabolised by adult female pigs and extracted from their plasma

362

C. Campagna et al. / Reproductive Toxicology 25 (2008) 361–366

would be physiologically relevant. This would allow the embryos to be exposed in vitro to the contaminants likely present in the oviduct in vivo. Hence, not only would the embryos be exposed to an environmentally relevant mixture of parent organochlorines, they would also be exposed to a mixture of organochlorine metabolites that are generated in vivo through biotransformation reactions. These metabolites could be embryotoxic by themselves or interact with the parent compounds to modulate their toxicity. To date, no report on the effect of mixtures of organochlorines on the preimplantation embryonic development is available. The first aim of this study was therefore to determine the effects of a mixture of organochlorines designed to resemble that present in the Arctic aquatic food chain [17] on the preimplantation development, using the pig as a model for human. The second aim of this study was to determine the effect of the mixture that has been metabolised in vivo, i.e. extracted from the sows to which the mixture was administered by the oral route during several months on embryonic development [8]. Taking into account the known embryotoxicity of PCBs and DDT [7,12–16], we hypothesized that the exposure of embryos to the native mixture of organochlorine compounds or to the metabolised mixture would reduce embryonic development and the quality of the developed embryos. 2. Materials and methods 2.1. Organochlorine mixture and metabolised extract The organochlorine mixture was designed to approximate the profile of organochlorines found in Arctic ringed seal blubber [17], which is frequently consumed by the Inuit population. Pure organochlorine compounds (15 components in total) or technical mixtures were dissolved in dimethyl sulfoxide (DMSO) as described previously [18] to obtain the proportions listed in Table 1, and a total PCB concentration of 42 mg/ml. The metabolised extract was isolated from the plasma of control sows and sows highly exposed to the organochlorine mixture were obtained from a previous study [8] and kept at −20 ◦ C. Frozen pooled plasma from controluntreated and -treated sows were processed in parallel for the extraction. The final extracts were dissolved in DMSO to obtain proportions summarized in Table 2. The extraction procedure as well as the complete composition of the extracts (controland organochlorine-treated sows) are described fully in a previous study [19].

Table 2 Concentrations (␮g/l) of the major organochlorines and their metabolites in plasma extract from sows orally exposed to the organochlorine mixture used in Experiment 2 [19] Substances

Abbreviation

Polychlorinated biphenyls congeners 2,2 ,6,6 -Tetrachlorobiphenyl 2,2 ,4,5,5 -Pentachlorobiphenyl 2,3 ,4,4 ,5-Pentachlorobiphenyl 2,2 ,3,4,4 ,5 -Hexachlorobiphenyl 2,2 ,4,4 5,5 -Hexachlorobiphenyl 2,2 ,3,3 ,4,4 ,5-Heptachlorobiphenyl 2,2 ,3,4,4 ,5,5 -Heptachlorobiphenyl 2,2 ,3,4,4 ,5 ,6-Heptachlorobiphenyl 2,2 ,3,4 ,5,5 ,6-Heptachlorobiphenyl 2,2 ,3,3 ,4,5 ,6,6 -Octachlorobiphenyl

PCB 54 PCB 101 PCB 118 PCB 138 PCB 153 PCB 170 PCB 180 PCB 183 PCB 187 PCB 201

695 442 835 1463 2,256 772 1,957 357 729 425

Hydroxy-PCBs 4-Hydroxy-2,3,3 ,4 ,5-pentachlorobyphenyl 4-Hydroxy-2,3,3 ,4 ,5,5 -hexachlorobiphenyl

4-OH-PCB 107 4-OH-PCB 162

3,270 705

Oxychlordane trans-Nonachlor p,p -Dichlorodiphenoldichloroethylene p,p -Dichlorodiphenyltrichloroethane Dieldrin Pentachlorophenol Hexachlorobenzene

p,p -DDE p,p -DDT

HBC

Concentration (␮g/l)

454 425 15,060 2,133 1,220 556 720

The complete compositions of the extracted metabolised mixture and of the control extract are presented in a previous study [19]. Concentrations of PCBs, OH-PCBs and p,p -DDE in the control extract do not exceed 1.2, 2.3 and 3.5 ␮g/l, respectively. Detection limit was 0.1 ␮g/l.

ON]) [18]. The pFF was collected from follicles (4–6 mm diameter) of unexposed prepubertal gilt ovaries as described earlier [18]. The medium used for in vitro fertilization (IVF) was a modified Tris-buffered medium (mTBM) containing 0.1% (w/v) BSA (fraction V; A4503) and 1 mM caffeine [18,21]. Sperm-washing medium was Beltsville Thawing Solution (BTS [22]). The culture medium (IVC) for embryonic development was NCSU 23 medium supplemented with 0.4% (w/v) BSA (A4503) [18].

