JOURNAL OF APPLIED TOXICOLOGY. VOL. I ] ( ?

) , 85-90 (1991)

Haemolytic Activity and Nephrotoxicity of

2-Hydroxy-l,4-naphthoquinonein Rats
R. Munday,? B.L. Smith and E.A. Fowke
Ruakura Animal Research Centre. Ministry of Agriculture and Fisheries. Private Bag, Hamilton. New Zealand

Key words: 3-hydroxy- I .4-naphthoquinone: lawsonc; haemolysis: rend tuhular necrosis

The short-term toxicity of 2-hydroxy-1,4-naphthoquinone(lawsone) and 2-methyl-1,4-naphthoquinone(menadione) has been compared in rats.
2-Methyl-I ,4-naphthoquinone has been shown previously to cause haemolytic anaemia in animals, and this
was confirmed in the present experiment. 2-Hydroxy-l,4-naphthoquinonewas found also to cause haemolysis,
in a dose-dependent manner, as reflected by decreased blood packed cell volumes and haemoglobin levels and
by histopathological changes in spleen, liver and kidney. With both naphthoquinones, the haemolysis was of
the oxidative type, characterized by the presence of Heinz bodies within erythrocytes.
In contrast,
Haemolysis was the only toxic change identified in rats dosed with 2-methyl-l,4-naphthoquinone.
2-hydroxy-l ,il-naphthoquinone was not only a haemolytic agent but also a nephrotoxin, causing renal
enlargement, elevated plasma levels of urea and creatinine and histologically-identified tubular necrosis, largely
confined to the distal segment of the proximal convoluted tubules.
The relationship between the in vivo toxic effects of these naphthoquinones and previously-reported data
on their in vitro cytotoxic action is discussed.

INTRODUCTION

MATERIALS AND METHODS

2-Hydroxy-1.4-naphthoquinone(lawsone) is a natural
product obtained from plants of the genera Luwsonici
and Imputiens. ' Henna, the dried powdered leaves
of Luwsotiiu ulhu, contains ca. 1% 2-hydroxy-l,4naphthoquinone; this material has been used for
colouring the hair and skin for more than 3000 years'
and has long been used in folk
More
recently. evidence for the clinical effectiveness of henna
in the treatment of amoebiasis and moniliasis has been
and the use o f 2-hydroxy-1.4-naphthoquinone in the treatment of sickle cell anaemia is under
investigation. "
Despite its long history of use, very little information
on the toxicity of 2-hydroxy- 1,4-naphthoquinone is
available. Other naphthoquinones, however, are
known to be toxic and alkyl and hydroxyalkyl derivatives, such as 2-methyl-, 2-hydroxy-3-methyl- and 2hydroxy-3-(3-methylbut-2-enyl)- 1 . 4-naphthoquinone,
have been shown to cause haemolytic anaemia in
experimental animals.7-"' In each case, the haemolysis
was of the oxidative type, characterized by the presence
of Heinz bodies within erythrocytes; red blood cells
were the only reported target tissue of these substances.
In the studies described in the present report, the
short-term toxicity of 2-hydroxy- 1.4-naphthoquinone
has been investigated in rats. and compared with that
of the 2-methyl derivative (menadione). Like other
naphthoquinones, 2-hydroxy- I ,4-naphthoquinone induced oxidative haemolysis in vivo. In addition. this
substance induced renal tubular necrosis, an effect that
appears to be unique among the naphthoquinone
derivatives investigated so far.

Chemicals

t Author t o whom corrcspondcncc should he addrcswd

2-Hydroxy- and 2-methyl-l,4-naphthoquinonewere

purchased from Aldrich Chemical Company Inc.,
Milwaukee, WI, USA.
Animals and maintenance

Ten- to eleven-week-old female rats of the Ruakura

colony ot 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
Ltd., Hamilton) and water ud libirirriz. The environment
was maintained at 21-24"C, with an artificially-controlled 12-h light/dark cycle.
The test compounds were administered, as suspensions in 7% Tween 80, to groups of six rats by
oral intubation o n six consecutive days. 2-Methyl-l,4naphthoquinone was given at a single dose level of
750 Fmol kg-lday I , whereas the hydroxy compound
was administered at 750, 500, 250. 125 and
62.5 pmol kg Iday-l. A further group of 12 rats,
dosed with 2% Tween 80 alone. served as control. The
volume of material administered was kept constant at
2 ml kg-I.
Necropsy, histology, haematology and clinical
chemistry

