Effects of anti-NADPH-cytochrome c reductase and anti-cytochrome b5 antibodies on the hepatic and pulmonary microsomal metabolism and covalent binding of the pulmonary toxin 4-ipomeanol.

An antibody prepared against purified rat liver NADPH-cytochrome $\text{c}$ reductase inhibited both the pulmonary and hepatic microsomal covalent binding of 4-ipomeanol as well as the respective NADPH-cytochrome $\text{c}$ reductase activities, findings which are consistent with previous studies which indicated the participation of cytochrome P450 in the metabolic activation of the toxin. An antibody prepared against purified rat liver cytochrome b₅, which strongly inhibited both the rat hepatic and pulmonary NADH-dependent cytochrome $\text{c}$ reductases, and was inactive against the respective NADPH-dependent cytochrome $\text{c}$ reductases, had little effect on metabolic activation of 4-ipomeanol by hepatic microsomes, but strongly inhibited both the NADH-supported and the NADPH-supported pulmonary microsomal metabolism and covalent binding of the compound. These results suggest that metabolic activation of 4-ipomeanol involves a two-electron transfer in which transfer of the second electron via cytochrome b₅ is rate-limiting in lung microsomes.     

Unscheduled DNA synthesis in isolated hepatic nuclei after treatment of rats with methylnitrosourea in vivo

Administration of the carcinogenic methylating agent, methylnitrosourea, to rats caused a significant increase in endogenous DNA synthesis assayed subsequently in isolated hepatic nuclei $\text{in}\text{vitro}$. DNA synthesis was related directly to the dose of carcinogen and inversely to the interval between treatment and isolation of nuclei. This synthesis appears to represent the continuation $\text{in}\text{vitro}$ of unscheduled, reparative DNA synthesis initiated in damaged cells $\text{in}\text{vivo}$.     

Studies on the hyperglycemia and hepatic glycogenolysis produced by alpha and beta adrenergic agonists in the cat

Previous studies in this laboratory showed that both alpha and beta receptors can mediate adrenergically-induced hyperglycemia in the cat. In the present study, the results of experiments on the isolated perfused cat liver provide affirmation that hepatic glycogenolysis in this species can be subserved by both types of receptors. Thus, the acute hepatic release of glucose induced by isoproterenol was found to be antagonized by propranolol but not by phentolamine or phenoxybenzamine. The opposite was found for the glycogenolytic action of phenylephrine. Experiments $\text{in}$$\text{vivo}$ showed that the hyperglycemic response to the beta agonist was associated with activation of hepatic phosphorylase and increased intracellular cAMP content while the hyperglycemia induced by the alpha agonist was associated with an activation of phosphorylase which was independent of cAMP.     

On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis.

The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD⁺ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio brought about by glucagon may be secondary to the increase in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ and $\text{[ATP]}\text{[ADP]}$ ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD⁺ system.     

Response of the mixed function oxidase system of rat hepatic microsomes to parathion and malathion and their oxygenated analogs

$\text{In}$$\text{vitro}$ inhibition of ethylmorphine metabolism in rat hepatic microsomes by parathion, malathion, malaoxon and paraoxon was not well correlated with their effects on NADPH oxidation, cytochrome C reduction or the reduction of cytochrome P-450. A parallel relationship was observed between inhibition of ethylmorphine metabolism by parathion, malathion and malaoxon and the binding affinity of these agents to microsomal cytochrome P-450 obtained from rats pretreated with an anticholinesterase agent, Bis-[?-nitrophenol] phosphate.     

Rapid inhibition by cycloheximide of rat hepatic nuclear free and engaged poly(A) polymerase activities

This paper reports the effect of cycloheximide on the activity of rat hepatic nuclear poly(A) polymerase. Three hours after cycloheximide treatment (3 mg/100 g body weight), total rat hepatic nuclear poly (A) polymerase activity was decreased to 50% of the normal level. This conclusion was reached when the enzyme activity was measured either in the whole nuclei $\text{in vitro}$ or with partly purified enzyme preparations. When examined at different times after a single injection of cycloheximide, it was observed that poly(A) polymerase activity decayed biphasically with an initial rapid decay phase reaching a minimum at one hour $\text{(}\text{t}\text{1}\text{2}\text{= 0.8}\text{hrs}\text{)}$, followed by a stable phase thereafter. These results have been interpreted to mean that poly(A) polymerase consists of either a mixture of two structurally distinct populations of enzymes with a different turnover rate, or of a single type of enzyme with a protein factor which is rapidly turning over and which is required for maximal enzyme activity.     

