High-affinity nitrosamine dealkylase system in rat liver microsomes and its induction by fasting

In order to elucidate the enzymic basis of nitrosamine metabolism, the in vitro metabolism of nitrosamines by rat liver microsomes and the effects of fasting on the microsomal enzymes have been studied. Fasting for 1 to 3 days causes a 2- to 3-fold enhancement of the reduced nicotinamide adenine dinucleotide phosphate-dependent nitrosodimethylamine demethylase (NDMAD) activity. The cytochrome P-450 content and the activities of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase and benzphetamine demethylase, however, are only modestly increased. Gel electrophoretic analysis reveals the induction of a 50,000-dalton protein band during fasting. The induction of this protein band as well as the enhancement of NDMAD activity are inhibited by CoCl2 and inhibitors of protein and RNA biosynthesis. The involvement of cytochrome P-450 in the NDMAD is supported by the fact that microsomal reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase is required for the demethylase activity. Kinetic analysis indicates that a low-Km form of NDMAD (apparent Km, 0.07 mM) is markedly induced by fasting. With microsomes of control rats, there are at least three apparent Km values (0.07, 0.38, and 38.6 mM) for NDMAD; but with microsomes of fasting rats, the low-Km (0.07 mM) form is predominant. These results suggest that rat liver microsomes contain a cytochrome P-450 isozyme which has high affinity for nitrosodimethylamine, and this isozyme is induced by fasting. In addition to nitrosodimethylamine, the oxidative demethylation of N-nitroso-N-methylethylamine, N-nitroso-N-methylbutylamine, N-nitroso-N-methylaniline, and N-nitroso-N-methylbenzylamine is also enhanced by fasting. The extent of enhancement and substrate dependency of these reactions, however, is different from that of NDMAD.     

Depression of the hepatic cytochrome P-450 monooxygenase system by treatment of mice with the antineoplastic agent 5-azacytidine

The effects of 5-azacytidine (5-AC) administration on the hepatic cytochrome P-450 systems of mice were studied. A single i.p. dose of 5-AC (25 mg/kg) to male Swiss-Webster mice caused about a 50% depression of microsomal cytochromes P-450 and b5 and of ethylmorphine N-demethylase and ethoxycoumarin O-deethylase activities. Depression was greatest 24 h after treatment; by 48 to 72 h, cytochromes and drug metabolism had returned to near control values. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity was also depressed by 5-AC, whereas reduced nicotinamide adenine dinucleotide-cytochrome c reductase was not. Incubation of 5-AC with microsomes produced no effect on drug metabolism. The prolongation of hexobarbital sleeping time by 5-AC showed that drug metabolism is also impaired by 5-AC in vivo. These studies may have important clinical implications when certain drugs are coadministered with 5-AC.     

Comparative anthracycline metabolism in rats: loss of marcellomycin fluorescence

The in vitro metabolism of marcellomycin by rat tissue fractions showed conversion of marcellomycin to 7-deoxypyrromycinone, bisanhydropyrromycinone, and an as yet unidentified compound by rat liver homogenate, microsomes, cytosol, and mitochondria, and purified hepatic reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase, under anaerobic conditions and in the presence of reduced nicotinamide adenine dinucleotide phosphate. All these fractions except the purified reductase subsequently induced a progressive loss of fluorescence. Mitochondria, however, were much less active than microsomes, cytosol, and homogenate in inducing this latter phenomenon. Marcellomycin was converted to 7-deoxyaglycones only partially by nuclei. No loss of fluorescence was observed with this subcellular fraction. No loss of fluorescence was observed when doxorubicin or daunorubicin were incubated under similar conditions. The appearance of a compound with distinct spectrophotometric properties was demonstrated by absorbance spectrometry. The formation of a compound with different fluorescent characteristics was excluded, as was the binding of the aglycones to subcellular components. The activity inducing the loss of fluorescence was studied in greater detail with cytosol. It predominated in the liver and required both an electron donor and anaerobic conditions. The optimal pH for the reaction was between 7.5 and 8.0. Our results suggest the existence of an enzymatic pathway capable of converting the fluorescent nucleus of marcellomycin to a nonfluorescent metabolite.     

