An experimental model for the determination of immunomodulating effects by volatile compounds.

An in vitro exposure system was developed to enable simultaneous exposure of primary cells or cell lines to defined concentrations of volatile organic compounds (VOC) without the necessity of a constant-flow exposure system. Toluene was used as model VOC and administered via the gas phase. CD3/CD28-stimulated human peripheral blood mononuclear cells (PBMC) were used as indicator cells. Vitality/proliferation of PBMC was tested using the MTT assay and their functional reactivity using cytokine ELISA for interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-13, and tumor-necrosis-factor-alpha (TNF-alpha). Chemical analysis using headspace gas chromatography confirmed that this new method guaranties reproducible VOC exposure (R2 = 0.995 for the correlation between external toluene concentration and toluene in the cell culture). While cytotoxic effects were not observed, dose-dependent toluene effects on functional reactivity of PBMC were found. The secretion of IFN-gamma, IL-4, and IL-13 was inhibited at concentrations of 72.5 g/m3 and above, whereas the TNF-alpha production was increased. Since the presented in vitro model ensures toluene exposure in concentrations comparable to the real situation, and allows the investigation of dose-dependent immunomodulatory toluene effects in concentrations without cytotoxicity, this method first described here is introduced as useful tool in analysis of VOC-triggered effects on immune cells.     

An experimental model of hepatic fibrosis induced by the administration of dibutyltin dichloride.

Dibutyltin dichloride (DBT) was investigated for its ability to induce hepatic fibrosis in rats. Since hydroxyproline does not appear in significant amounts in noncollagen protein and prolyl residues in collagen are not hydroxylated before they are in peptide-bound form, the conversion of isotopically labeled proline to hydroxyproline by prolyl hydroxylase was taken as one parameter reflecting the rate of collagen formation. In vitro collagen synthesis was determined by incubation of liver biopsies with labeled proline, after which incorporation of label into collagenase-digestible protein was compared to incorporation into collagenase-resistant protein. Hydroxyproline content served as a parameter of collagen accumulation. DBT, administered by oral intubation (10 and 20 mg/kg) daily for 4 days, produced inflammation in portal tracts and biliary damage at the end of this period. Prolyl hydroxylase activity was increased over control values in both treated groups, and in vitro collagen synthesis was increased in the higher dose group. DBT, administered by oral intubation (10 and 20 mg/kg) every other day for 12 days, produced extensive inflammation in portal tracts, biliary damage, fibrosis, necrosis, infarcted areas, and granulomatous lesions. In the higher dose group increases in hydroxyproline content, prolyl hydroxylase activity, and relative collagen synthesis in vitro were observed at the end of the 12-day period. DBT did not increase prolyl hydroxylase activity of L929 cells when added to culture media for 24 hr. These results suggest that DBT-induced fibrogenesis is a result of biliary damage and/or inflammatory processes rather than direct stimulation of fibroblasts.     

Validation of the hepatic blood flow rate model for verapamil first-pass metabolism.

The bioavailability of a new retard tablet formulation of verapamil was investigated in a randomized cross-over bioequivalence study on 12 healthy subjects. The drug was given in the form of a single 240-mg oral dose of a new retard tablet formulation, or as a standard retard tablet at the same dose to all subjects, followed by a single intravenous (i.v.) dose of 5 mg to 8 of the 12 subjects. Plasma verapamil concentrations were determined by a high performance liquid chromatography (HPLC) method. The bioavailability of the new peroral retard formulation was (20.00 +/- 4.30)% and was in reasonable agreement with that determined for the already registered verapamil retard formulation, i.e. (19.46 +/- 4.02)%, thereby indicating bioequivalence. For the prediction of systemic availability and estimation of the first-pass metabolism, only based on the data for peroral plasma levels, a hepatic blood flow rate limited model was used. In our experience, this model has been found to be extremely useful in providing reasonable estimates of verapamil first-pass effect.     

