Application of the PKCYP test to predict caffeine clearance mediated by CYP1A2 in a rat acute liver injury model.

We previously established a method for assessing in vivo drug-metabolizing capacity by pharmacokinetic estimation of the quantity of cytochrome P450 (CYP) in vivo (PKCYP test), in which an apparent liver-to-blood free concentration gradient in vivo (qg) is introduced (Matsunaga et al., Jpn. J.

Simultaneous assessment of the in vivo amount of CYP1A2 and CYP3A2 by the PKCYP-test using theophylline in rats.

Recently, we developed a method for assessing in vivo drug metabolism capacity by pharmacokinetic estimation of the quantity of cytochrome P450 (CYP) in vivo (PKCYP-test), in which an apparent liver-to-blood free concentration gradient in vivo (qg) is introduced. The qg value can be alternatively defined as the ratio of the in vivo-in vitro clearance by a single CYP isoform. In this study, we examined the application of the PKCYP-test to drugs metabolized by multiple CYP isoforms in a rat model with fluctuating CYP1A2 levels using theophylline as a model drug.

Application of the PKCYP-test to predict the amount of in vivo CYP2C11 using tolbutamide as a probe.

Previous reports have shown that the determination of drug metabolism capacity can be made by the pharmacokinetic estimation of the quantity of cytochrome P450 (CYP) in vivo (PKCYP-test), in which an apparent liver-to-blood free concentration gradient in vivo (qg) is introduced, which is useful for evaluating fluctuations of CYPIA2 in rats. The aim of the present study was to examine the application of the PKCYP-test to evaluate the quantity of in vivo CYP2C11 by using tolbutamide as a probe, to confirm its validity using a physiologically-based pharmacokinetic rat model. Rats treated with carbon tetrachloride (CCl4-treated rats) were used as a model for low levels of CYP2C11 in the liver.

Application of the PKCYP-test in cases of altered CYP1A2 for multiple CYP systems in rat models of disease.

Previously, we established a method to assess drug metabolism capacity based on a pharmacokinetic estimation of the quantity of cytochrome P450 (CYP) in vivo (PKCYP-test) by introducing an apparent liver-to-blood free concentration gradient in vivo (qg). The qg values were determined as the ratio of in vivo-in vitro clearance. In this study, we examined the application of the PKCYP-test to the clearance of acetanilide and caffeine mediated by CYP1A2 using rat models in which the levels of CYP enzymes were reduced.

Evaluation of stinging-inducing chemicals using cultured neuronal cells: an electrophysiological approach.

The effects of various cosmetic ingredients, including preservatives, humectants, ultraviolet absorbents and solvents, on the electrical properties of neuronal cells were investigated using rat phaeochromocytoma PC12 cells and cultured rat dorsal root ganglion (DRG) neurons. When membrane current was measured under whole-cell voltage-clamp, all nine test compounds inhibited voltage-activated K(+) current in PC12 cells. With the five compounds selected for further experiments with DRG neurons, two types of current responses were observed, namely inhibition of K(+) current by methyl or butyl p-hydroxybenzoate (MPHB or BPHB) and resorcinol, and induction of a cationic inward current by MPHB, BPHB, ethanol and dipropylene glycol.

Characterization of benzo[a]pyrene metabolism and related cytochrome P-450 isozymes in Syrian hamster livers.

Cytochrome P-450 monooxygenases of golden Syrian hamsters were characterized with respect to benzo[a]pyrene metabolism. Male hamsters were treated with phenobarbital, 3-methylcholanthrene, dexamethasone, benzoflavone, or ethanol, and the activity of aryl hydrocarbon hydroxylase and benzo[a]pyrene activation was determined by mutagenicity testing in hepatic microsomes. Aryl hydrocarbon hydroxylase activity was induced markedly by treatment with phenobarbital but not with 3-methylcholanthrene, nor with other chemicals.

A new cytochrome P450 form belonging to the CYP2D in dog liver microsomes: purification, cDNA cloning, and enzyme characterization.

A new form of cytochrome P450 (P450 DUT2) was purified from untreated male dog liver microsomes. The final preparation (a specific content of 19.1 nmol P450/mg protein) showed a single band with an apparent monomeric molecular weight of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but was further separated into two apoproteins (P450 DUT2a and P450 DUT2b) by reverse-phase HPLC.

Suppression of Na+ influx in ATP-depleted hepatocytes.

