RESUMO
Phenylmethylsulfonyl fluoride (PMSF) is a protease and esterase inhibitor that causes protection, or potentiation/"promotion," of organophosphorus delayed neuropathy (OPIDN), depending on whether it is dosed before or after an inducer of delayed neuropathy, such as mipafox. The molecular target of the potentiation/promotion of OPIDN has not yet been identified. The kinetic data of phenyl valerate esterase inhibition by PMSF were obtained with membrane chicken brain fractions, the animal model and tissue in which neuropathy target esterase (NTE) was first described. Data were analyzed using a kinetic model with a multienzymatic system in which inhibition, simultaneous chemical hydrolysis of the inhibitor and "ongoing inhibition" (inhibition during the substrate reaction) were considered. Three main esterase components were discriminated: two sensitive enzymatic entities representing 44 and 41 %, with I 50 (20 min) of 35 and 198 µM at 37 °C, respectively, and a resistant fraction of 15 % of activity. The estimated constant of the chemical hydrolysis of PMSF was also calculated (kh = 0.28 min(-1)). Four esterase components were globally identified considering also previously data with paraoxon and mipafox: EPα (4-8 %), highly sensitive to paraoxon and mipafox, spontaneously reactivates after inhibition with paraoxon, and resistant to PMSF; EPß (38-41 %), sensitive to paraoxon and PMSF, but practically resistant to mipafox, this esterase component has the kinetic characteristics expected for the PMSF potentiator target, even though paraoxon cannot be a potentiator in vivo due to high AChE inhibition; EPγ (NTE) (39-48 %), paraoxon-resistant and sensitive to the micromolar concentration of mipafox and PMSF; and EPδ (10 %), resistant to all the inhibitors assayed. This kinetic characterization study is needed for further isolation and molecular characterization studies, and these PMSF phenyl valerate esterase components will have to be considered in further studies of OPIDN promotion. A simple test for monitoring the four esterase components is proposed.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Inibidores Enzimáticos/farmacocinética , Esterases/metabolismo , Fluoreto de Fenilmetilsulfonil/farmacocinética , Animais , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Galinhas , Inibidores Enzimáticos/toxicidade , Esterases/antagonistas & inibidores , Isoflurofato/análogos & derivados , Isoflurofato/farmacologia , Cinética , Fluoreto de Fenilmetilsulfonil/toxicidadeRESUMO
Organophosphorus compounds (OPs) cause neurotoxic disorders through interactions with well-known target esterases, such as acetylcholinesterase and neuropathy target esterase (NTE). However, the OPs can potentially interact with other esterases of unknown significance. Therefore, identifying, characterizing and elucidating the nature and functional significance of the OP-sensitive pool of esterases in the central and peripheral nervous systems need to be investigated. Kinetic models have been developed and applied by considering multi-enzymatic systems, inhibition, spontaneous reactivation, the chemical hydrolysis of the inhibitor and "ongoing inhibition" (inhibition during the substrate reaction time). These models have been applied to discriminate enzymatic components among the esterases in nerve tissues of adult chicken, this being the experimental model for delayed neuropathy and to identify different modes of interactions between OPs and soluble brain esterases. The covalent interaction with the substrate catalytic site has been demonstrated by time-progressive inhibition during ongoing inhibition. The interaction of sequential exposure to an esterase inhibitor has been tested in brain soluble fraction where exposure to one inhibitor at a non inhibitory concentration has been seen to modify sensitivity to further exposure to others. The effect has been suggested to be caused by interaction with sites other than the inhibition site at the substrate catalytic site. This kind of interaction among esterase inhibitors should be considered to study the potentiation/promotion phenomenon, which is observed when some esterase inhibitors enhance the severity of the OP induced neuropathy if they are dosed after a non neuropathic low dose of a neuropathy inducer.
Assuntos
Inibidores Enzimáticos/toxicidade , Esterases/antagonistas & inibidores , Doenças do Sistema Nervoso/induzido quimicamente , Doenças do Sistema Nervoso/enzimologia , Compostos Organofosforados/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Domínio Catalítico , Galinhas , Esterases/química , Isoflurofato/análogos & derivados , Isoflurofato/toxicidade , Cinética , Paraoxon/toxicidade , Fluoreto de Fenilmetilsulfonil/toxicidade , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/enzimologia , SolubilidadeRESUMO
Phenylmethylsulfonyl fluoride (PMSF) is a protease and esterase inhibitor that causes protection or potentiation/promotion of organophosphorus delayed neuropathy (OPIDN) depending on whether it is dosed before or after an inducer of delayed neuropathy. The molecular target of promotion has not yet been identified. Kinetic data of esterase inhibition were first obtained for PMSF with a soluble chicken brain fraction and then analyzed using a kinetic model with a multienzymatic system in which inhibition occurred with the simultaneous chemical hydrolysis of the inhibitor and ongoing inhibition (inhibition during the substrate reaction). The best fitting model was a model with resistant fraction, Eα (28%), and two sensitive enzymatic entities, Eß (61%) and Eγ (11%), with I(50) at 20 min of 70 and 447 µM, respectively. The estimated constant of the chemical hydrolysis of PMSF was kh = 0.23 min(-1). Eα, which is sensitive to mipafox and resistant to PMSF, became less sensitive to mipafox when the preparation was preincubated with PMSF. Its Eα I(50) (30 min) of mipafox increased with the PMSF concentration used to preincubate it. Eγ is sensitive to both PMSF and mipafox, and after preincubation with PMSF, Eγ became less sensitive to mipafox and was totally resistant after preincubation with 10 µM PMSF or more. The sensitivity of Eα to paraoxon (I(50) 30 min from 9 to 11 nM) diminished after PMSF preincubation (I(50) 30 min 185 nM) and showed no spontaneous reactivation capacity. The nature of these interactions is unknown but might be due to covalent binding at sites other than the substrate catalytic center. Such interactions should be considered to interpret the potentiation/promotion phenomenon of PMSF and to understand the effects of multiple exposures to chemicals.