On the 7th day of the experiment. surviving rats

were anaesthetized with halothane and killed by
exsanguination. Blood was taken into EDTA-containing tubes from the posterior vena cava. At necropsy,

86

R. MUNDAY ET

livers, spleens and kidneys were weighed and portions

of these tissues were fixed in 4 % buffered formaldehyde. Paraffin wax sections were stained with haematoxylin and eosin and by Perls' Prussian Blue reaction
for examination by light microscopy. Selected tissues
were stained by the periodic acid-Schiff reaction for
glycogen.
Sections were examined without reference to treatment group. Splenic sinusoidal engorgement, renal
tubular necrosis, tubular eosinophilic casts and iron
deposition (as reflected by the intensity and distribution
of Prussian Blue staining) in spleen, liver and kidneys
were scored on an arbitrary scale of 0-5, with 0
indicating the absence of the specified parameter and
5 its presence to a severe degree.
The packed cell volume of blood samples was
determined by the microhaematocrit technique,
while the haemoglobic level was assessed using
the cyanmethaemoglobin method. I I Heinz bodies in
erythrocytes were stained supravitally with Nile Blue
sulphate in blood films. A quantitative assessment of
Heinz bodies was made by a turbidometric method"
in which the number and size of Heinz bodies in the
cells is estimated by the magnitude of the decrease in
optical density at 700 nm after centrifugation.
Plasma samples were analysed for activities of
aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH),
lactate dehydrogenase (LDH) and hydroxybutyrate
dehydrogenase (HBDH) and for levels of urea and
creatinine using Boehringer test-kits on a BoehringerMannheim Hitachi 705 random-access analyser. All
assays were conducted at 30C.

RESULTS
All rats remained in good health until the 5th day of
the study. After this time, aninials receiving 3-hydroxy1,4-naphthoquinone at 750 kmol kg- 'day- I rapidly
lost weight and two animals in this group died following
the 6th dose.
At necropsy, the spleens of rats receiving 2-methyl1.4-naphthoquinone were enlarged and dark, as were
those of animals dosed with 2-hydroxy- 1.4-naphthoquinone at 750 and 500 kmol kg 'day I . The inner
surface of the skin of rats in the latter two groups was
yellow-brown in colour.

AL

Significant increases in splenic weight were recorded

in rats dosed with 2-methyl-l,4-naphthoquinoneand
with the 2-hydroxy compound at the two highest
levels. Animals in the latter groups also showed renal
enlargement, and the livers of rats given 2-hydroxy-1,4naphthoquinone at 750 pmol kg- 'day- I were heavier
than those of the controls (Table 1).
Decreased blood packed cell volumes and haemoglobin levels were observed in rats given 2-methyl-1,
4-naphthoquinone and the higher levels of the 2hydroxy derivative. Heinz bodies were observed in
stained erythrocytes from these animals and the presence of these inclusion bodies was reflected also in the
increased turbidity of red cell lysates (Table 2). No
increase in haemoglobin level was observed in the
plasma of treated animals.
Pronounced changes in plasma biochemical parameters were recorded in animals dosed with 2hydroxy-1.4-naphthoquinone. with increases in ALT,
AST. GLDH, LDH, HBDH, urea and creatinine being
recorded at dose levels of 500 and/or 750 pmol kg- '
day-'. N o alterations in plasma biochemistry were
seen in rats receiving the 2-methyl derivative (Table
3).
Splenic sinusoidal engorgement was identified in
animals dosed with both of the naphthoquinones,
together with enhanced iron deposition in the spleen
and liver. Increased levels of splenic and hepatic iron
were the most sensitive indicators of 2-hydroxy-l,4naphthoquinone toxicity, a significant effect being
recorded in animals receiving 250 pmol kg- 'day-' of
this substance. Iron deposition was recorded in the
kidneys of rats given the higher levels of 2-hydroxy-1,
4-naphthoquinone. but not in those receiving the 2methyl derivative (Table 4). Renal tubular necrosis
was identified in rats dosed with 250 prnol kg-'day-'
or more of 2-hydroxy- 1.4-naphthoquinone. Necrosis
was largely confined to the cortical zone immediately
adjacent to the corticomedullary junction, the location
of the distal segments (S,) of the proximal convoluted
tubules; with the exception of a single rat given
750 pmol kg-'day-' of the quinone. in which severe
necrosis of the collccting tubules was observed, changes
in distal convoluted tubules and collecting tubules were
limited to intraluminal deposition of eosinophilic debris
(Table 5 ) . No degenerative changes were observed in
the livers of the test rats and no difference in hepatic
glycogen levels was observed between control animals

