In vivo and in vitro studies of the hepatotoxic effects of 4-chlorophenol in mice

4-Chlorophenol (4-CP) was studied for its toxicological effect on liver by using both in vivo and in vitro approaches. Male mice were administered 4-CP, 1.5 mmol/kg body weight, i.p., and were killed at 10, 20, 30 and 50 min after drug injection. Either i.p. or oral 4-CP administration significantly lowered total liver thiol levels by 20-30% after 30 min and 3 hr respectively. This time-dependent effect of 4-CP after i.p. treatment was enhanced when mice were pretreated with hepatic microsomal enzyme inducers (phenobarbital, 40 mg/kg body weight, b.i.d., 7 days; and beta-naphthoflavone, 80 mg/kg body weight once daily, 4 days). Further, the microsomal cytochrome P-450 inhibitor, SKF 525-A, 75 mg/kg body weight injected i.p. to mice 30 min prior to 4-CP administration, blocked the reduction of liver thiol content produced by 4-CP. The results suggest that a chemically reactive intermediate of 4-CP may be formed in liver which is responsible for the observed decrease in liver thiol content. Other investigations were done to characterize the in vitro irreversible binding of [14C]4-CP. [14C]4-CP was bound irreversibly to mouse liver microsomal proteins in a concentration-dependent manner. Binding was NADPH dependent and gave a maximal binding of 12.0 nmol/mg protein/20 min and an apparent binding constant of 0.222 mM. [14C]-Binding of 4-CP was increased by 155 and 127% in liver microsomes of phenobarbital- and beta-naphthoflavone. SKF 525-A, and CO:O2 (4:1, v/v)] and selected nucleophilic compounds (glutathione, L-cysteine or L-lysine) significantly reduced [14C]4-CP binding to mouse liver microsomes. An epoxide hydrolase inhibitor, cyclohexene oxide, did not alter the extent of irreversible binding, whereas scavengers of superoxide anions or agents that are reported to reduce accumulation of active semiquinone and quinone species (L-ascorbic acid, superoxide dismutase or epinephrine) decreased the binding of [14C]4-CP to mouse liver microsomal proteins by 56, 31 and 92% respectively. The data suggest that semiquinone and quinone species of 4-CP may be the chemically reactive intermediates leading to the in vivo reduction of liver thiol levels. Since 4-CP is a minor contaminant and possible metabolite of clofibrate and chemically related hypolipidemic agents, 4-CP and its metabolites may be partly responsible for some of the hepatotoxic effects seen after long-term administration of this therapeutic class of drugs.     

In vivo metabolism of norbormide in rats and mice

Norbormide's species-selective lethality displays 150-fold and 40-fold more sensitivity to rats than mice and guinea pigs, respectively. Our previous study revealed marked inter-species differences in rate and route of metabolism in liver preparations from different species, with hydroxylation the major route. To examine whether rapid metabolic clearance or species-dependent formation of a toxic metabolite play a role in the marked species-sensitivity, we initiated in vivo metabolic studies in rats and mice. After oral dosing, norbormide was detected in mouse but not rat blood. In contrast, liver analysis revealed that norbormide concentration was significantly higher in rat compared with mouse, and that it underwent extensive metabolism tentatively identified via hydroxylation in rat, whilst none was detected in mouse. Although an unidentified metabolite (M3) was detected in rat blood after oral dosing, no metabolites were detected 1min after intravenous dosing, which proved lethal at 0.5mg/kg. Taken together, the data indicate that the toxicity resides with the parent compound, rather species-dependent formation of a potent metabolite and that species sensitivity may be controlled at the pharmacodynamic level.     