2.2. Media Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). The oocyte in vitro maturation (IVM) medium was a BSA-free NCSU 23 (North Carolina State University [20]) supplemented with 25 ␮M ␤-mercaptoethanol (Bio-Rad, Hercules, CA), 0.1 mg/ml cysteine, 10% (v/v) porcine follicular fluid (pFF), 1 mM dibutyryl-cAMP and hormonal supplements (10 IU/ml hCG and 10 IU/ml PMSG [“Folligon” and “Chorulon”, Intervet, Whitby,

Table 1 Composition of the organochlorine mixture used in Experiment 1 [18] Substances Aroclor and congener neat mixa Technical chlordane p,p -Dichlorodiphenyldichloroethylene p,p -Dichlorodiphenyltrichloroethane Technical toxaphene ␣-Hexachlorocyclohexane Aldrin Dieldrin 1,2,4,5-Tetrachlorobenzene p,p -Dichlorodiphenyldichloroethane ␤-Hexachlorocyclohexane Hexachlorobenzene Mirex Lindane Pentachlorobenzene

Abbreviation

p,p -DDE p,p -DDT ␣-HCH

p,p -DDD ␤-HCH HCB ␥-HCH

% Relative weight 32.4 21.4 19.3 6.8 6.5 6.2 2.5 2.1 0.9 0.5 0.4 0.4 0.2 0.2 0.2

Origin and CAS number of each substance are presented in previous studies [18,25]. a Mix containing Aroclor 1260 (58.9%), Aroclor 1254 (39.3%), 2,4,4 trichlorobiphenyl (PCB 28; 1.0%), 2,2 ,4,4 -tetrachlorobiphenyl (PCB 47; 0.8%), 3,3 ,4,4 ,5-pentachlorobiphenyl (PCB 126; 0.02%), and 3,3 , 4,4 -tetrachlorobiphenyl (PCB 77; 0.004%).

2.3. In vitro maturation Cumulus–oocyte complexes (COCs) were obtained and prepared as described previously [18]. Briefly, slaughterhouse ovaries were collected from prepubertal gilts and transported to the laboratory within 1 h. Follicles (4–6 mm) were punctured, and COCs were aspirated and washed three times with Hepes-buffered Tyrode medium containing 0.1% polyvinyl alcohol (PVA-TL-HEPES) [23]. Those with an unexpanded, compact cumulus exhibiting uniform cytoplasm were selected for IVM. Selected COCs were washed three times in IVM medium and transferred into four-well multidishes (Nunc, Rosekilde, Denmark) containing 500 ␮l of the same medium covered with 500 ␮l mineral oil. COCs (30 per wells) were cultured for 20 h at 38.5 ◦ C in an atmosphere of 5% CO2 in air and 100% humidity. Complexes were then washed and transferred into fresh IVM medium without dibutyryl-cAMP or hormonal supplements and cultured for another 24 h [23].

2.4. In vitro fertilization After maturation, oocytes were mechanically denuded with IVM medium supplemented with 0.1% (w/v) hyaluronidase [18]. Denuded oocytes were washed three times with IVF medium and placed in 50 ␮l droplets of IVF medium covered with mineral oil as described previously [18]. Dishes were incubated (38.5 ◦ C, 5% CO2 and 100% humidity) until insemination (1 h). Boar semen was frozen in straws according to the method described previously [19]. A straw of frozen boar semen was thawed at 55 ◦ C for 12 s then transferred in 2 ml of sperm-washing medium at 37 ◦ C. The sperm solution was layered over a 65/70% Percoll gradient, centrifuged, and washed following an earlier procedure [18]. The sperm pellet was then resuspended in 0.5–1 ml IVF medium. Sperm motility and concentration were assessed using a Ceros computerassisted semen analysis system (Hamilton Thorne, Beverly, MA, USA) with the parameters set as previously described [24]. The sperm were diluted in IVF medium to a concentration of 300 000 motile sperm/ml. Aliquots (50 ␮l) of the sperm suspension were added to the drops containing the oocytes (15 per drop), for a final sperm concentration of 150 000 motile sperm/ml. Gametes were coincubated for 6 h.

