Microsomes From 3-methylcholanthrene Research Articles

Differential inhibition of hepatic microsomal alkoxyresorufin O-dealkylation activities by tetrachlorobiphenyls

Polychlorinated biphenyls (PCBs) elicit a spectrum of biochemical and toxic effects in exposed animals. In the present study, we assessed the effect of PCB structure, using four symmetrically-substituted PCBs, on cytochrome P450 (CYP)-mediated methoxy-, ethoxy- and benzyloxyresorufin O-dealkylase (MROD, EROD and BROD, respectively) activities. We found that 2,2′,4,4′-tetrachlorobiphenyl (PCB 47), 2,2′,5,5′-tetrachlorobiphenyl (PCB 52), 2,2′,6,6′-tetrachlorobiphenyl (PCB 54) and 3,3′,4,4′-tetrachlorobiphenyl (PCB 77) inhibited alkoxyresorufin O-dealkylase activities in hepatic microsomes from 3-methylcholanthrene (MC) or phenobarbital (PB)-treated rats. Measurement of the in vitro inhibitory potencies of the tetrachlorobiphenyls revealed that MROD, EROD and BROD activities were differentially inhibited and the degree of inhibition was determined by the chlorination pattern of the PCB. PCB 77 was more potent than PCB 47 or PCB 52 at inhibiting MROD and EROD activities in hepatic microsomes from MC-treated rats, while no inhibition of either activity was observed with PCB 54. In contrast, BROD activity measured in hepatic microsomes from PB-treated rats was inhibited by PCB 47, PCB 52 and PCB 54 but not by PCB 77. The mode of inhibition for each activity was also evaluated statistically. Inhibition of the alkoxyresorufin O-dealkylase activities could not be discerned in hepatic microsomes from corn oil-treated rats because the activities were inherently too low. No evidence for mechanism-based inhibition of MROD, EROD or BROD activities or an effect via CYP reductase was found. The results demonstrate that relatively coplanar PCBs such as PCB 77 preferentially inhibit EROD and MROD activities, whereas noncoplanar PCBs such as PCB 54 preferentially inhibit BROD activity.

Cytochrome P450 1A1 (CYP1A1) inhibitor alpha-naphthoflavone interferes with UDP-glucuronosyltransferase (UGT) activity in intact but not in permeabilized hepatic microsomes from 3-methylcholanthrene-treated rats: possible involvement of UGT-P450 interactions.

The effects of cytochrome P450 (P450, CYP) ligands and permeabilization of microsomes on 3-hydroxybenzo(a)pyrene [3-OH-B(a)P] glucuronidation mediated by rat hepatic microsomes were studied. While the UDP-glucuronosyltransferase (UGT) activity with non-permeabilized microsomes from 3-methylcholanthrene (MC)-treated rats was markedly reduced by alpha-naphthoflavone (NF), this inhibitor had hardly any effect when permeabilized microsomes were used in which the inhibitor was expected to have easy access to UGT. Kinetic analysis indicated that the inhibitory effect of alpha-NF is competitive. These results suggest that a UGT isoform(s) involved in 3-OH-B(a)P glucuronidation is interfered by a CYP1A inhibitor via a mechanism dependent on the intact nature of microsomal membranes in MC-treated rats. It is likely that P450 functions as a substrate transporter for some isoforms of UGT via possible interactions between UGT and P450.

Multiple Roles of Polyhalogenated Biphenyls in Causing Increases in Cytochrome P450 and Uroporphyrin Accumulation in Cultured Hepatocytes

