Enantioselective Metabolism and Interference on Tryptophan Metabolism of Myclobutanil in Rat Hepatocytes

Myclobutanil, (RS)‐2‐(4‐chlorophenyl)‐2‐(1H‐1, 2, 4‐triazol‐1‐ylmethyl) hexanenitrile is a widely used triazole fungicide. In this study, enantioselective metabolism and cytotoxicity were investigated in rat hepatocytes by chiral HPLC‐MS/MS and the methyl tetrazolium (MTT) assay, respectively. Furthermore, tryptophan metabolism disturbance in rat hepatocytes after myclobutanil exposure was also evaluated by target metabolomics method. The half‐life (t1/2) of (+)‐myclobutanil was 10.66 h, whereas that for (?)‐myclobutanil was 15.07 h. Such results indicated that the metabolic process of myclobutanil in rat hepatocytes was enantioselective with an enrichment of (?)‐myclobutanil. For the cytotoxicity research, the calculated EC₅₀ (12h) values for rac‐myclobutanil, (+)‐ and (?)‐myclobutanil were 123.65, 150.65 and 152.60 µM, respectively. The results of tryptophan metabolites profiling showed that the levels of kynurenine (KYN) and XA were both up‐regulated compared to the control, suggesting the activation effect of the KYN pathway by myclobutanil and its enantiomers which may provide an important insight into its toxicity mechanism. The data presented here could be useful for the environmental hazard assessment of myclobutanil. Chirality 27:643–649, 2015. © 2015 Wiley Periodicals, Inc.     

外源色氨酸对油菜幼苗色氨酸下游代谢网络及生长发育的影响

色氨酸是合成蛋白质的重要氨基酸,也是植物生长激素IAA和某些次生代谢产物的前体物质,对植物生长发育及病虫害防御有重要作用。为了探究色氨酸对白菜型油菜(Brassica rapa L)生长发育及防御物质累积的影响及其可能的机制,该研究采用外源色氨酸对油菜幼苗进行叶面喷施,分析了色氨酸对油菜幼苗生长发育及生长素IAA和次生代谢产物芥子油苷合成的影响。结果表明：(1)低浓度色氨酸(100 mg/L)处理可有效地促进油菜叶片与根系的发育,但随着浓度增高,促进作用逐渐减弱。(2)荧光定量PCR分析表明,外源色氨酸处理后,油菜幼苗叶片中生长素IAA的3条合成途径都被激活,IPA途径的BrTAA1和BrYUCCA8、IAM途径的BrAMI1及IAOx途径的BrCYP71A13和BrNIT2等关键酶基因的表达均受到强烈的诱导,因而导致IAA的含量显著提高。(3)外源色氨酸处理还激活了下游的吲哚族芥子油苷的合成途径调控因子基因BrMYB34、BrMYB51和BrMYB122以及合成酶基因BrCYP79B2、BrCYP79B3、BrCYP83B1、BrSUR1的表达,同时抑制了其降解酶基因BrTGG1、BrPEN2的表达,从而引起吲哚族芥子油苷的累积。研究发现,外源色氨酸处理可通过调控生长素IAA合成途径和吲哚族芥子油苷的合成途径相关基因表达,有效地促进油菜生长调节物质和生物防御物质的累积,从而增加生物量和提高潜在抗病能力。     

Effects of Dietary Pyrazinamide on the Metabolism of Tryptophan to Niacin in Streptozotocin-diabetic Rats

We investigated the effects of feeding with a diet containing pyrazinamide (PYR) on the metabolism of l-tryptophan (Trp) to nicotinamide in streptozotocin (STZ)-diabetic rats and whether the diabetic action of STZ is prevented by feeding with the PYR diet, which is known as an inhibitor of aminocarboxy-muconate-semialdehyde decarboxylase and poly(ADP)ribose synthetase and therefore, significantly increases the formation of nicotinamide from Trp in normal rats. As was expected, feeding with the PYR diet to the STZ-injected rats caused a significantly increased excretion of nicotinamide and its metabolites like that in normal rats. The body weight increased in the STZ-injected rats fed with the PYR diet, while it was lost in the STZ-injected rats fed with the non-PYR diet. However, the blood glucose level and the urinary excretion of glucose were not improved even when the rats were fed with the PYR diet. Therefore, it was suggested that chronically increasing the formation of nicotinamide from Trp could not completely prevent the STZ-diabetic action.     

