Rat Microsomal Preparations Research Articles

Molecular, cellular, and tissue impact of depleted uranium on xenobiotic-metabolizing enzymes

Enzymes that metabolize xenobiotics (XME) are well recognized in experimental models as representative indicators of organ detoxification functions and of exposure to toxicants. As several in vivo studies have shown, uranium can alter XME in the rat liver or kidneys after either acute or chronic exposure. To determine how length or level of exposure affects these changes in XME, we continued our investigation of chronic rat exposure to depleted uranium (DU, uranyl nitrate). The first study examined the effect of duration (1-18 months) of chronic exposure to DU, the second evaluated dose dependence, from a level close to that found in the environment near mining sites (0.2 mg/L) to a supra-environmental dose (120 mg/L, 10 times the highest level naturally found in the environment), and the third was an in vitro assessment of whether DU exposure directly affects XME and, in particular, CYP3A. The experimental in vivo models used here demonstrated that CYP3A is the enzyme modified to the greatest extent: high gene expression changed after 6 and 9 months. The most substantial effects were observed in the liver of rats after 9 months of exposure to 120 mg/L of DU: CYP3A gene and protein expression and enzyme activity all decreased by more than 40 %. Nonetheless, no direct effect of DU by itself was observed after in vitro exposure of rat microsomal preparations, HepG2 cells, or human primary hepatocytes. Overall, these results probably indicate the occurrence of regulatory or adaptive mechanisms that could explain the indirect effect observed in vivo after chronic exposure.

In Vitro Metabolism of Phenstatin: Potential Pharmacological Consequences

Phenstatin and its derivatives are potential anticancer drug candidates according to their inhibitory properties on tubulin polymerization, cell growth and antivascular activity. However, at the present time, neither pharmacological nor metabolic studies have been conducted in order to strengthen the relevance of phenstatine as a drug discovery candidate. In the present work, the metabolic fate of phenstatin in rat and human microsomal preparations was studied to investigate the stability of this tubulin polymerization inhibitor and any effects of the metabolites on polymerization and on PC3 cancer cell proliferation. The metabolites were separated by high-performance liquid chromatography and, after their synthesis, characterized by simultaneous LC-DAD-UV and LC-ESI-MS analyses. Thus, eight metabolites were identified. The major biotransformation pathways are carbonyl reduction, O-methylation at C-3', O-methylation after aromatic hydroxylation at the position C-2' on phenyl B ring and O-demethylation on A ring. Four of the identified metabolites were as active or more active, than phenstatin in vitro. Moreover, the better stability of phenstatin versus CA-4 and the lack of quinone formation could justify the design of new analogues which could include various substituents on phenyl rings or linker group in order to modulate the metabolism of phenstatin toward even more active metabolites and so up-regulate the pharmacological activity.

Effect of Intestinal Glucuronidation in Limiting Hepatic Exposure and Bioactivation of Raloxifene in Humans and Rats

Raloxifene (Evista) is a second generation selective estrogen receptor modulator used in the treatment of osteoporosis and for chemoprevention of breast cancer. It is bioactivated to reactive intermediates, which covalently bind to proteins and form GSH conjugates upon incubation with NADPH and GSH-supplemented human and rat liver microsomes. Despite these in vitro findings, no major raloxifene-related toxic events have been reported upon its oral administration to humans. This disconnect between safety of raloxifene and its in vitro bioactivation is attributed to its presystemic metabolism via glucuronidation. Current studies investigated the effect of hepatic and intestinal glucuronidation in modulating hepatic availability of raloxifene and its subsequent bioactivation, in vitro. The study design involved preincubation of raloxifene with intestinal microsomes followed by a sequential incubation with liver microsomes. The degree of bioactivation of raloxifene was assessed from the percentage of GSH conjugate formed in liver microsomal incubations or the amount of covalent binding of raloxifene-related material to liver microsomal proteins. The results indicated that human intestinal glucuronidation limited the hepatic exposure of raloxifene that underwent bioactivation in the liver. Similar experiments with rat microsomal preparations showed very little effect of intestinal glucuronidation. This effect of intestinal glucuronidation and the observed species difference were explained by comparing the efficiency (Cl(int)) of glucuronidation and oxidation in the two species. These findings suggested that even though the rate of bioactivation in the two species was similar, the Cl(int) of glucuronidation was 7.5-fold higher in the human intestine as compared to rats. These results support the hypothesis that intestinal glucuronidation modulates the amount of raloxifene undergoing bioactivation by liver and corroborate the importance of assessing other competitive metabolic pathways and species differences in metabolism prior to extrapolation of bioactivation results from rats to humans.