C. Campagna et al. / Reproductive Toxicology 25 (2008) 361–366 2.5. In vitro development At the end of the coincubation period, zygotes were washed three times in IVC medium and 15 zygotes were transferred randomly into a culture well of a 96-well ´ multidish (BD Falcon, VWR International, Montreal, Canada) containing 200 ␮l of IVC medium. To favour organochlorine contact with the embryos, no mineral oil was layered over the IVC medium and a total of 4 ml ultra-pure water was poured between and around the surrounding wells to prevent excess evaporation. Embryos were cultured for 8 days (38.5 ◦ C, 5% CO2 and 100% humidity). After 12 h of culture, two groups of control embryos (30 embryos in total) per repetition were fixed in 100 ␮l 4% paraformaldehyde for 15 min, then washed three times in 200 ␮l 0.5% Triton X-100 before being mounted over a drop of Mowiol gelatin containing 5 mg/ml Hoechst 33342 (Invitrogen Canada, Burlington, Canada) [18] and examined under ultraviolet illumination. Penetration (50–70%) and polyspermy (20%) rates (based on pronuclei formation) were calculated. The rates of blastocyst formation at Day 8 post-insemination were tabulated and expressed as percentages of penetrated oocytes. Expanded blastocysts were fixed and mounted, and their total number of cells was tabulated. 2.6. TUNEL assay Apoptosis in the organochlorine-exposed blastocysts was assessed using the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. After 8 days of development, blastocysts were washed three times in PBS supplemented with 3 mg/ml polyvinylalcohol (PBS-PVA). Embryos were then fixed in 4% paraformaldehyde in PBS for 1 h at room temperature (RT) and then stored until use in 1% paraformaldehyde in PBS at 4 ◦ C. Embryos were washed three times in PBS-PVA and permeabilized with 0.5% Triton X-100 in PBS (v/v) for 1 h at RT. The embryos were washed again twice in PBS-PVA and processed for TUNEL as per the In Situ Cell Death Detection Kit (#1684795 Roche Diagnostics Canada, Laval, Canada), with slight modification. Briefly, embryos were divided into five groups: positive controls, negative controls, unexposed embryos, DMSO-treated embryos and 1000× embryos. The positive controls were incubated in RQ1 DNase (50 U/ml, Promega, Fisher Scientific Ltd., Nepean, Canada) for 20 min at RT and washed twice in PBS-PVA. Negative controls were transferred to 10 ␮l droplets of the fluorescein-conjugated dUTP only. Positive controls and experimental embryos were transferred to 10 ␮l droplets of the terminal deoxynucleotidyl transferase enzyme and the fluorescein-conjugated dUTP (Roche Diagnostics Canada, Laval, Canada) in a 1:10 ratio. All embryos were incubated (38.5 ◦ C, 5% CO2 and 100% humidity) for 1 h. After, embryos were washed twice with 0.5% Triton in PBS, once with PBS-PVA and once with RNase buffer (40 mM Tris, 10 mM NaCl, 6 mM MgCl2 , pH 8.0). The embryos were then incubated in 0.1 mg RNase (Roche Diagnostics Canada, Laval, Canada) for 1 h at 38.5 ◦ C, washed twice in RNase buffer and counterstained with propidium iodide (5 ␮g/ml) for 45 min (38.5 ◦ C, 5% CO2 and 100% humidity). The embryos were then mounted onto microscope slides using FluoroGuard antifade reagent (Bio-Rad, Hercules, CA) and analyzed by fluorescent microscopy. Since high concentrations of organochlorines did not affect apoptosis in the blastocysts, the metabolite-exposed blastocysts were not processed for this assay. 2.7. Experiment 1: exposing embryos to an organochlorine mixture As for our previous reports using this mixture of organochlorines [18,19,25,26], dilutions of the stock organochlorine mixture were made in DMSO so that the IVC medium contains a final concentration of 0.1% DMSO with the following concentrations of the organochlorine mixture: 0×, 1×, 10×, 100×, 1000×, and 10 000×. The concentration referred to as the 1× concentration contains 4.2 ␮g/l total PCBs. Concentrations of other organochlorines can be calculated from proportions listed in Table 1. The 1× and 10× concentrations are within the range of concentrations of total PCBs in plasma samples measured in Inuit women of reproductive age from Nunavik (1.0–47.9 ␮g/l plasma [2]) and Greenland (3.0–95.3 ␮g/l plasma [3]). Treatment “0” contained vehicle only (0.1% DMSO). An untreated control (no DMSO) was also included.

363

0.3–13.3 ␮g/l p,p -DDE [2,27], respectively) and Greenland (0.5–29.9 ␮g/l p,p -DDE [3]). An untreated control (no DMSO) and a vehicle control (0.1% DMSO) were also included. 2.9. Statistical analyses Unless otherwise stated, experiments were repeated five times and in duplicate. Data for the blastocyst outcome were expressed as percentage of penetrated oocytes, while the cell death data were expressed as percentages. Those data were submitted to angular transformation (arcsine of square root) while the data for the number of cells per blastocyst were submitted to square root transformation. All were analyzed by the PROC MIXED procedure to detect overall treatment effects including controls. Normality and homogeneity of variance criteria were verified using Levene’s test and the univariate procedure, respectively. For models having p-values <0.05, the Duncan protected least significant difference test was applied for multiple comparisons among treatments. Dose–response effect of treatments was analyzed by simple regression excluding the untreated control (Experiment 1) or the untreated and vehicle controls (Experiment 2). The level of significance was set to p ≤ 0.05 while a p-value between 0.05 and 0.1 was considered as a trend or tendency. All statistical analyses were performed using SAS Software (v 8.0, SAS Institute, Cary, NC).

