S. D. Nelson

Cytochrome P-450 and NADPH cytochrome C reductase in rat brain: Formation of catechols and reactive catechol metabolites

Microsomes isolated from whole rat brain were found to contain cytochreme P-450 (0.025 to 0.051 nmoles/mg) and NADPH cytochrome c reductase activity (26.0 to 55.0 nmoles/mg/min). The oxidation of estradiol to a reactive metabolite that became covalently bound to rat brain microsomal protein was inhibited 63% by an atmosphere of CO:O2 (9:1), indicating the involvement of a cytochrome P-450 oxygenase. In contrast, this atmosphere had no effect on the binding of either the catechol estrogen, 2-hydroxyestradiol, or several catecholamines to rat brain microsomes. An antibody prepared against NADPH cytochrome c reductase was found to decrease significantly both the formation of 2-hydroxyestradiol from estradiol by rat brain microsomes and the covalent binding of the catechol estrogen and catecholamines to rat brain microsomal protein.

Cytochrome P-450-mediated oxidation of 2-hydroxyestrogens to reactive intermediates.

2-Hydroxyestradiol, 2-hydroxyestrone and 2-hydroxy-17α-ethynylestradiol, oxidation products of naturally occurring estrogens and synthetic estrogens in some oral contraceptives were found to be converted by rat liver microsomes to reactive metabolites that become irreversibly bound to microsomal protein. The irreversible binding required microsomes, oxygen and NADPH. The NADPH could be replaced by a xanthine-xanthine oxidase system which is known to generate superoxide anions. The irreversible binding was substantially inhibited by superoxide dismutase, 30% in those incubations containing NADPH and 98% in those incubations containing the xanthine-xanthine oxidase system. Further studies with 2-hydroxyestradiol showed that microsomal cytochrome P-450 was rate limiting in the NADPH-dependent irreversible binding, because the binding was inhibited 62% by an antibody against NADPH-cytochrome c reductase and 70% in an atmosphere of CO:O2 (9:1) when compared to an atmosphere of N2:O2 (9:1). Phenobarbital, a known inducer of cytochrome P-450, had no effect on the irreversible binding of 2-hydroxyestradiol, whereas another inducer of P-450, pregnenolone-16α-carbonitrile, markedly increased the irreversible binding. In contrast, cobaltous chloride, an inhibitor of the synthesis of cytochrome P-450, decreased both P-450 and the irreversible binding. These results are consistent with a mechanism for irreversible binding of estrogens and 2-hydroxyestrogens to microsomes that requires oxidation of the catechol nucleus by cytochrome P-450-generated superoxide anion.

Quantitative determination of the glutathione, cysteine, and N-acetyl cysteine conjugates of acetaminophen by high-pressure liquid chromatography.

Abstract A quantitative method for the estimation of the glutathione, cysteine, and N -acetyl cysteine conjugates of acetaminophen obtained from microsomal incubations has been developed using high-pressure liquid chromatography (HPLC). As little as 5 ng of the thiol conjugates of [ring- 14 C]acetaminophen can be measured with good precision from microsomal incubations with minimal sample preparation. High-pressure liquid chromatography offers distinct advantages over the standard separation techniques of thin-layer, paper, or gel filtration chromatography for the separation and quantitation of the polar thiol metabolites of acetaminophen.

Investigation of the mechanism of the metabolic activation of chloramphenicol by rat liver microsomes: Identification of a new metabolite

Abstract A new metabolite of chloramphenicol (CAP) has been isolated from incubation mixtures containing CAP and liver microsomes from phenobarbital-pretreated rats. This compound has been identified as the oxamic acid derivative of CAP by reverse isotope dilution, high pressure liquid chromatography, and isobutane chemical ionization mass spectroscopy. It has also been identified as a product of the chemical hydrolysis of radiolabeled microsomal protein. These results support the proposal that CAP is metabolically activated by liver microsomes to an oxamyl chloride reactive intermediate which either hydrolyzes to the oxamic acid or acylates protein.

Application of chemical ionization mass spectrometry and the twin-ion technique to better define a mechanism in acetylhydrazine toxicity

Chemical ionization mass spectrometry was used to show that N-acetylcysteine is formed in microsomal incubations containing NADPH, oxygen, cysteine and acetylhydrazine. The twin-ion technique was used to compare the ratio of hydrogen and deuterium in the N-acetylcysteine product with the initial ratio in a mixture of acetylhydrazine-trideuteroacetylhydrazine used as substrate. The results showed that the entire acetyl group was trapped by cysteine, thereby eliminating ketene as the reactive acylating agent formed during the oxidation of acetylhydrazine by liver microsomes.

