Changes in the expression and phosphorylation state of autophagy-related protein ATG4 in nervous tissues of hens treated with tri-ortho-cresyl phosphate / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the changes in the levels of authophagy-related proteins ATG4A and p-ATG4A in nervous tissue after treated with tri-ortho-cresyl phosphate and explore the possible pathogenesis of OPIDN.</p><p><b>METHODS</b>In the first experiment, thirty hens were randomly divided into control group and 1 d, 5 d, 10 d and 21d treated groups, hens in treated groups were treated with TOCP by gavage at a single dosage of 600 mg/kg. In the second experiment, other thirty hens were also randomly divided into control group and 1 d, 5 d, 10 d and 21 d treated groups, hens in treated group were pretreated with PMSF by subcutaneous at a single dosage of 90 mg/kg. 24 h later, hens in intervention group was treated with TOCP by gavage at a single dosage of 600 mg/kg. The hens were killed at the corresponding time points, and collected their tibial nerves. The levels of ATG4A and p-ATG4A were measured by immunoblotting.</p><p><b>RESULTS</b>compared with the control group, the levels of ATG4A decreased by36%, 43.7% and 41% at 1d, 5d and 10d in the intoxication groups (P < 0.05), the levels of p-ATG4A decreased by 22.5%, 25%and 21%at 1d, 5d and 10d in the intoxication group (P < 0.05). However, compared with the control group, there is no significant change in the levels of ATG4A and p-ATG4A in PMSF-pretreated groups.</p><p><b>CONCLUSION</b>The intoxication of TOCP influence the levels of autophagy-related proteins ATG4A and p-ATG4A, which might be associated with the inhibition of autophagy activity in neurons of OPIDN.</p>

Changes in autophagy-related protein levels in nervous tissues of hens with tri-ortho-cresyl phosphate-induced delayed neuropathy / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the changes in the levels of autophagy-related proteins, Atg1, Atg5, and Beclin1, in organophosphate-induced delayed neuropathy (OPIDN) caused by tri-ortho-cresyl phosphate (TOCP), and to investigate the molecular pathogenic mechanism of OPIDN.</p><p><b>METHODS</b>Thirty adult Roman hens were randomly and equally divided into control group and 1, 5, 10, and 21 d intoxication groups. Each hen in the intoxication group was administered TOCP by gavage at a single dose of 750 mg/kg, while each hen in the control group was administered the same volume of corn oil. The hens were killed at the corresponding time points, and their tibial nerves and spinal cords were collected. The levels of Atg1, Atg5, and Beclin1 in the tibial nerves and spinal cords were measured by immunoblotting.</p><p><b>RESULTS</b>Compared with those in the control group, the levels of Atg1 in tibial nerves decreased by 29.8%, 64.4%, 43.5%, and 19.8% at 1, 5, 10, and 21 d, respectively, after intoxication ((P < 0.05); the levels of Atg5 in tibial nerves decreased by 36.8%, 49.6%, 51.2%, and 31.5% at 1, 5, 10, and 21 d, respectively, after intoxication (P < 0.05); the levels of Beclin1 in tibial nerves decreased by 68.5%, 66.3%, and 32.2% at 1, 5, and 10 d, respectively, after intoxication (P < 0.05). Compared with those in the control group, the levels of Atg1 in spinal cords decreased by 23.5%, 48.7%, and 20% at 1, 5, and 10 d, respectively, after intoxication (P < 0.05); the levels of Atg5 in spinal cords decreased by 32.7%, 51.5%, 47.3%, and 39.6% at 1, 5, 10, and 21 d, respectively, after intoxication (P < 0.05); the levels of Beclin1 in spinal cords decreased by 28.9%, 50.2%, 43.2%, and 28.3% at 1, 5, 10, and 21 d, respectively, after intoxication (P < 0.05).</p><p><b>CONCLUSION</b>The intoxication of TOCP is associated with the significant changes in the levels of autophagy-related proteins in the nervous tissues of hens, which might be involved in the pathogenesis of OPIDN.</p>

