Exposure to low doses of formaldehyde during ...

Toxicology and Applied Pharmacology 278 (2014) 266–274

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Exposure to low doses of formaldehyde during pregnancy suppresses the development of allergic lung inflammation in offspring Marília Maiellaro a, Matheus Correa-Costa b, Luana Beatriz Vitoretti c, João Antônio Gimenes Júnior c, Niels Olsen Saraiva Câmara b, Wothan Tavares-de-Lima c, Sandra Helena Poliselli Farsky a, Adriana Lino-dos-Santos-Franco a,⁎ a b c

Department of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

a r t i c l e

i n f o

Article history: Received 5 March 2014 Revised 2 May 2014 Accepted 5 May 2014 Available online 15 May 2014 Keywords: Pollutants Asthma Cytokines Anaphylactic antibodies synthesis Programming mechanisms Oxidative stress

a b s t r a c t Formaldehyde (FA) is an environmental and occupational pollutant, and its toxic effects on the immune system have been shown. Nevertheless, no data are available regarding the programming mechanisms after FA exposure and its repercussions for the immune systems of offspring. In this study, our objective was to investigate the effects of low-dose exposure of FA on pregnant rats and its repercussion for the development of allergic lung inflammation in offspring. Pregnant Wistar rats were assigned in 3 groups: P (rats exposed to FA (0.75 ppm, 1 h/day, 5 days/week, for 21 days)), C (rats exposed to vehicle of FA (distillated water)) and B (rats non-manipulated). After 30 days of age, the offspring was sensitised with ovalbumin (OVA)-alum and challenged with aerosolized OVA (1%, 15 min, 3 days). After 24 h the OVA challenge the parameters were evaluated. Our data showed that low-dose exposure to FA during pregnancy induced low birth weight and suppressed the development of allergic lung inflammation and tracheal hyperresponsiveness in offspring by mechanisms mediated by reduced anaphylactic antibodies synthesis, IL-6 and TNF-alpha secretion. Elevated levels of IL-10 were found. Any systemic alteration was detected in the exposed pregnant rats, although oxidative stress in the uterine environment was evident at the moment of the delivery based on elevated COX-1 expression and reduced cNOS and SOD-2 in the uterus. Therefore, we show the putative programming mechanisms induced by FA on the immune system for the first time and the mechanisms involved may be related to oxidative stress in the foetal microenvironment. © 2014 Elsevier Inc. All rights reserved.

Introduction Formaldehyde (FA) is an occupational pollutant that is widely used in many products, including disinfectants, cosmetics, antiseptics and fungicides (Carlson et al., 2004). It is also used in the manufacture of plastics, resins and building materials, such as particle board, plywood, floor coverings and office furniture, and it is emitted by cigarette smoking (Fló-Neyret et al., 2001; Krakowiak et al., 1998). Thus, many individuals are potentially exposed to FA. The effect of FA on the development of asthma is still controversial. In this context, some studies associate exposure to FA with deterioration of asthma symptoms (Green-Mckenzie and Hudes, 2005; Mendell and Heath, 2005), while others show that FA impairs the development of an allergic lung response (Ezratty et al., 2007; Lino-dos-Santos-Franco ⁎ Corresponding author at: Department of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 580, BL 13B, 05508-900 São Paulo, Brazil. E-mail address: [email protected] (A. Lino-dos-Santos-Franco).

http://dx.doi.org/10.1016/j.taap.2014.05.003 0041-008X/© 2014 Elsevier Inc. All rights reserved.

et al., 2009). The reasons for the controversial data have not been elucidated, but differences in experimental protocols, resulting in different amounts of systemic FA, may be considered. It is well established that the development as well as exacerbation of airway inflammatory disorders such as asthma, emphysema and bronchitis has been correlated with exposure to a class of air pollutants including FA, particulate matter, ozone and others (Green-Mckenzie and Hudes, 2005; Habre et al., 2014; Wu et al., 2013, 2014). Moreover, associations have been found between exposure to several xenobiotics during pregnancy and increased risk factor for the development of inflammatory lung disease (Selgrade et al., 2013); however, no studies were found about FA exposure during pregnancy and its impact for development of allergic disease on offspring. Literature shows that prenatal as well postnatal exposure to cigarette smoke is associated with an increased risk of asthma during childhood (Raherison et al., 2007). In addition, exposure of mice to particulate matter during pregnancy increases lung inflammatory response of offspring when exposed to ozone (Austen et al., 2009). Other study found positive correlation between intrauterine exposure to polycyclic aromatic hydrocarbons, and

