Metabolite from mouse liver

Segall and coworkers described the in vitro mouse hepatic microsomal metabolism of the alkaloid senecionine (159) (Scheme 34). Several pyrrolizidine alkaloid metabolites were isolated from mouse liver microsomal incubation mixtures and identified (222, 223). Preparative-scale incubations with mouse liver microsomes enabled the isolation of metabolites for mass spectral and H-NMR analysis. Senecic acid (161) was identified by GC-MS comparison with authentic 161. A new metabolite, 19-hydroxysenecionine (160), gave a molecular ion consistent with the addition of one oxygen atom to the senecionine structure. The position to which the new oxygen atom had been added was made evident by the H-NMR spectrum. The three-proton doublet for the methyl group at position 19 of senecionine was absent in the NMR spectrum of the metabolite and was replaced by two signals (one proton each) at 3.99 and 3.61 ppm for a new carbinol methylene functional group. All other H-NMR spectral data were consistent for the structure of 160 as the new metabolite (222). 

In summary, it can be seen that Ai-THC is extensively transformed to a number of metabolites. Figure 10 shows a computer processed total ion chromatogram of the metabolites of A1-THC from mouse liver. The main point in showing this chromatogram is to emphasize the complexity of the metabolite picture. 

Little is knowm of the P450 isozyme specificity for either PBO inhibition, metabolite-inhibitory complex formation or type III spectrum formation. Studies involving five isoforms purified from mouse liver (Ijcvi and Hodgson. 19X5 Be nine I rt til., 1985 showed that epoxidation of aldrin by any of these isofonns was inhibited by PBO and that all isoforms appeared to form a stable inhibitory-complex, However, the form of the type III spectrum was variable, with little correlation between the extent of inhibition and the nature of the spectrum. More recently, Pappas and Franklin (1996 have provided indirect evidence that... 

Figure 2 (Top trace) TIC plot of the TMS derivatives of metabolites of A -tetrahydrocannabinol extracted from mouse liver. Metabolites appear in scans 160-360, fatty acids in scans 0-160, and bile acids in scans 360-480. (Lower trace) Single ion plot of the ion at m/z 145, diagnostic for hydroxylation at a specific site in the drug molecule. The metabolites producing peaks in the region of scans 250-320 contain this structural feature, while the other metabolite-related peaks do not. However, the ion is also present in the spectra of the fatty acids but not in those of the bile acids. The separation used a 2 m SE-30 packed column with a temperature-programmed run (2°min h-...

The in vivo tissue distribution, excretion, and hepatic metabolism of microcystins have been primarily investigated using variously radiolabeled ones. " The amounts of the injected microcystins were too small and the amounts of the contaminants in the tissues were too large to investigate the metabolites by instrumental analysis, such as HPLC with UV detection and LC/MS. Fig. 4 shows the HPLC profiles of a cytosolic extract from mouse liver spiked with 5 p.g each of microcystins-LR and -RR. When the cytosolic extract is prepared by the method described by Robinson et al., which consists of heat-denaturation, pronase digestion, and ODS silica gel treatment (Fig. 4a), the two spiked microcystins cannot be precisely analyzed because of a substantial amount of coexisting substances. When the cytosolic extract is further purified with the immunoaffinity column, the coexisting substances are effectively eliminated... 

Seven compounds were identified in fraction 4 from mouse liver (Fig. la). Four of them, including the two major metabolites 6a- ( 3, Table 1) and 7-hydroxy-A -THC (O have been reported previously. The mass spectrum of the TMS derivative of 6a-ljiydroxy--A -THC ( ) was characterized by ions m/e 459 ( / M-15 / Table 2) and a base peak at m/e 384 M-90 y ) whereas the spectrum of... 

FIG. 3. Acid metabolites present in fraction 5 from mouse liver (Fig. 2). The R groups in the formula show the possible positions of hydroxylation. Mono- and di-hydroxy metabolites are identified as shown in the key. 

Molinate has a low toxicity to rats, oral LDso=720 mg/kg, and is rapidly metabolized by plants to CO2 (1) (5) and naturally occurring plant constituents (1). Molinate is also readily metabolized by soil microorganisms (6). After incubation of molinate with Bacillus sp. 24, Nocardia sp. 119, and Micrococcus sp. 22r which were isolated from Russian garden soils and rice field drains (7,8), it was found that molinate was completely degraded into various hydroxy and oxidized products in the medium. Molinate can be metabolized to its corresponding sulfoxide in the mouse in vivo and by the microsome-NADPH system of mouse liver (9, 10). Hubbell et al. (11) and DeBaun et al. (12) also found molinate sulfoxide along with other polar and nonpolar metabolites in rat urine. 

Validation of the model. Validation of the model was performed using data from rat and mouse liver microsome preparations (Schlosser et al. 1993). The assumption that benzene and its metabolites compete for the same enzyme reaction site was supported in part by the observation of a lag time in the benzene-to-hydroquinone reaction as compared to the phenol-to-hydroquinone reaction. This lag could be explained by the fact that benzene is first hydrolyzed to phenol, which is then hydrolyzed to hydroquinone, and if all compounds are substrates for P-450 2E1, the kinetics of this pathway would be slowed compared to those of the direct phenol-to-hydroquinone pathway. The model also adequately predicted phenol depletion and concomitant hydroquinone formation resulting from phenol incubations. 

