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OBJECTIVE: To study the effect of brain derived neurotrophic factor( BDNF)-tyrosine kinase receptor B( Trk B)pathway on the learning and memory impairment in rats induced by benzo[a]pyrene( B[a]P). METHODS: Seventy-two specific pathogen free healthy male SD rats were randomly divided into 3 groups with 24 rats in each group: the control group,the solvent group and the B[a]P group. The control group received no treatment. The solvent group was given intraperitoneal injection of olive oil( 1. 00 mg / kg body weight),and the B[a]P group was given intraperitoneal injection of B[a]P( 2. 50 mg / kg body weight,dissolved in olive oil) every other day. The rats were given corresponding treatment for30,60 and 90 days. The learning and memory ability of rats was evaluated using Morris water maze test. Western-blot analysis was used to detect the relative expression of BDNF and Trk B protein in hippocampus of rats. RESULTS: The escape latency of rats in the B[a]P group was longer than those in the control group and the solvent group( P < 0. 01). The duration of first passing through the platform in 3 time points in rats of B[a]P group was longer than those at the same time point in the control group and the solvent group( P < 0. 01). The target quadrant residence time and the times of passing through platform in rats of the B[a]P group were less than those in the control group and the solvent group( P < 0. 01). The duration of first passing through the platform in rats of B[a]P group increased with the increasing time of B[a]P exposure,showing a time-effect relationship( P < 0. 05). Compared with the control group and the solvent group,the relative expression of BDNF protein in hippocampus of rats in the B[a]P group was lower than those at the same time points( P < 0. 01). The relative expression of BDNF protein at time points of 60 and 90 days was lower than those at time point of 30 days in the same group( P <0. 01). The relative expression of Trk B protein in hippocampus of rats of the B[a]P group was lower than those in the control group and the solvent group( P < 0. 01). CONCLUSION: The impairment of learning and memory in rats caused by B[a]P has a time-effect relationship,which might be related to the decreased expression of BDNF and Trk B protein.
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<p><b>OBJECTIVE</b>To compare the difference of polycyclic aromatic hydrocarbons (PAHs) levels in the urban air and the scores of Neonatal Behavioral Neurological Assessment (NBNA) between Taiyuan and Changzhi cities and to explore the effects of PAHs in the urban air during pregnancy on neonatal behavioral neurological development.</p><p><b>METHODS</b>High-performance liquid chromatography (HPLC) with subsequent fluorescence detection was used to determine the PAHs levels in the cooperational hospitals in Changzhi and Taiyuan cities and the urinary 1-hydroxypyrene levels of the 297 pregnant women living Changzhi and Taiyuan cities during Nov. 2009 to May 2010. NBNA was used to determine the development of neonatal neural behavior. The differences of PAHs levels in the urban air, the pregnant women urinary 1-hydroxypyrene levels and NBNA scores between Taiyuan and Changzhi were compared.</p><p><b>RESULTS</b>There are significant differences of levels of pyrene, benz [a] anthracene, Chrysene, benz [a] pyrene, dibenz [a, h] anthracene in the urban air between Taiyuan and Changzhi (P < 0.10). The median of urinary 1-hydroxypyrene levels in pregnant women of Taiyuan was 1.140 microg/mmolCr, (P25 was 0.457 microg/mmolCr, P75 was 2.678 microg/mmolCr), the median of urinary 1-hydroxypyrene levels in pregnant women of Changzhi was 0.761 microg/mmolCr, (P25 was 0.133 microg/mmolCr, P75 was 2.095 microg/mmolCr). There are significant differences of urinary 1-hydroxypyrene levels in pregnant women between Taiyuan and Changzhi (t = -3.140, P = 0.002). There are significant differences of the NBNA scores, capacity scores, passive muscle tension scores, active muscle tension scores and general assessment scores between Taiyuan and Changzhi (P < 0.10). There was correlation between NBNA scores and urinary 1-hydroxypyrene level in pregnant women.</p><p><b>CONCLUSION</b>The PAHs in the urban air during pregnancy may adversely affect the neonatal neurobehavioral development.</p>
