Masaharu Hirata

Fluorine containing amino acids and their derivatives. 7. Synthesis and antitumor activity of α- and γ-substituted methotrexate analogs

Abstract Three types of reactions of α-substituted malonates, difluoromethylation, alkylation with n,n,n-trifluoroalkyl sulfonates, and Michael addition to 2-substituted-acrylates, conveniently afforded a number of fluorine containing α amino acids such as β-fluorinated-alanines, 2-amino-n,n,n-trifluoroalkanoic acids, and fluorinated glutamic acids as well as other γ-heteroatom substituted glutamic acids. Here, an efficient enzymatic optical resolution using hog kidney acylase was conducted to obtain both optical isomers of 2-amino-n,n,n-trifluoroalkanoic acids. In addition, a novel sulfoxide rearrangement was observed in a base-catalyzed reaction of diethyl α-difluoromethyl-α-sulfoxymalonates. Finally, α- and γ-substituted glutamic acids obtained were used for chemical modification of the antitumor agent methotrexate to reveal remarkable structure-activity relationships. In particular, the significant effects of fluorine substitution on the in vivo antitumor activity were observed.

Adenyl cyclase of Brevibacterium liquefaciens

Abstract Adenyl cyclase has been purified about 100-fold from cell-free extracts of Brevibacterium liquefaciens . The purified enzyme preparation catalyzed the conversion of ATP (or dATP) to cyclic 3′,5′-AMP (or cyclic 3′,5′-dAMP) and pyrophosphate. In addition to Mg 2+ , pyruvate or another α-keto-monocarboxylic acid was required for the reaction. None of the other nucleoside triphosphates tested served as substrate.

Induction of γ-glutamylcysteine synthetase by prostaglandin A2 in L-1210 cells

Abstract Effects of prostaglandin A2 (PGA2) on glutathione (GSH) status in L-1210 cells were examined. When the cells were cultured in the presence of PGA2, a persistent rise of cellular GSH concentration was observed 6 h after the addition of PGA2. This stimulatory effect of PGA2 was abolished if the cells were pretreated with an enzyme inhibitor of GSH synthesis, buthionine sulfoximine. Subsequent study with cell free extract of cultured L-1210 has revealed that PGA2 stimulated the biosynthesis of γ-glutamylcysteine synthetase (EC 6.3.2.2). Actinomycin D inhibited this stimulatory effect of PGA2 on cultured cells. The optimal pH, Km value for glutamic acid and sensitivity to inhibitors of γ-glutamylcysteine synthetase from PGA2 treated and nontreated cells were virtually the same. Thus, our findings suggest that PGA2 induced γ-glutamylcysteine synthetase in cultured L-1210 cells which is responsible for the elevated level of GSH in these cells.

Spin trapping of a free radical intermediate formed during microsomal metabolism of hydrazine

A radical formed during oxidative metabolism of hydrazine in rat liver microsomes was spin-trapped with alpha-phenyl-t-butylnitrone. The trapped species was identified as hydrazine radical by comparison of its ESR parameters and mass spectrum with those of the adduct formed during CuCl2 catalyzed oxidation of hydrazine. The requirement for oxygen and NADPH in the microsomal oxidation and the occurrence of a typical binding spectrum by difference spectroscopy suggest the involvement of the participation of the cytochrome P-450 enzyme system in the formation of hydrazine radical which must be a precursor of diimide during microsomal oxidation of hydrazine.

Glucuronidation and sulfation of p-nitrophenol in isolated rat hepatocyte subpopulations. Effects of phenobarbital and 3-methylcholanthrene pretreatment

Parenchymal cells, isolated from untreated (control), phenobarbital(PB)-or 3-methylcholanthrene(3-MC)-treated rats, were separated into four subpopulations according to cell density, and glucuronidation and sulfation of p-nitrophenol (PNP) in the hepatocyte subpopulations were investigated. PB enhanced the glucuronidation almost 2-fold but not the sulfation, while 3-MC enhanced both glucuronidation (3-fold) and sulfation (2-fold) in the original cell suspensions. Some gradation trends were found in the conjugation activities among the hepatocyte subpopulations: In the control experiment, the extent of glucuronidation in four subpopulations was virtually the same but sulfation in high-density hepatocytes was slightly higher than in low-density ones. Both glucuronidation and sulfation were higher in low-density hepatocytes from PB-treated rats, though the gradation was very modest. Glucuronidation and sulfation tended to be slightly higher in middle-density hepatocytes in the 3-MC experiment. However, no definite correlation in conjugation activities vs. cell density, like those seen in cytochrome P-450s vs. cell density in the hepatocytes isolated from PB-treated rats, were found in the subpopulations from control or inducer-treated rats. Simultaneous studies on acetylation of p-aminobenzoic acid (PABA) revealed that the activities in the subpopulations were virtually the same and the inducers had little influence on the activity.

