Isolated hepatocytes are capable of excreting aflatoxin metabolites.

The intra- and extra-cellular concentrations of AFB1, AFM1, AFP1, AFQ1 and their conjugates were quantitatively determined after 60 min of incubation with [3H]-AFB1 (1500 pmol/10(8) cells). Comparing the total concentrations of water-soluble conjugates, the eight fold greater amounts found in the medium (718 pmol) than in the cell (86 pmol) indicate that these detoxication products were excreted soon after they were formed. When the cells were perturbed with butylated hydroxyanisole (BHA), a noncompetitive inhibitor of the mixed-function oxidases, accumulations of intracellular AFB1 and extracellular AFB1 were observed. In a cell-free microsomal system, the AFB1 was metabolized at a slower rate than in intact cells. When the activation of AFB1 is blocked, the accumulation of intracellular AFB1 and decreased internalization of AFB1 suggest that AFB1 uptake, translocation of AFB1 from site of entry to site of actions, oxidation by cytochrome P-450-dependent monoxygenase, metabolic detoxication and conjugation reactions, and excretion of water soluble metabolites are linked.

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

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/10(6) 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 (AFM1 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 microM, 40 microM, and 280 microM, 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 microM. In the absence of enzyme induction, the phenolic antioxidant, BHA, blocks the oxidative biotransformation of AFB1 in isolated hepatocytes.

Modification of protein synthetic components by aflatoxin B1.

Molecular sites of perturbation by the hepatocarcinogen aflatoxin B1 (AFB1) in the protein synthesis initiation complex were assessed using isolated hepatocytes and a cell-free activating system containing microsomes and cytoplasmic ribonucleoprotein complexes (cRPC). Ribosomal proteins showed no detectable modification by the toxin in either system. With hepatocytes, initiation factors demonstrated only slight modification by AFB1. RNAs from both hepatocytes and the cell-free system with microsomes and cRPC were modified, with poly(A)-containing RNA exhibiting at least a 5-fold higher modification than poly(A)-lacking RNA. The poly(A)-lacking RNAs were modified in the order 28S rRNA greater than 18S rRNA greater than 5-6S rRNA greater than 4S tRNA. Guanine was the target base of AFB1, but only 10% of the AFB1-GMP adducts were on guanines located in a poly(G) region. These results suggest that guanine modification in RNAs may be responsible for the observed inhibition of translational initiation by AFB1 to a greater extent than modification of either ribosomal intrinsic or associated proteins.

Inhibition of high-affinity choline uptake and acetylcholine synthesis by quinuclidinyl and hemicholinium derivatives.

Choline uptake into cholinergic neurons for acetylcholine (ACh) synthesis is by a specific, high-affinity, sodium- and temperature-dependent transport mechanism (HAChU). To assess the role of choline availability in regulation of ACh synthesis, the structure-activity relationships of several hemicholinium (HC) and quinuclidinyl analogs were evaluated in a dose response manner. As confirms previous studies, the HCs, e.g., HC-3, acetylsecohemicholinium, and HC-15 are potent inhibitors of HAChU, HC-3 being the most potent (I50 = 6.1 X 10(-8) M). In the present study, the most potent quinuclidinyl derivative was the N-methyl-3-quinuclidinone (I50 = 5.6 X 10(-7) M). This compound had approximately 100-fold greater inhibitory activity than the corresponding racemic alcohol, suggesting that the 3-hydroxyl functional group is not absolutely essential for activity. Increasing the size of the N-functional group from a methyl to an allyl in the alcohol led to a 10-fold increase in activity. However, removal of the quaternizing N-methyl group yielding the tertiary amine, 3-quinuclidinol hydrochloride, greatly reduced its capacity to inhibit HAChU. Of the 2-benzylidene-3-quinuclidinone derivatives studied, only the m-chloro derivative significantly reduced HAChU.