Assuntos
Encéfalo/enzimologia , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Doenças do Sistema Nervoso/prevenção & controle , Fluoreto de Fenilmetilsulfonil/farmacologia , Animais , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Galinhas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Hidrólise , Doenças do Sistema Nervoso/induzido quimicamente , Compostos Organofosforados/química , Fluoreto de Fenilmetilsulfonil/química , Fluoreto de Fenilmetilsulfonil/toxicidade , Solubilidade , Relação Estrutura-AtividadeRESUMO
Gallbladder bile from 2 fish species, mullet (Mugil liza) and tilapias (Tilapia rendalli), contain substantial matrix metalloproteinases (MMPs). Extensive purification studies were conducted in order to obtain workable samples for SDS-PAGE and zymography analysis. Proteinase activities were assayed by gelatin substrate zymography. Several protein bands were observed, corresponding to molecular weights of 200, 136, 43, 36, 34, 29, 23 and 14 kDa in mullet bile and 179, 97, 79, 61, 54, 45, 36, 33 and 21 kDa in tilapia bile. Specific inhibitor studies were conducted, in which MMPS were inhibited by EDTA and 1,10 phenanthroline, but not by serine and cysteine protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF) and transepoxysuccinyl-l-leucylamido-l-guanidino butane (E-64), confirming the proteinase identities as MMPs. Differences in proteinase expression were observed in fish from a contaminated and reference site. Some studies regarding MMPs in different fish tissues exist, however this is the first study conducted in fish bile, and their involvement in detoxification processes and organism protection against the effects of aquatic contaminants may be a possibility.
Assuntos
Bile/metabolismo , Monitoramento Ambiental/métodos , Metaloproteinases da Matriz/metabolismo , Poluentes Químicos da Água/toxicidade , Animais , Biomarcadores/metabolismo , Eletroforese em Gel de Poliacrilamida , Peixes , Leucina/análogos & derivados , Leucina/toxicidade , Fluoreto de Fenilmetilsulfonil/toxicidadeRESUMO
OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Assuntos
Cérebro/efeitos dos fármacos , Proteínas de Neurofilamentos/metabolismo , Fluoreto de Fenilmetilsulfonil/toxicidade , Tritolil Fosfatos/toxicidade , Animais , Encéfalo/metabolismo , Cérebro/metabolismo , Galinhas , Proteoma/análiseRESUMO
Neuropathy target esterase (NTE) is proven to act as a lysophospholipase (LysoPLA) in mice and phospholipase B (PLB) in cultured mammalian cells. In sensitive species, organophosphate (OP)-induced delayed neurotoxicity is initiated when NTE is inhibited by > 70% and then aged. It is hypothesized that homeostasis of phosphatidylcholine (PC) and/or lysophosphatidylcholine (LPC) in mice might be disrupted by the OPs since NTE and other phospholipases could be inhibited. To test this hypothesis, we treated mice using tri-o-cresyl phosphate (TOCP), which can inhibit and age NTE. Phenylmethylsulfonyl fluoride (PMSF), which inhibits NTE but cannot age, was used as a negative control. Effects on activity of NTE, LysoPLA, and PLB, the levels of PC, LPC, and glycerophosphocholine (GPC), and the aging of NTE in the brain, spinal cord, and sciatic nerve were examined. The results showed that the activities of NTE, NTE-LysoPLA, LysoPLA, NTE-PLB, and PLB were significantly inhibited in both TOCP- and PMSF-treated mice, and the inhibition of NTE and NTE-LysoPLA or NTE-PLB showed a high correlation coefficient. The NTE inhibited by TOCP was of the aged type, while nearly all NTE inhibited by PMSF was of the unaged type. Although the GPC level was remarkedly decreased, no significant change of PC and LPC levels was observed. However, the inhibition of these enzymes in mice by TOCP exhibited different characteristics from the TOCP-treated hens that we previously reported, which indicates that these enzymes were inhibited and then recovered more rapidly in mice than in hens. All results suggest that PC and LPC homeostasis was not disrupted in mice after exposure to TOCP. Differences in inhibition of NTE, LysoPLA, and PLB activities by TOCP between mice and hens may elucidate why these two species display different signs after exposure to the same neuropathic OPs.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Homeostase/efeitos dos fármacos , Lisofosfatidilcolinas/metabolismo , Fosfatidilcolinas/metabolismo , Tritolil Fosfatos/toxicidade , Análise de Variância , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Hidrolases de Éster Carboxílico/efeitos dos fármacos , Lisofosfolipase/metabolismo , Masculino , Camundongos , Síndromes Neurotóxicas/metabolismo , Fluoreto de Fenilmetilsulfonil/administração & dosagem , Fluoreto de Fenilmetilsulfonil/toxicidade , Fosforilação/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Tritolil Fosfatos/administração & dosagemRESUMO
