Reprint of: Recessive APC2 missense variants associated with epilepsies without neurodevelopmental disorders.

OBJECTIVES: The APC2 gene, encoding adenomatous polyposis coli protein-2, is involved in cytoskeletal regulation in neurons responding to endogenous extracellular signals and plays an important role in brain development. Previously, the APC2 variants have been reported to be associated with cortical dysplasia and intellectual disability. This study aims to explore the association between APC2 variants and epilepsy. METHODS: Whole-exome sequencing (WES) was performed in cases (trios) with epilepsies of unknown causes. The damaging effects of variants were predicted by protein modeling and in silico tools. Previously reported APC2 variants were reviewed to analyze the genotype-phenotype correlations. RESULTS: Four pairs of compound heterozygous missense variants were identified in four unrelated patients with epilepsy without brain malformation/intellectual disability. All variants presented no or low allele frequencies in the controls. The missense variants were predicted to be damaging by silico tools, and affect hydrogen bonding with surrounding amino acids or decreased protein stability. Patients with variants that resulted in significant changes in protein stability exhibited more severe and intractable epilepsy, whereas patients with variants that had minor effect on protein stability exhibited relatively mild phenotypes. The previously reported APC2 variants in patients with complex cortical dysplasia with other brain malformations-10 (CDCBM10; MIM: 618677) were all truncating variants; in contrast, the variants identified in epilepsy in this study were all missense variants, suggesting a potential genotype-phenotype correlation. SIGNIFICANCE: This study suggests that APC2 is potentially associated with epilepsy without brain malformation/intellectual disability. The genotype-phenotype correlation helps to understand the underlying mechanisms of phenotypic heterogeneity.

Recessive APC2 missense variants associated with epilepsies without neurodevelopmental disorders.

OBJECTIVES: The APC2 gene, encoding adenomatous polyposis coli protein-2, is involved in cytoskeletal regulation in neurons responding to endogenous extracellular signals and plays an important role in brain development. Previously, the APC2 variants have been reported to be associated with cortical dysplasia and intellectual disability. This study aims to explore the association between APC2 variants and epilepsy. METHODS: Whole-exome sequencing (WES) was performed in cases (trios) with epilepsies of unknown causes. The damaging effects of variants were predicted by protein modeling and in silico tools. Previously reported APC2 variants were reviewed to analyze the genotype-phenotype correlations. RESULTS: Four pairs of compound heterozygous missense variants were identified in four unrelated patients with epilepsy without brain malformation/intellectual disability. All variants presented no or low allele frequencies in the controls. The missense variants were predicted to be damaging by silico tools, and affect hydrogen bonding with surrounding amino acids or decreased protein stability. Patients with variants that resulted in significant changes in protein stability exhibited more severe and intractable epilepsy, whereas patients with variants that had minor effect on protein stability exhibited relatively mild phenotypes. The previously reported APC2 variants in patients with complex cortical dysplasia with other brain malformations-10 (CDCBM10; MIM: 618677) were all truncating variants; in contrast, the variants identified in epilepsy in this study were all missense variants, suggesting a potential genotype-phenotype correlation. SIGNIFICANCE: This study suggests that APC2 is potentially associated with epilepsy without brain malformation/intellectual disability. The genotype-phenotype correlation helps to understand the underlying mechanisms of phenotypic heterogeneity.

Effects of diurnal temperature range on mortality in Hefei city, China.

Although several studies indicated an association between diurnal temperature range (DTR) and mortality, the results about modifiers are inconsistent, and few studies were conducted in developing inland country. This study aims to evaluate the effects of DTR on cause-specific mortality and whether season, gender, or age might modify any association in Hefei city, China, during 2007-2016. Quasi-Poisson generalized linear regression models combined with a distributed lag non-linear model (DLNM) were applied to evaluate the relationships between DTR and non-accidental, cardiovascular, and respiratory mortality. We observed a J-shaped relationship between DTR and cause-specific mortality. With a DTR of 8.3 °C as the reference, the cumulative effects of extremely high DTR were significantly higher for all types of mortality than effects of lower or moderate DTR in full year. When stratified by season, extremely high DTR in spring had a greater impact on all cause-specific mortality than other three seasons. Male and the elderly (≥ 65 years) were consistently more susceptible to extremely high DTR effect than female and the youth (< 65 years) for non-accidental and cardiovascular mortality. To the contrary, female and the youth were more susceptible to extremely high DTR effect than male and the elderly for respiratory morality. The study suggests that extremely high DTR is a potential trigger for non-accidental mortality in Hefei city, China. Our findings also highlight the importance of protecting susceptible groups from extremely high DTR especially in the spring.