M. Maiellaro et al. / Toxicology and Applied Pharmacology 278 (2014) 266–274

increased risk of development of allergic respiratory disease in the first 5 years of life (Jedrychowski et al., 2005). Still, experimental study in mice showed that neonates of rats exposed to diesel particles during pregnancy showed bronchial hyperreactivity and pulmonary allergic inflammation (Fedulov et al., 2008). FA is an important inductor of lung inflammation by oxidative stressmediated mechanism in lung tissue (Lino-dos-Santos-Franco et al., 2009), and reactive oxygen species (ROS) generated during exposure to FA can cause injury to cellular structures such as proteins, lipids, DNA and others, leading to the increased generation of pro-inflammatory mediators. ROS are potent modifiers of genes and can directly interact with DNA base pairs, causing both genetic and epigenetic changes (Cerda and Weitzman, 1997; Thompson and Al-Hasan, 2012). Thus, oxidative stress may be a link between intrauterine insult and programming consequences after birth. Considering that ROS have been implicated in FA effects and many studies show that ROS modulated the onset of several diseases, including asthma, which can be initiated in utero (Searing and Rabinovitch, 2011; Wan and Winn, 2006), we aimed to investigate the effects of low-dose exposure of FA on pregnant rats and its repercussion for the development of allergic lung inflammation in offspring. Materials and methods Animals. Female and male 2-month-old Wistar rats (~200 g and 300 g respectively) were obtained from the Institute of Biomedical Sciences, University of São Paulo. The virgin females were caged overnight with a male, and vaginal smears were taken the following morning. Pregnancy was confirmed by vaginal smear. The rats were maintained in a light and temperature-controlled room (12/12-hour light–dark cycle, 21 ± 2 °C) with free access to food and water. The experiments were approved by the Institutional Animal Care Committee. Exposure to formaldehyde (FA) inhalation. The pregnant rats (5/chamber) were exposed to FA inhalation (0.75 ppm, 1 h/day, 5 days/week) during 21 days of gestation. For this purpose, we used a standard glass chamber (20 L) coupled to an ultrasonic nebuliser device (Icel®, Brazil) that produces an aerosol with particles of between 0.5 and 1 μm to generate a constant airstream in an aqueous solution of formalin (Lino dos Santos Franco et al., 2006, 2011, 2009). The dose of FA used in the present study was based on those which do not cause effects on human health (OSHA). However, pregnancy is a sensitive phase of life and very susceptible to the deleterious effects of xenobiotics. Thus, our objective was evaluated whether low doses of FA are also secure for offspring. The FA concentration was determined by UV–HPLC. The pregnant females were divided into 3 experimental groups: B (basal group, n = 5), rats non-manipulated; C (control group, n = 5), rats exposed to vehicle of FA (distilled water) and P (pollutant group, n = 5), rats exposed to FA. The weight of the mothers was determined during gestation. In a parallel set of experiments, group of pregnant rats (n = 5) was exposed or not to FA and the uterine tissue was removed at day 21 of gestation for PCR analyses. Allergic experimental model. After 30 days of age, male and female rats were sensitised with 10 μg of ovalbumin (OVA) and 10 mg of aluminium hydroxide by subcutaneous route. One week later, the rats received another injection of OVA-alum by the same route (booster). Seven days after the booster, the rats were submitted to an inhaled antigen challenge (aerosolised 1% OVA in PBS, 15 min) for three consecutive days. For this, we used an ultrasonic nebuliser device (Icel®, SP, Brazil) that produces an aerosol, with particles of between 0.5 and 1 μm coupled to a plastic inhalation chamber (20 L). The analyses were performed 24 h after the last challenge with OVA. Protocol of studies with offspring. After birth, the rats were left with their mothers for 21 days. After weaning, the rats were separated by gender