The change of cytosolic Na+ concentration was examined in ATP-depleted cultured rat hepatocytes. Cytosolic Na+ concentration was increased in the hepatocytes where ATP was more than 95% depleted by chemical hypoxia with 2.5 mM KCN and 0.

Modulation by alkyl p-hydroxybenzoates of voltage- and ligand-gated channels in peripheral neuronal cells.

Effects of alkyl p-hydroxybenzoates (APHBs), which are used as preservatives, on ion channels were investigated in rat pheochromocytoma PC12 cells. Methyl p-hydroxybenzoate (MPHB; 300 microM) and butyl p-hydroxybenzoate (BPHB; 300 microM) inhibited Ba2+ current passing through Ca2+ channels, and facilitated the inactivation of the Ba2+ current. K+ current obtained with a depolarizing voltage-step was also suppressed by 300 microM MPHB or 300 microM BPHB.

Endothelin-3 activates a voltage-gated Ca channel via a pertussis toxin sensitive mechanism leading to dopamine release from PC12 cells.

We have investigated the role of endothelin-3 (ET-3) in the stimulus-secretion coupling mechanism in rat pheochromocytoma PC12 cells. ET-3 (10-100 nM) evoked both dopamine (DA) release and an increase in intracellular Ca2+ concentration ([Ca]i). The ET-3-evoked DA release was partially inhibited by pretreatment with pertussis toxin (PTX; 2 ng/ml, 20 h).

Isolation and characterization of four cytochrome P450 isozymes from untreated and phenobarbital-treated beagle dogs.

Four different forms of cytochrome P450 (P450) were purified from liver microsomes of untreated or phenobarbital (PB)-treated male beagle dogs using HPLC techniques, and designated as DUT-1, DPB-1, DPB-2 and DPB-3, respectively. Specific contents of the purified DUT-1, DPB-1, DPB-2 and DPB-3 were 13.3, 9.

Inhibition of hepatocyte gap junctional communication by 25-hydroxycholesterol may be mediated through free radicals.

25-Hydroxycholesterol, an autoxidation product of cholesterol, has been shown to inhibit gap junctional communication between rat hepatocytes. In this study, we investigated whether free radicals are responsible for the effect of 25-hydroxycholesterol on hepatocytes. Addition of superoxide dismutase, hydroxyl radical scavenger mannitol, or the antioxidants diphenylphenylenediamine and alpha-tocopherol markedly decreased the inhibitory effect of 25-hydroxycholesterol.

Oxysterols inhibit gap junctional communication between rat hepatocytes in primary culture.

Several oxysterols were examined for their effect on gap junctional communication between rat hepatocytes in primary culture. 25-Hydroxycholesterol, 22(S)-hydroxycholesterol and 7 beta-hydroxycholesterol, in decreasing order of potency, markedly inhibited gap junctional communication. In contrast, 7-ketocholesterol showed no inhibitory effect.

Developmental toxicity of 2-chlorodibenzofuran in cultured post-implantation rat embryos.

The developmental toxicity of 2-chlorodibenzofuran (2-MCDF), a contaminant in tap water, was examined by using post-implantation rat embryo culture with or without a metabolic activation system. 3-Chlorodibenzofuran (3-MCDF) and dibenzofuran (DF) were examined for comparison. Rat embryos at day 9 of gestation were cultured for 48 hr in the presence of the test chemicals (0, 30, 100, 300 or 1000 mum).

Non-competitive antagonism by hirsuteine of nicotinic receptor-mediated dopamine release from rat pheochromocytoma cells.

Effects of hirsuteine, an indole alkaloid extracted from Uncaria genus, on nicotine- and high K-induced responses were investigated in rat pheochromocytoma PC12 cells. Hirsuteine (300 nM-10 microM) inhibited dopamine release evoked by 100 microM nicotine in a concentration-dependent manner. Hirsuteine did not produce a parallel shift of the concentration-response relationship curve for nicotine, but reduced maximal dopamine release.

Culture of postimplantation rat embryos in rabbit serum for the identification of the growth factor in fractionated rat serum.

The growth factor for postimplantation rat embryos was investigated on the basis of the serum species-specificity in supporting embryonic development in culture. We used rabbit serum as a basal medium for the culture of head-fold stage rat embryos, and examined the effects of various fractions of rat serum on their development. In rabbit serum alone, rat embryos developed poorly.

Dopamine receptor agonists and antagonists enhance ATP-activated currents.

