NADPH generating system

Analysis of reaction mixtures for 1-propanol and 2-propanol following incubation of NDPA with various rat liver fractions in the presence of an NADPH-generating system is shown in Table I ( ). Presence of microsomes leads to production of both alcohols, but there was no propanol formed with either the soluble enzyme fraction or with microsomes incubated with SKF-525A (an inhibitor of cytochrome P450-dependent oxidations). The combined yield of propanols from 280 ymoles of NDPA was 6.1 ymoles and 28.5 ymoles for the microsomal pellet and the 9000 g supernatant respectively. The difference in the ratio of 1- to 2-propanol in the two rat liver fractions may be due to differences in the chemical composition of the reaction mixtures (2) Subsequent experiments have shown that these ratios are quite reproducible. For comparison, Table I also shows formation of propanols following base catalyzed decomposition of N-propyl-N-nitrosourea. As expected (10,11), both propanol isomers were formed, the total yield in this case being almost quantitative. 

Forti, G.C., Paolini, M., Hrelia, P. et al. (1984) NADPH-generating system influence on microsomal monooxygenase stability during incubation for the liver microsomal assay with rat and mouse S9 fractions. Mutation Research, 129, 291-297. 

Reduced flavins (FADH2, FMNH2, and riboflavin) generated by flavin-dependent reductases have been hypothesized to reduce azo dyes in a nonspecific chemical reaction, and flavin reductases have been revealed to be indeed anaerobic azoreductases. Other reduced enzyme cofactors, for example, NADH, NADH, NADPH, and an NADPH-generating system, have also been reported to reduce azo dyes. Except for enzyme cofactors, different artificial redox mediating compounds, especially such as quinines, are important redox mediators of azo dye anaerobic reduction (Table 1). 

Incubation mixtures containing calf thymus DNA resulted in senecionine and a related compound being bound to a macromolecular liver fraction (224). The binding of the compounds was dependent on the presence of oxygen, the enzyme system, and an NADPH-generating system, thus demonstrating that metabolites such as those formed from senecionine (Scheme 34) may interact with macromolecules to result in alkaloid-associated toxicities. 

Biochemical studies with mussel tissue homogenates and microsomal fractions have been conducted using viscera (whole mussel minus adductor muscles), green gland, gill, and mantle. Using aerobic conditions and an NADPH-generating system, the metabolism of aldrin and p-nitroanisole have been observed. 

The electron transfer system has not been studied in detail in fish, but the metabolism of compounds such as biphenyl (37), benzo(a)pyrene (21) and 2,5-diphenyloxazole (38) by fish liver microsomes has been shown to require oxygen and NADPH generating system. The metabolism of BP (21), 2,5-diphenyloxazole (Ahokas, unpublished observation) and aldrin (27.) by fish liver microsomal enzyme system is inhibited strongly by carbon monoxide. This information and the fact that cytochrome P-1+50, as well as NADPH cytochrome c reductase system are present in fish, suggest strongly that fish have a cytochrome P-1+50 mediated monooxygenase system which is very similar to that described in mammals. 

S. Abu-El-Haj, R. Allahyari, E. Chavez, I. M. Fraser, A. Strother, Effects of an NADPH-Generating System on Primaquine Degradation by Hamster Liver Fractions , Xenobiotica 1988, 18, 1165 - 1178. 

These model studies permitted demonstration of metabolic a-hydroxylation of NPy by isolating 2-hydroxytetrahydrofuran as a metabolite of NPy. This was accoitplished by trapping 4-hydroxy-butyraldehyde as its 2,4-dinitrophenylhydrazone (DNP) derivative. For in vitvo studies, NPy-2,5-l C was incubated with rat liver microsomes, 02i and an NADPH generating system. After the incubation was complete, DNP reagent was added to the mixture and the products were extracted and examined by preparative TLC. A radioactive band corresponding to the DNP of 4-hydroxybutyralde-hyde was observed. This band was not present in controls in which NADPH was omitted or in which boiled enzyme was used. The mass spectrum was identical to that of a reference sairple. In addition, a minor metabolite with mass spectrum identical to that of the DNP of 2-butenal was also isolated. These results showed conclusively that NPy underwent metabolic a-hydroxylation in this in vitro system. 

