Toxicology Letters 119 (2001) 59–70

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Pharmacological interventions of cyanide-induced cytotoxicity

and DNA damage in isolated rat thymocytes and their
protective efficacy in vivo
R. Bhattacharya *, P.V. Lakshmana Rao
​ ​ision of Pharmacology and Toxicology​, ​Defence Research and De​6​elopment Establishment,​ ​Jhansi Road​,
Di6
Gwalior ​474 002 (​M​.​P​.​)​, ​India

Received 11 July 2000; received in revised form 13 November 2000; accepted 13 November 2000

Abstrac
t

Cyanide inhibits the mitochondrial respiratory chain enzyme cytochrome oxidase causing histotoxic hypoxia. It is
primarily considered as a neurotoxin but its other toxic manifestations are also well documented. Cyanide-induced apoptosis
in neuronal cells has also been demonstrated recently. At the same time we also reported that potassium cyanide (KCN)
produces extensive cytotoxicity and DNA fragmentation in rat thymocytes. The DNA damage was sensitive to elevated levels
of extracellular Ca​2+ ​and was attenuated by Zn​2+ ​(modulator of Ca​2+​-dependent endonuclease), ​N​-acetylcysteine (free radical
scavenger) and diltiazem (Ca​2+ ​channel blocker). In a continuation of this work, in the present study we have shown that the
cytotoxicity and DNA fragmentation induced by 5 mM KCN was preceded by loss of mitochondrial integrity (MTT assay and
rhodamine-123 staining) and nuclear viability (propidium iodide uptake) which were mediated by generation of reactive
oxygen species (DCHF-DA staining). The DNA damage was also accompanied by nuclear fragmentation (Hoechst 33342
staining), a phenomenon that characterises the ‘apoptotic’ type of cell death. The in vitro toxic insult of KCN was challenged
by pre-treatment (0.5 h), simultaneous treatment or post-treatment (0.5–3 h) of various pharmacological agents viz., Trolox​®
(antioxidant), EGTA (Ca​2+ ​modulator) and aurintricarboxylic acid (ATA; Ca​2+​/Mg​2+​-dependent endonuclease inhibitor). In
addition, Quercetin (antioxidant) was tested as simultaneous treatment alone and was found to be ineffective. On the basis of
various biochemical indices and DNA fragmentation (quantitative and qualitative), simultaneous treatment of Trolox​® ​was
found to be the most effective in attenuating cyanide toxicity in vitro. This protection can be attributed to interventions in
oxidative stress-mediated cell injury which is an early event preceding DNA damage.

Abbre6​ ​iations:​ ​ATA, aurintricarboxylic acid; DCF, 2​%​,7​%​-dichlorofluoroscein; DCFH-DA, 2​%​,7​%​-dichlorofluoroscin

diacetate; DCFH, 2​%​,7​%​-dichlorofluoroscin; EGTA, ethylene glycol-bis (​b​-aminoethyl ether)-​N​,​N,​ ​N​%​N​%​-tetra acetic acid; EMEM, Eagle’s
minimum essential medium; HBSH, Hank’s balanced salt solution–HEPES; HBSS, Hank’s balanced salt solution; HO342, Hoechst 33342;
KCN, potassium cyanide; KHB, Kreb’s–Hensleit buffer; LDH, lactate dehydrogenase; MTT, 3-4,5-dimethyl thiazol-​Z-​ yl)-
2,5-diphenyltetrazolium bromide; NAC, ​N-​ acetyl cysteine; PBS, phosphate buffered saline; PI, protection index; ROS, reactive oxygen
species.
* Corresponding author. Tel.: +91-751-340245, 341980; fax: +91-751-341148. ​E-​ ​mail
address:​ ​drde@gwr1.dot.net.in (R. Bhattacharya).

0378-4274/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII:
S0378-4274(00)00309-X

R.​ 60​Bhattacharya,​ ​P.​ ​V​. ​Lakshmana Rao ​/ ​Toxicology Letters 1

​ 19 (2001) 59–70 ​Both EGTA and ATA could not prevent this damage.

Trolox​® ​also increased the LD​50 of

​ KCN in mice 2.5-fold as compared to 1.8- and 1.6-fold for EGTA and ATA, respectively.
© 2001 Elsevier Science Ireland Ltd. All rights reserved.