Distribution of polybrominated diphenyl ethers in breast milk, cord blood and placentas: a systematic review.

Polybrominated diphenyl ethers (PBDEs) have been extensively used as flame retardants in consumer products. PBDEs rapidly bioaccumulate in the environment, food, wild animals and humans. In this review, we investigated the harmful effects of PBDEs on humans, especially in early life, and summarised the levels of PBDEs in human biological samples (breast milk, cord blood and placentas). In addition, we described the spatiotemporal distribution of PBDEs in this review. PBDE levels in breast milk, cord blood and placentas were generally higher in North America than in other regions, such as Asia, Europe, Oceania and Africa. However, high levels of PBDEs in human biological samples were detected at e-waste recycling sites in South China, East China and South Korea. This finding suggests that newborns living in e-waste regions are exposed to high levels of PBDEs during prenatal and postnatal periods. The time trends of PBDE concentration differed according to the region. Few studies have investigated PBDE levels in humans from 1967 to 2000, but they increased rapidly after 2000. PBDE concentration peaked at approximately 2006 globally. Compared with other PBDE congeners, BDE-47, BDE-153 and BDE-209 were the major components, but the detection rate of BDE-209 was lower than those of others. Future studies should focus on determining the BDE-209 concentration, which requires the implementation of different analytical approaches. Additionally, the levels of PBDEs in human samples and the environment should be monitored, especially in e-waste recycling regions. Graphical abstract The figures described the spatial distribution of the lowest (Fig. a1) and highest concentration of ∑PBDE (Fig. a2) in different countries by 2006 and described the spatial distribution of the lowest (Fig. b1) and highest concentration of ∑PBDE (Fig. b2) in different countries from 2007 to 2015. All the figures indicated that the levels of PBDEs in North America were substantially higher than those in many regions of Europe, Asia, Oceania, or Africa. Comparing Fig. a1-b1 or Fig. a2-b2, increasing trends were observed in some countries, especially in some regions in China, Korea and Canada.

Two follicle-stimulating hormone receptor polymorphisms and polycystic ovary syndrome risk: a meta-analysis.

The aim of this study was to explore the association between follicle stimulating hormone receptor (FSHR) Thr307Ala and Asn680Ser polymorphisms and susceptibility to polycystic ovary syndrome (PCOS). A comprehensive literature search for relevant studies was conducted on Google Scholar, PubMed, the Chinese National Knowledge Infrastructure (CNKI) and the Chinese Biomedical Literature Database (CBM). This meta-analysis was performed using the STATA 11.0 software and the pooled odds ratio (OR) with 95% confidence interval (CI) was calculated. Ten case-control studies were included in this meta-analysis. However, meta-analysis results showed no association between both FSHR Thr307Ala polymorphism and Asn680Ser polymorphism and susceptibility to PCOS. Stratified analysis of ethnicities also showed no association. In conclusion, the present study suggested that the FSHR polymorphisms were not associated with an increased risk of PCOS and larger-scale studies of populations are needed to explore the roles played by FSHR polymorphisms during the pathogenesis of PCOS.

CTLA-4 polymorphisms and systemic lupus erythematosus (SLE): a meta-analysis.

