Achievements in the Life Sciences 9 (2015) 51–56

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Achievements in the Life Sciences

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Glucose-6-phosphate Dehydrogenase Activity During

Nʹ-nitrosodiethylamine-induced Hepatic Damage
Devoshree Mukherjee, Riaz Ahmad ⁎
Biochemical and Clinical Genetics Laboratory, Section of Genetics, Department of Zoology, Aligarh Muslim University, Aligarh, 202002 U.P, India

a r t i c l e i n f o a b s t r a c t

Available online 17 June 2015 Glucose-6-phosphate dehydrogenase (G6PD), a key regulatory enzyme of the pentose phosphate
pathway, catalyses the first rate-limiting reaction to produce ribose-5-phosphate for nucleic acid
Keywords: synthesis and NADPH to use in reductive biosynthesis. The available studies indicate an antioxi-
Antioxidant dant role for G6PD and variation in its levels as a result of cellular insult. In this study, the activity
Glucose-6-phosphate dehydrogenase (G6PD) of G6PD was monitored during Nʹ-nitrosodiethylamine (NDEA)-induced hepatic damage in
Hepatic injury Wistar rats. NDEA generates hepatotoxicity and possesses mutagenic effects. To induce hepatic
Liver function damage, NDEA was administered at doses of 100 mg kg−1 body weight week−1 (i.p.) for
Nʹ-nitrosodiethylamine (NDEA) 14 days. The animals of the control and treated groups were sacrificed each week. The extent of
liver damage was ensured by LFT biomarkers, such as transaminases, ALP, bilirubin and the
hepato-somatic index (HSI) along with histopathological observations of H&E and Masson's
trichrome stained liver specimens. The results of the present study show that at the selected
doses, NDEA significantly elevates LFT proteins and bilirubin and damages the lobular architecture
in a dose-dependent manner. Software analysis of zymograms indicates maximum activity of the
hepatic G6PD levels in day-14 NDEA-treated animals. Our spectrophotometry data further sup-
port the above findings on hepatic G6PD levels and demonstrate an approximately 1.63× and
1.66× fold increase in day-7 and day-14 NDEA intoxicated animals (P b 0.05). It is concluded
that elevation in the G6PD activity is apparently the consequence of NDEA-induced intoxication
or oxidative stress, leading to hepatic damage to provide sufficient NADPH for microsomal detox-
ification and ribose-5-phosphate for DNA synthesis and repair, respectively, to maintain the cellu-
lar redox status.
© 2015 The Authors. Hosting by Elsevier B.V. on behalf of Far Eastern Federal University. This is an
open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Glucose-6-phosphate dehydrogenase (G6PD), also known as β-D-glucose-6-phosphate, is a housekeeping enzyme (EC 1.1.1.49) of
the pentose phosphate pathway (PPP) and is highly important in a variety of cellular processes (Ibrahim et al., 2014). In humans, the
monomer of G6PD contains 514 amino acids with a molecular weight of 59 kDa (Tian et al., 1998). This key regulatory enzyme, G6PD,
catalyses the first rate limiting reaction in the PPP, consequently producing ribose-5-phosphate for nucleic acid synthesis and NADPH

☆ Source of Funding: Council of Science and Technology (CST), U.P, India.

⁎ Corresponding author. Tel.: +91 571 2700920/3445.
E-mail address: riazzool@rediffmail.com (R. Ahmad).
Peer review under responsibility of Far Eastern Federal University.

http://dx.doi.org/10.1016/j.als.2015.05.007
2078-1520/© 2015 The Authors. Hosting by Elsevier B.V. on behalf of Far Eastern Federal University. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
52 D. Mukherjee, R. Ahmad / Achievements in the Life Sciences 9 (2015) 51–56

