John J. Ch'ih

Activation and deactivation of aflatoxin B1 in isolated rat hepatocytes.

Isolated rat hepatocytes took up [3H]-aflatoxin B1 during incubation with fifty percent of the aflatoxin B1 covalantly bound to cellular macromolecules. The amount of bound-aflatoxin B1 was proportional to the medium concentration of aflatoxin B1. The specific radioactivity (pmole/mg) of aflatoxin B1 found in the DNA fraction was 20 fold greater than that associated with protein. Metyrapone (0.75 mM) inhibited significantly the uptake and binding whereas 1,2-epoxy-3,3,3-trichloropropane (0.5 mM) enhanced 2-3 fold both the uptake and binding. Glutathione (0.25 mM) reduced these processes. Results indicate that a transformation of aflatoxin B1 is catalyzed by cytochrome P-450 mixed function oxidase and aflatoxin B1-2,3-epoxide so formed is primarily deactivated by epoxide hydrolase. In the isolated hepatocyte depletion of the epoxide by glutathione apparently has an insignificant role in aflatoxin detoxication.

Effect of inhibitors of microsomal enzymes on aflatoxin B1-induced cytotoxicity and inhibition of RNA synthesis in isolated rat hepatocytes.

Previous studies conducted in this laboratory demonstrated that AFB1 activation and deactivation was effectively inhibited by metyrapone and TCPO in isolated hepatocytes. The present study was undertaken to study the toxic effect of AFB1 on hepatocyte and RNA synthesis, and to assess the influence of the inhibitors on AFB1-induced cytotoxicity and AFB1-inhibited RNA synthesis. AFB1 at 50 microM was toxic and inhibited macromolecular synthesis by greater than 70% at 180 min of incubation whereas at lower concentrations of AFB1 (0.05-10 microM) dose-and time-dependent decreases in cell viability, protein and RNA synthesis were observed. Using [3H]-AFB1 (0.1.5 microM), the uptake and covalent binding of the toxin were also dose-and time-dependent. Initial rates of these processes to reach half-maximum was found to be 0.25 microM AFB1. In cells treated with AFB1 (5 microM) and metyrapone (1.0 mM) or SKF-525A (10 microM), the cell viability was similar to the control and [3H]-uridine incorporation was significantly higher than AFB1 treated cells. AFB1 and TCPO (0.5 mM) treated cells exhibited further decreases in cell viability and RNA synthesis. Results suggest that the binding of AFB1 to DNA and impairment of transcriptional activity may lead to cell death.

Studies on the biosynthesis of the DNA polymerase of rat liver mitochondria.

Abstract The biosynthesis of mitochondrial DNA polymerase was studied by determining the extent of isotope incorporated into the partially purified enzyme which had been isolated from mitochondria labeled in both in vivo and in vitro experiments. The DNA polymerase, isolated from mitochondria incubated with 14C-leucine or 14C-mixed l -amino acids in vitro, showed no incorporation of radioactivity under conditions where the residue fraction of mitochondria (membranes) from the enzyme isolation contained a level of radioactivity which approached the specific activity of whole mitochondria. Kinetic studies carried out in vivo showed that 3H-leucine was rapidly incorporated into the residue fraction of mitochondria and at time periods as short as 2 min, had a specific activity almost equal to that of whole mitochondria. The partially purified enzyme incorporated radioactivity more slowly and only after 60 min exposure to the 3H-leucine did the enzyme have a specific radioactivity equal to that of whole mitochondria. Moreover, the incorporation of radioactivity observed in the enzyme after 60 min was prevented by the prior injection of cycloheximide, an inhibitor of microsomal incorporation. These results tentatively support the hypothesis that mitochondrial DNA polymerase, like cytochrome c, may be synthesized extramitochondrially and transported into the organelle.

The distribution and intracellular translocation of aflatoxin B1 in isolated hepatocytes

The distribution and intracellular translocation of AFB1 in various subcellular fractions was investigated in isolated hepatocytes by pulse-chase experiments. After labeling the hepatocytes with [3H]-AFB1 (14.5 nM) for 15 min, the highest concentration of [3H]-AFB1 was found in the cytosolic fraction where 66% was bound noncovalently and 1.5% covalently. The lowest concentration of [3H]-AFB1 was found in the nuclear fraction; 36% and 4.9% were bound noncovalently and covalently respectively. When the [3H]-AFB1 loaded cells were chased with unlabeled AFB1 (1 microM), the radioactivity of [3H]-AFB1 in the cell lysate and cytosolic fraction decreased in time with an apparent rate of elimination (t1/2) of 93 min and 66 min, respectively. The levels of covalently bound AFB1 increased with time and reached a maximum at 60 min in nuclei (270%), and at 120 min in mitochondria (220%) and cytosol (430%) as compared to the zero time. Only in the microsomal fraction was there no significant increase with time in covalently bound AFB1. These results suggest that the toxin after activation by the microsomal mixed function oxidases was either detoxified or transported to other cellular organelles where covalent binding of macromolecules occurred.