Keywords​: ​Cyanide; Cytotoxicity; DNA fragmentation in vitro; Protection in vitro and in vivo
rat thymocytes in vitro; (2) interven- tion of these
ochemical processes by pre-treat-
1. simultaneous treatment or post-treatment of
Introduction s pharmacological agents, viz. Trolox​® ​and
etin (antioxidants), EGTA (Ca​2+ ​chelator) and
Numerous biochemical, morphological and ricarboxylic acid (Ca​2+​/ Mg​2+​-dependent
physiological studies have demonstrated that cya- uclease inhibitor); and (3) the effect of these
nide is a potent selective neurotoxin. Its toxicity is in challenging cyanide- induced acute toxicity
mediated through histotoxic hypoxia, consequent to o.
mitochondrial dysfunction. Although many other
effects of cyanide have been documented, till
recently its DNA damaging property remained terials and methods
cryptic (Ballantyne, 1987). In light of the fact that
cyanide produces various biochemical alterations
hemicals
which are associated with DNA damage, we re-
cently reported that cyanide induces extensive DNA
Trolox​®
damage in rat thymocytes in vitro. The DNA damage
droxy-2,5,7,8-tetramethylchro- man
was sensitive to extracellular Ca​2+ ​and was attenuated
oxylic acid), quercetin dihydrate (3-
by Zn​2+ ​(modulator of Ca​2+ ​/Mg​2+​-dependent
%​,5,7-pentahydroxy flavone), EGTA (ethylene
endonuclease), ​N-​ acetyl cys- teine (free radical
-bis (​b-​ aminoethyl ether)-​N​,​N,​ ​N​%N ​ %​ ​-tetra
scavenger) and diltiazem (Ca​2+ ​channel blocker). The
acid), aurintricarboxylic acid (ATA), MTT
DNA damage accompanied by cytotoxicity was also
-dimethyl thiazol-​Z-​ yl)-2,5-diphenyltetra-
observed in BHK-21 (baby hamster kidney) cell line
m bromide), ethidium bromide, propidium
(Bhattacharya and Lakshmana Rao, 1997).
, rhodamine 123, Hoechst 33342 (HO342),
Cyanide-induced lipid peroxidation in vitro and its
e type I-A and agarose were purchased from
challenge by antioxi- dants, namely ​N​-acetyl cysteine
, USA. DCFH-DA (2​%​,7​%-​ dichlor-
and curcumin, has also been published recently
oscin diacetate) was from Molecular Probes,
(Bhattacharya et al., 1999). The cascading effects of
and potassium cyanide (KCN) was from
cyanide-in- duced impairment of mitochondrial
k, Germany. Eagle’s Minimum Essen- tial
energy pro- duction include failure of ionic
um (EMEM), Hank’s balanced salt solu- tion
homeostasis, acidosis, elevated Ca​2+ ​levels and lipid
S), phosphate buffered saline (PBS), glutamine,
peroxida- tion leading to activation of proteases,
mycin and Eosin Y were from Hi Media India
lipases, xanthine oxidases, etc. (Maduh, 1989). All
Bombay and other chemicals of highest purity
these events and many others like activation of Ca​2+​/
from Merck or Qualigen India Ltd. Lactate
Mg​2+​-dependent endonucleases are implicated in
rogenase (LDH) diagnostic kit was purchased
DNA damage (McConkey et al., 1989; Sunder- man,
Ranbaxy India Ltd., India.
1995). In light of the above, the present study was
designed to address the following: (1) focus on
nimals
cyanide-induced mitochondrial and nu- clear
dysfunction, and implication of reactive oxy- gen
species preceding the cytotoxicity and DNA damage Male Wistar rats (6–8 weeks) and male
Swiss albino mice (25–30 g) were procured from the
animal facility of our establishment and main- tained
R.​ ​Bhattacharya​, ​P​.​V.​ ​Lakshmana Rao /​ ​Toxicology Letters 1
water ad libitum. Rats and mice were fasted for 12 h
prior to isolation of thymocytes and in vivo
experiments, respectively. This study has the ap-
proval of establishment’s ethical committee.
2​.​3​. ​Cell culture and treatment
Rats were killed by cervical dislocation and
thymus was isolated to prepare single cell suspen-
sion by gently teasing the gland in ice-cold HBSS, as
per the procedure described elsewhere (Bhat-
tacharya and Lakshmana Rao, 1997). The cells were
passed through a 100-​m​m nylon mesh and treated
with Tris buffered isotonic ammonium chloride to
lyse the red cells. The cells were resus- pended in
Kreb’s–Hensleit Buffer (KHB, pH 7.4),
supplemented with 10 mM Hepes, 15 mM glu- cose,
1 mM Ca Cl​2 ​and 100 ​m​g/ml gentamycin. Viability
of cells was determined by Eosin Y exclusion. Cell
suspension was diluted to a final density of 50×10​6
cells/ml and incubated (2 ml/ well) in 24-well plates
maintained at 37°C in a humidified atmosphere of
5% CO​2​+95% air; 5 mM KCN was added to cell
suspensions in 24- well plates and incubated for 6 h.
Selection of dose was based on our previous study
(Bhat- tacharya and Lakshmana Rao, 1997). For the
protection studies, Trolox​® ​(10 mM), EGTA (10 mM)
and ATA (100 ​m​M) were given as 0.5 h
pre-treatment, simultaneous treatment or post-
treatment. In the post-treatment experiments, the cells
treated with 5 mM KCN were subsequently
challenged by the above agents at 0.5, 1, 2 and 3 h.
The control consisted of triple distilled water. Cell
viability and DNA fragmentation were recorded after
a total of 6 h exposure to KCN. In the preliminary
experiments, quercetin (10 mM) was tested as
simultaneous treatment alone. Stock solution of
Trolox​® ​was prepared in 1 M NaHCO​3​, quercetin in
polyethylene glycol-300, and EGTA and ATA in 0.1
N NaOH. Further dilutions of the above agents and
stock solution of KCN were prepared in distilled
water.
e husk in polypropylene cages. Ani- mals were
llet diet (Lipton India Ltd.) and
​ 19 (2001) 59–70 ​61
4​. ​Determination of cytotoxicity
Aliquots of the cell suspension were drawn
1-, 2-, 4- and 6-h intervals to assess cell viability
sin Y exclusion and leakage of LDH into the
ellular medium. LDH was estimated
metrically at 340 nm using a commercial
ostic kit.
Mitochondrial integrity ​(​MTT test​)
Mitochondrial integrity was measured
- duction of mitochondrial enzyme succinate
drogenase by MTT. The MTT reduction was
