The effect of an NADPH-regenerating system on biphenyl metabolism in isolated rat hepatocytes.

Biphenyl 4-hydroxylation was studied in isolated rat hepatocytes. It was found that there was in inter-relationship between 4-hydroxylase activity and glucuronidase activity, removal of 4-hydroxybiphenyl by conjugation being necessary to stimulate a second phase of hydroxylation. Addition of an NADPH-regenerating system resulted in an initial depression of both processes, but later their activities were enhanced. This action could not be explained by the presence of non-viable cells.

Studies on the degranulation test for carcinogens.

The radiometric assay of degranulation of the hepatic endoplasmic reticulum by chemical carcinogens has been re-examined. Both 1,2,3,4,- and 1,25,6-dibenzanthracenes caused degranulation of rough membranes in vitro; with acetamidofluorenes and naphthylamines the carcinogenic analogues caused moderately greater degranulation. Degranulation by 1,2,3,4-dibenzantracene was rapid and was maximal after 5 min incubation. Pretreatment of animals with phenobarbital or methylcholanthrene increased the fraction of rough membranes, but these were not fully granulated. The assay is limited in specificity and sensitivity because the 1.35 M sucrose gradient does not effectively separate rough and smooth membranes, and sedimented membranes are contaminated with aggregates of free ribosomes.

Elimination of glyceryl trinitrate: effects of sex, age, species and route of administration.

1 Orally administered glyceryl trinitrate to rats undergoes extensive first pass metabolism leading to low bioavailability. 2 Sex differences in the plasma elimination of glyceryl trinitrate were seen in the rat, the female exhibiting the longer plasma half-life. No sex differences in this respect were detected in the rabbit. 3 The plasma half-life of glyceryl trinitrate was longer and the volume of distribution larger, in older animals. 4 The plasma elimination of glyceryl trinitrate was different in various animal species. There was a good correlation between plasma half-life and animal bodyweight.

Induction of the cytochrome P450 I and IV families and peroxisomal proliferation in the liver of rats treated with benoxaprofen. Possible implications in its hepatotoxicity.

Administration of the non-steroidal anti-inflammatory drug benoxaprofen to rats gave rise to significant increases in the hepatic O-dealkylations of ethoxyresorufin and methoxyresorufin and in the 12-hydroxylation of lauric acid but, in contrast, the N-demethylation of dimethylnitrosamine was inhibited. Immunoblot studies employing solubilized microsomes from benoxaprofen-treated rats revealed that benoxaprofen increased the apoprotein levels of P450 IA1 and A2 and of P450 IVA1. The same treatment with benoxaprofen increased the beta-oxidation of palmitoyl CoA determined in liver homogenates, and immunoblot analysis showed an increase in the apoprotein levels of the trans-2-enoyl CoA hydratase bifunctional protein. It is concluded that benoxaprofen is a peroxisomal proliferator which selectively induces the hepatic cytochrome P450 I and IV families. The possible implications of these findings to the well-known hepatotoxicity of this drug are discussed.

Molecular dimensions of the substrate binding site of cytochrome P-448.

The molecular geometries of specific substrates, inhibitors and inducers of cytochrome P-448 activity were determined using computer-graphic techniques for use in defining the molecular dimensions of the substrate binding site of this enzyme. Specific substrates of cytochrome P-448 are essentially planar molecules characterised by a small depth and a large area/depth ratio. In contrast, compounds that do not serve as substrates of cytochrome P-448 are bulky, non-planar molecules characterised by small area/depth ratios and greater flexibility in molecular conformation. Specific inhibitors of cytochrome P-448 whose effect is mediated through interaction with the haem still meet the dimensional criteria for substrates indicating that they must also interact with the substrate binding-site, which is probably located in proximity to the haem. Inducers of cytochrome P-448 activity exhibit similar molecular geometries to the substrates from which it may be inferred that the cytosolic receptor associated with the induction of cytochrome P-448 activity is structurally related to the active site of the cytochrome.

