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Maternal ethanol consumption reduces Se antioxidant function in placenta and liver of embryos and breastfeeding pups
Life Sciences 190 (2017) 1–6
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Maternal ethanol consumption reduces Se antioxidant function in placenta and liver of embryos and breastfeeding pups
MARK
Fátima Nogalesa,1, M. Luisa Ojedaa,1, Karick Jottyb, M. Luisa Murilloa, Olimpia Carrerasa,⁎ a b
Department of Physiology, Faculty of Pharmacy, Seville University, 41012 Seville, Spain Biology Program, Universidad de Cartagena, Cartagena, Colombia
A R T I C L E I N F O
A B S T R A C T
Keywords: Fetal alcohol syndrome disorders Selenium Glutathione peroxidase Embryos Placenta
Aim: The fetal alcohol exposition during pregnancy leads to different disorders in offspring, related to the oxidative stress generated by alcohol. It is well-documented that there is an impairment of the antioxidant selenoprotein Glutathione peroxidase (GPx) activity in ethanol offspring during the embryo period, although noone has described Selenium (Se) status. The aim is to analyze for the first time Se deposits in vivo and Se's biological implication in embryos and placenta after alcohol exposure and in offspring whose mothers continued to drink ethanol during lactation. Materials and methods: Se deposits, GPx and glutathione reductase (GR) activity, lipid and protein oxidation and the expression of GPx1 were measured in placenta and liver of both embryos (E-19) and breastfeeding pups (L21) in control and ethanol groups (20% v/v). Key findings: Ethanol consumption decreased Se deposits, GPx activity and GPx1 expression, while increasing biomolecular oxidation in placenta and in the liver of E-19 and L-21. The GR/GPx ratio decreased in placenta and in E-19, together with an increase in lipid oxidation, while increased in the liver of L-21 pups with protein oxidation. Ethanol also decreased the GPx1 expression/GPx activity ratio in the liver of E-19 and L-21, indicating that alcohol decreases GPx activity by both depleting Se deposits and promoting GPx inactivation. In placenta GPx activity is proportional to the GPx1 expression found, so the ethanol affects GPx activity in offspring more than in dams. Significance: Therefore, Se supplementation therapy in dams could contribute as an interesting antioxidant that prevents fetal alcohol syndrome.
1. Introduction Ethanol is one of the most common human teratogen drugs consumed, leading to serious adverse outcomes in the fetus, such as intrauterine growth retardation (IUGR), craniofacial malformations, physical and mental retardation, and cardiac septal defects. This is known as fetal alcohol syndrome (FAS). Sometimes a lesser degree of deformations, described as fetal alcohol spectrum disorders (FASDs), could be associated with FAS. Some offspring, however, have no symptoms (resilience) or only present partial FAS-related symptoms [1]. These effects could be even worse if mothers continue consuming ethanol during breastfeeding [2]. The cellular mechanisms by which ethanol induces damage in utero are not well understood, but induction of oxidative stress is believed to be one important mechanism [3]. The fetal injury by ethanol could be explained by a direct mechanism through feto-toxicity from ethanol
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and/or acetaldehyde, which is intimately related to oxidative stress induction and/or an indirect mechanism through ethanol-induced placenta injury and selective fetal malnutrition [4]. Insufficient supplies of essential trace elements with antioxidant properties such as selenium (Se) could contribute to the oxidative stress generated by ethanol exposure. Oxidation of the placenta itself is central to the pathogenesis of many disorders during pregnancy [5]. It has been demonstrated that ethanol exposure during lactation via maternal milk also increases protein and lipid oxidation in the liver of breastfeeding pups, decreasing endogenous antioxidant enzymes activities and hepatic glutathione (GSH) levels [6]. During embryo development there is a complex interplay among proliferation, differentiation and apoptotic cell events. Despite the fact that a low amount of reactive oxygen species (ROS) acting as primary or secondary messengers to promote cell growth or death are vital for maintaining this interplay, an excess of ROS levels are related to
Corresponding author at: Department of Physiology, Faculty of Pharmacy, Seville University, C/Profesor García González, n° 2, 41012 Sevilla, Spain. E-mail address: [email protected] (O. Carreras). Authors who have shared the main task of preparing the manuscript, and have dedicated the same effort, both should have the consideration of first author.
http://dx.doi.org/10.1016/j.lfs.2017.09.021 Received 14 July 2017; Received in revised form 8 September 2017; Accepted 18 September 2017 Available online 22 September 2017 0024-3205/ © 2017 Published by Elsevier Inc.
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during gestation and lactation [6]. Total kilocalories (kcal) was obtained by adding to the solid kcal the kcal derived from alcohol consumption. Solid kcal intake was calculated by multiplying the g of food ingested per day by 4.1 kcal, alcoholic kcal intake was determined by multiplying the ml of ethanol ingested per day by 7.1 kcal. Se intake was estimated by multiplying the g of food ingested per day by 0.1.
protein, lipid and DNA oxidation, leading to cell dysfunctions. Fortunately, antioxidants can obviate these effects by modifying gene expression, transcription factor signaling, and cell cycle alterations [7]. One of the main cellular protective systems that plays an important role during embryo development is the selenium-dependent glutathione peroxidases (GPxs) family [8]. These enzymes reduce the pro-oxidative ROS hydroperoxides to harmless water and oxygen. GPx1 is regarded as this family's major antioxidant enzyme. It does, however, depend on Se levels; if Se decreases, GPx1 expression falls [9]. Despite the fact that studies with GPx1 knockout mice have revealed that these animals compensate mild oxidative stress [10], GPx4 knockout mice are nonviable [11]. GPx4 is the only selenoprotein which protects cellular membranes and mitochondria from ROS, with mitochondria being related to apoptosis [12]. There are few studies related to ethanol consumption and Se alteration during pregnancy, but there are a lot which describe an impairment of GPx activity and/or expression in FASD in different tissues during the embryonic period [13,14,15,16] and even in placenta [17,5]. Using a whole embryo culture system exposed to 1 μl/ml ethanol in vitro, [18] described that mRNA levels of cytosolic GPx1, GPx4 and SelP decreased. Recent in vivo studies have found that ethanol exposure during gestation and lactation profoundly alters Se homeostasis and its body distribution by direct action in the pups [19]. This Se imbalance is linked to lower antioxidant GPx activity in tissues and a higher OE [6]. GPx4 liver expression, however, increased [20]. The aim of this study is to analyze Se levels, GPx activity and GPx1 expression in liver of embryos and placenta after chronic alcohol exposure in vivo for the first time. Moreover, these results will be compared to those of liver in offspring at the end of lactation period whose mothers continued drinking ethanol during lactation.