An understanding of flow- and diffusion-limited vs. Carrier-mediated hepatic transport: A simulation study

Concentration-dependent changes in the hepatic extraction ratio E and tissue accumulation of drugs were examined in a simulation study, wherein plasma protein binding, flow, and mode of entry were altered. A tubular flow model that described carrier-mediated (influx:K _ml =20 μM,V _max1=1000 nmol min⁻¹;effux,K _m2=200 μM,V _max2=250 nmol min⁻¹), flow-limited (influx clearanceCL _in=efflux clearanceCL _ef=50 ml min⁻¹), or diffusion-limited (CL _in=CL _ef=0.1 ml min⁻¹) hepatocytic entry was employed; drug removal was solelyvia biliary excretion (K _m3=100 μM,V _max3=1500 nmol min⁻¹). Other parameter space and the combination of carriermediated transport and passive diffusion were also explored. Increased plasma protein binding reduced the hepatic extraction of the substrate, and in some instances, constituted the ratecontrolling factor, especially at lower input concentrations for which tighter binding existed. Increased flow rate also brought about a reduction inE, affectingE almost inversely when values ofE were low (e.g., for the diffusion-limited case or at higher input concentration). Tissue accumulation patterns and the apparent tissue distribution equilibrium ratio, i.e., tissue to plasma unbound concentration ratioK _p, differed among the systems. The behavior ofK _p may be used as an identifier for the mode of drug transport: A declining (concave-down)K _p curve or a parabolicK _p that approached unity with input concentration (C _In) is associated with carrier-mediated entry; a risingK _p curve that approaches unity withC _In suggests flow limitation; and a waning concaveupK _p curve of very low magnitude represents diffusion limitation. Since the unbound tissue concentration (C _t) differs from the logarithmic average of the unbound input and output concentrations in plasma ( ) for carrier-mediated and diffusion-limited systems, excretion parameters may be obtained only upon fitting of the overall excretion ratevs.C _t in the Michaelis-Menten equation; whereas when data are fitted with , the rate-limiting step, influx, or deviations of influx, efflux, and excretion, will be obtained. WhenC _t equals , as in flow-limited systems, accurate excretion parameters will be provided with the fitting of data against eitherC _t or . This work was supported by the Medical Research Council of Canada (MA-9765, MA-9104) and the National Institutes of Health (GM-38250). Dr. W. P. Geng was supported by a postdoctoral fellowship in Basic Pharmacology from the Merck Sharp and Dohme Research Laboratories, West Point, PA. Karen Poon was supported by an Ontario Graduate Scholarship.     

Glycogen metabolism and glucose transport in experimental porphyria

Hexachlorobenzene (HCB) is a fungicide of well-known porphyrinogenic ability, which induces an experimental porphyria that resembles human porphyria cutanea tarda (PCT) in several animal species. It has been demonstrated that high glucose ingestion prevents porphyria development, and high-fat/high-protein diets enhance HCB porphyrinogenic ability. On the contrary, a diet rich in carbohydrates reduces HCB effects. The aim of this work was to study HCB effects on glycogen synthesis and degradation, as well as on glucose synthesis and transport, in order to elucidate whether would justify the beneficial use of carbohydrates in this porphyria. Rats were treated with HCB dissolved in corn oil (five daily doses 100mg/kg body weight). Results showed that: (1) HCB caused an increase in glycogen content; (2) glycogen synthase activity increased three times, and phosphorylase activity decreased about 40% due to fungicide intoxication. The effect of HCB on these two activities accounted for the higher glycogen content observed in treated animals; (3) three gluconeogenic enzymes were reduced 30-50%; (4) glucose uptake in the liver decreased in all weeks studied. The alterations found in glucose synthesis, its uptake in liver and other tissues, and its release from glycogen might contribute to the biochemical porphyria picture and would account for the effect of glucose above mentioned.     