Effect of ascorbic acid on heme metabolism in hepatic microsomes

Hepatic microsonal cytochrome P-450 levels are significantly decreased (46–68%) in ascorbic acid-deficient guinea pigs. Studies attempting to elucidate the mechanism responsible for decreased cytochrome P-450 demonstrated that ascorbic acid status did not influence the turnover $\text{(}\text{t}\text{1}\text{2}\text{)}$ or the degradation of hepatic cytochrome P-450 heme. Urinary excretion of Δ-aminolevulinic acid (ALA) and coproporphyrin was significantly decreased (30 and 69% respectively) in ascorbic acid-deficient guinea pigs. Injections (ip) of ALA into ascorbic acid-deficient guinea pigs were not effective in returning cytochrome P-450 levels to values found in ascorbic acid-supplemented guinea pigs. In addition, plasma and hepatic iron and blood heme were related directly, while hepatic copper and plasma copper or ceruloplasmin were related inversely, to the ascorbic-acid status of the guinea pig. These data, along with past investigations on heme synthesis in the ascorbic acid-deficient guinea pig, are consistent with mechanisms proposing that ascorbic acid may influence: 1) apocytochrome P-450 synthesis, 2) binding of heme and apo-cytochrome P-450 to form active cytochrome P-450, and/or 3) incorporation of Fe++ into the heme moiety of cytochrome P-450, perhaps via changes in copper metabolism.     

Main pathway for mutagenic activation of 2-acetylaminofluorene by guinea pig liver homogenates.

The activation pathway of 2-acetylaminofluorene (AAF) to N-hydroxy-2-amino-fluorene (N-OH-AF), a potent mutagen to $\text{Salmonella}$, by guinea pig liver postmitochondrial supernatant fraction (S-9 fraction) was studied. 2-Aminofluorene (AF), as well as N-hydroxy-2-acetylaminofluorene (N-OH-AAF, Takeishi et al., Mutation Res. in press), was detected as a metabolite of AAF. The mutagenicities of AF and N-OH-AAF comparable to that of AAF were inhibited by antiserum against NADPH-cytochrome $\text{c}$ reductase and by paraoxon, respectively. These data indicate that in the mutagenic activation of AAF, N-OH-AF can be produced by both N-hydroxylation of AF and deacetylation of N-OH-AAF. Furthermore, the data on the relative contribution of paraoxon-sensitive activation pathway to mutagenicities of AAF and N-OH-AAF led to a conclusion that deacetylation of AAF followed by N-hydroxylation to produce N-OH-AF is the main pathway for the mutagenic activation of AAF by guinea pig liver S-9 fraction.     

Depression of hepatic cytochrome P-450-dependent monooxygenase systems with administered interferon inducing agents.

Cytochrome P-450-dependent monooxygenase activities and cytochrome P-450 levels were depressed in hepatic microsomes from rats treated with 12 interferon inducing agents of various types: small molecules (e.g. tilorone), an RNA virus (Mengo), a fungal mycophage (statolon), liver RNA, a synthetic double-stranded polynucleotide (poly rI · poly rC), a bacterial lipopolysaccharide ($\text{E.}\text{coli}$ endotoxin) and an attenuated bacteria ($\text{B.}\text{pertussis}$ vaccine). The results suggest that the depression of hepatic cytochrome P-450-dependent monooxygenase systems may be a general property of interferon inducing agents.     

Serum glutathione S-transferases: Perinatal development,sex difference,and effect of carbon tetrachloride administration on enzyme activity in the rat

Glutathione $\text{S}$-transferase activity was determined in rat, rabbit, and guinea pig serum using styrene 7,8-oxide (SO) and benzo (a) pyrene 4,5-oxide (4,5-BPO) as substrates. Of the species tested, rat had the highest transferase activity (62.5 and 3.2 nmol/min/ml serum for SO and 4,5-BPO, respectively) and rabbit had the lowest activity. Glutathione $\text{S}$-transferase activity was 60% higher in serum from male rats than in female rats. In rats, serum enzyme specific activities (nmol/min/mg protein) were less than 1% of hepatic enzyme activities with SO, 4,5-BPO, 1,2-dichloro-4-nitrobenzene (DCNB), and 1-chloro-2,4-dinitrobenzene (DNCB). Glutathione $\text{S}$-transferase activity was also determined in rat serum during perinatal development. Serum from rats at 18 days of gestation or from 1- and 4-day-old animals had barely detectable transferase activity. Activity increased with age and reached a maximum in 140-day-old animals. The intraperitoneal administration of diethyl maleate (DEM) (0.8 ml/kg) or L-methionine-DL-sulfoximine (MS) (200 mg/kg) to male rats had no effect on serum or hepatic glutathione $\text{S}$-transferase activities 2 or 26 hr after dosing. Treatment with carbon tetrachloride (CCl₄) (1 m1/kg) caused an 11-fold increase in serum transferase activity and a 40% decrease in liver specific activities 24 hr after administration.     