Metabolism of Moexipril to Moexiprilat: Determination of In Vitro Metabolism Using HPLC-ES-MS

Moexipril is a long-acting, non-sulfhydryl angiotensine-converting enzyme inhibitor. It is used for treatment of arterial hypertension. Moexipril is the prodrug, yielding moexiprilat by hydrolysis of an ethyl ester group. Moexiprilat is the metabolite responsible for the pharmacological effect after moexipril administration. Samples of rat and human microsomal preparations exposed to moexipril treatment were analyzed by HPLC using octyl silica stationary phase and isocratic elution. To detect moexipril and moexiprilat the separation was monitored by both ultraviolet and mass specific detection. The rat liver microsomal preparation was more effective to in producing moexiprilat than the similar one derived from human liver cell lines. While additional potential metabolites of moexipril were suggested by computer-modeling, moexiprilat was the sole metabolite detected after microsomal treatment.

In vitro mircosomal metabolism of N-benzyl and N-benzoylnornicotine derivates by rat

The in vitro hepatic microsomal metabolism of N-1'-benzylnornicotine, N-l'-(p-chlorobenzyl)nornicotine, N-1'-benzoylnornicotine and N-1'-(p-chlorobenzoyl) nornicotine was studied using hepatic washed rat microsomal preparations fortified with NADPH. Substrates and their potential metabolites were synthesized, characterised by spectral methods, and separated using a reverse phase HPLC system consisted of a C18 column and a mobile phase composition of acetonitrile: phosphate buffer. Substrates and their potential metabolites were extracted from biological systems with dichloromethane. Metabolites detected were compared with retention times and uv spectra of authentic standards. Metabolic experiments indicated that oxidative dealkylation leading to the formation of nornicotine and the corresponding aldehydes was a major route of metabolism for N-alkylnornicotine substrates. In addition, N-1'-(p-chlorobenzyl)nornicotine produced the corresponding lactam and amide metabolites. N-Acylnornicotines were hydrolysed to nornicotine.

The stability studies andin vitro hepatic microsomal metabolism of some α-phenyl-N-substituted nitrones in rats

Nitrones are a very important class of synthetic chemicals as synthetic intermediates, antioxidant agents, and metabolic oxidation products of secondary amines and imines used drug, food, cosmetic and printing industry. In the present study, the stability experiments and in vitro metabolism studies using rat microsomal preparations fortified with NADPH were carried out using three different alpha-phenyl-N-substituted nitrones ie alpha-phenyl-N-tert-butylnitrone (PTBN), alpha-(2,6-dichlorophenyl)-N-phenylnitrone (DCPPN) and alpha-phenyl-N-adamantanylnitrone (PADN). The separation of these compounds from the potential degradation, isomerization and metabolic products were performed using a reverse phase HPLC system with a diodearray uv detection. Following stability experiments at 37 degrees C using methanolic nitrone solutions, it was observed that PTBN produced trace amounts of benzaldehyde and the corresponding amide. DCPPN also produced trace amounts of amide. After 12 hours, the amount of the amide significantly increased. PADN produced trace amount of benzaldehyde but not any amide. The proposed compounds were incubated with rat microsomal preparations fortified with NADPH, extracted into dichloromethane (DCM) and finally evaporated under nitrogen in the dark conditions. PTBN was metabolized into corresponding amide whereas DCPPN and PADN did not. With all of the substrates, the corresponding aldehydes are observed with both test and control tubes using denaturated microsomes and without co-factors.