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(5 rats per cage), and the animals were used with 30 days after birth. The rats were maintained in a light- and temperature-controlled room (12/12-hour light–dark cycle, 21 ± 2 °C) with free access to food and water. Thirty days after birth puppies' males and females of each experimental group, generated by different matrices were mixed and kept in litters of 10 animals for each one of experiments. These procedures were adopted in order to eliminate the formation of groups containing only puppies' brothers and a single mother, excluding the possibility of the results being influenced by individual susceptibility. Indeed, we mixed the results obtained from both sexes, because no differences we observed between male and female responses. The offspring was assigned into 3 groups: 1) B group, identified as non-sensitised offspring from non-manipulated mothers (n = 10); 2) C group, identified as OVA-sensitised offspring from mothers exposed to vehicle (n = 10); 3) P group, identified as OVA-sensitised offspring from mothers exposed to FA (n = 10). In parallel, we also determined several parameters after birth, such as birth weight, weight during 21 days after birth, the number of rats born and the sex of rats born. Evaluation of allergic lung inflammation by quantification of cells recruited in bronchoalveolar lavage (BAL). BAL fluids were harvested according to De Lima et al. (1992). Tracheae of rats (offspring and mother) were cannulated with a polyethylene tubing, and the lungs were flushed twice with PBS (10 ml total volume). The collected BAL was centrifuged (1500 rpm for 15 min at 20 °C), and the resulting cell pellet was resuspended in 1 ml of PBS. The cell suspensions (90 μl) were stained with 10 μl of 0.2% crystal violet, and the total cell number was determined microscopically using a Neubauer chamber. The differential cell counts were carried out by cytocentrifuge preparations (Cytospin, Fanem, Brazil) stained with instant-prov kit. Determination of cell mobilization and systemic effects by quantification of blood leukocytes and bone marrow cells. Blood aliquots obtained from aortic veins of offspring and mother were diluted (1:20) in Türk's fluid (3% acetic acid) for total white cell counts in a Neubauer chamber, whereas the differential leukocyte counts were performed in blood smears stained with a rapid-staining kit (Instant-prov, Brazil). Bone marrow cells were obtained by lavage of the femoral bone (5 ml). The fluid was centrifuged (1500 rpm for 15 min at 20 °C), the supernatant was discarded, and the pellet was resuspended in 1 ml of PBS. The cells were stained with crystal violet (0.2%) and quantified in a Neubauer chamber. Determination of anaphylactic antibodies synthesis. PCA reaction was used to indirectly measure anaphylactic antibody levels (Bach et al., 1971; Mota and Wong, 1969). Anaphylactic antibody-dependent cutaneous reaction was generated by sensitising the skin of non-manipulated rats with an intradermal injection (100 μl/site) of serially diluted (1:2 up to 1:256) sera from the C and P groups. Twenty-four hours after injection, the recipient rats received by intravenous (i.v.) solution of 500 μg OVA plus 2.5 mg of Evans blue dye dissolved in NaCl (0.9%). Thirty minutes later, the rats were euthanised by anaesthesia, the skin was removed and the diameter of the dye stain was measured at the inner surface of the skin. The PCA titres were represented by the highest dilution of the serum that resulted in a dye stain of N5 mm in diameter. Schultz–Dale reaction is an assay that demonstrates antigen specific airway response. Thus, tracheal tissue was suspended in an aerated organ bath (15 ml) filled with Krebs–Henseleit at 37 °C (95% O2 and 5% CO2). After equilibrium (30 min), the ovalbumin solution (4 mg/ml) was added to the organ bath, and the contraction force was measured using a force displacement transducer and a chart recorder (Powerlab®, Labchart Ad Instruments).

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Determination of ex-vivo tracheal responsiveness to methacholine (MCh). Tracheal tissue was mounted for the measurement of isometric force using two steel hooks in a 15-ml organ bath (De Lima et al., 1992). The force contraction was registered using a force displacement transducer and a chart recorder (Powerlab®, Labchart, AD Instruments). The tracheal tissue was suspended in a continuously aerated organ bath filled with Krebs–Hanseleit (KH) buffer at 37 °C (95% O2 and 5% CO2). After equilibrium (40 min), the tracheal tissue was adjusted to 0.5 g, and tissue viability was assessed by replacing KH solution with KCl buffer. Subsequently, cumulative dose–response curves to methacholine (MCh) were constructed according to Van Rossum (1963).