Screening the proteins of organophosphoms ester-induced delayed neurotoxicity in the cerebral tissue of hens exposed to tri-ortho-cresyl phosphate / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To screen the proteins with differential expression levels in the cerebral tissue of hens exposed to tri-ortho-cresyl phosphate (TOCP), and to provide target proteins for studying the mechanism of organophosphoms ester-induced delayed neurotoxicity (OPIDN).</p><p><b>METHODS</b>Thirty two adult Roman hens were randomly divided into four groups: TOCP group was exposed to 1000 mg/kg TOCP, PMSF group was exposed to 40 mg/kg PMSF, PMSF plus TOCP group was exposed to 40 mg/kg PMSF and after 24 h exposed to 1000 mg/kg TOCP, control group was exposed to normal saline. All hens exposed to chemicals by gastro-intestine for 5 days were sacrificed, and the cerebral tissue were dissected and homogenized in ice bath. Total proteins extracted from the cerebral tissue were separated by isoelectric focusing as the first dimension and SDS-PAGE as the second dimension. The 2-DE maps were visualized after silver staining and analyzed by Image Master 2D software. At last ,the expressed protein spots were identified by Mass spectrometry.</p><p><b>RESULTS</b>From total proteins in TOCP group, the PMSF plus TOCP group and PMSF group, 1185, 1294 and 1063 spots were detected, respectively. One thousand three hundred thirty two spots from total proteins in control group were detected. The match rates of protein spots in TOCP group, the PMSF plus TOCP group and PMSF group were 78.32 %, 79.56 % and 80.93%, respectively. There were 235 protein spots with differential expression levels between TOCP group and control group, which included 158 up regulation spots and 77 down regulation spots. According to the PMSF features, there were 102 spots with differential expression levels between TOCP group and control group and without differential expression levels between TOCP group and PMSF plus TOCP group, among them there were 13 spots with 4 fold differential expression levels between TOCP group and control group and without differential expression levels between TOCP group and PMSF group. Seven protein spots (homer-1b, Destrin, heat shock protein 70, eukaryotic translation initiation factors, proteasome alpha1 subunit, lactate dehydrogenase B, glutamine synthetase) were detected by Mass spectrometry.</p><p><b>CONCLUSION</b>There are 112 protein spots with differential expression levels of the cerebral tissue in TOCP group, which may be related to OPIDN, among them 13 protein spots with differential expression levels are associated closely with OPIDN. Seven protein spots detected by Mass spectrometry may be related to the mechanism induced by OPIDN.</p>

The differential expression of Stathmin in the spinal cord tissue of hens exposed to tri-o-cresyl phosphate / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To screen the differently expressed proteins related to regulating the depolymerization of microtubules in the spinal cord of hens exposed to tri-o-cresyl phosphate (TOCP) and to provide target protein evidence for exploring the mechanisms of the delayed neurotoxicology (OPIDN) induced by organophosphorus compounds (OPs).</p><p><b>METHODS</b>Forty two Roman hens were randomly divided into three groups, i.e. TOCP group treated with 1000 mg/kg TOCP; intervention group treated with 40 mg/kg phenylmethanesulfonyl fluoride (PMSF) before 1000 mg/kg TOCP treatment and control group treated with tap water. Four hens in each group were sacrificed on the 5th and 20th days after exposure, respectively. Spinal cords were separated and homogenates at low temperature, and the total proteins were extracted. The OPIDN symptoms observed and recorded in the remaining 6 hens in each group. The differently expressed proteins related to regulating the depolymerization of microtubules were screen by two-dimensional electrophoresis and mass spectroscopy (MS).</p><p><b>RESULTS</b>The OPIDN symptoms appeared on the 5th day after exposure in TOCP group, which were gradually serious with time. The results by two-dimensional electrophoresis and MS showed that the Stathmin expression was downregulated 3.4 times and 2.8 times in TOCP group, respectively, as compared with the control and PMSF intervention groups. However, there was no significant difference of the Stathmin expression between control group and PMSF intervention group.</p><p><b>CONCLUSION</b>The Stathmin expression in the spinal cord tissues of hens exposed to TOCP significantly downregulated. Moreover, the downregulated Stathmin expression may be related to excess polymerization of microtubules and the mechanism of OPIDN.</p>