Synthesis and identification of products derived from the metabolism of the carcinostatic 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by rat liver microsomes.

Liver microsomal metabolism of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea in the presence of reduced nicotinamide adenine dinucleotide phosphate and O2 was shown to produce seven metabolites that included the parent urea. A cytochrome P-450-dependent monohydroxylation of the cyclohexyl ring occurred in 3 positions, cis-3, trans-3, and cis-4, and on the methyl group to form a trans-4-hydroxymethyl derivative. In addition, monohydroxylation of the 2-chloroethyl carbon attached to the N-1 urea nitrogen yielded an alpha-hydroxy metabolite. A ring-hydroxylated derivative remained unidentified while the structures of all other such derivatives were established by comparison with compound synthesized, purified by high-pressure liquid chromatography, and characterized by mass spectral and nuclear magnetic resonance analyses. It was tentatively concluded that some parent urea is formed by a cytochrome P-450 dependent reaction because of a requirement for reduced nicotinamide adenine dinucleotide phosphate and inhibition by CO. Microsomes from rats pretreated with phenobarbital showed about a 3-fold increase in hydroxylation rate while phenobarbital-treated mice microsomes were induced 8-fold. However, in both species, the induced hydroxylation rate was about 4 nmol/min/mg protein. When microsomes from phenobarbital-induced rats were used, a mixture of 80% CO:20% O2 decreased the rate of formation of all metabolites to 14% of that in 80% N2:20% O2.     

Immunological identification and effects of 3-methylcholanthrene and phenobarbital on rat pulmonary cytochrome P-450

Rabbit antibodies to the phenobarbital (PB) inducible rat liver microsomal cytochrome P-450s b and e and to 3-methylcholanthrene (MC) inducible P-450c were used to examine the expression of these isozymes in rat lungs. Western blots of total lung microsomes demonstrated that about 40 pmol P-450b/mg protein (and no detectable P-450e) were present in lungs from control or MC treated rats and that pretreatment with PB caused a small but significant (P less than 0.05) increase in the expression of P-450b. Microsomes from control and PB treated lung contained minimal levels of P-450c, and MC induced this isozyme to 185 pmol/mg. Immunocytochemistry was used to demonstrate immunoreactivity to these isozymes in specific cell types. Neither P-450b nor P-450c was detectable in endothelial cells from control or PB treated lungs, but MC increased immunoreactivity to P-450c in pulmonary endothelial cells. Type II alveolar cells showed distinct immunoreactivity to P-450b and weak immunoreactivity to P-450c in control or PB treated rats. Individual Clara cells stained for either P-450c or P-450b in control, MC treated, and PB treated rats, and colocalization was observed in some cells. An increase in type II alveolar cell and Clara cell immunoreactivity to P-450c was observed after MC induction. Mast cells, identified by metachromatic Giemsa staining, appeared to react nonspecifically with both antisera. In conclusion, P-450c is highly inducible by MC in rat lung (detected in microsomes by Western blot), specifically in endothelial cells, Clara cells, and alveolar type II cells (as visualized by immunocytochemistry); and P-450b is present in rat lung microsomes, and immunoreactivity to this isozyme is localized in alveolar type II and Clara cells.     

Metabolism and activation of N-nitrosodimethylamine by hamster and rat microsomes: comparative study with weanling and adult animals

