Favism: effect of divicine on rat erythrocyte sulfhydryl status, hexose monophosphate shunt activity, morphology, and membrane skeletal proteins.

Favism is an acute anemic crisis that can occur in susceptible individuals who ingest fava beans. The fava bean pyrimidine aglycone divicine has been identified as a hemotoxic constituent; however, its mechanism of toxicity remains unknown. We have shown recently that divicine can induce a favic-like response in rats and that divicine is directly toxic to rat red cells. In the present study, we have examined the effect of hemotoxic concentrations of divicine on rat erythrocyte sulfhydryl status, hexose monophosphate (HMP) shunt activity, morphology, and membrane skeletal proteins. In vitro exposure of rat red cells to divicine markedly stimulated HMP shunt activity and resulted in depletion of reduced glutathione with concomitant formation of glutathione-protein mixed-disulfides. Examination of divicine-treated red cells by scanning electron microscopy revealed transformation of the cells to an extreme echinocytic morphology. SDS-PAGE and immunoblotting analysis of the membrane skeletal proteins indicated that hemotoxicity was associated with the apparent loss of skeletal protein bands 2.1, 3, and 4.2, and the appearance of membrane-bound hemoglobin. Treatment of divicine-damaged red cells with dithiothreitol reversed the protein changes, which indicated that the observed alterations were due primarily to the formation of disulfide-linked hemoglobin-skeletal protein adducts. The data suggest that oxidative modification of hemoglobin and membrane skeletal proteins by divicine may be key events in the mechanism underlying favism.

Primaquine-induced hemolytic anemia: formation and hemotoxicity of the arylhydroxylamine metabolite 6-methoxy-8-hydroxylaminoquinoline.

Primaquine is an important antimalarial agent because of its activity against exoerythrocytic forms of Plasmodium spp. However, methemoglobinemia and hemolytic anemia are dose-limiting side effects of primaquine therapy that limit its efficacy. These hemotoxicities are thought to be mediated by metabolites; however, the identity of the toxic species has remained unclear. Since N-hydroxy metabolites are known to mediate the hemotoxicity of several arylamines, the present studies were undertaken to determine whether 6-methoxy-8-aminoquinoline (6-MAQ), a known human metabolite of primaquine, could undergo N-hydroxylation to form a hemotoxic metabolite. When 6-MAQ was incubated with rat and human liver microsomes, a single metabolite was detected by high performance liquid chromatography (HPLC) with electrochemical detection. This metabolite was identified as 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH) by HPLC and mass spectral analyses. As measured by decreased survival of (51)Cr-labeled erythrocytes in rats, MAQ-NOH was hemolytic in vivo. Furthermore, in vitro exposure of (51)Cr-labeled erythrocytes to MAQ-NOH caused a concentration-dependent decrease in erythrocyte survival (EC(50) of 350 microM) when the exposed cells were returned to the circulation of isologous rats. MAQ-NOH also induced the formation of methemoglobin when incubated with suspensions of rat erythrocytes. These data indicate that 6-MAQ can be metabolized to MAQ-NOH by both rat and human liver microsomes and that MAQ-NOH has the requisite properties to be a hemotoxic metabolite of primaquine. The contribution of MAQ-NOH to the hemotoxicity of primaquine in vivo remains to be assessed.

Oxidative stress, glucose-6-phosphate dehydrogenase and the red cell.

