The superiority of UW solution for maintaining ATP concentrations during pulmonary preservation

We evaluated three solutions used for preserving lungs, namely, University of Wisconsin (UW), Euro-Collins (E-C), and low potassium dextran (LPD), by measuring the high energy phosphates in the preserved lung tissue. The left lungs of Sprague-Dawley rats were excised and flushed with 5 ml of one of the solutions at 10°C through the pulmonary artery, after which they were deflated and immersed in the solution at 10°C for 24 h. The tissue adenosine triphosphate (ATP) concentration in mol/g tissue wet weight after 24 h of storage was 2.55 ± 0.48 (n = 7) in the UW lungs, 1.98 ± 0.25 (n = 6) in the E-C lungs, and 1.53 ± 0.32 (n = 4) in the LPD lungs, being significantly higher in the UW lungs than in either the E-C or LPD lungs (P < 0.05). The histopathological findings of the E-C lungs were more deteriorated, with marked interstitial edema, septal hypertrophy, and perivascular hyaline degeneration, than either the UW or LPD lungs. Thus, the findings of this study indicate the superiority of UW solution for lung preservation.     

Activation of trans-l,2-dihydro-l,2-dihydroxy-6-aminochrysene to genotoxic metabolites by rat and human cytochromes P450

In order to address the hypothesis that 6-aminochrysene (6-AC)is converted to genotoxic products by cytochrome P450 enzymesvia two activation pathways (N-hydroxylation and epoxidation),the activation of 6-AC and trans-l,2-dihydro-l,2-dihydroxy-6-aminochrysene(6-AC-diol) to genotoxic metabolites was examined in rat andhuman liver microsomal cytochrome P450 enzymes using Salmonellatyphimurium TA1535/pSK1002 and TA1535/pSK1002/pNM12 (NM2009)as tester strains. The latter bacteria, an O-acetyl-transferase-overexpressingstrain, was highly sensitive to metabolites derived from activationof 6-AC, but not those from 6-AC-diol, using liver microsomesfrom phenobarbital-treated rats or a reconstituted monooxygenasesystem containing P4502B1 or -2B2, thus suggesting the rolesof P450 and acetyltransferase systems in the activation process.6-AC-diol, on the other hand, was activated very efficientlyby liver microsomes prepared from ß-naphthoflavone-treatedrats or a reconstituted system containing P4501A1 or -1A2; theactivation reaction is considered to proceed through diol-epoxideformation. The contribution of rat P4501A enzymes towards activationof 6-AC-diol was confirmed by the inhibitory effects on theactivation process of -naphthoflavone, a specific inhibitorof P4501 A-related activities, and antibodies raised againstpurified P4501A1 and -1A2. In humans, P4501A2 was found to bethe major enzyme involved in the activation of 6-AC-diol togenotoxic metabolites while the parent compound 6-AC was activatedmainly by P4503A4. Experiments using recombinant P450 proteinsexpressed in human lymphoblastoid cells lines showed that humanP4501A1 could also activate 6-AC-diol to reactive metabolitesat almost the same rate measured with P4501A2. In addition,P4502B6 was found to efficiently catalyze the activation of6-AC to genotoxic metabolites, and P4503A4 was active in theactivation of 6-AC-diol as well as 6-AC. Addition of purifiedrat epoxide hydrolase to the incubation mixture containing purifiedrat P4501A1 or microsomes expressing human P4501A1 caused inhibitionof activation of 6-AC-diol. These results suggest the existenceof different enzymatic activation pathways for 6-AC and 6-AC-diol.The former carcinogen may be N-hydroxylated principally by P4502Benzymes in rats and P4503A4 and -2B6 in humans and activationto its ultimate metabolites may proceed through esterificationof the N-hydroxy metabolites by an N-acetyltransferase. The6-AC-diol is metabolized to its ultimate diolepoxide productby P4501A enzymes in rat and human liver microsomes. P4503A4(humans) and P4503A2 (rats) may also contribute to some extentin the activation of 6-AC-diol, albeit at lower rates than thoseof P4501A enzymes.     

