Liver microsomal oxidative reactions

Iron complexes or microsomal nonheme iron are undoubtedly obligatory components in the microsomal oxidation of many organic compounds mediated by hydroxyl radicals. In 1980, Cohen and Cederbaum [27] suggested that rat liver microsomes oxidized ethanol, methional, 2-keto-4-thiomethylbutyric acid, and dimethylsulfoxide via hydrogen atom abstraction by hydroxyl radicals. Then, Ingelman-Sundberg and Ekstrom [28] assumed that the hydroxylation of aniline by reconstituted microsomal cytochrome P-450 system is mediated by hydroxyl radicals formed in the superoxide-driven Fenton reaction. Similar conclusion has been made for the explanation of inhibitory effects of pyrazole and 4-methylpyrazole on the microsomal oxidation of ethanol and DMSO [29],... 

Microsomal oxidative reactions constitute the most prominent phase I biotransformation pathway for a wide variety of structurally unrelated drugs (Table 1.4). Some drugs (e.g. amphetamine, diazepam, propranolol, lignocaine) simultaneously undergo more than one type of microsomal-mediated oxidative reaction. Microsomal enzymes are located primarily in liver cells, where they are associated with the smooth-surface (without ribosomes) endoplasmic reticulum (Fouts, 1961). Lipid solubility is a prerequisite for drug access to the... 

Since the results of our experiments with isolated rat liver fractions supported a reaction sequence Initiated by microsomal oxidation of the nitrosamine leading to formation of a carbonium ion, the results of the animal experiment suggested that in the intact hepatocyte, one of the earlier electrophilic intermediates (II, III or V, Figure 1) is intercepted by nucleophilic sites in DNA (exemplified here by the N7 position of guanine) before a carbocation is formed. 

Stndies of the antoxidation of carotenoids in liposomal suspensions have also been performed since liposomes can mimic the environment of carotenoids in vivo. Kim et al. stndied the antoxidation of lycopene," P-carotene," and phytofluene" " in liposomal snspensions and identified oxidative cleavage compounds. Stabilities to oxidation at room temperature of various carotenoids incorporated in pig liver microsomes have also been studied." The model took into account membrane dynamics. After 3 hr of reactions, P-carotene and lycopene had completely degraded, whereas xanthophylls tested were shown to be more stable. 

Screening is usually carried out with liver microsomes from humans, rats, mice, dogs and monkeys and liver S9 fraction from aroclor 1254-induced rats. The incubation is typically mn with a volume of 0.2-1. OmL in a microcentrifuge or a glass tube. Different incubation conditions are used for CYP and UGT reactions. The incubation mixture for formation of oxidative metabolites and/or GSH conjugates contains ... 

MgCl2 (10 mM) increased the apparent Km (83 to 173 /am) and reduced the Vjnax (3.4 to 2.4 min-1) of triazolam 4-hydroxylation by expressed CYP3A4 [21]. However, both MgCl2 (30 mM) and CaCl2 (30 mM) significantly increased reaction rates of testosterone 6/3-hydroxylation (approximately threefold) and nifedipine oxidation (three- to six-fold) by human liver microsomes (HLMs) or recombinant CYP3A4 (reconstituted with b5 and GSH) [15]. It was suggested that divalent cation stimulation on the activity was related to involvement of b5 in CYP 3A4 reaction. 

Second, metabolism of 6-fluoroBP by rat liver microsomes yields the same BP quinones obtained in the metabolism of BP (23). This suggests that these products are formed by an initial attack of a nucleophilic oxygen atom at C-6 in the 6-fluoroBP radical cation with displacement of the fluoro atom. In fact, when 6-fluoroBP is treated with the one-electron oxidant Mn(0Ac)3, the major products obtained are 6-acetoxyBP and a mixture of 1,6- and 3,6-diacetoxyBP (15), indicating that reaction occurs via an initial attack of acetate ion at C-6 of the 6-fluoroBP radical cation. On the other hand electrophilic substitution of 6-fluoroBP with bromine or deuterium ion shows no displacement of fluorine at C-6, although in both cases substitution occurs at C-l and/or C-3. These results indicate that... 

The above sequence mimics the proposed biosynthesis of Ervatamia alkaloids and in this context Thai and Mansuy (190) set out to determine whether an enzyme preparation would be able to promote the same transformation. By incubation of dregamine hydrochloride with a suspension of liver microsomes from a rat pretreated with phenobarbital (as a good inducer of P-450 cytochromes) in the presence of NADPH and 02, 20-epiervatamine (45) was formed together with the major metabolite Nl -demethyldregamine. It is well known that microsomal reaction on tertiary amines results in Af-oxide formation or N-deal-kylation. Thus it is likely that 45 was derived either from a rearrangement of dregamine JV4-oxide, catalyzed by the iron cytochrome P-450 or from one-electron oxidation of 30. 