It has been reported that hamster liver preparations are more effective for the metabolic activation of N-nitrosodimethylamine (NDMA) to a mutagen than rat liver preparations. The enzymatic basis for this phenomenon, however, has not been clearly elucidated. The present study was undertaken to examine the enzymology of NDMA metabolism by different hepatic subcellular fractions prepared from hamsters and rats of two different ages, and to investigate the correlation between the metabolism and the activation of NDMA to a mutagen for Chinese hamster V79 cells. The content of cytochrome P-450 was approximately 1.5-fold higher in hamster microsomes than in rat microsomes from both ages (1.19-1.38 versus 0.73-0.83 nmol P-450/mg protein). Weanling hamster microsomes exhibited multiple apparent Km values for NDMA metabolism as did weanling rat microsomes. The apparent Km I value of NDMA demethylase (NDMAd) in hamster microsomes was about one-half that in rat microsomes (36 versus 83 microM) with corresponding Vmax values of 2.09 and 2.57 nmol/min/nmol P-450. The Km I values for denitrosation did not differ from the corresponding values for NDMAd with Vmax values of 0.17 and 0.22 nmol/min/nmol P-450 for hamster and rat microsomes, respectively. These apparent Km values were affected neither by sonication nor by the presence of cytosolic proteins in S9 fractions. Adult rat liver microsomes showed less than one-half the NDMAd activity in weanling rat liver microsomes, whereas such age difference was not observed in hamster liver microsomes. This result was confirmed by Western blotting showing the levels of P-450ac (an acetone-inducible form of P-450) of these microsomes at comparable levels to their NDMAd activities. NDMAd was highly correlated to the metabolic activation of NDMA to a mutagen for V79 cells in an activation system mediated by microsomes prepared from hamsters and rats of different ages. The results from this study clearly demonstrate the enzymatic basis for the more effective metabolism of NDMA in adult hamsters than in adult rats.     

Prostaglandin synthetase and cytochrome P-450-dependent metabolism of (+/-)benzo(a)pyrene 7,8-dihydrodiol by enriched populations of rat Clara cells and alveolar type II cells

The metabolism of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol) by prostaglandin synthetase and cytochrome P-450-dependent monooxygenases was studied using enriched fractions of Clara cells and alveolar type II cells from rat lung. Arachidonic acid-fortified fractions enriched in Clara cells and alveolar type II cells metabolized BP-7,8-diol to the 7,10/8,9-tetrol of benzo(a)pyrene and the 7/8,9,10-tetrol of benzo(a)pyrene. These tetrols are formed upon solvolysis of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha- epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene (BP diol-epoxide I). Arachidonic acid-dependent metabolism of BP-7,8-diol to BP diol-epoxide I in enriched Clara cells and alveolar type II cells was completely inhibited by indomethacin, a classical inhibitor of prostaglandin synthetase. Enriched Clara cells and alveolar type II cells also metabolized BP-7,8-diol to BP diol-epoxide I in the presence of NADPH. Amounts of BP diol-epoxide I-derived tetrols formed from BP-7,8-diol by the prostaglandin synthetase-dependent and the cytochrome P-450-dependent pathways varied significantly between the two pulmonary cell fractions examined. In fractions enriched in Clara cells, cytochrome P-450-dependent BP-7,8-diol oxidation was higher than was prostaglandin synthetase-dependent BP-7,8-diol oxidation; while in fractions of alveolar type II cells, prostaglandin synthetase-dependent BP-7,8-diol oxidation to BP diol-epoxide I predominated. Pretreatment of rats with beta-naphthoflavone resulted in a 2- to 3-fold increase in BP diol-epoxide I formation by prostaglandin synthetase and cytochrome P-450-dependent monooxygenases in both enriched Clara cells and alveolar type II cells. These increases in BP-7,8-diol oxidation to BP diol-epoxide I appear to be due to induction of the two enzymatic pathways in both pulmonary cell types. No qualitative changes in the pattern of BP-7,8-diol metabolism by either enzymatic pathway in enriched Clara cells or alveolar type II cells were observed following beta-naphthoflavone treatment. The results suggest that pulmonary prostaglandin synthetase may serve as either an additional or an alternative bioactivating enzyme to the cytochrome P-450-dependent monooxygenases for the formation of reactive chemical carcinogens in the lung.     

