Microsomes with rabbit liver

The samples of l,6-T2-DBpD and l,6-T2-2,3,7,8-Cl4-DBpD are useful in metabolism and mode of action studies. For example, when incubated with rabbit liver microsomes, l,6-T.>-DBpD is extensively metabolized to polar product(s) but only when these preparations are fortified with reduced nicotinamide-adenine dinucleotide phosphate. Under the same conditions l,6-T2-2,3,7,8-Cl4-DBpD is completely resistant to metabolic attack. In some types of studies, a higher specific activity possibly is desirable i.e., >1 Ci/mmole), and this can be achieved, with the methodology already developed, by using larger amounts of tritium gas or working on a larger synthetic scale so that it is not necessary to add unlabeled materials to assist in crystallization steps where a certain minimum amount of compound is necessary. 

ENRICHMENT IN PARAOXON FOLLOWING INCUBATION OF PARATHION WITH RABBIT LIVER MICROSOMES ... 

Wildenauer DB, Oehlmann CE. 1982. Interaction of cyclophosphamide metabolites with membrane proteins An in vitro study with rabbit liver microsomes and human red blood cells. Effect of thiols. Biochem Pharmacol 31 3535-3541. 

Figure 9.3 Age-related changes in N-demethylation and N-oxidation of N,N-dimethylaniline in microsomes from rabbit liver. (From Devereux, T.R. and Fouts, J.R., Chem.-Biol. Interactions, 8, 91, 1974. With permission.)...

In the 5a series allochenodeoxycholate is 12a-hydroxylated to allocholate in the bile fistula rat [131] or with a hepatic microsomal preparation from rat, rabbit or human liver fortified with NADPH [152,153]. Kallner [133] noted that small amounts of more polar derivatives were present in rat bile, with largely unchanged allochenodeoxycholate. AUohyocholate was identified as a minor metabolite [154]. With rabbit liver microsomal preparations, allochenodeoxycholate is a competitive inhibitor for 12a-hydroxylation of 7a-hydroxy-cholest-4-en-3-one and 5a-choles-tane-3a,7a-diol, precursor of cholic and allocholic acids, respectively [155]. Allo-cholic acid has also been characterized as a metabolite of 3 8,7a-dihydroxy-5-cholenic acid after intraperitoneal injection into carp [156]. 

The in vivo administration of diethylni-trosamine (Et2N-N=0) to mice reportedly generates an alkylated porphyrin that was tentatively identified by mass spectrometry as A-(2-hydroxy-ethyl)protoporphyrin IX . In vitro studies with rabbit liver microsomes and purified P450 enzymes have independently shown that P450 oxidizes diethylnitrosamine to ethylene . Although not actually demonstrated, the proposed N-(2-hydroxyethyl) porphyrin adduct is likely to be derived from P450 enzymes inactivated by the ethylene metabolically generated from diethylnitrosamine (see Section 3.3.1). 

The grapefruit aroma, (-H)-nootkatone (2) was administered into rabbits to give 11,12-diol (6,7). The same metabolism has been found in that of biotransformation of nootkatone by miCTOorganisms as mentioned in the previous paragraph. Compounds (6, 7) were isolated from the urine of hypertensive subjects and named urodiolenone. The endogenous production of 6, 7 seem to occur inter-dentally from the administrative manner of nootkatone or grapefruit. Synthetic racemic nootkatone epoxide (14) was incubated with rabbit-liver microsomes to give 11,12-diol (6, 7) (Ishida, 2005). Thus, the role of the epoxide was clearly confirmed as an intermediate of nootkatone (2). 

Oae and coworkers oxidized several diaryl, dialkyl and alkyl aryl sulfides to their corresponding sulfoxides using purified cytochrome P-450 obtained from rabbit liver microsomes . In agreement with expectations, this enzyme did not exhibit much stereospecificity. Some examples including the observed e.e. values are shown by 121-125. A model was proposed to account for the absolute configurations of the sulfoxides produced (126). The sulfur atom is preferentially oxidized from the direction indicated. 

Dihydro-1,2-dihydroxybenzene (10.13) is oxidized by dihydrodiol dehydrogenase (EC 1.3.1.20) to catechol (10.15) (Chapt. 4 in [la]) [76], In a typical experiment in which 10.13 is incubated with phenobarbital-induced rabbit liver microsomes, phenol (10.14), catechol (10.15), and hydroquinone (10.16) represent 54, 39, and 1%, respectively, of the total metabolites detected [75]. In other words, neither benzene oxide (10.1) nor its hydration product l,2-dihydro-l,2-dihydroxybenzene (10.13) was detected. 

