Effect of particulates on metabolism and mutagenicity of benzo[a]pyrene

Mechanisms of co-carcinogenicity of particulates, such as iron oxide and asbestos, and benzo[a]pyrene (B[a]P) are not completely understood. Particulates dramatically alter rates of uptake of B[a]P into membranes, a factor which could account for co-carcinogenicity. However, B[a]P must be activated to reactive forms to be carcinogenic and mutagenic so alterations in metabolism of B[a]P by particulates also could result in co-carcinogenesis. To elucidate mechanisms of particulate-B[a]P co-carcinogenesis, we have correlated rates of uptake of B[a]P into microsomes with metabolism of B[a]P and with mutagenicity of B[a]P in the Ames test. In general, aryl hydrocarbon hydroxylase (AHH) activity paralleled rates of uptake of B[a]P, though some inhibition of AHH activity by particulates which was not attributable to availability of B[a]P was evident. This inhibition was studied further by assaying separately mixed function oxidase and epoxide hydrase activities in the presence of particulates. Both chrysotile and iron oxide inhibited O-deethylation of 7-ethoxyresorufin and hydration of B[a]P-4,5-oxide. To determine effects of this inhibition on activation of B[a]P to reactive forms, we studied profiles of metabolites of B[a]P and mutagenicity of B[a]P. The only alteration in profiles of B[a]P metabolites produced by particulates was that due to effects on rates of uptake. Similarly, mutagenicity of B[a]P was positively correlated with rates of uptake into microsomes. We conclude that the predominant effects of chrysotile and iron oxide are in altering rates of uptake of particle-adsorbed B[a]P. Changes in uptake rates then result in alterations of B[a]P metabolism and mutagenicity.     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

Effects of inducers on the regio- and stereoselective metabolism of benzo[a]pyrene by mouse tissue microsomes

The metabolism of benzo[a]pyrene (BP) by microsomal fractions of the skin, lungs and liver of the mouse, and the effects on this process of pretreatment with the xenobiotics phenobarbital (PB) and 3-methylcholanthrene (3-MC) were examined. Differences between the untreated tissues were found both in terms of the total amounts of diol recovered and in the relative proportions of the individual diols extracted following incubation. Induction with PB or 3-MC significantly altered the profiles of metabolic diols obtained with epidermal and hepatic microsomes compared with their respective controls. Pulmonary microsomes showed similar trends to those obtained with liver microsomes but these were not statistically significant. The optical purity of the BP-7,8-diol that was formed by each microsomal type was examined by direct resolution of the enantiomers on HPLC using a chiral stationary phase. In each case the (-)-7R,8R-enantiomer predominated. Pretreatment with 3-MC significantly decreased the optical purity of BP-7,8-diol recovered from incubations with skin microsomes, but significantly increased the optical purity of the diol extracted from incubations with lung and liver microsomes. In addition to the diols, an unidentified BP metabolite was found that eluted between BP-9,10- and 4,5-diol on a reverse-phase high-performance liquid chromatography (HPLC) system and which represented a major product in extracts of incubations of BP with both induced and uninduced skin and lung microsomal fractions.     

The metabolism of drugs and carcinogens in isolated subcellular fractions of Drosophila melanogaster. II. Enzyme induction and metabolism of benzo[a]pyrene