加替沙星聚氰基丙烯酸正丁酯纳米粒小鼠体内分布特征

目的:研究注射用加替沙星聚氰基丙烯酸正丁酯纳米粒(GPN)的体内分布特征。方法:雄性小鼠随机分为3组,分别尾缘静脉给予小鼠加替沙星生理盐水溶液(Ⅰ)、未经修饰的加替沙星GPN(Ⅱ)以及经聚山梨酯-80修饰的GPN(T-GPN,Ⅲ),高效液相法检测血浆、肝、肾、脾及肺脏组织内的浓度,评价GPN的体内分布特征。结果:Ⅰ,Ⅱ,Ⅲ组小鼠肺脏组织中的平均峰浓度分别为17.33,10.13和26.99μg.g-1;平均浓度-时间曲线下面积(AUC0~t)分别为85.99,100.00和242.34 mg.kg-1.h。结论:GNP经聚山梨酯-80修饰后,加替沙星肺脏组织分布量显著增加,具有一定的肺靶向性。     

Influence of 1-methyl-3-keto-4-phenylquinuclidinium bromide on rat tissue monoamine levels in vivo.

The effects of 1-methyl-3-keto-4-phenylquinuclidinium bromide (MA540) on tissue monoamine levels in male rats have been compared to those of guanethidine. Neither compound influenced norepinephrine (NE) or 5-hydroxytryptamine (5-HT) levels in brain or epinephrine (E) levels in adrenal medulla. MA540 is about twice as potent as guanethidine as a heart NE depleting agent at both 8 and 24 h after drug administration. The durations of action of the compounds are similar. Unlike guanethidine, MA540 does not deplete NE in small intestine, which suggests that the drug may not elicit guanethidine-like intestinal side effects.     

In vivo formation of a glutathione conjugate derived from thalidomide in humanized uPA-NOG mice

Metabolism of the teratogen thalidomide is proposed to be relevant to its toxicological action. We demonstrated the formation of the glutathione (GSH) conjugate of (R)-5-hydroxythalidomide in vivo in chimeric NOD-scid IL2Rg(null) mice with humanized livers (uPA-NOG mice). After an oral administration of racemic thalidomide (270 mg/kg), plasma concentrations of 5-hydroxythalidomide were significantly higher in humanized mice than in control mice. The GSH conjugate of 5-hydroxythalidomide was detected in the plasma. These results indicate that livers of humanized mice mediate thalidomide 5-hydroxylation and further oxidation leading to the GSH conjugate in vivo as well as in vitro and suggest that thalidomide activation occurs.     

In vivo micronucleus test in mice with 1-phenylethanol

1-Phenylethanol is one of the major primary phase-I metabolites of ethylbenzene. In principle it may yield an electrophilic intermediate by phase-II metabolism. Because of the extensive use of ethylbenzene as a solvent, 2-year carcinogenicity inhalation studies were carried out leading to renal hyperplasia and tubular neoplasms both in male and female rats and alveolar/bronchiolar neoplasms in male mice and hepatocellular neoplasms in female mice. Whereas the mechanism underlying the increased renal tumor incidences in rats has been clarified, the mechanism of tumor formation (genotoxic or nongenotoxic mode of action) in the lung and liver of mice is still unclear. The genotoxicity data available to date for 1-phenylethanol include in vitro studies using either bacteria (Salmonella reverse mutation assay, E. coli Pol A⁺/Pol A⁻ test) or mammalian cells (mouse lymphoma assay, chromosome aberration test and sister chromatid exchanges using CHO cells). These experiments, however, did not always follow current standard procedures and some of the data obtained are compromised and not always convincing. The present database thus does not allow a definitive assessment of the in vitro genotoxic potential of 1-phenylethanol. The in vitro database suggests that clastogenicity may be the most relevant genetic end point, and therefore an in vivo micronucleus assay in mouse bone marrow was carried out. The animals were given 1-phenylethanol in single oral doses up to the maximum tolerated dose of 750 mg/kg body weight. Bone marrow was sampled 24 and 48 h after treatment. Under the experimental conditions used, there was no evidence of increased micronuclei frequencies at any dose or sampling time. These findings indicate that 1-phenylethanol is not clastogenic in vivo. This information, together with other negative or inconclusive genotoxicity data available so far, suggests a nongenotoxic mode of action responsible for the lung and liver tumors observed in mice following 2 years of inhalation exposure to ethylbenzene.     