An in vitro transport model for rapid screening and predicting the permeability of candidate compounds at blood-brain barrier

The aim of this study was to design and develop a simple in vitro blood-brain barrier (BBB) permeation model for elementarily and rapidly predicting the permeability of candidate compounds at BBB and further evaluating whether P-glycoprotein (P-gp) affects them across BBB. The model was mainly composed of cultured rat brain microvascular endothelial cells (rBMECs), glass contraption, and micropore membrane. First, we evaluated the model by morphological observation. Second, the restriction effects of paracellular transport were verified by measuring marker probes transport, and monitoring transendothelial electrical resistance (TEER) and leakage. Finally, protein expression and activity of P-gp were confirmed by carrying out Western blot analysis and polarized transport of rhodamine-123 (Rho123) in rBMECs. The rBMECs retained both endothelial cells and BBB features. The rBMECs model reproducibly attained approximately 130 Ω cm2 on the steady-state TEER value, and displayed a barrier function to marker probes transport by decreasing the permeability. Protein band of 170 kDa manifested the existence of P-gp in the rBMECs, and the findings of cyclosporin A-sensitive decrease of Rho123 efflux confirmed the presence of P-gp activity. A simple, rapid, and convenient in vitro BBB permeation model was successfully established and applied to evaluate the BBB transport profiles of three natural flavonoids: quercetin, naringenin, and rutin.     

Liver slices in dynamic organ culture. I. An alternative in vitro technique for the study of rat hepatic drug metabolism

1. Precision-cut liver slices in dynamic organ culture, a novel in vitro technique, is described and applied to the study of hepatic drug metabolism in the rat. 2. These slices catalysed the oxidative O-deethylation of the substrate, 7-ethoxycoumarin, over 6 h incubation. In addition, the direct conjugation of 7-hydroxycoumarin with either sulphate or glucuronic acid was maintained over 6 h. 3. The formation of 7-hydroxycoumarin and the presence of the sulphate and glucuronide conjugates in slices exposed to 7-ethoxycoumarin demonstrated integrated phase I and phase II drug metabolizing activities in this system. 4. Minor modifications of the incubation system allowed for the metabolism of four volatile chlorinated benzenes: monochlorobenzene 1,2-, 1,3-, and 1,4-dichlorobenzenes to aqueous soluble metabolites. 5. The use of liver slices in dynamic organ culture as an alternative preparation for the study of xenobiotic metabolism is discussed.     

Eletriptan metabolism by human hepatic CYP450 enzymes and transport by human P-glycoprotein.

"Reaction phenotyping" studies were performed with eletriptan (ETT) to determine its propensity to interact with coadministered medications. Its ability to serve as a substrate for human P-glycoprotein (P-gp) was also investigated since a central mechanism of action has been proposed for this "triptan" class of drug. In studies with a characterized bank of human liver microsome preparations, a good correlation (r2 = 0.932) was obtained between formation of N-desmethyl eletriptan (DETT) and CYP3A4-catalyzed testosterone 6 beta-hydroxylation. DETT was selected to be monitored in our studies since it represents a significant ETT metabolite in humans, circulating at concentrations 10 to 20% of those observed for parent drug. ETT was metabolized to DETT by recombinant CYP2D6 (rCYP2D6) and rCYP3A4, and to a lesser extent by rCYP2C9 and rCYP2C19. The metabolism of ETT to DETT in human liver microsomes was markedly inhibited by troleandomycin, erythromycin, miconazole, and an inhibitory antibody to CYP3A4, but not by inhibitors of other major P450 enzymes. ETT had little inhibitory effect on any of the P450 enzymes investigated. ETT was determined to be a good substrate for human P-gp in vitro. In bidirectional transport studies across LLC-MDR1 and LLC-Mdr1a cell monolayers, ETT had a BA/AB transport ratio in the range 9 to 11. This finding had significance in vivo since brain exposure to ETT was reduced 40-fold in Mdr1a+/+ relative to Mdr1a-/- mice. ETT metabolism to DETT is therefore catalyzed primarily by CYP3A4, and plasma concentrations are expected to be increased when coadministered with inhibitors of CYP3A4 and P-gp activity.     