The effect of various pretreatments on the activities of several drug metabolizing enzymes was investigated in microsomes and postmicrosomal supernatant fractions isolated from whole body homogenates of Drosophila melanogaster larvae of different strains. Pretreatments of larvae with either phenobarbital (PB), β-naphthoflavone (BNF) or a mixture of polychlorinated biphenyls (Aroclor 1254, PCB) for 24 h increased microsomal benzo[a]pyrene (BP) monooxygenase activity 2- to 6-fold in all strains as compared to untreated larvae. A simultaneous increase in the contents of cytochrome P-450 occurred after pretreatment with PB and PCB. Comparison of the turnover rates of BP per molecule of cytochrome P-450 indicated that BP was a poor substrate for control cytochrome P-450 whereas BNF induced a most active hemoprotein for this metabolism. Marked differences in the qualitative pattern of BP metabolites were obtained between microsomes isolated from BNF-treated larvae or rat liver microsomes. 3-Hydroxy-BP (3-OH-BP) was the dominating metabolite with both preparations, while the BP dihydrodiols were formed in minor quantities in Drosophila as compared to rat liver. Metyrapone and SKF 525-A inhibited BP metabolism in microsomes isolated from untreated and BNF treated larvae of all strains. In contrast, α-naphthoflavone (ANF) stimulated the BP monooxygenase activity of microsomes isolated from untreated larvae approx. 3-fold but only slightly influenced the activity of microsomes from BNF treated larvae indicating that the latter species of cytochrome P-450 was less sensitive to ANF.In all strains, PCB and PB treatments approximately doubled microsomal epoxide hydrolase activity and increased cytosolic glutathione-S-transferase activity 25–60%, significant only in strain Berlin K after PB treatment. The activities of epoxide hydrolase and glutathione-S-transferase in control larvae were comparable in the different strains, whereas the content of cytochrome P-450 and BP monooxygenase activity was higher in the Hikone R strain. Variability in the induction response to the various pretreatment was observed among the three strains.     

Regioselectivity in rat microsomal metabolism of benzo[a]pyrene: evidence for involvement of two distinct binding sites

The metabolic profile of benzo[a]pyrene (BP) in cumene hydroperoxide-(CHP)-dependent reaction by male rat liver microsomes was dependent on CHP concentration. At 0.05 mM CHP, 3-hydroxy-BP was the major metabolite. Increase in CHP reduced 3-hydroxy-BP formation but increased BP quinone formation simultaneously. This change in metabolic profile was reversed by preincubation with pyrene. Pyrene (PY) selectively inhibited quinone formation but enhanced 3-hydroxy-BP formation. Naphthalene (NP) had no effect on BP quinone formation but inhibited BP 3-hydroxylation. Phenanthrene (PA) and benz[a]anthracene (BA) inhibited effectively 3-hydroxy-BP formation but only slightly quinone formation. BP binding to microsomal protein correlated to quinone formation and not BP 3-hydroxylation. BP metabolism by female rat liver microsomes also depended on CHP concentration but was much less efficient than the male. Quinones were consistently predominant metabolites and their formation was also inhibited by pyrene. Our data provide evidence that regioselectivity in BP metabolism involves at least two distinct binding sites. One site recognizes the benzo region of BP in BP 3-hydroxylation and the other recognizes the pyrene region in quinone formation. The different ratios of 3-hydroxy-BP to quinone formation by male and female rat liver microsomes suggest that the two binding sites are probably located at separate cytochrome P-450 isozymes.     

Mutagenicity of benzo[a]pyrene 7,8-dihydrodiol and 7,12-dimethylbenz[a]anthracene 3,4-dihydrodiol in S. typhimurium mediated by microsomes from rat liver and mouse skin

The mutagenic activities of trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP 7,8-diol) and of trans-3,4-dihydroxy-7,12-dimethylbenz[a]-anthracene (DMBA 3,4-diol) towards S. typhimurium TA100 were measured in assays that were carried out on a micro-scale in liquid medium in the presence of microsomal fractions prepared from mouse skin or rat liver. In the presence of an NADPH-generating system, microsomal enzymes converted both diols into mutagens that were probably the respective 'bay-region' diol-epoxides. The rate of the enzyme-catalysed conversion of the BP 7,8-diol into mutagens by microsomal preparations from mouse epidermis was similar to that occurring with microsomes from rat liver. Pretreatment of mice by the topical application of benz[a]anthracene (BA) or 7,12-dimethylbenz[a]-anthracene (DMBA) increased the mutagenic activity of BP 7,8-diol mediated by mouse skin microsomal preparations by 2-fold and this was paralleled by a 4-fold increase in epidermal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity. The results are discussed in relation to the high susceptibility of mouse skin to polycyclic aromatic hydrocarbon (PAH) carcinogenesis.     