for reductive biosynthesis (Shannon et al., 2000). Primarily, there are two types of regulation of the PPP: First in the liver, concentra-
tions of both G6PD and 6-phosphogluconate dehydrogenase change significantly with the nutritional state of the animal (Glock and
Mc Lean, 1955; Tepperman and Tepperman, 1958; Fitch et al., 1959; Rudack et al., 1971a,b). Second, the NADPH/NADP ratio changes
to regulate the G6PD levels in vitro (Holten et al., 1976), but not in vivo. It is evident that G6PD plays major roles in NADPH production
for the biosynthesis of the sugar moiety of nucleic acids and provides defence against oxidative stress (Pandolfi et al., 1995, Biagiotti
et al., 2000; Köhler and Noorden, 2003).
Because the liver is reported to contain the highest amount of G6PD activity (Ibrahim et al., 2014), any injury to the liver will even-
tually cause altered levels of the enzyme in the cells. Various studies have shown that enzyme levels may be a potential parameter to
assess hepatic damage because they are cytoplasmic and are subsequently released into the blood circulation following cellular dam-
age (Ahmad and Ahmad, 2012; Rezai et al., 2013). A number of studies have been performed to monitor the G6PD levels in liver cells
and reported the modulation of hepatic G6PD in response to external stimuli, such as growth factors, hormones, nutrients and oxida-
tive stress (Watanabe and Taketa, 1973; Katsurada et al., 1989; Kletzein et al., 1994; Sanz et al., 1997; Abd Ellah et al., 2004; Banu et al.,
2009). Surprisingly, there is an apparent disagreement among the above reports regarding the changes in the G6PD levels in the liver
in vivo. Few reports have correlated the G6PD levels with the antioxidant defence system of the animals and have suggested that there
is an enhanced activity of the enzyme during moderate or extreme toxic states (Wanatabe and Taketa, 1972; Taniguchi et al., 1999).
Therefore, we performed studies to monitor the levels of G6PD during Nʹ-nitrosodiethylamine (NDEA) induced hepatic damage in
male albino rats. Nʹ-Nitrosodiethylamine is a potential mutagen and is used in studies to induce hepatocarcinoma in laboratory
animals to investigate probable molecular mechanisms as well as potential drug targets of disease.

Material and Methods

Chemicals

Nʹ-nitrosodiethylamine (NDEA), acrylamide, bis-acrylamide, Tris, glycine, ammonium per sulphate (APS), N, N, N′, N′-
tetramethyletylenediamine (TEMED) and glucose-6-phosphate (disodium salt hydrated) were purchased from Sigma-Aldrich.
Bovine serum albumin (BSA), haematoxylin and eosin (H&E) stains, phenazine methosulphate (PMS), nicotinamide adenine di-
nucleotide phosphate-monosodium salt (NADP) and nitroblue tetrazolium (NBT) were purchased from SRL, India. All other
chemicals, reagents and salts used in this study were of AR grade.

Animal Care

Six to eight week old adult male albino rats (Rattus norvegicus) of the Wistar strain weighing 160 ± 10 g were used in the present
study. Experimental animals were housed under proper hygienic conditions in polycarbonate cages with a wire mesh top and a bed of
husk in an animal house facility with a 12 h light:dark cycle. Before the start of the experiment, the animals were acclimatized for one
week with regularly feeding of sterilized diet and water. Care of the experimental animals was in accordance with the guidelines of the
Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India.

Experimental Design

Animals were categorized into two groups comprising ten rats each. Group-I animals (control) were the saline control and re-
ceived normal saline. Group-II rats (NDEA treated) were given only Nʹ-nitrosodiethylamine (NDEA) at doses of 100 mg kg−1 body
weight (i.p.). The injections were administered weekly for two successive weeks on day-7 and day-14. Five animals from each
group were anaesthetized and sacrificed at the end of each week. The body and liver weights of the animals were monitored through-
out the experiment.

Liver Homogenate Preparation

Quickly excised liver samples were processed for homogenate preparation in ice cold 50 mM Tris–HCl buffer (pH, 7.5) according a
previously described procedure (Ahmad and Ahmad, 2014). In addition to the homogenates, a piece of liver tissue from each animal
was also stored in formalin (10%) for histopathological studies.

Total Protein Estimation

The protein quantity of the liver homogenates was estimated using the method of Lowry et al. (1951). Bovine serum albumin was
used as a standard, and the absorbance of the incubated samples was read at 660 nm on UV–VIS spectrophotometer (Bio Sync, India).

