Friedrich J. Wiebel

Aryl hydrocarbon (benzo(a)pyrene) hydroxylase in microsomes from rat tissues: differential inhibition and stimulation by benzoflavones and organic solvents.

Abstract The in vitro addition of 7,8-benzoflavone inhibits the aryl hydrocarbon (benzo[ a ]pyrene) hydroxylase 2 (AHH) 3 in hepatic microsomes from methylcholanthrene treated male rats, but not in hepatic microsomes from control or phenobarbital-treated animals. On the other hand, 7,8-benzoflavone inhibits microsomal hydroxylase from lung and kidney of both control and methylcholanthrene-treated rats; the inhibition is somewhat less in preparations from control animals. 7,8-Benzoflavone also inhibits the AHH from skin of control and benzanthracene-treated mice and from control and benzanthracene-treated hamster embryo cell cultures. In contrast to 7,8-benzoflavone, organic solvents such as monovalent alcohols or dimethylsulfoxide inhibit AHH in hepatic microsomes from control and phenobarbital treated rats, but have little inhibitory effect on methylcholanthrene-induced hepatic hydroxylase. The data indicate the existence of at least two forms of benzo[ a ]pyrene hydroxylating enzyme systems in rat tissues which can be differentially induced in vivo by administration of phenobarbital or methylcholanthrene.

Aryl hydrocarbon (benzo[a]pyrene) hydroxylases in liver from rats of different age, sex and nutritional status: Distinction of two types by 7,8-benzoflavone

Abstract Two types of aryl hydrocarbon (benzo[a]pyrene) hydroxylase have been distinguished in liver from rats of different sex and age by their sensitivity to the synthetic flavonoid, 7,8-benzoflavone. One type, which is stimulated by the 7,8-benzoflavone, is found in newborn rats and predominates in the liver of adult male rats. This type is inducible by phenobarbital. A second type, which is inhibited by 7,8-benzoflavone, comprises a larger fraction in the liver of adult female rats and is inducible by polycyclic hydrocarbons in immature and mature animals of either sex. The presence of this form in adult female liver is also indicated by the kinetics of the hydroxylase reaction. Removal of solid food for 18 hr not only decreases hepatic aryl hydrocarbon hydroxylase activity in female rats, but also lowers the degree of inhibition by 7,8-benzoflavone. Kinetic data suggest that at low concentrations 7,8-benzoflavone acts as a competitive inhibitor but at higher concentrations inhibits the hydroxylation reaction by a more complex mechanism.

Dimethylbenzanthracene Tumorigenesis and Aryl Hydrocarbon Hydroxylase in Mouse Skin: Inhibition by 7,8-Benzoflavone

Mouse skin contains aryl hydrocarbon hydroxylase which is highly inducible. The enzyme system is inhibited when 7,8-benzoflavone is added to homogenates of skin epidermis. 7,8-Benzoflavone also inhibits mouse skin tumorigenesis caused by repeated treatment with 9,10-dimethylbenzanthracene or by a single treatment with this chemical followed by weekly treatment with croton oil. These findings suggest that this enzyme system may be responsible for the activation of 9,10-dimethylbenzanthracene to its carcinogenic form.

Activation of xenobiotics by monooxygenases: cultures of mammalian cells as analytical tool

