J BIOCHEM MOLECULAR TOXICOLOGY Volume 29, Number 7, 2015

Mice Brain Tissue Injury Induced by Diisononyl Phthalate Exposure and the Protective Application of Vitamin E Ling Peng College of Chemistry and Bioengineering, Yichun university, Yichun 336000, People’s Republic of China; E-mail: [email protected] Received 24 October 2014; revised 16 February 2015; accepted 1 March 2015

ABSTRACT: As a widely used plasticizer in plastic industry, the data of diisononyl phthalate (DINP) toxicity due to exposure are insufficient. This work investigated the brain tissue injury induced by DINP exposure. Through oral exposure to DINP, oxidative stress, inflammatory responses, apoptosis, and hippocampus pathological alterations were found in the mice brain. And through the Morris water maze test, cognitive deficits were tested. Our data also showed that these exacerbations were counteracted by vitamin E. These results above indicated that oral exposure of mice to DINP induced brain damage, and oxidative stress, inflammation, and the consequential apoptosis jointly constituted the potential mechanisms of C 2015 Wiley Periodicals, Inc. J. such induced toxicity.  Biochem. Mol. Toxicol. 29:311–320, 2015; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21700

Diisononyl Phthalate (DINP); Cognitive Deficits; Oxidative Stress; Vitamin E

KEYWORDS:

INTRODUCTION Phthalates are a group of synthetic chemicals that are dialkyl or aryl/alkyl diesters of phthalic acid. In

Correspondence to: Ling Peng Contract Grant Sponsor: Jiangxi Department of Science and Technology. Contract Grant Number: 20132BBF60063. Contract Grant Sponsor: Youth Fund Project of Jiangxi Department of Education. Contract Grant Number: GJJ14709. Contract Grant Sponsor: Scientific Research Fund of Yichun Science, and Technology Bureau. Contract Grant Number: GJJ14709 (JXYC2012KSA001). Contract Grant Sponsor: Yichun Social Science Planning Fund. Contract Grant Number: YCSKL2013-081.  C 2015 Wiley Periodicals, Inc.

order to add soften and flexibility of polyvinyl chloride (PVC) products, phthalates have been widely used in the chemical industry in the manufacturing of polymers and a variety of consumer products, including food processing and medical applications since the 1920s [1]. According to the length of carbon chain backbone, phthalates are classified as “high-molecular-weight (HMW)” phthalates and “low-molecular-weight (LMW)” phthalates, HMW phthalates, such as di(2ethylhexyl) phthalate (DEHP) and diisononyl phthalate (DINP), which have been mainly used as plasticizers in polymer manufacturing [2]. Because phthalates are not chemically bound to the polymers, they can release out from the polymer matrix during usage, and then enter the food chain and the environment. Stringent government regulations in North America and Europe on the use of DEHP in consumer products due to its many adverse effects have resulted in its substitution with other less toxic phthalates, notably DINP [3]. DINP (CAS RN 68515-48-0) is a general-purpose plasticizer for PVC. The American Chemistry Council estimated the annual world production of DINP to be 1.5 million metric tons in 2013, assuming a 2.5% annual production growth during that period. More than 90% of DINP is used in PVC applications, including toy, constructions, and general consumer product markets [4, 5]. DINP is also not covalently bound to plastics. Humans may be exposed to DINP by the oral, dermal, and inhalation routes of exposure. Exposure of children to DINP through children’s products is a public concern [6]. The toxicity profiles and potency of other phthalates are varied. As a less-toxic phthalates, DINP has minimal acute toxicity, the oral LD50 is greater than 10 g/kg, and the dermal LD50 is greater than 3.16 g/kg. The acute inhalation toxicity exceeds the maximally attainable vapor concentration [7, 8]. It is minimally

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irritating to skin and eyes and does not cause allergic contact dermatitis [9]. However, in repeated-dose studies of DINP, usually carried out by oral administration, DINP also exhibits adverse effects. DINP also shows antiandrogenic activity like DEHP and can alter sexual differentiation of male rats by inhibiting fetal testicular testosterone synthesis [10]. Some studies reported that once the exposure level above 150 mg/kg/day DINP could induce liver and kidney enlargement and increased levels of peroxisomal enzymes, the peroxisomal proliferation also been confirmed by histologic evidence [11–13]. 100 mg/kg/day was considered as the no-effect level for all systemic effects [14]. Considering the widely applications and properties of DINP, humans are exposed to DINP in a variety of ways, and having a lower tolerance than rat or mice, humans may be more easily susceptible to damage [15]. Therefore, DINP is questionable relevance to human health. In this manuscript, the adverse effects of DINP exposure on mouse brain were evaluated by Morris water maze (MWM) test and histological assay. The indices of oxidative stress (reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) activity), inflammation (tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1)), and apoptosis (caspase-3 and caspase-3 activity) were examined to explore the possible mechanisms. The purpose of this study was to define the damage in mouse brains after DINP exposure and to investigate a method to avoid damage caused by exposure of DINP.