As discussed above, the process by which normal senescent red cells are selected for removal from the circulation is the subject of much ongoing research and is not yet well understood. This in turn creates a problem for studies on the enhanced removal that occurs in xenobiotic-induced hemolytic states; specifically, whether the enhanced removal should be considered as an increase in rate of the normal sequestration mechanism or as an unrelated process, in part or in whole. This difficulty bears directly on the interpretation of much of the mechanistic hemolytic literature. Because of its dual in vivo and in vitro hemolytic capability, and because of its capacity to induce frank lysis in the incubation mixture, phenylhydrazine has been used extensively as a model compound for mechanistic studies. These data have contributed heavily to our current concepts of how chemicals induce damage in the red cell. The comparison studies presented above cast doubt on the relevance of many of these phenylhydrazine studies for the in vivo hemolytic response. Phenylhydrazine, like divicine and DDS-NOH, shows an overwhelming predominance of uptake into the spleen, as distinct from removal by the RES system in general, as evidenced by relatively low liver uptake. This suggests strongly that damaged cells are removed intact by the spleen and do not lyse or fragment in the general circulation, at least to any significant extent. The studies with DDS-NOH indicate that neither Heinz body formation nor lipid peroxidation per se are essential steps in the process by which damaged red cells are removed from the circulation in the rat. It is not yet clear whether this lack of obligatory involvement of Heinz bodies and lipid peroxidation is peculiar to the arylhydroxylamine-induced hemolytic state or whether it will prove to be of general applicability. On the other hand, cysteamine failed to reverse the hemolytic damage caused by phenylhydrazine. Since cysteamine "rescued" DDS-NOH treated cells under the same experimental conditions, this observation raises the possibility that protein-thiol oxidation per se is also not an obligatory step in the sequence of events leading to premature sequestration. Clearly, the ratio of lipid to protein oxidation is markedly different in these three examples of hemotoxic compounds. DDS-NOH showed high protein oxidation with no discernible lipid oxidation, divicine showed both high protein and high lipid oxidation, and phenylhydrazine showed high lipid and low protein oxidation. While the significance of these markedly different patterns of injury is far from clear, it seems reasonable to conclude that there is more than one way by which chemicals damage the red cell. It is intriguing that these apparently different chemical insults within the red cell result in a common "message" on the outside of the cell, such that the cell appears as "prematurely" aged. Although the pattern of injury inside the cell may be significantly different, the process by which the three hemotoxic compounds enhance uptake by splenic macrophages may remain the same. That is, there may be a variety of insults sustained within the red cell that lead by different pathways to similar "recognition-specific" changes on the external surface of the red cell. Clearly, comparison of the effects of the three hemotoxic compounds will shed light on both the hemolytic process and on normal red cell sequestration mechanisms.

Favism: divicine hemotoxicity in the rat.

Favism is an acute hemolytic anemia known to occur in susceptible individuals who ingest fava beans. Susceptibility to favism is conferred by a genetic deficiency in erythrocytic glucose-6-phosphate dehydrogenase (G6PD) activity. Although the fava bean pyrimidine aglycones, divicine and isouramil, have been implicated in the onset of favism in humans, the lack of a well-defined experimental animal model for favism has hampered progress in elucidating the mechanism underlying hemotoxicity. We have examined whether a favic-like response could be provoked in G6PD-normal rats treated with synthetic divicine. Intraperitoneal administration of divicine to rats preloaded with 51Cr-tagged erythrocytes resulted in a severe, dose-dependent decrease in blood radioactivity (TD50 approximately 0.5 mmol/kg) within 24 h. The increased rate of removal of blood radioactivity was accompanied by a rapid decline in reduced glutathione levels in the blood, decreased hematocrits, marked hemoglobinuria, splenic enlargement, and reticulocytosis. In vitro exposure of 51Cr-tagged red cells to divicine before their re-administration to isologous rats also resulted in a sharp, concentration-dependent decrease in erythrocyte survival in vivo (TC50 approximately 1.5 mM), and these divicine-damaged red cells were removed from the circulation by the spleen. These data demonstrate that a favic response can be induced in G6PD-normal rats treated with divicine, and that hemolytic activity can be reproduced in isolated red cells under conditions that will allow a direct examination of the mechanism underlying this hemotoxicity.

Role of lipid peroxidation in dapsone-induced hemolytic anemia.

Dapsone hydroxylamine (DDS-NOH) is a direct-acting hemolytic agent responsible for dapsone-induced hemolytic anemia in the rat. The hemolytic activity of DDS-NOH is associated with the formation of disulfide-linked hemoglobin adducts on membrane skeletal proteins. We have postulated that this membrane protein "damage" is a consequence of DDS-NOH-induced oxidative stress within the red cell and that it serves as the trigger for premature removal of injured but intact red cells from the circulation by splenic macrophages. Oxidative stress has also been associated with the induction of lipid peroxidation, and it is possible that direct damage to the lipoidal membrane may play a role in the premature sequestration of the damaged cells in the spleen. To investigate this possibility, rat and human red cells were incubated with hemolytic concentrations of DDS-NOH and examined for evidence of lipid peroxidation using two independent assays: thiobarbituric acid-reactive substances formation and cis-paranaric acid degradation. Phenylhydrazine, which is known to induce lipid peroxidation in red cells, was used as a positive control. The extent of thiobarbituric acid-reactive substances formation and cis-paranaric acid degradation in DDS-NOH-treated rat and human red cells was not significantly different from that in control cells. In contrast, thiobarbituric acid-reactive substances formation and cis-paranaric acid degradation were significantly increased in red cells treated with hemolytic concentrations of the positive control, phenylhydrazine. These data suggest that lipid peroxidation is not involved in the mechanism underlying dapsone-induced hemolytic anemia.