d out by the method of Mosman (1983).
y, MTT was dissolved in culture medium at a
ntration of 0.5 mg/ml and filtered to re- move a
amount of insoluble residue. MTT containing
m was added to each well in a volume of 0.5
nd incubated for 3 h. There- after, the
natant was removed and 1.25 ml of a solution of
N HCl–isopropanol (1:24 v/v) was added to
t and solubilize the formazon. After 30 min at
temperature, the ab- sorbance of the formazon
ad at a wave- length of 570 nm.
Mitochondrial membrane potential
​ 23 ​test)​
amine 1
Rhodamine 123, a cationic fluorescent
whose mitochondrial fluorescence intensity de-
s quantitatively in response to dissipation of
itochondrial membrane potential, was used to
ate perturbations in mitochondrial mem- brane
ial (Jiang and Acosta, 1993). Cells were
ated with 5 ​m​g/ml of rhodamine 123 for 30 min
hen thoroughly washed three times with the
e medium. Thereafter, cells were suspended in
e media and diluted prior to fluorescence
rement at 485 nm excitation and 530 nm
on in a Shimadzu spectrofluorophotometer.
uclear integrity (​ ​propidium iodide staining​)
 d in 2 ml modified HBSS, adjusted to pH 7.4
Nuclear staining of cells was performed 0 mM Hepes (HBSH). Cell suspensions (1×10​6
by propidium iodide uptake (Koizumi et al., 1996). ml) were centrifuged at 50×​g f​ or 1
The media were decanted and the cells were sus-
R.​ 62​Bhattacharya,​ ​P​.​V.​ ​Lakshmana Rao ​/ ​Toxicology Letters 1
​ 19 (2001) 59–70 ​min
and the cells were resuspended in HBSH
containing 20 ​m​M propidium iodide and incu- bated at 37°C. Twenty minutes later, the fluores- cence intensity was
measured for each cell suspension. The cells were immediately lysed by 0.05% Triton X-100 for 20 min at 37°C, and
the fluorescence intensity was measured for each lysate (total staining). As cells lost viability, their nuclei were
labeled by propidium iodide and the fluorescent enhancement was measured at 535 and 614 nm excitation and
emission wavelengths, re- spectively. Values were expressed as percent control.
2​.​8​. ​Measurement of reacti6 ​ ​e oxygen species ​(​ROS)​
The assay is based on the fact that DCFH-DA, a non polar and non fluorescent compound can diffuse through the
cell membrane and be deacetylated by cytosolic esterases to yield polar, non-fluorescent DCFH
(2​%​,7​%​-dichlorofluoroscin). DCFH is trapped within the cytoplasm where it reacts with peroxides to form DCF
(2​%​,7​%​-dichlor- ofluorescein), which is fluorescent and can be monitored by a fluorometer (Koizumi et al., 1996;
Mills et al., 1996). Thymocytes (1×10​6 ​cells/ml) were suspended in 2 ml HBSH (pH 7.4) and centrifuged at 50×​g ​for
1 min. The cells were resuspended in HBS adjusted to pH 7.4 with 50 mM Tris–HCl and containing 1.6 ​m​M DCF
and incubated at 37°C for 2 min. At the end of the incubation, the fluorescence intensity was mea- sured by
spectrofluorophotometer at excitation and emission wavelengths of 500 and 520 nm, respectively. Results were
expressed as percent control.
2​.​9​. ​Nuclear fragmentation ​(​HO3​ 42 ​staining)​
The nuclear membrane of dead and apoptotic nuclei is permeable to HO342, which intercalates the fragmented DNA.
After removal of the cul- ture medium, thymocytes were washed once by PBS and stained with HO342 (8
m​g/ml)/PBS for 15 min at room temperature. The fragmented apoptotic nuclei were observed under Nikon epifl-
uorescence microscope (Maeda et al., 1996).
2​.​10​. ​DNA fragmentation analysis
The cells were lysed in ice-cold lysis buffer (10 mM Tris, 20 mM EDTA, 0.5% Triton X-100, pH 8.0) for 20 min
prior to centrifugation at 27 000×​g f​ or 30 min. Both pellet (intact chro- matin) and supernatant (DNA fragments)
frac- tions were assayed for DNA content fluorimetrically (Kizaki et al., 1988). The level of DNA fragmentation was
expressed as percentage value (Wyllie, 1980; Bhattacharya and Laksh- mana Rao, 1997).
2​.​11​. ​Agarose gel electrophoresis
DNA fragmentation in whole cells was assessed by gel electrophoresis (Prigent et al., 1993) using 1.2% agarose in
89 mM Tris, 89 mM boric acid and 2.5 mM EDTA (pH 8.0). ​Hind​ III digest and Eco-R1-​Hin​dIII digest of ​l ​phage
DNA served as molecular size standard (Bhattacharya and Lak- shmana Rao, 1997).
​ ​i6
2​.​12​. ​In 6 ​ ​o protection studies
Prophylactic efficacy of various treatment regi- mens was determined by calculating the protec- tion index (PI),
which is defined as the ratio of LD​50 of ​ KCN administered s.c. in protected mice to the LD​50 of
​ KCN in unprotected
mice. About 6–8 animals were used to determine LD​50 ​for each
​ group (Dixon, 1965). The treatment protocol for
®​
Trolox​ , EGTA and ATA was selected on the basis of preliminary experiments, where maximum protective efficacy
was obtained at a dose which did not produce any toxic signs per se. Animals were administered (i.p.) Trolox​® ​(10
mg/kg), and EGTA and ATA (5 mg/kg) 30 min prior to cyanide (s.c.) treatment and observed for mortal- ity up to 24
h. In another experiment, animals (​n=​ 4/treatment) were given KCN (LD​50​×8) in the
​ absence or presence of various
treatments and observed for mean survival time.
2​.​13​. ​Statistics
Results are indicated as mean​9​S.E. of three different experiments. Significance was determined
 R​. ​Bhattacharya,​ ​P​.​V.​ ​Lakshmana Rao ​/ ​Toxicology Letters 1
​ 19 (2001) 59–70 ​63
yanide-​ ​induced nuclear fragmentation
using Student’s ​t-​ test and the level of significance
was set at ​P​B​0.05. The cells undergoing apoptosis show
cter- istic morphological changes and their
me is subjected to internucleosomal DNA
enta- tion. Attempts were made to differentiate
3. Results
en normal intact nuclei of control cells (Fig.
and apoptotic nuclei observed in KCN-treated
3​.​1​. ​Time​- ​and dose-​ ​dependent effects of cyanide Fig. 1C,D), as shown by HO342 staining.