Uroporphyrin (URO) accumulation occurs in chick embryo hepatocytes treated with a number of polyhalogenated aromatic hydrocarbons (PHAHs) that are known inducers of cytochrome P4501As (CYP1A). Previous dose response studies had shown that URO accumulation does not begin until CYP1A, as indicated by ethoxyresorufin O-deethylase (EROD) activity, is maximally induced. The reason why the concentrations of PHAHs required for URO accumulation were higher than those required to induce EROD had not been explained. PHAHs, such as 3,3′,4,4′-tetrachlorobiphenyl (PCB77, IUPAC nomenclature, TCB) stimulate uroporphyrinogen (UROGEN) oxidation by microsomes from 3-methylcholanthrene (MC)-treated chick embryos. Here we used a new protocol to investigate whether the requirement for more TCB to stimulate in vitromicrosomal UROGEN oxidation extended to TCB-induced URO accumulation in intact cultured hepatocytes. Cultures were treated with increasing concentrations of TCB or other PHAHs to induce CYP1As, then with cycloheximide (CX) to prevent further P450 synthesis. The CX treatment was shown to block any further increases in CYP1A as determined by immunoblots. 5-Aminolevulinic acid and a high concentration of TCB (“postinduction TCB”) were then added to stimulate intracellular UROGEN oxidation. Using the protocol with postinduction TCB, the inducing concentrations of TCB which caused URO to begin to accumulate were now much lower than in the absence of postinduction TCB. Increases in CYP1A proteins, measured immunochemically, were detected at about the same inducing TCB concentrations that began to increase URO accumulation. The new protocol, with postinduction TCB, using URO accumulation as the end point, greatly increased the sensitivity of the culture system for detection of PHAHs with EC50s (n m) for 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), TCB, 3,3′,4,4′,5,5′-hexachlorobiphenyl, MC, and hexachlorobenzene being about 0.003, 0.11, 0.75, 3.5, and 30, respectively. As little as 2–4 fmol TCDD per culture dish caused detectible increases in URO accumulation. We conclude that URO accumulation in chick hepatocyte cultures is limited not only by the induction of CYP1A, but also by the stimulation of intracellular UROGEN oxidation.

Effect of Cytochrome P450 Inducers on Liver Microsomal Metabolism of Tetrachlorobiphenyls in Rats, Guinea Pigs and Hamsters.

Effect of cytochrome P450 (P450) inducers on liver microsomal metabolism of 3,4,3',4'-, 3,5,3',5'- and 2,5,2', 5'-tetrachlorobiphenyl (TCB) was studied using male Wistar rats, male Hartley guinea pigs and male Golden syrian hamsters. In metabolism of 3,4,3'4'- and 3,5,3',5'-TCB, liver microsomes from 3-methylcholanthrene (MC)- or 3,4,5,3',4'-pentachlorobiphenyl (PenCB)-treated hamsters showed hydroxylase activities for both TCB isomers, although the activities were much less than those of rats. In contrast, liver microsomes form untreated and phenobarbital (PB)-, MC- or PenCB-treated guinea pigs showed no hydroxylase activity. In 2,5,2',5'-TCB metabolism, 3-hydroxylase activity was observed in untreated guinea pigs and hamsters, but not in untreated rats. The activity pigs was induced by PB treatment in all three species, at rates of 324, 19 and 20 pmol/min/mg protein in rats, guinea and hamsters, respectively. This activity was not enhanced by treatment with either MC or PenCB. Only in hamsters was 4-hydroxylated metabolite formed in all microsomes used in addition to the 3-hydroxylated one, and the formation was accelerated 2.0-, 2.7- and 4.8-fold by treatment with PB, MC and PenCB, respectively. These results suggest that different P450 isoforms in hamster liver microsomes are involved in 3- and 4-hydroxylation of 2,5,2',5'-TCB. Thus, there are species differences in the basal ability to hydroxylate TCB isomers, and in the extent of effect of P450 inducers on the metabolism of these isomers among the three species.

Uroporphyrinogen oxidation catalyzed by reconstituted cytochrome P450IA2

Previous work suggested that the oxidation of uroporphyrinogen to uroporphyrin is catalyzed by cytochrome P450IA2. Here we determined whether purified reconstituted mouse P450IA1 and IA2 oxidize uroporphyrinogen. Cytochromes P450IA1 and IA2 were purified from hepatic microsomes from 3-methylcholanthrene (MC)-treated C57BL/6 mice, using a combination of affinity chromatography and high performance liquid chromatography. Reconstituted P450IA1 was more active than P450IA2 in catalyzing ethoxyresorufin- O-deethylase (EROD) activity, whereas P450IA2 was more active than P450IA1 in catalyzing uroporphyrinogen oxidation (UROX). Both reactions required NADPH, NADPH-cytochrome P450 reductase, and either P450IA1 or IA2. Ketoconazole competitively inhibited both EROD and UROX activities, in microsomes from MC-treated mice. Ketoconazole also inhibited UROX catalyzed by reconstituted P450IA2. In contrast, ketoconazole did not inhibit UROX catalyzed by xanthine oxidase in the presence of iron-EDTA. Superoxide dismutase, catalase, and mannitol inhibited UROX catalyzed by xanthine oxidase/ iron-EDTA, but did not affect UROX catalyzed by either microsomes or reconstituted P450IA2. These results suggest that UROX catalyzed by P450IA2 in microsomes and reconstituted systems does not involve free reactive oxygen species. Two known substrates of cytochrome P450IA2, 2-amino-3,4-dimethylimidazole[4,5- f]quinoline and phenacetin, were shown to inhibit the microsomal UROX reaction, suggesting that uroporphyrinogen binds to a substrate-binding site on the cytochrome P450.

