Developmental toxicity of hexachloronaphthalene in Wistar rats. A role of CYP1A1 expression

Reproductive Toxicology 58 (2015) 93–103

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Developmental toxicity of hexachloronaphthalene in Wistar rats. A role of CYP1A1 expression Anna Kilanowicz a,∗ , Piotr Czekaj b , Andrzej Sapota a , Malgorzata Skrzypinska-Gawrysiak a , Elzbieta Bruchajzer a , Adam Darago a , Ewa Czech c , Danuta Plewka b , Anna Wiaderkiewicz c , Krystyna Sitarek d a

Department of Toxicology, Faculty of Pharmacy, Medical University of Lodz, Poland Department of Cytophysiology, Chair of Histology and Embryology, School of Medicine in Katowice, Medical University of Silesia, Katowice, Poland c Department of Histology, Chair of Histology and Embryology, School of Medicine in Katowice, Medical University of Silesia, Katowice, Poland d Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, Lodz, Poland b

a r t i c l e

i n f o

Article history: Received 15 March 2015 Received in revised form 31 August 2015 Accepted 17 September 2015 Keywords: Polychlorinated naphthalenes Rat Embryotoxicity Developmental toxicity CYP1A1 Placenta

a b s t r a c t Hexachloronaphthalene (HxCN) is one of the most toxic congeners of polychlorinated naphthalenes (PCNs). This study assesses the prenatal toxicity of HxCN after daily administration at doses of 0.1–1.0 mg/kg b.w. to pregnant Wistar rats during organogenesis. We evaluated also the expression of CYP1A1 mRNA and protein in the livers of dams and fetuses, as well as the placenta. The results indicate that 0.3 mg/kg b.w. was the lowest HxCN toxic dose for dams (LOAEL) while a dose of 0.1 mg/kg b.w. was sufficient to impair the intrauterine development of embryos/fetuses without maternal toxicity. Regardless of the applied dose, HxCN generated embryotoxic effects. Dose-dependent fetotoxic effects were associated with HxCN exposure. HxCN was found to be a strong inducer of maternal and fetal CYP1A1. Expression of CYP1A1 mRNA in the placenta appears to be the most sensitive marker of HxCN exposure. © 2015 Elsevier Inc. All rights reserved.

1. Introduction The group of polychlorinated naphthalenes (PCNs) comprise 75 possible congeners in eight homologue groups, with one to eight chlorine atoms substituted around a planar aromatic naphthalene molecule. In the past, congener mixtures of PCNs were produced in several countries under the trade names Halowax, Nibren and Seekay waxes and Cerifal Materials [1]. PCNs were used mainly as flame retardants and dielectric fluids for capacitors. They also found an array of applications in industry, such as dye-making, fungicides in the wood, textile and paper industries, plasticizers, oil additives, casting materials for alloys and lubricants for graphite electrodes [1–3]. Despite the fact that the manufacture and application of PCNs have been formally restricted in the majority of countries, these compounds are still released into the environment, for example, as a result of thermal processing of plastics containing polychlorinated biphenyls (PCB), in which PCNs also occur as trace contaminants [1–4].

∗ Corresponding author at: Department of Toxicology, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90−151 Lodz, Poland. Fax: +48 42 677 91 48. E-mail address: [email protected] (A. Kilanowicz). http://dx.doi.org/10.1016/j.reprotox.2015.09.005 0890-6238/© 2015 Elsevier Inc. All rights reserved.

Owing to their chemical structure and physicochemical properties, i.e. their durability, weak water solubility and accumulation in all elements of the natural environment and total ecosystems, PCNs are candidates for inclusion in the Stockholm Convention on persistent organic pollutants (POPs) [5]. They can be transported over long distances, and have been detected in the air, water, and biota of many global locations, including the Arctic and Antarctic [1,3,6–9]. The consequence of contamination of the natural environment by PCNs is their presence in food. Higher chlorinated congeners of PCNs (especially from tetra- to heptaCN isomers) have been found in food of animal origin (milk, meat, eggs and fish) [10–16]. The consumption of even small, trace amounts of such lipophilic substances as PCNs results in their inevitable accumulation in the body. General population studies carried out in various regions of the globe have revealed the highest concentrations of PCNs (from tetra- to heptachloronaphthalene isomers) mainly in the adipose tissue [2,17,18]. Various pentachloronaphthalene isomers, as well as two major HxCN congeners, 1,2,3,4,6,7-hexachloronapththalene (PCN 66) and 1,2,3,5,6,7-hexachloronaphthalene (PCN 67), have been identified as dominant congeners in the adipose tissues of the general population [17]. As PCN66 and PCN67 cannot be readily separated in biological samples, their exposure assessments in food and human adipose tissue are usually estimated as a combined

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PCN66/PCN67 value [18,19]. The impact of PCNs on the human body in the context of potential health effects induced by environmental exposure, by ingestion, has not been practically elucidated. The only information concerning the toxicity of these compounds in humans applies to occupational exposure by inhalation. Symptoms associated with occupational exposure to PCN include, among others eye irritations, headache, anemia, impotency, anorexia and hepatotoxicity [2,6]. Scant experimental data also exists concerning the toxicity of PCNs. Due to their structural similarity to TCDD and affinity to the aryl hydrocarbon receptor (AhR), PCNs are frequently compared to dioxins, TCDD in particular, and are often referred to as dioxin-like compounds [20,21]. It was also shown that highly chlorinated naphthalenes (hepta- and oktaCNs) exert androgenic and anti-estrogenic properties and affect steroidogenesis in porcine ovarian follicles. These effects of PCNs in the ovary are similar to those caused by PCDDs [22,23]. Moreover, the similarity of the toxicities of the hexachloronaphthalenes (PCN66 and PCN67) and TCDD has been also confirmed by a number of studies, including that of Hooth et al. [19]. They noted that after repeated administration at very low doses to rats, both PCN congeners cause, characteristic of TCDD, hepatocellular hypertrophy and fatty change in the liver, together with the CYP1A1 induction, as well as histopathological changes in the thymus including atrophy, and changes in the lungs. Our own earlier studies carried out on rats indicate that PCNs induce anorectic effects such as wasting syndrome and neurotoxic effects, as well as hepatotoxicity; characteristic of TCDD [24–26]. Recently, more attention has been paid to the potential influence of the accumulation of lipophilic toxins such as PCNs in the adipose tissue of the maternal body on the safety of developing fetuses. However, only two studies regarding the potential prenatal toxicity of PCNs in mammals have been performed to date. Omura et al. [27] report that hexachloronaphthalene (HxCN) administered to pregnant female rats at a single dose not toxic to dams and developing fetuses (1 ␮g/kg b.w.) on days 14–16 of gestation accelerated the onset of spermatogenesis in male offspring. Another study notes that a mixture of different PCN congeners from tetraCN to heptaCN, approximately corresponding to Halowax 1013 and 1014, given to pregnant rats by gavage in the period of organogenesis, i.e. days 6–15 of gestation, generated embryo- and fetotoxic effects and teratogenicity that occurs in the absence of maternal toxicity [28]. In the present study, HxCN was given to pregnant female Wistar rats at three different daily doses during organogenesis, i.e. on days 6–15 of gestation. CYP1A1 expression was then assessed in dam and fetus livers and the placenta. The maternal toxicity, embryotoxic, fetotoxic and teratogenic effects were also assessed.