Phosphorylation of cytochrome-P-450-dependent monooxygenase components

Most chemical carcinogens require activation by polysubstratemonooxygenase. The phosphorylation of essential components ofthis cytochrome P-450 monooxygenase system, isolated from rabbitliver microsomes, cytochrome P-450 (LM₂) and cytochrome reductase,was tested using two different protein kinases. One of the kinases,a cyclic AMP-independent phosvitin kinase (kinase P), was inactivein all systems tested. However, the catalytic subunit of a cyclicAMP-dependent protein kinase (kinase C) catalyzed phosphorylgroup transfer to both proteins, but to different extents. CytochromeP-450 was phosphorylated when added as sole component and alsowhen in the presence of P-450 reductase and phosphatidylcholine.In contrast, the weak phosphorylation of P-450 reductase wasreduced considerably in a complete reconstituted system containingP-450 and phosphatidylcholine. The inclusion of kinase P didnot alter these results which excludes the possibility thatthese kinases participate in a sequential phosphorylation mechanism.The monooxygenase constituents themselves were without kinaseactivity. When hepatic microsomes were isolated in presenceof the phosphatase inhibitor sodium fluoride no significantchange in monooxygenase (7-ethoxycoumarin O-deethylation) activitywas observed, whilst after preincubation with either acid oralkaline phosphatase a significant reduction in monooxygenaseactivity was measured. Thus, cytochrome P-450 (LM₂) is phosphorylatableby protein kinase C and the catalytic activity of polysubstratemonooxygenase decreases after preincubation of microsomes withphosphatases.     

Oxidative metabolism of cyclophosphamide: identification of the hepatic monooxygenase catalysts of drug activation

Cytochrome P-450-catalyzed activation of cyclophosphamide to alkylating metabolites was studied in isolated rat liver microsomes and purified, reconstituted P-450 enzyme systems in order to identify the major enzymatic catalysts of drug activation in both uninduced and drug-induced liver tissue. P-450 form PB-4 (P-450 gene IIB1) activated cyclophosphamide with high efficiency [Vmax (app) = 18.2 nmol metabolite/min/nmol P-450; Km (app) = 0.16 mM] via the formation of 4-hydroxycyclophosphamide, which was quantitatively trapped as a bisulfite adduct then characterized following its conversion to cyano derivatives. Antibodies to P-450 PB-4 inhibited cyclophosphamide activation catalyzed by phenobarbital-induced adult male rat liver microsomes (specific activity, 5.4 nmol metabolite/min/mg liver microsomes) in a selective and near quantitative (greater than 80%) fashion; little or no inhibition was obtained using antibodies inhibitory towards six other rat hepatic P-450 forms. Cyclophosphamide activation catalyzed by uninduced adult male rat liver microsomes (specific activity, 0.68 nmol/min/mg), although not inhibited by anti-P-450 PB-4 antibodies, was partially inhibited (approximately 60%) by antibodies to P-450 PB-1 (gene IIC6) and more completely inhibited (greater than 95%) by antibodies reactive with both P-450 PB-1 and P-450 2c (gene IIC11). Consistent with these observations, P-450 PB-1 and P-450 2c both activated cyclophosphamide at moderate rates in reconstituted systems (turnover, 1.6-2.7 nmol metabolite/min/nmol P-450), while seven other purified hepatic P-450 forms exhibited significantly lower activities (turnover less than or equal to 0.5 nmol metabolite/min/nmol P-450). Further studies revealed that the changes in liver microsomal cyclophosphamide activation rates with age and sex and in response to in vivo administration of cisplatin primarily reflect changes in the levels of P-450 forms PB-1 and 2c. These studies establish that P-450 forms PB-1, 2c, and PB-4 are the major catalysts of cyclophosphamide activation in rat hepatic tissue and that the modulation of microsomal cyclophosphamide activation with development and in response to drug exposure largely reflects alterations in the levels of these three hepatic P-450 enzymes.     

The metabolism of benzo(alpha)pyrene in isolated rat liver cells.

Isolated rat liver cells catalyze the metabolism of benzo(alpha)pyrene (BP) with the resulting formation of phenols, dihydrodiols, and conjugates. The rate of the primary oxidative step in the process was similar to that catalyzed by isolated rat liver microsomes in the presence of a reduced nicotinamide adenine dinucleotide phosphate-generating system and responded similarly to various inhibitors, including 2-diethylaminoethyl-2,2-diphenylvalerate, metyrapone, alpha-naphthoflavone, and hexobarbital. The level of cytoplasmic, reduced nicotinamide adenine dinucleotide phosphate was not rate limiting in liver cells isolated from either fed or fasted animals. The conjugates and dihydrodiols formed were readily excreted, whereas low concentrations of phenols accumulated intracellularly. The pattern of metabolites of BP was the same in isolated rat liver cells and in the isolated perfused rat liver. 3-Methylcholanthrene treatment of the rats caused a marked increase in cellular BP metabolism as well as in cytochrome P-450 concentration. The induced hemoprotein revealed characteristics similar to those previously established with isolated liver microsomes, i.e., increase in high-spin form, enhanced affinity for BP as revealed by a lower Michaelis constant, and sensitivity to the inhibitory action of alpha-naphthoflavone. After 3-methylcholanthrene treatment, phenols and dehydrodiols constituted a larger percentage of the total metabolites, indicating a more pronounced stimulation of the oxidative than of the conjugative step of BP metabolism by induction, and the dihydrodiols now tended to accumulate intracellularly.     