Dietary fat inhibits the intestinal metabolism of the carcinogen benzo[a]pyrene in fish

Following the intestinal absorption of dietary benzo[a]pyrene (BP) by the killifish, this compound becomes incorporated along with dietary triglycerides into membrane-bound fat vacuoles within the intestinal epithelial cell (1985. J. Lipid Res. 26: 428-434). These vacuoles, arising from the smooth endoplasmic reticulum, are important transient structures involved in both the uptake and metabolism of dietary BP and, presumably, other lipophilic toxicants as well. In the present study we used subcellular fractions isolated from the intestines of spot (Leiostomus xanthurus), a teleost fish, to study factors that influence the metabolism of BP in a fat vacuole/microsomal system. Triglyceride-solubilized BP is capable of diffusion from fat vacuoles to microsomal enzymes. Increases in the concentration of fat vacuoles decrease the availability of BP to microsomal BP hydroxylase. The effect of fat vacuoles on the activity of BP hydroxylase becomes more pronounced as the concentration of BP in our test system decreases. Addition of cytosolic glutathione transferases to the fat vacuole/microsomal system enhances the activity of BP hydroxylase. Examination of binding of 3H-labeled BP to killifish (Fundulus heteroclitus) intestinal cytosolic proteins in vivo indicated that a large fraction of the radioactivity was associated within glutathione transferase. These results suggest that dietary fat inhibits metabolism of low levels of BP in the intestine. A consequence of this would be greater exposure of peripheral tissues to dietary carcinogens.     

Particulate enhanced membrane uptake of 1,2-benzanthracene observed by fluorescence spectroscopy: a possible role in co-carcinogenesis.

In recognition of the co-carcinogenic effects of particulate matter and chemical carcinogens, we investigated the effect of particulate silica on the rates of membrane uptake of 1,2-benzanthracene. The fluorescence emission spectra and the apparent quantum yields of benzanthracene and dependent upon adsorption to silica and upon the surface density of benzanthracene on the silica. The fluorescence spectral shifts which occur upon transfer of benzanthracene from the silica surface to phospholipid vesicles provided a convenient means to quantitate the membrane uptake of benzanthracene from particulates. The rate of benzanthracene uptake by dipalmitoyl-L-alpha-phosphatidylcholine vesicles was independent of the concentration of lipid, indicating that the rate-limiting step may involve its solubilization in the aqueous phase. These uptake rates were also independent of the surface density of benzanthracene on the silica, indicating that the benzathracene molecules are dispersed uniformly on the silica surface. Rates of membrane uptake of benzanthracene from the crystalline, microcrystalline, and the silica-absorbed states were compared, and are greatly enhanced by a reduction in crystal size. Silica-adsorbed benzanthracene had the most rapid rate of membrane uptake. Silica did not cause disruption of the lipid vesicles. These results indicate that particulates can enhance the cellular availability of the chemical carcinogens.     

The use of high pressure liquid chromatography to study chemically induced alterations in the pattern of benzo[a]pyrene metabolism.

The metabolism of radiolabeled benzo[a]pyrene (BP) by control, 3-methyl-cholanthrene (3-MC) induced, and 1,1,1-trichloropropene-2,3-oxide (TCPO)-inhibited rat liver microsomes was measured using fluorescence, radiometric, and high-pressure liquid chromatographic (HPLC) assays. Significant differences in the total measurable metabolism of BP by the three microsomal enzyme incubations resulted from the use of the three assay procedures. Appreciable differences in the concentration of the metabolite fractions after 3-MC induction and TCPO inhibition are clearly demonstrated. NMR analysis revealed that while the 3-hydroxy-BP fraction is greater than 90% pure, the 9-hydroxy fraction contains a number of metabolites having essentially identical retention times.     

