It has already been reported that muscle necrosis induced by various phenylenediamine derivatives are correlated with their autoxidation rate. Now in a more detailed investigation of the cytotoxic mechanism using a model system of isolated hepatocytes and ring-methylated structural isomer durenediamine (DD) we have shown that under aerobic conditions, phenylenediamine induced cytotoxicity and ROS formation were markedly increased by inactivating DT-diaphorase but were prevented by a subtoxic concentration of the mitochondrial respiratory inhibitor cyanide. This suggests that the H2O2 generation could be attributed to a futile two electron redox cycle involving oxidation of phenylenediamine to the corresponding diimine by the mitochondrial electron transfer chain and re-reduction by the DT- diaphorase. The subcellular organelle oxidative stress effects leading to cytotoxicity has not yet been identified. Hepatocyte mitochondrial membrane potential was only slightly decreased by phenylenediamine before cytotoxicity ensued. However phenylenediamine induced lysosomal damage and hepatocyte protease activation. Endocytosis inhibitors, lysosomotropic agents or lysosomal protease inhibitors also prevented phenylenediamine induced cytotoxicity.
Furthermore desferoxamine (a ferric chelator), antioxidants or ROS scavengers (catalase, mannitol, tempol or dimethylsulfoxide) prevented phenylenediamine cytotoxicity. It is concluded that H2O2 reacts with lysosomal Fe2+ to form “ROS” which causes lysosomal lipid peroxidation, membrane disruption, protease release and cell death.
It has already been reported that muscle
necrosis induced by various phenylenediamine derivatives are
correlated with their autoxidation rate. Now in a more detailed
investigation of the cytotoxic mechanism using a model system
of isolated hepatocytes and ring-methylated structural isomer
durenediamine (DD) we have shown that under aerobic conditions,
phenylenediamine induced cytotoxicity and ROS formation were
markedly increased by inactivating DT-diaphorase but were
prevented by a subtoxic concentration of the mitochondrial
respiratory inhibitor cyanide. This suggests that the H2O2 generation
could be attributed to a futile two electron redox cycle
involving oxidation of phenylenediamine to the corresponding
diimine by the mitochondrial electron transfer chain and
re-reduction by the DT- diaphorase. The subcellular
organelle oxidative stress effects leading to cytotoxicity has
not yet been identified. Hepatocyte mitochondrial membrane
potential was only slightly decreased by phenylenediamine
before cytotoxicity ensued. However phenylenediamine induced
lysosomal damage and hepatocyte protease activation.
Endocytosis inhibitors, lysosomotropic agents or lysosomal
protease inhibitors also prevented phenylenediamine induced
cytotoxicity.
Furthermore desferoxamine (a ferric
chelator), antioxidants or ROS scavengers (catalase, mannitol,
tempol or dimethylsulfoxide) prevented phenylenediamine
cytotoxicity. It is concluded that H2O2 reacts
with lysosomal Fe2+ to form “ROS” which causes
lysosomal lipid peroxidation, membrane disruption, protease
release and cell death.
Phenylenediamines are present in hair dye
constituents (1), photographic developing agents in photography
(2), vulcanizing agents in rubber industry (3) and as
industrial antioxidants (4). Rats given subcutaneous injections
of N, N, N’, N’-tetramethylphenylenediamine (TMPD)
developed degeneration and dissolution of skeletal muscle
fibers, necrotic lesions on the tongue, cytoplasmic vacuolation
in cardiac muscle and lesions in the gastrocnemius (5,6,7).
TMPD was also cytotoxic towards cultured rat myofibers (8). The
ring-methylated structural isomer durenediamine
(2,3,5,6-tetramethylphenylenediamine (DD)) also caused severe
skeletal muscle necrosis in rats (9,10). The target site
specificity of the various phenylenediamines may involve
mitochondrial oxidation of these compounds (7, 9, 11). However,
the effectiveness of various ring-methylated phenylenediamines
at inducing muscle necrosis is related with their autoxidation
rate (9).
Previously it was shown that incubation of
isolated rat hepatocytes with DD results in a nearly
stoichiometric oxidation of GSH to GSSG, which rapidly effluxes
the cell (12). The generation of H2O2 that oxidized GSH resulted from a two
electron redox cycle involving oxidation of DD to the
corresponding diimine and its subsequent reduction (12).