Little is known regarding early biochemical events in organophosphate-induced delayed neurotoxicity (OPIDN) except for the essential inhibition of neuropathy target esterase (NTE). We hypothesized that the homeostasis of lysophosphatidylcholine (LPC) and/or phosphatidylcholine (PC) in nervous tissues might be disrupted after exposure to the organophosphates (OP) which participates in the progression of OPIDN because new clues to possible mechanisms of OPIDN have recently been discovered that NTE acts as lysophospholipase (LysoPLA) in mice and phospholipase B (PLB) in cultured mammalian cells. To bioassay for such phospholipids, we induced OPIDN in hens using tri-o-cresyl phosphate (TOCP) as an inducer with phenylmethylsulfonyl fluoride (PMSF) as a negative control; and the effects on the activities of NTE, LysoPLA and PLB, the levels of PC, LPC, and glycerophosphocholine (GPC), and the aging of NTE enzyme in the brain, spinal cord, and sciatic nerves were examined. The results demonstrated that the activities of NTE, NTE-LysoPLA, LysoPLA, NTE-PLB and PLB were significantly inhibited in both TOCP- and PMSF-treated hens. The inhibition of NTE and NTE-LysoPLA or NTE-PLB showed a high correlation coefficient in the nervous tissues. Moreover, the NTE inhibited by TOCP was of the aged type, while nearly all of the NTE inhibited by PMSF was of the unaged type. No significant change in PC or LPC levels was observed, while the GPC level was significantly decreased. However, there is no relationship found between the GPC level and the delayed symptoms or aging of NTE. All results suggested that LPC and/or PC homeostasis disruption may not be a mechanism for OPIDN because the PC and LPC homeostasis was not disrupted after exposure to the neuropathic OP, although NTE, LysoPLA, and PLB were significantly inhibited and the GPC level was remarkably decreased.
Assuntos
Galinhas/fisiologia , Homeostase/efeitos dos fármacos , Lisofosfatidilcolinas/metabolismo , Síndromes Neurotóxicas/metabolismo , Fosfatidilcolinas/metabolismo , Tritolil Fosfatos/toxicidade , Animais , Química Encefálica/efeitos dos fármacos , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/metabolismo , Colina/metabolismo , Inibidores Enzimáticos/toxicidade , Feminino , Glicerilfosforilcolina/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lisofosfolipase/metabolismo , Fluoreto de Fenilmetilsulfonil/toxicidade , Fosfatos/metabolismo , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
Certain esterase inhibitors, such as carbamates, phosphinates and sulfonyl halides, do not cause neuropathy as some organophosphates, but they may exacerbate chemical or traumatic insults to axons. This phenomenon is called promotion of axonopathies. Given the biochemical and toxicological characteristics of these compounds, the hypothesis was made that the target of promotion is a phenyl valerate (PV) esterase similar to neuropathy target esterase (NTE), the target of organophosphate induced delayed polyneuropathy. However, attempts to identify a PV esterase in hen peripheral nerve have been, so far, unsuccessful. We tested several esters, other than PV, as substrates of esterases from crude homogenate of the hen peripheral nerve. The ideal substrate should be poorly hydrolysed by NTE but extensively by enzyme(s) that are insensitive to non-promoters, such as mipafox, and sensitive to promoters, such as phenyl methane sulfonyl fluoride (PMSF). When phenyl benzoate (PB) was used as substrate, about 65% of total activity was resistant to the non-promoter mipafox (up to 0.5 mM, 20 min, pH 8.0), that inhibits NTE and other esterases. More than 90% of this resistant activity was sensitive to the classical promoter PMSF (1 mM, 20 min, pH 8.0) with an IC(50) of about 0.08 mM (20 min, pH 8.0). On the contrary, the non-promoter p-toluene sulfonyl fluoride caused only about 10% inhibition at 0.5 mM. Several esterase inhibitors including, paraoxon, phenyl benzyl carbamate, di-n-butyl dichlorovinyl phosphate and di-isopropyl fluorophosphate, were tested both in vitro and in vivo for inhibition of this PB activity. Mipafox-resistant PMSF-sensitive PB esterase activity(ies) was inhibited by promoters but not by non promoters and neuropathic compounds.