Studies of effects of anticancer agents in combination with/without hyperthermia on metastasized human bladder cancer cells in chick embryos using the polymerase chain reaction technique

Cultivated T24 cells derived from a human bladder cancer were inoculated into the chorioallantoic membrane vein of chick embryos. Hyperthermic treatment was performed following injection of anticancer agents 3 days after the inoculation of the T24 cells. DNA samples were obtained from the livers of the chick embryos, and the polymerase chain reaction technique was used to amplify a DNA fragment specific to the human -globin gene. The Southern hybridization method was used to evaluate the inhibitory effects of anticancer agents in combination with/without hyperthermia on T24 cells metastasized to the liver. The hyperthermia exerted an inhibitory effect on the growth of the T24 cells in the livers of the chick embryos, and this was dependent on the thermal dose. The antitumor effects of hyperthermia performed at 42.5° C for 20 min and at 43.0° C for 10 min were evidenced by 69.2% an 82.0% inhibition of the growth of the metastasized T24 cells, respectively, as compared with the growth of untreated T24 cell. Hyperthermia performed at 42.5° C for 10 min alone produced 26.7% tumor growth inhibition, and these conditions for hyperthermia were subsequently used as a criterion for evaluating the effects of its combination with various anticancer agents. Adriamycin (20 g/egg) alone, mitomycin C (10 g/egg) alone, carboplatin (10 g/egg) alone, and cisplatin (10 g/egg) alone produced 13.5%, 58.9%, 27.3%, and 29.1% tumor growth inhibition, respectively. Adriamycin and mitomycin C applied in combination with hyperthermia showed additive inhibitory effects on the growth of the metastasized T24 cells in this chick embryo model.     

Protective effects of ONO-5046*Na, a specific neutrophil elastase inhibitor, on postperfusion lung injury

Background. Polymorphonuclear neutrophil elastase might contribute to postperfusion lung injury, so we evaluated the protective effect of ONO-5046·Na, a specific inhibitor of polymorphonuclear neutrophil elastase, against such an injury.

Methods. The study was done using 8 mongrel dogs that received ONO-5046·Na (15 mg/kg per hour) (group O) and 8 control dogs (group C), all of which had 1 hour of partial bypass and 5 hours of observation.

Results. The respiratory index showed no significant changes in group O, but increased significant in group C (1.4 ± 2.0 versus 5.1 ± 4.7, p = 0.0047). Pulmonary extravascular water volume increased markedly in group C but only slightly in group O (group C 20.6 ± 8.7, group O 11.2 ± 2.7 mL/kg; p = 0.0005). Blood concentrations of polymorphonuclear neutrophil elastase and interleukin-6 showed more than a tenfold increase in group C (PMN elastase, group C 12.9 ± 12.8, group O 2.4 ± 1.3 ng/mL; IL-b, group C 11.0 ± 9.3, group O 2.9 ± 3.8 pg/mL; p < 0.05) but were only slightly higher in group O. Histologic examination revealed interstitial and intraalveolar edema in group C, but group O was virtually normal.

Conclusions. ONO-5046·Na inhibits polymorphonuclear neutrophil elastase and maintains better pulmonary function, so it should reduce postperfusion lung injury.     

Effects of isatin, an endogenous MAO inhibitor, on dopamine (DA) and acetylcholine (ACh) concentrations in rats

Isatin (indole-2,3-dione), an endogenous inhibitor of monoamine oxidase (MAO), has several physiological properties for stress and anxiety. We previously identified isatin in the brain of stroke-prone spontaneously hypertensive rats (SHRSP) using gas-chromatography mass spectrometry. This study elucidated the effects of isatin on the ACh and DA levels of brain tissues in rats. Furthermore, we evaluated the effect of isatin on DA levels in a rat model of Parkinson's disease induced by Japanese encephalitis virus. Striatal ACh and DA levels significantly increased at 2 hours after isatin (50-200 mg/kg, i.p.) administration. Perfused through a microdialysis probe, isatin (10(-6)-10(-4) M) also produced a significant and concentration-dependent increase in the ACh and DA concentrations in the perfusate from the rat striatum. Furthermore, urinary isatin concentrations in patients with Parkinson's disease tend to increase according to the severity of disease. Isatin (100 mg/kg, i.p.) significantly increased striatal DA levels in a rat model of Parkinson's disease. These results suggest that urinary isatin may become a diagnostic marker for the clinical severity of Parkinson's disease and that endogenous isatin, a new biological modulator, may play a role in the regulation of the brain levels of ACh by increasing the level of DA under stress.     