Figure 2, Oxidation and other reactions of suljallate indicating mutagenic activities of the products in the S. typhimurium TA 100 assay (revertants/nmole without activation/with activation / designates no data available). All thio- and dithiocarbamates are formed from oxidations with MCPBA except for the a-hydroxy compound. 2-Chloroacrolein is a metabolite in the mouse liver microsome-NADPH system. The other compounds are potential metabolites. Several of the oxidized thio- and...

Crotonaldehyde was formed NADPH-dependently as a minor metabolite of butadiene (partial pressure of 48-52 cm Hg = 660 000 ppm) in microsomes obtained from liver, lung or kidney of male B6C3Fi mice (Sharer et al., 1992) or human liver (Duescher Elfarra, 1994), the formation rate being 20-50 times lower than that of epoxybutene. 3-Butenal was suggested as an intermediate metabolite. No crotonaldehyde formation was observed with microsomes from mouse testis or with microsomes of testis, liver, lung or kidney of male Sprague-Dawley rats (Sharer et al., 1992). 

DNA-protein cross-links caused by formaldehyde, a metabolite from the GST pathway, have been demonstrated in mice but not hamsters exposed to dichloromethane (Casanova et al., 1992). Similarly, in-vitro studies have not demonstrated DNA-protein cross-links in rat, hamster or human hepatocytes exposed to concentrations of dichloromethane of up to 5 mM. This is equivalent to the time-weighted average concentration predicted to occur in mouse liver during a 6-h inhalation exposure to a dichloromethane concentration of > 10 000 ppm [34 700 mg/m ] (Casanova et al., 1997). 

Boberg, E.W., Miller, E.C., Miller, J.A., Poland, A. Liem, A. (1983) Strong evidence from studies with brachymorphic mice and pentachlorophenol that I -sulfooxy safrole is the major ultimate electrophilic and carcinogenic metabolite of 1 -hydroxysafrole in mouse liver. Cancer Res., 43,5163-5173... 

The enzymic oxidative deamination of simple phenethylamines is exemplified by the reported bio transformations of mescaline (146) (114, 115) and ephedrine (148) (116). Mescaline is metabolized to 3,4,5-trimethoxy-phenylacetic acid by tissue homogenates of mouse brain, liver, kidney, and heart (114,115). 3,4,5-Trimethoxybenzoic acid is also formed as a minor metabolite. The formation of jV-acetylmescaline (147), a significant metabolite in vivo, was not observed in the in vitro studies. Both D-(—)-and L-(+)-ephedrine have been incubated with enzyme preparations from rabbit liver norephedrine (149), benzoic acid, and 1-phenyl-1,2-propanediol were characterized as metabolites (116). The D-(—)-isomer was the better substrate, being more rapidly converted. Similar results were previously reported with rabbit liver slices as the source of enzyme (153,154). The enzymic degradation of the side chain of /i-phenethylamines has been extensively investigated with nonalkaloid substrates such as amphetamine (151) and jV-methylamphetamine (150) (10,155-157), and the reader is referred to these studies for a more comprehensive coverage of this aspect of the subject. 

Data produced in vitro by mouse and rat liver microsomes also indicate species differences in benzene metabolism (Schlosser et al. 1993). Quantitation of metabolites from the microsomal metabolism of benzene indicated that after 45 minutes, mouse liver microsomes from male B6C3Fj mice had converted 20% of the benzene to phenol, 31% to hydroquinone, and 2% to catechol. In contrast, rat liver microsomes from male Fischer 344 rats converted 23% to phenol, 8% to hydroquinone, and 0.5% to catechol. Mouse liver microsomes continued to produce hydroquinone and catechol for 90 minutes, whereas rat liver microsomes had ceased production of these metabolites by 90 minutes. Muconic acid production by mouse liver microsomes was <0.04 and <0.2% from phenol and benzene, respectively, after 90 minutes. 

Figure 3 An example of the use of GC-MS for metabolic profiling. Left A section of the total ion chromatogram from the analysis of TMS-derivatized aqueous tissue extracts from the liver of PPAR- null mouse. Metabolites are identified from exact retention times and comparison of corresponding mass spectra with those in the NIST database. 97 metabolites were quantified. Right Summary of metabolite differences in the tissues of the PPAR-a null mouse. Red- increased relative to control, blue- decreased relative to control. The increased/decreased width of certain arrows reflects relative increased/decreased concentrations across these pathways, respectively.

Coprine, isolated from the Coprinus atramentarius mushroom, and identified as hydroxycyclopropyO-L-glutamine ", 135, is the first example of a natural product containing a cyclopropanone equivalent. Coprine inhibits mouse liver aldehyde dehydrogenase in vivo but not in vitro Cyclopropanone hydrate (170), which can be derived from coprine by hydrolysis to 169 (equation 39), inhibits the enzyme both in vivo and in vitro. Cyclopropanone hydrate has thus been proposed as a metabolite of coprine which is the active agent causing the toxic effects ... 

Isodrin is metabolized by biooxidation to endrin. Isodrin and its metabolite, endrin, have high fat water partition coefficients and, therefore, tend to accumulate in adipose tissue. At a constant rate of intake, however, the concentration of the insecticide in adipose tissue reaches an equilibrium and remains relatively constant. Following cessation of exposure, it is slowly eliminated from the body. In vitro studies have shown that mixed-function oxidase of mouse liver converts isodrin to endrin. Mice excrete 10% of the orally administered dose in urine. Four unidentified metabolites were present in urine, three probably are glucuronide or sulfate conjugates. Feces is... 

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