Biotransformation of allylbenzene analogues in vivo and in vitro through the epoxide-diol pathway

1. The epoxidation of allylbenzene, safrole, estragole, eugenol and eugenol methyl ether was investigated in rats pretreated with these compounds and also in vitro using hepatic microsomal preparations and adult rat liver cell cultures.

2. Dihydrodiols were detected in the urine and liver of rats pretreated with allylbenzene compounds. Similarly, incubation of allylbenzene epoxides with hepatic microsomal preparations and adult rat liver cell cultures gave rise to the formation of dihydrodiols.     

Variation of Commercial Diisopropyl Fluorophosphate Preparations in Toxicological Studies

ABSTRACT

The variation of three diisopropyl fluorophosphate (DFP) lots obtained from two sources was investigated. The effects of different lots of DFP on body weight, mortality, and brain acetylcholinesterase activity in rats varied significantly. The in vitro effects of these DFP preparations on braTn acetylcholinesterase activity also were markedly different. It appears that different commercial preparations of DFP possess different potencies and that the same apparent doses of different DFP preparations will not produce the same effects upon any physiological system under study.     

Cardiovascular effects of a labdenic diterpene isolated from Moldenhawera nutans in conscious,spontaneously hypertensive rats

Abstract

Context: The labdenic diterpene labd-8(17)-en-15-oic acid (labd-8) isolated from a methanolic extract of Moldenhawera nutans Queiroz & Alkin (Leguminosae) has hypotensive and tachycardiac properties in normotensive rats. A part of the hypotensive effect was due to a reduction in the sympathetic nerve drive to vessels, an event admittedly enhanced in spontaneously hypertensive rats (SHRs).

Objectives: We assessed whether the cardiovascular effects induced by labd-8 could be enhanced in SHRs.

Materials and methods: For in vivo experiments, arterial and venous catheters were implanted under anesthesia for blood pressure recording and drug administration, respectively. For in vitro experiments, thoracic aorta rings were suspended in organ baths containing warm (37?°C) perfusion medium that was continuously bubbled with carbogen.

Results: Intravenous injection of labd-8 (1, 3, 5, and 10?mg/kg) induced similar dose-dependent hypotension and tachycardia in both SHRs and Wistar–Kyoto rats (WKY). In SHRs, only the tachycardia response to labd-8 was significantly reduced by pretreatment with methylatropine or propranolol. However, both cardiovascular effects of labd-8 were reduced by hexamethonium while remained unchanged by l-NAME. In isolated aortic preparations from SHRs, labd-8 (1–1000?µg/mL) relaxed potassium-induced contractions with an IC₅₀ (geometric mean [95% confidence interval]) value (536.5 [441.0–631.9] µg/mL) significantly greater than that (157.6 [99.1–250.5] µg/mL) obtained in preparations from WKY rats.

Conclusion: In SHRs, the hypotension induced by labd-8 is associated with a reflex tachycardia and seems mediated partly through withdrawal of sympathetic vasomotor tone and partly through an active vasorelaxation. Its magnitude was not enhanced when compared with WKY rats likely because of impaired vasorelaxant effects of labd-8 in preparations from SHRs.     

Antidepressant-like effects of <Emphasis Type="Italic">Trichilia catigua</Emphasis> (Catuaba) extract: evidence for dopaminergic-mediated mechanisms

Rationale Currently available therapy for depression treatment is often associated with several undesirable side effects, and it is effective only in a certain portion of the population. Therefore, the identification of alternative therapeutic tools for the treatment of depression is still needed. Objective The present study analyzed the possible antidepressant-like effects of the Brazilian medicinal plant, Trichilia catigua, in rodents. Attempts were also made to investigate some of the possible mechanisms implicated in its actions. Methods The antidepressant-like effects of T. catigua extract were assessed in two species of rodents (mice and rats) by means of in vivo (forced swimming test) and in vitro (monoamine reuptake and release in synaptosomal preparations) approaches. Results Acute oral treatment with the extract of T. catigua produced antidepressant-like effects in the forced swimming model in both mice and rats. Anti-immobility actions of T. catigua extract in mice were significantly reversed by haloperidol or by chlorpromazine, but not by pimozide, ketanserin, spiroxatrine or p-chlorophenylalanine. In vitro, T. catigua extract concentration-dependently inhibited the uptake and increased the release of serotonin, and especially of dopamine, from rat brain synaptosomal preparations. Conclusions The present study provides convincing evidence for a dopamine-mediated antidepressant-like effect of the active principle(s) present in the hydroalcoholic extract of T. catigua in mice and rats when in vivo and in vitro strategies were employed. Therefore, a standardized T. catigua extract or its purified constituents could be of potential interest for the treatment of depressive disorders.     