Metabolic and chemical studies on N-(4-chlorobenzyl)- N′-benzoylhydrazine

The in vitro hepatic microsomal metabolism of N-(4-chlorobenzyl)- N′-benzoylhydrazine (CBBAH), a model compound representing N-alkyl substituted hydrazides, was studied using hepatic washed rat microsomal preparations fortified with NADPH to identify the possible N-oxidative, N-dealkylated and hydrolytic metabolites. CBBAH and its potential metabolites were prepared, characterized using spectroscopic techniques and then separated using a reversed phase HPLC system with UV detection at 254 nm. CBBAH was chemically converted to the corresponding hydrazone by m-chloroperbenzoic acid ( m-CPBA) oxidation. CBBAH was incubated with rat microsomal preparations in the presence of NADPH, extracted into dichloromethane and evaporated finally under nitrogen. The TLC and HPLC results from the metabolic experiments showed that CBBAH produced the corresponding hydrolytic and N-dealkylated metabolites together with the corresponding hydrazone.

Metabolism of thalidomide in human microsomes, cloned human cytochrome P-450 isozymes, and Hansen's disease patients.

Previous in vitro studies in rat microsomal preparations suggested that thalidomide is metabolized by the cytochrome P450 system (CYP). In this study, we examined the extent of thalidomide metabolism by preparations of pooled human microsomes, microsomes containing cloned human CYP isozymes (CYPIA2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4), and Hansen's disease patients. Results indicated that thalidomide was a poor substrate for CYP isozymes. Alteration of incubation buffer, pH, incubation time, and microsome and thalidomide concentrations did not increase the production of any metabolites. Thalidomide also did not inhibit metabolism of CYP-specific substrates and therefore any interactions with other drugs that are metabolized by the same enzyme system are unlikely. Hansen's patients were given a single oral dose of thalidomide (400 mg), and their blood and urine were collected at time points up to 72 hours, processed, and analyzed by tandem mass spectrometry. Although thalidomide was present in the plasma and urine, no metabolites were found in the plasma and very low amounts of the 5-OH thalidomide metabolite were present in the urine. These results suggest that thalidomide does not undergo significant metabolism by human CYP and that clinically important interactions between thalidomide and drugs that are also metabolized by this enzyme system are unlikely. The major route of thalidomide breakdown in humans and animals is through spontaneous hydrolysis with subsequent elimination in the urine.

The in vitro hepatic microsomal metabolism of N-benzyladamantanamine in rats.

The metabolism of N-benzyladamantanamine (NBAD) was studied in vitro using rat hepatic microsomal preparations. The substrate and proposed metabolites were synthesized and characterized using spectroscopic techniques and separated using a reverse phase HPLC system. NBAD was incubated with rat microsomal preparations, extracted into DCM in the presence of NaCl and evaporated under a stream of nitrogen. The results from HPLC studies showed that NBAD produced the corresponding nitrone and hydroxylamine. This experiment also revealed that dealkylation occurred. No metabolites were observed which corresponded to authentic amide or oxaziridine. The reactions required a microsomal enzyme source and NADPH as a cofactor. The results indicate that the nitrone observed as a metabolite of NBAD is not an intermediate leading to the formation of an oxaziridine and hence an amide, under careful experimental conditions excluding light.

The in vitro hepatic microsomal metabolism of 3,5-dimethyl-4-(phenylazo)-(1H)-pyrazole in rats.

The in vitro hepatic microsomal metabolism of 3,5-dimethyl-4-(phenylazo)-(1H)-pyrazole (DMPAP) was studied using washed rat hepatic microsomal preparations fortified with NADPH. The substrate, DMPAP, and its potential metabolites, i.e. the corresponding reduction product, 3,5-dimethyl-4-amino-(1H)-pyrazole (DMAP), and the oxidation product, 3,5-dimethyl-4-(phenylazoxy)-(1H)-pyrazole (DMAPO), were synthesized and their structures elucidated by use of their spectral characteristics. DMPAP and its potential metabolites were then separated using a reverse phase HPLC system consisting of a C18 column and a mobile phase of acetonitrile:water (50:50) at a flow rate of 1 ml/min with UV detection at 254 nm. DMPAP was incubated with rat microsomal preparations, extracted into DCM in the presence of NaCl, and finally evaporated under a stream of nitrogen. The results from HPLC studies showed that DMPAP was metabolised to the corresponding reduction and oxidation products in the presence of NADPH.

The in vitro hepatic microsomal metabolism of methyl 2-(2(3H)-benzoxazolone-3-yl)acetate in rats.