The effects of dopamine and related compounds on ATP-activated channels were investigated in pheochromocytoma PC12 cells. Dopamine (10 microM) enhanced an inward current activated by 100 microM ATP. A similar enhancement of the ATP-activated current was observed with apomorphine (10 microM), a non-selective dopamine receptor agonist, with (+)-SKF-38393 (10 microM), a selective dopamine D1 receptor agonist, and with (-)-quinpirole (10 microM), a selective dopamine D2 receptor agonist.

Neurochemical and histological analysis of motor dysfunction observed in rats with methylnitrosourea-induced experimental cerebellar hypoplasia.

Histological and neurochemical changes related to motor dysfunction observed in rats after neonatal treatment with nitrosoureas were examined. Neonatal rats received subcutaneous injections of methylnitrosourea (MNU: 0.125 mmol/kg, s.

Extracellular adenosine 5'-triphosphate-evoked glutamate release in cultured hippocampal neurons.

Characteristics of extracellular ATP-evoked electrical responses in rat hippocampal neurons were investigated. Extracellular ATP (100 microM) induced a rapid depolarization followed by repetitive firings of spikes in these cells under whole-cell current-clamp. In whole-cell voltage-clamp experiments, ATP activated 2 types of inward currents that were inhibited by P2-purinoceptor blocker suramin (300 microM).

N-nitrosodialkylamine dealkylation in reconstituted systems containing cytochrome P-450 purified from phenobarbital- and beta-naphthoflavone-treated rats.

Five cytochrome P-450 forms were purified from livers of rats pretreated with phenobarbital (PB) or beta-naphthoflavone (BNF), and the oxidative dealkylation of N-nitrosodialkylamines by the reconstituted cytochrome P-450 systems was measured. PB-II (P450IIB1) showed very high N-nitrosomethybutylamine (NMBA) debutylase activity, high NMBA demethylase activity and high N-nitrosomethyl-benzylamine (NMBeA) debenzylase activity, suggesting that the increase following PB treatment in hepatic microsomal NMBA debutylation and NMBeA debenzylation was due to the induction of PB-II. BNF-H (P450IA2) showed very high NMBA debutylase and high NMBeA debenzylase activities, and BNF-L (P450IA1) showed NMBA debutylase and high NMBeA debenzylase activities.

Indium inhibits gap junctional communication between rat hepatocytes in primary culture.

The effect of indium on gap junctional communication was investigated in primary cultured rat hepatocytes. Treatment of hepatocytes with indium chloride at concentrations of 100 microM to 1 mM for 2 h resulted in dose-dependent inhibition of gap junctional communication between hepatocytes. The effect of indium on hepatocytes was also evaluated using two indices for cell viability: lactate dehydrogenase (LDH) leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

Effect of tetrakis-mu-3,5-diisopropylsalicylatodiaquodicopper(II) on the status of reduced glutathione in freshly isolated hepatocytes.

Effects of different concentrations of tetrakis-mu-3,5-diisopropylsalicylatodiaquodicopper(II) (Cu(II)2(3,5-DIPS)4(H2O2)2) on the reduced status of glutathione (GSH), the major nonprotein thiol in tissues, were investigated using freshly isolated hepatocytes. Cu(II)2(3,5-DIPS)4 below 100 microM did not have any significant effects on either the GSH content or viability of the hepatocytes, but at 150-250 microM it decreased both parameters after 1 h of incubation. The decrease in cellular GSH was not followed by an increase in the oxidized form of GSH (GSSG) in the cell suspension.

[Studies on the teratogenic potential of p-tert-butylphenolformaldehyde resin in rats].

p-tert-Butylphenolformaldehyde resin, an adhesive, was given orally to pregnant Wistar rats by stomach intubation at the dose levels of 250, 500 and 1000 mg/kg body weight during days 7 to 17 of gestation, and the effects of the compound on dams and fetal developments were examined. No changes in general conditions, maternal body weight, food consumption, numbers of corpora lutea and implantation ratio were observed. There was no evidence of an increase in fetal death or of malformation attributable to the treatment with p-tert-butylphenolformaldehyde resin in any of dose levels examined.

Inhibition of ion channels by hirsutine in rat pheochromocytoma cells.

Effects of hirsutine, an alkaloid that produces a potent ganglion blocking effect, were investigated using rat pheochromocytoma PC12 cells. Hirsutine (1 to 10 microM) suppressed dopamine-release evoked by 100 microM nicotine. In voltage-clamped cells, hirsutine (1 to 10 microM) inhibited the inward current activated by 100 microM nicotine.