Effects of picloram on xenobiotic biotransformation in rat liver

1. The effect of picloram on model xenobiotic substrate biotransformation in vivo was studied in female and male rat liver.

2. Treatment with picloram had little effect on epoxide hydratase and glutathione S-transferase activity, but caused a dose-dependent increase in ethoxyresorufin-O-deethylase activity and a concomitant decrease in aldrin epoxidase activity in male rats.

3. Treatment of male rats with equivalent doses of 2-acetylaminofluorene, 2-amino-anthracene and picloram induced ethoxyresorufin-O-deethylase activity to the same degree.

4. Treatment of female rats with picloram resulted in dose-dependent increases in ethoxyresorufin and ethoxycoumarin-O-deethylation without decreasing aldrin epoxidase activity.

5. Picloram binds to liver microsomal preparations from rats pretreated with phenobarbitone and/or 3-methylcholanthrene, giving a type I spectrum.

6. The results indicate that picloram is a 3-methylcholanthrene-type inducer, and the implications are discussed.     

DIABETIC RAT AORTA RESPONSIVENESS TO D600 AND NIFEDIPINE

1. The responsiveness of aortic rings from 4 and 12 week streptozotocin-induced diabetic rats to D600 (Gallopamil) and nifedipine was studied. 2. The sensitivity and responsiveness to D600 were significantly enhanced (P < 0.05; 5-test, ANOVA; 9 d.f.) only in the 4 week diabetic preparations precontracted with noradrenaline. 3. Nifedipine-induced relaxations were significantly enhanced (P < 0.05–0.01; t-test, ANOVA; 8–12 d.f.) in all the diabetic (4 and 12 weeks) aortic preparations precontracted with both noradrenaline (10^-7 mol/L) and KCl (40 mmol/L) when compared with controls. 4. D600, unlike nifedipine, did not produce significant relaxation of diabetic aortic preparations precontracted with KCl (40 mmol/L) at both week 4 and 12 of the disease when compared with controls. 5. These results suggest that there is differential responsiveness of streptozotocin diabetic rat aorta to D600 and nifedipine.     

The action of azapropazone, oxyphenbutazone and phenylbutazone on lysosomes

Azapropazone, oxyphenbutazone and phenylbutazone have a stabilizing action on isolated lysosomes over a wide concentration range but a lytic action at high concentrations. The lytic action of phenylbutazone was greater than the other drugs. Phenylbutazone at a high concentration was found to accelerate the breakdown of lysosomes in isolated stomach preparations in vitro. Phenylbutazone was found to have a greater ulcerogenic action than azapropazone in rats and rabbits. Tissues removed from rats and rabbits dosed with phenylbutazone showed evidence of lysosomal damage when examined histochemically for acid phosphatase. In contrast, tissues from control and azapropazone-treated rats showed no evidence of lysosomal damage. Sections of rat gut incubated with azapropazone and phenylbutazone in vitro showed similar results. The possibility is discussed that drugs in high concentration may damage lysosomes in the gastrointestinal tract. It is suggested that lysosomal damage may contribute to the ulcerogenic action of the drugs in vivo.     

Antidiabetic Activity of Cow Urine and a Herbal Preparation Prepared Using Cow Urine