In the following, however, we provide
evidence that the cytotoxicity of durenediamine and other
phenylenediamines may involve oxygen activation and ROS
formation caused by futile intracellular redox cycling that
includes oxidation by the
mitochondrial respiratory chain and
subsequent rereduction by DT-diaphorase. Eventually, the
hepatocyte is unable to maintain redox homeostasis and all of
the DD becomes oxidized. The cytotoxic process that causes
plasma membrane disruption is probably mediated by lysosomal
membrane damage caused by the ROS formation and release of
deadly proteases.
Experimental
Chemicals
Durenediamine
(2,3,5,6-tetramethylphenylenediamine), collagenase (from
Clostridium histolyticum), bovine serum albumin (BSA), Hepes,
trypan blue, d mannitol, dimethylsulfoxide, catalase,
superoxide dismutase, chloroquine diphosphate, methylamine HCl,
3 methyl adenine, monensin sodium, leupeptin, pepstatin,
tempol, ethyleneglycol bis (p aminoethyl ether) N,NN',N' tetra
acetic acid (EGTA), sodium pentobarbital and heparin were
obtained from Sigma (St. Louis, MO, USA). Acridine orange and
dichlorofluorescin diacetate was purchased from Molecular
Probes (Eugene, Ore, USA). Desferoxamine was a gift from Ciba
Geigy Canada Ltd. (Toronto, ON, Canada). All chemicals were of
the highest commercial grade available.
Animals
Male Sprague-Dawley rats (280-300 g), fed
with a standard chow diet and given water ad libitum, were used in
all experiments.
Isolation and incubation of hepatocytes
Hepatocytes were obtained by collagenase
perfusion of the liver as described by Pourahmad and
O’Brien, 2000 (13). Approximately 85-90% of the
hepatocytes excluded trypan blue. Cells were suspended at a
density of 106 cells/ml in round bottomed flasks rotating in a
water bath maintained at 37°C in Krebs-Henseleit buffer (pH 7.4),
supplemented with 12.5 mM Hepes under an atmosphere of 10% O2, 85% N2, 5% CO2. Each
flask contained 10 ml of hepatocyte suspension. Hepatocytes
were preincubated for 30 min prior to addition of chemicals.
Stock solutions of all chemicals (×100 concentrated for
the water solutions or ×1000 concentrated for the
methanolic solutions) were prepared fresh prior to use. To
avoid either non toxic or very toxic conditions in this study
we used ED50 concentration for DD on the isolated
hepatocytes (140 mM). The ED50 of a chemical in hepatocyte cytotoxicity
assessment technique (with the total 3 h incubation period), is
defined as the concentration which decreases the hepatocyte
viability down to 50% following the 2 h of incubation (14). In
order to determine this value for the investigated compound
dose-response curves were plotted and then ED50 was
determined based on a regression plot of three different
concentrations (data and curves not shown). For the chemicals
which dissolved in water, we added 100 ml sample of its
concentrated stock solution (×100 concentrated) to one
rotating flask containing 10 ml hepatocyte suspension. For the
chemicals which were soluble in methanol we prepared methanolic
stock solutions (×1000 concentrated), and to obtain the
required concentration in the hepatocytes, we added 10 ml samples of the
stock solution to the 10 ml cell suspension. Ten microlitres of
methanol did not affect the hepatocyte viability after 3 h
incubation (data not shown).
Cell viability
The viability of isolated hepatocytes was
assessed from the intactness of the plasma membrane as
determined by the trypan blue (0.2% w/v) exclusion test (13).
Aliquots of the hepatocyte incubate were taken at different
time points during the 3 hour incubation period. At least
80-90% of the control cells were still viable after 3 hours.
Determination of reactive oxygen
species “ROS”
To determine the rate of hepatocyte
“ROS” generation, dichlorofluorescin diacetate was
added to the hepatocyte incubate as it penetrates hepatocytes
and becomes hydrolysed to non-fluorescent dichlorofluorescein
The latter then reacts with “ROS” to form the
highly fluorescent dichlorofluorescein which effluxes the cell.