Specific role of an α,β-unsaturated carbonyl group in γ-glutamylcysteine synthetase induction by prostaglandin A2

Abstract The effects of prostaglandins (PGs) on cellular glutathione (GSH) status in L-1210 cells were examined. PGA2 and J2, which have an α,β-unsaturated carbonyl group in the cyclopentane ring, elevated the GSH content, but PGB2, D2, E2 and F2α did not show the effect. When L-1210 cells were incubated with various 2-cyclopentenone derivatives, 4-hydroxy-2-cyclopentenone and some of related compounds elevated cellular GSH levels. Subsequent study with cell-free extract of cultured L-1210 cells revealed that PGA2 and 4-hydroxy-2-cyclopentenone induced γ-glutamylcysteine synthetase activity at the transcriptional level. This induction was also found in other cultured mammalian cells such as HeLa S3, NIH/3T3 and porcine aorta endothelial cells. When L-1210 cells were incubated with PGA2 in the presence of 4-hydroxy-2-cyclopentenone and its analogues, they inhibited the accumulation of PGA2 in cell nuclei. Our findings thus suggest that an α,β-unsaturated carbonyl moiety is responsible for enhancing the biosynthesis of γ-glutamylcysteine synthetase in cultured cells.

Characterization of the transport system of prostaglandin A2 in L-1210 murine leukemia cells.

Prostaglandin (PG) A2 has been shown to be actively incorporated into mammalian cells and transferred to the cell nucleus. To characterize the properties of the PGA2 transfer system, we examined the status of PGA2 in L-1210 cells with modified cellular glutathione (GSH) levels. PGA2 in the cytosol fraction of the cells existed in its free-form, the GSH conjugate-form and a macromolecule associate-form (protein bound-form). When the GSH level was lowered under culture conditions, the amount of free-form increased while that of the protein bound-form was unchanged. When PGA2-loaded cells were incubated in a salt solution, free- and conjugate-forms were emitted from the cells. A concomitant decrease and increase of protein bound PGA2 in cytosol and nuclei, respectively, were observed. Subsequent studies with isolated cellular fractions revealed that PGA2 bound to cytosolic protein was transported into the nuclear interior in a temperature-dependent manner. The binding of PGA2 to the protein and subsequent transport to the nuclei were inhibited by PGJ2 and 4-hydroxy-cyclopentenone, but not by PGB2, PGD2, PGE2, PGF2 alpha, arachidonic acid or oleic acid. N-Ethylmaleimide (NEM) and p-chloromercuribenzoate (PCMB) strongly interfered with these transfer processes, suggesting that sulfhydryl components are involved in the transport of PGA2. Subfractionation of cytosol by gel chromatography proved the presence of two proteins (100-150 kDa and 25-35 kDa) that are capable of transporting PGA2 to cell nuclei. Though 40-45 kDa proteins, which correspond to GSH S-transferases, bound to PGA2, they lacked the nuclear transport activities.

Induction of Cytochrome P-450 isoenzymes in cultured monkey hepatocytes

The effect of phenobarbital (PB), beta-naphthoflavone (beta-NF) and rifampicin (Rif) on the drug-metabolizing activity of cultured squirrel monkey hepatocytes was examined. The drug metabolizing activity (e.g. alkoxycoumarin dealkylase or steroid hydroxylase) gradually decreased during the culture period with 40-70% activity remaining at 72 hr. When 0.5 mM PB was added to the culture, the activities of 7-methoxycoumarin O-demethylase (MCOD) and 7-ethoxycoumarin O-deethylase (ECOD) increased to 6-7 fold higher level than those of control at 72 hr. Testosterone 6 beta-hydroxylase (6 beta-OH-T) and testosterone 16 beta-hydroxylase (16 beta-OH-T) activities were approx. 3-fold higher than those of the control. Addition of beta-NF significantly increased the activities of 7-ethoxyresorufin O-deethylase (EROD) and ECOD. Though statistically insignificant, Rif slightly increased 6 beta-OH-T activity. Western blot analysis indicated PB induced production of the CYP 2B and 3A subfamilies, while beta-NF and Rif induced that of the CYP 1A and the CYP 3A subfamily, respectively.

Protective effect of prostaglandin A2 against menadione-induced cell injury in cultured porcine aorta endothelial cells

Prostaglandin A2 (PGA2) stimulates the biosynthesis of gamma-glutamylcysteine synthetase and elevates glutathione (GSH) contents in cultured mammalian cells. To clarify the importance of gamma-glutamylcysteine synthetase induction in the defence of endothelial cells against oxidative stress, the effect of PGA2 on menadione (2-methyl-1,4-naphthoquinone)-induced cell injury was examined. Incubation of porcine aorta endothelial cells with menadione produced marked loss of cellular GSH and protein sulfhydryl groups, followed by leakage of lactic dehydrogenase (LDH) into the culture medium. The LDH leakage and modification of protein thiol was, however, completely prevented by pretreatment of the cells with PGA2. The protective effect of PGA2 was more potent than that of cysteine delivery agents such as methionine, N-acetylcysteine or 2-oxo-4-thiazolidine carboxylic acid (OTC). The results suggest that cellular GSH plays an important role in the defence against oxidative stress, and induction of gamma-glutamylcysteine synthetase is effective for protecting vascular endothelial cells.

Induction of 2-carboxybenzaldehyde reductase by phenobarbital in primary culture of rat hepatocytes

When rats were treated with phenobarbital (PB), the activity of CBA reductase, which catalyzes the conversion of 2-carboxybenzaldehyde (CBA) to 2-hydroxymethylbenzoic acid (HMB), in the liver was markedly enhanced. Likewise, addition of PB to the primary culture of rat hepatocytes increased the activity of CBA reductase. The enzyme recovered from cell lysate of cultured cells showed the same characteristics in molecular and catalytic properties as the enzyme purified from the livers of the rats treated with PB. Experiments with cycloheximide suggest that de novo synthesis of the enzyme protein is enhanced by PB in primary culture.