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Effects of butylated hydroxyanisole and dicoumarol on the toxicity of menadione to rats
Chemico-Biological Interactions 108 (1998) 155 – 170
Effects of butylated hydroxyanisole and dicoumarol on the toxicity of menadione to rats Rex Munday *, Barry L. Smith, Christine M. Munday AgResearch, Ruakura Agricultural Research Centre, Pri6ate Bag 3123, Hamilton, New Zealand Received 22 July 1997; received in revised form 28 October 1997; accepted 29 October 1997
Abstract The enzyme DT-diaphorase catalyses the 2-electron reduction of quinones. This reaction may facilitate the detoxification of such compounds, since the hydroquinone so formed can be converted into non-toxic conjugates. There is evidence for the involvement of DT-diaphorase in the detoxification of menadione (2-methyl-1,4-naphthoquinone) in a wide range of cells and tissues in vitro, but no information is available on the possible influence of this enzyme on the harmful effects of menadione in vivo. In animals, menadione is selectively toxic to erythrocytes, causing haemolytic anaemia. In the present study, rats were treated with dicoumarol, an inhibitor of DT-diaphorase, or butylated hydroxyanisole (BHA), a substance that increases the activity of this enzyme in vivo. They were then challenged with a toxic dose of menadione. Dicoumarol increased the severity of menadione-induced haemolytic anaemia while BHA decreased it, consistent with a role for DT-diaphorase in the detoxification of menadione in vivo, as previously described in vitro. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Menadione; Toxicity in vivo; Naphthoquinone toxicity; Butylated hydroxyanisole; Dicoumarol; DT-diaphorase
* Corresponding author. Tel.: +64 7 8385138; fax: + 64 7 8385012; e-mail: [email protected] 0009-2797/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 0 9 - 2 7 9 7 ( 9 7 ) 0 0 1 0 5 - 1
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1. Introduction The vitamin K analogue, 2-methyl-1,4-naphthoquinone (menadione) was once widely used in the prophylaxis of haemorrhagic disease of the newborn [1]. More recently, this substance has been investigated as an artificial electron carrier in the treatment of certain mitochondrial myopathies [2–4] and has shown promise as an adjunct to other drugs in cancer chemotherapy [5–11]. The toxicity of menadione has been extensively investigated in vitro, and harmful effects have been recorded in a wide range of cells and tissues [12–15]. In vivo, however, this substance is selectively toxic to erythrocytes, causing a Heinz body haemolytic anaemia when administered to rats [16–19], dogs [20–22], rabbits [22], cats [22], fish [23] and humans [1,5,7,9]. In the presence of reducing agents, menadione and other quinones undergo redox cycling, with formation of ‘active oxygen’ species [12–14]. Redox cycling by quinones may be either a 1- or 2-electron process. One-electron reduction yields semiquinones, which are very unstable substances that react rapidly with molecular oxygen, re-forming the quinone with concomitant generation of ‘active oxygen’ species [24,25]. One-electron reduction of quinones, mediated by flavoenzymes [26– 28] or by interaction with oxyhaemoglobin [19,29], is recognised as a toxification reaction and is held responsible for both the in vitro cytotoxicity of such substances [12,27,30] and their in vivo toxic effects [19]. Two-electron reduction of a quinone forms the corresponding hydroquinone. Although generally less reactive than semiquinones, hydroquinones also undergo autoxidation, again with production of ‘active oxygen’ species [31,32]. However, unlike semiquinones, hydroquinones can also undergo conjugation with glucuronide or sulphate [33]. The latter process is a detoxification reaction, since the conjugates so formed do not redox cycle and, being hydrophilic, are readily eliminated from the body [34]. Therefore, 2-electron reduction may lead either to toxification or detoxification and the balance between these processes will depend upon the relative rates of the 1-electron and 2-electron processes and the stability of the particular hydroquinone that is formed. If a quinone is rapidly reduced to a stable hydroquinone, 1-electron processes will be by-passed and conjugate formation will lead to detoxification. In contrast, a slowly reduced quinone that forms a very reactive hydroquinone will not be detoxified in this manner, since the quinone will not be rapidly excreted and its persistence in the body will permit 1-electron reactions to continue. Furthermore, ‘active oxygen’ species will be formed through autoxidation of the hydroquinone. The primary mediator of the 2-electron reduction of quinones is DT-diaphorase (NADPH:[quinone acceptor] oxidoreductase, E. C. 1.6.99.2). Menadione is readily reduced to the corresponding hydroquinone, menadiol, by DT-diaphorase [35,36] and menadiol is comparatively stable at neutral pH in the presence of diaphorase [32]. This substance is, therefore, a good candidate for detoxification via 2-electron reduction. If reduction is rate limiting, it would follow that an increased activity of diaphorase in cells and animals would decrease the toxicity of menadione while a reduction in diaphorase activity would lead to an increase in the severity of its harmful effects. In cellular systems in vitro, there is evidence for an association
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between enzyme levels and the severity of menadione toxicity. In cancer cells, the toxicity of menadione was shown to be directly proportional to cellular levels of DT-diaphorase [37], while dicoumarol, an inhibitor of this enzyme [38], increased the toxicity of this substance to hepatocytes [30,39–41], biliary epithelial cells [42], cardiomyocytes [43], hepatoma cells [44], fibroblasts [45,46], CHO cells [41] and a colon carcinoma cell line [47]. In contrast, hepatocytes isolated from rats treated with butylated hydroxyanisole (BHA), a powerful inducer of DT-diaphorase in animals [48], were more resistant to the harmful effects of menadione than control cells [49,50], as were cells from lines containing abnormally high levels of diaphorase [51,52]. Despite the large body of work on the effects of inducers or inhibitors of DT-diaphorase on menadione toxicity in vitro, no information is available on the effect of such manipulations on the haemolytic effect of menadione in vivo. In the present study, we have investigated the effects of BHA and dicoumarol on the haemolytic activity of menadione in rats.