Table 1. Relative organ weights of rats dosed with naphthoquinone derivatives for 6
days"
Naphthoquinone
derivative

Dose level
(pmol kg 'day ' )

Relative weight (g kg
Spleen
Liver

None (control)
2-Hydroxy
2-Hydroxy
2-Hydroxy
2-Hydroxy
2-Hydroxy
2-Methyl

2.17 2 0.06
2.23e0.12
2.3020.12
2.6420.13
4.42?0.31***
6.6920.74***
4.5420.38***

62.5
125
2 50
500
750
750

' body weight)

40.9 2 0.6
39.021.1
39.1 20.9
38.420.9
41.6? 1.7
50.02 1.9***
42.72 1.4

Kidneys
7.3020.16
7.61 20.21
7.6420.26
7.46 20.30
9.07?0.51***
11.9620.61* * *
6.8920.14

Results shown are the means and SEM of individual animal data. Values marked
with asterisks differ significantly (Student's t-test) from the control: * * * P i 0.001.

TOXICITY OF 7-HYDROXY-I .J-NAPHTHOQUINONE IN RATS

Table 2. Haematological parameters of rats dosed with naphthoquinone derivatives for 6 days
Naphthoquinone
derivative

Dose level
( k m o l kg day

None (control)
2-Hydroxy
2-Hydroxy
2-Hydroxy
2-Hydroxy
2-Hydroxy
2-Methyl

(%)

Parameters
Haemoglobin level
(g I l )

Heinz bodies
(OD700 m )

43.850.4
41.1 2 1.O
41.820.7
42.820.8
35.52 1.8***
22.5+2.4***
32.721.5***

15022
14423
14223
14323
117-+5***
81 212***
11424***

0.03020.003
0.02320.004
0.02220.004
0.027 20.002
0.03920.011
0.109 ?0.029***
0.09020.012***

Packed cell volume

l)

62.5
125
250
500
750
750

Results shown are the means and SEM of individual animal data. Values marked with asterisks differ
significantly (Students t-test) from the control: * * * P < 0.001.

Table 3. Plasma biochemical parameters of rats dosed with naphthoquinone derivatives for 6 days
Naphthoquinone
derivative
None (control)
2-Hydroxy
2-Hydroxy
2-H ydroxy
2-Hydroxy
2-Hydroxy
2-Methyl

Dose level
(kmol kg AST
day l )
(U I

l )

52+1
4622
4923
54+2
61 2 3
219258***
4522

62.5
125
250
500
750
750

ALT
(U I

1)

35.251.7
32.821.3
38.222.2
39.8-tl.6
39.222.2
41.021.7*
38.02 1.4

Parameters
HBDH
(U I l )
l)

GLDH
(U I 1 )

LDH
(U I

3.4+0.4
3.220.3
3.520.2
4.820.5
7.5+1.3***
8.5t0.5***
2.820.5

139213
130220
13029
133221
148212
2124?506***
148236

Urea
(mmol I

l)

32.52 2.4
5.720.3
30.224.0
5.420.1
29.522.6
4.520.3
3 1.8 2 4.6
5.920.3
34.3k3.4
10.522.0*
417.0~95.1*** 23.424.6***
34.826.7
5.020.4

Creatinine
(kmol I-)
38.822.5
45.32 1.1
40.720.6
45.822.1
74.0-+9.3***
103.5213.4**
45.52 1.5

