Frederick C. Kauffman

Biotransformation and Zonal Toxicity

Hepatotoxins are ubiquitous in nature. Chemical injury to the liver is dependent on the nature of the hepatotoxic agent and the circumstances of exposure (for a comprehensive review, see Zimmerman1). Products of plant, fungal, and bacterial metabolism, minerals,2–4 chemicals and pharmaceuticals, industrial byproducts, and waste materials can damage the liver.5 The types of hepatic injury that result from exposure to hepatotoxins are quite diverse. Some agents cause necrosis, fat accumulation, cirrhosis, or carcinoma,2 while others interfere with bile secretion, cause jaundice, and produce little or no injury to hepatocytes.2,5

Phenolic antioxidants: potent inhibitors of the (Ca2+ + Mg2+‐ATPase of sarcoplasmic reticulum

Bis(2‐hydroxy‐3‐tert‐butyl‐5‐methylphenyl)methane (bis‐phenol) is the most potent inhibitor of the (Ca2+ + Mg2+‐ATPase of skeletal muscle sarcoplasmic reticulum yet identified. The compound behaves as a reversible, tight‐binding inhibitor with apparant K i = 0.3 μM. Butylated hydroxytoluene, butylated hydroxyanisole, and 4‐nonylphenol are also effective inhibitors. These observations are of particular interest in light of the widespread use of such phenolic antioxidants and stabilizers in the food industry and in the manufacture of rubbers and plastics and the ease with which the compounds are extracted into organic solvents.

Co-regulation of the mixed-function oxidation of p-nitroanisole and glucuronidation of p-nitrophenol in the perfused rat liver by carbohydrate reserves.

Maximal rates of mixed-function oxidation of p-nitroanisole and the glucuronidation of p-nitrophenol in perfused livers from phenobarbital-treated rats varied directly with the nutritional state of the rat (i.e., fasted < fed < fasted-refed). Rates correlated with intracellular concentrations of NADPH, UDP-glucuronic acid, and glycogen but not with amounts of cytochrome P-450 or glucuronyltransferase activity. These data support the hypothesis that mixed-function oxidation and glucuronidation are coregulated in intact cells by carbohydrate-dependent cofactor synthesis.

Effects of organophosphates and nerve growth factor on muscarinic receptor binding number in rat pheochromocytoma PC12 cells

Muscarinic receptor binding in PC12 cells is influenced by both nerve growth factor (NGF) and organophosphates. Treatment of PC12 cells with a single dose of NGF (50 ng, 7S NGF/ml) increased [3H]N-methylscopolamine ([3H]-NMS) binding sites approximately two-fold at 48 hr but did not change the Kd for this ligand. Exposure of PC12 cells to soman, 50 microM, decreased [3H]-NMS binding in both undifferentiated and NGF-treated cells; however, decreases in muscarinic binding induced by the organophosphate were only minimal after the first hour after treatment and were maximal at about 24 hr. Other organophosphates including sarin, tabun, and VX as well as the carbamate, pyridostigmine, also reduced [3H]-NMS binding in PC12 cells measured 24-48 hr after treatment. The order of potency of organophosphates in lowering [3H]-NMS binding was soman greater than sarin greater than VX greater than tabun greater than DFP. High amounts of VX (200 microM) but not the other organophosphates inhibited [3H]-NMS binding when added to cells during the course of binding assays. Decreases in muscarinic receptor binding induced by the organophosphates differed markedly from that produced by carbamylcholine, which decreased [3H]-NMS binding maximally 30 min after addition to the cells. Decreases in [3H]-NMS binding produced by carbamylcholine were antagonized by atropine, but reductions in muscarinic binding produced by the organophosphates were not reversed by atropine. Thus, decreases in muscarinic receptor binding induced in PC12 cells by organophosphates occur via a novel mechanism that does not involve agonist-induced receptor desensitization.