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Feminino , Humanos , Recém-Nascido , Gravidez , Poluentes Atmosféricos , Urina , Aleitamento Materno , Desenvolvimento Infantil , China , Cidades , Exposição Materna , Hidrocarbonetos Policíclicos Aromáticos , UrinaRESUMO
<p><b>OBJECTIVE</b>To observe the effects of Benzo(a)pyrene (BaP) on apoptosis of neuronal cells and expression of Bcl-2 and Bax proteins and to explore the mechanism of neurotoxicity induced by BaP in rats.</p><p><b>METHODS</b>A total of 32 SD rats were divided randomly into 4 groups, i.e. 3 BaP (126.2, 63.1 and 31.5 µg/kg) groups and a solvent control (50 µg/kg olive oil) group. All rats were exposed to BaP or olive oil by lateral cerebral ventricle micro-injection 1 time a week for 3 weeks. The apoptosis of neuronal cells was detected with TdT-mediated dUTP-biotin nicked labeling (TUNEL) assay and the expression levels of Bcl-2 and Bax were measured with SABC immunohistochemistry in the cerebral cortex and hippocampus tissues of rats.</p><p><b>RESULTS</b>The results of TUNEL assay showed that apoptosis bodies on the surface of the neurons in the cerebral cortex and hippocampus were clearly observed and the number of apoptosis bodies increased with BaP. Apoptosis indexes (AIs) of the rat cerebral cortex and hippocampus in high exposure group were significantly higher than those in control group (P < 0.05 or P < 0.01). The analysis of immunohistochemistry showed that the Bcl-2 expression levels significantly decreased, the Bax expression levels obviously increased and the ratio of Bcl-2 to Bax decreased in the rat cerebral cortex and hippocampus of medium and high exposure groups, as compared with control group (P < 0.05 or P < 0.01). In the rat cerebral cortex and hippocampus, there were the negative correlation (r = -0.927, P < 0.01; r = -0.934, P < 0.01) between AI and Bcl-2, the positive correlation (r = 0.858, P < 0.01; r = 0.847, P < 0.01) between AI and Bax and the negative correlation (r = -0.939, P < 0.01; r = -0.942, P < 0.01) between AI and Bcl-2/Bax.</p><p><b>CONCLUSION</b>BaP could induce the apoptosis of neuronal cells in the rat cerebral cortex and hippocampus. Bcl-2 and Bax protein expression may play an important role in the apoptosis of neuronal cells induced by BaP.</p>
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Animais , Masculino , Ratos , Apoptose , Benzo(a)pireno , Toxicidade , Encéfalo , Metabolismo , Patologia , Neurônios , Metabolismo , Patologia , Proteínas Proto-Oncogênicas c-bcl-2 , Metabolismo , Ratos Sprague-Dawley , Proteína X Associada a bcl-2 , MetabolismoRESUMO
<p><b>OBJECTIVE</b>To study the effects of prenatal exposure to benzo[a]pyrene (B[a]P) on the physical development, early behavioral development, the adaptability to new environment and the learning and memory ability of rat offspring.</p><p><b>METHODS</b>Pregnant rats were randomly divided into five groups: control group, olive oil group, 3 exposure groups (25, 50 and 100 mg/kg B [a]P). The rats were exposed to B [a]P) by intraperitoneal injection on the 17th-19th days during gestation. The offspring were weighed on postnatal days (PND)1, PND 4, PND 7 and PND 28, the indices of physical development, reflective ability and sensory function were detected for offspring, the Morris water maze and Open-field tests were used to measure the ability of learning and memory and the adaptability to new environment of offspring.</p><p><b>RESULTS</b>The time of ear opening in middle and high-dose groups [(4.1 +/- 0.4),(5.0 +/-0.4) d] was posterior to that in untreated and solvent groups [(3.3 +/- 0.5), (3.4 +/- 0.6) d ](P < 0.01). The attainment rate (6.5%) of the surface righting reflex test in high-dose group on the 4th day was significantly lower than that (36.1%) in untreated group, the attainment rate (50.0%) in high-dose group on PND7 was significantly lower than those (81.3% and 79.3%) in untreated group and solvent group (P < 0.05). Compared to the untreated group, the time of forelimb hanging test in all exposure groups on PND12 and PND14 significantly decreased; compared to the solvent group the time of forelimb hanging