3. Results 3.1. Experiment 1: original organochlorine mixture No difference was observed between the untreated control versus the vehicle (0×) control on any of the observed embryonic parameters (p > 0.05). Exposing embryos to the organochlorine mixture significantly reduced embryonic development at Day 7 to blastocysts (Fig. 1; p < 0.0001). Blastocyst outcome was significantly reduced at the 10 000× concentration compared to the vehicle control (0×; p = 0.05). A significant quadratic regression (p < 0.0001; r2 = 0.77) was observed between the rate of blastocyst formation and the organochlorine concentration. Exposure of embryos to the organochlorine mixture reduced the total number of blastomeres, as demonstrated by a linear dose–response (p = 0.0376; r2 = 0.021; Fig. 2), but the overall model including the untreated control was not significant (p = 0.38). Exposure of embryos to the organochlorine mixture, however, had no effect on the number of apoptotic blastomeres per expanded blastocyst (TUNEL assay; p = 0.43; Fig. 3). 3.2. Experiment 2: metabolised organochlorine extract Exposing porcine embryos to the metabolised extract affected neither the blastocyst outcome (Fig. 4; p = 0.22) nor the number of

2.8. Experiment 2: exposing embryos to the metabolised extract Stock dilutions of the plasma extract containing the mixture of organochlorine metabolites were prepared every week in DMSO as previously described [19]. Stock extract from the control (unexposed) sow plasma was used as an extraction control and is designated as 0 ␮g/l hydroxy-PCBs (OH-PCBs). All stock dilutions were diluted to a final concentration of 0.1% (v/v) DMSO in the IVC medium the morning of experiment and left to equilibrate at 38.5 ◦ C, 5% CO2 in air and 100% humidity for at least 3 h. The concentrations of the metabolised extract in the IVC medium is expressed as the sum of OH-PCBs: 0.9, 1.8, 2.7, 3.6 and 4.5 ␮g/l. Concentrations of other organochlorines and metabolites can be calculated from proportions listed in Table 2. The range of OH-PCBs concentrations (0–4.5 ␮g/l) and other metabolites (0–15 ␮g/l p,p -DDE) used in this study corresponds to concentrations of organochlorines and metabolites found in plasma samples from Inuit men and women of reproductive age in Nunavik (0.12–11.6 ng/g whole blood wet weight OH-PCBs and

Fig. 1. Rate of development to blastocyst stage of fertilized oocytes during IVC in the presence of an environmentally relevant organochlorine mixture. The rates of blastocyst formation are expressed as percentages of penetrated oocytes. The 1× concentration corresponds to the mean concentration of PCBs present in plasma samples from Inuit women living in Nunavik (4.2 ␮g/l). Means with different letters are statistically different (p = 0.05 for overall treatment effect). A significant quadratic regression was noted in response of organochlorine level (p < 0.0001; r2 = 0.77). Each point represents the mean and S.E.M. of six repetitions in duplicate. The total number of embryos per treatment was 177 (control), 204 (0×), 179 (1×), 188 (10×), 208 (100×), 177 (1000×), and 200 (10 000×).

364

C. Campagna et al. / Reproductive Toxicology 25 (2008) 361–366

Fig. 2. Mean blastomere number per expanded blastocyst developed by IVC in the presence of an environmentally relevant organochlorine mixture. The 1× concentration corresponds to the mean concentration of PCBs present in plasma samples from Inuit women living in Nunavik (4.2 ␮g/l). The p-value (0.38) is for overall treatment effect. A linear regression was noted (r2 = 0.02; p = 0.0376). Each point represents the mean and S.E.M. of six repetitions in duplicate. The total number of blastocysts per treatment was 41 (control), 34 (0×), 37 (1×), 45 (10×), 23 (100×), and 20 (1000×).

Fig. 5. Mean blastomere number per expanded blastocyst developed by IVC in the presence of the metabolised organochlorine mixture. Each point represents the mean and S.E.M. of six repetitions in duplicate. There was no effect of treatment (p = 0.62). The total number of blastocysts per treatment was 18 (control), 16 (DMSO), 14 (0 ␮g), 10 (0.9 ␮g), 18 (1.8 ␮g), 15 (2.3 ␮g), 18 (3.6 ␮g), and 12 (4.5 ␮g). “Control” refers to the maturation control; “DMSO”, to the 0.1% vehicle control; and “0 ␮g/l OH-PCBs”, to the extraction control.