The in vivo disposition of aflatoxin B1 in rat liver

The disposition of a non-toxic i.p. dose of [3H]-aflatoxin B1 (0.70 micrograms/kg) in the blood, plasma, and liver was studied in male Wistar rats. Uptake into the blood, plasma, and liver was biphasic; there was an initial rapid rise (0-2 hr) followed by a second phase (2-12 hr) of a gradual increase. Most of the radioactivity in the blood was bound noncovalently to albumin. Distribution of radioactivity in the subcellular fractions of liver showed that the microsomes exhibited the highest labeling which increased over the time course; labeling of the cytosol reached a maximum at 2 hr then decreased to a new steady state, whereas the mitochondria and nuclei reached a plateau. When the content of aflatoxin B1 in the nuclear subfractions was examined, greater than 92% of the total radioactivity was found in the deoxyribonucleoprotein fraction, and 84% of this was bound noncovalently. These results suggest that aflatoxin B1 is transported from the site of injection through the blood to the liver and its subcellular and subnuclear fractions primarily in a noncovalent form.

Initial inhibition and recovery of protein synthesis in cycloheximide-treated hepatocytes

Previous studies conducted with intact rats had demonstrated that protein synthesis was reversibly inhibited by cycloheximide. Polysome aggregation occurred during inhibition with a return to normal during recovery. Suggesting that the block of translational activity involved termination and release of polypeptides. This study involving freshly isolated hepatocytes was undertaken to clarify the mechanism of the biphasic response to cycloheximide. Cycloheximide at 1 microM inhibited [3H]leucine incorporation into both cellular and secreted proteins by at least 86%, without having deleterious effects on membrane integrity as indicated by trypan blue uptake and lactate dehydrogenase (LDH) (EC 1.1.1.27) release. After removal of cycloheximide, incorporation of labeled amino acids into cellular protein and protein secreted into the medium returned to control levels. Kinetically, incorporation into secreted protein exhibited a lag of 30-45 min, indicating that a longer recovery period for restoration of proteosynthetic ability is required for membrane-bound polysomes. During the first 100 min of the recovery period, 30% of the cellular protein, which had been prelabeled during cycloheximide inhibition, was secreted into the medium; treated cells, however, secreted prelabeled protein at a lower initial rate. To elucidate the mechanism of action of cycloheximide, the content of the cytoplasmic ribonucleoprotein complexes (RPC), polysome size classes, and the distribution of radioactivity among the various ribosome classes were determined during inhibition and recovery. Larger size class polysomes (7+) were increased by cycloheximide treatment and remained increased during recovery. During inhibition, there was enhanced [3H]leucine labeling with increasing polysome size, implicating termination as the rate-limiting step, whereas during the recovery phase the labeled nascent polypeptides were removed from the ribonucleoprotein complex at a 3- to 4-fold greater rate than control, indicating an accelerated release of completed proteins.

Distribution of two mitochondrial populations in rabbit kidney cortex and medulla

Abstract Mitochondria from rabbit kidney were separated by isopycnic density centrifugation into two distinct bands with mean densities of 1.178 (M 1 ) and 1.163 (M 2 ). Cortex and medulla of rabbit kidney, separated surgically, yielded both the M 1 and M 2 population of mitochondria but in markedly different proportions. From whole kidney, M 1 fraction contained 70 to 75% of the total mitochondrial protein isolated, whereas from cortex, M 1 was 83–95% and from medulla 15 to 37% of the total mitochondrial protein. The rate of incorporation in vivo of 3 H-leucine into mitochondrial proteins from normal and renoprival kidneys indicated differences between the M 1 population and the M 2 population of the cortex and medulla. The results suggest that both the cortex and medulla of the kidney contain two distinct mitochondrial populations.