MATERIALS AND METHODS

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assay (ELISA) kit for tumor necrosis factor (TNF-а), interleukin-1 (IL-1), and caspase-3 were purchased from eBioscience (San Diego, CA, USA). The mouse caspase-3 activity assay kit was purchased from Beyotime (Nanjing, Jiangsu, People’s Republic of China). The mouse GSH and SOD activity assay kit was purchased from Jiancheng (Nanjing, Jiangsu, People’s Republic of China).

Preparation of DINP The highest dosage of DINP (150 mg/kg/day) was chosen according to the reported by Mckee et al [16]. The dosage was also similar to the European Chemicals Agency Report [2] and the European Union Risk Assessment Report [17]. The dosage of Vitamin E was 50 mg/kg/day. Different concentrations of DINP were prepared in Tween-80 (1:1, v/v) and diluted with sterile saline for the oral administrations [18].

Experimental Protocol Mice were divided randomly into six experimental groups of 10 animals. These groups were exposed to 0, 1.5, 15, and 150 mg/kg/day DINP, 150 mg/kg/day DINP + 50 mg/kg/day vitamin E (block group) [19], and 50 mg/kg/day vitamin E. DINP or vitamin E was administered for 9 days. During this period, MWM was used to investigate the cognitive ability of the mice and mice were euthanized at 10th day, 24 h later after the last DINP exposure, mouse brains were collected for other detections (Figure 1a).

Animals and Ethics Statement Specified pathogen-free (SPF) class male Kunming mice (5–6 weeks old, 22 ± 2 g) were purchased from the Hunan Slack King of Laboratory Animal Co. Ltd. (Changsha, People’s Republic of China) and housed in standard environmental conditions (12 h light-dark cycle, 50%–70% humidity, and 20°C–25°C). Food and water were provided ad libitum. Mice were quarantined for ࣙ7 days before study initiation. Protocols involving the use of animals were approved by the Changsha Animal Policy and Welfare Committee (approval documents: SCXK2011-0003).

Reagents and Kits DINP, 2’,7’-dichlorodihydrofluorescein (DCFHDA), 2-thiobarbituric acid (TBA), and vitamin E were purchased from Sigma–Aldrich (St Louis, MO). All other chemicals were the highest grade available commercially. A mouse-enzyme-linked immunosorbent

Morris Water Maze The MWM was conducted as previously described [20, 21]. Mice were placed into water (circular and opaque). There is an escape platform under the surface; mice were kept swimming until found the platform. The platform was in the center of SE quadrant. After days training, mice learned to swim directly onto the platform. The time of mice found the platform at the first to the seventh day was “escape latency” to ascertain the learning ability of mice after DINP exposure (hidden-platform acquisition test), and the swimming time in the platform area (Figure 1b) at ninth day was used to measure memory ability of mice (probe trial test). The mice were released into the water from the northeast (NE), northwest (NW), and southwest (SW) quadrants, facing the wall; each mouse had three trials per day. Each training session had a maximum duration of 60 s. The escape latency was recorded as 60 s, if mice could not find the platform within 60 s, then J Biochem Molecular Toxicology

DOI 10.1002/jbt

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BRAIN INJURY INDUCED BY DIISONONYL PHTHALATE

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FIGURE 1. (a) Experimental protocol. Six mice groups were exposure to DINP daily for 9 days. During the process, MWM was used to monitor the cognitive ability of mice. The 1st day to the 7th day was the hidden-platform acquisition test; the 8th day was the forget period; and the 9th day was the probe trial test. 24 h after the last exposure, on the 10th day, mice were sacrificed and their brains were collected for subsequent analysis. (b) Morris water maze devices. MWM was a pool, filled with water containing white paint (with negligible toxicity) to a depth of 20 cm. The platform submerged 0.5 cm below the surface of water, the pool was divided into four quadrants, the platform was in the center of the SE quadrant, all the devices at room temperature, a camera recorded the tracks of mice over the pool. The image signal through the ANY-MazeTM (Stoelting Co., Wood Dale, IL) software analysis to form the last data (All the figures in this paper are created by Ling Peng).

