Frequent loss of expression of the potential tumor suppressor gene DCC in ductal pancreatic adenocarcinoma.

The development of colon carcinomas is associated with allelic deletions on chromosomes 5q, 17p, and 18q. The DCC gene located on chromosome 18q21.3 codes for a potential tumor suppressor gene related to cellular adhesion receptors. We investigated the expression of this gene in several pancreatic carcinoma cell lines and in patients with ductal adenocarcinomas of the pancreas. In 8 of 11 cell lines and in 4 of 8 primary tumors a complete extinction of DCC gene expression was observed, whereas the c-Ki-ras gene was mutated at codon 12 in 7 of 8 tumors. A highly reduced or absent expression of DCC was found in all low or undifferentiated pancreatic tumor cell lines, whereas in the more differentiated ones DCC expression was conserved. These data suggest that loss of DCC gene expression is an important factor in the development or progress of pancreatic adenocarcinoma and may be linked to the differentiated phenotype of the pancreatic tumor cell.

Regulation of cytochrome P-450 CYPIA1 gene expression and proto-oncogene expression by growth factors in primary hepatocytes.

The effect of growth factors on the cytochrome P-450 (CYPIA1) gene expression was studied in primary mouse hepatocytes. Of the three growth factors used, i.e. epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and insulin, only EGF or TGF alpha completely blocked CYPIA1 expression in the presence of the CYPIA1 inducer 3-methylcholanthrene (3-MC). This repression was not linked to cell cycle progression of the hepatocyte because insulin was active to induce 'early immediate genes' and DNA replication as well as EGF/TGF alpha but failed to suppress CYPIA1 expression. A specific EGF/TGF alpha receptor-mediated function may repress CYPIA1 gene expression and contribute to the acquisition of a xenobiotic drug resistance phenotype.

Increased expression of alpha6-integrin receptors and of mRNA encoding the putative 37 kDa laminin receptor precursor in pancreatic carcinoma.

The expression of alpha6-integrin receptors (VLA-alpha6) and of mRNA encoding the putative 37 kDa laminin receptor precursor (37 LRP) was determined in ductal pancreatic adenocarcinoma and normal pancreatic tissue from the same patient. VLA-alpha6 expression was enhanced and redistributed in pancreatic carcinoma, and 37 LRP mRNA levels were elevated in carcinomatous pancreatic tissue as well as in five pancreatic tumor cell lines. The molecular weight of the major RNA species detected was higher in carcinoma tissue (1.9 kb) as opposed to cell lines (1.2 kb), possibly reflecting alternative splicing of 37 LRP mRNA in the primary tumor.

Induction of cytochrome P450 2B1 by pyrethroids in primary rat hepatocyte cultures.

Numerous xenobiotics are capable of inducing their own metabolism and by enzyme induction can also lead to enhanced biotransformation of other xenobiotics. In this project, we examined the influence of pyrethroids (permethrin, cypermethrin, and fenvalerate) on the expression and activity of the phenobarbital (PB)-inducible cytochrome P450 2B1 isoform (CYP2B1) in primary rat hepatocyte cultures. Incubation of hepatocyte cultures with pyrethroids resulted in a marked CYP2B1 induction. Among the tested pyrethroids, permethrin elicited the most pronounced induction of CYP2B1 mRNA, which exceeded maximal induction achieved by PB at concentrations approximately 10-fold higher. Furthermore, permethrin induced CYP3A1 mRNA expression, while the expression of the CYP1A1 isoform, which in vivo is not responsive to PB treatment, was not significantly affected by pyrethroids. Permethrin-dependent enhancement of CYP2B1 and CYP3A1 mRNA expression was repressed by the hepatotrophic cytokine epidermal growth factor, which is known to also inhibit PB-dependent induction of CYP2B1. Several metabolites of permethrin formed by hepatocytes (3-(2',2'-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzyl alcohol, and 3-phenoxybenzoic acid) were ineffective in inducing CYP2B1 mRNA. Furthermore, permethrin stimulated the expression of the luciferase reporter gene under control of the CYP2B1 promoter (comprising the PB-responsive enhancer module) in transiently transfected primary hepatocyte cultures. Thus, permethrin-stimulated gene expression occurred on the transcriptional level. Taken together, these results indicate that the pyrethroid permethrin is a PB-like inducer. Due to its superior potency in induction, permethrin appears as a useful substance for mechanistic studies to elucidate the mechanism of enzyme induction by phenobarbital.