Protein synthesis in two mitochondrial populations isolated by isopycnic density centrifugation from normal and renoprival kidney

Abstract During the 48 hours following mononephrectomy, there is a proliferation of mitochondria in the remaining kidney of the rat. Mitochondria from both normal and renoprival kidneys were separated by isopycnic density centrifugation into two distinct bands with mean densities of 1.178(M1) and 1.162 (M2). Using a dual-label procedure which reduces technical errors, the rates of mitochondrial protein synthesis of the fractions were compared. At 24, 48 and 72 hours post-mononephrectomy incorporation in vivo of leucine into M1 was increased 35%, 58% and 29% and into M2 50%, 85% and 14% over control. The results indicate a gradual shift in the rate of amino acid incorporation from M2 to M1 during mitochondrial formation.

Biogenesis of mammalian mitochondria in the renoprival kidney. I. Amino acid incorporation and respiratory activity.

Abstract Changes in the yield of mitochondrial protein, in the incorporation of leucine into mitochondrial proteins, and in the respiratory activity of isolated mitochondria were determined in the remaining kidney (renoprival kidney) of the rat during the first 72 hr postmononephrectomy. At 24, 48, and 72 hr the yield of mitochondrial protein isolated from the renoprival kidney increased 13, 23, and 34%, respectively, whereas renal mass increased 9, 14, and 19%. Incorporation of [ 3 H]-leucine in vivo into total mitochondrial protein was increased 96 and 130% over control at 12 and 24 hr, respectively. Incorporation of leucine in vitro by mitochondria was increased 27% over control at 24 hr; chloroamphenicol, but not cycloheximide, inhibited the in vitro incorporation. At 24 hr, the rate of O 2 consumption (state 3) was decreased, but P O ratios, respiratory control values, and glutamic and succinic dehydrogenase activities were unaltered. The content of pyridine nucleotide, cytochromes a , a 3 , and c was decreased at this time; these activities approached normal values by 72 hr. The results suggest that during the 24 and 48 hr postmononephrectomy there is an increase in mitochondrial protein and specific changes in mitochondrial function which may be indicative of a proliferation of mitochondria in the renoprival kidney. This system should be valuable in investigating the events involved in mitochondrial biogenesis in mammalian cells.

2(3)-tert-butyl-4-hydroxyanisole inhibits oxidative metabolism of aflatoxin B1 in isolated rat hepatocytes.

Abstract Previous studies indicate that dietary administration of phenolic antioxidants, 2(3)-tert-butyl-4-hydroxyanisole (BHA) and 3,5-di-tert-butyl-4-hydroxytoluene, inhibits the carcinogenic effect of a number of chemical carcinogens including aflatoxin B1 (AFB1). Induction of hepatic enzymes, such as glutathione S-transferase, UDP-glucuronyltransferase, and epoxide hydrolase, has been shown to be responsible for the reduction of AFB1 cytotoxic and carcinogenic effects. The effect of BHA on AFB1 activation was examined in vitro utilizing isolated rat hepatocytes and liver microsomes. In hepatocytes, the total AFB1 content and bound form of AFB1 were 3.4 and 1.4 pmol/106 cells, respectively. In the cell-free microsomal activating system, 2.2 pmol were activated per mg of microsomal protein during 60 min of incubation. BHA (0.1–0.5 mM) inhibited AFB1 activation and binding in both systems in a dose-dependent manner; in hepatocytes, 90% inhibition was observed at 0.5 mM. Analyzing various AFB1 adducts, BHA (0.25 mM)-treated hepatocytes contained a significantly reduced amount of AFB1 macromolecular adducts. The antioxidant neither stimulated nor inhibited the cytosolic glutathione S-transferase and microsomal UDP-glucuronyltransferase activities. Analysis of various hydroxylated (aflatoxins M1 and Q1 (AFM, and AFQ1)) and demethylated (aflatoxin P1 (AFP1)) metabolites of AFB1 in both the conjugated and unconjugated form indicated that there was a 30–50% reduction of unconjugated AFP1, AFQ1, and AFM1, whereas AFB1 was increased 3-fold. There was no significant change of conjugated metabolites. The effect of BHA on AFB1 activation in hepatocytes was compared with that of other cytochrome P-450 inhibitors; the ED50 values of SKF 525A, BHA, and metyrapone were 9 μM, 40 μM, and 280 μM, respectively. In the cell-free microsomal system, biotransformation of AFB1 to AFP1, AFM1, and AFQ1 was also inhibited. Kinetic analysis of p-nitroanisole O-demethylase activity of rat liver microsomes demonstrated that BHA inhibited noncompetitively with an apparent Ki of 90 μM. In the absence of enzyme induction, the phenolic antioxidant, BHA, blocks the oxidative biotransformation of AFB1 in isolated hepatocytes.