Maternal nicotine exposure leads to higher liver

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maternal nicotine exposure during lactation in liver oxidative status, insulin .... with nicotine free-base (Sigma, St Louis, MO, USA) diluted in isotonic saline (NaCl.
Food and Chemical Toxicology 78 (2015) 52–59

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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x

Maternal nicotine exposure leads to higher liver oxidative stress and steatosis in adult rat offspring E.P. Conceição, N. Peixoto-Silva, C.R. Pinheiro, E. Oliveira, E.G. Moura, P.C. Lisboa * Laboratory of Endocrine Physiology, Department of Physiological Sciences, Roberto Alcantara Gomes Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil

A R T I C L E

I N F O

Article history: Received 29 July 2014 Accepted 24 January 2015 Available online 3 February 2015 Keywords: Nicotine Oxidative stress Liver steatosis Insulin

A B S T R A C T

Early nicotine exposure causes future obesity and insulin resistance. We evaluated the long-term effect of the maternal nicotine exposure during lactation in liver oxidative status, insulin sensitivity and morphology in adult offspring. Two days after birth, osmotic minipumps were implanted in the dams: nicotine (N), 6 mg/kg/day for 14 days or saline (C). Offspring were killed at 180 days. Protein content of superoxide dismutase, glutathione peroxidase, catalase, nitrotyrosine, 4HNE, IRS1, Akt1 and PPARs were measured. MDA, bound protein carbonyl content, SOD, GPx and catalase activities were determined in liver and plasma. Hepatic morphology and triglycerides content were evaluated. Albumin and bilirubin were determined. In plasma, N offspring had higher catalase activity, and SOD/GPx ratio, albumin and bilirubin levels but lower MDA content. In liver, they presented higher MDA and 4HNE levels, bound protein carbonyl content, SOD activity but lower GPx activity. N offspring presented an increase of lipid droplet, higher triglyceride content and a trend to lower PPARα in liver despite unchanged insulin signaling pathway. Early nicotine exposure causes oxidative stress in liver at adulthood, while protect against oxidative stress at plasma level. In addition, N offspring develop liver microsteatosis, which is related to oxidative stress but not to insulin resistance. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Smoking is a serious health public problem that is associated with diseases such as cancer, pulmonary and cardiovascular diseases where nicotine, the main component of the cigarette, exerts an important effect of dependence (Benowitz et al., 2009). Also tobacco exposure can contribute to obesity development since several epidemiological studies have reported that maternal smoking during pregnancy and/or lactation can be a risk factor for child and teenager obesity and hypertension (Gao et al., 2005; Goldani et al., 2007; Hill et al., 2005; Von Kries et al., 2002; Wideroe et al., 2003). The relationship between environmental, nutritional and hormonal influences at critical windows of plasticity and several chronic adult diseases is named programming (Barker, 2003; de Moura et al., 2008). Therefore, maternal cigarette smoking or nicotine gum and patches

Abbreviations: CAT, catalase; C, control; CYP 2A6, cytochrome P450 2A6; FAS, fatty acid synthase; GPx, glutathione peroxidase; 4HNE, 4-hydroxy-2-nonenal; IRS1, insulin receptor substrate-1; MDA, malondialdehyde; NADPH, nicotinamide adenine dinucleotide phosphate-oxidase; N, nicotine; NAFLD, nonalcoholic fatty liver disease; PPAR, peroxisome proliferator-activated receptor; Akt1, RAC-alpha serine/threonineprotein kinase 1; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances. * Corresponding author. Departamento de Ciências Fisiológicas – 5o andar, Instituto de Biologia – Universidade do Estado do Rio de Janeiro, Av. 28 de Setembro, 87- Rio de Janeiro, RJ, 20551-031, Brazil. Tel.: +(5521) 28688334; fax: +(5521) 28688029. E-mail address: [email protected] (P.C. Lisboa). http://dx.doi.org/10.1016/j.fct.2015.01.025 0278-6915/© 2015 Elsevier Ltd. All rights reserved.