We have earlier demonstrated that cyanide
pro- duces dose- and time-dependent cytotoxicity and
DNA fragmentation in rat thymocytes in vitro
(Bhattacharya and Lakshmana Rao, 1997). In the
present study, we have validated that a dose of 5 mM
KCN was sufficient to elicit appreciable changes in
cell viability after 6 h exposure. The percent cells
excluding eosin Y, percent LDH leakage and percent
DNA fragmentation were 48.2​9​4.8, 66.7​9​4.2 and
45.8​9​2.4, respectively in 5 mM KCN treated cells as
compared to 89.2​9​6.7, 20.4​9​4.0 and 10.6​9​1.7,
respectively in control cells (not tabulated).
ffect of Trolox​®​, ​quercetin​, ​EGTA and ATA
anide​-​induced cytotoxicity and DNA damage
In the initial studies, 5 mM KCN was
lenged by simultaneous treatment of various
macological agents, viz. Trolox​®​, quercetin,
A and ATA. At this point, out of four agents
quercetin did not offer any protection at
s dose levels and was per se slightly toxic to
lls (data not shown). Therefore, quercetin was
d from the subsequent studies. In order

Fig. 1. (A) Hoechst 33342 staining of thymocytes showing normal control cells (×400); (B) Control cells (×800); (C) Cells exposed to KCN 5
mM for 6 h exhibiting nuclear fragmentation (×400); (D) Treated cells (×800).

R​. 64​Bhattacharya​, ​P​.​V.​ ​Lakshmana Rao /​ ​Toxicology Letters 1

​ 19 (2001) 59–70 ​Table 1 Effect
​ of pre-treatment (0.5 h) and simultaneous
treatment of Trolox​®​, EGTA and aurin tricarboxylic acid (ATA) on cyanide-in- duced cytotoxicity and DNA fragmentation in rat thymocytes after
6 h exposure​a
Treatment % Viability (eosin Y exclusion) LDH leakage (% of total) % DNA fragmentation
​ ontrol 89.2​9​6.7 20.4​9​4.0 10.6​91​ .7 KCN 48.2​9​4.8* 66.7​9​4.2* 45.8​9​2.4* KCN+Trolox​® ​90.1​9​4.0** 22.8​9​4.6** 23.4​9​4.2​*,**
Pre-​ ​treatment C
KCN+EGTA 82.4​9​6.0** 28.9​9​4.8** 29.3​9​0.3​*,** ​KCN+ATA 85.6​9​6.1** 28.0​9​5.6** 35.4​9​3.1* ​Simultaneous treatment ​KCN+Trolox​®
92.6​9​6.9** 23.4​9​1.9** 19.2​9​2.9** KCN+EGTA 76.8​94​ .9** 38.4​9​4.8​*,** ​16.0​9​3.2** KCN+ATA
60.1​9​6.9​*,** ​32.6​9​7.8** 30.7​9​3.8​*,** ​a ​Dose of KCN, Trolox​®​, EGTA and ATA were 5 mM, 10 mM, 10 mM and 100 ​m​M, respectively. Values are