Seasonally Dependent Effect of Ectopic Pituitary Grafts on 24-Hour Rhythms in Serum Prolactin and Gonadotropins in Rats

To assess to what extent the presence of an ectopic pituitary differentially affected circulating prolactin (PRL) and gonadotropin levels at different times of the year, rats kept under 12h light, 12h dark (12:12 LD) photoperi-ods and receiving a pituitary graft or a sham operation in summer or winter were examined 3 months later. In both male and female sham-operated rats, a circadian variation in serum PRL levels was found, with an acrophase varying from 21:53h to 00:54h and the mesor and amplitude higher in spring than autumn in males and higher in autumn than in spring in females. After grafting a pituitary, changes in serum PRL related to time of day were no longer observed. In pituitary-grafted male rats killed during spring, serum PRL levels were higher than controls at only a few time points throughout the 24h cycle, whereas in rats killed during autumn, there were no significant differences in PRL levels between grafted and control rats. Pituitary-grafted female rats killed during spring showed serum PRL levels significantly higher than those of sham-operated rats, while in female rats killed in autumn, PRL levels of pituitary-grafted and sham-operated rats did not differ. Significant variations of luteinizing hormone (LH) related to time of day were found in sham-operated male rats only, with acrophases at 23:52h and 00:24h for spring and autumn, respectively, and the mesor and amplitude of the rhythm significantly higher in autumn. Pituitary transplants suppressed 24h variations in circulating LH and depressed its levels during the two seasons examined. As far as follicle-stimulating hormone (FSH), pituitary grafts decreased circulating levels, with the extent of decrease higher during autumn than in spring. The results indicate that some endocrine consequences of the grafting of an ectopic pituitary are dependent on time of year.     

Seasonally Dependent Effect of Ectopic Pituitary Grafts on 24-Hour Rhythms in Serum Prolactin and Gonadotropins in Rats

To assess to what extent the presence of an ectopic pituitary differentially affected circulating prolactin (PRL) and gonadotropin levels at different times of the year, rats kept under 12h light, 12h dark (12:12 LD) photoperi-ods and receiving a pituitary graft or a sham operation in summer or winter were examined 3 months later. In both male and female sham-operated rats, a circadian variation in serum PRL levels was found, with an acrophase varying from 21:53h to 00:54h and the mesor and amplitude higher in spring than autumn in males and higher in autumn than in spring in females. After grafting a pituitary, changes in serum PRL related to time of day were no longer observed. In pituitary-grafted male rats killed during spring, serum PRL levels were higher than controls at only a few time points throughout the 24h cycle, whereas in rats killed during autumn, there were no significant differences in PRL levels between grafted and control rats. Pituitary-grafted female rats killed during spring showed serum PRL levels significantly higher than those of sham-operated rats, while in female rats killed in autumn, PRL levels of pituitary-grafted and sham-operated rats did not differ. Significant variations of luteinizing hormone (LH) related to time of day were found in sham-operated male rats only, with acrophases at 23:52h and 00:24h for spring and autumn, respectively, and the mesor and amplitude of the rhythm significantly higher in autumn. Pituitary transplants suppressed 24h variations in circulating LH and depressed its levels during the two seasons examined. As far as follicle-stimulating hormone (FSH), pituitary grafts decreased circulating levels, with the extent of decrease higher during autumn than in spring. The results indicate that some endocrine consequences of the grafting of an ectopic pituitary are dependent on time of year.     