2.3. Samples At the end of the first experimental period, on day 19 of gestation half of all dams were weighed and anesthetised with intraperitoneal 28% v/v urethane (0.5 ml/100 g of body weight). The abdomen was opened by a midline incision and whole livers were removed, debrided of adipose and connective tissue in ice-cold saline, weighed and stored to − 80 °C. The pregnant uterus was exposed via a mid-line incision and the anesthetized embryos were killed via spinal transaction. All fetuses (E19), their livers and their associated placentae were weighed and the samples were immediately stored at − 80 °C prior to biochemical determinations. At the end of the second experimental period, breastfeeding pups (L21) were anesthetised with intraperitoneal 28% v/v urethane (0.5 ml urethane/100 g of body weight). The abdomen was opened by a midline incision and whole livers were removed, debrided of adipose and connective tissue in ice-cold saline, weighed and stored at − 80 °C prior to biochemical determinations. 2.4. Gestation and lactating indexes Female fertility index was calculated as (number of pregnancies / number of mating) × 100; Gestation index as (number of successful births / number of pregnancies rats) × 100; Live-born index as (number of pups born alive / number of pups born) × 100; and Lactation survival index as (number of total offspring − number of dead offspring / number of total offspring) × 100. Hepatic somatic index (HSI) was calculated as (liver weight / body weight) × 100 and placental efficiency (PE) as (fetal weight / placenta weight).
2. Material and methods 2.1. Animals Male (n = 8) and female (n = 16) Wistar rats weighing about 150–200 g (Centre of Animal Production and Experimentation, ViceRector's office for Scientific Research, University of Seville), were randomized into two groups: control (C) and alcohol (A). Drinking water (with or without ethanol) and diets were given ad libitum during the whole experimental period: induction (7 weeks), gestation (3 weeks) and lactation (3 weeks). The diet was prepared in the lab according to The Council of the Institute of Laboratory Animal Resources (ILAR, 1979) and contained 0.1 ppm of Selenium. After the induction period male and female rats were mated to obtain the 1st generation offspring for each group (1 male plus 2 females per cage). The presence of a copulatory plug in the rat cage was considered to be the day 0 of pregnancy. After reproduction, the pregnant rats were housed singly in individual ventilated cages and continued their alcoholic or control treatment until the end of the gestation period. Half of all dams from each group were sacrificed to 19 day gestation to obtain their embryos (E-19). The rest of mothers gave birth to offspring rats at 21 days of gestation; they continue with their treatments and their progeny in their own cages until the end of breastfeeding. Offspring at the end of breastfeeding (L-21) were used to carry out the rest of experiments. The animals were kept at an automatically controlled temperature (22–23 °C) and a 12-h light-dark cycle (9:00 to 21:00). Every day liquid and solid intake was measured. Animal care complied with the ethical Seville University approved and with the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 1996).
2.5. Selenium analysis Se levels were determined by graphite-furnace atomic absorption spectrometry. Equipment: PerkinElmer AAnalyst™ 800 high-performance atomic absorption spectrometer with WinLab32 for AA software, equipped with a Transversely Heated Graphite Furnace (THGA) with longitudinal Zeeman-effect background corrector and AS-furnace autosampler (PerkinElmer, Ueberlingen, Germany). The source of radiation was a Se electrodeless discharge lamp (EDL). The instrumental operating conditions and the reagents are the same that we have used in the previous paper [21], with slight modifications in the mineralization step: ramp time and temperature were different between tissues depending on their matrix content. Samples: placenta and liver of E-19 and L-21 pups were weighed and digested in a sand bath heater (OVAN) with nitric acid during 72 h, and added perchloric acid and chloridric acid (6N). 2.6. Biochemical analysis In order to measure the activity of GPx and GR as well as the oxidation of lipids and protein, liver tissue samples were homogenized in a sucrose buffer (15 mM Tris/HCl, pH 7.4, 250 mM sucrose, 1 mM EDTA and 1 mM DTT) in an ice bath; the resulting supernatant was employed for biochemical assays. The degree of lipid peroxidation in the supernatant was evaluated by a colorimetric reaction at 535 nm with thiobarbituric acid (TBA) as described [22], the results were given as mol/ mg protein. Proteins oxidation was measured according to a method based on the spectrophotometric detection of the reaction of 2.4-dinitrophenylhydrazine (DNPH) with protein carbonyl (PC) to form protein hydrazones [23]. The level of PC was calculated at the maximum
2.2. Ethanol treatment Chronic progressive ethanol treatment was administered to dams in tap water at increasing concentrations until a level of 20% v/v was reached in the induction period, this concentration was maintained 2
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absorbance (366 nm) and the results were expressed as nmol/mg protein. The activity of the selenoprotein GPx was determined by NADPH coupled assay according to the method of [24], this enzyme catalyses the oxidation of glutathione by hydrogen peroxide. The oxidation of NADPH was followed spectrophotometrically at 340 nm. Specific activity was expressed as mU/mg protein where 1 mU is equal to the nanomoles of NADPH oxidized/min. Finally, the glutathione reductase (GR) activity was determined by using the methods of [25]. The protein content of the samples was determined by the method of [26], using bovine serum albumin as the standard.
Table 2 Reproductive, gestational and lactating index.
Female fertility index Gestation index Live–born index Lactation survival index Placenta weight Placental efficiency Body weight-E19 Liver weight-E19 HSI-E19 Body weight-L21 Liver weight-L21 HSI-L21
2.7. Western blot analysis Tissue homogenization to obtain protein lysates and subsequent sodium dodecyl sulphate-polyacrylamide gel electrophoresis and inmmunoblotting were performed. The specific primary antibodies were: rabbit polyclonal to glutathione peroxidase 1 (1:2500; Santa Cruz Biotechnology, Inc. GPx 1/2 H-151, sc-30147) and mouse monoclonal to β-actin (1:50,000; Sigma, St Louis, USA, code A5441). The horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz (anti-rabbit) and Sigma (anti-mouse), respectively. The quantification of the blots was performed by densitometry with PCBAS 2.08e software analysis (Raytest Inc., Straubenhardt, Germany). The results were expressed as percent arbitrary relative units, referring to values in control animals which were defined as 100%.
Control (C)
Alcohol (A)
100 100 100 100 0.34 ± 0.01* 4.03 ± 0.21 1.33 ± 0.03* 0.1 ± 0.003*** 0.077 ± 0.004** 31.6 ± 1*** 1.20 ± 0.07** 3.81 ± 0.07
81,8 66 100 89.36 0.29 ± 0.01 4.20 ± 0.26 1.20 ± 0.05 0.07 ± 0.003 0.058 ± 0.003 22 ± 1.7 0.81 ± 0.05 3.70 ± 0.07
The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of dams in each group during gestation is 8, and during lactation 4. Signification: C vs A: *p > 0.05; **p > 0.01; ***p < 0.001. HSI: hepatic somatic index.
2.8. Statistical methods Fig. 1. Selenium levels in placenta (PL) and liver of embryos (E-19) and offspring at the end of lactation period (L-21). The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group was PL (n = 4), E-19 (n = 8) and L-21 (n = 8). Signification: C vs A: *p > 0.05; **p > 0.01. PL: placenta, E-19: embryos of 19 d old, L-21: offspring at the end of lactation period (21 d old).
The results are expressed as means ± standard error of the mean (SEM). The effects of rats exposed to ethanol were determined by analysing the data obtained with a statistical program (GraphPad InStat 3, CA, USA) by analysis of variance (one-way ANOVA). The unpaired Student's t-test was used to prove the significance of the difference between the means of the values obtained from the experimental group and the values obtained from the control group. The statistical significance was established at p < 0.05.