during pregnancy and/or lactation are not desired since nicotine can be transferred through placenta and breast milk and can induce future metabolic disturbances, as a programming factor. Indeed, experimental studies of our group have evidenced that nicotine is an imprinting factor during lactation, acting as endocrine disruptor, which programs for overweight, higher visceral adiposity, hyperleptinemia and hypothyroidism in adulthood (Oliveira et al., 2010), as well as peripheral insulin resistance and central leptin resistance (de Oliveira et al., 2010), hypercorticosteronemia and higher catecholamine content in adrenal medulla (Pinheiro et al., 2011). Several studies have reported the relationship between nicotine and the high pro-oxidant status development, which triggers the oxidative stress. This process represents an unbalance between oxidant radical production and antioxidant molecule availability/ activity, resulting in an increase of pro-oxidant status, and consequent cell damage (Halliwell and Gutteridge, 2007). Indeed, the effects of acute nicotine administration upon oxidative stress can be observed in different tissues such as cardiomyocytes (Zhou et al., 2010), 3T3L1 adipocytes (An et al., 2007), aorta (Xiao et al., 2011), pancreas (Bruin et al., 2007, 2008) and liver (Sheng et al., 2001; Halima et al., 2010). The liver is a target for nicotine because its metabolism occurs mainly in this organ through the cytochrome CYP 2A6 (Benowitz et al., 2009), therefore contributing to ROS production (Kirby et al., 2011; Yamazaki et al., 1999). It has been already demonstrated that smoking contributes to nonalcoholic fatty liver disease (NAFLD), which is the most common form of liver diseases, representing a large spectrum of disorders (Liu et al.,

E.P. Conceição et al./Food and Chemical Toxicology 78 (2015) 52–59

2013). The NAFLD has two stages of establishment; the first is related to metabolic changes, which increases free fatty acids, and de novo lipogenesis, leading to steatosis. The second stage is associated with ROS deleterious effect at cell structures, insulin resistance, and release of proinflammatory cytokines, which promotes the progression of steatohepatitis. Thus, the NAFLD is correlated with decrease of antioxidant defenses (Conceição et al., 2013; Rolo et al., 2012; Videla et al., 2004). According to Valenca et al. (2008), oral chronic nicotine exposure in adult rats is associated with impairment of lipid metabolism and microsteatosis. However, these authors did not investigate the oxidative status in this model. It was already demonstrated that a moderate nicotine exposure via breast milk (administration of 2 mg nicotine/kg maternal body mass per day during lactation) induced lower body weight and liver pro-oxidant status at weaning (21 days old) and pubertal (45 days old) rats. In this experimental model, hepatic morphology was not studied (Halima et al., 2010). Therefore, considering the bare knowledge about the future consequences (at adulthood) of heavy nicotine exposure (6 mg nicotine/kg maternal body mass for 14 days of lactation), since it causes obesity and insulin resistance hypothyroidism (Oliveira et al., 2010), hypothalamic leptin resistance (de Oliveira et al., 2010), higher catecholamine and glucocorticoid levels (Pinheiro et al., 2011), the present study was designed to address the long lasting effects of maternal nicotine exposure upon the oxidative stress markers and antioxidant capacity in plasma and liver, associating with insulin sensitivity and hepatic morphology. 2. Materials and methods The experimental design was approved by the Animal Care and Use Committee of the Biology Institute of the State University of Rio de Janeiro (CEUA/017/ 2012), which based its analysis on the principles adopted and promulgated by the Brazilian Law (no. 11.794/2008) that concerns the rearing and use of animals in teaching and research activities in Brazil (Marques et al., 2009). Wistar rats were housed in a light-regulated (12 hour light cycle starting at 7 a.m.) and temperaturecontrolled room (25 ± 1 °C) over all period. Adult female rats were caged with male at the proportion of 3:1. After mating, each female was placed in an individual cage with water and chow ad libitum until delivery. 2.1. Experimental model of postnatal nicotine exposure Twenty lactating rats were used for this study. To avoid the prenatal litter size influence in the programming effect, only mothers with litter size of 10 ± 2 offspring were studied. At birth (PN 1), the litter size was adjusted to six males for dam to maximize the lactation performance. In the PN 2, the dams were randomly divided into two groups and were anesthetized with i.p. injection of thiopental sodium (40 mg/ kg body weight) to insert osmotic minipumps via s.c. (Alzet, 2ML2, Los Angeles, CA, EUA) on their back. For the nicotine group (N; n = 10), minipumps were prepared with nicotine free-base (Sigma, St Louis, MO, USA) diluted in isotonic saline (NaCl 0.9%) to release a dose of 6 mg/kg of nicotine throughout 14 days of lactation (Oliveira et al., 2010). For the control group (C; n = 10), dams were implanted with osmotic minipumps containing only saline solution. Since weaning until PN 180, offspring’s body weight was monitored every 4 days. 2.2. Euthanasia At PN 180, the animals were fasted for 12 h, killed by decapitation, and their blood was collected from the trunk. The plasma was obtained after centrifugation (1500 × g for 20 min at 4 °C) and was frozen (−20 °C) for further analyses. The visceral white adipose tissue (mesenteric, epididymal and retroperitoneal depots) was excised and weighed for evaluation of central adiposity. The liver was rapidly removed and its hydrostatic weight and volume obtained using the Scherle method. The hydrostatic liver weight was normalized by the right tibia length. The liver was sliced into several minor fragments and stored – frozen or fixed in freshly prepared fixative for 48 h at room temperature (Conceição et al., 2013). 2.3. Preparation of tissue extracts to oxidative status evaluation The liver extracts were obtained after homogenization with 20% (w/v) of KPE buffer (0.1 M KH2PO4, 0.1 M K2HPO4, and 10 mM EDTA, pH 7.5) by homogenizer PotterElvehjem (Marconi, São Paulo, Brazil). The homogenates were centrifuged (19.900 × g for 5 min at 4 °C), and the supernatants were stored at −80 °C for the enzymatic activity, lipid peroxidation assay and total protein bound carbonyl assays. The protein content was determined using the Bradford assay (1976).