Pesticide induced alterations in marrow physiology and depletion of stem and stromal progenitor population: An experimental model to study the toxic effects of pesticide

Long‐term exposure of agriculturally used organochloride and organophosphate pesticides have been shown to cause long‐lasting hematotoxicity and increased incidence of aplastic anemia in humans. The mechanisms involved in pesticide induced hematotoxicity and the features of toxicity that may play a major role in bone marrow suppression are not known. The aim of the present study was to investigate the hematological consequences of pesticide exposure in swiss albino mice exposed to aqueous mixture of common agriculturally used pesticides for 6 h/day, 5 days/week for 13 weeks. After the end of last exposure, without a recovery period, the strong hematotoxic effect of pesticide was assessed in mice with long‐term bone marrow explant culture (LTBMC‐Ex) system and cell colony forming assays. Bone marrow explant culture from the pesticide exposed group of mice failed to generate a supportive stromal matrix and did not produce adequate number of hematopoietic cells and found to contain largely the adipogenic precursors. The decreased cell colony numbers in the pesticide exposed group indicated defective maturational and functional status of different marrow cell lineages. As a whole, exposure of mice to the mixture of pesticides reduced the total number of bone marrow cells (granulocytes are the major targets of pesticide toxicity), hematopoietic, and non‐hematopoietic progenitor cells and most of the hematological parameters. Replication of primitive stem/progenitor cells in the marrow was decreased following pesticide exposure with G0/G1‐phase arrest of most of the cells. The progenitor cells showed decreased percentage of cells in S/G2/M‐phase. The increased apoptosis profile of the marrow progenitors (Increased CD95 expression) and primitive stem cells (High Annexin‐V positivity on Sca1+ cells) with an elevated intracellular cleaved caspase‐3 level on the Sca1+ bone marrow cells provided the base necessary for explaining the deranged bone marrow microenvironmental structure which was evident from scanning electron micrographs. These results clearly indicate a strong, long lasting toxic effect of pesticides on the bone marrow microenvironment and different microenvironmental components which ultimately leads to the formation of a degenerative disease like aplastic anemia. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 84–97, 2014.     

An in vivo rat model for assessment of extrahepatic metabolism.

INTRODUCTION: Xenobiotic metabolism in extrahepatic tissues has been extensively studied in vitro, but it is difficult to estimate in vivo the share of xenobiotic transformation in extrahepatic tissues for lack of a suitable approach. In this paper an in vivo rat model for assessment of extrahepatic metabolism is described, and the model was investigated using the conversion of lidocaine to monoethylglycinexylidide (MEGX). METHODS: The rats were anesthetized with ethyl ether inhalation. The liver was exposed, the liver artery ligated, and the portal vein was clamped at its distal end. The left hepatic lobe was partly excised along its inferior margin, and a heparinized silicone catheter, diameter 0.2 cm, was inserted into the portal and left hepatic veins to allow the recirculation of portal vein blood. A sham operation was performed in the control group. RESULTS: Phenol red test showed that hepatic blood supply was absolutely blocked in model rats. At 30 min after establishing the portal-cavum bypass, the renal function and electrolytes did not change, but serum glucose decreased by 64.4 +/- 30.4%; 30 min after intravenous administration of 1.0% lidocaine 2 mg x kg(-1), serum MEGX in model rats was 32.0 +/- 7.14% of that in the control group, which mostly existed in a free form and was not induced by phenobarbital pretreatment. DISCUSSION: The model is easy to establish and provides an in vivo method to study the extrahepatic metabolism of xenobiotics.     

An in vitro transport model for rapid screening and predicting the permeability of candidate compounds at blood–brain barrier