E-19 embryos; it did, however, increase significantly in ethanol-exposed L-21 pups during gestation and lactation. The glutathione antioxidant system was evaluated by the GR/GPx activities ratio. In this study this ratio is downregulated in placenta and in embryos and upregulated in L-21 pups (Fig. 3) after ethanol consumption. Alcohol exposure significantly increased lipid oxidation in placenta and in the liver of embryos, as well as increasing protein oxidation in the liver of L-21 pups (Fig. 4).Chronic ethanol consumption significantly decreased GPx1 expression in placenta and in the liver of E-19 and L-21 pups, this decrease being higher in embryos (Fig. 5). When GPx1 expression was compared to Se and to GPx activity, it was found that the GPx1 expression/GPx activity ratio decreased in the liver of their E-19 and L-21 pups, after their dams were exposed to ethanol, this decrease being higher in embryos (Fig. 6).
3. Results During gestation and lactation ethanol-exposed dams consumed the same energy (total kcal) as control ones. However, they consumed significantly less food-derived energy and Se. Lactating dams consumed a higher amount of food and ethanol than pregnant ones (Table 1). Alcohol consumption leads to a lower fertility and gestational index, and a significantly lower placental weight. The live births index was unaltered by ethanol while the lactation survival index decreased. Ethanol-exposed E-19 embryos had significantly lower body weight and HSI. Ethanol exposure during lactation profoundly decreased pups body weight and liver weight (Table 2). Chronic ethanol consumption significantly decreased Se deposits in placenta and in the liver of E-19 and L-21 pups, this decrease being higher in nursing rats (Fig. 1). Fig. 2 shows that GPx activity in placenta and in the liver of E-19 and L-21 pups decreased significantly after ethanol consumption, this decrease being higher in L-21 rats. GR activity also decreased in placenta and in
4. Discussion According to the bibliography, in this study chronic alcohol consumption induced an imbalance in the reproductive state; decreasing
Table 1 Kilocalories consumed and Se intake during gestation and lactation by dams. Period
Group
kcal/d (solid fed)
Gestation
Control (C) Alcohol (A) Control (C) Alcohol (A)
77.8 ± 3.61⁎⁎ 57.5 ± 3.4 127.1 ± 9.03⁎⁎ 77.9 ± 5.12
Breastfeeding
kcal/d (ethanol)
19.13 ± 0.9 37.14 ± 1.8
kcal total/d
Se intake (μg/día)
77.8 ± 3.61 76.6 ± 3.8 127.1 ± 9.03 115.1 ± 6.03
1.9 1.4 3.1 1.9
± ± ± ±
0.09⁎⁎ 0.09 0.18⁎⁎ 0.09
The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group during gestation is 8, and during lactation 4. Signification: C vs A: *p > 0.05; **p > 0.01.
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Fig. 2. GPx (A) and GR (B) activity in placenta (PL) and liver of embryos (E-19) and offspring at the end of lactation period (L-21). The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group was PL (n = 4), E-19 (n = 8) and L-21 (n = 8). Signification: C vs A: *p > 0.05; **p > 0.01; ***p < 0.001. PL: placenta, E-19: embryos of 19 d old, L-21: offspring at the end of lactation period (21 d old).
concentration [27]. Since ethanol reduced the suckling pressure and the frequency of rapid rhythmic sucks per minute [28], it is important to avoid ethanol consumption during gestation, when mothers do not ensure a correct supply of nutrients both via placenta and during lactation. Despite the fact that ethanol consumption did not affect dams' total energy intake during the whole experimental period, gestational alcoholic dams received 26% of their total kcal from ethanol, and lactating dams 32%. It is known that energy provided by alcohol is not accompanied by nutrients and is therefore called “empty kcal” [29]. Consequently, these animals received a lower amount of food and of essential elements such as Se. This lower Se intake during gestation and lactation alters Se tissue deposits; specifically in placenta where chronic ethanol consumption caused a depletion of Se deposits. This is the first time that this fact has been reported in vivo. Studies in vitro [5,17] have provided direct evidence that Se plays a crucial role in placenta since it regulates the glutathione system via GPx and the thioredoxin system via selenoprotein thioredoxin reductase. Catalase and superoxide dismutase systems are both of critical importance in placental tissues since they provide the first line of defense against ROS. These authors have proved that Se supplementation upregulated the endogenous antioxidant system and protects trophoblast cells from OE and, more specifically, from mitochondrial OS, emphasizing the importance of maintaining adequate Se deposits in placenta during gestation. After ethanol consumption, the Se depletion found in placenta could be acting as another important detrimental factor in the pathogenesis of alcohol consumption and OS, since trophoblast dysfunction has an important impact on pregnancy loss and fetal growth. In this context, [30] found that not only was heavy maternal alcohol drinking accompanied by an increase in maternal serum Se, but also by
Fig. 3. GR/GPx ratio in placenta and liver of embryos (E-19) and offspring at the end of lactation period (L-21). The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group was PL (n = 4), E-19 (n = 8) and L-21 (n = 8). Signification: C vs A: *p > 0.05; ***p < 0.001. PL: placenta, E-19: embryos of 19 d old, L-21: offspring at the end of lactation period (21 d old).
the capacity for pregnancy (34%) and conception (20%). Furthermore, alcohol exposure leads to a reduction in placenta development and in embryo size, this effect on body weight is even worse during lactation. This could, in part, be due to the fact that ethanol consumption during lactation and gestation provokes under-nutrition in dams. Moreover, during the breastfeeding period, ethanol-exposed dams intake a higher amount of ethanol than during gestation. This fact is also in agreement with the reduction of the lactation survival index found. Previous papers from our laboratory have reported that ethanol exposed-lactating pups suckled a lower amount of milk and that this milk had a lower Se
Fig. 4. Production of MDA (A) and PC (B) in placenta and liver of embryos (E-19) and offspring at the end of lactation period (L-21). The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group was PL (n = 4), E-19 (n = 8) and L-21 (n = 8). Signification: C vs A: *p > 0.05; **p < 0.01. PL: placenta, E-19: embryos of 19 d old, L21: offspring at the end of lactation period (21 d old).
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Fig. 5. Expression levels of GPx1 in placenta and liver of embryos (E-19) and offspring at the end of lactation period (L-21) (A). Representative Western blots of GPx1 (normalized to β-actin) (B). The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group was PL (n = 4), E-19 (n = 8) and L-21 (n = 8). Signification: C vs A: *p > 0.05; **p < 0.01. PL: placenta, E-19: embryos of 19 d old, L21: offspring at the end of lactation period (21 d old).