Assuntos
Benzoatos/toxicidade , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Inibidores Enzimáticos/toxicidade , Nervo Isquiático/efeitos dos fármacos , Animais , Galinhas , Isoflurofato/análogos & derivados , Isoflurofato/toxicidade , Fluoreto de Fenilmetilsulfonil/toxicidade , Nervo Isquiático/enzimologia , Especificidade por SubstratoRESUMO
Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester, which produces delayed neurotoxicity (OPIDN) in hens in 7-14 days. OPIDN is characterized by mild ataxia in its initial stages and severe ataxia or paralysis in about 3 weeks. It is marked by distal swollen axons, and exhibits aggregations of neurofilaments (NFs), microtubules, proliferated smooth endoplasmic reticulum, and multivesicular bodies. These aggregations subsequently undergo disintegration, leaving empty varicosities. Previous studies in this laboratory have shown an increased level of medium-molecular weight NF (NF-M) and decreased levels of high- and low-molecular weight NF (NF-H, NF-L) proteins in the spinal cord of DFP-treated hens. The main objective of this investigation was to study the effect of DFP administration on NF subunit levels when OPIDN is prevented or potentiated by pretreatment or post-treatment with phenylmethylsulfonyl fluoride (PMSF), respectively. Hens pretreated or post-treated with PMSF were killed 1, 5, 10, and 20 days after the last treatment. The alteration in NF subunit protein levels observed in DFP-treated hen spinal cords was not observed in protected hens. Estimation of NFs in the potentiation experiments, however, showed a different pattern of alteration in NF subunit levels. The results showed that an alteration in NF subunit levels in DFP-treated hens might be related to the development of OPIDN, since these changes were suppressed in PMSF-protected hens. However, results from PMSF post-treated hen spinal cords suggested that potentiation of OPIDN by PMSF was mediated by a mechanism different from that followed by DFP alone to produce OPIDN.
Assuntos
Isoflurofato/toxicidade , Proteínas de Neurofilamentos/metabolismo , Síndromes Neurotóxicas/metabolismo , Medula Espinal/efeitos dos fármacos , Telencéfalo/efeitos dos fármacos , Animais , Galinhas , Interações Medicamentosas , Proteínas de Neurofilamentos/imunologia , Fluoreto de Fenilmetilsulfonil/toxicidade , Inibidores de Proteases/toxicidade , Medula Espinal/metabolismo , Telencéfalo/metabolismoRESUMO
T614 (3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-o ne) is a member of the family of methanesulfonanilide non-steroidal anti-inflammatory drugs (mNSAIDs), most of which act as cyclooxygenase (COX)-2 inhibitors. L-leucine methyl ester (Leu-OME) is a reagent which has been shown to kill phagocytes following interaction with intracellular proteases. There are two pathways whereby Leu-OME becomes cytotoxic to phagocytes. Within lysosomes, Leu-OME is converted into free Leu, which causes disruption of the lysosomes and subsequent cell necrosis. The other is the conversion of Leu-OME into (Leu-Leu)(n)-OME, which is associated with the induction of apoptosis. In the present study, we examined the action of T614 on Leu-OME mediated killing of THP-1, a human monocytic cell line. We revealed that T614 and phenylmethyl sulfonyl fluoride (PMSF), a serine protease inhibitor, inhibited Leu-OME mediated killing of THP-1 cells. All the other mNSAIDs, including nimesulide (NIM-03), fluosulide (CGP28238), FK3311 and NS398, also rescued THP-1 from Leu-OME mediated killing, although to a lesser degree. Of the classical NSAIDs tested, a protective effect was observed with diclofenac at high concentration, but not with naproxen or indomethacin. Unlike conventional lysosomal inhibitors, such as chloroquine and ammonium chloride (NH(4)Cl), T614 and PMSF did not raise lysosomal pH, as measured by flow cytometry using fluorescein isothiocyanate dextran (FITC-dextran). Therefore, the mechanism whereby T614 and PMSF inhibit Leu-OME killing is distinct from that of chloroquine or NH(4)Cl. Based on the similarity of T614 and PMSF, we suggest that, besides their roles as COX-2 inhibitors, T614 and other mNSAIDs may act as lysosomal protease inhibitors.
Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Benzopiranos/toxicidade , Inibidores do Crescimento/antagonistas & inibidores , Inibidores do Crescimento/toxicidade , Leucina/antagonistas & inibidores , Leucina/toxicidade , Monócitos/efeitos dos fármacos , Sulfonamidas/toxicidade , Anti-Inflamatórios não Esteroides/metabolismo , Benzopiranos/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Dipeptídeos/toxicidade , Humanos , Concentração de Íons de Hidrogênio , Leucina/análogos & derivados , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Monócitos/enzimologia , Monócitos/metabolismo , Fluoreto de Fenilmetilsulfonil/toxicidade , Sulfonamidas/metabolismo , Células Tumorais Cultivadas , Células U937RESUMO
The serine/cysteine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) has been used both to promote and to protect against neuropathic events of organophosphorus-induced delayed neuropathy (OPIDN) in hens (Veronesi and Padilla, 1985; Pope and Padilla, 1990; Lotti et al., 1991; Pope et al., 1993; Randall et al., 1997). This study is the first to expand upon this work by using high resolution microscopy provided by epoxy resin embedding and thin sectioning to evaluate neuropathological manifestations of promotion and protection, and to correlate them with associated clinical modifications. To evaluate dose-related effects of OPIDN, single phenyl saligenin phosphate (PSP) dosages of 0.5, 1.0, or 2.5 mg/kg were administered to adult hens. PMSF (90 mg/kg) was given either 4 hours after (for promotion) or 12 hours prior to (for protection) PSP administration. Clinical signs and pathologic changes in the biventer cervicis nerve, which is uniquely sensitive to OPIDN (El-Fawal et al., 1988), were monitored. PSP alone, 2.5 mg/kg, caused severe OPIDN (terminal clinical score 7.5 +/- 1.0 [0-8 scale]; neuropathology score 2.7 +/- 0.3 [0-4 scale, based on myelinated fiber degeneration]). PMSF given 12 hours prior to PSP gave complete protection (clinical and neuropathology scores of 0; p<0.0001 compared to PSP alone). Signs and lesions of OPIDN were absent following 0.5 mg/kg PSP alone, but PMSF given 4 hours after PSP potentiated its neurotoxic effects (all hens had clinical scores of 4.0 and the average neuropathology score was 3.5 +/- 0.3; p<0.0001 compared to PSP alone). Although quantitative differences were noted, qualitative differences among nerves from hens with OPIDN were not evident, either with light or electron microscopy. At the time of sacrifice, there was a statistically linear relationship (r2 = 0.76) between the clinical scores on the last day of observation and the neuropathology scores (p<0.0001). This study demonstrates that the degree of peripheral nerve myelinated fiber degeneration correlates with clinical deficits in PMSF-induced potentiation of and protection against OPIDN.