Enhanced metabolism and mutagenesis of nitrosopyrrolidine in liver fractions isolated from chronic ethanol-consuming hamsters

The effect of chronic ethanol consumption on the ability of isolated liver fractions to metabolize the carcinogen N-nitrosopyrrolidine (NPY) was examined. Microsomal fractions of treated animals exhibited increased rates of alpha-hydroxylation of NPY. Similar increases in the specific activities of aniline hydroxylase, reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase, and the specific content of cytochrome P-450 were also observed. In contrast, no differences in the specific activities of benzo(a)pyrene hydroxylase or glucose-6-phosphatase were observed. Liver postmitochondrial supernatants from ethanol-consuming animals were able to produce 5 times more mutants than did control preparations. It is concluded that alpha-hydroxylation of NPY is probably the mechanism by which NPY is converted to a mutagen and that this pathway can be induced by ethanol.     

Binding of metabolically activated benzo(a)pyrene to nuclear macromolecules.

The binding of metabolically activated [3H]benzo(a)pyrene ([3H]BP) to the DNA, RNA, histones, and nonhistones of isolated rat liver and lung nuclei was studied. Conditions for optimal binding to the nuclear components were determined. Upon incubation with isolated liver nuclei and reduced nicotinamide adenine dinucleotide phosphate, [3H]BP was able to bind to nuclear components. The binding appeared to be covalent in nature. Treatment of the rats with 3-methylcholanthrene induced the nuclear aryl hydrocarbon hydroxylase (AHH) activity and also increased the level of carcinogen binding. The addition of rat liver microsomes to the incubation systems greatly enhanced the level of [3H]BP binding to the macromolecules in the nuclei from both the control and 3-methylcholanthrene-treated rats, and the maximal levels of binding obtained with these two types of nuclei were similar. The binding was inhibited by 7,8-benzoflavone or glutathione. Lung nuclei from control rats had very low AHH activity and did not exhibit appreciable carcinogen binding, whereas those from 3-methylcholanthrene-pretreated animals had slightly higher AHH activity and caused low levels of binding. The binding of [3H]BP to lung nuclei was greatly enhanced by liver microsomes but only slightly by lung microsomes, which had rather low AHH activity. Several lines of evidence indicate that, in the control experiments (no reduced nicotinamide adenine dinucleotide phosphate added), the radioactivity associated with the macromolecule fractions is probably a background value rather than due to the binding caused by a specific interaction between benzo(a)pyrene and cytochrome P-450. The present study clearly demonstrates that a carcinogen activated at the microsomes can enter into the nucleus and react with its macromolecules; the carcinogen can also be activated by the monoxygenase system of the nuclear envelope. It appears that both the endoplasmic reticulum and the nuclear envelope are potentially important sites of carcinogen activation.     

Metabolism of N-nitroso-2,6-dimethylmorpholine by isozymes of rabbit liver microsomal cytochrome P-450