Particulate enhanced membrane uptake of 1,2-benzanthracene observed by fluorescence spectroscopy: A possible role in co-carcinogenesis

In recognition of the co-carcinogenic effect of particulate matter and chemical carcinogens, we investigated the effect of particulate silica on the rates of membrane uptake of 1,2-benzanthracene. The fluorescence emission spectra and the apparent quantum yields of benzathracene are dependent upon adsorption to silica and upon the surface density of benzathracene on the silica. The fluorescence spectral shifts which occur upon transfer of benzathracene from silica surface to phospholipid vesicles provided a convenient means to quantitate the membrane uptake of benzanthracene from particulates.The rate of benzathracene uptake by dipalmitoyl-L-α-phosphatidylcholine vesicles was independent of the concentration of lipid, indicating that the rate-limiting step may involve its sulubilization in the aqueous phase. These uptake rates were also independent of the surface density of benzanthracene on the silica, indicating that the benzanthracene molecules are dispersed uniformly on the silica surface.Rates of membrane uptake of benzanthracene from the crystalline, microcrystalline, and the silica-absorbed states were compared, and are greatly enhanced by a reduction in crystal size. Silica-adsorbed benzanthracene had the most rapid rate of membrane uptake. Silica did not cause disruption of the lipid vesicles.These results indicate that particulates can enhance the cellular availability of chemical carcinogens.     

The DNA binding of benzo[alpha]pyrene metabolites catalysed by rat lung microsomes in vitro and in isolated perfused rat lung.

When [³H]benzo[a]pyrene is incubated in vitro together with DNA, NADPH and rat lung microsomes, covalent binding of benzo[a]pyrene (BP) metabolites to DNA occurs. These metabolite-nucleoside complexes can be resolved into several distinct peaks by elution of a Sephadex LH-20 column with a water-methanol gradient. 3-Methylcholanthrene (MC) pretreatment of animals induces the total covalent binding in vitro several-fold and increases the amounts of at least five metabolite-nucleoside complexes associated with the 7,8-diol-9,10-epoxidcs, the 7,8-oxide or quinones oxygenated further, the 4,5-oxide and phenols oxygenated further. These increases correspond well with the increases in the production of both non-K-region and K-region metabolites of BP by lung microsomes, as determined by highpressure liquid chromatography (HPLC). On the other hand, when [³H]BP is metabolized in isolated perfused rat lung, only the peak representing the 7,8-diol-9,10-epoxide bound to nucleoside(s) is readily detectable and then only in lungs from MC-treated animals. The extent of binding of BP metabolites to lung DNA is very low, about 0.0004% of the total dose applied to the perfusion medium; more than 60% of this can be accounted for by the binding of the 7,8-diol-9,10-epoxides to nucleoside(s). It is suggested that the further metabolism leading to metabolites not available to covalent binding, (e.g. conjugation) of primary BP metabolites in the intact tissue is responsible for the differences in the metabolite-nucleoside patterns observed in vivo, as compared with microsomal metabolism in vitro.     

Characteristics of microsomal reduction of benzo[a]pyrene 4,5-oxide

NADPH-reduction of benzo[a]pyrene 4,5-oxide (BP-4,5-oxide) to BP required four components from rat liver: cytochrome P-450, NADPH cytochrome P-450 reductase, phosphatidylcholine and a soluble, heat-sensitive factor which was present in 105 000 × g supernatant and was also released from microsomes by sonication. The requirement for this factor contrasts with recently reported results from Sugiura et al. (Cancer Res., 40 (1980) 2910). Oxide-reduction was 40 times faster under anaerobic conditions, but oxygen did not affect the stimulation factor. This stimulation was highest (× 15) at low concentrations of microsomal protein (<0.1 mg/ml) and was almost absent at high concentrations of microsomal protein (>1 mg/ml). Oxide-reduction activity was proportional to microsomal protein concentration in the presence of added 105 000 × g supernatant, but for microsomes alone (>0.1 mg/ml) exhibited a parallel plot with an intercept at 0.08 mg/ml microsomal protein. Stimulation was highest at high concentrations of BP-4,5-oxide and a linear plot of V⁻¹ vs. [BP-4,5-oxide]⁻¹ was only obtained in the presence of 105 000 × g supernatant (K_m = 3 μM, V_max = 3.3 nmol/mg/min). Microsomal hydration of BP-4,5-oxide (inhibited in reductase assays) was unaffected by 105 000 × g supernatant, suggesting that stimulation of oxide-reduction did not derive from solubilization of BP-4,5-oxide. Stimulation was observed in the initial rate of reaction and was independent of incubation time. Inhibition of lipid peroxidation, removal of peroxides and deoxygenation were all excluded as explanations of the stimulatory effect.     