2. Materials and methods
2.1. Chemicals Dicoumarol and menadione were purchased from Sigma. The latter substance was recrystallised from methanol before use. Butylated hydroxyanisole was bought from BDH Chemicals.
2.2. Animals and housing Female rats (11 – 12 weeks old) from the Ruakura colony of Sprague-Dawley derived animals were randomly allocated to treatment groups. The rats were housed in solid-bottomed cages containing bedding of wood shavings and were allowed food (NRM Feeds, Hamilton) and water ad lib. Room temperature was maintained at 21 – 24°C and a 12 h light/dark cycle was employed.
2.3. Time-course of menadione toxicity A total of 48 rats were dosed orally with menadione, as a suspension in 2% Tween 80, at a dose of 5000 mmol/kg. Six rats were killed 1, 2, 3, 4, 5, 6, 8 and 10 days after dosing. Six animals dosed only with 2% Tween were also sacrificed (‘Day 0’). The rats were anaesthetised with halothane and killed by exsanguination, blood being taken into EDTA-containing tubes from the posterior vena cava. At necropsy, splenic weights were recorded and portions of this tissue, together with portions of kidney and liver, were fixed in 4% buffered formaldehyde. Tissues were routinely processed and embedded in paraffin wax. Sections were stained with haematoxylin and eosin and by Perls’ Prussian blue reaction for examination by light microscopy. They were examined without reference to the treatment group.
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Splenic sinusoidal engorgement and ferric iron deposition in the spleen, liver and kidneys (as reflected by the intensity of Prussian blue staining) were scored on an arbitrary scale of 0 – 5, 0 indicating the absence of the specified histological feature and 5 its presence to a severe degree. Blood packed cell volumes were determined by the microhaematocrit technique and haemoglobin levels by the cyanmethaemoglobin method [53]. Heinz bodies were quantitated turbidometrically by measuring the change in optical density of red cell lysates at 700 nm following centrifugation [54]. Plasma samples were analysed for activities of aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase and for levels of urea and creatinine on a Boehringer-Mannheim Hitachi 705 random-access analyser at 30°C.
2.4. Experiments with BHA and dicoumarol BHA, as a solution in corn oil, was administered orally to groups of six rats at a dose of 500 mg/kg per day for 9 consecutive days. For examination of the effects of BHA per se, animals were sacrificed 5 days after the last dose of this substance. In the experiment on the effect of BHA on menadione toxicity, the naphthoquinone, at a dose of 6000 mmol/kg, was given by oral intubation 1 day after the last dose of BHA. The animals were killed 4 days later and in both these experiments the necropsy procedures and assays were as described above. In the study on the effect of BHA on hepatic DT-diaphorase activity, the animals were killed 24 h after the final dose and liver samples taken for immediate enzyme assay. Livers were homogenised in 0.25M sucrose and activities of DT-diaphorase determined by the method of Schor et al. [55], using an NADPH concentration of 75 mM. Experiments with dicoumarol were analogous to those with BHA, except that the test compound was administered by intraperitoneal injection twice, once in the afternoon and then again the following morning, as a suspension in corn oil, at a dose of 15 mg/kg. For measurement of DT-diaphorase activity, animals were killed 2 h after the second dose. In the experiment on the effect on menadione toxicity, the naphthoquinone, at a dose of 4000 mmol/kg, was given 2 h after the second dose of dicoumarol and the rats were killed 4 days later. In all experiments, an equal number of control animals were given corn oil alone according to the same dosing schedules as those employed for BHA and dicoumarol.
3. Results
3.1. Progression of the haemolytic anaemia induced by a single dose of menadione A single dose of 5000 mmol/kg of menadione led to a rapid increase in intra-erythrocytic Heinz bodies, as measured by the turbidity of lysed cells at 700 nm. Levels reached a maximum 2 days after dosing and then declined (Fig. 1A). Blood packed cell volumes (Fig. 1B) and haemoglobin levels (Fig. 1C) were
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significantly depressed 2 days after dosing and continued to decline, reaching a minimum at 4 – 6 days. Subsequently, levels rose again towards normal. Splenic weights were significantly increased 2 days after dosing and remained high throughout the experimental period (Fig. 1D). Splenic sinusoidal engorgement was evident 1 day after dosing (Fig. 2A), as were increased splenic levels of iron (Fig. 2B). Erythropoietic foci in the liver were not recorded until 3 days after menadione administration (Fig. 2C) although iron deposition in this organ was seen at 2 days (Fig. 2D). Iron deposition in the kidneys appeared much later, being first seen 5 days after dosing (Fig. 2E).
3.2. Effect of BHA and dicoumarol on hepatic le6els of DT-diaphorase Administration of BHA to rats increased hepatic DT-diaphorase levels by 275%. In contrast, dicoumarol decreased the hepatic activity of this enzyme by 65% (Table 1).
Fig. 1. Haematology and relative splenic weights of rats killed at various time intervals after a single dose of 5000 mmol/kg menadione. Data shown are the means and S.E.M. of the results from the six animals in each treatment group.
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Fig. 2. Histopathology of rats killed at various time intervals after a single dose of 5000 mmol/kg menadione. Lesions were scored on an arbitrary scale of 0 – 5, as described in Section 2. Data shown are the means and SE.M. of the scores for the six animals in each treatment group.
3.3. Effect of BHA and dicoumarol on haematological parameters and histology of rats Administration of BHA or dicoumarol to rats had no effect on blood packed cell volumes, haemoglobin levels, splenic weights or incidence of Heinz bodies and no histological changes were recorded in the spleen, liver or kidneys of the animals that
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would indicate a haemolytic effect (Table 2). No changes in plasma activities of aspartate aminotransferase, alanine aminotransferase or lactate dehydrogenase or plasma levels of urea and creatinine were recorded in the test animals and no degenerative changes were observed in the livers, spleens or kidneys of these animals (data not shown).
