David C. McMillan

DAPSONE-INDUCED HEMOLYTIC ANEMIA*

Dapsone, an old drug introduced and used almost exclusively for the treatment of leprosy, is now utilized in an increasing number of therapeutic situations. However, its hemotoxicity is potentially severe and is often dose limiting. Effective countermeasures, based on resolution of the mechanisms underlying dapsone-induced hemotoxicity, could significantly enhance the therapeutic value of the drug. In studies on rat red cells, we have established that the N-hydroxy metabolites of dapsone, DDS-NOH and MADDS-NOH, are direct-acting hemolytic agents, that they are formed in amounts sufficient to account for the hemotoxicity of the parent drug, and that the action of these toxic metabolites in the red cell induces premature sequestration by the spleen. Incubation of rat red cells with hemolytic concentrations of arylhydroxylamines leads to the generation of hydroxyl, glutathiyl, and hemoglobinthiyl radicals, and the formation of protein-glutathione mixed disulfides. Disulfide-linked adducts are also formed between membrane skeletal proteins and hemoglobin monomers, as well as between the monomeric hemoglobin units forming dimers, trimers, tetramers, and pentamers. Profound morphological changes are seen with change from normal discoidocity to an extreme nonspherocytic enchinocyte shape. Parallel studies with human red cells indicate that the response of human cells is qualitatively similar but that there are notable differences in regard to skeletal membrane effects. A working hypothesis for the mechanism underlying dapsone hemolytic activity is proposed.

Role of metabolites in propanil-induced hemolytic anemia☆

Hemolytic anemia and methemoglobinemia induced by exposure to certain arylamines, such as aniline and dapsone, are known to be mediated by their N-hydroxylamine metabolites. The arylamide propanil (3,4-dichloropropionanilide), a herbicide used extensively in rice fields, is also thought to induce methemoglobinemia through the action of metabolites. However, the hemolytic potential of this compound has not previously been reported. The present studies were undertaken to determine the hemolytic potential of propanil, and, if positive, the role of metabolites in this hemotoxicity. The survival of previously administered 51Cr-labeled erythrocytes in rats was reduced in a dose-dependent manner by ip administration of both propanil and its deacylated metabolite, 3,4-dichloroaniline (ED50 for both ca. 1.8 mmol/kg). When labeled erythrocytes were exposed in vitro to propanil or 3,4-dichloroaniline and then readministered to rats, no decrease in erythrocyte survival was observed, which indicated that these compounds were not direct-acting hemolytic agents. In contrast, erythrocyte survival was markedly reduced by ip administration or in vitro exposure to N-hydroxy-3,4-dichloroaniline. In addition, N-hydroxy-3,4-dichloroaniline was detected in the blood of propanil-treated rats in amounts sufficient to account for the hemolytic activity of the parent compound. These data indicate that N-hydroxy-3,4-dichloroaniline mediates propanil-induced hemolytic anemia, and that occupational exposure to propanil may result in an increased risk of hemolytic episodes.

Identification of free radicals produced in rat erythrocytes exposed to hemolytic concentrations of phenylhydroxylamine

Previous studies have shown that incubation of rat red blood cells in vitro with phenylhydroxylamine (50-300 microM) induces rapid splenic sequestration of the red cells on reintroduction to isologous rats. EPR and the spin trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), were utilized to determine if free radical species could be identified under these experimental conditions. Hemolytic concentrations of phenylhydroxylamine, in the presence of DMPO and 5-20% lysed or intact rat erythrocyte suspensions, gave rise to a four-line (1:2:2:1) EPR spectrum. No signal was obtained if phenylhydroxylamine, DMPO, or red cells was omitted. Comparison of the phenylhydroxylamine-induced signal with authentic hydroxyl radical- and GSH thiyl radical-DMPO standard adduct signals identified the phenylhydroxylamine-induced species as a GSH thiyl free radical. Removal of GSH from a red cell lysate abolished the GSH thiyl radical signal without the appearance of any other signal, while addition of exogenous GSH resulted in its return. When erythrocytes were exposed to concentrations of phenylhydroxylamine > or = 200 microM, a time-dependent transition of the GSH thiyl radical signal to a hemoglobin thiyl radical signal was observed. The data are consistent with the postulate that thiyl radical species, generated from the interaction of phenylhydroxylamine and oxyhemoglobin, play a key role in the development of hemolytic injury to the rat red cell.

Application of cryopreserved human hepatocytes in trichloroethylene risk assessment : Relative disposition of chloral hydrate to trichloroacetate and trichloroethanol

Background Trichloroethylene (TCE) is a suspected human carcinogen and a common ground-water contaminant. Chloral hydrate (CH) is the major metabolite of TCE formed in the liver by cytochrome P450 2E1. CH is metabolized to the hepatocarcinogen trichloroacetate (TCA) by aldehyde dehydrogenase (ALDH) and to the noncarcinogenic metabolite trichloroethanol (TCOH) by alcohol dehydrogenase (ADH). ALDH and ADH are polymorphic in humans, and these polymorphisms are known to affect the elimination of ethanol. It is therefore possible that polymorphisms in CH metabolism will yield subpopulations with greater than expected TCA formation with associated enhanced risk of liver tumors after TCE exposure. Methods The present studies were undertaken to determine the feasibility of using commercially available, cryogenically preserved human hepatocytes to determine simultaneously the kinetics of CH metabolism and ALDH/ADH genotype. Thirteen human hepatocyte samples were examined. Linear reciprocal plots were obtained for 11 ADH and 12 ALDH determinations. Results There was large interindividual variation in the Vmax values for both TCOH and TCA formation. Within this limited sample size, no correlation with ADH/ALDH genotype was apparent. Despite the large variation in Vmax values among individuals, disposition of CH into the two competing pathways was relatively constant. Conclusions These data support the use of cryopreserved human hepatocytes as an experimental system to generate metabolic and genomic information for incorporation into TCE cancer risk assessment models. The data are discussed with regard to cellular factors, other than genotype, that may contribute to the observed variability in metabolism of CH in human liver.