Formation of free radicals and protein mixed disulfides in rat red cells exposed to dapsone hydroxylamine.

The hemolytic activity of dapsone is well known to reside in its N-hydroxylamine metabolites. Addition of dapsone hydroxylamine (DDS-NOH) to red cell suspensions causes damage such that when reintroduced into the circulation of isologous rats, the injured cells are rapidly removed by the spleen. Hemolytic activity is associated with the extensive formation of disulfide-linked hemoglobin adducts on red cell membrane skeletal proteins. To determine if free radicals could be involved in this process, rat red cells were incubated with DDS-NOH in the presence of the spin trap, 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) and subjected to EPR analysis. Addition of DDS-NOH (25-50 microM) to a red cell suspension gave rise to a four-line (1:2:2:1) EPR spectrum with coupling constants identical to those of a DMPO-hydroxyl radical adduct (DMPO-OH) standard. No other radicals were detected; however, preincubation of red cells with cysteamine caused the DDS-NOH-generated DMPO-OH signal to be replaced by a cysteamine thiyl radical adduct signal. DDS-NOH-treated red cells were also found to contain ferrylhemoglobin, indicating the presence of hydrogen peroxide. Furthermore, DDS-NOH was found to stimulate salicylate hydroxylation in red cell suspensions, confirming the presence of oxygen radicals. These data support the hypothesis that oxygen radicals are involved in the mechanism underlying dapsone-induced hemolytic anemia.

Tolerance to nitroglycerin in vascular smooth muscle cells is not affected by the level of intracellular glutathione or L-cysteine.

A major hypothesis for the mechanism of tolerance to nitroglycerin (NTG) is that continued use causes a decrease in thiol donors within the vascular smooth muscle cell that are essential for the effect of NTG. We tested this idea directly in the target cell. NTG tolerance, measured as reduced formation of intracellular cyclic guanosine monophosphate (cGMP), was induced in pig coronary smooth muscle cells. The consequence of altering intracellular levels of the thiol donors, glutathione (GSH) and L-cysteine (L-cys), was determined. Incubating cells with 100 microM NTG for 1 h caused an 83% reduction in cGMP formation in response to acute readministration of 200 microM NTG for 2 min but was not associated with a reduction in intracellular GSH or L-cys. This result was not altered when intracellular GSH levels were increased three-fold by including 1 mM GSH in the incubation buffer. Also, recovery from tolerance was not affected by supplementation with GSH. Further, the response of cGMP to NTG was not altered by inhibiting the synthesis of GSH and lowering intracellular levels of GSH by 77%. Similar findings were made with supplemental L-cys or N-acetyl-L-cysteine. These results do not support the hypothesis that tolerance to NTG is the result of a reduction of the thiol donors GSH and L-cys within vascular smooth muscle cells.

Dapsone-induced hemolytic anemia: effect of dapsone hydroxylamine on sulfhydryl status, membrane skeletal proteins and morphology of human and rat erythrocytes.

Dapsone hydroxylamine is a direct-acting hemolytic agent responsible for dapsone-induced hemolytic anemia in the rat. In the present study, we compared the responsiveness of rat and human red cells to dapsone hydroxylamine-induced cellular changes. Dapsone hydroxylamine induced a rapid and concentration-dependent loss of erythrocytic reduced glutathione content with a concomitant increase in protein-glutathione mixed disulfide formation in both human and rat red cell suspensions. However, the rate of mixed disulfide formation in human cells was considerably slower than that in rat cells and was preceded by a transient increase in oxidized glutathione (glutathione disulfide) formation. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of membrane ghosts from human red cells revealed changes in skeletal proteins that in general were similar to those observed with rat cells, including a loss of protein band 2.1 and the appearance of membrane-bound hemoglobin. Notable differences were the resistance to loss of band 4.2 and a considerably higher amount of protein aggregation in human ghosts. Although the morphology of human red cells was altered, the incidence and degree of change were considerably less than those of rat red cells. Furthermore, the concentration of dapsone hydroxylamine required to induce damage in human red cells (175-750 microM) was significantly higher than that required for rat red cells (50-175 microM), suggesting that human cells are probably less sensitive than rat cells to dapsone hydroxylamine-induced oxidative damage.