mean​9​S.E. of three
​ different experiments. Please refer to Section 2 for details of treatment protocol.
* Statistically significant as compared to corresponding control at ​P​B​0.05, as determined by Student’s ​t-​ test. ** Statistically significant as
compared to KCN 5 mM at ​P​B​0.05, as determined by Student’s ​t​-test.
to maximise the protection, the remaining agents were screened as 0.5 h pre-treatment, simulta- neous treatment or
post-treatment (0.5, 1, 2 and 3 h) against cytotoxicity and DNA damage induced by 5 mM KCN for 6 h exposure.
Table 1 lists the effects of various pharmacolog- ical agents when given as pre-treatment or simul- taneous treatment.
The 5 mM KCN after 6 h exposure caused significant loss in cellular viabil- ity as evidenced by Eosin Y exclusion,
leakage of LDH and DNA fragmentation. Pre-treatment of Trolox​®​, EGTA and ATA significantly enhanced the cell
viability. Although, both Trolox​® ​and EGTA prevented the DNA fragmentation, the damage remained significant
over control. With respect to simultaneous treatment, Trolox​® ​pro- vided the maximum protection which was statisti-
cally comparable with control. No appreciable difference between the pre-treatment and simulta- neous treatment
was observed. However, simulta- neous treatment of Trolox​® ​could be considered marginally better than other
treatments, because protection in all the parameters was comparable with control. Fig. 2 illustrates the effects of vari-
ous pre-treatments on cyanide-induced DNA fragmentation, observed qualitatively on gel elec- trophoresis. One of
the hallmark features of apop- tosis is internucleosomal DNA fragmentation into multiples of 180 bp. On extraction,
these frag-
ments are visualized as a ‘ladder’ pattern on agarose gel. Thymocytes treated with 5 mM KCN for 6 h resulted in
pronounced DNA fragmenta- tion. This fragmentation was abolished by only Trolox​® ​but not EGTA or ATA.
However, when
Fig. 2. Agarose gel electrophoresis of DNA extracted from rat thymocytes receiving various pre-treatments and then exposed to cyanide for 6 h.
Hin​dIII digest of ​l ​phage DNA (1); Control (2); KCN 5 mM (3); KCN 5 mM+Trolox​® ​10 mM (4); KCN 5 mM+EGTA 10 mM (5); KCN 5
mM+ATA 100 ​m​m (6); 1 kb ladder (7).
R​. ​Bhattacharya​, ​P​.​V.​ ​Lakshmana Rao ​/ ​Toxicology Letters 1
​ 19 (2001) 59–70 ​65
Fig. 3. Agarose gel electrophoresis of DNA extracted from rat thymocytes receiving various treatments simultaneously with cyanide exposure for
6 h (b). ​Hind​ III digest of ​l ​phage DNA (1); Control (2); KCN 5 mM (3); KCN 5 mM+Trolox​® ​10 mM (4); KCN 5 mM+EGTA 10 mM (5); KCN 5
mM+ ATA 100 ​m​m (6).
3​.​4​. ​Effect of Trolox​®​, ​EGTA and ATA on cyanide​-​induced mitochondrial and nuclear dysfunction
The MTT assay assesses the viability of cells after incubation with toxicant and is based on the reduction of the
soluble yellow MTT tetrazolium salt to a blue insoluble MTT formazon product by mitochondrial succinate
dehydrogenase. Table 2 shows that there was a 65% reduction in the mitochondrial activity after 2 h of cyanide
insult. Although this was significantly ameliorated by simultaneous treatments of Trolox​®​, EGTA and
these agents were given as simultaneous treat- ment, the beneficial effects of EGTA could also be observed but the
DNA fragmentation in ATA- treated cells still persisted (Fig. 3).
Fig. 4 refers to effects of therapeutic interven- tion of Trolox​®​, EGTA and ATA on cyanide-in- duced cytotoxicity
and DNA damage. These agents were added 0.5, 1, 2 and 3 h after 5 mM KCN insult. Addition of Trolox​® ​at all the
time intervals was beneficial with regard to cell viabil- ity (Eosin Y exclusion), LDH leakage and DNA
fragmentation. The efficacy of EGTA in contain- ing the cytotoxicity declined when added 2 h after cyanide
treatment. However, it significantly an-
Fig. 4. Effect of 5 mM KCN on cell viability (eosin Y ​tagonised the DNA fragmentation regardless of
exclusion), LDH leakage and DNA fragmentation in rat thy- ​the time of addition. With the exception of LDH leakage (0.5 h), at
no time interval beneficial effects of ATA could be observed.
mocytes after 6 h exposure, in presence or absence of Trolox​® ​(10 mM), EGTA (10 mM) and aurin tricarboxylic acid (ATA, 100 ​m​m) added 0.5,
1, 2 and 3 h after KCN treatment. *Statistically significant to KCN at ​P​B​0.05.
R​. 66​Bhattacharya,​ ​P​.​V.​ ​Lakshmana Rao /​ ​Toxicology Letters ​119 (2001) 59–70 ​Table 2 ​Cyanide-induced changes in mitochondrial activity
(MTT), mitochondrial membrane potential (Rhodamine 123) and nuclear integrity (propidium iodide) in presence or absence of Trolox​®​, EGTA
and aurin tricarboxylic acid (ATA) given as simultaneous treatments​a
Treatment Mitochondrial activity (% Mitochondrial membrane potential (% Nuclear integrity (% of
control) control) total) Control – – 18.5​9​4.3 KCN 34.9​95​ .8* 50.0​9​4.2* 59.6​9​4.8* KCN+Trolox​® ​79.7​9​8.4​*,** ​70.7​9​6.9​*,** ​42.2​9​5.9* KCN+EGTA
66.2​9​5.7​*,** ​68.0​9​9.6* 48.2​9​4.9* KCN+ATA
​
58.8​9​6.9​*,** 60.3​ 9​7.0* 48.2​9​5.2* a​ ​Dose of KCN, Trolox​®​, EGTA and ATA were 5 mM, 10 mM, 10 mM and 100 ​m​M, respectively. MTT