test decreased in high-dose group on the 14th day significantly decreased (P < 0.01). The attainment rate (61.9%) of olfactory discrimination in high-dose group on PND12 was significantly lower than that (94.3%) in untreated group (P < 0.05). The results of Morris water maze test showed that the escape latency of different dose groups significantly increased, and the time of spatial probe and the times of traversing flat in high-dose group decreased significantly, as compared to the untreated and solvent groups (P < 0.01). The results of open-field test indicated that the center retention time in middle and high-dose groups significantly prolonged, the times of crossing lattice obviously reduced, and the rearing times decreased in high-dose group, as compared to untreated (P < 0.05).Compared to the solvent group, the times of crossing lattice in all exposure groups reduced significantly (P < 0.01 or P < 0.05).</p><p><b>CONCLUSION</b>The prenatal exposure to B[a]P could inhibit the physical development and early behavioral development, and influence the adaptability to new environment and learning and memory ability for offspring.</p>
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Animais , Feminino , Gravidez , Ratos , Benzo(a)pireno , Toxicidade , Aprendizagem , Aprendizagem em Labirinto , Memória , Atividade Motora , Síndromes Neurotóxicas , Efeitos Tardios da Exposição Pré-Natal , Ratos Sprague-DawleyRESUMO
<p><b>OBJECTIVE</b>To investigate the changes of mitochondria membrane potential and cytoplasma cytochrome C as the mechanism of neuron apoptosis induced by B(a)P.</p><p><b>METHODS</b>Primary neurons were dissociated from cerebral cortex of 1 - 3 days old SD rats and cultured with DMEM incubator at 37 degrees C. After 5 days' cultivation, the neurons were added S9 and B(a)P, and the concentrations of treated B(a)P were 0, 10, 20 and 40 micromol/L respectively. After administering of B(a)P, the neurons were cultivated for 40 hours. Apoptosis rate was measured by flow cytometry using Annexin V-FITC and propidium iodide (PI) staining, and the changes in mitochondrial potential (DeltaPsim) were tested with Rhodamine fluorescence (R2123) technique. Preparation of cytosolic extracts by centrifugation. Western blotting analysis was used to evaluate the level of cytochrome C of cytoplasm.</p><p><b>RESULTS</b>The apoptotic rate of neuron increased in both the middle dose group and the high dose group compared with controls, and had a dose-response tendency with the concentration of B(a)P. Moreover mitochondrial potential decreased in a dose dependent manner. There was a negative correlation between DeltaPsim and the apoptotic rate of neurons (r = -0.763, P < 0.05); Western blotting analysis showed cytoplasmic cytochrome C level increased significantly, which was positively related with neuron apoptosis (r = 0.831, P < 0.01).</p><p><b>CONCLUSION</b>Loss of mitochondria membrane potential and increase of cytoplasma cytochrome C may be the main cause of neuron apoptosis induced by B(a)P.</p>
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Animais , Ratos , Apoptose , Benzo(a)pireno , Toxicidade , Células Cultivadas , Citocromos c , Metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias , Metabolismo , Neurônios , Biologia Celular , Ratos Sprague-DawleyRESUMO
<p><b>OBJECTIVE</b>To observe the effect of nano-alumina on nerve cell viability through different detection kits of cell viability, using micro-alumina and nano-carbon as controls.</p><p><b>METHODS</b>Primary culturing nerve cells of mouse in vitro, which were exposed to 7 doses of 0 µmol/L, 62.5 µmol/L, 125.0 µmol/L, 250.0 µmol/L, 500.0 µmol/L, 1.0 mmol/L, 2.0 mmol/L concentrations of nano-alumina (nano-Al), micro alumina (micro-Al) and nano-carbon (nano-C), detecting cell viability (A(570) values) with CCK-8, MTT and LDH methods.