Induction of cytochrome P-4502B1-related mouse cytochrome P-450 and regulation of its expression by epidermal growth factor/transforming growth factor alpha in primary hepatocyte culture.

Phenobarbital-dependent induction of mouse cytochrome P-450 (Cyp) orthologous to rat CYP2B1 and its modulation by hepatotrophic growth factors were examined in primary hepatocyte cultures. Compared to rat hepatocytes, induction in mouse hepatocytes was more rapid and effective. Ligands of the EGF receptor, epidermal growth factor, and transforming growth factor alpha inhibited induction on the basis of protein expression and CYP2B-associated 7-pentoxyresorufin-O-depentylase activity. Furthermore, EGF led to repression of accumulation of corresponding mRNA under phenobarbital, an effect not blocked by inhibition of protein synthesis under cycloheximide. Ligands of the EGF receptor may contribute towards the decrease in hepatic CYP expression observed during (pre)neoplastic development and regeneration.

Kinetics of melphalan leakage during hyperthermic isolation perfusion in melanoma of the limb.

The kinetics of melphalan leakage into the peripheral blood were studied in 21 patients undergoing hyperthermic isolation perfusion of the upper or lower limb as an adjuvant treatment in high-risk melanoma; in 5 patients cisplatin was added. The melphalan concentrations in the peripheral blood rose predominantly during the first 20 min of perfusion and levelled out to an apparent steady state of about 0.28 micrograms/ml in upper extremity perfusions, and 0.34 (without cisplatin) and 0.37 micrograms/ml (with cisplatin) in lower extremity perfusion. Erythrocytes labelled with technetium Tc 99m, which were added concomitantly with melphalan to the perfusion medium, appeared in the systemic circulation of the patients at an almost constant rate of 0.32% (lower and upper limb perfusions without cisplatin and 0.37% (with cisplatin) of total tracer/min. This perfusate flow rate indicated by labelled erythrocytes completely explained the leakage of melphalan from the perfusion circuit into the peripheral blood. Peak concentrations of melphalan in the peripheral blood were observed immediately after reconstitution of normal hemodynamic conditions once isolation perfusion had been terminated. This fraction of melphalan might originate from tissue-binding sites, but also from vascular compartments; therefore, a thorough washing-out procedure might minimize this effect.

Binding of benzo(a)pyrene metabolites to cellular DNA in perfused rat lungs.

The influence of pretreatment with monooxygenase inducers on total irreversible binding of metabolically activated [3H]-benzo(a)pyrene to cellular DNA and the formation of benzo(a)pyrene metabolite-deoxyribonucleoside adducts after cytochrome P-448 induction was studied in perfused rat lungs. Pretreatment with the cytochrome P-448 inducer beta-naphthoflavone increasing binding by a factor of 23. In lungs of induced animals, 0.45 pmoles of benzo(a)pyrene equivalents were bound per mg DNA. Binding to RNA and to protein was also considerably induced by beta-naphthoflavone. Phenobarbital treatment did not significantly increase binding to cellular macromolecules of rat lung. Analysis of hydrolyzed DNA of lungs from beta-naphthoflavone-treated rats by Sephadex LH 20 chromatography revealed the formation of at least two nucleoside adducts with metabolically activated benzo(a)pyrene one of which is probably due to modification of the DNA with a benzo(a)pyrene-7, 8-dihydrodiol-9, 10-epoxide and the other to modification of DNA with secondary metabolites of benzo(a)pyrene phenols.