The aim of this study was to design and develop a simple in vitro blood–brain barrier (BBB) permeation model for elementarily and rapidly predicting the permeability of candidate compounds at BBB and further evaluating whether P-glycoprotein (P-gp) affects them across BBB. The model was mainly composed of cultured rat brain microvascular endothelial cells (rBMECs), glass contraption, and micropore membrane. First, we evaluated the model by morphological observation. Second, the restriction effects of paracellular transport were verified by measuring marker probes transport, and monitoring transendothelial electrical resistance (TEER) and leakage. Finally, protein expression and activity of P-gp were confirmed by carrying out Western blot analysis and polarized transport of rhodamine-123 (Rho123) in rBMECs. The rBMECs retained both endothelial cells and BBB features. The rBMECs model reproducibly attained approximately 130 Ω cm² on the steady-state TEER value, and displayed a barrier function to marker probes transport by decreasing the permeability. Protein band of 170 kDa manifested the existence of P-gp in the rBMECs, and the findings of cyclosporin A-sensitive decrease of Rho123 efflux confirmed the presence of P-gp activity. A simple, rapid, and convenient in vitro BBB permeation model was successfully established and applied to evaluate the BBB transport profiles of three natural flavonoids: quercetin, naringenin, and rutin.     

Structure-activity relationships in the induction of hepatic drug metabolism by azo compounds

1. Lipophilic azo compounds possessing 1-phenylazo-2-naphthol or 1-phenylazo-2-naphthylamine moieties induced cytochrome P-448 and related mono-oxygenase activities, UDP-glucuronyltransferase activity towards p-nitrophenol, glutathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene, aldehyde dehydrogenase, and menadione reductase activities. This pattern of induction by azo dyes is very similar to that by 3-methylcholanthrene.

2. None of the hydrophilic azo compounds tested and none of the other lipophilic azo compounds tested including 4-phenylazo-1-naphthol induced these activities.

3. It is suggested that the formation of a third six-membered ring fused to naphthalene in a phenanthrene-Iike arrangement by hydrogen bonding between the phenolic hydroxyl and azo nitrogen is required for induction.     

Structure-activity relationships in the induction of hepatic drug metabolism by azo compounds

Lipophilic azo compounds possessing 1-phenylazo-2-naphthol or 1-phenylazo-2-naphthylamine moieties induced cytochrome P-448 and related mono-oxygenase activities, UDP-glucuronyltransferase activity towards p-nitrophenol, glutathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene, aldehyde dehydrogenase, and menadione reductase activities. This pattern of induction by azo dyes is very similar to that by 3-methylcholanthrene. None of the hydrophilic azo compounds tested and none of the other lipophilic azo compounds tested including 4-phenylazo-1-naphthol induced these activities. It is suggested that the formation of a third six-membered ring fused to naphthalene in a phenanthrene-like arrangement by hydrogen bonding between the phenolic hydroxyl and azo nitrogen is required for induction.     

Studies in the metabolism of carcinogenic polycyclic heteroaromatic compounds. I. The hepatic microsomal metabolism of 7-methylbenz[c]acridine

An enzyme assay for the metabolism of the carcinogenic aza-aromatic polycyclic compound 7-methylbenz[c]acridine has been developed using a modification of a radiochemical assay described for the polycyclic aromatic hydrocarbon benzo[a]pyrene by DePierre et al. [J. W. DePierre, M. S. Moron, K. A. M. Johannesen and L. Ernster, Analyt. Biochem. 63, 470 (1975)]and Van Cantfort et al. [J. Van Cantfort, J. DeGraeve and J. E. Gielen, Biochem. biophys. Res. Commun. 79, 505 (1977)]. When the activities of control microsomes and microsomes of phenobarbital-, 3-methylcholanthrene-and 7-methylbenz[c]acridine-pretreated animals were compared, strong similarities were displayed toward oxidation of benzo[a]pyrene and 7-methylbenz[c]acridine. These similarities were seen in turnover numbers, Michaelis constants, and inducibility of both enzyme systems. 7-Methylbenz[c]acridine afforded a type I difference spectrum with 3-methylcholanthrene-pretreated microsomes. It is suggested that 7-methylbenz[c]acridine is oxidized by the same or a similar set of enzymes which is responsible for benzo[a]pyrene metabolism.     

