Joint toxic effect of formaldehyde and DEHP on learning and memory of mice
【Abstract】To explore the combined toxic effect of formaldehyde (FA) and DEHP on learning and memory of mice and the mechanism, Kunming mice were randomly divided into 14 groups: (1) the control group; (2) FA group: 0.5 mg/m3, 1 mg/m3, 3 mg/m3; (3) DEHP group: 5 mg/kg, 50 mg/kg, 500 mg/kg; (4) the combined exposure: 0.5 mg/m3 + 5 mg/kg, 1 mg/m3 + 50 mg/kg, 3 mg/m3 + 500 mg/kg; (5) VE group: saline + VE (100 mg/kg), 3.0 mg/m3 FA + VE, 500 mg/kg DEHP + VE, 3.0 mg/m3 FA + 500 mg/kg DEHP + VE. The mice of FA and combined exposure groups were exposed to gaseous FA for 8 h every day (continuous exposure for 5 days, interval for two days), and the mice of DEHP and combined exposure groups were subjected to gavage with DEHP solution. Besides, blocking groups were also subjected to gavage with VE solution (100 mg/kg) every day. Their behaviors of learning and memory were tested by Morris water maze test. Then the changes of biological indicators including oxidative damage, ROS, MDA, TNF-α, IL-β, 5-HT, etc., in the cerebral tissue were detected. Results showed that Morris water maze test in space training and learning indicated that escape latency significantly extended in 3.0 mg/m3 FA and 500 mg/kg DEHP treatment group, 1.0 mg/m3 + 50 mg/kg and 3.0 mg/m3 + 500 mg/kg of the combined exposure groups (P < 0.05). Compared with single exposure groups, escape latency significantly extended in the moderate and high dose of combined exposure groups (P < 0.05). In space exploration experiments, the proportions of time of the target quadrant in 1.0 mg/m3 and 3.0 mg/m3 FA treatment groups, the 500 mg/kg DEHP treatment group, and 1.0 mg/m3 + 50 mg/kg, 3.0 mg/m3 + 500 mg/kg of the combined exposure groups were less than the control group (P < 0.05). The proportions of time of the target quadrant in combined exposure groups were significantly decreased compared with single exposure (P < 0.05). Compared with control group, the content of ROS and MDA in 1.0 mg/m3 and 3.0 mg/m3 FA, 50 mg/kg and 500 mg/kg DEHP groups and all the combined exposure groups was increased, while the content of GSH decreased in 3.0 mg/m3 FA, 500 mg/kg DEHP groups and all the combined exposure groups decreased. Compared with formaldehyde or DEHP exposure groups, the content of GSH in combined exposure groups was significantly decreased (P < 0.01). The expression levels of inflammatory factors TNF-α and IL-β were also significantly increased, and caspase-3 was activated. The content of 5-HT in 3.0 mg/m3 + 500 mg/kg of combined exposure group was significantly decreased (P < 0.01). 0.5 mg/m3 FA had little effect on mice and their behaviors of learning and memory did not obviously change. This study shows that 1.0 mg/m3 and 3.0 mg/m3 FA and 500 mg/kg DEHP treatment group and 1.0 mg/m3 + 50 mg/kg, 3.0 mg/m3 + 500 mg/kg of the combined exposure groups could cause oxidative damage and inflammation of Kunming mice. The combined exposure to FA and DEHP has synergistic effect. And VE could protect brain tissue by reducing oxidative stress, inflammation, the level of caspase-3, and increasing the content of 5-HT.
【Keywords】 formaldehyde; DEHP; combined exposure; mice; learning and memory;
 Tang X, Bai Y, Duong A, et al. Formaldehyde in China: Production, consumption, exposure levels, and health effects [J]. Environment International, 2009, 35 (8): 1210–1224.
 Sul D, Kim H, Oh E, et al. Gene expression profiling in lung tissues from rats exposed to formaldehyde [J]. Archives of Toxicology, 2007, 81 (8): 589–597.
 Li H, Zhang Y C, Ke Y J, et al. Expression of proto-oncogene c-myc, MDM2 and anti-oncogene p53 induced by different concentrations of formaldehyde [J]. China Environmental Science, 2013, 33 (8): 1483–1486 (in Chinese).
 Ke Y J, Qin X D, Li L, et al. Toxic effect of formaldehyde on mouse bone marrow [J]. China Environmental Science, 2012, 32(6): 1129–1133 (in Chinese).
 Kerns W, Pavkov K, Donofrio D, et al. Carcinogenicity of formaldehyde in rats and mice after long-term inhalation exposure [J]. Cancer research, 1983, 43 (9): 4382–4392.
 Arts J, Rennen M, De Heer C. Inhaled formaldehyde: evaluation of sensory irritation in relation to carcinogenicity [J]. Regulatory Toxicology and Pharmacology, 2006, 44 (2): 144–160.