hippocampus of suckling pups exposed to a similar model of alcohol exposure. In contrast the ratio, in kidney decreased and lipid and protein oxidation took place [27]. However it is known that the oxidative stress and enzymatic antioxidant system differs among tissues [33]. It is known that in placenta ethanol exposure induces oxidative stress [34] and lipid peroxidation [35], both of which are related to placental apoptosis/necrosis. Moreover, Gundogan et al. [34] confirm that ethanol mediates part of its adverse effects during gestation by inhibiting the prolactin family of hormones. The decreased of prolactin secretion due to alcohol consumption is relevant to this study, since lactating dams and their pups appears to be specially affected by ethanol consumption. According to our data, Perez et al. [35], recorded that lipid oxidation in fetal liver samples was enhanced by ethanol. However, it is not clear how the ethanol does not affect lipid oxidation in pups during lactation while it does increase liver protein oxidation. It could be hypothesized that GR is playing a pivotal role in this event; that these pups are undernourished and have lower amounts of lipid, or that during this period after ethanol exposure GPx4 (the only GPx which prevents phospholipid oxidation) increases [20]. More studies need to be undertaken. Using a whole embryo culture system exposed to 1 μl/ml ethanol in vitro, Lee et al. [18] described that mRNA levels of cytosolic GPx1, GPx4 and SelP were lower. In this study chronic ethanol consumption significantly decreased GPx1 expression in placenta and in the liver of E-19 and L-21 pups. In all the tissues studied this decrease in GPx expression was related to Se tissue deposits in ethanol-exposed or nonexposed rats, as shown by the Se/GPx1expression ratio. This confirms that GPx1 is one of the lower-hierarchy selenoproteins within the selenoproteome [9], its expression depending on the amount of Se available. Thus, the synthesis of GPx1 appears to be a good indicator of depleted hepatic Se levels. In the liver of fetuses and weaning pups this GPx expression is not, however, directly related to GPx activity after
a decrease in umbilical cord Se concentration. Alcohol-damaged newborns did not, however, demonstrate any decrease in serum Se levels. It is also important to note that Se serum levels are not the only parameter related to Se status, since, as indicated [31], in order to establish a complete Se status it is necessary to obtain information upon its absorption, retention, distribution and how it functions. For this reason when liver Se deposits were measured in E-19 exposed to chronic ethanol, also for first time in vivo, these levels were found to be lower confirming that ethanol consumed by dams during pregnancy directly affects the Se status of their embryos. If dams continue drinking ethanol during breastfeeding, Se liver depletion in pups is even more accentuated. In order to determine if these tissue Se depletions have repercussions in biological Se function, we focused our attention on GPx activity and GSH antioxidant system. As expected, the reduction in Se levels after alcohol exposure was linked to a reduction in GPx1 activity, this being even greater in L-21 pups. Several studies reflected an impairment of GPx activity and/or expression in brain and liver in FASD during the embryo period [14,16], and in serum [15]. These authors also found higher lipid peroxidation and H202 levels, and lower GSH values in the tissues studied. Since GR activity increased in L-21 pups after ethanol exposition, the GR/GPx ratio increased during lactation. This only occurs during lactation, since the ratio was decreased in both placenta and embryos. To evaluate if this regulation is effective or not, lipids and protein oxidation were analyzed. As expected, lipid oxidation occurs in placenta and in embryos; it appears, therefore, that the GSH antioxidant system downregulation is related to tissue oxidation. In the liver of L-21 pups protein, but not lipid, oxidation occurred. Therefore despite the effort to increase GR activity in order to balance GSH levels, oxidation did indeed occur, since GPx activity decreased. Cesconetto et al. [32] have also found an upregulation of the GR/GPx ratio in the
Fig. 6. Selenium/GPx1 expression (A) and GPx1 expression/GPx activity (B) ratios in placenta and liver of embryos (E-19) and offspring at the end of lactation period (L-21). The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group was PL (n = 4), E-19 (n = 8) and L-21 (n = 8). Signification: C vs A: *p > 0.05; **p < 0.01. PL: placenta, E-19: embryos of 19 d old, L-21: offspring at the end of lactation period (21 d old).
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ethanol exposure. This shows that ethanol per se, apart from leading to a depletion of Se and GPx1 expression, inactivates GPx activity in offspring, but not in placenta. During its metabolic oxidation [20] ethanol consumes GSH and NADPH and these molecules are necessary for proper GPx activity [36]. These data support the idea that alcohol is a pro-oxidant substance that not only decreases GPx1 activity by depleting Se deposits and GPx1 expression, but also by promoting GPx inactivation, decreasing pups' antioxidant capacity. GPx activity in placenta is proportional to the GPx1 expression found, confirming that ethanol consumption affects GPx activity in offspring during pregnancy and weaning to a greater extent than it does in dams. In conclusion, for the first time in vivo it was found that Se is depleted after ethanol consumption in placenta and in the liver of E-19 embryos and that in both cases GPx activity depends on this element's concentration. In E-19 and L-21 pups, however, this GPx activity is also reduced by a direct action of ethanol metabolism. This decrease in GPx activity in placenta and fetuses are related to the oxidation of lipids and proteins in nursing pups, contributing to cell dysfunctions. For all of these reasons, in order to prevent oxidative stress and teratogen problems, administering a Se antioxidant therapy to dams consuming ethanol during gestation and/or lactation appears to be recommended. Acknowledgements This work was supported by a grant from Andalusian Regional Government (CTS-193) by supporting CTS-193 Research Group. References [1] C. Bosco, E. Diaz, Placental hypoxia and foetal development versus alcohol exposure in pregnancy, Alcohol Alcohol. 47 (2012) 109–117. [2] M.L. Murillo-Fuentes, R. Artillo, M.L. Ojeda, M.J. Delgado, M.L. Murillo, O. Carreras, Effects of prenatal or postnatal ethanol consumption on Zn intestinal absorption and excretion in rats, Alcohol 42 (2007) 3–10. [3] R. Cohen-Kerem, G. Koren, Antioxidants and fetal protection against ethanol teratogenicity. I. Review of the experimental data and implications to humans, Neurotoxicol. Teratol. 25 (2003) 1–9. [4] C. Bosco, V.R. Preedy, R.R. Watson, Alcohol and xenobiotics in placenta damage, Comprehensive Handbook of Alcohol Related Pathology, 2 Elsevier Science, London, 2005, pp. 921–935. [5] A. Khera, J.J. Vanderlelie, A.V. Perkins, Selenium supplementation protects trophoblast cells from mitochondrial oxidative stress, Placenta 34 (2013) 594–598. [6] M.L. Ojeda, M.J. Delgado-Villa, R. Llopis, M.L. Murillo, O. Carreras, Lipid metabolism in ethanol-treated rat pups and adults: effects of folic acid, Alcohol 43 (2008) 544–550. [7] P.A. Dennery, Effects of oxidative stress on embryonic development, Birth Defects Res. C Embryo Today. 81 (2007) 155–162. [8] C. Ufer, C.C. Wang, The roles of glutathione peroxidases during embryo development, Front. Mol. Neurosci. 4 (2011) 12. [9] R. Briguelius-Flohé, M. Maiorino, Glutathione peroxidases, Biochim. Biophys. Acta 1830 (2013) 3289–3303. [10] Y.S. Ho, J.L. Magnenat, R.T. Bronson, J. Cao, M. Gargano, M. Sugawara, C.D. Funk, Mice deficient in cellular glutathione peroxidase develop normally and show no increased sensitivity to hyperoxia, J. Biol. Chem. 272 (1997) 16644–16651. [11] H. Imai, F. Hirao, T. Sakamoto, K. Sekine, Y. Mizukura, M. Saito, T. Kitamoto, M. Hayasaka, K. Hanaoka, Y. Nakagawa, Early embryonic lethality caused by targeted disruption of the mouse PHGPx gene, Biochem. Biophys. Res. Commun. 305
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Maternal ethanol consumption reduces Se antioxidant function in placenta and liver of embryos and breastfeeding pups
MARK
Fátima Nogalesa,1, M. Luisa Ojedaa,1, Karick Jottyb, M. Luisa Murilloa, Olimpia Carrerasa,⁎ a b
Department of Physiology, Faculty of Pharmacy, Seville University, 41012 Seville, Spain Biology Program, Universidad de Cartagena, Cartagena, Colombia
A R T I C L E I N F O
A B S T R A C T
Keywords: Fetal alcohol syndrome disorders Selenium Glutathione peroxidase Embryos Placenta
Aim: The fetal alcohol exposition during pregnancy leads to different disorders in offspring, related to the oxidative stress generated by alcohol. It is well-documented that there is an impairment of the antioxidant selenoprotein Glutathione peroxidase (GPx) activity in ethanol offspring during the embryo period, although noone has described Selenium (Se) status. The aim is to analyze for the first time Se deposits in vivo and Se's biological implication in embryos and placenta after alcohol exposure and in offspring whose mothers continued to drink ethanol during lactation. Materials and methods: Se deposits, GPx and glutathione reductase (GR) activity, lipid and protein oxidation and the expression of GPx1 were measured in placenta and liver of both embryos (E-19) and breastfeeding pups (L21) in control and ethanol groups (20% v/v). Key findings: Ethanol consumption decreased Se deposits, GPx activity and GPx1 expression, while increasing biomolecular oxidation in placenta and in the liver of E-19 and L-21. The GR/GPx ratio decreased in placenta and in E-19, together with an increase in lipid oxidation, while increased in the liver of L-21 pups with protein oxidation. Ethanol also decreased the GPx1 expression/GPx activity ratio in the liver of E-19 and L-21, indicating that alcohol decreases GPx activity by both depleting Se deposits and promoting GPx inactivation. In placenta GPx activity is proportional to the GPx1 expression found, so the ethanol affects GPx activity in offspring more than in dams. Significance: Therefore, Se supplementation therapy in dams could contribute as an interesting antioxidant that prevents fetal alcohol syndrome.