Macrophage enhancement of galactosamine hepatotoxicity using a rat hepatocyte culture system.

Prior administration of endotoxin to rats is known to aggravate the hepatotoxicity of galactosamine. It has been proposed that this exacerbation occurs as a result of the release of cytokines and other humoral factors by resident macrophages (Kupffer cells). In order to study this phenomenon we have utilized a co-culture system consisting of rat activated peritoneal macrophages and rat hepatocytes. Peritoneal macrophages were isolated and cultured; LPS was added as a macrophage activator 16 hours later. Rat hepatocytes were isolated and plated in Transwell COL inserts, which were placed in wells with and without activated macrophages. Cytotoxicity was determined 24 hours later by measuring lactate dehydrogenase (LDH) leakage into the culture medium. In the presence of activated macrophages an approximate 3-fold increase in galactosamine-induced hepatocyte toxicity was observed, as compared to the toxicity in hepatocytes cultured alone. Using this co-culture system, we examined the role of leukotriene D4 (LTD4) and nitric oxide (NO) as mediators of this enhancement. Addition of either LTD4 or NO to hepatocytes cultured alone did not exacerbate galactosamine toxicity. Furthermore, addition of the LTD4 receptor antagonist SK & F 104353 (50 microM) or the NO synthase inhibitor N-monomethyl-L-arginine (1.0 mM) to macrophage/hepatocyte co-cultures did not attenuate the enhanced galactosamine hepatocyte toxicity in the co-cultures. Collectively, these data indicate that this co-culture system will be useful in examining the mechanism of macrophage enhancement of chemical-induced hepatoxicity and, further, suggest that LTD4 and NO may not be involved in the exacerbation of galactosamine toxicity to hepatocyte cultures.

Modulation of macrophage functioning abrogates the acute hepatotoxicity of acetaminophen.

Acetaminophen is a mild analgesic and antipyretic agent that is safe and effective when taken in therapeutic doses. Ingestion of overdoses, however, may lead to acute liver failure accompanied by centrilobular degeneration and necrosis. Although the toxicity of acetaminophen is generally thought to be caused by direct interaction of its reactive metabolites with cellular macromolecules, recent studies have suggested that nonparenchymal cells also may contribute to tissue injury indirectly through the release of cytotoxic mediators. We analyzed the potential role of hepatic macrophages in acetaminophen hepatotoxicity by examining the effects of modulating the activity of these cells on tissue injury. Treatment of male Long Evans Hooded rats with acetaminophen (800 mg/kg) was found to induce extensive centrilobular hepatic necrosis. Pretreatment of the rats with either dextran sulfate or gadolinium chloride, two inhibitors of hepatic macrophage functioning, completely blocked hepatic necrosis, as well as increases in serum transaminase levels induced by acetaminophen. Interestingly, treatment of rats with the macrophage activator, lipopolysaccharide (LPS), also reduced tissue injury induced by acetaminophen. To exclude the possibility that the effects of gadolinium chloride, dextran sulfate, or LPS were due to alterations in acetaminophen metabolism, we analyzed the effects of these agents on various pharmacokinetic properties of this analgesic. Dextran sulfate and gadolinium chloride had no effect on the half-life of a low dose of acetaminophen (20 mg/kg), or on the activity of any of its individual pathways of metabolism, including the formation of acetaminophen-mercapturic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Chemical analysis and hemolytic activity of the fava bean aglycon divicine.

