International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701

__________________________________________Review Article

Alloxan Induced Diabetes: Mechanisms and Effects

Ankur Rohilla* and Shahjad Ali

Department of Pharmaceutical Sciences, Shri Gopi Chand Group of Institutions, Baghpat,

Uttar Pradesh, India.
_________________________________________________________________________________
ABSTRACT
Diabetes mellitus is a metabolic disorder constituting a major health concern today whose prevalence has
continuously increased worldwide over the past few decades. Moreover, it has been considered as an
incurable metabolic disorder affecting about 2.8% of the global population. Alloxan-induced diabetes is one of
the widely used model to induce Type I diabetes mellitus in the the experimental animals. Alloxan has been
found to be selectively toxic to pancreatic beta cells as it preferentially accumulates in the beta cells as glucose
analogues. In addition, the cytotoxic action of alloxan is mediated mainly by the generation of reactive oxygen
species (ROS). Alloxan and the product of its reduction, dialuric acid, has been noted to establish a redox cycle
with the formation of superoxide radicals, which undergo dismutation to hydrogen peroxide (H2O2) and more
highly reactive hydroxyl radicals are formed by the Fenton reaction. Further, the massive increase in cytosolic
calcium concentration ultimately causes rapid destruction of beta cells of pancreatic islets. This review
decisively highlights about the mechanism of action of alloxan in order to induce diabetes mellitus in
experimental animals.

Key Words: Diabetes Mellitus, Alloxan, Dialuric acid.

INTRODUCTION uptake mechanism of the pancreatic beta-cells.12-13

Diabetes mellitus has been considered as one of the
major health concerns all around the world today.1-2
Experimental animal models are one of the best
strategies for the understanding of pathophysiology
of any disease in order to design and develop the
drugs for its treatment.3-4 Numerous animal models
have been developed for the past few decades for
studying diabetes mellitus and testing anti-diabetic
agents that include chemical, surgical and genetic
manipulations.5-6 One of the most potent methods
to induce experimental diabetes mellitus is
chemical induction by Alloxan.6 It is a well- known
diabetogenic agent that is used to induce Type I
diabetes in experimental animals.7 Alloxan is a urea
derivative which causes selective necrosis of the -
cells of pancreatic islets. In addition, it has been
widely used to produce experimental diabetes in
animals such as rabbits, rats, mice and dogs with
different grades of disease severity by varying the
dose of alloxan used.6,8 As it has been widely
accepted that alloxan selectively destroys the
insulin-producing beta-cells found in the pancreas,
hence it is used to induce diabetes in laboratory
animals. The toxic action of alloxan on pancreatic
beta cells involve oxidation of essential sulphydryl
(-SH groups), inhibition of glucokinase enzyme,
generation of free radicals and disturbances in
intracellular calcium homeostasis.9-11 The
underlying mechanism involves the selective
uptake of the compound due to its structural
similarity to glucose as well as highly efficient
The aim of the present review is to explicate the
mechanisms involved in alloxan induced induction
of experimental diabetes mellitus. Moreover, the
biological effects produced by alloxan have also
been discussed in the review article.
ETIOLOGY
Alloxan (2,4,5,6-tetraoxypyrimidine;2,4,5,6-
pyrimidinetetrone) is an oxygenated pyrimidine
derivative which is present as alloxan hydrate in
aqueous solution. Brugnatelli originally isolated
alloxan in 1818 and the name was given by Wohler
and Liebig in 1838.14 Moreover, the compound was
discovered by von Liebig and Wohler in 1828 and
has been regarded as one of the oldest named
organic compounds that exist. The name Alloxan
emerged from the merging of two words, i.e.,
Allantoin and Oxaluric acid. Allantoin is a product
of uric acid excreted by the foetus in the allantois
and oxaluric acid has been derived from oxalic acid
and urea that is found in urine. Additionally, the
alloxan model of diabetes induction was first
described in rabbits by Dunn, Sheehan and
McLetchie in 1943.9 Alloxan was originally
prepared by the oxidation of uric acid by nitric
acid. The monohydrate is simultaneously prepared
by oxidation of barbituric acid by chromium
trioxide.15 Moreover, alloxan has been regarded as
a strong oxidizing agent that forms a hemiacetal
with its reduced reaction product; dialuric acid, in
which a carbonyl group is reduced to a hydroxyl