AbstractThe present studies explore some of the limitations and possibilities of using cultured mammalian cells in the analysis of xenobiotic metabolism by microsomal monooxygenases.A large variety of cells in culture contain monooxygenase activity which is inducible severalfold by polycyclic hydrocarbons. Presently the application of cultured cells to studies of drug activation is restricted mainly by two factors:1)Cells in culture may contain forms of monooxygenases that differ from those predominating in liver. This is suggested by the metabolism of benzo(a)pyrene in fetal cells in culture and in liver microsomes of mouse and hamster. These cultured cells appear to oxygenate preferably the benzo ring of benzo(a)pyrene whereas microsomes of liver predominantly attack the pyrene side of the molecule. Earlier studies indicated that monooxygenases in cultured cells resemble in many respects those in extrahepatic tissues. The results suggest that at present cells in long term culture are unsuitable as model for the hepatic metabolism of xenobiotics and limited as tool for a general screening of potentially hazardous chemicals.2)Monooxygenase activity in many established cell lines is too low to be useful in studies of drug metabolism. In some selected cultures this may be overcome by the use of a cyclic nucleotide phosphodiesterase inhibitor which may increase polycyclic hydrocarbon induced monoxygenase activity 2 fold and in some cell lines more than 10 fold. Selection of cells with specific sets of enzymes involved in xenobiotic metabolism may be facilitated by somatic cell hybridization techniques. A beginning has been made with the preliminary assignment of a locus required for aryl hydrocarbon hydroxylase expression to human chromsome 2 in hybrids of RAG cells, an established mouse line, and normal human bone marrow cells.ZusammenfassungIn der vorliegenden Arbeit wird darüber berichtet, inwieweit Säugetierzellen in Kultur für Untersuchungen des oxidativen Metabolismus von Fremdstoffen geeignet sind. Viele Zellstämme enthalten Monooxygenaseaktivität, die sich durch polycyclische Kohlenwasserstoffe auf ein Mehrfaches induzieren läßt. Gegenwärtig lassen sich jedoch Zellkulturen aus folgenden Gründen nur beschränkt zum Studium der Fremdstoffaktivierung einsetzen:1)Zellen in Kultur enthalten anscheinend Monooxygenaseformen, die von den in der Leber vorherrschenden Formen verschieden sind. Dies spiegelt sich im unterschiedlichen Metabolismus des Benzpyrens wieder. Foetale Hamsterund Mauszellen scheinen vorwiegend Positionen am Benzo-Ring zu oxydieren, während Lebermikrosomen aus diesen Tierspezies in erster Linie den Pyren-Teil des Moleküls oxidativ angreifen. Schon früher war beobachtet worden, daß Monooxygenasen in Zellkulturen mehr denen in extrahepatischen Geweben ähneln. In Anbetracht der Befunde scheinen Zellen in Dauerkultur gegenwärtig ungeeignet zu sein, als Modell für den hepatischen Metabolismus von Fremdstoffen zu dienen, und nur begrenzt brauchbar zu sein zum generellen Screening gefährlicher Substanzen.2)In vielen Zellstämmen ist die Aktivität der Monooxygenasen so niedrig, daß der Fremdstoffmetabolismus nicht mehr analytisch erfaßt werden kann. Dem läßt sich in einigen spezifischen Zellstämmen durch Zusatz eines Hemmstoffes der Nucleotid-Phosphodiesterase entgegenwirken. Die durch polycyclische Kohlenwasserstoffe hervorgerufene Induktion kann dadurch um das 2-10fache gesteigert werden. Hybridisierung von somatischen Zellen sollte es ermöglichen, Zellen auszulesen, die sich durch ihre spezifische Ausstattung mit Fremdstoff-metabolisierenden Enzymen auszeichnen. Ein erster Schritt in dieser Richtung wurde mit der Hybridisierung von Mauszellen und menschlichen Knochenmarkzellen getan, die zur vorläufigen Zuordnung eines Gens, das für die Expression der Aryl-Hydrocarbon-Hydroxylase erforderlich ist, zum menschlichen Chromosom 2 führte.

Impairment of UDP-glucose dehydrogenase and glucuronidation activities in liver and small intestine of rat and guinea pig in vitro by piperine

The effects of piperine, a major ingredient of black pepper, on UDP-glucose dehydrogenase (UDP-GDH) and glucuronidation potentials of rat and guinea pig liver and intestine were studied. Piperine caused a concentration-related strong inhibition of UDP-GDH (50% at 10 microM) reversibly and equipotently, in both tissues. Partially purified rat liver UDP-GDH was used to obtain the kinetic values at pH optima of 9.4 and 8.6. At pH 9.4: KmUDP-glucose = 15 microM, Vmax = 5.2 nmol NADH/min/mg protein, Ki = 6 microM. With NAD, a Ki of 16 microM was obtained. At pH 8.6: Km = 35 microM, Vmax = 7.5 nmol, Ki = 15 microM. In all of these cases, piperine caused non-competitive inhibition. Data from structure-activity comparisons of piperine analogs indicated that the presence of conjugated double bonds in the side chain of the molecule is a factor in piperine inhibition. However, the UDP-glucuronic acid (UDPGA) contents were decreased less effectively by piperine in isolated rat hepatocytes compared with enterocytes of guinea pig small intestine. Piperine at 50 microM caused a marginal decrease of UDPGA in hepatocytes when the rate of glucuronidation of 3-hydroxybenzo[a]pyrene (3-OH-BP) decreased by about 40%. The decrease obtained at 10 microM piperine in intestinal cells was comparable to that obtained at 50-100 microM in hepatocytes. UDP-glucuronosyltransferase (UGT) activities towards 3-OH-BP (UGT1A1) and 4-OH-biphenyl (UGT2B1) were also determined. Piperine did not affect the rate of glucuronidation of 4-OH-biphenyl in rat liver, whereas that of 3-OH-BP was impaired significantly. In guinea pig small intestine, both these activities were inhibited significantly requiring less than 25 microM piperine to produce a more than 50% inhibition of UGT(s). The results suggested that (i) piperine is a potent inhibitor of UDP-GDH, (ii) inhibition is offered exclusively by the conjugated double bonds of the molecule, and (iii) piperine exerts stronger effects on intestinal glucuronidation than in rat liver.