Effect of nicotine or cotinine on metabolism of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in isolated rat lung and liver.

The scope of the present study was to investigate whether nicotine or cotinine will affect the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in isolated perfused rat lungs and livers and to study the effect of starvation on pulmonary metabolism of NNK. NNK metabolism was investigated in isolated perfused liver and lung of male F344 rats perfused with 35 nM [5-3H]NNK in presence of a 1400-fold excess of the main tobacco alkaloid nicotine and its metabolite cotinine. In perfused rat livers, nicotine and cotinine inhibited NNK elimination and metabolism and led to a substantial increase of elimination half-life from 14.6 min in controls to 25.5 min after nicotine and 36.6 min after cotinine co-administration, respectively. In parallel, the pattern of NNK metabolites was changed by nicotine and cotinine. The pathway of alpha-hydroxylation representing the metabolic activation of NNK was decreased to 77% and 85% of control values, whereas N-oxidation of NNK and glucuronidation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was increased 2.6- and 1.2-fold in presence of nicotine and cotinine, respectively. When isolated rat lungs were perfused with 35 nM NNK for 3 h neither the elimination nor the pattern of metabolites were substantially affected due to co-administration of 50 microM nicotine or cotinine. Cytochrome P450 2E1 is known to participate in the activation of NNK and can be induced by starvation. However, isolated rat lungs from male Sprague Dawley rats perfused with [1-14C]NNK at about 2 microM for 3 h, revealed only small differences in pulmonary elimination and pattern of NNK metabolites between fed and starved animals. These results suggest that nicotine and its main metabolite cotinine inhibit the metabolic activation of NNK predominantly in the liver whereas activation in lung, a main target organ of NNK induced carcinogenesis, remained almost unaffected.

Differential inhibition of biphenyl hydroxylation in perfused rat liver.

A differential inhibition of biphenyl hydroxylation by alpha-naphthoflavone and metyrapone was observed in isolated perfused rat liver. alpha-Naphthoflavone inhibited 2- and 4-hydroxylation in livers from beta-naphthoflavone-pretreated animals but had no effect on both reactions in livers from phenobarbital-pretreated animals. Metyrapone inhibited 2- and 4-hydroxylation in phenobarbital-stimulated livers, but only insignificant inhibition of 2-hydroxylation and a slight enhancement of 4-hydroxylation by metyrapone was observed in beta-naphthoflavone-stimulated livers. Conjugation of 2-hydroxybiphenyl and 4-hydroxybiphenyl by isolated perfused livers was also studied. 4-Hydroxybiphenyl preferentially formed sulphates in livers from untreated animals but after induction glucuronidation was as effective as sulphation or even exceeded sulphation. Only glucuronic acid conjugates of 2-hydroxybiphenyl were detected.

Effect of dietary antioxidants on benzo[a]pyrene metabolism in rat liver microsomes.

Feeding of rats with 1% ethoxyquin (EQ) and butylated hydroxytoluene (BHT) but not butylated hydroxyanisole (BHA) increases the formation rate of benzo[a]pyrene (BP)-4,5-dihydrodiol from BP in hepatic microsomes. The production of other BP-dihydrodiols and of BP phenols is decreased after treatment with EQ, BHT and BHA. EQ and BHT are more effective than BHA in inducing epoxide hydrolase (EH) activity towards styrene oxide as the substrate.

Inhibitors of mdr1-dependent transport activity delay accumulation of the mdr1 substrate rhodamine 123 in primary rat hepatocyte cultures.