Hepatocytes (1´106 cells/ml) were suspended in 10 ml modified
Hank’s balanced salt solution (HBS), adjusted to pH 7.4
with 10 mM Hepes (HBSH) and were incubated with DD at 37°C for 3 hours.
After centrifugation (50 ´ g. 1 min), the cells were resuspended in HBS
adjusted to pH 7.4 with 50 mM Tris-HCl and loaded with
dichlorofluorescein by incubating with 1.6ml
dichlorofluorescein diacetate for 2 min at 37°C. The fluorescence
intensity of the “ROS” product was measured using a
Shimadzu RF5000U fluorescence spectrophotometer. Excitation and
emission wavelengths were 500nm and 520 nm, respectively. The
results were expressed as fluorescent intensity per 106 cells
(15).
Lysosomal membrane stability assay
Hepatocyte lysosomal membrane stability
was determined from the redistribution of the fluorescent dye,
acridine orange (16). Aliquots of the cell suspension (0.5 ml)
that were previously stained with acridine orange 5 mM, were separated
from the incubation medium by 1 minute centrifugation at 1000
rpm. The cell pellet was then resuspended in 2 ml of fresh
incubation medium. This washing process was carried out for two
times to remove the fluorescent dye from the media. Acridine
orange redistribution in the cell suspension was then measured
fluorimetrically using a Shimadzu RF5000U fluorescence
spectrophotometer set at 495 nm excitation and 530 nm emission
wavelengths.
Determination of proteolysis
To determine the rate of hepatocyte
"ROS" generation, dichlorofluorescin diacetate was
added to the hepatocyte incubate as it penetrates hepatocytes
and becomes hydrolysed to non-fluorescent dichlorofluorescein
The latter then reacts with "ROS" to form the highly
fluorescent dichlorofluorescein which effluxes the cell.
Hepatocytes (1×106 cells/ml) were suspended in 10 ml
modified Hank's balanced salt solution (HBS), adjusted to pH
7.4 with 10 mM Hepes (HBSH) and were incubated with DD at
37°C for 3 hours. After centrifugation (50 × g. 1
min), the cells were resuspended in HBS adjusted to pH 7.4 with
50 mM Tris-HCl and loaded with dichlorofluorescein by
incubating with 1.6 l dichlorofluorescein diacetate for 2 min
at 37°C. The fluorescence intensity of the "ROS"
product was measured using a Shimadzu RF5000U fluorescence
spectrophotometer. Excitation and emission wavelengths were
500nm and 520 nm, respectively. The results were expressed as
fluorescent intensity per 106 cells (15).
Statistical analysis
The statistical significance of
differences between the control and treatment groups in these
studies was determined using a one-way analysis of variance
(ANOVA) and the Bartlett’s test for homogeneity of
variances. Results represent the mean±standard error of
the mean (SEM) of triplicate samples. The minimal level of
significance chosen was P < 0.001.
Results and Discussion
When hepatocytes were incubated with 140 mM DD the formation
of “ROS” was increased very rapidly (peak in about
60 minutes) in a concentration dependent fashion (Fig. 1, Table
1). The antioxidants, a-tocopheryl succinate, catalase, superoxide
dismutase (SOD) and “ROS” scavengers (18), tempol,
mannitol and dimethylsulfoxide (DMSO) protected the hepatocytes
against DD induced cytotoxicity as well as “ROS”
generation. On the other hand catalase inhibitors, sodium azide
(19) and cyanamide (20) significantly increased DD induced
cytotoxicity, and “ROS” formation (Table 1).
Neither of these agents showed any toxic effect on hepatocytes
at concentrations used (data not shown).
The DT-diaphorase inhibitor, dicumarol
(12) markedly increased DD induced cell lysis and
“ROS” generation (Table 1). However the CYP2E1
inhibitor phenylimidazole (18, 19) and P450 reductase
inhibitor, diphenyliodonium chloride (DPI) (18, 19) did not
show any significant effect on DD induced cell lysis and
“ROS” formation (data not shown). On the other hand
subtoxic concentration of mitochondrial cytochrome oxidase
inhibitor, potassium cyanide (300 mM) protected hepatocytes against DD induced
cytotoxicity and “ROS” formation (Table 1).