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2.4. Determination of the antioxidant enzyme activities Samples of liver homogenized in KPE buffer were used to superoxide dismutase (SOD) activity evaluation based on inhibition of epinephrine autoxidation in an alkaline medium (pH 10.2) during 3 minutes at 485 nm (Bannister and Calabrese, 1987). The catalase (CAT) activity evaluation was determined through the rate of hydrogen peroxide decomposition during 60 seconds at 240 nm (Aebi, 1984). The glutathione peroxidase (GPx) activity was estimated through oxidation of NADPH to NADP+ indicated by decrease in absorbance at 340 nm during 2 minutes (Flohé and Günzler, 1984). All absorbance were assayed on a spectrophotometer (Hidex Chameleon™, Turku, Finland) and enzyme activities were expressed as U/mg. 2.5. Oxidative damage Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) through thiobarbituric acid reactive substances (TBARS) method (Draper et al., 1993). Liver homogenate in KPE buffer and plasma were mixed with 10% trichloroacetic acid (1:5 v/v) and centrifuged 10 minutes at 8.300 × g. The supernatant was incubated with 0.67% thiobarbituric acid (1:1 v/v) for 30 minutes at 95 °C. The absorbance of the pink chromogen was measured at 532 nm (Hidex Chameleon™, Turku, Finland). The total MDA levels are expressed normalized by mg−1protein in 100 μL of sample. Protein oxidation by ROS, metal and aldehyde ROS-derived was evaluated in liver and plasma by total protein bound carbonyl content reacting with 2,4-dinitrophenylhydrazine (Sigma-Aldrich Co., St. Louis, MO, USA) (Levine et al., 1990). The absorbance were obtained at 380 nm (Hidex Chameleon™, Turku, Finland) and expressed as nMol/ mg protein. 2.6. Western blotting analysis Liver samples were homogenized in lysis buffer (50 mM Hepes, pH 6.4, 1 mM MgCl2, 10 mM EDTA and 1% Triton X-100, plus protease inhibitor cocktail) (Roche Diagnostics GmbH, Mannheim, Germany) using an Ultra-Turrax homogenizer (IKA Werke, Staufen, Germany). After centrifugation (7.500 × g for 5 min), homogenates were stored at −20 °C until the SDS-PAGE assay. The total protein content of homogenate was determined by the BCA protein assay kit (Pierce, Rockford, IL, USA). The liver protein content of glutathione peroxidase (GPx), Mn and Cu/Zn superoxide dismutase (SOD), catalase, nitrotyrosine-modified proteins, 4-hydroxy-2-nonenal (4-HNE) adducted proteins, insulin receptor substrate-1 (IRS1), phospho-IRS1, RAC-alpha serine/threonine-protein kinase 1 (Akt1), phospho-Akt1, peroxisome proliferator-activated receptor alpha and gamma (PPARα and PPARγ), and β-actin were evaluated using adequate primary antibody incubation (overnight; 1:1.000 antibody from Santa Cruz, CA, USA) followed by proper secondary antibody incubation (1 hour; 1:10.000 antibody from Sigma-Aldrich Co., St. Louis, MO, USA) and streptavidin (1 hour; 1:10.000; Zymed, CA, USA). The protein bands were visualized by chemiluminescence (Kit ECL plus, Amersham Biosciences) followed by exposure to auto radiographic film (Hyperfilm ECL, Amersham Biosciences). Area and density of the bands were quantified by Image J software (Media Cybernetics, Bethesda, MD, USA). The results were normalized by β-actin content and were expressed as relative (%) to the control group. 2.7. Plasma biochemical parameters The albumin and total bilirubin serum level were measured with colorimetric kit in accordance with manufacturer instructions (Bioclin, MG, Brazil). 2.8. Liver stereology After fixation and processing, random liver fragments obtained from all lobes were embedded in Paraplast plus (Sigma-Aldrich, St. Louis, MO, EUA), sectioned to 5 μm thick and stained with hematoxylin and eosin (HE) for visualization using light microscopy. The evaluation of hepatic steatosis was performed by point counting methods through a video microscope system and a test system composed of 36 test points (PT). The volume density (Vv) was estimated as: Vv [steatosis, liver] = PP [steatosis]/PT [liver]. Where PP is the number of points counting fat droplets on hepatic tissue (steatosis) and PT is the total test points (Catta-Preta et al., 2011). We highlight that several slices were performed and 10 microscopic fields per animal (n = 5) were analyzed at random (blind analysis). 2.9. Liver triglyceride content Total triglyceride was extracted from the liver following the adapted Folch and Sloane method (1957). Briefly, 50 mg of liver was homogenized in 1 mL of isopropanol (Vetec Química Fina, RJ, Brazil) and centrifuged at 1.000 × g for 10 min, at 4 °C. The triglyceride content was measured by colorimetric assay kit in accordance with manufacturer instructions (Bioclin, MG, Brazil). 2.10. Statistical analysis Data are presented as mean ± SEM. Differences between groups were analyzed using Student’s t-test. All statistical analyses were performed with GraphPad Prism