A quantitative structure-activity relationship study on a series of 10 para-substituted toluenes binding to cytochrome P4502B4 (CYP2B4), and their hydroxylation rates.

Molecular structural and molecular orbital calculations (AM1 method) are reported on a series of 10 para-substituted toluene derivatives and this structural information has been used to rationalize the differences between both rates of hydroxylation catalysed by cytochrome P4502B4 and binding to the same cytochrome P450, via the generation of quantitative structure-activity relationships (QSARs). It was found that the rate constant for hydroxylation can be described by a two-variable expression involving the dipole moment and volume of the solvent-accessible molecular surface (r = 0.98), whereas binding free energies are well characterized by combinations of molecular volume and various electronic frontier orbital parameters (r = 0.98 and 0.99). This study represents an advance on a previous evaluation by White and McCarthy (Arch Biochem Biophys 246: 19-32, 1986) who used empirical physico-chemical parameters to obtain similar results which were generally of lower statistical significance to those of the present work. The QSAR expressions suggest that both binding to P450 and metabolism for this series of compounds are dependent on the relative ability of the molecules to desolvate and occupy the heme binding site, together with electronic properties of the whole molecule and of the methyl group which undergoes hydroxylation.

Inhibition of gastrointestinal mucosal glycoprotein synthesis by the beta-adrenergic blocking drug, practolol.

The effect of administration of practolol and other beta-blocking agents on gastrointestinal mucosal glycoprotein synthesis was studied in the rat. Practolol, at a dose of 50 mg/kg, inhibited the incorporation of N-acetylglycosamine into gastric mucosal glycoproteins, while acebutolol, atenolol, pronethalol and propranolol had no inhibitory effect, even at a dose of 200 mg/kg. In addition, practolol inhibited the incorporation of N-acetylneuraminic acid, D-fucose and L-serine into gastric mucosal glycoproteins, while the other beta-blocking agents had no effect. Administration of practolol caused no significant change in the rate of incorporation of glycoprotein precursors into intestinal mucosal glycoproteins. These results indicate that of the beta-blocking drugs studied, inhibition of glycoprotein synthesis is associated only with practolol and is independent of its beta-blocking effect.

Interactions of imidazole antifungal agents with purified cytochrome P-450 proteins.

The imidazole N-substituted antifungal agents ketoconazole, miconazole and clotrimazole have been shown to be potent inhibitors of oxidative metabolism by both a phenobarbital-induced cytochrome P-450 (P-450b) and a 3-methylcholanthrene-induced cytochrome P-448-protein (P-450c) in reconstituted systems. All three compounds inhibited the cytochrome P-450b-dependent 7-pentoxyresorufin-O-dealkylase and the cytochrome P-450c-dependent 7-ethoxyresorufin-O-deethylase activities. When 7-benzyloxyresorufin and 7-ethoxycoumarin were employed as substrates with both cytochrome preparations, all three antifungal compounds exhibited selective inhibition of the cytochrome P-450b preparation; ketoconazole was always the weakest inhibitor. The three antifungal agents were also shown to elicit a type II difference spectral interaction with both isoenzymes, the magnitude of the spectral interaction being greater with the cytochrome P-450b preparation.

Induction of cytochrome P-448 activity as exemplified by the O-deethylation of ethoxyresorufin. Effects of dose, sex, tissue and animal species.

The effects of tissue, sex, animal species and dose on the induction of cytochrome P-448 activity by various inducing agents were investigated using O-ethoxyresorufin as a model substrate. The liver was by far more effective in catalysing the O-deethylation of ethoxyresorufin (EROD) than the lung and kidney. The extent of induction was also highest in the liver, with the exception of benzo(a)pyrene and 3-methylcholanthrene where inducibility was more pronounced in the kidney. The benzo(a)pyrene-induced hepatic EROD activity in the rat decayed to reach control levels four days after a single administration. Rat hepatic EROD activity was induced in both sexes but tended to be higher in the male. Marked species differences in the inducibility of hepatic EROD activity by various chemicals was observed, the rat being always more responsive when compared to the hamster or mouse. The induction of rat hepatic EROD activity by benzo(a)pyrene, 2-acetylaminofluorene and safrole was dose-dependent, maximum induction being achieved with single doses of 5, 2 and 5 mg/kg, respectively.

