Effects Of Dibutyl Phthalate On Neurobehavioral Development Of Rats And Mechanism Exploration

Dibutyl phthalate (DBP) is an important representative phthalate esters (PAEs) usedubiquitously nowadays, and is also an important endocine disruptor. DBP is widely used as plasticizerand flexibilizer in production of cellulose resin, polyvinyl chloride and rubbers, and is also onekind of raw material for many industrial products, such as lacquers, agglomerant, pharmaceuticals.Due to no tight binding to the matrix of these products, DBP can migrate from them into theenvironment, and may impair human health.The National Toxicology Program (NTP) estimated exposure level of DBP was 2-10μg/kgBW/day for general population from all sotirces. Once into the body, DBP is quickly metabolizedto monobutyl phthalate (MBP), which is responsible for the induction of developmental toxiceffects of DBP. The main impairments of DBP are reproductive and developmental toxicity,mechanism under which is the disruption of endocrine function. DBP has shown estrogenicactivtity in vitro and anti-androgenic activity in vivo, and can affect the activity of enzymecatalyzing sterides synthesis. However, the neurobehavioral effects of DBP still remain unclear.The central nevious system (CNS) is very sensitive to many hormones during development,and is susceptive to endocrine disruptors such as DBP. Epidemiological data suspected that DBPwas a neurotoxin; however, due to the combined exposure with other PAEs, the neuro effects ofDBP were not very certain. Neurobehaviors are sensitive indicators reflecting the effects of endocrinedisruptors on the nevious system and/or other systems. This study was design to investigate thetoxic effects on the development of neurobehaviors in rats, and to explore the undermining mechanismfor a better understanting of DBP's health effects. Wistar rats were treated with DBP in gestational and lactational periods, and their neurobehaviorswere evaluated by a battery of tests on different postnatal days. Multi-levels of dose, large sample sizeand cross-generation exposure are features of the present study. Due to the effects of DBP on spatiallearning and memory in the male offspring rats, the mechanism focused on hippocampal region of thebrain. The effects of DBP on the developent and function of hipocampal neurons were evaluated bymeans of detecting gene and protein expression using real time RT-PCR and/or Western Blot, anddetecting in vivo long-term potentiation, with an intent to explore the mechanism of neurotoxicityand provide experimental and theoretical data for DBP comtamination prevention and regulation.Partâ… Effects of DBP on Reproduction of F₀ Femal Rats and on Growth and Development of F₁Generation RatsObjective: To explore the effects of DBP on reproduction of F₀ female rats and on growth anddevelopment of F₁ generation rats.Methods: Sixty pregnant wistar rats were randomly assigned to control, low, sub-low,sub-high and high dose groups, each were treated with DBP at doses of 0%, 0.037%, 0.111%,0.333% and 1% in a powdered diet from GD 6 to PND 28. Body weight, food intake, symptoms ofintoxicication and reproductive effects were observed; Indexs of growth and development of F₁generation, including body length, tail length, anogenital distance (AGD), and ages at firstapperance of pinna detachement, incisor eruption and eye opening, were also measured or observed;Main organs of the F₁ generation were checked and weighted when the exposure expired onPND28 and organ coefficients were calculated.Results: (1) Food intake and body weight of the pregnant rats increased with the gestationaldays, but no alteration was observed as compared to control (p＞0.05). (2) No obvious toxicsymptoms were observed in the treatment group pregnant rats, and no significant difference wasfound in pregnancy failure rate, live pups per litter or sex ratio of the exposure g as compared tocontrol (p＞0.05), but the gestational period length of the high treatment group was prolongedslightly but significantly (p＜0.05). (3) Body weight of the pups (male and female respectively)exposed to high dose DBP was significantly less than that of control from PND0 to PND 28(p＜0.01). (4) AGD (adjusted by the cube root of body weight) of the male pups in the high dose group was significantly decreased, but that of the females was not changed (p＞0.05). (5) The