4. Discussion This study is the first to assess the effects of an environmentally relevant organochlorine mixture on in vitro preimplantation development. Results from the present experiments indicate that exposing porcine embryos to a mixture of organochlorines during in vitro preimplantation development reduced development to blastocysts and the number of blastomeres in the expanded blastocyst that were able to form. Exposing embryos to the metabolised extract at concentrations similar to those observed in the plasma of Arctic populations, however, did not induce any effect on the parameters assayed. Fig. 3. Mean rate of blastomere apoptosis (TUNEL assay) in expanded blastocysts developed by IVC in the presence of an environmentally relevant organochlorine mixture. The 1000× concentration corresponds to 1000-fold the mean concentration of PCBs present in plasma samples from Inuit women living in Nunavik (4.2 ␮g/l). Treatment did not affect apoptosis rate (p = 0.43). The total number of blastocysts per treatment was 17 (control), 10 (0×), and 12 (1000×).

blastomeres per expanded blastocyst (p = 0.62; Fig. 5). There was no effect of the control sow extract (0 ␮g/l OH-PCBs) or the vehicle control compared to the untreated control on the observed parameters (p > 0.05).

Fig. 4. Rate of development to blastocyst stage of fertilized oocytes during IVC in the presence of the metabolised organochlorine mixture. The rates of blastocyst formation are expressed as percentages of penetrated oocytes. Each point represents the mean and S.E.M. of six repetitions in duplicate. There was no effect of treatment (p = 0.22). The total number of embryos per treatment was 105 (control), 142 (DMSO), 140 (0 ␮g/l), 140 (0.9 ␮g/l), 141 (1.8 ␮g/l), 120 (2.3 ␮g/l), 118 (3.6 ␮g/l), and 121 (4.5 ␮g/l). “Control” refers to the maturation control; “DMSO”, to the 0.1% vehicle control; and “0 ␮g/l OH-PCBs”, to the extraction control.

4.1. The organochlorine mixture induces embryotoxicity The results from Experiment 1 using the organochlorine mixture support previous studies demonstrating the detrimental effect of several organochlorines on early embryonic development [12–14,16,28]. In the rabbit, in vitro exposure of in vivo-derived morulae to mixtures of coplanar (PCB-77, -126 and -169) and noncoplanar (PCB-28, -52, -118, -138, -153 and -180) PCB congeners reduced blastocyst formation and cell proliferation at relatively high concentrations (30 and 6 mg/l PCBs, respectively) [12]. Similar results (reduced blastocyst formation and cell proliferation) were obtained when exposing those embryos to 5 mg/l Aroclor 1260, a commercial PCB mixture [13]. In our experiments, a concentration of 0.4 mg/l PCBs corresponds to the 100× concentration while 42 mg/l PCBs corresponds to the 10 000× concentration that inhibited blastocyst formation (Table 1; PCBs account for 32.4% of our organochlorine mixture). In the mouse, in vitro exposure to the PCB mixture Aroclor 1254 reduced development to four-cell-stage embryos and blastocysts at a concentration of 0.1 mg/l PCB [14], while exposure to DDT and lindane reduced blastocyst outcome at concentrations of 3.6 and 7.2 mg/l, respectively [16]. In vivo exposure of rabbit does to Aroclor 1260 (4 mg/kg body weight) increased blastocyst loss by 20% [28]. Together, the results from these studies and our data from Experiment 1 with the organochlorine mixture (Figs. 1–3) reveal that organochlorine compounds clearly induce embryotoxicity at concentrations only slightly greater than reported in human populations in the Arctic. Many of the compounds present in the native organochlorine mixture are known endocrine-disrupting compounds (PCB, DDT, DDE, and lindane) [29]. In Fig. 1, the 10× concentration seems to enhance blastocyst outcome compare to

C. Campagna et al. / Reproductive Toxicology 25 (2008) 361–366

DMSO (0×), whereas the highest concentration of the mixture completely inhibited it. This kind of inverted U-shaped curve is often associated with endocrine active chemicals [30,31]. In the porcine embryo, the estrogen receptor is expressed in the early and late cell cycles of preimplantation development [32,33] and exposure to the anti-estrogen nafoxidine (15 ␮g/ml) or to 5% estradiol-17␤antiserum reduced blastocyst outcome [34]. The reduced rate of blastocyst formation caused by the organochlorine treatment in our study does not involve the apoptosis pathway (Fig. 3) and thus may imply delayed or faulty cell division, as confirmed by the lower number of blastomeres in the treated blastocyst (Fig. 2). In a previous study, we showed that the same organochlorine mixture reduced maturation and developmental competence of porcine oocytes in vitro [18]. We hypothesized that those effects were mainly due to a disruption of intercellular communication. The role of gap junctional communication during preimplantation development and in blastocysts, however, is still controversial (for review, see [35]). Another possible toxicity pathway would be through the activation of the aryl hydrocarbon receptor (AhR), which is known to mediate the toxic effects induced by coplanar PCBs and dioxins (for review, see [36]). On the other hand, the implication of AhR activation during preimplantation is still unclear, since PCBs (both coplanar and non-coplanar) induced transcriptional changes in AhR target genes in exposed rabbit blastocysts without involving the AhR signal transduction pathway [37]. Recently, a study reported AhR-independent elevated expression of CYP 1A1, CYP 1B1, VEGFR2 and COX-2 by PCBs (at 0.1 ␮g/l and/or 1 mg/l) in the rabbit blastocyst [38]. Those particular genes are essential for metabolising xenobiotics (CYP 1A1 and 1B1), vascularization (VEGFR) and implantation (COX) [38]. 4.2. Low concentrations of metabolised extract do not affect embryonic development