Protective effect of sulfhydryl compounds on acute toxicity of morphinone

The ability of sulfhydryl compounds to provide protection against the acute toxicity of morphinone was investigated in mice. Subcutaneous administration of morphinone produced a reduction of hepatic non-protein sulfhydryl concentration. Pretreatments of mice with glutathione or cysteine significantly increased the survival rate of mice given a lethal dose of morphinone, whereas morphinone lethality was markedly potentiated by diethyl maleate. On the other hand, the administration of morphine produced a dose dependent reduction of hepatic non-protein sulfhydryl contents. However, neither glutathione nor cysteine protected mice from the acute toxicity of morphine. A possible explanation for these observations was proposed as follows: morphine is oxidized by morphine 6-dehydrogenase to morphinone, and the morphinone thus produced decreases the sulfhydryl contents in the liver. This mechanism is supported by the fact that morphinone reacts easily with glutathione and cysteine $\text{in vitro}$.     

Protection of acetaminophen induced mitochondrial dysfunctions and hepatic necrosis via Akt-NF-kappaB pathway: role of a novel plant protein

Oxidative stress is a major cause of drug induced hepatic diseases. The present study aims to investigate the antioxidative signaling mechanism of a protein isolated from the herb, Cajanus indicus against acetaminophen induced necrotic cell death. We found that incubation of hepatocytes with the protein prevented acetaminophen-induced loss in cell viability, reduction in glutathione level and enhancement of reactive oxygen species generation. Treatment of mice with the protein before administration of acetaminophen also reduced serum nitrite and TNF-α formation. Moreover, it counteracted acetaminophen-induced loss in mitochondrial membrane potential, loss in adenosine tri phosphate and rise in intracellular calcium. Investigating the cell signaling pathways, we found that the protein exerts its protective action via the activation of NF-κB and Akt and deactivation of STAT-1. Surprisingly, no role of ERK1/2 or STAT-3 was found in the protein-mediated protection of hepatocytes during acetaminophen exposure. Finally, we found that acetaminophen introduces necrosis as the primary phenomena of cell death and protein treatment decreased the necrotic process as evident from the DNA fragmentation and flow-cytometry studies. In addition, administration of the protein to mice before acetaminophen application showed fewer number of TUNEL positive cells. Combining, data suggest that the protein possesses cytoprotective activity against acetaminophen-induced oxidative cellular damage and prevents hepatocytes from necrotic death.     

Investigations into the hepatic metabolism of dimethylnitrosamine in the rat.

In this paper we report the detection and identification of methanol as an intermediate formed during both the $\text{in vivo}$ and the $\text{in vitro}$ metabolism of dimethylnitrosamine (DMN) in the rat. Methanol was formed in both hepatic 10,000 $\text{g}$ av. supernatant and washed microsomal fractions over a wide range of nitrosamine substrate concentrations. Furthermore the total amounts of methanol and formaldehyde formed largely accounted for the metabolic fate of both methyl moieties of DMN. Although a number of inhibitors of alcohol metabolism profoundly inhibited the hepatic metabolism of DMN they had little effect on the activities of two mixed function oxidase dependent enzymes. The results suggest that DMN and possibly other dialkylnitrosamines are degraded by enzymic pathway(s) not dependent on cytochrome P-450.     