The in vitro hepatic microsomal metabolism of methyl 2-(2(3H)-benzoxazolone-3-yl)acetate (I) was studied using hepatic washed rat microsomal preparations fortified with NADPH. The substrate (I) and its potential hydrolytic metabolite 2-(2(3H)-benzoxazolone-3-yl)acetic acid (II) and 2(3H)-benzoxazolone (III), a potential dealkylation metabolite, were separated using a reverse phase HPLC system which consisted of a C18 column and a mobile phase of acetonitrile: 0.02 M phosphate buffer (30:70, final pH 7) at a flow rate of 1 ml/min with UV detection at 254 nm. The substrate (I) was incubated with rat microsomal preparations, extracted into DCM, and finally evaporated under nitrogen. The results from HPLC studies showed that (I) was metabolised to (II) and (III) by rat microsomes in the presence of NADPH.

The in vitro hepatic metabolism of 1-phenyl-2-(2-benzothiazolinone-3-yl)ethanone and their reduced derivatives

The in vitro hepatic microsomal metabolism of 1-phenyl-2-(2-benzothiazolinone-3-yl)ethanone (I) and (+/-)-1-Phenyl-2-(2-benzothiazolinone-3-yl)ethanol (II) was studied using both rat microsomal preparations and soluble fractions fortified with NADPH. These two substrates and their potential dealkylation metabolite 2-benzothiazolinone were synthesized and their structures were elucidated by spectral methods. The results showed that (I) was metabolised to (II), the corresponding alcohol by reduction. More alcohol metabolite was observed with microsomes than those obtained with the soluble fractions. No dealkylation of (I) was observed in the experiments using both microsomes and soluble fractions. (II) was metabolised to (I), the corresponding ketone by oxidation. However, compared to the metabolism of (I), more ketone metabolite was observed with soluble fractions than microsomes. (II) was also dealkylated to (III) with only soluble fractions but no dealkylated metabolites were found in the microsomes. In addition, a common unknown metabolite was observed with each substrates.

In vitro hepatic microsomal metabolism of N-benzyl- N-methylaniline

In the present study, the in vitro microsomal metabolism of a tertiary aniline, N-benzyl- N-methylaniline (NBNMA) was studied to determine whether this compound produces an amide derivative (benzoyl) together with N-dealkylation and C- and N-oxidation products as metabolites. The preparations of the corresponding potential metabolites were undertaken and were separated using TLC and HPLC. Incubations were performed using rat microsomal preparations fortified with NADPH. The substrate and its potential metabolites were extracted into dichloromethane in the presence of NaCl and examined by TLC and HPLC–UV. The results indicated that NBNMA did not produce the corresponding amide (benzoyl derivative) or N-oxide metabolite but was dealkylated to the corresponding secondary amine. Two p-hydroxylated phenolic metabolites were also observed. These findings support the concept that nitrones are essential intermediate metabolites for the formation of amides from secondary aromatic amines (chemical rearrangement to amide via an oxaziridine intermediate). The carbinolamine produced from NBNMA does not seem stable enough to allow further oxidation to the amide and therefore this intermediate is broken down to the dealkylation products. N-Dealkylations and p-hydroxylations are major metabolic reactions following in vitro hepatic microsomal metabolism of the benzylic tertiary aniline, NBNMA.

IN VITRO METABOLIC FATE OF N-SUBSTITUTED DIBENZYLAMINES : INVESTIGATION OF BIOLOGICAL OXIDATION SITE ON NITROGEN COMPOUNDS WITH DIFFERENT pKa

The in vitro rnicrosomal metabolism of four model tertiary amines ie. Nethyldibenzylamine (EDBA), tribenzylamine (TBA), N,N-dibenzylaniline (DBAN) and N,N-dibenzylbenzarnide (BZDBA) was studied to establish whether the corresponding N- and C-oxidation products such as the corresponding arnides, dealkylation products, N-oxides and phenols formed. These compounds were selected as substrates to establish the effect of the varying pK, and logP characteristics on their iiz vitro metabolism. The substrates and their potential metabolites were synthesized and then separated using HPLC. Incubations were performed using rat microsomal preparations fortified with NADPH. The substrates and their potential metabolites were extracted into dichloromethane and examined by HPLC. Results from HPLC analysis indicated that all these substrates failed to produce the corresponding amides as metabolites. However, the dealkylation metabolites were observed with all the substrates used as major metabolites. The substrates EDBA and TBA produced the corresponding N-oxide metabolites: Three phenolic. metabolites of DBAN were also demonstrated. These findings support the concept that nitrones are essential metabolic intermediates for the formation of arnides and this reaction reguees,a secondary mine as substrate. In addition, the metabolic N-oxidation of these substrates depends on the pKa of the constituent nitrogen of the molecule. Key words :Tertiary amines, in vitro metabolism, the effect of pKa.