3.4. Effect of pre-treatment with BHA and dicoumarol on the toxicity of menadione to rats Severe haemolysis was observed in control rats killed 4 days after a dose of 6000 mmol/kg of menadione. Significant protection was given, however, by pre-treatment with BHA (Table 3). Blood packed cell volumes and haemoglobin levels were significantly higher in the treated animals, while Heinz body formation and splenic enlargement were lower. Less erythropoietic activity was recorded in the livers of animals pre-treated with BHA and both hepatic and renal iron storage was decreased. Menadione, at a dose of 4000 mmol/kg, produced a moderate degree of haemolysis in rats. Pre-treatment with dicoumarol increased the severity of the erythrocyte destruction, as reflected by significantly greater decreases in blood packed cell volumes and haemoglobin levels. Erythropoietic activity in the liver was also increased in animals receiving dicoumarol, as was the level of iron deposition in this organ (Table 4). The effects of menadione were restricted to erythrocytes. No plasma biochemical changes or histological alterations indicative of tissue damage was observed in the livers, kidneys or spleens of animals dosed with menadione alone or in those pre-treated with BHA or dicoumarol (data not shown).
4. Discussion In previous studies on the haemolytic action of menadione in animals, the test material was administered to the animals over a period of days or weeks [16–23]. However, in a study designed to test the effects of BHA and dicoumarol on menadione toxicity, prolonged dosing is not feasible, since it would be necessary to Table 1 Effect of BHA and dicoumarol on hepatic levels of DT-diaphorase in rats Treatment
Hepatic DT-diaphorase activity (U/g)
None (control) BHA Dicoumarol
42.0 9 5.5 157.7 9 10.6 14.89 1.5
Rats were dosed with BHA and dicoumarol as described in Section 2. Data shown are the means and S.E.M. of the results from the six animals in each treatment group.
43.391.0 45.1 9 0.6 42.89 0.8 42.79 0.3
Control DIC Control BHA
0.218 9 0.016 0.220 90.009
0.031 90.004 0.028 9 0.004 0.035 9 0.002 0.034 9 0.003
141 92 146 92 138 92 137 91
0.255 90.008 0.264 90.009
Relative splenic weight (g/ 100g body weight)
Haemoglobin Heinz bodies level (g/l) (DOD 700)
0.0 0.0
0.0 0.0
Splenic sinusoidal engorgementa
0.0 0.0
0.0 0.0
Hepatic erythropoiesisa
0.58 90.15 0.50 9 0.13
0.50 9 0.12 0.58 9 0.08
Splenic level of irona
0.0 0.0
0.0 0.0
Hepatic level of irona
0.0 0.0
0.0 0.0
Renal level of irona
Dicoumarol (DIC) and BHA were dosed according to the schedule described in Section 2. The corresponding control animals received the vehicle (corn oil) alone. Data shown are the means and S.E.M. of the results from the six animals in each treatment group. a Scored on an arbitrary scale of 0 – 5, as described in Section 2.
Packed cell volume (%)
Treatment group
Table 2 Haematology, relative splenic weights and hepatic, splenic and renal histology in control rats and in animals treated with BHA or dicoumarol
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Packed cell volume (%)
63 95
0.677 90.050
0.83 9 0.17*
1.67 9 0.33
Hepatic erythropoiesisa
2.679 0.21
2.839 0.31
1.839 0.17**
3.339 0.33
Splenic level Hepatic level of irona of irona
0.0
1.17 9 0.54
Renal level of irona
Rats were pre-treated with a solution of BHA in corn oil or with corn oil alone according to the dosing schedule described in Section 2. They were then challenged with a single dose of menadione at 6000 mmol/kg. Data shown are the means and S.E.M. of the results from the six animals in each treatment group. Figures marked with asterisks are significantly different from those of the control group, not pre-treated with BHA. * PB0.05; ** PB0.01; *** PB0.001. a Scored on an arbitrary scale of 0 – 5, as described in Section 2.
4.33 90.21
4.17 90.31
Relative splenic Splenic sinuweight (g/100g soidal enbody weight) gorgementa
0.343 90.028* 0.529 9 0.028*
0.438 9 0.010
Haemoglobin Heinz bodies level (g/l) (DOD 700)
BHA+ 32.19 1.1*** 101 94*** menadione
Control+ 22.1 91.3 menadione
Treatment group
Table 3 Effect of pre-treatment with BHA on the haemolytic activity of menadione
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37.391.3 31.291.3**
Control+ menadione DIC+ menadione
0.184 9 0.016 0.459 90.015 3.67 9 0.21
100 94*
Splenic sinusoidal engorgementa
0.171 90.019 0.444 90.030 3.50 90.22
Relative splenic weight (g/ 100g body weight)
113 9 4
Haemoglobin Heinz bodies level (g/l) (DOD 700)
1.58 90.15*
0.83 90.28
Hepatic erythropoiesisa
3.17 9 0.28
2.67 9 0.17
Splenic level of irona
2.41 9 0.15*
1.41 9 0.30
Hepatic level of irona
0.25 9 0.17
0.00
Renal level of irona
Rats were pre-treated with a solution of dicoumarol (DIC) in corn oil or with corn oil alone according to the dosing schedule described in Section 2. They were then challenged with a single dose of menadione at 4000 mmol/kg. Data shown are the means and SEM of the results from the six animals in each treatment group. Figures marked with asterisks are significantly different from those of the group not pre-treated with dicoumarol. * PB0.05; ** PB0.01. a Scored on an arbitrary scale of 0 – 5, as described in Section 2.