The aim of this study was to summarize results on the association of cytotoxic T-lymphocyte antigen-4 (CTLA-4) promoter exon-1 +49 and 1722T/C polymorphism with systemic lupus erythematosus (SLE) susceptibility by using the meta-analysis. We searched all the publications about the association between CTLA-4) promoter exon-1 +49 and 1722T/C polymorphism and SLE from PubMed, Elsevier Science Direct, Chinese Biomedical Literature Database (CBM), Chinese National Knowledge Infrastructure (CNKI), and Wanfang (Chinese). Previous CTLA-4 association studies with SLE, however, have produced inconsistent results. We have performed a meta-analysis to better assess the purported associations. A total of 17 independent studies (to June 2012) testing association between one or more CTLA-4 polymorphisms and SLE were used in this analysis. We have compared allele and genotype frequencies at two polymorphic sites found in exon-1 (at +49) and the promoter region (at -1722). The data demonstrate that the exon-1 +49 polymorphism is associated with SLE susceptibility in Asian population. The overall risk, measured by odds ratio (OR), stratification by ethnicity indicates the exon-1 +49 GG+GA genotype is associated with SLE, at least in Asians (OR = 0.85, 95 % CI = 0.73-0.99, P = 0.04 for GG+GA vs. AA; OR = 0.85, 95 % CI = 0.72-1.00, P = 0.05 for AG vs. AA). Similar trends are found in allele-specific risk estimates and disease association. Overall, there was significant association between the 1722T/C polymorphism and overall SLE risks (OR = 0.78, 95 % CI = 0.63-0.97, P = 0.04 for GG+GA vs. AA, OR = 0.87, 95 % CI = 0.76-0.99, P = 0.04 for G vs. A) in Asian population.In summary, this meta-analysis demonstrates that the CTLA-4 promoter +49A/G and promoter -1722C/T polymorphism may confer susceptibility to SLE, especially in Asian-derived population.

No association between COMT Val158Met polymorphism and prostate cancer risk: a meta-analysis.

OBJECTIVE: Results from published studies on the association between the catechol-O-methyltransferase (COMT) Val158Met polymorphism and prostate cancer (PCa) risk are inconsistent. To derive a more precise estimate, we performed a meta-analysis. METHODS: We searched in the PubMed and Elsevier Science Direct database for studies on the association between the COMT Val158Met polymorphism and PCa. We used the odds ratio with 95% confidence interval for PCa risk, detected potential sources of heterogeneity with the Chi-square-based Q-test, performed sensitivity analysis of studies adapted to the Hardy-Weinberg equilibrium. RESULTS: We found seven case-control studies included 2292 patients and 2158 controls. Fix-effects meta-analysis failed to explore any significant association of PCa with the genetic model and the allelic model of COMT Val158Met. We also did not detect any association in the subgroup analysis by ethnicity in all genetic models. The gene-based analysis suggested that the genetic polymorphism in COMT is not associated with PCa. CONCLUSIONS: There is no association between the COMT Val158Met polymorphism and PCa.

Meta-analysis of MCP-1 promoter -2518 A/G polymorphism and SLE susceptibility.

The aim of this meta-analysis was to summarize results on the association of monocyte chemoattractant protein-1(MCP-1) promoter -2518 A/G polymorphism with systemic lupus erythematosus (SLE) susceptibility. We searches all the publications about the association between MCP-1 promoter -2518 A/G polymorphism and SLE from Pubmed, Elsevier Science Direct, Chinese Biomedical Literature Database, Chinese National Knowledge Infrastructure. The meta-analysis was performed for genotypes AA verse GG, AA+AG verse GG, AA verse AG+GG, and A allele verse G allele in a fixed/random effect model. A total of 14 studies (2,333 cases and 2,391 controls) were included in the meta-analysis. When all groups were pooled, we had not observed significant association between A allele and G allele (OR = 0.94, 95 %CI = 0.79-1.12, P = 0.50). When analysis were restricted to more ethnically homogeneous populations, the similar results were found in European population and Asian population (OR = 1.05, 95 %CI = 0.75-1.46, P = 0.80; OR = 1.00, 95 %CI = 0.86-1.17, P = 0.99). However, we had not detected a significant association between MCP-1 promoter -2518 A/G polymorphism and SLE when examining the genotypes AA verse GG, AA+AG verse GG, AA verse AG+GG. The meta-analysis did not demonstrate the association between MCP-1 promoter -2518 A/G polymorphism and SLE.

PDTC attenuate LPS-induced kidney injury in systemic lupus erythematosus-prone MRL/lpr mice.