In vivo electrophysiological examination of 5-HT2 responses in 5-HT2C receptor mutant mice

The present study used 5-HT2C receptor mutant mice and their wild-type littermates to characterize the 5-HT2 receptor using the 5-HT2 agonists (+/-)-2-dimethoxy-4-iodoamphetamine hydrochloride (DOI) and 1-(3-chlorophenyl)piperazine (mCPP) applied locally in the orbitofrontal cortex (OFC) and head of the caudate nucleus. Microiontophoretically-applied 5-HT, DOI and mCPP induced current-dependent inhibition of neuronal firing activity in both brain regions. There was no difference between 5-HT2C receptor mutants and wild-type mice in the ability of 5-HT or DOI to inhibit neuronal firing at any current used. In contrast, there was a reduced ability of mCPP to inhibit firing activity in the OFC when ejected at 10 nA. Unexpectedly, there was a small but significant increase in mCPP-induced inhibition in the caudate nucleus of mutant mice. In the OFC, the 5-HT2A antagonist MDL 100907 (2 mg/kg, i.p.) significantly antagonized the effect of both DOI and mCPP. In contrast, the non-selective 5-HT antagonist clozapine (10 mg/kg, i.p.) significantly antagonized only mCPP in the wild-type mice. However, neither MDL 100907 nor clozapine antagonized DOI or mCPP in the caudate nucleus. Finally, it required significantly less quisqualate to activate neurons in the 5-HT2C receptor mutants than in the wild-type mice, suggesting that 5-HT2C receptors serve a tonic inhibitory role in membrane excitability. The present results indicate that the inhibitory action of DOI is predominantly mediated by the 5-HT2A receptor in the OFC. mCPP, when applied locally, inhibits OFC firing activity by acting on both 5-HT2A and 5-HT2C receptors. However, DOI and mCPP might be acting in the caudate nucleus through an atypical 5-HT2 receptor yet to be characterized.     

The role of 2-keto-4-pentenoic acid in seizures induced by allylglycine.

2 Keto-4-pentenoic acid (2K4PA) was synthesised from l-allylglycine (L-AG) using purified l-amino acid oxidase and isolated relatively free from allylglycine (AG) contamination. 2 keto-4-pentenoic acid is a potent inhibitor of cerebral glutamic acid decarboxylase (GAD) activity, being one to two thousand times more effective than AG. Simultaneous addition of 2K4PA and substrate to GAD gave classical competitive inhibition plots, with a if K_i of 10^?6 M. Preincubation of GAD with 2K4PA for 30 min before addition of substrate produced non-competitive kinetic plots. In the absence of substrate, maximal inhibition of GAD activity was seen within 2–5 min and was only partially reversed by dialysis. The activities of cerebral DOPA decarboxylase, glutamine synthetase and GABA-transaminase in vitro were unchanged compared to control activities at concentrations of 2K4PA which produced marked inhibition of GAD activity. In vivo, 2K4PA was twenty two and fifty five times more effective than l-allylglycine and d-allylglycine respectively, at inducing seizures in mice after intracerebroventricular injection. The demonstration that 2K4PA is both a more potent convulsant and a more potent GAD inhibitor than the parent compound, AG, is consistent with the metabolic conversion of AG to 2K4PA in vivo and rationalises previously observed difference between the effects of AG on GAD activity in vitro and in vivo.     

In vivo fate of folate-BSA in non-tumor- and tumor-bearing mice

KB tumor cells exhibit an increased number of folate receptors on their membrane. This receptor has been proposed as a promising target for tumor drug targeting. Therefore, the disposition of folate-conjugated bovine serum albumin (folate-BSA) was examined as a model system for drug targeting. Nude mice which had received KB tumor cell transplants were given bolus intravenous administration of either 111In-labeled folate-BSA (111In-folate-BSA; 1 mg/kg) or unmodified 111In-BSA (111In-BSA; 1 mg/kg). The disposition characteristics and pharmacokinetics of 111In-folate-BSA were compared with those of the 111In-BSA as a control. The half-life of the beta-phase of 111ln-folate-BSA in plasma was 140 min. The tumor uptake rate index for 111In-folate-BSA was 0.46 microL/min/g, and that for 111In-BSA was 0.32 microL/min/g. This index of 111In-folate-BSA was slightly higher than that of 111In-BSA in vivo, by a factor of 1.4. In vivo experiments showed folate-BSA has a relatively long plasma duration. 111In-folate-BSA also showed selective distribution to tumors, but not as great as recent results from in vitro experiments. Therefore, the low vascular permeability of BSA into solid tumor tissue and inhibition of folate-mediated 111In-folate-BSA uptake by tumor cells from the blood may be the rate-limiting factor of distribution.     

