高果糖喂养对大鼠骨骼肌内长链酯酰辅酶A含量的影响

目的：观察高果糖饮食喂养对大鼠骨骼肌内长链酯酰辅酶A( LCACoAs)含量的影响,并探讨高果糖饮食喂养大鼠肌细胞内LCACoAs与胰岛素抵抗( IR)的关系。方法 Wistar雄性大鼠40只随机分为对照组及高果糖组,每组20只。对照组进食普通饲料,高果糖组饮食中果糖占总热量34.5%。喂养8周,测定各组大鼠空腹血糖( FBG)、空腹胰岛素( FINS)、肌肉甘油三酯( TG)及肌细胞内LCACoAs的含量,正常葡萄糖高胰岛素钳夹技术测定葡萄糖输注率(GIR),并用RT-PCR法测定脂酰辅酶A合成酶1(ACS1)的基因表达。结果与对照组相比,高果糖组的FBG和FINS浓度、肌内TG和LCACoAs含量、ACS1的基因表达均明显增高,而GIR明显降低( P<0．01);TG和LCA-CoAs含量与GIR均呈负相关(P<0．01)。结论高果糖饮食可引起机体IR及肌肉LCACoA的堆积,肌肉LCACoA增多与ACS基因表达增高有关。     

Chronic Treatment with Naltrexone Prevents Memory Retention Deficits in Rats Poisoned with the Sarin Analog Diisopropylfluorophosphate (DFP) and Treated with Atropine and Pralidoxime

Humans and rats poisoned with sarin develop chronic neurological disabilities that are not prevented with standardized antidotal therapy. We hypothesized that rats poisoned with the sarin analogue diisopropylfluorophosphate (DFP) and resuscitated with atropine and pralidoxime would have long-term memory deficits that were preventable with naltrexone treatment. Long Evans rats (250–275 g) were randomized to: DFP (N = 8): single subcutaneous (SC) injection of DFP (5 mg/kg). Treatment (N = 9): DFP (5 mg/kg) followed by chronic naltrexone (5 mg/kg/day × 12 weeks). Control (N = 12): single SC injection of isopropyl alcohol, (DFP vehicle) followed by chronic naltrexone (5 mg/kg/day). If toxicity developed after injection, antidotal therapy was initiated with atropine (2 mg/kg) and pralidoxime (25 mg/kg) and repeated as needed. After 12 weeks, rats underwent testing for place learning (acquisition) across 5 days of training using the Morris Water Maze. On day 6 a memory retention test was performed. Statistical analysis was performed using IBM SPSS Statistics. Rats receiving DFP rapidly developed toxicity requiring antidotal rescue. No differences in acquisition were seen between the DFP vs. DFP + naltrexone rats. During memory testing, DFP-poisoned rats spent significantly less time (29.4 ± 2.11 versus 38.5 ± 2.5 s, p < 0.05) and traveled less distance (267 ± 24.6 versus 370 ± 27.5 cm, p < 0.05) in the target quadrant compared to the treatment group. Treatment rats performed as well as control rats (p > 0.05) on the test for memory retention. Poisoning with DFP induced impaired memory retention. Deficits were not prevented by acute rescue with atropine and pralidoxime. Chronic naltrexone treatment led to preserved memory after DFP poisoning.     

Safety of Administration of Human Butyrylcholinesterase and its Conjugates with Soman or VX in Rats

Abstract: We evaluated the effects of conjugated enzyme‐nerve agent product resulting from the inhibition of bioscavenger human serum butyrylcholinesterase (Hu BChE) by nerve agents soman or VX. Rats were trained on a multiple Fixed‐Ratio 32, Extinction 30 sec. (FR32, Ext30) schedule of food reinforcement and then injected (i.m.) with Hu BChE (30 mg/kg), equivalent amounts of Hu BChE–soman conjugate (GDC), Hu BChE–VX conjugate, oxotremorine (OXO) (0.316 mg/kg) or vehicle (n = 8, each group). On the day of injection and on 10 subsequent daily sessions, performance was evaluated on the FR32, Ext30 schedule. Neither conjugates nor Hu BChE produced a performance deficit under the schedule. OXO produced a substantial decrease in responding on the day of administration, with complete recovery observed on subsequent sessions. None of the treatments affected circulating acetylcholinesterase (AChE) activity when evaluated 24–72 hr after injection. The dose of Hu BChE produced a 20,000‐fold increase above baseline in circulating BChE activity. Pathological evaluation of organ systems approximately 2 weeks following administration of conjugates or Hu BChE alone did not show toxicity. Taken together, these results suggest that Hu BChE – nerve agent conjugates produced following bioscavenger protection against nerve agents soman and VX do not appear to be particularly toxic. These results add to the safety assessment of Hu BChE as a bioscavenger countermeasure against nerve agent exposure.     