Effect of phenylmethylsulfonyl fluoride pretreated on neurofilament subunits in spinal cords of hens administrated with tri-o-cresyl phosphate / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic changes of neurofilaments (NFs) proteins in spinal cords of hens with phenylmethylsulfonyl fluoride (PMSF) pretreatment for exploring the mechanism of tri-o-cresyl phosphate (TOCP)-induced delayed neuropathy (OPIDN).</p><p><b>METHOD</b>Adult Roman hens were randomly divided into three groups, control, TOCP and PMSF + TOCP. Birds in PMSF + TOCP set were pretreated with PMSF, 24 hours later, hens in both TOCP group and PMSF + TOCP group were administrated with TOCP at a single dosage of 750 mg/kg. Then all animals were sacrificed on the corresponding time-points of 1, 5, 10, and 21 days respectively after dosing of 750 mg/kg TOCP. The spinal cords were dissected, homogenized, and centrifuged at 100,000 x g. The levels of high molecular neurofilament (NF-H), medium molecular neurofilament (NF-M) and low molecular neurofilament (NF-L) in both pellet and supernatant fractions of spinal cords were determined by SDS-PAGE and Western-blotting.</p><p><b>RESULTS</b>The hens in TOCP group showed paralysis gait at the end of 21-day experimental period. The levels of NFs proteins in spinal cords changed obviously. Compared with control, the NFs in pellet showed a dramatic decrease on day 10 and then followed by a recovery. In the supernatant, the NFs proteins showed similar changes, which decreased significantly on day 10 and almost recovered control on day 21. Such as, NF-L, NF-M and NF-H decreased by 51%, 86% and 38% on day 10. The OPIDN signs were not observed in PMSF + TOCP group, and imbalances of NFs were obviously alleviated. Compared with control, only NF-M in pellet increased by 21% (P < 0.05) on day 21, others remained no changes; The levels of NF-H and NF-M in supernatant respectively increased by 19% and 35% on day 21, others were no significant statistical differences.</p><p><b>CONCLUSION</b>TOCP may induce imbalance of NFs levels in progress of OPIDN, and PMSF pretreatment may protect animals from OPIDN by reducing above changes, which may explain that TOCP-induced imbalance of NFs may be connected with the occurrence and development of OPIDN.</p>

Tri-ortho-cresyl phosphate induced oligodendrocyte vacuolation and process degeneration in cultured hen oligodendrocytes / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate whether tri-ortho-cresyl phosphate (TOCP) and organophosphate compound that could produce organophosphate-induced delayed neuropathy (OPIDN) in hen and other sensitive species, directly affect oligodendrocytes, the myelin-forming cell of the central nervous system.</p><p><b>METHODS</b>This was achieved by a combination of measurements of cell viability (MTT) cell pathological observation in the presence and absence of the compound cultured hen brain oligodendrocytes were prepared and treated with various concentrations of TOCP.</p><p><b>RESULTS</b>In a time-course experiment TOCP showed a cytotoxic effect to oligodendrocytes and led to the oligodendrocyte processes disintegrated and membranous blebs, cytoplasmic vacuolation following exposure time of 24 h or longer, it showed a dose-depended and time-depended manner cytotoxic effect to oligodendrocytes at dose levels of 0.5 approximately 1.5 microg/ml (1.35 approximately 4.05 mol/L) concentrations of TOCP for 24 - 72 h exposure. MTT experiment indicated that TOCP inhibited cell viability by dose-depended manner at dose levels of 0.5 approximately 1.5 microg/ml (1.35 approximately 4.05 mol/L) concentrations of TOCP for an 24 h exposure.</p><p><b>CONCLUSIONS</b>TOCP is cytotoxic to oligodendrocytes and leads to the oligodendrocyte processes disintegrated and membranous blebs, vacuolar degeneration, which suggests that this oligodendrocyte degeneration may involve in the pathogenesis mechanism for OPIDN.</p>