The cis isomer of N-nitroso-2,6-dimethylmorpholine (NNDM), a pancreatic carcinogen for the Syrian golden hamster, is metabolized by hamster liver microsomes to yield N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) as the major product. Rabbit liver microsomes catalyze the metabolism of cis-NNDM to HPOP at a rate slower than that observed with hamster microsomes, but significantly faster than that obtained with rat microsomes. Pretreatment of rabbits with phenobarbital results in a 6-fold increase in the cis-NNDM hydroxylase activity of the rabbit microsomes to levels equal to that observed with the hamster; pretreatment of rabbits with other xenobiotics had no effect on the hydroxylation of cis-NNDM. The role of rabbit liver microsomal cytochrome P-450 in the metabolism of the cis isomer of NNDM was studied in the reconstituted system consisting of NADPH:cytochrome P-450 reductase, phospholipid, and cytochrome P-450. Cytochrome P-450LM2, which is induced by pretreatment with phenobarbital, exhibited the highest activity for the metabolism of cis-NNDM. The Vmax for the formation of HPOP was 1.78 nmol/min/nmol cytochrome P-450LM2, and the apparent Km was 360 microM. Cytochrome P-450LM3a also catalyzed the metabolism of NNDM to HPOP at a significant rate (0.25 nmol/min/nmol cytochrome P-450). Of the four other isozymes of cytochrome P-450 (forms 3b, 3c, 4, and 6) tested in the reconstituted system, only forms 3b and 3c exhibited measurable activities (approximately 0.04 nmol of HPOP formed/min/nmol cytochrome P-450). The addition of antibodies to isozyme 2 to microsomes from phenobarbital-treated rabbits resulted in approximately 95% inhibition of the metabolism of NNDM, while the addition of antibodies to LM3a inhibited NNDM metabolism by only 7%. In microsomes from untreated rabbits, inhibition by anti-LM2 and anti-LM3a antibodies was 50 and 64%, respectively. The addition of antibodies to isozyme 3a to microsomes isolated from ethanol-treated rabbits caused approximately 90% inhibition of the metabolism of NNDM. These data conclusively demonstrate that several forms of cytochrome P-450 can catalyze the metabolism of cis-NNDM and that isozymes 2 and 3a play important roles in the rabbit hepatic metabolism of NNDM to HPOP, the proximate carcinogenic metabolite.     

Glutathione S-transferase-catalyzed conjugation of bioactivated aflatoxin B(1) in human lung: differential cellular distribution and lack of significance of the GSTM1 genetic polymorphism.

Epidemiological studies suggest that aflatoxin B(1) (AFB(1)), a mycotoxin produced by certain Aspergillus species, may play a role in human respiratory cancers in occupationally-exposed individuals. AFB(1) requires bioactivation to the corresponding exo-8,9-epoxide for carcinogenicity, and glutathione S-transferase (GST)-catalyzed conjugation of the epoxide with glutathione (GSH) is a critical determinant of susceptibility to AFB(1). Of the purified human GST enzymes studied, the polymorphic hGSTM1-1 has the highest activity towards AFB(1) exo-epoxide. The influence of the GSTM1 polymorphism on AFB(1)-GSH formation, as well as the abilities of cytosols from preparations enriched in different isolated lung cell types to conjugate AFB(1)-epoxides, were examined. In whole-lung cytosols from patients undergoing clinically indicated lobectomy, GSTM1 genotype correlated with GSTM1 phenotype as determined by [(3)H]trans-stilbene oxide conjugation: GSTM1-positive = 295 +/- 31 pmol/mg/h (n = 6); GSTM1-negative = 92.8 +/- 23.3 pmol/mg/h (n = 4) (P < 0.05). In contrast, conjugation of microsome-generated [(3)H]AFB(1)-epoxides with GSH was low and variable between patients, and did not correlate with GSTM1 genotype: GSTM1-positive = 11.9 +/- 8.1, 111 +/- 66 and 510 +/- 248 fmol/mg/h (n = 6); GSTM1-negative = 15.3 +/- 16.7, 167 +/- 225 and 540 +/- 618 fmol/mg/h (n = 4) (for 1, 10 and 100 microM [(3)H]AFB(1), respectively). GSH conjugates of AFB(1) exo-epoxide and the much less mutagenic stereoisomer AFB(1) endo-epoxide were produced in a ratio of approximately 1:1 in cytosols from both whole lung and isolated cells. Total cytosolic AFB(1)-epoxide conjugation was significantly higher in fractions enriched in alveolar type II cells (3.07 +/- 1.61 pmol/mg/h) than in unseparated lung cells (0.143 +/- 0.055 pmol/mg/h) or fractions enriched in alveolar macrophages (0. 904 +/- 0.319 pmol/mg/h; n = 4) (P < 0.05). Furthermore, AFB(1)-GSH formation and percentage of alveolar type II cells in different cell fractions were correlated (r = 0.78, P < 0.05). These results demonstrate that human lung GSTs exhibit very low conjugation activity for both AFB(1)-8,9-epoxide stereoisomers, and that this activity is heterogeneously distributed among cell types, with alveolar type II cells exhibiting relatively high activity. Of the GSTs present in human peripheral lung which contribute to AFB(1) exo- and endo-epoxide detoxification, hGSTM1-1 appears to play at most only a minor role.     