Piperine inhibits aflatoxin B1-induced cytotoxicity and genotoxicity in V79 Chinese hamster cells genetically engineered to express rat cytochrome P4502B1

We have investigated the potential of piperine for inhibiting the activity of cytochrome P4502B1 and protecting against aflatoxin B1 (AFB1) in V79MZr2B1 (r2B1) cells, i.e. V79 Chinese hamster cells engineered for the expression of rat CYP4502B1. The cells were found to contain high activities of 7-methoxycoumarin demethylase (MOCD). Piperine inhibited MOCD in preparations of r2B1 cells with an IC50 of approximately 10 microM. The cells in culture dealkylated 7-methoxycoumarin (MOC) to 7-OH-coumarin linearly, at least for 12 h, where piperine produced concentration-dependent inhibition with IC50 < 30 microM. The time required for maximal inhibition was approximately 8 h with both 30 and 60 microM concentrations of piperine used. AFB1 at 0.1-20 microM caused a concentration dependent decrease in the amount of DNA and an increase in the formation of micronuclei (MN). The mycotoxin at 10 microM reduced DNA by approximately 30% and increased MN appearance by 20-fold against the background level of 7 MN per 500 nuclei. Piperine at 60 microM completely counteracted cytotoxicity and formation of MN by 10 microM AFB1 and reduced the toxic effects of 20 microM AFB1 by > 50%. The results suggest that: (i) Piperine is a potent inhibitor of rat CYP4502B1 activity; (ii) AFB1 is activated by r2B1 cells to cytotoxic and genotoxic metabolites; and (iii) piperine counteracts CYP4502B1 mediated toxicity of AFB1 in the cells and might, therefore, offer a potent chemopreventive effect against procarcinogens activated by CYP4502B1.

Piperine, a major ingredient of black and long peppers, protects against AFB1-induced cytotoxicity and micronuclei formation in H4IIEC3 rat hepatoma cells

We studied the effect of piperine on the cytotoxicity and genotoxicity of aflatoxin B1 (AFB1) in rat hepatoma cells H4IIEC3/G-(H4IIE) using cellular growth and formation of micronuclei as endpoints. Piperine was earlier shown to inhibit cytochrome P-450-dependent aryl hydrocarbon hydroxylase and 7-methoxycoumarin demethylase activities in preparations of these cells with 1/2 maximum inhibition at 30-50 microM (Singh J. and Reen R.K., Current Science, 66, 365-369, 1994). The results of the present study showed that AFB1 inhibited the growth of H4IIE cells with an ED50 of 15 nM. Piperine markedly reduced the toxicity of the mycotoxin. Thus at 100 microM piperine largely restored the rate of growth of the cells. Likewise, piperine reduced the AFB1-induced formation of micronuclei in a concentration-dependent manner. Piperine itself was not toxic to the cells up to a concentration of almost 100 microM. The results suggest, that piperine is capable of counteracting AFB1 toxicity by suppressing cytochromes P-450 mediated bioactivation of the mycotoxin.