Results shown are the means and SEM of individual animal data. Values marked with asterisks differ significantly (Students ftest) from the control: * P < 0.05; * * * P < 0.001,

Table 4. Splenic sinusoidal engorgement and levels of iron in spleen, liver and kidneys of rats dosed with
naphthoquinone derivatives for 6 days
Naphthoquinone
derivative

Dose level

None (control)
2-Hydroxy
2-Hydroxy
2-Hydroxy
2-Hydroxy
2-Hydroxy
2-Methyl

(Fmol kg day

62.5
125
250
500
750
750

l)

Splenic sinusoidal
engorgement

0.020.0
0.020.0
O.O?O.O
0.020.0
1.020.6
3.820.6***
4.3+0.3***

Spleen

Iron level
Liver

0.920.1
0.920.2
1.220.3
1.7?0.3**
3.220.3***
4.2 +0.3***
3.920.2***

0.020.0
0.020.0
0.1 20.1
0.4?0.2*
2.0?0.3***
3.720.4***
2.5?0.2***

Kidney
0.020.0

o.o-ro.0
0.020.0
O.O?O.O
0.220.1
1.3-t0.4***
O.O?O.O

Splenic sinusoidal engorgement was assessed in sections stained with haematoxylin and eosin, iron deposition
in sections stained by Perls Prussian Blue reaction. These parameters were scored on an arbitrary scale of 0-5
and results shown are the means and SEM of individual animal data. Values marked with asterisks differ
significantly (randomization test) from the control: * P < 0.05; * * P < 0.01; * * * P < 0,001.
a

and those receiving 750 pmol kg- Iday- I of 2-hydroxy1,4-naphthoquinone.

DISCUSSION
The results of the present study confirm the previously
reported haemolytic activity of 2-methyl-] ,4-naphthoquinone7. and show that the 2-hydroxy derivative is
also a haemolytic agent in rats. In neither case was

there evidence of haemoglobinaemia, indicating that

the haemolysis was not intravasculiir but was due to
destruction of damaged erythroctyes by cells of the
reticuloendothial system. l 3 The spleen is the primary
site of erythroclasis in the
and this organ also
plays a major role in erythropoiesis.l5.Ih Accelerated
splenic erythropoiesis leads to engorgement of red pulp
sinusoids, associated with increased splenic weight
and darkening of the
as observed in the
naphthoquinone-dosed rats of the present experiment.
The iron deposition recorded in the spleens and livers

88

R. M U N D A Y E T A L

Table 5. Histological changes in the kidneys of rats dosed with naphthoquinone

derivatives for 6 days"
Naphthoquinone
derivative

Dose level
(pmol kg day^')

Tubular
necrosis

Tubular
eosinophilic debris

None (control)
2-Hydroxy
2-Hydroxy
2-Hydroxy
2-Hydroxy
2-Hydroxy
2-Methyl

62.5

o.o*o.o

O.OtO.0
O.O?O.O

125
250
500
750
750

o.o*o.o

o.oto.0
0.5?0.2**
2.2?0.4***

3.3?0.6***

o.o+o.o

o.oto.0
0.2tO.1
1.1 ?0.4***
2.0+0.5***
O.OtO.0

Tubular necrosis and debris was scored on an arbitrary scale of 0-5 and results
shown are the means and SEM of individual animal data. Values marked with
asterisks differ significantly (randomization test) from the control: * * P < 0.01,
* * * P < 0.001.
a