</p><p><b>RESULTS</b>(1) The results of CCK-8 kit showed that, in doses of 250.0 µmol/L - 2.0 mmol/L, the cell viability values of nano-alumina (the values of A(570) were 0.878 ± 0.009, 0.823 ± 0.016, 0.647 ± 0.008, 0.594 ± 0.013, respectively) were significantly lower than that of micro-Al (the values of A(570) were 0.960 ± 0.008, 0.951 ± 0.036, 0.833 ± 0.008, 0.708 ± 0.012, respectively) and nano-C (the values of A(570) were 0.977 ± 0.003, 0.973 ± 0.002, 0.924 ± 0.006, 0.891 ± 0.023, respectively). While, comparing nano-Al with the same dose of micro-Al, there was significant difference (the t values were -0.082, -0.128, -0.186, -0.114, respectively, P < 0.01), and so as to the comparison of nano-Al with the same dose of nano-C (the t values were -0.099, -0.150, -0.277, -0.297, respectively, P < 0.01). (2) MTT results showed that in the doses of 500.0 µmol/L and 1.0 mmol/L, the cell viability of nano-Al (the values of A(570) were 0.648 ± 0.095 and 0.575 ± 0.061) were lower than that of micro-Al (the values of A(570) were 0.830 ± 0.044 and 0.816 ± 0.014) and nano-C (the values of A(570) were 0.889 ± 0.009 and 0.765 ± 0.049), and the differences were significant (nano-Al compared with the same dose of micro-Al, the t values were -0.183 and -0.242, P < 0.01; nano-Al compared with the same dose of nano-C, the t values were -0.241 and -0.190, P < 0.01). (3) LDH results showed that in the dose from 125.0 µmol/L to 2.0 mmol/L, the LDH release of nano-Al group (the values of A(570) were 1.862 ± 0.102, 1.905 ± 0.066, 1.930 ± 0.037, 1.946 ± 0.033, 1.967 ± 0.068, respectively) were higher than that of nano-C (the values of A(570) were 1.484 ± 0.110, 1.559 ± 0.039, 1.663 ± 0.014, 1.732 ± 0.076, 1.765 ± 0.073, respectively), and the differences were significant (the t values were -0.377, 0.346, 0.266, 0.213, 0.202, respectively, P < 0.01). In the dose from 125.0 µmol/L to 1.0 mmol/L, the LDH release of nano-Al group were higher than that of micro-Al (the values of A(570) were 1.578 ± 0.011, 1.639 ± 0.025, 1.727 ± 0.024, 1.808 ± 0.020, respectively), and the differences were significant (the t values were 0.284, 0.266, 0.202, 0.172, respectively, P < 0.01).</p><p><b>CONCLUSION</b>The toxicity of nano-Al is greater than nano-C and micro-Al on the viability of nerve cells; LDH is more suitable for detecting changes of cell viability after the effect of nano-materials than CCK-8 and MTT.</p>
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Animais , Camundongos , Óxido de Alumínio , Toxicidade , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Nanopartículas Metálicas , Toxicidade , Camundongos Endogâmicos , Neurônios , Cultura Primária de CélulasRESUMO
<p><b>OBJECTIVE</b>To study the associations of CYP1A1 gene polymorphisms with levels of urinary 1-hydroxypyrene among coke oven workers.</p><p><b>METHODS</b>223 male workers from a coke plant (76, 82 and 65 workers in oven top group, oven-side group and oven-bottom group respectively) and 119 controls without occupational polycyclic aromatic hydrocarbons exposure were selected. The MspI gene polymorphism in CYP1A1 3' flanking region and the genotypes at I462V site in exon 7 of CYP1A1 were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele specific amplification (ASA).</p><p><b>RESULTS</b>The urinary 1-hydroxypyrene of coke oven workers in oven-top, oven-side and oven-bottom (3.77+/-0.64, 3.57+/-0.49, 3.26+/-0.80 micromol/mol Cr) were significantly higher than controls (2.80+/-1.02 micromol/mol Cr) (P<0.01). The urinary 1-hydroxypyrene was not significantly different among MspI genotypes in CYP1A1 3' flanking region (P>0.05). In oven-top group and oven-side group, the subjects with Val/Val genotype in exon 7 of CYP1A1 had significantly higher urinary 1-hydroxypyrene levels than those with Ile/Val or Ile/Ile genotype, and urinary 1-hydroxypyrene of Ile-Val genotype were also significantly higher than Ile/Ile genotype (P<0.01). Multivariate logistic regression analysis showed that the coke oven workers (OR in oven top group, oven-side group and oven-bottom group was 24.926, 4.226 and 6.729 respectively) and subjects with m2/m2 genotype in CYP1A1 3' flanking region (OR=4.031) or with Val/Val or Ile/Val genotype in exon 7 of CYP1A1 (OR were 5.524 and 3.811) had elevated urinary 1-hydroxypyrene (greater than 95 percentile of control group, 3.876 micromol/mol Cr).</p><p><b>CONCLUSION</b>BAP concentration of work environment contributes to the elevated urinary 1-hydroxypyrene levels, and the exposed BAP levels were regulated by the CYP1A1 MspI and I462V genotypes. Genetic polymorphism of CYP1A1 gene could be a susceptible biomarker in coke oven workers which was involved in the individual susceptibility on metabolism of PAHs.</p>