Assuntos
Galinhas/fisiologia , Doenças do Sistema Nervoso/induzido quimicamente , Compostos Organofosforados/toxicidade , Fluoreto de Fenilmetilsulfonil/toxicidade , Inibidores de Proteases/toxicidade , Animais , Encéfalo/patologia , Encéfalo/ultraestrutura , Feminino , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Fibras Nervosas/patologia , Fibras Nervosas/ultraestrutura , Doenças do Sistema Nervoso/patologia , Inclusão em Plástico , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Fixação de TecidosRESUMO
This study investigated the neurotoxic potentiation and metabolic interaction between p-bromophenylacetylurea (BPAU) and phenylmethanesulfonyl fluoride (PMSF). The results showed that F344 rats given two successive daily doses of 150 mg/kg BPAU developed a moderate degree of ataxia. When rats were coadministrated a single intraperitoneal dose of 100 mg/kg PMSF either 1 day before, or 4 h or 1 day after the two daily doses of BPAU, the severity of ataxia was significantly increased. No such effect was observed when PMSF was given 4 days after BPAU, although this time point was still prior to the development of the neuropathy. The enhancement or potentiation of neuropathy by PMSF was thus seen only at times when parent BPAU was present in the target tissues. A pharmacokinetic study showed that PMSF increased the concentrations of BPAU and its metabolite, N'-hydroxy-p-bromophenylacetylurea (M1), in tissues and decreased the concentration of the metabolite 4-(4-bromophenyl)-3-oxapyrrolidine-2,5-dione (M2) in serum. This indicated that PMSF inhibited the M2 pathway and more BPAU was metabolized via the M1 pathway. This increased both BPAU and M1 levels in tissues and hence would have increased BPAU-induced neurotoxicity. We conclude that PMSF does not need to act directly on target sites to potentiate BPAU-induced neurotoxicity, since its interference with BPAU metabolism was sufficient to account for the increase in BPAU neurotoxicity. Thus a metabolic interaction underlies the neurotoxic potentiation between these two compounds rather than the target site interaction seen between PMSF and neuropathic organophosphates. This study is the first to demonstrate that interference with the metabolism of BPAU is an important aspect of the potentiation of BPAU-induced neurotoxicity.
Assuntos
Fluoreto de Fenilmetilsulfonil/toxicidade , Ureia/análogos & derivados , Animais , Ataxia/induzido quimicamente , Comportamento Animal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Sinergismo Farmacológico , Feminino , Fluoreto de Fenilmetilsulfonil/metabolismo , Fluoreto de Fenilmetilsulfonil/farmacocinética , Ratos , Ratos Endogâmicos F344 , Distribuição Tecidual , Ureia/metabolismo , Ureia/farmacocinética , Ureia/toxicidadeRESUMO
Certain esterase inhibitors such as O-(2-chloro-2,3,3-trifluorocyclobutyl) O-ethyl S-propyl phosphorothioate (KBR-2822) and phenylmethanesulfonyl fluoride (PMSF) cause exacerbation (promotion) of toxic and traumatic axonopathies. Although these chemicals are capable of inhibiting neuropathy target esterase (NTE), which is the target for organophosphate induced delayed neuropathy, the target for promotion is unlikely to be NTE. Experiments were aimed to ascertain if neuropathy is caused by repeated dosing with a promoter not causing NTE inhibition and in the absence of deliberate injury to axons. Hens were treated with KBR-2822 (0.2 or 0.4 mg/kg per day) by gavage for 90 days and observed for clinical signs up to 21-23 days after treatment when histopathological examination was carried out. NTE and acetylcholinesterase (AChE) were measured at intervals and mean percentages of inhibition at steady state of inhibition/resynthesis (on day 20) were as follows: mean inhibition NTE was < or = 8% in the 0.2 mg/kg group and between 15 and 18% in the 0.4 mg/kg group in brain, spinal cord and peripheral nerve; mean AChE inhibition in brain was 31 and 57% in the two experimental groups, respectively. Controls treated with paraoxon (not neuropathic or a promoter and given at 0.05 mg/kg per day by gavage) showed 45% mean AChE inhibition and no NTE inhibition. Neither clinical nor morphological signs of neuropathy were observed in any group. To ascertain whether subclinical lesions were produced by the repeated treatment with KBR-2822, hens were given KBR-2822 (0.2 mg/kg per day) for 21 days by gavage followed by PMSF (120 mg/kg s.c. 24 h after the last dose of KBR-2822). A control group of hens was treated with the neuropathic DFP (0.03 mg/kg s.c. daily for 21 days causing 40-50% NTE inhibition) followed by PMSF (120 mg/kg s.c.). After PMSF, the KBR-2822 treated hens did not develop neuropathy whereas DFP treated hens did. Lack of neuropathy after repeated treatment with KBR-2822 indicates that a continuous promoting 'pressure' on hen axons is harmless in the absence of a concurrent biochemical or neurotoxic injury.