Effect of Vitamin E Deficiency on Rat Brain Monoamine Metabolism

We investigated the effects of vitamin E deficiency on the monoamine metabolism in the rat brain. Male Wistar rats fed on the vitamin E deficient diet for 24 weeks were analyzed. At 28 weeks, they showed a reduced growth rate (52% of reduction), muscle atrophy, a motor weakness of hind limbs and disturbance of gait. The concentrations of monoamines, their precursors and metabolites in the brain were simultaneously determined using high performance liquid chromatography (HPLC) coupled with a coulometric detection with electrode array system. In addition, tryptophan hydroxylase activity was measured. The dopamine (p = 0.009) and serotonin (p = 0.04) levels in the brain stem of vitamin E deficients rats were significantly lower than in the controls, whereas their precursors tyrosine (p = 0.0009) and tryptophan (p = 0.0065) levels in the brain stem were significantly higher than in the controls. Moreover, tryptophan hydroxylase activity (p = 0.0005) in the brain stem of vitamin E deficient brains was significantly lower than in the controls. All statistical comparisons were done using non-parametric tests (Mann-Whitney U test). These results suggest that vitamin E deficiency may play a role in the disturbance of monoamine metabolism in rat brain.     

High Doses of Vitamin B6 in the Rat Are Associated with Inhibition of Hepatic Tryptophan Metabolism and Increased Uptake of Tryptophan into the Brain

Abstract: Acute administration of vitamin B₆ to rats (10 mg/kg body weight) led to reduced urinary excretion of N ¹-methyl nicotinamide and methyl pyridone carboxamide, indicating inhibition of the oxidative metabolism of tryptophan. There was a considerable reduction in the production of ¹⁴CO₂ from [ ring -2-¹⁴C]tryptophan, and a significant inhibition of hepatic tryptophan oxygenase when measured in liver homogenates, together with an increase in the concentration of tryptophan in plasma. There was an increase in both the concentration of tryptophan in the brain and the uptake into the brain of peripherally administered [³H]tryptophan, accompanied by a small increase in the rate of synthesis of 5-hydroxy-tryptamine in the brain. It is suggested that this increase in the uptake of tryptophan into the brain following a relatively large dose of vitamin B₆ may explain the beneficial action of the vitamin in some cases of depressive illness.     

Circadian Variation in the Uptake of Tryptophan by Cortical Synaptosomes of the Rat Brain

The kinetics of the high affinity uptake system for L-tryptophan (L-Try)have been measured over 24 hr in cortical synaptosome preparations of rat brain. Both the K_m and V_max, of the uptake process showed a statistically significant 24 hr variation. The highest K_m value, 6.71 ± 10^-5 M, was measured at the beginning of the light phase and the lowest value, 4.23 ± 10^-5 M, 6 hr into the dark phase. V_max was highest at the end of the dark phase (10.43 nmol/mg/5 min) and lowest (4.80 nmol/mg/5 min) 3 hr into the dark phase. In contrast, there was no variation over 24 hr in the V_max/K_m ratio. These results suggest that the high affinity uptake process serves to ensure a constant rate of L-tryptophan entry into the neuron in the face of circadian or ultradian variations in extracellular concentration of tryptophan.     

Circadian Variation in the Uptake of Tryptophan by Cortical Synaptosomes of the Rat Brain

The kinetics of the high affinity uptake system for L-tryptophan (L-Try)have been measured over 24 hr in cortical synaptosome preparations of rat brain. Both the K_m and V_max, of the uptake process showed a statistically significant 24 hr variation. The highest K_m value, 6.71 ± 10^-5 M, was measured at the beginning of the light phase and the lowest value, 4.23 ± 10^-5 M, 6 hr into the dark phase. V_max was highest at the end of the dark phase (10.43 nmol/mg/5 min) and lowest (4.80 nmol/mg/5 min) 3 hr into the dark phase. In contrast, there was no variation over 24 hr in the V_max/K_m ratio. These results suggest that the high affinity uptake process serves to ensure a constant rate of L-tryptophan entry into the neuron in the face of circadian or ultradian variations in extracellular concentration of tryptophan.     

The Effect of Tryptophan Present in Plant Root Exudates on the Phytostimulating Activity of Rhizobacteria

Aseptic tomato and radish roots were found to exude, respectively, 2.8–5.3 and 290–390 ng tryptophan per seedling per day. The inoculation of radish plants with rhizosphere pseudomonads increased the root biomass by 1.4 times. The inoculation of tomato plants with the same pseudomonads was ineffective. The beneficial effect of bacterial inoculation on the radish plants can be explained by the fact that the introduced rhizobacteria produce the plant growth–stimulating hormone indole-3-acetic acid. In pot experiments, the addition of this phytohormone to the soil increased the mass of radish roots by 36%. The phytohormonal action of the rhizosphere microflora was found to be efficient provided that the concentration of tryptophan in the rhizosphere is sufficiently high.     