Effects of ether anaesthesia and fasting on various cytochromes P450 of rat liver and kidney.

Fed and fasted, male, Wistar albino rats exposed to light ether anaesthesia and killed immediately or after 30 or 120 min recovery were compared with non-anaesthetized rats for changes in liver and kidney cytochrome P450 (CYP) activities. In fed rats, liver total CYP (nmol/mg protein) decreased by 30% immediately after ether, but was restored to normal levels after 30 min recovery; in fasted rats, liver total CYP increased by 20% by fasting alone, then decreased by 65% immediately after ether, and recovered to only 70% of control at 2 hr after ether. Rat liver cytochrome P4501A (CYP1A; 7-ethoxyresorufin O-deethylase or EROD activity) and cytochrome P4502B (CYP2B; 7-pentoxyresorufin O-dealkylase or PROD activity) were decreased after ether anaesthesia, similar to those for total CYP. In contrast, rat liver cytochrome P4502E1 (CYP2E1), determined by p-nitrophenol hydroxylation, increased by 40% by ether anaesthesia alone, 70% by fasting alone and 140% by ether plus fasting; these increases were confirmed by the CYP2E1-mediated activation of nitrosopyrrolidine and by immunoblot analysis using antibody to CYP2E1. In rat kidney, losses of total CYP, CYP1A and CYP2B, and increases of CYP2E1, induced by ether anaesthesia, were much more marked in fasted (90% loss in total CYP, 30% increase in CYP2E1) than in fed rats (slight loss in total cytochrome P450, 30% increase in CYP2E1). As maximum losses of total CYP in liver of fasted rats exposed to ether occurred at the time of maximum increase of CYP2E1 and maximum rate of generation of reactive oxygen species (ROS), it is suggested that the increase of CYP2E1, resulting from its stabilization by fasting and ether, leads to generation of ROS, increase in lipid peroxidation and consequent loss of total CYP, associated with the hepatic and renal necrosis seen in ether intoxication and surgical trauma.

Effect of deuteration of imipramine on its pharmacokinetic properties in the rat.

Imipramine was specifically deuterated in either both aromatic rings or in the N-methyl group, or in both positions, and the pharmacokinetic properties of the products were determined in the rat and compared with those of the non-deuterated analogue. Deuteration of imipramine resulted in a small but significant isotope effect on N-demethylation while aromatic hydroxylation was unaffected. This isotope effect led to a slower rate of systemic clearance, a longer half-life and, when orally administered, enhanced bioavailability. Urinary excretion of didesmethylimipramine-d4, following oral administration of imipramine-d7, was significantly lower than the excretion of didesmethylimipramine following administration of unlabelled imipramine, indicating inhibited demethylation. Similarly, the urinary excretion of desmethylimipramine-d4, didesmethylimipramine-d4 and 2-hydroxydesmethylimipramine-d4 were lower than for the corresponding unlabelled or d7-analogues, indicating the stability of the N-CD3 group. Deuteration had no effect on the pharmacological properties of imipramine as determined in this study.

The mechanism of action of phenformin in starved rats.

The ability of phenformin to lower the blood glucose concentration after an intraperitoneal glucose load, with a concomitant increase in blood lactate concentration, indicated that the drug was increasing the rate of anaerobic glycolysis. The results of experiments in which glucose and gluconeogenic precursors were given to starved rats were explained by a hypothesis for the mechanism of action of phenformin involving inhibition of certain NAD+-dependent dehydrogenases. Substrates with NAD+-linked oxidations could be discriminated from those, like succinate, with FAD-linked oxidations, and succinate may be of use in the treatment of clinical lacticacidosis caused by biguanide drugs.

Personal reflections on 50 years of study of benzene toxicology.