The in vitro metabolism of norcotinine and related biotransformation products by microsomal preparations.

Since norcotinine and 4-(3-pyridyl)-4-oxobutyramide (POBAM) are probable metabolites of nicotine and cotinine, it was of interest to investigate the further in vitro metabolism of these compounds. We now report our preliminary findings using rat microsomal preparations (induced and/or non-induced with phenobarbitone) fortified with NADPH. Following norcotinine metabolism, two compounds, i.e. the corresponding ketoamide (POBAM) and another product, were detected. The latter metabolite has an identical HPLC retention time as that of nicotinamide. Both metabolites showed identical UV spectra when compared to authentic compounds using a multi-array UV detector linked to a HPLC system. The structures of these metabolites were also confirmed by mass spectral analyses. The amount of POBAM was significantly increased when phenobarbitone induced rat microsomes was used. It indicates that the ketoamide is formed via a phenobarbitone inducible isozyme of CYP450. Following the metabolism of POBAM, two compounds, i.e. the corresponding acid (POBA) and an unidentified product, were detected. The uncharacterised compound had an identical HPLC retention time as nicotinamide. Both metabolites gave a UV spectrum identical to the authentic compounds. The detection of nicotinamide in incubates of norcotinine and POBAM suggests that it is released from NADP(H) by a stimulatory effect on glycohydrolase by the substrates. Further studies on the enzymology of these processes are in progress.

The In Vitro Hepatic Microsomal Metabolism of Benzoic Acid Benzylidenehydrazide

The in vitro hepatic microsomal metabolism of benzoic acid benzylidenehydrazide was studied using rat microsomal preparations fortified with NADPH. The substrate and its potential metabolites were synthesised and their structures were elucidated by use of their UV, IR, ¹H-NMR and mass spectral characteristics. The results showed this compound produced hydrolytic metabolites, i.e. benzoic hydrazide and benzaldehyde, together with a phenolic metabolite, benzoic acid phydroxybenzylidene hydrazide.

Metabolic activation of pyrrolizidine alkaloids by human, rat and avocado microsomes

Two pyrrolizidine alkaloids and one pyrrolizidine alkaloid-N-oxide were incubated with microsomal preparations from humans, rat and avocado and the product profiles examined. The alkaloids were converted to dehydroretronecine, the putative toxic metabolite, by both rat and human microsomal preparations. In addition, alkaloid-N-oxides, the major detoxication products from pyrrolizidine alkaloids, were also formed. The pyrrolizidine alkaloid-N-oxide was converted to both dehydroretronecine and the parent alkaloid. This suggests that the toxicity of pyrrolizidine alkaloid-N-oxides could be greater than suggested hitherto as a result of conversion to the toxic metabolite via the parent alkaloid. Quantitative differences in the proportions of products formed by the different microsomal preparations may be of significance in the extrapolation of toxicological data from animal models such as the rat to humans.

Specific prorenin/renin binding (ProBP). Identification and characterization of a novel membrane site.