Lipopolysaccharide (LPS) from bacteria can accelerate and exacerbate lupus nephritis (LN) and induce infiltrating inflammatory cells in kidney in animal models. Pyrrolidine dithiocarbamate (PDTC) is known to exert anti-inflammatory effects. Monocyte chemoattractant protein-1(MCP-1) is upregulated by various stimuli, including LPS, high glucose, and hyperosmolality. However, the molecular mechanisms of transcriptional regulation of the MCP-1 protein expression with LPS are poorly understood. Expression of MCP-1 was examined by western blot and enzyme-linked immunosorbent assay, respectively. The activity of nuclear factor (NF)-kappaB was measured by western blot. These mice have uncontrolled proliferation of T cells, an impaired response to T cell mitogen and produce autoantibodies against nuclear antigens, including DNA. We found that after LPS treatment for 14 weeks, LPS increased MCP-1 protein expression in kidney, which was significantly suppressed by antioxidant PDTC. The expression of NF-κB, pERK, pJNK and MCP-1 were increased, pp38 expression was decreased significantly, concomitantly with sera anti-dsDNA, MCP-1 and the acceleration of severity of autoimmune kidney injury. LPS induce markedly neutrophil infiltration in the glomerulus, especially around the mesangial region. PDTC reduced the number of infiltrating inflammatory cells and severity of kidney injury via inhibiting NF-κB and p38 MAPK activity. They also markedly prevented LPS-induced pJNK and MCP-1. Therefore, MCP-1 may be responsible for the recruitment and activation of leukocytes in diseased kidneys in female MRL/lpr mice. In this study, the long-term administration of PDTC had impacts on the prevention of end-stage organ damage induced by LPS treated. We demonstrated that PDTC inhibited LPS-induced monocyte migration and attenuated LPS-induced p38 MAPK activation. Based on these data we infer that PDTC inhibits LPS-induced MCP-1 expression, secretion and function through inhibition of NF-κB and p38 MAPK activity. Our study suggests that MAPK is an important therapeutic target of monocyte recruitment and accumulation within the glomerulus in inflammatory renal disease. These results suggest that PDTC protects against kidney inflammation of SLE at least in part via NF-κB and MAPK signaling pathways induction, and that inhibitory action on anti-dsDNA may be associated with the protective mechanism of PDTC. In summary, PDTC pretreatment attenuates LPS-induced kidney injury in female MRL/lpr mice through regulating NF-κB and MAPK signaling pathways. Our results indicate that LPS induces MCP-1 mainly through activating NF-κB and its downstream MAPK, and that such effect was inhibited by PDTC, suggesting the efficacy of PDTC in preventing kidney fibrosis in lupus-prone mice. Therefore, appropriate inhibition of NF-κB activation may attenuate the kidney injury in lupus-prone mice.

[Studying the lipid peroxidation index, morphology and apoptosis in testis of male BALB/c mice exposed to polybrominated diphenyl ether (BDE-209)].

OBJECTIVE: To explore the lipid peroxidation and the testicular morphological change induced by decabrominated diphenyl ether (BDE-209) in male BALB/c mice. METHODS: Twenty one male BALB/c mice were randomly divided into three groups: the high exposure group (500 mg/kg BDE-209), the low exposure group (200 mg/kg BDE-20) and control group (normal saline). The mice were exposed by gavage one time a day for 6 weeks, then were sacrificed. Body weight, testis weight, malonyldialdehyde (MDA), total superoxide dismutase (T-SOD) and glutathione (GSH) in testis were examined. The morphological alteration of testis was observed. TUNEL assay was used to detect the apoptosis in testicular cells. RESULTS: Body weight and testis weight in high and low exposure groups were (21.6140 +/- 2.3550) g, (20.8000 +/- 1.7630) g and (0.1859 +/- 0.0349) g, (0.1718 +/- 0.0266) g, respectively, which were significantly lower than those (27.7570 +/- 1.2880) g and (0.2302 +/- 0.0335) g in the control group (P < 0.05); the testis coefficient in high exposure group was (0.8640% +/- 0.1706%), which was significantly higher than that (0.8329 +/- 0.1386%) in the control group (P < 0.05). The GSH level and SOD activities of testis in 2 BDE-209 groups were 0.044 +/- 0.006, 0.039 +/- 0.005 nmol/mg prot, and 0.735 +/- 0.179, 0.907 +/- 0.198 U/mg prot, respectively, which were significantly lower than those (0.052 +/- 0.067) mol/mg and (1.161 +/- 0.188) U/mg in the control group (P < 0.05). The levels of MDA in 2 BDE-209 groups were (2.365 +/- 0.339) and (1.752 +/- 0.366) nmol/mg prot, which were significantly higher than that (1.173 +/- 0.232 nmol/mg prot) in control group (P < 0.05). there were significant differences of SOD and MDA levels between high exposure group and low exposure group (P < 0.05). Histological examination showed that the number of spermatogenic cells and layer were decreased significantly in 2 exposure groups as compared with control group. TUNEL assay showed that apoptosis cells appeared in 2 exposure groups. CONCLUSION: BDE-209 changed lipid peroxidation in male BALB/c mice testis and caused toxic effects on the testis.

