Microsomal preparations

Mutagenicity. The AJ-nitrosamines, in general, induce mutations in standard bacterial-tester strains (117). As with carcinogenicity, enzymatic activation, typically with Hver microsomal preparations, is required. Certain substituted A/-nitrosamine derivatives (12) induce mutations without microsomal activation (31,33,34). Because the a-acetoxy derivatives can hydroly2e to the corresponding a-hydroxy compounds, this is consistent with the hypothesis that enzymatic oxidation leads to the formation of such unstable a-hydroxy intermediates (13) (118). However, for simple /V-nitrosamines, no systematic relationship has been found between carcinogenicity and mutagenicity (117,119—123). 

Two important examples of reductive metabolism of xenobiotics are the reductive dehalogenation of organohalogen compounds, and the reduction of nitroaromatic compounds. Examples of each are shown in Figure 2.13. Both types of reaction can take place in hepatic microsomal preparations at low oxygen tensions. Cytochrome P450 can catalyze both types of reduction. If a substrate is bound to P450 in the... 

The cytochrome P-450-dependent metabolism of trichloroethylene was studied in hepatic microsomal fractions from 23 different humans (Lipscomb et al. 1997). CYP2E1 was the predominant form of P-450 responsible for the metabolism of trichloroethylene in humans. Incubations of trichloroethylene with the microsomal preparations resulted in hyperbolic plots consistent with Michaelis-Menton kinetics. The values ranged from 12 to 55.7 pM, and were not normally distributed, and the values range from 490 to 3,455 pmol/min/mg protein and were normally distributed. The study authors concluded that the human variability in metabolism of trichloroethylene via P-450-dependent pathways was within a 10-fold range. 

Further experiments on the metabolism of NDPA by rat liver fractions have also provided support for the 3-oxidation mechanism of Kruger. In addition to the products of a-oxidation, we have isolated and characterized NHPPA as a major product of the microsomal oxidation of NDPA (18). We have also shown that NHPPA is further oxidized to NOPPA by microsomal preparations from rat liver (18), Finally, with NOPPA as substrate, we have shown that metabolism takes place principally by reduction with the microsomal or soluble fraction of rat liver to yield NHPPA, although microsomal a-oxidation also takes place to some extent (19). 

Miller, K.J. Anderholm, D.C. and Ames, M.M. Metabolic activation of the serotonergic neurotoxin para-chloroamphetamine to chemically reactive intermediates by hepatic and brain microsomal preparations. Biochem Pharmacon>5 mi- lA2, 1986. 

An alternative to chemical synthesis is to use human CYP enzymes to generate the desired human drug metabolites. Various means of making human P450s have been used, all with certain drawbacks [81]. The most common source is pooled HLMs, which has been described in detail previously, but these microsomal preparations contain a mixture of many different enzymes, and their cost, batch-to-batch variability in activity and restrictions on availability can limit the usefulness of HLMs for preparative synthetic work. These limitations can become particularly acute when the required amount of a pure metabolite exceeds 5-10 mg. 

The aim of the present example was to investigate whether the assessment of an in silico model of metabolic stability from a training set of several hundred drugs or drug-like compounds in human CYP3A4 cDNA-expressed microsomal preparations, would offer a suitable approach to predict the metabolic stability of external compounds. 

Each entry is an average of data obtained from two separate experiments using different microsomal preparations. Enantiomeric composition was determined by CD spectral data (18) and by CSP- HPLC (19,20). 

BA trans-3.4-dihvdrodiol cannot be separated from BA trans-8.9-dihydrodiol in several HPLC conditions (27-29). Quantification of BA trana-3,4-dihydrodiol by HPLC can only be accomplished after converting the 3,4-dihydrodiol to its diacetate (25.26). The BA trans-3.4-dihydrodiol formed in BA metabolism by liver microsomes from pheno-barbital-treated rats was determined to have a 3R,4R/3S,4S enantiomer ratio of 69 31 (30). Recently we have determined the optical purity of the BA trans-3.4-dihvdrodiol formed in the metabolism of BA by three liver microsomes prepared from untreated rats and rats that had been pretreated with an enzyme inducer. As shown in Table II, cytochrome P-450 isozymes contained in liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats had similar stereoselectivity toward the 3,4-double bond of BA. BA trans-3.4-dihydrodiol is formed via the 3,4-epoxide intermediate (31). 

In contrast to the metabolism of BA and BaP, the 5,6-dihydrodiols formed in the metabolism of DMBA by liver microsomes from untreated, phenobarbital-treated, and 3-methylcholanthrene-treated rats are found to have 5R,6R/5S,6S enantiomer ratios of 11 89, 6 94, and 5 95, respectively (7.49 and Table II). The enantiomeric contents of the dihydrodiols were determined by a CSP-HPLC method (7.43). The 5,6-epoxide formed in the metabolism of DMBA by liver microsomes from 3MC-treated rats was found to contain predominantly (>97%) the 5R,6S-enantiomer which is converted by microsomal epoxide hydrolase-catalyzed hydration predominantly (>95%) at the R-center (C-5 position, see Figure 3) to yield the 5S,6S-dihydrodiol (49). In the metabolism of 12-methyl-BA, the 5S,6S-dihydrodiol was also found to be the major enantiomer formed (50) and this stereoselective reaction is similar to the reactions catalyzed by rat liver microsomes prepared with different enzyme inducers (unpublished results). Labeling studies using molecular oxygen-18 indicate that 5R,68-epoxide is the precursor of the 5S,6S-dihydrodiol formed in the metabolism of 12-methyl-BA (51). 