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in N offspring (−35%; p = 0.02) but the total protein bound carbonyl level was not different between the groups. The content of endogenous antioxidants was higher in N offspring compared with C offspring (albumin: +11% and bilirubin total +132%, p < 0.05).

Software (GraphPad Prism version 5.03 for Windows; GraphPad Software, San Diego, CA, USA). Differences were considered significant if p values ≤ 0·05.

3. Results 3.1. The effects of neonatal nicotine exposure on biometric parameters

3.3. The effects of neonatal nicotine exposure on liver REDOX status As shown in Table 2, SOD activity was increased (+50%, p = 0.02) and GPx activity was decreased (−43%; p = 0.03) in liver of adult N offspring despite no change in MnSOD, Cu/ZnSOD and GPx protein contents (Fig. 1A, 1B and 1C). Both CAT activity and SOD/GPx ratio (Table 2) were not different between groups as well as CAT protein contents (Fig. 1D). In PN 180, N offspring showed higher oxidative damage on lipid membranes, estimated by MDA level (+24%, p = 0.02; Table 2) as well as by 4-HNE adducted proteins (+25%, p = 0.005; Fig. 2A). Additionally, the hepatic data showed higher protein damage in N group, estimated by higher levels of total bound protein carbonyl (+100%, p = 0.01; Table 2); however, no difference in the nitrotyrosine content (Fig. 2B) was observed.