The metabolism of benzene is reviewed, and the objectives of a quantitative balance study begun in 1945 are outlined; problems of toxicology and metabolism research of some 50 years ago are considered. The quantitative metabolism of 14C-benzene in the rabbit is annotated and compared with that of unlabeled benzene quantified by nonisotopic methods. The anomalies of phenylmercapturic acid and trans-trans-muconic acid as metabolites of benzene are examined in detail by isotopic and nonisotopic methods; these compounds are true but minor metabolites of benzene. Oxygen radicals are involved in both the metabolism of benzene and its toxicity; the roles of CYP2E1, the redox cycling of quinone metabolites, glutathione oxidation, and oxidative stress in the unique radiomimetic, hematopoietic toxicity of benzene are discussed. Differences between the toxicity of benzene and the halobenzenes are related to fundamental differences in their electronic structures and to the consequent pathways of metabolic activation and detoxication.

The cytochromes P450 and mechanisms of chemical carcinogenesis.

This article reviews mechanisms of chemical carcinogenesis, from metabolic activation and generation of reactive oxygen species by cytochromes P4511 and P4502E to DNA damage, activation of protein kinase C and ocogenes, hyperplasia, and proteoglycan changes in the cell glycocalyx and lysosomal enzymes which mediate invasion and metastasis.

Cytochromes P450 and species differences in xenobiotic metabolism and activation of carcinogen.

The importance of cytochrome P450 isoforms to species differences in the metabolism of foreign compounds and activation of procarcinogens has been identified. The possible range of P450 isozymes in significant variations in toxicity exhibited by experimental rodent species may have a relevance to chemical risk assessment, especially as human P450s are likely to show changes in the way they metabolize xenobiotics. Consequently, in the safety evaluation of chemicals, we should be cautious in extrapolating results from experimental animal models to humans. This paper focuses on examples in which species differences in P450s lead to significant alterations in carcinogenic response, and includes a discussion of the current procedures for toxicity screening, with an emphasis on short-term tests.

COMPACT and molecular structure in toxicity assessment: a prospective evaluation of 30 chemicals currently being tested for rodent carcinogenicity by the NCI/NTP.

A new series of 30 miscellaneous National Toxicology Program chemicals has been evaluated prospectively for carcinogenicity and overt toxicity by COMPACT (Computer Optimised Molecular Parametric Analysis for Chemical Toxicity. CYP1A and CYP2E1). Evaluations were also made by Hazardexpert, and for metal ion redox potentials; and these, together with COMPACT, were compared with results from the Ames test for mutagenicity in Salmonella, the micronucleus test, and 90-day subchronic rodent pathology. Seven of the 30 chemicals (nitromethane, chloroprene, xylenesulphonic acid, furfuryl alcohol, anthraquinone, emodin, cinnamaldehyde) were positive for potential carcinogenicity in the COMPACT evaluation; xylenesulphonic acid and furfuryl alcohol were only equivocally positive. Four of the 30 chemicals-scopolamine, D&C Yellow No. 11, citral, cinnamaldehyde-were positive by Hazardexpert; 6 of 30-D&C Yellow No. 11, 1-chloro-2-propanol, anthraquinone, emodin, sodium nitrite, cinnamaldehyde-were positive in the Ames test; 2 of 30-phenolphthalein and emodin-were positive in the in vivo cytogenetics test; and 3 of 30-molybdenum trioxide, gallium arsenide, vanadium pentoxide-were metal compounds with redox potentials of the metal/metal ion indicative of possible carcinogenicity. The overall prediction for carcinogenicity was positive for 12 of 30 chemicals: nitromethane, chloroprene, D&C Yellow No. 11, molybdenum trioxide, 1-chloro-2-propanol, furfuryl alcohol, gallium arsenide, anthraquinone, emodin, sodium nitrite, cinnamaldehyde, vanadium pentoxide). This overall prediction has been made on the basis of the results of the computer tests and from consideration of the information from bacterial mutagenicity, together with likely lipid solubility and pathways of metabolism and elimination.

A retrospective evaluation of COMPACT predictions of the outcome of NTP rodent carcinogenicity testing.