氯胺酮对抑郁大鼠模型抗抑郁作用研究

目的:探讨在抑郁大鼠模型中单次氯胺酮可产生快速持久地抗抑郁作用。方法:实验一:32只Wistar大鼠随机分为四组(n=8),药物干预前1 d大鼠强迫游泳15 min,药物干预当天,分别腹腔注射相同容积的生理盐水(S组)、5 mg/kg氯胺酮(K5组)、10 mg/kg氯胺酮(K10组)、15 mg/kg氯胺酮(K15组)。30 min后记录大鼠运动能力及不动时间。实验二:20只Wistar大鼠随机分为两组(n=10),所有大鼠均经历21天慢性不可预知应激试验。第22天大鼠分别腹腔注射相同容积生理盐水及10 mg/kg氯胺酮,于干预前、干预后1 h、2 h、6 h、1 d、4 d、7 d分别进行敞箱试验,并记录大鼠水平运动及垂直运动得分。结果:与S组相比,K5、K10及K15组大鼠运动能力无明显变化(P>0.05)且强迫游泳不动时间均显著减少(P<0.01);与干预前生理盐水组相比,生理盐水干预后1 h、2 h、6 h、1 d、4 d及7 d组大鼠敞箱试验水平运动及垂直运动均无明显差异(P>0.05);与干预前氯胺酮组相比,生理盐水干预后1 h、2 h、6 h、1 d、4 d及7 d组大鼠敞箱试验水平运动及垂直运动有明显差异(P<0.05)。结论:在抑郁症大鼠模型中氯胺酮可产生快速且持久的抗抑郁作用。     

Carbon Balance Studies of Glucose Metabolism in Rat Cerebral Cortical Synaptosomes

Synaptosomes were isolated from rat cerebral cortex and incubated with [U-14C]-, [1-14C]- or [6-14C]glucose. Glucose utilization and the metabolic partitioning of glucose carbon in products were determined by isotopic methods. From the data obtained a carbon balance was constructed, showing lactate to be the main product of glucose metabolism, followed by CO2, amino acids and pyruvate. Measuring the release of 14CO2 from glucose labelled in three different positions allowed the construction of a flow diagram of glucose carbon atoms in synaptosomes, which provides information about the contribution of the various pathways of glucose metabolism. Some 2% of glucose utilized was calculated to be degraded via the pentose phosphate pathway. Addition of chlorpromazine, imipramine or haloperidol at concentrations of 10(-5) M reduced glucose utilisation by 30% without changing the distribution pattern of radioactivity in the various products.     

Chronic Antidepressant Administration Alters the Subcellular Distribution of Cyclic AMP-Dependent Protein Kinase in Rat Frontal Cortex

The influence of chronic administration of antidepressants on cyclic AMP-dependent protein kinase activity was examined in rat frontal cortex. Chronic administration of imipramine, tranylcypromine, or electroconvulsive seizures decreased cyclic AMP-dependent protein kinase activity in soluble fractions by approximately 25%, whereas enzyme activity was increased in the particulate fractions by approximately 20%. In contrast, enzyme activity in crude homogenates was not altered. This effect appears to be specific to antidepressant drugs, because representatives of several other classes of psychotropic drugs-namely, haloperidol, morphine, and diazepam--failed to alter either soluble or particulate levels of cyclic AMP-dependent protein kinase activity in this brain region following chronic administration. When the total particulate fraction was subfractionated, it was found that chronic imipramine treatment significantly increased the activity of cyclic AMP-dependent protein kinase in crude nuclear fractions but not in crude synaptosomal or microsomal fractions. Taken together, the data raise the possibility that chronic antidepressant treatments may stimulate the translocation of cyclic AMP-dependent protein kinase from the cytosol to the nucleus. This effect would represent a novel action of antidepressants that could contribute to the long-term adaptive changes in brain thought to be essential for the clinical actions of these treatments.     