1. Introduction Ethanol is one of the most common human teratogen drugs consumed, leading to serious adverse outcomes in the fetus, such as intrauterine growth retardation (IUGR), craniofacial malformations, physical and mental retardation, and cardiac septal defects. This is known as fetal alcohol syndrome (FAS). Sometimes a lesser degree of deformations, described as fetal alcohol spectrum disorders (FASDs), could be associated with FAS. Some offspring, however, have no symptoms (resilience) or only present partial FAS-related symptoms [1]. These effects could be even worse if mothers continue consuming ethanol during breastfeeding [2]. The cellular mechanisms by which ethanol induces damage in utero are not well understood, but induction of oxidative stress is believed to be one important mechanism [3]. The fetal injury by ethanol could be explained by a direct mechanism through feto-toxicity from ethanol
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1
and/or acetaldehyde, which is intimately related to oxidative stress induction and/or an indirect mechanism through ethanol-induced placenta injury and selective fetal malnutrition [4]. Insufficient supplies of essential trace elements with antioxidant properties such as selenium (Se) could contribute to the oxidative stress generated by ethanol exposure. Oxidation of the placenta itself is central to the pathogenesis of many disorders during pregnancy [5]. It has been demonstrated that ethanol exposure during lactation via maternal milk also increases protein and lipid oxidation in the liver of breastfeeding pups, decreasing endogenous antioxidant enzymes activities and hepatic glutathione (GSH) levels [6]. During embryo development there is a complex interplay among proliferation, differentiation and apoptotic cell events. Despite the fact that a low amount of reactive oxygen species (ROS) acting as primary or secondary messengers to promote cell growth or death are vital for maintaining this interplay, an excess of ROS levels are related to
Corresponding author at: Department of Physiology, Faculty of Pharmacy, Seville University, C/Profesor García González, n° 2, 41012 Sevilla, Spain. E-mail address: [email protected] (O. Carreras). Authors who have shared the main task of preparing the manuscript, and have dedicated the same effort, both should have the consideration of first author.
http://dx.doi.org/10.1016/j.lfs.2017.09.021 Received 14 July 2017; Received in revised form 8 September 2017; Accepted 18 September 2017 Available online 22 September 2017 0024-3205/ © 2017 Published by Elsevier Inc.
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during gestation and lactation [6]. Total kilocalories (kcal) was obtained by adding to the solid kcal the kcal derived from alcohol consumption. Solid kcal intake was calculated by multiplying the g of food ingested per day by 4.1 kcal, alcoholic kcal intake was determined by multiplying the ml of ethanol ingested per day by 7.1 kcal. Se intake was estimated by multiplying the g of food ingested per day by 0.1.
protein, lipid and DNA oxidation, leading to cell dysfunctions. Fortunately, antioxidants can obviate these effects by modifying gene expression, transcription factor signaling, and cell cycle alterations [7]. One of the main cellular protective systems that plays an important role during embryo development is the selenium-dependent glutathione peroxidases (GPxs) family [8]. These enzymes reduce the pro-oxidative ROS hydroperoxides to harmless water and oxygen. GPx1 is regarded as this family's major antioxidant enzyme. It does, however, depend on Se levels; if Se decreases, GPx1 expression falls [9]. Despite the fact that studies with GPx1 knockout mice have revealed that these animals compensate mild oxidative stress [10], GPx4 knockout mice are nonviable [11]. GPx4 is the only selenoprotein which protects cellular membranes and mitochondria from ROS, with mitochondria being related to apoptosis [12]. There are few studies related to ethanol consumption and Se alteration during pregnancy, but there are a lot which describe an impairment of GPx activity and/or expression in FASD in different tissues during the embryonic period [13,14,15,16] and even in placenta [17,5]. Using a whole embryo culture system exposed to 1 μl/ml ethanol in vitro, [18] described that mRNA levels of cytosolic GPx1, GPx4 and SelP decreased. Recent in vivo studies have found that ethanol exposure during gestation and lactation profoundly alters Se homeostasis and its body distribution by direct action in the pups [19]. This Se imbalance is linked to lower antioxidant GPx activity in tissues and a higher OE [6]. GPx4 liver expression, however, increased [20]. The aim of this study is to analyze Se levels, GPx activity and GPx1 expression in liver of embryos and placenta after chronic alcohol exposure in vivo for the first time. Moreover, these results will be compared to those of liver in offspring at the end of lactation period whose mothers continued drinking ethanol during lactation.
