Disulfiram and Its Copper Chelate Attenuate Cisplatin-Induced Acute Nephrotoxicity in Rats Via Reduction of Oxidative Stress and Inflammation

Article abstract of DOI:10.1007/s12011-019-01683-w

The use of cisplatin (CP) in chemotherapy of resistant cancers is limited due to its dose-dependent nephrotoxicity. Disulfiram (DSF), the aversion therapy for alcoholism, has recently emerged as an anticancer and chemopreventive agent. Its anticancer activity is potentiated in the presence of copper. However, such use of copper leads to several adverse effects. In the present study, the protective effect of DSF and its copper chelate (Cu-DEDC) against CP-induced nephrotoxicity in rats was evaluated. Nephrotoxicity was induced by a single intraperitoneal injection of CP (5?mg/kg). The treatment groups included control (vehicle treated), CP (CP-treated), CP + DSF (CP followed by DSF), CP + DSF + Cu (CP followed by DSF and CuCl₂), CP + Cu-DEDC (CP followed by Cu-DEDC), and CP + AMF (amifostine pre-treated and CP-treated). The DSF, Cu-DEDC, and CuCl₂ were administered orally at 50?mM/kg/day dose for 5?days post CP injection. AMF served as a standard chemo protectant, administered intravenously 30?min prior to CP. The markers of oxidative stress, inflammation, and kidney function estimated on the 6th day revealed that both DSF and Cu-DEDC significantly attenuated the CP-induced rise in the serum/urine creatinine and blood urea nitrogen (BUN). The CP-induced rise in serum alkaline phosphatase (ALPase) was reversed by these drugs. Both drugs reduced the levels of malondialdehyde and nitric oxide (NO) in kidney tissues. These drugs reversed CP-induced depletion of SOD, catalase, and GSH in the kidneys. There was a significant reduction in the CP-induced TNF-α and IL-1β production along with prevention of histological alterations. Above observations indicate that DSF and Cu-DEDC may have significance as adjuvants to protect against CP-induced nephrotoxicity.

Full text of DOI:10.1007/s12011-019-01683-w

Biological Trace Element Research
https://doi.org/10.1007/s12011-019-01683-w
Disulfiram and Its Copper Chelate Attenuate Cisplatin-Induced Acute
Nephrotoxicity in Rats Via Reduction of Oxidative
Stress and Inflammation
Shraddha I. Khairnar1 & Umesh B. Mahajan1 & Kalpesh R. Patil2 & Harun M. Patel3 & Sachin D. Shinde1
Sameer N. Goyal1,4 & Sateesh Belemkar5 & Shreesh Ojha6 & Chandragouda R. Patil1
&
Received: 22 September 2018 /Accepted: 19 February 2019
#
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
The use of cisplatin (CP) in chemotherapy of resistant cancers is limited due to its dose-dependent nephrotoxicity. Disulfiram (DSF),
the aversion therapy for alcoholism, has recently emerged as an anticancer and chemopreventive agent. Its anticancer activity is
potentiated in the presence of copper. However, such use of copper leads to several adverse effects. In the present study, the protective
effect of DSF and its copper chelate (Cu-DEDC) against CP-induced nephrotoxicity in rats was evaluated. Nephrotoxicity was
induced by a single intraperitoneal injection of CP (5 mg/kg). The treatment groups included control (vehicle treated), CP (CP-
treated), CP + DSF (CP followed by DSF), CP + DSF + Cu (CP followed by DSF and CuCl2), CP + Cu-DEDC (CP followed by Cu-
DEDC), and CP + AMF (amifostine pre-treated and CP-treated). The DSF, Cu-DEDC, and CuCl2 were administered orally at
50 mM/kg/day dose for 5 days post CP injection. AMF served as a standard chemo protectant, administered intravenously 30 min
prior to CP. The markers of oxidative stress, inflammation, and kidney function estimated on the 6th day revealed that both DSF and
Cu-DEDC significantly attenuated the CP-induced rise in the serum/urine creatinine and blood urea nitrogen (BUN). The CP-induced
rise in serum alkaline phosphatase (ALPase) was reversed by these drugs. Both drugs reduced the levels of malondialdehyde and
nitric oxide (NO) in kidney tissues. These drugs reversed CP-induced depletion of SOD, catalase, and GSH in the kidneys. There was
a significant reduction in the CP-induced TNF-α and IL-1β production along with prevention of histological alterations. Above
observations indicate that DSF and Cu-DEDC may have significance as adjuvants to protect against CP-induced nephrotoxicity.
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Keywords Cisplatin Cucl2 Cu-DEDC Cytokines Disulfiram Nephrotoxicity
* Chandragouda R. Patil
Introduction
xplore.remedies@gmail.com
1
Cisplatin (CP) is widely used as a chemotherapeutic agent in
Department of Pharmacology, R. C. Patel Institute of Pharmaceutical
Education and Research, Shirpur, Dist. Dhule, Maharashtra 425405,
India
the treatment of several cancers including head, neck, testis,
ovary, breast, bladder, esophageal, and cervical cancers.
However, its clinical use is restricted due to its adverse effects
like nephrotoxicity, ototoxicity, and neurotoxicity [1–4].