2. Materials and methods 2.1. Chemicals Hexachloronaphthalene was synthesized according to Auger et al. [29] and obtained from the Institute of Radiation, Faculty of Chemistry, Technical University of Lodz (Poland) as a mixture of HxCN isomers. The purity of the HxCN sample was over 94% and it contained mainly 1,2,3,5,6,7-hexaCN (∼81%). An Agilent-6890 gas chromatograph, with an electronic pressure programmer and a split/splitless injector was used to analyze the HxCN sample. Isotope Dilution HRGC/HRMS was used to analyze the tetra- through octa-chlorinated dioxin and furan content of the HxCN administered to the animals [26,30]. The analysis demonstrated that the content of dioxins and furans was <0.1 pg/100 ␮g of the investigated sample. All chemicals used for preparation and staining of fetuses were obtained from Sigma (St. Louis, MO).

2.2. Animals exposure Wistar rats were obtained from the breeding colony of the Nofer Institute of Occupational Medicine, Lodz, Poland (Imp: WIST rats). They were housed with controlled temperature (22 ± 1 ◦ C), relative humidity (45–55%), a 12-h light/dark cycle and maintained on commercial pelleted chow (Food Factory, Motycz, Poland) and tap water. Food and water were supplied ad libitum throughout the study. Nulliparous female rats, aged approximately 10 weeks and weighing 185–220 g, after 10 days of acclimatization, were mated (2:1) overnight with 14-week-old males and examined by vaginal smear for the presence of sperm the following morning. The day on which sperm was observed in the vaginal smear was designated as day 0 of gestation. Mated females were assigned to four experimental groups (19–21 dams per group). The animals were kept in cages (two females in each) in the same quarters as mentioned above. The HxCN preparation was dissolved in sunflower oil and administered to females by stomach tube at single daily doses of 0.1, 0.3 or 1.0 mg/kg body weight (b.w.) at the same time for 10 consecutive days during organogenesis (days 6–15 of gestation). The dose given to animals was based on a recent determination of individual body weight, all dams were weighed every day just before administration, hence the absolute dose of HxCN was in agreement with current body weight of pregnant females on a given gestation day. The animals were given 0.3 ml of test solution per 100 g b.w. Controls received an equivalent volume of oil. The doses of HxCN were chosen on the basis of earlier prenatal toxicity studies [28]. In line with the OECD guidelines and UE law, the doses should be properly chosen so that the highest dose could cause developmental toxicity and/or toxic symptoms in females (body weight loss), at least one medium dose should induce minimum toxic effects, and the lowest dose should not cause any developmental toxicity or toxicity in dams. The choice of two extreme doses was intentional to analyze CYP1A1 mRNA and protein expression as the aim of the study was to investigate the effects in the absence of maternal toxicity (dose 0.1 mg/kg b.w.), and in the presence of maternal toxicity manifested by a significant decrease in body weight gain during pregnancy (dose 1.0 mg/kg b.w.). The studies were performed in accordance with Polish law on the protection of animals and with the permission of the Local Ethical Committee for Experimentation on Animals of Lodz, Poland (No 42/LB478/2009 and 28/LB478/211). 2.3. Maternal and fetal toxicity The general behavior of the dams was observed daily. The pregnant females were weighed every day from 0 to 20 gestation days (GD) always at the same time of the day. Based on weights determined on days 0, 6, 10, 15 and 20 of gestation; body weight gain was calculated. Food and water consumption were measured on days 1, 6, 10, 15 and 20 of gestation. Final GD 20 body weights were corrected for gravid uterine weights (adjusted final body weight), and gestation body weight change and adjusted body weight change from day 0 (final body weight GD 20 minus gravid uterine weight minus GD 0 body weight) were calculated. All females were sacrificed by decapitation on day 20 of gestation. The maternal liver, kidneys, adrenals, ovaries, spleen, brain and gravid uterine were removed and weighed. Live fetuses from each litter were weighed, sexed, and tagged. Maternal blood, placenta, whole maternal and fetal liver were collected for assays. The following indicators of maternal toxicity were used: the concentrations of reduced glutathione (GSH) and malondialdehyde (MDA) in the liver and the activity of alanine aminotransferase

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Table 1 Primers for PCR reaction used for the evaluation of CYP1A1 and GAPDH mRNA expression in the liver. Gene CYP1A1 GAPDH

Primer sequences 



F) 5 - GAT GCT GAG GAC CAG AAG ACC GCR) 5 -CAG GAG GCT GGA CGA GAA TGC F) 5 -GTG AAC GGA TTT GGC CGT ATC G R) 5 -ATC ACG CCA CAG CTT TCC AGA GG

