A variant of ornithine aminotransferase from mouse small intestine.

The ornithine aminotransferase (OAT) activity of mouse was found to be highest in the small intestine. The mitochondrial OAT from mouse small intestine was purified to homogeneity by the procedures including heart treatment, ammonium sulfate fractionation, octyl-Sepharose chromatography, and Sephadex G-150 gel filtration. Comparing to the amino acid sequence of mouse hepatic OAT, six N-terminal amino acid residues have been deleted in intestinal OAT. However, the subsequent sequence was identical with that of hepatic OAT. The molecular weights of both intestinal and hepatic OAT were estimated as 46 kDa by SDS-gel electrophoresis and as 92 kDa by gel filtration, indicating that both native OATs are dimeric. Biochemical properties of intestinal OAT, such as molecular weight, pH optimum and K(m) values for L-ornithine and alpha-ketoglutarate, were similar to those of hepatic OAT. However, intestinal OAT was more labile than hepatic OAT to tryptic digestion.

Glycosylation, dimerization, and heparin affinity of lipoprotein lipase in 3T3-L1 adipocytes.

The relationship between glycosylation, dimerization, and heparin affinity of lipoprotein lipase (LPL) was studied in 3T3-L1 adipocytes. Three forms of LPL subunits were found in normal cells; totally endo H-resistant (57 kDa), partially sensitive (54 kDa), and totally sensitive (51 kDa) forms. LPL in normal cells was active, dimeric, and showed high affinity for heparin. LPL in cells treated with tunicamycin, preventing the transfer of N-linked oligosaccharide chain, was unglycosylated (51 kDa) and inactive. LPL proteins were found as an aggregate, and had low affinity for heparin. After treatment with castanospermine, an inhibitor of ER glucosidase I, 80% of LPL activity was inhibited. Most of LPL proteins were totally endo H-sensitive, present as an aggregate, and had low affinity for heparin. LPL in cells treated with deoxymannojirimycin, an inhibitor of Golgi mannosidase I, was active, dimeric, and had high affinity for heparin as in normal cells. But LPL subunits were all endo H-sensitive. These results suggest that core glycosylation and subsequent removal of glucose residue is required, but processing after Golgi mannosidase I is not necessary for dimerization and acquisition of high heparin affinity of LPL.

N-nitroso compounds in two nitrosated food products in southwest Korea.

Gastric cancer is the commonest malignant neoplasm in Southwest Korea. The possibility of carcinogenic dietary factors led to the investigation of exposure to N-nitroso compound precursors among residents of the City of Chonju and of two outlying rural townships in North Cholla Province. Two traditional and widely consumed home-prepared food products, salted pickled cabbage (kimchi) and salted seafood sauce (chut-kal) were analysed (a) for nitrite, nitrate, total secondary amines and pH in these food products prior to nitrite incubation and (b) for volatile nitrosamines and total N-nitroso compounds before and after incubation with nitrite in simulated human stomach conditions. Nitrate levels were significantly higher in kimchi (median 1550 mg/kg) than in chut-kal (median 140 mg/kg) (P < 0.001). Secondary amine levels in non-nitrosated samples of kimchi (median 5.5 mg/kg) were significantly lower than secondary amine levels in non-nitrosated chut-kal (median 56 mg/kg) (P = < 0.001). Analyses of nitrite-incubated kimchi revealed high levels of total N-nitroso compounds (median 1173 micrograms/kg); the increase with nitrosation was significant (P = 0.001). The concentration of N-nitroso compounds in nitrite-incubated kimchi was significantly greater than that found in nitrite-incubated chut-kal (P = 0.015). The combination of high levels of nitrate in the kimchi, the demonstration of high levels of total N-nitroso compounds in this food after nitrosation, and the volume of kimchi consumed in the traditional diet suggest that salted pickled cabbage may play a role in gastric carcinogenesis in Southwest Korea.

Aflatoxin B1-DNA binding and aflatoxin B1-glutathione conjugation with isolated hepatocytes from rats and hamsters.

Binding of aflatoxin B1 (AFB1) to DNA and AFB1-glutathione conjugation during the metabolism of AFB1 have been examined with freshly isolated hepatocytes from male Fischer rats and Syrian hamsters. Even though there was no significant difference in cytochrome P450 and glutathione contents, there were marked differences in the metabolism of AFB1 (33 nM) in hepatocytes from these two species. Thus, AFB1-DNA binding was six-fold higher in the rat than in hamster hepatocytes, whereas AFB1-glutathione conjugation was 12-fold higher in hamster than in rat hepatocytes. The addition of 0.5 mM diethylmaleate had no significant effect in rats, whereas its presence produced a nine-fold increase in AFB1-DNA binding with 85% inhibition of thiol conjugation in hamster hepatocytes. Styrene oxide (1 mM) produced 50% and 25-fold increases in AFB1-DNA binding in rat and hamster hepatocytes, respectively, with corresponding decreases in thiol conjugation. Triethyltin bromide (50 microM) inhibited both processes by 50% in rat hepatocytes, whereas it produced a nine-fold increase in AFB1-DNA binding with a concomitant decrease in thiol conjugation in hamster hepatocytes. These results suggest that glutathione S-transferases play a more significant role in modulating AFB1-DNA binding in hamster than in rat hepatocytes.

