Helena Vadi

Metabolism of benzo(a)pyrene-3,6-quinone and 3-hydroxybenzo(a)pyrene in liver microsomes from 3-methylcholanthrene-treated rats: A possible role of DT-diaphorase in the formation of glucuronyl conjugates

Abstract Benzo( a )pyrene (BP) quinones and 3-OH-BP are the metabolites preferentially converted to glucuronyl conjugates when BP is metabolized by microsomes from 3-methyl-cholanthrene (MC)-treated rats in the presence of NADPH, O 2 , and UDP-glucuronic acid (UDPGA). No glucuronyl conjugates of [ 14 C]BP-3,6-quinone are formed in the absence of NAD(P)H, indicating that BP quinones must be reduced prior to glucuronylation. The observations that NADH can replace NADPH in BP-3,6-quinone glucuronylation and that these reactions are equally sensitive to dicoumarol, a potent inhibitor of DT-diaphorase, suggest that the reduction of BP-3,6-quinone preceding glucuronylation is catalyzed by DT-diaphorase. Furthermore, trypsin-treated microsomes, which have unchanged DT-diaphorase activity but less than 5% of original NADPH-cytochrome c reductase activity, exhibit unaltered capacity for the conversion of BP-3,6-quinone to glucuronyl conjugates. Analysis of ethyl acetate extracts from incubations with [ 14 C]BP-3,6-quinone by high pressure liquid chromatography reveals that BP-3,6-quinone can be further metabolized by MC-induced microsomes to more polar but still water-insoluble products. These metabolites are not formed in the absence of NADPH or in trypsin-treated microsomes, which have no detectable aryl hydrocarbon monooxygenase (AHM) activity, indicating that the further metabolism of BP-3,6-quinone proceeds through the AHM system. The rate of this reaction and of glucuronylation of BP-3,6-quinone is very similar. The glucuronylation of 3-OH-BP by MC-induced microsomes is also inhibited by dicoumarol, whereas that of p -nitrophenol, methylumbelliferone or phenolphtalein is not. Trypsin treatment of microsomes strongly enhances 3-OH-BP glucuronylation. Evidence is presented suggesting that the dicoumarol effect is probably not due to an inhibition of UDP-glucuronosyltransferase, and the possibility is considered that a DT-diaphorase dependent rearrangement of 3-OH-BP prior to glucuronylation may be responsible for the observed dicoumarol sensitivity.

Cytochrome P-450-linked activation of 3-hydroxybenzo(a)pyrene

Summary Incubation of 3-hydroxybenzo(α)pyrene with rat lung microsomes in the presence of NADPH and oxygen results in the formation of a metabolite which binds covalently to DNA. The reaction is inhibited by carbon monoxide and a-naphthoflavone and markedly induced by pretreatment of the rats with 3-methylcholanthrene.

Formation of DAN-binding products from isolated benzo[a]pyrene metabolites in rat liver nuclei

Liver nuclei from 3-methylcholanthrene-treated rats in the presence of NADPH metabolized 3- and 9-hydroxybenzo[a]pyrene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene to products that bound to DNA. Maximal binding was obtained with the dihydrodiol which was approximately 3-fold that with 9-hydroxybenzo[a]pyrene, and 60-fold that with 3-hydroxybenzo[a]pyrene, as substrates. Both 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene and 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene were also extensively metabolized by the nuclear fraction but did not give rise to DNA-binding products. The available evidence suggests that the DNA binding species derived from 9-hydroxy-benzo[a]pyrene is 9-hydroxy-benzo[a]pyrene-4,5-oxide and from 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, as previously observed in different systems, 7,8-dihydro-7,8-dihydroxy-benzo[a]pyrene-9,10-oxide.

Spectral studies on the rapid uptake and subsequent binding of drugs to cytochrome P-450 in isolated rat liver cells

Summary The rate of formation of the type I spectral change upon drug addition to a suspension of isolated rat liver cells was used to study factors that influence drug uptake by the hepatocytes. Although considerably slower than in liver homogenates and microsomes, drug combination with cellular cytochrome P-450 was still rapid and occurred within a few seconds. The effects of varying temperature and concentration and lipid solubility of the drugs studied as well as the lack of effect of preincubation of the cells with rotenone on the rate of formation of the type I spectral change, lead us to suggest that drug uptake into the hepatocytes occurs by a non-energy requiring diffusion process.

Drug metabolism in isolated rat liver cells.

Although considerable knowledge has been gathered on the functional aspects of microsomal monooxygenation, comparatively little has so far been known about the intracellular regulation of this process. For such studies, we have found the isolated rat liver cell system to be a very useful model, combining the convenience of an in vitro system with the access to the complex mechanisms of the intact in vivo system. This model has the advantage over the perfused liver that it readily lends itself to the study of rapid reaction sequences and makes quantitation of short-term drug metabolic reactions easier. It is also superior to liver slices which often show considerable leakage of adenine and pyridine nucleotides and where substrate penetration and oxygen diffusion may present problems depending on the relative thickness of the slice.

THE ROLE OF LIVER NUCLEI IN THE FORMATION OF DNA BINDING PRODUCTS FROM BENZO(a) PYRENE

Publisher Summary This chapter discusses the role of liver nuclei in the formation of DNA binding products from benzo(a)pyrene (BP). It is generally assumed that the metabolic activation of the parent compound to electrophilic species that can interact with DNA is an early and critical event in the mutagenic and carcinogenic effect of BP and related compounds. Such electrophilic metabolites are produced by monooxygenation through the cytochrome P-450 system and include both primary epoxides and dihydrodiol epoxides. The nuclear BP monooxygenase can convert BP, and metabolites of BP preformed in the microsomes, to DNA binding products. A direct transfer of electrophilic microsomal metabolites seems to have contributed to the total incorporation of BP products into nuclear DNA. The relative importance of these mechanisms under physiological conditions remains to be established although it is tempting to speculate that the formation of the DNA binding species in the close vicinity of the nucleus would permit them to escape trapping by the various cytoplasmic defense systems and thus facilitate their binding to DNA.