Packed cell volume (%)
Treatment group
Table 4 Effect of pre-treatment with dicoumarol on the haemolytic activity of menadione
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dose the naphthoquinone and the potential modifier simultaneously, with the possibility of interactions between them. Furthermore, dicoumarol itself is toxic when given over a prolonged period [56,57]. In order to avoid such problems, it was decided to pre-treat the animals with the modifiers and then challenge with a single dose of menadione. Since anaemia takes some time to develop after toxic challenge, it was necessary to establish the time course of menadione toxicity after dosing so that the most appropriate time for examining the effects of the modifiers could be established. Animals were therefore dosed with menadione and killed at various time intervals thereafter. Menadione exerts its toxic effect in animals through oxidative damage to erythrocytes, initiated by ‘active oxygen’ species generated through autoxidation of the semiquinone [19]. Oxidation of haemoglobin leads to the formation of granules of denatured haemoglobin (Heinz bodies) within erythrocytes. This, together with other oxidative changes within the cells, causes sequestration of the damaged erythrocytes within the splenic cords, where they are destroyed by phagocytic cells [58– 60]. Iron released from lysed erythrocytes is stored in the form of haemosiderin. The spleen is the primary site of storage, but when the capacity of this organ is exceeded, iron is taken up by the liver and kidneys and haemosiderin deposition, as reflected by Prussian blue staining, is observed in these tissues. In response to a decrease in the number of circulating erythrocytes, compensatory erythropoiesis occurs. In the rat, this takes place primarily in the spleen, although in severe anaemia, hepatic erythropoiesis also occurs. The increased activity of the spleen in both destroying and manufacturing erythrocytes results in enlargement of this organ. Such changes were clearly seen in the time course of menadione haemolysis. Soon after dosing, Heinz bodies appeared in the erythrocytes and blood packed cell volumes and haemoglobin levels began to fall as damaged cells were removed from the circulation. Splenic iron levels increased as iron released from the cells was stored and the weight of the spleen increased. The weight gain was associated with sinusoidal engorgement, attributable to increased levels of red pulp, which is involved in both erythroclasis and erythropoiesis. At later time intervals, Heinz body levels declined, as the oxidatively damaged cells were destroyed, and the compensatory erythropoiesis was very effective in reversing the decline in packed cell volumes and haemoglobin levels. The liver contributed to this process, since foci of erythropoiesis were observed. Iron storage in the liver and kidney was recorded toward the end of the experimental period, reflecting saturation of the storage capacity of the spleen. The maximum effects on blood packed cell volumes and haemoglobin levels were recorded between days 4 and 6. The effects of the possible modulating agents were therefore studied in animals pre-treated with these agents and killed 4 days after challenge with menadione. As expected from the results of previous studies [48,55] dicoumarol significantly decreased hepatic levels of DT-diaphorase while BHA caused a marked increase. Before investigating the effects of these substances on the haemolytic action of menadione, however, it was necessary to establish that these compounds themselves had no effect on red blood cells. Neither BHA nor dicoumarol when administered
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alone had any effect on blood packed cell volumes, haemoglobin levels or Heinz body production and there were no changes in splenic weight and no histological evidence of a toxic effect in spleen, liver or kidneys. As measured by several parameters of haemolysis, dicoumarol increased the toxicity of menadione to erythrocytes while BHA decreased the haemolytic effect of this substance. The toxicity of menadione can thus be modulated in vivo in the same way that it is in vitro. This is in accord with the observation that menadione is excreted as conjugates of menadiol in rats [34] and changes in the rate of reduction would be expected to change the rate of elimination of menadione. However, the relative rates of reduction and conjugation are not known and whether the observed modulation of menadione toxicity is due solely to effects upon DT-diaphorase activity cannot be stated with certainty. Although BHA and dicoumarol have been widely used as modulators of DT-diaphorase activity, their effects in animals are not restricted to this enzyme. It is possible that induction or inhibition of the enzymes responsible for hydroquinone conjugation (UDP glucuronosyl transferases and sulphotransferases) could contribute to the observed effects on menadione toxicity. It is known that BHA increases tissue activities of UDP glucuronosyl transferase in rats [61,62], but no information on the in vivo effects of dicoumarol on conjugating enzymes is available and results with this substance in vitro are equivocal. Dicoumarol inhibited the glucuronidation of 3-hydroxybenzpyrene, but it had no effect upon the conjugation of several other aglycones [63]. Studies on the pharmacokinetics of menadione under the influence of BHA and dicoumarol, together with an investigation of the rate-limiting step of menadione detoxification, are required to resolve this point. Experiments with other substances that are known to increase tissue levels of DT-diaphorase, such as butylated hydroxytoluene [61], disulfiram [64], dimethyl fumarate [65] and ethoxyquin [65] would also be valuable. Like BHA, none of these compounds is absolutely specific in its effect, but consistent protection against menadione toxicity by all these substances would provide further evidence for the involvement of DT-diaphorase in the detoxification of menadione. Haemolytic anaemia is commonly observed in patients treated with menadione in the therapy of cancer [5,7,9] and this limits the dose of the compound that can be administered. Although no direct information on the metabolism of menadione in humans is available, the observation that related naphthoquinones are excreted largely as glucuronides suggests that reduction to the hydroquinone does occur in man as in rats [66]. It is possible, therefore, that the problem of menadione toxicity in cancer patients could be ameliorated by pre-treatment with compounds that stimulate quinone reduction and conjugation. Conversely, it would be expected that concomitant treatment with the diaphorase inhibitor, Warfarin, as used in combination with menadione and other drugs in some treatment regimens [5,11] would increase the severity of menadione toxicity to red blood cells. The results of the present study show that BHA, which is known to increase tissue levels of DT-diaphorase, protects against the toxicity of menadione to rats. This may not, however, be a general phenomenon with all naphthoquinone derivatives. As discussed previously, the rapid reduction of menadione and the
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comparative stability of menadiol make this compound ideally suited to detoxification by 2-electron reduction. In vitro, redox cycling of this substance is progressively inhibited by increasing concentrations of DT-diaphorase [32]. In contrast, 2-hydroxy-1,4-naphthoquinone is but slowly reduced by DT-diaphorase and this substance forms a very unstable hydroquinone. Redox cycling of this substance is not inhibited by DT-diaphorase, but increases with increasing levels of the enzyme in vitro [32]. It is possible, therefore, that the toxicity of 2-hydroxy-1,4-naphthoquinone could be increased by increasing tissue levels of DT-diaphorase. Studies on this possibility are in progress.