(ALT, E.C.2.6.1.2) and sorbitol dehydrogenase (SDH, E.C.1.1.1.14) in serum. The concentration of MDA was determined in hepatic homogenates according to Uchiyama and Mihara [31]. Lipid peroxidation products (mainly MDA), which reacted with thiobarbituric acid were measured spectrophotometrically at 532 nm. Reduced glutathione (GSH) was determined spectrophotometrically, according to Sedlak and Lindsay [32] by a reaction with 5,5 -dithiobis-(2-nitrobenzoic acid). The activity of ALT was determined colorimetrically with 2,4-dinitrophenyl-hydrazine [33]. The amount of pyruvate (␮mol/ml serum) formed during a one-hour incubation at 37 ◦ C was accepted as the activity unit [U]. Sorbitol dehydrogenase was determined spectrophotometrically; using the optic test with the molar absorbance coefficient at 340 nm, according to the following equation: IU (25 ◦ C) = A/min 645 ␮mol min−1 1−1 [34]. The ovaries were removed and the corpora lutea were counted immediately under a dissecting microscope. The removed and weighed uterus was opened and the numbers of total implantations and the number of live and dead fetuses was recorded, together with the number of early and late resorption sites. Uteri from females that appeared non-gravid and individual uterine without visible implantations were examined with 10% ammonium sulfide solution. A site was judged as a late resorption when macroscopic discrimination between fetal and placental residues was possible. When this discrimination could not to be made, the site was judged as an early resorption. Total implantation was calculated as the sum of the number of fetuses and resorption sites in each pregnant female rat. Preimplantation losses and postimplantation losses were determined as given below: No. of preimplantation losses per litter = No. of corpora lutea per litter− minus no. of implantations per litter, No. of postimplantation losses per litter = No. of implantations minus no. of live fetuses per litter. Live fetuses from each litter were measured for body weight and crown-rump length, sexed and examined for external malformations. Approximately one-half of the live fetuses from each litter were preserved in 95% ethanol for subsequent skeletal examination after staining with Alizarin-S [35]. The remaining fetuses were fixed in Bouin’s solution for visceral examination [36]. 2.3.1. Morphological studies Excised sections of liver left lobe, fetal liver and placenta were fixed in 10% formaldehyde, dehydrated and embedded in paraffin. The obtained paraffin blocks were cut into 4 ␮m slices, which were then deparaffinized, rehydrated and stained with hematoxylin and eosin (H–E) by standard procedure. 2.3.2. Reverse transcription-polymerase chain reaction (RT-PCR) For evaluation of CYP1A1 expression, small tissue sections (100 mg) were taken from the liver left lobe, fetal liver and the central part of the placenta. Total cellular RNA was isolated by acid guanidinium thiocyanate/phenol/chloroform extraction [37] using a commercially available kit (TriReagent, Sigma) and 3 ␮g of RNA was reverse-transcribed into cDNA. The reverse transcription reaction was carried out at 42 ◦ C for 1 h. The PCR reaction mixture contained primers specific for rat CYP1A1 and the GAPDH reference gene (Table 1). Amplification was carried out by 24 repeated cycles at 94 ◦ C for 1 min, 65–69 ◦ C for 1.5 min, and 72 ◦ C for 1 min on Programmable Thermal Controller PTC-200 (MJ Research, Inc.,

Hybridization temp. (◦ C)

Product size (bp)

References

65 69

680 543

[38] [39]

Watertown, MA, USA). An aliquot of each reaction mixture was subjected to electrophoresis in 2% agarose gels, following which, the gels were stained with ethidium bromide and analyzed qualitatively (bp) and semi-quantitatively by densitometry (One D-scan software, Scanalytics). 2.3.3. Western blotting In order to assess CYP1A1 protein expression; the microsomal fraction was isolated from individual maternal livers and pools of 6–9 fetal livers or placentas according to Dallner [40]. The protein content was determined according to Bradford [41]. The samples (0.1–80 ␮g of protein) were then subjected to polyacrylamide gel (10%) electrophoresis in the presence of sodium dodecyl sulphate [42], and blotted electrophoretically onto PVDF membrane (Millipore). Following this, microsomal CYP1A1 was identified with specific antibodies (diluted 1:1000). The polyclonal antibodies to CYP1A1 (Millipore) were developed in rabbits. Bound primary antibodies were detected with goat anti-rabbit secondary antibody conjugated with alkaline phosphatase and the reaction was developed with a BCIP/NBT liquid substrate system (Sigma), according to the manufacturer’s instructions. Molecular weight markers were purchased from Bio Rad. For quantification of CYP1A1 expression, the intensities of the stained bands on the membranes were analyzed with One D-scan software (Scanalytics). Microsomes from ␤-naphthoflavone-induced adult females (BNF) were used as positive controls: These rats were administered ␤-naphthoflavone twice at a daily intraperitoneal (i.p.) dose of 50 mg/kg b.w. 2.3.4. Immunohistochemistry The localization of CYP1A1 protein in liver acini zones was visualized with the ABC method according to Hsu et al. [43]. Tissue sections were taken from the dam’s liver left lobe, fetal liver and the central part of the placenta. They were fixed overnight in 10% neutral-buffered formalin, subsequently passed through alcohol and xylene solutions, and finally embedded in paraffin blocks. Slices of 5 ␮m thickness were cut, deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 0.3% H2 O2 for 30 min. Before incubation with the primary antibody, the sections were pretreated with 1% BSA and 2.5% goat serum for 30 min to block places of nonspecific binding of antibodies. Appropriate primary antibody (ab 1247, Millipore) was added, and the sections were incubated for 22 h at 4 ◦ C. This step was followed by incubation with proper biotinylated secondary antibody. Appropriate negative controls were created as sections without primary antibodies. The primary antibody was replaced with IgG delivered from the same host and diluted to match the protein concentration of the primary antibody. The dilutions of primary antibodies used for immunohistochemistry are shown in Table 2. Moreover, sections were done in which immediately after endogenous peroxidase quenching, its absence in the sections was confirmed by the use of DAB and hydrogen peroxide. Table 2 Primary antibody dilutions used for immunohistochemistry. Tissue

Control

0.1HxCN

1.0HxCN

Maternal liver Fetal liver Placenta

800x 600x

1600x

2400x

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Final visualization was achieved by using the avidin-biotinperoxidase complex kit (Vectastain ABC Kit, Vector Laboratories), freshly prepared DAB and hydrogen peroxide according to the protocol provided by the manufacturer. If the antigen-antibody complex is not present in the tissue, the product of the reaction with DAB is not formed and a result is negative (usually, there is not brown stain and the specimen is more or less blue). Immunohistochemical reactions were documented with a SSC-DC58AP camera (Sony) coupled with an Eclipse E600 optical microscope (Nikon).