Indicine IV-oxide (169) (Scheme 36) is a clinically important pyrrolizidine alkaloid being used in the treatment of neoplasms. The compound is an attractive drug candidate because it does not have the acute toxicity observed in other pyrrolizidine alkaloids. Indicine IV-oxide apparently demonstrates increased biological activity and toxicity after reduction to the tertiary amine. Duffel and Gillespie (90) demonstrated that horseradish peroxidase catalyzes the reduction of indicine IV-oxide to indicine in an anaerobic reaction requiring a reduced pyridine nucleotide (either NADH or NADPH) and a flavin coenzyme (FMN or FAD). Rat liver microsomes and the 100,000 x g supernatant fraction also catalyze the reduction of the IV-oxide, and cofactor requirements and inhibition characteristics with these enzyme systems are similar to those exhibited by horseradish peroxidase. Sodium azide inhibited the TV-oxide reduction reaction, while aminotriazole did not. With rat liver microsomes, IV-octylamine decreased... 

By analogy to N- and O-dealkylation reactions, one might expect esters and amides to be susceptible to P450-catalyzed oxidative attack at the a-carbon to oxygen (esters) or a to nitrogen (amides). This is indeed the case and was first established (132) by demonstration that the pyridine diester (Fig. 4.66) was oxidatively cleaved by rat-liver microsomes to yield the monoacid as shown. 

Beaty NB, Ballou DP. The oxidative half-reaction of liver microsomal FAD-containing monooxygenase. J Biol Chem 1981 256(9) 4619—4625. 

Estabrook, R.W., Hildebrandt, A.G., Baron, J., Netter, K.J. and Leibman, K. (1971) A new spectral intermediate associated with cytochrome P-450 function in liver microsomes. Biochemical and Biophysical Research Communications, 42 (1), 132-139. Pompon, D. and Coon, M.J. (1984) On the mechanism of action of cytochrome P-450. Oxidation and reduction of the ferrous dioxygen complex of liver microsomal cytochrome P-450 by cytochrome b5. Journal of Biological Chemistry, 259 (24), 15377-15385. Hildebrandt, A. and Estabrook, R.W. (1971) Evidence for the participation of cytochrome b 5 in hepatic microsomal mixed-function oxidation reactions. Archives of Biochemistry and Biophysics, 143 (1), 66-79. 

The antihypertensive agent tripamide (4.267), when incubated with rat liver microsomes or partially purified microsomal arylamidase, was extensively hydrolyzed to 4-chloro-3-sulfamoylbenzoic acid (4.268) [171]. This metabolite seems to be produced by direct hydrolysis, since the other metabolites formed by oxidation of the cycloalkyl moiety remained unchanged when incubated with rat liver microsomes. The mechanism of hydrolysis of tripamide has not yet been fully elucidated. The inhibition of the reaction by O-ethyl 0-(4-nitro-phenyl) phenyl phosphothionate indicates that amidases may be involved. 

An important drug in the present context is the mineralocorticoid receptor antagonist spironolactone (7.74, Fig. 7.12). Among its many metabolic reactions, spironolactone is readily hydrolyzed at the thioester bond (Fig. 7.12, Reaction a) to form deacetyl-spironolactone (7.75, Fig. 7.12), a metabolite found in a variety of tissues [155 -157]. This thiol compound, which is also a potent mineralocorticoid antagonist, promotes the mechanism-based inactivation of hepatic, adrenal, and testicular cytochrome P450 isozymes. There is now good evidence to indicate that this behavior is the result of microsomal 5-oxidation (see Chapt. 7 in [7]). When spironolactone was incubated with liver microsomes from rats pretreated with dexamethasone (an inducer of CYP3A), the sulfinic and sulfonic acid derivatives were characterized [158]. Perhaps the importance of the 5-deacetylation of spironolactone... 

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,... 

Interestingly, there is a marked species difference in the in vitro hydrolysis of carbamazepine 10,11-epoxide, such that the reaction was observed only in liver microsomes from humans but not in liver microsomal or cytosolic preparations from dogs, rabbits, hamsters, rats, or mice [181][196], Thus, carbamazepine appears to be a very poor substrate for EH, in analogy with the simpler analogues 10.129 (X = RN, RCH, or RCH=C). The human enzyme is exceptional in this respect, but not, however, in the steric course of the reaction. The diol formed (10.131, X = H2NCON) is mostly the trans-(10.S, 11. S )-enaniiomer [196], In other words, the product enantioselectivity of the hydration of carbamazepine epoxide catalyzed by human EH is the same as that of di benzol a,oxide catalyzed by rabbit microsomal EH, discussed above. 

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. 

Although it has been assumed for years that -oxidation involves cytochrome P450 there is no good evidence for this (10. Differences in the species and sex specificity of the -oxidation and the N-de-ethylation of cyanatryn catalysed by liver microsomes suggested that different enzymes may be involved in the two reactions. Relevant to this problem was the isolation of a cytochrome P450-free enzyme (10) which catalyses the -oxidation of 2-ethyl-4-thioisonicotinamide. Compounds of similar structure, the thioureylenes, of which methimazole (l-methyl-2-... 

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