The covalent binding of acetaminophen to protein. Evidence for cysteine residues as major sites of arylation in vitro

Covalent binding of the reactive metabolite of acetaminophen has been investigated in hepatic microsomal preparations from phenobarbital-pretreated mice. Low molecular weight thiols (cysteine and glutathione) were found to inhibit this binding, whereas several other amino acids which were tested did not. Bovine serum albumin (BSA), which contains a single free sulfhydryl group per molecule and which thus represents a macromolecular thiol compound, inhibited covalent binding of the reactive acetaminophen metabolite to microsomal protein in a concentration-dependent manner. The acetaminophen metabolite also became irreversibly bound to BSA in these experiments, although this binding was reduced by approx. 47% when the thiol function of BSA was selectively blocked prior to incubation. Covalent binding of the acetaminophen metabolite to bovine alpha s1-casein, a soluble protein which does not contain any cysteine residues, was found to occur to an extent of 37% of that which became bound to native BSA. These results were taken to indicate that protein thiol groups are major sites of covalent binding of the reactive metabolite of acetaminophen in vitro. The covalent binding characteristics of synthetic N-acetyl-p-benzoquinoneimine (NAPQI), the putative electrophilic intermediate produced during oxidative metabolism of acetaminophen, paralleled closely those of the reactive species generated metabolically. These findings support the contention that NAPQI is indeed the reactive arylating metabolite of acetaminophen which binds irreversibly to protein.     

Glutathione peroxidase,glutathione reductase,and thiobarbituric acid-reactive products in muscles of chickens and mice with genetic muscular dystrophy

The specific activities of glutathione peroxidase and glutathione reductase, and the level of thiobarbituric acid-reactive products in muscles from chickens and mice with genetic muscular dystrophy were increased over those of control animals. These findings indicate the occurrence of lipid peroxidation and the activation of an $\text{in}$$\text{vivo}$ lipid peroxidation protective system in these dystrophic animals. A partial explanation for some of the metabolic changes that occur in dystrophic species is provided by this work.     

Photoaffinity labelling by S-(p-azidophenacyl)-glutathione of glyoxalase II and glutathione S-transferase

S-(p-azidophenacyl)-glutathione, $\text{l}$, is a linear competitive inhibitor at pH 7.40 of beef liver glyoxalase II with K_i = 7.96 × 10^?4 M. On irradiation at 340 nm it covalently inhibits glyoxalase II to a level of 42 ± 5% inhibition. This photoaffinity labelling is prevented by the presence of a glyoxalase II competitive inhibitor (the hemimercaptal of glutathione and methylglyoxal). A crude preparation of sheep liver glutathione S-transferases is also irreversibly inactivated (86% ± 5% inhibition) by irradiation at 320 nm in the presence of $\text{l}$.     

Regulation of the basal and cyclic AMP-stimulated rates of glycogen synthesis in Escherichia coli by an intermediate of purine biosynthesis

In $\text{Escherichia}\text{coli}$an abrupt increase in the rate of glycogen synthesis occurs at the onset of total nitrogen starvation. We present here both $\text{in}\text{vivo}$ and $\text{in}\text{vitro}$ data indicating that this increase occurs because of the loss of a nitrogen-containing intermediate of purine biosynthesis (apparently 5-aminoimidazole-4-carboxamide ribonucleotide) that inhibits glycogen synthesis. We also show that this inhibitory intermediate antagonizes the stimulation of glycogen synthesis by 3′,5′-cyclic AMP. The uncovering of the regulation of glycogen synthesis by this inhibitor apparently provides the first link in understanding the 23-year-old observation of a reciprocal relationship between growth rate and glycogen accumulation in $\text{E.}\text{coli}$.     

Acceleration of pentobarbital metabolism in tolerant mice induced by pentobarbital pellet implantation

The time course of inductions of N-demethylation and pentobarbital hydroxylation of hepatic drug metabolizing system in continuous pentobarbital administration by pentobarbital pellet implantation in the mouse is presented. The results also demonstrate that hepatic microsomal drug-metabolizing enzymes in the mouse could be induced much faster by a single pentobarbital pellet implantation than by the ordinary parenteral administration technique. The reduction of pentobarbital half-life ($\text{T}\text{1}\text{2}$) in plasma, brain and liver of the animals which had been implanted with a pentobarbital pellet also substantiates the acceleration of pentobarbital metabolism in the mouse by the pellet implantation method. The results show that the $\text{T}\text{1}\text{2}$ of pentobarbital in plasma, brain and liver of pentobarbital pellet implanted groups is only $\text{1}\text{2}\text{,}\text{1}\text{6}\text{and}\text{1}\text{9}$ of that of the placebo control group, respectively. The studies on urinary excretion of pentobarbital and its metabolites also reveals that pentobarbital pellet implantation induced much faster rate of metabolism of pentobarbital in the mouse.