Effects of arachidonic acid, prostaglandins, retinol, retinoic acid and cholecalciferol on xenobiotic oxidations catalysed by human cytochrome P450 enzymes

1. Effects of arachidonic acid, prostaglandins, retinol, retinoic acid and cholecalciferol on xenobiotic oxidations catalysed by 12 recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes have been investigated. 2. Arachidonic acid (50 microM) significantly inhibited CYP1A1- and 1A2-dependent 7-ethoxycoumarin O-deethylations, CYP2C8-dependent taxol 6alpha-hydroxylation and CYP2C19-dependent R-warfarin 7-hydroxylation. This chemical also inhibited slightly the xenobiotic oxidations catalysed by CYP1B1, 2B6, 2C9, 2D6, 2E1 and 3A4 in recombinant enzyme systems. 3. Retinol, retinoic acid and cholecalciferol were strong inhibitors for xenobiotic oxidations catalysed by recombinant CYP1A1, 2C8 and 2C19. 4. Dixon plots of inhibitions of CYP1A1-, 1A2-, 2C8- and 2C19-dependent xenobiotic oxidations by arachidonic acid, of CYP1A1-, 2B6- and 2C19-dependent activities by retinol, and of CYP1A1- and 2C19-dependent activities by cholecalciferol indicated that these chemicals inhibit P450 activities mainly through a competitive mechanism. 5. In human liver microsomes, arachidonic acid inhibited CYP1A2-dependent theophylline hydroxylation, CYP2C8-dependent taxol 6alpha-hydroxylation and CYP2C19-dependent omeprazole 5-hydroxylation. Taxol 6alpha-hydroxylation was also inhibited by retinol and retinoic acid, and omeprazole 5-hydroxylation was inhibited by retinol in human liver microsomes. 6. These results suggest that xenobiotic oxidations by P450 enzymes are affected by endobiotic chemicals and that the endobiotic-xenobiotic interactions as well as drug-drug interactions may be of great importance when understanding the basis for pharmacological and toxicological actions of a number of xenobiotic chemicals.     

Effect of dexamethasone on the intestinal first-pass metabolism of indinavir in rats: evidence of cytochrome P-450 3A [correction of P-450 A] and p-glycoprotein induction .

Indinavir, a potent and specific inhibitor of HIV protease, is a known substrate of cytochrome P-450 (CYP) 3A and p-glycoprotein. The purpose of this study is to investigate and compare the inducing effect of dexamethasone (DEX) on CYP3A and p-glycoprotein in the hepatic and intestinal first-pass metabolism of indinavir in rats. Pretreatment of rats with DEX had little effect on the pharmacokinetics (Cl and T(1/2)) after i.v. administration of indinavir, whereas DEX markedly altered the peak concentration (C(max)) and bioavailability of indinavir after oral dosing. The C(max) decreased from 2.8 microM in control rats to 0.28 microM in DEX-treated rats, and bioavailability decreased from 28 to 12.4%. The decreased bioavailability after DEX pretreatment was due mainly to an increase in first-pass metabolism. Intestinal first-pass metabolism (E(G)) increased from 6% in control rats to 34% in DEX-treated rats, and hepatic first-pass metabolism (E(H)) increased from 65 to 82%. Analysis of in vitro kinetic data revealed that the increased intestinal and hepatic metabolism by DEX was attributed to an increase in the V(max), as a result of CYP3A induction, without a significant change in the K(m) values. DEX pretreatment also induced p-glycoprotein in the intestine and liver of rats. p-Glycoprotein appeared to increase the intestinal metabolism of indinavir whereas it had little effect on the hepatic metabolism of indinavir. Although it has been suggested that the role of intestinal metabolism for some drugs is quantitatively greater than that of hepatic metabolism in the overall first-pass metabolism, the contribution of intestinal metabolism to the overall first-pass metabolism of indinavir in rats is not quantitatively as important as the hepatic metabolism, regardless of DEX induction.     