140

Body weight gain (g)

120 100

Control

80

0.1 mg/kg b.w.

60

0.3 mg/kg b.w.

*

40

*

20

2.4. Statistical evaluation of the results

exposure period

0 6

The unit for statistical measurement was the pregnant female or the litter, except CYP1A1 mRNA analysis. A two-way ANOVA (groups × measurements) for repeated measures (GLM model) was employed to assess differences occurring over time: specifically, to evaluate the effect of HxCN on daily food and water consumption; maternal body weight gain, adjusted body weight, gestation body weight changes, and adjusted body weight change from day 0. A significant interaction identified by the analysis was followed by simple effects assessment [44]. One-way analysis of variance with Tukey’s test for pairwise comparisons was used for evaluating fetal body weight, uterus weight, sex ratio, fetal/placental weight ratio, fetus length, and number of fetuses, corpora lutea, resorptions, implants, pre- and post-implantation losses per litter. The same tests were used to evaluate absolute and relative weights of the internal organs of the mothers. Frequency data (female deaths, litters totally resorbed, litters with resorptions, external, skeletal and visceral findings) were analyzed with Fisher’s exact probability test [45]. A one-way ANOVA was used for parametric analysis of the obtained CYP1A1 expression data. If normal distributions could not be assumed, the Mann–Whitney U-test was used for independent samples or Wilcoxon tests were used for related samples. Categorical data was analyzed using 2 tests. Pearson’s correlation tests were used to identify possible relationships between outcome measures. In all statistical analyses, a value of p < 0.05 was considered as indicating statistical significance. The calculations were performed using SPSS Statistics 17.0 software.

a)

b) Daily water consumpon (ml)

Daily food consumpon (g)

30 25 20 15

*

5 exposure period

0

6

10

20

resorptions were found in its uterus. The necropsy did not reveal any macroscopic lesions in the investigated internal organs. Figs. 1 and 2 show integral toxicity indicators for female rats: body weight gain during pregnancy, daily consumption of water and food on the chosen days of gestation. On days 15–20 of gestation, a significantly decreased body weight gain of about 50% was observed in female rats exposed to the highest HxCN dose (1.0 mg/kg b.w.). On day 20 of gestation, the weight gains were 48.62 ± 20.36 g in the 1.0 mg/kg b.w. group and 99.93 ± 23.14 g in the control group (Fig 1). As regards two other groups of females receiving HxCN a body weight gain on individual days of gestation was comparable to that of the control group. In addition adjusted body weight change and gravid uterine weight were significantly reduced in females exposed to the highest dose of HxCN (1.0 mg/kg b.w.) in comparison to the control group (Table 4). Daily consumption of food (Fig. 2a) and water (Fig. 2b) by females given the highest dose of the compound (1.0 mg/kg b.w.) was significantly lower than that of controls from day 10 of gestation until administration of the compound was terminated. No significant differences were observed between the controls and female rats exposed to HxCN doses with regard to the absolute weights of their internal organs (Table 3). Only the mean weight of the placenta was significantly lower in females exposed to the highest dose of HxCN (1.0 mg/kg b.w.) than in the control group and the females exposed to lower doses (Table 4). However; differences were identified in relative weights of the liver, kidneys, adrenal glands, spleen and brain, being significantly elevated in females exposed to 1.0 mg/kg b.w. compared to controls on day 20 of gestation (Table 3). The increase in the relative weight of organs

In the course of the experiment carried out in the groups of inseminated female rats (19–21 per group), only one female was found to be dead on day 18 of gestation. The female, from the 0.1 mg/kg b.w. group, was pregnant and five fetuses and six late

0

15

Fig. 1. Body weight gain (g) of pregnant females exposed to HxCN on days 6–15 of gestation. All values are expressed as means ± SD. * Significantly different from the control group, p < 0.05.

3.1. Assessment of toxic effect on pregnant rats

*

10

Gestaon day

3. Results

10

1.0 mg/kg b.w.

15

Gestaon day

20

50 45 40 35 30 25 20 15 10 5 0

Control 0.1 mg/kg b.w.

* *

*

0.3 mg/kg b.w. 1.0 mg/kg b.w.

exposure period

0

6

10

15

20

Gestaon day

Fig. 2. Daily consumption of food (a) and water (b) on selected days of gestation in females rats exposed to HxCN during organogenesis. All values are expressed as means ± SD. * Significantly different from the control group, p < 0.05.

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Table 3 Final body weight; absolute and relative (per 100 g b.w.) organ weights and selected biochemical parameters of maternal toxicity determined on day 20 of gestation. All values are expressed as means ± SD. Daily dose of hexachloronaphtalene (HxCN)

Control n = 18

0.1 mg/kg n = 17

0.3 mg/kg n = 19

1.0 mg/kg n = 17

306.4 ± 26.3

299.0 ± 17.6

300.8 ± 22.8

254.5 ± 32.9a

12.18 ± 1.00 3.98 ± 0.32 1.36 ± 0.15 0.44 ± 0.05 64.94 ± 7.64 21.24 ± 2.72 93.21 ± 18.10 30.46 ± 5.63 0.66 ± 0.08 0.22 ± 0.02 1.60 ± 0.12 0.52 ± 0.06

11.85 ± 0.93 3.98 ± 0.27 1.29 ± 0.10 0.43 ± 0.04 63.96 ± 9.85 21.46 ± 4.17 85.96 ± 15.53 28.84 ± 4.86 0.61 ± 0.06 0.20 ± 0.03 1.60 ± 0.08 0.54 ± 0.04

12.62 ± 1.19 4.24 ± 0.22 1.37 ± 0.11 0.46 ± 0.03 63.80 ± 5.36 21.34 ± 2.44 89.02 ± 12.94 29.77 ± 3.89 0.62 ± 0.09 0.21 ± 0.04 1.64 ± 0.13 0.55 ± 0.06

12.41 ± 2.68 4.89 ± 0.52a 1.37 ± 0.18 0.54 ± 0.08a 66.22 ± 11.86 26.12 ± 9.00a 81.55 ± 13.31 32.17 ± 5.89 0.74 ± 0.24 0.30 ± 0.07a 1.58 ± 0.07 0.62 ± 0.08a

