Biochemical mechanism of resistance of cultured sarcoma 180 cells to 6-thioguanine

A 6-thioguanine (6TG) resistant clone (S180/TG) of the murine neoplastic cell line Sarcoma 180 (S180) synthesized significantly less 6-thioguanosine 5′-monophosphate (6TGMP) than parent sensitive S180 cells when exposed to 6TG in culture. The decrease in the intracellular level of 6TGMP in S180/TG cells did not appear to be due to a change in the (a) rate of uptake of 6TG (or guanine), (b) synthesis and accumulation of 5-phosphoribosyl 1-pyrophosphate (PRPP) from glucose, (c) capacity to enzymatically convert 6TG (or guanine) to its corresponding 5′-mononucleotide in the presence of exogenous PRPP, or (d) rate of hydrolysis of phosphate groups from newly synthesized 6TGMP. Contrasting with the decreased conversion of 6TG to 6TGMP by S180/TG cells was the finding of significantly more guanine conversion to guanine nucleotides by intact S180/TG cells, a phenomenon that resulted in approximately 4- and 2-fold greater incorporation of [8-¹⁴C]guanine into RNA and DNA, respectively, in resistant cells than in the parent sensitive subline. The findings suggest that the mechanism by which S180/TG cells achieve insensitivity to the 6-thiopurine was through decreased synthesis of analog nucleotide; this phenomenon appears to be the result of altered purine metabolism in resistant cells which selectively permits the conversion of guanine to guanine nucleotides, while suppressing the anabolism of 6TG to 6TGMP.     

Selectivity of 1-phenylimidazole as a ligand for cytochrome P-450 and as an inhibitor of microsomal oxidation

Equilibrium dialysis studies established that 1-[4′-(³H)-phenyl]imidazole (PI) was bound to hepatic microsomal suspensions from control, phenobarbital (PB)- and 3-methylcholanthrene (3MC)-treated rats and that the binding was directly related to the cytochrome P-450 content. Computer-assisted Scatchard plot analysis of the binding data indicated the existence of two major types of microsomal binding sites in both control and induced rats, one with a high affinity (K_a ～ 1.5 × 10⁷ M^?1) and the other with a low affinity (K_a ～ 5 × 10⁵ M^?1) for PI. The binding of PI to the highly purified, individual cytochrome P-450s that constituted the major forms from the PB- and β-naphthoflavone (βNF)-induced rats exhibited affinities similar to the high and low affinity binding sites observed in microsomal suspensions. The two types of PI binding sites were characteristics of two classes of cytochrome P-450, and the major cytochrome induced by PB and 3MC (or βNF) were each associated with one of these two classes. In concurrence with this, it was shown that, although PI was an excellent inhibitor of aromatic hydrocarbon hydroxylase (AHH) activity in PB-induced rats, it exhibited little or no inhibitory activity towards AHH activity in 3MC-induced animals.     

Acetaminophen hepatotoxicity. An alternative mechanism

Alcohol-fed hamsters were used to study the mechanism by which acetaminophen initiates hepatotoxicity. Animals maintained on an ethanol-containing diet (Group B) exhibited an increased mortality rate after administration of acetaminophen (400 mg/kg) as compared to control hamsters (Group A). However, in those animals in which the ethanol-containing diet had been replaced by the control diet 24 hr before receiving acetaminophen (Group C), significant protection against acetaminophen toxicity was observed as compared to control animals (Group A). This observation correlates well with the finding that Group C hamsters had higher levels of glutathione and catalase than was found in either Group A or Group B animals. It was also demonstrated that acetaminophen was oxidized by cytochrome P-450, producing acetaminophen free radical and hydrogen peroxide. The free radical in the presence of oxygen was found to generate superoxide and presumably N-acetyl-p-benzoquinone imine. Microsomal lipid peroxidation was found to be stimulated markedly in the presence of acetaminophen. The role of glutathione in protecting hamsters from acetaminophen-mediated hepatotoxicity is discussed.     