365

4.3. Are embryos more resistant to contaminants than oocytes? From these results, the porcine blastocysts showed signs of embryotoxicity at much higher concentrations (10 000×; 42 mg/l PCBs) than we observed for oocytes in an earlier study (100×; 0.42 mg/l PCBs [18]). An in vivo mouse study showed no signs of embryotoxicity resulting from lindane exposure during the preimplantation period at a dose (25 mg/kg/day)) that increased the number of degenerated embryos if the exposure period extended from ovulation to fertilization [40]. Rabbit embryos are sensitive to 5 mg/l PCBs, but not to 0.5 mg/l, which is consistent with our results [13]. Reduced blastocyst formation was also observed in the same model following 6–60 mg/l PCBs [12]. No data are available concerning embryotoxicity in higher animal models such as bovine, porcine and ovine. This difference in sensitivity between the oocyte and the embryo is not clear. Up to the third cell cycle, porcine embryos have not initiated their RNA transcription [41], and might therefore be less vulnerable to the environment. Moreover, blastocysts seem to express more biotransformation enzymes (CYP 1A1, CYP 1A2, CYP 1B2, glutathione S-transferase, UDPglucuronosyl transferase, NADH ubiquinone oxydoreductase [37]) than the oocyte (CYP 1A1 and glutathione S-transferase [42,43]); this may therefore increase the resistance of embryos to the environment compared to the oocyte. The absence of detectable embryotoxicity in our experimental conditions, however, does not necessarily imply that the concentrations tested have no adverse effect. In vivo-produced rabbit blastocysts exposed 4 h to mixtures of PCBs showed no signs of embryotoxicity, but had detectable variation in the transcripts of genes that are essential for implantation and early development [38]. Such modifications at the cellular level can therefore affect the health of the future individual without inducing early embryotoxicity [44]. 4.4. Summary and conclusion

The results obtained from Experiment 2 using the metabolised organochlorine extract do not support our hypothesis of a detrimental effect induced by the organochlorine metabolites on embryonic development. The range of OH-PCBs concentrations tested in this study (up to 4.6 ␮g/l) encompasses those found in the plasma of Inuit women in Nunavik (0.12–11.6 ng/g whole blood wet weight OH-PCBs [2]). The strength of this study is that the metabolites were extracted from the plasma of sows highly exposed to the original organochlorine mixture [8], although this is at the same time its weakness, since we were restricted in the range of concentrations that we could use without increasing the solvent concentration (0.1% DMSO). It would have been interesting to test higher concentrations of metabolites, but the increased concentrations of DMSO might also affect embryonic development [39]. Specifically, the highest level of the metabolite mixture (4.5 ␮g/l OH-PCBs) only approximates the 1× concentration of the original organochlorine mixture, which also did not adversely affect the embryos. It is therefore impossible to conclude that the metabolised mixture is less toxic than the native organochlorine mixture, because the levels tested are so different. We would have liked to test the impact of higher levels of the metabolised extract, however, we were limited by the starting concentration of our stock. Perhaps higher levels of the metabolised organochlorine extract would have a direct toxic effect on the embryos. Moreover, preimplantation development is not the only indication of harmful effects. For instance, it is possible that other developmental parameters could be affected later on, such as implantation, placental formation, sex determination and postnatal development and function.

In summary, this study indicates that exposure of porcine embryos to a high concentration of an environmentally relevant complex organochlorine mixture harms embryonic development. In contrast, exposing porcine embryos to the metabolised extract did not cause any observable effect on blastocyst development. This study, therefore, confirms the embryotoxicity potential of organochlorines and also indicates a certain tolerance of preimplantation embryos to xenobiotics that is not apparent in oocytes. Acknowledgments The authors thank Kristina Braun and Dr. W. Allan King for performing the TUNEL experiment and Pierre Dumas from the Laboratoire de toxicologie of the Institut national de sant´e publique du Qu´ebec for preparing the plasma extracts and performing the organochlorine analyses. We are also grateful to the Centre d’ins´emination porcine du Qu´ebec for donating semen and Salaisons Brochu slaughterhouse for the ovaries. This research was supported by the Northern Contaminants Program of Indian and Northern Affairs of Canada and by the Toxic Substances Research Initiative Program of Health Canada. C. Campagna was a recipient of an NSERC Ph.D. fellowship. References [1] AMAP. AMAP assessment 2002: persistent organic pollutants in the Arctic. Oslo, Norway: Arctic Monitoring and Assessment Programme (AMAP); 2004, xvi + 310. [2] Muckle G, Ayotte P, Dewailly E, Jacobson SW, Jacobson JL. Prenatal exposure of the northern Quebec Inuit infants to environmental contaminants. Environ Health Perspect 2001;109:1291–9.