Effect of vinblastine on neural regulation of metabolism in rat skeletal muscle

Abstract Chronic application of vinblastine, a substance known to disrupt axoplasmic flow, to nerves innervating the fast extensor digitorum longus and slow soleus muscles of the rat, produces electrophysiological signs of denervation (depolarization and extrajunctional acetylcholine sensitivity), but does not alter motor activity. We therefore examined the effects of vinblastine treatment on those metabolites and enzymes that are known to change after denervation of fast and slow skeletal muscle. A silastic cuff containing 0.1% vinblastine was placed around sciatic nerves of adult rats for 5 days. Glucose-6-P decreased 68% in the extensor, but did not change in soleus muscles. Phosphocreatine also decreased slightly, but significantly, in the extensor. Thus, intracellular levels of glucose-6-P and phosphocreatine in the extensor may be controlled, in part, by a factor (s) transported to the muscle by axoplasmic flow. Other metabolites known to change 5 days after denervation, namely glucose, glycogen, lactate, and α-ketoglutarate, were not altered in extensor and soleus muscles innervated by vinblastine-treated nerves. The activities of glucose-6-phosphate dehydrogenase and hexokinase increased in denervated extensor muscles, but not in denervated soleus muscles. Thus, metabolism in extensor muscles may be more readily altered after disruption of neural influences than is metabolism in soleus muscles. In contrast to denervation, exposure of sciatic nerves to vinblastine did not alter enzyme activities. These results provide evidence that certain metabolic processes, as well as membrane properties in skeletal muscle, are influenced by separate and distinct neural factors.

Stimulation of hepatic microsomal β-glucuronidase by calcium

Abstract Hydrolysis of 3-methylumbelliferyl glucuronide by liver microsomal β-glucuronidase is enhanced about 2-fold by micromolar concentrations of Ca2+; half-maximal stimulation occurs with 0.35 μM Ca2+. Dissociation of the enzyme from microsomal membranes by various treatments increases basal β-glucuronidase activity and markedly decreases the sensitivity of the enzyme to Ca2+. Under similar conditions, the soluble lysosomal form of the enzyme is insensitive to Ca2+. Ca2+ stimulation was unaltered by addition of calmodulin inhibitors or exogenous calmodulin. Thus, interaction of cytosolic Ca2+ with membrane bound β-glucuronidase may modulate glucuronidation in intact hepatocytes via a novel, calmodulin-independent mechanism.

Regulation of NADPH-dependent mixed-function oxidation in perfused livers: Comparative studies with sorbitol and ethanol☆

Sorbitol and ethanol were shown to have opposite effects on p-nitroanisole O-demethylation in perfused livers from fasted, phenobarbital-treated rats. Sorbitol (2 mM) stimulated drug metabolism by 50% while ethanol (20 mM) caused 80% inhibition. Both sorbitol and ethanol infusion decreased the NAD+/NADH ratio and increased fluorescence of pyridine nucleotides monitored from the liver surface; however, the NADP+/NADPH ratio was decreased by sorbitol but tended to be increased by ethanol. Stimulation of drug metabolism by sorbitol was abolished by pretreatment of fasted rats with 6-aminonicotinamide, an inhibitor of the pentose phosphate shunt, but was not affected by aminooxyacetate, a transaminase inhibitor. These results indicate that sorbitol stimulated p-nitroanisole metabolism by providing NADPH via the pentose phosphate shunt. Ethanol and sorbitol caused changes in intracellular concentrations of NADPH in livers from fasted rats which correlated directly with changes in hepatic levels of citrate and aspartate. Furthermore, aspartate infusion reduced the inhibition of p-nitroanisole O-demethylation by ethanol. This inhibition was also reversed partially by sorbitol in livers from 6-aminonicotinamide-treated rats. It is concluded that ethanol inhibits mixed-function oxidation primarily by decreasing the concentrations of citric acid cycle intermediates which leads to depletion of cytosolic NADPH.