reduction, Rhodamine 123 ​ and propidium iodide staining were performed 2 h after KCN treatment. Values are mean​9​S.E. of three different
experiments. Please refer to Section 2 for details of treatment protocol.
* Statistically significant as compared to corresponding control at ​P​B​0.05, as determined by Student’s ​t-​ test. ** Statistically significant as
compared to KCN 5 mM at ​P​B​0.05, as determined by Student’s ​t​-test.
ATA, the deleterious effects of KCN prevailed over control. Rhodamine 123 is a cationic fluores- cent dye whose
fluorescence intensity decreases quantitatively in response to dissipation of the mitochondrial transmembrane
potential. A 50% impairment of mitochondrial membrane potential was observed in KCN-treated cells which could
be attenuated by Trolox​® ​alone. Both EGTA and ATA exhibited only marginal effects. Propidium iodide binds to the
nuclei of non viable cells but cannot readily enter viable cells. Binding to dou- ble stranded nucleic acids causes
enhancement of fluorescence. KCN exposed cells after 2 h showed about a threefold increase in fluorescence which
could not be effectively challenged by any of the above treatments.
3​.​5​. ​Effect of Trolox​®​, ​EGTA and ATA on cyanide​-​induced ROS generation
To examine the role of ROS in mediating cya- nide toxicity, levels of intracellular peroxides were measured in
KCN-treated cells in the presence or absence of Trolox​®​, EGTA and ATA (Table 3). After 30 min of 5 mM KCN
exposure, significant enhancement in microfluorescence was observed depicting about a 1.5 times increase in
accumu- lated peroxide levels which marginally increased with time. This could not be prevented by EGTA or ATA
treatments but Trolox​® ​had significant effects in blunting the effects of KCN. The ROS
observed in Trolox​® ​protected cells were com- parable to control cells.
A marginal decrease in ROS levels was ob- served in EGTA protected cells at 1 h post exposure.
3​.​6​. ​Effect of Trolox​®​, ​EGTA and ATA on cyanide toxicity in 6
​ ​i​6​o
The acute (24 h) LD​50 of ​ KCN in male mice was 8.0 mg/kg, which was augmented by Trolox​®​,
Table 3 Generation of peroxides (DCHF-DA) by cyanide in presence or absence of Trolox​®​, EGTA and aurin tricarboxylic acid (ATA) given as
simultaneous treatments​a
Treatment Peroxide levels (% control)
0.5 h 1 h
KCN 252.1​9​14.9* 279.6​9​22.6* KCN+Trolox​® ​122.5​9​13.8** 100.2​9​9.2** KCN+EGTA 203.9​9​16.2* 212.6​9​16.7* KCN+ATA 245.0​9​14.9*
252.4​9​11.8* ​a ​Dose of KCN, Trolox​®​, EGTA and ATA were 5 mM, 10 ​mM, 10 mM and 100 ​m​M, respectively. DCFH-DA staining was
performed 0.5 and 1 h after KCN treatment. Values are mean​9​S.E. of three different experiments. Please refer to Section 2 for details of treatment
protocol.
* Statistically significant as compared to corresponding con- trol at ​P​B​0.05, as determined by Student’s ​t-​ test.
** Statistically significant as compared to KCN 5 mM at ​PB ​ ​0.05, as determined by Student’s ​t​-test.
R​. ​Bhattacharya​, ​P​.​V​. ​Lakshmana Rao ​/ ​Toxicology Letters 1​ 19 (2001) 59–70 ​67
Table 4 Cyanide antagonism in mice by pre-treatments of Trolox​®​, EGTA or aurin tricarboxylic acid (ATA)
Treatment LD​50 ​of KCN (mg/kg)​a ​(fiducial limits) Protection Index​b ​(fiducial limits) Mean survival time (min)​c
KCN 8.0 (7.1–9.4) – 2.5​9​0.87 KCN+Trolox​® ​20.2 (17.4–33.6) 2.5 (2.2–4.2) 4.6​9​1.80 KCN+EGTA 14.2 (10.2–16.9) 1.8 (1.3–2.1) 2.9​9​0.98
KCN+ATA

13.1 (9.5–15.6) 1.6 (1.2–2.0) 3.1​9​1.11 ​a ​Acute (24 h) LD​50 (s.c.)