In vitro metabolism of N-methyl-dibenzo [ c, g]carbazole a potent sarcomatogen devoid of hepatotoxic and hepatocarcinogenic properties

The metabolism of N-methyl substituted 7 H-dibenzo[ c, g]carbazole ( N-Me DBC) was investigated in vitro using liver microsomes from 3-methylcholanthrene (MC)-, benzo[ c]carbazole (BC) and Arochlor-pretreated mice and rats. N-Me DBC is a potent sarcomatogen devoid of hepatotoxicity and liver carcinogenic activity. The ethyl acetate-extractable metabolites were separated by high performance liquid chromatography (HPLC) and most of them were identified by proton magnetic resonance (PMR), mass spectrometry (MS) and comparison with synthetically prepared specimens. Mouse and rat microsomes gave rise to the same metabolites. The major metabolites were 5-OH- N-Me DBC (50%), N-hydroxymethyl (HMe) DBC (25–30%) and 3-OH- N-Me DBC (10%). Addition of 1,1,1-trichloropropene-2,3-oxide (TCPO) to the standard incubation medium permitted the identification of two dihydrodiols among the minor metabolites. No metabolite of DBC was observed after incubation of N-Me DBC, or its major metabolite N-HMe DBC, with either mouse or rat microsomes, but the possibility of a slight demethylation cannot be totally excluded. The lack of biotransformation at the nitrogen atom site may explain the lack of hepatotoxicity and liver carcinogenic activity of N-Me DBC. The modulation of metabolism by epoxide hydrolase, cytosol and glutathione was also investigated. The results are discussed in the light of data previously obtained with hepatotoxic and hepatocarcinogenic DBC.

Light reversal of carbon monoxide inhibition of 2-acetylaminofluorene- N- and ring-hydroxylations by liver microsomes from 3-methylcholanthrene pretreated hamsters

Light reversal of carbon monoxide inhibition of 2-acetylaminofluorene (AAF) N- and ringhydroxylations is examined with liver microsomes from 3-methylcholanthrene (MC) pretreated hamsters. Photochemical action spectra indicate that carbon monoxide inhibition of ring-hydroxylation is readily reversed by light of 440 nm while that of N-hydroxylation is not reversed. These data suggest that these mixed function oxidations are catalyzed by CO combining enzymes with different properties.

Antibody inhibition of oxidative activation and inactivation of the carcinogen 2-acetylaminofluorene by purified hepatic cytochrome P-450 from 3-methylcholanthrene pretreated rats

Immunochemical studies on metabolic N- and ring hydroxylation of 2-acetylaminofluorene (AAF) were performed with total cytochrome P-450 isozymes and with highly purified P-450 D isozyme isolated from liver microsomes from 3-methylcholanthrene (MC)-pretreated rats. In a reconstituted system with rat P-450 D, addition of antibodies against β-naphthoflavone (BNF) induced rat P-450 B at 15 mg IgG/nmol P-450 inhibited both oxidations completely. With total P-450 isozymes in a reconstituted system, antibody against BNF-rat- P-450 B at 20 mg IgG/nmol P-450 inhibited both oxidations up to 70–80% only. At these concentrations, preimmune antibody or phenobarbital (PB) induced antibody against rat- P-450 B showed no inhibtion of AAF oxidations. These results suggest that P-450 D is the predominant cytochrome P-450 isozyme responsible for AAF N- and ring-oxidations in liver microsomes from MC-pretreated rats. Other P-450 isozymes are also suggested to be involved in AAF oxidations.