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 International Journal of Research in Pharmaceutical and Biomedical Sciences
group, that is called alloxantin.16 The drug has been
noted to exert its diabetogenic action when
administered parenterally, i.e., intravenously,
intraperitoneally or subcutaneously. Furthermore,
the dose of alloxan required for inducing diabetes
depends on the animal species, route of
administration and nutritional status.17 Moreover,
alloxan has been demonstrated to be non-toxic to
the human beta-cells, even in very high doses, the
reason of which may be attributed to the differing
glucose uptake mechanisms in humans and
rodents.18-19
PHASES OF DIABETES INDUCTION
Alloxan has been used to induce experimental
diabetes due to the selective destruction of the
insulin-producing pancreatic beta-islets. Alloxan
induces a multiphasic blood glucose response when
injected into to an experimental animal, which is
accompanied by corresponding inverse changes in
the plasma insulin concentration followed by
sequential ultrastructural beta cell changes
ultimately leading to necrotic cell death. The first
phase that comes into view within the first minutes
after alloxan injection is transient hypoglycemic
phase that lasts maximally for 30 minutes.12,20 This
little hypoglycemic response has been noted to be
the result of a transient stimulation of insulin
secretion that was confirmed by an increase of the
plasma insulin concentration.21 The underlying
mechanism of this transient hyperinsulinemia may
be attributed to a temporary increase in ATP
availability due to inhibition of glucose
phosphorylation through glucokinase inhibition.
The 2nd phase appearing one hour after
administration of alloxan leads to rise in blood
glucose concentration. Moreover, the plasma
insulin concentration has been noted to decrease at
the same time. This is the first hyperglycemic
phase after the first contact of the pancreatic beta
cells with the toxin.12,22-24 This hyperglycemic
phase lasts for 2-4 hours which is accompanied by
decreased plasma insulin concentrations. These
changes are a result of inhibition of insulin
secretion from the pancreatic beta cells that is
attributed to the induction due to their beta cell
toxicity.
The 3rd phase is again a hypoglycemic phase that
is noted 4-8 hours after the alloxan injection, which
lasts for several hours.12,23-25 The flooding of
circulation with insulin occurs as a result of the
alloxan-induced secretory granule and cell
membrane rupture resulting in severe transitional
hypoglycemia.26-27 In addition, other subcellular
organelles are also ruptured that include cisternae
of rough endoplasmic reticulum and the golgi
complex. Moreover, the outer and inner
membranes of the mitochondria loose structural
integrity in this particular phase.28-29 These changes
are irreversible and highly characteristic for a
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ISSN: 2229-3701
necrotic cell death of pancreatic islets. The last and
the 4th phase of the blood glucose response is the
final permanent diabetic hyperglycemic phase
during which complete degranulation and loss of
the integrity of the beta cells within 24-48 h after
administration of the alloxan takes place.12,22-24,29
Surprisingly, the non-beta cells and other endocrine
and non-endocrine islet cell types alongwith
extrapancreatic parenchyma remain intact,
providing the evidence of selective toxic action of
alloxan.28,30 Thus, alloxan injection has been noted
to induce an insulin-dependent type I like diabetes
syndrome and all the morphological features of
beta cell destruction are characteristic for a necrotic
cell death.12,28,31
MECHANISM OF ACTION
Alloxan-induced diabetes has been commonly
employed as an experimental model of insulin
dependent diabetes mellitus. The mechanism of
alloxan action has been thoroughly studied which
currently can be characterized quite well. Several
experimental studies have demonstrated that
alloxan evokes a sudden rise in insulin secretion in
the presence or absence of glucose which appeared
just after alloxan treatment.32-33 This particular
alloxan-induced insulin release occurs for short
duration followed by the complete suppression of
the islet response to glucose even when high
concentrations of glucose were used.21 Further, the
alloxan action in the pancreas is preceded by its
rapid uptake by pancreatic beta cells that have been
proposed to be one of the important features
determining alloxan diabetogenicity. Moreover, in
pancreatic beta cells, the reduction process occurs
in the presence of different reducing agents like
reduced glutathione (GSH), cysteine, ascorbate and
protein-bound sulfhydryl (-SH) groups.34-35 Alloxan
reacts with two -SH groups in the sugar binding
site of glucokinase resulting in the formation of the
disulfide bond and inactivation of the enzyme. As a
result of alloxan reduction, dialuric acid is formed
which is then re-oxidized back to alloxan
establishing a redox cycle for the generation of
reactive oxygen species (ROS) and superoxide