An herbal preparation prepared by the traditional healers of Mandsaur using cow urine and Gymnema sylvestre R. Br. (Asclepiadaceae), Momordica charantia L. (Cucurbitaceae), Eugenia jambolana Lam. (Myrtaceae), Aegle marmelos Correa (Rutaceae), Cinnamomum tamala Buch.-Ham. (Lauraceae), Aloe barbadensis Linn. (Liliaceae), and Trigonella foenum-graecum L. (Leguminosae) is being used in the treatment of diabetes. In order to scientifically appraise the claim, this preparation was studied for antidiabetic activity and also compared with the herbal preparation prepared using water. Fresh cow urine was also used in the study to identify the synergistic effect. The preparations were tested for antidiabetic activity in alloxan-induced diabetic rats at two dose level, 200 and 400 mg/kg, respectively. The study was done for a period of 21 days. The activity was compared with reference standard, insulin (1 unit/kg, i.p.) and control. The herbal preparations significantly (P < 0.05, P < 0.01) lowered the blood sugar level of hyperglycemic rats in a dose-dependent manner. Comparatively, the cow urine preparation showed better activity than did the preparation prepared using water. Fresh cow urine also exhibited significant antidiabetic effect. This study supports the claim of the local traditional healers.     

Biotransformation of ketamine, (Z)-6-hydroxyketamine,and (E)-6-hydroxyketamine by rat,rabbit, and human liver microsomal preparations

1. (Z)- and (E)-6-Hydroxyketamine have been synthesized and their metabolism by hepatic microsomal preparations studied to elucidate the metabolism of ketamine.

2. Both 6-hydroxyketamines are exclusively converted to 6-hydroxy-norketamines by N-demethylation. The g.1.c. retention properties and mass spectral characteristics of these 6-hydroxy-norketamines were used to confirm the structures of ketamine metabolites.

3. Ketamine is converted to norketamine, 4-, 5- and 6-hydroxynorketamines and possibly 4- and 6-hydroxyketamines in hepatic microsomal preparations from rats, rabbits and man. Norketamine is the major metabolite in all species tested.

4. 6-Hydroxynorketamine is the major hydroxylated metabolite and is found only in the (Z)-form in the species examined.

5. The metabolism of ketamine and the 6-hydroxy-ketamines is greatly increased after phenobarbital pretreatment of rats and rabbits.     

Influence of gallic acid esters on drug-metabolizing enzymes of rat liver

The effect of three antioxidants, propyl, octyl and dodecyl gallate, on hepatic drug metabolism in male rats was studied in vivo and in vitro. When fed at a dietary concentration of 1% for 14 days, only dodecyl gallate increased relative liver weight. Cytochrome P-450 content was not influenced, but a slight increase in cytochrome b₅ content was observed after the feeding of propyl gallate. Monooxygenase activity (benzo[a]pyrene-hydroxylase and ethoxycoumarin-deethylase activities) was not affected by propyl or octyl gallate. but a significant decrease in benzo[a]pyrene-hydroxylase activity was apparent in rats fed dodecyl gallate. Study of benzo[a]pyrene-metabolite formation in liver microsome preparations from control and propyl gallate-treated rats showed an overall decrease in metabolite production following gallate treatment, the decrease being statistically significant for the formation of the 9,10-dihydrodiol. Epoxide-hydratase activity was enhanced by a factor of 1·5 in rats fed propyl gallate; glutathione-transferase activity was unaffected. In vitro, the gallates proved to be potent inhibitors of ethoxycoumarin deethylation in liver microsomes from untreated and phenobarbital-treated rats; however, when cytochrome P-448 had been induced by pretreatment with 3-methylcholanthrene, ethoxycoumarin deethylase was less sensitive to the inhibitory action of the gallates.     

Effect of early stage of experimental diabetes on vascular functions in isolated perfused kidneys

1 The present study investigates the renal vascular responsiveness to vasoactive agents in diabetic rats which present an early stage of renal failure. 2 Adult male Wistar rats were administered alloxan (150 mg kg^–1, s.c.). Seven days later the right kidneys were isolated and perfused. Renal perfusion pressure was measured continuously. Concentration-response curves were plotted for noradrenaline (NA), sodium nitroprusside (SNP) and carbachol. 3 In basal conditions, kidneys from diabetic rats presented a decreased vascular resistance compared with those from control rats. 4 The vasoconstrictor response to NA showed decreased EC₅₀ values in preparations from diabetic rats compared with control ones (EC₅₀ nmols, control: 2.03 ± 0.44, n = 8; diabetic: 0.84 ± 0.18, n = 6, P < 0.05). This enhanced sensitivity to NA could be in line with the decreased glomerular filtration rate and cortical renal plasma flow previously described in vivo in our laboratory ( Garcia et al. 1998 ) . Vasoconstrictor responses to phenylephrine were not however, different between diabetic and control rat kidneys. This suggests that the increased sensitivity to NA was due to impaired neuronal uptake since phenylephrine is not a substrate for neuronal uptake. 5 After precontraction with phenylephrine, both endothelium-dependent (carbachol) and endothelium independent (SNP) vasodilator agents caused similar response in the preparations taken from the two groups of animals. So, the enhanced sensitivity to NA is not associated with a deficient dilator responsiveness of the renal vasculature. 6 The vasodilator response to carbachol was the same in absence or presence of l -arginine in the perfusate, suggesting no alteration in its availability at this stage of diabetes. 7 Diabetic animals showed increased plasma level of fructosamine and glycosylated haemoglobin (Hb A_1c), indicating the presence of early glycated products at this stage of diabetes, which could be involved in a possible structural alteration of the vessels.     