Cyclohexene oxide (1,2-epoxycyclohexane, 10.5, Fig. 10.29) has received particular attention as a substrate for EH (see Table 10.1). The compound has a meso-cis geometry like d.v-stilbcnc oxide (10.7), and, like the latter, is hydrated preferentially to the chiral (/ ,/ )-/ran.y-cyclohexane-1,2-diol (10.124) [185], There was a difference, however, between the activities of the rabbit liver microsomal and cytosolic EHs. The former was ca. 20-fold more active than the latter toward this substrate also, it formed the (R,R)-diol with 94% enantiomeric excess (ee) compared to only 22% ee for the cytosolic EH. 

Resistance to hydration was elucidated with tricyclic model compounds that lack the side chain and, hence, pharmacological activity. In this context, a useful comparison has been made between two meso compounds, namely 5W-dibenz.oja, dIcycloheplene 10,11-oxide (10.130, X = CH2) and d.v-slilbcnc oxide (10.7) [195]. The former compound proved to be a very poor substrate for rabbit liver microsomal EH, with a Km value comparable to that of cis-stilbene oxide, but Emax ca. 100-fold lower. This indicates that the two compounds have a comparable affinity for the enzyme, but that nucleophilic attack in the catalytic step is much less efficient for dibcnzo[ // cycloheplcnc 10,11-oxide than for d.v-slilbcnc oxide. This implies that the former compound acts better as an inhibitor than as a substrate of microsomal EH. Furthermore, there was also a fundamental steric difference in the reaction course of the two substrates, since the predominant stereoisomer formed from dibenzo //]cyclohep(ene 10,11-oxide had the (I OS, 11. -configuration,... 

The -oxygenation of cyanatryn occurred readily with liver microsomes and 10,000 g supernatant (the latter was fortified with GSH and the observed product was the glutathione conjugate [5]). The reaction could be detected in 2% liver homogenates but not in homogenates of kidney, lung, intestine, or caecal content. The reaction was readily catalyzed by microsomes from the livers of male and female rats, male and female rabbits and a male human (Table 1)(9). The rat sex difference was much larger for N-de-ethylation than for -oxidation. Typically, microsomes from male rats were more active than those from females. 

Formaldehyde generated from nitromethane was found only in trace amounts after incubation with microsomes from from Fischer 344 rat liver, but none was found after incubation with rat nasal microsomes (Dahl Hadley, 1983). Nitromethane inhibited rabbit liver cytochrome P450 activity, apparently competing for the same ferrohaemo-chrome-binding sites as carbon monoxide (Wade et al, 1977). 

Urinary metabolites are S-methylthioacetic acid sulfoxide, V-acetyl-.S -methyl-L-cysteine and /-(methylthioacetyl)glycine, which are metabolites of Y-methyl-i-cysteine and 5-methylglutathione. These last two compounds were found after incubation of methyl chloride with rodent liver, kidney and brain homogenates. The methyl group of methyl chloride is metabolized via -methyl-L-cysteine to formate which is found in urine and blood of rats, whereas formaldehyde is found in rat liver microsomes and blood of mice and rabbits (lARC, 1986). 

In an attempt to correlate pharmacological activity with the rate of biotransformation, Beckett and Morton have studied the metabolism of a series of indole alkaloids and related model systems by a variety of enzyme preparations of mammalian origin (39, 40, 57-59). Of the systems studied, rabbit liver microsomes were effective in performing O-demethylation liver microsome preparations from rat and guinea pig were not very effective in transforming alkaloids 4-7 and 9-19 (but vide infra) (40). In the case of corynantheidine (4), the product of metabolism was identified as the 0-( 17)-demethyl compound 8 by TLC comparison with authentic material. 

Beckett and Morton did not comment on the inability of their rabbit liver microsomal preparation to metabolize the closed ring E alkaloids 14-19. They found no correlation of partition coefficients or pK0 values with the degree or type of metabolism of the corynantheidine-type alkaloids 4 -7 and 9-13, but explained the observed differences on the basis of the preferred conformations of the members of this series, noting that significant metabolism by a route other than O-demethylation occurred only with the pseudo... 

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