the mice was led to place on the platform for 30 s; this training continued until the seventh day. On the eighth day, the water maze was not used, allowing a period for the mice to forget the location of the platform. On the ninth day, the platform was removed from the pool to detect the memory ability of the mice. The mice were put into the pool from the NE, NW, and SW quadrants for the probe trial test also, and the camera recorded their tracks for 60 s.

embedded in paraffin, sectioned into 10 μm slices, and separate pieces stained with H & E and Nissl, as previously described [22]. Stained pieces were observed using a DM 4000B microscope (Leica, Berlin, Germany). The average optical density (OD) of each slice was determined using Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD) software. A nonstained region was selected and set as the background. All tissue sections were examined qualitatively by two experienced pathologists in a blinded fashion.

Brain Histological Assay 24 h after the oral administration on the ninth day, the mice were sacrificed by cervical dislocation and the brains collected. All samples were fixed in 10% formalin solution for 24 h at room temperature, J Biochem Molecular Toxicology

DOI 10.1002/jbt

Brain Sample Preparation The collected brains were weighed and placed in 10 mL/g ice-cold 1 ×phosphate-buffered saline (PBS, pH 7.5) and homogenized using a glass homogenizer.

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Homogenate was centrifuged at 10, 000 rpm for 10 min at 4°C. The supernatant was collected and frozen at −70°C until assayed; the protein concentration of supernatant was determined using the Lowry assay [23].

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kits according to the manufacturer’s instructions. The sensitivities of the ELISA kits were 8 pg/mL for TNF-α, 80 pg/mL for IL-1, and 0.5 pg/mL for caspase-3.

Analysis of Caspase-3 Activity Detection of ROS ROS levels were detected using DCFH-DA. Briefly, the supernatants were diluted 10-fold in PBS (pH 7.5), 100 μL was removed to microplate, and 100 μL of 10 μmol/L DCFH-DA was added, kept in the dark for 30 min at 37°C. Fluorescence intensity was then measured at an excitation wavelength of 488 nm and an emission wavelength of 525 nm by a fluorescence reader [24].

Measurement of MDA MDA contents were measured by TBA. Briefly, 2 mL 0.6% TBA solution was added into 0.5 mL supernatant and incubated in boiling water for 15 min. The mixture was cooled and centrifuged at 10, 000 rpm for 5 min at 4°C. The supernatant was collected, and absorbance measured at 532, 600, and 450 nm. The concentration of MDA was calculated as MDA (μmol/L) = 6.45 × (OD532 – OD600 ) – 0.56 × OD450 [25].

GSH Depletion Assay GSH contents were detected by an assay kit according to the manufacturer’s instructions. GSH can react with 3-carboxy-4-nitrophenyl disulfide (DTNB) in the dark and produces 2-nitro-5-thiobenzoic acid (TNB). Experimental and standard samples were analyzed using a microplate reader at a wavelength of 412 nm.

Analysis of SOD Activity SOD activity was determined with a SOD assay kit according to the manufacturer’s instructions. The xanthine and xanthine oxidase reaction systems were used to produce superoxide anions, which can oxidize hydroxylamine to form nitrites. With the action of the chromogenic agent, the nitrites turn purple. SOD inhibits the superoxide anion and decreases the production of nitrites, thus reducing absorbance.

Analysis of TNF-α, IL-1, and Caspase-3 Content The supematant in “Brain sample preparation” was used to detect the concentration of TNF-α, IL-1, and caspase-3. The contents were measured by ELISA

Caspase-3 activity was determined with a caspase3 activity assay kit according to the manufacturer’s instructions. This assay is based on spectrophotometric detection of the chromophore p-nitroanilide (pNA) after its cleavage from the labeled substrate acetyl-AspGlu-Val-Asp P-nitroanilide (DEVD-pNA). Caspase-3 activities were determined according to the manufacturer’s instructions. Absorbance of the chromophore pNA produced was measured at 405 nm.

Statistical Analysis Analysis of variance (ANOVA) was applied for statistical analysis. The data were presented as the mean ± standard error of the mean. Statistical analyses were carried out using SPSS (SPSS, Chicago, IL) software, version 13.0. A repeated measures ANOVA followed by a post-hoc test named as Tukey was used for the MWM escape latency analyses; and all other data were analyzed by a one-way ANOVA followed by Tukey test. A P-value of