Effects of Pyridostigmine and Cholinolytics on Cholinesterasx and Acetylcholine in Soman Poisoned Rats

ABSTRACT

Soman reduced blood and brain cholinesterase (ChE) activity to less than 15% and increased cerebral acetylcholine (ACh) levels to 137.4% of control. When pyridostigmine (P) was used as a prophylatic adjunct, it reduced blood ChE activity to 31.6% of control, failed to significantly alter brain ChE activity, and protected more than 70% of the blood (but not brain enzyme) from phosphonylation by Soman

Benactyzine (B) was more effective than atropine (A) in reducing cerebral ACh concentrations, while a combination of the two was more effective than either alone. A prophylaxis of P + A + B was effective in controlling ACh levels in rats poisoned with one LD50 dose of Soman, Since P did not diminish the effects of the cholinolytics on cerebral ACh, this (together with the enzyme data) suggests that the two cholinolytics alone provided the central protection.     

Effects of diisopropylfluorophosphate (DFP) on renal function in the rat

Numerous studies have suggested a role for the nervous system in renal function. Several cholinergic agents have been examined for effects in vivo and in renal slices. Effects mediated through the vascular system and also direct effects have been reported. In this study an irreversible cholinesterase inhibitor, diisopropylfluorophosphate (DFP) in doses of 1-4 mg/kg, was tested on renal function. A single dose of DFP (2, 3 or 4 mg/kg) caused an increased flow of urine of low osmolality over the 6 h after the administration of the drug with essentially a return to control status by 24 h after either of the lower doses. Twenty-four hours after 4 mg/kg, urine volume was less and urine osmolality greater than control. Renal and brain cholinesterases remained depressed 24 h after DFP. In acute experiments on anesthetized animals, inulin clearance was increased by the lower doses and decreased by the highest dose of DFP. Renal blood flow measured with an electromagnetic flow meter showed a similar dose-response relationship. However, urine flow increased at all doses. The increased urine flow associated with decreased inulin clearance (4 mg/kg) and renal blood flow (3 or 4 mg/kg) suggest a direct effect of DFP on renal tubular function. These effects do not appear to be related to inhibition of cholinesterase.     

Parathion and diisopropylfluorophosphate (DFP) toxicity in partially hepatectomized rats

The toxic effects of parathion and DFP in male rats either increased or remained unchanged after partial hepatectomy. LD₅₀ values and blood cholinesterase activities were used as indices of toxicity. These results suggest that parathion toxicity is most likely not due to hepatic conversion of parathion to paraoxon.     

On the binding of diisopropyl-phosphoro-fluoridate (DFP) to plasma proteins

Archives of Toxicology - Sephadex® gel filtration has been employed for the quantitative determination of the binding of3H-labeIled diisopropyl-phosphoro-fluoridate (DFP) in guinea-pig plasma.     

Interaction of cycloheximide and diisopropylphosphorofluoridate (DFP) during subchronic administration in rat