In vivo osteoarthritis target validation utilizing genetically-modified mice

Osteoarthritis (OA) is a progressive disease of cartilage degradation that significantly impacts quality of life. There are currently no effective treatments and, while a large number of potential therapeutic targets exist, most have not been validated in vivo. The range of OA models in the mouse has dramatically expanded in the last decade, beyond spontaneous models, to include genetically modified transgenic, knockout (KO) and knock-in (KI) mice that can develop premature cartilage degeneration reminiscent of OA. In addition, instability models of OA, either induced by intra-articular (IA) collagenase or surgery, are providing a set of tools to assist in the identification of disease-modifying OA drug (DMOAD) targets. These models are now vital tools to dissect the pathways essential to the pathogenesis of OA. Two targets, ADAMTS (a disintegrin and metalloproteinase with thrombospondin-like motifs)-5 and IL-1beta (interleukin-1 beta), have been validated in the surgical destabilization of the medial meniscus model (DMM) in KO mice. Other potential targets evaluated in instability models, either showed no disease modification or a worsening of disease, suggesting that those targets have no role, a protective role or that other, more destructive enzymes etc., can overcompensate. Development of small molecule or protein antagonist inhibitors of therapeutic targets require many years to bring to clinical trials and often confront potency and safety issues which impede successful progress. Validation, or confirmation of therapeutic targets in vivo is most clearly and efficiently obtained by using KO studies, than by creating potent and selective DMOADs to multiple potential targets. While the results in the mouse will not always transpose to the human condition, the track record of mouse knockouts corresponding to the human phenotype have been excellent. These results indicate that the evaluation of genetically modified mice will become increasingly important as we unravel the genes contributing to OA.     

In vivo antibacterial activity of cefoperazone in intraperitoneal infections in mice

The in vivo antibacterial activity of cefoperazone (CPZ) against systemic infections was studied in comparison with that of cefotiam (CTM) using beta-lactamase non-producing bacteria and producing bacteria. In vitro antibacterial activity of CPZ against Gram-positive bacteria was slightly inferior to that of CTM, but there was no significant difference between their in vivo activities. On the other hand, the therapeutic effect of CPZ against Gram-negative bacteria was nearly parallel to in vitro antibacterial activity and particularly was superior to that of CTM against cephalosporinase (CSase) producing bacteria. The ascitic levels of CPZ in mice infected with CSase producing bacteria were persisted longer, but those of CTM disappeared quickly after administration. This result appeared to reflect on the therapeutic effects of both drugs.     

In vivo metabolism and kinetics of ethylene glycol monobutyl ether and its metabolites, 2-butoxyacetaldehyde and 2-butoxyacetic acid,as measured in blood,liver and forestomach of mice

1. Ethylene glycol monobutyl ether (EGBE) causes forestomach hyperplasia and neoplasia in mice when administered chronically by inhalation.

2. The study was initiated to test the physiologically based pharmacokinetic (PBPK) model prediction that 2-butoxyacetaldehyde (BAL), a transient, labile intermediate in the oxidation of EGBE to butoxyacetic acid (BAA), is unlikely to achieve concentrations sufficient to cause DNA damage in target tissues.

3. Male and female B6C3F1 mice were administered a high oral dose of EGBE (600?mg?kg^?1), and tissues were collected at 5, 15, 45 and 90?min following the dose. The tissues were processed for determination of EGBE, BAL and BAA by gas chromatography-mass spectrometry.

4. BAL was detected at low concentrations in all tissues sampled and at all time points following EGBE administration (about 0.3–33?μM). BAL concentrations were highest in the initial samples (5?min) in all tissues and declined from that point.

5. BAL concentrations in liver and forestomach tissues corresponded to the peak concentrations predicted by an already published PBPK model, and are higher than BAL concentrations that could be achieved by inhalation exposure to EGBE.