6.09 ± 0.32

5.99 ± 0.42

5.41 ± 0.55

6.03 ± 0.14

105.9 ± 15.02

180.7 ± 63.51

375.8 ± 41.35 a

560.3 ± 65.24 a

2.67 ± 0.34 3.09 ± 0.76

2.98 ± 0.12 3.17 ± 0.67

2.22 ± 0.43 4.06 ± 0.75

2.08 ± 0.40 4.12 ± 0.99

Final body weight (g) on 20 GD Absolute and relative (per 100 g b.w.) organ weights Liver g g% Kidneys g g% Adrenals mg mg% Ovaries mg mg% Spleen g g% Brain g g% Chosen biochemical parameters of maternal toxicity GSH in liver (␮mol/g tissue) MDA in liver (nmol/g tissue) ALT (U) SDH (IU)

n – number of animals per group. a Significantly different from the control group (p < 0.05).

Table 4 Effect of hexachloronaphtalene (HxCN) administered on days 6–15 of gestation on fertility/reproduction and offspring development. Endpoints

No. of females inseminated No. of dead females No. of pregnant females No. of non-pregnant females No. of live fetuses per litterb No. of litters totally resorbed No. of litters with resorptions No. of litters with early resorptions No. of litters with late resorptions No. of early resorptions per litterb No. of late resorptions per litterb No. of implantations per litterb No. of corpora lutea per litterb No. of preimplantation losses per litterb No. of postimplantation losses per litterb Placental weight (g) c Fetal sex ratio (% males per litter)c Fetal/placental weight ratio (sexes combined)c Fetal crown-rump length (sexes combined) (cm)c Males (cm)c Females (cm)c Body weight of live fetuses (sexes combined)(g)c Males (g)c Females (g)c Gravid uterine weight (g)b Adjusted final body weight (g)b Adjusted weight change from day 0 (g)b

Control

20 0 (90.0%) 18 2 11.67 ± 4.10 0 3 0 3 0 0.21 ± 0.43 11.93 ± 4.12 14.36 ± 1.98 2.43 ± 2.87 0.21 ± 0.43 0.55 ± 0.10 48.09 ± 8.25 6.38 ± 0.75 4.02 ± 0.12 4.04 ± 0.13 3.98 ± 0.15 3.35 ± 0.23 3.39 ± 0.29 3.32 ± 0.20 59.18 ± 6.90 247.23 ± 17.77 40.75 ± 9.45

Daily dose of hexachloronaphtalene (HxCN) 0.1 mg/kg

0.3 mg/kg

1.0 mg/kg

19 1d (94.4%) 17 1 11.06 ± 2.96 0 8a 5a 6 0.36 ± 0.33 0.63 ± 0.96 12.27 ± 2.49 13.81 ± 1.80 1.50 ± 2.03 1.25 ± 2.02 0.51 ± 0.05 51.67 ± 12.04 6.47 ± 0.51 3.81 ± 0.11a 3.82 ± 0.12 3.80 ± 0.11 3.30 ± 0.26 3.35 ± 0.25 3.25 ± 0.23 56.04 ± 7.73 242.08 ± 15.29 38.35 ± 10.65

21 0 (90.5%) 19 2 12.07 ± 2.63 0 10a 4a 6 0.60 ± 1.18 0.93 ± 1.49 13.60 ± 1.72 14.67 ± 1.88 1.07 ± 0.96 1.63 ± 1.45 0.49 ± 0.04 51.24 ± 10.35 6.32 ± 0.41 3.63 ± 0.16a 3.64 ± 0.17a 3.63 ± 0.14a 3.12 ± 0.27a 3.19 ± 0.26a 3.10 ± 0.21a 58.69 ± 8.21 241.54 ± 15.85 35.89 ± 6.89

20 0 (85.0%) 17 3 8.91 ± 3.94 2 8a 5a 7a 1.00 ± 1.96 2.62 ± 3.99a 12.54 ± 2.15 13.92 ± 1.73 1.23 ± 1.87 3.62 ± 4.59a 0.42 ± 0.04a 41.09 ± 9.78 6.37 ± 0.48 3.38 ± 0.09a 3.38 ± 0.09a 3.38 ± 0.10a 2.65 ± 0.26a 2.69 ± 0.24a 2.61 ± 0.33a 37.52 ± 10.39a 216.48 ± 22.51 a 11.31 ± 5.08a

No. of preimplantation losses per litter b = No. of corpora lutea per litter− minus No. implantations per litter No. of postimplantation losses per litter b = No. of implantations minus No. of live fetuses per litter Adjusted final body weight = final body weight (g) minus gravid uterine weight (g) Adjusted final body weight change = final body weight (g) minus gravid uterine weight (g) minus Gestation Day 0 body weight (g). a Significantly different from the control group (p < 0.05). b Mean ± SD. c Mean of litters ± SD. d Females that died were not taken into account in the analysis of results.