Male Sprague-Dawley rats daily treated with DFP (0.5 mg/kg/day, sc) exhibited signs of cholinergic toxicity such as tremors and muscle fasciculations between Days 3 and 5 comparable to those observed 15 min after a single acute signs-producing dose (1.5 mg/kg, sc). Further administration of DFP (0.5 mg/kg/day, sc) for 6-14 days led to tolerance development as evidenced by disappearance of the described toxicity signs. The protein synthesis inhibitor cycloheximide, when given in a nontoxic dose (0.5 mg/kg/day, sc) 1 hr before DFP (0.5 mg/kg/day, sc) administration, potentiated the DFP toxicity and rats died after the fifth injection. DFP-tolerant rats developed toxicity signs when subsequently treated with cycloheximide (0.5 mg/kg/day, sc) and DFP (0.5 mg/kg/day, sc). Each drug when given alone for 4 days caused 30-50% reduction of [14C]valine uptake in vivo into the free amino acids pool as well as its incorporation into proteins of brain and skeletal muscles. A combination of these drugs caused a significantly greater inhibitory effect on [14C]valine incorporation into proteins. Cycloheximide (0.5 mg/kg/day, sc) administered for 4 days did not significantly alter the levels of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), or carboxylesterase (CarbE) activities but potentiated the DFP-induced inhibition of the activities of these enzymes. It is concluded that the cycloheximide pretreatment potentiates DFP toxicity by a mechanism that is related to inhibition of the synthesis of proteins such as AChE, BuChE, and CarbE.     

Subchronic and Reproduction Studies with Dibutyl Phenyl Phosphate in Sprague-Dawley Rats

Dibutyl phenyl phosphate (DBPP) was administered to male andfemale. Sprague-Dawley rats in their diets in separate subchronic(91-day) and two-generation reproduction studies. Dose levelsof DBPP were 5, 50, and 250 mg/kg/day in both studies. In thereproduction study, cross-fostering was performed between somehigh-exposure and control litter offspring and dams followinga second mating of F₀ animals. Compared to control animals,body weights were consistently lower in high-exposure adultanimals in both studies; this observation was less consistentin mid-exposure adult rats. High-exposure rats in the subchronicstudy had decreased erythrocyte counts and hematocrit and hemoglobinlevels. They also had increased absolute and/or relative liverweights with concomitant decreased hepatocytic vacuolation andincreased fatty accumulation. In the reproduction study, matingand fertility indices were comparable among the parental animalsin both generations, but survivability among high-exposure pupsreared by control dams appeared to be decreased. Urinary bladderhistopathologic changes, consisting of mononuclear cell infiltrationand transitional epithelial hyperplasia, were noted in mid-and high-exposure rats from both studies. The no observableadverse effect level in both of these studies was 5 mg/kg/ day.     

Effect of diisopropyl phosphorofluoridate (DFP) on the somatosensory evoked potentials in rats

In rats anaesthetised with pentobarbitone, somatosensory evoked potentials, produced by stimulation of contralateral or ipsilateral forepaws were used to measure the activity in different neuronal populations. Computer derived averages of 32 consecutive responses yielded a stable and consistent measurement of the evoked potentials. The drug was applied to the cerebral cortex by using the cortical cup technique and changes were measured in the evoked potentials. Cortical application of DFP increased the amplitude of the repetitive afterdischarges without affecting the primary complex of the evoked potentials. It appears that the afferent pathways responsible for these repetitive afterdischarges involve cholinergic synapses.The author wishes to express sincere thanks to Dr. B. S. Meldrum of the M. R. C. Laboratories, Carshalton, for his valuable guidance and criticism. The author acknowledges the receipt of a Fellowship from the Wellcome Trust.     

Roles of mu-opioid receptors in development of tolerance to diisopropylfluorophosphate (DFP)

Anatomical evidence indicates that cholinergic and opioidergic systems are co-localized and acting on the same neuron. However, the regulatory mechanisms between cholinergic and opioidergic system have not been well characterized. In the present study, the potential involvement of mu-opioid receptors in mediating the changes of toxic signs and muscarinic receptor binding after administration of irreversible anti-acetylcholinesterase diisopropylfluorophosphate (DFP) was investigated. DFP (1 mg/kg/day, subcutaneous injection, s.c.)-induced tremors and chewing movements were monitored during the 28-day treatment period in mu-opioid receptor knockout and wild type mice. Autoradiographic studies of total, M1, and M2 muscarinic receptors were conducted using [(3)H]-quinuclidinyl benzilate, [(3)H]-pirenzepine, and [(3)H]-AF-DX384 as ligands, respectively. DFP-induced tremors in both mu-opioid receptor knockout and wild type mice showed tolerance development. However, DFP-induced tremors in mu-opioid receptor knockout mice showed delayed tolerance development than that of DFP-treated wild type controls. DFP-induced chewing movements in both mu-opioid receptor knockout and wild type mice failed to show development of tolerance after four weeks of treatment. M2 muscarinic receptor binding of DFP-treated mu-opioid receptor knockout mice was significantly decreased than that of the DFP-treated wild type controls in the striatum, but not in the cortex and hippocampus. However, there were no significant differences in total and M1 muscarinic receptor binding between DFP-treated mu-opioid receptor knockout and wild type mice in the cortex, striatum and hippocampus. These studies indicate that mu-opioid receptors play an important role through the striatal M2 muscarinic receptors to regulate the development of tolerance to DFP-induced tremors.     