age atfirst appearance of pinna detachement, incisor eruption or eye opening of neither male of femalepups was affected by DBP exposure (p＞0.05). (6) On PND 28, liver-to-body weight of both maleand female pups exposed to high dose DBP increased significantly (p＜0.01); spleen-to-body weightof female pups in high and sub-high dose groups decreased significantly (p＜0.01); testes-to-bodyweight of male pups in high dose group also decreased significantly (p＜0.01); however, none ofbrain-, heart-, lung-, kidneys- or womb-to-body weight was altered obviously (p＞0.05) in anytreatment group (p＞0.05).Conclusions: DBP treatments in the present study exhibited no significant toxic effect on thereproductive ability of the F₀ male rats, but the high treatment affected the growth and developmentof the F₁ generation rats and showed overt organ toxicity, including decreased body weight,shortened AGD and altered liver- spleen- and testes-to-body weight.Partâ…¡Effects of DBP on Neurobehaioral Development of F₁ Generation RatsObjective: To evaluate the effects of DBP on neurobehaioral development in the F₁ generationrats.Methods: The treatment was the same as in Partâ… . Pups were selected to perform air rightingon PNDs 4 and 7, negative geotaxis on PNDs 4 and 7, cliff avoidance on PND 7, forepaw grip timeon PND 10 and air righting on PND 16 for evaluation of the Pups at the age of 4 weeks were aslosubmitted to a open field tests to measure the motional activity; Learning and memory capacity wasmeasureed by Morris water maze tests in the F₁ generation pups at the age of 5 weeks.Results: (1) Early neurobehaioral development: Male pups in the high-dose group had lowerscores in the surface righting reflex on PND 7 than control with significant difference (p＜0.05), andmale pups in both the low and the high-dose groups showed shortened forepaw grip time on PND 10(p＜0.05), but for the surface righting on PND 7, negative geotaxis on PNDs 4 and 7, cliff avoidanceon PND 7 and air righting on PND 16, no significant treatment effects were found in either themale or the female pups. (2) Open field test: No significant treatment effect or sex effect was foundin the numbers of crossings, rearrings or retentions for more than 5 seconds (p＞0.05), and still notreatment effect was found when the upper data were collaped across sex (p＞0.05). (3) Morris water maze:â‘ 5-day hidden platform trial: Sex X Day interaction were found significant (p＜0.01),male pups in the high-dose group exhibited better performances in spatial acquisition in the last 3days (p＜0.05), but male pups in the low-dose group showed depressed spatial acquisition learningin the last 2 days (p＜0.05). However, no treatment effect was found in the female pups.â‘¡One-dayprobe trial: Male pups in the low- and sub-low-dose groups exhibited less preference for the targetzone, indicating worse spatial memory (p＜0.05), however, there still no treatment effect on thepreference was found in the female pups.â‘¢Visible platform trial: No significant treatment effectson either the escape latency or swimming path length was witnessed in either the male or femalepups (p＞0.05).Conclusions: Under the experiment conditions as ours, DBP exposure altered a fewneurobehavioral parameters and spatial learning and memory capacity in the F₁ generation malepups, but scarcely affected that of the females, indicating significant sex effect and non-monotonicdose-related responses.Partâ…¢Effects of DBP on the Expression of Development-related Genes and Proteins in theHippocampus of the Male PupsObjective: To investigate the effects of DBP on the expression of develpment-related genesand proteins for exploration into the neurotoxic mechanism.Methods: Pregant Wistar rats were randomly derided into five groups, each containing 8～10rats. They were treated daily by lavage with corn oil, 25, 75,225 or 675 mg/kg BW DBP from GD6 to PND 21; Male offspring rats aged 1, 7 and 21 days were sacrificed for determinating theexpression of hippicampal genes, including c-fos,BDNF(all variants),Spinophilin,PSD95,Synaptophysin,CREST,BDNF variantâ…¢, and of hippicampal proteins (PND 21 only), such asBDNF, p-CREB, spinophilin and aromatase.Results: (1) Effects on hippocampal gene expression:â‘ Immediate early genes related withcell growth and differentiation and learing and memory: BDNF expression in the high-dose group(PND21) increased by about 30%, and the differences were significant as compared to control(p＜0.01), but the expression of c-fos gene was not significantly altered by the treatment,â‘¡Genesrelated with the structure and function of dendrite and synapse: spinophilin expression in the low-dose group (PND21) increased by 35.7% with significant differences as compaired to control(p＜0.05). However, the expression of PSD95 and synaptophysin genes was scarcely affected by thetreatment.