Accumulating evidences suggest a need for the development
of therapeutic strategies to prevent the CP-associated organ
toxicities while retaining its anticancer activity. Intravenous
administration of amifostine (AMF) prior to CP injection is
a currently available therapy against CP-induced nephrotoxi-
city. Therefore, research to identify and develop suitable
nephroprotective adjuvants to chemotherapy is warranted.
Disulfiram (DSF) is in use since the last five decades as an
aversion therapy for alcoholism [5]. Recently, DSF is re-
emerging as an anticancer and chemopreventive agent for
the treatment of various cancers [6, 7]. DSF has been reported
2
3
Department of Pharmacology, H. R. Patel Institute of Pharmaceutical
Education and Research, Shirpur, Dist. Dhule, Maharashtra 425405,
India
Department of Pharmaceutical Chemistry, R. C. Patel Institute of
Pharmaceutical Education and Research, Shirpur, Dist. Dhule,
Maharashtra 425405, India
4
5
Shri Vile Parle Kelavani Mandal’s Institute of Pharmacy,
Dhule, Maharashtra 424001, India
Department of Pharmacology, School of Pharmacy & Technology
Management, SVKM’s NMIMS, Shirpur, India, Shirpur, Dist.
Dhule, Maharashtra 425405, India
6
Department of Pharmacology and Therapeutics, College of Medicine
and Health Sciences, United Arab Emirates University,
P.O. Box 17666, Al Ain, Abu Dhabi, United Arab Emirates

Khairnar et al.
for its anticancer effects in breast, colon, prostate, and ovarian
cancers along with leukemia, melanoma, and glioblastoma
[6]. It functions as a copper ionophore and induces copper-
dependent oxidative stress exerting antitumor effects in in-
flammatory breast cancer [8]. Anticancer activity of DSF is
potentiated in the presence of copper. The interaction between
copper and DSF offers an insight into its cytotoxic mecha-
nisms [9]. Simultaneous administration of copper salt with
DSF enhances its cytotoxicity [10]. However, such a concur-
rent administration of DSF and copper salts results in the
occurrence of hematological and immunological toxicities
[11–13].
The results of a phase-I clinical study conducted to inves-
tigate the nephroprotective effect of simultaneous administra-
tion of DSF and CP concluded that DSF reduced the CP-
induced nephrotoxicity [14]. However, no significant differ-
ences in the response rate and survival between CP alone
versus CP with DSF-treated group were noted in randomized
phase-II clinical studies. Moreover, the increased incidences
of ototoxicity and neurotoxicity were observed in the CP with
DSF-treated group. This study concluded the lack of
nephroprotective effect of DSF in phase-II studies [14–16].
DSF forms a complex with copper known as copper
(II) N,N-diethyldithiocarbamate (Cu-DEDC). The Cu-
DEDC activates the nuclear factor erythroid 2 (NFE2)–
related factor 2 (Nrf2) signaling in the cultured vascular
endothelial cells [17]. The activation of Nrf2 by Cu-
DEDC reduces oxidative stress. Among the various metal
chelates of DSF, the Cu-DEDC is more potent and prom-
ising Nrf2 inducer. However, it remains to be established
whether Cu-DEDC exerts any organ toxicities or protects
the vital organs against the chemotherapy-induced toxic-
ities. Therefore, the present study was executed to evalu-
ate the effects of Cu-DEDC in the CP-induced nephrotox-
icity in rats. We evaluated the nephroprotective effects of
DSF, DSF + CuCl2, and Cu-DEDC against animal models
of CP-induced nephrotoxicity.
Animals
Experimental procedures involving the use of laboratory ani-
mals were approved (protocol approval no. IAEC/CPCSEA/
RCPIPER/2017-15) by the Institutional Animal Ethics
Committee (IAEC) of the R. C. Patel Institute of
Pharmaceutical Education and Research, Shirpur, Dist.
Dhule (M.S.), India (reg. no. 651/PO/ReBi/S/02/CPCSEA)
constituted under the BPrevention of Cruelty to the Animals
Act- 1960.^ All the experiments were carried out according to
the guidelines prescribed by the Committee for the Purpose of
Control and Supervision of Experiments on Animals
(CPCSEA), Government of India. Healthy male Wistar rats
(170–200 g) were used for the study. Animals were kept under
standard conditions (12:12 h light:dark cycles, 55–70% rela-
tive humidity) at 22 2 °C with free access to water and stan-
dard pellet feed (Nutrimix Std-1020) obtained from Nutrivet
Life Sciences, India.
Experimental Protocol
Synthesis of Cu-DEDC (Fig. 1)
The Cu-DEDC complex was prepared by mixing the aqueous
solution of CuCl2 (3 M) and the DSF (1 M) in equal quantities
(DSF was dissolved in water using DMSO as a co-solvent).
Then, the reaction mixture was stirred at room temperature for
12 h in a conical flask with a magnetic stirrer and the reaction
was monitored by TLC. The precipitate formed was separated,
washed with distilled water, and dried. It was recrystallized by
using chloroform. The characterization of the copper complex
of DSF (Cu-DEDC) was done by the MASS analysis, where
we got the characteristic peak at 358 corresponding to the Cu-
DEDC complex.
Animal Study
Wistar rats were randomly divided into six groups (n = 6) and
were administered with the following treatments:
Materials and Method
Group I: control, treated with vehicle (0.5% of tween-80
solution; 2 ml/kg, p.o.) for 5 days.