Histopathological Evaluation

Formalin fixed liver specimens were processed to prepare 5-μm thick liver sections as per the protocol of Ahmad et al. (2009).
Staining of the liver sections was performed with haematoxylin–eosin (H&E) and Masson's trichrome (MT). The best slides were
photographed under a Nikon microscope with an LCD attachment (Model: 80i).
 D. Mukherjee, R. Ahmad / Achievements in the Life Sciences 9 (2015) 51–56 53

Polyacrylamide Gel Electrophoresis

Non-denaturing vertical slab polyacrylamide gel electrophoresis (PAGE) was performed (80 × 100 × 1 mm) according to the pro-
tocol of Ahmad and Hasnain (2005). In-gel visualization of the G6PD enzyme and subsequent processing was performed using
established protocols (Shaw and Prasad, 1970; Ahmad and Hasnain, 2006). Enzyme bands were developed in the dark by soaking
the gel in the reaction mixture overnight at 25 °C. The replica gels were subsequently stained with CBBR-250 to ensure the quality
of the runs. Zymograms were documented under visible range illumination using a SONY Cyber shot digital camera
(14.1 Megapixel, 4× optical zoom). G6PD activity was also monitored for one hour spectrophotometrically at 540 nm. The reaction
mixture (2 ml) was prepared by adding constant volumes of liver homogenates (25 μl) to the following: NADP (1.08 × 10−4 M),
NBT (6 × 10−4 M), PMS (17 × 10−3 mM), Na2G6.PO4.H2O (16 × 10−4 M) and Tris–HCl (125 mM).

Assessment of the Zymograms

Gel-Pro (Media Cybernetics, USA) and Scion Imaging (Scion Corporation: Beta release-4.0) software programs were used for
quantitative estimation of the enzyme activity in the zymograms.

Statistical Analysis

The differences between the control and treatment groups were evaluated by Student's t-test. The values were considered
statistically significant at P b 0.05.

Results and Discussion

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme that catalyses the first step of the pentose phosphate pathway to
produce NADPH and ribose-5-phosphate (Ulusu and Tandogan, 2006). This regulatory house-keeping enzyme is localized to the cy-
tosol and mitochondria and is an antioxidant that preserves the cytosolic redox status by producing the cellular reductant, NADPH
(Jain et al., 2003; Frederiks et al., 2003; Stanton, 2012). Because the activity of G6PD is highest in the liver, studies on the changes
in the levels of this enzyme during the progression of N′-nitrosodiethylamine (NDEA)-induced hepatic damage may be of significance.
Because of the action of the enzyme cytochrome P450, NDEA becomes metabolically active to produce reactive oxygen species (ROS)
that result in oxidative stress and cellular insult, consequently leading to hepatic injury (Nitha et al., 2014).
The liver is structurally heterogeneous and therefore executes various important functions, such as detoxification, hydrolysis of
fats; production of urea and cholesterol; storage of vitamins, glycogen and minerals; processing of nutrients; and filtration of

A B C

D E F

Fig. 1. Photomicrographs of liver specimens stained with H&E (A–C) and Masson' strichrome (D–F) during NDEA-induced liver injury in rats. (A & D) Control liver with a
normal lobular architecture. (B & E) Day-7 NDEA-treated liver specimens. (C & F) Day-14 NDEA-treated liver specimens.
54 D. Mukherjee, R. Ahmad / Achievements in the Life Sciences 9 (2015) 51–56

Fig. 2. 3-D surface view of the G6PD zymograms analysed using the Scion Imaging software program. Inset shows zymograms of the corresponding lanes of hepatic
G6PD developed by substrate staining. (Symbols − & + shows the direction of the electrophoretic run and the arrow indicates the activity zone of G6PD).