Influence of hypoxia on the metabolism and excretion of misonidazole by the isolated perfused rat liver--a model system

The isolated perfused rat liver was evaluated as a model system for the characterization of misonidazole metabolism under hypoxic conditions. Misonidazole metabolism by livers perfused under aerobic conditions was also examined. The clearance of misonidazole was more than three times greater under anaerobic compared to aerobic conditions (4.94 +/- 1.56 vs 1.27 +/- 0.22 ml/min; means +/- S.D., N = 3). Misonidazole metabolites were detected only in the bile. Analysis of these metabolites by reverse-phase high performance liquid chromatography (HPLC) demonstrated that misonidazole metabolism was also qualitatively changed when anaerobic conditions were employed. Misonidazole beta-glucuronide was the major metabolite detected under aerobic conditions, but it was a minor metabolite in anaerobically perfused livers. The three major metabolites produced under anaerobic conditions were not characterized, but desmethyl misonidazole (RO-07-9963) and the 2-amino-imidazole derivative of misonidazole (1-[2-aminoimidazol-1-yl]-3-methoxy-2-propanol) were excluded as possible structures.     

Inorganic mercury secretion into bile as a low molecular weight complex

Previous studies on the biliary secretion of inorganic mercury have concluded that inorganic mercury in bile is bound almost exclusively to substances of high molecular weight (HMW). In contrast, our results showed that inorganic mercury in bile is bound predominantly to a substance of low molecular weight (LMW), which is most likely glutathione (GSH). The previously reported binding of inorganic mercury to bile proteins is now explained as a postsecretory in vitro artifact resulting from the rapid oxidation of endogenous GSH which occurs during the collection and storage of bile samples. Gel filtration on Sephadex G-75 of freshly collected bile from rats treated with 203HgCl2 or of control bile supplemented in vitro with 203HgCl2 showed that most of the mercury was in the LMW fraction. On Sephadex G-25, the biliary mercury peak co-eluted with the mercury-GSH standard. However, when bile was allowed to stand at room temperature, there was a time-dependent shift of the mercury towards the HMW fraction. The rate of this shift was proportional to the rate of oxidation of GSH in bile. When GSH oxidation was inhibited by collecting bile in EDTA at 4 degrees, the mercury remained associated with the LMW fraction. At a given GSH concentration in bile, the fraction of mercury bound to the HMW fraction was independent of mercury concentration, in the range of 0.05 to 5.0 microM HgCl2. These results suggest that the inorganic mercury was secreted into bile complexed with a LMW substance. This LMW substance has been tentatively identified as GSH.     

Effects of hypoxia on atrial muscarinic cholinergic receptors and cardiac parasympathetic responsiveness

Chronic exposure of rats to hypoxia resulted in a lower resting heart rate and a supranormal increase in heart rate in response to parasympathetic blockade by atropine. The density of muscarinic cholinergic receptors labeled by the antagonist [³H]quinuclidinyl benzilate was elevated significantly in the atria of animals kept hypoxic for 2–4 weeks. Chronic hypoxia did not change the affinity of the receptor for [³H]quinuclidinyl benzilate, the weight of the atria, or the amount of protein per atrial pair. Thus, the decrease in resting heart rate may be explained by the increase in the density of atrial muscarinic cholinergic receptors.     

On the mechanism of butylated hydroxytoluene-induced hepatic toxicity in rats

The relations between serum transaminase activity and the hepatic contents of glutathione and lipid peroxide were examined following oral administration to rats of butylated hydroxytoluene (BHT; 500 or 1000 mg/kg). The glutathione level rapidly diminished and reached a minimum at 6 hr after BHT administration. The period of depletion was dependent on dose: restoration of the glutathione level took longer in high-dose rats than in low-dose rats. The content of hepatic lipid peroxide was not markedly changed by BHT throughout the experimental period. The activity of glutathione S-transferase was not affected until 12 hr after BHT administration but, thereafter, it increased with time and was accompanied by elevation of the glutathione level. Though the activities of serum glutamate-oxaloacetate transsminase and glutamate-pyruvate transaminase were not affected by low-dose BHT, they increased rapidly in the high-dose rates after a lag period of about 6 hr and reached a maximum at 24 hr after administration; at that time, the livers of the high-dose rats showed centrilobular necrosis. The results indicate that acute hepatic injury was induced by the high-dose BHT. Pretreatment with cobaltous chloride inhibited the increase in the activities of the serum transaminases produced by the high-dose of BHT accompanying the depletion of microsomal cytochrome P-450 content and the induction of glutathione content. These observations suggest that hepatic damage was associated with prolonged depletion of glutathione rather than with lipid peroxidation in the liver, and that the activated metabolites of BHT rather than the parent compound induced the tissue damage.     