[Effects of mitogen-activated protein kinases signaling pathway proteins on kidney injury in mice exposed subchronically to cadmium].

OBJECTIVE: To explore the effects of mitogen activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK) on kidney injury in female BALB/c mice exposed to cadmium. METHOD: Twenty-one female BALB/c mice were randomly divided into 3 groups, i.e. control group, low Cd exposure group (2.5 µmol/kg) and high Cd exposure group (10 µmol/kg) were exposed to normal saline, 2.5, 10 µmol/kg Cd, respectively, 3 times a week for 14 weeks. The kidney slice were stained by HE, PAS and Masson staining to observe the morphological changes. The expression levels of pERK, ERK, pp38, p38, pJNK and JNK proteins in kidneys were tested by Western blot assay. RESULTS: The ratios of pERK/ERK, pp38/p38, pJNK/JNK in high Cd group were higher than those in the control group (P < 0.05). The ratio of pERK/ERK in low Cd group was higher than control group (P < 0.05). The expression levels of bcl-2, bax proteins and the ratio of bcl-2 to bax in Cd exposure groups decreased significantly, as compared with the control group (P < 0.05). The impairment of renal glomeruli and tubules were observed in HE, PAS and Masson staining slices of kidneys in mice exposed to Cd. CONCLUSION: CdCl2 may induced renal injury by affecting the expression levels of MAPK.

[Effect of exposure to higher decabrominated diphenyl ether (PBDE-209) on learning and memory functions of BALB/c mice].

OBJECTIVE: To investigate the effects of exposure to decabrominated diphenyl ether (PBDE-209) on learning and memory of BALB/c mice. METHODS: Eighteen female BALB/c mice were randomized divided into 3 groups and gavaged with peanut oil in the control groups and 300, 1500 mg x kg(-1)xd(-1) PBDE-209 in peanut oil daily in two exposed groups respectively for 4 weeks. The learning and memory ability of mice were tested by the Morris water maze and the shuttling box respectively. The body weight and organs index were measured and the acetylcholinesterase and butyrylcholinesterase activity in brain were determined. The liver histopathological examination was performed. RESULTS: The heart index in high dose PBDE-209 group was higher than that of the low dose PBDE-209 group (P < 0.05). The results of Morris water maze showed that escape latency period was significantly shorter than the control group (F = 3.134, P < 0.05). The swimming time in the second quadrant of low dose PBDE-209 group was (15.78 +/- 10.92) s, significantly shorter compared with the swimming time in the second quadrant of the control group's [(28.80 +/- 8.67) s] (P < 0.05). There was no significant difference in the times of active avoidance in the shuttling between three groups (F = 3.423, P = 0.06). There were no significant differences in acetylcholinesterase and butyrylcholinesterase activity in brain of PBDE-209 groups compared with the control group (P > 0.05). Histologically liver damages in structure such as adipose degeneration and swelling were observed in PBDE groups. CONCLUSION: Exposure to PBDE-209 slightly impairs the space learning and memory ability of BALB/c mice, and it has some hepatotoxicity.

Simultaneous determination of phthalates and adipates in human serum using gas chromatography-mass spectrometry with solid-phase extraction.

A gas chromatography-mass spectrometry assay was developed and validated for the simultaneous determination of phthalates and adipates in human serum. The phthalates and adipates studied were dimethyl phthalate, diethyl phthalate, dibutyl phthalate, benzylbutyl phthalate, di-2-ethylhexyl phthalate, di-n-octyl phthalate, diethyl adipate, dibutyl adipate, diisobutyl adipate, bis(2-butoxyethyl) adipate and di-2-ethylhexyl adipate, with diisooctyl phthalate as internal standard. The extraction and cleaning up procedure was carried out with solid-phase extraction cartridges containing dimethyl butylamine groups, which showed extraction efficiencies over 88% for each analyte and the internal standard. The calibration curves obtained were linear with correlation coefficients greater than 0.98. For all analytes, the assay gave CV% values for intra-day precision from 4.9 to 13.3% and mean accuracy values from 91.4 to 108.4%, while inter-day precision was 5.2-13.4% and mean accuracy 91.0-110.2%. The limits of detection for the assay of phthalates and adipates were in the range 0.7-4.5 ng/mL. The method is simple, sensitive and accurate, and allows for simultaneous determination of nanogram levels of phthalates and adipates in human serum. It was successfully applied to an investigation on the level of phthalates and adipates in a non-occupationally exposed population.