Cytochrome P-450-catalyzed stereoselective epoxidation at the K region of benz[a]anthracene and benzo[a]pyrene

The enantiomers of K-region benz[a]anthracene (BA) 5,6-epoxide and benzo[a]pyrene (BP) 4,5-epoxide were resolved by chiral stationary-phase high-performance liquid chromatography (CSP-HPLC). The K-region epoxides formed in the metabolism of BA by liver microsomes from untreated (control), phenobarbital (PB)-treated, and 3-methylcholanthrene (MC)-treated male Sprague-Dawley rats were determined by CSP-HPLC to have a 5R,6S/5S,6R enantiomer ratio of 25:75, 21:79, and 4:96, respectively. The K-region 4,5-epoxide formed in the metabolism of BP by the same rat liver microsomal preparations contained a 4R,5S/4S,5R enantiomer ratio of 48:52 (control), 40:60 (PB), and 5:95 (MC), respectively. The results indicate that various cytochrome P-450 isozymes of rat liver exhibit different stereoselective properties in catalyzing the epoxidation reactions at the K region of BA and of BP.     

Comparison of the formation of benzo[a]pyrene diolepoxide-DNA adducts in vitro by rat and human microsomes: evidence for the involvement of P-450IA1 and P-450IA2

The involvement of cytochrome P-450 isozymes in the activation of benzo[a]pyrene (BP) by human placental and liver microsomes was studied in vitro using monoclonal antibodies (Mab) toward the major 3-methylcholanthrene (MC)-inducible and phenobarbital-inductible rat liver P-450 isozymes (Mab 1-7-1 and Mab 2-66-3, respectively). Microsomes from human placenta and liver and rat liver were incubated with BP and DNA, and BP-diolepoxide-DNA (BPDE-DNA) adducts were measured by synchronous fluorescence spectrophotometry (SFS). The only BP metabolite giving the same fluorescence peak as chemically modified BPDE-DNA was BP-7,8-dihydrodiol. Five (smokers) out of 29 human placentas (smokers and nonsmokers), and five out of nine human livers were able to metabolically activate BP to BPDE-DNA adducts in this system. The Mab 1-7-1 totally inhibited the formation of BPDE-DNA adducts in placental microsomal incubations. Inhibition using rat or human liver microsomes was 50-60% and about 90%, respectively. The Mab 2-66-3 had no effect in any of the microsome types. Adduct formation was inhibited more strongly and at lower concentrations of Mab 1-7-1 compared with the inhibition of AHH activity. This study is a clear indication of the major role of P-450IA1 (P-450c) in human placenta and probably P-450IA2 (P-450d) in human liver in BP activation, while other isozymes also take part in the activation in rat liver. Furthermore, this clearly indicates that AHH activity and BP activation are not necessarily associated.     