Quantification of cytokines in fluid of bronchoalveolar lavage. Interleukin levels were determined in the BAL supernatants samples. The results are expressed as pg of cytokine produced per ml. IL-6, IL-4, IL-10 and TNF-alpha were quantified using ELISA kits purchased from R&D Systems (Minneapolis, MN). Determinations were made in duplicate for every sample using standard curves according to the manufacturer's specifications.

Evaluation of gene expression of cytokines in lung tissue and uterus. Lung or uterus samples of mothers and lung samples of offspring were snap-frozen in liquid nitrogen. Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, Calif) and following the protocol according to Invitrogen. RNA concentrations were determined by spectrophotometer readings at an absorbance of 260 nm. Firststrand cDNAs was synthesised using the MML-V reverse transcriptase (Promega, Madison, Wisc). RT-PCR was performed using the Taqman real-time PCR assay (Applied Biosystem, USA) for the following molecules: IL-4 (Rn01456865_m1), IL-6 (Rn00561420_ m1), IL-10(Rn00563409_m1), IFN-γ(Rn01649192_m1), catalase (Rn00560930_m1), Sod2(Rn00690587_m1), Sod1(Rn00566938_m1), COX2(Rn01483828_m1), COX-(Rn00566881_m1), iNOS(Rn02132634_ m1), cNOS (Rn00561646_m1) and HPRT (Rn01527838_g1). The cycling conditions were as follows: 10 min at 95 °C followed by 45 cycles of 20 s at 95 °C, 20 s at 58 °C, and 20 s at 72 °C. Sequence Detection Software 1.9 (SDS) was used for the analysis, and mRNA expression was normalised to HPRT expression.

Statistical analysis. Data are expressed as the means ± SEM, and comparisons among the experimental groups were analyzed by one-way ANOVA followed by the Student's Newman–Keuls test for multiple comparisons using the GraphPad software V.5. P-values less than 0.05 were considered statistically significant.

Results Effects of FA exposure on cellular recruitment and gene expression of cytokines in lung tissue of pregnant rats To evaluate the impact of FA exposure on the mother, several parameters were investigated, and pollutant exposure did not appear to cause systemic alterations. The changes in body weight during gestation were equivalent between FA- and vehicle exposed mothers (Fig. 1 A). Panels B and C show that exposure to FA during pregnancy (group P) did not induce alterations in total cells and differential cells present in the blood in relation to the control group. Similarly, the number of cells recruited to the BAL in pregnant rats exposed to FA did not differ from pregnant rats exposed to the vehicle (C group) (Panels D and E). In addition, we also observed that exposure to FA did not alter the gene expression of IL-6, IL-4 and IL-10 when compare to the group exposed to the vehicle (Panels F, G and H).

Effects of FA exposure during pregnant on the weight of offspring Fig. 2 (Panels A and B) shows that exposure to FA during pregnancy decreased the birth weight of offspring, and this reduction in weight was maintained for 21 days (period of weaning) when compared to the B and C groups. No differences were observed in the number of rats born or the sex of the rats born between P, C and B groups (Panels C and D). Effects of FA exposure during pregnancy on cellular mobilization in the lung, blood and bone marrow of offspring Fig. 3 (Panel A) shows that exposure to FA during pregnancy (group P) caused a marked reduction in the total number of leukocytes recruited to the lung when compared to offspring of mothers exposed to the vehicle (group C). The C group had an increased total number of cells in the BAL when compared to the B group. Moreover, no differences were found in the B and P groups. Panel B shows the differential cells recruited in BAL. Our data showed that the exposure to FA caused a significant reduction in the number of mononuclear cells, neutrophils and eosinophils recruited to the BAL in relation to the C group and did not differ from the B group. Moreover, offspring of mother exposed to vehicle (C group) showed a significant increase in mononuclear cells, neutrophils and eosinophils when compared to the offspring of mother non-manipulated (B group). The offspring of mother exposed to FA (P group) and offspring of mother exposed to vehicle (C group) decreased the number of total cells in the bone marrow when compared to the offspring of mother non-manipulated (B group) (Panel C). Moreover, we showed that FA exposure caused an additional reduction in bone marrow cells when compared to the C group. A significant increase in the number of mononuclear cells, neutrophils and eosinophils was observed in the peripheral blood of animals of the C group when compared to the B group (Panel D). Conversely, FA exposure during pregnancy led to a decrease in the number of mononuclear cells, neutrophils and eosinophils in the peripheral blood in relation to animals of the C group and did not differ from the B group. Effects of FA exposure during pregnancy on anaphylactic antibodies synthesis and tracheal responsiveness of offspring. Exposure to FA during pregnancy significantly decreased the anaphylactic antibodies synthesis of offspring sensitised with OVA (Fig. 4, Panel A). The PCA values were lower in these animals compared to the offspring of vehicle-exposed mothers. There was a reduction in tracheal contractile response after OVA administration in the offspring of mother exposed to FA (P group) compared to the offspring of mother exposed to vehicle (C group) (Panel B). As a consequence of both parameters depicted above, the exposure to FA during pregnancy decreased the tracheal responsiveness to methacholine of offspring when compared to the offspring of C group. In addition, no differences were observed between the P and B groups (Panel C). Role of FA exposure during pregnancy on cytokines released in the BAL fluid of offspring The data presented in Fig. 5 show that no differences were observed in the level of IL-4 in the BAL fluid between groups of study (Panel A). In Panel B we observed a decreased level of IL-6 in the BAL fluid of the P group in relation to the C group. In addition, we also observed that C group showed a significant increase in IL-6 levels when compared to the B group. The P group had increased levels of IL-10 in relation to the C group, while the C group had decreased levels of IL-10 in relation to the B group (Panel C). No differences were observed between the P and B groups.