Assuntos
Axônios/patologia , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Inibidores da Colinesterase/toxicidade , Doenças do Sistema Nervoso/induzido quimicamente , Organotiofosfatos/toxicidade , Fluoreto de Fenilmetilsulfonil/toxicidade , Inibidores de Proteases/toxicidade , Acetilcolinesterase/metabolismo , Animais , Axônios/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Galinhas , Feminino , Injeções Subcutâneas , Doenças do Sistema Nervoso/enzimologia , Organotiofosfatos/administração & dosagem , Sistema Nervoso Periférico/efeitos dos fármacos , Sistema Nervoso Periférico/enzimologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/enzimologiaRESUMO
The lung is highly susceptible to ethyl carbamate (EC)-induced tumorigenesis. Our goal in this study was to investigate the in vitro isozyme-selective metabolism of EC in lung microsomes by cytochrome P450 and carboxylesterase enzymes. Our results showed that incubations with EC produced significant reduction in p-nitrophenol (PNP) hydroxylation and N-nitrosodimethylamine (NDMA) demethylation; there were no alterations in 7-pentoxyresorufin- and 7-ethoxyresorufin O-dealkylase activities. Reaction of microsomes with an inhibitory CYP2E1 antibody and subsequent reaction with EC abolished the EC-induced diminution in NDMA demethylase activity. Carboxylesterase activity, as assessed by hydrolysis of p-nitrophenyl acetate, was significantly decreased in microsomes incubated with EC. Reactions with EC in conjunction with the carboxylesterase inhibitors, paraoxon (PAX) or phenylmethylsulfonyl fluoride (PMSF), abolished the EC-induced decrease in carboxylesterase activity; PAX is a broad-spectrum carboxylesterase inhibitor, whereas PMSF is a specific inhibitor of hydrolase A, a carboxylesterase isozyme. Incubations of EC in combination with either PAX or PMSF exacerbated the EC-induced reduction in PNP hydroxylase and NDMA demethylase activities. Alterations in immunodetectable CYP2E1 protein levels were not apparent in microsomes incubated with EC alone, but the amounts were decreased in reactions with EC in conjunction with either PAX or PMSF. Immunoblotting with antibodies for the carboxylesterase isozymes, hydrolase A and B, revealed loss of immunodetectable hydrolase A in microsomes incubated with EC, PAX, or PMSF. However, immunodetectable hydrolase B was only decreased in microsomes reacted with PAX but not with PMSF or EC. These findings correlated with our covalent binding data, which showed that levels of binding of [14C-ethyl]EC to lung microsomes were significantly higher in incubations conducted in conjunction with PAX or PMSF, compared with control levels. Antibody inhibition of the CYP2E1 enzyme significantly reduced the extent of binding. Our results demonstrated that EC metabolism in lung microsomes, as estimated from magnitudes of covalent binding, is mediated by the P450 isozyme CYP2E1 and the carboxylesterase isozyme hydrolase A.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Carcinógenos/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Isoenzimas/metabolismo , Pulmão/enzimologia , Uretana/metabolismo , Animais , Ligação Competitiva , Biotransformação , Carboxilesterase , Carcinógenos/toxicidade , Inibidores da Colinesterase/toxicidade , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Feminino , Hidrólise , Hidroxilação , Immunoblotting , Inativação Metabólica , Pulmão/efeitos dos fármacos , Camundongos , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Nitrofenóis/química , Paraoxon/toxicidade , Fluoreto de Fenilmetilsulfonil/toxicidade , Inibidores de Proteases/toxicidade , Uretana/toxicidadeRESUMO
Clinical manifestations of mild organophosphorus compound-induced delayed neurotoxicity (OPIDN) produced by diisopropylphosphorofluoridate (DFP) in adult hens are potentiated by posttreatment with phenylmethanesulfonyl fluoride (PMSF). The purpose of this study was to assess whether potentiation of mild OPIDN produces a pattern of axonal lesions in the central and peripheral nervous system similar to that seen in severe OPIDN. Groups of 6 hens each were given the following priming/challenge doses sc at 0 and 4 h, respectively: 0.20 ml/kg corn oil/0.50 ml/kg glycerol formal (GF) (control); 0.50 mg/kg DFP/GF (low-dose DFP); 0.50 mg/kg DFP/60 mg/kg PMSF (potentiated DFP); 60 mg/kg PMSF/GF (PMSF alone); 60 mg/kg PMSF/1.5 mg/kg DFP (protected DFP); and 1.5 mg/kg DFP/GF (high-dose DFP). Two hens from each group were used to assay brain neurotoxic esterase (NTE) 24 h after the challenge dose, and the remaining hens were scored for deficits in walking, standing, and perching ability on d 18. Three hens from each group were perfusion-fixed on d 22 and neural tissues were prepared for histologic evaluation. DFP and/or PMSF caused > 88% brain NTE inhibition in all treated groups, compared to control. Protected DFP yielded no clinical deficits and a distribution and frequency of axonal lesions similar to control. PMSF alone produced a small increase in the frequency of lesions in the cervical spinal cord and peripheral nerves compared to control. Low-dose DFP caused minimal ataxia and increased frequency of axonal lesions in dorsal and lateral cervical spinal cord, ventral lumbar spinal cord, and inferior cerebellar peduncles (ICP) compared to control. Potentiated DFP and high-dose DFP produced maximal ataxia and essentially identical increases in the frequency of lesions in dorsal and ventral thoracic spinal cord, lateral lumbar spinal cord, and peripheral nerves compared to low-dose DFP. The results indicate that PMSF potentiation of mild OPIDN induced in adult hens by low-dose DFP results in an overall pattern of axonal degeneration like that produced by a threefold higher dose of DFP alone, and support the hypothesis that potentiation causes an increase in the frequency of axonal lesions in central and peripheral loci normally affected by OPIDN.