Chemicals
Group II: CP, treated with cisplatin (5 mg/kg, i.p.) only on
the first day of the study followed by once daily oral
administration of vehicle for 5 days.
Group III: CP + AMF, treated with 100 mg/kg amifostine
intravenously 90 min prior to the cisplatin injection
(5 mg/kg, i.p.) on the first day of the study followed by
once daily oral administration of vehicle for 5 days.
Group IV: CP + DSF, received single dose of cisplatin
(5 mg/kg, i.p.) on the first day followed by DSF (50 mM/
kg/day, p.o.) for 5 days.
CP (CAS number: 15663-27-1) was purchased from Sigma
Aldrich, USA. DSF (purity > 99%) was procured from
Unidrug Innovative Pharma Technologies Ltd., India. Cupric
chloride and thiobarbituric acid were obtained from Loba
Chemie, Pvt. Ltd., India. Creatinine and urea/blood urea ni-
trogen (BUN) estimation kits were obtained from Erba
Manheim, Germany. Albumin and protein determination kits
were purchased from Span Diagnostic Ltd., India. Other
chemicals used for this study were of analytical grade.

Disulfiram and Its Copper Chelate Attenuate Cisplatin-Induced Acute Nephrotoxicity in Rats Via Reduction of...
Fig. 1 Scheme for synthesis of
Cu-DEDC
Group V: CP + DSF + Cu, received a single dose of
cisplatin (5 mg/kg, i.p.) on the first day followed by di-
sulfiram (50 mM/kg/day, p.o.) along with CuCl2 (50 mM/
kg/day, p.o.) for 5 days.
Group VI: CP + Cu-DEDC, received a single dose of
cisplatin (5 mg/kg, i.p.) on the first day followed by Cu-
DEDC (50 mM/kg/day, p.o.) for 5 days.
percentage change in kidney weight was calculated as the ratio
of kidney weight change to the body weight on the sixth day.
Biochemical Estimations
The blood samples and/or kidney homogenates were analyzed
for markers of renal function. Creatinine, albumin, alkaline
phosphatase (ALPase), blood urea nitrogen (BUN), and albu-
min were estimated using commercially available kits according
to the manufacturers’ instructions [18–21]. Fresh urine samples
collected within first 5 h following drug treatments on the fifth
day were used for the determination of urine biochemistry.
On the fifth day, after the administration of respective drug
treatments, the rats were individually kept in metabolic cages
for collection of urine up to 24 h; sufficient food and water
were provided during urine collection. On the sixth day, fol-
lowing urine collection, the blood samples were collected
from anesthetized rats through retro-orbital puncture under
light anesthesia. Rats were sacrificed by the overdose of an-
esthetic. The kidneys were excised and processed for the bio-
chemical and histological examinations. The details of drug
treatments and evaluation parameters are shown in Fig. 2.
Oxidative Stress Parameter
Preparation of Renal Homogenate
Immediately, following the scarification, the left kidney from
each rat was dissected and rinsed with ice-cold phosphate
buffered saline, blotted on filter paper, and weighed. The kid-
ney was minced with scissors and 10% w/v homogenate was
prepared in ice-cold phosphate buffered saline (0.1 mol/L, pH
7.4) using a homogenizer. This homogenate was centrifuged
at 2000 rpm for 5 min and the supernatant was used for the
estimation of various biochemical parameters such as lipid
peroxidation (MDA), oxidative stress (reduced glutathione,
Observational Parameter
Body weights of rats were recorded before the administration
of CP and on the sixth day. The changes in the body weights
were calculated. At the end of the experiment, the kidneys
were excised from each sacrificed rat and were separately
weighed. The average kidney weight was recorded. Relative
Fig. 2 Drug treatments and
protocol executed for cisplatin-
induced nephrotoxicity in rats
Only Vehicle (0.5% of tween-80 solution; 2 ml/kg, p.o.)
Control
+ Vehicle (0.5% of tween-80 solution; 2 ml/kg, p.o.)
+ AMF (100 mg/kg, i.v.)
CP
CP+AMF
CP+DSF
CP+DSF+Cu
CP+Cu-DEDC
+ DSF (50 mM/kg/day, p.o.)
+ DSF (50 mM/kg/day) + CuCl2 (50 mM/kg/day, p.o.)
+ Cu-DEDC (50 mM/kg/day, p.o.)
Body & Kidney
weight
Blood
Urine
Kidney
Oxidative Stress &
Biochemical Estimations
(Creatinine, ALPase, BUN)
Biochemical Estimations
(Creatinine, Albumin, BUN)
Cytokine Estimation
(GSH, Catalase, SOD, NO,
MDA, TNF-α and IL-1β)
Histopathology

Khairnar et al.
catalase, and superoxide dismutase), and nitrosative stress
(NO).
Estimation of Nitric Oxide
The tissue homogenate supernatant was mixed with equal
quantity of the Griess reagent and the absorbance was deter-
mined at 540 nm using microplate spectrophotometer. Sodium
nitrite was used as a standard for the preparation of calibration
curve. The nitric oxide (NO) concentration was expressed as
μM/L of NO [27].
Estimation of Lipid Peroxidation
Lipid peroxidation (LPO) was measured as a malondialdehyde
(MDA) concentration, according to the earlier method [22]. The
kidney homogenate supernatant was treated with 1% phosphoric
acid solution and aqueous solution of 0.6% thiobarbituric acid.