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expressed as g (or mg) per 100 g b.w. results from the decreased body weight gain during pregnancy; and thus about 20% decrease in absolute weight of exposed females as compared with controls. The final body weight of females on 20 GD is presented in Table 3. The macroscopic assessment of internal organs in controls and female rats exposed to HxCN at doses of 0.1 and 0.3 mg/kg b.w. did not reveal significant pathological lesions. However, in 4/17 female rats exposed to 1.0 mg/kg b.w., the surface of the liver showed a marble pattern and beige color. The level of MDA was used as an indicator of lipid peroxidation (Table 3). Statistically significant increases in liver MDA concentration were found: 355% of control values for the 0.3 mg/kg b.w. dose and 533% for the 1.0 mg/kg b.w. dose. However, no significant changes in liver GSH level were found in female rats exposed to any HxCN dose. In addition, serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities, indicators of acute liver necrosis after repeated intragastric administration of HxCN, were determined. No significant changes in the activity of these indicator enzymes were observed, regardless of HxCN dose (Table 3). 3.2. Morphological studies The advanced morphological changes caused by HxCN were noted in the maternal liver after administration of a higher HxCN dose (1.0 mg/kg b.w.). Distinct microvesicular fatty changes were revealed in hepatocytes within zone 3 of some liver acini. Macrovesicular steatosis associated with a single cell necrosis was also observed. There were no morphological changes in the fetal liver and placenta which could result from the damage of these organs caused by HxCN (data not shown). 3.3. Assessment of embryotoxic, fetotoxic and teratogenic effect The percentage of pregnant females among those inseminated, in both controls and the group exposed to HxCN, amounted from 85 to 94% (Table 4). No significant differences were observed between the control group and the groups exposed to HxCN with regard to the number of implantations, corpora lutea, preimplantation losses and the average number of live fetuses in the litter (Table 4). HxCN increased the intrauterine mortality of embryos and fetuses. The number of litters with resorption was significantly higher in all groups exposed to HxCN than in controls. The average number of late resorptions and post-implantation losses in litters of females given HxCN at the highest dose (1.0 mg/kg b.w.) was significantly higher than in controls; with completely resorbed litters being found in two females in the group (Table 4). There was no effect on the fetal sex ratio (Table 4). The body length of fetuses in all exposed groups was significantly lower compared to controls and was dependent on HxCN dose. Significantly reduced body weights of fetuses were revealed only in the groups of females exposed to HxCN at doses of 0.3 and 1.0 mg/kg b.w. compared to controls (Table 4). The mean male and female fetal body weights were comparable within the groups (Table 4). The ratio of fetal weight to placental weight in the groups administered with HxCN was not significantly different to that of the control group (Table 4). To confirm that HxCN exerted an impact on pregnant mothers and their offspring; the results of some tested parameters were also analyzed for correlation in each experimental group. High correlations (p < 0.05) were obtained between the final body weight of pregnant females (for all pregnant females, controls and administered HxCN), and mean fetal body weight per litter (r = 0.52) or litter weight (r = 0.81). The offspring of 18 control female rats and of 15–19 females exposed to HxCN at doses of 0.1, 0.3 and 1.0 mg/kg b.w. was assessed. The respective numbers of assessed fetuses were 220, 188, 228 and 147 in the control; 0.1, 0.3 and 1.0 mg/kg b.w. groups

(Table 5). HxCN did not generate congenital skeleton defects. However, the frequency of fetuses and litters with delayed ossification was found to be significantly higher in the offspring of female rats exposed to 1.0 mg/kg b.w. than controls (Table 5). In the majority of cases; this pathology was manifested by the lack of one or two ossification centers of the sternum and in one litter by the enlargement of the fontanelle (Table 5). In single fetuses of females exposed to 0.3 mg/kg b.w., short supernumerary ribs were observed. No congenital or developmental defects were revealed by soft tissue assessment in controls or females exposed to HxCN at all doses. 3.4. CYP1A1 expression and immunolocalization In control animals, constitutive expression of CYP1A1 mRNA was very weak in the maternal liver and placenta, and undetectable in the fetal liver (Fig. 3). The bands in the immunoblots corresponding to maternal and fetal hepatic (but not placental) CYP1A1 protein were poorly visible after loading 80 ␮g protein per well (Fig. 4). These observations were confirmed by immunohistochemistry (IHC) staining. A very weak positive reaction for CYP1A1 was found in a few layers of hepatocytes surrounding the central veins (zone 3 of the liver acini) (Fig. 5). HxCN increased CYP1A1 mRNA expression by up to more than 20 times the control values and CYP1A1 protein expression up to 265 times in maternal livers in a dose-dependent manner (Figs. 3 and 4). In the 0.1 mg/kg b.w. group, a strong, positive IHC reaction for CYP1A1 was localized in the hepatocytes of zone 3 of the liver acinus. In the 1.0 mg/kg b.w. group, a positive reaction was enhanced and localized in all hepatocytes of liver acini (Fig. 5). In fetal livers, HxCN significantly elevated CYP1A1 mRNA expression and induced CYP1A1 protein expression (about 235fold) when administered at 1.0 mg/kg b.w., but not at 0.1 mg/kg b.w. (Figs. 3 and 4). The protein expression was confirmed by IHC staining (Fig. 5). A greater than 13-fold increase in placental CYP1A1 mRNA was observed in both the 0.1 mg/kg and 1.0 mg/kg b.w. groups (Fig. 3). In these groups, the expression of CYP1A1 protein was detectable but very low, being twice as high in the 1.0 mg/kg b.w. group than the 0.1 mg/kg b.w. group (Fig. 4). Greater CYP1A1 expression was observed in IHC, in the cells of both the maternal and fetal parts of the placenta for all HxCN-treated groups (Fig. 5). 4. Discussion The development of the embryo is one of the most complex processes in life, and it is a period of extreme sensitivity to the action of toxic substances. However, limited experimental data exists on the toxicity of PCNs. Regulatory guidelines for developmental toxicity studies require that the highest dose level should induce overt maternal toxicity, usually manifested in the form of decreased maternal body weight gain greater than 20% [46]. Therefore, reduced maternal body weight gain and/or food intake and fetal body weight are usually considered the most sensitive endpoints in developmental toxicity assays, especially for environmental chemicals [46,47]. In our study, such decreased body weight gain during pregnancy (by about 50%) was observed only in females exposed to the highest dose of HxCN (1.0 mg/kg b.w.). Significantly decreased (by about 20%) body weight in these females on day 20 of gestation, compared with controls, was due to a lower daily intake of food and water, which indicates anorectic action. The anorectic effect is a very characteristic symptom of the PCN toxicity observed in both humans [2,48] and animals [2,6,24,25,26,28,30] exposed to this compound. The observed significant increases in the relative

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99

Table 5 Morphological examinations in fetuses of rats administered hexachloronaphtalene (HxCN) on days 6–15 of gestation. Daily dose of hexachloronaphtalene (HxCN) Control External examination Total no. 220/18 of fetuses/litters examined Total no. of fetuses/litters with external abnormalities Internal examination Total no. of fetuses/litters examined Total no. of fetuses/litters with dilatation of renal pelvis Skeletal examination Total no. of fetuses/litters examined Total no. of fetuses/litters with variations -absence of one or two ossification centers of the sternum -enlargement of fontanelle -short supernumerary ribs