​ of KCN determined by Dixon’s Up and Down Method in the presence or absence
of Trolox​® ​(10 mg/kg), EGTA (10 mg/kg) or ATA (5 mg/kg) administered (i.p.) 30 min prior to KCN.
b​
Protection Index=LD​50 ​of KCN in protected mice/LD​50 ​of KCN in unprotected mice. ​c ​Time of mortality was recorded in animals receiving a

lethal dose (LD​50​×8) of KCN in the presence or absence of above agents. Values are mean​9​S.E. (​n​=4).
EGTA and ATA to 20.2, 14.2 and 13.1 mg/kg, resulting in a PI of 2.5, 1.8 and 1.6, respectively (Table 4). The mean
survival time of mice exposed to a lethal dose (LD​50​×8) of KCN was 2.5 min which could not be significantly
extended by any of the treatments. The efficacy of various treatments were in the following order: Trolox​®​\​EGTA
ATA.
4. Discussion
Impairment of mitochondrial energy production, failure of ionic homeostasis, acidosis, elevated cellular Ca​2+ ​levels
and lipid peroxidation culmi- nating in activation of proteases, lipases, xanthine oxidases, etc. are some of the
important biochem- ical events known to precede cyanide-induced cell death (Maduh, 1989; Borowitz et al., 1991).
Cya- nide also causes surface blebbing and cytoarchitec- tural defects of neuronal cells as a consequence of
Ca​2+​-activated phospholipases and proteases (Nicotera et al., 1989). Implication of oxidative stress and activation of
endonucleases in oligonu- cleosomal cleavage of DNA is well documented (Trump and Berezesky, 1995). ROS as
mediators of programmed cell death (PCD) has been reviewed recently, where cyanide is listed as one of the
inducers of PCD in mammalian neuronal and plant suspension-cultured cells (Jabs, 1999). Although DNA damaging
potential of cyanide has not yet been fully characterized, the documented evidence is suggestive of such a
phenomenon. Cyanide being predominantly a neurotoxin, most of its effects
have been demonstrated on neuronal cells or cen- tral nervous system. Cyanide-induced apoptosis has been reported
earlier in terminally differenti- ated PC12 cells (Mills et al., 1996). However its similar effects on other cell systems
have been elusive. Thymocytes are a widely used experimental model for studying DNA damage and apoptosis
(Stifanelli et al., 1995). We have earlier employed this system to show cyanide-induced DNA frag- mentation and its
attenuation by Zn​2+​, NAC and diltiazem (Bhattacharya and Lakshmana Rao, 1997). Severe disruption of
mitochondrial function can trigger apoptosis in mammalian cells and this phenomenon has been observed with a
number of agents including the mitochondrial respiratory chain inhibitors like KCN, rotenone, antimycin A, etc.
Since, implication of mitochondrial modifica- tions has been described in apoptosis of rat thymo- cytes (Cossarizza
et al., 1994), we used this system to show the role of mitochondrial and nuclear dysfunction, and generation of ROS
prior to cya- nide-induced cell death. In the present study, cya- nide-induced cytotoxicity and DNA damage were
challenged by various pharmacological agents like Trolox​® ​(antioxidant), EGTA (Ca​2+ ​modulator) and ATA
(Ca​2+​/Mg​2+​-dependent endonuclease inhibitor).
In our previous study, we have established that the internucleosomal DNA fragmentation (IDF) was Ca​2+​-dependent
(Bhattacharya and Laksh- mana Rao, 1997). Ca​2+ ​overload has also been responsible for activation of endonucleases
which are associated with oligonucleosomal cleavage of DNA or ‘laddering’, a typical feature of apoptosis (Trump
and Berezesky, 1995). In the present
R.​ 68​Bhattacharya,​ ​P​.​V.​ ​Lakshmana Rao /​ ​Toxicology Letters ​119 (2001) 59–70 ​study
we have demonstrated that a dose of 5 mM
KCN was sufficient to cause extensive cytotoxicity and DNA damage in thymocytes after 6 h expo- sure. At this
time the cleavage of DNA at internu- cleosomal sites correlated with nuclear fragmentation as depicted by HO342
staining. Although, we observed the nuclear fragmentation after 6 h KCN exposure, this event might have occurred
as early as 2 h post exposure, at which time nuclear integrity was significantly impaired. Morphological changes
during apoptosis are char- acterized by membrane blebbing, chromatin con- densation and margination to the nuclear
membrane, nuclear fragmentation and disintegra- tion, decrease in cell volume and dilatation of endoplasmic
reticulum, etc. (Cohen, 1993). In the present study, cyanide-induced nuclear fragmenta- tion was also accompanied
by mitochondrial dys- function, which was characterized by a 65% decline in mitochondrial MTT assay and a 50%
decrease in mitochondrial membrane potential as compared to control. Cyanide is a mitochondrial poison and
mitochondria are generally susceptible to damage from oxidants and electrophiles. The mitochondria contain small
genomes which are essential for mitochondrial function and not well protected against DNA damage. Correlation be-
tween toxicant-induced oxidative stress and DNA damage is well established (Jabs, 1999). In the present study,