Phenobarbital induces cytochrome P-450- and cytochrome P-448-dependent monooxygenases in rat hepatoma cells

The induction by phenobarbital (PB) of aldrin epoxidase (AE) and aryl hydrocarbon hydroxylase (AHH), markers of cytochrome P-450- and cytochrome P-448-dependent monooxygenases, was studied in cell lines derived from Reuber H35 rat hepatoma which differ widely in their degree of differentiation. The following results were obtained: (1) PB induced AE 2-6-fold and AHH 2-4-fold in the differentiated clones, Fao, 2sFou, and C2Rev7 during an exposure period of 72 h. The barbiturate increased AHH but not AE in the dedifferentiated clone H5, the poorly differentiated line H4IIEC3/T, and in the well differentiated line H4IIEC3/G-. (2) Continuous presence of the barbiturate was required for maintaining the induction of the two monooxygenase activities in C2Rev7 cells. (3) Maximum induction of AE was observed at a PB concentration of 1.5-3.0 mM. (4) The effects of 7,8-benzoflavone on AHH-activities induced by phenobarbital in C2Rev7 and H5 cells suggested that they are mediated by cytochrome P-450- and cytochrome P-448-dependent monooxygenase forms, respectively. Thus, the flavonoid had only a slight inhibitory effect on PB-induced AHH in C2Rev7 cells, but strongly inhibited PB-induced AHH in H5 cells. The induction of AE and of 7,8-benzoflavone-inhibitable AHH in 2sFou cells indicated that PB is capable of inducing cytochromes P-450 and cytochrome P-448 in the same cell.

Monooxygenase and UDP-glucuronyltransferase activities in established cell cultures

Cells in culture were investigated for the expression of monooxygenase and UDP-glucuronyltransferase activities as representatives of activating and inactivating pathways of drug metabolism. Most established cell lines express monooxygenase activity that is detected by the oxygenation of polycyclic hydrocarbons and appears to be a function of cytochrome P-448-dependent enzyme forms (Wiebel et al., 1977). In the hepatoma cell line, H-4-II-E, dexamethasone is capable of increasing the level of benzo(a)pyrene-monooxygenation about 10-fold and of potentiating its induction by benz(a)anthracene. The enzyme activities elicited by dexamethasone and the polycyclic hydrocarbon did not significantly differ in their response to 7,8-benzoflavone, an inhibitor of cytochrome P-448-dependent monooxygenases. Observations on the pattern of benzo(a)pyrene metabolites formed in benz(a)anthracene-treated H-4-II-E and BHK21(C13) cells indicate that established cell cultures may contain different forms of monooxygenases of the cytochrome P-448 type.The majority of cell lines tested express UDP-glucuronyltransferase activity directed toward the substrate, 3-hydroxybenzo(a)pyrene. No clear correlation appears to exist between the presence and level of monooxygenase and glucuronyltransferase activities in the various cell lines, i.e., the cultures may express any one or both enzymes. The ratio of the two enzyme levels can be modified by selective induction. Thus, at present there is a choice of established cells in culture exhibiting widely differing ratios of activating and inactivating enzymes to analyse the metabolic pathways of selected classes of foreign compounds, such as polycyclic hydrocarbons, and to determine their toxicological significance. Further efforts are likely to yield cell lines that express the enzymic functions lacking in the cultures currently used and will be suitable to study the full spectrum of foreign compounds.

Dexamethasone-mediated potentiation of P450IA1 induction in H4IIEC3/T hepatoma cells is dependent on a time-consuming process and associated with induction of the Ah receptor

The synergistic effect of dexamethasone (DEX) and polycyclic aromatic hydrocarbons on the induction of cytochrome P450IA1 (P450IA1) was examined in H4IIEC3/T Reuber hepatoma cells. P450IA1 activity was determined by the hydroxylation of benzo[a]pyrene (AHH) and deethylation of 7-ethoxyresorufin (EROD). The amount of Ah receptor, i.e. the specific cytosolic binding protein of 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in H4IIEC3/T cells was characterized and quantitated by high performance gel filtration. Benz[a]anthracene and TCDD induced AHH and EROD activities, respectively, about 20-fold within 4 h. The increase was about 100-fold when cells were pretreated with DEX. The glucocorticoid alone induced P450IA1 activities 3-4 fold. DEX elicited half maximum AHH induction at a concentration of 20 nM in the presence or absence of benz[a]anthracene. Maximal potentiation of AHH induction required treatment with DEX for at least 32 h prior to the exposure to benz[a]anthracene. Treatment of H4IIEC3/T cells with DEX for 20 h caused a 2-3-fold increase in the amount of Ah receptor. The results suggest that the synergistic effect of DEX and polycyclic aromatic hydrocarbons on P450IA1 induction involves a time-consuming process which may consist of the synthesis or modification of a factor, possibly the Ah receptor.