of these animals is attributable to storage of surplus

metal following breakdown of haemoglobin released
from erythrocytes in the reticuloendothelial system. " ' . I 7
The iron deposition in the kidneys of rats given 2hydroxy-l,4-naphthoquinonemay be of the same origin, consistent with the higher haemolytic activity of
the former compound, although a n association with
the nephrotoxic action of this substance cannot be
ruled out. The haemolysis induced by both quinones
was associated with Heinz body formation, indicative
of oxidative damage to the erythrocyte. I" There is
evidence that such oxidative damage, by decreasing
cellular deformability, is the cause of erythrocyte
destruction in viva."'.''
Renal damage was recorded in rats receiving the
higher dose levels of 2-hydroxy- I ,4-naphthoquinone,
associated with increased kidney weights and elevated
plasma concentrations of urea and creatinine. The
observed increase in plasma AST activity is also
consistent with nephrotoxicity, and the elevated activities of LDH and HBDH may. likewise. have been of
renal origin. Histologically, necrosis of the distal
portion of the proximal convoluted tubule was
observed, accompanied by tubular deposition of eosinophilic material; such changes are similar to those
recorded with other nephrotoxic substances, such a s
bromobenzene," acetaminophen.'3 unsaturated aliphatic halides'4,'5 and p-aminophenol.'" In animals
receiving 750 pmol kg 'day-' of 2-hydroxy-l,4-naphthoquinone. renal damage was severe and renal failure
is considered to be the most likely cause of the two
deaths in this group. At this same dose level, the 2methyl derivative was without effect upon the kidney.
The livers of rats dosed with 2-hydroxy- 1,4-naphthoquinone at 750 pmol kg-.'day-' were enlarged and
slight increases in the plasma activities of the comparatively liver-specific enzymes ALT and GLDH were
recorded. On histological examination, however, there
was no evidence of degenerative change, glycogen
storage or cellular hypertrophy in these livers and no
explanation can be offered for the observed weight
and biochemical changes.
Haemolysis appears to be a common toxic effect of
naphthoquinones, although whether the same niechanism of toxicity applies in all cases is not known. The
question also arises as to why the 2-hydroxy derivative
~

is different, this being the only naphthoquinone

reported so far to cause renal damage.
It? vitro studies have identified two major mechanisms
of quinone cytotoxicity. The importance of oxidative
damage in initiating the harmful effects of such
compounds has been s t r e s ~ e d ; ' ~ .such
' ~ damage arises
from generation of 'active oxygen' species (superoxide
radical, hydrogen peroxide and hydroxyl radical)
through 1-electron redox cycling of the quinone.
Secondly, reaction of quinones with cellular nucleophiles, particularly thiols. may be involved in their
cytotoxic action and both the arylation of protein thiol
groups'" and the formation of glutathione conjugates3"
may contribute to quinone toxicity.
The haemolysis induced ill v i w by 2-methyl-l.4naphthoquinone can be rationalized in terms of a direct
oxidative effect of the quinone upon erythrocytes.31
Haemoglobin mediates the 1-electron reduction of this
substance, initiating a redox cycle for the production
of 'active oxygen' species.3' Hydrogen peroxide is
detected readily in erythrocytes incubated with low
levels of 2-methyl-] .4-naphthoquinone in vitro,32 and
such cells also suffer oxidative damage, reflected by
methaemoglobin production, Heinz body formation
and glutathione
In contrast, 2-hydroxyI .4-naphthoquinone causes little or no oxidative damage to erythrocytes in vitro. No haemoglobin oxidation
was recorded in erythrocytes incubated with this
quinone, even at high concentration, and little hydroeen peroxide formation or glutathione depletion was
kbserved..3"The haemolytic activity of the latter compound does not. therefore. result from a direct effect
upon erythrocytes. However. as in the case of the 2methyl derivative, the haemolysis induced by 2hydroxy-l,4-naphthoquinone is of the oxidative type,
and it is therefore likely that 'active oxygen' species
are again involved; the inertness of this substance in
vitro suggests that it undergoes metabolic activation to
a n oxidant compound it7 \ii!o. One possible candidate is
1.2.4-trihydroxynaphthalene, the 2-electron reduction
product of the quinone. 2-Electron reduction of naphthoquinones is generally considered to be a detoxication
reaction. yielding comparatively stable hydroquinones
that can be conjugated and excreted?' 1.2.4-Trihydroxynaphthalene, however, is a very unstable substance that readily undergoes autoxidation. with