As expected, N group showed higher body weight (+18.5%; C: 370.5 g ± 10.5 vs N: 439.0 g ± 14.9; p < 0.05) and visceral fat mass (+27%, C: 0.029 ± 0.003 g/g of body mass vs N: 0.037 ± 0.001 g/g of body mass; p < 0.05) than controls in PN 180. 3.2. The effects of neonatal nicotine exposure on serum REDOX status As depicted in Table 1, N offspring had no change in SOD and GPx activities but had higher CAT activity (+33%, p = 0.02) and SOD/ GPx ratio (1.8 fold increase, p = 0.001). The MDA content was lower

C 150

150

GPx (% of control)

MnSOD (% of control)

A 100

50

100

50

0

0 C

C

N

B

N

150

150

Catalase (% control)

Cu/Zn SOD (% of control)

D

100

50

100

50

0

0 C

C

N

E

C

N

C

N

N

MnSOD

25 kDa

Cu/ZnSOD

23 kDa

GPx

23 kDa

Catalase

60 kDa

β acn

42 kDa

Fig. 1. Quantification of protein expression of antioxidant enzymes by Western blotting in the liver at PN 180. (A) MnSOD; (B) Cu/ZnSOD; (C) GPx; (D) Catalase and (E) the representative Western blotting of each protein with their correspondent molecular weight. C – control group; N – nicotine group; SOD – superoxide dismutase; GPx – glutathione peroxidase. The protein contents were quantified by scanning densitometry of the bands and normalized through the β-actin loading control and were expressed in % of control. Data are presented as the mean ± SEM. p < 0.05. n = 7 per group.

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Table 1 Blood oxidative stress parameters of rat offspring at PN 180 whose mothers were saline or nicotine exposed during lactation.

Antioxidant enzymes SOD (U/mg of protein) CAT (U/mg of protein) GPx (U/mg of protein) SOD/GPx ratio Non-enzymatic antioxidants Albumin (g/dL) Total bilirubin (mg/dL) Oxidation products MDA (nMol/mg protein) Carbonyl protein (U/mg of protein)

C

N

p

123.3 ± 13.33 0.46 ± 0.05 12.73 ± 2.78 5.91 ± 1.9

130.5 ± 25.96 0.61 ± 0.03* 15.48 ± 2.45 16.85 ± 0.5*

0.82 0.02 0.48 0.001

2.28 ± 0.1 0.021 ± 0.002

2.56 ± 0.1* 0.049 ± 0.012*

0.01 0.04

0.067 ± 0.006 0.76 ± 0.07

0.043 ± 0.007* 0.76 ± 0.13

0.02 0.90

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Table 2 Liver oxidative stress parameters of rat offspring at PN 180 whose mothers were saline or nicotine exposed during lactation. C

Results are expressed as means ± S.E.M. of 10 rats per group. C – control group; N – nicotine group; SOD – superoxide dismutase; CAT – catalase; GPx – glutathione peroxidase; MDA – malondialdehyde. * vs C group.

3.4. The effects of neonatal nicotine exposure on liver insulin signaling pathway, morphology and PPARs Adult N offspring had no alteration of IRS1, phospho-IRS1, Akt1, and phospho-Akt1 protein content in liver. There were no differences at phospho-IRS1/ IRS1 and phospho-Akt1/Akt1 ratios between the groups (Fig. 3A and 3B, respectively). The liver weight was not different between offspring (C: 0.1896 ± 0.0074 g/mm vs N: 0.2026 ± 0.0043 g/mm); however, the

Antioxidant enzymes SOD (U/mg of protein) CAT (U/mg of protein) GPx (U/mg of protein) SOD/GPx ratio Oxidation products MDA (nMol/mg protein) Carbonyl protein (U/mg of protein)