十三种中药及民間草药对大鼠蛋白性及甲醛性关节炎的影响

用大鼠蛋白性及甲醛性关节炎的病理模型,对十三种中医临床已用或祖国医学文献上记载的中药和从地方搜集的民间单方进行了抗关节炎作用的研究.发现对大鼠蛋白性关节炎的抑制作用,以腰骨藤茎叶及白花地胆草为最突出,生姜、六月雪及腰骨藤根等次之,防风及细辛等则稍弱.对大鼠甲醛性关节炎的治疗作用则以怀牛膝酒剂及地胆草酒剂为最明显,细辛、薏仁及六月雪亦有一定作用.     

Kinetik der Dihydrofolsäurereduktase aus Rattenleber und -gehirn mit 3-APADPH+H+ als Wasserstoffdonator

Summary An enzyme preparation capable to reduce dihydrofolic acid to tetrahydrofolic acid, was prepared from liver and brain of rats. Using a spectrophotometric method the reaction rates of these preparations were determined. For rat liver dihydrofolic acid reductase K _m was found to 2×10^–6 M for NADPH + H⁺ and 1.8×10^–6 M for FH₂. Rat brain dihydrofolic acid-reductase has a K _m of 8×10^–6 M for NADPH + H⁺. Folic acid was shown to be a competitive inhibitor of the conversion of dihydrofolate to tetrahydrofolate. With 3-APADPH + H⁺ which is formed in brain of rats after application of 3-acetylpyridine, the enzyme activity of both preparations was found to be significantly decreased. These observations are discussed in relation to the functional impairment of rats treated with 3-acetylpyridine.

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Mit 4 Textabbildungen

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Ein Teil der Ergebnisse wurde auf der 5. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft vorgetragen.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

A STUDY OF THE EFFECTS OF HYPOTHYROIDISM INDUCED BY PROPYLTHIOURACIL ON ADRENOCEPTORS AND NORADRENALINE LEVELS IN THE VAS DEFERENS OF THE RAT

1. The anti-thyroid drug, propylthiouracil (0.05% w/v in drinking water) was administered to rats for 3–4 weeks. This treatment decreased the rate at which rats gained body weight but did not affect the weight of the vasa deferentia. Circulating levels of thyroxine and triiodothyronine were markedly decreased. 2. The potencies of postjunctional α-adrenoceptor agonists, 1-noradrenaline and 1-phenylephrine, in eliciting contraction of the epididymal end of the vas deferens were unaffected by propylthiouracil treatment. In contrast, the potency of the β-adrenoceptor agonist, 1-isoprenaline, in inhibiting field stimulation-induced twitches of the prostatic end of the vas deferens was decreased in tissues from propylthiouracil-treated rats. Thus hypothyroidism induced by propylthiouracil produced an apparent change in the properties of postjunctional β-adrenoceptors without a concomitant reciprocal change in the properties of postjunctional α-adrenoceptors. 3. Treatment of rats with propylthiouracil led to a small increase in the concentration of noradrenaline in the vas deferens, and a concomitant small increase in the rate of accumulation of ³ H-noradrenaline in in vitro experiments. Extra-neuronal uptake of ³H-isoprenaline was unaffected by propylthiouracil treatment. 4. The potencies of the prejunctional α-adrenoceptor agonists, xylazine and 1-noradrenaline, in producing inhibition of twitches evoked by field stimulation of preparations of the prostatic end of the vas deferens, were similar in preparations from propylthiouracil-treated and control rats, although the slope of the log dose-response curve to xylazine was decreased in the former group. Thus unequivocal changes in the properties of prejunctional α-adrenoceptors did not accompany the other prejunctional effects of propylthiouracil upon endogenous noradrenaline levels and neuronal uptake of ³H-noradrenaline in this organ.     