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Fig. 1. Exposure to FA did not alter the weight and the number of cells in the lung and in the blood as well as cytokines in lung tissue of pregnant rats. Group of rats were exposed to FA (0.75 ppm) or vehicle during 21 days of pregnancy (1 h/day for 5 days/week). The weight of pregnant rats was determined during gestation (Panel A). At 21 days of gestation the analyses of cells recruited in BAL (Panels D and E) and blood (Panels B and C) were performed. The expression of IL-6, IL-4 and IL-10 in lung tissue was also evaluated (Panels F, G and H). Data are mean ± SEM of 5 mothers per group. ⁎P b 0.05 when compared to C group.

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Fig. 2. Exposure to FA during pregnancy reduces weight of offspring. Group of rats were exposed to FA (0.75 ppm) or vehicle during 21 days of pregnancy (1 h/day for 5 days/week). The weight of offspring was evaluated immediately after the birth (Panel A) and during period of weaning (Panel B). Number of born (Panel C) and sex (Panel D) were also evaluated. Data are mean ± SEM of 5 mothers per group. ⁎P b 0.05 when compared to C group.

There was a decreased level of TNF-α in the BAL fluid of the P group in relation to the C group (Panel D). Furthermore, the C group showed a significant increase in the level of TNF-α when compared to the B group.

rats of the control group. However, we observed decreased expression of IL-10 (Panel D) in pregnant rats of the P group when compared to the C group.

Effects of FA exposure on gene expression of cytokines in the uterus of pregnant rats

Effects of FA exposure on gene expression of oxidants and antioxidants enzymes in the uterus of pregnant rats

In Fig. 6 we observed that the gene expression of IL-6, IL-4 and IFN in the uterus of pregnant rats exposed to FA did not differ from pregnant

Fig. 7 (Panel A) shows that FA exposure increased COX-1 gene expression in the uterus of pregnant rats in relation to pregnant rats

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Fig. 3. Exposure to FA during pregnancy decreases cellular recruitment into the lung, blood and bone marrow of offspring. Group of rats were exposed to FA (0.75 ppm) or vehicle during 21 days of pregnancy (1 h/day for 5 days/week). After 30 days of birth, the rats were sensitised and challenged with OVA. The BAL (Panels A and B), blood (Panel D) and bone marrow (Panel C) were collected 24 h after the OVA challenge. Data are mean ± SEM of 5 mothers per group (puppies n = 10). ⁎P b 0.05 when compared to B group; and θP b 0.05 in relation C group.