The reaction mixture was heated at 80 °C for 45 min. The mix-
ture was cooled immediately in an ice bath and extracted with
4.0 mL of n-butanol. It was vortexed and centrifuged at
5000 rpm for 10 min. The n-butanol layer was separated and
the absorbance of the formed pink complex was recorded at
535 nm [23].
Estimation of Pro-Inflammatory Cytokines
The cytokines were estimated in kidney homogenate superna-
tant as described earlier (Vasaikar et al. 2018). Briefly, the
animals were sacrificed and the kidneys were harvested and
stored in liquid nitrogen at − 80 °C until the execution of
assay. The pro-inflammatory cytokine levels including
TNF-α and IL-1β were determined by using sandwich
ELISA methods according to the manufacturer’s protocol,
and the values were calculated from the optical density [26].
Estimation of Reduced Glutathione
The reduced glutathione (GSH) content in the kidney homog-
enate supernatants was determined by using the earlier method
with some modifications [24]. Briefly, the homogenate was
precipitated with 4% sulfosalicylic acid. The sample was cen-
trifuged at 1200 g at 4 °C for 15 min. Assay mixture consisted
of 0.5 mL supernatant and 4.5 mL of 1 mM/L of 5-5-dithio
bis-(2-nitrobenzoic acid) in 0.1 Mol/L phosphate buffer, pH
8.0. The optical density of the resulting yellow color was
measured at 412 nm using a microplate spectrophotometer
(Powerwave XS, Biotek, USA) [25].
Protein Quantification and Alkaline Phosphatase
Protein concentration was determined by the method of
Bradford using bovine serum albumin as the standard when-
ever needed [28]. The ALPase was estimated by using com-
mercial kit (Beacon Diagnostic Pvt. Ltd., Navsari, India) as
per the manufacturer’s instructions [29].
Estimation of Catalase Activity
Histopathology
Catalase activity was assayed according to the earlier method
with some modifications [26]. Briefly, the kidney homogenate
supernatant (20 μL) was added to 1 mL of 10 mM hydrogen
peroxide (H2O2) solution in the quartz cuvette. The reduction
in optical density of this mixture was measured in UV mode at
240 nm by using a spectrophotometer. The rate of decrease in
the optical density within 3 min from the addition of kidney
homogenate was taken as an indicator of the catalase activity
present in the homogenate.
Kidney tissues fixed in buffered formalin were embedded in
paraffin, and serial sections (3 μm thick) were cut using mi-
crotome (Leica RM 2125, Germany). The representative sec-
tions were stained with hematoxylin and eosin and examined
under light microscope (Leica, Germany). The histopatholog-
ical data was objective and the sections were screened from a
pathologist blinded to the treatments.
Estimation of Superoxide Dismutase Activity
Results
The kidney homogenate supernatant was added to a mixture
of 500 mM/L of Na2CO3, 0.3% Triton X-100, 1.0 mM/L of
EDTA, 10 mM/L of hydroxylamine, and distilled water. To
this mixture, 20 μL of 240 μM/L of NBT was added and
optical density was measured at 560 nm in kinetic mode for
3 min at the interval of 1 min. The change in optical density
was determined as an indicator of the superoxide dismutase
(SOD) activity [23, 26].
Characterization of Cu-DEDC
The characteristics of Cu-DEDC were confirmed using
mass spectroscopy, FTIR, and differential scanning cal-
orimetry (Fig. 3).
Fig. 3 Characterization of Cu-DEDC. a Mass spectroscopy. b FTIR
graph. c Differential scanning calorimetry
„

Disulfiram and Its Copper Chelate Attenuate Cisplatin-Induced Acute Nephrotoxicity in Rats Via Reduction of...
a
b
c

Khairnar et al.
Cu-DEDC Protected Rats from CP-Induced Changes
in the Body Weight and Kidney Weight
catalase levels in CP group were significantly (p < 0.001)
lowered, as compared with the control group. The levels of
GSH (p < 0.05), SOD (p < 0.001), and catalase (p < 0.001) in
Cu-DEDC-treated group appeared significantly higher than
compared with CP injected rats. Similarly, AMF-treated group
revealed a significant increase in GSH (p < 0.001), SOD
(p < 0.001) and catalase (p < 0.01) activity, when compared
with CP control group. The DSF and DSF + Cu–treated
groups also showed significant (p < 0.001) elevation in SOD
and catalase levels. However, an increase in GSH level in
these groups was statistically non-significant as compared
with the CP-treated group (Fig. 4).
CP-treated rats had a significant (p < 0.001) decrease in the
body weight and relative kidney weight as compared with
normal rats. However, the rats treated with CP + DSF, CP +
DSF + Cu, CP + Cu-DEDC, and CP + AMF had significant
(p < 0.001) protection from the reduction in the body weight
and relative kidney weight. Moreover, the urine output was
significantly increased in the CP-treated rats as compared with
the control group, while the treatment with Cu-DEDC effi-
ciently reduced the CP-induced rise in urine volume shown
in Table 1.