Activity Rhythms and Masking Response in the Diurnal Fat Sand Rat Under Laboratory Conditions

Daily rhythms are heavily influenced by light in two major ways. One is through photic entrainment of a circadian clock, and the other is through a more direct process, referred to as masking. Whereas entraining effects of photic stimuli are quite similar in nocturnal and diurnal species, masking is very different. Laboratory conditions differ greatly from what is experienced by individuals in their natural habitat, and several studies have shown that activity patterns can greatly differ between laboratory environment and natural condition. This is especially prevalent in diurnal rodents. We studied the daily rhythms and masking response in the fat sand rat (Psammomys obesus), a diurnal desert rodent, and activity rhythms of Tristram’s jird (Meriones tristrami), a nocturnal member of the same subfamily (Gerbillinae). We found that most sand rats kept on a 12?h:12?h light-dark (LD) cycles at two light intensities (500 and 1000?lux) have a nocturnal phase preferences of general activity and higher body temperature during the dark phase. In most individuals, activity was not as stable that of the nocturnal Tritram’s jirds, which showed a clear and stable nocturnal activity pattern under the same conditions. Sand rats responded to a 6-h phase advance and 6-h phase delay as expected, and, under constant conditions, all tested animals free ran. In contrast with the nocturnal phase preference, fat sand rats did not show a masking response to light pulses during the dark phase or to a dark pulse during the light phase. They did, however, have a significant preference to the light phase under a 3.5?h:3.5?h LD schedule. Currently, we could not identify the underlying mechanisms responsible for the temporal niche switch in this species. However, our results provide us with a valuable tool for further studies of the circadian system of diurnal species, and will hopefully lead us to understanding diurnality, its mechanisms, causes, and consequences.     

The Effect of Endurance Training on Regional Serotonin Metabolism in the Brain During Early Stage of Detraining Period in the Female Rat

1. The effect was examined of a single bout of nonexhaustive endurance exercise on tryptophan (Try), serotonin (5-HT), 5-hydroxyindolacetic acid (5-HIAA), and tryptophan hydroxylase (TpH) levels in different parts of rat brain (brain cortex, cerebellum, hypothalamus, midbrain striatum, medulla) on the last day of endurance training and 48 h later (detraining period).2. Female rats were subjected to a 6-week endurance training programme. The effectiveness of the training was evaluated by measuring anaerobic threshold (AT). High performance liquid chromatography (HPLC) was used to determine regional Try, 5-HT, and 5-HIAA contents in the brain, and thin layer chromatography followed by gas-liquid chromatography was used to determine blood levels of free fatty acids. Regional TpH levels were measured by Western blot analysis.3. In the two rat groups subjected to endurance training, in all brain regions studied but cerebellum, 5-HT content was significantly lower after the last bout of nonexhaustive endurance exercise than in resting control rats that were not subjected to the training. Similarly, the cortical and striatal, but not cerebellar, 5-HT/Try ratios were significantly lower in the trained rats at the end of the last training session and at the end of a single bout of nonexhaustive exercise administered after a 48-h detraining period than in the controls. TpH protein level was decreased by 15–25% after the last bout of exercise either during the training process or after the and 1 h bout of endurance exercise performed 48 h after cessation of endurance training in brain cortex and striatum but not cerebellar.4. These results indicate that the reduction in 5-HT level was the adaptive response to endurance training. The lowered 5-HT/Try ratio and lowered TpH protein level attained after the training process suggests and that this change may be, at least partially, attributed to downregulation of TpH activity.     

The Effect of Old Age on the Free-Running Period of Circadian Rhythms in Rat

The free-running period is regarded to be an exclusive feature of the endogenous circadian clock. Changes during aging in the free-running period may therefore reflect age-related changes in the internal organization of this clock. However, the literature on alterations in the free-running period in aging is not unequivocal. In the present study, with various confounding factors kept to a minimum, it was found that the free-running periods for active wakefulness, body temperature, and drinking behavior were significantly shorter (by 12-17 min) in old than in young rats. In addition, it was found that the day-to-day stability of the different sleep states was reduced in old rats, whereas that of the drinking rhythm was enhanced. Transient cycles were not observed, nor were there any age-related differences in daily totals of the various sleep-wake states. The amplitudes of the circadian rhythms of active wakefulness, quiet sleep, and temperature were reduced, whereas those of paradoxical sleep and quiet wakefulness remained unchanged.