The fundamentals of tissue engineering: new scaffolds

The ability to regenerate new bone for skeletal use is a major clinical need. In this study, two novel porous calcium phosphate materials pure HA and biphasic HA/beta-Tricalcium phosphate (HA/beta -TCP) were evaluated as potential scaffolds for cell-seeded bone substitutes using human osteoblast-like cells (HOS) and primary human mesenchymal stem cells (hMSCs). A high rate of proliferation was observed on both scaffolds. A greater increase in alkaline phosphatase (ALP- an indicator of osteoblast differentiation) was observed on HA/beta -TCP compared to HA. This observation indicates that HA/TCP may play a role in inducing osteoblastic differentiation. Although further evaluation is required both materials show potential as innovative synthetic substitutes for tissue engineered scaffolds.

Hydroxapatite and tricalcium phosphate prepared by precipitation method

A mixture with different compositions of HA and TCP were synthesize in this work by precipitation method using Ca(NO3)2 4H2 and (NH4)2HPO4 as the starting materials. A mixture with HA and TCP phases in different ratios were produced. The powders were sintered from 1000 degrees C to 1250 degrees C. The phase compositions of the mixtures were then studied via XRD. This work shows that the pH value determines the different phase compositions of the HA-TCP mixture. Chemical analyses were carried out by FTIR. The microstructure was observed under SEM.

Spinal cord neuronal apoptosis induced by triorthocresyl phosphate poisoning in hens / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the role of neuronal apoptosis in organophosphorus poisoning-induced delayed neuropathy (OPIDN) and its dynamic pathological changes.</p><p><b>METHODS</b>To establish OPIDN animal model, triorthocresyl phosphate (TOCP)was given to hens with a single dose (1 000 mg/kg, im). Changes of neuropathology, number of neurons and apoptotic cells in the third lumbar spinal cord were observed by HE, Nissl and TUNEL methods 3, 5, 7, 10, 14, 18 days after injection.</p><p><b>RESULTS</b>The hens showed OPIDN typical signs (progressive ataxia and hypotonia) about 9 days after TOCP exposure. HE staining revealed dark red nucleus in neurons of anterior horn of lumbar spinal cord 5 days after exposure, but this phenomenon disappeared 18 days later. Nissl method showed that the number of neurons in anterior horn of spinal cord decreased [from (82 +/- 4) cell/mm(2) to (66 +/- 6) cell/mm(2)]. TUNEL positive cells began to appear [(22 +/- 2) cell/mm(2)] 5 days after TOCP exposure, and reached the peak [(27 +/- 3) cell/mm(2)] 7 days later, and disappeared 18 days later.</p><p><b>CONCLUSION</b>Neuronal apoptosis in anterior horn of spinal cord of hens appeared in OPIDN, suggesting that cellular apoptosis may play an important role in the pathogenesis of OPIDN.</p>

Investigation of Indoor Air Pollution by Chlorpyrifos: Determination of Chlorpyrifos in Indoor Air and 3,5,6-Trichloro-2-pyridinol in Residents\rq Urine as an Exposure Index