The 8,9- and 10,11-dihydrodiols formed in the metabolism of BA and DMBA respectively are all highly enriched (>90%) in R,R enantiomers (Table III). Labeling experiments using molecular oxygen-18 in the in vitro metabolism of the respective parent compounds and subsequent mass spectral analyses of dihydrodiol metabolites and their acid-catalyzed dehydration products indicated that microsomal epoxide hydrolase-catalyzed hydration reactions occurred exclusively at the nonbenzylic carbons of the metabolically formed epoxide intermediates (unpublished results). These findings indicate that the 8,9- and 10,11-epoxide intermediates, formed in the metabolism of BA and DMBA respectively, contain predominantly the 8R,9S and 10S,11R enantiomer, respectively. These stereoselective epoxidation reactions are relatively insensitive to the cytochrome P-450 isozyme contents of different rat liver microsomal preparations (Table III). 

A potentially powerful probe for sorting out the contribution of hydroperoxide-dependent and mixed-function oxidase-dependent polycyclic hydrocarbon oxidation is stereochemistry. Figure 9 summarizes the stereochemical differences in epoxidation of ( )-BP-7,8-dihydrodiol by hydroperoxide-dependent and mixed-function oxidase-dependent pathways (31,55,56). The (-)-enantiomer of BP-7,8-dihydrodiol is converted primarily to the (+)-anti-diol epoxide by both pathways whereas the (+)-enantiomer of BP-7,8-dihydrodiol is converted primarily to the (-)-anti-diol epoxide by hydroperoxide-dependent oxidation and to the (+)-syn-diol epoxide by mixed-function oxidases. The stereochemical course of oxidation by cytochrome P-450 isoenzymes was first elucidated for the methycholanthrene-inducible form but we have detected the same stereochemical profile using rat liver microsomes from control, phenobarbital-, or methyl-cholanthrene-induced animals (32). The only difference between the microsomal preparations is the rate of oxidation. 

Most work on the SR and diseased smooth muscle has concerned vascular smooth muscle in hypertensive animals, and bladders from animal models of outflow obstruction. The tools used to study SR function are mainly indirect, and include recording tension or intracellular [Ca2+] with fluorescent probes, measuring Ca2+ fluxes with 45Ca, and investigating the effects of drugs known to block SERCA or activate store release. More directly, some measurement of the activity of SERCA in microsomal preparations has been undertaken (e.g. Zderic et al 1996). 

Recent work in our laboratories has confirmed the existence of a similar pathway in the oxidation of vindoline in mammals (777). The availability of compounds such as 59 as analytical standards, along with published mass spectral and NMR spectral properties of this compound, served to facilitate identification of metabolites formed in mammalian liver microsome incubations. Two compounds are produced during incubations with mouse liver microsome preparations 17-deacetylvindoline, and the dihydrovindoline ether dimer 59. Both compounds were isolated and completely characterized by spectral comparison to authentic standards. This work emphasizes the prospective value of microbial and enzymatic transformation studies in predicting pathways of metabolism in mammalian systems. This work would also suggest the involvement of cytochrome P-450 enzyme system(s) in the oxidation process. Whether the first steps involve direct introduction of molecular oxygen at position 3 of vindoline or an initial abstraction of electrons, as in Scheme 15, remains unknown. The establishment of a metabolic pathway in mammals, identical to those found in Strep-tomycetes, with copper oxidases and peroxidases again confirms the prospective value of the microbial models of mammalian metabolism concept. 

Hepatic microsome preparations from 12 human subjects differed in the rate at which HFC-134a was metabolized. In a study that utilized microsomes from human subjects with relatively high P-450 2E1 levels, HFC-134a was metabolized at rates 5-fold to 10-fold greater than in microsomes of individuals with lower levels of this... 

The above differences disappear when induced microsomal preparations are used. A number of enzyme inducers have been used the most popular being Aroclor 1254,... 

Ashwood-Smith, M.J. (1980). Stability of frozen microsome preparations for use in the Ames Salmonella mutagenicity assay. Mutation Res. 69 199-200. 

With carcinogenic nitrosamines and DMPO, microsomal preparations give (DMPO—HO ) and an unidentified adduct derived from the nitrosamine (Floyd et al., 1978). Metabolic activation of CC14 has also been examined (Ingall et al., 1978 Poyer et al., 1978). 

Whole Homogenate and Microsome Preparation, Enzyme Assays. 

Thawed microsomal preparations (500-700 mg protein) from little skates were digested with sodium cholate (1 mg/mg protein) in 10 mM potassium phosphate buffer (pH 7.7) to make a final concentration of 10 mg protein/ml, in the presence of 0.1 mM EDTA,... 

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