N

p

0.866 ± 0.07 1.59 ± 0.05 42.85 ± 6.28 3.448 ± 0.34

1.296 ± 0.15* 1.64 ± 0.15 24.63 ± 3.36* 6.305 ± 1.36

0.02 0.79 0.03 0.09

0.041 ± 0.002 0.0065 ± 0.002

0.051 ± 0.004* 0.0132 ± 0.002*

0.02 0.01

Results are expressed as means ± S.E.M. of 10 rats per group. C – control group; N – nicotine group; SOD – superoxide dismutase; CAT – catalase; GPx – glutathione peroxidase; MDA – malondialdehyde. * vs C group.

morphological study evidenced a greater accumulation of lipid drops in the hepatocytes of the N offspring (96%, p < 0.05, Fig. 4A and Fig. 4B) featuring a microesteatosis, concomitantly with higher triglyceride content (+48%, p < 0.01, Fig. 4C). Although there is no statistical significance, N group had a lower PPARα protein content (−32%, p = 0.2, Fig. 5A) and normal PPARγ (Fig. 5B). 4. Discussion Here, we confirm that nicotine exposure during lactation is able to induce an increase of body weight and visceral fat mass in the

A 4HNE (% of control)

150

* 100

50

0 C

N

C

N

Nitrotyrosine (% of control)

B 150

100

50

0

Fig. 2. Quantification of protein ROS modification by Western blotting in the liver at PN 180. (A) 4-hydroxy-2-nonenal (4-HNE) adducted proteins; (B) nitrotyrosinemodified proteins; (C) the representative Western blotting of each protein with their correspondent molecular weight. C – control group; N – nicotine group. The protein contents were quantified by scanning densitometry of the bands and normalized through the β-actin loading control and were expressed in % of control. Data are presented as the mean ± SEM. p < 0.05. n = 7 per group.

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phospho-IRS1/IRS1 (% of control)

A

1.5

1.0

0.5

0.0 C

N

C

N

phospho-Akt/Akt (% of control)

B 2.5 2.0 1.5 1.0 0.5 0.0

C

Fig. 3. Quantification of protein expression for insulin signaling by Western blotting in the liver at PN 180. (A) phospho-IRS1/IRS1; (B) phospho-Akt1/Akt1 and (C) the representative Western blotting of each protein analyzed. C – control group; N – nicotine group. The protein contents were quantified by scanning densitometry of the bands, and normalized through the β-actin loading control and were expressed in % of control. Data are presented as the mean ± SEM. p < 0.05. n = 7 per group.

male offspring at adulthood. Our group has a great interest in the study of metabolic programming as a cause of obesity and other metabolic disorders during adulthood. Different models of nutritional or environmental changes during the lactation period have been studied in rats and sex-related differences were already detected (Guarda et al., 2014; Pinheiro et al., 2011; Troina et al., 2010, 2012). Regarding the model of maternal nicotine exposure during lactation, we previously showed that the male offspring whose mothers were exposed to nicotine during lactation are programmed for overweight, higher visceral and total adiposity, hyperleptinemia and adrenal hormone dysfunction at adulthood, while the female offspring did not present any change of these parameters (Pinheiro et al., 2011). Then, since females were not programmed for obesity