Effect of butylated hydroxyanisole pretreatment on aflatoxin B1-DNA binding and aflatoxin B1-glutathione conjugation in isolated hepatocytes from rats.

The effect of 2(3)-tert-butyl-4-hydroxyanisole (BHA) pretreatment of rats on both aflatoxin B1 (AFB1)-DNA binding and AFB1-glutathione has been examined with isolated hepatocytes and in intact rats. Young male F344 rats were fed AIN-76A diet with or without 0.75% BHA for 2 weeks. Even though there were no significant differences in either cytochrome P-450 or reduced glutathione contents, there were marked differences in AFB1 metabolism in isolated hepatocytes from these two groups. Thus, at the 33 nM AFB1 level, AFB1-DNA binding was 3-fold higher in control compared to BHA-treated hepatocytes whereas AFB1-glutathione conjugation was 5-fold higher in treated compared to controls. Even at higher AFB1 concentrations (2 and 10 microM), DNA binding was 4-6-fold higher in controls whereas thiol conjugation was 5-9-fold higher in treated compared to control hepatocytes. Addition of 0.5-1.0 mM diethylmaleate did not have any significant effect in control hepatocytes whereas its presence produced about 70-100% increase in DNA binding with 65-80% inhibition of thiol conjugation in treated hepatocytes. Addition of 1 mM styrene oxide caused 75-100% and 4-8-fold increase in AFB1-DNA binding in control and treated hepatocytes, respectively, with corresponding decreases in thiol conjugation. In intact rats, BHA treatment reduced hepatic AFB1-DNA binding to 15% of controls with concomitant increase in biliary excretion of AFB1-reduced glutathione conjugate. It appears that the induced cytosolic GSH S-transferases after BHA treatment of rats play a significant role in inhibiting hepatic AFB1-DNA binding and AFB1 hepatocarcinogenesis presumably by inactivation of the reactive AFB1-epoxide.

A mechanism of inhibition of aflatoxin B1-DNA binding in the liver by phenobarbital pretreatment of rats.

The effect of phenobarbital (PB) pretreatment of rats on both hepatic aflatoxin B1 (AFB1)-DNA binding and AFB1-glutathione (AFB1-SG) conjugation have been examined in studies in vivo and in vitro. Male Sprague-Dawley rats fed a commercial diet with 0.1% PB in their drinking water for 1 week had total wet liver weight and microsomal protein content about 27% and 38% higher, respectively, than controls. Hepatic cytochrome P-450 content, microsomal cytochrome P-450 mediated AFB1 binding to exogenous DNA and formation of hydroxy metabolites of AFB1 were also about threefold higher in PB-treated rats and cytosolic reduced glutathione S-transferase activities were about doubled. Microsome-mediated AFB1-DNA binding, when examined at 2 microM and 10 microM levels of AFB1, was inhibited two-to threefold more by cytosols of treated rats whereas AFB1-SG conjugation was two- to threefold higher by cytosols of treated rats. In reconstitution experiments with 2 microM AFB1, with intact nuclei serving as a source of endogenous DNA, addition of microsomes from either group generated a large amount of AFB1-DNA binding (68-105 pmol) and a smaller amount of AFB1-SG conjugate (12-21 pmol). The presence of cytosol from the controls reduced AFB1-DNA binding to a much lesser extent than the cytosol from the treated group whereas AFB1-SG conjugation was much higher with the cytosol from the treated group. These results are in agreement with the studies in vivo. In isolated hepatocytes at 33 nM, 2 microM and 10 microM AFB1 levels, AFB1-DNA binding was decreased 50 to 70% by prior PB-treatment whereas AFB1-SG conjugation was two- to threefold higher in treated compared to control hepatocytes. In hepatocytes, addition of 1 mM diethylmaleate increased DNA binding two- to threefold with a corresponding decrease in AFB1-SG conjugation. Addition of 1 mM styrene oxide caused 5- to 10-fold increases in AFB1-DNA binding at levels of AFB1 of 33 nM and 2 microM; but at 10 microM AFB1, increases in AFB1-DNA binding were two- to threefold. In intact rats, PB treatment reduced hepatic AFB1-DNA binding to 30% of controls with concomitant increase in biliary excretion of AFB1-SG conjugate. It appears that the induced cytosolic GSH S-transferases after PB treatment of rats plays a significant role in inhibiting hepatic AFB1-DNA binding and hepatocarcinogenesis presumably by inactivation of the reactive AFB1-epoxide.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of butylated hydroxyanisole pretreatment on in vitro hepatic aflatoxin B1-DNA binding and aflatoxin B1-glutathione conjugation in rats.