Effects of model traumatic injury on hepatic drug metabolism in the rat. II. In vivo metabolism of hexobarbital and zoxazolamine

A previously validated small mammal trauma model, hind-limb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to further investigate the effects of traumatic injury on hepatic drug metabolism in vivo. The pharmacokinetic sequelae of the previously observed post-traumatic decrease in cytochrome P-450 content were demonstrated, using hexobarbital and zoxazolamine as model cytochrome P-450-oxidized drugs. Sleeping times after ip or iv administration of these drugs was prolonged by model injury. Longer sleeping times appeared to be caused by a slower elimination of the drugs from the bloodstream rather than to an increased sensitivity to their central nervous system effects or to an alteration in plasma protein-binding. Detailed pharmacokinetic analyses by the two-compartmental open system model revealed that the major alteration in post-traumatic drug metabolism was decreased in vivo elimination rather than a shift in distribution. These studies further confirm the pharmacokinetic utility of this convenient small mammal trauma model in the study of post-traumatic derangements in hepatic drug metabolism and emphasize the toxicologic importance of these derangements.     

肝部分切除危险性预测的实验研究

本实验采用新西兰种大白兔制成慢性肝损伤模型，进行肝脏部分切除术，将术前肝功能储备情况分为Ａ、Ｂ、Ｃ三级。对照观察术前、术后肝功能，肝功除量与预后的关系。结果发现：在慢性肝损伤的动物，肝切除的量越大，术后死亡率越高。当肝切除量在４０％时，术后死亡率为４４．４％，而肝切除量在５０％时，死亡率为１００％。术前肝功能储备越差，术后死亡率越高。     

Effects of model traumatic injury on hepatic drug metabolism in the rat. IV. Glucuronidation

A previously validated small mammal trauma model, hind-limb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to investigate the effects of traumatic injury on hepatic glucuronidation activity. As was previously observed with hepatic oxidative drug metabolism, model trauma resulted in a significant decrease in the in vivo glucuronidation of chloramphenicol, with a 23% drop in clearance of this drug. The effect on in vivo pharmacokinetics appeared to result from a complex interaction between trauma's differential influences on conjugating enzyme(s), deconjugating enzyme(s), and hepatic UDP-glucuronic acid levels, as well as the relative physiological importance of these variables. Hepatic UDP-glucuronyltransferase activities towards both p-nitrophenol and chloramphenicol were elevated (44-54%) after model injury when measured in native hepatic microsomes. However, microsomes which had been "activated" by treatment with Triton X-100 showed no significant difference between control and traumatized animals. Serum beta-glucuronidase activities were elevated by 58%, while hepatic beta-glucuronidase rose by about 16%. Nevertheless, in vivo deconjugation showed no significant change. Model trauma also resulted in a 46% decrease in hepatic UDP-glucuronic acid content. Thus, the observed post-traumatic depression of in vivo chloramphenicol glucuronidation could be due either to a diminished availability of a necessary cofactor (UDP-glucuronic acid) or to an alteration in enzyme kinetics or function in vivo.     

骨髓间充质干细胞异体移植对大鼠肝细胞增殖影响的实验研究

目的探讨骨髓间充质干细胞治疗急性肝衰竭的机制。方法全骨髓贴壁法培养扩增原代SD大鼠骨髓干细胞,传代培养,形态学、生长曲线及免疫表型鉴定骨髓间充质干细胞,移植治疗D-氨基半乳糖联合脂多糖致肝衰竭大鼠,观察干预治疗后1d、3d、5d、7d肝组织病理学改变、肝组织PCNA蛋白、CyclinDlmRNA表达量。结果原代培养大鼠骨髓间充质干细胞传代后,形态较均一,为梭形或纺锤形,高表达CD90（94．3％）,CD29（99．4％）,不表达CD11b／c（5．7％）,CD45（3．2％）。骨髓间充质干细胞移植减轻肝脏组织病理损伤（P〈0．05）。BMSCs治疗组PCNA的蛋白表达高于对照组（P〈0．01）。干预后不同时间点治疗组细胞周期蛋白D1的mRNA表达高于对照组（1．182±0．248VS0．468±0．053、0．858±0．114vs0．218±0．178）,差异有统计学意义（P〈0．01）。结论骨髓间充质干细胞移植减轻肝脏病理损伤,上调细胞周期蛋白DlmRNA的表达,促进肝再生,对急性肝衰竭有治疗作用。