blood. Any insult by chemicals, viruses or parasites causes hepatic injury that directs the transdifferentiation of hepatic stellate cells
(HSCs) into myofibroblast-like cells. These activated HSCs are characterized by the marked loss of lipid droplets as well as increased
cell proliferation to induce the disproportionate synthesis of extra cellular matrix (ECM) and consequent expression of α-smooth
muscle actin (α-SMA) (Ahmad and Ahmad, 2012). Prolonged or repeated exposure to chemicals, such as NDEA or NDMA, produces
hepatocarcinoma, which is also characterized by the deregulation of the normal healing process and significant structural and func-
tional changes in the liver (Farazi and De Pinho, 2006; Wills et al., 2006; Zhang et al., 2012; Linza et al., 2013). We used NDEA to induce
hepatic damage in experimental animals by administering a single dose of 100 mg kg−1 body weight wk−1 for two weeks. The dam-
age to the liver was assessed using routine LFT parameters, such as ALP, AST, ALT, γGT, and bilirubin; the hepatosomatic index (HSI);
and the histopathology of liver sections using H&E and Masson's trichrome staining.
Our results based on the biochemical parameters and HSI are consistent with previous reports in which NDEA or NDMA shows a
significant increase in LFT proteins and altered HSI due to hepatic intoxication (Kumar and Kuttan, 2000; George et al., 2001, 2004;
Rezai et al., 2013; Ahmad et al., 2009, 2011, 2014; Ahmad and Ahmad, 2012, 2014; Latha and Latha, 2014). These data clearly demon-
strate that NDEA damages the lobular architecture in a dose-dependent manner. The photomicrograph record of control liver sections
shows a normal lobular architecture with central veins and radiating hepatic cords (Fig. 1 A and D). Noticeable haemorrhage, dilated
central veins, and lymphocyte infiltration were the features of day-7 NDEA treated specimens, whereas day-14 treated animal spec-
imens showed intense neutrophil infiltration, severe haemorrhage and amassing of collagen (Fig. 1 B–C and E–F). The chronic toxicity
due to NDEA is attributed to its conversion into an active ethyl radical metabolite by cytochrome P450 (CYP 2E1), which reacts with
DNA to cause mutations and carcinogenesis (Anis et al., 2001). Therefore, it is proposed that NDEA, similar to other nitroso com-
pounds, such as NDMA, disturbs the redox potential of the cell and may use a similar molecular mechanism to induce hepatic damage
in rats.
The results of this study further show that the levels of G6PD significantly vary between the control and NDEA treated groups.
Upon increasing the contrast of the gels, the software analysis demonstrates the highest levels of G6PD in the day-14 NDEA-treated
group, which received a dose of 100 mg kg−1 b.wt. week−1 for two weeks (Fig. 2). Our spectrophotometry data also support the
above findings based on the software analysis of G6PD zymograms and show an approximately 1.63 × and 1.66× fold increase in
the day-7 and day-14 NDEA-treated groups, respectively. A few studies reported interesting findings in which there is apparent con-
troversy for the levels of G6PD under conditions of oxidative stress (Sanz et al., 1997; Abd Ellah et al., 2004; Banu et al., 2009;
Taniguchi et al., 1999). It has been shown that in AFB1 administered rats (state of oxidative stress), the levels of glutathione metab-
olizing enzymes, such as G6PD, decrease as a result of the deficient flow of G-6-P through the hexose monophosphate shunt and de-
creased supply of reduced NADPH for the conversion of GSSG to GSH, thereby causing a switch in the NADP+/NADPH ratio in favour of
NADP+ (Banu et al., 2009).
The literature suggests that under proliferative conditions, G6PD gene expression increases both in foetal hepatocytes (Molero
et al., 1994) and in cultured mature liver cells (Stanton et al., 1991). However, the glucose level in rat livers was decreased during
the development of liver cirrhosis if routed for fatty acid and pyruvate synthesis. The glucose channelled to pentose phosphates
and reducing equivalents supports DNA synthesis, DNA repair and detoxification reactions (Sanz et al., 1997). Our results indicate a
significant quantitative differential increase (Fig. 3) in the activity of G6PD during NDEA treatment, which may be a consequence
of one or both roles of the enzyme: (1) G6PD is required to provide NADPH for microsomal detoxifying mechanisms and/or
(2) G6PD provides ribose-5-phosphate for DNA synthesis and repair. Further studies are warranted to evaluate the molecular mech-
anism of the changes in the G6PD levels under oxidative assault and xenobiotics biotransformation in the damaged liver.
 D. Mukherjee, R. Ahmad / Achievements in the Life Sciences 9 (2015) 51–56 55

0.25
* *

0.2

Optical density
0.15

0.1

0.05

0
CONTROL DAY-7 DAY-14

Fig. 3. The bar diagram illustrates the spectrophotometry-based quantitative differences in the levels of G6PD between control and treated groups during NDEA-in-
duced hepatic damage (*P b 0.05).

Conflict of Interest

None.

Acknowledgements

Financial assistance from Council of Science & Technology [CST/SERPD/D-368], UP to RA is gratefully acknowledged for performing
this work. The authors sincerely thank the Chairman, Department of Zoology for the necessary facilities.

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