and metabolic alterations that could affect the oxidative stress and liver function, in the present paper, only male offspring were studied. Interestingly, the nicotine group had decreased plasma MDA content. This result can be due to the higher catalase activity and SOD/GPx activity ratio. Previous reports showed that SOD/GPx activity ratio is considered a better enzymatic antioxidant indicator than the absolute enzyme activity (de Haan et al., 1995; Dittmar et al., 2008), which can contribute to protect the nicotine group against lipid peroxidation. Moreover, the N offspring showed an increase of plasma albumin and total bilirubin, reinforcing the protection system against the lipid peroxidation. Plasma albumin is an important target of ROS, working as free radical-trapping. Also, due to its abundance, high binding capacity of oxidative molecules as copper and iron cations, their availability to hydroxyl radical production through the Fenton reaction is thus reduced (Roche et al., 2008). Moreover, the albumin-bound bilirubin acts as a lipid peroxidation inhibitor (Neuzil and Stocker, 1993). Thus, we suggest that early nicotine exposure induces a rearrangement of antioxidant molecules in the adult life of the offspring and consequently improves antioxidant defenses in plasma. During pregnancy, maternal nicotine exposure (4 μg s.c./kg/ min since 4th of gestational period) induces deleterious effects in the vascular system of Sprague-Dawley male offspring at 150 days old, represented by increased oxidative stress and high blood pressure (Xiao et al., 2011). This long-term effect of nicotine was associated with the increase of NADPH oxidase Nox2/gp91 protein expression in aorta, and consequently, higher ROS production. Those animals had reduced SOD activity, increased lipid and protein damage in aorta, and increased vasoconstriction. The contractile impairments were abolished by vessel treatment with tempol (superoxide dismutase mimetic) and apocynin (NADPH oxidase inhibitor). Nicotine exposure from intrauterine life until weaning also impaired the vascular system of animals at 182 days old through an increase of the oxidative unbalance, resulting in higher damage of blood vessel function compared to nicotine exposure during lactation (Gao et al., 2005). In this sense, specifically concerning the plasma level, we suggest that the impact of maternal nicotine exposure during lactation is less severe than in gestational period. Concerning the hepatic findings, it suggests that early nicotine exposure is able to influence the antioxidant liver enzymes in the offspring at 180 days old. Interestingly, the nicotine offspring showed an increase of hepatic SOD activity and a decrease of GPx activity. This imbalance can cause oxidative cell damage due to higher production of H2O2. Moreover, programmed animals presented an increase of MDA, increased total bound protein carbonyl content (although unchanged nitrotyrosine), indicating an oxidative damage. Taken together, these data suggest that even after nicotine withdrawal, the pro-oxidative status at liver level remains in animals at PN 180. In fact, according to Halima et al. (2010), maternal nicotine exposure during lactation (2 mg i.p./kg per day) caused a decrease in liver thiol concentration, SOD and CAT activities, an increase in liver MDA content, an increase in serum AST and ALT activities (biomarkers of hepatocyte cytolysis) in the offspring at weaning. At 45 days old, the nicotine offspring still had lower liver CAT activity, higher MDA content and higher serum ALT, indicating a longstanding nicotine pro-oxidative effect on liver. Additionally, the 4-HNE content, an important marker of lipid peroxidation, is higher in nicotine offspring, therefore demonstrating that oxidative stress is a potential mechanism to morphological and functional disturbances in the liver. Indeed, another study has already demonstrated that 4-HNE is involved in oxidative stress in liver samples of patients with NAFD (Seki et al., 2005). Corroborating the increased pro-oxidative status in the liver, the hepatocyte morphology demonstrates that nicotine offspring displays greater accumulation of lipid drops, characterizing a microsteatosis associated with higher triglyceride content.

E.P. Conceição et al./Food and Chemical Toxicology 78 (2015) 52–59

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A

C Live r triglyce ride s conte nt (m g/μg of liver protein)

B

80

*

60 40 20 0 C

N

Fig. 4. Hepatic evaluations at PN 180. (A) Liver photomicrographs with same magnification (×60) and stained with hematoxylin and eosin (HE), demonstrating a normal appearance in control group with minimal focus of lipids and a microvesicular steatosis widely distributed in hepatic tissue of the nicotine group; (B) % of steatosis performed by point counting method; (C) Liver triglycerides content. C – control group; N – nicotine group. Data are presented as the mean ± SEM. p < 0.05. n = 5–7 per group.