Rat liver microsomal metabolites of 7-methylbenz[ c]acridine

The metabolism of the weak carcinogen 7-methylbenz[ c]acridine (7MBAC) was examined in rat liver microsomes from 3-methylcholanthrene(MC)-induced animals by the use of mixed 14C- and 2H-labelled substrate. The three metabolites identified by spectroscopic and chromatographic examination were 7-OHMBAC and two dihydrodiols. The dihydrodiols were assigned structures consistent with attack on the 8,9- and 5,6- or K-region of the aromatic system.

Specific labelling of microsomal proteins by reactive intermediates generated from 2-acetylaminofluorene in vitro

Incubation of 2-[9- 14C] acetylaminofluorene (2-[9- 14C]AAF) in vitro with rat liver microsomes, leads to covalent binding of label to microsomal proteins. The binding is NADPH-dependent, increases linearly with time, and is inhibited by SKF-525A and 7,8-benzoflavone (7,8-BF). Binding is increased more than 8-fold in microsomes from 3-methylcholanthrene(MC)-pretreated rats, but only less than 2-fold in those from phenobarbital(PB)-pretreated rats. In the presence of cytosolic proteins, there is slight enhancement of the labelling of microsomes and some labelling of the cytosolic proteins. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional gel electrophoresis indicate that covalent labelling by 2-AAF derivatives is concentrated in specific proteins. The pattern of labelling varies between microsomes from animals pretreated with PB, MC and 2-AAF. Factors which may contribute to the specificity of labelling are discussed.

Absolute stereochemistry of the trans-dihydrodiols formed from benzo[a]anthracene by liver microsomes

Through application of the exciton chirality method, absolute stereochemistry has been assigned to the (+)- and (−)-enantiomers of four of the five metabolically possible trans-dihydrodiols of the polycyclic hydrocarbon benzo[ a]anthracene (BA). The (+)- and (−)-enantiomers of each of these dihydrodiols can be separated as their diastereomeric bis-esters with (−)-α-methoxy-α-trifluoromethylphenylacetic acid by high pressure liquid chromatography (HPLC). BA 3,4-, 5,6-, 8,9- and 10,11-dihydrodiol are formed in 38%, 36%, 78% and 66% enantiomeric purity, respectively, by liver microsomes from phenobarbital-treated rats, whereas the liver microsomes from 3-methylcholanthrene(MC)-treated rats form BA 5,6-, 8,9- and 10,11-dihydrodiols with higher optical purity (62%, 96% and 96%, respectively). BA 3,4-dihydrodiol is formed from (±)-BA 3,4-oxide by microsomal epoxide hydrase in very high enantiometric purity (78%). The major enantiomer of the BA dihydrodiols formed by liver enzymes has R,R absolute stereochemistry in each case. In parallel with previous studies on the metabolism of benzo[ a]pyrene, the more tumorigenic (−)-enantiomer is the predominant isomer of BA 3,4-dihydrodiol formed by liver microsomes from BA.

The in vitro metabolism and covalent binding of benzo[ a]pyrene to dna catalysed by trout liver microsomes

Summary Aryl hydrocarbon ( benzo[ a ]pyrene) hydroxylase activity (EC 1.14.14.2) and the binding of benzo[ a ]pyrene metabolites to deproteinized DNA in vitro and both about 3–4 times higher in trout liver microsomes than in control rat liver microsomes. The hydroxylase activity and the DNA binding with liver microsomes from 3-methylcholanthrene (MC) treated rats are about 10 and 36 times, respectively, greater than in control rat liver microsomes. The liver microsomes from another fresh-water fish (roach) are relatively inactive, the enzyme activity about 7% and the DNA binding 11% of that in control rat liver microsomes. Several BP metabolite-nucleoside complexes were resolved with a Sephadex LH-20 column chromatography in samples with rat and trout liver microsomes. There are no discernible peaks with roach liver microsomes. With rat liver microsomes, more than ten peaks are detectable, the most prominent being peaks associated with benzo[ a ]pyrene-diol-epoxide and benzo[ a ]pyrene-phenols. The same peaks are highest with trout liver microsomes. The benzo[ a ]pyrene metabolite patterns with trout or 3-MC-induced rat liver microsomes show a predominance of non-K-region metabolites, compared with control rat liver microsomes. These results show that the formation of different reactive benzo[ a ]pyrene intermediates which bind covalently to DNA is very similar between 3-MC-treated rat and untreated trout liver microsomes.

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

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 [ 14C]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 [ 14C]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.