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Adulto , Humanos , Masculino , Pessoa de Meia-Idade , Benzo(a)pireno , Coque , Citocromo P-450 CYP1A1 , Genética , Exposição Ocupacional , Polimorfismo Genético , Pirenos , Farmacocinética , Urina , QuímicaRESUMO
<p><b>OBJECTIVE</b>To study the effects of benzo[a]pyrene (B[a]P) on capability of learning and memory and the content of amino acid neurotransmitters in hippocampus of rats.</p><p><b>METHODS</b>Thirty-two healthy, male SD rats were randomly divided into 4 groups according to their weights after intubated into ventricles: the solvent control group, 2.5, 5.0 and 10.0 mmol/L groups. 10 microl of B[a]P olive oil solutions, of different concentrations 2.5, 5.0 and 10.0 mmol/L, were injected into rats' lateral ventricles, respectively. Rats in the solvent control group were injected into the same volume of olive oil as that in B[a]P group. Rats' capability of learning and memory was tested by Morris water maze. The content of amino acid neurotransmitters in rats' hippocampus were determined by high performance liquid chromatogram with a fluorescence detector.</p><p><b>RESULTS</b>Compared with the controls, the performances of learning and memory of rats decreased significantly in B[a]P treated groups (P<0.01). Levels of glutamate (Glu) were lower significantly in treated groups than that in controls (P<0.01). No significant differences were found in contents of aspartic acid (Asp), glycine (Gly) and aminobutyric acid (GABA) among the four groups.</p><p><b>CONCLUSION</b>B[a]P can damage rats' spatial learning and memory, and which could be related to decreased contents of excitatory amino acids in hippocampus.</p>
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Animais , Masculino , Ratos , Aminoácidos , Metabolismo , Benzo(a)pireno , Toxicidade , Hipocampo , Metabolismo , Aprendizagem em Labirinto , Memória , Neurotransmissores , Metabolismo , Ratos Sprague-DawleyRESUMO
<p><b>OBJECTIVE</b>To study the relationship between polymorphism of aryl hydrocarbon receptor (AhR) gene in G1661A and the level of urinary 1-hydroxypyrene among coke oven workers.</p><p><b>METHODS</b>295 male subjects were studied, including 214 workers working in coke oven plant and 81 controls working in raw material plant who were not generally exposed to polycyclic aromatic hydrocarbons occupationally. General in-formation of subjects were collected in a specific questionnaire including age, smoking and drinking habits, the history of occupation and so on. The AhR genotypes were detected by allele specific amplification (ASA), and the levels of urinary 1-hydroxypyrene were measured by high performance liquid chromatography with a fluorescence detector.</p><p><b>RESULTS</b>The frequencies of G/G, G/A and A/A genotype were 52.8% (113/214), 27.6% (59/214) and 19.6% (42/214) in exposed group and 67.9% (55/81), 19.8% (16/81) and 12.3% (10/81) in control group, respectively. No significant difference was found in three genotypes between the exposed and control group. Allele frequencies of G and A were 66.6% (285/428) and 33.4% (143/428) in exposed group and 77.8% (126/162) and 22.2% (36/162) in control group, and no statistical differences were found in allele frequency between exposed and control group. After the length of service and external exposure orders in general linear model were adjusted, results of covariance analysis showed that logarithmic transformed urinary 1-hydroxypyrene levels were (3.62 +/- 0.12), (3.43 +/- 0.12) and (3.44 +/- 0.08) micromol/mol Cr in individuals with A/A, G/A and G/G, respectively. The logarithmic transformed urinary 1-hydroxypyrene levels were (3.24 +/- 0.09) and (3.43 +/- 0.10) micromol/mol Cr in individuals with allele of G and A. No statistical differences were found in level of 1-hydroxypyrene among A/A, G/A and G/G genotype individuals, and between allele G and allele A after external exposure orders and length of service were adjusted.</p><p><b>CONCLUSION</b>The polymorphism of aryl hydrocarbon receptor G1661A has no significant impact on levels of urinary 1-hydroxypyrene.</p>