Divicine is an unstable aglycon metabolite of the fava bean pyrimidine beta-glucoside vicine. Divicine has long been thought to be a mediator of an acute hemolytic crisis, known as favism, in susceptible individuals who ingest fava beans (Vicia faba). However, a recent report has questioned the chemical identity of the divicine that was used in most of the studies on divicine hemotoxicity. The present study was undertaken to examine the hemolytic potential of synthetic divicine. Divicine was synthesized and its identity and purity were confirmed by HPLC, mass spectrometry, and NMR spectroscopy. The stability and redox behavior of divicine, under physiological conditions, were examined by HPLC and cyclic voltammetry. The data indicate that divicine is readily oxidized under aerobic conditions; however, it was sufficiently stable at pH 7.4 to permit its experimental manipulation. When 51Cr-labeled rat erythrocytes were exposed in vitro to the parent glucoside, vicine (5 mM), and then readministered to rats, no decrease in erythrocyte survival was observed. In contrast, erythrocyte survival was dramatically reduced by in vitro exposure to divicine (1.5 mM). These data demonstrate that divicine is a direct-acting hemolytic agent and thus may be a mediator of the hemolytic crisis induced by fava bean ingestion.

Lack of in vivo evidence of a cytochrome P450 metabolite participating in aminoglycoside nephrotoxicity.

Recent in vitro evidence has suggested that the cytotoxicity of aminoglycosides may be mediated by a metabolite generated by the hepatic cytochrome P450 drug-metabolizing system. This postulate has been tested by pretreating rats with cobalt protoporphyrin IX (CoP) to suppress hepatic P450 levels prior to administration of gentamicin. CoP pretreatment was observed to suppress antipyrine clearance markedly but not to alter gentamicin nephrotoxicity.

Dapsone-induced hemolytic anemia: effect of N-hydroxy dapsone on the sulfhydryl status and membrane proteins of rat erythrocytes.

Dapsone hydroxylamine (DDS-NOH), a known metabolite of dapsone, has recently been shown to be a direct-acting hemotoxin responsible in part for dapsone-induced hemolytic anemia in the rat. The effect of DDS-NOH on the morphology, sulfhydryl status, and membrane skeletal proteins of the rat red cell has been investigated. Exposure of rat red cells to a TC50 of DDS-NOH induced transformation of about 50% of the cells to an extreme echinocyte morphology. Reduced glutathione content of the cells was rapidly lost with concomitant increase in the formation of mixed disulfide between glutathione and the soluble protein of the cell. Oxidized glutathione content of the cells did not increase at any time during exposure to DDS-NOH. Examination of the skeletal membrane proteins by SDS-PAGE indicated that DDS-NOH caused the apparent loss of band 4.2, decrease in peaks 1, 2.1, and 3, and the appearance of new bands at about 16, 27, 40, and 54 kDa. Bands 4.1 and 7 appeared unchanged. Treatment of DDS-NOH altered proteins with dithiothreitol, reversed the protein changes, and indicated that the observed alterations were due to the formation of disulfide-linked adducts between hemoglobin and the various skeletal proteins as well as between hemoglobin monomers. The possible significance of the parallel changes in cell morphology and in membrane skeletal proteins for the premature splenic sequestration of the injured rat red cells is discussed.

Galactosamine hepatotoxicity: effect of galactosamine on glutathione resynthesis in rat primary hepatocyte cultures.

The effect of galactosamine on the resynthesis of glutathione in rat primary hepatocyte cultures was investigated. Cultured rat hepatocytes were treated with galactosamine (4 mM) 1.5 hr prior to concurrent with, or 1.5 hr after cell attachment; total cellular glutathione was then measured over time. Addition of galactosamine at any of these times suppressed methionine-enhanced glutathione resynthesis in the cultures after a lag period of about 120 min. The lag period was not due to slow uptake of galactosamine by the cultured cells, since cellular UTP levels fell to less than 10% of controls within 60 min, a time frame comparable to that observed in vivo. Neither was the lag period a result of interference with cellular uptake of methionine or with conversion of methionine to cysteine, since the phenomenon was observed regardless of whether methionine or cysteine was used to promote glutathione resynthesis. Addition of uridine, which protects against galactosamine hepatotoxicity in vivo by replenishing hepatic UTP levels, did not prevent the suppression of glutathione resynthesis. The data indicate that (a) galactosamine inhibits the time-dependent resynthesis of glutathione in primary hepatocyte cultures, (b) a lag period exists for this response, and (c) this effect is not directly related to depletion of cellular UTP stores.

The role of N-hydroxyphenetidine in phenacetin-induced hemolytic anemia.