菟丝子黄酮对雷公藤多苷片致生精细胞周期阻滞、凋亡及相关蛋白表达降低的影响

目的 研究菟丝子黄酮对雷公藤多苷片致大鼠睾丸生精细胞周期阻滞、凋亡及相关蛋白表达降低的影响。方法 SD雄性大鼠随机分为5组：对照组,雷公藤多苷片高、低剂量（12、9 mg/kg）组,雷公藤多苷片高、低剂量（12、9 mg/kg）+菟丝子黄酮（生药100 mg/kg）组,每组6只。每天ig给药1次,连续给药7 d,对照组大鼠ig给予等体积0.5% CMC-Na溶液;腹主动脉采血,制备含药血清。通过体外培养大鼠睾丸生精细胞,给予含药血清干预24 h,采用流式细胞仪检测生精细胞周期及凋亡率;采用Western blotting技术检测生精细胞中SCF/c-kit、C-myc和CREM蛋白表达。结果 与对照组比较,雷公藤多苷片高剂量组使G₀/G₁期生精细胞百分率显著降低、S期细胞百分率显著升高（P < 0.01）,低、高剂量组均可显著升高生精细胞凋亡率（P < 0.01）;与等剂量雷公藤多苷片单给药组比较,配伍菟丝子黄酮后,显著降低S期细胞百分率、生精细胞凋亡率（P < 0.01）。雷公藤多苷片低、高剂量组均使生精细胞中SCF/c-kit、C-myc和CREM蛋白表达显著降低（P < 0.01）;配伍菟丝子黄酮后显著增加蛋白表达量（P < 0.01）。结论 通过合理配伍菟丝子黄酮可显著改善雷公藤多苷片对生精细胞的损伤作用。     

Brain muscarinic cholinergic receptor binding in Roman high- and low-avoidance rats

This experiment sought to determine whether the behavioral differences between the Roman high-(RHA/Verh) and low-avoidance (RLA/Verh) lines of rats could be related to differences in the number and/or affinity of brain muscarinic cholinergic receptors. The binding of the specific muscarinic antagonist ³H-quinuclidinyl benzilate to crude membrane preparations from the cerebral cortex, hippocampus and striatum was determined. There were no significant differences between the two rat lines for the number of muscarinic binding sites (B _max) or the apparent dissociation constant (K _D) as determined by Scatchard analysis of the saturation isotherms. These data indicate that the behavioral differences between RHA/Verh and RLA/Verh rats cannot be accounted for by differences in the number or affinity of brain muscarinic cholinergic receptors.     

Aflatoxin B1 metabolism in the rat: Polyhalogenated biphenyl enhanced conversion to aflatoxin M1

1. The effects of polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs) on the formation in vitro of aflatoxin Q1 and aflatoxin M1 from aflatoxin B1 by rat-liver microsomes were investigated.

2. AFB1 metabolism by hepatic microsomes from PBB- and PCB-treated rats resulted in 16- and 30-fold increases, respectively, in levels of aflatoxin M1. The enhanced formation of aflatoxin M1 did not correlate with PBB and PCB stimulation of benzo[a]pyrene hydroxylase (AHH) activity.

3. Studies in vivo clearly demonstrated enhanced secretion of aflatoxin M1 by female lactating rats with prior exposure to PCBs. PCB pretreatment enhanced the activity of mammary as well as hepatic tissue microsomal preparations in converting aflatoxin B1 to aflatoxin M1.

4. Our findings indicate that PCB exposure increases the production of aflatoxin M1 in vitro and also increases the levels of aflatoxin M1 released into the milk.     