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-Log [M] methacholine Fig. 4. FA exposure during pregnancy suppresses the anaphylactic antibodies synthesis and reduces tracheal hyperresponsiveness. Group of rats were exposed to FA (0.75 ppm) or vehicle during 21 days of pregnancy (1 h/day for 5 days/week). After 30 days of birth, the rats were sensitised and challenged with OVA. After the OVA challenge the sera of animals were collected and used to PCA (Panel A) and tracheal responsiveness was evaluated (Panel C). In parallel, another group of rats were sensitised and fourteen days later the Schultz–Dale reaction (Panel B) was performed by administration of OVA directly in bath chamber. Data are mean ± SEM of 5 mothers per group (puppies n = 10). ⁎P b 0.05 when compared to C group.

of the C group. No differences were observed in COX-2, iNOS, catalase and SOD-1 gene expression between the P and C groups (Panels B, D, E and F). However, in Panels C and G we observed a significant decrease in cNOS and SOD-2 gene expression in the uterus of pregnant rats exposed to FA in relation to pregnant rats of the C group. Discussion To our knowledge, this is the first demonstration of the programming mechanisms induced by FA exposure on the immune system. We found that FA exposure during pregnancy suppresses the development of the allergic lung response in offspring, as shown by impaired cellular responses and muscular reactivity. We have utilised FA, an indoor and outdoor pollutant, to investigate the role of pollutants on the development of asthma. The dose of FA used in the present study was based on dose that does not cause effects on human health. In fact, our data showed that this concentration of FA (0.75 ppm/1 h day) did not alter body weight during pregnancy, did not

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alter the number of blood and lung leukocytes, and did not affect lung cytokine gene expression. It is important to mention that exposure to FA did not alter the balance of oxidant and antioxidant system in the lung tissue of pregnant rats (data not shown). Based on these unaltered parameters, we can infer that the dose and period of FA exposure does not cause a perceptive toxic effect in pregnant rats, which could affect the offspring. Nevertheless, FA exposure of mothers clearly affected the offspring as detected by lower birth weight. The effect was persistent, as pups presented lower body weights during the breastfeeding period. However, at the day of analyses no significant differences were observed in the weight of animals (~6 g). These results indicate the possible programming mechanisms induced by FA. The mechanism responsible for these toxic effects will be further investigated. Asthma is an allergic disease that can be triggered by many factors, including exposure to pollutants. Pollutants can result in oxidative stress, leading to modifications in genes that may be responsible for the development of several diseases. In previous studies, we showed that the effects of FA are mediated by oxidative stress (Lino dos Santos Franco et al., 2011). Environmental exposure in utero has been proposed as an important driver of gene programming that determines physiological changes, culminating in the development of many diseases, such as asthma, Alzheimer, hypertension and others. Considering that FA exposure causes oxidative stress and, when it occurs prior to immune sensitization, reduces allergic lung inflammation by mast cells-, NO- and adhesion molecule-mediated mechanisms (Lino-dos-Santos-Franco et al., 2009), we evaluated the programming mechanism involved in FA exposure. Our data showed that the offspring of rats exposed to FA during pregnancy had reduced cell recruitment to the lungs, blood and bone marrow after OVA sensitisation. This result showed that FA exposure during the intrauterine phase modified the immune system, thereby suppressing the development of allergic response in offspring. Another study showed the suppression of allergic sensitisation with OVA in offspring whose mothers were exposed to TCDD during gestation (Tarkowski et al., 2010). The reduced lung inflammation observed in the present study may be a reflex for the suppressor effect of FA exposure in the bone marrow. We noted that both C and P groups had reduced numbers of cells in the bone marrow; however, the reduction in the C group was likely a consequence of cellular mobilization, as we recorded increased numbers of cells in the blood. Conversely, we observed reduced cell numbers in both the bone marrow and blood in the offspring of mothers was exposed to FA. Thus, we did not attribute the reduced number of cells in the bone marrow to mobilization process. The production and delivery of leukocytes from the bone marrow into the blood is dependent on multi, complex mechanisms, which will be further investigated by our group. It is well established that the allergy depends on IgE, FCεRI expression and involves mast cell participation (Galli et al., 2005). Our data showed that exposure to FA during pregnancy significantly reduced anaphylactic antibodies synthesis in offspring sensitised with OVA. These results indicate that FA inhalation modifies the synthesis of anti-OVA anaphylactic antibodies. Thus, it is conceivable that the reduced cell migration found in the P group might be associated to the impaired ability of anaphylactic antibody to bind to mast cell surfaces. Moreover, we found that FA exposure reduced the contractile response after OVA administration, as seen in the Schultz–Dale reaction. This result reinforces those obtained with PCA. Thus, we can suppose that this reduction in contractile response after OVA is due minor anaphylactic antibodies synthesis accompanied by reduced capacity of mast cells from offspring to degranulate. Our data argue that FA exposure in utero modifies mechanisms involving the induction of allergic immune response. Another important characteristic observed in asthma is hyperresponsiveness. In our study, we showed that exposure to FA during pregnancy reduced tracheal hyperresponsiveness to cholinergic