Assuntos
Axônios/efeitos dos fármacos , Encefalopatias/induzido quimicamente , Encéfalo/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Isoflurofato/toxicidade , Sistema Nervoso Periférico/efeitos dos fármacos , Fluoreto de Fenilmetilsulfonil/toxicidade , Animais , Ataxia/induzido quimicamente , Ataxia/fisiopatologia , Axônios/patologia , Biomarcadores , Encéfalo/enzimologia , Encéfalo/patologia , Encefalopatias/patologia , Hidrolases de Éster Carboxílico/metabolismo , Galinhas , Sinergismo Farmacológico , Feminino , Sistema Nervoso Periférico/enzimologia , Sistema Nervoso Periférico/patologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Nervo Tibial/efeitos dos fármacos , Nervo Tibial/patologiaRESUMO
Phenylmethylsulfonyl fluoride (PMSF), a nonneuropathic inhibitor of neurotoxic esterase (NTE), is a known potentiator of organophosphorus-induced delayed neurotoxicity (OPIDN). The ability of PMSF posttreatment (90 mg/kg, sc, 4 hr after the last PSP injection) to modify development of delayed neurotoxicity was examined in 2-, 5-, and 8-week-old White Leghorn chickens treated either one, two, or three times (doses separated by 24 hr) with the neuropathic OP compound phenyl saligenin phosphate (PSP, 5 mg/kg, sc). NTE activity was measured in the cervical spinal cord 4 hr after the last PSP treatment. Development of delayed neurotoxicity was measured over a 16-day postexposure period. All PSP-treated groups exhibited > 97% NTE inhibition regardless of age or number of OP treatments. Two-week-old birds did not develop clinical signs of neurotoxicity in response to either single or repeated OP treatment regimens nor following subsequent treatment with PMSF. Five-week-old birds were resistant to the clinical effects of a single PSP exposure and were minimally affected by repeated doses. PMSF posttreatment, however, significantly amplified the clinical effects of one, two, or three doses of PSP. A single exposure to PSP induced slight to moderate signs of delayed neurotoxicity in 8-week-old birds with more extensive neurotoxicity being noted following repeated dosing. As with 5-week-old birds, PMSF exacerbated the clinical signs of neurotoxicity when given after one, two, or three doses of PSP in 8-week-old birds. Axonal degeneration studies supported the clinical findings: PMSF posttreatment did not influence the degree of degeneration in 2-week-old chickens but resulted in more severe degeneration (relative to PSP only exposure) in cervical cords from both 5- and 8-week-old birds. The results indicate that PMSF does not alter the progression of delayed neurotoxicity in very young (2 weeks of age) chickens but potentiates PSP-induced delayed neurotoxicity in the presence of 0-3% residual NTE activity in older animals. We conclude that posttreatment with neuropathic or nonneuropathic NTE inhibitors, following virtually complete NTE inhibition by either single or repeated doses of a neuropathic agent in sensitive age groups, can modify both the clinical and morphological indices of delayed neurotoxicity. This study further supports the hypothesis that potentiation of OPIDN occurs through a mechanism unrelated to NTE.