Endocytosis inhibitors including lysosomotropic agents,
chloroquine (21), methylamine (22), monensin a Na+ ionophore
that inhibits hepatocyte endosomal acidification (23) and
3-Methyladenine, an inhibitor of hepatocyte autophagy (24) also
protected the hepatocytes against DD induced cell lysis and
“ROS” formation (Table 1). Neither of these agents
showed any toxic effect on hepatocytes at concentrations used
(data not shown).
When hepatocyte lysosomes were preloaded
with acridine orange, a release of acridine orange into the
cytosolic fraction ensued within 60 minutes after treating the
loaded hepatocytes with 140 mM DD (Table 2). The DD induced acridine orange
release was prevented by dimethylsulfoxide, mannitol, catalase,
superoxide dismutase (SOD) or the ferric chelator desferoxamine
(Table 2). All endocytosis inhibitors and potassium cyanide
(KCN) also inhibited DD induced acridine orange release (Table
2). On the other hand DT-diaphorase inhibitor, dicumarol
significantly increased DD induced acridine orange release
(Table 2).
Hepatocyte proteolysis as determined by
the release of the amino acid tyrosine into the extracellular
medium over 120 minutes was markedly increased when hepatocytes
were incubated with DD (Table 3). The DD induced tyrosine
release was completely prevented by the lysosomal protease
inhibitors; leupeptin (25) and pepstatin (26) (Table 3).
Dimethylsulfoxide, mannitol, catalase, SOD, deferoxamine,
potassium cyanide (KCN), 3-methyladenine, methylamine and
chloroquine also inhibited DD induced tyrosine release (Table
3). On the otherhand DT-diaphorase inhibitor, dicumarol
significantly increased DD induced tyrosine release (Table 3).
ROS formation contributes to DD induced
cell lysis was markedly increased following the treatment of
hepatocytes with DD and the antioxidants and “ROS”
scavengers prevented both DD induced “ROS”
formation and cytotoxicity (Table 1).
It was already suggested that an
oxidative stress without “ROS” generation is
responsible for DD induced cytotoxicity (12). However in our
study, we determined huge increase in “ROS”
formation following the treatment of hepatocytes with DD (Fig.
1). As shown here pretreatment of hepatocytes with subtoxic
concentration of KCN (an inhibitor of mitochondrial cytochrome
oxidase) prevented DD induced cytotoxicity, “ROS”
formation, lysosomal membrane damage and proteolysis (Tables 1,
2, and 3), which suggests that mitochondrial respiratory chain
cytochrome oxidase activates DD.
DD induced hepatocyte lysosomal disruption
ensued within 60 min following its addition (Table 2). A
similar release of acridine orange occurred when acridine
orange loaded hepatocytes were treated with hydrogen peroxide
generated by glucose/glucose oxidase (23, 27, 28) or
nitrofurantoin (29). Hepatocyte proteolysis markedly
increased following lysosomal disruption by DD which was
inhibited by the lysosomal protease inhibitors leupeptin and
pepstatin (Table 3). Furthermore, DD induced cytotoxicity was
also prevented by leupeptin or pepstatin (data not shown).
Besides, DD induced cytotoxicity, “ROS” formation,
lysosomal membrane damage and proteolysis were also prevented
by the hepatocyte endocytosis inhibitors; methylamine,
chloroquine, monensin and 3-methyladenine (Tables 1, 2, and 3).
Methylamine or chloroquine or the ferric
chelator desferoxamine also prevented hepatocyte cytotoxicity
induced by hydrogen peroxide generated by glucose/glucose
oxidase (23, 30), xanthine/xanthine oxidase (31) and
nitrofurantoin (29).
In conclusion as shown in Fig. 2 these
results suggest that DD induced hepatocyte toxicity involves
oxidation by mitochondrial respiratory chain cytochrome oxidase
to form the corresponding diimine which undergoes futile redox
cycling resulting in the formation of H2O2 which
is the cause of GSH oxidation. Then, H2O2
diffuses inside the lysosomes and interacts with lysosomal Fe2+/Cu+ leading
to hydroxyl radical formation (Haber-weiss reaction). Hydroxy
radicals cause lysosomal membrane damage and deadly protease
release.