Phenacetin is well known to cause hemolytic anemia and methemoglobinemia in humans. Early mechanistic studies clearly established a causal role for active/reactive drug metabolites in the process but did not unequivocally identify these metabolite(s) or resolve the question of whether these two hemotoxicities are mechanistically linked. As part of ongoing studies on the mechanism underlying arylamine-induced hemotoxicities, we have recently shown that the arylhydroxylamine metabolites of aniline and dapsone mediate the hemolytic activity of aniline and dapsone, respectively. The present study was undertaken to determine if N-hydroxyphenetidine (PNOH), the known arylhydroxylamine metabolite of phenacetin, is responsible for phenacetin-induced hemolytic anemia. As measured by decreased survival of 51Cr-labeled erythrocytes in rats, phenacetin, p-phenetidine, and PNOH were all hemolytic in vivo, with PNOH being significantly the most potent of the three. In vitro exposure of 51Cr-tagged erythrocytes to PNOH, followed by transfusion into isologous rats, resulted in a concentration-dependent reduction in erythrocyte survival, indicating that PNOH is a direct-acting hemolytic agent. Phenacetin and p-phenetidine were inactive. Phenacetin, p-phenetidine, and PNOH all produced dose-dependent methemoglobinemia in rats. In parallel in vitro studies, PNOH elevated methemoglobin levels, p-phenetidine and phenacetin did not. However, attempts to identify PNOH in the blood of phenacetin- and p-phenetidine-treated rats were unsuccessful, despite the use of a highly sensitive analytical method. Hemotoxic concentrations of PNOH were found to be highly unstable in the presence of red cells, though relatively stable in the buffer vehicle alone. Inhibitors of acetylation (p-aminobenzoic acid [PABA]) and deacetylation (bis-[p-nitrophenyl]phosphate [BNPP]), used to alter the cyclic interconversion of phenacetin and p-phenetidine, caused changes in phenacetin hemotoxicity that indicated the hemotoxin was a deacetylated metabolite distal to p-phenetidine. These data are consistent with the hypothesis that PNOH, formed during the metabolic clearance of phenacetin, mediates phenacetin-induced hemolytic anemia and methemoglobinemia through direct toxic actions in the erythrocyte.

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.

Role of glutathione in the in vitro synergism between 4-hydroperoxy-cyclophosphamide and cisplatin in leukemia cell lines.

To explain the sequence-dependent in vitro cytotoxic synergism between 4-hydroperoxycyclophosphamide (4-HC) and cisplatin in the K-562 human leukemia cell line, we have hypothesized that 4-HC decreases cellular glutathione (GSH) levels and that the resulting diminution of the cellular protective effect of GSH leads to the increased cytotoxicity of cisplatin. Exposure of K-562 cells to 4-HC resulted in a concentration- and time-dependent depletion of cellular GSH. To determine the effect of modulation of GSH levels on the toxicity of cisplatin, K-562 cells were exposed to buthionine sulfoximine (BSO) and/or GSH ethyl esters. Depletion of GSH to approximately 10% of control values by BSO potentiated the cytotoxicity of cisplatin, while rapid replenishment of GSH to within normal levels by GSH esters abolished the potentiation of BSO. Doubling cellular GSH by incubation with GSH esters protected against cisplatin cytotoxicity. Of importance, pretreatment of K-562 cells with BSO, in addition to increasing the cytotoxicity of 4-HC and cisplatin, abolished the synergism between the two drugs. The working hypothesis was also tested in two other cell lines in which the cytotoxic synergism between 4-HC and cisplatin was exhibited: the Raji cell line, a human lymphoblastic cell line, and the L1210-CPA cell line, a subclone of the murine L1210 leukemia with resistance to 4-HC. GSH levels in these two cell lines were not altered by incubation with concentrations of 4-HC at which the synergism was observed. In conclusion, the data for the K-562 cell line, indicating that (a) 4-HC depletes cellular GSH levels, (b) the lowering of cellular GSH levels enhances the toxicity of cisplatin, and (c) intact GSH stores are required for the synergism, strongly support the postulate that the cytotoxic synergism between 4-HC and cisplatin is modulated by GSH levels in this cell line. However, the lack of 4-HC-mediated depletion of GSH at concentrations of 4-HC resulting in cytotoxic synergism in the Raji and L1210-CPA cell line indicates that mechanisms other than modulation of GSH levels by 4-HC are responsible for the synergism in these cells.