2.3. Samples At the end of the first experimental period, on day 19 of gestation half of all dams were weighed and anesthetised with intraperitoneal 28% v/v urethane (0.5 ml/100 g of body weight). The abdomen was opened by a midline incision and whole livers were removed, debrided of adipose and connective tissue in ice-cold saline, weighed and stored to − 80 °C. The pregnant uterus was exposed via a mid-line incision and the anesthetized embryos were killed via spinal transaction. All fetuses (E19), their livers and their associated placentae were weighed and the samples were immediately stored at − 80 °C prior to biochemical determinations. At the end of the second experimental period, breastfeeding pups (L21) were anesthetised with intraperitoneal 28% v/v urethane (0.5 ml urethane/100 g of body weight). The abdomen was opened by a midline incision and whole livers were removed, debrided of adipose and connective tissue in ice-cold saline, weighed and stored at − 80 °C prior to biochemical determinations. 2.4. Gestation and lactating indexes Female fertility index was calculated as (number of pregnancies / number of mating) × 100; Gestation index as (number of successful births / number of pregnancies rats) × 100; Live-born index as (number of pups born alive / number of pups born) × 100; and Lactation survival index as (number of total offspring − number of dead offspring / number of total offspring) × 100. Hepatic somatic index (HSI) was calculated as (liver weight / body weight) × 100 and placental efficiency (PE) as (fetal weight / placenta weight).
2. Material and methods 2.1. Animals Male (n = 8) and female (n = 16) Wistar rats weighing about 150–200 g (Centre of Animal Production and Experimentation, ViceRector's office for Scientific Research, University of Seville), were randomized into two groups: control (C) and alcohol (A). Drinking water (with or without ethanol) and diets were given ad libitum during the whole experimental period: induction (7 weeks), gestation (3 weeks) and lactation (3 weeks). The diet was prepared in the lab according to The Council of the Institute of Laboratory Animal Resources (ILAR, 1979) and contained 0.1 ppm of Selenium. After the induction period male and female rats were mated to obtain the 1st generation offspring for each group (1 male plus 2 females per cage). The presence of a copulatory plug in the rat cage was considered to be the day 0 of pregnancy. After reproduction, the pregnant rats were housed singly in individual ventilated cages and continued their alcoholic or control treatment until the end of the gestation period. Half of all dams from each group were sacrificed to 19 day gestation to obtain their embryos (E-19). The rest of mothers gave birth to offspring rats at 21 days of gestation; they continue with their treatments and their progeny in their own cages until the end of breastfeeding. Offspring at the end of breastfeeding (L-21) were used to carry out the rest of experiments. The animals were kept at an automatically controlled temperature (22–23 °C) and a 12-h light-dark cycle (9:00 to 21:00). Every day liquid and solid intake was measured. Animal care complied with the ethical Seville University approved and with the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 1996).
2.5. Selenium analysis Se levels were determined by graphite-furnace atomic absorption spectrometry. Equipment: PerkinElmer AAnalyst™ 800 high-performance atomic absorption spectrometer with WinLab32 for AA software, equipped with a Transversely Heated Graphite Furnace (THGA) with longitudinal Zeeman-effect background corrector and AS-furnace autosampler (PerkinElmer, Ueberlingen, Germany). The source of radiation was a Se electrodeless discharge lamp (EDL). The instrumental operating conditions and the reagents are the same that we have used in the previous paper [21], with slight modifications in the mineralization step: ramp time and temperature were different between tissues depending on their matrix content. Samples: placenta and liver of E-19 and L-21 pups were weighed and digested in a sand bath heater (OVAN) with nitric acid during 72 h, and added perchloric acid and chloridric acid (6N). 2.6. Biochemical analysis In order to measure the activity of GPx and GR as well as the oxidation of lipids and protein, liver tissue samples were homogenized in a sucrose buffer (15 mM Tris/HCl, pH 7.4, 250 mM sucrose, 1 mM EDTA and 1 mM DTT) in an ice bath; the resulting supernatant was employed for biochemical assays. The degree of lipid peroxidation in the supernatant was evaluated by a colorimetric reaction at 535 nm with thiobarbituric acid (TBA) as described [22], the results were given as mol/ mg protein. Proteins oxidation was measured according to a method based on the spectrophotometric detection of the reaction of 2.4-dinitrophenylhydrazine (DNPH) with protein carbonyl (PC) to form protein hydrazones [23]. The level of PC was calculated at the maximum
2.2. Ethanol treatment Chronic progressive ethanol treatment was administered to dams in tap water at increasing concentrations until a level of 20% v/v was reached in the induction period, this concentration was maintained 2
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absorbance (366 nm) and the results were expressed as nmol/mg protein. The activity of the selenoprotein GPx was determined by NADPH coupled assay according to the method of [24], this enzyme catalyses the oxidation of glutathione by hydrogen peroxide. The oxidation of NADPH was followed spectrophotometrically at 340 nm. Specific activity was expressed as mU/mg protein where 1 mU is equal to the nanomoles of NADPH oxidized/min. Finally, the glutathione reductase (GR) activity was determined by using the methods of [25]. The protein content of the samples was determined by the method of [26], using bovine serum albumin as the standard.
Table 2 Reproductive, gestational and lactating index.
Female fertility index Gestation index Live–born index Lactation survival index Placenta weight Placental efficiency Body weight-E19 Liver weight-E19 HSI-E19 Body weight-L21 Liver weight-L21 HSI-L21
2.7. Western blot analysis Tissue homogenization to obtain protein lysates and subsequent sodium dodecyl sulphate-polyacrylamide gel electrophoresis and inmmunoblotting were performed. The specific primary antibodies were: rabbit polyclonal to glutathione peroxidase 1 (1:2500; Santa Cruz Biotechnology, Inc. GPx 1/2 H-151, sc-30147) and mouse monoclonal to β-actin (1:50,000; Sigma, St Louis, USA, code A5441). The horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz (anti-rabbit) and Sigma (anti-mouse), respectively. The quantification of the blots was performed by densitometry with PCBAS 2.08e software analysis (Raytest Inc., Straubenhardt, Germany). The results were expressed as percent arbitrary relative units, referring to values in control animals which were defined as 100%.
Control (C)
Alcohol (A)
100 100 100 100 0.34 ± 0.01* 4.03 ± 0.21 1.33 ± 0.03* 0.1 ± 0.003*** 0.077 ± 0.004** 31.6 ± 1*** 1.20 ± 0.07** 3.81 ± 0.07
81,8 66 100 89.36 0.29 ± 0.01 4.20 ± 0.26 1.20 ± 0.05 0.07 ± 0.003 0.058 ± 0.003 22 ± 1.7 0.81 ± 0.05 3.70 ± 0.07
The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of dams in each group during gestation is 8, and during lactation 4. Signification: C vs A: *p > 0.05; **p > 0.01; ***p < 0.001. HSI: hepatic somatic index.
2.8. Statistical methods Fig. 1. Selenium levels in placenta (PL) and liver of embryos (E-19) and offspring at the end of lactation period (L-21). The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group was PL (n = 4), E-19 (n = 8) and L-21 (n = 8). Signification: C vs A: *p > 0.05; **p > 0.01. PL: placenta, E-19: embryos of 19 d old, L-21: offspring at the end of lactation period (21 d old).
The results are expressed as means ± standard error of the mean (SEM). The effects of rats exposed to ethanol were determined by analysing the data obtained with a statistical program (GraphPad InStat 3, CA, USA) by analysis of variance (one-way ANOVA). The unpaired Student's t-test was used to prove the significance of the difference between the means of the values obtained from the experimental group and the values obtained from the control group. The statistical significance was established at p < 0.05.