References
(1) International Agency for Research on
Cancer. IARC Monographs on the
Evaluation of the Carcinogenic Risk of Chemicals to Man, Vol. 16, IARC, Lyon (1978)
(2) Liden C. Occupational dermatoses at a
film laboratory. Contact
Dermatitis (1984) 10: 77-87
(4) Rhee CK, Lattimer RP and Layer RW.
Antiozonants In: Kroschwitz JI. (Ed.) Encyclopedia of Polymer Science and Engineering, Wiley, New York (1985) 91-99
(5) Elias EA and Meijer AEFH. The increase
in activity of glucose-6-phosphate dehydrogenase and
6-phosphogluconate dehydrogenase in skeletal muscles of rats
after subcutaneous administration of N, N'-dimethyl-p-phenylenediamine.
Histochemistry (1981) 71: 543-558
(6) Jasmin G and Gareau R.
Histopathological study of muscle lesions produced by p-phenylenediamine
in rats. Br. J. Exp. Pathol. (1961) 42: 592-596
(7) Munday R, Manns E, Fowke EA and Hoggard
GK. Muscle necrosis by N-methylated p-phenylenediamines
in rats: structure-activity relationships and correlation with
free-radical production in vitro. Toxicology (1989)
57: 303-314
(8) Harauchi T and Hirata M. Effects of
p-phenylenediamines
and adriamycin on primary culture of rat skeletal muscle cells.
Toxicol. Lett. (1993) 66: 35-46
(9) Munday R, Manns E, Fowke EA and Hoggard
GK. Structure-activity relationships in the myotoxicity of
ring-methylated p-phenylenediamines in rats and correlation
with autoxidation rates in vitro.
Chem. Biol. Interact. (1990)
76: 31-45
(10) Draper RP, Waterfield CJ, York MJ and
Timbrell JA. Studies on the muscle toxicant 2, 3, 5,
6-tetramethyl p-phenylenediamine: effects on various biomarkers
including urinary creatine and taurine. Arch. Toxicol. (1994) 69: 111-117
(11) Munday R. Mitochondrial oxidation of
p-phenylenediamine
derivatives in vitro: structure-activity relationships and
correlation with myotoxic activity in vivo.Chem. Biol. Interact. (1992) 82: 165-179
(12) Sood C, Khan S and O'Brien PJ.
Phenylenediamine induced hepatocyte cytotoxicity redox. Cycling
mediated oxidative stress without oxygen activation. Biochim. Biophys. Acta (1997) 1335: 343-52
(13) Pourahmad J and O'Brien PJ. Contrasting
role of Na+ ions in modulating Cu2+ or Cd2+
induced hepatocyte cytotoxicity. Chem.
Biol. Interact. (2000) 126:
159-69
(14) Galati G, Teng S, Moridani MY, Chan TS
and O'Brien PJ. Cancer chemoprevention and apoptosis mechanisms
induced by dietary polyphenolics. Drug Metabol. Drug Interact. (2000) 17:311-49
(15) Shen HM, Shi CY, Shen Y and Ong CN.
Detection of elevated reactive oxygen species level in cultured
rat hepatocytes treated with aflatoxin B1. Free Radic. Biol. Med. (1996) 21: 139-146
(16) Pourahmad J, Ross S and O'Brien PJ.
Lysosomal involvement in hepatocyte cytotoxicity induced by Cu2+ but
not Cd2+. Free Radic.
Biol. Med. (2001) 30: 89-97
(17) Novak RF, Kharasch ED and Wendel NK.
Nitrofurantoin-stimulated proteolysis in human erythrocytes: a
novel index of toxic insult by nitroaromatics. J. Pharmacol. Exp. Ther. (1988) 247: 439-44
(18) Siraki AG, Pourahmad J, Chan TS, Khan S
and O`Brien PJ. Endogenous and endobiotic induced reactive
oxygen species formation by isolated hepatocytes. Free Radic. Biol. Med. (2002) 32: 2-10
(19) Hallinan T, Gor J, Rice-Evans CA,
Stanley R, O'Reilly R and Brown D. Lipid peroxidation in
electroporated hepatocytes occurs much more readily than does
hydroxyl-radical formation. Biochem.