Maintenance of the Adult Rat Superior Cervical Ganglion In Vitro: Comparison of Organ and Explant Culture Systems

Abstract: Concentrations of selected intermediates of energy metabolism whole rat superior cervical ganglia maintained in vitro by an organ culture technique were compared with values measured in small slices of this maintained under essentially the same conditions. Rates of incorporation [3H]leucine into trichloroacetic acid‐precipitable material in whole ganglia mained constant for at least 48 h: however, the oxidation‐reduction state tissue as indexed by (NAD):(NADH) ratios calculated from measured amounts of lactate and pyruvate decreased more than 50% within 3h in vitro. Ganglion explants prepared by cutting the tissue into 300‐pm transverse sections played (NAD):(NADH) ratios that were about three times greater than noted in whole ganglia maintained in vitro for the same period of time. explants contained significantly higher concentrations of pyruvate and α‐ketoglutarate than whole ganglia maintained in culture. Maintenance of vorable metabolic state may support the extensive growth of neurites seen explant cultures of superior cervical ganglia. Outgrowth of processes containing catecholamines could be detected readily in explant cultures of ganglia adult rats; however, this was somewhat slower and less consistent than growth observed in explants from neonatal rats. Outgrowth of neurites adult ganglia was minimal without the addition of Nerve Growth Factor.

Inhibition of mixed-function oxidation of p-nitroanisole in perfused rat liver by 2,4-dinitrophenol☆

Abstract The effect of dinitrophenol (52 μ m ), an uncoupler of oxidative phosphorylation, on p-nitroanisole O-demethylation in the perfused rat liver was examined. Dinitrophenol inhibited p-nitroanisole metabolism 70% in perfused livers from fasted, phenobarbital-treated rats, and 30% in livers from normal rats, but had no effect on this reaction in isolated microsomes. Rates of p-nitroanisole O-demethylation in livers from fed, phenobarbitaltreated rats were not inhibited by dinitrophenol unless the pentose phosphate shunt was first inhibited by 6-aminonicotinamide pretreatment. Dinitrophenol diminished cellular concentrations of ATP and NADPH 30 and 50%, respectively. Since mixed-function oxidation requires NADPH, these data are consistent with the hypothesis that dinitrophenol interrupts the synthesis and/or transfer of reducing equivalents from the mitochondria into the extramitochondrial space by interfering with energy-dependent NADPH synthesis and substrate shuttle mechanisms. In addition, dinitrophenol diminished conjugation reactions 57 and 89% in all metabolic states studied, most likely because it decreased UDP-glucose levels considerably (40 to 60%).

Physiological, biochemical and histological changes in skeletal muscle, neuromuscular junction and spinal cord of rats rendered paraplegic by subarachnoidal administration of 6-aminonicotinamide

Summary Exposure of rats to the nicotinamide analog, 6-aminonicotinamide, produces an irreversible rigid paralysis of the hind limbs. The present study describes electrophysiological alterations that occur in extensor digitorum longus (extensor) and soleus muscles of rats treated with a single subarachnoid injection of 6-aminonicotinamide and compares these changes with cellular and biochemical changes that occur in the spinal cord. Injection of 50 μ of 6-aminonicotinamide into the subarachnoid space at L2–L4 of adult female rats produced rigid paralysis of both hind limbs in 24–36 h. In extensor muscles, the membrane potential decreased significantly about 48 h after injection, but this partial depolarization took somewhat longer in the soleus muscles. Spontaneous transmitter release, as studied by frequency and amplitude of miniature endplate potentials, was significantly affected from day 7 after injection. At the onset of paralysis, 6-phosphogluconate levels were increased more than 1000-fold in the lumbar region of the spinal cord and returned to control levels by 14 days after injection. This metabolite thus serves as a biochemical marker for the presence of the 6-aminonicotinamide analog of NADP in tissues. Beginning at 3 days an increase in small phagocytic cells was noted in both the cervical and lumbar regions of the cord. These proliferating cells were highly localized to the anterior horn region where maximal numbers were detected at 8 days. Few viable motor neurons were present in the lumbar region of the cord at this time. There was a close correlation between the accumulation of small cells and the increased β-glucuronidase activity. Despite the marked cellular alterations in the spinal cord at 8 days there were no changes in the total extractable lipid in gray and white matter. Cellular changes were maintained in the lumbar region for as long as 540 days. At this time most of the electrophysiological properties of the extensor muscle were restored to normal, suggesting that collateral sprouts from the residual motor neurons may have reinnervated the denervated muscle fibers.