P-glycoproteins (P-gps) encoded by mdr1 (multidrug resistance) genes mediate extrusion of numerous lipophilic xeno- and endobiotics through the plasma membrane. Rhodamine 123 (Rh123), a fluorescent dye which is accumulated by mitochondria, is a mdr1 substrate and a well-established tool to study mdr1 transport activity. Inhibitors of mdr1-dependent transport such as verapamil or cyclosporin A have been found to decrease Rh123 efflux from mdr1-expressing cells. Mdr1b gene expression increases with time in primary rat hepatocyte culture. In hepatocytes cultured for 4 days and expressing high levels of P-gp, intracellular Rh123 accumulation was enhanced in the presence of mdr1 inhibitors (cyclosporin A, 8 and 80 microM, verapamil, 8 and 80 microM, or triton X-100, 8 microM). Surprisingly, in hepatocytes expressing low levels of P-gp (after 1 day of culture), time-dependent Rh123 accumulation was not enhanced, but delayed by cyclosporin A, verapamil or triton X-100. In these cells orthovanadate (50 microM), an inhibitor of P-glycoprotein ATPase activity, suppressed Rh123 accumulation, while tetraethylammonium (200 microM), an organic cation transporter (OCT) substrate, had no effect. The paradoxical delay in Rh123 accumulation by verapamil and cyclosporin A occurred eventhough these compounds decreased dye extrusion from Rh123 pre-loaded cells. These observations suggest that a hitherto unknown mechanism which is sensitive to modulators of mdr1-activity contributes to Rh123 uptake or accumulation in primary rat hepatocytes.

Metabolism of benzo[a]pyrene in the combined rat liver--lung perfusion system.

The influence of the insertion of a liver into the perfusion circuit of a lung on the availability of benzo[a]pyrene and benzo[a]pyrene metabolites to the lung was examined. Perfused lungs from 5,6-benzoflavone pretreated rats release high quantities of free benzo[a]pyrene metabolites and conjugates into the perfusion medium. The insertion of a liver taken from an untreated rat reduces the concentration of unmetabolized substrate and of free diol, quinone and phenol metabolites to less than 20% of the concentrations found in the absence of the liver. When the liver of a 5,6-benzoflavone-pretreated rat is used, substrate depletion is not much greater than in the experiments with control livers; however, the concentration of free metabolites is further reduced to one third. In lung tissue, only very low levels of benzo[a]pyrene and greatly reduced levels of free and conjugated metabolites are found when a 5,6-benzoflavone-induced liver had been present during perfusion. These findings can explain the protective effect of the liver on covalent binding of benzo[a]pyrene metabolites to pulmonary macro-molecules observed in previous experiments with the combined liver-lung perfusion model [Klaus et al., Biochem. Biophys. Res. Commun., 105 (1982) 596].

Monitoring of cytochrome P-450 1A activity by determination of the urinary pattern of caffeine metabolites in Wistar and hyperbilirubinemic Gunn rats.

Various studies suggest that induction of cytochrome P-450 1A (CYP1A) might be a valuable therapeutic modality for reducing the hyperbilirubinemia of infants with Crigler-Najjar syndrome type I (CNS-I), a severe form of congenital jaundice. To evaluate inducers of CYP1A as possible tools in the treatment of hyperbilirubinemia, a novel assay was established, based on the analysis of the urinary pattern of caffeine metabolites in rats. Wistar rats received [1-Me-(14)C]-caffeine (10 mg/kg i.p.), before and 48h after administration of the potent CYP1A inducer 5,6-benzoflavone (BNF) (80 mg/kg, i.p.). A substantial increase in the fractions of the terminal caffeine metabolites 1-methyluric acid (1-U), 1-methylxanthine (1-X), and a concomitant decrease in the caffeine demethylation product 1,7-dimethylxanthine (1,7-X) was observed after application of BNF. The ratio of the caffeine metabolites (1-U+1-X)/1,7-X may serve as an index of CYP1A activity in rats in vivo. Hyperbilirubinemic, homozygous (jj) Gunn rats are an accepted model for human CNS-I. In male jj Gunn rats treated with BNF or with indole-3-carbinol (I3C, 80 mg/kg, oral gavage), the inducing effect of BNF and 13C on CYP1A activity was confirmed by the urinary pattern of caffeine metabolites, and was parallelled by a decrease in plasma bilirubin levels. These data demonstrate the usefulness of the established caffeine assay for the evaluation of inducers of CYP1A as tools for reducing hyperbilirubinemia and further confirm the potential value of I3C in the treatment of CNS-I.