[Oxidative stress of decabromodiphenylether in mice brain tissue].

OBJECTIVE: To study the oxidative stress induced by decabromodiphenylether (PBDE-209) in the cerebral cortex, hippocampus, cerebellum and striatum of mice. METHODS: Twenty-eight male BALB/c mice were randomized divided into four groups with seven mice in each: solvent control, blank control, low (200 mg/kg) and high (500 mg/kg) dose groups. Test substances were administered by gavage and mice were sacrificed 6 weeks after treatment. Malonyldialdehyde (MDA), total superoxide dismutase (T-SOD) and glutathione (GSH) in cerebral cortex, hippocampus, cerebellum and striatum were examined. RESULTS: The content of MDA in cerebral cortex, cerebellum, striatum and hippocampus in high dose group was (92.25 ± 36.64), (4.24 ± 1.15), (12.92 ± 4.30), (12.12 ± 6.39) nmol/mg pro respectively, higher than that in blank group [(56.713 ± 6.44), (2.42 ± 1.41), (4.05 ± 2.23), (4.91 ± 1.60) nmol/mg pro] and the difference was statistically significant (P < 0.05); T-SOD activity in cerebral cortex, cerebellum and striatum in low dose group was (182.48 ± 11.59), (6.67 ± 1.56), (35.48 ± 21.98) U/mg pro respectively, lower than that in blank group [(277.76 ± 106.70), (18.02 ± 16.40), (63.57 ± 20.83) U/mg pro] and the difference was statistically significant (P < 0.05); in high dose group the T-SOD activity in hippocampus was(59.26 ± 37.09) U/mg pro, lower than that in blank group [(93.28 ± 21.75) U/mg pro] and the difference was statistically significant (P < 0.05); The content of GSH in cerebral cortex, cerebellum and striatum in high dose group was (40.98 ± 13.19), (3.55 ± 1.55), (24.46 ± 11.30) mg/g pro respectively, lower than that in blank group [(75.79 ± 26.51), (8.01 ± 3.23), (44.52 ± 13.15) mg/g pro and the difference was statistically significant (P < 0.05); while the content of GSH in hippocampus was not decreased significantly compared with the blank group (P > 0.05). CONCLUSION: PBDE-209 could induce oxidative stress in nervous tissue. The tissue oxidative damage might be one of the primary mechanisms of neurotoxicity of PBDE-209.

[Studies on fluoride concentration and cholinesterase activity in rat hippocampus].

OBJECTIVE: To study the accumulation of fluoride in rat hippocampus and its effect on cholinesterase activity. METHODS: Rats were subchronically exposed to NaF, and fluoride concentration and cholinesterase activity in rat hippocampus were determined. RESULTS: Fluoride concentration in rat hippocampus was significantly correlated with the dosage of fluoride, and there were significant differences among high dosage group [(13.03 +/- 1.79) micro g/g], low dosage group [(9.83 +/- 0.92) micro g/g] and control [(8.27 +/- 1.11) micro g/g], P < 0.01. Acetylcholinesterase activities among three groups [(0.111 +/- 0.031) micro mol/mg, (0.143 +/- 0.025) micro mol/mg, (0.183 +/- 0.027) micro mol/mg] were also significantly different (P < 0.01), which was negatively correlated with fluoride concentration in rat hippocampus (r = -0.700, P < 0.01). The activity of butylcholinesterase in high dosage group [(0.041 +/- 0.010) micro mol/mg] was different from that of control [(0.067 +/- 0.025) micro mol/mg, P < 0.05], but the activity was not significantly related with fluoride concentration in rat hippocampus (r = -0.317, P = 0.094). CONCLUSION: Fluoride may go through the blood-brain barrier and accumulate in rat hippocampus, and inhibit the activity of cholinesterase.