Cu-DEDC Treatment Prevented CP-Induced
Alterations in Serum Biomarkers
Cu-DEDC Alerted the CP-Induced Changes
in Pro-Inflammatory Cytokines and NO
Serum creatinine and BUN levels were significantly
(p < 0.001) increased in CP-treated control rats as com-
pared with the control rats. Animals treated with DSF,
DSF + Cu, and Cu-DEDC prevented the CP-induced
elevation of the creatinine and BUN levels as compared
with the CP-treated animals. Interestingly, favorable ef-
fects of Cu-DEDC treatment against BUN and ALPase
serum levels were more prominent than the DSF or
DSF + Cu treatment.
The levels of pro-inflammatory cytokines, TNF-α, and IL-1β
in kidney tissues were estimated by ELISA. Significant
(p < 0.001) increase in cytokine levels was observed in CP-
treated group as compared with control group (TNF-α, 62
0.95 vs 26 0.88 pg/mg and IL-1β, 55 1.0 vs 19 1.0 pg/
mg). The administration of Cu-DEDC significantly reduced
the level of pro-inflammatory cytokines (TNF-α, 27
0.99 pg/mg and IL-1β, 20 0.58 pg/mg) as well as nitric
oxide in kidney homogenates. The treatment of rats with
Cu-DEDC and AMF restored the elevated cytokines and NO
levels in those observed in the control group rats. The admin-
istration of DSF or DSF + Cu exerted similar effects on cyto-
kine and nitric oxide levels; however, the effect of Cu-DEDC
was more prominent than the effect of either DFS or DSF + Cu
treatment (Fig. 5).
Cu-DEDC Protected the Rats against CP-Induced
Changes in the Urine Biomarkers
Urine creatinine and BUN concentrations in the CP
group had a significant (p < 0.001) increase as compared
with the control group rats. However, the treatment of
rats with DSF, DSF + Cu, or Cu-DEDC offered signif-
icant (p < 0.001) protection against the rise in the urine
creatinine and BUN. Urine levels of albumin were sig-
nificantly (p < 0.001) increased in the CP-treated group
as compared with the normal rats. Treatment with Cu-
DEDC significantly (p < 0.01) reversed these changes
induced by CP as shown in Table 2.
Cu-DEDC Ameliorated CP-Induced Kidney Toxicity
in Rats
Histological alterations in kidney tissues of various
treatment groups are shown in Fig. 6. The data was
shown from representative sample and the observations
were objective. The vehicle-treated control group had
regular morphology of the kidney with renal corpuscles
and the tubuli with normal appearance of parietal and
visceral layers of the glomerulus (Fig. 6a). The admin-
istration of CP (5 mg/kg, i.p.) caused severe degenera-
tion of glomerulus structure (red arrow), widening of
urinary space (black arrow) with damage in the parietal
and visceral layers, inflammatory cell infiltration (blue
arrowhead), and necrosis in the kidney (Fig. 6b). In
contrast, the administration of DSF, DSF + Cu, Cu-
DEDC, and AMF prevented histological changes evoked
by CP (Fig. 6c–f). The treatment with Cu-DEDC
showed normalization of kidney architecture (Fig. 6e).
Cu-DEDC Prevented Lipid Peroxidation
and Alterations in Oxidative Stress Biomarkers
CP induced a state of marked oxidative stress in the kidney
tissues. It was evident from the several-fold increase in MDA
levels and a simultaneous decrease in the levels of antioxidant
enzyme systems (SOD and catalase) and GSH as compared
with the control group. The administration of DSF, DSF + Cu,
or Cu-DEDC significantly (p < 0.001) reduced the CP-
induced lipid peroxidation by decreasing the MDA content.
This effect of Cu-DEDC was more prominent than the indi-
vidual effects of DSF or DSF + Cu. The GSH, SOD, and

Disulfiram and Its Copper Chelate Attenuate Cisplatin-Induced Acute Nephrotoxicity in Rats Via Reduction of...
Table 1 Effect of Cu-DEDC on
body weight, relative kidney
Treatment
Change in bodyweight (g)
Relative change in kidney weight
Urine output (mL/5 h)
weight, and urine output changes
Control
2.7 0.01
7.7 0.09
2.1 0.1
CP
− 8.0 0.02###
− 2.8 0.09***
− 4.7 0.01***
− 4.1 0.01***
− 3.0 0.01***
− 5.7 0.07###
7.5 0.20***
7.0 0.10***
7.1 0.10***
7.6 0.10***
9.5 0.1###
2.9 0.7***
5.4 0.9***
7.3 0.1**
4.0 0.7***
CP + AMF
CP + DSF
CP + DSF + Cu
CP + Cu-DEDC
Data represent mean SEM. Statistical analysis was performed by one-way ANOVA followed by the Bonferroni
multiple comparison test. p < 0.05 was considered as statistically significant. ### p < 0.001 as compared with the
normal control group. **p < 0.01, ***p < 0.001 as compared with the CP-treated group. CP, cisplatin; AMF,
amifostine; DSF, disulfiram; Cu, CuCl2; Cu-DEDC, copper (II) diethyldithiocarbamate
Discussion
platinum levels, restricts the loss of copper from the kidney,
and reduces the toxicity of platinum compounds like CP to-
The CP-induced nephrotoxicity limits its utility in the treat-
ment of chemotherapy-resistant cancers [30]. There is a need
for adjuvant therapies which could reduce the severity of CP-
induced organ toxicity, while increasing the sensitivity of the
cancer cells to its cytotoxic effects. Several studies have been
undertaken to evaluate the effectiveness of an adjuvant thera-
py to reduce the CP-induced toxicities [31–34]. CP accumu-
lates in the renal cells and induces severe nephrotoxicity [30].