Reproductive Toxicity of Butylated Triphenyl Phosphate and Tricresyl Phosphate Fluids in F344 Rats

The effects of tricresyl phosphate (TCP) and butylated triphenylphosphate (BTP)-based hydraulic fluid on reproduction were studiedin F344 rats using a modification of the National ToxicologyProgram's Continuous Breeding Protocol. Groups of breeding pairsreceived single daily oral doses of an equal volume of either0, 0.6, 1.0 g BTP/kg or 0.4 g TCP/kg in sesame oil or 1.7 gneat BTP/kg for up to 135 days. A naive control group allowedto breed, but not dosed or handled daily, demonstrated thatdaily dosing and handling of the rats had no effect on reproduction.The fertility index and number of litters born were significantlydecreased in rats exposed to 1.0 and 1.7 g BTP/kg and 0.4 gTCP/kg. The number of pups per litter was significantly decreasedin the TCP group. A crossover mating experiment using 0.4 gTCP/kg/day and 1.0 g BTP/kg/day groups, each mated with vehiclecontrols, demonstrated that TCP caused 100% infertility in malerats but did not affect reproduction in females. BTP causeda significant decline in reproduction in female rats characterizedby low mating and fertility indices, decreased number of litters,and abnormal estrous cycles. Fertility was decreased in theBTP-dosed male rats. Both sexes of rats in the crossover experimentwith TCP and BTP had significant decreases in terminal bodyweights and increases in adrenal gland and liver weights. OnlyTCP-dosed male rats had significantly decreased testicular andepididymal weights. TCP-dosed female rats had increased ovarianweights, while BTP-dosed females had significantly lower uterineweights. The results of this study indicate that BTP and TCPare reproductive toxicants in F344 rats.     

Prevention of diisopropylphosphorofluoridate (DFP)-induced skeletal muscle fiber lesions in rat

The objective of the present investigation was to assess the comparative efficacy of prophylactic treatment with d-tubocurarine (d-TC) (0.075 mg/kg), atropine sulfate (16 mg/kg), and atropine methylnitrate (16 mg/kg), employed singly or in combination against the diisopropylphosphorofluoridate (DFP)-induced myopathy in rat. DFP (1.5 mg/kg, s.c.) produced signs of cholinergic toxicity with predominantly peripheral involvement manifest as severe muscle fasciculations beginning within 5-7 min and persisting in excess of 4-6 h. Maximal muscle fiber necrosis was observed within 24 h. Rats were protected against the apparent behavioural and morphological changes as well as electrophysiological signs of neuromuscular toxicity by all pretreatment agents. Combined pretreatment with d-TC (0.075 mg/kg, s.c.) and atropine methylnitrate (16 mg/kg, s.c.) was found to be most effective in attenuating DFP-induced muscle fiber necrosis as evidenced by complete absence of lesions and the prevention of DFP-induced hyperactivity in nerve and muscle. Significant protection was afforded by all pretreatment agents when given alone. It is suggested that the pretreatment agents act presynaptically by preventing drug-induced backfiring and muscle fasciculations possibly by reducing the release of acetylcholine (ACh). The protective drugs in the concentrations used had no significant effect on the normal characteristics of conduction and transmission.     