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Effects of butylated hydroxyanisole and dicoumarol on the toxicity of menadione to rats Rex Munday *, Barry L. Smith, Christine M. Munday AgResearch, Ruakura Agricultural Research Centre, Pri6ate Bag 3123, Hamilton, New Zealand Received 22 July 1997; received in revised form 28 October 1997; accepted 29 October 1997
Abstract The enzyme DT-diaphorase catalyses the 2-electron reduction of quinones. This reaction may facilitate the detoxification of such compounds, since the hydroquinone so formed can be converted into non-toxic conjugates. There is evidence for the involvement of DT-diaphorase in the detoxification of menadione (2-methyl-1,4-naphthoquinone) in a wide range of cells and tissues in vitro, but no information is available on the possible influence of this enzyme on the harmful effects of menadione in vivo. In animals, menadione is selectively toxic to erythrocytes, causing haemolytic anaemia. In the present study, rats were treated with dicoumarol, an inhibitor of DT-diaphorase, or butylated hydroxyanisole (BHA), a substance that increases the activity of this enzyme in vivo. They were then challenged with a toxic dose of menadione. Dicoumarol increased the severity of menadione-induced haemolytic anaemia while BHA decreased it, consistent with a role for DT-diaphorase in the detoxification of menadione in vivo, as previously described in vitro. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Menadione; Toxicity in vivo; Naphthoquinone toxicity; Butylated hydroxyanisole; Dicoumarol; DT-diaphorase
* Corresponding author. Tel.: +64 7 8385138; fax: + 64 7 8385012; e-mail: [email protected] 0009-2797/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 0 9 - 2 7 9 7 ( 9 7 ) 0 0 1 0 5 - 1
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1. Introduction The vitamin K analogue, 2-methyl-1,4-naphthoquinone (menadione) was once widely used in the prophylaxis of haemorrhagic disease of the newborn [1]. More recently, this substance has been investigated as an artificial electron carrier in the treatment of certain mitochondrial myopathies [2–4] and has shown promise as an adjunct to other drugs in cancer chemotherapy [5–11]. The toxicity of menadione has been extensively investigated in vitro, and harmful effects have been recorded in a wide range of cells and tissues [12–15]. In vivo, however, this substance is selectively toxic to erythrocytes, causing a Heinz body haemolytic anaemia when administered to rats [16–19], dogs [20–22], rabbits [22], cats [22], fish [23] and humans [1,5,7,9]. In the presence of reducing agents, menadione and other quinones undergo redox cycling, with formation of ‘active oxygen’ species [12–14]. Redox cycling by quinones may be either a 1- or 2-electron process. One-electron reduction yields semiquinones, which are very unstable substances that react rapidly with molecular oxygen, re-forming the quinone with concomitant generation of ‘active oxygen’ species [24,25]. One-electron reduction of quinones, mediated by flavoenzymes [26– 28] or by interaction with oxyhaemoglobin [19,29], is recognised as a toxification reaction and is held responsible for both the in vitro cytotoxicity of such substances [12,27,30] and their in vivo toxic effects [19]. Two-electron reduction of a quinone forms the corresponding hydroquinone. Although generally less reactive than semiquinones, hydroquinones also undergo autoxidation, again with production of ‘active oxygen’ species [31,32]. However, unlike semiquinones, hydroquinones can also undergo conjugation with glucuronide or sulphate [33]. The latter process is a detoxification reaction, since the conjugates so formed do not redox cycle and, being hydrophilic, are readily eliminated from the body [34]. Therefore, 2-electron reduction may lead either to toxification or detoxification and the balance between these processes will depend upon the relative rates of the 1-electron and 2-electron processes and the stability of the particular hydroquinone that is formed. If a quinone is rapidly reduced to a stable hydroquinone, 1-electron processes will be by-passed and conjugate formation will lead to detoxification. In contrast, a slowly reduced quinone that forms a very reactive hydroquinone will not be detoxified in this manner, since the quinone will not be rapidly excreted and its persistence in the body will permit 1-electron reactions to continue. Furthermore, ‘active oxygen’ species will be formed through autoxidation of the hydroquinone. The primary mediator of the 2-electron reduction of quinones is DT-diaphorase (NADPH:[quinone acceptor] oxidoreductase, E. C. 1.6.99.2). Menadione is readily reduced to the corresponding hydroquinone, menadiol, by DT-diaphorase [35,36] and menadiol is comparatively stable at neutral pH in the presence of diaphorase [32]. This substance is, therefore, a good candidate for detoxification via 2-electron reduction. If reduction is rate limiting, it would follow that an increased activity of diaphorase in cells and animals would decrease the toxicity of menadione while a reduction in diaphorase activity would lead to an increase in the severity of its harmful effects. In cellular systems in vitro, there is evidence for an association
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between enzyme levels and the severity of menadione toxicity. In cancer cells, the toxicity of menadione was shown to be directly proportional to cellular levels of DT-diaphorase [37], while dicoumarol, an inhibitor of this enzyme [38], increased the toxicity of this substance to hepatocytes [30,39–41], biliary epithelial cells [42], cardiomyocytes [43], hepatoma cells [44], fibroblasts [45,46], CHO cells [41] and a colon carcinoma cell line [47]. In contrast, hepatocytes isolated from rats treated with butylated hydroxyanisole (BHA), a powerful inducer of DT-diaphorase in animals [48], were more resistant to the harmful effects of menadione than control cells [49,50], as were cells from lines containing abnormally high levels of diaphorase [51,52]. Despite the large body of work on the effects of inducers or inhibitors of DT-diaphorase on menadione toxicity in vitro, no information is available on the effect of such manipulations on the haemolytic effect of menadione in vivo. In the present study, we have investigated the effects of BHA and dicoumarol on the haemolytic activity of menadione in rats.