J. (1991) 277: 767-71
(20) DeMaster EG, Redfern B, Shirota FN and
Nagasawa HT. Differential inhibition of rat tissue catalase by
cyanamide. Biochem. Pharmacol.
(1986) 35: 2081-5
(21) Graham RM, Morgan EH and Baker E.
Characterisation of citrate and iron citrate uptake by cultured
rat hepswcytes. J. Hepatol. (1998) 29: 603-13
(22) Luiken LL, Amts LM and Meijer AJ. The
role of the intralysosomal pH in the control of autophagic
protedytic flux in rat hepatocytes. Eur. J. Biochem. (1996)
235: 564-73
(23) Brunk UT, Zhang H, Roberg K and
Ollinger K. Lethal hydrogen peroxide toxicity involves
lysosattd iron-catalyzed reactions with membrane damage. Redox Report(1995) 1: 267-277
(24) Seglen PO. DNA ploidy and autophagic
protein degradation as determinants of hepatoceitular growth
and survival. Cell. Biol.
Toxicol. (1997) 13: 301-15
(25) Fengsrud M, Roos N, Berg T, LiouW, Slot
JW, and Seglen PO. Ultrastructural and immunocytochemical
characterization of autophagic vacuoles in isolated hepatocytes:
effects of vinblastine and asparagine on vacuole
distributions. Exp. Cell. Res. (1995) 221: 504-19
(26) Olav Berg T, Fengsrud M, Stromhaug PE,
Berg T and Seglen PO. Isolation and characterization of rat
liver amphisomes: fvidence for fusion of autophagosomes with
both early and late endosomes. J.
Biol. Chem. (1998) 273:
21883-92
(27) Garberg P and Hogberg J. Studies on Se
incorporation in selenoproteins; effects of peroxisome
proliferators and hydrogen peroxide generating system. Chem. Biol. Interact. (1992) 81: 291-306
(28) Latour I, Pregaldien JL and
Buc-Calderon P. Cell death and lipid peroxidation in isolated
hepatocytes incubated in the presence of hydrogen peroxide and
iron salts. Arch.Toxicol. (1992) 66: 743-9
(29) Pourahmad J, Khan S and O'Brien PJ.
Lysosomal oxidative stress cytotoxicity induced by
nitrofurantoin redox cycling in hepatocytes. Adv. Exp. Med. Biol. (2001)
500: 261-5
(30) Starke P, Gilbertson JD and Farber LL.
Lysosomal origin of the ferric iron required for cell lolling
by hydrogen peroxide. Biochem.
Biophys. Res. Commum. (1985)
133: 371-9
(31) Motoyama S, Saito S, Saito R, Minamiya
Y, Nakamura M, Okuyama M, Imano H and Ogawa J. Hydrogen
peroxide-dependent declines in Bcl-2 induces apoptosis in
hypoxic liver. J. Surg. Res. (2003) 110: 211-6
Pourahmad, J., Kobarfard, F., Shakoori, A. (2010). Lysosomal Oxidative Stress Cytotoxicity Induced By Para-phenylenediamine Redox Cycling In Hepatocytes. Iranian Journal of Pharmaceutical Research, Volume 3(Number 4), 193-199. doi: 10.22037/ijpr.2010.601
MLA
J Pourahmad; F Kobarfard; A Shakoori. "Lysosomal Oxidative Stress Cytotoxicity Induced By Para-phenylenediamine Redox Cycling In Hepatocytes". Iranian Journal of Pharmaceutical Research, Volume 3, Number 4, 2010, 193-199. doi: 10.22037/ijpr.2010.601
HARVARD
Pourahmad, J., Kobarfard, F., Shakoori, A. (2010). 'Lysosomal Oxidative Stress Cytotoxicity Induced By Para-phenylenediamine Redox Cycling In Hepatocytes', Iranian Journal of Pharmaceutical Research, Volume 3(Number 4), pp. 193-199. doi: 10.22037/ijpr.2010.601
VANCOUVER
Pourahmad, J., Kobarfard, F., Shakoori, A. Lysosomal Oxidative Stress Cytotoxicity Induced By Para-phenylenediamine Redox Cycling In Hepatocytes. Iranian Journal of Pharmaceutical Research, 2010; Volume 3(Number 4): 193-199. doi: 10.22037/ijpr.2010.601
)