It includes multipronged mechanisms, and diverse treatments
have been reported to ameliorate the CP toxicities [35, 36]. In
the present investigation, we attempted to explore the use of
DSF, DSF with copper chloride, and DSF-copper complex,
i.e., Cu-DEDC as a protective adjuvant therapy against CP-
induced nephrotoxicity.
Preclinical studies have revealed that DSF and its copper
chelate (Cu-DEDC) sensitize the chemotherapy-resistant can-
cer cells to CP and increase its therapeutic index. CP admin-
istration causes loss of copper from the kidney tissues and this
contributes to the development of nephrotoxicity [37]. The
diethyldithiocarbamate (DEDC)—a metabolite of DSF—is
known to reduce the CP nephrotoxicity even after a single-
dose administration. The DEDC effectively controls kidney
wards the kidney [24].
DSF is emerging as an anticancer and chemopreventive agent
in various types of cancers. The interaction of DSF with CuCl2 in
solution has been studied using a range of spectroscopic tech-
niques. In situ evidences have proved a rapid formation of bis
(N,N-diethyldithiocarbamate) copper complex (Cu-DEDC) [9].
DSF functions as a copper ionophore to induce oxidative stress
and this ability of DSF facilitates chemosensitization of tumor
cells to chemotherapeutic agents [8]. Obviously, the anticancer
activity of DSF is potentiated in the presence of copper [38].
Considering this synergism, the approach of simultaneous ad-
ministration of DSF and copper has been evaluated for the
chemosensitizing potential of this combination. It was also hy-
pothesized that the use of copper salts may offer kidney protec-
tion by compensating and preventing copper loss from the kid-
neys. The DSF, combination of DSF and copper salts, and Cu-
DEDC are reported to sensitize cancer cells to the chemotherapy.
Out of these, Cu-DEDC is known to enhance the Nrf2 transcrip-
tion factor expression and hence exert antioxidative effects [17].
To determine whether the Cu-DEDC offers any kidney pro-
tection better than DSF, we studied the effects of DSF alone, DSF
+ CuCl2, and Cu-DEDC against CP-induced nephrotoxicity. For
Table 2 Effect of Cu-DEDC on serum and urine indices of CP-induced nephrotoxicity in rats
Treatment
Serum parameters
Creatinine (mg/dL)
Urine parameters
BUN (mg/dL)
ALPase (U/L)
Creatinine (mg/dL)
BUN (mg/dL)
Albumin (g/dL)
Control
0.73 0.06
34.0 0.36
75.0 1.30
2.7 0.08
49.0 0.88
0.13 0.05
CP
3.50 0.15###
0.84 0.12***
1.40 0.06**
0.57 0.03***
0.91 0.13***
73.0 0.34###
35.0 0.27***
55.0 0.22***
50.0 0.26***
44.0 0.31***
213.0 1.30###
86.0 0.97***
131.0 1.70***
126.0 1.30***
103.0 1.40***
13.0 0.44###
2.8 0.19***
4.4 0.45***
3.2 0.09***
2.9 0.15***
129.0 3.10###
62.0 2.40***
85.0 5.60***
59.0 1.90***
53.0 0.77***
0.45 0.04###
0.21 0.01***
0.29 0.01*
0.27 0.02*
0.25 0.02**
CP + AMF
CP + DSF
CP + DSF + Cu
CP + Cu-DEDC
Data represent mean SEM. Statistical analysis was performed by one-way ANOVA followed by the Bonferroni multiple comparison test. p < 0.05 is
considered as statistically significant. ### p < 0.001 as compared with the control group. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with the CP-
treated group. CP, cisplatin; AMF, amifostine; DSF, disulfiram; Cu, CuCl2; Cu-DEDC, copper (II) diethyldithiocarbamate

Khairnar et al.
Fig. 4 Effect of Cu-DEDC on
cisplatin-induced oxidative stress
in rats. a MDA. b GSH. c SOD. d
Catalase. Data represents mean
SEM. Statistical analysis was
performed by one-way ANOVA
followed by the Bonferroni mul-
tiple comparison test. p < 0.05 is
considered as statistically signifi-
cant. ###p < 0.001 as compared
with the normal control group.
*p < 0.05, **p < 0.01, ***p <
0.001 as compared with CP-
treated control group. ns, non-
significant, CP, cisplatin; AMF,
amifostine; DSF, disulfiram; Cu,
CuCl2; Cu-DEDC, copper (II)
diethyldithiocarbamate
20
15
10
5
a
b
100
75
50
25
0
###
*
*
# # #
***
***
***
***
0
l
F
S
u
+C
CP
Cu
DC
ro
trol
CP
MF
t
AMF
E
D
A
EDC
F+
+
F
on
+
D
S
P
P
C
Con
CP+DSF
C
C
CP+
+DS
+Cu-D
P+D
P
C
C
CP
CP+Cu-
c
d
6
4
2
0
4
3
2
1
0
***
***
***
***
***
***
***
***
###
###
l
ro
F
P
C
u
l
SF
F
M
u
C
t
o
D
r
A
SF
EDC
CP
M
C
D
t
+
on
D
D
A
P+
P
+
C
+
CP
DE
u-
u-
DSF+C
P+
C
C
Con
C
CP
+
+DSF+
C
P+
P
C
P
C
C
C
the comparison of the nephroprotective effects, we used equimo-
lar doses (50 mM) of all these three drugs.
associated enzymes, and subsequent production of toxic metab-
olites [39, 40]. Increased MDA level was noted in the CP-
injected rats, and these results are in line with the observations
of earlier studies [19, 33, 41–43]. Although the treatment of
animals with DSF and DSF + Cu reduced the extent of lipid
peroxidation, most prominent protection was observed in the
Cu-DEDC-treated rats.