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Adulto , Humanos , Masculino , Pessoa de Meia-Idade , Coque , Genótipo , Polimorfismo Genético , Pirenos , Farmacocinética , Receptores de Hidrocarboneto Arílico , Genética , Urina , QuímicaRESUMO
<p><b>OBJECTIVE</b>To explore the effect of polycyclic aromatic hydrocarbons on the neurobehavioral function of coke oven workers.</p><p><b>METHODS</b>200 healthy adult male coke oven workers were selected from a coke plant of a state-owned steel enterprise in Taiyuan City. 88 controls occupationally unexposed to polycyclic aromatic hydrocarbons (PAHs) were selected from the same enterprise. All the subjects participated in this investigation voluntarily in their consent. Concentration of B(a)P in the working environment was monitored by High Performance Liquid Chromatography (HPLC). Urine samples were sampled immediately after working shifts. The level of urinary 1-hydroxypyrene was determined by HPLC. General information of workers correlated with the investigation was collected in a questionnaire according to the same criteria by well-trained investigators. Neurobehavioral core test battery (NCTB) recommended WHO was performed on coke oven workers and controls to test the neurobehavioral changes and the mood state.</p><p><b>RESULTS</b>the concentration of B(a)P at oven bottom,oven side and oven top were 0.0195 microg/m3, 0.186 microg/m3 and 1.624 microg/m3 respectively, that at oven side and oven top being higher than the one stipulated by the occupational hygiene criterion. Urinary 1-hydroxypyrene was significantly different between the exposure group (3.42 +/- 0.98 micromol/mol creatinine) and control group (2.75 +/- 1.09 micromol/mol creatinine). No significant differences were found between exposure group and control group of age, working years, smoking, drinking and unhealthy food consumption; however, compared to the controls, the scores of total digital span, the forward digital span, and right dotting in the coke oven workers were lower, but that of total dotting was higher, with a statistical significance. According to urinary 1-hydroxypyrene concentration, all the subjects were divided into three groups. (<3.10 micromol/mol creatinine, 3.10 micromol/mol creatinine, >3.87 micromol/mol creatinine). Significant differences of the total digital span, the forward digital span, backward digital span, digit symbol and Benton visual retentions existed in different urinary 1-hydroxypyrene concentration groups and showed a dose-response tendency. Results of multiple stepwise regression analysis and correlation analysis showed that the level of urinary 1-hydroxypyrene affected memory and perception of coke oven workers and negative correlations between the level of urinary 1-hydroxypyrene and changes in neurobehavioral function were found.</p><p><b>CONCLUSION</b>PAHs mainly causes decrease of memory and perception in coke oven workers.</p>
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Adulto , Humanos , Masculino , Poluentes Ocupacionais do Ar , Coque , Memória , Testes Neuropsicológicos , Exposição Ocupacional , Hidrocarbonetos Policíclicos Aromáticos , UrinaRESUMO
<p><b>OBJECTIVE</b>To explore the coincidence of lipid peroxidation and neurobehavioral function changes in coke oven workers.</p><p><b>METHODS</b>One hundred and thirty-four coke oven workers were divided into three groups: 35 in the oven-bottom group, 49 in the oven-side group and 50 in oven-top group. WHO recommended NCTB was performed on coke oven workers and 36 controls from material conservation department; The contents of total superoxide dismutases (T-SOD), glutathione (GSH) and malondialdehyde (MDA) in blood were determined by test kits.</p><p><b>RESULTS</b>Compared with the controls, the coke oven workers showed lower levels of T-SOD and GSH (P < 0.01), significantly higher MDA levels in blood (P < 0.01), higher score on negative mood state, lower scores on positive mood state, and poorer performance in NCTB test (P < 0.05). Further analysis revealed that there was a weak positive correlation between neurobehavioral function changes and the level of lipid peroxidation with a coefficient lower than 0.25.</p><p><b>CONCLUSION</b>The level of lipid peroxidation in coke oven workers' blood increased and coincided with neurobehavioral function impairment.</p>