Differential activation of the c-Ki-ras-2 proto-oncogene in human colorectal carcinoma.

In colorectal carcinoma, c-Ki-ras-2 mutations predominantly occur in codon 12 and, to a considerably lesser extent, in codon 13. To our knowledge, involvement of codon 61 in c-Ki-ras-2 has been reported only once among the large number of colon cancers investigated altogether. In this study, five human primary colorectal carcinomas were analyzed for the presence of activating c-Ki-ras-2 point mutations in codon 12, 13, and 61. Tumor DNAs were amplified by PCR and subsequently hybridized to a panel of synthetic oligonucleotides representing the complete spectrum of possible mutations. In two of the five tumors, mutations involving codons 13 and 61, respectively, were detected. These data extend previous findings that point mutation of codon 61 may be an improbable yet possible event leading to activation of c-Ki-ras-2 in colorectal carcinoma.

Pharmacokinetics of low doses of benzo[a]pyrene in the rat.

Intestinal absorption, bioavailability, hepatic and pulmonary extraction and elimination of low doses of benzo[a]pyrene (BP; 0.7-4.4 nmol) were studied in the rat using [G-3H]BP. The hepatic extraction ratio was 0.4 both in a liver perfusion model and in vivo as determined by comparison of intravenous and intraportal infusion experiments in anaesthetized rats. The pulmonary extraction ratio in vivo was 0.11 in control rats and 0.16 in rats pretreated with an inducer of cytochrome P-448. Analysis of BP concentrations in atrial blood and in the bile after continuous BP infusion into the duodenum of anaesthetized rats indicated that at least 30% of the dose must have been absorbed from the gut. Studies have also been performed in conscious rats given BP either as an intravenous bolus or by gavage. The bioavailability was determined to be about 10% in these experiments. Elimination proceeded in a triphasic manner with a half-life of 16.6 hr for the terminal phase.

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.

Determination of 2-hydroxyphenylacetic acid (2HPAA) in urine after oral and parenteral administration of coumarin by gas-liquid chromatography with flame-ionization detection.

The urinary excretion of 2-hydroxyphenylacetic acid (2HPAA) was studied in human volunteers after oral and parenteral doses of coumarin. The presence of 2HPAA in the urine was confirmed by gas chromatography mass spectroscopy (GC MS). Mass spectra of reference material and samples are presented. The determination of 2HPAA was carried out by GC with flame-ionization detection. Prior to analysis samples were extracted into ethyl ether and the analytes were derivatized with trimethlyphenylammonium hydroxide. A calibration range from 0.3 to 150 micrograms ml-1 was established using 3-hydroxyphenyl acetic acid (3HPAA) as an internal standard. On average less than 10% of the coumarin administered were excreted into the urine in the form of 2HPAA.

[Ethanol ingestion following Antabus overdose: acetaldehyde-induced cardiological emergency]. / Ethanolaufnahme nach Antabus-Uberdosierung: Acetaldehyd-induzierter kardiologischer Notfall.

The differential diagnosis of chest pain is challenging, when the clinical presentation appears pathognomonic, yet conventional diagnostic tests fail to reveal the suspected cause. We report the case of a 38-year-old patient who had an acetaldehyde intoxication (antabuse syndrome) in the setting of disulfiram overdose and ethanol ingestion. The patient presented with severe angina pectoris. Coronary artery disease was suspected, because the patient had risk factors and electrocardiographic repolarization changes were present. During the further investigation it became evident that symptoms were solely caused by acetaldehyde intoxication following disulfiram and alcohol ingestion. Toxic levels of acetaldehyde were found in the patient's serum. Coronary artery disease was ruled out by cardiac catheterization.