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Fig. 5. FA exposure during pregnancy interferes in cytokines released in BAL fluid of offspring. Group of rats were exposed to FA (0.75 ppm) or vehicle during 21 days of pregnancy (1 h/day for 5 days/week). After 30 days of birth, the rats were sensitised and challenged with OVA. The BAL fluid was collected 24 h after the OVA challenge and stored for cytokines analyses. The cytokines investigated were IL-4 (Panel A), IL-6 (Panel B), IL-10 (Panel C) and TNF (Panel D). Data are mean ± SEM of 5 mothers per group (puppies n = 10). ⁎P b 0.05 in relation B group; and θP b 0.05 in relation C group.

stimulus of offspring. Thus, we again noted the suppressor effect of FA on asthma symptoms. This result may be explained by reduced anaphylactic antibodies synthesis and possible reduced capacity of mast cells to degranulate as well as reduced lung inflammation. Cytokines, including IL-4, IL-6, TNF-alpha and IL-10, are important in inflammatory diseases, such as asthma, by contributing to the recruitment and activation of leukocytes and bronchial hyperresponsiveness.

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Fig. 6. FA exposure reduces anti-inflammatory cytokine gene expression in the uterus of pregnant rats. Group of rats were exposed to FA (0.75 ppm) or vehicle during 21 days of pregnancy (1 h/day for 5 days/week). The uterus was collected at day 21 of gestation for cytokines analyses. The cytokines investigated were IL-6 (Panel A), IL-4 (Panel B), IFN (Panel C) and IL-10 (Panel D). Data are mean ± SEM 5 mothers per group. ⁎P b 0.05 in relation C group.


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1.5 1.0 0.5 0.0

P

G

2.0

2.0 1.5 1.0 0.5

*

0.0 C

P

Fig. 7. FA exposure causes an imbalance in oxidant and antioxidant enzyme gene expression in uterus of pregnant rats. Group of rats were exposed to FA (0.75 ppm) or vehicle during 21 days of pregnancy (1 h/day for 5 days/week). The uterus was collected at day 21 of gestation for enzymes analyses. The enzymes investigated were COX-1 (Panel A), COX-2 (Panel B), cNOS (Panel C), iNOS (Panel D), catalase (Panel E), SOD-1 (Panel F) and SOD-2 (Panel G). Data are mean ± SEM of 5 mothers per group. ⁎P b 0.05 in relation C group.

ongoing inflammation in the lung (Rincon and Irvin, 2012). Moreover, IL-6 controls CD4+CD25+ survival as well as the initial stages of Th2 development in the lung (Doganci et al., 2005). Confirming reduced inflammation, the levels of pro-inflammatory cytokines in the BAL (IL-6 and TNF-alpha) were reduced. Nevertheless, elevated concentrations of the anti-inflammatory cytokine IL-10 were observed, suggesting that the profile of activated cells in the BAL is different. IL-10 is mainly secreted by T lymphocytes and polarised M2 macrophages, which act to resolve the inflammation. Elevated levels of IL-10 in the BAL were associated with altered bone marrow and blood leukocyte profiles. It is plausible that FA exposure to the mother may modify the pattern of tissue leukocytes and reactivity.

In order to elucidate the mechanisms responsible for the programming mechanisms, we sought to clarify the effect of FA exposure on the uterine microenvironment. A possible local inflammatory response associated with elevated ROS activity cannot be excluded, as reduced levels of the anti-inflammatory cytokine IL-10 and an imbalanced anti- and prooxidant enzyme expression was detected in the uterus of FA-exposed mothers. Oxidative stress occurs when the generation of reactive species exceeds the capacity of the antioxidant defence mechanisms. A number of studies have identified a variety of intracellular sources for the production of ROS, such as the enzymes nitric oxide synthase (NOS), cyclooxygenase (COX), lipoxygenase and NADPH-oxidase (Wolin, 2000).