In tune with the reduced lipid peroxidation, other indicators of
the oxidative stress like depletion of the GSH and reduction in
the catalase and SOD activities were prevented by the treatment
of DSF, DSF and CuCl2, and Cu-DEDC. The DSF has been
reported to exert organ protective effects against multiple
xenobiotics.
Single-dose CP (5 mg/kg, i.p.) induced observable nephrotox-
icity evident as perturbed kidney function, histological alterna-
tions like ruptured glomeruli, widening of tubular space, and
infiltration of inflammatory cells. There was a significant de-
crease in the body weight and a reduction in the relative kidney
weight in CP-treated rats. The DSF, DSF + CuCl2, and Cu-
DEDC treatments significantly inhibited the CP-induced neph-
rotoxicity. The effects of Cu-DEDC were more potent than the
DSF or the combination of DSF and CuCl2.
CP-induced nephrotoxicity is associated with increased lipid
peroxidation. The lipid peroxidation induces alteration and dis-
continuation in the biological membrane. It leads to the destruc-
tion of membrane transport proteins, inactivation of membrane-
Nitric oxide (NO) is a recognized mediator and regulator of
inflammatory responses. Increased NO levels in response to
80
c
800
a
b
60
50
40
30
20
10
0
###
# # #
# # #
60
40
20
0
600
***
***
***
400
***
***
***
***
***
***
***
***
***
200
0
l
F
u
l
l
tro
C
F
F
P
C
o
r
CP
tro
S
CP
Cu
F+
DC
DE
DS
ED
n
D
+Cu
F
AMF
n
Co
+
o
D
-
+
CP
+
CP
DEDC
SF+C
P
P+AMF
-
Cu
C
Cont
C
C
CP+DSF
CP+AM
Cu
+DS
CP
P+D
+
P+DS
C
C
CP+
CP
CP+Cu-
Fig. 5 Effect of Cu-DEDC on cisplatin-induced NO and the release of
cytokine in rats. a TNF-α. b IL-1β. c Nitric oxide (NO). Data represents
mean SEM. Statistical analysis was performed by one-way analysis of
variance (ANOVA) followed by the Bonferroni multiple comparison test.
p < 0.05 is considered as statistically significant. ###p < 0.001 as com-
pared with the normal control group. ***p < 0.001 as compared with
CP-treated control group. CP, cisplatin; AMF, amifostine; DSF, disulfi-
ram; Cu, CuCl2; Cu-DEDC, copper (II) diethyldithiocarbamate

Disulfiram and Its Copper Chelate Attenuate Cisplatin-Induced Acute Nephrotoxicity in Rats Via Reduction of...
Fig. 6 Effect of Cu-DEDC on cisplatin-induced histological changes in
rat kidney. a Normal kidney tissue showed normal glomerulus structure
and urinary space. b Cisplatin (5 mg/kg, i.p.)-treated tissue, had ruptured
glomeruli (red arrow), widening of urinary space (black arrow), and in-
filtration of inflammatory cells (blue arrowhead). c AMF-treated tissue
showed less glomerulus damage and decreased presence of inflammatory
cells. d DSF-treated tissue showed decreased urinary space and presence
of inflammatory cells. e DSF + Cu–treated tissue showed least rupturing
of glomerulus structure and less inflammatory cells. f Cu-DEDC-treated
showed the normal glomerulus structure, urinary space, and few inflam-
matory cells
inflammatory stimuli have pro-inflammatory and destructive ef-
fects on host tissue [44]. CP treatment increases calcium-
independent nitric oxide synthase (NOS) activity in rat kidney.
This can lead to an increase in the serum and tissue NO levels
[45, 46]. The upregulation of NOS and peroxynitrite formation
after CP treatment causes inflammatory alterations in the renal
tissues [47]. The inhibition of iNOS reduces nitrosative stress and
hence the CP-induced nephrotoxicity [48]. The DSF, combina-
tion of DSF + CuCl2, and Cu-DEDC treatments prevented the
elevation in the nitric oxide content in the rat kidney tissues.
Along with these antioxidative effects, the DSF, DSF and
CuCl2, and Cu-DEDC were found to exert significant inhibition
of the pro-inflammatory cytokine release in the kidney tissues.
Inflammatory alternations are implicated in the pathogenesis of
CP-induced kidney injury [35]. The nuclear factor kappa beta
(NF-κB) is a ubiquitous protein complex that regulates various
cell signals in the body. Earlier studies showed that cisplatin
activates the NF-κB pathway and promotes the transcription of
genes that encode a series of inflammatory cytokines including
TNF-α and IL-1β [49, 50]. TNF-α is a prototype inflammatory
cytokine that induces apoptosis of epithelial cell and plays a
major role in mediating renal injury [35]. Cu-DEDC treatment
markedly suppressed the CP-induced elevation in the inflamma-
tory cytokines including TNF-α and IL-1β. Interestingly, the
effect of Cu-DEDC treatment against TNF-α and IL-1β was
more significant than the effects of other treatment groups
(DSF, DSF + Cu, and AMF).