2. Materials and methods
2.1. Chemicals Dicoumarol and menadione were purchased from Sigma. The latter substance was recrystallised from methanol before use. Butylated hydroxyanisole was bought from BDH Chemicals.
2.2. Animals and housing Female rats (11 – 12 weeks old) from the Ruakura colony of Sprague-Dawley derived animals were randomly allocated to treatment groups. The rats were housed in solid-bottomed cages containing bedding of wood shavings and were allowed food (NRM Feeds, Hamilton) and water ad lib. Room temperature was maintained at 21 – 24°C and a 12 h light/dark cycle was employed.
2.3. Time-course of menadione toxicity A total of 48 rats were dosed orally with menadione, as a suspension in 2% Tween 80, at a dose of 5000 mmol/kg. Six rats were killed 1, 2, 3, 4, 5, 6, 8 and 10 days after dosing. Six animals dosed only with 2% Tween were also sacrificed (‘Day 0’). The rats were anaesthetised with halothane and killed by exsanguination, blood being taken into EDTA-containing tubes from the posterior vena cava. At necropsy, splenic weights were recorded and portions of this tissue, together with portions of kidney and liver, were fixed in 4% buffered formaldehyde. Tissues were routinely processed and embedded in paraffin wax. Sections were stained with haematoxylin and eosin and by Perls’ Prussian blue reaction for examination by light microscopy. They were examined without reference to the treatment group.
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Splenic sinusoidal engorgement and ferric iron deposition in the spleen, liver and kidneys (as reflected by the intensity of Prussian blue staining) were scored on an arbitrary scale of 0 – 5, 0 indicating the absence of the specified histological feature and 5 its presence to a severe degree. Blood packed cell volumes were determined by the microhaematocrit technique and haemoglobin levels by the cyanmethaemoglobin method [53]. Heinz bodies were quantitated turbidometrically by measuring the change in optical density of red cell lysates at 700 nm following centrifugation [54]. Plasma samples were analysed for activities of aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase and for levels of urea and creatinine on a Boehringer-Mannheim Hitachi 705 random-access analyser at 30°C.
2.4. Experiments with BHA and dicoumarol BHA, as a solution in corn oil, was administered orally to groups of six rats at a dose of 500 mg/kg per day for 9 consecutive days. For examination of the effects of BHA per se, animals were sacrificed 5 days after the last dose of this substance. In the experiment on the effect of BHA on menadione toxicity, the naphthoquinone, at a dose of 6000 mmol/kg, was given by oral intubation 1 day after the last dose of BHA. The animals were killed 4 days later and in both these experiments the necropsy procedures and assays were as described above. In the study on the effect of BHA on hepatic DT-diaphorase activity, the animals were killed 24 h after the final dose and liver samples taken for immediate enzyme assay. Livers were homogenised in 0.25M sucrose and activities of DT-diaphorase determined by the method of Schor et al. [55], using an NADPH concentration of 75 mM. Experiments with dicoumarol were analogous to those with BHA, except that the test compound was administered by intraperitoneal injection twice, once in the afternoon and then again the following morning, as a suspension in corn oil, at a dose of 15 mg/kg. For measurement of DT-diaphorase activity, animals were killed 2 h after the second dose. In the experiment on the effect on menadione toxicity, the naphthoquinone, at a dose of 4000 mmol/kg, was given 2 h after the second dose of dicoumarol and the rats were killed 4 days later. In all experiments, an equal number of control animals were given corn oil alone according to the same dosing schedules as those employed for BHA and dicoumarol.
3. Results
3.1. Progression of the haemolytic anaemia induced by a single dose of menadione A single dose of 5000 mmol/kg of menadione led to a rapid increase in intra-erythrocytic Heinz bodies, as measured by the turbidity of lysed cells at 700 nm. Levels reached a maximum 2 days after dosing and then declined (Fig. 1A). Blood packed cell volumes (Fig. 1B) and haemoglobin levels (Fig. 1C) were
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significantly depressed 2 days after dosing and continued to decline, reaching a minimum at 4 – 6 days. Subsequently, levels rose again towards normal. Splenic weights were significantly increased 2 days after dosing and remained high throughout the experimental period (Fig. 1D). Splenic sinusoidal engorgement was evident 1 day after dosing (Fig. 2A), as were increased splenic levels of iron (Fig. 2B). Erythropoietic foci in the liver were not recorded until 3 days after menadione administration (Fig. 2C) although iron deposition in this organ was seen at 2 days (Fig. 2D). Iron deposition in the kidneys appeared much later, being first seen 5 days after dosing (Fig. 2E).
3.2. Effect of BHA and dicoumarol on hepatic le6els of DT-diaphorase Administration of BHA to rats increased hepatic DT-diaphorase levels by 275%. In contrast, dicoumarol decreased the hepatic activity of this enzyme by 65% (Table 1).
Fig. 1. Haematology and relative splenic weights of rats killed at various time intervals after a single dose of 5000 mmol/kg menadione. Data shown are the means and S.E.M. of the results from the six animals in each treatment group.
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Fig. 2. Histopathology of rats killed at various time intervals after a single dose of 5000 mmol/kg menadione. Lesions were scored on an arbitrary scale of 0 – 5, as described in Section 2. Data shown are the means and SE.M. of the scores for the six animals in each treatment group.
3.3. Effect of BHA and dicoumarol on haematological parameters and histology of rats Administration of BHA or dicoumarol to rats had no effect on blood packed cell volumes, haemoglobin levels, splenic weights or incidence of Heinz bodies and no histological changes were recorded in the spleen, liver or kidneys of the animals that
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would indicate a haemolytic effect (Table 2). No changes in plasma activities of aspartate aminotransferase, alanine aminotransferase or lactate dehydrogenase or plasma levels of urea and creatinine were recorded in the test animals and no degenerative changes were observed in the livers, spleens or kidneys of these animals (data not shown).