Objects: We carried out an investigation to clarify the real state of indoor air pollution by chlorpyrifos (termiticide) and exposure to chlorpyrifos of residents by measuring its urinary metabolite 3,5,6-trichloro-2-pyridinol (TCP) as an exposure index, such as biological monitoring. Methods: The investigation was conducted in 43 individual houses with termiticide application (whether the termiticide was chlorpyrifos is uncertain) and 3 control houses without any termiticide application in Kagawa, Japan. Urine samples were collected from 46 healthy adult residents of the aforementioned houses. Results: Chlorpyrifos in indoor air in the control houses was not detected (ND<1 ng/m3, n=3), while 41 of 43 houses with termiticide application showed 1−350 ng/m3. Although the chlorpyrifos concentrations in these 41 houses did not exceeded the indoor air quality guideline of 1000 ng/m3, but 3 houses were higher than the guideline 100 ng/m3 for children in Japan. Urinary TCP concentrations of 0.1−7.8 ng/mg·creatinine were detected in 41 residents from the 41 houses where chlorpyrifos had been detected. The chlorpyrifos concentration and the urinary TCP revealed a positive correlation (r=0.5468, p<0.01, n=41). Conclusions: The immediate health hazard from air born chlorpyrifos in the examined houses was negligible, but the findings suggest that it is necessary to monitor chemicals which may contaminate indoor air and to assess the risk of prolonged exposure to such chemicals. The measuring of urinary metabolite TCP of chlorpyrifos via biological monitoring would be useful, allowing comprehensive evaluation of the exposure to chlorpyrifos in indoor air.

Tri-ortho-cresyl phosphate induced neurotarget esterase inhibition in brain and peripheral nerve effectively monitored by lymphocytes.

Tri-ortho-cresyl phosphate ester (TOCP: 10-100 mg/kg)--an organophosphorus compound (OP) inhibited brain, peripheral nerve and lymphocyte neurotarget esterase activities in a dose related and time-dependent manner. There was a good correlation of inhibition between brain, peripheral nerve and lymphocyte tissues taken from adult hens treated with 3 different doses of TOCP and sacrificed 24 and 48 hr after exposure. The results suggest that lymphocyte can effectively monitor OP compounds for investigating inhibition of neurotarget esterase activity in brain and peripheral nerve tissues.

Acute oral toxicity of insect repellent N,N-diethylphenylacetamide in mice, rats and rabbits and protective effect of sodium pentobarbital.

Median lethal dose (LD50) of undiluted liquid insect repellent N,N-diethylphenylacetamide (DEPA) in male mice, rats and rabbits was 900, 825 and 635 mg/kg respectively when administered by gavage. Signs of DEPA intoxication point to stimulation of central nervous system (CNS). Acetazolamide (10 mg/kg), sodium bicarbonate (40 mg/kg), and atropine (5 mg/kg) when injected (ip) 5 min after a lethal oral dose of DEPA (1700 mg/kg) did not prevent mortality, while sodium pentobarbital (SPB; 20 mg/kg) when injected 5 min after or 15 min before DEPA provided greater protection to the animals. SPB pretreatment elevated the LD50 of DEPA to 1780 and 1535 mg/kg in mice and rats respectively and 85% rats survived when SPB was injected 5 min after acute oral exposure to DEPA (1000 mg/kg). Carboxylesterase (CaE) inhibition is not a factor in the protection mechanism of SPB. DEPA (1000 mg/kg) when given orally elevated blood PCO2 and reduced pH, O2 content and per cent O2 saturation, while administration of SPB after the same dose of DEPA reduced the degree of acidosis and raised PCO2, and increased the O2 content and per cent O2 saturation to near normal status. The CNS depressant action of SPB may be a crucial factor in protection of rats from DEPA poisoning.

Effect of tri-ortho-cresyl phosphate on hen nervous system.

Correlation of tri-ortho-cresyl phosphate (TOCP) toxicity with clinical symptoms and inhibition patterns of neurotoxic esterase (NTE) in different regions of the central nervous system was studied in hens. The total content of NTE in spinal cord and peripheral nerves was relatively much lower than that of the brain, about 29 and 4.8 per cent respectively. Brain and spinal cord NTE was inhibited to 75 and 67 per cent respectively on day 1 post-TOCP treatment, and 91 and 84 per cent respectively on day 2, while the activity of the enzyme in peripheral nerve was inhibited to 92 per cent on day 1. However, the clinical manifestations of paralysis appeared after 7-8 days of TOCP treatment. It can be concluded that the presence of NTE in very low quantities in the peripheral nerves and its almost complete inhibition as compared to that of brain and spinal cord, could be of great significance in the development of neurotoxicity.