Generation of reactive oxygen radicals through bioactivation of mitomycin antibiotics

Mitomycin C (MC) is a naturally occurring anticancer agent which has been shown to be more cytotoxic to hypoxic tumor cells than to their aerobic counterparts. The mechanism of action of this agent is thought to involve biological reductive activation, to a species that alkylates DNA. A comparison of the cytotoxicity of MC to EMT6 tumor cells with that of the structural analogues porfiromycin (PM), N-(N',N'-dimethylaminomethylene)amine analogue of mitomycin C (BMY-25282), and N-(N',N'-dimethylaminomethylene)amine analogue of porfiromycin (BL-6783) has demonstrated that PM is considerably less cytotoxic to aerobic EMT6 cells than MC, whereas BMY-25282 and BL-6783 are significantly more toxic. The relative abilities of each of these compounds to generate oxygen free radicals following biological activation were measured. Tumor cell sonicates, reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase, xanthine oxidase, and mitochondria were used as the biological reducing systems. All four mitomycin antibiotics produced oxygen radicals following biological reduction, a process that may account for the aerobic cytotoxicity of agents of this class. The generation of relative amounts of superoxide and hydroxyl radical were also measured in EMT6 cell sonicates. BMY-25282 and BL-6783 produced significantly greater quantities of oxygen free radicals with the EMT6 cell sonicate, reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase, and mitochondria than did MC and PM. In contrast, BMY-25282 and BL-6783 did not generate detectable levels of free radicals in the presence of xanthine oxidase, whereas this enzyme was capable of generating free radicals with MC and PM as substrates. MC consistently produced greater amounts of free radicals than PM with all of the reducing systems. BMY-25282, BL-6783, and MC all generated hydroxyl radicals, while PM did not appear to form these radicals. The findings indicate that a correlation exists between the ability of the mitomycin antibiotics to generate oxygen radicals and their cytotoxicity to aerobic EMT6 tumor cells.     

Biotransformations of daunorubicin aglycones by rat liver microsomes

Daunorubicin is biotransformed anaerobically by rat liver microsomes with a reduced nicotinamide adenine dinucleotide phosphate-generating system to form a series of aglycones. The first reaction, reductive cleavage of daunosamine (at C-7 in ring A) to form the 7- deoxyaglycone , is followed by reduction of the C-13 keto group. The 7- hydroxyaglycone may also form by hydrolytic cleavage of the amino sugar followed then by the same C-13 keto reduction. These reactions are not inhibited by beta- diethylaminoethyldiphenylpropyl acetate, whereas subsequent reactions in the D ring of the aglycones can be completely blocked by this cytochrome P-450 inhibitor: reductive and hydrolytic cleavage of the C-4 methoxy group. Thus, five reactions at three sites are described and theoretical pathways are proposed for the expected 12 aglycones from daunorubicin.     