radicals.36-37 The superoxide radicals liberate ferric
ions from ferritin and reduce them to ferrous and
ferric ions.38 In addition, superoxide radicals
undergo dismutation to yield hydrogen peroxide
(H2O2) in the presence of superoxide dismutase. As
a result, highly reactive hydroxyl radicals are
formed according to the Fenton reaction in the
presence of ferrous and H2O2.
Another mechanism that has been reported is the
effect of ROS on the DNA of pancreatic islets. The
fragmentation of DNA takes place in the beta cells
exposed to alloxan that causes DNA damage,
which stimulates poly ADP-ribosylation, a process
participating in DNA repair. Antioxidants like
superoxide dismutase, catalase and the non-
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 International Journal of Research in Pharmaceutical and Biomedical Sciences
enzymatic scavengers of hydroxyl radicals have
been found to protect against alloxan toxicity. 39 In
addition, the disturbance in intracellular calcium
homeostasis has also been reported to constitute an
important step in the diabetogenic action of
alloxan. It has been noted that alloxan elevates
cytosolic free Ca2+ concentration in the beta cells of
pancreatic islets.40 The calcium influx is resulted
from the ability of alloxan to depolarize pancreatic
beta cells that further opens voltage dependent
calcium channels and enhances calcium entry into
pancreatic cells. The increased concentration of
Ca2+ ion further contributes to supraphysiological
insulin release that alongwith ROS has been noted
to ultimately cause damage of beta cells of
pancreatic islets.6,10,12
BIOLOGICAL EFFECTS
Alloxan is a hydrophilic and unstable chemical
compound which has similar shape as that of
glucose, which is responsible for its selective
uptake and accumulation by the pancreatic beta
cell.41 Similarity in the shape allows it to transports
into the cytosol by the glucose transporter
(GLUT2) in the plasma membrane of beta cell.41-42
Another biological effect of alloxan has been
attributed to the thiol group reactivity that allows
selective inhibition of glucose-induced insulin
secretion through inhibition of glucokinase. This
inhibition of glucose-induced insulin secretion has
been regarded as the major pathophysiological
effect of alloxan, which results from the thiol group
reactivity of alloxan. The thiol groups of
glucokinase, the glucose phosphorylating enzyme,
are particularly sensitive to oxidation by alloxan.43
Glucokinase inhibition reduces glucose oxidation
and ATP generation that further suppresses
glucose-induced insulin secretion.43-44 Moreover,
the insulin biosynthesis is also inhibited by alloxan
through the same mechanism. Alloxan inhibits
many cellular functions at higher concentrations
such as the ability to oxidize thiol groups of many
functionally important enzymes like hexokinase,
phosphofructokinase, calmodulin-dependent
protein kinase, aconitase and other proteins.12,45
Hence, it is evident that the inhibition of glucose-
induced insulin secretion by alloxan is the result of
the thiol reactivity of the glucokinase. Another
biological effect of alloxan is pancreatic beta cell
toxicity and diabetogenicity that may be attributed
to alloxan-induced redox cycling and ROS
generation. The mechanism underlying the
cytotoxic action of alloxan to insulin-producing
cells may be ascribed as the reduction by
interaction with intracellular thiols such as
glutathione.46 The resultant formation of cytotoxic
ROS is the result of a cyclic reaction between
alloxan and its reduction product, dialuric acid,
which by autoxidation generates superoxide
radicals, hydroxyl radicals and H2O 2.36,47 Induction
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ISSN: 2229-3701
of diabetes in the laboratory animals by alloxan
injection is the result of selective uptake of alloxan
via GLUT2 into a pancreatic beta cell.42,48 The
effective prevention of redox cycling and
generation of ROS can prevent pancreatic beta cell
death and counteract the development of alloxan
diabetes in vivo.12,46,49-50 Hence, it can be
summarized that the alloxan-induced pancreatic
beta cell toxicity and the resultant diabetogenicity
is due to the redox cycling and the toxic ROS
generation in combination with the hydrophilicity
and the glucose similarity of the molecular shape of
alloxan.
CONCLUSION
The chemical induction of diabetes appears to be
the most popularly used procedure in inducing
diabetes mellitus in experimental animals. The
foremost drug-induced diabetic model is the
alloxan diabetes that is capable of inducing type I
diabetes mellitus in experimental animals. The
surgical and genetic methods of diabetes induction
are associated with a high percentage of animal
morbidity and mortality. Hence, alloxan induced
diabetes model appears to be the most reliable and
easily reproducible method of inducing diabetes
mellitus in experimental animals. So, efforts should
be made towards upbringing and uplifting the
model of alloxan induced diabetes mellitus in the
experimental animals.
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