Renin can be detected in cardiovascular and other tissues but it disappears after bilateral nephrectomy indicating that tissues can take up or bind renal renin from the circulation. If renin uptake is the result of specific binding, plasma prorenin may be a natural antagonist of tissue directed renin-angiotensin systems. To investigate if specific prorenin/renin uptake occurs in rat tissues, binding studies were performed, with rat microsomal membrane preparations using recombinant rat prorenin metabolically labeled with 35S-methionine as a probe. A high affinity binding site for both renin and prorenin was identified. Affinities for prorenin and renin were approximately 200 and 900 pmol/L, respectively. Binding was reversible, saturable, and pH and temperature dependent. The relative binding capacities of membranes from various rat tissues were as follows (fmol/mg): renal cortex (55), liver (54), testis (63), lung (31), brain (18), renal medulla (15), adrenal (17), aorta (7), heart (4), and skeletal muscle (1). Bound prorenin was displaced by rat and human renin or prorenin but not by the prosequence of rat prorenin, angiotensin I or II, rat or human angiotensinogen, the renin inhibitor SQ30697, atrial natriuretic factor, amylase, insulin, bovine serum albumin, hemoglobin, heparin, lysozyme, ovalbumin, cytochrome C, pepsin, pepsinogen, ribonuclease A, mannose-6-phosphate, alpha-methyl mannoside, gonadotropin releasing hormone, or an antibody to hog renin binding protein. these results demonstrate specific binding of prorenin to a site in rat tissues, herein named ProBP, that also binds renin. It is possible that differences in prorenin/renin binding capacity determine the activity of tissue-directed renin-angiotensin systems and that prorenin is a natural antagonist. Alternatively, a prorenin/renin receptor may have been identified that may function by transducing an intracellular signal.

Metabolism of the Catharanthus alkaloids: from Streptomyces griseus to monoamine oxidase B.

More than three decades after their discovery and implementation in medicine, essentially nothing is known about the metabolism or the implications of metabolism in mechanism of action or toxicity of the Catharanthus alkaloids. The frustrating paucity of information about pathways of metabolism has limited a major source of structure-activity relationship information and has blocked a critical avenue necessary for the logical development of new and more useful Catharanthus alkaloids. Microbial transformations, peroxidases, copper oxidases, mouse and rat cytochrome P-450 systems, and mouse brain and bovine liver monoamine oxidase (MAO) preparations have been explored in the study of Catharanthus alkaloid metabolism. In this report, we present results which have clarified the involvement of enzymatic and chemically catalyzed one-electron oxidations that yield nitrogen-centered cation radicals, iminium, and carbinolamine intermediates, all of which explain how new carbon-carbon and carbon-oxygen bonds form, or break and rearrange. The dimeric Catharanthus alkaloids are recalcitrant to oxidations catalyzed by monoamine oxidases and to both normal and induced P-450 rat microsomal preparations. However, the Catharanthus alkaloids appear to be selective reversible inhibitors of MAO-B. Chemical and biochemical aspects of the metabolic transformations of dimeric Catharanthus alkaloids are reviewed together with the implications of our findings.

Comparative in vitro metabolism of T-2 toxin by hepatic microsomes prepared from phenobarbital-induced or control rats, mice, rabbits and chickens

Hepatic microsomes were prepared from phenobarbital (PB)-treated and control rats, mice, rabbits and chickens and were incubated with T-2 toxin (100 μg/mg microsomal protein). Additional microsomes from PB-induced animals were incubated with T-2 toxin and the esterase inhibitor paraoxon (PA) at 2.5 nmol/mg microsomal protein. The major metabolite in microsomal preparations from both control and PB-induced rats, rabbits and mice was HT-2. In microsomes isolated from PB-treated chickens, 3′-hydroxy T-2 was the major metabolite, but 30 and 79% of the added T-2 toxin remained unmetabolized at 60 min in incubations from PB-induced and control birds, respectively. The percentage of hydroxylated metabolites formed in the microsomal preparations of the four species studied was significantly increased following PB treatment compared with the non-treated controls. The addition of PA to the incubation system effectively inhibited the hydrolysis of the ester groups in T-2 toxin, resulting in 1.4- and 1.25-fold increases in the percentage of 3′-hydroxy T-2 in the mouse and rat microsomal samples, respectively. In the rabbit microsomal preparations, 3′-hydroxy T-2, which was not detected in the absence of PA, represented 11% of the added substrate in the PB/PA incubation samples. Addition of PA did not cause a significant change in the amount of 3′-hydroxy T-2 formed in chicken microsomal samples, since competition between hydrolysis and hydroxylation pathways for the T-2 toxin substrate was not an important factor in this species. Two new metabolites, designated RLM-2 and RLM-3 were detected in chicken, rat and mouse microsomal preparations. On the basis of gas chromatography/mass spectrometry data, the compounds were tentatively identified as isomers of 3′-hydroxy T-2.