Biotransformation of aflatoxin B1 in human lung

In addition to being a potent hepatocarcinogen, aflatoxin B₁(AFB₁) is a pulmonary carcinogen in experimental animals, andepidemiological studies have shown an association between AFB₁exposure and lung cancer in humans. This study investigatedAFB₁ bioactivation and detoxification in human lung tissue obtainedfrom patients under-going clinically indicated lobectomy. [³H]AFB₁was bioactivated to a DNA binding metabolite by human wholelung cytosols in a time-, protein concentration-, and AFB₁ concentration-dependentmanner. Cytosolic activation of [³H]AFB₁ correlated with lipoxygenase(LOX) activity and was inhibited by the LOX inhibitor nordihydroguaiareticacid (NDGA; 100 µM), indicating that LOXs were largelyresponsible for the observed cytosolic activation of AFB₁. Inwhole lung microsomes, low levels of indomethacin inhibitableprostaglandin H synthase (PHS)-mediated [³H]AFB₁-DNA bindingand cytochrome P-450 (P450)-mediated [³H]AFB₁-DNA binding wereobserved. Cytosolic glutathione S-transferase (GST)-catalyzeddetoxification of AFB₁–8,9-epoxide, produced by rabbitliver microsomes, was minimal at 1 and 10 µM [³H]AFB₁.With 100 µM [³H]AFB₁, [³H]AFB₁–8, 9-epoxide conjugationwith reduced glutathione was 0.34 ± 0.26 pmol/mg/h (n= 10). In intact, isolated human lung cells, [³H]AFB₁ bindingto cellular DNA was higher in cell fractions enriched in macrophagesthan in either type II cell-enriched fractions or fractionscontaining unseparated cell types. Indomethacin produced a 63–100%decrease in [³H]AFB₁-DNA binding in macrophages from five ofseven patients, while NDGA inhibited [³H]AFB₁ -DNA adduct formationby 19, 40 and 56% in macrophages from three of seven patients.In alveolar type O cells, NDGA decreased [³H]AFB₁-DNA bindingby 30–100% in cells from three patients and indomethacinhad little effect. SKF525A, an isozyme non-selective P450 inhibitor,enhanced [³H]AFB₁ binding to cellular DNA in unseparated cells,macrophages, and type II cells, suggesting that P450-mediatedbioactivation of AFB₁ is not a major pathway by which AFB₁–8,9-epoxideis formed in human lung cells. Overall, these studies suggestthat P450 has a minor role in the bioactivation of AFB₁ in humanlung. Rather, LOXs and PHS appear to be important bioactivationenzymes. Co-oxidative bioactivation of AFB₁, in combinationwith the low conjugating activity displayed by human lung cytosolicGSTs, likely contributes to human pulmonary susceptibility toAFB₁.     

Characterization of benzo(a)pyrene hydroxylase of trout liver.

Trout liver microsomes contained as 0.40 nmole of cytochrome P-450 per mg of protein and a NADPH-cytochrome c reductase activity of 23 nmoles of cytochrome c reduced per mg of protein per min at 22 degrees. Associated with these was a high benzo(a)pyrene hydroxylase activity, which required NADPH and O2 and was inhibited by CO. With thin-layer chromatography, at least five metabolites could be identified (including dihydrodiols, phenols, and quinones of benzo(a)pyrene). Inhibitors such as 2-diethylaminoethyl-2,2-diphenylvalerate, aminopyrine, metyrapone, pyridine, n-octylamine, and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane were relatively ineffective in inhibiting trout benzo(a)pyrene hydroxylase. Typical inhibitors of 3-methylcholanthrene-induced cytochrome (P-448), such as alpha-naphthoflavone, zoxazolamine, and testosterone, were effective, however. With benzo(a)pyrene it was possible to induce type I spectral change in trout cytochrome P-450. In spite of the many enzymatic characteristics of cytochrome P-448, trout cytochrome P-450 had maximum absorbance at 450.6 nm. when in reduced form and complexed with CO. the ethyl isocyanide gave an interaction spectrum with reduced trout liver cytochrome P-450 resembling that of control rat.     

Role of N-methylolpentamethylmelamine in the metabolic activation of hexamethylmelamine

Hexamethylmelamine (HMM) is metabolized by rat hepatic microsomal preparations to reactive species which covalently bind to microsomal protein and to calf thymus DNA added to microsomal incubation mixtures. Covalent binding to macromolecules is dependent on the presence of molecular oxygen and reduced nicotinamide adenine dinucleotide phosphate and is catalyzed by cytochrome P-450 monooxygenases. Reduced nicotinamide adenine dinucleotide-dependent covalent binding of [methyl-14C]HMM to microsomal protein is greater than that of [ring-14C]HMM. Reduced nicotinamide adenine dinucleotide phosphate-dependent covalent binding of [ring-14C]HMM and [methyl-14C]HMM to calf thymus DNA added to microsomal incubation mixtures are approximately equal. The [ring-14C]-labeled carbinolamine intermediate in HMM demethylation, N-methylolpentamethylmelamine, covalently binds to microsomal protein and, to a much greater extent, to calf thymus DNA.