Metabolism of benzo(a)pyrene by duck liver microsomes

The metabolism of benzo(a)pyrene [BP], a model carcinogenic PAH, by hepatic microsomes of two duck species, mallard (Anas platyrhynchos) and common merganser (Mergus merganser americanus) collected from chemically-contaminated and relatively non-contaminated areas was investigated. The rate of metabolism of BP by liver microsomes of common merganser and mallard collected from polluted areas (2,650 +/- 310 and 2,200 +/- 310 pmol/min per mg microsomal protein, respectively) was significantly higher than that obtained with liver microsomes of the two species collected from non-polluted areas (334 +/- 33 and 231 +/- 30 pmol/min per mg microsomal protein, respectively). The level of cytochrome P-450 1A1 was significantly higher in the liver microsomes of both duck species from the polluted areas as compared to the ducks from the non-polluted areas. The major BP metabolites, including BP-9, 10-diol, BP-4, 5-diol, BP-7, 8-diol, BP-1, 6-dione, BP-3, 6-dione, BP-6, 12-dione, 9-hydroxy-BP and 3-hydroxy-BP, formed by liver microsomes of both duck species from polluted and non-polluted areas, were qualitatively similar. However, the patterns of these metabolites were considerably different from each other. Liver microsomes of ducks from the polluted areas produced a higher proportion of benzo-ring dihydrodiols than the liver microsomes of ducks from the non-polluted areas, which converted a greater proportion of BP to BP-phenols. The predominant enantiomer of BP-7,8-diol formed by hepatic microsomes of the two duck species had an (-)R,R absolute stereochemistry. The data suggest that duck and rat liver microsomal enzymes have different regioselectivity but similar stereoselectivity in the metabolism of BP.     

Inhibition of epidermal metabolism and DNA-binding of benzo[a]pyrene by ellagic acid

Ellagic acid, a common plant phenol, was shown to be a potent inhibitor of epidermal microsomal aryl hydrocarbon hydroxylase (AHH) activity in vitro, and of benzo[a]pyrene (BP)-binding to both calf thymus DNA in vitro and to epidermal DNA in vivo. The in vitro addition of ellagic acid (0.25-2.0 microM) resulted in a dose-dependent inhibition of AHH activity in epidermal microsomes prepared from control or carcinogen-treated animals. The I50 of ellagic acid for epidermal AHH was 1.0 microM making it the most potent inhibitor of epidermal AHH yet identified. In vitro addition of ellagic acid to microsomal suspensions prepared from control or coal tar-treated animals resulted in 90% inhibition of BP-binding to calf thymus DNA. Application of ellagic acid to the skin (0.5-10.0 mumol/10 gm body wt) caused a dose-dependent inhibition of BP-binding to epidermal DNA. Our results suggest that phenolic compounds such as ellagic acid may prove useful in modulating the risk of cutaneous cancer from environmental chemicals.     

The kinetics of benzo(a)pyrene anti-7,8-dihydrodiol 9, 10-epoxide formation from benzo(a)pyrene and regulatory membrane effects

r-7,c-10,t-8,t-9-Tetrahydroxybenzo(a)pyrene (7,10/8,9-tetrol), which is the principal hydrolysis product of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-diol-epoxide), was resolved and measured by HPLC in organic extracts of incubations which contained induced rat liver microsomes and BP. Kinetic analyses showed that: (a) following a 5- to 7-min lag period, anti-diol-epoxide formation was linear, and (b) levels of anti-diol-epoxide formed were highly dependent upon the starting BP concentration. anti-Diol-epoxide production increased at starting BP concentrations of 0–12 μm and decreased in incubations containing 12–25 μm BP. However, between 25 and 100 μm BP, anti-diol-epoxide formation was stable at a level representing 65% of the peak production which occurred at a starting BP concentration of 12 μm. BP oxidation was competitively inhibited by (?)-trans-BP-7,8-dihydrodiol and about five times less effectively by the (+)-trans-BP-7,8-dihydrodiol. The inability of a severalfold excess of BP (25–100 μm) to totally inhibit BP-7,8-dihydrodiol oxidation was explained by the presence of a microsomal substrate compartment which was saturated at only 6–8 μm BP, the remaining BP present as aggregates in the aqueous compartment. Purification of microsomes by Sepharose 2B gel filtration after reaction with [³H]BP also indicated that BP-7,8-dihydrodiol was preferentially concentrated in the microsome compartment leading to a net increase in the ratio of BP-7,8-dihydrodiol to BP in the microsomal compartment, which favored BP-7,8-dihydrodiol oxidation to yield the biologically active anti-diol-epoxide.     