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Oxidative stress can be regulated by mechanisms of cellular defence that include superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) (Halliwell and Gutteridge, 1989). It is known that FA causes a disruption of the physiological balance between oxidant and antioxidant enzymes in lung tissue (Lino dos Santos Franco et al., 2011). Similarly, the present study shows that FA exposure increased COX-1 gene expression in the uterus of pregnant rats. In parallel, SOD-2 and cNOS gene expression were significantly reduced in pregnant rats when exposed to FA. The reduced SOD-2 gene expression may reflect excessive consumption or degradation under conditions of high oxidative stress. On the other hand, COX-1 gene expression was enhanced, indicating the generation of oxidant species. These data show that FA exposure caused an imbalance in oxidant/ antioxidant systems in the uterine environment, and such effects can result in severe injury during foetal formation. Foetal origins of adult disease are associated with several changes in the uterine environment, which are dependent on several conditions, including stress (Hanson and Gluckman, 2008). Moreover, studies have shown an interaction between NOS and COX in the regulation of physiopathological events during pregnancy (Cella et al., 2006). In these studies, the authors showed that the production of prostaglandins inhibited NOS activity. Similarly, we found elevated levels of COX gene expression and low expression of NOS in the present study. In conclusion, our study revealed that low doses of FA exposure during pregnancy suppresses the development of allergic lung inflammation and tracheal hyperresponsiveness in offspring by mechanisms mediated by reduced anaphylactic antibodies synthesis, IL-6 and TNF-alpha secretions. Elevated levels of IL-10 may contribute to reduced allergic responses. Oxidative stress in the uterine environment was evident at the moment of the delivery as quantified by elevated COX-1 expression and reduced cNOS and SOD-2 in the uterus. Thus, the data obtained in this study may contribute to the understanding the programming mechanisms of pollutants during pregnancy and its repercussion for the development of allergic diseases. Acknowledgments This study was sponsored by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP no. 11/51711-9). Adriana Lino dos Santos Franco is a fellow researcher of FAPESP. References Adner, M., Rose, A.C., Zhang, Y., Swärd, K., Benson, M., Uddman, R., Shankley, N.P., Cardell, L.O., 2002. An assay to evaluate the long-term effects of inflammatory mediators on murine airway smooth muscle: evidence that TNFalpha up-regulates 5-HT(2A)-mediated contraction. Br. J. Pharmacol. 137, 971–982. Amrani, Y., Chen, H., Panettieri Jr., R.A., 2000. Activation of tumor necrosis factor receptor 1 in airway smooth muscle: a potential pathway that modulates bronchial hyperresponsiveness in asthma? Respir. Res. 1, 49–53. Austen, R.L., Potts, E.N., Mason, S.N., Fischer, B., Huang, Y., Foster, W.M., 2009. Maternal exposure to particulate matter increases potnatal ozone-induced airway hyperreactivity in juvenile mice. Am. J. Respir. Crit. Care Med. 180 (12), 1218–1226. Bach, M.K., Bloch, K., Austen, K.F., 1971. IgE and IgG2a antibody-mediated release of histamine from rat peritoneal cells. Optimum conditions for in vitro preparation of target cells with antibody and challenge with antigen. J. Exp. Med. 133, 752. Carlson, R.M., Smith, M.C., Nedorost, S.T., 2004. Diagnosis and treatment of dermatitis due to formaldehyde resins in clothing. Dermatitis 15, 169–175. Cella, M., Aisemberg, J., Sordelli, M.S., Billi, S., Farina, M., Franchi, A.M., Ribeiro, M.L., 2006. Prostaglandins modulate nitric oxide synthase activity early in time in the uterus of estrogenized rat challenged with lipopolysaccharide. Eur. J. Pharmacol. 18;534 (1–3), 218–226. Cerda, S., Weitzman, S.A., 1997. Influence of oxygen radical injury on DNA methylation. Mutat. Res. 386 (2), 141–152. De Lima, W.T., Sirois, P., Jancar, S., 1992. Immune-complex alveolitis in the rat: evidence for platelet activating factor and leukotrienes as mediators of the vascular lesions. Eur. J. Pharmacol. 213, 63–70. Doganci, A., Sauer, K., Karwot, R., Finotto, S., 2005. Pathological role of IL-6 in the experimental allergic bronchial asthma in mice. Clin. Rev. Allergy Immunol. 28 (3), 257–270.

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