3.4. Effect of pre-treatment with BHA and dicoumarol on the toxicity of menadione to rats Severe haemolysis was observed in control rats killed 4 days after a dose of 6000 mmol/kg of menadione. Significant protection was given, however, by pre-treatment with BHA (Table 3). Blood packed cell volumes and haemoglobin levels were significantly higher in the treated animals, while Heinz body formation and splenic enlargement were lower. Less erythropoietic activity was recorded in the livers of animals pre-treated with BHA and both hepatic and renal iron storage was decreased. Menadione, at a dose of 4000 mmol/kg, produced a moderate degree of haemolysis in rats. Pre-treatment with dicoumarol increased the severity of the erythrocyte destruction, as reflected by significantly greater decreases in blood packed cell volumes and haemoglobin levels. Erythropoietic activity in the liver was also increased in animals receiving dicoumarol, as was the level of iron deposition in this organ (Table 4). The effects of menadione were restricted to erythrocytes. No plasma biochemical changes or histological alterations indicative of tissue damage was observed in the livers, kidneys or spleens of animals dosed with menadione alone or in those pre-treated with BHA or dicoumarol (data not shown).
4. Discussion In previous studies on the haemolytic action of menadione in animals, the test material was administered to the animals over a period of days or weeks [16–23]. However, in a study designed to test the effects of BHA and dicoumarol on menadione toxicity, prolonged dosing is not feasible, since it would be necessary to Table 1 Effect of BHA and dicoumarol on hepatic levels of DT-diaphorase in rats Treatment
Hepatic DT-diaphorase activity (U/g)
None (control) BHA Dicoumarol
42.0 9 5.5 157.7 9 10.6 14.89 1.5
Rats were dosed with BHA and dicoumarol as described in Section 2. Data shown are the means and S.E.M. of the results from the six animals in each treatment group.
43.391.0 45.1 9 0.6 42.89 0.8 42.79 0.3
Control DIC Control BHA
0.218 9 0.016 0.220 90.009
0.031 90.004 0.028 9 0.004 0.035 9 0.002 0.034 9 0.003
141 92 146 92 138 92 137 91
0.255 90.008 0.264 90.009
Relative splenic weight (g/ 100g body weight)
Haemoglobin Heinz bodies level (g/l) (DOD 700)
0.0 0.0
0.0 0.0
Splenic sinusoidal engorgementa
0.0 0.0
0.0 0.0
Hepatic erythropoiesisa
0.58 90.15 0.50 9 0.13
0.50 9 0.12 0.58 9 0.08
Splenic level of irona
0.0 0.0
0.0 0.0
Hepatic level of irona
0.0 0.0
0.0 0.0
Renal level of irona
Dicoumarol (DIC) and BHA were dosed according to the schedule described in Section 2. The corresponding control animals received the vehicle (corn oil) alone. Data shown are the means and S.E.M. of the results from the six animals in each treatment group. a Scored on an arbitrary scale of 0 – 5, as described in Section 2.
Packed cell volume (%)
Treatment group
Table 2 Haematology, relative splenic weights and hepatic, splenic and renal histology in control rats and in animals treated with BHA or dicoumarol
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Packed cell volume (%)
63 95
0.677 90.050
0.83 9 0.17*
1.67 9 0.33
Hepatic erythropoiesisa
2.679 0.21
2.839 0.31
1.839 0.17**
3.339 0.33
Splenic level Hepatic level of irona of irona
0.0
1.17 9 0.54
Renal level of irona
Rats were pre-treated with a solution of BHA in corn oil or with corn oil alone according to the dosing schedule described in Section 2. They were then challenged with a single dose of menadione at 6000 mmol/kg. Data shown are the means and S.E.M. of the results from the six animals in each treatment group. Figures marked with asterisks are significantly different from those of the control group, not pre-treated with BHA. * PB0.05; ** PB0.01; *** PB0.001. a Scored on an arbitrary scale of 0 – 5, as described in Section 2.
4.33 90.21
4.17 90.31
Relative splenic Splenic sinuweight (g/100g soidal enbody weight) gorgementa
0.343 90.028* 0.529 9 0.028*
0.438 9 0.010
Haemoglobin Heinz bodies level (g/l) (DOD 700)
BHA+ 32.19 1.1*** 101 94*** menadione
Control+ 22.1 91.3 menadione
Treatment group
Table 3 Effect of pre-treatment with BHA on the haemolytic activity of menadione
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37.391.3 31.291.3**
Control+ menadione DIC+ menadione
0.184 9 0.016 0.459 90.015 3.67 9 0.21
100 94*
Splenic sinusoidal engorgementa
0.171 90.019 0.444 90.030 3.50 90.22
Relative splenic weight (g/ 100g body weight)
113 9 4
Haemoglobin Heinz bodies level (g/l) (DOD 700)
1.58 90.15*
0.83 90.28
Hepatic erythropoiesisa
3.17 9 0.28
2.67 9 0.17
Splenic level of irona
2.41 9 0.15*
1.41 9 0.30
Hepatic level of irona
0.25 9 0.17
0.00
Renal level of irona
Rats were pre-treated with a solution of dicoumarol (DIC) in corn oil or with corn oil alone according to the dosing schedule described in Section 2. They were then challenged with a single dose of menadione at 4000 mmol/kg. Data shown are the means and SEM of the results from the six animals in each treatment group. Figures marked with asterisks are significantly different from those of the group not pre-treated with dicoumarol. * PB0.05; ** PB0.01. a Scored on an arbitrary scale of 0 – 5, as described in Section 2.