The effect of 3(2)-tert-butyl-4-hydroxyanisole (BHA) pretreatment of rats on both in vitro hepatic aflatoxin B1 (AFB1)-DNA binding and AFB1-glutathione (AFB1-SG) conjugation has been examined. For these studies, young male F344 rats were fed AIN-76 A diet with or without 0.75% BHA for 2 weeks. There were no significant differences either in microsomal cytochrome P-450 content or microsome-mediated exogenous DNA binding to AFB1 with cytochrome P-450 from control or BHA-treated animals. There were large differences in reduced glutathione S-transferase activity with treated cytosols showing 2.5-fold higher activity than the controls. Hepatic reduced glutathione levels were 25% higher in treated than in controls. Kinetics of cytosolic inhibition of microsome-mediated AFB1-DNA binding and formation of AFB1-SG conjugate when examined at two levels of AFB1 (2 and 10 microM) and a 4-fold range of cytosolic concentrations showed that inhibition of AFB1-DNA binding was greater with cytosol from the treated compared to the controls. However, AFB1-SG conjugation was 3- to 4-fold greater in treated than in controls. Inhibition of AFB1-DNA binding by cytosol was reversed in the presence of 1 mM level of various epoxides with concomitant inhibition of AFB1-SG conjugation. In reconstitution studies with 2 microM AFB1, intact nuclei alone from either group did not yield significant amounts of either DNA binding or AFB1-SG conjugation. However, addition of microsomes from either group to these nuclei generated a large amount of AFB1-DNA binding (82-111 pmol) and a smaller amount of AFB1-SG conjugate (9-28 pmol). The presence of cytosols from the control group reduced AFB1-DNA binding to a much lesser extent than the cytosols from the treated group. However, AFB1-SG conjugation was much higher with the cytosol from treated than with the controls. These reconstitution studies with endogenous DNA show more AFB1-DNA binding with the control than with BHA-treated animals and are in agreement with the studies in vivo. It appears that induced levels of cytosolic reduced glutathione S-transferase modulate AFB1-DNA binding and AFB1 hepatocarcinogenesis.

Effect of butylated hydroxyanisole on in vivo and in vitro hepatic aflatoxin B1-DNA binding in rats.

Butylated hydroxyanisole (BHA) pretreatment of male rats has been examined for its effect on in vivo and in vitro hepatic aflatoxin B1-DNA binding (AFB1-DNA) in these animals. No difference either in cytochrome P-450 content or microsome-mediated AFB1-DNA was observed between livers from control and treated rats. However, cytosols from treated animals showed severalfold more inhibition of microsome mediated AFB1 binding to either exogenous or endogenous DNA than cytosols from controls. Presence of 1 mM level of either trichloropropene oxide or styrene oxide partially reversed the cytosolic inhibition of binding. Intraperitoneal administration of AFB1 2h before killing produced 50% less AFB1 binding to nuclear DNA in treated than in control animals. The role of induced glutathione S-transferases in treated rats in modulating hepatic AFB1-DNA binding is discussed.

Modulation of microsome-mediated aflatoxin B1 binding to exogenous and endogenous DNA by cytosolic glutathione S-transferases in rat and hamster livers.

Microsome mediated aflatoxin B1 (AFB1) binding to exogenous and endogenous DNA and its modulation by cytosolic glutathione (GSH) S-transferases have been examined in rat and hamster livers. Kinetic studies over a wide range of cytosol concentrations indicate that cytosol from the hamster is several-fold more effective than that from the rat in inhibiting AFB1 binding to exogenous calf thymus DNA mediated by microsomes from either species. Low concentrations of GSH (0.1-0.2 mM) are required for 50% inhibition of AFB1-DNA binding by cytosol. With exogenous DNA, combined microsome-cytosol fractions from the hamster give more AFB1-DNA binding than those from the rat. However, with nuclei as a source of endogenous DNA, AFB1-DNA binding is less with combined microsome-cytosol fractions from the hamster than those from the rat. Cytosolic inhibition of AFB1-DNA binding is almost completely reversed in the presence of 1 mM levels of either trichloropropene oxide or styrene oxide. Quantitation of AFB1-DNA binding and AFB1-GSH conjugation indicate an inverse relationship between these two processes. Cytosol from the rat has less capacity than that from the hamster to form an AFB1-GSH conjugate. Hepatic GSH levels are about equal (6-7 mM) in both species. I.p. administration of [14C]AFB1 2 h before sacrifice gives more AFB1 binding to hepatic nuclear DNA in rats than in hamsters. However, depletion of hepatic GSH levels by 80% by i.p. administration of diethylmaleate (600 mg/kg) increases AFB1-DNA binding 2- to 3-fold in both species. The role of cytosolic GSH S-transferases in modulating hepatic AFB1-DNA binding in rats and hamsters is discussed.

Inhibition of microsome-mediated binding of aflatoxin B1 to DNA by glutathione S-transferase.

Hamster liver microsome-mediated [14C]aflatoxin B1 binding to DNA is inhibited by 105,000 X g liver supernatant fraction. Absolute requirement of glutathione for the inhibitory activity of the dialyzed supernatant and irreversible loss of the inhibitory activity by the heat-treated supernatant indicate involvement of glutathione S-transferase. Concomitant with inhibition of aflatoxin B1-DNA binding, the formation of an aflatoxin B1-glutathione conjugate is indicated.