Characterization of eight biogenic indoleamines as substrates for type A and type B monoamine oxidase

Tryptamine, N-methyltryptamine, N,N-dimethyltryptamine, 5-hydroxytryptamine (5-HT), 5-hydroxy-N-methyltryptamine, bufotenine, 5-methoxytryptamine, and 5-methoxy-N,N-dimethyltryptamine were characterized as substrates for type A and type B monoamine oxidase (MAO) in rat liver mitochondria. Experiments on sensitivity to clorgyline and to deprenyl, using two substrate concentrations, showed that tryptamine and its N-methylated and N,N-dimethylated derivatives were common substrates for both types of MAO at a substrate concentration of 20.0 μM; at 1000 μM, tryptamine and N-methyltryptamine were common, but N,N-dimethyltryptamine became specific for type B MAO. All the 5-hydroxy- or 5-methoxy-indole derivatives were almost completely specific for type A MAO at a substrate concentration of 20.0 μM; when the concentration was 1000 μM, some of the MAO activity was due to type B MAO for 5-HT, bufotenine and 5-methoxytryptamine. The rat liver mitochondrial enzyme was pretreated with 10^?7M clorgyline and 10^?7M deprenyl to obtain, respectively, the type B-rich and the type A-rich enzyme. These enzyme preparations were subjected to kinetic analyses for the eight amines. From the kinetic analyses, together with data on inhibitor sensitivity, the following phenomena can be described. N-Methylation of tryptamine or of 5-HT did not cause appreciable changes in the specificity of the substrates toward each type of MAO, but it elevated the K_mvalue of type B MAO when the values for tryptamine and N-methyltryptamine were compared. N,N-Dimethylation of tryptamine and 5-HT tended to increase the specificity for type B MAO. All the dimethylated compounds had very low activities with either type A or type B MAO. Either the 5-hydroxy- or the 5-methoxy-group contributed to the specificity of the substrates for type A MAO.     

Spectrophotometric assay for mammalian cytosolic epoxide hydrolase using trans-stilbene oxide as the substrate

A continuous spectrophotometric assay based on the differences in the ultraviolet spectra of trans-stilbene oxide (TSO) and its reaction product l,2-diphenyl-l,2-ethanediol is described for the measurement of mammalian cytosolic epoxide hydrolase activity. Rates of TSO hydration determined by this method were similar to those obtained by independent analytical methods, and the assay is rapid, reproducible and relatively free from interference.     

Phospholipase inhibition and the mechanism of angiotensin-induced prostacyclin release from rat mesenteric vasculature

Generation of prostaglandins by arterial vasculature of rats was measured by perfusing the isolated mesenteric arterial vascular bed with Krebs-Henseleit solution. The effluent directly superfused bioassay tissues in cascade, and aliquots were collected for subsequent chromatography and radioimmunoassay. Injection of arachidonate (1–10 μg) or angiotensin II (0.1–0.5 μg) through the mesentery caused release of a PGI₂-like substance. After extraction and Chromatographic separation of the mesenteric effluent, it was confirmed by radioimmunoassay that 6-oxo-PGF_1α (the hydration production of PGI₂) is the predominant prostanoid generated from exogenous arachidonate. Release of 6-oxo PGF_1α and PGE₂ from mesentery was also stimulated by injection of angiotensin II (0.05–0.5 μg). Treatment of the mesentery with indomethacin (1 μg/ml) abolished all effects of angiotensin II and arachidonate. Perfusion of the mesentery with dexamethasone (3 μg/ml) or mepacrine (33 μg/ml) both of which have been reported to inhibit phospholipase A₂ activity, reduced PGI₂ release induced by angiotensin II, but did not affect conversion of exogenous arachidonate. It is concluded that PGI₂ is the major prostanoid generated in perfused mesenteric arterial vasculature of rats, and angiotensin II releases PGI₂ by activation of a phospholipase.     