cyanide was also found to generate peroxide levels 0.5 h post treatment which did not increase further up to 1 h. This
suggests that generation of ROS is an early event underlying the mitochondrial dysfunction followed by nu- clear
fragmentation. Oxidative stress can lead to either necrosis or apoptosis, depending on the cell model and the level of
oxidative insult; whether cell manifests as apoptosis or necrosis may de- pend on the severity and duration of the
oxidative injury (Nicotera et al., 1997). Recently cyanide-in- duced neurodegeneration in different brain areas was
 found to involve both apoptotic and non- apoptotic cell death. This was attributed to selec- tive vulnerability of these
brain areas to cyanide associated with variable susceptibility to oxidative stress (Mills et al., 1999). The present
findings also suggest that cell death in cyanide-exposed thymo- cytes could be of apoptotic nature.
Pharmacological interventions of cyanide-medi- ated cytotoxicity is linked to mobilization of in- tracellular Ca​2+
levels leading to generation of ROS and nitric oxide (NO). These oxidant species then initiate peroxidation of
cellular lipids (Gu- nasekar et al., 1996). Consequent to oxidative stress, widespread DNA fragmentation leading to
apoptotic type of cell death has been documented in cyanide poisoning (Mills et al., 1996, 1999). Hydrogen peroxide
(H​2​O​2​) is one of the main ROS known to cause lipid peroxidation and DNA damage in cells. ROS-mediated DNA
damage and cell death is usually accompanied by a depletion of intracellular reduced glutathione (GSH), whose
depletion lowers a cell’s capacity to buffer against endogenous oxidants (Slater et al., 1995). We have earlier
demonstrated that cyanide depletes the GSH contents in BHK-21 (baby hamster kid- ney) cell line which could be
challenged by antiox- idants like curcumin and NAC (Bhattacharya et al., 1999). In the present study, we observed
simi- lar cytoprotective effects of Trolox​®​, a water solu- ble derivative of vitamin E that penetrates biomembranes
and protects mammalian cells from oxidative damage and DNA fragmentation (McClain et al., 1995). It is
considered to be more potent than its parent compound vitamin E. In the present study, Trolox​® ​conferred additional
protection against DNA fragmentation and mito- chondrial and nuclear dysfunction by virtue of attenuating the
peroxide levels. Similarly, a free radical scavenger like NAC was earlier found to be very effective against
cyanide-induced cytotoxi- city and DNA damage (Bhattacharya and Laksh- mana Rao, 1997). Antioxidant protection
by Trolox​® ​has also been shown against adriamycin- induced ROS in cardiomyocyte (DeAtley et al., 1999).
Similarly, antioxidant properties of quercetin (a polyphenolic natural bioflavonoid) are also documented (Kuhlman et
al., 1998a,b). However, in our experiments, quercetin did not exhibit any cytoprotective effect which is related to its
ability to interrupt membrane lipid peroxi- dation rather than the scavenging of oxygen free radicals which is
sufficiently generated in cyanide toxicity. It is less likely to protect in the absence of lipid peroxidation (Kuhlman et
al., 1998a). The contention that cyanide-induced nuclear Ca​2+
R.​ ​Bhattacharya,​ ​P​.​V​. ​Lakshmana Rao /​ ​Toxicology Letters 1
​ 19 (2001) 59–70 ​69
nclusion, this study in addition to the mechanistic
deregulation is responsible for activation of Ca​2+ quences of cyanide toxicity in vitro and their
-endonuclease activity leading to DNA fragmen- armacological interventions, reveals the
tation prompted us to evaluate two more agents in the erapeutic implications of Trolox​® ​in cyanide
present study. EGTA, which chelates Ca​2+ ​and xicity. The role of various antioxidants in cya- nide
prevents endonuclease activation has been shown to isoning needs to be exploited.
prevent cytotoxicity and DNA fragmen- tation in
acetaminophen-treated hepatocytes (Shen et al.,
1992). Similarly, triphenylmethane dye ATA inhibits cknowledgement
Ca​2+​-endonuclease activity in the nucleus and also
blocks DNA cleavage and cell death, independent of
changes in total Ca​2+ ​levels (McConkey et al., 1989; The authors would like to express their
Shen et al., 1992; Mills et al., 1996). In our ati- tude to Dr R.V. Swamy, Director and Dr R.
experiments, both EGTA and ATA were not as jayaraghavan, Head of Pharmacology and Tox-
effective as Trolox​®​, particularly in respect of DNA ology Division, Defence Research and Develop-
fragmentation. The protective efficacy in vitro is also ent Establishment, Gwalior for their encouragement
supported by in vivo data, where Trolox​® ​was found d support.
to confer maximum protection as compared to the
other agents tested. Possibly, cyanide-mediated
extensive generation of ROS which appears to be an eference
early event if not blunted appropriately, exac- erbates
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