The supportive parameters like changes in relative kidney
weight and urine output were reversed by Cu-DEDC treatment
more efficiently than DSF and DSF + CuCl2. Acute increase in
urine output after a single dose of CP has been reported earlier as
an indication of CP toxicity [51]. Our results are in congruence
with this report and the reversal of relative kidney weight and
urine output further confirms the protective efficacy of Cu-
DEDC against CP-induced nephrotoxicity.
The renal function parameters estimated in the pres-
ent study including serum and urine BUN, creatinine,
and ALPase indicated that the DSF, DSF + CuCl2, and
Cu-DEDC significantly inhibited the CP-induced
nephrotoxicity.
Certain in vivo studies have reported that chronic administra-
tion of copper salts twice a day for 4 weeks at doses above
7.5 mg/kg exerts numerous adverse effects including hematolog-
ical malignancies, immunotoxicity, cell cycle arrest, and
clastogenic effects on the bone marrow chromosomes [11–13].
In the present study, we evaluated 50 mM/kg (7 mg/kg, once a
day) dose of CuCl2 once a day for 5 days. The results of the study
conducted by Mitra et al. [11] indicate that this dose and the

Khairnar et al.
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duration of treatment are lesser than the doses at which immu-
nological toxicities are evident. As our focus was to study wheth-
er Cu-DEDC protects the kidneys from CP-induced toxicities
relied on the dose level mentioned in the study conducted by
Mitra et al. [11], we used the once-daily dose of 50 mM/kg for
5 days. It cannot be ruled out that even at this dose, CuCl2 alone
or in combination with DSF may still exert toxicities. It will be
interesting to further investigate the effects of equimolar doses of
Cu-DEDC, CuCl2, and DSF + CuCl2 on the immune functions
to determine whether Cu-DEDC has reduced the
immunotoxicity. Considering the Nrf2-inducing effect of Cu-
DEDC, it can be postulated that Cu-DEDC may exert lesser
immunotoxicity as compared with the CuCl2 and DSF +
CuCl2. The present study aimed at determining the
nephroprotective effects of Cu-DEDC and hence, the outcomes
of our study cannot be extrapolated to comment on
immunotoxicity of these treatments.
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J (2009) Copper ions regulate cytotoxicity of disulfiram to myeloid
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Considering the intravenous administration of AMF, the ef-
fects of orally administered Cu-DEDC appear to have an advan-
tage. The Cu-DEDC is a better antioxidative compound with a
favorable activity profile as compared with DSF. Further inves-
tigations on the nephroprotective potential of Cu-DEDC may
establish its use as an organ-protective adjuvant in CP
chemotherapy.
Compliance with Ethical Standards
13. Agarwal K, Sharma A, Talukder G (1990) Clastogenic effects of
copper sulphate on the bone marrow chromosomes of mice in vivo.
Mut Res Lett 243(1):1–6
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bination of disulfiram with cisplatin. Am J Clin Oncol 10(6):517–
519
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antioxidant micronutrients and chemotherapy-induced toxicity in
cancer patients treated with cisplatin-based chemotherapy: a
randomised, double-blind, placebo-controlled study. Euro J
Cancer 40(11):1713–1723
Experimental procedures involving the use of laboratory animals were
approved (protocol approval no. IAEC/CPCSEA/RCPIPER/2017-15) by
the Institutional Animal Ethics Committee (IAEC) of the R. C. Patel
Institute of Pharmaceutical Education and Research, Shirpur, Dist.
Dhule (M.S.), India (reg. no. 651/PO/ReBi/S/02/CPCSEA) constituted
under the BPrevention of Cruelty to the Animals Act- 1960.^ All the
experiments were carried out according to the guidelines prescribed by
the Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA), Government of India.
16. Verma S, Stewart DJ, Maroun JA, Nair RC (1990) A randomized
phase II study of cisplatin alone versus cisplatin plus disulfiram.
Am J Clin Oncol 13(2):119–124
Conflict of Interest The authors declare that they have no conflict of
interest
17. Fujie T, Murakami M, Yoshida E, Tachinami T, Shinkai Y, Fujiwara
Y, Yamamoto C, Kumagai Y, Naka H, Kaji T (2016) Copper
diethyldithiocarbamate as an activator of Nrf2 in cultured vascular
endothelial cells. JBIC. J Biol Inorg Chem 21(2):263–273
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(2014) Protective effect of quercetin on cisplatin-induced nephro-
toxicity in rats. Facta Univ Ser Med Biol 16(2):71–75
Publisher’s Note Springer Nature remains neutral with regard to juris-
dictional claims in published maps and institutional affiliations..
19. Bami E, Ozakpınar OB, Ozdemir-Kumral ZN, Köroglu K, Ercan F,
Cirakli Z, Sekerler T, Izzettin FV, Sancar M, Okuyan B (2017)
Protective effect of ferulic acid on cisplatin induced nephrotoxicity
in rats. Environment Toxicol Pharmacol 54:105–111
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