A role for phospholipids in the binding and metabolism of drugs by hepatic microsomes. Use of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulphonate

1. The pretreatment of rat liver microsomes with phospholipase C or D decreased the N-demethylation of (+)-benzphetamine. The hydroxylation of aniline was essentially unchanged by pretreatment of microsomes with phospholipase C. 2. Some components of the microsomal mixed-function oxidase system were impaired by phospholipases. 3. The fluorescence of 1-anilinonaphthalene-8-sulphonate (ANS) was greatly enhanced by microsomes. Phospholipase C or D markedly decreased ANS–microsome fluorescence. Quantum yield of ANS–microsome fluorescence appeared to be related directly to phospholipid content of microsomes. 4. Most of the drugs studied enhanced ANS–microsome fluorescence. Warfarin, however, displaced ANS fluorescence competitively from microsomes. The latter effect was postulated as being due to warfarin competing with ANS for the cationic site on microsomal phosphatidylcholine. 5. ANS fluorescence was also increased by the presence of phospholipid micelles. The fluorescence of ANS–phosphatidylcholine micelles was modified by warfarin and (+)-benzphetamine in a manner similar to that observed with microsomes. Warfarin decrease of fluorescence was absent when ANS was bound to phosphatidic acid, which lacks a cationic site. 6. Trypsin pretreatment of microsomes did not modify ANS–microsome fluorescence, including drug-induced changes. 7. It was postulated that phospholipids have a permissive role in the metabolism of most drugs by hepatic microsomes and that the ANS probe might reflect interactions of compounds with microsomal membrane phospholipids.     

Effects of 1-ethynylpyrene and related inhibitors of P450 isozymes upon benzo[a]pyrene metabolism by liver microsomes

The effects of three aryl acetylenes, 1-ethynylpyrene (EP), 2-ethynylnaphthalene (EN) and 3-ethynylperylene (EPE), upon the metabolism of benzo[a]pyrene (BaP) by microsomes isolated from rat liver were investigated. These aryl acetylenes all inhibited the total metabolism of BaP. Formation of BaP 7,8-dihydrodiol and BaP tetrol products by microsomal preparations from rats that had been pretreated with 3-methylcholanthrene (3MC) were preferentially inhibited. The effects of EP upon the metabolism of BaP 7,8-dihydrodiol by microsomes from rat liver were also studied. This aryl acetylene strongly inhibited the formation of BaP tetrols from BaP 7,8-dihydrodiol by liver microsomes both from untreated rats and from rats pretreated with 3MC, but enhanced the conversion of the BaP dihydrodiol into other metabolites.     

Unusual patterns of benzo[a]pyrene metabolites and DNA-benzo[a]pyrene adducts produced by human placental microsomes in vitro

Human placental microsomes were incubated with [³H]benzo[a]pyrene (BP) and Salmon sperm DNA and the resulting metabolite-nucleoside complexes resolved by Sephadex LH-20 chromatography. The metabolite pattern was analyzed by high-pressure liquid chromatography (HPLC). The incubates were also co-chromatographed with extracts obtained from incubates with rat liver microsomes and [¹⁴C]BP. Phenols, quinones and 7,8-dihydrodiol were detected in the placental incubates. Both 9,10- and 4,5-dihydrodiols were very low as compared with control rat liver samples. Placental microsomes catalyzed the binding of BP metabolites to DNA in vitro, giving rise to two main complexes which co-chromatographed with rat liver-produced peaks attributable to 7,8-diol-9,10-epoxide and 7,8-oxide and/or quinones when metabolized further. The nucleoside metabolite peaks attributable to 4,5-oxide and 9-phenol-4,5-oxide were lacking when compared with the binding pattern catalyzed by rat liver. Both the total binding and specific metabolite-nucleoside adducts in the placenta correlated with fluorometrically measured aryl hydrocarbon hydroxylase (AHH) activity and with the amount of dihydrodiol formed. The results demonstrate that both the metabolite pattern and the nucleoside-metabolite complexes formed by the placental microsomes in vitro differed greatly from those produced by rat liver microsomes. These studies also suggest that it is not possible to predict specific patterns of DNA binding from AHH measurements or even from BP metabolite patterns, especially when comparing different tissues and species.