366

C. Campagna et al. / Reproductive Toxicology 25 (2008) 361–366

[3] Bjerregaard P, Hansen JC. Organochlorines and heavy metals in pregnant women from the Disko Bay area in Greenland. Sci Total Environ 2000;245:195–202. [4] Van Oostdam JC, Dewailly E, Gilman A, Hansen JC, Odland JO, Chashchin V, et al. Circumpolar maternal blood contaminant survey, 1994–1997 organochlorine compounds. Sci Total Environ 2004;330:55–70. [5] Dewailly E, Ayotte P, Bruneau S, Gingras S, Belles-Isles M, Roy R. Susceptibility to infections and immune status in Inuit infants exposed to organochlorines. Environ Health Perspect 2000;108:205–11. [6] Foster WG, Younglai EV, Boutross-Tadross O, Hughes CL, Wade MG. Mammary gland morphology in Sprague–Dawley rats following treatment with an organochlorine mixture in utero and neonatal genistein. Toxicol Sci 2004;77:91–100. [7] Bowers WJ, Nakai JS, Chu I, Wade MG, Moir D, Yagminas A, et al. Early developmental neurotoxicity of a PCB/organochlorine mixture in rodents after gestational and lactational exposure. Toxicol Sci 2004;77:51–62. [8] Bilrha H, Roy R, Wagner E, Belles-Isles M, Bailey JL, Ayotte P. Effects of gestational and lactational exposure to organochlorine compounds on cellular, humoral, and innate immunity in swine. Toxicol Sci 2004;77:41–50. [9] Lindenau A, Fischer B, Seiler P, Beier HM. Effects of persistent chlorinated hydrocarbons on reproductive tissues in female rabbits. Hum Reprod 1994;9:772– 80. [10] Trapp M, Baukloh V, Bohnet HG, Heeschen W. Pollutants in human follicular fluid. Fertil Steril 1984;42:146–8. [11] Younglai EV, Foster WG, Hughes EG, Trim K, Jarrell JF. Levels of environmental contaminants in human follicular fluid, serum, and seminal plasma of couples undergoing in vitro fertilization. Arch Environ Contam Toxicol 2002;43:121– 6. [12] Kuchenhoff A, Eckard R, Buff K, Fischer B. Stage-specific effects of defined mixtures of polychlorinated biphenyls on in vitro development of rabbit preimplantation embryos. Mol Reprod Dev 1999;54:126–34. [13] Lindenau A, Fischer B. Embryotoxicity of polychlorinated biphenyls (PCBs) for preimplantation embryos. Reprod Toxicol 1996;10:227–30. [14] Kholkute SD, Rodriguez J, Dukelow WR. Reproductive toxicity of Aroclor-1254: effects on oocyte, spermatozoa, in vitro fertilization, and embryo development in the mouse. Reprod Toxicol 1994;8:487–93. [15] Greenlee AR, Quail CA, Berg RL. Developmental alterations in murine embryos exposed in vitro to an estrogenic pesticide, o,p -DDT. Reprod Toxicol 1999;13:555–65. [16] Alm H, Tiemann U, Torner H. Influence of organochlorine pesticides on development of mouse embryos in vitro. Reprod Toxicol 1996;10:321–6. [17] Muir DC, Riget FF, Cleemann M, Skaare J, Kleivane L, Nakata H, et al. Circumpolar trends of PCBs and organochlorine pesticides in the Arctic marine environment inferred form levels in ringed seals. Environ Sci Technol 2000;34:2431–8. [18] Campagna C, Sirard MA, Ayotte P, Bailey JL. Impaired maturation, fertilization, and embryonic development of porcine oocytes following exposure to an environmentally relevant organochlorine mixture. Biol Reprod 2001;65:554–60. [19] Campagna C, Ayotte P, Sirard MA, Arsenault G, Laforest JP, Bailey JL. Effect of an environmentally relevant metabolized organochlorine mixture on porcine cumulus-oocyte complexes. Reprod Toxicol 2007;23:145–52. [20] Petters RM, Wells KD. Culture of pig embryos. J Reprod Fertil Suppl 1993;48:61–73. [21] Abeydeera LR, Wang WH, Cantley TC, Prather RS, Day BN. Presence of betamercaptoethanol can increase the glutathione content of pig oocytes matured in vitro and the rate of blastocyst development after in vitro fertilization. Theriogenology 1998;50:747–56. [22] Johnson LA, Weitze KF, Fiser P, Maxwell WM. Storage of boar semen. Anim Reprod Sci 2000;62:143–72. [23] Funahashi H, Cantley TC, Day BN. Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization. Biol Reprod 1997;57:49– 53.