Evidence for metabolic a-hydroxylation of NNN was first obtained through in vitro experiments. NNN-2 -l C was incubated with rat liver microsomes, O2 and an NADPH generating system. The resulting mixtures were added to DNP reagent and analyzed by preparative TLC or HPLC. The DNPs of the keto alcohol, 4-hydroxy-l-(3-pyridyl)-l-butanone (0.6% from NNN) and of 4—hydroxy-4-C3-pyr— idyDbutanal (0.3% from NNN) were both identified by comparison of their mass spectra to reference samples. These products, which were not present in controls, resulted from 2 -hydroxylation and 5 -hydroxylation of NNN, respectively (see Figure 12). Another product of 2 -hydroxylation, myosmine (0.6% from NNN) was identi-by GLC-MS analysis of incubation mixtures, after extraction with chloroform. 

Various mammalian-tissue preparations were also found224 to catalyze the formation of GDP-Fuc from GDP-Man both NAD+ and an NADPH-generating system were necessary for maximum activity. 

A reduction and activation of HjOj by other one-electron donors, like semiquinones, has also to be considered. This follows from a study of the ethylene production from methionine in the presence of pyridoxal phosphate, a reaction characteristic for OH radicals or for Fenton-type oxidants. The ethylene production in the presence of dioxygen, anthraquinone-2-sulfonate, and an NADPH-generating system in phosphate buffer pH 7.6 was inhibited by SOD and by catalase, but stimulated by scavengers of OH radicals, like 0.1 mM mannitol, a-tocopherol, and formiate... 

Incubation of 1,1,2,2-tetrachloroethane with hepatic microsomes and an NADPH-generating system results in the production of chlorinated metabolites, the major ones being mono- and dichloroacetate (Ivanetich Van Den Honert, 1981). 

Male Fischer 344 rats were exposed by inhalation to 1% 2-chloro-1,1,1 -trifluoroethane for 2 h and then urine was collected for 24 h. Urinary metabolites identified by 19F nuclear magnetic resonance and gas chromatography/mass spectrometry were 2,2,2-trifluoroethyl glucuronide (16%), trifluoroacetic acid (14%), trifluoroacetaldehyde hydrate (26%), trifluoroacetaldehyde-urea adduct (40%) and inorganic fluoride (3%). A minor, unidentified metabolite was also detected. No covalent binding of fluorine-containing metabolites was observed in the liver and kidney from the exposed rats (Yin et al., 1995). In-vitro incubation of 2-chloro-1,1,1-trifluoroethane with rat liver microsomes and an NADPH-generating system has been shown to involve a dechlorination reaction (Salmon et al., 1981) that produced trifluoroacetaldehyde hydrate as the only metabolite (Yin et al., 1995). 

Nutritional and nutritional status markedly influence xenobiotic metabolism in laboratory animals. Microsomes were prepared from the livers of rats which had been fed chow or modified AIN-76 diets with or without oxidized or unoxidized sulfur amino acids for 7 days. The pattern of benzo(a)pyrene (BaP) metabolites formed by each microsomal preparation in the presence of a NADPH-generating system was determined using high performance liquid chromatography (HPLC). The results indicate that oxidized sulfur amino acids induce different forms of cytochromes P-450 in rat liver Which are reflected by different BaP metabolic profiles. 

Protein concentrations are less than 0.1 mg/mL in two cases to avoid over-metabolism of the substrate. The buffer consists of potassium phosphate (50 mM, pH 7.4), MgCl2 (3 mM), EDTA (1 mM), and the NADPH-generating system [NADP (1 mM), glucose-6-phosphate (5 mM), and glucose-6-phosphate dehydrogenase (1 U/mL)]. The incubation time is five minutes for all assays. 