The chronic administration of nicotine also leads to deleterious effects in hepatic tissue. Adult Wistar rats treated daily for 10 days with 5 mg of nicotine in drinking water showed varied size of hepatocytes with different histoarchitecture; increase of fat drops in cytoplasm and reduction of hepatocyte number (Valenca et al., 2008). Liver lesions were also observed in pups of 10 days submitted to prenatal exposure to nicotine (4.3 ± 0.3 mg/kg/day at drinking water), demonstrating that during hepatic organogenesis, nicotine induced a hepatic focal necrosis in utero that persist for 10 days without reversion after nicotine withdrawal. Interestingly, the lesion intensity was higher in the pups breastfed by nicotine exposed dams, although the postnatal nicotine exposure alone was unable to promote histological changes in the liver pups, despite its association with lower SOD activity (Sheng et al., 2001). Therefore, nicotine dose, administration via and period of exposure can influence the offspring’s outcome. In the present study, despite the presence of extensive hepatic microsteatosis observed in nicotine offspring, the unchanged insulin signaling suggests that, at least in this moment, the morphological alteration is caused by pro-oxidative status in the liver tissue instead of insulin resistance. Previous studies confirm the correlation between insulin resistance and hepatic steatosis; however, it is not fully understood whether insulin resistance causes the ectopic fat accumulation in liver or whether the contrary also is true, making triglyceride accumulation the cause of insulin resistance in liver (Postic and Girard, 2008). Despite the increase of lipid storage in liver, the nicotine offspring did not present change of PPARγ expression, which is often associated with liver steatosis, through stimulation of lipogenic gene transcription (Inoue et al., 2005; Perfield et al., 2013). However, the nicotine offspring showed a trend to lower PPARα expression (−32%) which, despite not being significant when together with other changes in liver function, can contribute to liver

mitochondrial dysfunction. This transcription factor is related to energy expenditure, increasing mitochondrial biogenesis and β-oxidation (Seo et al., 2008). In addition, the reduction of hepatic PPARα content has been associated with NAFLD induced by high fat diet and in other obesity models (Abdelmegeed et al., 2011; Yeon et al., 2004). Thus it is possible that adult nicotine offspring developed a dysfunction in the beta-oxidation mechanism, which could contribute to worsening of the liver microsteatosis. In summary, epigenetic mechanisms, such as DNA methylation, histone acetylation/deacetylation or miRNAs, induced by perinatal environmental factors, may lead to a higher risk of metabolic disturbances in the adult life of the rat progeny (de Moura et al., 2008). This explanation may help to understand the mechanisms involved in the long-lasting findings of oxidative stress and steatosis induced by nicotine transfer through the milk. One limitation of our study is that the mitochondrial function or epigenetic changes in liver were not evaluated. Notwithstanding, whether nicotine can make nursing infants exposed to cigarette smoke more susceptible to pro-oxidant damage and fat liver disease in adulthood deserves more studies. 5. Conclusions Here we evidenced that early postnatal nicotine exposure, although without altering the plasma pro-oxidative status due to compensatory better antioxidant capacity in other tissues, was capable of programming the hepatic oxidative damage that was responsible for the emergence of microsteatosis in the adult life, which preceded the liver insulin resistance. Thus, the liver impairment seems to be a primary target of this kind of programming and this study helps to elucidate the initial steps that lead to liver microsteatosis. New experiments are being carried out in our laboratory to evaluate the oxidative stress in other tissues, such as

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A PPARα (% control)

150

100

50

0 C

N

B

References

150

PPARγ (% control)

Desenvolvimento Científico e Tecnológico – CNPq), Coordination for the Improvement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES) and State of Rio de Janeiro Carlos Chagas Filho Research Foundation (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro – FAPERJ). All authors are grateful to Dr. Jose Firmino Nogueira Neto for lipid measurements (LabLip – State University of Rio de Janeiro) as well as to Mr. Ulisses Risso Siqueira for technical assistance. EPS Conceição and N Peixoto-Silva conceived, carried out experiments and wrote the paper. CR Pinheiro was responsible for animal programming, carried out experiments and analyzed data. EG Moura, E Oliveira and PC Lisboa conceived and were involved in writing and revising the paper. All authors contributed to and approved the final manuscript.

100

50

0 C

N

C

Fig. 5. Quantification of protein expression for PPARα and PPARγ by Western blotting in the liver at PN 180. (A) PPARα; (B) PPARγ; and (C) the representative Western blotting of each protein with their correspondent molecular weight. C – control group; N – nicotine group; PPARα – peroxisome proliferator-activated receptor alpha; PPARγ – peroxisome proliferator-activated receptor gamma. The protein contents were quantified by scanning densitometry of the bands and normalized through the β-actin loading control and were expressed in % of control. Data are presented as the mean ± SEM. p < 0.05. n = 7 per group.

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