E-19 embryos; it did, however, increase significantly in ethanol-exposed L-21 pups during gestation and lactation. The glutathione antioxidant system was evaluated by the GR/GPx activities ratio. In this study this ratio is downregulated in placenta and in embryos and upregulated in L-21 pups (Fig. 3) after ethanol consumption. Alcohol exposure significantly increased lipid oxidation in placenta and in the liver of embryos, as well as increasing protein oxidation in the liver of L-21 pups (Fig. 4).Chronic ethanol consumption significantly decreased GPx1 expression in placenta and in the liver of E-19 and L-21 pups, this decrease being higher in embryos (Fig. 5). When GPx1 expression was compared to Se and to GPx activity, it was found that the GPx1 expression/GPx activity ratio decreased in the liver of their E-19 and L-21 pups, after their dams were exposed to ethanol, this decrease being higher in embryos (Fig. 6).
3. Results During gestation and lactation ethanol-exposed dams consumed the same energy (total kcal) as control ones. However, they consumed significantly less food-derived energy and Se. Lactating dams consumed a higher amount of food and ethanol than pregnant ones (Table 1). Alcohol consumption leads to a lower fertility and gestational index, and a significantly lower placental weight. The live births index was unaltered by ethanol while the lactation survival index decreased. Ethanol-exposed E-19 embryos had significantly lower body weight and HSI. Ethanol exposure during lactation profoundly decreased pups body weight and liver weight (Table 2). Chronic ethanol consumption significantly decreased Se deposits in placenta and in the liver of E-19 and L-21 pups, this decrease being higher in nursing rats (Fig. 1). Fig. 2 shows that GPx activity in placenta and in the liver of E-19 and L-21 pups decreased significantly after ethanol consumption, this decrease being higher in L-21 rats. GR activity also decreased in placenta and in
4. Discussion According to the bibliography, in this study chronic alcohol consumption induced an imbalance in the reproductive state; decreasing
Table 1 Kilocalories consumed and Se intake during gestation and lactation by dams. Period
Group
kcal/d (solid fed)
Gestation
Control (C) Alcohol (A) Control (C) Alcohol (A)
77.8 ± 3.61⁎⁎ 57.5 ± 3.4 127.1 ± 9.03⁎⁎ 77.9 ± 5.12
Breastfeeding
kcal/d (ethanol)
19.13 ± 0.9 37.14 ± 1.8
kcal total/d
Se intake (μg/día)
77.8 ± 3.61 76.6 ± 3.8 127.1 ± 9.03 115.1 ± 6.03
1.9 1.4 3.1 1.9
± ± ± ±
0.09⁎⁎ 0.09 0.18⁎⁎ 0.09
The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group during gestation is 8, and during lactation 4. Signification: C vs A: *p > 0.05; **p > 0.01.
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Fig. 2. GPx (A) and GR (B) activity in placenta (PL) and liver of embryos (E-19) and offspring at the end of lactation period (L-21). The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group was PL (n = 4), E-19 (n = 8) and L-21 (n = 8). Signification: C vs A: *p > 0.05; **p > 0.01; ***p < 0.001. PL: placenta, E-19: embryos of 19 d old, L-21: offspring at the end of lactation period (21 d old).
concentration [27]. Since ethanol reduced the suckling pressure and the frequency of rapid rhythmic sucks per minute [28], it is important to avoid ethanol consumption during gestation, when mothers do not ensure a correct supply of nutrients both via placenta and during lactation. Despite the fact that ethanol consumption did not affect dams' total energy intake during the whole experimental period, gestational alcoholic dams received 26% of their total kcal from ethanol, and lactating dams 32%. It is known that energy provided by alcohol is not accompanied by nutrients and is therefore called “empty kcal” [29]. Consequently, these animals received a lower amount of food and of essential elements such as Se. This lower Se intake during gestation and lactation alters Se tissue deposits; specifically in placenta where chronic ethanol consumption caused a depletion of Se deposits. This is the first time that this fact has been reported in vivo. Studies in vitro [5,17] have provided direct evidence that Se plays a crucial role in placenta since it regulates the glutathione system via GPx and the thioredoxin system via selenoprotein thioredoxin reductase. Catalase and superoxide dismutase systems are both of critical importance in placental tissues since they provide the first line of defense against ROS. These authors have proved that Se supplementation upregulated the endogenous antioxidant system and protects trophoblast cells from OE and, more specifically, from mitochondrial OS, emphasizing the importance of maintaining adequate Se deposits in placenta during gestation. After ethanol consumption, the Se depletion found in placenta could be acting as another important detrimental factor in the pathogenesis of alcohol consumption and OS, since trophoblast dysfunction has an important impact on pregnancy loss and fetal growth. In this context, [30] found that not only was heavy maternal alcohol drinking accompanied by an increase in maternal serum Se, but also by
Fig. 3. GR/GPx ratio in placenta and liver of embryos (E-19) and offspring at the end of lactation period (L-21). The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group was PL (n = 4), E-19 (n = 8) and L-21 (n = 8). Signification: C vs A: *p > 0.05; ***p < 0.001. PL: placenta, E-19: embryos of 19 d old, L-21: offspring at the end of lactation period (21 d old).
the capacity for pregnancy (34%) and conception (20%). Furthermore, alcohol exposure leads to a reduction in placenta development and in embryo size, this effect on body weight is even worse during lactation. This could, in part, be due to the fact that ethanol consumption during lactation and gestation provokes under-nutrition in dams. Moreover, during the breastfeeding period, ethanol-exposed dams intake a higher amount of ethanol than during gestation. This fact is also in agreement with the reduction of the lactation survival index found. Previous papers from our laboratory have reported that ethanol exposed-lactating pups suckled a lower amount of milk and that this milk had a lower Se
Fig. 4. Production of MDA (A) and PC (B) in placenta and liver of embryos (E-19) and offspring at the end of lactation period (L-21). The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group was PL (n = 4), E-19 (n = 8) and L-21 (n = 8). Signification: C vs A: *p > 0.05; **p < 0.01. PL: placenta, E-19: embryos of 19 d old, L21: offspring at the end of lactation period (21 d old).
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Fig. 5. Expression levels of GPx1 in placenta and liver of embryos (E-19) and offspring at the end of lactation period (L-21) (A). Representative Western blots of GPx1 (normalized to β-actin) (B). The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group was PL (n = 4), E-19 (n = 8) and L-21 (n = 8). Signification: C vs A: *p > 0.05; **p < 0.01. PL: placenta, E-19: embryos of 19 d old, L21: offspring at the end of lactation period (21 d old).
