Toxic Effect Of High Dose Phenylalanine On Mice Brain

Objective:Hyperphenylalaninemia（HPA）is an autosomal recessive metabolic disease.Due to the lack of phenylalanine hydroxylase activity in the liver,phenylalanine（Phe）and its by-pass metabolites are accumulated in large amounts in the blood and tissues,thereby causing direct or indirect damage to brain function.Phe levels that are too high during brain development can cause irreversible brain damage.If the newborn has not been diagnosed early or has not been treated early after the illness,it will seriously affect the development of the infant’s intelligence and appear psychiatric symptoms such as irritability,anxiety,and restlessness.Under normal circumstances,limiting Phe intake for a long period of time is difficult for children,and limiting dietary Phe intake is the best way to treat this disease.Therefore,studying the toxicological mechanism of Phe and finding ways to reduce the toxic effects of Phe are important for preventing and treating this disease.In this study,mice were intragastrically administered with phenylalanine to observe their effects on various indicators of brain tissue in mice,and the damage mechanism of mice brain tissue under high phenylalanine status was explored.Methods:Forty C57 male mice were randomly divided into four groups of 10 mice each.The four groups were high-dose phenylalanine group（4.20 mmol/kg）,middle-dose phenylalanine group（2.10 mmol/kg）,low-dose phenylalanine group（1.05 mmol/kg）,and control group（distilled water）.After phenylalanine was intragastrically administered for28 days,phenylalanine and tyrosine contents in serum and brain tissues of mice were measured,and oxidative damage indexes such as ROS,8-OHdG,and MDA,SOD,CAT,GSH-Px,GSH and other antioxidant capacity indicators,DA,5-HT,NE,Ach（whole blood）and other neurotransmitters,AchE,DBH,MAO and other neurotransmitter-related enzymes,NAA,NSE,SM and other neuronal cell damage markers,MBP,NFP,GM1markers of myelin sheath damage,PI,PC,PE and other cell membrane phospholipid components,Na⁺-K⁺-ATP enzyme,Ca²⁺-Mg²⁺-ATPase and other cell membrane ATP enzyme were detected in brain tissue.Experimental data is expressed as±s.Statistical analysis of the experimental results was performed using one-way analysis of variance（ANOVA）in SPSS 22.0 software.The LSD method was used to compare each group and the significance test levelα=0.05.Results:1.General condition of mice and change of body weight and brain coefficientDuring the gavage,mice in each group were normal and did not die.There was no significant difference in the body weight between the mice in the phenylalanine group and the control group（P>0.05）.The brain coefficient of high-dose phenylalanine group increased compared with the control group（P<0.05）.2.The contents of Phe,Tyr and Phe/Tyr in serum and brain tissueThere was no significant difference in serum Phe content between the groups（P>0.05）.The Tyr content in the low-dose phenylalanine group was higher than that in the high-and middle-dose phenylalanine group and the control group（P<0.05）.The Phe/Tyr values in the high,medium,and low doses of phenylalanine were lower than those in the control group（P<0.05）.The Phe/Tyr values in the high and middle doses of phenylalanine were higher than those in the low-dose phenylalanine group（P<0.05）.There was no significant difference in Phe content in mouse brain tissue between groups（P>0.05）.The Tyr content in the high-dose phenylalanine group was lower than that in the low-dose phenylalanine group and the control group（P<0.05）.There was no significant difference in Phe/Tyr between groups（P>0.05）.3.Brain tissue oxidative damage measurement resultsThe ROS content in phenylalanine group decreased compared with the control group.The ROS content in the high-dose phenylalanine group was lower than that in the medium-dose phenylalanine group.The ROS content in the middle-dose phenylalanine group was lower than that in the low-dose phenylalanine group（P<0.05）.8-OHdG content in the phenylalanine group was significantly lower than that in the control group,the high-dose phenylalanine group was higher than the middle-dose phenylalanine group,and the medium-dose phenylalanine group was higher than the low-dose phenylalanine group（P<0.05）.The MDA content in the high-and middle-dose phenylalanine group was lower than the low-dose phenylalanine group and the control group（P<0.05）.The activity of SOD was significantly lower in the high-dose phenylalanine group than in the other groups（P<0.05）.The CAT activity in the phenylalanine group decreased compared with the control group,and the CAT activity in the high-dose phenylalanine group was significantly lower than the low-dose phenylalanine group（P<0.05）.GSH-Px activity in phenylalanine group decreased compared with control group.GSH-Px activity in high-dose phenylalanine group was lower than that in low-dose phenylalanine group（P<0.05）.The levels of The GSH content in the phenylalanine group were lower than those in the control group.The GSH content in the high-dose