Treating inflammation: some (needless) difficulties for gaining acceptance of effective natural products and traditional medicines

The quality of so-called 'natural medicines' is extraordinarily variable. Lack of resolute pharmacological assays contributes to this hiatus. More stringent evaluation of anti-inflammatory and anti-pyretic activities in rats can help resolve some of the uncertainties surrounding (a) preparations of some herbal products including so-called 'nature's aspirin' (e.g. willowbark, ginger), cat's claw, celery seed, etc., and (b) some animal lipids (e.g. Lyprinol® (NZ Mussel), emu and fish oils). These animal products can be a remarkable resource for supplementing conventional/allopathic therapy for inflammatory disease, e.g. providing lipoxygenase inhibitors. Beyond the verifiable science, the healing professions and the general public still need to examine more carefully criteria for QUALITY(S) in any alternative medicine—to ensure the good (= both reputations and products) are not destroyed by the bad—in essence counteracting Gresham's Law which states: the bad tends to displace the good.     

Absorption of interferon alpha from patches in rats

Interferon alpha (IFN-α), patch preparations composed of three layers, water-insoluble backing layer, drug containing layer with absorption enhancer and surface layer containing pH-dependent polymer were prepared. As absorption enhancer, three surfactants, Gelucire44/14 (Lauroyl macrogol-32 glycerides), Labrasol (Caprylocaproyl macrogol-8 glycerides) and HCO-60 (polyoxyethylated hydrogenerated castor oil) were used in preparing IFN-α patch preparations. The intestinal absorption of IFN-α was studied after the administration of test patch preparations into the rat jejunum, 50,000 IU/kg. The serum IFN-α levels were measured by an ELISA method and both C _max and AUC were determined as the index of absorption of IFN-α. Gelucire44/14 preparation including Pharmasol for the stable solidification showed the higher C _max, 7.66 ± 0.82 IU/ml, and AUC, 12.85 ± 1.49 IU h/ml, than Labrasol (6.51 ± 0.89 and 8.30 ± 1.34 IU h/ml) and HCO-60 (6.02 ± 1.14, 7.53 ± 1.84 IU h/ml) preparations, respectively. By comparing to the AUC obtained after s.c. injection of the same dose of IFN-α to rats, bioavailability (BA) was estimated to be 7.8% in Gelucire44/14 preparation. In vitro release study showed that the T50%s, the time when half of the formulated IFN-α is released from the patches, were 3.4 ± 0.1 min for HCO-60, 7.8 ± 0.1 min for Gelucire44/14 and 11.4 ± 0.1 min for Labrasol preparations. To study the effect of absorption site, Gelucire44/14 preparation was administered into the rat duodenum and ileum. However, there were not significant differences on AUC among the three absorption sites. By reducing the IFN-α dose from 50,000 to 25,000 IU/kg, the serum IFN-α levels vs time profile showed a tendency of dose-dependency. When the histological examination of small intestinal mucosa was carried out in this study, the small intestinal mucosa after the Gelucire44/14 patches administered and before it was administered, could not recognize impaired. From these results, the usefulness of oral patch system for the oral delivery of IFN-α has been proved in rats.     

Intraduodenal delivery of intrinsically and extrinsically labelled CaCO3 in the rat: effect of solubilization on calcium bioavailability*

Abstract— The dissolution of CaCO₃ before intraduodenal administration was found to be an important factor determining calcium (Ca) bioavailability. Extrinsically and intrinsically labelled ⁴⁷CaCO₃ preparations were sequentially dissolved by serial additions of HCl. Aliquots of these preparations were collected before (no HCl added) and during the solubilization process and administered intraduodenally to rats. Whole body ⁴⁷Ca retention 72 h post-dose was used as a measure of Ca bioavailability. Although dissolution of CaCO₃ significantly increased Ca bioavailability (p < 0·001), Ca from both intrinsically and extrinsically labelled CaCO₃ was absorbed and retained to some extent without prior acid dissolution. Due to a disproportionately high concentration of ⁴⁷Ca on the particle surface, extrinsically labelled ⁴⁷CaCO₃ overestimated bioavailability when unsolubilized or partially solubilized CaCO₃ preparations were used (P < 0·05). These data indicate that dissolution is a determining factor for Ca bioavailability from CaCO₃. Incomplete dissolution will significantly limit but not completely prevent Ca bioavailability. The disintegration and dissolution characteristics of commercial CaCO₃ preparations, which vary widely, may produce important differences in Ca absorption.