The carcinogenic potentials of 40 National Toxicology Program chemicals previously predicted by Computer Optimised Molecular Parametric Analysis for Chemical Toxicity (COMPACT), based on the identification of potential substrates of cytochromes P4501A and 2E (CYP1A and CYP2E), have been compared with new rodent carcinogenicity results. The COMPACT predictions have also been compared with published Ames mutagenicity data and with our own Hazardexpert predictions for carcinogenicity. Concordance evaluations between rodent carcinogenicity (1/4 segments positive) and predictions by COMPACT or Hazardexpert were 64% for COMPACT (CYP1A only), 72% for COMPACT (CYP1A plus CYP2E), 70% for Hazardexpert alone, and 86% for COMPACT (CYP1A plus CYP2E) plus Hazardexpert. Sensitivities of the predictions were for COMPACT, 75%; Hazardexpert, 60%; and Ames, 54%. Positive predictivities were for COMPACT, 75%; Hazardexpert, 78%; and Ames 81%. Negative predictivites were for COMPACT, 62%; Hazardexpert, 52%; and Ames, 42%.

Quantitative structure-activity relationships in a series of endogenous and synthetic steroids exhibiting induction of CYP3A activity and hepatomegaly associated with increased DNA synthesis.

The results of a quantitative structure-activity relationship (QSAR) study on a total of 14 steroids exhibiting induction of a CYP3A-associated activity and increase in liver weight/DNA synthesis is reported. It is found that different, but related, structural descriptors correlate with increase in ethylmorphine N-demethylase activity (r=0.92) and with the increase in liver weight (r=0.78) and DNA synthesis (r=0.78). Although there is a strong correlation between increase in liver weight and DNA content (r=0.999), neither of these correlated with ethylmorphine N-demethylase activity. These findings are discussed in the light of CYP3A induction, substrate specificity and inhibition; a proposed model of human CYP3A4 based on sequence homology with CYP102, a bacterial P450 of known crystal structure, demonstrates the possible mode of interaction between substrates and inhibitors within the putative active site.

Computer modelling in predicting carcinogenicity.

The cytochrome P450-dependent mixed-function oxidases are the most important enzyme system in the oxidation of chemicals to their reactive intermediates which then interact with cellular components to provoke toxicity and carcinogenicity. These enzymes comprise a multifamily of proteins, two families of which, namely CYP1A and CYP2E, activate planar and small molecular weight compounds, respectively. A computer graphic procedure (COMPACT) has been developed which, based on the molecular shape and electronic structure of the chemical, determines whether the chemical will interact with these two particular cytochrome P450 families and thus be metabolized to toxic and carcinogenic intermediates. As the basal levels of these enzyme families are low, the ability of the chemical to induce them selectively, on repeated administration, is an important determinant of its carcinogenic potential. Inductive capability may be determined in short-term experiments (ENACT) utilizing a small number of animals. Thus the combination of COMPACT and ENACT provides a rapid and inexpensive means for the preliminary screening of chemicals, before the long term and expensive rodent lifetime bioassays are undertaken.

Effect of dietary substitution of sucrose and its constituent monosaccharides on the activity of aromatic hydroxylase and the level of cytochrome P-450 in hepatic microsomes of growing rats.

The effect of dietary substitution of starch by sucrose, glucose, fructose or an equimolar mixture of glucose and fructose on the activity of biphenyl hydroxylase and the level of the major terminal mixed function oxygenase, cytochrome P-450, has been studied in hepatic microsomal preparations from growing rats. The substitution of sucrose for starch depressed the contration and total activity per liver of biphenyl 4-hydroxylase and the concentration, but not the total amount, of cytochrome P-450 in weanling rats. The absolute amount of these enzymes in whole liver was not, however, similarly depressed by the constituents of sucrose, namely glucose and fructose, either when given alone or in equimolar mixture. All these sugars, however, depressed the total activity of biphenyl 2-hydroxylase. The activity of biphenyl 4-hydroxylase in the liver of weanling rats, but not of adults, was reduced when the level of sucrose in the diet was reduced from 60 to 10%.