phenylalanine group was lower than that in the medium-dose phenylalanine group,and the medium-dose phenylalanine was lower than that in the low-dose phenylalanine group（P<0.05）.4.Determination of neurotransmitters and neurotransmitter related enzymesThe DA content in the high and middle dose phenylalanine group was lower than that in the control group,and the DA content in the high-dose phenylalanine group was lower than the low-dose phenylalanine group and the control group（P<0.05）.The content of 5-HT in the high and medium dose phenylalanine group was lower than that in the low-dose phenylalanine group and the control group.The 5-HT content in the low-dose phenylalanine group was higher than that in the control group（P<0.05）.The content of NE in the high-dose phenylalanine group was significantly lower than other groups.The NE content of the middle-dose phenylalanine group was lower than that of the low-dose phenylalanine group and the control group（P<0.05）.The Ach content in the high-dose phenylalanine group was significantly lower than other groups.The Ach content in the middle-dose phenylalanine group was lower than that in the low-dose phenylalanine group and the control group（P<0.05）.The activity of AchE in high-dose phenylalanine group was lower than that in other groups.The activity of AchE in middle-dose phenylalanine group was lower than that in low-dose phenylalanine group and control group（P<0.05）.The activity of DBH in high-dose phenylalanine group was lower than that of low-dose phenylalanine group and control group（P<0.05）.MAO activity in high-and medium-dose phenylalanine group was higher than that in low-dose phenylalanine group and control group（P<0.05）.5.Measurement results of markers of neuronal cells and myelin damageThe NAA content in the high-dose phenylalanine group was lower than that in the low-dose phenylalanine group and the control group,and the NAA content in the middle-dose phenylalanine group was lower than that in the control group（P<0.05）.The content of NSE in each dose phenylalanine group was higher than that in control group.The content of NSE in the low-dose phenylalanine group was higher than that in the control group.The NSE content in the middle-dose phenylalanine group was higher than that in the low-dose phenylalanine group,the high-dose phenylalanine group was higher than the medium-dose phenylalanine group,and the medium-dose phenylalanine group was higher than the low-dose phenylalanine group（P<0.05）.The SM content of each phenylalanine groups were lower than that of the control group（P<0.05）.There was no significant difference in SM content between the phenylalanine groups（P>0.05）.The MBP content in the high-dose phenylalanine group was lower than that in the middle and low-dose phenylalanine group and the control group.The MBP content in the middle-dose phenylalanine group was lower than the low-dose phenylalanine group.The MBP content of the low-dose group was higher than that of the control group（P<0.05）.The NFP content in the high-dose phenylalanine group was lower than other groups.The NFP content in the medium-dose phenylalanine group was lower than the low-dose phenylalanine group and the control group（P<0.05）.The content of GM1 in the high and medium-dose phenylalanine group was higher than that in the low-dose phenylalanine group and the control group（P<0.05）.6.Determination of phospholipid components and ATPase activity in nerve cell membranesThe content of PI in high-dose phenylalanine group was lower than the middle and low-dose phenylalanine group and the control group（P<0.05）,and the low-dose phenylalanine group was lower than the control group（P<0.05）.There was no significant difference of the PC content among the groups（P>0.05）.The PE content of the phenylalanine group was lower than that of the control group（P<0.05）.There was no significant difference between the each dose phenylalanine groups（P>0.05）.The activity of Na⁺-K⁺-ATPase in the high and middle dose phenylalanine group was lower than that in the low dose phenylalanine group and the control group（P<0.05）.The activity of Ca²⁺-Mg²⁺-ATPase in the each phenylalanine groups was lower than that in the control group,the activity of Ca²⁺-Mg²⁺-ATPase in the high and medium dose phenylalanine group was lower than that in the low dose phenylalanine group.The Ca²⁺-Mg²⁺-ATPase activity in the high dose phenylalanine group was higher than that in the medium dose phenylalanine group（P<0.05）.Conclusion:After high-phenylalanine gavages in mice,high phenylalanine can inhibit the oxidative metabolism of brain tissue.Reduce the synthesis of neurotransmitters DA,5-HT,NE,Ach.Reduce the neurotransmitter-related enzymes AchE,DBH Activity,increased MAO enzyme activity.Decreased neuronal cell injury markers NAA,SM content,increased NSE content.Decreased myelin damage markers MBP,NFP,GM1 content.Reduced neuronal membrane phospholipids components PC,PI content,the content of PE had no effect.Decreased the activity of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in ATPase of cell membrane.