Assuntos
Hidrolases de Éster Carboxílico/antagonistas & inibidores , Inibidores Enzimáticos/toxicidade , Isoflurofato/análogos & derivados , Compostos Organofosforados/toxicidade , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Fluoreto de Fenilmetilsulfonil/toxicidade , Medula Espinal/efeitos dos fármacos , Fatores Etários , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Galinhas , Sinergismo Farmacológico , Feminino , Isoflurofato/toxicidade , Medula Espinal/enzimologia , Medula Espinal/patologiaAssuntos
Corpo Carotídeo/enzimologia , Inibidores Enzimáticos/farmacologia , Hipóxia/enzimologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Fluoreto de Fenilmetilsulfonil/farmacologia , Fosfatidilinositóis/fisiologia , Respiração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fosfolipases Tipo C/antagonistas & inibidores , Animais , Corpo Carotídeo/efeitos dos fármacos , Corpo Carotídeo/ultraestrutura , Gatos , Ativação Enzimática , Inibidores Enzimáticos/toxicidade , Hiperventilação/etiologia , Hiperventilação/fisiopatologia , Hiperventilação/prevenção & controle , Hipóxia/complicações , Proteínas do Tecido Nervoso/farmacologia , Fluoreto de Fenilmetilsulfonil/toxicidade , Respiração/fisiologia , Fosfolipases Tipo C/fisiologiaRESUMO
This paper reports studies in hens showing that diisopropyl phosphorofluoridate (DFP) neuropathy is promoted by PMSF when initiated either in central (spinal cord) or peripheral nervous system. Moreover, the critical site for promotion is in peripheral nerve axons rather than in their cell bodies. Selective promotion in peripheral nerves was achieved by giving PMSF into sciatic artery monolaterally (7 mg/kg) to birds where neuropathy was initiated by DFP, either systematically (0.3 mg/kg s.c.) or intra-arterially (0.04 mg/kg in the same artery). Birds developed monolateral neuropathy in the leg where PMSF was delivered. Promotion of spinal cord neuropathy was achieved by giving PMSF (120 mg/kg s.c.) to birds where neuropathy was initiated selectively in spinal cord. This was obtained by protecting peripheral axons with intra-arterial bilateral injections of PMSF (0.55 x 2 mg/kg) followed by DFP (0.3, 0.4 or 0.7 mg/kg s.c.). The resulting syndrome was characterized by spastic ataxia.
Assuntos
Isoflurofato/toxicidade , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Fluoreto de Fenilmetilsulfonil/toxicidade , Doenças da Medula Espinal/induzido quimicamente , Animais , Ataxia/induzido quimicamente , Axônios/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Galinhas , Interações Medicamentosas , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/toxicidade , Feminino , Isoflurofato/administração & dosagem , Nervos Periféricos/efeitos dos fármacos , Nervos Periféricos/enzimologia , Fluoreto de Fenilmetilsulfonil/administração & dosagem , Medula Espinal/efeitos dos fármacos , Medula Espinal/enzimologiaRESUMO
Little is known regarding early biochemical events in organophosphate-induced delayed neurotoxicity (OPIDN) except for the essential inhibition of neurotoxic esterase (NTE). We hypothesized that a trophic factor may be produced in situ shortly after exposure to the OP which participates in the progression of OPIDN. To bioassay for such a growth-modulating factor(s), we treated chickens with the neuropathic agents diisopropylfluorophosphate (DFP) or cyclic phenyl saligenin phosphate (PSP), with or without phenylmethylsulfonyl fluoride (PMSF, a chemical which markedly modifies OPIDN). Soluble extracts of cervical spinal cord (a region of the nervous system which degenerates with OPIDN) were collected 24 h later and these were incubated with human neuroblastoma SY5Y cells in culture. The cells were allowed to grow for another 6 days and observed for changes in morphology and growth. After 3 days in culture, tissue extracts from OP-treated chickens caused SY5Y cells to begin to elongate and extend processes (neurites), similar to cells treated with nerve growth factor (1 microgram/ml). Extracts from chickens not receiving OP had no or minimal effects on cell morphology. In addition, extracts from chickens in which OPIDN was prevented by pretreatment with PMSF did not cause the marked extension of cell processes exhibited after exposure of SY5Y cells to extracts from chickens given regimens known to cause OPIDN. In parallel-treated animals. DFP and PSP caused clinical dysfunction characteristic of OPIDN, PMSF posttreatment markedly amplified the clinical deficits and PMSF pretreatment prevented OPIDN. In vivo DFP treatment also caused a marked reduction in the activity of the growth-related enzyme ornithine decarboxylase (ODC) in spinal cord but DFP was without effect on ODC activity in vitro (up to 1 mM final concentration). Characterization of this growth-modulating factor(s) may aid in the elucidation of pathological mechanisms of OPIDN.
Assuntos
Fatores de Crescimento Neural/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Sistema Nervoso/efeitos dos fármacos , Compostos Organofosforados/toxicidade , Animais , Galinhas , Feminino , Humanos , Isoflurofato/toxicidade , Masculino , Ornitina Descarboxilase/metabolismo , Fluoreto de Fenilmetilsulfonil/farmacologia , Fluoreto de Fenilmetilsulfonil/toxicidade , Células Tumorais CultivadasRESUMO
Phenyl di-n-pentylphosphinate (PPP) is a potent inhibitor of neuropathy target esterase (NTE) with negligible effect on acetylcholinesterase: I50S at 37 degrees C for 20 min and pH 8, respectively are 0.2 microM and > 2mM. PPP is not neuropathic. This is compatible with the fact that inhibited NTE is autopsy material from hens dosed with PPP can always be reactivated in vitro, presumably because no 'aging' reaction has occurred. PPP (10 mg/kg s.c.) given to hens up to 4 days before severely neuropathic doses (1.7 mg/kg) of diisopropylphosphorofluoridate (DFP) prevented neuropathic but not cholinergic effects of DFP. Hens given PPP 3 days after a sub-neuropathic dose of DFP (0.4 mg/kg) developed severe clinical neuropathy (clinical scores of 7 and 5 compared with DFP-plus-solvent scores 0,1,3). These prophylactic and promoting effects are similar to those exerted by phenylmethanesulphonyl fluoride (PMSF) at doses which inhibit NTE. In 3 out of 4 birds a pre-dose with PMSF (15 mg/kg) prevented the promoting effect of 120 mg/kg PMSF given after DFP.