Packed cell volume (%)
Treatment group
Table 4 Effect of pre-treatment with dicoumarol on the haemolytic activity of menadione
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dose the naphthoquinone and the potential modifier simultaneously, with the possibility of interactions between them. Furthermore, dicoumarol itself is toxic when given over a prolonged period [56,57]. In order to avoid such problems, it was decided to pre-treat the animals with the modifiers and then challenge with a single dose of menadione. Since anaemia takes some time to develop after toxic challenge, it was necessary to establish the time course of menadione toxicity after dosing so that the most appropriate time for examining the effects of the modifiers could be established. Animals were therefore dosed with menadione and killed at various time intervals thereafter. Menadione exerts its toxic effect in animals through oxidative damage to erythrocytes, initiated by ‘active oxygen’ species generated through autoxidation of the semiquinone [19]. Oxidation of haemoglobin leads to the formation of granules of denatured haemoglobin (Heinz bodies) within erythrocytes. This, together with other oxidative changes within the cells, causes sequestration of the damaged erythrocytes within the splenic cords, where they are destroyed by phagocytic cells [58– 60]. Iron released from lysed erythrocytes is stored in the form of haemosiderin. The spleen is the primary site of storage, but when the capacity of this organ is exceeded, iron is taken up by the liver and kidneys and haemosiderin deposition, as reflected by Prussian blue staining, is observed in these tissues. In response to a decrease in the number of circulating erythrocytes, compensatory erythropoiesis occurs. In the rat, this takes place primarily in the spleen, although in severe anaemia, hepatic erythropoiesis also occurs. The increased activity of the spleen in both destroying and manufacturing erythrocytes results in enlargement of this organ. Such changes were clearly seen in the time course of menadione haemolysis. Soon after dosing, Heinz bodies appeared in the erythrocytes and blood packed cell volumes and haemoglobin levels began to fall as damaged cells were removed from the circulation. Splenic iron levels increased as iron released from the cells was stored and the weight of the spleen increased. The weight gain was associated with sinusoidal engorgement, attributable to increased levels of red pulp, which is involved in both erythroclasis and erythropoiesis. At later time intervals, Heinz body levels declined, as the oxidatively damaged cells were destroyed, and the compensatory erythropoiesis was very effective in reversing the decline in packed cell volumes and haemoglobin levels. The liver contributed to this process, since foci of erythropoiesis were observed. Iron storage in the liver and kidney was recorded toward the end of the experimental period, reflecting saturation of the storage capacity of the spleen. The maximum effects on blood packed cell volumes and haemoglobin levels were recorded between days 4 and 6. The effects of the possible modulating agents were therefore studied in animals pre-treated with these agents and killed 4 days after challenge with menadione. As expected from the results of previous studies [48,55] dicoumarol significantly decreased hepatic levels of DT-diaphorase while BHA caused a marked increase. Before investigating the effects of these substances on the haemolytic action of menadione, however, it was necessary to establish that these compounds themselves had no effect on red blood cells. Neither BHA nor dicoumarol when administered
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alone had any effect on blood packed cell volumes, haemoglobin levels or Heinz body production and there were no changes in splenic weight and no histological evidence of a toxic effect in spleen, liver or kidneys. As measured by several parameters of haemolysis, dicoumarol increased the toxicity of menadione to erythrocytes while BHA decreased the haemolytic effect of this substance. The toxicity of menadione can thus be modulated in vivo in the same way that it is in vitro. This is in accord with the observation that menadione is excreted as conjugates of menadiol in rats [34] and changes in the rate of reduction would be expected to change the rate of elimination of menadione. However, the relative rates of reduction and conjugation are not known and whether the observed modulation of menadione toxicity is due solely to effects upon DT-diaphorase activity cannot be stated with certainty. Although BHA and dicoumarol have been widely used as modulators of DT-diaphorase activity, their effects in animals are not restricted to this enzyme. It is possible that induction or inhibition of the enzymes responsible for hydroquinone conjugation (UDP glucuronosyl transferases and sulphotransferases) could contribute to the observed effects on menadione toxicity. It is known that BHA increases tissue activities of UDP glucuronosyl transferase in rats [61,62], but no information on the in vivo effects of dicoumarol on conjugating enzymes is available and results with this substance in vitro are equivocal. Dicoumarol inhibited the glucuronidation of 3-hydroxybenzpyrene, but it had no effect upon the conjugation of several other aglycones [63]. Studies on the pharmacokinetics of menadione under the influence of BHA and dicoumarol, together with an investigation of the rate-limiting step of menadione detoxification, are required to resolve this point. Experiments with other substances that are known to increase tissue levels of DT-diaphorase, such as butylated hydroxytoluene [61], disulfiram [64], dimethyl fumarate [65] and ethoxyquin [65] would also be valuable. Like BHA, none of these compounds is absolutely specific in its effect, but consistent protection against menadione toxicity by all these substances would provide further evidence for the involvement of DT-diaphorase in the detoxification of menadione. Haemolytic anaemia is commonly observed in patients treated with menadione in the therapy of cancer [5,7,9] and this limits the dose of the compound that can be administered. Although no direct information on the metabolism of menadione in humans is available, the observation that related naphthoquinones are excreted largely as glucuronides suggests that reduction to the hydroquinone does occur in man as in rats [66]. It is possible, therefore, that the problem of menadione toxicity in cancer patients could be ameliorated by pre-treatment with compounds that stimulate quinone reduction and conjugation. Conversely, it would be expected that concomitant treatment with the diaphorase inhibitor, Warfarin, as used in combination with menadione and other drugs in some treatment regimens [5,11] would increase the severity of menadione toxicity to red blood cells. The results of the present study show that BHA, which is known to increase tissue levels of DT-diaphorase, protects against the toxicity of menadione to rats. This may not, however, be a general phenomenon with all naphthoquinone derivatives. As discussed previously, the rapid reduction of menadione and the
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comparative stability of menadiol make this compound ideally suited to detoxification by 2-electron reduction. In vitro, redox cycling of this substance is progressively inhibited by increasing concentrations of DT-diaphorase [32]. In contrast, 2-hydroxy-1,4-naphthoquinone is but slowly reduced by DT-diaphorase and this substance forms a very unstable hydroquinone. Redox cycling of this substance is not inhibited by DT-diaphorase, but increases with increasing levels of the enzyme in vitro [32]. It is possible, therefore, that the toxicity of 2-hydroxy-1,4-naphthoquinone could be increased by increasing tissue levels of DT-diaphorase. Studies on this possibility are in progress.
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