TOXICITY OF 2-HYDROXY-I .J-NAPHTHOOUINONE I N RATS

concomitant production of superoxide radical and

hydrogen peroxide.3" Studies on the it1 vitro and in
vivo toxicity of this substance would be of interest.
The comparatively high degree of ionization of 2hydroxy-l,4-naphthoquinone may facilitiate its uptake
by the kidney, although selective uptake cannot explain
fully the renal damage caused by this substance.
Nephrotoxicity commonly involves binding of toxins
to renal macromolecule^^^ and, in the case of benzoquinones and aliphatic halides, this has been shown to
occur following intrarenal metabolism of the corresponding glutathione conjugates.3".30,3' 2-Hydoxy-l,4naphthoquinone does not, however, react with cellular
nucleophiles,4' nor does it undergo conjugate formation
with g1utathione.j' Furthermore, whereas 2-methyl-1,
4-naphthoquinone is very toxic to isolated kidney cells
and hepatocytes, the 2-hydroxy compound is relatively
harmless.",34 Again, metabolic activation is likely, and
studies in this area are in progress.

89

In the present study, the no-effect level of 2-hydroxy1,4-naphthoquinone was 125 pmol kg- 'day-', equivalent to 21.8 mg kg-lday-'. Longer term studies with
this compound, to define more accurately the safety
factors, would be advisable before any extension of
cosmetic or therapeutic use is considered. Toxicity
studies on 2-hydroxy-3-alkyl-l,4-naphthoquinones,
which are under investigation as pesticidesJ5.j6 and as
p h a r m a ~ e u t i c a l s , ~ and
~ - ~ on
" other hydroxynaphthoquinones would be valuable also in order to establish
structure-activity relationships and to determine if any
of these, like the parent compound. are nephrotoxic
in vivo.
Acknowledgements
The assistance of Gavin Hoggard, Pat Paul, Glenda Smith, Basil
Young and Isahcllc Gravctt is gratefully acknowledged.

REFERENCES

1. R. H. Thomson, Naturally Occurring Quinones. 2nd Edn,

pp. 198-366. Academic Press, London (1971).
2. G. R. Hughes, The cosmetic arts in ancient Egypt. J. SOC.
Cosmet. Chem. 10, 159-176 (1959).
3. J. A. Duke, CRC Handbook of Medicinal Herbs, p. 274. CRC
Press, Boca Raton, FL (1985).
4. M. E. Hanke and S. M. Talaat, The biochemistry and
physiology of henna (Lawsonia alba): its use as a remedy
for intestinal amoebiasis. froc. R. SOC. Trop. Med. Hyg.
55, 56-62 (1961).
5. S. M. Talaat, Henna for intestinal moniliasis. Br. Med. J.
2, 944-945 (1960).
6. H. Chang and S. E. Suzuka, Lawsone (2-OH-1.4naphthoquinone) derived from the henna plant increases
the oxygen affinity of sickle cell blood. Biochem. Biophys.
Res. Commun. 107, 602-608 (1982).
7. S. Ansbacher, W. C. Corwin and B. G. H. Thomas, Toxicity
of menadione, menadiol and esters. J. Pharm. Exp. Ther.
75, 111-124 (1942).
8. E. H. Hatton, A. Dodds, H. C. Hodge and L. S. Fosdick, The
effect of prolonged ingestion of synthetic vitamin K in rats.
J. Dent. Res. 24, 283-295 (1945).
9. H. Molitor and H. J. Robinson, Oral and parenteral toxicity
of vitamin K,, phthiocol and 2-methyl-l,4-naphthoquinone.
Proc. SOC.Exp. Biol. Med. 43, 125-128 (1940).
10. R. K. Morrison, D. E. Brown, J. J. Oleson and D. A. Cooney,
Oral toxicology studies with lapachol. Toxicol. Appl. Pharmacol. 17, 1-11 (1970).
11. K. A. Evelyn and H. T. Malloy, Microdetermination of
oxyhemoglobin, methemoglobin, and sulfhemoglobin in a
single sample of blood. J. Biol. Chem. 126, 655662 (1938).
12. C. C. Winterbourn, Protection by ascorbate against acetylphenylhydrazine-induced Heinz body formation in glucose6-phosphate dehydrogenase deficient erythroctyes. Br. J.
Haematol. 41, 245252 (1979).
13. R. A. Cooper and J. H. Jandl, Destruction of erythrocytes.
In Hematology, 3rd Edn, ed. by W. J. Williams, pp. 377-388.
McGraw Hill, New York (1983).
14. E. A. Azen and R. F. Schilling, Extravascular destruction of
acetylphenylhydrazine-damaged erythrocytes in the rat. J.
Lab. Clin. Med. 63, 122-136 (1964).
15. E. A. Azen and R. F. Schilling, Role of the spleen in
acetylphenylhydrazine (APH) anemia in rats. J. Lab. Clin.
Med. 62, 59-71 (1963).
16. B. I. Lord, Erythropoietic cell proliferation during recovery
from acute haemorrhage. Br. J. Haematol. 13, 160-167
(1967).
17. F. P. Jenkins, J. A. Robinson, J. B. M. Gellatly and G. W.
A. Salmond, The no-effect dose of aniline in human subjects
and a comparison of aniline toxicity in man and the rat.
Food Cosmet. Toxicol. 10, 671-679 (1972).