Typical experimental procedures are as follows The test drug candidate is incubated with pooled human liver microsomes (e.g., 1 mg protein/mL) that were previously preincubated with ABT (1 or 2 mM) for 30 minutes at (37 1)°C in the presence of an NADPH-generating system. Incubations of the drug candidate in the absence of ABT serve as controls. For hepatocytes, suspensions of freshly isolated or cryopreserved hepatocytes (lx 106 cells/ mL) are preincubated with 100-pM ABT for 30 minutes in 0.25 mL of Krebs-Henseleit buffer or Waymouth s medium (without phenol red) supplemented with FBS (4.5%), insulin (5.6 pg/mL), glutamine (3.6 mM), sodium pyruvate (4.5 mM), and dexamethasone (0.9 pM) at the final concentrations indicated. After the preincubation, the drug candidate is added to the incubation and the rate of metabolism of the drug candidate is compared in hepatocytes or microsomes with and without ABT treatment. A marked difference in metabolism caused by ABT is evidence that CYP plays a prominent role in the metabolism of the drug candidate. 

Figure 18 Effect of nonionic detergent (Triton X-100) on benzydamine N-oxygenation (FMO) and N-demethylation (CYP) by human liver microsomes. Benzydamine (500 pM) was incubated with pooled human liver microsomes (1.0 mg protein/mL) in tricine buffer (50 mM, pH 8.5 at 37°C) with or without Triton X-100 [1% (v/v)]. Reactions were initiated by the addition of an NADPH-generating system and stopped after 10 minute by the addition of an equal volume (500 pL) of methanol. Precipitated protein was removed by centrifugation, and an aliquot (25 pL) of the supernatant fraction was analyzed by HPLC with fluorescence detection. Abbreviations FMO, flavin monooxygenase CYP, cytochrome P450.

Figure 9.85 Normal phase HPLC profiles of the reaction product of the cholesterol side chain cleavage system. Peaks were identified on the basis of their retention times. (i4) Without cholesterol oxidase treatment. Cholesterol (100 nmol) was incubated with cytochrome P450scc (70 pmol) in the presence of adrenodoxin, adrenodoxin reductase, and an NADPH-generating system. Monitoring was at 214 nm. Peaks 1, cholesterol 2, pregnenolone 3, deoxycorticosterone acetate (internal standard) (B) The reaction mixture of (A) was further incubated with cholesterol oxidase at 37°C for 10 minutes. Monitoring was at 240 nm. Peaks 1, cholestenone 2, progesterone 3, deoxycorticosterone acetate (internal standard). (From Sugano et al., 1989.)...

Complete NADPH-generating system under Nj (Control) 100... 

Figure 2 Effect of age on the 6ft-, la-, and 16a-hydroxylation of testosterone by rat liver microsomes. Liver microsomes equivalent to 333 mg of wet-weight liver from male rats were incubated with 700 m/xmoles of testosterone-4-C14 in a final volume of 5.7 ml for 7.5 min at 37°C in the presence of an NADPH-generating system. Formation of 6ft-hydroxytestosterone (6/f-OH), 7a-hydroxytestosterone (7a-OH), and 16a-hydroxytestosterone (16a-OH) was measured. Each value represents the average SE of three to six values where each value was obtained with the pooled livers from three to six rats. Taken from Ref. (36).

Melanin formation via microsomes in presence of an NADPH-generating system and molecular oxygen Study of melanin formation via polyphenol oxidase... 

OH-A -THC were incubatedin an in vitro rat liver microsomal NADPH-generating system ( ). Despite the low yield, the presence of the fatty acid-conjugated cannabinoids in the incubation mixture led our laboratory to assume that this commonly used microsomal mixed function oxidase system was involved in the conjugating process. Soon, however, it was confirmed that an energy-dependent... 

Figure 12. ESR spectra of nitro-anion free radicals from the microsomal reduction of nitro compounds, a. Radicals from the anaerobic incubation of 2 mM nitrobenzene with an NADPH-generating system and 1.5 mg/ml hepatic microsomes b, radicals from the anaerobic incubation of 1 mM nitrofurantoin with an NADPH-generating system and 1 mg/ml hepatic microsomes. From [88,169], with permission.

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