[24] Dube C, Beaulieu M, Reyes-Moreno C, Guillemette C, Bailey JL. Boar sperm storage capacity of BTS and Androhep Plus: viability, motility, capacitation, and tyrosine phosphorylation. Theriogenology 2004;62:874–86. [25] Campagna C, Guillemette C, Paradis R, Sirard MA, Ayotte P, Bailey JL. An environmentally relevant organochlorine mixture impairs sperm function and embryo development in the porcine model. Biol Reprod 2002;67:80–7. [26] Campagna C, Bailey JL, Sirard MA, Ayotte P, Maddox-Hyttel P. An environmentally-relevant mixture of organochlorines and its vehicle control, dimethylsulfoxide, induce ultrastructural alterations in porcine oocytes. Mol Reprod Dev 2006;73:83–91. [27] Sandau CD, Ayotte P, Dewailly E, Duffe J, Norstrom RJ. Analysis of hydroxylated metabolites of PCBs (OH-PCBs) and other chlorinated phenolic compounds in whole blood from Canadian Inuit. Environ Health Perspect 2000;108:611–6. [28] Seiler P, Fischer B, Lindenau A, Beier HM. Effects of persistent chlorinated hydrocarbons on fertility and embryonic development in the rabbit. Hum Reprod 1994;9:1920–6. [29] National Research Council US. Hormonally active agents in the environment. Committee on hormonally active agents in the environment. Washington, DC: National Research Council US; 1999. [30] Li L, Andersen ME, Heber S, Zhang Q. Non-monotonic dose–response relationship in steroid hormone receptor-mediated gene expression. J Mol Endocrinol 2007;38:569–85. [31] Calabrese EJ, Baldwin LA. The frequency of U-shaped dose responses in the toxicological literature. Toxicol Sci 2001;62:330–8. [32] Ying C, Hsu WL, Hong WF, Cheng WT, Yang Y. Estrogen receptor is expressed in pig embryos during preimplantation development. Mol Reprod Dev 2000;55:83–8. [33] Kowalski AA, Graddy LG, Vale-Cruz DS, Choi I, Katzenellenbogen BS, Simmen FA, et al. Molecular cloning of porcine estrogen receptor-beta complementary DNAs and developmental expression in periimplantation embryos. Biol Reprod 2002;66:760–9. [34] Niemann H, Elsaesser F. Evidence for estrogen-dependent blastocyst formation in the pig. Biol Reprod 1986;35:10–6. [35] Houghton FD. Role of gap junctions during early embryo development. Reproduction 2005;129:129–35. [36] Safe SH. Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic responses, and implications for risk assessment. Crit Rev Toxicol 1994;24:87–149. [37] Kietz S, Fischer B. Polychlorinated biphenyls affect gene expression in the rabbit preimplantation embryo. Mol Reprod Dev 2003;64:251–60. [38] Clausen I, Kietz S, Fischer B. Lineage-specific effects of polychlorinated biphenyls (PCB) on gene expression in the rabbit blastocyst. Reprod Toxicol 2005;20:47–56. [39] Hallare AV, Kohler HR, Triebskorn R. Developmental toxicity and stress protein responses in zebrafish embryos after exposure to diclofenac and its solvent, DMSO. Chemosphere 2004;56:659–66. [40] Scascitelli M, Pacchierotti F. Effects of lindane on oocyte maturation and preimplantation embryonic development in the mouse. Reprod Toxicol 2003;17:299–303. [41] Viuff D, Greve T, Holm P, Callesen H, Hyttel P, Thomsen PD. Activation of the ribosomal RNA genes late in the third cell cycle of porcine embryos. Biol Reprod 2002;66:629–34. [42] Pocar P, Augustin R, Fischer B. Constitutive expression of CYP1A1 in bovine cumulus oocyte-complexes in vitro: mechanisms and biological implications. Endocrinology 2004;145:1594–601. [43] Rabahi F, Brule S, Sirois J, Beckers JF, Silversides DW, Lussier JG. High expression of bovine alpha glutathione S-transferase (GSTA1, GSTA2) subunits is mainly associated with steroidogenically active cells and regulated by gonadotropins in bovine ovarian follicles. Endocrinology 1999;140:3507–17. [44] Lane M, Gardner DK. Understanding cellular disruptions during early embryo development that perturb viability and fetal development. Reprod Fertil Dev 2005;17:371–8.