Oxidative stress mediates dibutyl phthalateinduced anxiety-like behavior in Kunming mice

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Highlights

  • Dibutyl phthalate can induce anxiety-like behavior in mice.

  • Oxidative stress mediates the anxiety-like behavior induced by DBP.

  • Mangiferin, as an antioxidant, can attenuate the anxiety-like behavior.

Abstract

Among all phthalate esters, dibutyl phthalate (DBP) is only second to di-(2-ethylhexyl) phthalate (DEHP) in terms of adverse health outcomes, and its potential cerebral neurotoxicity has raised concern in recent years. DBP exposure has been reported to be responsible for neurobehavioral effects and related neurological diseases. In this study, we found that neurobehavioral changes induced by DBP may be mediated by oxidative damage in the mouse brain, and that the co-administration of Mangiferin (MAG, 50 mg/kg/day) may protect the brain against oxidative damage caused by DBP exposure. The results of ethological analysis (elevated plus maze test and open-field test), histopathological examination of the brain, and assessments of oxidative stress (OS) in the mouse brain showed that there is a link between oxidative stress and anxiety-like behavior produced by DBP at higher doses (25 or 125 mg/kg/day). Biomarkers of oxidative stress encompass reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA) and DPC coefficients (DPC). MAG (50 mg/kg/day),administered as an antioxidant,can attenuatetheanxiety-like behavior of the tested mice.

Introduction

Phthalic acid esters (PAEs) are widely used as plasticizers in a range of industrial applications and consumer products, such as polyvinyl chloride (PVC) or vinyl, medical equipment and toys (Net et al., 2015, Wormuth et al., 2006). The total global consumption of PAEs is estimated to exceed 30 million tons per annum (Rivera-Utrilla et al., 2012). The current consumption of PAEs in Europe accounts for approximately 1 million tons (Net et al., 2015), while Chinese consumption has increased to 6 million tons/year since the 0.87 million tons consumed in 2006 (Zhang et al., 2013, Xu et al., 2014, He et al., 2015). Incredibly, these applications and products do not require the labeling to specify whether PAEs are present (Dodson et al., 2012). It is noteworthy that PAEs are easily emitted since they are not tightly bound to the polymer matrix. Millions of pounds of PAEs are discharged into the environment every year (Net et al., 2015, Tong, 2014).

The most widely used PAEs include di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DBP or DnBP), butylbenzyl phthalate (BBP or BBzP), diisoheptyl phthalate (DiHP), diisononyl phthalate (DiNP), and diethyl phthalate (DEP). DBP is the primary plasticizer currently used in China, and has been listed as a priority environmental pollutant by the China National Environmental Monitoring Centre and United States Environmental Protection Agency (EPA) (Wang et al., 2016). DBP is second only to DEHP in terms of adverse health effects (Zeng et al., 2013). Exposure to PAEs through daily diet, inhalation, and dermal contact can lead to health problems, including developmental and reproductive toxicity (Swan, 2008). PAEs are endocrine disruptors that contribute to estrogen-like activity and have been shown to affect normal brain development in laboratory animals (Li et al., 2014). Recently, the neurotoxicity of DBP has attracted increasing attention since it is suspected of being involved in mental conditions linked to endocrine disruption (Cho et al., 2010, Chopra et al., 2014, Miodovnik et al., 2014).

Toxicological studies in animals support the epidemiological data. Male rat pups exposed to oral DBP in utero and via lactation at low (30–55 mg/kg/day) and medium (100–165 mg/kg/day) dosages exhibited inhibited spatial learning and memory on the Morris water maze (MWM) test (Li et al., 2009). Boberg also found a dose-dependent effect in spatial memory testing of female adult rats exposed to DINP (Boberg et al., 2011). Another investigation of school-age children showed an inverse relationship between phthalate metabolites and IQ scores (Cho et al., 2010).Although there are some reports on the toxic effects of DBP induced oxidative stress on animals (Zhou et al., 2010, Zuo et al., 2014, Aly H et al., 2015), there are few studies to ascertain whether neurological effects such as depressive mood or anxiety-like behavior are affected by oxidative stress. We therefore undertook a study to determine whether neurotoxicity and anxiety-like behavior isinduced by DBP via oxidative stress in Kunming mice (Fig. 1).

In our study, after exposure to DBP, neurobehavioral changes (anxiety-like behavior) of mice were determined using the elevated plus maze (EPM) and the open-field test (OFT). Oxidative stress was evaluated through observing reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA) and DPC coefficients (DPC) to explore the key upstream events. Histopathological examination of the brainwas used for identifying oxidative damage.Biomarkers such as brain viscera coefficients (BVC) and brain histological assays (BHA) were examined. In addition, mangiferin (MAG), an antioxidant that protects cells from oxidative damage, was used in a 50 mg/kg/day dose, since it has been shown to improve memory and reduce learning deficits in rodents. This experiment enabled us to elucidate the role of MAG in a model of oxidative stress/damage (Márquez et al., 2012).

Section snippets

Materials and methods

All experimental procedures were approved by the Office of Scientific Research Management of Hubei University of Science and Technology (Xianning, China) with a Certificate on Application for the Use of Animals dated 26 February 2016 (approval ID: HBUST-IACUC-2016-001).

Anxiety-like behavior results

The anxiety-like effect on the mice after DBP exposure is shown in Fig. 2. Significant (Ptrend < 0.0001) dose-dependent increaseswere observed. In the EPM testing (Fig. 2A), mice treated with DBP (5, 25, 125 mg/kg/day) in a dose-dependent manner exhibited a significant decrease in the number of open-arm entries and in the percentage of time spent in the open arms as compared to the vehicle control (Fig. 2B).The OFT, (Fig. 2(C–D))showed that after DBP exposure, the mice traveled shorter distances

Discussion

Although many epidemiological studies on humans suggest that DBP exposure is associated with many potential risks (Koo and Lee, 2004, Jurewicz and Hanke, 2011), very few studies have focused on DBP neurotoxicity, and in particular on neurobehavioral changes (Tang et al., 2015). Recent experimental evidence suggests that several phthalates may disrupt endocrine pathways resulting in abnormal reproductive outcomes (Miodovnik et al., 2014). Damage to the central nervous system is also reported to

Conclusion

The main findings of this study include: (1) A statistical association exists between DBP oral exposure and anxiety-like effects as determined by the EPM and OFT of Kunming mice. (2) A statistical association is also found between DBP oral exposure (at 25 and 125 mg/kg/d levels) and oxidative stress in mouse brain tissue. (3) Co-administration of MAG (50 mg/kg/day) diminishes, to a certain extent, the elevation of ROS, MDA, and DPC induced by DBP (125 mg/kg/d) from significant to non-significant.

Conflict of interest

The authors have no conflict of interests.

Acknowledgements

This work was funded by the Science and Technology Research Foundation from Hubei province Education Department (Q20152801) and the Scientific Research Foundation for the PhD Start-up of Hubei University of Science and Technology (BK1412).

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These authors contributed equally to this work.

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