6. Mouse inhalation exposure to EGBE is therefore unlikely to generate BAL concentrations in tissues sufficient to initiate a carcinogenic response.

     

In vivo metabolism and kinetics of ethylene glycol monobutyl ether and its metabolites, 2-butoxyacetaldehyde and 2-butoxyacetic acid, as measured in blood, liver and forestomach of mice

1. Ethylene glycol monobutyl ether (EGBE) causes forestomach hyperplasia and neoplasia in mice when administered chronically by inhalation. 2. The study was initiated to test the physiologically based pharmacokinetic (PBPK) model prediction that 2-butoxyacetaldehyde (BAL), a transient, labile intermediate in the oxidation of EGBE to butoxyacetic acid (BAA), is unlikely to achieve concentrations sufficient to cause DNA damage in target tissues. 3. Male and female B6C3F1 mice were administered a high oral dose of EGBE (600mgkg(-1)), and tissues were collected at 5, 15, 45 and 90min following the dose. The tissues were processed for determination of EGBE, BAL and BAA by gas chromatography-mass spectrometry. 4. BAL was detected at low concentrations in all tissues sampled and at all time points following EGBE administration (about 0.3-33 microM). BAL concentrations were highest in the initial samples (5 min) in all tissues and declined from that point. 5. BAL concentrations in liver and forestomach tissues corresponded to the peak concentrations predicted by an already published PBPK model, and are higher than BAL concentrations that could be achieved by inhalation exposure to EGBE. 6. Mouse inhalation exposure to EGBE is therefore unlikely to generate BAL concentrations in tissues sufficient to initiate a carcinogenic response.     

In vivo opiate binding unchanged in tolerant/dependent mice

The in vivo receptor binding was measured for an agonist and antagonist in the brains of naive mice and of mice made tolerant to and dependent on opiates by morphine pretreatment. There were significant differences between the receptor binding of these substances in the naive and in the tolerant/dependent animals. However, these differences disappeared 8 h after interruption of the morphine supply, although tolerance showed no decline. Therefore, the differences in the binding were obviously not produced by changes in the opiate receptors related to tolerance. Rather, the differences were probably caused by morphine in the brain of the tolerant/dependent mice pretreated with morphine and disappeared because the concentration of the morphine in the brains of these animals dropped during abrupt withdrawal. The fact that tolerance persisted for hours beyond this time indicated that tolerance is a time-dependent phenomena, not directly dependent on receptor occupation by an agonist.     

In vivo antioxidant activity of total flavonoids from indocalamus leaves in aging mice caused by D-galactose

Objective: The aim of this thesis is to explore antioxidant activity of total flavonoids extracted from indocalamus leaves. Method: Aging mice model was established by D-galactose induction. Three groups of mice were treated with total flavonoids extracted from indocalamus leaves at doses of 20, 40 and 80 mg/kg d bw respectively. The antioxidant status in the aging mice was measured by determining the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and total anti-oxidant capability (T-AOC) in the serum and liver and malondialdehyde (MDA) content in the serum, liver and brain. Results: Compared with control group, extracts of indocalamus leaves significantly enhanced activities of SOD, GSH-Px, CAT in the serum and liver, and decreased MDA content in the serum, liver and brain at the tested doses. Conclusion: Total flavonoids extracted from indocalamus leaves demonstrated the potent antioxidant activity.     

In vivo binding of naloxone to opioid receptors in morphine-dependent mice

In vivo binding assay of opioid receptors for naloxone was tested in morphine-dependent mice. The content of naloxone in the brain (whole brain minus cerebellum) and cerebellum was determined as the total binding and non-specific binding, respectively, 20 min after the intravenous injection of [3H]-naloxone. Acute treatment with 8 mg/kg of morphine sulfate markedly enhanced the non-specific binding of naloxone. In contrast, withdrawal for 6 hr after 72 hr of implantation of morphine decreased the non-specific binding of naloxone. The Scatchard analysis of the specific binding revealed two binding sites. The apparent low affinity Kd values of both morphine-tolerant and morphine-withdrawn mice were significantly decreased when compared to their respective controls. The apparent high affinity Kd and the high and low Bmax values were not altered by the morphine dependent-tolerant state.