18. J. F. Schmedtje and W. Andrew, Effect of phenylhydrazine

on the rabbit appendix and spleen. Toxicol. Appl. PharmaC O / . 13, 43-49 (1968).
19. J. D. Harley and A. M. Mauer, Studies on the formation of
Heinz bodies. II. The nature and significance of Heinz
bodies. Blood 17, 418-433 (1961).
20. J. Palek and S. E. Lux, Red cell membrane skeletal defects
in hereditary and acquired hemolytic anemias. Semin.
Hematol. 20, 189-224 (1983).
21. W. H. Reinhart, L. A. Sung and S. Chien, Quantitative
relationship between Heinz body formation and red blood
cell deformability. Blood 68, 1376-1383 (1986).
22. A. F. Casini, M. Ferrali, A. Pompslla, E. Maellaro and
M. Comporti, Lipid peroxidation and cellular damage in
extrahepatic tissues of bromobenzene-intoxicated mice.
Am. J. Pathol. 123, 520-531 (1986).
23. R. J. McMurtry, W. R. Snodgrass and J. R. Mitchell, Renal
necrosis, glutathione depletion, and covalent binding after
acetaminophen. Toxicol. Appl. fharmacol. 46, 87-100
(1978).
24. J. Ishmael, I. Pratt and E. A. Lock, Necrosis of the pars
recta (S, segment) of the rat kidney produced by hexachloroI-3-butadiene. J. Pathol. 138, 99-113 (1982).
25. D. Reichert, D. Ewald and D. Henschler, Generation and
inhalation toxicity of dichloroacetylene. Food Cosmet. Toxi c ~ /13,
. 511-515 (1975).
26. C. R. Green, K. N. Ham and J. D. Tange, Kidney lesions
induced in rats by p-aminophenol. Br. Med. J. 1, 162-164
(1969).
27. M. T. Smith, C. G. Evans, H. Thor and S. Orrenius, Quinoneinduced oxidative injury to cells and tissues. In Oxidative
Stress, ed. by H. Sies, pp. 91-113. Academic Press, London
( 1985).
28. G. Powis, Metabolism and reactions of quinoid anticancer
agents, fharmacol. Ther. 35, 57-162 (1987).
29. T. W. Gant, D. N. Ramakrishna Rao, R. P. Mason and G. M.
Cohen, Redox cycling and sulphydryl arylation; their relative
importance in the mechanism of quinone cytotoxicity to
isolated hepatocytes. Chem.-Biol. Interact. 65, 157-173
(1988).
30. T. J. Monks and S. S. Lau, Sulphur conjugate-mediated
toxicity. In Reviews in Biochemical Toxicology, Vol. 10, ed.
by E. Hodgson, J. R. Bend and R. M. Philpot, pp. 41-90.
Elsevier, New York (1989).
31. B. Godlberg and A. Stern, Production of superoxide anion
during the oxidation of hemoglobin by menadione. Biochim.
Biophys. Acta 437, 628-632 (1976).
32. G. Cohen and P. Hochstein, Generation of hydrogen peroxide in ervthrocvtes bv hemolvtic
aqents.
Biochemistrv 3,
.
895-900 (1964). '
33. J. A. Mezick, C. T. Settlemire, G. P. Brierley, K. P. Barefield,

90

34.
35.
36.
37.