Effects of Soman (Pinacolyl Methylphosphonofluoridate) on Coronary Blood Flow and Cardiac Function in Swine

The effects of soman (pinacolyl methylphosphonofluoridate) oncoronary blood flow, the electrocardiogram, and cardiac functionwere measured in -chloralose-anesthetized swine. Coronary bloodflow (CBF), mean arterial blood pressure (MAP), peak systolicleft ventricular pressure (LVP), maximum rate of left ventricularpressure development (dP/dt_max), cardiac output, and the ECGwere monitored continuously. A dose of 2x LD50 of soman (1 LD50= 4.6 .µg/kg) was given at 1 LD50/min in the femoral vein,which produced an increase in coronary sinus plasma acetylcholine(ACh) from a control of 0.7±0.01 nmol/ml to a maximum314% of control at 15 mm and a decrease in CBF from a controlof 99±13 ml/min/100 g to a minimum 55% of control at15 mm. The increase in ACh in the coronary sinus was significantlycorrelated with a decrease in CBF (r=–0.87, p<0.001).The fall in CBF was accompanied by concomitant decreases inIVP, MAP, and dP/dt_max, with S-T segment elevation and ventricularfibrillation. The increase in coronary sinus acetylcholine concentration was significantly correlated with a 10-fold fall incoronary sinus acetylcholinesterase levels from a control of2.47±0.97 mol acetylcholine hydrolyzed/ml blood/min andwas consistent with the time course for the reduced hemodynamicmeasurements. These studies support the hypothesis that acetylcholineincreases following soman toxicity may decrease coronary bloodflow, thereby initiating ischemic electrocardiographic changesand reducing cardiac function.     

Inhibition of a soman- and diisopropyl phosphorofluoridate (DFP)-detoxifying enzyme by Mipafox

Mipafox, N,N'-diisopropylphosphordiamidofluoridate, has been found to be a reversible competitive inhibitor of a diisopropyl phosphorofluoridate hydrolyzing enzyme (DFPase) isolated from hog kidney and Escherichia coli. Heretofore, this DFPase was characterized by its more rapid hydrolysis of Soman (1,2,2-trimethylpropyl methylphosphonofluoridate), its stimulation by Mn2+, and its wide distribution. In sharp contrast, Mipafox did not inhibit the DFPase found only in cephalopod nerve, hepatopancreas, and saliva, and further characterized by its more rapid hydrolysis of DFP than of Soman, and its indifference to Mn2+. Neither of these two DFPases hydrolyzed Mipafox.     

Acute effects of diisopropyl fluorophosphate (DFP) on autonomic and behavioral thermoregulatory responses in the Long-Evans rat

Experiments were designed to assess the mechanisms of diisopropyl fluorophosphate (DFP)-induced changes in thermoregulation of the rat. In one study, male rats of the Long-Evans strain were injected with DFP (s.c.) at doses ranging from 0 to 2.0 mg/kg while maintained at an ambient temperature (Ta) of 20--24 degrees C. Body (Tb) and tail skin (Tt) temperatures were recorded for 5 h post-injection. DFP doses of greater than or equal to 1.0 mg/kg resulted in significant decreases in Tb lasting up to 5 h and increases in Tt lasting up to 1 h post-injection. In a second study, metabolic rate (MR), evaporative water loss (EWL), motor activity (MA), Tb, and Tt were measured at 2 h post-injection of 0, 0.5, 1.0, and 1.5 mg/kg DFP (s.c.) at Ta values of 10, 20, and 30 degrees C. DFP treatment resulted in hypothermia at all three Ta values, but the effect was attenuated at 30 degrees C. MR was significantly reduced at a Ta of 20 degrees C following 1.5 mg/kg, unaffected by DFP at a Ta of 30 degrees C, and stimulated at 10 degrees C following 0.5 mg/kg DFP. EWL was significantly elevated at 30 degrees C following 1.5 mg/kg DFP. MA was significantly reduced following greater than or equal to 1.0 mg/kg DFP at 20 and 30 degrees C and 1.5 mg/kg at 10 degrees C. Tt was elevated and reduced by DFP at Ta values of 30 and 10 degrees C, respectively. In a third study, rats were injected with DFP and placed in a temperature gradient for 1 to 2 h post-injection while selected Ta and Tb were monitored. While both control and DFP-treated rats remained in the cool end of the gradient, rats administered DFP at doses of 1.0 and 1.5 mg/kg were significantly hypothermic. It was also found that Ta values of 10, 20, and 30 degrees C had no effect on DFP-induced inhibition of cholinesterase activity of plasma and erythrocyte fractions of whole blood. Overall, these data support the hypothesis that acute DFP may lower the set-point for the control of body temperature in the rat and demonstrates that the toxicity of DFP is modified by changes in Ta.