Oxidation of troglitazone to a quinone-type metabolite catalyzed by cytochrome P-450 2C8 and P-450 3A4 in human liver microsomes.

Troglitazone, a new oral antidiabetic drug, is reported to be mostly metabolized to its conjugates and not to be oxidized by cytochrome P-450 (P-450) enzymes. Of fourteen cDNA-expressed human P-450 enzymes examined, CYP1A1, CYP2C8, CYP2C19, and CYP3A4 were active in catalyzing formation of a quinone-type metabolite at a concentration of 10 microM troglitazone, whereas CYP3A4 had the highest catalytic activity at 100 microM substrate. In human liver microsomes, rates of the quinone-type metabolite formation (at 100 microM) were correlated well with rates of testosterone 6beta-hydroxylation (r = 0.98), but those at 10 microM troglitazone were not correlated with any of several marker activities of P-450 enzymes. Quercetin efficiently inhibited quinone-type metabolite formation (at 10 microM troglitazone) in human samples that contained relatively high levels of CYP2C, whereas ketoconazole affected these activities in liver microsomes in which CYP3A4 levels were relatively high. Anti-CYP2C antibodies strongly inhibited quinone-type metabolite formation (at 10 microM troglitazone) in CYP2C-rich human liver microsomes (by approximately 85%); the intensity of this effect depended on the human samples and their P-450 status. The results suggest that in human liver both CYP2C8 and CYP3A4 have major roles in quinone-type metabolite formation and that the hepatic contents of these two P-450 forms determine which P-450 enzymes play major roles in individual humans. CYP3A4 may be expected to play a role in formation of quinone-type metabolite from troglitazone even at a low concentration in humans.     

Prediction of human liver microsomal oxidations of 7-ethoxycoumarin and chlorzoxazone with kinetic parameters of recombinant cytochrome P-450 enzymes.

Different roles of individual forms of human cytochrome P-450 (CYP) in the oxidation of 7-ethoxycoumarin and chlorzoxazone were investigated in liver microsomes of different human samples, and the microsomal activities thus obtained were predicted with kinetic parameters obtained from cDNA-derived recombinant CYP enzymes in microsomes of Trichoplusia ni cells. Of 14 forms of recombinant CYP examined, CYP1A1 had the highest activities (V(max)/K(m) ratio) in catalyzing 7-ethoxycoumarin O-deethylation followed by CYP1A2, 2E1, 2A6, and 2B6, although CYP1A1 has been shown to be an extrahepatic enzyme. With these kinetic parameters (excluding CYP1A1) we found that CYP1A2 and 2E1 were the major enzymes catalyzing 7-ethoxycoumarin; the contributions of these two forms were dependent on the contents of these CYPs in liver microsomes of different humans. Similarly, chlorzoxazone 6-hydroxylation activities of liver microsomes were predicted with kinetic parameters of recombinant human CYP enzymes and it was found that CYP3A4 as well as CYP1A2 and 2E1 were involved in chlorzoxazone hydroxylation, depending on the contents of these CYP forms in the livers. Recombinant CYP2A6 and 2B6 and CYP2D6 had considerable roles (V(max)/K(m) ratio) for 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation, respectively; however, these CYP forms had relatively minor roles in the reactions, probably due to low expression in human livers. These results support the view that the roles of individual CYP enzymes in the oxidation of xenobiotic chemicals in human liver microsomes could be predicted by kinetic parameters of individual CYP enzymes and by the levels of each of the CYP enzymes in liver microsomes of human samples.