Characterization of N-methylphenylethylamine and N-methylphenylethanolamine as substrates for type A and type B monoamine oxidase

N-Methylphenylethylamine (MPEA) and N-methylphenylethanolamine (MPEOA) were characterized as substrates for type A and type B monoamine oxidase (MAO) in rat brain mitochondria. The inhibition experiments with clorgyline and deprenyl showed that the inhibition patterns with MPEA as substrate were dependent on substrate concentrations but that this amine was a common substrate for both types of MAO at all substrate concentrations tested. When MPEOA was used as substrate, the inhibition patterns differed markedly at different substrate concentrations; at 10.0 /smM, MPEOA acted as a specific substrate for type B MAO, but at 100 and 1000, μM it became a common substrate for both types. Kinetic analyses were carried out for MPEA and MPEOA with the uninhibited, the clorgyline-treated (type B MAO), and the deprenyl-treated enzyme (type A MAO). With the uninhibited enzyme, there were downward deflections in the curves of Lineweaver-Burk plots for both MPEA and MPEOA, suggesting the existence of different affinity components derived from type A and type B MAO. By means of the double-reciprocal plots, using the clorgyline- and the deprenyl-treated enzyme, it was confirmed that the high affinity corresponded to that for type B MAO and the low affinity to that for type A MAO for both MPEA and MPEOA. Therefore, the changes in the inhibition pattern at different substrate concentrations may be due to different affinities of the substrate for both types. By comparing the K_m and V_max values of both types observed for MPEA and MPEOA, it was pointed out that the β-hydroxylation of MPEA tended to increase the K_m value for type A MAO and to decrease the V_max values for both types.     

Further studies of stereospecificity at carbon 6 for membrane transport of tetrahydrofolates: Diastereoisomers of 5-methyltetrahydrofolates as competitive inhibitors of transport of methotrexate in L1210 cells

The unnatural d diastereoisomer at carbon 6 of 5-methyltetrahydrofolate was only slightly less effective than the natural l diastereoisomer as a competitive inhibitor of the carrier-mediated membrane transport of [³H]methotrexate into L1210 murine leukemia cells. The apparent K_i for a mixture containing equal amounts of both natural and unnatural diastereoisomers was not significantly different from that found for the unnatural form. These results show that the reduced folate carrier system in these cells has a strong affinity for the unnatural stereoisomer, a finding in contrast to that obtained with the corresponding diastereoisomer of 5-formyltetrahydrofolate.     

Changes in apomorphine-induced stereotypy as a result of subacute neuroleptic treatment correlates with increased D-2 receptors, but not with increases in D-1 receptors

Administration of haloperidol (5 mg/kg i.p.), cis-flupenthixol (2.5 mg/kg i.p.) or sulpiride (2 X 100 mg/kg i.p.) daily for 21 days followed by a 3-day drug withdrawal period caused equivalent cerebral dopamine receptor supersensitivity as judged by enhanced apomorphine-induced stereotypy. These treatments also produced equivalent rises in the number of adenylate cyclase-independent dopamine receptors (D-2) in both striatal and mesolimbic tissue as assessed by specific [3H]spiperone and [3H]N,n-propylnorapomorphine (NPA) binding. No change in the dissociation constant (KD) was apparent in response to neuroleptic treatment. However, only repeated administration of cis-flupenthixol caused an increase in the number of adenylate cyclase-linked dopamine receptors (D-1) in striatum as assessed by enhanced [3H]piflutixol binding and increased dopamine-stimulated cyclic AMP formation. The dissociation constant for [3H]piflutixol binding was unchanged by cis-flupenthixol administration. No change in D-1 receptor numbers or dopamine stimulation of adenylate cyclase occurred in mesolimbic tissue. Repeated treatment with sulpiride or haloperidol was without effect on either [3H]piflutixol binding to D-1 receptors or cyclic AMP formation. In conclusion, increased apomorphine-induced stereotypy following subacute neuroleptic treatment correlates with changes in D-2 receptor numbers, but not with changes in D-1 receptors.     

Erythromycin binding to human serum

Erythromycin binding to human serum was measured under conditions of binding equilibrium. The binding is sensitive to pH changes, decreasing at acid pH. Over a great range of serum dilution, the bound fraction is semilogarithmically related to serum concentration. Binding is shown to be completely reversible. With increasing erythromycin concentration a specific part of binding is saturable and specifically displaceable by erythromycin in excess, whereas a nonspecific part linearly increases with total concentration. Erythromycin is specifically bound to a single class of noninteracting binding sites with an apparent dissociation constant K_d=5.9 μM (38°C). The kinetic and thermodynamic parameters at 25° are: K_d = 8.4 μM, ΔH° = +4.4 × 10³ cal per mole, ΔG° = ?6.9 × 10³ cal per mole, ΔS°= +38 e.u.