 Xiao Y, Chen J Q, Zhou H S, et al. Eytotoxicity of volatile organic compounds on Chinese hamster lung cells [J]. Chinese Journal of Public Health, 2006, 22(9): 1095–1096 (in Chinese).
 Ren R. 警惕邻苯二甲酸酯污染 [J]. University Chemistry, 2003, 18(6): 33–36 (in Chinese).
 Takashima K, Ito Y, Gonzalez F, et al. Different mechanisms of DEHP-induced hepatocellular adenoma tumorigenesis in wild-type and Pparα-null mice [J]. Journal of Occupational Health, 2008, 50 (2): 169–180.
 Manikkam M, Tracey R, Guerrero-Bosagna C, et al. Plastics derived endocrine disruptors (BPA, DEHP and DBP) induce epigenetic transgenerational inheritance of obesity, reproductive disease and sperm epimutations [J]. Plo S One, 2013, 8 (1): 583–587.
 Lu X, Liu Q, Jin C, et al. Study on neurotoxicity and lipid peroxidation of brain after exposure to DEHP in rats [J]. Journal of Shenyang Medical College, 2008, 4 (2): 198–201.
 Pitten F, Kramer A, Herrmann K, et al. Formaldehyde neurotoxicity in animal experiments [J]. Pathology-Research and Practice, 2000, 196 (3): 193–198.
 Malek F, Möritz K, Fanghänel J. Effects of a single inhalative exposure to formaldehyde on the open field behavior of mice [J]. International Journal of Hygiene and Environmental Health, 2004, 207 (2): 151–158.
 Cheng J F, Shang S, He G B, et al. Decreased learning and memory ability caused by the oxidative stress induced by DEHP and the protection effect of vitamin E in mice [J]. Journal of Medical Research, 2012, 12(5): 24–28 (in Chinese).
 Act C E P. Priority substances list assessment report: formaldehyde [R]. Environment Canada, Health Canada, 2007.
 Ke Y J. Exposure to formaldehyde, cell activity, and endogenous formaldehyde content changes in cultured cells [J]. Wuhan: Journal of Central China Normal University, 2013 (in Chinese).
 Li X Y, Lan X F. Effects of granulocyte colony-stimulating factor on neural cell apoptosis in the hippocampus of vascular dementia rats [J]. Chinese Journal of Tissue Engineering Research, 2011, 15(27): 5044–5047 (in Chinese).
 Halliwell B. Oxidative stress and neurodegeneration: where are we now? [J]. Journal of Neurochemistry, 2006, 97 (6): 1634–1658.
 Reviews of environmental contamination and toxicology [M]. Springer, 2012.
 Irmak M, Fadillioglu E, Sogut S, et al. Effects of caffeic acid phenethyl ester and alpha‐tocopherol on reperfusion injury in rat brain [J]. Cell Biochemistry and Function, 2003, 21 (3): 283–289.
 Das M, Babu K, Reddy N, et al. Oxidative damage of plasma proteins and lipids in epidemic dropsy patients: alterations in antioxidant status [J]. Biochimica et Biophysica Acta (BBA)-General Subjects, 2005, 1722 (2): 209–217.
 Gałażyn-Sidorczuk M, Brzóska M, Jurczuk M, et al. Oxidative damage to proteins and DNA in rats exposed to cadmium and/or ethanol [J]. Chemico-Biological Interactions, 2009, 180 (1): 31–38.
 Xiao G, Wang M, Li N, et al. Use of proteomics to demonstrate a hierarchical oxidative stress response to diesel exhaust particle chemicals in a macrophage cell line [J]. J.Biol.Chem., 2003, 278 (50): 50781–50790.
 Brown D, Donaldson K, Borm P, et al. Calcium and ROS mediated activation of transcription factors and TNF-αcytokine gene expression in macrophages exposed to ultrafine particles [J]. American Journal of Physiology-Lung Cellular and Molecular Physiology, 2004, 286 (2): L344–L353.
 Shankar E, Vykhovanets E, Vykhovanets O, et al. High‐fat diet activates pro‐inflammatory response in the prostate through association of Stat-3and NF-κB [J]. The Prostate, 2012, 72 (3): 233–243.
 Metz B, Kersten G, Hoogerhout P, et al. Identification of formaldehyde-induced modifications in proteins reactions with model peptides [J]. Journal of Biological Chemistry, 2004, 279 (8): 6235–6243.
 Gurel A, Coskun O, Armutcu F, et al. Vitamin E against oxidative damage caused by formaldehyde in frontal cortex and hippocampus: biochemical and histological studies [J]. Journal of Chemical Neuroanatomy, 2005, 29 (3): 173–178.