â‘¢Genes related with CREB singal pathway: the expression of BDNF variantâ…¢(percaentage among all BDNF variants decreased in the high-dose group) and CREST was notaffected by DBP treatment. (2) Effects on hippocampal protein expression: the content of BDNFmature molecule, spinophilin and aromatase in the high-dose group (PND21) increasedsignificantly (p＜0.01, p＜0.05 and p＜0.01), however, the relative level of p-CREB in thehippocampus was not altered by the treatment.Conclusions: In utero and lactational exposure to high-dose DBP increased the expression ofBDNF, spinophilin and aromatase, indicating that DBP may promote the growth and differentiationof hippocampal nerve cells and increase dendrite spine abundance. The increase in BDNFexpression may be unrelated with the upper stream regulation of p-CREB. DBP treatment may notaffect the synaptic structure and function in the hippocampus of the male rats.Partâ…£Effects of DBP on learning and memory, serum biochemical indicators andhippocampal LTP in the adult male ratsObjective: To investigate the effects of DBP on learning and memory in adult male rats, onserum biochemical indicators and on hippocampal LTP.Methods: Male offsping rats, one/litter/group, were those remained in stuty Partâ…¢, whichcontinuted to receive the same DBP treatment from PND 21 (wearing) to PND 28. Spatial learningand memory capacity was determined twice by water maze at the age of one and two monthsrespectively. The animals in the high- and low-dose groups then continued to receive DBPtreatment till 3 months old, which were then detrained for serum biochemical indicators and forhippocampal LTP.Results: (1) Water maze tests: male rats exposed to high-dose DBP demonstrated betteracquisition of spatial information (p＜0.05) at the age of one month, and still exhibited improvedmemory (p＜0.05) after a one-month exposure intermission. (2) Serum biochemical indicators: ALBand TP were decreased by high-dose DBP treatment in the male offspring rats, but the activities ofAST and CHE were significantly increased by the same treatment (both p＜0.05). (3) LTP in hippocampal dentate gyrus: achievement ratio of LTP induction was more than 70%, andamplification of both PS wave and EPSP slope rate was about 100%. However, these indicatorswere not significantly affected by the high- or low-dose DBP exposure as compared to control (allp＞0.05)Conclusions: The male rats exposed to high-dose DBP still exhibited better learning andmemory capacity when grown tip, which may resulted from the alterations in the nervous systemdevelopment. Tiffs high-dose DBP also showed obvious hepatic toxicity to the adult F₁ generationmale rats. Exposure to this high-dose DBP scarcely affected the function of synaptic transmissionin the hippocampus dentate gyrus of the male rats.Collectively, under the present experimental conditions, the high-dose DBP exposure showedsignificant reproductive, developmental and hepatic toxicities. As for the early neurobehavioralreflexes, DBP treatment exhibited few influences on these indicators of the female pups, butaffected some reflexes of the males, such as surface righting and forepaw grip time. The toxichigh-dose DBP, however, promoted spatial learning and memory of the nlale pups, which may berelated with the improved expression of genes and protein accounting for neuro-development.While the low-dose DBP exhibited no overt toxicity of reproduction and development, it decreasedthe forepaw grip time and spatial learning and memory capacity of the male pups. In theexploration for mechanism, no appropriate explanation was derived for the effects caused by thelow-dose exposure; however, this level was lower than the reported NOAEL (50mg/kg/day) basedon morphological lesions, indicating neuro-behaviors, especially learning and memory are moresusceptive to DBP exposure and should be paid special consideration when been evaluated.