0.1 mg/kg

0.3 mg/kg

1.0 mg/kg

188/17

228/19

147/15

♀

♂

♀

♂

♀

♂

♀

♂

0

0

0

0

0

0

0

0

61/18

48/18

40/17

51/17

57/19

54/18

48/15

26/15

0

0

0

0

0

0

0

54/18

57/18

51/17

46/17

54/19

63/19

38/15

2/1 (7.7/6.7) 35/15

4/3 (7.4/16.7) 4/3 (7.4/16.7)

3/1 (5.3/5.6) 3/1 (5.6/5.9)

1/1 (2.0/5.9) 1/1 (2.0/5.9)

0

8/4 14.8/21.1 3/3 (5.6/15.8)

5/5 7.9/26.3 4/4 (6.3/21.1)

12a /11a (34.6a /73.3a ) 11a /10a (28.9a /66.7a )

11a /8a (31.4a /53.3a ) 10a /7a (28.5a /46.6a )

0

0

0

0

0

0

0

0

0

4/2 (7.4/10.5) 1/1 (1.9/5.3)

1/1 (2.6/6.7) 0

2/1 (5.7/6.7) 0

0

1/1 (1.6/5.3)

In parentheses—the percentage of fetuses and litter. a Significantly different from the control group (p < 0.05).

Fig. 3. Relative CYP1A1 mRNA expression in the maternal and fetal livers, as well as in the placenta of rats administered placebo (C) or HxCN. Representative samples taken from individual adult and fetal livers and placentas, are presented. CYP1A1 mRNA was normalized with GAPDH mRNA level for individual samples. R.E.L.—Relative Expression Level, the mean values (n = 6) are relative expressions to appropriate detectable controls (C = 1.00). Control values for fetal liver mRNA were not detectable. *Statistically significant as compared to controls, p < 0.05.

weight of many internal organs free of significant exposure-related macroscopic changes were most likely the consequence of significantly decreased body weight on day 20 of gestation observed in females exposed to HxCN at a dose of 1.0 mg/kg b.w. Nevertheless, after administration of HxCN at the highest dose of 1.0 mg/kg b.w. histological changes in the liver, such as microvesicular fatty changes in zone 3 of the liver acini and macrovesicular steatosis in single hepatocytes were observed. Although maternal toxicity, manifested by reduced maternal weight/gain, is generally adopted and recommended in conventional teratology study, it frequently appears to be an “incomplete” indicator because it tells us only a little about the underlying cause of toxicity [49]. Additional endpoints, indicators of maternal toxicity, such as gravid uterine weight, adjusted body weight and adjusted body weight changes„ were also significantly reduced, but only in females exposed to the highest dose of HxCN (1.0 mg/kg b.w.). The biological pathway by which HxCN induces toxicity has not as yet been recognized (thus far only single studies have addressed this issue). It is thought that its major mechanism of action is related to the activation of the Ah receptor (AhR) [20,21,23]. Although the physiological role of AhR has not yet been fully elucidated, it is known to play an important role in prenatal development and reproduction/fertility [50,51]. Furthermore, AhR controls the expression of a diverse set of genes, including cytochrome P450CYP1A1 [52]. Modulation of cytochrome P450 iso-

form expression could be partially responsible for developmental toxicity [53]. As indicated by our studies, as well as by those carried out by other authors [19], HxCN is a very strong CYP1A1 inductor. However, the role of CYP1A1 induction in the toxicity of this compound still remains unclear; especially in view of the fact that HxCN is not metabolized in the liver, as evidenced by the excretion of HxCN with feces in an unchanged form [2]. Moreover, in our earlier experiment [54], it was indicated that HxCN showed a slow turnover rate in the rats, and 5 days after its single intragastric administration, the retention in adipose tissue was over 26%, whereas in the liver it was still about 16%. Therefore, strong systemic accumulation of HxCN, especially in the liver, may lead to CYP1A1 induction and lipid peroxidation which is likely to be responsible for toxic effects. Based on the analysis of generally adopted indicators of maternal toxicity, we conclude that a dose of 1.0 mg/kg .bw. was the only dose toxic to dams. However, the MDA level in the liver, previously determined in our earlier work as an indicator of lipid peroxidation, reveals that toxic effects of HxCN on pregnant females occur at doses ranging from 0.3 to 1.0 mg/kg b.w. The observed dose-dependent increase in liver MDA level suggests that this compound generates oxidative stress. Moreover, previous studies also imply that PCNs may induce oxidative stress in experimental animals [55]. Providing that the increased MDA level in the liver induces the toxic effect to dams, a dose of 0.3 mg/kg b.w. can be adopted as the lowest observed adverse effect level (LOAEL). Induc-

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Fig. 4. Relative CYP1A1 protein expression in hepatic and placental microsomes. Representative samples taken from individual adult livers; pools of 6–9 fetal livers or placentas are presented. Presented R.E.L. values are means obtained from 6 independent samples. Microsomes from ␤-naphthoflavone-induced adult females (BNF) were used as positive controls. CYP1A1 was not identified in control (C) placentas. The amount of microsomal protein per lane is also shown. Mm: molecular weight marker. The position of the 53 kDa molecular weight marker is indicated. *Statistically significant as compared to appropriate controls, p < 0.05.

Fig. 5. CYP1A1 protein immunolocalization in the maternal and fetal livers, as well as in the placental labyrinth of HxCN-treated rats. Tissue sections were taken on day 20 of gestation. Appropriate negative controls (sections without primary antibody) are also presented. I and III: first and third zones of the liver acinus, respectively. Magnification: maternal liver C and Negative Control × 100, maternal liver − 0.1 and 1.0HxCN –x40, fetal liver and placenta – x100.

tion of oxidative stress and impairment of the antioxidant defense system are considered to be promoters of several prenatal developmental disorders (e.g. embryonic mortality, abortion, intrauterine fetal death or low birth weight) generated by various chemical compounds known as environmental pollutants [47].