Primaquine-Induced Hemolytic Anemia: Formation of Free Radicals in Rat Erythrocytes Exposed to 6-Methoxy-8-hydroxylaminoquinoline

Primaquine is an important antimalarial drug that is often dose-limited in therapy by the onset of hemolytic anemia. We have shown recently that an N-hydroxy metabolite of primaquine, 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH), is a direct-acting hemolytic agent in rat red cells and that the hemolytic activity of this metabolite is associated with GSH oxidation and oxidative damage to both membrane lipids and skeletal proteins. To determine whether the formation of free radicals may be involved in this process, rat red cells (40% suspensions) were incubated with hemolytic concentrations of MAQ-NOH (150–750 μM) and examined by EPR spectroscopy using 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) as a spin trap. Addition of MAQ-NOH to red cell suspensions containing 10 mM EMPO gave rise to an EPR spectrum with hyperfine constants consistent with those of an EMPO-hydroxyl radical adduct standard. Of interest, formation of EMPO-OH was constant for up to 20 min and dependent on the presence of erythrocytic GSH. Although no other radical adduct signals were detected in the cells by EPR, spectrophotometric analysis revealed the presence of ferrylhemoglobin, which indicates that hydrogen peroxide is generated under these experimental conditions. The data support the hypothesis that oxygen-derived and possibly other free radicals are involved in the mechanism underlying MAQ-NOH-induced hemolytic anemia.

Detection of trichloroethylene-protein adducts in rat liver and plasma☆

Trichloroethylene is an industrial chemical with widespread occupational exposure and is a major environmental contaminant. In a Western blot using antiserum that recognizes trichloroethylene covalently bound to protein, a single 50 kDa microsomal adduct was detected in the livers of trichloroethylene-treated Sprague-Dawley rats. To determine if trichloroethylene-protein adducts could be detected in blood, plasma proteins were immunoaffinity purified using an antidichloroacetyl column. A single 50 kDa protein was detected in the affinity-purified fraction in a Western blot using dichloroacetyl antiserum. This protein was also immunochemically reactive with anti-cytochrome P450 2E1 antibodies. The 50 kDa trichloroethylene-protein adduct may be formed in the liver and released into the blood following exposure to trichloroethylene. The significance of adduct formation with respect to trichloroethylene toxicity remains to be established; however, the data suggest that this approach may be useful in the investigation of trichloroethylene-protein adducts and adverse effects following exposure.

Contribution of 3,4-Dichlorophenylhydroxylamine in Propanil-Induced Hemolytic Anemia

The methemoglobinemia and hemolytic anemia observed in experimental animals given aniline has been shown to be mediated by its N-oxidation metabolite, phenylhydroxylamine (Harrison, J.H. et al., 1987; Harrison, J.H. et al., 1986). The aniline derivative, propanil (3,4-dichloropropionanilide), is a widely used arylamide herbicide that has been shown to induce methemoglobinemia in experimental animals following conversion of the parent amide to one or more oxidized metabolites (Singleton, S > D. et al., 1973; Chow, A.Y.K. et al., 1975). Two methemoglobinemic metabolites of propanil have been identified, 3,4-dichlorophenylhydroxylamine (N-hydroxy-3,4-dichloroaniline) and 6-hydroxy-3,4-dichloroaniline (McMillan, D.C. et al., 1990). In view of the hemolytic activity of phenylhydroxylamine, we have examined the hemolytic potential of propanil and its metabolites in rats.

Arylamine-Induced Hemolytic Anemia: Electron Spin Resonance Spectrometry Studies

A variety of arylamine derivatives (e.g. dapsone, primaquine) are known to produce hemolytic anemia in man and experimental animals. Extensive studies in the 1950’s and 60’s established that hemolytic drugs induced methemoglobinemia and loss of erythrocytic reduced glutathione (GSH), and that individuals deficient in erthrocytic glucose-6-phosphate dehydrog-enase displayed enhanced susceptibility to drug-induced hemolytic anemia (for review, see E. Beutler, 1969; E. Beutler, 1972). Since drugs such as primaquine were active in vivo but not in vitro, the concept arose that the drugs were metabolized in the liver to active/reactive metabolites which, on entry to the red cell, produced a state of “oxidative stress”. The resulting oxidative damage to critical sites within the red cell has been considered to lead to their “premature aging” and premature removal from the circulation by the spleen (A.R. Tarlov, et al., 1962; F.C. Gooden-Smith, et al., 1974; G. Cohen et al., 1964; A. Miller et al., 1970; R.W. Carrell, et al., 1975). The nature of the oxidant stress and the identity of the critical sites, however, are still unclear. Since the oxidation of hemoglobin to methemoglobin is known to be associated with the reduction of oxygen, much work has centered around the role of active oxygen species and free radicals as molecules capable of attacking cellular components (G. Cohen, et al., 1964; R.W. Carrell, et al., 1975; H.P. Misra et al., 1976; H.A. Itano, et al., 1977; B. Goldberg, et al., 1977). We have recently shown that phenylhydroxylamine (PHA) is a direct acting hemotoxin, capable of damaging the red blood cell during in vitro incubation such that, when readministered to isologous rats, the cells are rapidly sequestered by the spleen (J.H. Harrison, et al., 1986). This communication describes spin trap studies aimed at determination of whether or not free radical specie(s) are formed in the red cell in response to PHA, and if so, the identification of these specie(s).