hippocampus of suckling pups exposed to a similar model of alcohol exposure. In contrast the ratio, in kidney decreased and lipid and protein oxidation took place [27]. However it is known that the oxidative stress and enzymatic antioxidant system differs among tissues [33]. It is known that in placenta ethanol exposure induces oxidative stress [34] and lipid peroxidation [35], both of which are related to placental apoptosis/necrosis. Moreover, Gundogan et al. [34] confirm that ethanol mediates part of its adverse effects during gestation by inhibiting the prolactin family of hormones. The decreased of prolactin secretion due to alcohol consumption is relevant to this study, since lactating dams and their pups appears to be specially affected by ethanol consumption. According to our data, Perez et al. [35], recorded that lipid oxidation in fetal liver samples was enhanced by ethanol. However, it is not clear how the ethanol does not affect lipid oxidation in pups during lactation while it does increase liver protein oxidation. It could be hypothesized that GR is playing a pivotal role in this event; that these pups are undernourished and have lower amounts of lipid, or that during this period after ethanol exposure GPx4 (the only GPx which prevents phospholipid oxidation) increases [20]. More studies need to be undertaken. Using a whole embryo culture system exposed to 1 μl/ml ethanol in vitro, Lee et al. [18] described that mRNA levels of cytosolic GPx1, GPx4 and SelP were lower. In this study chronic ethanol consumption significantly decreased GPx1 expression in placenta and in the liver of E-19 and L-21 pups. In all the tissues studied this decrease in GPx expression was related to Se tissue deposits in ethanol-exposed or nonexposed rats, as shown by the Se/GPx1expression ratio. This confirms that GPx1 is one of the lower-hierarchy selenoproteins within the selenoproteome [9], its expression depending on the amount of Se available. Thus, the synthesis of GPx1 appears to be a good indicator of depleted hepatic Se levels. In the liver of fetuses and weaning pups this GPx expression is not, however, directly related to GPx activity after
a decrease in umbilical cord Se concentration. Alcohol-damaged newborns did not, however, demonstrate any decrease in serum Se levels. It is also important to note that Se serum levels are not the only parameter related to Se status, since, as indicated [31], in order to establish a complete Se status it is necessary to obtain information upon its absorption, retention, distribution and how it functions. For this reason when liver Se deposits were measured in E-19 exposed to chronic ethanol, also for first time in vivo, these levels were found to be lower confirming that ethanol consumed by dams during pregnancy directly affects the Se status of their embryos. If dams continue drinking ethanol during breastfeeding, Se liver depletion in pups is even more accentuated. In order to determine if these tissue Se depletions have repercussions in biological Se function, we focused our attention on GPx activity and GSH antioxidant system. As expected, the reduction in Se levels after alcohol exposure was linked to a reduction in GPx1 activity, this being even greater in L-21 pups. Several studies reflected an impairment of GPx activity and/or expression in brain and liver in FASD during the embryo period [14,16], and in serum [15]. These authors also found higher lipid peroxidation and H202 levels, and lower GSH values in the tissues studied. Since GR activity increased in L-21 pups after ethanol exposition, the GR/GPx ratio increased during lactation. This only occurs during lactation, since the ratio was decreased in both placenta and embryos. To evaluate if this regulation is effective or not, lipids and protein oxidation were analyzed. As expected, lipid oxidation occurs in placenta and in embryos; it appears, therefore, that the GSH antioxidant system downregulation is related to tissue oxidation. In the liver of L-21 pups protein, but not lipid, oxidation occurred. Therefore despite the effort to increase GR activity in order to balance GSH levels, oxidation did indeed occur, since GPx activity decreased. Cesconetto et al. [32] have also found an upregulation of the GR/GPx ratio in the
Fig. 6. Selenium/GPx1 expression (A) and GPx1 expression/GPx activity (B) ratios in placenta and liver of embryos (E-19) and offspring at the end of lactation period (L-21). The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way ANOVA) followed by the Student's t-test. The number of animals in each group was PL (n = 4), E-19 (n = 8) and L-21 (n = 8). Signification: C vs A: *p > 0.05; **p < 0.01. PL: placenta, E-19: embryos of 19 d old, L-21: offspring at the end of lactation period (21 d old).
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ethanol exposure. This shows that ethanol per se, apart from leading to a depletion of Se and GPx1 expression, inactivates GPx activity in offspring, but not in placenta. During its metabolic oxidation [20] ethanol consumes GSH and NADPH and these molecules are necessary for proper GPx activity [36]. These data support the idea that alcohol is a pro-oxidant substance that not only decreases GPx1 activity by depleting Se deposits and GPx1 expression, but also by promoting GPx inactivation, decreasing pups' antioxidant capacity. GPx activity in placenta is proportional to the GPx1 expression found, confirming that ethanol consumption affects GPx activity in offspring during pregnancy and weaning to a greater extent than it does in dams. In conclusion, for the first time in vivo it was found that Se is depleted after ethanol consumption in placenta and in the liver of E-19 embryos and that in both cases GPx activity depends on this element's concentration. In E-19 and L-21 pups, however, this GPx activity is also reduced by a direct action of ethanol metabolism. This decrease in GPx activity in placenta and fetuses are related to the oxidation of lipids and proteins in nursing pups, contributing to cell dysfunctions. For all of these reasons, in order to prevent oxidative stress and teratogen problems, administering a Se antioxidant therapy to dams consuming ethanol during gestation and/or lactation appears to be recommended. Acknowledgements This work was supported by a grant from Andalusian Regional Government (CTS-193) by supporting CTS-193 Research Group. References [1] C. Bosco, E. Diaz, Placental hypoxia and foetal development versus alcohol exposure in pregnancy, Alcohol Alcohol. 47 (2012) 109–117. [2] M.L. Murillo-Fuentes, R. Artillo, M.L. Ojeda, M.J. Delgado, M.L. Murillo, O. Carreras, Effects of prenatal or postnatal ethanol consumption on Zn intestinal absorption and excretion in rats, Alcohol 42 (2007) 3–10. [3] R. Cohen-Kerem, G. Koren, Antioxidants and fetal protection against ethanol teratogenicity. I. Review of the experimental data and implications to humans, Neurotoxicol. Teratol. 25 (2003) 1–9. [4] C. Bosco, V.R. Preedy, R.R. Watson, Alcohol and xenobiotics in placenta damage, Comprehensive Handbook of Alcohol Related Pathology, 2 Elsevier Science, London, 2005, pp. 921–935. [5] A. Khera, J.J. Vanderlelie, A.V. Perkins, Selenium supplementation protects trophoblast cells from mitochondrial oxidative stress, Placenta 34 (2013) 594–598. [6] M.L. Ojeda, M.J. Delgado-Villa, R. Llopis, M.L. Murillo, O. Carreras, Lipid metabolism in ethanol-treated rat pups and adults: effects of folic acid, Alcohol 43 (2008) 544–550. [7] P.A. Dennery, Effects of oxidative stress on embryonic development, Birth Defects Res. C Embryo Today. 81 (2007) 155–162. [8] C. Ufer, C.C. Wang, The roles of glutathione peroxidases during embryo development, Front. Mol. Neurosci. 4 (2011) 12. [9] R. Briguelius-Flohé, M. Maiorino, Glutathione peroxidases, Biochim. Biophys. Acta 1830 (2013) 3289–3303. [10] Y.S. Ho, J.L. Magnenat, R.T. Bronson, J. Cao, M. Gargano, M. Sugawara, C.D. Funk, Mice deficient in cellular glutathione peroxidase develop normally and show no increased sensitivity to hyperoxia, J. Biol. Chem. 272 (1997) 16644–16651. [11] H. Imai, F. Hirao, T. Sakamoto, K. Sekine, Y. Mizukura, M. Saito, T. Kitamoto, M. Hayasaka, K. Hanaoka, Y. Nakagawa, Early embryonic lethality caused by targeted disruption of the mouse PHGPx gene, Biochem. Biophys. Res. Commun. 305
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