It is difficult to delineate clearly the role of maternal and direct embryo/fetal toxicity on in utero development [49]. Although in this study HxCN concentrations were not measured in dam livers, fetuses and the placenta, the induction of CYP1A1 expression in the fetal liver and placenta could be considered as an indicator that HxCN reaches the placenta and fetal liver and would show bio-

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logical activity in those sites. The direct effect of the compound on developing fetuses was indicated by the observed embryotoxic effects, and to a lesser extent, by fetotoxic effect. HxCN given at doses of 0.1–1.0 mg/kg b.w. to pregnant female rats during organogenesis induced a dose-independent embryotoxic effect: in all exposed groups, the number of litters with resorptions was significantly higher than in controls. The dose-dependent fetotoxic effect of HxCN was demonstrated by significantly decreased crownrump length in a whole range of the studied doses and fetal body weight at doses of 0.3–1.0 mg/kg b.w. It should be emphasized that the observed fetotoxic effects occurred already after exposure to HxCN at doses of 0.1 and 0.3 mg/kg b.w., which did not cause significant decrease in body weight gain or intake of food and water. On the other hand, in females exposed to the highest dose, maternal toxicity would have been of significant importance because the decreased fetal weight is frequently a consequence of decreased maternal weight gain [46]. Our calculation of the correlations for the whole range of administered doses reveal a strong relationship between the final body weight relative to both litter weight and mean fetus weight in the litter. Hence, adverse developmental effects in fetuses cannot be associated with maternal toxicity [46,56]. Maternal toxicity, defined as the decreased body weight gain during pregnancy, which is frequently connected with the decreased intake of food [57], may play an important role in the interpretation of findings concerning the ossification delay in fetuses. Incomplete ossification or its delay is related to maternal toxicity and maternal factors, such as nutritional status. It is thought that maternal malnutrition or reduced food intake is a common mechanism by which fetal growth and ossification can be decreased in toxicity studies [58]. Our study reveals that in the offspring of female rats exposed to HxCN at the two high doses (0.3 and 1.0 mg/kg b.w.); fetuses and litters with delayed ossification, manifested mostly by the absence of one or two ossification centers of the sternum and the enlargement of fontanelle in one litter, were also found more frequently. However, delayed ossification (absence of ossification centers of the sternum) became apparent only in fetuses exposed in utero to the highest dose (1.0 mg/kg b.w.): the dose most toxic to dams. This effect was observed in about 29% of fetuses of the 1.0 mg/kg b.w. group, while in the control group, the frequency of this effect was about 7%. Historical control data provides other means of characterizing the normal pattern of skeletogenesis that are important for interpreting delayed ossification [58]. It should be noted, however, that in the control used in this study, as well as in past (historical) controls in our laboratory, numbering a few thousand fetuses, similar frequencies of fetuses with delayed ossification have been detected. The delayed ossification included mainly delayed ossification of the sternum (about 12%) or skull bones (∼3%) [28,59,60]. On account of the fact that the observed effect – delayed ossification – in fetuses exposed to HxCN was significant only after exposure of dams to the highest dose toxic to them, it cannot be excluded that this resulted from both the maternal toxicity and direct toxicity of the compound. Factors other than malnutrition of dams may also play a significant role in ossification disorders in fetuses, e.g. disturbed metabolism of steroid and/or thyroid hormones [58,61]. The significance and the role of CYP1A1 expression in the development of fetal toxicity seem to constitute a very complex issue and the role of CYP1A1 induction in dam liver due to HxCN exposure remains unclear. Interestingly, while enhanced dose-dependent expression was observed for both CYP1A1 mRNA and protein in the dam liver, this expression was significant in the fetus liver only after exposure to a dose toxic to the dam (1.0 mg/kg b.w.). As a result, after exposure of pregnant females to a toxic dose (1.0 mg/kg b.w.),

101

the increases in CYP1A1 protein expression were almost identical to those observed in the livers of dams (over 265-fold) and their fetuses (about 235-fold). It seems that the mechanism of CYP1A1 induction in the fetal liver is not so sensitive to the inducer; what means that higher doses of inducer are needed for activation of CYP1A1 synthesis as compared to adult liver. Compared to the liver, HxCN was found to have a significantly weaker impact on CYP1A1 expression in the placenta, which may result from the immaturity of the CYP1A1 induction mechanisms in the syncytiotrophoblast [62,63]. The influence of HxCN on CYP1A1 mRNA expression in the placenta was found to be not dosedependent: CYP1A1 mRNA levels increased over 13-fold regardless of whether female rats received a nontoxic dose (0.1 mg/kg b.w.) or the most toxic dose (1.0 mg/kg b.w.) of HxCN. Similarly, no significant difference was observed between the two HxCN doses regarding CYP1A1 protein expression. However, it should be noted that in the placenta, HxCN-induced CYP1A1 expression was very weak. In conclusion, HxCN induces embryotoxic effects at all doses from 0.1 to 1.0 mg/kg b.w., when administered to pregnant female rats during organogenesis, as indicated by the number of resorption and postimplantation losses. On the other hand, the amount and intensity of fetotoxic effects were dose-dependent. A value of 0.3 mg/kg b.w. can be adopted as the lowest toxic dose (LOAEL) for pregnant female rats. Although HxCN at a dose of 0.1 mg/kg b.w is non-toxic to dams, it is, however, toxic to their progeny. HxCN was found to be a strong inducer of maternal hepatic CYP1A1. HxCN permeates to the fetus, causing CYP1A1 induction in the placenta and the fetal liver. Taking into consideration that it is probably not metabolized, it cannot be ruled out that fetotoxic effects are due to HxCN itself, rather than its metabolites reaching the fetus from a strongly inducted dam liver. The CYP1A1 induction, especially at the protein level, is limited in the placenta, which may reflect the immaturity of the mechanism of CYP expression. Nevertheless, the practical significance of these observations could be that placental induction of CYP1A1 mRNA expression is not specific, but a sensitive marker of HxCN exposure. It reflects the exposure of the fetus to even small doses of the compound, but not its embryo/fetotoxicity. Although there is a lack of data regarding prenatal exposure to PCNs in the human population; reports describe some postnatal disturbances in children born to pregnant mothers who had consumed fish from the Great Lakes (North America) [64,65], in which various chloroorganic compounds, including PCNs, have been identified [7,10,13]. Bearing in mind that HxCN was found to be strongly bioaccumulated and persistent, with half-lives of several years in humans [2], the possible effects on developmental as well as postnatal toxicity in humans are of major concern. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgment The study was supported by the XNational Science Centre, Grant No. NN 404 27 1240, Poland. References

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