38.

39.
40.

41.

R. M U N D A Y E T A L
W. N. Jensen and D. G. Cornwell, Erythrocyte membrane
interactions with menadione and the mechanism of menadione-induced hemolysis. Biochirn. Biophys. Acta 219,
361-371 (1970).
A. Miller and H. C. Smith, The intracellular and membrane
effects of oxidant agents on normal red cells. Br. J.
Haernatol. 19, 417-428 (1970).
L. Magos, Globin-haemochromogen formation caused by
quinones. Biochirn. Biophys. Acta 90, 55-61 (1964).
E. Kruger-Zeitzer, S. G . Sullivan, A. Stern and R. Munday,
Effects of 1.4-naphthoquinone derivatives on red blood cell
metabolism. J. Appl. Toxicol. 10, 129-133 (1990).
C. Lind, P. Hochstein and L. Ernster, DT-Diaphorase as
a quinone reductase: a cellular control device against
semiquinone and superoxide radical formation. Arch.
Biochern. Biophys. 216, 178-185 (1982).
E. Cadenas, D. Mira, A. Brunmark, C. Lind, J. Segura-Aguilar
and L. Ernster, Effect of superoxide dismutase on the
autoxidation of various hydroquinones-a possible role
of superoxide dismutase as a superoxide: semiquinone
oxidoreductase. Free Radical Biol. Med. 5, 71-79 (1988).
G . F. Rush, J. H. Smith, J. F. Newton and J. B. Hook,
Chemically induced nephrotoxicity: role of metabolic activation. CRC Crit. Rev. Toxicol. 13, 9S160 (1984).
S. S. Lau, B. A. Hill, R. J. Highet and T. J. Monks, Sequential
oxidation and glutathione addition to 1,4-benzoquinone:
correlation of toxicity with increased glutathione substitution. Mol. Pharrnacol. 34, 829-836 (1988).
S. S. Lau and T. J. Monks, The in vivo disposition of 2br~mo-[~C]hydroquinoneand the effect of y-glutamyl

transpeptidase inhibition. Toxicol. Appl. Pharrnacol. 103,

121-132 (1990).
42. M. dArcy Doherty, A. Rodgers and G. M. Cohen, Mechanisms of toxicity of 2- and 5-hydroxy-l,4-naphthoquinone;
absence of a role for redox cycling in the toxicity of 2hydroxy-1,4-naphthoquinone to isolated hepatocytes. J.
Appl. TOX~CO~.
7, 123-129 (1987).
43. G. D. Buffinton. K. Ollinger, A. Brunmark and E. Cadenas,
DT-diaphorase-catalysed reduction of 1,4-naphthoquinone
derivatives and gluthathionyl-quinone conjugates. Effect of
substituents on autoxidation rates. Biochem. J. 257,561-571
(1989).
44. T. W. Jones, H. Thor and S. Orrenius, In vitro studies
of mechanisms of cytotoxicity. Food Chern. Toxicol. 24,
769-773 (1986).
45. N. Jacobsen and L. K. Pedersen, Activity of 2-(l-alkenyl)3-hydroxy-1,4-naphthoquinones and related compounds
against Musca dornestica. Pestic. Sci. 17, 51 1-516 (1986).
46. N. Jacobsen and A. Wengel, Fungicidal activity of 2(1alkenyl)-3-hydroxy-l,4-naphthoquinones and related cornpounds. Pestic. Sci. 17, 686-690 (1986).
47. J. L. Vennerstrom and J. W. Eaton, Oxidants, oxidant drugs
and malaria. J. Med. Chem. 31, 1269-1277 (1988).
48. R. Docampo and S. N. J. Moreno, Free-radical intermediates
i n the antiparasitic action of drugs and phagocytic cells. In
free Radicals in Biology, Vol. 6, ed. by W. A. Pryor,
pp. 243-288. Academic Press, New York (1984).
49. G. Powis, Free radical formation by antitumor quinones.
free Radical Biol. Med. 6, 63-101 (1989).