Metabolic Activation of Hydrazines to Highly Reactive Hepatotoxic Intermediates

Substituted hydrazine derivatives are receiving considerable attention in chemical toxicology because of their widespread use as therapeutic agents for the treatment of depression, tuberculosis, and cancer, as herbicides, as intermediates in chemical syntheses, and even as rocket fuels. Hydrazines can produce many toxic responses, including methemoglobinemia, cell necrosis, mutagenesis, and carcinogenesis. We would like to report on our work concerning the possible mechanism of hepatotoxicity caused by the tuberculostatic drug isoniazid and related hydrazine compounds.

Application of Chemical Ionization Ms and the Twin-Ion Technique in the Analysis of Reactive Intermediates in Drug Metabolism

Since the pioneering work of the Millers (1) and Magee and Barnes (2), it has become increasingly evident that many stable chemicals are metabolized to electrophilic intermediates that alkylate and arylate tissue macromolecules. This laboratory (3–5) has shown that drugs, such as acetaminophen (paracetamol), phenacetin, furosemide (frusemide), isoniazid, and iproniazid, also are oxidatively activated by microsomal enzymes to electrophilic intermediates that covalently bind to tissue macromolecules and cause massive tissue necrosis.

Covalent Binding of 2,4-Diaminoanisole and 2,4-Diaminotoluene in vivo

We have studied the activation of 2,4-diaminoanisole (2,4-DAA), a mutagenic hair-dye component, and 2,4-diaminotoluene (2,4-DAT), a hepatocarcinogen, to products which blind covalently to tissue macromolecules. Four hours after a dose of 100 mg/kg ring-labeled 3H-2,4-DAA, 0.30 nmol is found covalently bound per mg liver protein. This amount is increased by 83% after phenobarbital pretreatment, and by 43% after beta-naphthoflavone-pretreatment. Almost the same degree of binding is seen in kidneys. Subcellular fractionation of livers shows that most of the bound material is in the microsomal fraction. Similar levels of covalent protein binding is seen after administering ring-labeled 3H-2,4-DAT. No significant binding to DNA in vitro or in vivo could be demonstrated using 3H-2,4-DAA or 3H-2,4-DAT, whereas 3H-2,4-DAT is found to covalently bind to hepatic RNA.

Metabolic Activation of Methyldopa by Cytochrome P450-Generated Superoxide Anion

Renewed interest in the hepatic injury produced by methyldopa (MD) has been stimulated by recent reports that the antihypertensive drug may initiate chronic active liver disease, occasionally with a fatal outcome (1–3). The hepatic damage has been attributed to hypersensitivity rather than to direct toxicity, but careful review reveals that the syndrome is similar to that produced by isoniazid (4). Most individuals fail to show constitutional features indicative of an allergic response and usually demonstrate hepatic injury upon rechallenge only after lengthy reexposure to MD. Moreover, MD produces mild, clinically covert hepatic injury in more than 15% of recipients when liver function tests are monitored (5), and thus the injury is not restricted to rare, idiosyncratic individuals. To determine if the toxicity produced by MD might be due to a reactive intermediate, [3H] MD was incubated with liver microsomes.

Metabolic Activation of Methyldopa and Other Catechols

The antihypertensive agent methyldopa (MD) is reported to produce mild, clinically covert, hepatic injury in 14% of recepients when liver function tests are monitored. Recent reports have shown that MD also may initiate chronic active liver disease, occasionally with a fatal outcome. We have studied metabolic activation of MD as a possible mechanism of MD hepatitis. Human and rodent liver microsomes convert MD to a reactive metabolite that binds covalently to protein in the presence of NADPH and O2. The binding is inhibited by a CO: O2 atmosphere, as well as by superoxide dismutase, ascorbic acid, ethylene diamine and glutathione, suggesting that MD is being oxidized by cytochrome P-450-generated superoxide anion to a reactive semiquinone and/or quinone. Emerging evidence indicate that synthetic estrogens may induce neoplastic changes in the liver. Hydroxylated estrogens are also activated to covalently bound products by liver microsomes in the presence of NADPH and O2, presumably via oxidation of the catechol nucleus by cytochrome P-450-generated superoxide anion. Oxyhemoglobin of red blood cells can be visualized as a bound form of superoxide anion. Human red blood cells activate MD to covalently bound products, this reaction may be responsible for immunological disorders such as a positive Coombs test and immune hemolysis.